Purpose For AMG 317, a fully human monoclonal antibody to interleukin receptor IL-4Rα, we developed a population pharmacokinetic (PK) model by fitting data from four early phase clinical trials of intravenous and subcutaneous (SC) routes simultaneously, investigated important PK covariates, and explored the relationship between exposure and IgE response. Methods Data for 294 subjects and 2183 AMG 317 plasma concentrations from three Phase 1 and 1 Phase 2 studies were analyzed by nonlinear mixed effects modeling using first-order conditional estimation with interaction. The relationship of IgE response with post hoc estimates of exposure generated from the final PK model was explored based on data from asthmatic patients.Results The best structural model was a two-compartment quasi-steady-state target-mediated drug disposition model with linear and non-linear clearances. For a typical 80-kg, 40-year subject, linear clearance was 35.0 mL/hr, central and peripheral volumes of distribution were 1.78 and 5.03 L, respectively, and SC bioavailability was 24.3%. Body weight was an important covariate on linear clearance and central volume; age influenced absorption rate. A significant treatment effect was observable between the cumulative AUC and IgE response measured.Conclusion The population PK model adequately described AMG 317 PK from IV and SC routes over a 60-fold range of doses with two dosing strengths across multiple studies covering healthy volunteers and patients with mild to severe asthma. IgE response across a range of doses and over the sampling time points was found to be related to cumulative AMG 317 exposure.

Protein L-isoaspartyl methyltransferase (PIMT) has been implicated in the repair or metabolism of proteins containing atypical L-isoaspartyl peptide bonds. The repair hypothesis is supported by previous studies demonstrating in vitro repair of isoaspartyl peptides via formation of a succinimide intermediate. Utilization of this mechanism in vivo predicts that PIMT modification sites should exhibit significant racemization as a side reaction to the main repair pathway. We therefore studied the D/L ratio of aspartic acid at specific sites in histone H2B, a known target of PIMT in vivo. Using H2B from canine brain, we found that Asp 25 (the major PIMT target site in H2B) was significantly racemized, exhibiting D/L ratios as high as 0.12, whereas Asp 51 , a comparison site, exhibited negligible racemization (D/L < 0.01). Racemization of Asp 25 was independent of animal age over the range of 2-15 years. Using H2B from 2-3-week mouse brain, we found a similar D/L ratio (0.14) at Asp 25 in wild type mice, but substantially less racemization (D/L ‫؍‬ 0.035) at Asp 25 in PIMT-deficient mice. These findings suggest that PIMT functions in the repair, rather than the metabolic turnover, of isoaspartyl proteins in vivo. Because PIMT has numerous substrates in cells, these findings also suggest that D-aspartate may be more common in cellular proteins than hitherto imagined and that its occurrence, in some proteins at least, is independent of animal age. Protein L-isoaspartyl methyltransferase (PIMT)1 selectively methylates atypical L-isoaspartyl sites in proteins (1-4). The formation of isoaspartate (see Fig. 1) is a common occurrence at certain Asn-Xaa and Asp-Xaa sites in proteins, especially when such sites fall in a flexible domain (5-7). This mechanism explains the propensity and sequence dependence of spontaneous protein deamidation at asparagine residues under mild conditions (8). The formation of isoaspartyl sites at aspartyl residues is also quite common (5, 7, 9) but has not been well appreciated because isomerization of Asp-Xaa linkages alters neither the mass nor the charge (at neutral pH) of a polypeptide.The formation of isoaspartate in proteins is generally viewed as a deleterious event associated with protein aging (10). It has been suggested that PIMT-dependent methylation of isoaspartyl sites serves to either repair the damaged sites or to tag the damaged proteins for degradation (11,12). A repair function is supported by in vitro studies using PIMT and defined polypeptide substrates (13-15). These studies indicate the pathway shown in Fig. 2 whereby methylation serves to activate the atypical ␣-carboxyl of the isoaspartyl site. This activation promotes rapid, spontaneous demethylation via formation of the same succinimide intermediate that occurs during isoaspartate formation. Each cycle of methylation/demethylation converts ϳ15-30% of the isoaspartyl linkage to a normal aspartyl linkage; however, after multiple cycles of methylation/demethylation, a majority of isoaspartyl sites are converted to normal asp...

Abstract. The availability of highly sensitive immunoassays enables the detection of antidrug antibody (ADA) responses of various concentrations and affinities. The analysis of the impact of antibody status on drug pharmacokinetics (PK) is confounded by the presence of low-affinity or low-concentration antibody responses within the dataset. In a phase 2 clinical trial, a large proportion of subjects (45%) developed ADA following weekly dosing with AMG 317, a fully human monoclonal antibody therapeutic. The antibody responses displayed a wide range of relative concentrations (30 ng/mL to >13 μg/mL) and peaked at various times during the study. To evaluate the impact of immunogenicity on PK, AMG 317 concentration data were analyzed following stratification by dose group, time point, antibody status (positive or negative), and antibody level (relative concentration). With dose group as a stratifying variable, a moderate reduction in AMG 317 levels (<50%) was observed in antibody-positive subjects when compared to antibody-negative subjects, but the difference was not statistically significant in all dose groups. The most significant reduction in AMG 317 levels was revealed when antibody data was stratified by both time point and antibody level. In general, high ADA concentrations (>500 ng/mL) and later time points (week 12) were associated with significantly (up to 97%) lower trough AMG 317 concentrations. The use of quasi-quantitative antibody data and appropriate statistical methods was critical for the most comprehensive evaluation of the impact of immunogenicity on PK.

Immunogenicity assays incorporating epitope determination may provide additional information about the characteristics of induced antitherapeutic antibodies, including the magnitude and timing of the various types of antibody responses.

Background: Titer methods are commonly used to characterize the magnitude of an antidrug antibody response. Assay S/N is an appealing alternative, but the circumstances under which use of signal-to-noise (S/N) is appropriate have not been well defined. Results: We validated both titer and S/N-based methods for several therapeutics. S/N correlated strongly with titer both in aggregate and when examined on a per subject basis. Analysis of impact of antibody magnitude on pharmacokinetics yielded the same result using either method. Each assay demonstrated excellent precision, good linearity, and adequate drug tolerance. Conclusion: Under these circumstances, assay S/N is a valid alternative to titer for assessment of the magnitude of an antidrug antibody response.