Poster #055: Use of an In Vitro Model of Human Immunity to Evaluate the Innate Immune Profile of Originator and Biological Copies of Insulin Glargine

摘要

Statement of Purpose, Innovation or Hypothesis: Because the manufacture of insulin analogs require sophisticated production procedures, it is possible that originator and biological copies of insulin glargines might have varied immunogenicity profiles based on differences in the structure (protein aggregation, de-naturation), purity (contamination with endotoxins or others) and/or other characteristics of these products. Previously, it had been shown 30 nM/L of some lots of glargine copies induced a significant secretion of IL-8 and IL-6 in an in vitro model of human immunity, termed the MIMIC~? system. The fact that numerous lots of the originator glargine displayed no effect shows the robustness of the production process of this product. The aim of this study was to dissect the biological mechanism(s) mediating the observed effect. Description of Methods and Materials: We used an in vitro model of immunity, termed the MIMIC system, to study antigen-presenting cell activation, cy-tokine secretion and insulin-receptor signaling pathways induced by a range of doses of originator and biological copies of insulin glargine. We also examined the purity of the compounds using a toll-like receptor (TLR) reporter cell line and native PAGE and LCMS analyses. Data and Results: In a series of studies aimed at addressing the mechanisms leading to differential IL-6 and IL-8 by various insulin glargines in an in vitro human immune model, we report the following observations: 1) no TLR/NON-like receptor (NLR) activators were detected in any glargine formulations, suggesting the inflammatory response observed in vitro was not mediated by bacterial contaminants in the preparations; 2) no evidence of product aggregates was detected using gel electrophoresis, though the presence of some high molecular-weight polypeptides in the im-munostimulatory preparations, as detected by LCMS, argues for the presence of insulin dimers or other contaminants in the formulations; and 3) using various inhibitors of insulin signaling pathways, we found IL-8 secretion was driven by the insulin receptor (IR) and mitogen-activated protein kinase (MAPK) (MEK) pathways. Following up on the latter point, we found preservative-free insulins induced a dose-dependent secretion of cytokines and, importantly, the removal of insulin from the immunostimulatory formulations significantly reduced their capacity to induce IL-8 secretion in the in vitro assays. Interpretation, Conclusion or Significance: These data suggest that insulin is the driver of the observed in vitro innate response, providing preliminary evidence that changes in the biochemical composition (dimers, impurities) of the insulin glargine copy formulations might be responsible for their immunostimulatory potential.