Development of a mAb Binding a Conformational Epitope on Human and Rodent Insulin Receptor Using a Naive Antibody Yeast Display Library

摘要

The insulin receptor (IR) is a key player in the homeostasis of blood glucose via its constant response to the shifting insulin levels in the blood. Antibody based assays are often used to study the activation of the IR upon development of new or improved insulin analogues. However, lack of antibodies binding the rodent versions of the IR is strongly hampering the translational aspects of the rodent diabetes disease models. Here, we present an anti-IR antibody developed using the Adimab yeast display antibody platform. Due to the naive nature of this library we were able to develop an anti-IR antibody binding a conformational epitope conserved between human, mouse, rat and pig. The antibody binds the alpha-subunit of the IR with nM affinity and does not interfere with the binding of insulin to its receptor. Importantly, the antibody can be used to capture IR from tissue derived samples in a sandwich ELISA based assay where we measure activation of the receptor using phosphospecific mAbs directed against the intracellular tyrosine kinase domain of the beta-subunit. In addition, immunoprecipitation and flow cytometry studies using HCT116 wt and HCT116 IR-ko cells show high specificity of the antibody and no cross-reactivity to the closely related insulin-like growth factor 1 receptor. In conclusion, we have developed a tool antibody which enables detailed analyses of the in vitro and in vivo activation status of the insulin receptor after dosing of insulin or insulin analogues in mouse and rat models which are crucial for the development of improved insulin based diabetes therapies.