Development of an ‘equinised’ antibody to block the equine insulin-like growth factor-1 receptor (IGF-1R):A potential immunotherapy for endocrinopathic equine laminitis

© 2019 Niveditha VathsangamLaminitis is a common and debilitating disease of horses’ feet that results in lameness, and in severe cases, may require euthanasia. Insulin toxicity appears to have a key role in the pathogenesis of the disease, and research has indicated that hyperinsulinemia may cause lamellar damage through excessive stimulation of insulin-like growth factor-1 receptors (IGF-1R). Inhibition of dysfunctional receptor activation has been the target of therapeutic (anti-cancer) monoclonal antibody (mAb) treatments for humans. Although common in human medicine, only a handful of therapeutic mAbs are under development for diseases in companion animals (dogs and cats), and none are available for horses. This approach demonstrated promise as a potential treatment of endocrinopathic laminitis. Therefore, the aim of this project was to convert a clinically-tested human IGF-1R-neutralizing mAb with high-affinity for the equine IGF-1R, into a horse-specific mAb while avoiding immuno-intolerance when administered in vivo. Development of the equine antibody was challenging due to the limited availability of immunoglobulin sequences (EquCab 2.0 database), in addition to the predominance of circulating light chain isotypes (Lambda), unlike the case in humans. Based on structural isotype characteristics, high binding affinity for the IGF-1R and minimal binding to insulin receptors, the parental antibody (IMC-A12) was chosen for chimerisation studies. Chimeric versions of this antibody were generated by combining the VH (Variable Heavy) and VL (Variable Light) domains of the human antibody to equine constant Fc regions. The mAbs were further engineered to abolish effector functions (activation of complement or the immune system) while retaining receptor binding functionality. Chimeric versions of IMC-A12 expressed at similar levels to the human parental mAb and bound to the equine receptor at similar affinities to human IMC-A12. The full ‘equinisation’ of chimeric mAbs was carried out using a novel speciation process known as “PETisation” and was adopted to generate thirteen candidate equine mAbs. Amongst three of the strongest equine mAb candidates (mAb8, mAb10, and mAb11), mAb11 displayed the highest expression yield purity (98 percent) and strong binding characteristics (KD of 0.2-0.3 nM and an EC50 of 127 pM, by BIAcore and ELISA, respectively). Cell proliferation assays demonstrated that mAb11 significantly inhibited the concentration-dependent proliferative effect of insulin in cultured lamellar cells (p