Development of an ‘equinised’ antibody to block the equine insulin-like growth factor-1 receptor (IGF-1R):A potential immunotherapy for endocrinopathic equine laminitis
AffiliationVeterinary and Agricultural Sciences Collected Works
Document TypePhD thesis
Access StatusThis item is embargoed and will be available on 2022-02-07.
© 2019 Niveditha Vathsangam
Laminitis 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<0.01). Further immunoassay assessment of lamellar growth factor signalling under hyperinsulinemic conditions demonstrated a significant reduction (p less than 0.05) in the phosphorylation of Ribosomal protein s6 (RPS6), Ras/extracellular signal-regulated kinase (ERK (1/2)) and Protein kinase B (AKT) after 24 h, in the presence of mAb11. As a novel approach, mAb therapies are an emerging option in the veterinary market. As far as I am aware, the fully-equine antibody mAb11, with its high-affinity for the target receptor (IGF-1R), is the first of its kind in the world and could open avenues for research on other equine indications and treatments. While promising results have been observed in vitro, further characterisation of mAb11 is required to establish its viability as a treatment for equine laminitis. A successful immunotherapy for equine laminitis would reduce the economic and welfare costs of this destructive disease while advancing our understanding of antibody therapeutics in the horse.
KeywordsEquine; Antibody Therapy; Laminitis; Veterinary Immunology; PETisation; Equine antibody; Monoclonal antibody therapy; Insulin-like growth factor-1 receptor
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