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ItemDevelopment of an ‘equinised’ antibody to block the equine insulin-like growth factor-1 receptor (IGF-1R):A potential immunotherapy for endocrinopathic equine laminitisVathsangam, Niveditha ( 2019)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.
ItemResponse of subchondral bone and articular cartilage to joint surface incongruity: an equine osteochondral defect modelThomas, Megan Elizabeth ( 2019)Many diseases and injuries of synovial joints result in a focal loss of articular surface congruity. These conditions are known to lead to degenerative changes within the affected joint and subsequent osteoarthritis (OA). Incongruity due to loss of articular cartilage is also a feature of OA itself. However there is little work specifically investigating how the tissues of a synovial joint respond to loss of congruity. The aim of this study was to observe how articular cartilage and subchondral bone (SCB) respond to a focal loss of surface congruity using an equine model. It was hypothesised that the degree of remodelling and bone volume of SCB is dependent on local loading of the overlying joint surface and that intact cartilage would respond to changes in local surface pressure in a way that attempts to restore congruity. An osteochondral defect was created in one surface of the midcarpal joint of six adult horses. Two weeks post-operatively the animals began an eight-week treadmill training program. Following this, osteochondral samples were collected from the site of the defect and immediately adjacent to it in the radial carpal bone (Cr), and from the unloaded site directly opposing the defect and a loaded site immediately adjacent to this in the third carpal bone (C3). Control samples were collected from equivalent sites in the sham-operated contralateral limb and from un-operated, untrained control animals. Undemineralised samples were imaged with microCT and backscattered scanning electron microscopy (BSEM) and stained with Masson’s Goldner trichrome to measure cartilage thickness and osteoid. Decalcified cryosections were stained for TRAP to identify osteoclasts. Cartilage was examined with routine histology and differential interference contrast microscopy. In SCB immediately below the Cr lesion in treated joints bone volume fraction (BV/TV) was 32% lower and bone formation and erosion were markedly increased, whereas in the deeper bone formation was increased, erosion activity became less prominent and BV/TV did not change. In the unloaded region of C3 opposing the defect in treated joints the hyaline cartilage (HC) was 50% thicker, while SCB displayed a mild increase in remodelling activity with a net decrease in BV/TV of 5.3%. In the loaded region adjacent to the lesion in Cr HC was 30% thinner in treated joints while there was no change in SCB parameters. At the loaded site of C3 the HC thickness was unchanged while the calcified cartilage in treated joints was 40% thicker than in controls. In the underlying SCB in treated joints there was a mild reduction in remodelling activity and unchanged BV/TV. In this equine osteochondral defect model the articular cartilage and SCB of adult synovial joints responded in a focal manner to changes in joint surface congruity. Local unloading due to a loss of surface contact resulted in thickened HC and, in the underlying SCB, increased remodelling activity and loss of bone volume. Focally increased loading resulted in thickened calcified cartilage and decreased SCB bone remodelling activity. These focal responses may play a role in normal joint homeostasis and in the pathogenesis of OA.