Elezanumab, a clinical stage human monoclonal antibody that selectively targets repulsive guidance molecule A to promote neuroregeneration and neuroprotection in neuronal injury and demyelination models.

Repulsive guidance molecule A (RGMa) is a potent inhibitor of axonal growth and a regulator of neuronal cell death. It is up-regulated following neuronal injury and accumulates in chronic neurodegenerative diseases. Neutralizing RGMa has the potential to promote neuroregeneration and neuroprotection. Previously we reported that a rat anti-N terminal RGMa (N-RGMa) antibody r5F9 and its humanized version h5F9 (ABT-207) promote neuroprotection and neuroregeneration in preclinical neurodegenerative disease models. However, due to its cross-reactivity to RGMc/hemojuvelin, ABT-207 causes iron accumulation in vivo, which could present a safety liability. Here we report the generation and characterization of a novel RGMa-selective anti-N-RGMa antibody elezanumab, which is currently under Phase 2 clinical evaluation in multiple disease indications. Elezanumab, a human monoclonal antibody generated by in vitro PROfusion mRNA display technology, competes with ABT-207 in binding to N-RGMa but lacks RGMc cross-reactivity with no impact on iron metabolism. It neutralizes repulsive activity of soluble RGMa in vitro and blocks membrane RGMa mediated BMP signaling. In the optic nerve crush and optic neuritis models, elezanumab promotes axonal regeneration and prevents retinal nerve fiber layer degeneration. In the spinal targeted experimental autoimmune encephalomyelitis (EAE) model, elezanumab promotes axonal regeneration and remyelination, decreases inflammatory lesion area and improves functional recovery. Finally, in the mouse cuprizone model, elezanumab reduces demyelination, which is consistent with its inhibitory effect on BMP signaling. Taken together, these preclinical data demonstrate that elezanumab has neuroregenerative and neuroprotective activities without impact on iron metabolism, thus providing a compelling rationale for its clinical development in neurodegenerative diseases.

Quantification of Hepcidin-related Iron Accumulation in the Rat Liver.

Hepcidin was originally detected as a liver peptide with antimicrobial activity and it functions as a central regulator in the systemic iron metabolism. Consequently suppression of hepcidin leads to iron accumulation in the liver. AbbVie developed a monoclonal antibody ([mAb]; repulsive guidance molecule [RGMa/c] mAb) that downregulates hepcidin expression by influencing the RGMc/bone morphogenetic protein (BMP)/neogenin receptor complex and causes iron deposition in the liver. In a dose range finding study with RGMa/c mAb, rats were treated with different dose levels for a total of 4 weekly doses. The results of this morphometric analysis in the liver showed that iron accumulation is not homogenous between liver lobes and the left lateral lobe was the most responsive lobe in the rat. Quantitative hepcidin messenger RNA analysis showed that the left lateral lobe was the most responsive lobe showing hepcidin downregulation with increasing antibody dose. In addition, the morphometric analysis had higher sensitivity than the chemical iron extraction and quantification using a colorimetric assay. In conclusion, the Prussian blue stain in combination with semi-quantitative and quantitative morphometric analysis is the most reliable method to demonstrate iron accumulation in the liver compared to direct measurement of iron in unfixed tissue using a colorimetric assay.

Anti-repulsive Guidance Molecule C (RGMc) Antibodies Increases Serum Iron in Rats and Cynomolgus Monkeys by Hepcidin Downregulation.

High levels of hepcidin, the main regulator of systemic iron metabolism, lead to various diseases. Targeting hepcidin and lowering its concentration is a possible form of intervention in order to treat these diseases. High turnover rate of hepcidin is a major drawback of therapies directly targeting this peptide. We developed two monoclonal antibodies ABT-207 and h5F9-AM8 which inhibit hemojuvelin/repulsive guidance molecule C (RGMc) and downregulate hepcidin. We conducted single-application and dose response studies to understand the antibodies' mechanism and subchronic toxicology studies to exclude safety-related concerns. Investigation was carried out at different biological levels through qPCR, Affymetrix, liquid chromatography coupled with mass spectrometry (LC-MS/MS), histopathology, serum iron, unsaturated iron binding capacity (UIBC), and drug concentration measurements. After a single application of these antibodies, hepcidin expression in liver and its serum protein levels were reduced. Serum iron increased for several weeks. The RGMc antibodies show a pronounced dose response relationship in rats with h5F9-AM8 having an IC50 (UIBC) of approximately 80-fold higher than ABT-207. When hepcidin levels were downregulated, iron deposition in the liver was visible histologically 1 week post application. These antibody-mediated iron depositions were not associated with any adverse toxicologically relevant effect at the doses and time points evaluated. Iron depositions seen after 14 weekly treatments with ABT-207 were reversible in rats and in cynomolgus monkeys. Due to their long-lasting effects and excellent safety profile, both RGMc-blocking antibodies ABT-207 and h5F9-AM8 are favorable clinical candidates for diseases characterized by high serum hepcidin levels like anemia of chronic disease.