Preclinical Evaluation of Anti-Fibrin Immunotherapy for Cerebral ALD: Leveraging a Novel Mouse Model to Guide Clinical Trial Design

X-linked adrenoleukodystrophy (ALD) is a devastating genetic disorder, caused by mutations in a peroxisomal gene (ABCD1), in which two-thirds of affected males develop cerebral ALD (cALD), a progressive, inflammatory brain condition that is often fatal. Currently, the only available treatments are hematopoietic stem cell transplantation (HSCT) and ex vivo gene correction (FDA-approved in 2022). However, these therapies are limited to a subset of cALD patients based on factors such as lesion stage, age, donor availability, and access to advanced healthcare facilities. Consequently, most boys and men worldwide who develop cALD die from it. We propose using a novel cALD mouse model to test a first-in-class, monoclonal antibody therapy targeting fibrin, a potent pro-inflammatory protein that is highly expressed in cALD brain lesions. Fibrin, a hallmark of blood-brain barrier (BBB) disruption, plays a crucial role in neuroinflammation and demyelination by activating macrophages, microglia, and astrocytes, and impairing oligodendrocyte maturation. Our approach leverages our recently developed mouse model which combines cuprizone (CPZ) diet and MOG injection (EAE model) in Abcd1-knockout mice to induce a cALD phenotype. Our model replicates key features of human cALD, including demyelination, axonal damage, BBB disruption, oxidative stress, and fibrin deposition. We will evaluate the efficacy of 5B8, an antibody targeting fibrin's pro-inflammatory epitope (amino acids 377-395), in alleviating neurological disability and reducing pathological manifestations in cALD mice. We will compare the effect of therapy in early (presymptomatic) and late (symptomatic) stages of the disease and against sham IgG control. The project comprises three aims: In Aim 1, we will investigate whether anti-fibrin immunotherapy ameliorates behavioral symptoms by assessing motor function using the open field test and cognitive function using the Barnes maze. In Aim 2, we will examine whether anti-fibrin immunotherapy improves BBB disruption in the brain using T1- weighted MRI and increases cerebral blood flow using Arterial Spin Labeling MRI in the cALD mouse model. We will also analyze plasma biomarkers of BBB disruption and neuroinflammation, including neurofilament light chain, interleukin-18, vascular cell adhesion molecule 1, and other markers. In Aim 3, we will confirm target engagement and evaluate whether anti-fibrin therapy reduces histological brain lesions by performing immunostaining to measure BBB integrity, immune cell infiltration, and axonal damage. The successful completion of these aims will generate necessary preclinical data and identify relevant biomarkers to facilitate the design of a Phase 2/3 human clinical trial for anti-fibrin immunotherapy in cALD. Success could lead to a novel treatment option for this devastating disorder, addressing a serious unmet medical need and offering hope for patients currently ineligible for existing therapies.