Brain delivery and activity of a lysosomal enzyme using a blood-brain barrier transport vehicle in mice

Abstract

Most lysosomal storage diseases (LSDs) involve progressive central nervous system (CNS) impairment, resulting from deficiency of a lysosomal enzyme. Treatment of neuronopathic LSDs remains a considerable challenge, as approved intravenously administered enzyme therapies are ineffective in modifying CNS disease because they do not effectively cross the blood-brain barrier (BBB). We describe a therapeutic platform for increasing the brain exposure of enzyme replacement therapies. The enzyme transport vehicle (ETV) is a lysosomal enzyme fused to an Fc domain that has been engineered to bind to the transferrin receptor, which facilitates receptor-mediated transcytosis across the BBB. We demonstrate that ETV fusions containing iduronate 2-sulfatase (ETV:IDS), the lysosomal enzyme deficient in mucopolysaccharidosis type II, exhibited high intrinsic activity and degraded accumulated substrates in both IDS-deficient cell and in vivo models. ETV substantially improved brain delivery of IDS in a preclinical model of disease, enabling enhanced cellular distribution to neurons, astrocytes, and microglia throughout the brain. Improved brain exposure for ETV:IDS translated to a reduction in accumulated substrates in these CNS cell types and peripheral tissues and resulted in a complete correction of downstream disease-relevant pathologies in the brain, including secondary accumulation of lysosomal lipids, perturbed gene expression, neuroinflammation, and neuroaxonal damage. These data highlight the therapeutic potential of the ETV platform for LSDs and provide preclinical proof of concept for TV-enabled therapeutics to treat CNS diseases more broadly.

Differential expression of receptors mediating receptor-mediated transcytosis (RMT) in brain microvessels, brain parenchyma and peripheral tissues of the mouse and the human

“…TfR is highly expressed in reticulocytes causing on-target toxicity of effector-function competent antibodies [38]; high-a nity TfR antibodies also exhibit poor pharmacokinetics due to peripheral target-mediated clearance [42,53]. Due to lack of species cross-reactivity of TfR antibodies, the development of transgenic mouse expressing human TfR extracellular domain [7,12,13,37], use of surrogate antibody or of transgenic mouse expressing human TfR [14,15,37] were necessary in pre-clinical studies. The translational PK-PD models developed based on these studies [54] did not take into account differences in TfR abundance between mouse and human.…”

“…A ligand binding to a receptor on the luminal surface of brain endothelial cells (BEC) triggers ligand-receptor complex endocytosis, routing through various intracellular endosomal compartments where cargo is detached from the receptor and released on the abluminal side, while the receptor recycles 'back' to accept additional cargo molecules [7][8][9][10][11]. This pathway has been particularly well described for the transferrin receptor, which undergoes constitutive recycling and has been considered a 'prototypical' trigger of the RMT pathway [7][8][9][10][11][12][13].…”

“…Antibodies and peptide ligands against various endocytosing BBB receptors have been developed as potential molecular Trojan horses or shuttles to deliver therapeutic cargos across the BBB [2,8,9,[12][13][14][15][16]. Discovery strategies utilizing molecular ('omics') analyses of the BBB and screening of antibody libraries to select antibody species that can transmigrate the BBB [17][18][19][20][21][22] have yielded an experimental proof of concept for novel RMT-antibody pairs.…”

“…Discovery strategies utilizing molecular ('omics') analyses of the BBB and screening of antibody libraries to select antibody species that can transmigrate the BBB [17][18][19][20][21][22] have yielded an experimental proof of concept for novel RMT-antibody pairs. The literature survey of BBB targets shown to mediate transcytosis of ligands or antibodies in various model systems in vitro and in vivo, identi ed the following broad classes of RMT transporters: a) iron transporters: transferrin receptor (TFRC) [3][4][5][6]12,13]; b) insulin and insulin-like growth factors receptors: INSR, IGF1R, and IGF2R [8,16,23,24]; c) lipid transporters: lowdensity lipoprotein receptor (LDLR), LDLR-related protein 1 (LRP1), LRP8, and transmembrane protein 30A (TMEM30A/CDC50A) [8,9,[25][26][27][28][29][30][31][32]; d) solute carrier family transporters: glucose transporter-1 (GLUT-1)/SLC2A1 and SLC3A2/CD98hc [33,34]; and e) neuropeptide receptors: leptin receptor (LEPR) [35]. Structure/function relationships, ligand interactions and signaling, and specialized biology of these receptors is vastly different (summarized brie y in Supplementary Table 1), expanding the spectrum of potential RMT pathways across the BBB beyond the 'canonical' mechanism of TfR recycling.…”

Metabolism of Glycosphingolipids and Their Role in the Pathophysiology of Lysosomal Storage Disorders

“…ERTs for many LSDs have been developed for mice and humans [10,168,169]. The enzyme must be targeted to the lysosome, which is typically accomplished with a Man-6-P tag, and usually does not cross the BBB.…”