Brain delivery of therapeutic proteins using an Fc fragment blood-brain barrier transport vehicle in mice and monkeys

Abstract

Effective delivery of protein therapeutics to the central nervous system (CNS) has been greatly restricted by the blood-brain barrier (BBB). We describe the development of a BBB transport vehicle (TV) comprising an engineered Fc fragment that exploits receptor-mediated transcytosis for CNS delivery of biotherapeutics by binding a highly expressed brain endothelial cell target. TVs were engineered using directed evolution to bind the apical domain of the human transferrin receptor (hTfR) without the use of amino acid insertions, deletions, or unnatural appendages. A crystal structure of the TV-TfR complex revealed the TV binding site to be away from transferrin and FcRn binding sites, which was further confirmed experimentally in vitro and in vivo. Recombinant expression of TVs fused to anti–β-secretase (BACE1) Fabs yielded antibody transport vehicle (ATV) molecules with native immunoglobulin G (IgG) structure and stability. Peripheral administration of anti-BACE1 ATVs to hTfR-engineered mice and cynomolgus monkeys resulted in substantially improved CNS uptake and sustained pharmacodynamic responses. The TV platform readily accommodates numerous additional configurations, including bispecific antibodies and protein fusions, yielding a highly modular CNS delivery platform.

Differential expression of receptors mediating receptor-mediated transcytosis (RMT) in brain microvessels, brain parenchyma and peripheral tissues of the mouse and the 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 (TfR), which undergoes constitutive recycling and has been considered a 'prototypical' trigger of the RMT pathway [7][8][9][10][11][12][13].…”

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.…”