Delivery of β-Galactosidase to Mouse Brain via the Blood-Brain Barrier Transferrin Receptor

Abstract

Enzyme replacement therapy of lysosomal storage disorders is complicated by the lack of enzyme transport across the bloodbrain barrier (BBB). The present studies evaluate the delivery of a model enzyme across the BBB following enzyme conjugation to a BBB receptor-specific monoclonal antibody (mAb). Bacterial ␤-galactosidase (116 kDa) was conjugated to the rat 8D3 mAb to the rat transferrin receptor (TfR) via a streptavidin-biotin linkage. The unconjugated ␤-galactosidase or the ␤-galactosidase-8D3 conjugate was injected intravenously in adult mice, and enzyme activity was measured at 1 and 4 h in brain and peripheral organs (liver, spleen, kidney, and heart). Unconjugated ␤-galactosidase was rapidly removed from the blood compartment owing to avid uptake by liver and spleen. There was minimal uptake of the unconjugated ␤-galactosidase by brain. Following conjugation of the enzyme to the 8D3 TfRmAb, there was a 10-fold increase in brain uptake of the enzyme based on measurement of enzyme activity. Histochemistry of brain showed localization of the enzyme in the intraendothelial compartment of brain following intravenous injection of the enzyme-mAb conjugate. The capillary depletion technique showed that more than 90% of the enzyme-8D3 conjugate that entered into the endothelial compartment of brain passed through the BBB to enter brain parenchyma. In conclusion, high molecular weight enzymes, such as bacterial ␤-galactosidase, can be conjugated to BBB targeting antibodies for effective delivery across the BBB in vivo. Fusion proteins comprised of BBB targeting antibodies and recombinant enzymes could be therapeutic in the treatment of the brain in human lysosomal storage disorders.Lysosomal storage disorders are treated with recombinant enzyme replacement therapy. The majority of lysosomal storage disorders affect the brain (Cheng and Smith, 2003). A major limitation in the enzyme replacement therapy of lysosomal storage disorders is the lack of transport of the therapeutic enzyme across the brain capillary wall, which forms the blood-brain barrier (BBB). The involvement of the central nervous system is generally severe in lysosomal storage disorders (Cheng and Smith, 2003), and it is important to develop BBB drug delivery strategies for therapeutic enzymes. Recombinant proteins as large as 40,000 Da have been delivered across the BBB in vivo with molecular Trojan horses that access endogenous BBB receptor-mediated transport systems (Pardridge, 2001). A peptidomimetic monoclonal antibody (mAb) to the BBB transferrin receptor (TfR) mediated the delivery of several peptides and recombinant proteins across the BBB with in vivo central nervous system pharmacological effects following intravenous administration (Pardridge, 2002). The recombinant protein is attached to the TfRmAb via avidin-biotin technology. In this approach, the nontransportable protein drug is monobiotinylated in parallel with the production of a TfRmAb-streptavidin (SA) conjugate. Owing to the very high affinity of SA binding of biotin, the...

Inhibition of brain tumor growth by intravenous poly(β- l -malic acid) nanobioconjugate with pH-dependent drug release

“…The nanoconjugate actively passed the endothelial barrier by a well-established mechanism of transcytosis (11,40,41). BTB permeation was additionally facilitated by EPR effect (42).…”

“…Although the microvascular BTB is more permissible than the blood-brain barrier (BBB), it still retains BBB characteristics (7,8), hampering drug delivery to brain tumors. Antibodies to certain cell surface proteins, including transferrin receptor (TfR), can pass the BBB and BTB by endothelial transcytosis and then direct carrier systems with attached drugs into tumor cells by receptor-mediated endocytosis (9)(10)(11)(12)(13).…”

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“…Drug delivery to the cerebral vasculature is an important and challenging goal (Zhang and Pardridge, 2005). Some endothelial antigens potentially useful for targeting, such as angiotensin-converting enzyme (Muzykantov et al, 1996b), are readily internalized (Muzykantov et al, 1996a), a downside from the standpoint of localizing drugs intended to act within the vascular lumen.…”

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“…An antibody against a receptor expressed on the BBB is bound to the targeted protein in vitro before i.v. delivery (22)(23)(24). This method allows delivery of the targeted protein to neurons; however, it does not allow for continuous delivery of the targeted protein without repeated i.v.…”