The Trojan Horse Liposome Technology for Nonviral Gene Transfer across the Blood-Brain Barrier

The application of blood-borne gene therapy protocols to the brain is limited by the presence of the blood-brain barrier (BBB). Viruses have been extensively used as gene delivery systems. However, their efficacy in brain is limited by the lack of transport across the BBB following intravenous (IV) administration. Recent progress in the “Trojan Horse Liposome” (THL) technology applied to transvascular non-viral gene therapy of the brain presents a promising solution to the trans-vascular brain gene delivery problem. THLs are comprised of immunoliposomes carrying nonviral gene expression plasmids. The tissue target specificity of the THL is provided by peptidomimetic monoclonal antibody (MAb) component of the THL, which binds to specific endogenous receptors located on both the BBB and on brain cellular membranes, for example, insulin receptor and transferrin receptor. These MAbs mediate (a) receptor-mediated transcytosis of the THL complex through the BBB, (b) endocytosis into brain cells and (c) transport to the brain cell nuclear compartment. The expression of the transgene in brain may be restricted using tissue/cell specific gene promoters. This manuscript presents an overview on the THL transport technology applied to brain disorders, including lysosomal storage disorders and Parkinson's disease

Rapid Sequestration and Degradation of Somatostatin Analogues by Isolated Brain Microvessels

Somatostatin (SRIF) is a putative peptide neurotransmitter that may interact with brain capillaries following neurosecretion of the peptide. The present studies investigate the binding and metabolism of SRIF analogues in isolated bovine brain microvessels. 125 I [Tyr 1 ]SRIF was rapidly degraded by capillary aminopeptidase with a half-time of approximately 3 min at 23°C. The microvessel aminopeptidase had a low affinity and high capacity for the peptide, K m = 76 Μ M and V max = 74 nmol min −1 . 125 I-[Tyr 11 ]SRIF was converted to free iodotyrosine at a much slower rate, presumably by a lower-activity endopeptidase. 125 I-[Tyr 11 ]SRIF was rapidly bound by microvessels, whereas another basic peptide, [Tyr 8 ]bradykinin, or an acidic peptide, CCK8, or a neutral peptide, leucine enkephalin, were bound to a considerably less extent. The binding of 125 I-[Tyr 11 ]SRIF to the capillaries was nonsaturable up to a concentration of 1 Μg/ml of unlabeled peptide, and the binding reaction was extremely rapid, reaching equilibrium within 5 s at either 0°C or 37°C. Approximately 20% of the SRIF bound by the microvessels was resistant to acid wash and presumably represented internalized peptide. In addition, the 125 I-[Tyr 11 ]SRIF bound rapidly to the endothelial cytoskeleton remaining after a 1% Triton X-100 extraction of the microvessels. The peptide-cytoskeletal binding reaction was nonsaturable up to 1 Μg/ml of unlabeled [Tyr 11 ]SRIF, but it was inhibited by 0.5% polylysine or 0.8 M KC1 and was stimulated by 1 m M dithiothreiotol. These studies suggest that brain microvessels rapidly sequester and degrade SRIF analogues and that this may represent one mechanism for rapid inactivation of the neuropeptides subsequent to neurosecretion.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/66285/1/j.1471-4159.1985.tb08741.x.pd

Brain Protection from Stroke with Intravenous TNFα Decoy Receptor-Trojan Horse Fusion Protein

Tumor necrosis factor (TNF)-α is produced in brain in response to acute cerebral ischemia, and promotes neuronal apoptosis. Biologic TNF inhibitors (TNFIs), such as the etanercept, cannot be developed as new stroke treatments because these large molecule drugs do not cross the blood–brain barrier (BBB). A BBB-penetrating biologic TNFI was engineered by fusion of the type II human TNF receptor (TNFR) to each heavy chain of a genetically engineered chimeric monoclonal antibody (MAb) against the mouse transferrin receptor (TfR), designated as cTfRMAb-TNFR fusion protein. The cTfRMAb domain of the fusion protein acts as a molecular Trojan horse to deliver the fused TNFR across the BBB. Etanercept or the cTfRMAb-TNFR fusion protein (1 mg/kg) was administered intravenously in adult mice subjected to 1-hour reversible middle cerebral artery occlusion up to 90 minutes after the occlusion. Neuroprotection was assessed at 24 hours or 7 days after occlusion. The cTfRMAb-TNFR fusion protein treatment caused a significant 45%, 48%, 42%, and 54% reduction in hemispheric, cortical, and subcortical stroke volumes, and neural deficit, respectively. Intravenous etanercept had no therapeutic effect. Biologic TNFIs can be reengineered for BBB penetration, and the IgG-TNFR fusion protein is therapeutic after delayed intravenous administration in experimental stroke

Plasma Pharmacokinetics of High-Affinity Transferrin Receptor Antibody-Erythropoietin Fusion Protein is a Function of Effector Attenuation in Mice

Erythropoietin (EPO) is a potential therapeutic for Alzheimer’s disease (AD); however, limited blood–brain barrier (BBB) penetration reduces its applicability as a CNS therapeutic. Antibodies against the BBB transferrin receptor (TfRMAbs) act as molecular Trojan horses for brain drug delivery, and a fusion protein of EPO and TfRMAb, designated TfRMAb-EPO, is protective in a mouse model of AD. TfRMAbs have Fc effector function side effects, and removal of the Fc N-linked glycosylation site by substituting Asn with Gly reduces the Fc effector function. However, the effect of such Fc mutations on the pharmacokinetics (PK) of plasma clearance of TfRMAb-based fusion proteins, such as TfRMAb-EPO, is unknown. To examine this, the plasma PK of TfRMAb-EPO (wild-type), which expresses the mouse IgG1 constant heavy chain region and includes the Asn residue at position 292, was compared to the mutant TfRMAb-N292G-EPO, in which the Asn residue at position 292 is mutated to Gly. Plasma PK was compared following IV, IP, and SQ administration for doses between 0.3 and 3 mg/kg in adult male C57 mice. The results show a profound increase in clearance (6- to 8-fold) of the TfRMAb-N292G-EPO compared with the wild-type TfRMAb-EPO following IV administration. The clearance of both the wild-type and mutant TfRMAb-EPO fusion proteins followed nonlinear PK, and a 10-fold increase in dose resulted in a 7- to 11-fold decrease in plasma clearance. Following IP and SQ administration, the Cmax values of the TfRMAb-N292G-EPO mutant were profoundly (37- to 114-fold) reduced compared with the wild-type TfRMAb-EPO, owing to comparable increases in plasma clearance of the mutant fusion protein. The wild-type TfRMAb fusion protein was associated with reticulocyte suppression, and the N292G mutation mitigated this suppression of reticulocytes. Overall, the beneficial suppression of effector function via the N292G mutation may be offset by the deleterious effect this mutation has on the plasma levels of the TfRMAb-EPO fusion protein, especially following SQ administration, which is the preferred route of administration in humans for chronic neurodegenerative diseases including AD

Eliminating Fc N-linked Glycosylation and Its Impact on Dosing Consideration for a Transferrin Receptor Antibody-Erythropoietin Fusion Protein in Mice

Erythropoietin (EPO), a hematopoietic growth factor and a promising therapy for Alzheimer’s disease, has low permeability across the blood–brain barrier. The transferrin receptor antibody fused to EPO (TfRMAb-EPO) is a chimeric monoclonal antibody that ferries EPO into the brain via the transvascular route. However, TfRMAbs have Fc-effector function-related adverse effects including reticulocyte suppression. To overcome this, we recently developed an effectorless TfRMAb-EPO fusion protein, designated TfRMAb-N292G-EPO, by eliminating the Fc N-linked glycosylation site at position 292 of the antibody heavy chain. The mutant fusion protein showed enhanced plasma clearance and dramatically reduced plasma concentrations compared with the wild-type (WT) nonmutant fusion protein. This increased clearance of the aglycosylated TfRMAb is expected to increase the injection dose of the mutant fusion protein. To provide a basis for future therapeutic uses of this IgG-neurotrophin fusion protein, the current study aimed to characterize the pharmacokinetic profile of this effectorless TfRMAb-N292G-EPO at different doses following different routes of administration in the mouse. Adult C57BL/6J male mice were injected with a single dose (3, 6, 9, or 20 mg/kg; n = 3–6 per dose) of TfRMAb-N292G-EPO through either the subcutaneous (SQ) or intraperitoneal (IP) route. TfRMAb-N292G-EPO plasma concentrations were determined using an enzyme-linked immunosorbent assay. Mice were sacrificed 24 h after injection, and terminal blood was used for a complete blood count. Brain concentrations in the WT- and mutant fusion protein-treated mice were compared. We observed stark differences in the plasma pharmacokinetics of TfRMAb-N292G-EPO between the IP and SQ routes of administration. Dose escalation from 3 to 20 mg/kg increased the plasma Cmax only 3.5-fold for the SQ route, compared with a 35-fold increase for the IP route. The plasma Cmax was 15.0 ± 2.0, 21.3 ± 4.1, 21.3 ± 6.4, and 52.8 ± 27.9 ng/mL following SQ injection and 288 ± 47, 389 ± 154, 633 ± 194, and 10,066 ± 7059 ng/mL following IP injection for 3, 6, 9, and 20 mg/kg doses, respectively. The plasma Cmax following the SQ route was therefore 19- to 190-fold lower than that following the IP route. This finding is consistent with a 31-fold higher apparent clearance following the SQ route compared with the IP route at the highest dose administered. The brain concentrations in the mice treated with a 3 mg/kg dose of the mutant fusion protein were lower than those in the nonmutant WT-treated mice. No reticulocyte suppression was observed at the 3 mg/kg SQ dose of TfRMAb-N292G-EPO. However, reticulocyte suppression increased with an increase in dose and area under the plasma concentration–time curve (AUC) for both the IP and SQ routes. Overall, elimination of Fc N-linked glycosylation, to mitigate TfRMAb effector function side effects, has a profound effect on the plasma exposure of TfRMAb-N292G-EPO at therapeutic as well as high doses (3–20 mg/kg). This effect is more pronounced following SQ injection. The low plasma concentrations of the mutant fusion protein following a 3 mg/kg dose resulted in negligible brain uptake. The beneficial rescue of reticulocyte reduction by the N292G mutation is a function of AUC and is negated at high doses of the N292G mutant

Acute and Chronic Dosing of a High-Affinity Rat/Mouse Chimeric Transferrin Receptor Antibody in Mice

Non-invasive brain delivery of neurotherapeutics is challenging due to the blood-brain barrier. The revived interest in transferrin receptor antibodies (TfRMAbs) as brain drug-delivery vectors has revealed the effect of dosing regimen, valency, and affinity on brain uptake, TfR expression, and Fc-effector function side effects. These studies have primarily used monovalent TfRMAbs with a human constant region following acute intravenous dosing in mice. The effects of a high-affinity bivalent TfRMAb with a murine constant region, without a fusion partner, following extravascular dosing in mice are, however, not well characterized. Here we elucidate the plasma pharmacokinetics and safety of a high-affinity bivalent TfRMAb with a murine constant region following acute and chronic subcutaneous dosing in adult C57BL/6J male mice. Mice received a single (acute dosing) 3 mg/kg dose, or were treated for four weeks (chronic dosing). TfRMAb and control IgG1 significantly altered reticulocyte counts following acute and chronic dosing, while other hematologic parameters showed minimal change. Chronic TfRMAb dosing did not alter plasma- and brain-iron measurements, nor brain TfR levels, however, it significantly increased splenic-TfR and -iron. Plasma concentrations of TfRMAb were significantly lower in mice chronically treated with IgG1 or TfRMAb. Overall, no injection related reactions were observed in mic

Pharmacokinetics and Brain Uptake in the Rhesus Monkey of a Fusion Protein of Arylsulfatase a and a Monoclonal Antibody Against the Human Insulin Receptor

Metachromatic leukodystrophy (MLD) is a lysosomal storage disorder of the brain caused by mutations in the gene encoding the lysosomal sulfatase, arylsulfatase A (ASA). It is not possible to treat the brain in MLD with recombinant ASA, because the enzyme does not cross the blood-brain barrier (BBB). In the present investigation, a BBB-penetrating IgG-ASA fusion protein is engineered and expressed, where the ASA monomer is fused to the carboxyl terminus of each heavy chain of an engineered monoclonal antibody (MAb) against the human insulin receptor (HIR). The HIRMAb crosses the BBB via receptor-mediated transport on the endogenous BBB insulin receptor, and acts as a molecular Trojan horse to ferry the ASA into brain from blood. The HIRMAb-ASA is expressed in stably transfected Chinese hamster ovary cells grown in serum free medium, and purified by protein A affinity chromatography. The fusion protein retains high affinity binding to the HIR, EC50 = 0.34 ± 0.11 nM, and retains high ASA enzyme activity, 20 ± 1 units/mg. The HIRMAb-ASA fusion protein is endocytosed and triaged to the lysosomal compartment in MLD fibroblasts. The fusion protein was radio-labeled with the Bolton–Hunter reagent, and the [125I]-HIRMAb-ASA rapidly penetrates the brain in the Rhesus monkey following intravenous administration. Film and emulsion autoradiography of primate brain shows global distribution of the fusion protein throughout the monkey brain. These studies describe a new biological entity that is designed to treat the brain of humans with MLD following non-invasive, intravenous infusion of an IgG-ASA fusion protein

Pharmacokinetics and Brain Uptake of an IgG-TNF Decoy Receptor Fusion Protein Following Intravenous, Intraperitoneal, and Subcutaneous Administration in Mice

Tumor necrosis factor (TNF)-α is a proinflammatory cytokine active in the brain. Etanercept, the TNF decoy receptor (TNFR), does not cross the blood–brain barrier (BBB). The TNFR was re-engineered for BBB penetration as a fusion protein with a chimeric monoclonal antibody (mAb) against the mouse transferrin receptor (TfR), and this fusion protein is designated cTfRMAb-TNFR. The cTfRMAb domain of the fusion protein acts as a molecular Trojan horse and mediates transport via the endogenous BBB TfR. To support future chronic treatment of mouse models of neural disease with daily administration of the cTfRMAb-TNFR fusion protein, a series of pharmacokinetics and brain uptake studies in the mouse was performed. The cTfRMAb-TNFR fusion protein was radiolabeled and injected into mice via the intravenous, intraperitoneal (IP), or subcutaneous (SQ) routes of administration at doses ranging from 0.35 to 10 mg/kg. The distribution of the fusion protein into plasma following the IP or SQ routes was enhanced by increasing the injection dose from 3 to 10 mg/kg. The fusion protein demonstrated long circulation times with high metabolic stability following the IP or SQ routes of injection. The IP or SQ routes produced concentrations of the cTfRMAb-TNFR fusion protein in the brain that exceed by 20- to 50-fold the concentration of TNFα in pathologic conditions of the brain. The SQ injection is the preferred route of administration, as the level of cTfRMAb fusion protein produced in the brain is comparable to that generated with intravenous injection, and at a much lower plasma area under the concentration curve of the fusion protein as compared to IP administration

Hematologic Safety of Chronic Brain-Penetrating Erythropoietin Dosing in APP/PS1 Mice

Introduction: Low blood-brain barrier (BBB) penetration and hematopoietic side effects limit the therapeutic development of erythropoietin (EPO) for Alzheimer\u27s disease (AD). A fusion protein of EPO and a chimeric monoclonal antibody targeting the mouse transferrin receptor (cTfRMAb) has been engineered. The latter drives EPO into the brain via receptor-mediated transcytosis across the BBB and increases its peripheral clearance to reduce hematopoietic side effects of EPO. Our previous work shows the protective effects of this BBB-penetrating EPO in AD mice but hematologic effects have not been studied. Herein, we investigate the hematologic safety and therapeutic effects of chronic cTfRMAb-EPO dosing, in comparison to recombinant human EPO (rhu-EPO), in AD mice. Methods: Male APPswe PSEN1dE9 (APP/PS1) mice (9.5 months) were treated with saline (n = 11), and equimolar doses of cTfRMAb-EPO (3 mg/kg, n = 7), or rhu-EPO (0.6 mg/kg, n = 9) 2 days/week subcutaneously for 6 weeks, compared to saline-treated wild-type mice (n = 10). At 6 weeks, exploration and memory were assessed, and mice were sacrificed at 8 weeks. Spleens were weighed, and brains were evaluated for amyloid beta (Aβ) load and synaptophysin. Blood was collected at 4, 6 and 8 weeks for a complete blood count and white blood cells differential. Results: cTfRMAb-EPO transiently increased reticulocyte counts after 4 weeks, followed by normalization of reticulocytes at 6 and 8 weeks. rhu-EPO transiently increased red blood cell count, hemoglobin and hematocrit, and significantly decreased mean corpuscular volume and reticulocytes at 4 weeks, which remained low at 6 weeks. At 8 weeks, a significant decline in red blood cell indices was observed with rhu-EPO treatment. Exploration and cognitive deficits were significantly worse in APP/PS1-rhu-EPO mice. Both cTfRMAb-EPO and rhu-EPO decreased 6E10-positive brain Aβ load; however, cTfRMAb-EPO and not rhu-EPO selectively reduced brain Aβ1-42 and elevated synaptophysin expression. Discussion: Chronic treatment with cTfRMAb-EPO results in better hematologic safety, behavioral, and therapeutic indices compared with rhu-EPO, supporting the development of this BBB-penetrable EPO analog for AD. therapeutic indices compared with rhu-EPO, supporting the development of this BBB-penetrable EPO analog for AD