Central nervous system (CNS) exposure to blood-borne biotherapeutics is limited by the restrictive nature of the brain vasculature. In particular, tightly sealed endothelial cells of the blood–brain barrier (BBB) prevent the uptake of protein and gene medicines. An approach to increase the bioavailability of such therapeutics is harnessing the BBB endothelial cells’ own receptor-mediated transcytosis (RMT) mechanisms. Key to this process is a targeting ligand that can engage a BBB-resident RMT receptor. We recently identified an antibody, named 46.1, that accumulates in the mouse brain after intravenous injection. To further characterize the brain targeting and penetrating properties of clone 46.1, we conjugated neurotensin (NT) to an scFv-Fc form of the antibody (46.1-scFv-Fc-LongLinker-NT). While centrally administered NT decreases the core body temperature and locomotor activity, effects attributed to two spatially segregated brain areas, systemically administered NT has limited effects. Hence, NT can be used as a model therapeutic payload to evaluate the brain penetration of BBB-targeting antibodies and their capability to accumulate in discrete brain areas. We demonstrate that intravenously administered 46.1-scFv-Fc-LL-NT can elicit transient hypothermia and reduce drug-induced hyperlocomotion, confirming that 46.1 can deliver drug cargo to the CNS at pharmacologically relevant doses. Interestingly, when two intravenous administration routes in mice, retro-orbital and tail vein, were compared, only retro-orbital administration led to transient hypothermia. We further explored the retro-orbital route and demonstrated that the 46.1-scFv-Fc-LL-NT could enter the brain arterial blood supply directly from the retro-orbital/cavernous sinus. Taken together, the 46.1 antibody is capable of transporting drug cargo into the CNS, and at least of a portion of its CNS accumulation occurs via the cavernous sinus–arterial route.
Drug delivery into the brain remains the rate limiting step in the development of new therapies whose targets lie within the central nervous system (CNS). In particular, the passage of newer biologic therapeutics (antibodies, peptides, nucleic acids, etc.) from the systemic circulation into the brain is substantially restricted by the blood–brain barrier (BBB). As a result, various technologies are being developed to increase the brain bioavailability. Despite known limitations, those that co-opt endogenous receptor-mediated transcytosis (RMT) systems in BBB endothelial cells hold particular promise. The transport of a drug payload from the blood into the brain tissue by RMT is mediated by a BBB-targeting motif that recognizes a cognate receptor on the blood-side endothelial membrane and initiates transcytosis. In preceding work, we identified antibodies capable of BBB transcytosis using a phenotypic transcytosis screen of a large phage displayed human single-chain antibody (scFv) library. The lead molecule, scFv 46.1, bound mouse and human BBB in tissue sections and accumulated in the mouse brain parenchyma after intravenous administration.
A key step in the preclinical evaluation of BBB-targeting motifs is demonstrable transport of a drug payload into the brain. Advanced disease models in translational research such as those examining β-amyloid clearance have been used to demonstrate the uptake of pharmacologically relevant doses of the therapeutic using RMT-directed brain drug delivery approaches. Other strategies that are more focused on validating the RMT-targeted antibody as capable of mediating drug uptake into the CNS, rather than a therapeutic outcome, have also been used to validate RMT-targeting antibodies. One such approach employs the conjugation of the RMT-targeting antibody to neurotensin (NT), a 13 amino acid peptide with a myriad of physiological functions. If administered peripherally, NT has a very low BBB permeability and does not elicit profound effects in the CNS. However, if NT is introduced into the CNS, it can interact with NT receptors expressed on brain cells (NTSR1 and NTSR2) in different brain regions. For example, NT and its analogs inhibit food intake in arcuate nucleus, modulate pain response, and mitigate addiction behavior in nucleus accumbens. In addition, NT integrates with dopamine neurotransmission, acting as an endogenous neuroleptic, leading to decreased drug-induced hyper- and spontaneous locomotor activity. Upon release in the median preoptic nucleus (MnPO), NT activates its cognate receptors NTSR1 and NTSR2, which results in decreased core body temperature. Since the CNS effects of NT are limited to central local release or central administration, fusion of NT to BBB-targeting antibodies can be used to test the BBB-permeation of the complex. Notably, given the anatomically distinct effects of NT, it can also be used as a proxy for verifying brain uptake in different brain regions. For instance, in this study, we measured the effects of 46.1-NT fusions on the body temperature in mice (i.e., NT response from the MnPO), and additionally measured their drug-induced locomotor activity (i.e., response from the striatum) to demonstrate that 46.1 could mediate the uptake of pharmacologically relevant levels of NT in the CNS.
It has been shown that the pharmacokinetic profile of intravenous therapeutic antibodies can be independent of the route of intravenous administration. Nevertheless, viral particles with CNS tropism seem to distribute differently upon tail vein, facial vein, or retro-orbital vein injection, a phenomenon speculated to arise in the cavernous sinus (Sin. Cavern.). The cavernous sinus consists of trabeculated cavities formed by splitting of the layers of the dura mater that are covered by endothelial cells. Located in the base of the skull, right below the hypophyseal gland, the cavernous sinus collects venous blood from the retro-orbital sinus, ophthalmic veins, superficial, and interior cerebral veins. The internal carotid artery spans the Sin. Cavern. before branching and entering the brain in a segment known as the cavernous portion of the internal carotid artery. Thus, in the Sin. Cavern., venous blood is separated from the arterial blood by the wall of the internal carotid artery, a multilayered structure with an innermost endothelial cell layer, covered by internal elastic lamina, smooth muscle cell layer, and adventia. In this study, we additionally examined the effects of the route of intravenous administration (retro-orbital versus tail vein) on the hypothermic response in mice and demonstrated that the 46.1-NT fusion could enter the internal carotid artery directly from the retro-orbital/cavernous sinus and be transported throughout the brain via the microvasculature.
A previously described pIRES vector with the test and control scFvs fused to the rabbit Fc region was used to generate the neurotensin fusion construct. Long linker (G 4 S)2 and mouse neurotensin sequences (NM_024435) were subcloned at the C-terminal of the rabbit Fc region between the custom inserted BamHI and NotI restriction sites. The following oligonucleotide was used: 5′-GGATCCGGTGGTGGCGGCTCTGGTGGCGGTGGCAGCCAGCTGTATGAAAATAAACCCAGAGGCCCTACATTCTCTGAGCGGCCGC-3′. The oligonucleotide duplex was inserted with standard restriction cloning. The sequence identity was verified at the UW-Madison sequencing facility and had the final annotation Clone ID-scFv-Fc-LL-NT. Fused proteins were produced and purified exactly as previously described. The functional activity of neurotensin was monitored for each batch with the Path-Hunter eXpress NTSR1 CHO-K1 β-Arrestin GPCR assay (Eurofins/DiscoverX, #93-0280E2CP0L, Fremont, CA, USA), according to the manufacturer’s protocol.
IPSC-derived brain microvascular endothelial cells (iPSC-BMEC-like cells) were purified on Lab Tek II chamber slides (Nunc #154917, Thermo Fisher Scientific, Madison, WI, USA) or on 0.4 µm Transwell filters, as described previously. Cells grown on chamber slides were washed once and the media exchanged with prewarmed PBS++ (PBS supplemented with Ca 2+ and Mg 2+). Antibody 46.1-scFv-Fc-LL-NT was added to the PBS++ to a final concentration of 5 µg/mL. Cells were kept at 37 °C for 45 min to allow for antibody binding and internalization. Afterward, cells were washed with ice cold PBS++ and media exchanged with ice cold PBS++ plus 10% goat serum (PBSG). Secondary anti-rabbit AlexaFluor555 (Invitrogen #A21428, Thermo Fisher Scientific, Madison, WI, USA)-conjugated antibody of a final dilution 1:1000 was added to the live cells to label the membrane bound fraction. After an additional 20 min on ice, the cells were washed three times with cold PBS++, fixed with ice cold 4% paraformaldehyde (PFA) for 15 min, permeabilized with 0.2% Triton X for 2 min, and washed with PBS++. Anti-rabbit AlexaFluor488 (Invitrogen #A11008, Thermo Fisher Scientific, Madison, WI, USA) diluted 1:1000 in PBSG was added to the chamber slides to label the internalized fraction and incubated at RT for an additional 20 min. The IPSC-BMECs were washed and mounted with ProLong Gold anti-fade reagent with DAPI (Invitrogen, P36935, Thermo Fisher Scientific, Madison, WI, USA). Images were acquired on a Zeiss Axio Imager Z2 Upright microscope (Carl Zeiss Microscopy, LLC, White Plains, NY, USA) and processed with ImageJ (Version 1.53e, Wayne Rasband and contributors, NIH, USA).
The TEER value of iPSC-BMECs, grown on Transwell filters (n = 3) was measured and on the day of the experiment averaged ~1600 Ω/cm 2. Cells were washed and media exchanged with prewarmed PBS++. Antibody 46.1-scFv-Fc-LL-NT was added to the basolateral compartment to a final concentration of 5 µg/mL. The IPSC-BMEC-like cells were kept at 37 °C for 45 min. After a brief washing step with ice cold PBS++ and PBSG, the apical and the basolateral compartment solution was exchanged with ice cold PBSG containing secondary anti-rabbit antibodies in a 1:1000 dilution, AlexaFluor647 (Invitrogen #A-21235, Thermo Fisher Scientific, Madison, WI, USA) in the apical compartment to label the apical membrane associated 46.1-scFv-Fc-LL-NT and AlexaFluor555 (Invitrogen #A21428, Thermo Fisher Scientific, Madison, WI, USA) in the basolateral compartment to label the basolateral membrane bound 46.1-scFv-Fc-LL-NT. The live, not-permeabilized cells were kept on ice for 20 min. The filter was washed quickly 3x with cold PBS++. Cells were fixed with ice cold 4% PFA for 15 min and permeabilized with 0.2% Triton X for 2 min, applied on both sides of the filter. Anti-rabbit AlexaFluor488 (Invitrogen #A11008, Thermo Fisher Scientific, Madison, WI, USA) in 1:1000 dilution was added to both sides of the filter to label the internalized 46.1-scFv-Fc-LL-NT and incubated at RT for an additional 20 min. Cells were washed and mounted with the ProLong Gold mounting media. Images were acquired on a Zeiss Axio Imager Z2 upright microscope and processed with ImageJ.
Animal studies were approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Wisconsin-Madison and performed in compliance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Mice were group housed in an AAALAC accredited vivarium on a standardized light cycle (lights on: 8 a.m.–8 p.m.) with ad libitum access to food and water.
Mice C57BL6 (Harlan Laboratories, Inc., Envigo Bioproducts, Inc., Madison, WI, USA), male, ~18 g were anesthetized with 100/10 mg/kg ketamine/xylazine (Vetaket C-III (N), Akorn, Inc., #2010020, Lake Forest, IL, USA). Under aseptic conditions, an incision was made in the
