The NewroBus platform: engineered humanized anti-TfR1 nanobodies for efficient brain delivery.

INTRODUCTION Delivery of therapeutic agents to the brain is constrained by the blood-brain barrier (BBB). Previous work (Yarnitsky) suggested BBB permeability was increased with stimulation of the sphenopalatine ganglion (SPG). However, their model looked at FITC-dextran signal in CSF superfusate, a reflection of epithelial tight junctions at the blood-CSF barrier, and quantified BBB permeability using Evans blue, a marker insufficient for this role (Saunders). METHODS Experiments were conducted in Sprague-Dawley rats using 70 kDa FITC-dextran as a marker to quantify BBB permeability. Once anesthetized, the right femoral vein was exposed and catheterized. Next, SPG fibers were exposed behind the right eye and an electrode was hooked around those fibers. Stimulation occurred in blocks of 90 seconds of “on” time at 5 volts and 10 Hz followed by 60 seconds of “off” time. Injection of 0.1 mL of a 100 mg/mL concentration of FITC coincided with each “on” stimulation cycle; a total of 1.0 mL was injected per animal. Control animals were not stimulated, but had the same injection protocol as test animals. 90 seconds after the final injection, a blood sample was collected and the cerebrovasculature was flushed. Each brain was removed, equally divided, and homogenized. Post-centrifugation supernatant from blood and tissue homogenates was analyzed using fluorescent spectrometry. FITC concentrations were calculated using known standards. An uptake ratio was calculated by dividing sample FITC concentration by blood FITC concentration. RESULTS >Data from 4 control animals and 4 test animals demonstrated a significantly greater uptake ratio in test brains compared with control brains (P = 0.001). A significantly increased uptake ratio was also observed in both right and left hemispheres of test animals compared with controls (right, P = 0.03; left, P = 0.01). CONCLUSION Stimulation of SPG fibers significantly increased BBB permeability in both cerebral hemispheres of test animals when compared with controls.

The delivery of therapeutics into the brain is highly limited by the blood-brain barrier (BBB). Although this is essential to protect the brain from potentially harmful material found in the blood, it poses a great challenge for the treatment of diseases affecting the central nervous system (CNS). Substances from the periphery that are required for the function of the brain must rely on active mechanisms of entry. One such physiological pathway is called receptor-mediated transcytosis (RMT). In this process, ligands bind to specific receptors expressed at the luminal membrane of endothelial cells composing the BBB leading to the internalization of the receptor-ligand complex into intracellular vesicles, their trafficking through various intracellular compartments and finally their fusion with the abluminal membrane to release the cargo into the brain. Targeting such RMT receptors for BBB crossing represents an emerging and clinically validated strategy to increase the brain permeability of biologicals. However, the choice of an appropriate receptor is critical to achieve the best selectivity and efficacy of the delivery method. Whereas the majority of work has been focused on transferrin (Tf) receptor (TfR), the search for novel receptors expressed in brain endothelial cells (BECs) that can deliver protein or viral vector cargos across the BBB has yielded several novel targets with diverse molecular/structural properties and biological functions, and mechanisms of transcytosis. In this review, we summarize well-studied RMT pathways, and explore mechanisms engaged in BBB transport by various RMT receptors. We then discuss key criteria that would be desired for an optimal RMT target, based on lessons-learned from studies on TfR and accumulating experimental evidence on emerging RMT receptors and their ligands.

Liver failure is often associated with hepatic encephalopathy, due to dyshomeostasis of the central nervous system (CNS). Under physiological conditions, the CNS homeostasis is precisely regulated by the blood-brain barrier (BBB). The BBB consists of brain microvessel endothelial cells connected with a junctional complex by the adherens junctions and tight junctions. Its main function is to maintain brain homoeostasis via limiting the entry of drugs/toxins to brain. The brain microvessel endothelial cells are characterized by minimal pinocytotic activity, absent fenestrations, and highly expressions of ATP-binding cassette (ABC) family transporters (such as P-glycoprotein, breast cancer resistance protein and multidrug resistance-associated proteins). These ABC transporters prevent brain from toxin accumulation by pumping toxins out of brain. Accumulating evidences demonstrates that liver failure diseases altered the expression and function of ABC transporters at The BBB, indicating that the alterations subsequently affect drugs’ brain distribution and CNS activity/neurotoxicity. ABC transporters also mediate the transport of endogenous substrates across the BBB, inferring that ABC transporters are also implicated in some physiological processes and the development of hepatic encephalopathy. This paper focuses on the alteration in the BBB permeability, the expression and function of ABC transporters at the BBB under liver failure status and their clinical significances.

BackgroundAntibody‐based PET ligands are desirable due to their high specificity; however, their brain uptakes are limited by the blood‐brain barrier (BBB). We previously demonstrated that transport of antibody across BBB can be facilitated through interaction with the transferrin receptor (TfR)1. We report here the first in vivo PET imaging studies using BBB permeable F‐18 radiolabeled bispecific antibodies specific to tau protein in mice.MethodWe performed multiple PET scans using each of the radio‐probes synthesized via conjugation of [18F]SFB with one of the four bispecific antibody constructs: 1) 6B2G12‐ScFv8D3 (full‐size Tau antibody conjugated with TfR fragment (ScFv8D3); abbreviated as TAUb), 2) ScFv235‐ScFv8D3 (small Tau‐TfR; TAUs), 3) 3D6‐ScFv8D3 (full‐size Aβ‐TfR; Aβb) or 4) ScFv3D6‐ScFv3D6 (small Aβ‐TfR; Aβs). We used a matched control design to compare a WT with one of the two types of transgenic (tg) tauopathy mice [PS19 (P301S) or JNPL3 (P301)] in each scan. Subjects were scanned on a MOLECUBES PET/CT scanner. An initial low‐dose (244 ± 54 μCi) dynamic scan (30 min) was performed, followed by two scans at 8hr and 12hr (20 min each static scans) after injection of a high dose (1832 ± 447 μCi) of the same probe. PMOD and FireVoxel were used for imaging processing. After co‐registration of PET/CT/atlas, data were quantified as SUV and SUVR [CB as a reference], and clearance was estimated as %change/hr.ResultTAUs probe displayed highest brain uptake with more specific binding retained in the brain, as compared to TAUb and Aβs. TAUs probe showed faster brain penetration in tau‐tg mice than in WT, with differences in amygdala, basal forebrain, and hypothalamus of 66%, 62% and 85%, respectively, at 8hr post‐injection (Fig. 1). Further, higher brain uptakes (TAUs > Aβs) in the same tau‐tg mouse (Fig. 2) suggest the specific binding of TAUs probe, while Aβs probe can serve as a control due to its lack of specific binding in tau‐tg mice.ConclusionWe have successfully synthesized and evaluated four novel F‐18 bispecific antibodies via in vivo PET. TAUs probe showed most promising BBB permeability and binding specificity to tau antigen with a reasonable clearance.

Therapeutic antibodies have been successfully used to treat several diseases, such as cancers and autoimmune diseases. However, the utility of conventional antibodies for neurological conditions is limited by the blood-brain barrier (BBB). Several strategies to address this issue have been reported, including receptor-mediated transcytosis (RMT) of antibodies using transferrin receptors. We hypothesize that this strategy could be further improved by the use of single-domain antibodies (sdAbs), such as the variable domain of heavy-chain-only antibodies (HCAbs), or variable new antigen receptors (VNARs), which are significantly smaller, and therefore could be used to more efficiently transport drugs of interest across the BBB. To this end, we developed anti-transferrin receptor 1 (TFR1) HCAbs utilizing our fully human heavy-chain-only antibody mice (RenNano®). We immunized RenNano® mice with recombinant TFR1 proteins, isolated the B cells from spleen and lymph nodes, and performed single B cell antibody screening using the Beacon® Optofluidic system. Most of the antibodies tested were cross-reactive to human and monkey TFR1. Furthermore, even though the antigen specificity relies on the VHH domain instead of a conventional antibody variable domain, the affinity of these HCAbs can reach 10−8-10−9 (KD). Of the 7 HCAbs tested, 6 were internalized into the human brain microvascular endothelial cell line, hCMEC/D3. To assess brain penetration of these antibodies in vivo, mice expressing human TFR1 (hTFR1 mice) received a tail vein injection with either isotype control, positive control pabinafusp alfa (a BBB penetrating anti-TFR1 monoclonal antibody enzyme conjugate) analog or RenNano®-derived HCAbs. After 0.5, 6, 24, and 72 h of exposure, mice brains were dissected for the quantification of HCAbs and for immunohistochemical analyses. The levels of anti-TFR1 HCAbs in the parenchyma was significantly higher than isotype controls and pabinafusp alfa analog. In brain sections, HCAbs were clearly observed in the parenchyma, and were colocalized with TFR1-expressing cells. These results demonstrate that HCAbs developed from RenNano® mice are able to penetrate the BBB. Taken together, these data highlight the tremendous potential for HCAbs and its variable domain sdAbs for transporting cargo across the BBB. Due to their smaller size and simpler structure, sdAbs could ultimately provide therapeutic benefit for neurodegenerative diseases, and offer promising potential for tumor penetration. Citation Format: Yiqing Hu, Lijun Zhang, Wenying Wang, Huizhen Zhao, Jiawei Yao, Chunhui Lv, Yunsheng Yao, Li Hui, Qingcong Lin, Taolin Liu, Yuelei Shen. Discovery of RenNano®-derived human heavy-chain-only antibodies that cross the blood-brain barrier [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB211.

The homeostasis of the central nervous system is maintained by the blood–brain barrier (BBB). Angiopoietins (Ang-1/Ang-2) act as antagonizing molecules to regulate angiogenesis, vascular stability, vascular permeability and lymphatic integrity. However, the precise role of angiopoietin/Tie2 signaling at the BBB remains unclear. We investigated the influence of Ang-2 on BBB permeability in wild-type and gain-of-function (GOF) mice and demonstrated an increase in permeability by Ang-2, both in vitro and in vivo. Expression analysis of brain endothelial cells from Ang-2 GOF mice showed a downregulation of tight/adherens junction molecules and increased caveolin-1, a vesicular permeability-related molecule. Immunohistochemistry revealed reduced pericyte coverage in Ang-2 GOF mice that was supported by electron microscopy analyses, which demonstrated defective intra-endothelial junctions with increased vesicles and decreased/disrupted glycocalyx. These results demonstrate that Ang-2 mediates permeability via paracellular and transcellular routes. In patients suffering from stroke, a cerebrovascular disorder associated with BBB disruption, Ang-2 levels were upregulated. In mice, Ang-2 GOF resulted in increased infarct sizes and vessel permeability upon experimental stroke, implicating a role of Ang-2 in stroke pathophysiology. Increased permeability and stroke size were rescued by activation of Tie2 signaling using a vascular endothelial protein tyrosine phosphatase inhibitor and were independent of VE-cadherin phosphorylation. We thus identified Ang-2 as an endothelial cell-derived regulator of BBB permeability. We postulate that novel therapeutics targeting Tie2 signaling could be of potential use for opening the BBB for increased CNS drug delivery or tighten it in neurological disorders associated with cerebrovascular leakage and brain edema.Electronic supplementary materialThe online version of this article (doi:10.1007/s00401-016-1551-3) contains supplementary material, which is available to authorized users.

Angiogenesis, the growth of new blood vessels, is a natural defense mechanism helping to restore oxygen and nutrient supply to the affected brain tissue following an ischemic stroke. By stimulating vessel growth, angiogenesis may stabilize brain perfusion, thereby promoting neuronal survival, brain plasticity, and neurologic recovery. However, therapeutic angiogenesis after stroke faces challenges: new angiogenesis-induced vessels have a higher than normal permeability, and treatment to promote angiogenesis may exacerbate outcomes in stroke patients. The development of therapies requires elucidation of the precise cellular and molecular basis of the disease. Microenvironment homeostasis of the central nervous system is essential for its normal function and is maintained by the blood-brain barrier (BBB). Tight junction proteins (TJP) form the tight junction (TJ) between vascular endothelial cells (ECs) and play a key role in regulating the BBB permeability. We demonstrated that after stroke, new angiogenesis-induced vessels in peri-infarct areas have abnormally high BBB permeability due to a lack of major TJPs in ECs. Therefore, promoting TJ formation and BBB integrity in the new vessels coupled with speedy angiogenesis will provide a promising and safer treatment strategy for improving recovery from stroke. Pericyte is a central neurovascular unite component in vascular barriergenesis and are vital to BBB integrity. We found that pericytes also play a key role in stroke-induced angiogenesis and TJ formation in the newly formed vessels. Based on these findings, in this article, we focus on regulation aspects of the BBB functions and describe cellular and molecular special features of TJ formation with an emphasis on role of pericytes in BBB integrity during angiogenesis after stroke.

Chimeric human/chicken transferrin receptors have been constructed using the polymerase chain reaction. Different regions of the 671-residue external domain of the human transferrin receptor were replaced by the corresponding sequences from the chicken transferrin receptor. As chicken transferrin receptors do not bind human transferrin, functional analysis of such chimeric receptors provides an approach to define the ligand-binding site of the human transferrin receptor. Four of 16 chimeric human/chicken transferrin receptors expressed in chick embryo fibroblasts were efficiently transported to the plasma membrane and displayed on the cell surface. Studies of the four chimeric receptors indicated that binding of human transferrin was abolished if the carboxy terminal 192 amino acids of the human transferrin receptor (residues 569-760) were replaced with the corresponding region from the chicken transferrin receptor. Further, a chimeric receptor in which the carboxy-terminal 72 residues were derived from the chicken transferrin receptor exhibited a 16-fold decrease in binding affinity for human transferrin. In contrast, analysis of the other two chimeric receptors showed that 340 amino acids of the human transferrin receptor external domain more proximal to the transmembrane region (residues 151-490) could be replaced with the corresponding region from the chicken transferrin receptor without loss of high-affinity ligand binding. In contrast, two mAbs against the human transferrin receptor external domain, B3/25 and D65.3, that do not compete with transferrin binding, do not bind the chimeric transferrin receptors in which the membrane proximal part is replaced by chicken sequences, while they do bind the two other chimeric transferrin receptors with high affinity. These data indicate that sequence differences in the carboxy-terminal region of human and chicken transferrin receptor external domains are important for the species specificity of transferrin binding and imply that this portion of the human transferrin receptor is critical for ligand binding.

The blood-brain barrier (BBB), while being the gatekeeper of the central nervous system (CNS), is a bottleneck for the treatment of neurological diseases. Unfortunately, most of the biologicals do not reach their brain targets in sufficient quantities. The antibody targeting of receptor-mediated transcytosis (RMT) receptors is an exploited mechanism that increases brain permeability. We previously discovered an anti-human transferrin receptor (TfR) nanobody that could efficiently deliver a therapeutic moiety across the BBB. Despite the high homology between human and cynomolgus TfR, the nanobody was unable to bind the non-human primate receptor. Here we report the discovery of two nanobodies that were able to bind human and cynomolgus TfR, making these nanobodies more clinically relevant. Whereas nanobody BBB00515 bound cynomolgus TfR with 18 times more affinity than it did human TfR, nanobody BBB00533 bound human and cynomolgus TfR with similar affinities. When fused with an anti-beta-site amyloid precursor protein cleaving enzyme (BACE1) antibody (1A11AM), each of the nanobodies was able to increase its brain permeability after peripheral injection. A 40% reduction of brain Aβ1-40 levels could be observed in mice injected with anti-TfR/BACE1 bispecific antibodies when compared to vehicle-injected mice. In summary, we found two nanobodies that could bind both human and cynomolgus TfR with the potential to be used clinically to increase the brain permeability of therapeutic biologicals.

The beta-amyloid precursor protein (APP) is proteolytically processed to generate beta-amyloid protein, the principal protein component of neuropathological lesions characteristic of Alzheimer's disease. To investigate potential sorting signals in the cytoplasmic tail of APP, we transplanted APP cytoplasmic tail sequences into the cytoplasmic tail of the human transferrin receptor (TR) and showed that two sequence motifs from the APP cytoplasmic tail promote TR internalization. One sequence, GYENPTY, is related to the low density lipoprotein receptor internalization signal, FDNPVY, but also involves a critical glycine residue; the other, YTSI, conforms to the 4-residue tyrosine-based internalization signal consensus sequence. Furthermore, a chimeric molecule (APP-TR) consisting of the cytoplasmic domain of APP and the transmembrane and external domains of TR was rapidly internalized enabling the transport of iron into the cell at approximately 50% the rate of wild-type TR. Alanine scanning mutations indicated that the two sequences identified in transplantation experiments were required for internalization of the chimera. Metabolic pulse-chase experiments showed that the APP-TR chimeras were degraded in a post-Golgi membrane compartment within 2-4 h following normal glycosylation. Degradation was partially dependent upon the two internalization signals and was inhibited by ammonium chloride. A fraction of APP-TR chimeras traffic to a degradative endocytic compartment after appearing on the cell surface. Comparison of soluble APP released from cells expressing either full-length human APP or mutant APP with the sequence YENPTY deleted indicated that this sequence is required for sorting of full-length APP along similar trafficking pathways as the APP-TR chimera.

The central nervous system is protected by the blood-brain barrier (BBB). The tight junction (TJ) proteins claudin-5 and zonula occludens-1 (ZO-1) as well as the cytoskeletal component F-actin play key roles in maintaining homeostasis of the BBB. Increases in BBB permeability may be beneficial for the delivery of pharmacological substances into the brain. Therefore, here, we assessed the use of ultrasound to induce transient enhancement of BBB permeability. We used fluorescein isothiocyanate (FITC)-dextran 40 to detect changes in the membrane permeability of bEnd.3 cells during ultrasound treatment. Ultrasound increased FITC-dextran 40 uptake into bEnd.3 cells for 2-6 h after treatment; however, normal levels returned after 24 h. An insignificant increase in lactate dehydrogenase (LDH) leakage also occurred 3 and 6 h after ultrasound treatment, whereas at 24 h, LDH leakage was indistinguishable between the control and treatment groups. Expression of claudin-5, ZO-1, and F-actin at the messenger RNA (mRNA) and protein levels was assessed with real-time polymerase chain reaction and western blotting. Ultrasound induced a transient decrease in claudin-5 mRNA and protein expression within 2 h of treatment; however, no significant changes in ZO-1 and F-actin expression were observed. Claudin-5, ZO-1, and F-actin immunofluorescence demonstrated that the cellular structures incorporating these proteins were transiently impaired by ultrasound. In conclusion, our ultrasound technique can temporarily increase BBB permeability without cytotoxicity to exposed cells, and the method can be exploited in the delivery of drugs to the brain with minimal damage.

Neuronal survival, electrical signaling and synaptic activity require a well-balanced micro-environment in the central nervous system. This is achieved by the blood-brain barrier (BBB), an endothelial barrier situated in the brain capillaries, that controls near-to-all passage in and out of the brain. The endothelial barrier function is highly dependent on signaling interactions with surrounding glial, neuronal and vascular cells, together forming the neuro-glio-vascular unit. Within this functional unit, connexin (Cx) channels are of utmost importance for intercellular communication between the different cellular compartments. Connexins are best known as the building blocks of gap junction (GJ) channels that enable direct cell-cell transfer of metabolic, biochemical and electric signals. In addition, beyond their role in direct intercellular communication, Cxs also form unapposed, non-junctional hemichannels in the plasma membrane that allow the passage of several paracrine messengers, complementing direct GJ communication. Within the NGVU, Cxs are expressed in vascular endothelial cells, including those that form the BBB, and are eminent in astrocytes, especially at their endfoot processes that wrap around cerebral vessels. However, despite the density of Cx channels at this so-called gliovascular interface, it remains unclear as to how Cx-based signaling between astrocytes and BBB endothelial cells may converge control over BBB permeability in health and disease. In this review we describe available evidence that supports a role for astroglial as well as endothelial Cxs in the regulation of BBB permeability during development as well as in disease states.

IntroductionGlioblastoma multiforme (GBM) is one of the deadliest and most invasive brain/glioma cancers, and there is currently no established way to treat this disease. The treatment of GBM typically involves intracranial surgery sometimes followed by chemotherapy and/or radiation therapy. Intracranial treatment is difficult to accomplish; however, as the delivery of the treatment drug is impeded by the blood‐brain barrier (BBB). The BBB is a semipermeable barrier constructed by the brain tissue that prevents certain substances in the bloodstream from entering the brain.ChallengeThe treatment drug happens to be a substance that is prevented entry into the brain by the BBB and cannot pass through the BBB unassisted, which severely impedes its ability to treat GBM.ActionIn this study, we use a nanobubble (NB) embedding chemotherapeutic drug alongside a high‐intensity focused ultrasound (HIFU) oscillation to generate a cavitation impact on the BBB, which helps the drug bypass the BBB and reach the brain. The HIFU is a non‐invasive therapeutic technique that can be used to assist the delivery of drugs into the brain. Being an HIFU reagent, NBs have been proven by recent studies to generate an even more powerful cavitation effect due to the difference in composition and temporarily remove the BBB even more effectively due to the difference composition of NBs between the internal gaseous cavity and the liquid environment, which allows the drugs to pass through the BBB and reach the brain. Additionally, NBs also serve as carriers for functional nanoparticles to be loaded into the hydrophobic core. FePt nanoparticles are functional nanoparticles that produce high resolution images when observed under a T2‐weighted magnetic resonance imaging (MRI).ResolutionLoading FePt nanoparticles into the hydrophobic core of NBs forms a bubble‐based drug delivery system (FePt@NB), and enables MRI to track the changes in GBM. The FePt@NB nanocomposite developed in this study represents a potential breakthrough in GBM treatment, through improved biological imaging and drug delivery into the brain tissue.Support or Funding InformationThis research was supported by Academia Sinica [AS‐SUMMIT‐108] to MH.FePt has excellent T2‐weighted MRI imaging capabilities. As a hard magnetic material in a magnetic material, it has ferromagnetism and high magnetocrystalline anisotropy. The superparamagnetism of FePt NPs has made them attractive candidates to be used as MRI/CT scanning agents and high‐density recording material. This figure shows an MRI image evaluation experimental design of mice with FePt@NB material.Figure 1Protective barriers of the brain. The collective term “blood‐brain barrier” is used to describe four main interfaces between the central nervous system and the periphery. This research has demonstrated that FePt@NB can introduce a sensitive fluorescence signal in the brain. FePt@NB shows great MRI imaging and GBM therapy.Figure 2

Receptor-mediated transcytosis of nanobodies targeting the heparin-binding EGF-like growth factor in human blood-brain barrier models.

Most therapeutic agents are excluded from entering the central nervous system by the blood-brain barrier (BBB). Receptor mediated transcytosis (RMT) is a common mechanism used by proteins, including transferrin (Tf), to traverse the BBB. Here, we prepared Tf-containing, 80-nm gold nanoparticles with an acid-cleavable linkage between the Tf and the nanoparticle core to facilitate nanoparticle RMT across the BBB. These nanoparticles are designed to bind to Tf receptors (TfRs) with high avidity on the blood side of the BBB, but separate from their multidentate Tf-TfR interactions upon acidification during the transcytosis process to allow release of the nanoparticle into the brain. These targeted nanoparticles show increased ability to cross an in vitro model of the BBB and, most important, enter the brain parenchyma of mice in greater amounts in vivo after systemic administration compared with similar high-avidity nanoparticles containing noncleavable Tf. In addition, we investigated this design with nanoparticles containing high-affinity antibodies (Abs) to TfR. With the Abs, the addition of the acid-cleavable linkage provided no improvement to in vivo brain uptake for Ab-containing nanoparticles, and overall brain uptake was decreased for all Ab-containing nanoparticles compared with Tf-containing ones. These results are consistent with recent reports of high-affinity anti-TfR Abs trafficking to the lysosome within BBB endothelium. In contrast, high-avidity, Tf-containing nanoparticles with the acid-cleavable linkage avoid major endothelium retention by shedding surface Tf during their transcytosis.