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. 2026 Aug 28;18(1):2724587. doi: 10.1080/19420862.2026.2724587

Efficient brain delivery and sustained retention of antibodies via dual targeting of blood–brain barrier and intra-brain proteins

Yuki Noguchi a,✉, Naoka Hironiwa b, Kazuki Sato a, Masaki Yamazaki c, Masashi Kawashima c, Nishiki Nagaya b, Keitaro Nakagawa a, Momoko Okuda d, Atsuhiko Kato c, Feng Shu b, Kenta Haraya a, Taichi Kuramochi a
PMCID: PMC13532014  PMID: 42665535

ABSTRACT

Efficient delivery of therapeutic antibodies to the brain has been increasingly achieved using receptor-mediated transcytosis (RMT) approaches such as transferrin receptor (TfR)-mediated shuttle systems. Moreover, a strategy for enhancing brain retention has recently emerged as a focus of attention to overcome the rapid clearance of TfR shuttles from the brain. Here, we propose a novel approach that combines binding to intra-brain proteins, such as myelin oligodendrocyte glycoprotein (MOG), cell adhesion molecule 3, and chondroitin sulfate proteoglycan 5, for sustained retention in the brain with binding to blood–brain barrier (BBB) proteins, such as TfR and insulin-like growth factor 1 receptor, for enhanced BBB permeability. A mouse pharmacokinetic study demonstrated that the anti-MOG/TfR antibody reached a higher maximum concentration and maintained higher concentrations in the brain for 4 months than anti-MOG and anti-TfR antibodies. In immunohistochemistry and brain 3D-imaging study, the anti-MOG/TfR antibody distributed throughout the whole brain, suggesting it penetrates the BBB across the whole brain parenchyma and is retained there. Finally, this antibody technology was applied to brain delivery of neprilysin, an enzyme that degrades amyloid beta, demonstrating that it can enhance both the brain exposure and the pharmacodynamic effect of the potential therapeutic molecule. In conclusion, the concept of achieving sustained high brain exposure by combining BBB transport with enhanced brain retention was shown to be broadly applicable. This novel antibody technology platform is expected to deliver and retain various therapeutic molecules efficiently within the brain, addressing critical challenges in drug development for the central nervous system.

KEYWORDS: Antibody, blood–brain barrier (BBB), brain, pharmacokinetics (PK), retention

Introduction

Delivering a therapeutic antibody to the brain remains a significant challenge in treating central nervous system (CNS) diseases such as Alzheimer’s disease. The main obstacle is the limited permeability of an antibody on the blood–brain barrier (BBB), which prevents antibodies from penetrating into the brain. In recent research, receptor-mediated transcytosis (RMT) has shown promise in enhancing BBB transport of antibodies in mice, non-human primates (NHP) and humans. Several RMT targets that enhance antibody transport across the BBB, including transferrin receptor (TfR),1–4 CD98 heavy chain (CD98hc or SLC3A2),5,6 and insulin-like growth factor receptor 1 (IGF-1 R), have been identified.7 Among these, the TfR-mediated transcytosis approach, which can enhance the maximum brain concentration of an antibody by approximately 10- to 30-fold compared with conventional antibodies, is the most advanced.1,8 However, antibodies transported into the brain using this method are rapidly eliminated through cerebrospinal fluid (CSF)/interstitial fluid (ISF) bulk flow, reverse transcytosis from the brain to the blood, and internalization and degradation in neural cells, making it difficult to maintain long-term antibody exposure in the brain after a single dose.1,8,9 Anti-TfR antibodies that bind to target antigens with slow turnover, such as amyloid beta (Aβ) plaques, have been reported to exhibit slow brain clearance because of target binding within the brain.10 However, when target antigens turn over rapidly or when anti-TfR antibodies do not bind to an intra-brain target, additional strategies are required to maintain antibody exposure in the brain.

Several approaches for overcoming this issue have been recently reported. One is the use of CD98hc. In addition to increasing maximum brain concentrations, anti-CD98hc antibodies show longer half-life in the brain than anti-TfR antibodies in mice and non-human primates (NHP) because anti-TfR antibodies appear to be internalized and degraded in brain parenchymal cells more rapidly than anti-CD98hc antibodies.5,6 Furthermore, antibodies that bind to both TfR and CD98hc have been shown to exhibit additive effects on brain antibody concentrations.11 Another approach involves binding to myelin oligodendrocyte glycoprotein (MOG). MOG is known to be a component of the myelin sheath formed by oligodendrocytes around neurons,12 and its turnover is quite slow.13 Anti-MOG antibodies are reported to accumulate in the brain over time, although the brain concentrations at early time points after dosing are as low as those of control antibodies.8 Combining binding to both TfR and MOG has been found to extend half-life of antibodies in the brain.14 However, several aspects remain to be elucidated, including long-term antibody retention over periods of several months, distribution in CNS tissues beyond the brain such as the spinal cord and optic nerve, the effects of combining alternative BBB transport and retention moieties other than TfR and MOG, and the application to the brain delivery of the therapeutic molecule.

In this study, we aimed to enhance BBB transport and retention of antibodies in the brain through the dual targeting of BBB and intra-brain proteins (Figure 1). To demonstrate the versatility of this concept, brain exposure of antibodies binding to TfR and IGF-1 R as RMT targets and to MOG, cell adhesion molecule 3 (CADM3), and chondroitin sulfate proteoglycan 5 (CSPG5) as retention targets was evaluated. CADM3, also known as nectin-like molecule 1 (Necl-1), is reported to be a neural tissue-specific cell–cell adhesion molecule.15 CSPG5, also known as neuroglycan C (NGC), is a chondroitin sulfate proteoglycan localized in the brain.16,17 These neural proteins are known to be expressed in neurons and astrocytes and are involved in neural network formation. For the antibody binding to TfR and MOG, intra-brain and peripheral tissue distribution were also examined. The application of this technology to the delivery of neprilysin (NEP), an Aβ-degrading enzyme,18 into the brain was also investigated.

Figure 1.

Dual targeting of BBB proteins and intra-brain proteins enables both efficient brain penetration and prolonged retention of therapeutic molecules, resulting in sustained exposure within the brain.

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Concept of a dual-targeting antibody that binds both the blood–brain barrier and intra-brain proteins.

The antibody comprises three functional moieties: a transport moiety, a retention moiety, and a functional moiety. It is efficiently delivered into the brain via receptor-mediated transcytosis across the blood–brain barrier, facilitated by the transport moiety. Once inside the brain, the antibody binds to its intra-brain target, promoting its retention within the brain parenchyma. The functional moiety may consist of therapeutic agents such as antibodies, peptides, enzymes, neurotrophic factors, or cytokines. BBB, blood–brain barrier; ISF, interstitial fluid; RMT, receptor-mediated transcytosis.

Results

Plasma and brain pharmacokinetics of the antibodies binding to TfR and MOG in mice

To demonstrate the effect of binding to TfR and MOG on the plasma and brain pharmacokinetics (PK), a bivalent anti-keyhole limpet hemocyanin antibody (Bi KLH, negative control IgG), a bivalent anti-MOG antibody (Bi MOG), an anti-KLH antibody fused with an anti-TfR Fab at the C-terminus (Bi KLH/TfR), and an anti-MOG antibody fused with an anti-TfR Fab at the C-terminus (Bi MOG/TfR) were administered intravenously to mice and their concentrations in plasma and brain were measured. The binding affinity for each target, measured by surface plasmon resonance (SPR), is shown in Table 1, and the corresponding sensorgrams are provided in Supplementary Figure S1. The plasma PK profiles of the antibodies are shown in Figure 2a. Similar PK profiles in plasma were observed for both Bi KLH and Bi MOG antibodies. In contrast, Bi KLH/TfR and Bi MOG/TfR antibodies showed lower concentrations starting from day 1 and were eliminated much faster than Bi KLH and Bi MOG antibodies. As shown in Table 2, the clearance and the volume of distribution (Vd) of Bi KLH/TfR and Bi MOG/TfR antibodies were more than 10-fold higher than those of Bi KLH and Bi MOG antibodies. These results suggest that antibodies with the anti-TfR domain decreased from plasma rapidly due to uptake and degradation via systemically expressed TfR.

Table 1.

Binding affinities of antibodies against mouse MOG and mouse TfR.

Antibody Antigen Temperature (°C) ka (M−1·s−1) kd (s−1) KD (M)
MOG/TfR Mouse MOG 25 5.39 × 105 ±1.18 × 104 2.05 × 10−2 ±5.86 × 10−4 3.80 × 10−8 ±4.62 × 10−10
KLH/TfR Mouse MOG 25 N.D.
MOG/TfR Mouse TfR 37 1.01 × 105 ±5.85 × 103 3.76 × 10−2 ±4.73 × 10−4 3.72 × 10−7 ±2.61 × 10−8
KLH/TfR Mouse TfR 37 1.01 × 105 ±5.69 × 103 3.78 × 10−2 ±8.54 × 10−4 3.76 × 10−7 ±2.25 × 10−8
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Kinetic parameters were obtained by global 1:1 fitting of double‑referenced SPR sensorgrams. Measurements were performed in triplicate, and values are presented as mean ± SD. N.D. means not determined.

Figure 2.

Dual targeting of TfR and MOG produces synergistic enhancement of brain exposure, providing both high brain concentrations and long-term retention compared with antibodies targeting either molecule alone.

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Plasma and brain PK of the antibodies binding to TfR and MOG.

The Bi KLH, Bi MOG, Bi KLH/TfR, and Bi MOG/TfR antibodies were administered intravenously at 2 mg/kg (13.8 nmol/kg for Bi KLH and Bi MOG, 10.2 nmol/kg for Bi KLH/TfR, 10.3 nmol/kg for Bi MOG/TfR) to mice, and the concentrations in plasma and brain were measured until day 112. (a) Plasma PK profiles of the antibodies. (b) Brain PK profiles of the antibodies. (c) Brain/plasma concentration ratio. (d) Percent of injected dose per gram of brain tissue. (e) Cumulative brain area under the curve. Data shows mean ± SD (n = 6). ***p < 0.001 vs all other groups at day 1 and day 112, respectively.

Table 2.

Plasma PK parameters of the antibodies.

Antibody Half-life AUC (0-inf) Clearance Vd
day pmol*day/mL mL/day/kg mL/kg
KLH 21.2 3.46 × 103 3.99 1.36 × 102
MOG 22.7 4.14 × 103 3.34 1.11 × 102
KLH/TfR 21.7 1.80 × 102 56.9 1.30 × 103
MOG/TfR 22.9 1.96 × 102 52.4 1.03 × 103
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Non-compartment model analysis of the plasma PK profiles was performed, and the PK parameters, half-life, area under the curve (AUC), clearance, and volume of distribution (Vd) were estimated. The AUC (0-inf) represents AUC from time zero to infinity.

The brain concentration-time profiles of these antibodies are shown in Figure 2b. At day 1 after dosing, Bi KLH antibody showed a low concentration in brain, but this represented the maximum concentration (Cmax) during the study period. Bi MOG antibody showed a similar concentration to Bi KLH antibody at day 1, whereas the concentration of Bi KLH/TfR antibody was higher than Bi KLH and Bi MOG antibodies and reached Cmax during the study period. Interestingly, Bi MOG/TfR antibody showed an approximately 2-fold higher concentration in brain than Bi KLH/TfR antibody at day 1. A similar tendency was also observed in the brain-to-plasma ratio and the percent of injected dose per gram of brain tissue (%ID/g brain), as shown in Figure 2c,d. These results indicate that combination of the transport moiety (anti-TfR) and the retention moiety (anti-MOG) contributes to increased antibody concentration in the brain within a short duration after antibody injection.

From day 7 to day 112 after dosing, the brain concentrations and the percent of dose of Bi KLH antibody decreased gradually, while the brain-to-plasma ratio was almost constant (Figure 2b-d). The brain concentrations, brain-to-plasma ratio, and the percent of dose of Bi KLH/TfR antibody decreased rapidly. The concentrations after day 14 fell below that of Bi KLH antibody, and the concentrations at day 28 and thereafter were below the limit of quantification.

In contrast, Bi MOG antibody accumulated in the brain in a time-dependent manner due to binding to MOG after transport into the brain. The concentration continued to increase, ultimately reaching Cmax at day 112. Notably, Bi MOG/TfR antibody maintained high concentrations in the brain from day 1 until at least day 112. The Cmax was 3.1 μg/g, corresponding to 7.0%ID/g brain over the study period (Figure 2b-d). These data suggest that the combination of the transport moiety (anti-TfR) and retention moiety (anti-MOG) provides high and long-lasting antibody concentrations in the brain. The cumulative brain area under the curve (AUC), which represents the total antibody exposure in the brain over a given time interval, further supports the synergistic effect of this combination on the brain antibody exposure (Figure 2e).

Intra-brain distribution of the antibodies against TfR and MOG in mice by immunohistochemistry and 3D fluorescence imaging

The distribution of antibodies in the brain was evaluated by immunohistochemistry (IHC) to reveal the localization of the antibodies in each brain region (Figure 3a). At day 1 after dosing, Bi KLH and Bi MOG antibodies were localized around the lateral ventricles including the choroid plexus and hippocampal fimbria, whereas both the Bi KLH/TfR and Bi MOG/TfR antibody were distributed throughout the whole brain. At day 7, the Bi KLH antibody showed little signal, but the Bi MOG antibody showed strong localization, particularly in the hippocampal fimbria and the commissure of the fornix. Interestingly, the Bi KLH/TfR antibody signals almost disappeared, whereas the Bi MOG/TfR antibody signals were still strongly distributed throughout the whole brain. The Bi MOG/TfR antibody localized to nerve fibers, presumably representing MOG-expressing regions. Notably, significant localization was observed in the fornix commissure, parts of the external and internal capsules, striatum, thalamus, and the median eminence of the hypothalamus. Higher magnification images of the Bi MOG/TfR antibody were also shown in the Supplementary Figure S2. These data suggest that the Bi MOG/TfR antibody, similar to the Bi KLH/TfR antibody, penetrates the BBB across the whole brain parenchyma and is furthermore retained throughout the whole brain over a prolonged period.

Figure 3.

Brain imaging demonstrates that TfR-mediated transport promotes widespread distribution throughout the brain, while additional MOG binding enables prolonged retention of the antibody across brain regions.

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Intra-brain distribution of the antibodies against binding to TfR and MOG in mice by IHC and 3D-imaging.

(a) Immunohistochemistry (IHC) of the antibodies against TfR and MOG in the brain. The anti-KLH, anti-MOG, anti-KLH/TfR, and anti-MOG/TfR antibodies were administered intravenously at 20 mg/kg (138 nmol/kg for bivalent KLH and bivalent MOG, 102 nmol/kg for bivalent KLH/TfR, 103 nmol/kg for bivalent MOG/TfR) to mice, and the imaging was performed at day 1 and 7. The signal was analyzed using Halo AI, with increasing signal intensity indicated by yellow, orange, and red. The nuclei were stained with hematoxylin and are shown in blue-purple. Representative images of antibody distribution in the mouse cerebrum are shown. The experiment was conducted in n = 3, and one representative image was shown. (b) 3D imaging of the antibodies against TfR and MOG in the brain. The Alexa Fluor 647-labeled anti-KLH, anti-MOG, anti-KLH/TfR, and anti-MOG/TfR antibodies were administered intravenously at 20 mg/kg (138 nmol/kg for bivalent KLH and bivalent MOG, 102 nmol/kg for bivalent KLH/TfR, 103 nmol/kg for bivalent MOG/TfR) in mice, and the fluorescence of the cleared brain tissue was observed by a light-sheet microscope. White signals indicate the administered antibody. The animal experiment was conducted in n = 4, and one representative image was shown.

3D fluorescence imaging was also performed to characterize the spatial localization of the antibodies. Alexa Fluor 647-labeled antibodies were administered into mice, and brains were collected at days 1 and 7. For non-treated controls, vehicle (PBST) was administered and brains were collected at day 1. Brain tissues were fixed and cleared then imaged using light-sheet microscopy. As shown in Figure 3b, Bi KLH and Bi MOG antibodies exhibited limited localization within the brain, especially around the thalamus and hippocampus. In contrast, Bi KLH/TfR and Bi MOG/TfR antibodies demonstrated widespread distribution throughout the entire brain. Notably, the Bi MOG/TfR antibody showed strong signals in the brain even at day 7, while the Bi KLH/TfR antibody did not. The spatial distribution of antibodies throughout the entire brain parenchyma is further illustrated in a rotating 3D video (Supplementary Video S1), and the regional distribution of antibodies in the brain is provided in Supplementary Figure S3.

Tissue distribution of the antibodies against TfR and MOG in mice

The transport and retention of the antibodies in CNS tissues and peripheral nervous system (PNS) tissues were investigated in mice. In PNS tissues, including liver, lung, muscle, and spleen, no accumulation of any of the antibodies through day 7 was observed (Figure 4). Especially in the liver and spleen, higher concentrations of Bi KLH/TfR and Bi MOG/TfR antibodies than Bi KLH and Bi MOG antibodies were found at day 1, but the concentration had largely decreased by day 7. In the retina, where oligodendrocytes do not exist in mice and humans, no retention of the antibodies through day 7 was observed. These findings suggest that antibodies containing the retention moiety do not persist in PNS tissues, likely due to the absence of MOG-expressing oligodendrocytes in these tissues.

Figure 4.

MOG-targeting antibodies preferentially accumulate in central nervous system tissues. Combining MOG and TfR targeting further increases exposure in the brain, optic nerve, spinal cord, and other CNS tissues while limiting persistence in peripheral tissues.

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Tissue distribution of the antibodies binding to TfR and MOG in mice.

The anti-KLH, anti-MOG, anti-KLH/TfR, and anti-MOG/TfR antibodies were administered intravenously at 10 mg/kg (69.0 nmol/kg for bivalent KLH, 69.1 nmol/kg for bivalent MOG, 51.2 nmol/kg for bivalent KLH/TfR, 51.3 nmol/kg for bivalent MOG/TfR) to mice, and the antibody concentrations in following tissues were measured at day 1 and 7: plasma, brain, liver, muscle, spleen, lung, retina, optic nerve, spinal cord, olfactory bulb, and medulla oblongata. Data shows mean ± SD (n = 4). ***p < 0.001; ns, not significant vs KLH/TfR antibody at day 7, respectively. ###p < 0.001 ##p < 0.01, and #p < 0.05; ns, not significant vs MOG antibody at day 7, respectively.

In contrast, Bi MOG and Bi MOG/TfR antibodies accumulated in the CNS tissues such as the optic nerve, spinal cord, olfactory bulb, and medulla oblongata through day 7, whereas Bi KLH and Bi KLH/TfR antibodies decreased over the same period (Figure 4). The Bi MOG/TfR antibody showed higher concentrations than the other antibodies even at day 1, with continued accumulation until day 7. These results indicate that antibodies with the anti-MOG retention moiety bind to MOG on oligodendrocytes and are retained in these CNS tissues, and that the combination of the transport and retention moieties has synergistic effects on antibody concentrations in these CNS tissues from as early as day 1.

Plasma and brain PK of the antibodies binding to other BBB transport targets and retention targets in mice

To investigate whether target molecules other than TfR and MOG can also confer high BBB penetration and sustained retention similar to the anti-MOG/TfR antibody, IGF-1 R was evaluated as a BBB transport molecule, and CADM3 and CSPG5 were evaluated as retention molecules. The anti-KLH/MOG bispecific (Mono MOG), anti-KLH/CADM3 bispecific (Mono CADM3), and anti-CSPG5 bivalent (Bi CSPG5) antibodies were used as retention-only controls, whereas the anti-KLH/TfR (Mono TfR) and anti-KLH/IGF-1 R (Mono IGF-1 R) antibodies were served as BBB transport-only controls. The anti-MOG/TfR bispecific, anti-CADM3/TfR bispecific, anti-CSPG5 IgG fused with anti-TfR Fab (Bi CSPG5/TfR), and anti-MOG/IGF-1 R bispecific antibodies were used to evaluate the dual-targeting strategy. Compared to antibodies binding to retention targets only, the antibodies binding to both TfR and retention targets such as CADM3 and CSPG5 showed faster elimination from plasma until day 7 (Figure 5a). Conversely, antibodies binding to both TfR and retention targets showed higher brain concentrations at day 1 and were well retained in the brain at day 7 compared to the Mono TfR or anti-retention target antibodies (Figure 5b). The Mono CADM3 and Bi CSPG5 exhibited higher brain concentrations at day 7 than at day 1. On the other hand, the CADM3/TfR bispecific and Bi CSPG5/TfR antibodies showed much higher concentrations compared to the retention-only molecules, further demonstrating the synergistic potency of combining BBB transport with brain-retention mechanisms.

Figure 5.

Enhanced brain exposure is not limited to TfR and MOG. Combining different BBB transport targets and brain-retention targets consistently increases brain concentrations and duration compared with either strategy alone.

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Plasma and brain PK of the antibodies binding to the other BBB targets and retention targets.

The anti-MOG, anti-CADM3, and anti-CSPG5 antibodies, with or without TfR or IGF-1 R binding, were administered intravenously at 2 mg/kg (10.2–13.8 nmol/kg for CADM3 and CSPG5 evaluation, 17.7–17.8 nmol/kg for IGF-1 R evaluation) to mice, and the concentrations in plasma and brain were measured at day 1 and 7. (a) Plasma and (b) brain concentrations of the anti-MOG, anti-CADM3, and anti-CSPG5 antibodies, with or without TfR binding. (c) Plasma and (d) brain concentrations of the anti-MOG antibody, with or without IGF-1 R binding. Data shows mean ± SD (n = 3). ***p < 0.001 and **p < 0.01 vs Mono TfR or Mono IGF-1 R antibody at day 7, respectively. ###p < 0.001 and #p < 0.05; ns, not significant vs each retention only antibody at day 7, respectively.

IGF-1 R was also investigated as a BBB transport moiety in combination with a retention moiety. No clear differences were found in plasma PK among antibodies; however, the brain concentrations at day 7 increased for the antibodies binding to both IGF-1 R and MOG compared to the Mono IGF-1 R or the Mono MOG antibodies (Figure 5c,d). Together with the TfR and MOG findings, these results suggest that the combination of the binding to a BBB transport moiety and a retention moiety has a synergistic effect, enabling long-lasting high concentrations of the antibodies in the brain compared with antibodies possessing a BBB transport moiety or retention moiety alone.

Brain delivery of neprilysin using the antibodies against TfR and MOG in mice

The anti-MOG/TfR antibody was utilized to deliver neprilysin (NEP), an enzyme responsible for degrading an amyloid-beta (Aβ). The structure of the NEP-fused antibody is shown in Figure 6a. Following antibody administration, the concentrations of both the antibodies and Aβ were monitored in plasma and brain. Mono KLH/TfR-NEP and mono MOG/TfR-NEP antibodies exhibited rapid clearance from plasma compared to Bi KLH and mono KLH-NEP antibodies (Figure 6b). In contrast, the mono MOG/TfR-NEP antibody maintained the highest concentration in the brain throughout the study period, whereas the mono KLH/TfR-NEP antibody was eliminated more rapidly from the brain (Figure 6c). Moreover, the mono MOG/TfR-NEP antibody significantly reduced brain Aβ levels compared to the mono KLH/TfR-NEP antibody (Figure 6d), suggesting that dual targeting of TfR and MOG enhances the delivery and retention of NEP in the brain.

Figure 6.

Dual targeting of MOG and TfR improves brain delivery and retention of NEP, resulting in greater reduction of brain Aβ1-40 levels than a TfR-only delivery approach.

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PK/PD of the neprilysin-fused antibodies against MOG and TfR.

The neprilysin-fused antibodies were administered intravenously at 50 nmol/kg in mice, and the concentrations of the antibodies and Aβ1-40 in plasma and brain were measured at day 1 and 3. (a) Illustration of the neprilysin-fused antibodies. (b) Plasma concentrations of the antibodies. (c) Brain concentrations of the antibodies. (d) Brain concentrations of Aβ1-40. Data shows mean ± SD (n = 6). ***p < 0.001 vs all other groups at day 1 and day 3, respectively.

Discussion

In this study, we demonstrated that the combination of RMT target binding for enhanced BBB transport and intra-brain protein binding for robust retention works synergistically to achieve long-lasting high antibody concentrations in the brain and distribution throughout the whole brain. Specifically, CADM3 and CSPG5 are effective for retention in the brain in addition to MOG.

TfR-mediated BBB transcytosis is one of the most promising approaches for enhancing the brain delivery of antibodies. Abundant expression of TfR on the BBB and fast internalization kinetics into brain endothelial cells are considered to contribute to the efficient delivery of antibodies to the brain.19 However, several PK challenges remain for anti-TfR antibodies, including rapid clearance from both plasma and brain. The abundant peripheral expression of TfR contributes to the accelerated elimination of anti-TfR antibodies from plasma, thereby reducing their availability for the brain delivery. As shown in Figure 2a, the Bi MOG/TfR antibody exhibited rapid clearance from plasma, similar to the Bi KLH/TfR antibody. This suggests that binding to TfR – rather than MOG – primarily influences plasma clearance, likely due to the CNS-specific expression of MOG. A similar tendency was also found for anti-CADM3/TfR bispecific and anti-CSPG5/TfR (Bi CSPG5/TfR) antibodies (Figure 5a). While rapid plasma clearance is a common feature of TfR-targeting antibodies, antibodies that bind to both the receptors on the BBB and intra-brain proteins achieved sustained brain exposure. This profile likely minimizes systemic side effects while maintaining therapeutic efficacy within the brain.

In the brain, the Bi MOG/TfR antibody exhibited higher concentration at day 1 compared to the Bi KLH/TfR antibody, and this elevated level was maintained through day 112 (Figure 2b). TfR is also expressed on the abluminal side of BBB endothelial cells, facilitating reverse transcytosis of anti-TfR antibodies from the brain back into the plasma.20,21 It is also expressed on neural cells, which promotes the internalization and degradation of anti-TfR antibodies.22,23 Large ISF/CSF bulk flow also influences the brain clearance of antibodies.24 The supply of antibodies from plasma to brain continues to decrease due to rapid plasma clearance, as discussed above. Taken together, these mechanisms likely contribute to the rapid clearance of anti-TfR antibodies from the brain. In contrast, MOG exhibits a very slow turnover, with approximately 14% remaining after 6 months.13 Therefore, once the antibody binds to MOG, it is expected to remain associated with MOG-expressing oligodendrocytes, contributing to the sustained high concentrations observed in the brain. Although the Bi MOG antibody gradually accumulates in the brain, its concentration ultimately remains lower than that of the Bi MOG/TfR antibody. These findings suggest that dual targeting of TfR and MOG compensates for the low initial brain exposure of the anti-MOG antibody and the reduced exposure of the anti-TfR antibody at later time points.

The concept of dual targeting of the BBB and intra-brain proteins was further demonstrated using the antibodies that bind to IGF-1 R as a BBB-penetrating molecule and to CADM3 (Necl-1) or CSPG5 (neuroglycan C) as retention molecules. At day 7, brain concentrations were higher for the antibody targeting both IGF-1 R and MOG compared to those of anti-IGF-1 R and anti-MOG antibodies (Figure 5d). These results suggest that BBB transport molecules other than TfR can also enhance the brain delivery and retention when combined with intra-brain retention targets. The retention molecules, CADM3 and CSPG5, are expressed extracellularly on neurons and astrocytes and function as structural components that support the neural network. Consequently, their turnover is presumed to be relatively slow. Antibodies targeting both TfR and either CADM3 or CSPG5 (CADM3/TfR bispecific and Bi CSPG5/TfR antibodies) showed higher brain concentrations at day 7 compared to Mono TfR, Mono CADM3, and Bi CSPG5 antibodies (Figure 5b). However, the brain retention of CADM3 and CSPG5 antibodies was weaker than the MOG antibody at day 7. It is likely that the faster turnover of these molecules compared to MOG affects their retention. These findings further indicate that brain retention molecules other than MOG can also be utilized to enhance drug exposure in the brain.

Dual targeting of the BBB and intra-brain proteins has a significant impact on the distribution in the brain as well as the brain concentration of the antibodies. In Figure 3a, IHC revealed that the control (Bi KLH) and Bi MOG antibodies were localized to the choroid plexus in the lateral ventricle and hippocampal fimbria, while both the Bi KLH/TfR and Bi MOG/TfR antibodies were distributed throughout the whole brain. These data suggest that the anti-MOG/TfR antibody (Bi MOG/TfR), similar to the anti-TfR antibody (Bi KLH/TfR), penetrates the BBB across the whole brain parenchyma via TfR, while the non-RMT antibodies (i.e., Bi KLH and Bi MOG antibodies) would enter the brain mainly through the blood-cerebrospinal fluid barrier (BCSFB). The restricted distribution of the Bi MOG antibody to the periventricular region may lead to local saturation of MOG binding sites and flow out from CSF, resulting in lower whole brain exposure compared to the Bi MOG/TfR antibody, as shown in Figure 2b. Furthermore, at day 7 post-administration, the Bi MOG/TfR antibody appears to be retained in MOG-expressing oligodendrocytes, whereas the Bi KLH/TfR antibody does not (Figure 3a and Supplementary Figure S2). At day 1, asymmetric antibody distribution was observed in the Bi KLH/TfR antibody, not in the Bi MOG/TfR antibody; however, this is considered to be due to procedural factors such as brain perfusion and staining, rather than biological laterality because it was not observed in 3D fluorescence imaging shown in Figure 3b. These data further support the sustained retention of the anti-MOG/TfR antibody in the brain.

Tissue clearing-based whole brain imaging further supports the impact of dual targeting on antibody brain distribution (Figure 3b, Supplementary Figure S3, and Supplementary Video S1). The Bi MOG antibody exhibited a wing-shaped localization in the central brain, suggesting distribution in areas adjacent to CSF flow, such as the thalamus and hippocampus. This restricted distribution pattern indicates the difficulty of achieving whole brain delivery of antibodies using a single targeting approach. In contrast, while anti-TfR antibodies (i.e., Bi KLH/TfR and Bi MOG/TfR antibodies) distributed throughout the entire brain, their fluorescence signals vanished by day 7. Although almost no signal was detected for the Bi KLH/TfR antibody at day 7, it was detected in the antibody quantification of brain lysates (Figure 2b), which is likely due to differences in measurement sensitivity. Notably, the dual targeting antibody acted synergistically to overcome these individual limitations, demonstrating sustained and widespread distribution throughout the whole brain.

As described above, the intra-brain localization of the anti-MOG/TfR antibody was evaluated in detail by IHC and 3D fluorescence imaging; however, other retention molecules such as CADM3 and CSPG5 were not assessed in this study. Because these molecules are reported to be expressed in neurons and astrocytes,15–17 unlike MOG, they may exhibit intra-brain distribution patterns distinct from that of the anti-MOG/TfR antibody. Clarifying this point will be an important subject for future investigation. The Bi MOG/TfR antibody demonstrated enhanced transfer and retention not only in the brain, but also in other CNS tissues such as the spinal cord, medulla oblongata, and optic nerve (Figure 4). CNS tissues contain oligodendrocytes that express MOG and surround neuronal axons. In contrast, PNS tissues, including the liver, muscle, and lung, contain Schwann cells instead of oligodendrocytes around peripheral neurons.25 Therefore, the Bi MOG/TfR antibody is expected to preferentially remain in CNS tissues, including the brain, while minimizing exposure in peripheral tissues. This dual targeting of the BBB and intra-brain proteins is anticipated to enable the efficient delivery and sustained retention of therapeutic molecules within CNS tissues including the spinal cord and optic nerve over the brain, while potentially reducing peripheral side effects. These experimental findings suggest the potential applicability of this concept to the treatment of spinal cord injury and ophthalmic diseases, addressing a broad range of CNS disorders.

Finally, the therapeutic potential of the dual targeting strategy was evaluated using the anti-MOG/TfR antibody conjugated with NEP (Figure 6). At the terminal time point, higher concentrations of the mono MOG/TfR-NEP antibody and lower Aβ levels were observed in the brain compared to the mono KLH/TfR-NEP antibody, suggesting that the dual targeting antibody enhances both the delivery and retention of NEP in the brain, thereby facilitating the reduction of brain Aβ. Compared with a previous report,26 the mono MOG/TfR-NEP antibody appears to have better antibody concentrations and Aβ reduction. Moreover, because mono MOG/TfR-NEP antibody showed better PK/PD than mono KLH/TfR-NEP antibody through day 3, longer term studies are expected to further clarify the advantage of this antibody. While this study focused on enzyme delivery, the dual targeting strategy could also be applied to other therapeutic modalities, including peptides, neurotrophic factors, and cytokines. For antibodies targeting intra-brain antigens, the applicability and added value of this technology should be considered in light of the properties of the target antigen. For antigens with slow turnover, such as Aβ plaques, antibody binding itself may promote prolonged retention of antibodies in the brain. However, if free (therapeutic target-unbound) antibodies can be retained longer through binding to a retention target such as MOG, they may subsequently bind to newly generated target antigens, potentially leading to prolonged efficacy. In contrast, for antigens with rapid turnover, this approach is expected to be beneficial for improving antibody retention in the brain. In the future, broad applications of dual targeting antibodies for the treatment of CNS disorders are highly anticipated and warrant further investigation.

From the perspective of safety concerns associated with targeting MOG, MOG is generally known to be involved in autoimmune diseases mediated by anti-MOG autoantibodies, such as myelin oligodendrocyte glycoprotein antibody-associated disease (MOGAD). In this study, however, we used an Fc domain that does not bind to Fc gamma receptors and therefore lacks effector function. Accordingly, the safety risk associated with MOG targeting is presumed to be low, and indeed no abnormalities were detected in the PK studies. Nevertheless, it will be important to further evaluate safety at higher doses, as well as in monkeys and humans. Regarding the clinical translatability of this concept, MOG is similarly expressed in human oligodendrocytes and constitutes the myelin sheath. Therefore, it is expected to exhibit high expression levels and a long half-life in the human brain. By dually targeting TfR and MOG, this approach is anticipated to achieve both enhanced BBB penetration and sustained retention in humans. Further studies using in vitro assays and NHP are warranted to evaluate the clinical translatability of this approach in humans. In addition, PK/PD evaluation was conducted using male normal mice in this study. The effects of factors, such as sex and age, and the pathology on disease models of brain retention should be further investigated in future studies. Importantly, the dose used in this study was within the range of those evaluated in clinical trials of therapeutic antibodies utilizing TfR-mediated brain delivery, such as pabinafusp alfa and trontinemab.3,27 In comparison with previous studies of anti-Aβ antibodies, a universal threshold of brain antibody concentrations and brain Aβ reduction that predicts therapeutic effect has not been established; however, previous studies of anti-Aβ antibodies suggest that lowering of pathogenic Aβ species can be associated with functional improvement.28,29 The antibody brain concentrations at 2 mg/kg in our study (Figure 2b) are within the range reported for antibodies associated with pharmacological activity and therapeutic benefit. Therefore, the retained brain concentrations observed in this study are considered pharmacologically relevant and support the clinically translational potential of our approach. However, the antibody brain concentrations, pharmacodynamic responses, and therapeutic efficacy depend on the disease, target, and mechanism of action. Further studies are required to establish the quantitative relationship between brain exposure, pharmacodynamic effects, and therapeutic efficacy in disease model mice.

In conclusion, the combination of RMT protein binding for enhanced BBB transport and intra-brain protein binding for robust retention acts synergistically to achieve sustained, high antibody concentrations in the brain. This dual targeting strategy holds promise for the effective delivery and prolonged retention of various therapeutic molecules within the brain, thereby addressing key challenges in CNS drug development.

Materials and methods

Animals

C57BL/6J mice (male, 6–8 weeks) were purchased from Charles River Laboratories Japan, Inc (Japan).

Animal experiments

The animal studies were performed in compliance with the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines (https://arriveguidelines.org/). All experiments were approved by the Institutional Animal Care and Use Committee (IACUC) in Chugai Pharmaceutical Co., Ltd, which is accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC), under protocol number 21–327. All experiments were conducted in Chugai Pharmaceutical Co., Ltd.

Mice were group-housed, typically three animals per cage, under standard laboratory conditions. Animals were randomly allocated to each experimental group. The number of animals per group was determined based on prior experience, published literature, and consideration of expected experimental variability. All data obtained from individual animals were included for analysis. No blinding was performed during group allocation, conduct of the experiment, outcome assessment, or data analysis.

For intravenous administration, test articles were administered as a bolus injection via the tail vein. Blood samples were collected from the jugular vein. For terminal tissue sampling, mice were anesthetized with isoflurane and euthanized by exsanguination during transcardial perfusion in accordance with institutional animal welfare guidelines.

Preparation of antibodies and NEP-fused antibodies

Recombinant antibodies were transiently expressed in Expi293F cells (Thermo Fisher Scientific) or stably expressed by CHO K1 cells using plasmids for immunoglobulin heavy and light chains and purified via Protein A chromatography, followed by multimodal cation exchange chromatography and/or size exclusion chromatography to remove variants. Anti-MOG/TfR (Bi MOG/TfR), anti-KLH/TfR (Bi KLH/TfR), and anti-CSPG5/TfR (Bi CSPG5/TfR) antibodies feature a single-chain Fab targeting the transferrin receptor at the Fc C-terminal. Bispecific antibody with monovalent anti-TfR single-chain Fab (clone 8D3) was generated using knobs-into-holes engineering: one heavy chain carried a CH3 knobs mutation with the anti-TfR Fab linked via a 19-amino-acid Gly-Ser linker, and another heavy chain contained a CH3 holes mutation; three plasmids encoding these components were transfected and purified as above.

Bispecific antibodies with anti-TfR were made using controlled Fab-Arm Exchange,30 where parental homodimers were produced, mixed in equal molar ratios, reconstituted with 25 mmol/L 2-MEA, and dialyzed to remove 2-MEA. Bispecific antibodies targeting IGF-1 R were prepared as VHH/Fab bispecifics via knobs-into-holes engineering, introducing holes mutation into CH3 regions of the anti-IGF-1 R or irrelevant VHH-Fc antibody, and knobs mutation into anti-KLH or anti-MOG antibody; three respective plasmids were used.

NEP-fused antibodies, designed to deliver NEP (human neprilysin, UniProt P08473 aa 56–750) to the brain, were similarly constructed, fusing NEP to the light‑chain C-terminus via a 10-amino-acid Gly-Ser linker. Knobs and holes mutations were introduced as described. All constructs were transfected into Expi293F cells and purified following the same protocol.

The antibody sequences against mouse TfR (clone 8D3), MOG, CADM3, CSPG5, and IGF-1 R (clone IGF1R5) are from Boado et al.,31 WO2018123979, WO2020004492, WO2020004490, and the report from Yogi et al.,32 respectively. All antibodies have Fc domains that do not bind to Fc gamma receptors and therefore lack effector function.

Binding affinity measurement

Binding affinities of antibodies to recombinant mouse MOG were measured by SPR using a Biacore 8K (Cytiva) in an antibody-captured assay format. Anti-Human IgG (Fc) antibody (Cytiva) was immobilized on all flow cells of a Series S Sensor Chip CM4 (Cytiva) using an Amine Coupling Kit (Cytiva). The assay temperature was set to 25°C. All antibodies and analytes were diluted in running buffer consisting of 20 mM ACES, 150 mM NaCl, 0.05% Tween 20, 0.005% NaN3, pH 7.4. Antibodies were captured on an active flow cell at 160–210 RU before analyte injection, while the other flow cell served as a reference without captured antibody. Recombinant mouse MOG protein (R&D Systems, 8536-MO; 12.5–200 nM, two-fold serial dilution) was injected at 10 μL/min for 120 seconds, followed by a 120 second dissociation phase. The surface was regenerated with two injections of 3 M MgCl2 at 30 μL/min for 18 seconds each.

Binding affinities to recombinant mouse TfR were determined at 37°C on a Biacore 8K+ (Cytiva) using the biotin CAPture kit (Cytiva). Antibodies and analytes were diluted in the same ACES-based running buffer described above. For each cycle, biotin CAPture reagent was injected followed by recombinant mouse TfR (Acrobiosystems, TFR-M82E3), resulting in immobilization levels of 80–95 RU. Antibodies (62.5–1000 nM, two-fold serial dilution) were injected at 10 μL/min for 120 seconds, followed by 120 seconds dissociation. The sensor surface was regenerated each cycle according to the kit instructions.

Sensorgrams were processed using double referencing, subtracting both the reference flow cell and buffer‑only injections, and were globally fitted to a 1:1 binding model using Biacore Insight Evaluation Software (Cytiva, 5.0.18.22102). Association rate constant (ka, M−1·s−1), dissociation rate constant (kd, s−1), and dissociation constant (KD, M) were obtained from global fitting of the concentration series.

PK study of the antibodies in mice

The antibodies were administered in mice intravenously at a dose of 2 mg/kg. Blood was collected at each time point, and plasma was obtained by centrifugation (12000 rpm, 4°C, 5 min). After blood sampling, phosphate-buffered saline (PBS) was perfused from the heart by inserting a catheter to remove blood, and then brain was collected. 50 mg of the minced whole brain was dissolved by PBS containing NP-40 alternative detergent (Millipore, 492,018-50MLCN) and Complete Mini Protease Inhibitor Cocktail (Roche, 11,836,153,001) with 5 mm stainless beads and was homogenized with a TissueLyser (Qiagen, Germany). After rotation at 4°C for 60 min, samples were centrifuged (15000 rpm, 4°C, 20 min) and the supernatant was collected. To correct obtained antibody concentrations based on the recovery of antibody-spiked control samples, antibody-spiked brain samples were processed in parallel using the same procedure.

Tissue distribution study in mice

The antibodies were intravenously administered at a dose of 10 mg/kg into C57BL/6J mice (male, 6–8 weeks). Blood was collected on day 1 and day 7 after dosing, and then plasma was obtained by centrifugation (12000 rpm, 4°C, 5 min). After blood sampling at each time point, PBS was perfused from the heart by inserting a catheter to remove blood, and then the following tissues were collected: brain, liver, muscle, spleen, lung, retina, optic nerve, spinal cord, olfactory bulb, and medulla oblongata. A part of the minced whole tissues was dissolved by cell extraction buffer (Invitrogen, FNN0011) containing Complete Protease Inhibitor Cocktail (Roche, 11,697,498,001) in a tube with 5 mm stainless beads and was homogenized with a TissueLyser (Qiagen, Germany). Then, the samples were centrifuged (15000 rpm, 4°C, 20 min) and the supernatant was collected.

Measurement of the antibody concentrations in plasma and tissues

The concentrations of the antibodies in plasma, brain, and other tissues were determined by the electro chemiluminescence-immunoassay (ECL-IA). As a capture antibody, an anti-human IgG Fab’2 antibody (LifeSpan BioSciences, LS-C61358) was applied in the ECL plate (Meso Scale Discovery, L15XA or L21XA). After PBS with Tween20 (PBST, Sigma-Aldrich, P3563) containing 1% bovine serum albumin (BSA; Sigma-Aldrich, A7030) was added as blocking solution, diluted plasma and tissue homogenate samples were applied. As a detection antibody, a biotin-labeled anti-human IgG antibody (Bethyl Laboratories, A80-319B) was added. Finally, a streptavidin labeled with SULFO-tag (Meso Scale Discovery, R32AD) was added, followed by the addition of Read buffer (Meso Scale Discovery, R92TC). Signals were detected by MESO SECTOR S 600 (Meso Scale Discovery, United States). The representative standard curves for quantifying antibody concentrations in the plasma and the brain are provided in Supplementary Figure S4.

Monitoring the Aβ1–40 concentrations in brain after dosing of NEP-fused antibodies

NEP-fused antibodies were administered intravenously in mice at a dose of 50 nmol/kg. At 1 day and 3 days after antibody administration, PBS was perfused from the heart by inserting a catheter to remove blood, and then brain was collected. A mouse whole brain was dissolved by 5 M Guanidine (Wako, 070–01825) and homogenized with stainless beads by TissueLyser (Qiagen, Germany). Homogenates were diluted with 1% BSA-PBST containing Complete Mini Protease Inhibitor Cocktail (Roche, 11,697,498,001) and the supernatants were collected after centrifugation (15000 rpm, 4°C, 20 min). Concentrations of Aβ1–40 were determined by commercially available ECL kit (Meso Scale Discovery, K15199E2).

PK analysis of the mouse PK data

Since the long-term PK study employed destructive sampling (one animal per time point), average concentrations were calculated from six individual animals at each time point to generate the concentration-time profiles. The concentration-time profiles of the antibodies in plasma and brain were analyzed by non-compartment model using Phoenix WinNonlin (ver. 8.3, Certara, United States) to reveal the AUC, clearance, the volume of distribution (Vd), and half-life. The brain/plasma ratio was calculated by dividing the antibody concentrations in brain by those in plasma. The percent of injected dose per gram of brain tissue (%ID/g) was calculated by dividing the concentrations in brain by dose amount.

IHC in mouse brain

The antibodies were administered in mice intravenously at a dose of 20 mg/kg. At day 1 and 7, PBS was perfused from the heart by inserting a catheter to remove blood, and furthermore 10% neutral-buffered formalin (NBF; Wako, 062–01661) was perfused. After perfusion, the brain was collected, following fixing it by NBF for overnight.

Trimming was performed at level 3 according to the report by Deepa et al.,33 including the cerebral cortex, hippocampus, and hypothalamus, and formalin-fixed paraffin-embedded (FFPE) blocks were prepared according to standard procedures. FFPE blocks were sectioned, and IHC was performed using anti-human IgG (1.0 µg/mL, OriGene, R1336P). ImmPRESS-VR-HRP Horse anti-Rabbit IgG (Vector Laboratories, MP-6401) was used as the secondary antibody, and 3,3’-Diaminobenzidine Tetrahydrochloride (DAB, FUJIFILM Wako Pure Chemical Corporation, 045–22833) was used for visualization. The stained slides were scanned using a slide scanner (NanoZoomer S360, Hamamatsu Photonics K.K., Japan) and converted to whole slide images. Positive areas stained with anti-human IgG antibody were detected using Area Quantification v2.3.1 in HALO AI™ (Indica Labs, United States) image analysis software.

3D imaging of the antibodies in the brain

The antibodies were labeled with AlexaFluor647 using the labeling kit (Thermo Fisher Scientific, A20173). The labeled antibodies were administered to mice intravenously at a dose of 20 mg/kg. At days 1 and 7, perfusion was performed and the collected brain tissues were fixed with 4% paraformaldehyde (nacalai tesque, 09154–85). Tissue clearing, staining, and imaging were performed by CUBICStars, Inc. according to previous reports.34,35 Briefly, tissues were delipidated using CUBIC-L reagent (CUBICStars, CSCR001), stained with the CUBIC-HV2 staining kit (CUBICStars, CSSR003), and subsequently refractive-index matched using CUBIC-R+(N) reagent (CUBICStars, CSCR002) to achieve optical clearing. Anti-CD31 antibody (R&D, AF3628) and anti-goat antibody (Jackson Immuno Research, AB_2632580) were used for staining vessels, and anti-MOG antibody (Abcam, ab32760) and anti-rabbit antibody (Jackson Immuno Research, AB_2632462) were used for staining MOG. The tissues were observed using a light-sheet microscope (QUBICStars, Japan). Image segmentation was performed to extract the signals of the injected antibody, based on the fluorescence of nuclei, CD31, and MOG.

Statistical analysis

Statistical analyses were performed using GraphPad Prism (ver. 10.1.2, GraphPad Software, United States). Differences among multiple groups were analyzed by one-way analysis of variance (one-way ANOVA), followed by Tukey’s multiple comparisons test.

Supplementary Material

Supplementary information_Cleaned.docx
KMAB_A_2724587_SM5293.docx (1.7MB, docx)

Acknowledgments

The authors wish to thank all research assistants at Chugai Pharmaceutical Co. Ltd. and Chugai Pharmabody Research Pte. Ltd. for their extraordinary experimental assistance. The authors also wish to thank the members of Chugai Research Institute for Medical Science, Inc. for their assistance with the mouse PK studies and IHC. Additionally, we wish to thank Jacob Davis for his help in writing the English manuscript.

Funding Statement

This study was sponsored by Chugai Pharmaceutical Co., Ltd. and Chugai Pharmabody Research Pte. Ltd.

Disclosure statement

Y.N., K.S., M.Y., M.K., K.N., M.O., A.K., K.H., and T.K. are employees of Chugai Pharmaceutical Co., Ltd. N.H., N.N., and F.S. are employees of Chugai Pharmabody Research Pte. Ltd.

Data availability statement

The datasets generated in this study are available from the corresponding author on reasonable request.

Supplementary material

Supplemental data for this article can be accessed online at https://doi.org/10.1080/19420862.2026.2724587

References

  • 1.Grimm HP, Schumacher V, Schäfer M, Imhof-Jung S, Freskgård P-O, Brady K, Hofmann C, Rüger P, Schlothauer T, Göpfert U, et al. Delivery of the Brainshuttle™ amyloid-beta antibody fusion trontinemab to non-human primate brain and projected efficacious dose regimens in humans. Mabs-austin. 2023;15(1):2261509. doi: 10.1080/19420862.2023.2261509. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2.Kariolis MS, Wells RC, Getz JA, Kwan W, Mahon CS, Tong R, Kim DJ, Srivastava A, Bedard C, Henne KR, et al. Brain delivery of therapeutic proteins using an Fc fragment blood-brain barrier transport vehicle in mice and monkeys. Sci Transl Med. 2020;12(545). doi: 10.1126/scitranslmed.aay1359. [DOI] [PubMed] [Google Scholar]
  • 3.Okuyama T, Eto Y, Sakai N, Nakamura K, Yamamoto T, Yamaoka M, Ikeda T, So S, Tanizawa K, Sonoda H, et al. A phase 2/3 trial of Pabinafusp Alfa, IDS fused with anti-human transferrin receptor antibody, targeting neurodegeneration in MPS-II. Mol Ther. 2021;29(2):671–17. doi: 10.1016/j.ymthe.2020.09.039. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Sonoda H, Minami K.. IZCARGOⓇ: the world’s first biological drug applied with brain drug delivery technology. Drug Delivery Sys. 2023;38:68–74. doi: 10.2745/dds.38.68. [DOI] [Google Scholar]
  • 5.Chew KS, Wells RC, Moshkforoush A, Chan D, Lechtenberg KJ, Tran HL, Chow J, Kim DJ, Robles-Colmenares Y, Srivastava DB, et al. CD98hc is a target for brain delivery of biotherapeutics. Nat Commun. 2023;14(1):5053. doi: 10.1038/s41467-023-40681-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Pornnoppadol G, Bond LG, Lucas MJ, Zupancic JM, Kuo Y-H, Zhang B, Greineder CF, Tessier PM. Bispecific antibody shuttles targeting CD98hc mediate efficient and long-lived brain delivery of IgGs. Cell Chem Biol. 2024;31(2):361–372.e368. doi: 10.1016/j.chembiol.2023.09.008. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Shin JW, An S, Kim D, Kim H, Ahn J, Eom J, You W-K, Yun H, Lee B, Sung B, et al. Grabody B, an IGF1 receptor-based shuttle, mediates efficient delivery of biologics across the blood-brain barrier. Cell Rep Methods. 2022;2(11):100338. doi: 10.1016/j.crmeth.2022.100338. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Nakano R, Takagi-Maeda S, Ito Y, Kishimoto S, Osato T, Noguchi K, Kurihara-Suda K, Takahashi N. A new technology for increasing therapeutic protein levels in the brain over extended periods. PLOS ONE. 2019;14(4):e0214404. doi: 10.1371/journal.pone.0214404. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.Schwinghamer K, Siahaan TJ. Enhancing antibody exposure in the central nervous system: mechanisms of uptake, clearance, and strategies for improved brain delivery. J Nanother. 2023;4(4):463–479. doi: 10.3390/jnt4040020. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Hultqvist G, Syvänen S, Fang XT, Lannfelt L, Sehlin D. Bivalent brain shuttle increases antibody uptake by monovalent binding to the transferrin receptor. Theranostics. 2017;7(2):308–318. doi: 10.7150/thno.17155. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Khoury N, Pizzo ME, Discenza CB, Joy D, Tatarakis D, Todorov MI, Negwer M, Ha C, De Melo GL, Sarrafha L, et al. Fc-engineered large molecules targeting blood-brain barrier transferrin receptor and CD98hc have distinct central nervous system and peripheral biodistribution. Nat Commun. 2025;16(1):1822. doi: 10.1038/s41467-025-57108-x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Johns TG, Bernard CC. The structure and function of myelin oligodendrocyte glycoprotein. J Neurochem. 1999;72(1):1–9. doi: 10.1046/j.1471-4159.1999.0720001.x. [DOI] [PubMed] [Google Scholar]
  • 13.Toyama BH, Savas J, Park S, Harris M, Ingolia N, Yates J, Hetzer M. Identification of long-lived proteins reveals exceptional stability of essential cellular structures. Cell. 2013;154(5):971–982. doi: 10.1016/j.cell.2013.07.037. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Cuypers ML, Jaspers T, Clerckx J, Leekens S, Cawthorne C, Bormans G, Cleeren F, Geukens N, De Strooper B, Dewilde M. Increasing brain half-life of antibodies by additional binding to myelin oligodendrocyte glycoprotein, a CNS specific protein. Fluids Barriers CNS. 2025;22(1):11. doi: 10.1186/s12987-025-00624-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Kakunaga S, Ikeda W, Itoh S, Deguchi-Tawarada M, Ohtsuka T, Mizoguchi A, Takai Y. Nectin-like molecule-1/TSLL1/SynCAM3: a neural tissue-specific immunoglobulin-like cell-cell adhesion molecule localizing at non-junctional contact sites of presynaptic nerve terminals, axons and glia cell processes. J Cell Sci. 2005;118(6):1267–1277. doi: 10.1242/jcs.01656. [DOI] [PubMed] [Google Scholar]
  • 16.Aono S, Keino H, Ono T, Yasuda Y, Tokita Y, Matsui F, Taniguchi M, Sonta S-I, Oohira A. Genomic organization and expression pattern of mouse neuroglycan C in the cerebellar development. J Biol Chem. 2000;275(1):337–342. doi: 10.1074/jbc.275.1.337. [DOI] [PubMed] [Google Scholar]
  • 17.Watanabe E, Maeda N, Matsui F, Kushima Y, Noda M, Oohira A. Neuroglycan C, a novel membrane-spanning chondroitin sulfate proteoglycan that is restricted to the brain. J Biol Chem. 1995;270(45):26876–26882. doi: 10.1074/jbc.270.45.26876. [DOI] [PubMed] [Google Scholar]
  • 18.Morito T, Hashimoto S, Takamura R, Watamura N, Kakiya N, Fujioka R, Mihara N, Sekiguchi M, Watanabe-Iwata K, Kamano N, et al. The role of neprilysin and insulin-degrading enzyme in the etiology of sporadic Alzheimer’s disease. J Neurosci. 2025;45(23):e2152242025. doi: 10.1523/jneurosci.2152-24.2025. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Ito S, Oishi M, Ogata S, Uemura T, Couraud P-O, Masuda T, Ohtsuki S. Identification of cell-surface proteins Endocytosed by human brain microvascular endothelial cells in vitro. Pharmaceutics. 2020;12(6):579. doi: 10.3390/pharmaceutics12060579. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Johnsen KB, Burkhart A, Thomsen LB, Andresen TL, Moos T. Targeting the transferrin receptor for brain drug delivery. Prog Neurobiol. 2019;181:101665. doi: 10.1016/j.pneurobio.2019.101665. [DOI] [PubMed] [Google Scholar]
  • 21.Zhang Y, Pardridge WM. Rapid transferrin efflux from brain to blood across the blood-brain barrier. J Neurochem. 2001;76(5):1597–1600. doi: 10.1046/j.1471-4159.2001.00222.x. [DOI] [PubMed] [Google Scholar]
  • 22.Abe E, Fuwa TJ, Hoshi K, Saito T, Murakami T, Miyajima M, Ogawa N, Akatsu H, Hashizume Y, Hashimoto Y, et al. Expression of transferrin protein and messenger RNA in neural cells from mouse and human brain tissue. Metabolites. 2022;12(7):594. doi: 10.3390/metabo12070594. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Veire JL, Lucas MJ, Bond LG, Tripu DR, Tessier PM, Greineder CF. Comparative radiotracing quantifies brain cellular uptake and catabolism of bispecific antibodies targeting transferrin receptor and CD98hc. ACS Chem Neurosci. 2025;16(7):1264–1274. doi: 10.1021/acschemneuro.4c00552. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Noguchi Y, Kato M, Ozeki K, Ishigai M. Pharmacokinetics of an intracerebroventricularly administered antibody in rats. Mabs-austin. 2017;9(7):1210–1215. doi: 10.1080/19420862.2017.1345834. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Chen CZ, Neumann B, Förster S, Franklin RJM. Schwann cell remyelination of the central nervous system: why does it happen and what are the benefits? Open Biol. 2021;11(1):200352. doi: 10.1098/rsob.200352. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Campos CR, Kemble AM, Niewoehner J, Freskgård PO, Urich E. Brain shuttle neprilysin reduces central amyloid-β levels. PLOS ONE. 2020;15(3):e0229850. doi: 10.1371/journal.pone.0229850. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Smith J, Mummery CJ, Cummings JL, Rabinovici GD, Salloway S, Sperling RA, Zetterberg H, Thanasopoulou A, Lane C, Delmar P, et al. TRONTIER 1 and TRONTIER 2: pivotal trials of trontinemab in early symptomatic Alzheimer’s disease. Alzheimers Dement. 2025;21(S5). doi: 10.1002/alz70859_104294. [DOI] [Google Scholar]
  • 28.Leinenga G, Koh WK, Götz J. A comparative study of the effects of aducanumab and scanning ultrasound on amyloid plaques and behavior in the APP23 mouse model of alzheimer disease. Alzheimers Res Ther. 2021;13(76). doi: 10.1186/s13195-021-00809-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Sevigny J, Chiao P, Bussière T, Weinreb PH, Williams L, Maier M, Dunstan R, Salloway S, Chen T, Ling Y, et al. The antibody aducanumab reduces Aβ plaques in Alzheimer’s disease. Nature. 2016;537(7618):50–56. doi: 10.1038/nature19323. [DOI] [PubMed] [Google Scholar]
  • 30.Labrijn AF, Meesters JI, Priem P, de Jong RN, van den Bremer ETJ, van Kampen MD, Gerritsen AF, Schuurman J, Parren PWHI. Controlled Fab-arm exchange for the generation of stable bispecific IgG1. Nat Protoc. 2014;9(10):2450–2463. doi: 10.1038/nprot.2014.169. [DOI] [PubMed] [Google Scholar]
  • 31.Boado RJ, Zhang Y, Wang Y, Pardridge WM. Engineering and expression of a chimeric transferrin receptor monoclonal antibody for blood-brain barrier delivery in the mouse. Biotechnol Bioeng. 2009;102(4):1251–1258. doi: 10.1002/bit.22135. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Yogi A, Hussack G, van Faassen H, Haqqani AS, Delaney CE, Brunette E, Sandhu JK, Hewitt M, Sulea T, Kemmerich K, et al. Brain delivery of IGF1R5, a single-domain antibody targeting insulin-like growth factor-1 receptor. Pharmaceutics. 2022;14(7):1452. doi: 10.3390/pharmaceutics14071452. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.Bolon B, Garman RH, Pardo ID, Jensen K, Sills RC, Roulois A, Radovsky A, Bradley A, Andrews-Jones L, Butt M, et al. STP position paper: recommended practices for sampling and processing the nervous system (brain, spinal cord, nerve, and eye) during nonclinical general toxicity studies. Toxicol Pathol. 2013;41(7):1028–1048. doi: 10.1177/0192623312474865. [DOI] [PubMed] [Google Scholar]
  • 34.Matsumoto K, Mitani TT, Horiguchi SA, Kaneshiro J, Murakami TC, Mano T, Fujishima H, Konno A, Watanabe TM, Hirai H, et al. Advanced CUBIC tissue clearing for whole-organ cell profiling. Nat Protoc. 2019;14(12):3506–3537. doi: 10.1038/s41596-019-0240-9. [DOI] [PubMed] [Google Scholar]
  • 35.Susaki EA, Shimizu C, Kuno A, Tainaka K, Li X, Nishi K, Morishima K, Ono H, Ode KL, Saeki Y, et al. Versatile whole-organ/body staining and imaging based on electrolyte-gel properties of biological tissues. Nat Commun. 2020;11(1):1982. doi: 10.1038/s41467-020-15906-5. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary information_Cleaned.docx
KMAB_A_2724587_SM5293.docx (1.7MB, docx)

Data Availability Statement

The datasets generated in this study are available from the corresponding author on reasonable request.

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