The blood–brain barrier (BBB) is a highly selective, semipermeable physiological interface that preserves central nervous system homeostasis by regulating the transport of circulating substances and limiting the penetration of blood-borne compounds, including even small molecule drugs. Given the significant challenges associated with delivering biologics across the blood-brain barrier, there is a growing need for molecular platforms capable of enabling efficient central nervous system targeting while maintaining specificity and stability. VHH have emerged as promising candidates in this context due to their small size, structural stability, and amenability to engineering. Accumulating preclinical data shows that VHHs can facilitate the transport of imaging agents and therapeutic cargos across the BBB, be incorporated into gene therapy delivery systems, and function directly as target-specific therapeutic agents within the central nervous system (CNS).

The BBB challenge

The blood–brain barrier (BBB) is a highly selective physiological barrier that protects the brain by regulating the transport of circulating substances and limiting the penetration of blood-borne compounds. At a structural level, this barrier is established by brain capillary endothelial cells interconnected by tight junctions and supported by further specialized cells, forming a highly restrictive interface that limits the passage of most polar and large molecules [1]. As a result, nearly all large and hydrophilic therapeutics are effectively excluded: it is estimated that approximately 98% of small molecule drugs and virtually 100% of large biomolecules — including antibodies, proteins, viral vector — cannot penetrate an intact BBB [1]. This exclusion represents a major obstacle for treating central nervous system (CNS) disorders, as conventional biologics rarely reach their intended targets.

For instance, peripherally administered monoclonal antibodies targeting amyloid-β (Aβ) in Alzheimer’s disease achieve less than 0.1% of the injected dose in brain tissue, illustrating the severe limitations imposed by the BBB [2]. Clinically meaningful target engagement, particularly significant reduction of amyloid-β plaques as measured by PET, can be achieved with repeated high-dose intravenous (IV) anti-amyloid antibody regimens (e.g., ~10–15 mg/kg every 2–4 weeks) [3]. However this approach introduces significant financial disadvantages as well as high incidence of dose-dependent amyloid-related imaging abnormalities (ARIA) [3]. To overcome risks of high-dose IV regimens, specialized delivery methods are in various stages of clinical use and development. MRI-guided focused ultrasound (MRgFUS), an image-guided approach that transiently increases BBB permeability, has demonstrated the ability to enhance local delivery of therapeutics in Alzheimer’s disease. But this method introduces procedural complexity, increased cost, and carries minor safety risks (including microhemorrhage and edema), while questions remain regarding the repeatability and durability of BBB disruption across multiple treatments [4]. Consequently, the BBB continues to represent a major bottleneck for the delivery of many biologics, highlighting the need for engineered therapeutic formats capable of efficiently and selectively crossing this barrier.

Considerable effort has focused on developing therapeutic strategies capable of engaging with endogenous BBB transport systems to achieve efficient delivery to the central nervous system. Under normal physiological conditions, essential nutrients cross into the brain via specific carrier-mediated or receptor-mediated transport. Glucose and amino acids cross the BBB primarily via carrier-mediated transport systems expressed on brain capillary endothelial cells, including facilitative transporters such as GLUT1 for glucose and other carrier-mediated transporters (CMT) for large amino acids [5]. Macromolecules like transferrin and insulin traverse the BBB via receptor-mediated transcytosis (RMT) where ligand binding to receptors on the endothelial surface triggers vesicular uptake and transport across the cell, releasing the cargo into the brain. Scientists increasingly seek to exploit these pathways to shuttle biologics into the CNS [6].

Figure 1 – Transportation methods to cross the BBB. The blood-brain barrier (BBB) forms a highly selective interface between the bloodstream and the central nervous system, primarly through tightly connected brain endothelial cells. Only certain molecules are able to cross this barrier via specialised transport mechanisms. Small lipophilic compounds may enter the brain through passive diffusion, while essential nutrients such as glucose and amido acids are transported by carrier-mediated transport (e.g., GLUT1 and amino acid transporters). Larger biomolecules, including peptides and proteins, can cross through vesicular pathways such as receptor-mediated transcytosis, which involves receptors (e.g., transferrin or insulin receptor), or adsorptive-mediated transcytosis driven by electrostatic interactions with the endothelial surface. Understanding these transport pathways is critical for the design of brain-targeted therapeutics, including engineered antibody formats such as VHHs that can be developed to exploit receptor-mediated transport systems to access CNS targets.

Each approach presents trade-offs. For instance, CMT systems enable efficient nutrient flux into the brain but exhibit strict substrate specificity and are largely incompatible with large biologics. In contrast, RMT pathways, such as insulin or transferrin receptors permit the transport of macromolecules. However, their exploitation is constrained by receptor saturation, competition with endogenous ligands, retention resulting from high-affinity binding, and peripheral side effects [7]. Designing effective BBB shuttles therefore requires the development of therapeutics with optimized receptor affinity and avidity. In short, the BBB remains a formidable gatekeeper: without specialized shuttles, nearly all systemically delivered biologics fail to reach brain targets.

Figure 2 – Shuttling biologics into the CNS via receptor-mediated transcytosis (RMT), using transferrin receptors.

How VHH antibodies can help with CNS delivery

A VHH is a single-domain antibody derived from heavy-chain-only immunoglobulins naturally found in camelids including camels and llamas. These VHH domains are exceptionally small (12–15 kDa), approximately one‐tenth the size of a conventional IgG antibody. Their compact, single-domain structure confers favorable biophysical properties including high solubility, robust thermal and chemical stability, and efficient recombinant expression at high yield in bacteria or yeast. Importantly, their extended antigen binding loop enables VHHs to access recessed or sterically occluded epitopes, such as enzyme active sites or receptor clefts, that are often inaccessible to conventional antibodies.

These properties are particularly advantageous for BBB delivery applications The compact size of VHH reduces steric constraints during receptor engagement and intracellular trafficking, facilitating more efficient transcytosis across brain endothelial cells compared with full-length antibodies. Their small size also facilitates rapid tissue penetration and fast systemic clearance, which is ideal for imaging and diagnostic applications. The monovalent architecture of VHHs enables precise control over receptor affinity and valency. In addition, their small size and modular structure allow incorporation into multi-specific formats and with minimal increase in overall size. For example, bi-specific constructs can be generated by combining a VHH targeting a BBB transport receptor, such as the transferrin receptor (TfR) with a second VHH directed against a therapeutic target within the central nervous system. VHHs can similarly be fused or conjugated to small-molecule drugs, imaging agents, or oligonucleotides as “VHH drug conjugates” or targeted payloads. In each case, the small VHH scaffold makes the overall molecule much smaller than a traditional IgG-based fusion, a feature that improves tissue penetration and could enhance brain delivery.

Another advantage of VHHs is their amenability to rapid in vitro discovery and engineering. Large synthetic VHH libraries can be screened at high-throughput using platforms such as phage display to isolate binders against virtually any target, including human BBB receptors like the TfR or insulin-like growth factor 1 receptor (IGF1R). This approach circumvents the time, variability, and species-specific constraints associated with animal immunization and allows rapid humanization and affinity maturation of lead candidates.

In practice, researchers can select VHHs with precise binding characteristics, such as moderate affinity or pH-dependent kinetics, directly at the bench, enabling control over transcytosis efficiency and receptor engagement. This de novo approach meshes well with modern protein engineering: once identified, a VHH can be further optimized through iterative design or site-directed mutagenesis without introducing size penalties. In short, VHH antibodies combine small size, robustness and engineering ease to form an ideal platform for BBB carrier molecules [6].

Mechanisms of BBB transport

Current strategies for delivering biologics to the brain predominantly exploit receptor-mediated transcytosis (RMT) across brain endothelial cells. Among these, the transferrin receptor (TfR) is the most widely targeted, due to its high expression on brain capillaries and its native role in shuttling iron into the central nervous system. Transferrin-bound ferric iron (Fe3+) binds to TfR, triggering internalization into acidified endosomes where iron is released, after which the apotransferrin-TfR complex is recycled back to the cell surface [8].

Therapeutic molecules can exploit this pathway by ‘piggybacking’ on the TfR-mediated transport pathway. The first approved BBB shuttle therapy uses this approach: pabinafusp alpha (Idursulfase–Tb antibody fusion) is a TfR-binding IgG fused to the lysosomal enzyme iduronate-2-sulfatase for the treatment of Hunter syndrome [9]. Following TfR binding, the fusion protein is internalized and transported across the BBB, allowing the enzyme to reach the CNS and degrade accumulated substrate, thereby demonstrating the translational potential of TfR-targeted delivery.

Besides TfR, other RMT targets are being explored. The insulin receptor [10] and the IGF1R [11] are expressed on the BBB and have been targeted by antibody shuttles. More recently, the heavy chain of the large neutral amino acid transporter (CD98hc) [12] and a phospholipid flippase (TMEM30) [6] have emerged as promising shuttling receptors. In principle, any receptor capable of active transcytosis can be used to carry therapeutic cargo, although in practice the binding and release kinetics must be carefully optimized to avoid endothelial retention. For instance, high-affinity or bivalent binders to TfR frequently become sequestered within the endothelium, whereas monovalent or moderate-affinity binders often achieve better transport across the BBB [13].

Crucially, preclinical studies show that engineered VHHs can harness these RMT pathways. A recent example is a llama-derived VHH that binds the mouse TfR, called M1. By inserting histidine residues that weaken binding at low pH, researchers made a pH-sensitive M1 variant. When fused to a pair of anti-P2X7 receptor VHHs as cargo, this modified VHH delivered ~3.5% of the injected dose per gram of brain tissue within 4h after IV injection in mice [6] – a much higher brain uptake than accomplished with unconjugated VHHs.

When the VHHs were fused to an anti-BACE1 antibody (1A11AM) and administered peripherally in mice, they enhanced brain permeability of the bispecific construct; in these animals, brain Aβ1–40 levels were reduced by approximately 40% compared to vehicle-treated controls [6].

Other RMT examples include bispecific antibodies where one arm binds TfR (or other receptors) and the other binds a brain target. For instance, a TfR-scFv fusion delivered an anti-BACE1 antibody into mice brain, lowering brain Aβ and enzyme levels [13]. At least one bi-specific anti-TfR/anti-Aβ antibody (trontinemab, also called Brainshuttle™) has entered clinical trials. In non-human primates, trontinemab penetrated the brain 4 to 18x more efficiently than a conventional Aβ antibody [14]. Notably, it also appeared to cause fewer vascular side effects, perhaps by avoiding excess anti-Aβ binding in vessels [15]. In humans, a phase III trial of trontinemab in Alzheimer’s patients is currently ongoing [16].

Other BBB transport strategies are being combined with VHHs. For example, focused ultrasound transiently opens the barrier to allow larger agents through, and viral vectors (like AAV) are being engineered to reach the brain. Recently, new AAV capsids have been created with peptides that bind human TfR, dramatically enhancing CNS delivery. In one study, an engineered AAV (CAP-B10) showed enriched brain uptake in both mice and marmosets [17]. Similarly, AAV variants carrying “cell-penetrating peptide” inserts (CPP.16 and CPP.21) also achieved improved brain transduction in mice and even macaques [18]. These innovations hint at future synergies: an AAV could encode a brain-targeting VHH, or a VHH could transport a viral particle.

In summary, by leveraging RMT pathways (especially TfR-mediated transcytosis), researchers are now achieving clinically meaningful CNS delivery of biologics. Crucial to this effort is finding VHHs that bind the chosen receptor without blocking it or getting stuck – something enabled by phage display optimization. The emerging data, from mice to monkeys, demonstrate that tailored VHH shuttles can overcome the BBB bottleneck to shuttle cargo into the brain, from antibodies to enzymes and even imaging probes.

Emerging applications

The unique properties of VHHs are spurring a wave of new CNS applications in therapeutics, diagnostics, and delivery platforms.

Therapeutics

VHH-based formats are being explored for treating brain diseases from Alzheimer’s to glioblastoma. One promising avenue is using VHHs to target amyloid and tau in Alzheimer’s [19]. In the lab, VHHs are directed to Aβ and phospho-Tau bound plaques and tangles in transgenic mouse models after intravenous injection [19].

VHHs can also be configured for delivery of various payloads. Because of their small size and multiple terminal residues, VHHs can be linked to potent small molecules (toxins or radionuclides) or oligonucleotides for targeted brain tumor therapy [20]. Although full-scale brain tumor VHH-ADCs remain preclinical.

Imaging and diagnostics

The rapid blood clearance and excellent tissue penetration of VHHs make them attractive as brain imaging agents. VHHs can be labeled with radionuclides or MRI contrast moieties to create highly specific tracers. For instance, VHHs against Aβ or phospho‑Tau have been engineered as imaging probes, including technetium‑99m‑labeled VHHs that detect Aβ deposits in APP/PS1 transgenic mice, and an anti‑Aβ VHH (R3VQ) conjugated to gadolinium for MRI contrast to visualize amyloid plaques [21].

Furthermore, VHH-based immuno-PET tracers produce images of superior quality to those achieved with full IgGs, thanks to better diffusion and lower background signal in non-target areas [22].

Gene and viral delivery platforms

Since VHH genes are very short (~350 base pairs), they fit easily into AAV or lentiviral vectors, and there have been numerous examples of using VHHs in cell and gene therapy applications e.g. as CAR-Ts and ‘intrabodies’ expressed inside cells. In the brain, a single intravenous dose of AAV encoding an anti-BACE1 VHH yields sustained production of the VHH in brain tissues, with beneficial effects lasting over a year [23]. In that Alzheimer’s model, the AAV-driven VHH lowered amyloid pathology, reduced neuroinflammation, and improved cognitive performance for 12+ months after one dose [24].

Viral vectors can also be retargeted by VHHs. For example, recent AAV capsids have been designed with inserted peptides to bind human TfR1, enhancing brain tropism in primates. One study reported an AAV variant (CAP-B10) that, when administered via IV, yielded high brain transduction in mice and marmosets [17].

Conclusions

As preclinical and clinical data accumulate, they show that VHHs can shuttle imaging agents and therapeutics across the BBB, be encoded by gene therapy vectors, or even serve as on-target drugs themselves. Each use exploits VHH’s unique qualities – size, stability, modularity, and high specificity – in a different way. And importantly, all efficacy claims are supported by experiments in relevant models.

For instance, brain-penetrant VHH carriers have demonstrated clear pharmacodynamic effects (amyloid reduction, hypothermia via neurotensin, etc.) in vivo [6]. As this field advances, one can imagine VHHs used as radiotracers for PET/MRI, bispecific immunotherapies for Alzheimer’s or brain cancer, and even VHH-vaccines or CAR-T cells delivered to the CNS.

Isogenica’s experience with synthetic VHH libraries and in vitro display can help accelerate any CNS program. By screening our diverse synthetic VHH repertoires against BBB targets, such as human TfR, or intracellular aggregate targets, we can identify lead VHHs that naturally engage transport pathways or directly target disease-causing pathways in the brain. These leads can then be matured or engineered (for example, tuning affinity or pH-dependence) entirely in vitro. The result is a highly customized VHH ready for fusion to your therapeutic payload or genetic delivery, without the need for animal immunization.

If you are interested in advancing brain-penetrant biologics, we encourage you to book a discovery call with our team. We can discuss how to apply our VHH platform and selection workflows to your CNS targets. Explore how synthetic VHHs could open the door across the BBB in your next project.

References

1. Rust, R., Yin, H., Achón Buil, B., Sagare, A.P. & Kisler, K. (2025). The blood–brain barrier: A help and a hindrance. Brain, 148(7): 2262–2282. https://doi.org/10.1093/brain/awaf068
2. Julku, U., Xiong, M., Wik, E., Roshanbin, S., Sehlin, D. & Syvänen, S. (2022). Brain pharmacokinetics of mono- and bispecific amyloid-β antibodies in wild-type and Alzheimer’s disease mice measured by high cut-off microdialysis. Fluids and Barriers of the CNS, 19(1): 99. https://doi.org/10.1186/s12987-022-00398-w
3. Cummings, J., Osse, A.M.L., Cammann, D., Powell, J. & Chen, J. (2024). Anti-Amyloid Monoclonal Antibodies for the Treatment of Alzheimer’s Disease. Biodrugs, 38(1): 5–22. https://doi.org/10.1007/s40259-023-00633-2
4. Fishman, P.S. & Fischell, J.M. (2021). Focused Ultrasound Mediated Opening of the Blood-Brain Barrier for Neurodegenerative Diseases. Frontiers in Neurology, 12, 749047. https://doi.org/10.3389/fneur.2021.749047
5. Zaragozá, R. (2020). Transport of Amino Acids Across the Blood-Brain Barrier. Frontiers in Physiology, 11: 973. https://doi.org/10.3389/fphys.2020.00973
6. Esparza, T.J., Su, S., Francescutti, C.M., Rodionova, E., Kim, J.H. & Brody, D.L. (2023). Enhanced in Vivo Blood Brain Barrier Transcytosis of Macromolecular Cargo Using an Engineered pH-sensitive Mouse Transferrin Receptor Binding Nanobody. bioRxiv: 2023.04.26.538462. https://doi.org/10.1101/2023.04.26.538462
7. Sehlin, D. & Syvänen, S. (2019). Engineered antibodies: New possibilities for brain PET? European Journal of Nuclear Medicine and Molecular Imaging, 46(13): 2848–2858. https://doi.org/10.1007/s00259-019-04426-0
8. Shen, X., Li, H., Zhang, B., Li, Y. & Zhu, Z. (2025). Targeting Transferrin Receptor 1 for Enhancing Drug Delivery Through the Blood–Brain Barrier for Alzheimer’s Disease. International Journal of Molecular Sciences, 26(19): 9793. https://doi.org/10.3390/ijms26199793
9. Sonoda, H., Morimoto, H., Yoden, E., Koshimura, Y., Kinoshita, M., Golovina, G., Takagi, H., Yamamoto, R., Minami, K., Mizoguchi, A., Tachibana, K., Hirato, T. & Takahashi, K. (2018). A Blood-Brain-Barrier-Penetrating Anti-human Transferrin Receptor Antibody Fusion Protein for Neuronopathic Mucopolysaccharidosis II. Molecular Therapy: The Journal of the American Society of Gene Therapy, 26(5): 1366–1374. https://doi.org/10.1016/j.ymthe.2018.02.032
10. Boado, R.J. & Pardridge, W.M. (2017). Brain and Organ Uptake in the Rhesus Monkey in Vivo of Recombinant Iduronidase Compared to an Insulin Receptor Antibody-Iduronidase Fusion Protein. Molecular Pharmaceutics, 14(4): 1271–1277. https://doi.org/10.1021/acs.molpharmaceut.6b01166
11. Yogi, A., Hussack, G., van Faassen, H., Haqqani, A.S., Delaney, C.E., Brunette, E., Sandhu, J.K., Hewitt, M., Sulea, T., Kemmerich, K. & Stanimirovic, D.B. (2022). Brain Delivery of IGF1R5, a Single-Domain Antibody Targeting Insulin-like Growth Factor-1 Receptor. Pharmaceutics, 14(7): 1452. https://doi.org/10.3390/pharmaceutics14071452
12. Zuchero, Y.J.Y., Chen, X., Bien-Ly, N., Bumbaca, D., Tong, R.K., Gao, X., Zhang, S., Hoyte, K., Luk, W., Huntley, M.A., Phu, L., Tan, C., Kallop, D., Weimer, R.M., Lu, Y., Kirkpatrick, D.S., Ernst, J.A., Chih, B., Dennis, M.S. & Watts, R.J. (2016). Discovery of Novel Blood-Brain Barrier Targets to Enhance Brain Uptake of Therapeutic Antibodies. Neuron, 89(1): 70–82. https://doi.org/10.1016/j.neuron.2015.11.024
13. Yu, Y.J., Zhang, Y., Kenrick, M., Hoyte, K., Luk, W., Lu, Y., Atwal, J., Elliott, J.M., Prabhu, S., Watts, R.J. & Dennis, M. S. (2011). Boosting brain uptake of a therapeutic antibody by reducing its affinity for a transcytosis target. Science Translational Medicine, 3(84): 84ra44. https://doi.org/10.1126/scitranslmed.3002230
14. Grimm, H.P., Schumacher, V., Schäfer, M., Imhof-Jung, S., Freskgård, P.-O., Brady, K., Hofmann, C., Rüger, P., Schlothauer, T., Göpfert, U., Hartl, M., Rottach, S., Zwick, A., Seger, S., Neff, R., Niewoehner, J. & Janssen, N. (2023). Delivery of the BrainshuttleTM amyloid-beta antibody fusion trontinemab to non-human primate brain and projected efficacious dose regimens in humans. mAbs, 15(1): 2261509. https://doi.org/10.1080/19420862.2023.2261509
15. ALZFORUM (no date). Unlocking Blood-Brain Barrier Boosts Immunotherapy Efficacy, Lowers ARIA. Available at: https://www.alzforum.org/news/conference-coverage/unlocking-blood-brain-barrier-boosts-immunotherapy-efficacy-lowers-aria
16. Roche (no date). Roche presents new insights in Alzheimer’s disease research across its diagnostics and pharmaceutical portfolios at AAIC. Available at: https://www.roche.com//media/releases/med-cor-2025-07-28
17. Goertsen, D., Flytzanis, N.C., Goeden, N., Chuapoco, M.R., Cummins, A., Chen, Yijing, Fan, Y., Zhang, Q., Sharma, J., Duan, Y., Wang, L., Feng, G., Chen, Yu, Ip, N.Y., Pickel, J. & Gradinaru, V. (2022). AAV capsid variants with brain-wide transgene expression and decreased liver targeting after intravenous delivery in mouse and marmoset. Nature Neuroscience, 25(1): 106–115. https://doi.org/10.1038/s41593-021-00969-4
18. Słyk, Ż., Stachowiak, N. & Małecki, M. (2024). Recombinant Adeno-Associated Virus Vectors for Gene Therapy of the Central Nervous System: Delivery Routes and Clinical Aspects. Biomedicines, 12(7): 1523. https://doi.org/10.3390/biomedicines12071523
19. Zheng, F., Pang, Yucheng, Li, L., Pang, Yuxing, Zhang, J., Wang, X. & Raes, G. (2022). Applications of nanobodies in brain diseases. Frontiers in Immunology, 13: 978513. https://doi.org/10.3389/fimmu.2022.978513
20. Bannas, P., Hambach, J. & Koch-Nolte, F. (2017). Nanobodies and Nanobody-Based Human Heavy Chain Antibodies As Antitumor Therapeutics. Frontiers in Immunology, 8: 1603. https://doi.org/10.3389/fimmu.2017.01603
21. Nabuurs, R. J. A., Rutgers, K. S., Welling, M. M., Metaxas, A., de Backer, M. E., Rotman, M., Bacskai, B.J., van Buchem, M.A., van der Maarel, S.M. & van der Weerd, L. (2012). In Vivo Detection of Amyloid-β Deposits Using Heavy Chain Antibody Fragments in a Transgenic Mouse Model for Alzheimer’s Disease. PLoS ONE, 7(6): e38284. https://doi.org/10.1371/journal.pone.0038284
22. Erreni, M., Schorn, T., D’Autilia, F. & Doni, A. (2020). Nanobodies as Versatile Tool for Multiscale Imaging Modalities. Biomolecules, 10(12): 1695. https://doi.org/10.3390/biom10121695
23. Danis, C., Dupré, E., Zejneli, O., Caillierez, R., Arrial, A., Bégard, S., Mortelecque, J., Eddarkaoui, S., Loyens, A., Cantrelle, F.-X., Hanoulle, X., Rain, J.-C., Colin, M., Buée, L. & Landrieu, I. (2022). Inhibition of Tau seeding by targeting Tau nucleation core within neurons with a single domain antibody fragment. Molecular Therapy: The Journal of the American Society of Gene Therapy, 30(4): 1484–1499. https://doi.org/10.1016/j.ymthe.2022.01.009
24. Marino, M., Zhou, L., Rincon, M.Y., Callaerts‐Vegh, Z., Verhaert, J., Wahis, J., Creemers, E., Yshii, L., Wierda, K., Saito, T., Marneffe, C., Voytyuk, I., Wouters, Y., Dewilde, M., Duqué, S.I., Vincke, C., Levites, Y., Golde, T.E., Saido, T.C., et al. (2022). AAV‐mediated delivery of an anti‐BACE1 VHH alleviates pathology in an Alzheimer’s disease model. EMBO Molecular Medicine, 14(4): e09824. https://doi.org/10.15252/emmm.201809824