TfR1 Brain Delivery Track Key Domain: Apical Domain

The central nervous system (CNS) is one of the most densely vascularized organ systems in the human body. Brain blood vessels and associated brain cells together form the neurovascular unit; this structure couples neural activity with blood flow dynamics and participates in the formation and maintenance of the blood-brain barrier (BBB)^[1]. The BBB is a specialized physiological barrier that separates the blood from brain tissue. It is primarily composed of brain microvascular endothelial cells, astrocytes, pericytes, the basement membrane, and junctional complexes including tight junctions (TJs) and adherens junctions (AJs). The BBB heavily restricts the cross-barrier transport of the vast majority of macromolecules in the blood, representing the core challenge for delivering peripherally administered protein drugs to the CNS^[2]. Currently, CNS drug delivery strategies include receptor-mediated transcytosis (RMT), transient BBB disruption, local therapies, and CSF-mediated delivery. RMT-based delivery technology offers the advantage of non-invasive administration. It utilizes highly expressed cell surface receptors at the BBB interface (such as TfR1/CD71, CD98hc, IGF1R, etc.) to deliver macromolecular drugs to the brain parenchyma^[3]. At present, research on targeted drug delivery to the brain frequently focuses on TfR1 as the core target.

Structure and Function of TfR1

Structure of TfR1

Transferrin receptor 1 (TfR1, also known as CD71) is a homodimeric type II transmembrane glycoprotein. It consists of an N-terminal intracellular segment, a transmembrane region, and an extracellular region. The extracellular region is composed of a stalk region, an apical domain, a protease-like domain, and a helix domain. Two cysteines in the stalk region (Cys89, Cys98) covalently cross-link the two large homodimeric extracellular domains into an intact receptor via disulfide bonds. The extracellular segment is responsible for binding its ligand, transferrin (Tf), and mediating its endocytosis into the cell to complete iron ion delivery. The N-lobe of Tf interacts with the receptor's protease-like domain, while the C-lobe binds to the receptor's helix domain. 

Schematic Diagram of the Tf-TfR1 Complex^[4]

TfR1 Participates in Cellular Iron Uptake

TfR1 plays a core, irreplaceable role in cellular iron uptake and is the primary channel for cells to take up iron from the blood. Iron in the blood binds tightly to Tf in the form of ferric iron (Fe³⁺). Iron-loaded transferrin (diferric transferrin, holo-Tf) binds to the TfR1 homodimer on the cell membrane surface. The resulting complex aggregates in clathrin-coated pits, which invaginate to initiate the endocytosis process. The Tf-TfR1 complex internalizes to form clathrin-coated vesicles, which mature into early endosomes. The acidic environment of the endosome (pH around 5.5) induces conformational changes in Tf and TfR1, causing Fe³⁺ to be released from the Tf protein. The released Fe³⁺ is reduced to Fe²⁺ by the STEAP ferrireductase and transported to the cytoplasm by divalent metal transporter 1 (DMT1). It is then used to supply heme, ribonucleotide reductase (the rate-limiting enzyme in DNA synthesis), etc., to exert their functions, or it is stored in ferritin. After iron release, apo-transferrin (apo-Tf) remains bound to TfR1 within the low-pH endosome. Subsequently, the apo-Tf-TfR1 complex is recycled back to the cell membrane surface. In the extracellular neutral pH environment, the two dissociate; apo-Tf is released into the blood circulation, and TfR1 re-participates in the next round of iron uptake^[5][6]. 

The Complete Process of Cellular Iron Uptake^[5]

TfR1 Can Serve as a Delivery Target for Crossing the BBB

TfR1 is widely expressed on the surface of hepatocytes, erythrocytes, and proliferating cells^[7]. Furthermore, it is highly expressed on the luminal membrane of brain microvascular endothelial cells—the gatekeepers of the blood-brain barrier—at levels much higher than those found on peripheral endothelial cells. This distribution characteristic makes TfR1 an ideal target for the delivery of macromolecules across the blood-brain barrier. Research on Alzheimer's disease-related models has further highlighted the advantages of this target: under pathological conditions, the expression level of TfR1 in brain endothelial cells is significantly upregulated, whereas the expression of other barrier receptors such as LRP1, GLUT1, and insulin receptors is downregulated as the disease progresses, substantially weakening their delivery efficiency^[8]. 

TfR1-Based BBB-Crossing Drugs

Several Technology Platforms Based on TfR1 for Crossing the BBB^[9]

Currently, multiple pharmaceutical companies have established delivery technology platforms using TfR1 as the delivery target to mediate drug crossing of the BBB. Representative platforms include Denali's Transport Vehicle™ (TV), Roche's Brainshuttle™, JCR Pharma's J-Brain Cargo®, and the Modular Delivery (MODEL™) platform developed in collaboration between Aliada and Abbvie. From the structural information disclosed by each platform, it is evident that the design strategies for the TfR1 delivery module differ significantly. The mainstream approach, based on the requirements of the indication, fuses various suitable therapeutic modules (such as antibodies, enzymes, oligonucleotides, proteins, etc.) with a TfR1-targeting antibody. One end of the drug molecule acts on the therapeutic target of the disease, while the other end specifically targets TfR1, relying on TfR1-mediated endocytosis to achieve highly efficient drug transport across the blood-brain barrier. The TV delivery platform developed by Denali represents a differentiated route. This platform engineers the loop region of the human IgG1 Fc domain, embedding a TfR1 binding epitope and optimizing its affinity. The TV architecture has a simple design with no appended sequences. While maintaining the native structure of IgG1 and its binding activity to FcRn, it can flexibly conjugate with antibodies, enzymes, or oligonucleotides to construct Antibody Transport Vehicles (ATV), Enzyme Transport Vehicles (ETV), and Oligonucleotide Transport Vehicles (OTV), respectively, supporting the highly efficient intracranial delivery of different types of biological macromolecules^[10]. 

For the TfR1-mediated delivery drugs developed at this stage, their targeting components primarily recognize the apical domain and the protease-like domain of TfR1, with R&D pipelines targeting the apical domain currently dominating. 

J-Brain Cargo® Technology Platform and Representative Drugs

Pabinafusp alfa (JR-141), a product empowered by JCR Pharma's J-Brain Cargo® technology platform, is currently the fastest-advancing TfR1-based BBB-crossing drug. This drug has been approved for marketing in Japan and is in phase III globally. It is a fusion protein capable of penetrating the blood-brain barrier, composed of the complete human IDS enzyme fused to an anti-TfR1 antibody. Its indication is Mucopolysaccharidosis Type II (MPS II, also known as Hunter syndrome, a genetic disorder where the lack of iduronate-2-sulfatase (IDS enzyme) leads to the accumulation of carbohydrates in the body, especially in the CNS). The anti-TfR1 antibody portion of this drug specifically targets the apical domain of TfR1 and does not overlap with the binding site of Tf, thereby avoiding any impact on the iron uptake function of TfR1 while triggering TfR1-mediated transcytosis^[11]. The IDS enzyme portion carries a polysaccharide chain containing mannose 6-phosphate (M6P), which can recognize the mannose 6-phosphate receptor (M6PR). After Pabinafusp alfa binds to TfR1, it undergoes endocytosis alongside TfR1, is transported intracellularly to the opposite side, and enters the brain parenchyma via exocytosis. Subsequently, it is recognized and taken up by M6PR receptors on the surface of neurons, astrocytes, and pericytes, thereby delivering the IDS enzyme into the brain to produce a therapeutic effect.

Structure of Pabinafusp Alfa and Schematic of Its Cellular Uptake Pathway^[12]

Transport Vehicle™ (TV) Technology Platform and Representative Drugs

Representative Drugs of the TV Technology Platform^[13]

Denali has developed several CNS therapeutic drugs targeting TfR1 relying on its proprietary TV platform. The core innovation of the TV technology is the protein engineering of the CH3 domain of IgG1 Fc to introduce a TfR1 binding epitope. To screen for the Fc mutant with optimal performance, the team constructed a high-throughput yeast surface directed evolution library. They sequentially carried out soft mutagenesis, NNK walk scanning, and multiple rounds of iterative affinity optimization of focused libraries, ultimately selecting two mature candidate sequences: TV35.21.16 and TV35.21.17. During the screening process, the research team used a circularly permutated apical domain as the screening antigen. They removed the flexible loop within the native apical domain and rearranged the N/C termini of the protein to prepare a homogeneous monomeric antigen. Compared to the native dimeric full-length TfR1, this structure reduces the multivalent binding effects that occur on the yeast cell surface, substantially improving the rigor of the screening, effectively avoiding false-positive clones, and precisely enriching TV mutant clones capable of binding both human and cynomolgus monkey TfR1. Crystal structures confirmed that the binding region between TV and TfR1 is completely independent of the native binding sites for Tf and FcRn, ensuring it does not interfere with the body's normal iron transport and protein recycling functions.

Trividenotusp alfa/DNL310 is the first candidate drug developed by Denali based on the TV platform. This drug is an IDS enzyme-Fc fusion protein indicated for MPS II, and a BLA application was submitted in 2025. This fusion protein can specifically bind to TfR1 on brain endothelial cells, penetrate the blood-brain barrier via receptor-mediated transcytosis, and deliver the IDS enzyme to the brain parenchyma to degrade the carbohydrates accumulated in the CNS of MPS II patients, thereby alleviating the neurocognitive and physical symptoms caused by MPS II. 

TV Screening Process and Special Design of the Apical Domain Antigen^[14]

Brainshuttle™ Technology Platform and Representative Drugs

Trontinemab is a bispecific 2+1 formatted anti-Aβ antibody developed by Roche based on its proprietary Brainshuttle™ technology platform. This technology fuses an anti-Aβ monoclonal antibody with a TfR1 shuttle module (possessing one TfR1 binding site). Due to its unique structural design, Trontinemab can efficiently cross the BBB, target aggregated forms of Aβ, and clear amyloid plaques in the brain. It achieves CNS exposure at low doses, rapidly reduces Aβ in Alzheimer's patients, and delays disease progression^[15]. At the 2026 AAIC conference, Roche presented new long-term safety, amyloid clearance, and biomarker data from the trontinemab Brainshuttle™ AD Phase Ib/IIa open-label extension study. Based on models established from this study, the dosing regimens have been determined for two currently ongoing Phase III clinical trials, TRONTIER1 and TRONTIER, for early symptomatic Alzheimer's disease^[16]. 

Mechanism of Action of Trontinemab^[17]

KACTUS Supplies High-Quality Apical Domain and TfR1 ECD Proteins

As various TfR1 brain shuttle delivery technologies rapidly mature, the bottlenecks in CNS drug R&D are progressively being broken, and related clinical pipelines have experienced explosive growth in recent years. Because the Apical Domain does not compete with endogenous Tf for the TfR1 target, it has become the mainstream targeting site. 
KACTUS can provide two types of recombinant apical domain proteins: wild type and circularly permutated, which are tailored for different R&D scenarios such as animal immunization and molecular screening, respectively. Among them, the circularly permutated apical domain is a core, essential antigen for high-throughput yeast/phage screening, as it can effectively circumvent false-positive interference caused by multivalent binding. Concurrently, the company also supplies TfR1 ECD proteins, providing comprehensive support for the R&D process of TfR1-based CNS drugs. 

Product Data

Immobilized Human Transferrin R Apical Domain, Llama IgG2b Fc Tag at 0.5μg/ml (100μl/well) on the plate. Dose response curve for Anti-Transferrin R Antibody, hFc Tag with the EC50 of 9.4ng/ml determined by ELISA.

Immobilized Biotinylated Human Transferrin R Apical Domain, His Avi Tag at 1μg/ml (100μl/well) on the streptavidin precoated plate (5μg/ml). Dose response curve for Anti-Transferrin R Antibody, hFc Tag with the EC50 of 4.2ng/ml determined by ELISA. 

Immobilized Biotinylated Human Circularly Permuted Apical Domain, His Avi Tag at 1μg/ml(100μl/well) on the streptavidin precoated plate(5μg/ml). Dose response curve for Anti-Transferrin R Antibody, hFc Tag with the EC50 of 3.4ng/ml determined by ELISA.

Immobilized Human Transferrin R, His Tag at 2μg/ml (100μl/well) on the plate. Dose response curve for Biotinylated Human Transferrin, His Avi Tag with the EC50 of 14.3ng/ml determined by ELISA. 

Loaded Anti-Transferrin R Antibody, hFc Tag on ProA-Biosensor can bind Human Transferrin R, His Tag with an affinity constant of 0.65 nM as determined in BLI assay (Gator® Prime).

Related Product

References

[1]    Zuchero YJ, Silverman A, Dennis M, et al. Brain delivery of therapeutic proteins using an Fc fragment blood-brain barrier transport vehicle in mice and monkeys Science Translational Medicine. 2020;12(545). doi:10.1126/scitranslmed.aay1359
[2]    Liu, S., Jin, X., Ge, Y. et al. Advances in brain-targeted delivery strategies and natural product-mediated enhancement of blood–brain barrier permeability. J Nanobiotechnol 23, 382 (2025). https://doi.org/10.1186/s12951-025-03415-w
[3]    Gampa, G., Vadlakonda, R., Stefanich, E. et al. Bridging the blood-brain barrier: strategies to improve delivery of biologics to tumors in the brain. Fluids Barriers CNS 23, 25 (2026). https://doi.org/10.1186/s12987-026-00760-2
[4]    Wessling-Resnick, Marianne. (2018). Crossing the Iron Gate: Why and How Transferrin Receptors Mediate Viral Entry. Annual Review of Nutrition. 38. 431-458. 10.1146/annurev-nutr-082117-051749. 
[5]    Sabo, S.L., & Buxbaum, J.D. (2002). The role of the transferrin-transferrin-receptor system in drug delivery and targeting.
[6]    Shen, X.; Li, H.; Zhang, B.; Li, Y.; Zhu, Z. Targeting Transferrin Receptor 1 for Enhancing Drug Delivery Through the Blood–Brain Barrier for Alzheimer’s Disease. Int. J. Mol. Sci. 2025, 26, 9793. https://doi.org/10.3390/ijms26199793
[7]    Visser CC, Voorwinden LH, Crommelin DJ, Danhof M, de Boer AG. Characterization and modulation of the transferrin receptor on brain capillary endothelial cells. Pharmaceutical Research. 2004 May;21(5):761-769. DOI: 10.1023/b:pham.0000026425.69874.8e. PMID: 15180331.
[8]    Shen, X.; Li, H.; Zhang, B.; Li, Y.; Zhu, Z. Targeting Transferrin Receptor 1 for Enhancing Drug Delivery Through the Blood–Brain Barrier for Alzheimer’s Disease. Int. J. Mol. Sci. 2025, 26, 9793. https://doi.org/10.3390/ijms26199793
[9]    https://investors.denalitherapeutics.com
[10]    Chew KS, Wells RC, Moshkforoush A, Chan D, Lechtenberg KJ, Tran HL, Chow J, Kim DJ, Robles-Colmenares Y, Srivastava DB, Tong RK, Tong M, Xa K, Yang A, Zhou Y, Akkapeddi P, Annamalai L, Bajc K, Blanchette M, Cherf GM, Earr TK, Gill A, Huynh D, Joy D, Knight KN, Lac D, Leung AW, Lexa KW, Liau NPD, Becerra I, Malfavon M, McInnes J, Nguyen HN, Lozano EI, Pizzo ME, Roche E, Sacayon P, Calvert MEK, Daneman R, Dennis MS, Duque J, Gadkar K, Lewcock JW, Mahon CS, Meisner R, Solanoy H, Thorne RG, Watts RJ, Zuchero YJY, Kariolis MS. CD98hc is a target for brain delivery of biotherapeutics. Nat Commun. 2023 Aug 19;14(1):5053. doi: 10.1038/s41467-023-40681-4. Erratum in: Nat Commun. 2023 Sep 7;14(1):5516. doi: 10.1038/s41467-023-41355-x. PMID: 37598178; PMCID: PMC10439950.
[11]    Yamamoto R, Yoden E, Tanaka N, Kinoshita M, Imakiire A, Hirato T, Minami K. Nonclinical safety evaluation of pabinafusp alfa, an anti-human transferrin receptor antibody and iduronate-2-sulfatase fusion protein, for the treatment of neuronopathic mucopolysaccharidosis type II. Mol Genet Metab Rep. 2021 Apr 18;27:100758. doi: 10.1016/j.ymgmr.2021.100758. PMID: 33981582; PMCID: PMC8081988.
[12]    Fukatsu T, Morio H, Furihata T, Sonoda H. Transferrin receptor-targeting property of pabinafusp alfa facilitates its uptake by various types of human brain-derived cells in vitro. Front Drug Deliv. 2023 Jul 3;3:1082672. doi: 10.3389/fddev.2023.1082672. PMID: 40838062; PMCID: PMC12363330.
[13]    https://investors.denalitherapeutics.com/static-files/bacd8b61-af8a-4f84-91fa-2c20ce531548
[14]    Kariolis MS, Wells RC, Getz JA, Kwan W, Mahon CS, Tong R, Kim DJ, Srivastava A, Bedard C, Henne KR, Giese T, Assimon VA, Chen X, Zhang Y, Solanoy H, Jenkins K, Sanchez PE, Kane L, Miyamoto T, Chew KS, Pizzo ME, Liang N, Calvert MEK, DeVos SL, Baskaran S, Hall S, Sweeney ZK, Thorne RG, Watts RJ, Dennis MS, Silverman AP, Zuchero YJY. Brain delivery of therapeutic proteins using an Fc fragment blood-brain barrier transport vehicle in mice and monkeys. Sci Transl Med. 2020 May 27;12(545):eaay1359. doi: 10.1126/scitranslmed.aay1359. PMID: 32461332.
[15]    Roche presents novel therapeutic and diagnostic advancements in Alzheimer’s at AD/PD 2025
[16]    Roche presents new data in Alzheimer’s disease from across its integrated pharmaceutical and diagnostics portfolio at AAIC
[17] Brainshuttle™ AD: New interim results of a randomized, placebo-controlled Phase Ib/IIa proof-of-concept study with trontinemab, a novel anti-amyloid monoclonal bispecific antibody for the treatment of Alzheimer’s disease - CTAD-2025-presentation-kulic-brainshuttle-tm-ad-new-interim-results.pdf