TFR1: Target for Drug Delivery Across the BBB

• TfR1: Receptor for transferrin, mediates iron transport via transcytosis, and maintains brain iron homeostasis
• Highly enriched on brain endothelial cells in the BBB
• Serves as a target for enhancing drug delivery across the BBB

• Highly expressed on brain endothelial cells, enabling selective engagement for CNS delivery.
• Facilitates transcytosis, offering a mechanism to transport therapeutic agents across the BBB.
• Demonstrated potential in preclinical models for enhancing CNS penetration of biologics without compromising BBB integrity.

• Gene Information: Human IGF1R maps to chromosome 15q26.3,containing 21 exons encoding a heterotetrameric receptor precursor.
• Protein Expression: IGF1R is ubiquitously expressed across mosthuman tissues, highly abundant in brain, lung, epithelium andtumor cells. It maintains low basal levels in mature organs but ismarkedly upregulated in proliferative, fibrotic and malignantlesions for cell survival
• Signaling Pathway: Upon IGF-1/IGF-2 binding, IGF1R activatesPI3K/Akt and RAS/MAPK cascades, driving cell proliferation, anti-apoptosis and matrix remodeling.
• Therapeutic Inhibition: Anti-IGF1R neutralizing antibodies, small-molecule kinase inhibitors and IGF ligand traps block receptoractivation to suppress tumor growth and tissue fibrosis. Combinedchemo- or targeted therapy improves efficacy, while drug resistanceremains a key clinical limitation under investigation.

TFR1：

The exons 4 19 of mouse Tfr1 gene that encode extracellular domain are replaced by human counterparts.

The genomic region of mouse Tfr1 gene that encodes cytoplasmic portion is retained. The promoter, 5’UTR and 3’UTR region of the mouse gene are also retained.

The chimeric TFR1 expression is driven by endogenous mouse Tfr1 promoter, while mouse Tfr1 gene transcription and translation will be disrupted.

CD98HC：

The exons 2 10 of mouse CD98HC gene that encode extracellular domain are replaced by human CD98HC exons 4 12.

The genomic region of mouse CD98HC gene that encodes cytoplasmic portion is retained. The promoter, 5’UTR and 3’UTR region of the mouse gene are also retained.

The chimeric CD98HC expression is driven by endogenous mouse CD98HC promoter, while mouse CD98HC gene transcription and translation will be disrupted.

IGF1R：

A chimeric CDS that encodes mouse Igf1r signal peptide and human extracellular domain, human transmembrane and mouse cytoplasmic domain, as well as mouse 3’UTR regionwere inserted right after mouse Igf1r exon 2 to replace the exon 2 of mouse Igf1r gene.

The chimeric IGF1R protein expression will be driven by endogenous mouse Igf1r promoter, while mouse Igf1r gene transcription and translation will be disrupted.

B-hTFR1/hCD98HC/hIGF1R plus mice were obtained by mating B-hTFR1 mice (110861), B-hCD98HC mice (110983) and B-hIGF1R mice plus (111974).

• TFR1 and IGF1R were both detected in wild-type mice and humanized B-hTFR1/hCD98HC/hIGF1R plus mice, as the antibody was cross-reactive between human and mouse. 
• CD98HC was exclusively detected in humanized B-hCD98HC/hIGF1R plus mice and homozygous B-hTFR1/hCD98HC/hIGF1R plus mice.

Strain-specific TFR1, CD98HC and IGF1R expression analysis in wild-type C57BL/6JNifdc mice, homozygous B-hCD98HC/hIGF1R plus mice and homozygousB-hTFR1/hCD98HC/hIGF1R plus mice. Cortex lysates were collected from wild-type C57BL/6JNifdc mice (+/+), homozygous B-hCD98HC/hIGF1R plus mice andhomozygous B-hTFR1/hCD98HC/hIGF1R plus mice, and then analyzed by western blot with anti-transferrin receptor antibody (abcam, ab214039), anti-CD98antibody (abcam, ab307587) and anti-IGF1R antibody (CST, 3027). 30 μg total proteins were loaded for western blotting analysis.