Background:

• TFR1, a membrane protein expressed across various cell and tissue types in the human body, plays a crucial role in facilitating iron transport and metabolism. TFR1 is markedly expressed in numerous types of cancer cells. Studies have demonstrated that targeting TFR1 can effectively suppress tumor growth and metastasis. Moreover, TFR1 is also implicated in other conditions such as anemia and neurodegenerative disorders, etc.
• CD98HC (also known as SLC3A2) plays a crucial role in maintaining the integrity of the blood-brain barrier (BBB) and in nutrient transport, including amino acid transport, integrin signaling, and cellular stress responses.
• Given its high expression of TFR1 and CD98HC on brain endothelial cells, TFR1 and/or CD98HC can be leveraged as a receptor for receptor-mediated transcytosis (RMT), which allows for the efficient transport of large molecules across the BBB. This makes it an ideal target for delivering biotherapeutics to the central nervous system (CNS).
• Biocytogen's TFR1/CD98HC double humanized mice express human TFR1 and CD98HC under physiological regulatory control, enabling in vivo evaluation of human-specific antibodies and biologics targeting BBB shuttle pathways. This model supports quantitative assessment of brain penetration, head-to-head comparison of TFR1- versus CD98HC-mediated transport, and translational safety profiling to inform optimal CNS delivery strategies.

Verification:

• Human TFR1 protein and CD98HC protein were exclusively detectable in B-hTFR1/hCD98HC mice but not in wild-type mice. Humanization of TFR1 and CD98HC did not change the overall frequency or distribution of immune cell types in spleen, blood and lymph nodes.

Key Advantages

• Dual-target humanized model covering both TFR1 (CD71) and CD98HC (SLC3A2)
• Preserved immune system development and physiological stability
• Enables evaluation of human-specific antibodies, bispecifics, and other biologics
• Supports quantitative brain penetration and PK/biodistribution analysis
• Suitable for oncology, immunology, and BBB-penetrating CNS therapeutic research

Applications

• Validation of BBB shuttle antibodies and biologics targeting TFR1 (TFRC/CD71) or CD98HC (SLC3A2)
• Head-to-head comparison of receptor-mediated transcytosis strategies for CNS drug delivery
• Quantitative evaluation of brain penetration, biodistribution, and PK/PD relationships
• Oncology research, including tumor metabolism, nutrient transport, and immune modulation
• Immuno-oncology therapeutic efficacy studies in a human-relevant context
• Safety, target biology, and pharmacodynamic assessment of human-specific antibodies and biologics

TFR1 humanization (B-hTFR1 mice):

• The extracellular domain of the mouse Tfr1 gene (exons 4–19) is replaced with the corresponding human sequence.

CD98HC humanization (B-hCD98HC mice):

• The extracellular domain of the mouse Cd98 gene (exons 2–10) is replaced with human CD98 exons 4–12.

• Mouse TFR1 was detected only in erythrocytes of wild-type mice
• Human TFR1 was detected only in erythrocytes of homozygous TFR1 humanized mice and TFR1/CD98HC humanized mice.

Strain-specific TFR1 expression was analyzed by flow cytometry in bone marrow erythrocytes from wild-type C57BL/6JNifdc mice (+/+), homozygous TFR1 humanized mice (H/H), and homozygous TFR1/CD98HC humanized mice (H/H, H/H). Bone marrow cells were analyzed with anti-mouse TFR1 antibody (BioLegend, 113808) and anti-human TFR1 antibody (BioLegend, 334108). Mouse TFR1 was detected only in erythrocytes of wild-type mice, whereas humanized TFR1 was exclusively detected in erythrocytes of homozygous TFR1 humanized mice and TFR1/CD98HC humanized mice, but not in wild-type mice.

• Mouse TFR1 was detected only in erythrocytes of wild-type mice
• Human TFR1 was detected only in erythrocytes of homozygous TFR1 humanized mice and TFR1/CD98HC humanized mice.

Brain cells were collected from wild-type C57BL/6JNifdc mice (+/+), homozygous TFR1 humanized mice (H/H), and homozygous TFR1/CD98HC humanized mice (H/H, H/H). Flow cytometry was performed using anti-mouse TFR1 antibody (BioLegend, 113808) and anti-human TFR1 antibody (BioLegend, 334108). Mouse TFR1 was detectable only in brain endothelial cells of wild-type mice, whereas humanized TFR1 was exclusively detected in brain endothelial cells of homozygous TFR1 humanized mice and TFR1/CD98HC humanized mice.

• TFR1 was detected in the spinal cord, cerebral cortex, hippocampus, and cerebellum from both wild-type C57BL/6JNifdc mice and TFR1/CD98HC humanized mice, as the antibody was cross-reactive between human and mouse.

Western blot analysis was performed on tissue lysates from heart, liver, spleen, lung, kidney, stomach, colon, muscle, and brain of wild-type C57BL/6JNifdc mice (+/+) and homozygous TFR1 humanized mice (H/H). Samples were probed with anti-TFR1 antibody (Abcam, ab214039), which is cross-reactive between mouse and human TFR1. TFR1 protein was detectable in all examined tissues from both genotypes. GAPDH was used as a loading control. M: marker. Total protein loaded per lane: 40 μg.

• TFR1 was detected in the spinal cord, cerebral cortex, hippocampus, and cerebellum from both wild-type C57BL/6JNifdc mice and TFR1/CD98HC humanized mice, as the antibody was cross-reactive between human and mouse.

Protein expression of TFR1 in the spinal cord, cerebral cortex, hippocampus, and cerebellum was analyzed by western blot in wild-type C57BL/6JNifdc mice (+/+) and homozygous TFR1/CD98HC humanized mice (H/H). Tissue lysates were probed with anti-transferrin receptor antibody (Abcam, ab214039), which is cross-reactive between species. TFR1 was detectable in CNS tissues from both wild-type and humanized mice. (A) Male. (B) Female. Total protein loaded per lane: 40 μg.

• TFR1 was detected in the uterus, ovary, vagina, breast, testis, epididymis, prostate, and seminal vesicle from both wild-type C57BL/6JNifdc mice and homozygous TFR1/CD98HC humanized mice, as the antibody was cross-reactive between human and mouse.

Western blot analysis of uterus, ovary, vagina, breast, testis, epididymis, prostate, and seminal vesicle was performed in wild-type C57BL/6JNifdc mice (+/+) and homozygous TFR1/CD98HC humanized mice (H/H) using anti-transferrin receptor antibody (Abcam, ab214039). TFR1 protein was detectable in all examined reproductive organs from both genotypes due to antibody cross-reactivity. Total protein loaded per lane: 30 μg.

• Mouse CD98HC was detectable only in T cells, B cells and NK cells of wild-type mice.
• Human CD98HC was detectable only in T cells, B cells and NK cells of homozygous B-hTFR1/hCD98HC mice.

Strain specific CD98HC expression analysis in wild-type C57BL/6JNifdc and homozygous B-hTFR1/hCD98HC mice by flow cytometry. Blood cells were collected from wild-type C57BL/6JNifdc (+/+) and homozygous B-hTFR1/hCD98HC mice (H/H, H/H) and analyzed by flow cytometry with anti-mouse CD98HC antibody (Biolegend, 128211) and anti-human CD98HC antibody (231161-CD98BBBB-h1.L produced in-house).

• Mouse CD98HC was detectable only in neutrophils and macrophages of wild-type mice.
• Human CD98HC was detectable only in neutrophils and macrophages of homozygous B-hTFR1/hCD98HC mice.

Strain specific CD98HC expression analysis in wild-type C57BL/6JNifdc and homozygous B-hTFR1/hCD98HC mice by flow cytometry. Blood cells were collected from wild-type C57BL/6JNifdc (+/+) and homozygous B-hTFR1/hCD98HC mice (H/H, H/H) and analyzed by flow cytometry with anti-mouse CD98HC antibody (Biolegend, 128211) and anti-human CD98HC antibody (231161-CD98BBBB-h1.L produced in-house).

• Mouse CD98HC was detectable only in brain endothelial cells of wild-type mice.
• Human CD98HC was detectable only in brain endothelial cells of homozygous B-hTFR1/hCD98HC mice.

Strain specific CD98HC expression in wild-type C57BL/6JNifdc and homozygous B-hTFR1/hCD98HC mice by flow cytometry. Brain cells were collected from wild-type C57BL/6JNifdc (+/+) and homozygous B-hTFR1/hCD98HC mice (H/H, H/H) and analyzed by flow cytometry with anti-mouse CD98HC antibody (Biolegend, 128211) and anti-human CD98HC antibody (231161-CD98BBBB-h1.L produced in-house).

• Human CD98 is detected in the spleen, lung, kidney, stomach, colon, spinal cord, skin and brain of homozygous B-hCD98HC mice.

Western blot analysis of hCD98HC protein expression in homozygous B-hCD98HC mice.

Various tissue lysates were collected from wild-type C57BL/6 and homozygous B-hCD98HC mice (H/H), and then analyzed by western blot with species-specific anti-human CD98 antibody (Abcam, ab307587). 50 μg total proteins were loaded for western blotting analysis. M, marker.

• Human CD98HC was exclusively detected in spinal cord, cerebral cortex, hippocampus and cerebellum from B-hTFR1/hCD98HC mice.

Protein expression analysis of CD98HC in homozygous B-hTFR1/hCD98HC mice. Various tissue lysates were collected from wild-type C57BL/6JNifdc mice (+/+) and homozygous B-hTFR1/hCD98HC mice (H/H), and then analyzed by western blot with anti-CD98 antibody (abcam, ab307587). 40 μg total protein was loaded for western blotting analysis. (A) Male. (B) Female.

• Human CD98HC was exclusively detected in uterus, ovary, vagina, breast, testis, epididymis, prostate and seminal vesicle from homozygous B-hTFR1/hCD98HC mice.

Protein expression analysis of CD98HC in homozygous B-hTFR1/hCD98HC mice. Various tissue lysates were collected from wild-type C57BL/6JNifdc mice (+/+) and homozygous B-hTFR1/hCD98HC mice (H/H), and then analyzed by western blot with anti-CD98 antibody (Abcam, ab307587). 30 μg total protein was loaded for western blotting analysis.

• Humanization of TFR1 and CD98HC does not alter the frequency or distribution of immune cell types in spleen, blood and lymph nodes.

Values are expressed as mean ± SD.

• B-hTFR1/hCD98HC mice enable uptake of intravenously administered anti-human TFR1 or anti-human CD98HC antibodies.
• B-hTFR1/hCD98HC mice can be used to compare penetration efficacy of shuttle molecules targeting TFR1 vs. CD98HC.

In vivo PK evaluation and comparison of anti-human TFR1 and anti-CD98HC antibody. B-hTFR1/hCD98HC mice (n=2, female, 8-week-old) were injected with control IgG (10 mpk) anti-human TFR1 antibody (TFR1 Ab, JR-141 analog, monovalent, produced in house, 12.56 mpk) and anti-human CD98HC antibody (CD98HC Ab, CD98BBBB-h1.L analog, monovalent, produced in house, 13.3 mpk) via tail vein. Brain were taken for in vivo PK evaluation after dosing 18 h and  3, 5, 14, 21 days. Brain concentrations (A) and % of injection/gram brain (B) were quantified. As shown in panel, anti-human TFR1 antibody exhibited higher brain exposure in 24 h after dose, while anti-CD98HC antibody exhibited higher brain exposure in 72 h after dose. The results confirmed that B-hTFR1/hCD98HC mice enables uptake of an intravenously administered anti-human TFR1 antibody or anti-human CD98HC antibody, and this mice can be used for the comparison of penetration efficacy of shuttle molecules targeting TFR1 or CD98HC. Graphs represent mean ± SEM.