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

• The exons 4–19 of the mouse Tfr1 gene encoding the extracellular domain were replaced with the corresponding human TFR1 sequences in B-hTFR1 mice. The genomic region encoding the cytoplasmic domain of mouse Tfr1 is retained. The promoter, 5′ UTR, and 3′ UTR regions of the mouse gene are also preserved. Expression of the chimeric TFR1 protein is driven by the endogenous mouse Tfr1 promoter, while native mouse Tfr1 transcription and translation are disrupted.
• The exons 2-10 encoding the extracellular domain of the mouse Slc3a2 (Cd98hc) gene were replaced with the corresponding human CD98HC (SLC3A2) exons in B-hCD98HC mice.
• B-hTFR1/hCD98HC double humanized mice were generated by crossing B-hTFR1 mice (110861) with B-hCD98HC mice (110983).

• 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 TFR1/CD98HC humanized mice

Blood cells were collected from wild-type C57BL/6JNifdc mice (+/+) and homozygous TFR1/CD98HC humanized mice (H/H, H/H) and analyzed by flow cytometry. Cells were stained 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 T cells, B cells, NK cells, neutrophils, and macrophages of wild-type mice, whereas humanized CD98HC was exclusively detected in corresponding leukocyte populations of homozygous TFR1/CD98HC humanized 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). mCD98HC was only detectable in wild-type mice, and hCD98HC was exclusively detectable in homozygous mice but not in wild-type mice.

• Mouse CD98HC was detectable only in neutrophils and macrophages of wild-type mice
• Human CD98HC was detectable only in neutrophils and macrophages of homozygous TFR1/CD98HC humanized mice

• Mouse CD98HC was detectable only in brain endothelial cells of wild-type mice
• Human CD98HC was detectable only in brain endothelial cells of homozygous TFR1/CD98HC humanized mice

Brain cells were isolated from wild-type C57BL/6JNifdc mice (+/+) and homozygous TFR1/CD98HC humanized mice (H/H, H/H). Flow cytometry was performed using species-specific antibodies against mouse and human CD98HC. Mouse CD98HC was detected only in brain endothelial cells of wild-type mice, while human CD98HC was exclusively detected in brain endothelial cells of homozygous TFR1/CD98HC humanized mice.

Western blot analysis was performed to evaluate human CD98HC protein expression in homozygous CD98HC humanized mice (H/H). Tissue lysates were collected from wild-type C57BL/6 mice and homozygous CD98HC humanized mice, and analyzed by western blot using a species-specific anti-human CD98 antibody (Abcam, ab307587). A total of 50 μg protein was loaded per lane. Human CD98HC protein was detectable in spleen, lung, kidney, and brain tissues from homozygous CD98HC humanized mice, but not in wild-type mice.

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

Protein expression of human CD98HC in the central nervous system was analyzed by western blot in wild-type C57BL/6JNifdc mice (+/+) and homozygous TFR1/CD98HC humanized mice (H/H). Tissue lysates from spinal cord, cerebral cortex, hippocampus, and cerebellum were collected and analyzed using anti-CD98 antibody (Abcam, ab307587). Human CD98HC protein was exclusively detected in all examined CNS regions of TFR1/CD98HC humanized mice, but not in wild-type controls. (A) Male mice. (B) Female mice. A total of 40 μg protein was loaded per lane.

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

Protein expression of human CD98HC in reproductive organs was evaluated by western blot in wild-type C57BL/6JNifdc mice (+/+) and homozygous TFR1/CD98HC humanized mice (H/H). Tissue lysates were collected from uterus, ovary, vagina, breast, testis, epididymis, prostate, and seminal vesicle, and analyzed using anti-CD98 antibody (Abcam, ab307587). Human CD98HC protein was exclusively detected in all examined reproductive tissues of TFR1/CD98HC humanized mice, but not in wild-type mice. A total of 30 μg protein was loaded per lane.

• Percentages of T cells, B cells, NK cells, DCs, monocytes, macrophages, and neutrophils are similar between TFR1/CD98HC humanized mice and C57BL/6 mice
• Humanization of TFR1 and CD98HC does not alter the frequency or distribution of major immune cell types in the spleen, blood, and lymph nodes.

Splenocytes were isolated from wild-type C57BL/6JNifdc mice (female, n=3, 7-week-old) and homozygous TFR1/CD98HC humanized mice (female, n=3, 7-week-old). Flow cytometry analysis was performed to assess leukocyte subpopulations. Percentages of T cells, B cells, NK cells, dendritic cells, neutrophils, monocytes, macrophages, CD4⁺ T cells, CD8⁺ T cells, and Tregs were comparable between genotypes. Similar distributions were observed in blood and lymph nodes (data not shown). Values are expressed as mean ± SEM. Statistical significance was determined by two-way ANOVA (*P < 0.05, **P < 0.01, ***P < 0.001). These results demonstrate that humanization of TFR1 and CD98HC does not alter immune cell development or distribution.

• Anti-human TFR1 antibody (JR-141 analog) showed higher brain exposure at 24 hours post-dose
• Anti-human CD98HC antibody (CD98BBBB-h1.L analog) showed higher brain exposure at 72 hours post-dose
• TFR1/CD98HC humanized mice enable the uptake of intravenously administered anti-human TFR1 or CD98HC antibodies
• TFR1/CD98HC humanized mice support a comparative evaluation of the brain penetration efficacy of shuttle molecules targeting TFR1 or CD98HC

Homozygous TFR1/CD98HC humanized mice (female, 8-week-old, n=2) were intravenously injected with control IgG (10 mpk), anti-humanized TFR1 antibody (JR-141 analog, monovalent, produced in-house, 12.56 mpk), or anti-humanized CD98HC antibody (CD98BBBB-h1.L analog, monovalent, produced in-house, 13.3 mpk). Brain samples were collected for in vivo pharmacokinetic evaluation. Brain concentration and percentage of injected dose per gram of brain tissue were quantified. Anti-humanized TFR1 antibody showed higher brain exposure at 24 hours post-dose, whereas anti-humanized CD98HC antibody showed higher brain exposure at 72 hours post-dose. These results demonstrate that TFR1/CD98HC humanized mice enable uptake of intravenously administered antibodies targeting either TFR1 or CD98HC and support comparative evaluation of brain penetration efficiency for shuttle molecules. Data are shown as mean ± SEM.

Q1. What makes TFR1 / CD98HC humanized mice unique?

They co-express human TFR1 and CD98HC, enabling validation of BBB shuttle antibodies and biologics targeting TFR1 (TFRC/CD71) or CD98HC (SLC3A2).

Q2. Are these mice suitable for antibody validation in vivo?

Yes, the model is designed for direct testing of human-specific antibodies and biologics.

Q3. Does humanization affect immune system development?

No, immune cell composition and distribution remain comparable to wild-type mice.

Q4. What disease areas can be studied using this model?

BBB-crossing drugs for brain diseases, oncology, immunology, autoimmune diseases, and metabolic disorders.

Q5. Can this model be used for long-term studies?

Yes, normal growth and immune homeostasis support chronic dosing and longitudinal studies.