• 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.

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 detected exclusively in T cells, B cells, and NK cells of wild-type C57BL/6JNifdc mice.
• Human CD98HC was detected exclusively in T cells, B cells, and NK cells of homozygous TFR1/CD98HC humanized mice.

Strain-specific analysis of mouse and human CD98HC protein expression in wild-type C57BL/6JNifdc mice and TFR1/CD98HC humanized mice by flow cytometry. Blood cells were collected from wild-type C57BL/6JNifdc mice (+/+) and homozygous TFR1/CD98HC humanized mice (H/H; H/H). Mouse and human CD98HC protein expression was analyzed by flow cytometry using anti-mouse CD98HC (BioLegend, 128211) and anti-human CD98HC (231161-CD98BBBB-h1.L, produced in-house) antibodies.

• 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.

Strain-specific analysis of mouse and human CD98HC protein expression in wild-type C57BL/6JNifdc mice and TFR1/CD98HC humanized mice by flow cytometry. Blood cells were collected from wild-type C57BL/6JNifdc mice (+/+) and homozygous TFR1/CD98HC humanized mice (H/H; H/H). Mouse and human CD98HC protein expression was analyzed by flow cytometry using anti-mouse CD98HC (BioLegend, 128211) and anti-human CD98HC (231161-CD98BBBB-h1.L, produced in-house) antibodies.

• Mouse CD98HC was detected only in brain endothelial cells of wild-type C57BL/6JNifdc mice.
• Human CD98HC was detected only in brain endothelial cells of homozygous TFR1/CD98HC humanized mice.

Strain-specific analysis of mouse and human CD98HC protein expression in brain endothelial cells of wild-type C57BL/6JNifdc mice and TFR1/CD98HC humanized mice by flow cytometry. Brain cells were collected from wild-type C57BL/6JNifdc mice (+/+) and homozygous TFR1/CD98HC humanized mice (H/H; H/H). Mouse and human CD98HC protein expression was analyzed by flow cytometry using anti-mouse CD98HC (BioLegend, 128211) and anti-human CD98HC (231161-CD98BBBB-h1.L, produced in-house) antibodies.

• Human CD98HC protein was detected in the spleen, lung, kidney, stomach, colon, spinal cord, skin, and brain of homozygous CD98HC humanized mice.

Western blot analysis of human CD98HC protein expression in multiple tissues of CD98HC humanized mice. Tissue lysates were collected from wild-type C57BL/6 mice and homozygous CD98HC humanized mice (H/H), including the spleen, lung, kidney, stomach, colon, spinal cord, skin, and brain. Human CD98HC protein expression was analyzed by Western blot using a species-specific anti-human CD98 antibody (Abcam, ab307587). A total of 50 μg of protein was loaded for Western blot analysis. M: protein marker.

• Human CD98HC protein was detected exclusively in the spinal cord, cerebral cortex, hippocampus, and cerebellum of TFR1/CD98HC humanized mice and was not detected in wild-type C57BL/6JNifdc mice.

Western blot analysis of human CD98HC protein expression in central nervous system tissues of TFR1/CD98HC humanized mice. Tissue lysates were collected from the spinal cord, cerebral cortex, hippocampus, and cerebellum of wild-type C57BL/6JNifdc mice (+/+) and homozygous TFR1/CD98HC humanized mice (H/H). Human CD98HC protein expression was analyzed by Western blot using an anti-CD98 antibody (Abcam, ab307587). A total of 40 μg of protein was loaded for Western blot analysis. (A) Male mice. (B) Female mice.

• Human CD98HC was exclusively detected in uterus, ovary, vagina, breast, testis, epididymis, prostate and seminal vesicle from homozygous TFR1/CD98HC humanized mice.

Western blot analysis of human CD98HC protein expression in reproductive tissues of TFR1/CD98HC humanized mice. Tissue lysates were collected from the uterus, ovary, vagina, mammary gland, testis, epididymis, prostate, and seminal vesicle of wild-type C57BL/6JNifdc mice (+/+) and homozygous TFR1/CD98HC humanized mice (H/H). Human CD98HC protein expression was analyzed by Western blot using an anti-CD98 antibody (Abcam, ab307587). A total of 30 μg of protein was loaded for Western blot 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.

• TFR1/CD98HC humanized mice support the evaluation of brain uptake following intravenous administration of anti-human TFR1 or anti-human CD98HC antibodies.
• TFR1/CD98HC humanized mice provide a preclinical model for comparing the brain penetration and pharmacokinetic profiles of antibody shuttle molecules targeting TFR1 versus CD98HC.

In vivo pharmacokinetic (PK) evaluation and comparison of brain exposure to anti-human TFR1 and anti-human CD98HC antibodies in TFR1/CD98HC humanized mice. Female TFR1/CD98HC humanized mice (8 weeks old, n = 2) received intravenous injections via the tail vein of control IgG (10 mg/kg), an anti-human TFR1 antibody (TFR1 Ab, JR-141 analog, monovalent, produced in-house, 12.56 mg/kg), or an anti-human CD98HC antibody (CD98HC Ab, CD98BBBB-h1.L analog, monovalent, produced in-house, 13.3 mg/kg). Brain tissues were collected at 18 hours and on days 3, 5, 14, and 21 after dosing for in vivo pharmacokinetic analysis. (A) Brain antibody concentrations. (B) Percentage of injected dose per gram of brain tissue (%ID/g). The anti-human TFR1 antibody exhibited higher brain exposure at the early time point (within 24 hours after dosing), whereas the anti-human CD98HC antibody exhibited higher brain exposure at 72 hours after dosing. These results demonstrate the utility of TFR1/CD98HC humanized mice for evaluating brain uptake and comparing the pharmacokinetic profiles of antibody shuttle molecules targeting human TFR1 and CD98HC. Data are presented as mean ± SEM.