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.
• Given its high expression of TFR1 on brain endothelial cells, TFR1 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).

Validation:

• Human TFR1 was exclusively detectable in bone marrow erythrocytes and brain endothelial cells.
• Human TFR1 was detectable on brain microvascular endothelium of homozygous TFR1 humanized mice, but not in wild-type C57BL/6 mice.
• Human TFR1 was detectable in heart, liver, spleen, lung, kidney, brain, colon, skeletal muscle and eyes in homozygous TFR1 humanized mice.
• Leukocytes cell subpopulation analysis:
• Humanization of TFR1 does not change the overall frequency or distribution of immune cell types in spleen, blood, lymph nodes and thymus.
• In vivo PK analysis showed that TFR1 humanized mice enables brain uptake of an intravenously administered anti-human TFR1 BsAbs.
• TFR1 and DMPK dual-target humanized mice enables muscle uptake of an intravenously administered AOC drug targeted DMPK.

Application:

• This product is utilized to evaluate the pharmacodynamics and safety of therapeutic agents targeting tumors and neurodegenerative diseases, and to assess potential of drugs to cross the blood-brain barrier.

Targeting strategy of TFR1 humanized mice. The exons 4-19 of mouse Tfr1 gene that encode the extracellular domain were replaced by human TFR1 exons 4-19 in TFR1 humanized mice.

Human TFR1 was exclusively detectable in blood of homozygous TFR1 humanized mice, but not in wild-type C57BL/6 mice, and its expression level is independent of gender.

Human TFR1 was exclusively detectable in blood of homozygous TFR1 humanized mice, but not in wild-type C57BL/6 mice, and its expression level is independent of gender.

Human TFR1 was exclusively detectable in blood of homozygous TFR1 humanized mice, but not in wild-type C57BL/6 mice, and its expression level is independent of gender.

Human TFR1 was detectable on brain microvascular endothelium of homozygous TFR1 humanized mice, but not in wild-type C57BL/6 mice.

Immunohistochemical (IHC) analysis of TFR1 protein expression in wild-type C57BL/6 mice and homozygous TFR1 humanized mice. Mouse tissues were collected from wild-type C57BL/6 mice and homozygous TFR1 humanized mice (female, 8-week-old, n=3). Protein expression was analyzed with anti-TFR1 antibody by IHC. Human TFR1 was detected in the heart, liver, spleen, lung, kidney, brain, colon and stomach of TFR1 humanized mice, which is similar to the expression pattern of mouse TFR1 in wild-type C57BL/6 mice. Red arrow: positive cells expressing TFR1.

Western blot analysis of TFR1 protein expression in wild-type C57BL/6JNidc mice and homozygous TFR1 humanized mice by WB. Various tissues were collected from wild-type C57BL/6JNifdc mice (+/+) and homozygous TFR1 humanized mice (H/H), and then analyzed by western blot with anti-TFR1 antibody (abcam, ab214039). 40 μg total proteins were loaded for western blotting analysis. GAPDH were detected as internal control. TFR1 was detectable in heart, liver, spleen, lung, kidney, stomach, colon, muscle and brain from both C57BL/6JNifdc and homozygous TFR1 humanized mice, as the antibody was cross-reactive between human and mouse. M, marker.

TFR1 expression analysis in eyeball. The expression of human TFR1 was observed in corneal epithelial cells, endothelial cells, and lens epithelial cells, while it was highly expressed in the retina in TFR1/CD98HC humanized mice, but not in wild-type C57BL/6 mice.

TFR1 mRNA expression analysis in homozygous TFR1 humanized mice. Brain (A), skeletal muscle (B), eyeball (C) and heart (D) RNA were isolated from wild-type C57BL/6 mice (+/+) (female, 7-week-old, n=3; male, 7-week-old and 24-week-old, n=3) and homozygous TFR1 humanized mice (H/H) (female, 7-week-old, 11-week-old, 15-week-old, n=3; male, 7-week-old, 11-week-old, 15-week-old, 24-week-old, n=3) ), then cDNA libraries were synthesized by reverse transcription, followed by PCR with TFR1 primers. TFR1 expression level in female homozygous TFR1 humanized mice is similar to those in wild-type C57BL/6 mice and remains consistent across different ages of mice (up). TFR1 expression level in male homozygous TFR1 humanized mice is similar to those in brain, heart and eyeball in wild-type C57BL/6 mice (down). Values are expressed as mean ± SEM.

Humanization of TFR1 does not change the overall frequency or distribution of immune cell types in spleen, blood and lymph nodes. Values are expressed as mean ± SD.

Serum iron levels in wild-type C57BL/6 and homozygous TFR1 humanized mice. Serum were collected from C57BL/6 and TFR1 humanized mice (n=3, 6 week old, female), and then analyzed by Iron Assay Kit (abcam, ab83366). Serum iron levels in homozygous TFR1 humanized mice were similar to those in the wild-type C57BL/6 mice. Values are expressed as mean ± SEM. Significance was determined by two-way ANOVA test.  *P < 0.05, **P < 0.01, ***p < 0.0001.

Liver complement component 3 levels in wild-type C57BL/6 and homozygous TFR1 humanized mice. Liver were collected from C57BL/6 and TFR1 humanized mice (n=3, 6 week old, female), then cDNA libraries were synthesized by reverse transcription, followed by qPCR with mouse complement component 3 primers. The mRNA expression of mouse complement component 3 in homozygous TFR1 humanized mice was similar to those in the wild-type C57BL/6 mice. Values are expressed as mean ± SEM. Significance was determined by unpaired t-test.  *P < 0.05, **P < 0.01, ***P < 0.001.

Growth curve of TFR1 humanized mice. Select mice aged 3 to 8 weeks, and randomly sample and weigh 50 males and 50 females from each age group. The minimum and maximum weights of the mice in the table are calculated as the average ± SD. The growth curve follows a normal distribution, with a 68% probability that random errors fall within the ± SD range.

Hematoxylin and eosin (H&E) staining of femur. The femur from wild-type C57BL/6 mice and TFR1 humanized mice (female, 10 week-old, n=10) were collected and analyzed by H&E staining. Representative results of WT mice (A) and homozygous TFR1 humanized mice (B) are shown, and no abnormalities were found in the bone marrow of both mice. The results indicated that humanization of TFR1 does not change the normal morphology of mouse bone marrow.

TFR1 humanized mice were intravenously injected with AF680-conjugated control hIgG1 or anti human TFR1 antibodies ab1 and ab2 (provided by a client). After 2 days or 3 days post-injection, the mice were perfused and their brains were collected for analysis. (A) Mouse brain images under imaging system. (B) Fluorescence intensity of mouse brain under imaging system. The results indicate that the uptake of anti-human TFR1 antibody ab2 in the brain of TFR1 humanized mice was higher than that of anti-human TFR1 antibody ab1.

In vivo pharmacokinetic (PK)  evaluation of anti-human TFR1 bispecific antibodies (BsAbs). TFR1 humanized mice were injected with control IgG (10 mpk) and anti-human TFR1 BsAbs (10.9 mpk) provided by a client via tail vein. Brain and serum were taken for in vivo PK evaluation. Brain concentrations (A), serum concentrations (B), and brain-to-serum ratio (C) of anti-human TFR1 BsAbs were quantified. As shown in panel, anti-human TFR1 BsAbs exhibited higher serum clearance and enhanced brain exposure after dose. The results confirmed that brain of TFR1 humanized mice enables uptake of an intravenously administered anti-human TFR1 BsAbs and TFR1 humanized mice provide a powerful preclinical model for in vivo evaluation of effective delivery of protein therapeutics to the central nervous system (CNS). Graphs represent mean ± SEM.

Note: This experiment was performed by the client using TFR1 humanized mice. All the other materials were provided by the client.

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 (JR-141 analog, monovalent, produced in house, 12.56 mpk) and anti-human CD98HC antibody (CD98BBBB-h1.L analog, monovalent, produced in house, 13.3 mpk) via tail vein. Brain were taken for in vivo PK evaluation. 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.

The inhibitory efficiency of the antibody oligonucleotide conjugates drug against human DMPK in heterozygous TFR1/DMPK humanized mice. The antibody oligonucleotide conjugates drug (in-house), naked antibody (in-house) and PBS were administered to the heterozygous TFR1/DMPK humanized mice individually on day 0. The mice were sacrificed on day 7, and the liver, gastrocnemius muscle and tibialis anterior muscle were collected to detect the expression level of human DMPK mRNA by qPCR. The human DMPK mRNA in the treatment groups (antibody oligonucleotide conjugates drug) were significantly reduced compared to the control groups (naked antibody and PBS) in tibialis anterior muscle, demonstrating that TFR1/DMPK humanized mice provide a powerful preclinical model for in vivo evaluation of human DMPK targeted antibody oligonucleotide conjugates drug. Values are expressed as mean ± SEM.

Humanization of TFR1 does not alter hematological parameters. Values are expressed as mean ± SD.

Biochemical test of B-hTFR1 mice. Values are expressed as mean ± SD.

Q1: What is TFR1 and why is it important?

A1: TFR1 (CD71) is a transferrin receptor essential for iron uptake and cell proliferation. It is implicated in cancer growth, immune regulation, and iron metabolism disorders.

Q2: Why use B-hTFR1 mice instead of wild-type mice?

A2: B-hTFR1 mice express human TFR1 protein, making them suitable for antibody validation and translational research, which cannot be achieved in wild-type mice.

Q3: What research areas can benefit from B-hTFR1 mice?

A3: They are valuable for oncology drug development, autoimmune disease studies, and preclinical safety evaluation of TFR1-targeting therapies.

Q4: Can B-hTFR1 mice be used for antibody validation in vivo?

A4: Yes, they are specifically designed for in vivo validation of antibodies targeting human TFR1.