Fig.1 Detection of TFR1 expression in liver by RT-PCR.?

Wild type: only one band at 243 bp with primers F1/R1(mTfr1);

Homozygous:? only one band at 279 bp with primers F2/R2(hTFR1).

Abbr. M, DNA marker; HO, homozygous; WT, wild type.

Fig2. Detection of human TFR1 expression in hTFR1 mice by WB.?

Abbr. HO, homozygous; WT, wild type.

Fig3. Expression characterization of human TFR1 on activated T cells in spleen in hTFR1 knockin mice.

Fig4. Detection of human TFR1 expression on erythroid cells in hTFR1 knockin mice-derived bone marrow.?

Fig5. Pattern of expression of activation markers overtime on activated CD4⁺ and CD8⁺ T cells upon anti-mCD3/mCD28 treatment.?

Note: The splenocytes were stimulated with anti-mCD3/mCD28 antibody in vitro.

Fig6. Detection of hTFR1 expression on endothelial cells in hTFR1 knokcin mice by immunofluorescence staining.?

Fig7. In vivo evaluation of TFR1-targeting therapeutic agents in hTFR1 knockin mice.

In vivo knock-down efficiency of hTFR1-targted siRNA in liver and heart in hTFR1 knockin mice.

Fig8.?In vivo?evaluation of TFR1-targeting therapeutic agents in hTFR1 knockin mice.

hTFR1/CD71 knockin mice were injected with a control antibody (10mg/kg) and an antibody (10 mg/kg) against human TFR1 via i.v. injection.?

Brain tissues and serums were taken after 18 hr post administration. Brain concentrations, serum concentrations and brain-to-serum ratio of antibodies were quantified. As shown in the figure, hTfr binding antibody exhibited higher serum clearance and enhanced brain exposure.

Fig.9 In vivo evaluation of TFR1-targeting therapeutic agents in hTFR1 knockin mice.

hTFR1/CD71 knockin mice were injected with tested antibody and hIgG1 control antibody? (10mg/kg) via i.v. injection.?

Brain tissues and serums were taken at indicated timepoint post administration. Brain concentrations, serum concentrations and brain-to-serum ratio of antibodies were quantified. As shown in the figure, hTfr binding antibodies are capable of targeting and penetrating BBB. (n=3)