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14 Humanized TFR1/CD71 knockin mouse model enables in vivo assessment of TFR1-targeted antibody therapies for cancer and beyond to across the blood-brain barrier
  1. Hechun Ma1,
  2. Yi Li1,
  3. Ping Yang1,
  4. Renxing Zhang1,
  5. Dongxiao Feng2,
  6. Jian Fei1,
  7. Ruilin Sun1 and
  8. Daniel X He1,2
  1. 1Shanghai Model Organisms Center, Inc., Shanghai, China
  2. 2Shanghai Model Organisms Center, Inc., Houston, TX, USA
  • Journal for ImmunoTherapy of Cancer (JITC) preprint. The copyright holder for this preprint are the authors/funders, who have granted JITC permission to display the preprint. All rights reserved. No reuse allowed without permission.


Background The development of antibody-based therapeutics for the neurological diseases and glioma is largely hampered due to the blood-brain barrier (BBB). It is appealing to identify more promising targets that are highly selectively expressed on brain endothelial cells (BECs) which could be leveraged to transport antibodies across the BBB into the brain.1 2 TFR1 (transferrin receptor 1), also known as CD71, is such an example which has been most extensively studied for receptor-mediated transcytosis (RMT) for drug delivery to the brain.3 4 However, actively proliferating cells, reticulocytes as well as tumor cells can also express TFR1. The increased expression of TFR1 is associated with the poor prognosis in various cancer types, for example, NSCLC and esophageal squamous cell carcinoma. Thus, TFR1 can also be a therapeutic target against tumor by developing either TFR1-targeted antibody drug conjugations (ADC) via RMT or TFR1 mAb-mediated ADCC.5 6 Taken together, we developed a humanized TFR1 knockin mouse model, which could be a powerful preclinical model to enable assessment of the efficacy, safety and PK/PD of therapeutic antibodies specifically against human TFR1/CD71 in vivo.

Methods We developed the humanized TFR1 knockin mouse model by insertion of whole human TFR1 coding sequence plus WPRE/polyA stop cassette into mouse Cd71 gene locus in C57BL/6 background via ES cell-based gene targeting. To characterize the hTFR1 knockin mice, firstly, we verified hTFR1 expression on the brain endothelial cells by immunohistochemistry (IHC) and then we performed flow cytometry analysis to characterize hTFR1 expression on activated T cells after in vitro stimulation by PHA-L and anti-mCD3/mCD28 for various exposure time. Additionally, the hTFR1 expression on bone marrow-derived erythroid cells was confirmed by FACS. Finally, we treated the hTFR1 knockin mice with an anti-human TFR1 antibody and control antibody via i.v. injection to evaluate the in vivo PK and brain uptake capability across the BBB.

Results We observed increased expression of hTFR1 on activated T lymphocytes upon stimulation. Brain tissues and serums were taken from the hTFR1 knockin mice i.v. injected with a human-specific TFR1 antibody (10 mg/kg) and a control antibody (10mg/kg) as a single dose 18 hours post treatment for PK analysis. The antibody concentrations in brain and serum were quantified by ELISA. Human-specific TFR1-binding antibody exhibited higher serum clearance and enhanced brain uptake against blood-brain barrier.

Conclusions Our humanized TFR1 knockin mice provides a powerful tool to evaluate the in vivo efficacy, safety and PK/PD of the therapeutic antibodies targeting human TFR1/CD71 in the preclinical investigations.


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Ethics Approval All studies were conducted following an approved IACUC protocol (SMOC IACUC No. 2022–0008). Although this study was not conducted in accordance with the FDA Good Laboratory Practice regulations, 21 CFR Part 58, all experimental data management and reporting procedures were in strict accordance with applicable Shanghai Model Organisms Center, Inc. Guidelines. Guidelines and Standard Operating Procedures. The methods and results in this study accurately reflect the raw data generated during the execution of the study.

This is an open access article distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited, appropriate credit is given, any changes made indicated, and the use is non-commercial. See

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