PO.PR02.01 · 预防研究
人源化TFR1敲入小鼠：评估TFR1靶向脑穿梭体的强大平台
Humanized TFR1 knock-in mice: A powerful platform for evaluating TFR1-targeting brain shuttles
作者与单位 Authors & Affiliations
摘要 Abstract
中文摘要
背景：血脑屏障（BBB）的存在极大地阻碍了针对神经系统疾病和胶质瘤的治疗性抗体和基于抗体的诊断方法的开发。虽然BBB限制了大多数大分子，但它通过选择性转运机制允许必需营养物质的转运，从而为利用脑穿梭技术将药物递送至中枢神经系统创造了机会。转铁蛋白受体1（TFR1）在脑内皮细胞（BEC）上表达，是研究最广泛的受体介导的胞吞转运（RMT）靶点之一，并已被验证为脑药物递送的门户。为支持TFR1靶向抗体或基于肽的脑穿梭体的开发，我们生成了一种人源化TFR1敲入小鼠模型（hTFR1），作为评估TFR1靶向治疗药物疗效、安全性和PK/PD特征的稳健临床前平台。
方法：通过基于ES细胞的基因打靶，将全长人TFR1编码序列加上WPRE/polyA终止盒插入C57BL/6背景的小鼠Cd71基因位点，生成hTFR1敲入小鼠模型，其中全长人TFR1蛋白表达于靶细胞表面。为表征hTFR1敲入小鼠中人TFR1的表达，我们通过免疫荧光（IF）染色评估了其在脑内皮细胞上的定位，并进行流式细胞术分析以表征其在活化T细胞上的表达。最后，我们通过静脉注射向hTFR1小鼠给予Trontinemab和通过我们的SmocMab平台生成的抗人TFR1抗体，以评估其PK特征和跨BBB的脑摄取能力。
结果：hTFR1小鼠中人TFR1的表达在脑微血管中得到确认，并在活化的T淋巴细胞上上调。对于PK分析，在单次静脉给药（10 mg/kg）人特异性TFR1抗体或对照抗体后24小时采集脑组织和血清样本。通过ELISA定量抗体浓度。与对照抗体相比，人特异性TFR1结合抗体表现出加速的血清清除和增加的脑摄取，证明了其增强的BBB穿透能力。
结论：我们的人源化TFR1敲入小鼠为TFR1靶向治疗性抗体的临床前评估提供了强大工具。
查看英文原文 English abstract
Background: The development of therapeutic antibodies and antibody-based diagnostics for the neurological disorders and gliomas is largely hampered by the presence of blood-brain barrier (BBB). While the BBB restricts most large molecules, it permits the transport of essential nutrients through selective transport mechanisms, creating opportunities to delivery drugs into the central nervous system using brain shuttle technologies. Transferrin receptor 1 (TFR1), which is expressed on brain endothelial cells (BECs), is one of the most extensively studied targets for receptor-mediated transcytosis (RMT) and has been validated as a gateway for brain drug delivery. To support the development of TFR1-targeting antibody or peptide-based brain shuttles, we generated a humanized TFR1 knock-in mouse model (hTFR1), serving as a robust preclinical platform for evaluating the efficacy, safety, and PK/PD profiles of TFR1-targeted therapeutics.
Methods: The hTFR1 knock-in mouse model was generated by inserting the full-length human TFR1 coding sequence plus WPRE/polyA stop cassette into mouse Cd71 gene locus in C57BL/6 background via ES cell-based gene targeting, in which full-length human TFR1 proteins are expressed on the surface of targeted cells. To characterize the human TFR1 expression in the hTFR1 knockin mice, we assessed its localization on the brain endothelial cells by immunofluorescence (IF) staining and performed flow cytometry analysis to characterize its expression on activated T cells. Finally, we administered Trontinemab and the anti-human TFR1 antibodies generated via our SmocMab platform to hTFR1 mice through i.v. injection to evaluate th PK characteristics and brain uptake capability across the BBB.
Results: Human TFR1 expression in hTFR1 mice was confirmed in brain microvessels and was upregulated on activated T lymphocytes. For PK analysis, brain tissues and serum samples were collected 24 hours after a single intravenous dose (10 mg/kg) of the human-specific TFR1 antibodies or a control antibody. Antibody concentrations were quantified by ELISA. The human-specific TFR1-binding antibody showed accelerated serum clearance and increased brain uptake compared with the control antibody, demonstrating its enhanced ability to penetrate the BBB.
Conclusions: Our humanized TFR1 knockin mice provides a powerful tool for preclinical evaluation of TFR1-targeting therapeutic antibodies.
利益披露 Disclosure
L. Feng,
Shanghai Model Organisms Center, Inc. Other, Parent Company.
S. Li,
Shanghai Model Organisms Center, Inc. Other, Parent Company.
Z. Cao,
Shanghai Model Organisms Center, Inc. Other, Parent Company.
Y. Li,
Shanghai Model Organisms Center, Inc. Other, Parent Company.
D. Feng,
Shanghai Model Organisms Center, Inc. Other, Parent Company.
R. Sun,
Shanghai Model Organisms Center, Inc. Other, Parent Company.