PO.ET02.05 · 实验与分子治疗
免疫缺陷背景的FcRn人源化小鼠可用于评估抗体药物的药代动力学同时避免抗药抗体
FcRn humanized mice with an immunodeficient background can be used to evaluate the pharmacokinetics of antibody drugs while avoiding anti-drug antibodies
作者与单位 Authors & Affiliations
摘要 Abstract
中文摘要
引言：FcRn是一种pH依赖性受体，可结合并循环利用IgG，防止其被溶酶体降解，从而延长血浆半衰期。另一方面，Rag2对淋巴细胞成熟过程中的V(D)J重组至关重要，其缺失会导致缺乏功能性T细胞和B细胞，从而阻止抗药抗体（ADA）的形成。百奥赛图（Biocytogen）的B-hFcRn、Rag2 KO小鼠模型是一种用于人IgG治疗药物PK/PD/安全性评估的工具，通过T细胞和B细胞缺陷规避ADA。
方法：·B-hFcRn小鼠（110001）通过将小鼠Fcgrt基因的外显子2-4替换为编码全长人FCGRT的编码序列（CDS）而生成，同时小鼠Fcgrt基因的转录和翻译被破坏。B-Rag2 KO小鼠（110809）通过对Rag2基因外显子3及3'UTR区的靶向敲除而产生。该敲除导致Rag2失活。为生成B-hFcRn、Rag2 KO小鼠，将B-hFcRn小鼠（110001）与B-Rag2 KO小鼠（110809）交配。·为进行蛋白表达分析，从野生型C57BL/6N小鼠（+/+）、纯合B-hFcRn小鼠（H/H）以及纯合B-hFcRn、Rag2 KO小鼠（H/H, -/-）中采集脾脏、肺组织裂解物，然后用物种特异性抗FcRn抗体进行蛋白质印迹分析。·在B-hFcRn、Rag2 KO小鼠中分析了白细胞亚群的频率。使用流式细胞术对脾脏、血液和胸腺中的免疫细胞（包括T细胞和B细胞）进行定量，并与野生型C57BL/6小鼠比较。·在C57BL/6、B-hFcRn、B-Rag2 KO以及B-hFcRn、Rag2 KO小鼠中，经尾静脉注射并连续采血后，评估了YTE突变抗体和对照抗体的体内PK。
结果：·蛋白质印迹分析结果表明，在B-hFcRn、Rag2 KO小鼠的脾脏和肺中检测到了人FcRn。·流式细胞术分析显示，在纯合B-hFcRn、Rag2 KO小鼠的脾脏、血液和胸腺中未检测到T细胞和B细胞。·PK数据提示，在免疫功能正常的小鼠中形成了针对我们YTE抗体的ADA，而在Rag2 KO小鼠中未形成，且半衰期延长效应仅限于hFcRn人源化小鼠。
结论：B-hFcRn、Rag2 KO小鼠模型缺乏T细胞和B细胞但表达人FcRn，为评估在免疫功能正常小鼠中易受ADA干扰的抗体候选物的PK/PD提供了一种有价值的工具。
查看英文原文 English abstract
Introduction: FcRn is a pH-dependent receptor that binds and recycles IgG, preventing its lysosomal degradation to extend plasma half-life. Separately, Rag2 is essential for V(D)J recombination in lymphocyte maturation, and its absence results in a lack of functional T and B cells, preventing the formation of Anti-Drug Antibodies (ADA).Biocytogen's B-hFcRn, Rag2 KO mouse model is a tool for PK/PD/safety evaluation of human IgG therapeutics that circumvents ADA via T and B cell deficiency.
Methods: l B-hFcRn mice (110001) were generated by replacing exons 2-4 of the mouse Fcgrt gene with a coding sequence (CDS) that encodes full-length human FCGRT , while the mouse Fcgrt gene transcription and translation will be disrupted.B-Rag2 KO mice (110809) were produced through targeted knockout of exon 3 and the 3' UTR region of the Rag2 gene. This knockout leads to the inactivation of Rag2.To generate B-hFcRn, Rag2 KO mice, B-hFcRn mice (110001) were mated with B-Rag2 KO mice (110809).l For protein expression analysis, spleen, lung tissue lysates were collected from wild-type C57BL/6N mice (+/+), homozygous B-hFcRn mice (H/H), and homozygous B-hFcRn, Rag2 KO mice (H/H, -/-), and then analyzed by western blot with species-specific anti-FcRn antibody. l The frequency of leukocyte subpopulations was analyzed in B-hFcRn, Rag2 KO mice. Using flow cytometry, immune cells (including T and B cells) in the spleen, blood, and thymus were quantified and compared to wild-type C57BL/6 mice.l The in vivo PK of YTE-mutated and control antibodies was assessed in C57BL/6, B-hFcRn, B-Rag2 KO, and B-hFcRn, Rag2 KO mice following tail vein injection and serial blood collection.
Results: l Western blot analysis results indicated that human FcRn was detected in the spleen and lung of B-hFcRn, Rag2 KO mice.l Flow cytometry analysis revealed that T cells and B cells were undetectable in the spleen, blood, and thymus of homozygous B-hFcRn, Rag2 KO mice.l PK data suggest ADA formation against our YTE antibody in immunocompetent mice, its absence in Rag2 KO mice, and a half-life-extending effect that was confined to hFcRn humanized mice.
Conclusions: The B-hFcRn, Rag2 KO mouse model, deficient in T and B cells yet expressing human FcRn, provides a valuable tool for assessing the PK/PD of antibody candidates that are susceptible to ADA interference in immunocompetent mice.
利益披露 Disclosure
H. Horton, None..
S. Zhao, None..
Q. Xu, None..
X. Zhou, None.