摘要
目的:旨在测定人生长激素-Fc融合蛋白注射液(recombinant human growth hormone-Fc fusion protein,rhGH-Fc)与不同种属(人、食蟹猴、犬、大鼠、小鼠)生长激素受体(growth hormone receptor,GHR)的结合力,并通过动力学参数分析,为非临床研究中动物模型的选择提供科学依据。方法:采用表面等离子共振(surface plasmon resonance,SPR)技术,利用Biacore 1K分子互作仪进行多循环动力学检测。实验采用氨基偶联法将GHR以及Fc受体(neonatal Fc receptor,FcRn)固定至CM5芯片表面,经偶联优化后,检测rhGH-Fc与不同种属GHR及FcRn的结合动力学参数,包括结合速率常数(ka)、解离速率常数(kd)及平衡解离常数(KD),采用捕获法进行亲和力分析。数据分析使用BiacoreInsight Evaluation软件完成。并通过NCBI数据库及SnapGene等工具,进行氨基酸序列同源性及进化树分析。结果:实验结果显示,rhGH-Fc与人及食蟹猴GHR和FcRn的结合力较高,而与大鼠结合力较低。结合动力学参数表明,食蟹猴受体与人类最为相似,验证其作为非临床研究相关动物模型的合理性。氨基酸序列同源性分析显示,恒河猴和食蟹猴生长激素(growth hormone,GH)与人类的同源性均为96.31%,GHR分别为94.2%和94.67%;犬、大鼠、小鼠GH同源性较低,分别为67.47%、64.98%和66.82%。IgG4 Fc段及FcRn区域的分析表明,恒河猴和食蟹猴与人类IgG4 Fc的同源性较高,分别为89%和87%,而FcRn同源性,恒河猴和食蟹猴分别为88%和89%,犬较低(77%),大鼠和小鼠最低(68%)。进化树分析显示,食蟹猴和恒河猴与人类亲缘性最高,犬次之,大鼠和小鼠亲缘性最远。结论:rhGH-Fc在食蟹猴中的结合动力学特征与人类高度相似,支持其作为非临床研究相关种属的合理性。相比之下,大鼠因GHR和FcRn结合力较低,不适合作为模拟人类药效和药代动力学的理想模型。本研究为非临床研究中动物模型的选择提供了重要参考依据。
Objective:This study aimed to determine the binding affinity of recombinant human Growth Hormone-Fc Fusion Protein injection(rhGH-Fc)to growth hormone receptors(GHR)of different species(human,cynomolgus monkey,dog,rat,and mouse)and to provide a scientific basis for selecting animal models in non-clinical studies through kinetic parameter analysis.Methods:Surface plasmon resonance(SPR)technology was used to perform multi-cycle kinetic assays with a Biacore 1K instrument.GHR and the neonatal Fc receptor(FcRn)were immobilized on the surface of a CM5 chip using amine coupling.After optimization of the coupling,the binding kinetic parameters of rhGH-Fc with GHR and FcRn from different species,including the association rate constant(ka),dissociation rate constant(kd),and equilibrium dissociation constant(KD),were determined.Affinity analysis was performed using a capture method.Data analysis was performed using BiacoreInsight Evaluation software.Amino acid sequence homology and phylogenetic tree analysis were performed using the NCBI database and SnapGene software.Results:The experimental results showed that rhGH-Fc had a higher binding affinity to human and cynomolgus monkey GHR and FcRn,while it had a lower binding affinity to rat GHR and FcRn.The binding kinetic parameters indicated that the cynomolgus monkey receptor was most similar to the human receptor,validating its suitability as an animal model for related non-clinical studies.Amino acid sequence homology analysis showed that the homology of growth hormone(GH)in rhesus monkeys and cynomolgus monkeys to human GH was 96.31%,while GHR homology was 94.2%and 94.67%,respectively.The homology of GH in dogs,rats,and mice was lower,at 67.47%,64.98%,and 66.82%,respectively.Analysis of the IgG4 Fc segment and FcRn region showed that the homology of rhesus monkeys and cynomolgus monkeys to human IgG4 Fc was higher,at 89%and 87%,respectively.The homology of FcRn in rhesus monkeys and cynomolgus monkeys was 88%and 89%,respectively,while it was lower in dogs(77%)and lowest in rats and mice(68%).Phylogenetic tree analysis showed that cynomolgus monkeys and rhesus monkeys were most closely related to humans,followed by dogs,with rats and mice being the most distantly related.Conclusion:The binding kinetic characteristics of rhGH-Fc in cynomolgus monkeys are highly similar to those in humans,supporting its suitability as a species for non-clinical studies.In contrast,rats are not suitable as an ideal model for simulating human pharmacodynamics and pharmacokinetics due to the lower binding affinity of GHR and FcRn.This study provides an important reference for the selection of animal models in non-clinical studies.
作者
尉建
李卓男
徐晓明
高健
马媛媛
张宇
刘云南
YU Jian;LI Zhuonan;XU Xiaoming;GAO Jian;MA Yuanyuan;ZHANG Yu;LIU Yunnan(Changchun Institute of Biological Products,Jilin Changchun 130000,China)
出处
《中国药物评价》
2025年第3期211-217,共7页
Chinese Journal of Drug Evaluation
作者简介
尉建,男,硕士研究生,研究方向:长效生长激素,分子检测等。
