Studies on molecular basis of the pharmacokinetic regulation of protein drugs consisting of immunoglobulin Fc region

新生儿Fc受体（FcRn）是一种保护IgG免于分解代谢的受体，在维持高血清抗体水平方面很重要。为了阐明FcRn在含Fc结构域蛋白药物的药代动力学中的作用，本研究的重点是(1)建立表面等离子体共振（SPR）分析方法，测量含Fc结构域蛋白药物与FcRn的亲和力；(2)通过SPR分析评价FcRn与含Fc结构域蛋白药物的亲和力；(3)建立体外测定FcRn蛋白循环的方法；(4)炎症因子对FcRn表达的调控。(1)建立了以FcRn胞外结构域为配体的SPR分析方法。(2)含Fc结构域蛋白药物与FcRn的亲和力几乎与人血清半衰期相关，提示FcRn在调节这些药物的血清半衰期中的重要作用。所用分析物为人抗体（阿达木单抗）、人源抗体（Daclizumab和Omalizumab）、嵌合抗体（英夫利昔单抗）和铁融合蛋白（依那西普和Alefacept）。(3)建立了0.1 ~ 10ng/ml生物素化英夫利昔单抗的ELISA定量方法。测定生物素化英夫利昔单抗在脉冲标记的人脐静脉内皮细胞（HUVECs）培养上清中的浓度。(4)在huvec中，FcRn的表达被TNFα、IL-1β或IL-6抑制。提示这些炎性因子可能调控FcRn的表达水平。

The neonatal Fc receptor (FcRn) is a receptor that protects IgG from catabolism and is important in maintaining high serum antibody levels. In order to elucidate the role of FcRn in pharmacokinetics of Fc domain-containing protein drugs, this study focused on (1) establishment of Surface Plasmon Resonance (SPR) analysis that can measure the affinity of Fc domain-containing protein drugs to FcRn; (2) evaluation of the affinity between FcRn and Fc domain-containing protein drugs by SPR analysis; (3) establishment of in vitro method to measure the protein recycling via FcRn; and (4) regulation of FcRn expression by inflammatory cytokines.(1) SPR analysis method using extracellular domain of FcRn as the ligand was established. (2)The affinity of Fc domain-containing protein drugs to FcRn was almost correlated with the serum half lives in humans, suggesting the importance of FcRn in regulating serum half lives of these drugs. The analytes used were human antibody (Adalimumab), humanized antibodies (Daclizumab and Omalizumab), chimeric antibody (Infliximab), and Fe-fusion proteins (Etanercept and Alefacept). (3) ELISA method to quantify the 0.1〜10ng/ml of biotinylated Infliximab was established. Concentrations of biotinylated Infliximab in culture supernatant of human umbilical vein endothelial cells (HUVECs) those were pulse-labeled with it was measured. (4) In HUVECs, FcRn expression was suppressed by TNFα, IL-1β, or IL-6. The possibility of regulating the FcRn expression level by these inflammatory cytokines was suggested.