Large quantities of NPC can be readily obtained in the process of purifying hepatocytes from liver, but this fraction is usually discarded. In this study, we propose an alternative method using NPC (Fig. Thus, a method for determining the binding and uptake of an antibody by LSEC is an important prerequisite for understanding and/or predicting the elimination process of an antibody. Also, during the isolation and culturing processes, the phenotype and biological activity are lost when they are cultured in the absence of other types of feeding cells 15. 1) since, especially for monkeys and humans, preparing LSEC involves multiple steps including isolation and purification 4, 13, 14. However, it is difficult to use in vitro LSEC, especially in monkeys and humans, for binding and uptake assays of an antibody (Fig. Therefore, evaluating the binding and uptake of an antibody in FcγRIIB-expressing cells in NPC, and determining the PK parameters related to FcγRIIB-mediated elimination is essential in terms of understanding the PK of an antibody. Thus, the binding of the Fc region of an antibody against FcγRIIB has a large influence on antibody clearance 12. LSEC and KC, which express FcγRIIB in NPC, play a key role in the clearance of an antibody and an immune complex particularly 5, 6. NPC, in which various scavenger receptors are expressed, play an important role in the elimination of an antibody, an antigen–antibody complex (immune complex) 5, 6, an oligonucleotide 7, 8, a proteglycan 9, 10, and a virus 11 from the blood circulation. Thus, an in vitro assay which is capable of evaluating both binding and uptake via the neonatal Fc receptor (FcRn) and the Fc fragment of the IgG receptor (FcγR) IIB, which play important roles in antibody clearance would be highly desirable 3.Īpproximately 40% of hepatic cells are non-parenchymal cells (NPC), and are a mixture of various cells including liver sinusoidal endothelial cells (LSEC), kupffer cells (KC), stellate cells, among others 4. This is a problem in terms of the 3Rs (replacement, reduction and refinement of experimental animals) principle in the early stage of drug discovery. However, monitoring plasma concentrations typically requires a long period of time (generally several days to months), and a large number of experimental animals, especially monkeys, are needed to evaluate the PK of an antibody in vivo.
These parameters are generally used to determine the human PK of an antibody 1, 2. The in vivo fitting of the PK profile is the gold standard in antibody PK assays, since it permits the PK parameters to be determined separately for non-specific and target-dependent elimination. The findings presented herein demonstrate that the in vitro liver NPC assay using flow cytometry is a useful tool to determine the binding and uptake of biologics and to predict the PK. The concentration-dependent binding and uptake was measured to determine the PK parameters using monkey NPC, the FcγRIIB-expressing fraction of which was identified by CD31 and CD45. This approach was also extended to NPC from monkeys. A titration assay was performed to determine the PK parameters, and the obtained parameter was comparable to that determined by the fitting of the in vivo PK. In mice, the FcγRIIB-expressing cells were identified in the CD146-positive and CD45-negative fraction by flow cytometry. This study reports on the development of a cell-based assay for evaluating the binding and uptake of such an antibody using liver NPC of mice and monkeys.
However, there is currently no in vitro quantitative assay using NPC. Evaluation of the binding and uptake of an antibody in liver non-parenchymal cells (NPC), including liver sinusoidal endothelial cells, is important for revealing its pharmacokinetic (PK) behavior, since NPC has important roles in eliminating an antibody from the blood via the Fc fragment of IgG receptor IIB (FcγRIIB).
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