Department of Bioanalytics, R&D Protein Analytics, Biologics Research, Pharma Research and Early Development Penzberg, Roche Diagnostics GmbH, Nonnenwald 2, 82377 Penzberg, Germany.
J Pharm Biomed Anal. 2013 Jan;72:208-15. doi: 10.1016/j.jpba.2012.08.023. Epub 2012 Sep 7.
Generic immunoassay formats in animal serum have been described for pharmacokinetic (PK) analysis of human full-length antibodies, but not of human antigen binding fragment (Fab) proteins. Here we characterize two murine monoclonal antibodies (mAb) raised against human immunoglobulin G (IgG) which bind to unique epitopes in the Fab region of human IgG. mAb M-1.7.10 is directed against the constant domain of the kappa light chain and mAb M-1.19.31 binds to the constant domain 1 (CH1) of the heavy chain. Surface plasmon resonance analysis showed that mAb M-1.7.10 does not cross-react with sera from mouse, rat, rabbit, dog, marmoset, rhesus macaque, baboon and cynomolgus monkey, but binds to human and chimpanzee serum (dissociation constant K(D) of 6.8 × 10(-12) and 3.1 × 10(-11)M, respectively). mAb M-1.19.31 shows a higher K(D) for human and chimpanzee IgG (2.0 × 10(-9)M and 5.8 × 10(-10)M, respectively), but also does not bind to serum of the other species. Therefore, mAb M-1.7.10 was used as capture and mAb M-1.19.31 as detection reagent in a generic enzyme linked immunosorbent assay (ELISA) to quantify the human anti-IGF-1R Fab in mouse serum. The generic human Fab assay showed a limit of detection of 31.5 ng/mL anti-IGF-1R Fab. Intra- and inter-assay precision was less than 12% and the accuracy range for all controls was within ±20% of the target concentration. The generic human Fab ELISA was applied to determine serum levels of human anti-IGF-1R Fab after intravenous (iv) administration of 10mg/kg to mice. The resulting concentration-time profile was nearly identical to that obtained by analysis with a validated specific ELISA for anti-IGF-1R Fab. The mean relative concentration of anti-IGF-1R Fab analyzed by the generic assay was 82-118% of that of the specific assay. This equivalence was confirmed in a cynomolgus monkey study with the full length human mAb anti-TROP-2 IgG. Both specific ELISAs used mAb M-1.7.10 as detection reagent and their targets for capturing. In conclusion, the two murine anti-human Fabs are versatile tools as capture and detection reagents for human antibodies in generic and specific PK ELISA formats for animal studies. Their use in specific ELISAs as detection reagents allows the usage of Fc-fusion proteins as capture reagents.
通用免疫分析方法已被用于分析人源全长抗体的药代动力学（PK），但不能用于分析人源抗原结合片段（Fab）蛋白。本研究中，我们鉴定了两种针对人免疫球蛋白 G（IgG）的鼠源单克隆抗体（mAb），它们可以结合 IgG 的 Fab 区域的独特表位。mAb M-1.7.10 针对κ轻链的恒定区，mAb M-1.19.31 结合重链的恒定区 1（CH1）。表面等离子体共振分析表明，mAb M-1.7.10 不与来自小鼠、大鼠、兔、狗、狨猴、恒河猴、狒狒和食蟹猴的血清发生交叉反应，但与人和黑猩猩的血清发生反应（解离常数 K(D)分别为 6.8×10(-12)和 3.1×10(-11)M）。mAb M-1.19.31 与人源和黑猩猩 IgG 的 K(D)更高（分别为 2.0×10(-9)M 和 5.8×10(-10)M），但也不与其他物种的血清发生反应。因此，mAb M-1.7.10 被用作捕获抗体，mAb M-1.19.31 被用作检测抗体，建立了通用酶联免疫吸附试验（ELISA），以定量检测小鼠血清中的人源 IGF-1R Fab。该通用的人源 Fab 检测方法的检测下限为 31.5ng/mL 抗 IGF-1R Fab。该方法的批内和批间精密度均小于 12%，所有对照的准确度均在目标浓度的±20%范围内。该通用的人源 Fab ELISA 用于分析静脉注射（iv）10mg/kg 体重的人源 IGF-1R Fab 后，在小鼠血清中的浓度-时间曲线。该方法得到的浓度-时间曲线与使用抗 IGF-1R Fab 特异性 ELISA 分析的结果几乎相同。通过通用检测方法分析的抗 IGF-1R Fab 的平均相对浓度为特异性检测方法的 82-118%。该等效性在食蟹猴的研究中得到了验证，该研究使用全长人源 mAb 抗 TROP-2 IgG。两种特异性 ELISA 均使用 mAb M-1.7.10 作为检测抗体，使用相同的抗体作为捕获抗体。总之，这两种鼠源抗人 Fab 是用于动物研究的通用和特异性 PK ELISA 中分析人源抗体的多功能工具，可作为捕获和检测抗体。在特异性 ELISA 中作为检测抗体使用时，它们可以与 Fc 融合蛋白一起作为捕获抗体。
