Kaufman E N, Jain R K
Department of Chemical Engineering, Carnegie Mellon University, Pittsburgh, Pennsylvania 15213-3890.
Cancer Res. 1992 Aug 1;52(15):4157-67.
The affinity of a monoclonal antibody for its tumor-associated antigen is one of several parameters governing in vivo monoclonal antibody distribution. However, there is a lack of apparent correlation between the affinity of a bivalent monoclonal antibody measured using equilibrium binding experiments and its in vivo delivery. Furthermore, differences in the reported affinity for identical antibody/antigen pairs are quite common in the literature. In this paper, both of these discrepancies are addressed in terms of variation in avidity due to bivalent interaction. The enhancement of avidity afforded by bivalent attachment is addressed theoretically by extending the model of Crothers and Metzger (Immunochemistry, 9: 341-357, 1972). Theoretical assessment of Lineweaver-Burk, Scatchard, Steward-Petty, Langmuir, fluorescence recovery after photobleaching, and Sips models demonstrates quantitatively that the measured affinity using equilibrium binding experiments may vary over four orders of magnitude with similar variation in experimental cellular antigen density. Further, the measured affinity is a function of the experimental protocol. Predictions of avidity enhancement were confirmed experimentally using fluorescence recovery after photobleaching. These experiments measured the equilibrium binding constant and concentration of binding sites for an immunoglobulin G monoclonal antibody and its F(ab) fragment directed against the rabbit VX2 carcinoma cell line. Bivalent binding data agree quantitatively with those predicted by the bivalent model with no adjustable parameters. It is concluded that bivalent equilibrium binding constants are useful only in antibody screening, where experimental conditions are identical for all series. They must be used with caution in predicting in vivo antibody distribution, and it is recommended that the intrinsic, monovalent affinity be measured in tandem with any bivalent antibody study as a standard reference.
单克隆抗体对其肿瘤相关抗原的亲和力是决定体内单克隆抗体分布的几个参数之一。然而，使用平衡结合实验测量的二价单克隆抗体的亲和力与其体内递送之间缺乏明显的相关性。此外，文献中报道的相同抗体/抗原对亲和力的差异相当常见。在本文中，从二价相互作用导致的亲和力变化方面解决了这两个差异问题。通过扩展Crothers和Metzger的模型（《免疫化学》，9: 341 - 357, 1972）从理论上探讨了二价结合所带来的亲和力增强。对Lineweaver - Burk、Scatchard、Steward - Petty、Langmuir、光漂白后荧光恢复和Sips模型的理论评估定量地表明，使用平衡结合实验测量的亲和力可能在四个数量级范围内变化，而实验细胞抗原密度也有类似变化。此外，测量的亲和力是实验方案的函数。通过光漂白后荧光恢复实验从实验上证实了对亲和力增强的预测。这些实验测量了针对兔VX2癌细胞系的免疫球蛋白G单克隆抗体及其F(ab)片段的平衡结合常数和结合位点浓度。二价结合数据与二价模型预测的数据在无可调参数的情况下定量相符。得出的结论是，二价平衡结合常数仅在抗体筛选中有用，此时所有系列的实验条件相同。在预测体内抗体分布时必须谨慎使用，建议在任何二价抗体研究中同时测量内在的单价亲和力作为标准参考。
