Idusogie E E, Presta L G, Gazzano-Santoro H, Totpal K, Wong P Y, Ultsch M, Meng Y G, Mulkerrin M G
Departments of Analytical Chemistry, Immunology, QC Clinical Development, BioAnalytical Technology, and Protein Engineering, Genentech, South San Francisco, CA 94080, USA.
J Immunol. 2000 Apr 15;164(8):4178-84. doi: 10.4049/jimmunol.164.8.4178.
Rituxan (Rituximab) is a chimeric mAb with human IgG1 constant domains used in the therapy of non-Hodgkin's B cell lymphomas. This Ab targets B cells by binding to the cell-surface receptor, CD20. In our investigation of the mechanism of B cell depletion mediated by Rituximab, we first constructed mutants of Rituximab defective in complement activation but with all other effector functions intact. Our results demonstrate that the previously described C1q binding motif in murine IgG2b constituting residues E318, K320, and K322 is not applicable to a human IgG1 when challenged with either human, rabbit, or guinea pig complement. Alanine substitution at positions E318 and K320 in Rituximab had little or no effect on C1q binding and complement activation, whereas alanine substitution at positions D270, K322, P329, and P331 significantly reduced the ability of Rituximab to bind C1q and activate complement. We have also observed that concentrations of complement approaching physiological levels are able to rescue >60% of the activity of these mutant Abs with low affinity for C1q. These data localize the C1q binding epicenter on human IgG1 and suggest that there are species-specific differences in the C1q binding site of Igs.
美罗华（利妥昔单抗）是一种具有人IgG1恒定区的嵌合单克隆抗体，用于治疗非霍奇金B细胞淋巴瘤。该抗体通过与细胞表面受体CD20结合来靶向B细胞。在我们对利妥昔单抗介导的B细胞耗竭机制的研究中，我们首先构建了在补体激活方面有缺陷但所有其他效应功能均完整的利妥昔单抗突变体。我们的结果表明，先前描述的鼠IgG2b中构成残基E318、K320和K322的C1q结合基序，在用人、兔或豚鼠补体攻击时不适用于人IgG1。利妥昔单抗中E318和K320位点的丙氨酸取代对C1q结合和补体激活几乎没有影响，而D270、K322、P329和P331位点的丙氨酸取代显著降低了利妥昔单抗结合C1q和激活补体的能力。我们还观察到，接近生理水平的补体浓度能够挽救这些对C1q亲和力低的突变抗体>60%的活性。这些数据将C1q结合中心定位在人IgG1上，并表明Ig的C1q结合位点存在种属特异性差异。
