Blood Cancer J. 2011 Apr;1(4):e15. doi: 10.1038/bcj.2011.11. Epub 2011 Apr 8.
C-terminal mutations of CD20 constitute part of the mechanisms that resist rituximab therapy. Most CD20 having a C-terminal mutation was not recognized by L26 antibody. As the exact epitope of L26 has not been determined, expression and localization of mutated CD20 have not been completely elucidated. In this study, we revealed that the binding site of L26 monoclonal antibody is located in the C-terminal cytoplasmic region of CD20 molecule, which was often lost in mutated CD20 molecules. This indicates that it is difficult to distinguish the mutation of CD20 from under expression of the CD20 protein. To detect comprehensive CD20 molecules including the resistant mutants, we developed a novel monoclonal antibody that recognizes the N-terminal cytoplasm region of CD20 molecule. We screened L26-negative cases with our antibody and found several mutations. A rituximab-binding analysis using the cryopreserved specimen that mutation was identified in CD20 molecules indicated that the C-terminal region of CD20 undertakes a critical role in presentation of the large loop in which the rituximab-binding site locates. Thus, combination of antibodies of two kinds of epitope permits the identification of C-terminal CD20 mutations associated with irreversible resistance to rituximab and may help the decision of the treatment strategy.
CD20 的 C 末端突变构成了抵抗利妥昔单抗治疗的机制的一部分。大多数具有 C 末端突变的 CD20 未被 L26 抗体识别。由于 L26 的确切表位尚未确定，因此突变 CD20 的表达和定位尚未完全阐明。在这项研究中，我们揭示了 L26 单克隆抗体的结合位点位于 CD20 分子的 C 末端胞质区域，该区域在突变的 CD20 分子中经常丢失。这表明很难区分 CD20 的突变与 CD20 蛋白的低表达。为了检测包括耐药突变体在内的全面 CD20 分子，我们开发了一种识别 CD20 分子 N 末端胞质区域的新型单克隆抗体。我们用我们的抗体筛选了 L26 阴性病例，并发现了几个突变。使用在 CD20 分子中鉴定出突变的冷冻保存标本进行的利妥昔单抗结合分析表明，CD20 的 C 末端区域在展示位于利妥昔单抗结合位点的大环中起着关键作用。因此，两种表位的抗体的组合可以识别与利妥昔单抗不可逆耐药相关的 C 末端 CD20 突变，并有助于治疗策略的决策。
