Hu Jian, Huang Xiaodong, Ling Chang-Chun, Bundle David R, Cheung Nai-Kong V
Department of Pediatrics, Memorial Sloan-Kettering Cancer Center, New York, NY 10065, USA.
J Immunol. 2009 Nov 1;183(9):5748-55. doi: 10.4049/jimmunol.0901409. Epub 2009 Oct 7.
Ab affinity maturation in vivo is always accompanied by negative selection to maintain Ag specificity. In contrast, in vitro affinity maturation can lead to epitope spread, resulting in loss of specificity. Anti-ganglioside-GD2 mAbs are clinically effective against neuroblastoma; pain and neuropathy are major side effects. We used structural relatives of GD2 to define epitope spread during in vitro affinity maturation of an anti-GD2 single-chain variable fragment (scFv) called 5F11-scFv. Clonal dominance identified by polyclonal sequencing was confirmed by analyzing individual clones. Affinity-matured mutations were introduced into scFv-streptavidin for functional studies. Without a negative selector, 19-fold affinity improvement (clone Q, where Q is the symbol for glutamine) was associated with strong cross-reactivity with GM2 and GD1b and moderate cross-reactivity with GD3, resulting in positive immunohistochemical staining of all 13 non-neural normal human tissues, in contrast to none of 13 tissues with parental clone P. With GM2 as a negative selector, clone Y (where Y is the symbol for tyrosine) was generated with only weak cross-reactivity with GD1b, adrenal and thyroid glands, and no staining of other non-neural normal tissues. Even though there was only a 3-fold affinity improvement, clone Y showed significantly higher tumor uptake over parental clone P (134%, p = 0.04), whereas clone Q was inferior (54% of clone P; p = 0.05) as confirmed by tumor-to-normal tissue ratios across 16 organs (41% of clone P; p < 0.0001). Using the less efficient negative selector GD3, a clone mixture (Q, V, and Y, where V is the symbol for valine) emerged. We conclude that epitope spread during affinity maturation can be reduced by negative selection. Furthermore, efficiency of the negative selector depends on its cross-reactive affinity with the matured scFv.
体内的抗体亲和力成熟总是伴随着阴性选择以维持抗原特异性。相比之下，体外亲和力成熟可能导致表位扩展，从而导致特异性丧失。抗神经节苷脂-GD2单克隆抗体在临床上对神经母细胞瘤有效；疼痛和神经病变是主要的副作用。我们使用GD2的结构类似物来定义一种名为5F11-单链可变片段（scFv）的抗GD2 scFv在体外亲和力成熟过程中的表位扩展。通过分析单个克隆证实了通过多克隆测序确定的克隆优势。将亲和力成熟的突变引入scFv-链霉亲和素中进行功能研究。在没有阴性选择剂的情况下，亲和力提高了19倍（克隆Q，其中Q是谷氨酰胺的符号），与GM2和GD1b有强烈的交叉反应，与GD3有中等程度的交叉反应，导致13种非神经正常人组织均出现阳性免疫组化染色，而亲本克隆P的13种组织均未出现染色。以GM2作为阴性选择剂，产生了克隆Y（其中Y是酪氨酸的符号），它与GD1b、肾上腺和甲状腺仅有微弱的交叉反应，对其他非神经正常组织无染色。尽管亲和力仅提高了3倍，但克隆Y在肿瘤摄取方面比亲本克隆P显著更高（134%，p = 0.04），而克隆Q较差（为克隆P的54%；p = 0.05），这通过16个器官的肿瘤与正常组织比率得到证实（为克隆P的41%；p < 0.0001）。使用效率较低的阴性选择剂GD3，出现了一个克隆混合物（Q、V和Y，其中V是缬氨酸的符号）。我们得出结论，亲和力成熟过程中的表位扩展可以通过阴性选择来减少。此外，阴性选择剂的效率取决于其与成熟scFv的交叉反应亲和力。
