Division of Stem Cell Transplantation and Immunotherapy, Department of Medicine II, Zoological Institute, Christian-Albrechts-University, Kiel, Germany.
PLoS One. 2012;7(8):e42426. doi: 10.1371/journal.pone.0042426. Epub 2012 Aug 3.
CD96, a cell surface antigen recently described to be preferentially expressed on acute myeloid leukemia (AML) leukemic stem cells (LSC) may represent an interesting target structure for the development of antibody-based therapeutic approaches. The v-regions from the CD96-specific hybridoma TH-111 were isolated and used to generate a CD96-specific single chain fragment of the variable regions (scFv). An affinity maturated variant resulting in 4-fold enhanced CD96-binding was generated by random mutagenesis and stringent selection using phage display. The affinity maturated scFv CD96-S32F was used to generate bivalent mini-antibodies by genetically fusing an IgG1 wild type Fc region or a variant with enhanced CD16a binding. Antibody dependent cell-mediated cytotoxicity (ADCC) experiments revealed that Fc engineering was essential to trigger significant effector cell-mediated lysis when the wild type scFv was used. The mini-antibody variant generated by fusing the affinity-maturated scFv with the optimized Fc variant demonstrated the highest ADCC activity (2.3-fold enhancement in efficacy). In conclusion, our data provide proof of concept that CD96 could serve as a target structure for effector cell-mediated lysis and demonstrate that both enhancing affinity for CD96 and for CD16a resulted in mini-antibodies with the highest cytolytic potential.
CD96 是一种新近被描述为在急性髓性白血病 (AML) 白血病干细胞 (LSC) 上优先表达的细胞表面抗原，可能代表着开发基于抗体的治疗方法的一个有趣的靶结构。从 CD96 特异性杂交瘤 TH-111 中分离出 v 区，并用于生成 CD96 特异性可变区单链片段 (scFv)。通过随机诱变和噬菌体展示进行严格选择，生成了亲和力成熟的变体，其 CD96 结合能力增强了 4 倍。亲和力成熟的 scFv CD96-S32F 用于通过基因融合 IgG1 野生型 Fc 区或增强 CD16a 结合的变体来生成二价 mini 抗体。抗体依赖性细胞介导的细胞毒性 (ADCC) 实验表明，当使用野生型 scFv 时，Fc 工程对于触发显著的效应细胞介导的裂解是必不可少的。通过将亲和力成熟的 scFv 与优化的 Fc 变体融合生成的 mini 抗体变体显示出最高的 ADCC 活性（功效提高了 2.3 倍）。总之，我们的数据提供了概念验证，表明 CD96 可以作为效应细胞介导的裂解的靶结构，并表明提高对 CD96 和 CD16a 的亲和力都会导致具有最高细胞毒性潜力的 mini 抗体。
