Chair of Genetics, University of Erlangen-Nuremberg, Erwin-Rommel-Strasse 3, Erlangen, Germany.
Br J Haematol. 2010 Mar;148(6):879-89. doi: 10.1111/j.1365-2141.2009.08033.x. Epub 2010 Jan 8.
Four new single-chain Fv antibody fragments (scFvs) specific for the human leucocyte surface antigen CD123 (interleukin-3 receptor alpha) were generated to achieve preferential targeting of leukaemia stem cells (LSCs) in acute myeloid leukaemia (AML). The scFvs were isolated from a phage display library generated with spleen RNA from mice, immunized with a fusion protein consisting of the extracellular domain of CD123 and the Fc domain of a human immunoglobulin G1. The scFvs displayed CD123-specific binding on tumour cells (binding constants (K(D)) 4.5-101 nmol/l). The scFv with the highest affinity was used to design two cell death-inducing molecules. First, an immunotoxin, a fusion protein with truncated Pseudomonas Exotoxin A, induced potent apoptosis of AML-derived MOLM-13 and SKNO-1 cells at nanomolar concentrations. Second, the fusion to another scFv, specific for the low affinity Fcgamma-receptor III (CD16), created a bispecific single chain Fv (bsscFv). This bsscFv [123 x ds16] mediated potent lysis of AML-derived MOLM-13, THP-1 and SKNO-1 cells in antibody-dependent cellular cytotoxicity (ADCC) reactions at picomolar concentrations. The recruitment of CD16-positive effector cells for the lysis of AML cells via CD123 represents a novel combination with attractive prospects for future clinical testing.
四种针对人白细胞表面抗原 CD123（白细胞介素 3 受体 α）的新型单链 Fv 抗体片段（scFv）被生成，以实现对急性髓系白血病（AML）中的白血病干细胞（LSCs）的优先靶向。scFv 从用 CD123 的细胞外域和人免疫球蛋白 G1 的 Fc 域融合蛋白免疫的小鼠脾 RNA 生成的噬菌体展示文库中分离。scFv 在肿瘤细胞上显示出 CD123 特异性结合（结合常数（K(D))4.5-101nmol/l）。具有最高亲和力的 scFv 用于设计两种诱导细胞死亡的分子。首先，免疫毒素，一种与截短的假单胞菌外毒素 A 融合的蛋白，在纳摩尔浓度下诱导 AML 衍生的 MOLM-13 和 SKNO-1 细胞发生强烈的凋亡。其次，与另一种特异性结合低亲和力 Fcγ 受体 III（CD16）的 scFv 融合，产生了双特异性单链 Fv（bsscFv）。这种 bsscFv [123 x ds16] 在 picomolar 浓度下通过抗体依赖性细胞毒性（ADCC）反应介导对 AML 衍生的 MOLM-13、THP-1 和 SKNO-1 细胞的有效溶解。通过 CD123 募集 CD16 阳性效应细胞来裂解 AML 细胞代表了一种具有吸引力的未来临床测试前景的新型组合。
