Pauza M E, Doumbia S O, Pennell C A
Department of Laboratory Medicine and Pathology, University of Minnesota Medical School, Minneapolis 55455, USA.
J Immunol. 1997 Apr 1;158(7):3259-69.
To develop novel therapeutic agents for treatment of human T cell malignancies, we constructed two single-chain Fv (sFv) immunotoxins specific for the T cell-associated Ag CD7. The sFv fragments were derived from the murine hybridomas 3A1e and 3A1f and were expressed as soluble proteins in Escherichia coli. Surface plasmon resonance analyses demonstrated that the purified 3A1e and 3A1f sFv fragments specifically bound CD7 with high affinity, 8.1 and 1.8 nM, respectively. The difference in affinity is chiefly due to a slower dissociation rate for the 3A1f sFv fragment. Despite this difference, both monovalent sFv fragments were comparably internalized by CD7+ human T leukemic cells within 30 min. These data support findings of previous studies suggesting that CD7 internalization does not require cross-linking. The sFv immunotoxins were assembled by linking ricin toxin A chain to the C termini of the sFv fragments via disulfide bonds. Both sFv immunotoxins were comparably potent in their ability to inhibit protein synthesis in vitro in CD7+ Jurkat cells (50% inhibiting concentration = 15 pM). Further preclinical studies on the use of the 3A1e and 3A1f sFv immunotoxins to treat human T cell diseases therefore appear warranted.
为开发用于治疗人类T细胞恶性肿瘤的新型治疗药物，我们构建了两种对T细胞相关抗原CD7具有特异性的单链Fv（sFv）免疫毒素。sFv片段源自鼠杂交瘤3A1e和3A1f，并在大肠杆菌中作为可溶性蛋白表达。表面等离子体共振分析表明，纯化的3A1e和3A1f sFv片段分别以8.1 nM和1.8 nM的高亲和力特异性结合CD7。亲和力的差异主要是由于3A1f sFv片段的解离速率较慢。尽管存在这种差异，但两种单价sFv片段在30分钟内均被CD7 +人T白血病细胞同等程度地内化。这些数据支持了先前研究的结果，表明CD7内化不需要交联。通过二硫键将蓖麻毒素A链连接到sFv片段的C末端来组装sFv免疫毒素。两种sFv免疫毒素在体外抑制CD7 + Jurkat细胞中蛋白质合成的能力相当（50％抑制浓度= 15 pM）。因此，对使用3A1e和3A1f sFv免疫毒素治疗人类T细胞疾病进行进一步的临床前研究似乎是有必要的。
