Thirion S, Motmans K, Heyligen H, Janssens J, Raus J, Vandevyver C
Dr L Willems-Instituut, Diepenbeek, Belgium.
Eur J Cancer Prev. 1996 Dec;5(6):507-11.
Especially when dealing with solid cancers, single-chain antibody fragments (scFvs) have a lot of advantages. Due to their small size (27 kDa), these proteins clear more rapidly from the blood, and penetrate faster and deeper into tissues, than whole antibodies. Furthermore, the lack of constant regions ensures that they are not retained in tissues such as the liver and kidney. This reduces possible toxic side-effects. Single-chain construction is normally done by polymerase chain reaction (PCR). To decrease the overall cost of oligonucleotide primer synthesis, time-consuming primer design, multiple PCR reactions and individual PCR optimization, we designed a universal single-step overlap extension PCR protocol using hybridoma cDNA as a template. To overcome the lack of effector function, bispecific scFvs, consisting of an scFv produced against a tumour-associated antigen fused to a T cell marker-specific scFv, are being created, starting from already assembled scFv, by means of two additional PCR reactions. In this paper we describe both PCR methods that were successfully used to create scFvs against the human transferrin receptor, the human interleukin-2 receptor, the human CD3 molecule, a breast tumour-associated antigen and an anti-transferrin-anti-CD3 bispecific scFv.
特别是在处理实体癌时，单链抗体片段（scFv）具有很多优势。由于其尺寸较小（27 kDa），与完整抗体相比，这些蛋白质从血液中清除得更快，并且能更快、更深地渗透到组织中。此外，由于缺乏恒定区，它们不会滞留在肝脏和肾脏等组织中。这减少了可能的毒副作用。单链构建通常通过聚合酶链反应（PCR）完成。为了降低寡核苷酸引物合成的总体成本、耗时的引物设计、多次PCR反应以及单独的PCR优化，我们设计了一种通用的单步重叠延伸PCR方案，以杂交瘤cDNA为模板。为了克服效应子功能的缺失，正在通过另外两次PCR反应，从已经组装好的scFv开始，构建双特异性scFv，其由针对肿瘤相关抗原产生的scFv与T细胞标志物特异性scFv融合而成。在本文中，我们描述了两种成功用于制备针对人转铁蛋白受体、人白细胞介素-2受体、人CD3分子、一种乳腺肿瘤相关抗原的scFv以及一种抗转铁蛋白-抗CD3双特异性scFv的PCR方法。
