Shaw D M, Embleton M J, Westwater C, Ryan M G, Myers K A, Kingsman S M, Carroll M W, Stern P L
CRC Immunology Group, Paterson Institute for Cancer Research, Christie Hospital, Manchester, UK.
Biochim Biophys Acta. 2000 Dec 15;1524(2-3):238-46. doi: 10.1016/s0304-4165(00)00165-3.
The oncofoetal antigen 5T4 is a 72 kDa glycoprotein expressed at the cell surface. It is defined by a monoclonal antibody, mAb5T4, that recognises a conformational extracellular epitope in the molecule. Overexpression of 5T4 antigen by tumours of several types has been linked with disease progression and poor clinical outcome. Its restricted expression in non-malignant tissue makes 5T4 antigen a suitable target for the development of antibody directed therapies. The use of murine monoclonal antibodies for targeted therapy allows the tumour specific delivery of therapeutic agents. However, their use has several drawbacks, including a strong human anti-mouse immune (HAMA) response and limited tumour penetration due to the size of the molecules. The use of antibody fragments leads to improved targeting, pharmacokinetics and a reduced HAMA. A single chain antibody (scFv) comprising the variable regions of the mAb5T4 heavy and light chains has been expressed in Escherichia coli. The addition of a eukaryotic leader sequence allowed production in mammalian cells. The two 5T4 single chain antibodies, scFv5T4WT19 and LscFv5T4, described the same pattern of 5T4 antigen expression as mAb5T4 in normal human placenta and by FACS. Construction of a 5T4 extracellular domain-IgGFc fusion protein and its expression in COS-7 cells allowed the relative affinities of the antibodies to be compared by ELISA and measured in real time using a biosensor based assay. MAb5T4 has a high affinity, K(D)=1.8x10(-11) M, as did both single chain antibodies, scFv5T4WT19 K(D)=2.3x10(-9) M and LscFv5T4 K(D)=7.9x10(-10) M. The small size of this 5T4 specific scFv should allow construction of fusion proteins with a range of biological response modifiers to be prepared whilst retaining the improved pharmacokinetic properties of scFvs.
癌胚抗原5T4是一种表达于细胞表面的72 kDa糖蛋白。它由单克隆抗体mAb5T4所界定，该抗体识别分子中的一种构象性细胞外表位。多种类型肿瘤中5T4抗原的过表达与疾病进展及不良临床预后相关。其在非恶性组织中的局限性表达使5T4抗原成为抗体导向治疗开发的合适靶点。使用鼠单克隆抗体进行靶向治疗可实现治疗剂的肿瘤特异性递送。然而，其应用存在若干缺点，包括强烈的人抗鼠免疫（HAMA）反应以及由于分子大小导致的肿瘤穿透受限。使用抗体片段可改善靶向性、药代动力学并降低HAMA反应。包含mAb5T4重链和轻链可变区的单链抗体（scFv）已在大肠杆菌中表达。添加真核前导序列可使其在哺乳动物细胞中产生。两种5T4单链抗体scFv5T4WT19和LscFv5T4，在正常人胎盘和通过荧光激活细胞分选术（FACS）检测时，显示出与mAb5T4相同的5T4抗原表达模式。构建5T4细胞外结构域-IgGFc融合蛋白并在COS-7细胞中表达，可通过酶联免疫吸附测定（ELISA）比较抗体的相对亲和力，并使用基于生物传感器的测定法进行实时测量。mAb5T4具有高亲和力，解离常数K(D)=1.8x10(-11) M，两种单链抗体scFv5T4WT19的K(D)=2.3x10(-9) M和LscFv5T4的K(D)=7.9x10(-10) M也是如此。这种5T4特异性scFv的小尺寸应允许构建与一系列生物反应调节剂的融合蛋白，同时保留scFv改善的药代动力学特性。
