Dunn R D, Weston K M, Longhurst T J, Lilley G G, Rivett D E, Hudson P J, Raison R L
Department of Cell and Molecular Biology, University of Technology Sydney, NSW, Australia.
Immunotechnology. 1996 Sep;2(3):229-40. doi: 10.1016/s1380-2933(96)00055-3.
The majority of immunotoxins studied to date incorporate toxins that act in the cytosol and thus need to be endocytosed by the target cell. An alternative strategy for immunotoxin development is the use of membrane active toxins, such as the pore-forming proteins. Melittin, a 26 amino acid cytolytic peptide from bee venom, is such a protein.
We report here the construction, production and functional analysis of a recombinant immunotoxin obtained by fusion of genes which encode an antibody fragment (scFv) with an oligonucleotide encoding melittin.
The antibody fragment was derived from a murine monoclonal antibody, K121, which recognises a specific epitope (KMA) expressed on the surface of human kappa myeloma and lymphoma cells, and on human free kappa Bence Jones protein (BJP). Melittin is a 26-amino acid, membrane-lytic peptide which is a major component of bee venom. The scFv of K121 was constructed by PCR to link VH and VL genes via an oligonucleotide which encodes a flexible, hydrophilic peptide. An oligonucleotide encoding melittin and the peptide marker sequence FLAG was fused to the scFv construct using a similar linker peptide. The gene construct (scFv-mel) was inserted into the secretion vector pPOW and expressed in Escherichia coli (TOPP2).
Expression of the recombinant scFv-mel gene and purification of the protein product was monitored by Western blot analysis. Following purification by anti-FLAG affinity chromatography, the recombinant immunotoxin (scFv-mel) was assessed for antigen binding and for cytotoxic activity by flow cytometry using antigen-expressing and non-expressing cell targets. The scFv-mel was found to exhibit binding and killing properties consistent with the specificity of the original K121 antibody. Moreover, the cytolytic activity of the scFv-mel was significantly greater on a molar basis than that of native melittin alone.
The data presented here constitute the first report of a melittin-based recombinant immunotoxin and demonstrate that such a membrane active immunotoxin can be synthesised in a bacterial expression. Linking of melittin to an antibody fragment overcame the non-specific toxicity of melittin as the recombinant immunotoxin exhibited specific toxicity towards antigen-bearing target cells. The observation that the immunotoxin exhibited enhanced cytotoxic activity over the free toxin indicates the potential of this approach for the development of an effective therapeutic agent.
迄今为止研究的大多数免疫毒素都包含作用于细胞质溶胶的毒素，因此需要被靶细胞内吞。免疫毒素开发的另一种策略是使用膜活性毒素，例如成孔蛋白。蜂毒溶血肽是一种来自蜂毒的由26个氨基酸组成的溶细胞肽，就是这样一种蛋白。
我们在此报告一种重组免疫毒素的构建、生产及功能分析，该重组免疫毒素是通过将编码抗体片段（单链抗体片段）的基因与编码蜂毒溶血肽的寡核苷酸融合而获得的。
抗体片段源自鼠单克隆抗体K121，该抗体识别在人κ骨髓瘤细胞和淋巴瘤细胞表面以及人游离κ本斯·琼斯蛋白（BJP）上表达的特定表位（KMA）。蜂毒溶血肽是一种由26个氨基酸组成的膜溶解肽，是蜂毒的主要成分。通过聚合酶链反应构建K121的单链抗体片段，通过编码柔性亲水性肽的寡核苷酸连接重链可变区（VH）和轻链可变区（VL）基因。使用类似的连接肽将编码蜂毒溶血肽和肽标记序列FLAG的寡核苷酸与单链抗体片段构建体融合。将基因构建体（单链抗体片段-蜂毒溶血肽）插入分泌载体pPOW并在大肠杆菌（TOPP2）中表达。
通过蛋白质免疫印迹分析监测重组单链抗体片段-蜂毒溶血肽基因的表达和蛋白质产物的纯化。通过抗FLAG亲和层析纯化后，使用表达抗原和不表达抗原的细胞靶标通过流式细胞术评估重组免疫毒素（单链抗体片段-蜂毒溶血肽）的抗原结合和细胞毒性活性。发现单链抗体片段-蜂毒溶血肽表现出与原始K121抗体的特异性一致的结合和杀伤特性。此外，单链抗体片段-蜂毒溶血肽的溶细胞活性在摩尔基础上明显高于单独的天然蜂毒溶血肽。
此处呈现的数据构成了基于蜂毒溶血肽的重组免疫毒素的首次报道，并证明这种膜活性免疫毒素可以在细菌表达中合成。将蜂毒溶血肽与抗体片段连接克服了蜂毒溶血肽的非特异性毒性，因为重组免疫毒素对携带抗原的靶细胞表现出特异性毒性。免疫毒素比游离毒素表现出增强的细胞毒性活性这一观察结果表明这种方法在开发有效治疗剂方面的潜力。
