Tripathi P K, Qin H, Deng S, Xu C, Bhattacharya-Chatterjee M, Foon K A, Chatterjee S K
Department of Internal Medicine, and The Lucille Parker Markey Cancer Center, University of Kentucky Medical Center, Lexington 40536-0096, USA.
Mol Immunol. 1998 Sep;35(13):853-63. doi: 10.1016/s0161-5890(98)00072-8.
For the therapy of cancer patients whose disease is positive for Carcinoembryonic Antigen (CEA), we developed an active specific immunotherapy based on the idiotypic network. The anti-idiotype monoclonal antibody (mAb), 3H1 was generated by immunization of mice with the anti-CEA mAb, 8019. 3H1 mimics CEA both functionally and structurally and acts as a surrogate for CEA. To define the minimum structural requirements for antigen mimicry by 3H1, we constructed plasmid vectors for expression of single chain Fv (scFv) variants of 3H1 in Escherichia coli. Variable heavy (VH) and variable light (VL) chain domains of 3H1 were linked by a 15 amino acid linker (Ln), (Gly4Ser)3 in two constructs, VH-Ln-VL and VL-LnVH. Ln was omitted in two constructs, VH-VL and VL-VH. Each of the scFv constructs has a tag of six His [(His)6 tag] for purification by metal chelate affinity chromatography and detection by enzyme-linked immunoabsorbent assay (ELISA). Comparisons of the binding of 8019 to purified scFv proteins by ELISA and immunoblot experiments showed that only VH-Ln-VL had significant activity. VH-Ln-VL also showed maximum inhibition of binding of 8019 to CEA. Immunization of mice with naked VH-Ln-VL and VH-Ln-VL conjugated to keyhole limpet hemocyanin induced anti-CEA antibodies in mouse sera. Sera from immunized mice inhibited the binding of 8019 to 3H1 as well as CEA. Induction of anti-CEA antibodies in the immunized mice was confirmed by flow cytometric analysis using CEA positive MC-38cea cells. These results demonstrate that for antigen mimicry of 3H1 scFv, the presence of Ln is necessary and the domain order should be VH followed by VL.
对于癌胚抗原（CEA）呈阳性的癌症患者的治疗，我们基于独特型网络开发了一种主动特异性免疫疗法。抗独特型单克隆抗体（mAb）3H1是通过用抗CEA单克隆抗体8019免疫小鼠产生的。3H1在功能和结构上模拟CEA，并作为CEA的替代物。为了确定3H1模拟抗原的最小结构要求，我们构建了质粒载体，用于在大肠杆菌中表达3H1的单链Fv（scFv）变体。在两个构建体VH-Ln-VL和VL-LnVH中，3H1的可变重链（VH）和可变轻链（VL）结构域通过15个氨基酸的接头（Ln），即（Gly4Ser）3连接。在两个构建体VH-VL和VL-VH中省略了Ln。每个scFv构建体都有一个六个组氨酸的标签[（His）6标签]，用于通过金属螯合亲和色谱法纯化并通过酶联免疫吸附测定（ELISA）进行检测。通过ELISA和免疫印迹实验比较8019与纯化的scFv蛋白的结合，结果表明只有VH-Ln-VL具有显著活性。VH-Ln-VL还显示出对8019与CEA结合的最大抑制作用。用裸VH-Ln-VL和与钥孔血蓝蛋白偶联的VH-Ln-VL免疫小鼠，可诱导小鼠血清中产生抗CEA抗体。免疫小鼠的血清抑制了8019与3H1以及CEA的结合。使用CEA阳性的MC-38cea细胞通过流式细胞术分析证实了免疫小鼠中抗CEA抗体的诱导。这些结果表明，对于3H1 scFv的抗原模拟，Ln的存在是必要的，结构域顺序应为VH后接VL。
