摘要
抗体是肿瘤导向治疗的重要工具，用抗体将药物导向病灶能够显著降低药物的副作用。目前抗体已经达到临床应用阶段，不过在抗体药物治疗应用中始终存在着很多局限。首先完整抗体本身是大分子药物，分子量较大，难以穿透到达实体瘤内部。另外，单克隆抗体的鼠源性会诱发人抗鼠抗体反应，而小型化抗体能够同时解决上述两个问题，因此在抗体药物研究领域药物分子小型化成为新的研究趋势。在抗体的小型化中研究人员主要着眼于F(ab’)2、Fab、scFv以及单域抗体(dAbs)等。单域抗体的分子大小只有全抗的1／12，同时它的免疫原性很低。除此之外，单域抗体和其它抗体小型化分子一样具有较高的肿瘤穿透率，用单域抗体作为小分子导向药物的“载体”携带高效药物能够构建强效的抗体药物。本研究中，我们构建并制备大鼠抗肝癌单克隆抗体3A5衍生的单域抗体以及单域抗体和力达霉素的强化融合蛋白，并对其生物活性进行了研究。
1．大鼠mAb 3A5的免疫亲和性检测
大鼠抗肝癌单克隆抗体3A5是用人肝癌Bel 7402细胞免疫LOU／C大鼠后取其脾细胞和IR983F浆细胞融合的杂交瘤细胞所产生的。我们采用免疫细胞组化的方法对mAb 3A5进行的检测。免疫荧光分析结果表明mAb 3A5主要存在于Bel 7402的细胞膜上。同时Western blot分析表明mAb 3A5特异的与Bel 7402细胞的40kDa和60kDa蛋白相结合。这些结果表明mAb 3A5可以在抗癌导向治疗中作为一个有效的抗癌药物“载体”。
2．制备、构建以及表征大鼠抗肝癌轻链单域抗体VL以及单域抗体与力达霉素强化融合蛋白VL-LDP-AE
单域抗体(single domain antibody)是抗体的最小功能结合单位，相当于人抗体的重链或轻链可变区(VH／VL)，是继全抗、F(ab’)2、Fab、scFv之后的新一代的治疗抗体。单域抗体具有传统抗体和小分子药物的双重优势，作为肿瘤靶向药物的载体有着诱人的前景。大分子肽类抗肿瘤抗生素力达霉素(lidamycin，LDM，
Antibody is an important tool in cancer targeting therapy. The specific delivery of drugs to the targeting site can significantly enhance the efficacy and decrease the side effects of the drug. As an appealing approach, the use of antibody has entered clinical stages. However, there still have many limitations in the field of antibody-based therapy. Because of macromolecular size, the intact antibody molecule poorly penetrates into the solid tumor. In addition, the monoclonal antibody of mouse origin can induce human anti-mouse antibody response. Therefore, one of the trends in development of monoclonal antibody therapeutics is the molecule-downsizing of the drug. The antibody miniature studies highlight on the fragments of F(ab')2, Fab, scFv and single antibody domain (dAbs). The single antibody domain only has the 1/12 molecular size of entire antibody and shows low immunogenicity. Like other minimizations, dAbs offer the advantages of increasing rates of tumor penetration. Using the antibody single domain as the guided vehicle to combine with extremely potent drug as the "warhead" agent may lead to constructing highly potent antibody-based therapeutics with down-sized molecule.
In this study, we constructed an energized fusion proteins consisting of LDM and single domain antibodies derived from rat mAb 3A5 directed against hepatocarcinoma and further studied its biological activity. 1. Immunoaffinity determination of the rat mAb 3A5
mAb 3A5, an anti-hepatocarcinoma mAb, was secreted by hybridoma cells that were obtained by immunize LOU/C rats with Bel 7402 cells (human liver caner cells), followed by fusion of spleen cells and rat IR983F myeloma cells. The immunofluorescence assay results showed that rat mAb 3A5 stained mainly on the
引文
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