力达霉素构建的小型化单克隆抗体免疫偶联物的抗肿瘤作用

目的研制一种具有高效、小型化特点的抗肿瘤单克隆抗体(单抗)免疫偶联物.方法以化学偶联法制备抗肿瘤抗生素力达霉素(LDM)与单抗Fab＇片段的偶联物Fab＇LDM.以非还原型SDS-PAGE法确定偶联物的相对分子质量;ELISA法、四氮唑蓝(MTT)法、克隆形成法及小鼠移植性结肠癌26(C26)模型观察Fab＇-LDM偶联物的抗肿瘤作用.结果 Fab＇-LDM偶联物的相对分子质量(Mr)约为65 000,Fab＇与LDM的偶联分子比为11;偶联物与BEL-7402及C26细胞均呈免疫学阳性反应,但与KB细胞无反应;Fab＇-LDM对靶细胞BEL-7402的杀伤作用比游离LDM强13倍,对C26细胞的杀伤作用比LDM强5.5倍,但对非靶细胞KB的作用与LDM相当.动物试验结果表明,0.05 mg/kg、0.1 mg/kg和0.2 mg/kg剂量Fab＇-LDM偶联物对小鼠移植性结肠癌26的抑瘤率分别达80%、92%和94%,而0.1 mg/kg剂量游离LDM的抑瘤率为77%;同时偶联物治疗组动物生存时间较对照组延长.偶联物的抑瘤作用强于等剂量游离LDM(P＜0.01).结论以高效抗肿瘤抗生素LDM为"弹头"药物,可与单抗活性片段构建成具有良好抗肿瘤作用的小型化单抗导向药物.

Antitumor effect of lidamycin-containing monoclonal antibody immunoconjugate with downsized-molecule

OBJECTIVE: To develop a novel monoclonal antibody (mAb) immunoconjugate with downsized-molecule and highly potent antitumor effects. METHODS: The mAb conjugate was prepared by linking lidamycin (LDM), an antitumor antibiotic with extremely potent cytotoxicity, to mAb Fab' fragment. The molecular weight of Fab'-LDM was determined by non-reduced SDS-PAGE. The immunoreactivity of Fab'-LDM conjugate with cancer cells was detected by ELISA. The antitumor activity of the conjugate was determined by MTT assay, clonogenic assay, and animal model of transplantable colon carcinoma 26' (C26) in mice. RESULTS: The relative molecular mass of Fab'-LDM conjugate was approximately 65,000, suggesting a molar ratio of 1:1 between Fab' fragment and LDM in the conjugate. Fab'-LDM was reactive with hepatoma BEL-7402 and colon carcinoma 26 cells, but not reactive with KB cells. The cytotoxicity of Fab'-LDM conjugate to BEL-7402 cells, the antigen relevant cancer cells, was 13-fold more potent than that of free LDM, while the cytotoxicity of Fab'-LDM conjugate against C26 cells was 5.5-fold more potent than that of free LDM. However, the cytotoxicity of Fab'-LDM conjugate to KB cells, the antigen irrelevant cells, was similar to that of free LDM. Given by 3 intravenous injections, on day 1, 4 and 7, Fab'-LDM conjugate at doses of 0.05 mg/kg, 0.1 mg/kg and 0.2 mg/kg markedly suppressed the growth of colon carcinoma 26 in mice by 80%, 92% and 94%, respectively, whereas free LDM at 0.1 mg/kg suppressed the growth by 77%. The survival time of tumor-bearing mice was also increased by Fab'-LDM conjugate treatment. Fab'-LDM conjugate was more effective than equivalent unconjugated LDM (P < 0.01). CONCLUSIONS: The immunoconjugate composed of LDM and Fab' fragment that is characterized by downsized-molecule shows remarkable effectiveness against tumor growth.