Mount P F, Sutton V R, Li W, Burgess J, McKEnzie I F, Pietersz G A, Trapani J A
Austin Research Institute, Austin Hospital, Heidelberg, Australia.
Cancer Res. 1994 Dec 1;54(23):6160-6.
The mouse monoclonal antibody, m30.6 (IgG2b), detects an antigenic determinant expressed predominantly on the surface of colorectal adenocarcinoma cells and has been shown previously to be a potentially useful therapeutic and diagnostic reagent for human colon cancer. We report the production and characterization of a mouse/human chimeric antibody, c30.6, with potent in vitro and in vivo antitumor activity. The genes encoding the variable domains for heavy and light chains were amplified by thermal cycling using degenerate oligonucleotide primers complementary to conserved immunoglobulin framework sequences. The gene segments were sequenced, subcloned into eukaryotic expression vectors containing human constant region genes (IgG1 and kappa), and cotransfected into nonsecreting Sp2/0 mouse myeloma cells. There were significant differences in the biological activities of the murine and chimeric antibodies. The i.p. administration of c30.6 but not of m30.6 produced a marked growth inhibition of s.c. 30.6+ COLO 205 tumors in scid/scid mice (approximately 40% reduction in tumor size, measured 21 days after tumor inoculation). Reduced tumor growth was not due to altered binding characteristics of c30.6 because: (a) the chimeric antibody was shown by flow cytometry to bind exclusively to cell lines that expressed the 30.6 determinant; (b) c30.6 was able to completely inhibit the binding of m30.6 on 30.6+ cells; and (c) the affinity of binding of the two antibodies was the same (Ka, approximately 1.50 x 10(8)). Up to 15% of the total injected antibody dose/g tissue was localized in 30.6+ tumors at 24 h, approximately 13% was present in the tumors at 48 h, and approximately 10% was present at 72 h. Furthermore, c30.6 demonstrated a shorter circulating half-life (53 h; m30.6, 72 h) when given i.p. to C57BL6 x BALB/cF1 mice. Unlike m30.6, c30.6 was also strongly active in antibody-dependent cell-mediated cytotoxicity against a range of 30.6+ tumor target cells in vitro. Up to 80% specific 51Cr release was achieved using either freshly isolated human peripheral blood mononuclear cells or 2-day-old interleukin 2-stimulated human peripheral blood mononuclear cells as effectors. The enhanced antitumor activity of c30.6 suggests that it might be a useful immunotherapeutic reagent for colorectal carcinoma.
小鼠单克隆抗体m30.6（IgG2b）可检测主要在结肠直肠腺癌细胞表面表达的一种抗原决定簇，此前已证明它是一种对人类结肠癌具有潜在治疗和诊断价值的试剂。我们报告了一种具有强大体外和体内抗肿瘤活性的小鼠/人嵌合抗体c30.6的制备及特性。使用与保守免疫球蛋白框架序列互补的简并寡核苷酸引物通过热循环扩增编码重链和轻链可变区的基因。对基因片段进行测序后，亚克隆到含有人类恒定区基因（IgG1和κ）的真核表达载体中，并共转染到不分泌的Sp2/0小鼠骨髓瘤细胞中。小鼠抗体和嵌合抗体的生物学活性存在显著差异。腹腔注射c30.6而非m30.6可使scid/scid小鼠体内皮下接种的30.6 + COLO 205肿瘤显著生长抑制（肿瘤接种21天后测量，肿瘤大小减少约40%）。肿瘤生长减缓并非由于c30.6结合特性改变，原因如下：（a）流式细胞术显示嵌合抗体仅与表达30.6决定簇的细胞系结合；（b）c30.6能够完全抑制m30.6在30.6 + 细胞上的结合；（c）两种抗体的结合亲和力相同（Ka约为1.50×10⁸）。注射的抗体总量中，每克组织中高达15%在24小时时定位于30.6 + 肿瘤中，48小时时约为13%，72小时时约为10%。此外，腹腔注射给C57BL6×BALB/cF1小鼠时，c30.6的循环半衰期较短（53小时；m30.6为72小时）。与m30.6不同，c30.6在体外对一系列30.6 + 肿瘤靶细胞的抗体依赖性细胞介导的细胞毒性中也具有很强的活性。使用新鲜分离的人外周血单个核细胞或培养2天的白细胞介素2刺激的人外周血单个核细胞作为效应细胞时，特异性⁵¹Cr释放率高达80%。c30.6增强的抗肿瘤活性表明它可能是一种对结肠直肠癌有用的免疫治疗试剂。
