摘要
AIM： To construct and produce a recombinant bispecific humanized single-chain Fv （sFv） /Interleukin-2 （IL-2） fusion protein by using mammalian cells. METHODS： The sFv/IL-2 protein was genetically engineered, and transfected to mammalian cells to determine whether the mammalian protein folding machinery can produce and secrete active sFv/IL-2 with high efficiency. RESULTS： The fusion protein was constructed and high efficiently expressed with yields up to 102 ±4.2 mg/L in culture supernatant of the stably transfected 293 cell line. This recombinant fusion protein consisted of humanized variable heavy （VH） and light （VL） domains of monoclonal antibody （mAb） 520C9 directed against the human HER-2/neu （c-erbB2） proto-oncogene product p185, and human IL-2 connected by polypeptide linker. The fusion protein was shown to retain the immunostimulatory activities of IL-2 as measured by IL- 2-dependent cell proliferation and cytotoxicity assays. In addition to its IL-2 activities, this fusion protein also possessed antigen-binding specificity against p185, as determined by indirect ELISA using p185 positive SKOV 3ip1 cells. CONCLUSION： The large-scale preparation of the recombinant humanized sFv antibody/IL-2 fusion protein is performed with 293 cells. The recombinant humanized sFv antibody/IL-2 fusion protein may provide an effective means.of targeting therapeutic doses of IL-2 to p185 positive tumors without increasing systemic toxicity or immunogenicity.
瞄准：为了构造并且生产 recombinant 双性人 specific，使单个链的 Fv (sFv ) 人性化由使用哺乳动物的房间的 /Interleukin-2 (IL-2 ) 熔化蛋白质。方法：sFv/IL-2 蛋白质遗传上被设计，并且 transfected 到哺乳动物的房间到决定合拢机械的哺乳动物的蛋白质是否能与高效率生产并且藏匿活跃 sFv/IL-2。结果：熔化蛋白质被构造并且高效地高与收益表示了直到 102 +/- 在文化上层清液的 4.2 mg/L 稳定地 293 房间衬里的 transfected。这 recombinant 熔化蛋白质由人性化的变量组成了重(V (H)) 并且光(V (L)) 520C9 对人的 HER-2/neu (c-erbB2 ) proto-oncogene 产品 p185 指导了的单音的同种细胞的抗体(mAb ) 的域，和人的 IL-2 由多肽连接器连接了。熔化蛋白质被显示保留由 IL-2-dependent 房间增长和细胞毒性试金测量了的 IL-2 的 immunostimulatory 活动。除了它的 IL-2 活动，这熔化蛋白质也对 p185 拥有了抗原绑定特性，由使用 p185 的间接 ELISA 决定了积极 SKOV 3ip1 房间。结论：recombinant 的大规模准备使 sFv antibody/IL-2 人性化熔化蛋白质与 293 个房间被执行。recombinant 使熔化蛋白质可以提供的 sFv antibody/IL-2 人性化指向 IL-2 的治疗学的剂量到 p185 的一个有效工具没有增加全身的毒性或 immunogenicity 的积极肿瘤。
