抗体-白细胞介素2融合蛋白183B2scFv-Interleukin 2的表达及活性检测

目的在哺乳动物细胞内表达抗体细胞因子融合蛋白183B2scFv Interleukin2,为卵巢癌提供一种新的治疗方法。方法通过基因工程的方法将两段基因IL2和183B2scFv开放读框的编码序列克隆在一起,在CHO K1细胞内人巨细胞病毒启动子的作用下表达可溶性融合蛋白,并检测其对肿瘤细胞的靶向性。结果采用哺乳动物细胞表达,系统表达的这种小分子融合蛋白,既保留了与卵巢癌OC183B2抗原结合的特性,又保持了IL2的生物学活性。融合蛋白在细胞培养上清中保持稳定,更重要的是融合蛋白将IL2靶向表达OC183B2抗原的卵巢癌细胞表面化的同时,又能刺激IL2依赖细胞株的增殖,从而诱发局部有效的抗肿瘤反应。既提高了融合蛋白的穿透组织能力和肿瘤组织部位的浓聚,又避免了大剂量全身应用细胞因子引起的副作用。结论真核表达系统表达的融合蛋白具有很好的生物学活性。

THE STUDY OF EXPRESSION AND FUNCTION OF ANTIBODY-CYTOKINE FUSION PROTEIN 183B2scFv-INTERLEUKIN 2

Objective To provide a new tool for the immunotherapy of human ovarian carcinoma, we constructed a fusion protein between interleukin-2 and the single-chain Fv(scFv) of 183B2, and and to investigate if the antibody-cytokine fusion protein 183B2scFv-interleukin 2 was expressed in mammalian cells. Methods 183B2 was a monoclonal antibody directed against ovarian cancer associated antigen OC183B2, known to be overexpressed on human epithelia ovarian carcinoma. This construction was accomplished by fusing the coding sequences in a single open reading frame and expressing the IL-2/183B2scFv chimera under the control of the human cytomegalovirus promoter in CHO-K1 cells. The bioactivity in vitro of the fusion protein was tested several times by CTLL-2 proliferation and tumor cells targeting assays. Results These novel molecules retainld both antibody and cytokine associated functions. The IL-2/183B2 scFv was stable on the cell supernatant and was biological active. Importantly, this construct was able to target IL-2 onto the surface of OC183B2 antigen overexpressing tumor cells and stimulate the proliferation of the IL-2 dependent cell. The design allowed the construction of a small molecule combining the specificity of 183B2 with the immunostimulatory activity of IL-2. This might improve the tissue penetration and distribution of the fusion protein within the tumor, reduce its immunogenicity, and avoid the toxicity related to the systemic administration of IL-2. These properties suggest the Ab-IL-2 fusion proteins targeting cancer cells may be effective for cancer treatment.Conclusion Mammalian cells are well suited for recombinant protein expression because they can be correctly processed.;