HPV ONCOPROTEIN ABLATION VIA SINGLE CHAIN ANTIBODIES

描述(申请人的描述)这项计划的长期目标 研究项目是确定人乳头瘤病毒类型(HPV)16的作用 E6和E7癌蛋白在宫颈癌发生中的作用人乳头瘤病毒 16型E6和E7是病毒癌蛋白，被认为是主要的 在宫颈肿瘤发生发展中的作用。来自我们以前的数据 研究表明，抗E7细胞内单链抗体 单链抗体对HPV16阳性细胞增殖的抑制作用 人宫颈癌细胞株85%至95%，并强烈 下调HPV16E7特异性蛋白水平。HPV16阴性细胞 线路没有受到影响。 HPV16E6在宫颈癌的发生发展中也起着重要作用，并可能 是一个理想的敲除靶基因。这里要检验的假设是 初步研究是使用单链抗体对HPV16E6和E7进行敲除 癌蛋白会抑制宫颈癌细胞的增殖和 在体外下调这些癌蛋白的表达。第一 目的是构建针对HPV16E6癌蛋白的单链抗体，并 利用单链抗体a)直接在细胞内表达抗HPV16E6 人宫颈癌细胞株中的抗体，以及b)敲除的HPV16 HPV阳性细胞株中的E6癌蛋白。第二个目标是使 由以下成分组成的细胞内单链双特异性抗体[bs(ScFv)2] 抗E6和抗E7同时敲除E6和E7癌蛋白 体外培养。假设bs(ScFv)2将比任何一种都更有效 单链抗体对细胞增殖有抑制作用。 HPV感染是90%以上宫颈癌的致病因素 在世界范围内，HPV的致癌作用已被分配给E6和 E7基因。抑制这些癌蛋白表达的能力将 从而有望降低宫颈癌的发病率。如果在 建议使用抗HPV16E6和E7细胞内的体外研究 单链抗体是成功的，它们将提供支持数据 用于将来在裸鼠身上使用这些抗体的体内研究，以及 旨在抑制宫颈肿瘤的未来临床和营养研究 通过抗基因治疗取得进展。

DESCRIPTION (Applicant's Description) The long-term objective of this research project is to define the role of human papillomavirus type (HPV) 16 E6 and E7 oncoproteins in cervical carcinogenesis. Human papillomavirus type 16 E6 and E7 are viral oncoproteins that are believed to play a major role in the development of cervical neoplasia. Data from our previous studies demonstrated that anti-E7 intracellular single-chain antibodies (scFvs) have the ability to inhibit cell proliferation in HPV 16-positive human cervical carcinoma cell lines by 85 to 95 percent, and to strongly downregulate the level of HPV 16 E7-specific protein. HPV 16-negative cell lines were unaffected. HPV 16 E6 also plays a major role in development of cervical cancer, and may be an ideal target gene to knock out. The hypothesis to be tested in this pilot study is that knock-out of HPV 16 E6 and E7 using scFvs against these oncoproteins will inhibit cervical cancer cell proliferation and down-regulate expression of these oncoproteins in vitro. The first objective will be to construct scFvs against the HPV 16 E6 oncoprotein, and to use the scfvs to a) direct expression of intracellular anti-HPV 16 E6 antibodies in human cervical carcinoma cell lines, and b) knock-out HPV 16 E6 oncoprotein in HPV-positive cell lines. The second objective is to make an intracellular single-chain bispecific antibody [bs(scFv)2] composed of anti-E6 and - anti-E7 to simultaneously knock out E6 and E7 oncoproteins in vitro. It is hypothesized that bs(scFv)2 will be more effective than either single scFv in inhibiting cell proliferation. HPV infection is a causative factor in over 90 percent of cervical cancers worldwide, and the oncogenic function of HPV has been assigned to the E6 and E7 genes. The ability to inhibit expression of these oncoproteins would thus be expected to decrease the incidence of cervical cancer. If the in vitro studies proposed here employing anti-HPV 16 E6 and E7 intracellular single-chain antibodies are successful, they will provide supporting data for future in vivo studies using these antibodies in nude mice, as well as future clinical and nutritional studies aimed at inhibiting cervical tumor progression via anti-gene therapy.