HPV ONCOPROTEIN ABLATION VIA SINGLE CHAIN ANTIBODIES

描述（申请人的描述）本申请的长期目标 研究项目是确定人乳头瘤病毒16型（HPV）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是病毒的癌蛋白，据信它们在 在宫颈癌的发展中起作用。 数据来自我们以前的 研究表明，抗E7细胞内单链抗体 scFv具有抑制HPV 16阳性细胞增殖的能力。 人宫颈癌细胞系85%至95%， 下调HPV 16 E7特异性蛋白水平。 HPV 16阴性细胞 线不受影响。 HPV 16 E6在宫颈癌的发展中也起着重要作用， 是一个理想的目标基因敲除。 在这个实验中要检验的假设 初步研究是使用针对HPV 16 E6和E7的scFv敲除这些E6和E7， 癌蛋白将抑制宫颈癌细胞增殖， 在体外下调这些癌蛋白的表达。 第一 目的是构建针对HPV 16 E6癌蛋白的scFv， a）指导细胞内抗HPV 16 E6的表达 人宫颈癌细胞系中的抗体，和B）敲除HPV 16 HPV阳性细胞系中的E6癌蛋白。 第二个目标是使 一种细胞内单链双特异性抗体[bs（scFv）2]，其由以下组成： 抗E6和抗E7，以同时敲除E6和E7癌蛋白， 体外 据推测，bs（scFv）2将比 单一scFv抑制细胞增殖。 HPV感染是90%以上宫颈癌的致病因素 在世界范围内，HPV的致癌功能已被分配给E6， 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 本文提出的体外研究采用抗HPV 16 E6和E7细胞内 单链抗体是成功的，他们将提供支持数据， 用于将来在裸鼠中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.