) 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.
