In vivo antitumor effect of an intracellular single‐chain antibody fragment against the E7 oncoprotein of human papillomavirus 16

Abstract

Human papillomavirus (HPV)-associated tumors still represent an urgent problem of public health in spite of the efficacy of the prophylactic HPV vaccines. Specific antibodies in single-chain format expressed as intracellular antibodies (intrabodies) are valid tools to counteract the activity of target proteins. We previously showed that the M2SD intrabody, specific for the E7 oncoprotein of HPV16 and expressed in the endoplasmic reticulum of the HPV16-positive SiHa cells, was able to inhibit cell proliferation. Here, we showed by confocal microscopy that M2SD and E7 colocalize in the endoplasmic reticulum of SiHa cells, suggesting that the E7 delocalization mediated by M2SD could account for the anti-proliferative activity of the intrabody. We then tested the M2SD antitumor activity in two mouse models for HPV tumors based respectively on TC-1 and C3 cells. The M2SD intrabody was delivered by retroviral vector to tumor cells before cell injection into C57BL/6 mice. In both models, a marked delay of tumor onset with respect to the controls was observed in all the mice injected with the M2SD-expressing tumor cells and, importantly, a significant percentage of mice remained tumor-free permanently. This is the first in vivo demonstration of the antitumor activity of an intrabody directed towards an HPV oncoprotein. We consider that these results could contribute to the development of new therapeutic molecules based on antibodies in single-chain format, to be employed against the HPV-associated lesions even in combination with other drugs.Human papillomaviruses (HPVs) belonging to 15 genotypes defined as "high risk," are the recognized cause of an increasing number of malignancies among which cervical cancer (CC) is the most represented in women worldwide.1,2 Despite the high protective efficacy of the HPV vaccine, expected to reduce the number of HPV-associated cancers in the coming decades, non-invasive therapies are urgently needed for avoiding overtreatment of pre-tumor lesions and preventing or treating metastatic lesions in the case of established tumors, particularly for the treatment of immunosuppressed people. 3The E6 and E7 oncoproteins of the high risk genotypes are tumor-specific antigens expressed in tumors and precursor lesions; they contribute to viral immunoevasion and act in concert to promote tumor development through the interaction with multiple cellular proteins. The E7 mainly affects factors involved in proliferation and cell cycle regulation, such as the retinoblastoma (pRb) and the whole pocket proteins family, the p21 and p27 cyclin-dependent kinase inhibitors, and the cyclins A and E, whereas the E6 binds to the p53 tumor suppressor through the E6-AP and to Bak proteins to ensure hampering of cell apoptosis.4,5 Therefore, E6 and E7 represent ideal targets for antitumor therapeutic interventions. Several approaches were and are currently explored to counteract the oncoproteins at gene or protein level. 6 In the last decades, emerged the possibility of designing drugs based on antibodies again...

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