Purification and Characterization of Antibodies in Single-Chain Format against the E6 Oncoprotein of Human Papillomavirus Type 16

Abstract

In Human Papillomaviruses- (HPV-) associated carcinogenesis, continuous expression of the E6 oncoprotein supports its value as a potential target for the development of diagnostics and therapeutics for HPV cancer. We previously reported that the I7 single-chain antibody fragment (scFv) specific for HPV16 E6, expressed as an intrabody by retroviral system, could inhibit significantly the growth of cervical cancer cells in vitro and was even able to reduce tumor development in experimental HPV-related cancer models. Nevertheless, for the development of therapeutic tools to be employed in humans, it is important to achieve maximum safety guarantee, which can be provided by the protein format. In the current study, two anti-16E6 scFvs derived from I7 were expressed in E. coli and purified in soluble form by affinity chromatography. Specificity, sensitivity and stability in physiologic environment of the purified scFvs were demonstrated by binding studies using recombinant 16E6 as an antigen. The scFvs functionality was confirmed by immunofluorescence in cervical cancer cells, where the scFvs were able to recognize the nuclear E6. Furthermore, an antiproliferative activity of the scFvI7nuc delivered in protein format to HPV16-positive cell lines was observed. Our results demonstrate that functional anti-16E6 scFvs can be produced in E. coli, suggesting that such purified antibodies could be used in the diagnosis and treatment of HPV-induced malignancies.

Pathogenic role of exosomes and microRNAs in HPV‐mediated inflammation and cervical cancer: A review

“…Previous studies had shown that single‐chain anti‐HPV‐16‐E6 and anti‐HPV‐16‐E7 antibodies had antiproliferative activities against HPV‐positive cells in vitro , and therapeutic efficiency against HPV‐positive tumors in animal models . Initial testing showed that the exosomes derived from cells transfected with plasmids expressing an Nefmut‐anti‐16E7 scFv fusion protein, contained the antibody in an active E7‐binding conformation which performed the same as scFv delivered as DNA or protein . These antibody‐containing exosmes could be used to treat lesions situated not only on the cervix, but also on the anogenital region or in the oropharynx.…”

“…154 Initial testing showed that the exosomes derived from cells transfected with plasmids expressing an Nefmut-anti-16E7 scFv fusion protein, contained the antibody in an active E7-binding conformation which performed the same as scFv delivered as DNA or protein. 154,155 These antibody-containing exosmes could be used to treat lesions situated not only on the cervix, but also on the anogenital region or in the oropharynx. Although these findings are promising, more widespread clinical application will require improved standardized techniques to purify the exosomes and monitor the exosomal contents.…”

Role of Extracellular Vesicles in Human Papillomavirus-Induced Tumorigenesis

“…Anti-HPV16-E6 and anti-HPV16-E7 antibodies in single-chain format were previously shown to exert antiproliferative activity in HPV-positive cells in vitro and therapeutic efficacy against HPV-positive tumors in animal models [98]. Preliminary experiments show that exosomes purified from cells transfected with plasmids expressing the Nef mut -anti-16E7 scFv fusion protein contain an antibody in an active conformation able to bind to the target E7 (Accardi et al, unpublished), exactly as what occurs for the scFv delivered as DNA or protein [98,99]. In view of the translational application of these antibodies in the clinic for the treatment of preneoplastic HPV-related lesions localized in the anogenital area or even in the oropharynx, exosomes can represent a safe and easy delivery tool.…”

Epitope Mapping and Computational Analysis of Anti-HPV16 E6 and E7 Antibodies in Single-Chain Format for Clinical Development as Antitumor Drugs

“…The extraction of scFvs and of the E6 and E7 proteins was performed from the respective transformed bacteria, and the proteins were purified using protein A-Sepharose CL-4B agarose beads (Amersham Biosciences) for the scFvs, and Ni-NTA agarose beads (Qiagen) for the oncoproteins, as previously reported [ 22 , 23 , 39 , 40 ].…”

“…The anti-16E7 scFvs were all inserted in the pDN332 phagemid, also allowing expression in the prokaryotic systems [28]. Interestingly, the coding sequences of the anti-16E6 scFvI7, which had been selected as an intrabody, were subcloned into the scFvExCyto-SV5 eukaryotic vector for expression in cell cytoplasm, and into the pQE30 prokaryotic vector for protein expression [25,39].…”