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.
Highlights
In the immune system, each antibody recognizes a specific target antigen through the antibody-antigen binding site, formed by 3 structurally hypervariable loops in the light (VL) and heavy chain (VH) variable regions of an Immunoglobulin (Ig), called complementarity determining regions (CDRs).Antibodies for biomedical purposes can be produced in different formats thanks to the recombinant DNA technology [1]
Expression, and purification of single-chain antibody fragments directed towards 16E6
We demonstrated the ability of the purified antibody fragments to detect 16E6 in different assays
Summary
Each antibody recognizes a specific target antigen through the antibody-antigen binding site, formed by 3 structurally hypervariable loops in the light (VL) and heavy chain (VH) variable regions of an Immunoglobulin (Ig), called complementarity determining regions (CDRs).Antibodies for biomedical purposes can be produced in different formats thanks to the recombinant DNA technology [1]. One of the most used formats is the single-chain variable antibody fragment (scFv), including the Ig VH and VL domains linked by a flexible polypeptide linker. A scFv retains the specificity towards its target and has increased tissue penetrance, faster blood plasma clearance, and lower immunogenicity with respect to the parental antibody [2, 3]. The ease of manipulation and expression on a large scale make the scFv an ideal antibody format for diagnostic and therapeutic application even in the oncologic field [4]. A number of human cancers are etiologically related to persistent infection with high-risk Human Papillomaviruses (HR HPVs). Approximately 600,000 cases of HPV-related diseases occur per year, which still represents a serious problem of public health. The HPVassociated head and neck cancers increased considerably over the past two decades, especially in developed countries [8, 9]
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