A novel intracellular antibody against the E6 oncoprotein impairs growth of human papillomavirus 16-positive tumor cells in mouse models

Abstract

Single-chain variable fragments (scFvs) expressed as “intracellular antibodies” (intrabodies) can target intracellular antigens to hamper their function efficaciously and specifically. Here we use an intrabody targeting the E6 oncoprotein of Human papillomavirus 16 (HPV16) to address the issue of a non-invasive therapy for HPV cancer patients.A scFv against the HPV16 E6 was selected by Intracellular Antibody Capture Technology and expressed as I7nuc in the nucleus of HPV16-positive SiHa, HPV-negative C33A and 293T cells. Colocalization of I7nuc and recombinant E6 was observed in different cell compartments, obtaining evidence of E6 delocalization ascribable to I7nuc. In SiHa cells, I7nuc expressed by pLNCX retroviral vector was able to partially inhibit degradation of the main E6 target p53, and induced p53 accumulation in nucleus. When analyzing in vitro activity on cell proliferation and survival, I7nuc was able to decrease growth inducing late apoptosis and necrosis of SiHa cells.Finally, I7nuc antitumor activity was demonstrated in two pre-clinical models of HPV tumors. C57BL/6 mice were injected subcutaneously with HPV16-positive TC-1 or C3 tumor cells, infected with pLNCX retroviral vector expressing or non-expressing I7nuc. All the mice injected with I7nuc-expressing cells showed a clear delay in tumor onset; 60% and 40% of mice receiving TC-1 and C3 cells, respectively, remained tumor-free for 17 weeks of follow-up, whereas 100% of the controls were tumor-bearing 20 days post-inoculum. Our data support the therapeutic potential of E6-targeted I7nuc against HPV tumors.

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

“…The I7nuc sequence was amplified by specific primers from the I7nucpGEMT previously described [18] and cloned in pQE30 BamHI/SacI restriction sites, obtaining I7nucQE. The plasmids obtained were checked by sequencing.…”

“…The I7nuc sequences were PCR amplified as above from the I7nucpGEMT plasmid previously described [18] using the sense primer anti-E6Dir reported above and the I7nucSacRev antisense primer to introduce the BamHI and SacI restriction sites, with sequence as follows:…”

“…Cell viability was determined 48 hours after delivery by MTS assay (CellTiter 96® AQ ueous One Solution Cell Proliferation Assay, Promega, Madison, USA) and densitometry analysis according to manufacturer's instructions. Alternatively, 48 h after scFv delivery, cells survival was analyzed by cell forming assay (CFA) as previously described [18]. …”

“…This effect can be ascribed to p53 rescue. A remarkable hindering effect on tumor onset due to I7nuc expression was observed also in two preclinical mouse models for HPV-associated tumors [18]. …”

“…Due to the selection method and previous experiments [18], we know that I7 and I7nuc are specific and can bind to the 16E6 when expressed in eukaryotic environment. We do not know whether the same binding capacity is retained by the scFv produced in prokaryotes as a recombinant protein.…”