Background: Intrabodies are defined as antibody molecules which are ectopically expressed inside the cell. Such intrabodies can be used to visualize or inhibit the targeted antigen in living cells. However, most antibody fragments cannot be used as intrabodies because they do not fold under the reducing conditions of the cell cytosol and nucleus.

Cervical cancer is caused by high-risk types of human papillomaviruses (HPV) that encode the E6 and E7 oncogenes. Silencing of E6 gene expression in HPVpositive cell lines by transfection of small interfering RNA (siRNA) with cationic lipids restores the dormant p53 tumor suppressor pathway. Because cationic lipids can also be used for intracytoplasmic delivery of proteins, we tested whether the delivery of monoclonal antibodies that bind to HPV16 E6 and neutralize its biological activity in vitro could restore p53 function in tumor cells. Here, we show that the 4C6 antibody is efficiently delivered into the cell cytoplasm using a lipidic reagent used for siRNA transfection. The delivery of 4C6 resulted in the nuclear accumulation of p53 protein in CaSki and SiHa cells but not in HeLa cells. Furthermore, the antibody-mediated p53 response was dramatically increased when a peptide corresponding to the 4C6 epitope and bearing a COOHterminal cysteine residue was added to the transduction mixture. We found that a fraction of the added peptides were dimers that allowed the formation of antibody polymers adsorbed onto the lipidic matrix. With this system, the proliferation of CaSki and SiHa cells was strongly diminished, but no apoptosis was detectable. Remarkably, cell growth was almost totally suppressed by the addition of E6-specific siRNA to the transduction complex. The results indicate that the activity of E6 oncoprotein can be down-regulated in vivo by lipidmediated antibody delivery and that antibodies and siRNA act synergistically when codelivered. This novel targeting strategy is simple to implement and may find therapeutic applications.

Intracellular antibodies or intrabodies are antibody fragments used inside cells for interaction with targets. In case intrabodies interfere with antigen function, they can mediate cell killing following binding (recently reviewed in [1]). Whilst a variety of cell types including plant cells, fungal cells and even bacteria [2] have been described as hosts, mammalian cells are the most commonly used target cells [3], implying that intrabodies with functional ablation capabilities might be a useful antibody-based format for disease-specific reagents. Until now, the preferred intrabody is the recombinant single-chain Fv antibody fragment (scFv), expressed from a single cDNA and composed of an antibody variable heavy-chain (VH) sequence tethered to a variable light-chain (VL) sequence by a flexible linker. ScFv carries the specificity inherent in the antibody combining site, namely the three hypervariable complementary-determining regions (CDRs) of each variable (V) region that form the antigen-binding pocket. The ectopic expression of antibody fragments inside mammalian cells (intrabodies) is a challenging approach for probing and modulating target activities. We previously described the shuttling activity of intracellularly expressed Escherichia coli b-galactosidase conferred by the single-chain Fv (scFv) fragment 13R4 equipped with nuclear import ⁄ export signals. Here, by appending to scFvs the proteolytic PEST signal sequence (a protein region rich in proline, glutamic acid, serine and threonine) of mouse ornithine decarboxylase, we tested whether short-lived or destabilized intrabodies could affect the steady-state level of target by redirecting it to the proteasomes. In the absence of antigen, the half-life of the modified scFv 13R4, relative to untagged molecules, was considerably reduced in vivo. However, after coexpression with either cytoplasmic or nuclear antigen, the destabilized 13R4 fragments were readily maintained in the cell and strictly colocalized with b-galactosidase. Analysis of destabilized site-directed mutants, that were as soluble as 13R4 in the intracellular context, demonstrated that binding to antigen was essential for survival under these conditions. This unique property allowed specific detection of b-galactosidase, even when expressed at low level in stably transformed cells, and permitted isolation by flow cytometry from a transfected cell mixture of those living cells specifically labeled with bound intrabody. Altogether, we show that PESTtagged intrabodies of sufficient affinity and solubility are powerful tools for imaging the presence and likely the dynamics of protein antigens that are resistant to proteasomal degradation in animal cells.Abbreviations b-gal, b-galactosidase; CDR, complementary-determining region; (E)GFP, (enhanced) green fluorescent protein; mODC, mouse ornithine decarboxylase; ODC, ornithine decarboxylase; scFv, single-chain Fv antibody fragment; VH, variable heavy-chain; VL, variable light-chain; V, variable.

<div>Abstract<p>Cervical cancer is caused by high-risk types of human papillomaviruses (HPV) that encode the E6 and E7 oncogenes. Silencing of E6 gene expression in HPV-positive cell lines by transfection of small interfering RNA (siRNA) with cationic lipids restores the dormant p53 tumor suppressor pathway. Because cationic lipids can also be used for intracytoplasmic delivery of proteins, we tested whether the delivery of monoclonal antibodies that bind to HPV16 E6 and neutralize its biological activity <i>in vitro</i> could restore p53 function in tumor cells. Here, we show that the 4C6 antibody is efficiently delivered into the cell cytoplasm using a lipidic reagent used for siRNA transfection. The delivery of 4C6 resulted in the nuclear accumulation of p53 protein in CaSki and SiHa cells but not in HeLa cells. Furthermore, the antibody-mediated p53 response was dramatically increased when a peptide corresponding to the 4C6 epitope and bearing a COOH-terminal cysteine residue was added to the transduction mixture. We found that a fraction of the added peptides were dimers that allowed the formation of antibody polymers adsorbed onto the lipidic matrix. With this system, the proliferation of CaSki and SiHa cells was strongly diminished, but no apoptosis was detectable. Remarkably, cell growth was almost totally suppressed by the addition of E6-specific siRNA to the transduction complex. The results indicate that the activity of E6 oncoprotein can be down-regulated <i>in vivo</i> by lipid-mediated antibody delivery and that antibodies and siRNA act synergistically when codelivered. This novel targeting strategy is simple to implement and may find therapeutic applications. [Mol Cancer Ther 2007;16(5):1728–35]</p></div>