IJC International Journal of Cancer

In vivo antitumor effect of an intracellular single-chain antibody fragment against the E7 oncoprotein of human papillomavirus 16 Luisa Accardi1, Francesca Paolini2, Angela Mandarino1, Zulema Percario3, Paola Di Bonito1, Valentina Di Carlo1, Elisabetta Affabris3, Colomba Giorgi1, Carla Amici4 and Aldo Venuti2 1

Department of Infectious, Parasitic and Immune-mediated Diseases, Istituto Superiore di Sanita, viale Regina Elena, 299, Rome, Italy Regina Elena National Cancer Institute, Laboratory of Virology and HPV-Unit, via delle Messi d’Oro 156, Rome, Italy 3 Department of Science, University Roma Tre, Viale G. Marconi 446, Rome, Italy 4 Department of Biology, University of Rome Tor Vergata, Via della Ricerca Scientifica 1, Rome, Italy 2

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.

Short Report

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 Key words: scFv, intrabodies, human papillomaviruses, cancer therapy, E7 oncoprotein Abbreviations: CC: cervical cancer; ER: endoplasmic reticulum; HPV: Human papillomavirus; NLS: nuclear localization signal; pRb: retinoblastoma protein; RF-E7: red fluorescent E7; scFvs: single-chain antibody fragments Grant sponsor: Ministry of Health; Grant number: AIDS project 2009 3H/01 (to L.A.); Grant sponsor: AIRC IG; Grant number: 12916 (to A.V.) DOI: 10.1002/ijc.28604 History: Received 16 July 2013; Accepted 7 Nov 2013; Online 13 Nov 2013 Correspondence to: Dr Luisa Accardi, Section of Molecular Pathogenesis, Department of Infectious, Parasitic and Immunemediated Diseases, Istituto Superiore di Sanita, Viale Regina Elena, 299, Rome 00161, Italy, Tel.: 139-06-4990-3265, Fax: 139-06-49902082, E-mail: [email protected]

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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.3 The 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 against virtually any antigen of interest, whether tumor or not. Nevertheless, recombinant non-human antibodies, mainly obtained by Hybridoma technology, show some drawback due to the possible induction of an immune response and to the large size which hinders adequate exposure of solid tumors to drugs. Single-chain

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What’s new? Intrabodies, or antibodies that act intracellularly, could be effective for counteracting oncoproteins, such as the E7 oncoprotein of human papillomavirus (HPV). This study shows that the intrabody M2SD co-localizes intracellularly with E7 of HPV16, likely accounting for the intrabody’s antiproliferative effects. In vivo antitumor efficacy of M2SD was demonstrated in two HPV animal models, in which tumor onset was delayed. The findings pave the way for the development of a new, intrabody-based therapeutic approach against HPV.

Material and Methods Clones, cell lines, transfection, retroviral transduction, MTS and confocal microscopy analysis

The plasmids expressing (scFv43 M2SD) or non-expressing the M2SD intrabody (scFvExpressSD), used for cell transfection and the M2SD-encoding pLNCX retroviral construct (M2SD-pLNCX) were previously described (14 and Fig. 2, panel a). The plasmid expressing E7 fused to the Red Fluorescent protein (pRF-E7) was kindly provided by David Pim (ICGEB, Trieste). SiHa cells (ATCC HTB-35) used for transfection, Phoenix cells (ATCC 3444) used for production of retroviral particles, TC-1 and C57BL/6 (C3) tumor cells were cultured under standard conditions. TC-1 cells are primary lung epithelial cells of C57BL/6 mice co-transformed with HPV16 E6/E7 and c-Ha-ras.15 C3 cells were derived from embryonic mouse cells transformed with the full HPV16 genome and activated ras C 2013 UICC Int. J. Cancer: 134, 2742–2747 (2014) V

oncogene.16 Both cell lines are able to establish subcutaneous tumors in C57BL/6 mice, which are used as models of human HPV16-associated neoplasms. The TC-1 and C3 cells utilized in our experiments are passages of the original clones and were routinely checked for the presence of HPV sequence and for resistance to the G418 antibiotic selection (0.4 mg/ml). Cell transfection was performed by jetPEIV transfection reagent (Polyplus transfection, Illkirch, France) according to the manufacturer’s recommendations. Retroviral production and transduction were performed as previously described14 with the following modifications. Forty-eight hours after transfection of Phoenix packaging cells, the virus-containing cell culture supernatant was concentrated 1003 by RetroX concentrator (Clontech, CA) and used to infect C3 or TC-1 cells at 60% of confluence. The neomycin concentration was increased to 0.8 mg/ml for transgene selection, 24 hr post-infection. Cell proliferation was investigated by MTS assay (CellTiter 96V AQueous One Solution Cell Proliferation Assay, Promega, Madison, WI) in exponentially growing cell cultures 24, 48 and 72 hr post-infection. The assay was carried out three times in triplicate and statistical analysis of the results performed by Student’s t test for unpaired data. Staining of M2SD-expressing cells was as previously reported.14 For staining of the ER, permeabilized cells were incubated with rabbit polyclonal anti-calnexin antibody (SPA865, Stressgen, Belgium) followed by Cy5-conjugated goat antirabbit IgG (Jackson Immunoresearch Laboratories, Inc.). Images were acquired using a Leica TCS SP5 confocal microscope and LAS AF version 1.6.3 software (Leica microsystems). R

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Mouse models

Six-week-old female C57BL/6 mice were obtained from Charles River (Como, Italy) and maintained under specific pathogen-free conditions at the Experimental Animal Department of the Regina Elena National Cancer Institute (Rome, Italy). Animal experiments described in this study were performed according to the Institutional animal use guidelines and the Italian law DL 116/92. Before injection into mice, TC-1 or C3 cells were trypsin/EDTA treated, washed, resuspended in saline solution and adjusted to the right density for injection. Groups of C57BL/6 mice (5–8 mice/treatment) were injected subcutaneously into the right inner flank with 5 3 104 M2SD-infected TC-1 (M2SD-TC-1) or 5 3 105 M2SD-infected C3 (M2SD-C3) cells. The different number of

Short Report

antibody fragments (scFvs), derived from the IgG repertoire by genetic engineering, represent the smallest molecules (27 KDa) able to specifically bind to an antigen, and find multiple applications in diagnosis and therapy.7,8 ScFvs can be easily tailored to modify pharmacokinetics, immunogenicity, specificity and effectors functions in a cost-effective-way, can be expressed and produced in E. coli, or delivered to cells as intracellular antibodies (intrabodies) to alter the function of specific targets, thus representing a powerful alternative to methods of gene inactivation.9–11 We previously selected by Phage display technology some scFvs against the E7 protein of HPV16 (16E7), and reported the intracellular expression and anti-proliferative activity of one of them (scFv43) in the HPV16-positive SiHa cells.12 The scFv43 stability was then improved by site-directed mutagenesis, obtaining scFv43M2.13 Recently, the scFv43M2 and scFv51, expressed either in nucleus or in endoplasmic reticulum (ER) of SiHa cells by retroviral system, were shown to exert a specific anti-proliferative activity.14 Interestingly, the most efficacious intrabodies resulted to be 43M2SD (M2SD) and 51SD, both localized in ER. Here, we analyze the M2SD localization in relation to the E7 intracellular distribution, and assess, for the first time in vivo, the ability of an anti-16E7 intrabody to inhibit tumorigenicity of TC-1 and C3 cells in two preclinical models for HPV tumors.

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Figure 1. M2SD intrabody expression alters intracellular localization of the E7 protein. Representative confocal microscopy images showing intracellular distribution of the RF-E7 protein expressed in SiHa cells alone (panel a) or in the presence of M2SD intrabody (panel b), 48 hr post-transfection. Polyclonal anti-calnexin antibody followed by Cy5-conjugated secondary antibody was used to stain ER (blue). The M2SDintrabody was detected by anti-myc antibody and FITC-conjugated secondary antibody (green). In the absence of M2SD, E7 accumulates in the nucleus (panel a). Co-localization of E7 with M2SD (yellow) and localization in ER of M2SD (cyan) and of E7 (magenta) are shown in the merge images. Scale bar 5 10 mm.

TC-1 and C3 cells injected corresponds to the minimum dosage known to cause tumor onset within 2 weeks in the two experimental models. Uninfected or retrovirus-infected (pLNCX) tumor cells were used as negative controls. Mice were observed daily until tumor onset and, after that, tumor burden was evaluated by palpation twice a week for about 5 months. The experiments were repeated three times; data were analyzed by Log-Rank test among Kaplan-Meier curves and considered significant when p < 0.005.

Results Intracellular localization of M2SD and E7 protein

The anti-proliferative effect of anti-E7 intrabodies expressed in the ER of HPV16-positive cell lines12,14 raised the issue to envisage a mechanism of action compatible with the E7 heterogeneous intracellular distribution.17–19

Therefore, we studied the E7 localization in relation to the anti-E7 intrabodies expression by performing transient single and double transfection of SiHa cells with pRF-E7 and scFv43 M2SD.14 When transfection was with pRF-E7 only, the Red-Fluorescent E7 (RF-E7) was detected mainly in cell nucleus after 48 hr (Fig. 1, panel a). At the same time, cells transfected with scFv43 M2SD showed the typical ER staining pattern previously described.14 Interestingly, colocalization analysis by confocal microscopy performed 48 hr after double transfection, highlighted the presence of RF-E7 and M2SD in the same cell compartment, identified as ER by staining through a specific antibody (Fig. 1, panel b). These findings suggest that the RF-E7 accumulates in the ER compartment after binding to the intrabody, which would be able to delocalize the oncoprotein from its main usual compartment.

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Expression of the M2SD intrabody in tumor cells and in vitro effect on cell proliferation

In view of next experiments in vivo, the M2SD expression in TC-1 and C3 cells was investigated after delivery by retroviral system. The recombinant pLNCX vector used for production of the M2SD-expressing retroviruses (M2SD-pLNCX) is schematically represented in Figure 2, panel a. TC-1 and C3 cells infected with M2SD-pLNCX were analyzed by immunofluorescence confocal microscopy at different times. In Figure 2, panel b, the expression of M2SD in the ER of TC-1 cells 48 hr after infection is shown. The effect of M2SD on the TC-1 and C3 cell growth was then tested by MTS assay 24, 48 and 72 hr post-infection. As shown in Figure 2, panel c, infection with the empty pLNCX virus only does not affect the replicative potential of tumor cells as compared with uninfected C3 cells, 3 days postinfection. Conversely, M2SD expression caused significant inhibition of cell growth, with a reduction greater than 50%. Similar results were obtained for the infected TC-1 cells (data not shown). Notably, the levels of proliferation inhibition observed in both tumor cell lines are in agreement with the results previously obtained in SiHa cells,12,14 supporting the notion that also in TC-1 and C3 tumor cells the expression C 2013 UICC Int. J. Cancer: 134, 2742–2747 (2014) V

Antitumor effect of M2SD in HPV tumor mouse models

The potential antitumor activity of M2SD was tested in vivo in 2 preclinical models of HPV tumors. TC-1 or C3 cells were infected with the M2SD-expressing retrovirus, and M2SD expression was monitored by immunofluorescence microscopy 48 hr after transduction. Tumor cells were injected subcutaneously in groups of C57BL/6 mice, only if the intrabody was expressed in a percentage of cells higher than 40%, which corresponds to the average percentage of transduction obtainable in these cell systems. TC-1 or C3 cells non infected or infected with retrovirus only (pLNCX) represented the negative controls. Mice were then monitored for tumor onset, and those showing a delay of tumor development were observed for a further 4 months after suppression of the mice of the control groups for ethical reasons. Interestingly, tumor onset and growth had similar trends in both the TC-1 and C3 models, with slight differences. In fact, all the mice of the control groups, injected with pLNCX-infected or uninfected tumor cells, developed subcutaneous tumors 2 weeks post-injection. Tumors did not develop in 60% of the mice injected with M2SD-TC-1 cells whereas in the residual 40% of mice grew with a significant delay with respect to the control groups of mice (Fig. 3, panel a). The effect of M2SD-C3 cells was less evident; in fact, although a significant delay of tumor onset was observed, only 40% of mice remained tumor-free during all the time of monitoring (Fig. 3, panel b). Log-Rank test among KaplanMeier curves indicated statistically significant differences between the M2SD-TC-1- and M2SD-C3-injected mice and the controls, with p 5 0.0035 and p 5 0.0027, respectively. Furthermore, we observed that tumors developed from the M2SD-infected cells were overall without ulceration and smaller than those developed in the controls. These results support the anti-E7 intrabody ability to inhibit tumorigenicity

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Figure 2. M2SD intrabody reduces proliferation of HPV16-positive tumor cells in vitro. (a) Schematic representation of the recombinant pLNCX retroviral vector expressing the scFv43 M2 intrabody with localization in the ER (M2SD) under the control of CMV promoter. The intrabody has a signal peptide for secretion (SEC), a myc-tag for detection and the ER retention signal (SEKDEL), as indicated. (b) Representative confocal microscopy images of TC-1 cells expressing the M2SD-intrabody, 48 hr post-infection, detected as described in the legend of Figure 1. Differential interference contrast image (DIC) is also shown. Scale bar 5 10.1 mm. (c) C3 tumor cell proliferation evaluated by MTS assay 72 hr after the infection with either retrovirus only (pLNCX) or M2SD-expressing retrovirus (M2SD). Cell proliferation is expressed as a percentage of that obtained in pLNCX-infected cells, considered as the reference value (100%). Each bar represents the mean value 6 standard deviation of three independent experiments. The star (*) indicates significance of the results (p < 0.01).

of anti-E7 intrabodies in ER can efficaciously hamper pathways affected by E7. To further test the specificity of the anti-proliferative effect of M2SD in murine cells, MTS assay was performed 48 hr after transfection of TC-1 cells with either the scFv43 M2SD, expressing the M2SD intrabody, or the scFvExpressSD plasmid, expressing a polypeptide carrying the signals for localization and retention in ER but not containing the scFv sequence.14 Cells treated with transfection reagent were used as a control. Average transfection was evaluated by IF to be about 30%. The growth potential of TC-1 cells was not affected by transfection with scFv43ExpressSD (OD490 5 1.53 6 0.34) as compared to the control cells (OD490 5 1.39 6 0.29). On the contrary, transfection with scFv43 M2SD significantly reduced the TC-1 cells proliferation (OD490 5 0.82 6 0.19; p < 0.01). These results allowed to exclude the possibility that the anti-proliferative effect of the M2SD intrabody was dependent on an aberrant ER stress response induced by the recombinant protein retained in the ER membrane.20

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Figure 3. M2SD intrabody expression inhibits tumor growth in vivo. Kaplan-Meier analysis indicating time-to-tumor development of C57BL/6 mice injected subcutaneously in the right leg with M2SDexpressing tumor cells. Mice were injected with 5 3 104 TC-1 (panel a) or with 5 3 105 C3 tumor cells (panel b). Both cell lines were untreated, infected with retrovirus only (pLNCX) or with M2SD-expressing retrovirus (M2SD) before injection in mice. LogRank test among Kaplan-Meier curves indicate statistically significant differences (p < 0.005).

of HPV-positive cells, and could represent a start point for an intrabody-based therapeutical approach of HPV cancer.

Short Report

Discussion We explored the use of scFv intrabodies specific for the 16E7 oncoprotein to address the issue of HPV cancer therapy. We presumed that the M2SD intrabody, previously shown effective in inhibiting proliferation of the HPV16-positive SiHa cells in vitro, could revert the tumor phenotype of HPV16positive tumor cells, and evaluated its antitumor efficacy in two preclinical models for HPV tumors based on TC-1 and C3 cells, respectively. Both cell lines are co-transformed with ras oncogene, but continued expression of the HPVl6 E6 and E7 oncoproteins is required for maintenance of their transformed phenotype.21 In C3 cells the E6/E7 genes are under the control of their natural promoter, whereas in TC-1 cells they are controlled by a retroviral promoter. This difference may affect the expression level of the HPV proteins which, in turn, may influence specific therapies targeting the viral oncoproteins.22 However, Q-RT PCR for E7-containing transcripts did not show any significant difference between TC-1 and C3 cells.23 In both the TC-1 and C3 preclinical models formerly used, tumor cell challenge in C57BL/6 mice was typically utilized to evaluate a therapeutic intervention in terms of both safety and induction of HPV-specific immune responses able

In vivo Intrabody Efficacy Against HPV Tumors

to eradicate a transplanted tumor. In contrast, in our study, such models were employed to assess, in environmental conditions more appropriate than in vitro cultures, an antitumor activity not based on the immunological response of the host but rather due to a specific binder (M2SD) that blocks a protein involved in tumor development. Indeed, the therapeutic scFv was delivered as an intrabody to tumor cells before their injection into animals. In this regard, it should be noted that we have identified the proper time to inject the mice between 24 and 48 hr after infection, because in this time interval there is high intrabody expression but at the same time the infected cells are still proliferating and countable. The possibility to revert tumor phenotype of TC-1 and C3 cells by downregulating the oncoproteins has been assessed with different methods.24 Here we show, for the first time, that tumorigenicity of these cells can be reduced by an E7binder. Kaplan-Meyer analysis of time-to-tumor development showed statistically significant differences between the tumors derived from cells expressing or not expressing the M2SD intrabody. Our results show that both the M2SD-expressing cell lines either were unable to cause tumors or caused tumor onset with a significant delay with respect to the controls. Notably, the M2SD resulted to be more efficacious in TC-1 than in C3 cells. This occurrence does not seem to be correlated to the different levels of E7 expression, which are comparable in the two cell lines even though slightly lower in TC-1 cells.23 The efficacy difference may be ascribed to a diverse biological behavior of these cell lines, which were obtained by different transfection procedures and have different origin, namely embryonic for C3 and epithelial for TC-1 cells. The demonstration of the M2SD efficacy is unequivocal in our experimental system, and the data indicate that the M2SD-induced alteration of cell proliferation, demonstrated in vitro, occurs also in vivo, where the cells are exposed to the proper proliferation stimuli. Furthermore, we believe that our in vivo results strictly depend on the M2SD expression level obtained in each experiment, and efficacy could be therefore underestimated. The anti-proliferative effect of M2SD, which is expressed in the ER, was expected in view of previous findings in vitro and also because the ER environment, while having a strict quality control system, promotes proper folding, stability and thus activity of an intrabody.20,25 Nevertheless, it is necessary to take into account that the intracellular localization of E7 is not yet fully known. The E7 protein has both nuclear import (NLS) and export signals allowing it to shuttle between the two compartments.18 Also, the E7 intracellular localization was shown to vary with cell confluence19 and phases of cell cycle,18 that is consistent with the E7 ability to target proteins located in different cell compartments. In our study, we observed by confocal microscopy the colocalization of plasmid-encoded E7 and M2SD in the ER of SiHa cells. In view of previous findings highlighting the E7 C 2013 UICC Int. J. Cancer: 134, 2742–2747 (2014) V

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interaction with acid alpha-glucosidase localized in ER26 and the trafficking of different E7 isoforms in the ER-Golgi compartment,27 it could be hypothesized that M2SD sequesters E7 in the ER thus hampering the nuclear and cytoplasmic pathways in which it is involved. However, further knowledge on the intracellular trafficking will be required to predict how and where E7 and M2SD can meet within the cell. Interestingly, we previously showed in vitro that the preferential binding site of scFv 43M2 on E7 maps in the aminoacids 10–2114 contained in the stretch 1–37, which represents the main E7 NLS.17 As the major E7 activity is to favor the entry of cells into cell cycle, the observed inhibition of cellular tumorigenesis may result from the intrabody binding to the E7 NLS, which would hamper the E7 translocation to the nucleus and interaction with its nuclear targets. Furthermore, this is consistent with our previous studies suggesting that the mechanism of action of M2SD is pRB-independent.14 The evaluation of M2SD in a therapeutic setting is ongoing. The intrabody will be delivered directly to mice by injection in already established tumors, and its capability to revert tumors will be evaluated. However, for a successful translation of this or other intrabodies to clinics, the issue of

an efficacious and safe delivery to patients will need to be addressed. The options relate to DNA and protein delivery by different methods: safe viral particles, electroporation/ sonoporation, fusogenic or cell penetrating peptides, liposomes/nanoparticles or even new emerging approaches such as exosomes.28–30 Of note, in this case, the task is facilitated by the possibility to obtain highly specific antitumor intrabodies working only in the presence of their oncoprotein target. In conclusion, this is the first report of the in vivo antitumor efficacy of an intrabody directed against an HPV oncoprotein, and suggests that such intrabody may be profitably employed in the therapy of HPV-associated early lesions, tumors and metastatic lesions, even in combination with the conventional chemotherapeutic agents currently in use for CC.31

Acknowledgements The authors kindly thank Dr David Pim for providing the RF-E7 plasmid and they are grateful to Dr Alessandra Care` for critical reading of the article. Many thanks are also due to Mr Walter Tranquilli for computer artwork.

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Zur Hausen H. Papillomavirus infections: a major cause of human cancer. In: zur Hausen H, ed. Infections causing human cancer. Weinheim: WILEY-VCH Verlag GmbH & Co. KGaA, 2006. 145–243. 2. Forman D, de Martel C, Lacey CJ, et al. Global burden of human papillomavirus and related diseases. Vaccine 2012;30:F12–23. 3. Stanley MA. Genital human papillomavirus infections: current and prospective therapies. J Gen Virol 2012;93:681–91. 4. Moody CA, Laimins A. Human papillomavirus oncoproteins: pathways to transformation. Nature 2010;10:550–60. 5. Ghittoni R, Accardi R, Hasan U, et al. The biological properties of E6 and E7 oncoproteins from human papillomaviruses. Virus Genes 2010;40:1–13. 6. Tan S, de Vries EG, van der Zee AG, et al. Anticancer drugs aimed at E6 and E7 activity in HPV-positive cervical cancer. Curr Cancer Drug Targets 2012;12:170–84. 7. Holliger P, Hudson PJ. Engineered antibody fragments and the rise of single domains. Nat Biotechnol 2005;23:1126–36. 8. Accardi L, Di Bonito P. Antibodies in singlechain format against tumour-associated antigens: present and future applications. Curr Med Chem 2010;17:1730–55. 9. Lener M, Horn IR, Cardinale A, et al. Diverting a protein from its cellular location by intracellular antibodies. The case of p21Ras. Eur J Biochem 2000;267:1196–205. 10. Perez-Martinez D, Tanaka T, Rabbitts TH. Intracellular antibodies and cancer: new technologies offer therapeutic opportunities. Bioessays 2010;32:589–98. 11. Hong CW, Zeng Q. Awaiting a new era of cancer immunotherapy. Cancer Res 2012;2:3715–29. 12. Accardi L, Dona MG, Di Bonito P, et al. Intracellular anti-E7 human antibodies in single-chain format inhibit proliferation of HPV16-positive

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References