A novel, broad spectrum therapeutic HPV vaccine targeting the E7 proteins of HPV16, 18, 31, 45 and 52 that elicits potent E7-specific CD8T cell immunity and regression of large, established, E7-expressing TC-1 tumors

Vaccine 29 (2011) 7857–7866

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A novel, broad spectrum therapeutic HPV vaccine targeting the E7 proteins of HPV16, 18, 31, 45 and 52 that elicits potent E7-specific CD8T cell immunity and regression of large, established, E7-expressing TC-1 tumors Darin A. Wick, John R. Webb ∗ British Columbia Cancer Agency, Trev and Joyce Deeley Research Centre, 2410 Lee Avenue, Victoria, Canada V8R 6V5

a r t i c l e

i n f o

Article history: Received 20 January 2011 Received in revised form 8 July 2011 Accepted 20 July 2011 Available online 2 August 2011 Keywords: CD8 T cells Human papillomavirus TLR3 TLR9 Vaccine

a b s t r a c t Persistent infection by high risk genotypes of human papillomavirus (HPV) is the cause of cervical cancer, which remains one of the most common cancers among women worldwide. In addition, there is a growing appreciation that high risk HPVs are associated with a number of other cancers including anogenital cancers as well as a subset of head and neck cancers. Recently, prophylactic HPV vaccines targeting the two most prevalent high risk HPVs (HPV16 and HPV18) have been deployed in large-scale vaccination campaigns. However, the extent to which these prophylactic vaccines confer protection against other high risk HPV genotypes is largely unknown and prophylactic vaccines have been shown to be ineffective against pre-existing infection. Thus there continues to be an urgent need for effective therapeutic vaccines against HPV. The E7 protein of HPV16 has been widely studied as a target for therapeutic vaccines in HPVassociated cancer settings because HPV16 is the most prevalent of the high risk HPV genotypes. However, HPV16 accounts for only about 50% of cervical cancers and there are at least 15 other high risk HPVs that are known to be oncogenic. We have developed a novel, broad-spectrum, therapeutic vaccine (Pentarix) directed at the E7 proteins from five of the most prevalent high-risk genotypes of HPV worldwide (HPV16, 18, 31, 45 and 52) that together account for more than 80% of all HPV-associated cancers. Pentarix is a recombinant protein-based vaccine that elicits strong, multi-genotype specific CD8 T cell immunity when administered to mice in combination with adjuvants comprised of agonists of the TLR3 or TLR9 family of innate immune receptors. Furthermore, large, established E7-expressing TC-1 tumors undergo rapid and complete regression after therapeutic vaccination of mice with Pentarix. Together, these data suggest that Pentarix may be of clinical value for patients with E7-positive, HPV-associated precancerous lesions or malignant disease. © 2011 Elsevier Ltd. All rights reserved.

1. Introduction Cervical cancer is caused by infection with high risk genotypes of human papillomavirus (HPV) and is the second most common cancer among women with 500,000 new cases every year worldwide [1]. Although many HPV infections are thought to be self self-limiting, persistent infection can lead to cervical dysplasia and ultimately to cancer (for review see [2]). In addition to cervical cancer, HPV infection is also associated with a variety of anogenital cancers [3] as well as a subset of head and neck cancers [4,5]. Recently, an association between HPV and breast cancer has also been proposed [6]. HPV comprises a group of more than 100 genetically distinct, ‘genotypes’ of virus, which can be broadly broken down into low-risk and high-risk types. Over the last several years, prophylactic HPV vaccines targeting the two most prevalent of the

∗ Corresponding author. Tel.: +1 250 519 5706; fax: +1 250 519 2040. E-mail address: [email protected] (J.R. Webb). 0264-410X/$ – see front matter © 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.vaccine.2011.07.090

high risk HPVs (HPV16 and HPV18) have entered the market place [7,8]. These vaccines are reported to provide excellent protection against new HPV16/18 infections and are thus being administered to young women prior to the age of onset of sexual activity in a growing number of large-scale vaccination campaigns. Although there have been a handful of studies suggesting that these prophylactic vaccines offer some cross-protection against some other genotypes [9–11], the actual extent of cross-protection is largely unknown. Furthermore, prophylactic vaccines have been shown to be ineffective against pre-existing infection [12]. Thus, therapeutic HPV vaccines continue to be an area of intense investigation as a treatment modality for HPV-associated cancers (for review see [13–16]). Two oncogenic viral proteins, E6 and E7, are of particular interest in this regard as they are required for the transformation of infected cells and their continuous expression is required to maintain cells in a transformed state [17]. Indeed E6 and E7 are often the only viral genes that continue to be expressed in cancerous cells [7] thus they represent ideal targets for immunotherapy of

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cervical cancer. Accordingly, numerous methodologies to elicit strong anti-E6/E7 cellular immunity have been explored including peptide immunization [18–22], DNA immunization [23–26], immunization with recombinant, E7-expressing Vaccinia virus [27,28], adenovirus [29–31], Salmonella typhimurium [32,33] or Listeria monocytogenes [34,35], E7-pulsed dendritic cells [22,36–39] or E7-containing virus-like particles (VLP) [40–43]. Concurrent treatment of tumors with CpG oligonucelotide plus chemotherapy or surgical resection has also been previously reported to be effective at regressing TC1 tumors in mice [44]. Although a number of these approaches have been assessed in early stage clinical trials, none have yet to advance beyond phase II [45]. Herein, we describe the development of a new broad-spectrum, therapeutic vaccine (Pentarix) directed at the E7 proteins from five of the most prevalent high-risk genotypes of HPV (HPV16, 18, 31, 45 and 52) that together account for more than 80% of all HPVassociated cancers worldwide. Pentarix is a recombinant fusion protein-based vaccine that contains the E7 oncoproteins of HPV genotypes 16, 18, 31, 45 and 52, linked together as a single contiguous protein. Although immunization with whole exogenous protein has not historically been considered to be an efficient means of eliciting MHC class I-restricted CD8 T cell responses [46], we have recently shown that full length protein can elicit profound CD8 T cell immunity when combined with the TLR3 agonist poly(I:C) and delivered via a ‘cluster’ immunization strategy [47]. In the current study we demonstrate that Pentarix is capable of eliciting dramatic expansion of multi-genotype reactive, anti-E7 CD8 T cell immunity in mice when delivered in combination with adjuvants comprised of either the TLR3 agonist poly(I:C) or the TLR9 agonist CpG DNA. Furthermore, mice vaccinated with Pentarix are capable of fully regressing large, pre-established E7-expressing TC1 tumors. Together these data suggest that Pentarix may comprise a valuable therapeutic vaccine reagent in the setting of cervical cancer and/or pre-cancerous cervical neoplasia, as well as other HPV-associated cancers.

2. Materials and methods 2.1. Cloning and expression of recombinant Pentarix protein A single contiguous DNA comprising the complete E7 protein from each of HPV16, 18, 31, 45 and 52 plus an amino-terminal 6×HIS affinity TAG and thrombin cleavage site was produced as a synthetic DNA construct. The multi-gene sequence was subsequently cloned into the expression vector pET24a (Invitrogen) and full length protein (Pentarix) with a cleavable 6×HIS affinity TAG was expressed in E. coli genotype BL21 (DE3) pLysS. Briefly, 5 ml of LB media containing kanamycin (34 ␮g/ml) plus chloramphenicol (34 ␮g/ml) was inoculated with a single colony of recombinant E. coli and allowed to grow to saturation overnight. Five ml of saturated overnight culture was then used to inoculate 1000 ml of LB media containing kanamycin (34 ␮g/ml) plus chloramphenicol (34 ␮g/ml). When growth in the 1000 ml culture reached OD600 of between 0.2 and 0.4, IPTG was added to the culture to a final concentration of 2 mM and growth was continued for another 2–3 h to allow for expression of recombinant protein. Bacteria were subsequently pelleted by centrifugation and resuspended in 30 ml lysis buffer (20 mM sodium phosphate, pH 7.4, 500 mM sodium chloride, 10 mM imidazole, 6 M urea, 1 mM DTT). Bacteria were lysed by two successive cycles of freezing and thawing followed by sonication. Debris and insoluble protein were removed by centrifugation for 15 min at 15,000 rpm to render a urea soluble lysate solution. Pentarix protein was purified from urea soluble lysate by passage over an affinity column (HisTrap HP, GE Healthcare) using an AKTA column chromatography system. After extensive washing of the

column with lysis buffer, Pentarix protein was eluted from the column using lysis buffer containing 500 mM imidazole. Elution fractions containing Pentarix protein were then pooled and dialyzed against 4 changes of sterile, tissue culture grade phosphate buffered saline to render the final solution of Pentarix protein used for studies described herein. Protein expression and purification was monitored by running various in process and final fractions on SDS-PAGE and visualizing proteins via Coomassie Blue staining or Western blot using anti-6×HIS Tag antibody (ABM) or antiHPV16E7 antibody (Invitrogen). 2.2. Animals and reagents Eight to twelve week old female C57Bl/6 mice were obtained from Charles River Canada and were maintained under specific pathogen-free conditions. HLA-A2 transgenic mice (HLA-A2/Db [48]) were obtained from Jackson Labs (stock # 004191). Agematched animals were used in all experiments. All animal experiments were approved by the University of Victoria Animal Care Committee. Recombinant Pentarix protein was expressed as described above, aliquoted and stored at −20 ◦ C. Purified HPV16E7 protein with an amino-terminal 6×HIS tag was prepared by Genscript and used as a positive control for Western blotting. Poly(I:C) was purchased from GE Healthcare. CpG oligonucleotide 2395 was purchased from Invivogen. The HPV16 E749–57 peptide was obtained from Anaspec and the HPV31 E749–57 peptide was obtained from Genscript. A peptide library comprised of overlapping 15mers (overlapping by 11 a.a.) spanning each of the E7 proteins within Pentarix was purchased from Genscript. PE-conjugated H-2Db tetramer containing 16E749–57 was kindly provided by the Ludwig Institute for Cancer Research. Cells were analyzed on a BD FACSCalibur collecting a minimum of 50,000 events. 2.3. Immunizations Mice were immunized subcutaneously in the scruff of the neck with Pentarix protein admixed with poly(I:C) or CpG oligonucleotide, as indicated, combined in a total volume of 300 ␮l of PBS. Mice receiving multiple immunizations were immunized at approximately 24 h intervals. 2.4. IFN- ELISPOT analyses Mice were euthanized and spleens were excised at various times post-immunization as indicated. Single cell suspensions of splenocytes were prepared in 10 ml of cRPMI (RPMI 1640, 10% FBS, 2 mM l-glutamine, 50 ␮M 2-mercaptoethanol, 10 mM HEPES, 10 mM MEM non-essential amino acids, 10 mM sodium pyruvate and 50 ␮g/ml gentamicin) by mashing spleens through a 70 ␮M filter using the plunger from a 5 ml syringe followed by RBC depletion by ACK lysis. Where indicated, CD4 cells were depleted from bulk splenocytes using magnetic depletion. Briefly, bulk splenocytes were stained with PE-conjugated anti-CD4 antibody (clone L3T4; BD Biosciences) and labeled cells were depleted using antiPE microbeads according to manufacturer’s instructions (Miltenyi). ELISPOT plates (MSIP, Millipore) were pre-coated overnight with 10 ␮g/ml anti-IFN-␥ capture antibody (AN18-Mabtech) and then blocked for 2 h at 37 ◦ C with cRPMI. Splenocytes (3 × 105 cells per well) were plated in triplicate in the absence of any stimulus (media only) or in the presence of the indicated peptide (10 ␮g/ml). In those instances where spot density was too high for accurate automated counting, splenocytes from immunized mice were diluted 1:3 or 1:9 with splenocytes from normal naive mice, keeping the final total cell density at 3 × 105 cells per well. After overnight incubation at 37 ◦ C ELISPOT plates were washed and incubated

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for 2 h at 37 ◦ C with 1 ␮g/ml biotinylated anti-mouse IFN-␥ (mAb R4-6A2, Mabtech) followed by development with Vectastain ABC Elite kit and Vectastain AEC substrate reagent according to manufacturer’s instructions (Vector Labs). Plates were evaluated using a commercial ELISPOT evaluation service (ZellNet) and results are reported as the number of spot-forming cells (SFC) per 106 splenocytes.

2.5. TC-1 tumor challenge experiments TC-1 tumor cells were grown in cRPMI containing 0.4 mg/ml G418 to 60–80% confluency and were harvested by a brief exposure to 0.25% trypsin followed by neutralization with cRPMI. TC-1 tumor cells (1 × 105 per mouse) were implanted subcutaneously into the left flank of naive C57Bl/6 mice and tumor growth was monitored by measuring the tumor every two to three days using electronic calipers. Tumor volumes were calculated using the formula width2 × length × 0.5. Tumor-bearing mice were euthanized when the tumor volume exceeded 2000 mm3 according to the CCAC (Canadian Council on Animal Care) guidelines.

3. Results 3.1. Immunization with exogenous Pentarix protein plus the TLR3 agonist poly(I:C) or the TLR9 agonist CpG elicits robust HPV16 E7-specific CD8 T cell immunity Sequence alignment of the E7 proteins from five of the most prevalent high risk HPV genotypes (HPV16, 18, 35, 41 and 52) reveals that E7 proteins are rather poorly conserved across this group (Fig. 1A). Thus, specific targeting of HPV16 E7 via vaccination is unlikely to confer significant cross-reactive immunity against other high risk genotypes. To overcome this obstacle we have developed a novel multi-genotype therapeutic HPV vaccine called ‘Pentarix’, that is a recombinant protein-based vaccine comprised of single fusion protein encompassing the E7 proteins from HPV16, 18, 35, 41 and 52 (Fig. 1B and C). Recombinant Pentarix protein is produced in E. coli with an amino terminal 6×His tag and is purified using nickel-based affinity chromatography. The purified protein is fully soluble in PBS and migrates on SDS-PAGE gel in accordance with its predicted molecular weight of 59,037 Da (Fig. 1C). Mice immunized with a single dose of whole exogenous Pentarix protein admixed with an adjuvant comprised of either poly(I:C) (TLR3 agonist) or CpG DNA (TLR9 agonist) mounted a robust CD8 T cell response against the well-characterized H-2Db -restricted epitope HPV16 E749–57 as measured at 7 days post-immunization by IFN-␥ ELISPOT (Fig. 2). We also assessed the effector function of CD8 T cells elicited by a single immunization with Pentarix protein plus these adjuvants in terms of their ability to regress established, E7-expressing tumors. HPV16 E7-expressing TC-1 tumors were implanted subcutaneously in naive recipient mice and were allowed to grow until they reached a volume of approximately 200 mm3 . Animals immunized subcutaneously with Pentarix protein admixed with either poly(I:C) or CpG oligonucleotide began to regress these tumors, generally within one week of immunization (Fig. 3). Indeed all animals immunized with Pentarix protein plus adjuvant had complete tumor regression by three weeks post-immunization and remained tumor free for at least three months. In contrast, mice that were either untreated or that were treated with adjuvant only or Pentarix protein only displayed progressive tumor growth and were euthanized (generally within 28 days of tumor implantation) due to excessive tumor burden.

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3.2. Repeated daily immunization with exogenous Pentarix protein plus the TLR3 agonist poly(I:C) results in dramatic enhancement of CD8 T cell immunity Although significant numbers of antigen-specific CD8 T cells were elicited after a single immunization with Pentarix plus poly(I:C) or CpG, we also explored whether the strength of the response could be augmented through the use of homologous prime-boosting. In particular, we recently demonstrated that repeated daily ‘cluster’ immunization with soluble exogenous OVA protein combined with poly(I:C) results in profound expansion of effector CD8 T cells [47]. As was previously observed with OVA protein, we found that immunization with whole exogenous Pentarix protein plus poly(I:C) for four consecutive days evoked a marked increase in the frequency of HPV16 E749–57 -specific T cells compared to mice receiving a single immunization (Fig. 4A). Indeed, in mice receiving 4 successive doses of Pentarix protein plus poly(I:C) up to 11% of CD8 T cells in peripheral blood and up to 22% of CD8 T cells in the spleen stained positively with H-2Db HPV16 E749–57 tetramer (Fig. 4B). In contrast, no HPV-specific T cells were detectable in the spleens of mice immunized with 4 doses of Pentarix protein only, confirming a requirement for adjuvant for CD8 T cell expansion. Importantly, the very high level of E7-specific CD8 T cells evoked by cluster vaccination also conferred an improved ability to regress E7-expresing TC1 tumors, which was evident when tumors were allowed to grow to a larger size than normal prior to the initiation of treatment (Fig. 4C). Mice that harbored tumors with an average volume of 350 mm3 at time of treatment were immunized with either a single dose or 4 successive daily doses of Pentarix plus poly(I:C). Five of 8 mice receiving a single dose of vaccine exhibited transient (but incomplete) tumor regression and significantly improved time of survival compared to untreated mice or mice treated with poly(I:C) only. However, all mice receiving a single dose of vaccine eventually succumbed to progressive tumor growth. In contrast, of mice that received 4 successive doses of Pentarix plus poly(I:C), 100% (8 of 8) fully regressed these large tumors to the point that they were no longer palpable. Although some tumors began to recur 4–5 weeks after treatment, 75% of mice (6 of 8) in the 4-dose cohort were still alive at day 38 and 50% remained tumor free. All mice in all other cohorts had been euthanized due to progressive tumor growth by this time point. 3.3. Identification of novel CD8 epitopes of Pentarix in C57Bl/6 mice Because there is a paucity of information regarding the identity of E7 CD8 T cell epitopes for HPV genotypes other than HPV16, we also took advantage of the very high levels of immunity elicited by cluster vaccination to assess the scope of the cellular immune response elicited by Pentarix. Bulk splenocytes and CD4-depleted splenocytes from mice immunized with Pentarix plus poly(I:C) were analyzed directly ex vivo by ELISPOT with a library of overlapping 15mer peptides that spanned the entire Pentarix protein sequence. As shown in Fig. 5A, the response elicited in C57Bl/6 mice by Pentarix encompassed all five of the HPV strains contained within the vaccine. Peptides containing the well-characterized H2Db -restricted epitope HPV16 E749–57 (RAHYNIVTF) comprised the strongest response in terms of absolute numbers of antigen-specific CD8 T cells. The next strongest response was elicited by 15mer peptides from HPV31 encompassing a related peptide (TSNYNIVTF) that is predicted by algorithm analyses to be an even stronger binder to H-2Db than HPV16 E749–57 (data not shown). A variety of 15mer peptides from other HPV E7 protein sequences also elicited responses of varying intensity from both bulk and CD4-depleted splenocytes, confirming that Pentarix is capable of eliciting a broad scope cellular immune response, even in inbred mice with a limited

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Fig. 1. (A) Alignment of the amino acid sequences of the E7 proteins from the high risk HPV genotypes HPV16, 18, 31, 45 and 52. Positions that are identical across 3, 4 or 5 genotypes are indicated by increasing levels of shading. (B) Schematic representation of the Pentarix protein showing the position of the E7 proteins from HPV16, 18, 31, 45 and 52 as well as the amino-terminal Tag containing a 6×His affinity tag and a thrombin cleavage site. (C) Expression of recombinant Pentarix protein in E. coli and example of a typical purification using nickel affinity purification. Total protein contained within a lysate of IPTG-induced E. coli before and after passage over a HisTrap column (GE Healthcare) and protein eluted from the column (Fr # 1–12) in the presence of increasing concentrations of imidazole were detected by Coommassie Blue staining (left panel). Identity of purified Pentarix protein was confirmed by Western blot analysis of lysates from un-induced and induced cultures as well as purified Pentarix and HPV16 E7 proteins using anti-6×His tag antibody (middle panel) or anti-HPV16E7 antibody (right panel).

Fig. 2. E7-specific CD8 T cell responses are rapidly elicited after a single injection with Pentarix protein admixed with either TLR3 or TLR9 agonists. Naive C57Bl/6 mice were immunized (s.c.) with 100 ␮g Pentarix protein combined with 10 ␮g poly(I:C) or 10 ␮g poly(I:C) only (left panel) or 100 ␮g Pentarix protein combined with 10 ␮g CpG oligonucleotide or 10 ␮g CpG oligonucleotide only (right panel). Seven days post-immunization mice were euthanized and bulk splenocyte preparations were assessed by IFN-␥ ELISPOT. Briefly, splenocytes (3 × 105 per well, triplicate wells per condition) from individual animals were stimulated overnight with either media alone or with HPV16 E749–57 peptide (10 ␮g/ml) or irrelevant control peptide (KAVYNFATM). Results from naive (unimmunized) mice are included for comparison. Results are reported as the number of IFN-␥ spot-forming cells per 1 × 106 splenocytes ± SD for each triplicate. The data is representative of three experiments.

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Fig. 3. Immunization with whole, soluble Pentarix protein combined with poly(I:C) or CpG oligonucleotide induces regression of established TC-1 tumors. Naive C57Bl/6 mice (indicated number of mice per cohort) were implanted subcutaneously with 1 × 105 E7-expressing TC-1 tumor cells. Once tumors reached an average volume of 200 mm3 mice were treated (as indicated) with either a single dose of Pentarix (100 ␮g) plus poly(I:C) (10 ␮g), a single dose of Pentarix (100 ␮g) plus CpG oligonucleotide (10 ␮g) or poly(I:C) (10 ␮g), CpG oligonucleotide (10 ␮g) or Pentarix protein (100 ␮g) alone or were left untreated. Tumors were measured every 2 to 4 days with electronic calipers, and data are presented as tumor volume over time for individual animals within each cohort (upper 6 panels) or as survival for all mice within a cohort (lower 2 panels). Tumor-bearing mice were euthanized when the tumor volume exceeded approximately 2000 mm3 or when mice became moribund or lost >20% body weight). p values were calculated using the log rank (Mantel–Cox) test.

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Fig. 4. Sequential daily immunization with whole, soluble Pentarix protein plus poly(I:C) boosts CD8 T cell immunity and the ability to rapidly regress very large established TC1 tumors. (A) Naive C57Bl/6 mice (3 per cohort) were left untreated (naive) or were immunized (s.c.) either once or daily for 4 successive days with 100 ␮g Pentarix protein combined with 10 ␮g poly(I:C). Eight days post-immunization mice were euthanized and bulk splenocyte preparations were assessed by IFN-␥ ELISPOT. Splenocytes (3 × 105 per well, triplicate wells per condition) from individual animals were stimulated overnight with either media alone or with HPV16 E749–57 peptide (10 ␮g/ml) or irrelevant control peptide (KAVYNFATM). Results are reported as the number of IFN-␥ spot-forming cells per 1 × 106 splenocytes ± SD for each triplicate. (B) Lymphocytes in spleen and peripheral blood of a mouse that was immunized for 4 successive days with 100 ␮g Pentarix protein plus 10 ␮g poly(I:C) (left two panels) or 100 ␮g Pentarix protein only (right panel) were stained with FITC-conjugated anti-CD8 and PE-conjugated Db /16 E749–57 tetramer and analyzed by flow cytometry. Events shown are gated on CD8 lymphocytes and are representative of 4 such animals. (C) Naive C57Bl/6 mice (8 per cohort) were implanted subcutaneously with 1 × 105 E7-expressing TC-1 tumors cells. Once tumors reached an average volume of 350 mm3 mice were treated with either a single dose of Pentarix (100 ␮g) plus poly(I:C) (10 ␮g), 4 successive daily doses of Pentarix (100 ␮g) plus poly(I:C) (10 ␮g), 4 successive daily doses of poly(I:C) only (10 ␮g per dose) or were left untreated. Tumors were measured every 2–4 days with electronic calipers and tumor-bearing mice were euthanized when the tumor volume exceeded approximately 2000 mm3 or when mice became moribund or lost >20% body weight. Data are presented as average tumor volume for all mice within a cohort (left panel) or survival (right panel).

repertoire of MHC molecules. In addition, HLA-A2 transgenic mice (HLA-A2/Db , [48]) were also immunized with Pentarix plus poly(I:C) and assessed by ELISPOT using the same library of overlapping 15mer peptides. Interestingly, although the general strength of the response was greater in C57Bl/6 than HLA-A2/Db mice, the overall complexity of the response was very similar (Fig. 5B), suggesting that an H2b -restricted response had been elicited but that an HLA-A2 restricted response had not. Furthermore, as has been observed in a number of other studies using HPV16 E7 antigen

[49–51], we were unable to detect a response against the HLA-A2 restricted 11–20 or 86–93 minimal peptide epitopes of HPV16 E7 in mice immunized with Pentarix plus poly(I:C) (data not shown). To confirm that TSNYNIVTF was the precise minimal epitope within the strongly reactive HPV31 15mer peptides (AEPDTSNYNIVTFCC and TSNYNIVTFCCQCKS) splenocytes from mice immunized with Pentarix plus poly(I:C) were assessed by ELISPOT and were found to be responsive to this 9mer minimal peptide (Fig. 5C).

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Fig. 5. Immunization with whole, soluble Pentarix protein plus poly(I:C) elicits immune responses against multiple genotypes of HPV. C57Bl/6 (A) or HLA-A2/Db transgenic mice (B) were immunized (s.c.) daily for 4 successive days with 100 ␮g Pentarix protein combined with 10 ␮g poly(I:C). Eight days post-immunization mice were euthanized and bulk (A and B) or CD4-depleted splenocyte preparations (A only) were analyzed by IFN-␥ ELISPOT (CD4 depletion was >99% as measured by FACS analysis post-depletion). Bulk and CD4-depleted splenocyte preparations were stimulated overnight with a panel of overlapping 15mer peptides (overlapping by 11 amino acids) that spanned the entire Pentarix protein. (C) Splenocytes from C57Bl/6 mice immunized (s.c.) with Pentarix protein combined with 10 ␮g poly(I:C) were stimulated overnight with either media alone or with the minimal peptide epitopes HPV16 E749–57, HPV31 E749–57 or irrelevant control peptide (KAVYNFATM) and analyzed by IFN-␥ ELISPOT. Results are reported as the number of IFN-␥ spot-forming cells per 1 × 106 splenocytes and are representative of 3 such experiments.

4. Discussion In the current study, we report a novel therapeutic vaccine candidate directed against 5 high risk genotypes of HPV. When combined with a recently developed CD8-promoting cluster immunization strategy [47], this vaccine is capable of eliciting powerful and broad scope anti-HPV CD8 T cell immunity with potent therapeutic effects against E7-expressing tumor cells. Because the E6 and E7 proteins of high risk HPV are often the only viral genes that

continue to be expressed in cancerous cells [4] they represent highly rational targets for immune therapy of HPV-associated malignancy. In particular, significant effort has been focused upon methodologies for inducing E7-specific CD8 T cell immunity. Traditionally, peptides comprising minimal MHC class I epitopes have been considered the simplest and most convenient approach for eliciting CD8 T cell responses. However, immunization against minimal peptide epitopes is not an optimal strategy as their use is limited to individuals expressing the appropriate HLA allele and

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it is relatively easy for an infectious agent to escape an immune response that is directed towards a single epitope. Whole antigens or even long synthetic peptides may bypass these constraints and thus represent superior alternatives for vaccines designed to elicit CD8 immunity. Although vaccination with whole exogenous protein antigen is not generally considered to be an efficient means of eliciting MHC class I-restricted CD8 T cell responses [46], immunization with recombinant [52] or synthetic [53] full length E7 protein has been reported to elicit CD8 T cell immunity when delivered in combination with either QuilA or CpG-containing oligonucleotides, respectively. Likewise, various fusion protein approaches have been reported to drive CD8 immunity to E7 including fusion to heat shock proteins [54–56], E7-containing virus like particles [57,58], E7/listeriolysin fusions [34,59], or fusions between E7 and various immunomodulatory or intracellular trafficking domains [30,60,61]. In addition, a variety of DNA and viral vector approaches that result in the direct loading of antigens into the MHC class I processing pathway have been explored. However, in the latter two approaches there has been concern about the safety of introducing E7-encoding genes into host cells as E7 is itself an oncogene. If E7-encoding plasmid or virus was to somehow become stably integrated into the host genome then this would potentially provide a perpetual source of E7 protein. In contrast, delivery of E7 protein in extracellular protein form is anticipated to have no transforming capability. This is because (1) extracellular protein is not expected to have ready access to the interior of cells where it could have oncogenic potential, (2) because the vaccine is delivered in protein form, the E7 protein will present only transiently, and transformation of HPV-infected cells is dependent upon the continuous expression of E6 and E7 (i.e. transformation does not occur via a single ‘hit’ type mechanism). Together these factors suggest that delivery of any E7 protein-based vaccine, including Pentarix, presents very little risk in terms of potential E7-associated oncogenicity. Recently, adjuvants comprised of TLR3 and TLR9 agonists have been shown to be particularly efficacious in terms of promoting the cross-presentation of whole exogenous protein antigens into the MHC class I pathway and eliciting strong CD8 immune responses [47,62–65]. Herein, we describe an immunization approach that elicits strong CD8 T cell responses against the E7 antigen by immunization with whole exogenous recombinant protein and the TLR3 agonist poly(I:C) or the TLR9 agonist CpG. Moreover, although readily detectable numbers of E7-specific CD8 T cells are elicited after a single immunization with Pentarix plus poly(I:C) or Pentarix plus CpG, this number can be dramatically increased by repeated daily homologous prime-boost immunization. Although both poly(I:C) and CpG were effective as adjuvants using the cluster vaccination strategy (data not shown for CpG), we noted that mice receiving multiple consecutive doses of CpG adjuvant, either alone or in combination with protein antigen, occasionally presented with profound splenomegaly at time of euthanasia. TLR9-induced splenomegaly has been noted by other groups [66–68] and although it is reported to be a transient phenomenon, we have chosen to focus upon poly(I:C) as a preferred adjuvant as it does not elicit any evidence of splenomegaly. We also demonstrate that the strong E7-specific immune responses elicited by cluster vaccination with Pentarix plus poly(I:C) are capable of driving complete regression of large, established E7-expressing TC-1 tumors, at a time when they are normally fatal to mice within days. This finding suggests that the massive expansion of HPVspecific T cells achieved through repeat dose-immunization may be capable of overcoming the various immuno-inhibitory mechanisms that tumors utilize, including production of inhibitory cytokines or induction of regulatory T cells (for review see [69]). As such, we anticipate that the Pentarix vaccine used in conjunction with the CD8-inducing cluster vaccination strategy [47] could have

broad applicability for the treatment of patients in a variety of HPV disease settings. Indeed, the potential clinical applicability of Pentarix as a therapeutic vaccine is supported by recent success in the treatment of HPV16+ vulvar intraepithelial neoplasia using a long synthetic peptide-based vaccine [70]. Retrospective analysis of responders in this trial revealed that tumor regression correlated with the development of vaccine-induced effector T cell responses [71] demonstrating that HPV-induced tumors are amenable to immunotherapeutic treatment. Unfortunately, until now, most therapeutic HPV vaccines have targeted one or at most two genotypes of HPV, thereby potentially ‘missing’ a large proportion of the ‘at risk’ population. Indeed, a number of studies are beginning to reveal that in many cases of advanced cervical dysplasia [72], and even in cervical cancer [73] there are multiple genotypes of high risk HPV present in the infected tissue. Obviously, targeting a single genotype of HPV in this scenario would likely be ineffective as a therapeutic approach. Our studies in mice reveal that Pentarix is capable of eliciting broad immunity against the E7 proteins of multiple genotypes of HPV. Indeed we have identified a novel class I-restricted epitope from HPV31 E749–57 (TSNYNIVTF) and are in the process of characterizing additional epitopes that are contained within the 15mer peptides identified as immunoreactive in immunized C57Bl/6 mice using ELISPOT analysis. Interestingly, in our study, immunization of HLA-A2 transgenic mice (HLA-A2/Db ) failed to elicit a cellular immune response to ‘known’ HLA-A2-restricted epitopes of HPV16E7. However, this is consistent with previous results showing that HLA-A2-restricted epitopes of HPV16E7 are immunogenic in HLA-A2 transgenic mice when delivered in the form of minimal peptides, but that they may not be properly processed by the murine MHC class I processing machinery when delivered in longer forms [49–51]. Alternatively, the lack of a detectable HLA-A2-directed response may be partially due to the overwhelming strength of the response against the two strongest epitopes RAHYNIVTF and TSNYNIVTF. Indeed the response against RAHYNIVTF is so strong that it occupies greater than 22% of the entire CD8 compartment as measured by tetramer analysis. A single response of this magnitude may be so immunodominant that it simply out-competes less immunogenic epitopes. This scenario would be consistent with prior work showing that in a DNA immunization setting, HLA-A2 transgenic mice respond robustly to the H-2Db -restricted epitope RAHYNIVTF but fail to respond to HLAA2-specific epitopes unless the RAHYNIVTF epitope is deleted [50]. Likewise a single response of this magnitude may actually pose a technical hurdle to the detection of more subtle immune responses as there is limitation to the number of cells that can be loaded into each well of an ELISPOT assay plate. In future studies, immunization of HLA transgenic mice lacking in endogenous class I expression (HHD mice) with Pentarix may help to identify new human epitopes of the E7 proteins from these high risk HPV genotypes, an area for which there is currently little information available. However, the true extent of multi-species reactivity that can be provided by Pentarix will ultimately only be known through clinical trials wherein the spectrum of responses would likely be highly dependent upon the HLA haplotype of study participants. Lastly we would like to emphasize that although current prophylactic vaccines are predicted to have a major impact on HPVassociated malignancies in the future, their full effect is not likely to be realized for many years [7,74]. Thus there continues to be an urgent need for effective therapeutic approaches for treating HPVassociated cancers and precancerous dysplasias. Despite a large number of clinical trials testing various therapeutic HPV candidates, there is currently no vaccine approved for use in this setting. Pentarix represents a novel protein-based vaccine that is simple to produce, highly effective in preclinical studies and with the potential to confer protective immunity against multiple high risk types

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