Therapeutic hpv16 vaccines

The present invention relates to the field of drugs, and more particularly to nucleic acid constructs and polypeptides that can be used in therapeutic vaccines against human papillomavirus type 16.

The human papillomavirus (HPV) family consists of over 100 types (also called subtypes) that can infect skin or mucosal keratinocytes. Over 40 types of HPV are typically transmitted through sexual intercourse, and HPV infection in the anogenital area is extremely common in both men and women. Some sexually infectious HPV types can cause genital warts. Persistent infections with "high-risk" HPV types (eg, types 16, 18, 31, 45) (different from those that cause skin warts) include, for example, cervix, vulva, vagina, penis, oropharyngeal and It can progress to precancerous lesions of the anus and invasive cancer. Most HPV infections are voluntarily cleared within 1-2 years of infection. In healthy individuals, circulating Th1 and Th2 type CD4 + T cells specific for the early viral proteins E2, E6 and E7 of HPV-16, and E6 specific CD8 + T cells migrate to the skin during antigen loading. , This indicates that effective defense against HPV-16 infection is generally associated with systemic effector T cell responses to these early viral antigens. In a minority of infected individuals (about 1%), HPV infection persists, eventually resulting in genital neoplastic lesions. HPV16 and HPV18 among high-risk HPVs are the leading causes of cervical cancer and both cause about 70% of cases, and these two types also have other HPVs such as anal and oropharyngeal cancers. It plays a major role in induced cancer. Globally, HPV is one of the most important infectious agents that cause cancer.

Vaccination against HPV appears to be a method capable of reducing the incidence or efficacy of HPV infection (van der Burg and Melief, 2011, Curr Opinion Immunol 23: 252-257).

Preventive HPV vaccines based on virus-like particles (VLPs) formed by HPV 16 and 18 (enveloped) proteins L1 are extremely efficient in preventing persistent infections and related diseases with HPV 16 and HPV 18. These vaccines are thought to provide sterile immunity through the induction of neutralizing antibodies against the L1 protein. The addition of L1-based VLPs from additional high-risk HPV types may further enhance the broad protection provided by such vaccines.

However, while such vaccines can prevent early infections (ie, they provide prophylaxis), they are therapeutic vaccines against HPV, as there is no evidence of a beneficial effect on the established genital lesions caused by HPV16 and HPV18. Not considered (Hildesheim et al., 2007, JAMA 298: 743-53).

Despite the introduction of these prophylactic vaccines, many people are already at or at additional risk of having a persistent high-risk HPV infection and are therefore at risk of developing cancer. Therapeutic vaccines for the eradication of established HPV infections and related diseases are an urgent medical demand that has not yet been addressed.

Some attempts to meet this demand have been described. For example, a variety of different vaccination methods, eg, a fusion protein consisting of heat shock protein (Hsp) and HPV-16 E7 from Mycobacterium bovis or E6 from HPV-16 and HPV-18, Fusion protein consisting of E7 and L2, chimeric L1-E7 VLP, HPV-16 and HPV-18 E6 and E7 or recombinant vaccinia virus, HPV expressing either bovine papillomavirus E2 DNA vaccines expressing the E6 and E7 CTL epitopes of -16 and HPV-18, attenuated live Listeria monocytogenes (Lm) secreting the HPV-16 E7 antigen, and HPV-16 Clinical trials are being conducted with synthetic long peptides (SLPs) containing E6 and E7 peptides. While some, but not limited to, some of these methods have shown clinical efficacy, most have been unsuccessful, indicating that existing methods need improvement.

Integration of the early HPV proteins E6 and E7 is a necessary step in the process from infection to cancer and requires sustained expression of E6 and E7 to maintain the neoplastic phenotype of cervical cancer cells. .. Therefore, E6 and E7 are considered good targets for therapeutic vaccination. As mentioned earlier, some studies have shown that therapeutic vaccination of women infected with high-risk HPV can induce regression of existing lesions. Using SLPs and adjuvants derived from HPV16 E6 and E7 proteins as therapeutic vaccines, Kenter et al. Showed persistent and complete regression in 47% of patients with vulvar intraepithelial neoplasia (VIN) (Kenter et al). ., 2009, N Engl J Med 361: 1838-47). Similarly, a study combining a protein-based vaccine (consisting of a fusion protein of TA-CIN, HPV16 E6, E7 and L2) with local immunomodulation in 2/3 of patients with VIN was complete in 63% of patients. Retraction was shown (Daayana et al., 2010, Br J Cancer 102: 1129-36). Possible drawbacks of synthetic long peptides as vaccines include large-scale manufacturability and associated costs, demand for potentially reactive adjuvants and associated immune-related adverse effects (particularly pain and swelling). Can be mentioned. SLP is unlikely to be used in early-stage disease if the rate of spontaneous clearance is still high due to high levels of discomfort. Similarly, due to the demand for topical imiquimod treatment in the case of TA-CIN treatment, the majority of women experience topical and systemic side effects that persist over the duration of imiquimod treatment, which is part of their daily activities. Tolerability is an important issue as it can have an impact.

A possible alternative is to use nucleic acid-based vaccinations such as DNA vaccines or viral vaccines that encode the HPV E6 and / or E7 proteins for vaccination.

However, HPV E6 and E7 proteins have carcinogenic potential, and therefore vaccination with vaccines containing nucleic acids encoding these proteins results in cell transformation due to the potential for sustained expression of the antigen. Raise the risk of inducing.

Therefore, in the case of gene vaccination, non-carcinogenic / detoxified versions of E6 and / or E7 can be used to eliminate any risk of cell transformation due to vaccination. Reduced carcinogenic potential of wild-type E6 and E7 is generally achievable by deletion and / or substitution of residues known to be important for the function of these proteins (eg, Smahel et al. , 2001, Virology 281: 231-38; Yan et al., 2009, Vaccine 27: 431-40; Wieking et al., 2012, Cancer Gene Ther 19: 667-74; International Publication No. 2009/106362). However, the disadvantage of these approaches is that they carry the risk of removing important T cell epitopes from the protein and / or introducing new and unwanted T cell epitopes into the protein, and are therefore desired. It may not result in an immune response.

Other methods for eliminating the carcinogenic potential of HPV16 E6 and E7 have been to construct mixed versions of the E6 and E7 proteins (ie, re-ordered polypeptides of wild-type protein fragments). (For example, Oelschlaeger et al., 2006, Vaccine 24: 2880-93; Oosterhuis et al., 2011, Int J Cancer 129: 397-406; Oosterhuis et al., 2012, Hum Gen Ther 23: 1301-12). However, these approaches require additional preparation, formulation, and administration of multiple molecules to ensure inclusion of all possible epitopes of both E6 and E7 proteins, resulting in suboptimal logistics and relatively high costs. Bringing, the method described further introduces potentially potent unnatural epitopes that are not present in E6 and E7, and the immune response can be diverted from the associated E6 / E7 epitope to such unnatural epitope. Therefore, the constructs described may not have optimal immunological properties.

<p> Therefore, there remains a demand in the art for therapeutic vaccines against HPV, preferably the methods described above, which have fewer drawbacks.</p>

<p> The present invention contains fragments of the E6 and E7 proteins that have been rearranged despite containing essentially all possible T cell epitopes of the HPV16 neoplastic proteins E6 and E7, while at the same time unwanted neoepitope. By having a minimum number, provides a nucleic acid molecule encoding a polypeptide having significantly reduced (compared to wild-type E6 and E7) undetectable transforming activity. This contrasts with the molecules previously presented by others.</p><p> The present invention provides a nucleic acid molecule encoding a polypeptide comprising a sequence as set forth in SEQ ID NO: 1.</p><p> The coding polypeptide may further comprise a leader sequence.</p><p> In certain embodiments, the coding polypeptide further comprises at least one epitope of a human papillomavirus (HPV) E2 protein, such as the HPV16 E2 protein. The E2 protein may be mutated, for example by deletion or mutation within its DNA binding domain, to reduce DNA binding. In certain embodiments, the coding polypeptide comprises a sequence as set forth in SEQ ID NO: 3 or SEQ ID NO: 5.</p><p> In certain embodiments, the nucleic acid sequence is codon-optimized, for example for expression in human cells.</p><p> In certain embodiments, the nucleic acid sequence comprises a sequence as set forth in SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6.</p><p> The invention also provides a vector comprising a nucleic acid molecule according to the invention, wherein the sequence encoding the polypeptide is operably linked to a promoter.</p><p> In certain embodiments, the vector is a DNA vector, such as a plasmid. In other embodiments, the vector is a viral vector, such as an MVA vector or a recombinant adenovirus vector. In certain preferred embodiments, the vector is recombinant adenovirus.</p><p> In certain embodiments, the promoter in the vector is operably linked to the repressor operator sequence and the repressor protein binds to it in order to suppress expression of the promoter in the presence of the repressor protein. Can be done. In certain embodiments, the repressor operator sequence is a TetO sequence or a CuO sequence.</p><p> The invention also provides a vaccine composition comprising a vector according to the invention and a pharmaceutically acceptable excipient.</p><p> The present invention also provides a method of inducing an immune response against HPV, particularly HPV16, in a subject, comprising the step of administering to the subject a vaccine composition according to the invention. The present invention also provides a vaccine according to the present invention intended for use in inducing an immune response against HPV, particularly HPV16.</p><p> In certain embodiments, the vaccine is administered to the subject more than once.</p><p> The present invention also presents persistent HPV infection (particularly persistent HPV16 infection), genital intraepithelial neoplasia (VIN), cervical intraepithelial neoplasia (CIN), vaginal intraepithelial neoplasia (VaIN), anal intraepithelial neoplasia (in particular) in subjects. AIN), cervical cancer (cervical flat epithelial cancer (SCC), nasopharyngeal cancer, penis cancer, vaginal cancer or anal cancer, etc. A method comprising administering to a subject a vaccine according to the invention is provided. The invention also provides persistent HPV infection (particularly persistent HPV16 infection), vulvar intraepithelial neoplasia (VIN), cervical intraepithelial neoplasia in the subject. (CIN), Vaginal intraepithelial neoplasia (VaIN), Anal intraepithelial neoplasia (AIN), Cervical cancer (cervical flat epithelial cancer (SCC), etc., nasopharyngeal cancer, penis cancer, vaginal cancer or Provided is a vaccine according to the present invention intended for use in the treatment of any of the anal cancers.</p><p> The present invention also provides polypeptides comprising the sequences set forth in SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 5. The present invention also provides:</p><p>[1] A nucleic acid molecule encoding a polypeptide comprising SEQ ID NO: 1.</p><p>[2] The nucleic acid molecule according to [1], wherein the coding polypeptide further comprises a leader sequence.</p><p>[3] The nucleic acid molecule according to [1] or [2], wherein the coding polypeptide further comprises at least one epitope of the human papillomavirus (HPV) E2 protein.</p><p>[4] The nucleic acid molecule according to [3], wherein the coding polypeptide comprises an HPV16 E2 protein having a deletion or mutation in the DNA binding domain.</p><p>[5] The nucleic acid molecule according to [4], wherein the coding polypeptide comprises SEQ ID NO: 3 or SEQ ID NO: 5.</p><p>[6] The nucleic acid molecule according to any one of [1] to [5], which is codon-optimized.</p><p>[7] The nucleic acid molecule according to any one of [1] to [6], which comprises SEQ ID NO: 2.</p><p>[8] The nucleic acid molecule according to any one of [1] to [7], which comprises SEQ ID NO: 4 or SEQ ID NO: 6.</p><p>[9] The vector containing the nucleic acid molecule according to any one of [1] to [8], wherein the sequence encoding the polypeptide is operably linked to a promoter.</p><p>[10] The vector according to [9], which is a recombinant adenovirus.</p><p>[11] The promoter is operably linked to the repressor operator sequence, and the repressor protein can bind to it to suppress expression of the promoter in the presence of the repressor protein, [11] The vector according to 9] or [10].</p><p>[12] A vaccine composition comprising the vector according to any one of [9] to [11] and a pharmaceutically acceptable excipient.</p><p>[13] A method of inducing an immune response against HPV in a subject, comprising the step of administering to the subject the vaccine composition according to [12].</p><p>[14] The method according to [13], comprising the step of administering the vaccine according to [12] to the subject more than once.</p><p>[15] Persistent HPV infection, genital intraepithelial neoplasia (VIN), cervical intraepithelial neoplasia (CIN), vaginal intraepithelial neoplasia (VaIN), anal intraepithelial neoplasia (AIN), cervical cancer ( A method for treating cervical flat epithelial cancer (SCC), nasopharyngeal cancer, penis cancer, vaginal cancer or anal cancer, and the vaccine described in [12] is applied to the subject. A method comprising the step of administering.</p><p>[16] A polypeptide comprising SEQ ID NO: 1.</p><p>[17] The polypeptide according to [16], comprising SEQ ID NO: 3 or SEQ ID NO: 5.</p>

<figref num="1">Expression of fusion proteins of HPV16 E6 and E7. A DNA vector expressing the transgene shown in the upper part of the figure was transiently introduced into HEK-293T cells. Twenty-four hours after gene transfer, cells were collected and cell extracts analyzed by SDS-PAGE and Western blotting with antibodies against HPV16 E7 (upper panel). A loading control showing NF-kB (bottom panel) confirms a similar loading of cell solubilized products in all lanes. The molecular weight marker is shown on the left. The expected size of the fusion protein is about 38 kDa for E6E7SH; about 75 kDa for E2E6E7SH and E6E7E2SH, and about 78 kDa for LSE2E6E7SH.</figref><figref num="2">Colonization in soft agar. A) Schematic diagram of how the soft agar assay works. B) Representative microscopic images of cells in agar at 40x magnification 6 weeks after sowing. White arrows highlight the colonies found in E7wt transgenic cells. C) Quantification of colonies in agar, 6 weeks after sowing, using Gelcount and related software.^*: p &lt;0.05 (Poisson regression model);^**: Non-inferior (generalized linear model with 5% non-inferiority margin).</figref><figref num="3">E6E7SH has lost E6 and E7 activity. A) Representative Western blot showing lack of p53 degradation by E6E7SH. A plasmid expressing p53 was co-transfected into human p53 null NCI-H1299 cells in combination with a plasmid expressing HPV16 E6 wild-type, E6 E7SH or empty vector. Non-TF indicates non-transgenetic cells. Twenty-four hours after gene transfer, cell solubilized products were prepared and 30 μg of total protein was loaded onto the gel. Upper panel-p53 stain, intermediate panel-E6 stain, lower panel-NF-kB stain (loading control). (B) Quantification of p53 levels in four independent assays. The p53 signal was normalized to the NF-κB signal. C) Western blot showing lack of pRb degradation by E6E7SH. HPV16 is a plasmid that expresses pRb in pRb null Saos-2 cells. The gene was introduced in combination with a plasmid expressing the E7 wild type, E6E7SH or empty vector. Non-TF indicates non-transgenetic cells. Twenty-four hours after gene transfer, cell solubilized products were prepared and 10 μg of total protein was loaded onto the gel. Upper panel-pRb stain, intermediate panel-E7 stain, lower panel-NF-κB stain (loading control). D) Quantification of pRb levels in four independent assays. The pRb signal was normalized to the NF-κB signal.^*: p &lt;0.05 (ANOVA model);^**: Non-inferiority (study was based on 95% CI from ANOVA model; non-inferiority margin was set to 75%).</figref><figref num="4">E6E7SH does not immortalize primary human epithelial keratinocytes. Primary human epithelial keratinocytes were transduced with an open reading frame (E6E7wt) encoding wild-type E6 and E7 of HPV16, a lentivirus encoding either the E6E7SH sequence or eGFP. Non-transduced donor cells were used as controls. At about 200 days, only E6E7wt expression induces (not described) primary keratinocyte immortalization, as indicated by longevity extension and hTERT activation. The cross symbol indicates that the cells died due to aging and could not be further cultured. See Example 2 for details. Similar results were obtained with two additional donors (not listed).</figref><figref num="5">Immune response induced by E6E7SH after DNA immunization-IFNγ ELISPOT analysis. A. Immune scheme. CB6F1 mice were immunized with a DNA plasmid expressing E6E7SH or a plasmid not expressing the transgene (control). Two weeks after immunization, mice were sacrificed and isolated splenocytes were stimulated overnight in an E7-corresponding 15mer peptide pool. B. E7-specific immune response in individual mice, as measured by the IFNγ ELISPOT assay, 10⁶It is given as a spot-forming unit (SFU) per splenocytes.</figref><figref num="6">Immunogenicity of E6E7SH-IFNγ ELISPOT analysis. (A) Immune scheme. Mice were immunized with an adenovector with the inserts as specified. E7-specific responses after 2 weeks (B) and 8 weeks (C) were analyzed by IFNγ ELISPOT (10).⁶Represented as a spot-forming unit (SFU) per splenocytes). Black circles are 1x10¹⁰Represents mice immunized at a dose of vp, white circles are 5x10⁹Represents a mouse immunized with vp. The black bar represents the geometric mean of the response. The dotted line indicates the lower limit of detection in the ELISPOT assay. ANOVA Post-hoc Bonferroni statistical analysis was performed on the log conversion data (ANOVA Post-hoc Bonferroni statistical analysis).^*: p &lt;0.05). See Example 3 for details.</figref><figref num="7">Immunogenicity of E2E6E7SH-E7 tetramer staining. (A) Immune scheme. 1 × 10 in which CB6F1 mice express the transgene as specified¹⁰Immunized with vp adenovector. Two weeks after immunization, the mice were sacrificed and isolated splenocytes were released at E7._49-57The presence of CD8 + cells capable of interacting with the -H2-Db tetramer was analyzed. (B) Percentages of E7 tetramer-positive CD8 + T cells are shown on the y-axis. Post-ANOVA Bonferroni statistical analysis was performed on the log conversion data and the differences between the different E6E7SH variants were not statistically significant.</figref><figref num="8">Immunogenicity of E2E6E7SH-IFNγ ELISPOT analysis. (A) Immune scheme. CB6F1 mice were immunized with an adenovector expressing the transgene shown at the bottom of panels B and C. Two weeks after immunization, mice were sacrificed and isolated splenocytes were stimulated overnight in a 15mer peptide pool corresponding to E2 (B), E6 (not described) or E7 (C). Response is 10⁶It is given as SFU per splenocytes. A post-ANOVA Bonferroni statistical analysis was performed on the log conversion data. The E2 response induced by the adenovector encoding E2 alone is higher than the response induced by the polypeptides of the invention containing the E6 and E7 fragments. Differences between E2 vs E2E6E7SH and E2 vs E6E7E2SH are significant (^*: p &lt;0.05). A post-ANOVA Bonferroni statistical analysis was performed on the log conversion data.</figref><figref num="9">Sustained response in immune mice. (A) Immune scheme. 1 × 10 in which CB6F1 mice express mutants LSE2E6E7SH, E2E6E7SH, E6E7SH¹⁰It was immunized with the vp Ad35 vector or an adenovector (Emtpy) that does not express the transgene. Blood samples were taken every two weeks to measure the percentage of E7-specific CD8 + T cells by tetramer staining. (B) Immune response 2 weeks after immunization. Vectors containing a leader sequence elicited a higher response than vectors without a leader sequence. LSE2E6E7SH vs E2E6E7SH (^*: p &lt;0.05). (C) Response dynamics. Post-ANOVA Bonferroni statistical analysis was performed on the log conversion data of the second week dataset. The E7 response induced by E2-containing molecules tends to be higher than that of E2-free molecules, but the results were not statistically significant.</figref><figref num="10">Use of different adenoviral vectors to boost the immune response. (A) Immune scheme. CB6F1 mice were immunized with the Ad26 vector (HPV16-Tx) expressing HPV16 E2E6E7SH or the Ad26 vector (empty) not expressing the transgene. After 2 weeks, immunization was repeated using an Ad35-based vector as shown at the bottom of the figure. Four weeks after the second immunization, the mice were sacrificed and blood samples were used to measure the percentage of E7-specific CD8 + T cells by tetramer staining. (B)^*Shows a comparison of Ad26.HPV16-Tx / Ad35.HPV16-Tx vs. Ad26.HPV16-Tx / Ad35.Empty (p &lt;0.05) (Student's t-test for log conversion data with α = 0.01 in multiple comparisons).</figref><figref num="11">Cellular immunogenicity of E2E6E7SH in rhesus macaques. (A) Immune scheme. Rhesus macaques were immunized on day 0, ie, by intramuscular immunization (im), 8 animals received Ad26.HPV16-E2E6E7SH and 2 control animals received Ad26.Empty. Boost immunity (Ad26.HPV16-E2E6E7SH or Ad26.Empty) was given after 8 weeks. After 16 weeks, animals received a second boost immunization with an Ad35 vector expressing the same E2E6E7SH, while control animals received Ad35.Empty. Adenovector dose is 1 x 10¹¹It was vp / immunity. Blood was drawn several times. (B) The cellular immune response in PBMC was measured by IFNγ ELISPOT. PBMCs are stimulated with peptide pools corresponding to HPV16 E2, E6 or E7, 1 × 10⁶The number of spot formation units (SFUs) in each PBMC is illustrated. Empty control animals (n = 2) showed no detectable response. See Example 4 for details.</figref><figref num="12">Therapeutic effect of an adenovector expressing HPV16-E2E6E7SH. (A) TC-1 injection and immune scheme. Day 0, 1x10 on CB6F1 mouse⁵TC-1 cells were injected subcutaneously. Six days later, when the tumor was palpable, mice were given 5 nmol of ODN1826-CpG (B) or Ad26.HPV16-E2E6E7SH (C) in a final volume of 200 μl in 0.9% saline, 150 μg of HPV16 E6 And E7 immunodominant epitopes (ie HPV16 E6, aa41-65 (KQQLLRREVYDFAFRDLCIVYRDGN; SEQ ID NO: 18) and HPV16 E7, aa43-77 (GQAEPDRAHYNIVTFCCKCDSTLRLCVQSTHVDIR; SEQ ID NO: 19)) were immunized with two SLPs. Control mice received either CpG alone (D) or Ad26.Empty (E). All mice received boost immunization on day 20. Mice that received the Ad26 vector in prime immunization were then immunized with the corresponding Ad35 vector. Other mice received SLP immunostimulated with CpG or CpG alone, such as in prime immunization. (B ~ E) Tumor measurement in TC-1 injected mice. Tumor volume is (width)²× Length) / 2 was calculated. Tumor volume is 1000 mm³Above, the mice were sacrificed. Two mice had to be sacrificed due to weight loss of more than 20% (indicated by aster risk). (F ~ G) Close-up of panels B and C for the first 35 days. (H) Survival after TC-1 injection. Survival of mice treated with Ad.HPV16-E2E6E7SH was significantly increased compared to mice immunized with SLP and CpG (log rank test p &lt;0.05). Three mice immunized with Ad.HPV16-E2E6E7SH were tumor-free at the end of the experiment (day 92).</figref><figref num="13">Adenovirus vectors carrying a transgene encoding either HPV Ag or LSE2E6E7SH show increased virus yield in cells capable of suppressing transgene expression. A) Virus yield assay in Ad35 vector. PER.C6, PER.C6 / CymR, and PER.C6 / TetR cells were infected with an Ad35 vector carrying a transgene encoding GFP-Luc or HPVAg. These transgenes were driven by either CuO or TetO-containing CMV promoters. Virus yield was measured 4 days after infection by an Ad35 hexone-specific qPCR-based method. B) Virus yield assay in Ad26 vector. PER.C6 and PER.C6 / TetR cells were infected with an Ad26 vector carrying a transgene encoding GFP-Luc, HPVAg, or LSE2E6E7SH, all driven by the TetO-containing CMV promoter. Virus yield was measured 3 days after infection by an Ad26 hexone-specific qPCR-based method. See Example 6 for details.</figref><figref num="14">The use of a repressor system to suppress transgene expression during vector production prevents the instability of the transgene cassette in transgene vectors carrying the transgene encoding HPVAg. The Ad35 vector expressing HPVAg under the control of CMVCuO was rescued by DNA gene transfer in either the PER.C6 or PER.C6 / CymR cell line. The resulting viral plaque was harvested at 5 cells / cell line and used over continuous infection in each cell line. A) PCR analysis of the integrity of the vector / transgene cassette region after 10 viral passages. PCR products from viral isolates passaged with PER.C6 and PER.C6 / CymR are shown in the middle and right panels, respectively. PCR products appearing to be full length obtained in virus isolates 1, 2, 4, and 5 passaged with PER.C6, and isolates 1-5 passaged with PER.C6 / CymR. What was found was analyzed by Sanger DNA sequencing. Chromatogram trace analysis (not shown) showed that all isolates grown on PER.C6 (not those grown on PER.C6 / CymR) had a small frameshift deletion in the coding sequence in HPVAg. Or it has been shown to have either a stop mutation in the middle. B) Analysis of the ability of the vector to express HPVAg after 7 viral passages. A549 cells were transduced with virus isolates grown on PER.C6 and PER.C6 / CymR and HPVAg expression was analyzed by Western blot using HPV16 E7 specific antibody. The predicted size in HPVAg is 83 kDa. See Example 6 for details.</figref>

The present invention provides a nucleic acid molecule encoding a polypeptide comprising SEQ ID NO: 1. A polypeptide is a fusion polypeptide, sometimes referred to herein as the polypeptide of the invention, or fusion polypeptide of the invention. This polypeptide is useful in providing an immune response of HPV16 to the E6 and E7 proteins, and thus the nucleic acid molecule can be used as a therapeutic vaccine to prevent persistent HPV16 infection and associated diseases.

The polypeptides of the invention are in the form of fragments that have been rearranged and partially overlapped so that (essentially) all T cell epitopes of the HPV16 E6 and E7 proteins are present, virtually HPV16. A carefully designed molecule containing the complete E6 and E7 amino acid sequences of (it lacks only one amino acid from the C-terminus of the native HPV16 E6 protein). Earlier molecules with some potential as HPV vaccines have been described by others (eg, Kenter et al., 2009, N Engl J Med 361: 1838-47; Daayana et al., 2010). , Br J Cancer 102: 1129-36; Smahel et al., 2001, Virology 281: 231-38; Yan et al., 2009, Vaccine 27: 431-40; Oehlschlaeger et al., 2006, Vaccine 24: 2880- 93; Oosterhuis et al., 2011, Int J Cancer 129: 397-406; European Patent No. 1183368, WO 2013/083287 (Pamphlet), but each of these molecules has one or more drawbacks. The designer polypeptide molecule of the present invention is advantageous over at least one, typically several embodiments, over the aforementioned techniques. In particular, the advantages of the molecules and / or vectors of the invention are: (i) they are desired because they have significantly reduced undetectable transforming activity (compared to native E6 and E7 proteins). (Ii) They are single nucleic acid molecules that are easy to produce on an industrial scale to be economically profitable, and pose a logistical challenge unlike multi-molecule methods. Do not; (iii) Code polypeptide is natural HPV16 Containing essentially all T cell epitopes of the E6 and E7 proteins; (iv) Due to the design of the coding polypeptide, unwanted potential potent neoepitope (ie, epitopes that are not present in the native E6 and E7 proteins) The introduction of these is minimized; and (v) in certain embodiments, they do not rely on highly reactive adjuvants to produce the desired immune response. Therefore, the molecules of the invention represent a major step forward from combining various advantageous properties in a single design and are also excellent candidates primarily for therapeutic vaccination against HPV16. These molecules could probably also act as prophylactic vaccines against HPV16, which means they are likely to prevent persistent infection with HPV16 in vaccinated subjects.

In certain embodiments, careful design allows for predicted binding affinities for the 20 most common HLA-A, the 20 most common HLA-B and the 20 most common HLA-C alleles. The number of 9 amino acid long neoepitope with 50 nM was minimized to only one. A single shuffled E6 protein already has more than 30 such neoepitope, and constructs have several more neos in the sequences added to these constructs to prevent the lack of epitopes. This is a significant improvement over constructs described by others, as it is more likely to contain epitopes (Oehlschlaeger et al., 2006, Vaccine 24: 2880-93). Therefore, the constructs of the invention are significantly improved compared to methods described by others, as the chances of an immune response being modified in the molecules of the invention are minimized compared to native E6 and E7. Has immunological properties.

Those skilled in the art use conventional techniques to influence polypeptide sequences encoded by polynucleotides described to reflect codon use in any particular host organism in which the polypeptide is to be expressed. Nucleotide substitutions can be made so as not to give. Thus, unless otherwise specified, a "nucleotide sequence encoding an amino acid sequence" is a degenerate version of each other and includes all nucleotide sequences encoding the same amino acid sequence. Nucleotide sequences encoding proteins and RNA may include introns.

In a preferred embodiment, the nucleic acid encoding the polypeptide according to the invention is codon-optimized for expression in mammalian cells, preferably human cells. Methods for codon optimization are known and have been described in advance (eg, WO 96/09378). A sequence is considered codon-optimized if at least one unfavorable codon is replaced with a more preferred codon compared to the wild-type sequence. In the present specification, an unfavorable codon is a codon that is used less frequently than another codon that encodes the same amino acid in an organism, and a more preferred codon is a codon that is used more frequently than an unfavorable codon in an organism. Is. The frequency of codon use in a particular organism can be found in the codon frequency table, eg http://www.kazusa.or.jp/codon. Preferably, two or more unfavorable codons, such as more than 10%, 40%, 60%, 80% unfavorable codons, preferably maximal (eg at least 90%) or all unfavorable codons are more preferred. Replaced by codons. Preferably, the most frequently used codons in the organism are used in codon-optimized sequences. Substitution with the preferred codon generally results in higher expression.

Nucleic acid sequences can be cloned using conventional molecular biology techniques or newly created by DNA synthesis, which is a service company operating in the field of DNA synthesis and / or molecular cloning (eg, GeneArt). , GenScripts, Invitrogen, Eurofins) can be performed using the usual method.

It will be appreciated by those skilled in the art that proteins can be modified, for example, by substitution, deletion, addition, etc. of amino acids, eg, using conventional molecular biological methods. In general, conservative amino acid substitutions may be applied as long as they are not associated with a functional or immunogenic deficiency of the polypeptide. This can be considered according to conventional methods well known to those skilled in the art.

In certain embodiments, the coding polypeptide according to the invention further comprises a leader sequence, also referred to as a signal sequence or signal peptide. This is a short (typically 5-30 amino acid long) peptide present at the N-terminus of most newly synthesized proteins destined for the secretory pathway. The presence of such sequences can result in enhanced expression and immunogenicity. As non-limiting examples that can be used, IgE leader peptide (see, eg, US Pat. No. 6,733,994; eg, having sequence MDWTWILFLVAAATRVHS (SEQ ID NO: 7)) or HAVT20 leader peptide (eg, having sequence MACPGFLWALVISTCLEFSMA (SEQ ID NO: 9)). Can be mentioned. One of these can optionally be added to the N-terminus of the polypeptide of the invention. In other embodiments, the polypeptide according to the invention does not contain a leader sequence.

Various HPV types are present (more than 120 types have been identified and are referenced by number), and type-specific antigens are generally incorporated into the vaccine for each type that needs to be included by the vaccine. Although it may be necessary, there may be some cross-reactivity for a particular antigen. Types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, 73, and 82 are carcinogenic "high-risk" sexually infectious HPV and uterine It can cause the development of cervical intraepithelial neoplasia (CIN), vulvar intraepithelial neoplasia (VIN), vaginal intraepithelial neoplasia (VaIN), penile intraepithelial neoplasia (PIN), and / or anal intraepithelial neoplasia (AIN). The HPV according to the present invention (ie, the HPV from which the E6 and E7 fragments in the coding polypeptide are derived) is HPV16. It can be used for subjects infected with HPV16. It may also be suitably combined with vaccines against other HPV types in certain embodiments. In certain embodiments, this combination involves a vaccine against the identified high-risk HPV, such as a vaccine against HPV18. In other embodiments, the vaccines of the invention are HPV-18, -31, -33, -35, -39, -45, -51, -52, -56, -58, -59, -68,-. Combined with a vaccine against one or more of 73, or -82. Such a combination can be used, for example, if the exact type of HPV infection has not yet been determined, or if an immune response with a prophylactic effect is desired for two or more HPV types. Combinations of the vaccines of the invention with vaccines against HPV types that cause genital warts, such as HPV6 and / or HPV11, are also envisioned. The sequences of these HPV types and the proteins encoded by them (eg, E6, E7, E2) can be found in public databases, such as the National Center for biotechnology.

A polypeptide according to the invention comprises SEQ ID NO: 1, and in one embodiment, a nucleic acid molecule according to the invention comprises SEQ ID NO: 2.

The sequences herein are presented in the 5'to 3'direction or from the N to the C-terminus, as is customary in the art.

The polypeptide according to the present invention is HPV16. Contains epitopes of E6 and E7 proteins. In certain embodiments, the polypeptide according to the invention further comprises at least one additional antigen or epitope of such additional antigen (and thus the nucleic acid encoding the polypeptide further encodes them). Such additional antigens preferably belong to the same HPV type, ie HPV16, as the HPV antigen, preferably the E6 and E7 proteins in the polypeptide. Thus, such additional antigens can be HPV proteins or immunogenic fragments thereof, and in certain embodiments include E2 proteins or fragments thereof that contain at least one epitope of HPV, preferably HPV16-derived E2. Such additional antigens or epitopes can be placed internally between two fragments of E6 and / or E7 in the polypeptide containing SEQ ID NO: 1, but preferably the N-terminus of the E6 / E7 polypeptide containing SEQ ID NO: 1. Fused to the terminus or C-terminus. Alternatively or in addition, there may be amino acid sequences that stimulate the immune response. Thus, in certain embodiments, the invention provides a nucleic acid molecule according to the invention encoding a polypeptide comprising SEQ ID NO: 1, wherein the polypeptide is here at least one other antigen, such as the HPV E2 protein or a protein thereof. It further comprises at least one epitope (preferably more epitopes). One advantage of the addition of the E2 antigen in the present invention is that E2 is known to be expressed early during infection / in low grade lesions where E6 and E7 expression is still very low. During the development of cervical cancer, E2 expression is reduced, resulting in elevated E6 and E7 levels (Yugawa and Kiyono, 2009, Rev Med Virol 19: 97-113). By combining E2, E6, and E7-derived epitopes in a single vaccine, treatment in a broad target population of patients ranging from persistent infections to invasive cervical cancer (or other HPV16-induced cancers) Becomes possible. In certain embodiments, the E2 protein is a wild-type E2 protein. In certain other embodiments, the E2 protein has a deletion or one or more mutations within its DNA binding domain (compared to the wild-type E2 protein). The sequence of the HPV16 E2 protein (NP_041328.1) can be found in the NCBI protein database (www.ncbi.nlm.nih.gov/protein) under the number NP_041328.1. Several single amino acid changes in E2, such as G293V, K299M, or C300R, at the C-terminal portion of this protein are known to suppress DNA binding. The advantage of using a variant or fragment of E2 that lacks DNA binding ability is that it can block unpredictable transcriptional changes through direct binding to host cell DNA within the cell in which it is expressed. It is a point. The E2 protein or a portion or variant thereof can be added internally, but preferably to the N-terminus or C-terminus of the polypeptide of the invention having SEQ ID NO: 1. In one embodiment, the nucleic acid molecule of the invention encodes a polypeptide comprising SEQ ID NO: 3. In one embodiment, the nucleic acid molecule of the invention comprises SEQ ID NO: 4. In another embodiment, the nucleic acid molecule of the invention encodes a polypeptide comprising SEQ ID NO: 5. In one embodiment, the nucleic acid molecule of the invention comprises SEQ ID NO: 6. It can be found under the number NP_041328.1 in ncbi.nlm.nih.gov/protein). Several single amino acid changes in E2, such as G293V, K299M, or C300R, at the C-terminal portion of this protein are known to suppress DNA binding. The advantage of using a variant or fragment of E2 that lacks DNA binding ability is that it can block unpredictable transcriptional changes through direct binding to host cell DNA within the cell in which it is expressed. It is a point. The E2 protein or a portion or variant thereof can be added internally, but preferably to the N-terminus or C-terminus of the polypeptide of the invention having SEQ ID NO: 1. In one embodiment, the nucleic acid molecule of the invention encodes a polypeptide comprising SEQ ID NO: 3. In one embodiment, the nucleic acid molecule of the invention comprises SEQ ID NO: 4. In another embodiment, the nucleic acid molecule of the invention encodes a polypeptide comprising SEQ ID NO: 5. In one embodiment, the nucleic acid molecule of the invention comprises SEQ ID NO: 6. It can be found under the number NP_041328.1 in ncbi.nlm.nih.gov/protein). Several single amino acid changes in E2, such as G293V, K299M, or C300R, at the C-terminal portion of this protein are known to suppress DNA binding. The advantage of using a variant or fragment of E2 that lacks DNA binding ability is that it can block unpredictable transcriptional changes through direct binding to host cell DNA within the cell in which it is expressed. It is a point. The E2 protein or a portion or variant thereof can be added internally, but preferably to the N-terminus or C-terminus of the polypeptide of the invention having SEQ ID NO: 1. In one embodiment, the nucleic acid molecule of the invention encodes a polypeptide comprising SEQ ID NO: 3. In one embodiment, the nucleic acid molecule of the invention comprises SEQ ID NO: 4. In another embodiment, the nucleic acid molecule of the invention encodes a polypeptide comprising SEQ ID NO: 5. In one embodiment, the nucleic acid molecule of the invention comprises SEQ ID NO: 6. Can be found under 1. Several single amino acid changes in E2, such as G293V, K299M, or C300R, at the C-terminal portion of this protein are known to suppress DNA binding. The advantage of using a variant or fragment of E2 that lacks DNA binding ability is that it can block unpredictable transcriptional changes through direct binding to host cell DNA within the cell in which it is expressed. It is a point. The E2 protein or a portion or variant thereof can be added internally, but preferably to the N-terminus or C-terminus of the polypeptide of the invention having SEQ ID NO: 1. In one embodiment, the nucleic acid molecule of the invention encodes a polypeptide comprising SEQ ID NO: 3. In one embodiment, the nucleic acid molecule of the invention comprises SEQ ID NO: 4. In another embodiment, the nucleic acid molecule of the invention encodes a polypeptide comprising SEQ ID NO: 5. In one embodiment, the nucleic acid molecule of the invention comprises SEQ ID NO: 6. Can be found under 1. Several single amino acid changes in E2, such as G293V, K299M, or C300R, at the C-terminal portion of this protein are known to suppress DNA binding. The advantage of using a variant or fragment of E2 that lacks DNA binding ability is that it can block unpredictable transcriptional changes through direct binding to host cell DNA within the cell in which it is expressed. It is a point. The E2 protein or a portion or variant thereof can be added internally, but preferably to the N-terminus or C-terminus of the polypeptide of the invention having SEQ ID NO: 1. In one embodiment, the nucleic acid molecule of the invention encodes a polypeptide comprising SEQ ID NO: 3. In one embodiment, the nucleic acid molecule of the invention comprises SEQ ID NO: 4. In another embodiment, the nucleic acid molecule of the invention encodes a polypeptide comprising SEQ ID NO: 5. In one embodiment, the nucleic acid molecule of the invention comprises SEQ ID NO: 6.

Further fusion of the designer polypeptides of the invention with additional proteins such as so-called carrier proteins such as calreticulin, Mycobacterium Tuberculosis heat shock protein 70, IP10, or thetanus toxin fragment C is also possible. Yes (see Oosterhuis et al., Human Gene Ther, 2012 (above) for further examples), in which case the immune response to HPV E6 and E7 (and optionally E2) polypeptides can be further enhanced. .. Accordingly, the invention also provides such additional fusion proteins, and nucleic acids encoding them.

In certain embodiments, nucleic acid molecules according to the invention are incorporated into the vector. A "vector", as used herein, is typically a medium for artificially transporting a foreign genetic material to another cell in which it can replicate and / or express, according to the invention. , Can be any nucleic acid molecule that incorporates the nucleic acid molecule according to the present invention. These can be prepared according to conventional molecular biology techniques such as cloning. Typically, such vectors can be grown in at least one type of suitable host, such as bacteria, yeast, insect cells, mammalian cells. The four major types of vectors are plasmids, viral vectors, cosmids, and artificial chromosomes. The vector itself is generally an insert (transgene; In the present invention, it is a DNA sequence consisting of a nucleic acid encoding the fusion polypeptide of the present invention) and a sequence that contributes as a "skeleton" of a vector. The purpose of a vector to transfer genetic information to another cell is typically to isolate, proliferate, or express the insert within the target cell. Preferably, the sequence encoding the polypeptide is operably linked to a promoter in the vector. The term "operably linked" means that the nucleotide sequence of interest is linked to the promoter in a manner that allows expression of the nucleotide sequence (eg, within the host cell when the vector is introduced into the host cell). Is intended to mean. The expression control sequence can be operably linked to the transgene. In certain embodiments, the vector is designed with the intention of expressing the introduced gene in a target cell and generally has a promoter sequence that drives the expression of the introduced gene. In certain embodiments, one or more of the commonly used vector elements such as transcription terminator sequences, polyadenylation tail sequences, Kozak sequences, UTRs, replication origins, multiple cloning sites, genetic markers, antibiotic resistance, and additional sequences It may be present and can be used by those skilled in the art to replicate the vector, eg, for propagation and proliferation in a particular cell, and for expression of the vector's transgene in the target cell into which the vector is introduced. Therefore, it can be designed to have the desired properties. A vector containing a nucleic acid encoding a fusion polypeptide according to the invention, preferably designed for expression in mammalian cells, is suitable as a vaccine according to the invention. In certain embodiments, the vectors according to the invention are plasmids, cosmids, yeast artificial chromosomes, bacterial artificial chromosomes, viral vectors and the like. One of ordinary skill in the art recognizes that various promoters can be used to obtain expression of a gene in a host cell. Some well-known and commonly used promoters for expression in eukaryotic cells are promoters derived from viruses such as adenovirus, such as adenovirus. E1A promoter, cytomegalovirus (CMV) -derived promoter, such as the CMV earliest (IE) promoter (referred to herein as the CMV promoter) (eg, available from pcDNA, Invitrogen), salvirus 40. (Simian Virus 40) (SV40) -derived promoters (eg, pIRES, catalog number 631605, available from BD Sciences) and the like. Suitable promoters are also derived from eukaryotic cells, such as metallothioneine (MT) promoter, elongation factor 1α (EF-1α) promoter, ubiquitin C or UB6 promoter, actin promoter, immunoglobulin promoter, heat shock promoter and the like. (See, for example, International Publication No. 2006/048459 pamphlet). As a non-limiting example of a promoter suitable for obtaining expression in eukaryotic cells, nt from the CMV promoter (US Pat. No. 5,385,839), such as the CMV earliest promoter, eg, the CMV earliest gene enhancer / promoter. Examples include those comprising .-735 to +95, such as the CMV promoter as provided herein, having the sequence set forth in SEQ ID NO: 13. A polyadenylation signal, such as bovine growth hormone polyA signal (US Pat. No. 5,122,458), may be present after the transgene. Includes promoters from Invitrogen), Simian Virus 40 (SV40), etc. (eg, pIRES, catalog number 631605, available from BD Sciences). Suitable promoters are also derived from eukaryotic cells, such as metallothioneine (MT) promoter, elongation factor 1α (EF-1α) promoter, ubiquitin C or UB6 promoter, actin promoter, immunoglobulin promoter, heat shock promoter and the like. (See, for example, International Publication No. 2006/048459 pamphlet). As a non-limiting example of a promoter suitable for obtaining expression in eukaryotic cells, nt from the CMV promoter (US Pat. No. 5,385,839), such as the CMV earliest promoter, eg, the CMV earliest gene enhancer / promoter. Examples include those comprising .-735 to +95, such as the CMV promoter as provided herein, having the sequence set forth in SEQ ID NO: 13. A polyadenylation signal, such as bovine growth hormone polyA signal (US Pat. No. 5,122,458), may be present after the transgene. Includes promoters from Invitrogen), Simian Virus 40 (SV40), etc. (eg, pIRES, catalog number 631605, available from BD Sciences). Suitable promoters are also derived from eukaryotic cells, such as metallothioneine (MT) promoter, elongation factor 1α (EF-1α) promoter, ubiquitin C or UB6 promoter, actin promoter, immunoglobulin promoter, heat shock promoter and the like. (See, for example, International Publication No. 2006/048459 pamphlet). As a non-limiting example of a promoter suitable for obtaining expression in eukaryotic cells, nt from the CMV promoter (US Pat. No. 5,385,839), such as the CMV earliest promoter, eg, the CMV earliest gene enhancer / promoter. Examples include those comprising .-735 to +95, such as the CMV promoter as provided herein, having the sequence set forth in SEQ ID NO: 13. A polyadenylation signal, such as bovine growth hormone polyA signal (US Pat. No. 5,122,458), may be present after the transgene. Examples include those comprising -735 to +95, such as the CMV promoter as provided herein, having the sequence set forth in SEQ ID NO: 13. A polyadenylation signal, such as bovine growth hormone polyA signal (US Pat. No. 5,122,458), may be present after the transgene. Examples include those comprising -735 to +95, such as the CMV promoter as provided herein, having the sequence set forth in SEQ ID NO: 13. A polyadenylation signal, such as bovine growth hormone polyA signal (US Pat. No. 5,122,458), may be present after the transgene.

In addition, control sequences may also be added. The term "regulatory sequence" is used interchangeably herein as a "regulatory element" and typically, but is not limited to DNA, regulates the transcription of the nucleic acid sequence of interest to which it is operably linked. Refers to a segment of nucleic acid and therefore acts as a post-transcriptional modifier. The control sequence is a transcriptional binding domain and often comprises a nucleic acid binding domain of a transcription protein and / or a transcription factor, a nucleic acid sequence recognized by an enhancer or repressor or the like. For example, it is possible to operably bind a repressor sequence to a promoter, where the repressor sequence reduces the expression of the transgene in a repressor protein, a production cell line that expresses this repressor protein. Or can be blocked). This may improve the genetic stability and / or expression level of the nucleic acid molecule during passage and / or when it is produced in large quantities in the producing cell line. Such systems are described in the art. For example, the control sequence can contain one or more tetracycline operon operator sequences (tetO), so expression is inhibited in the presence of the tetracycline operon repressor protein (tetR). In the absence of tetracycline, the tetR protein can bind to the tetO site and suppress transcription of genes that are operably linked to the tetO site. However, in the presence of tetracycline, conformational changes in the tetR protein prevent it from binding to the operator sequence and resulting in transcription of the operably linked gene. In certain embodiments, the nucleic acid molecule of the invention, eg, when present in a recombinant adenovirus vector, can optionally contain tetO operably linked to a promoter, thus introducing one or more. Gene expression is inhibited in recombinant adenovirus produced in producer cell lines in which the tetR protein is expressed. Subsequently, the recombinant adenovirus does not express the subject or tetR protein. When introduced into cells, expression will not be inhibited (eg, International Publication No. 07/073513 Pamphlet of International Patent Application). In certain other embodiments, the nucleic acid molecules of the invention may optionally include a cumate gene switch system, for example when present in recombinant adenovirus, where regulation of expression is carried out by a repressor (CymR). , Mediated by binding to an operator site (CuO) located downstream of the promoter (eg, Mullick et al. BMC Biotechnol. 2006 6: 43). As used herein, the term "repressor" is an entity having the ability to inhibit, interfere with, delay, and / or suppress the production of heterologous protein products of recombinant expression vectors (eg, proteins). Or other molecule). For example, by interference with the binding site at an appropriate position along the expression vector in the expression cassette. Examples of repressors include tetR, CymR, lac repressors, trp repressors, gal repressors, λ repressors, and other suitable repressors known in the art. Examples of use of the tetO / tetR operator / repressor system and the CuO / CymR operator / repressor system are provided herein. Suppression of vector / transgene expression during vector transmission can prevent the instability of the transgene and increase the yield during production of the vector carrying the transgene of the invention. Therefore, in some embodiments, the vectors of the invention are represented by, for example, a repressor operator sequence (eg, in a non-limiting embodiment, a TetO-containing sequence, eg, SEQ ID NO: 11) by binding of a repressor protein. , Or a promoter that can be suppressed by having a promoter that operably binds to a CuO-containing sequence, such as that shown in SEQ ID NO: 12, and is shown by a repressor protein (eg, TetR protein, eg, SEQ ID NO: 15). A CymR protein, such as one having an amino acid sequence as set forth in SEQ ID NO: 17, can bind to it.

In certain embodiments, the vector is a plasmid DNA molecule, or fragment thereof. These can be used for DNA vaccination. Other platforms, such as the attenuated live double-deleted Listeria monocytogenes strain, can also be used as vectors.

In other embodiments, the vector is a recombinant viral vector, which may be replication competent or replication deficient. In certain embodiments, the viral vector comprises a recombinant DNA genome. In certain embodiments, the vectors according to the invention are, for example, recombinant adenovirus, recombinant retrovirus, recombinant pox virus, eg vaccinia virus (eg, modified). Vaccinia Ankara (MVA), recombinant alphavirus, eg semliki forest virus, recombinant paramyxovirus, eg recombinant measles virus, or Another recombinant virus. In certain embodiments, the vector according to the invention is an MVA vector.

In a preferred embodiment, the vector according to the invention is recombinant adenovirus. The advantages of adenovirus for use as a vaccine are ease of operation, large scale, based on years of experience in research, development, manufacturing and clinical trials with the vast number of reported adenovirus vectors. Includes good manufacturability and excellent safety records. Adenoviral vectors used as vaccines generally provide a good immune response to the protein encoded by the transgene, eg, a cellular immune response. The adenovirus vector according to the present invention may be based on any type of adenovirus, in certain embodiments a human adenovirus, which may belong to any serotype. In other embodiments, it is a monkey adenovirus, such as a chimpanzee or gorilla adenovirus, which can belong to any serotype. In certain embodiments, the vectors according to the invention belong to human adenovirus serotypes 5, 26 or 35. Preparation of recombinant adenovirus vectors is well known in the art. In certain embodiments, the adenovirus vector according to the invention lacks at least one essential gene function in the E1 region of the adenovirus genome, eg, the E1a region and / or the E1b region, which is required for viral replication. In certain embodiments, the adenovirus vector according to the invention lacks at least a portion of the non-essential E3 region. In certain embodiments, the vector lacks at least one essential gene function in the E1 region and at least a portion of the non-essential E3 region.

Adenovirus vectors, methods of construction thereof and methods for their transmission are well known in the art and are described, for example, in US Pat. No. 5,559,099, US Pat. No. 5,837,511, US Pat. No. 5,846,782, US Pat. No. 5,851,806, US Pat. No. 5,994,106, US Pat. No. 5,994,128, US Pat. No. 5,965,541, US Pat. No. 5,981,225, US Pat. No. 6,040,174, US Patent. 6,020,191 and US Pat. No. 6,113,913, as well as Thomas Shenk, "Adenoviridae and their Replication," MS Horwitz, "Adenoviruses", Chapters 67 and 68 (each), Virology, BNFields et al., Eds., 3d ed., Raven Press, Ltd., New It is described in York (1996) and other references described herein. Typically, the construction of adenoviral vectors is based on standard molecular biology techniques, such as Sambrook et al., Molecular Cloning, a Laboratory Manual, 2d ed., Cold Spring Harbor Press, Cold Spring Harbor, NY. (1989), Watson et al., Recombinant DNA, 2d ed., Scientific American Books (1992), and Ausubel et al., Current Protocols in Molecular Biology, Wiley Interscience Publishers, NY (1995), and herein. Includes the use of those described in other references described.

A particularly preferred serotype for recombinant adenovirus is human serotype 35 or human serotype 26. The preparation of the rAd26 vector is described, for example, in WO 2007/104792 and in Abbink et al., 2007 Virology 81: 4654-63. An exemplary genomic sequence of Ad26 is found in Jenbank Receipt No. EF153474 and SEQ ID NO: 1 in WO 2007/104792. The preparation of the rAd35 vector is described, for example, in US Pat. No. 7,270,811, WO 00/70071, and Vogels et al., 2003, J Virol 77: 8263-71. An exemplary genomic sequence of Ad35 is found in Jenbank Receipt No. AC_000019 and Figure 6 of WO 00/70071.

In certain embodiments, the adenovirus is replication deficient, eg, because it has a deletion in the E1 region of the genome. As is known to those skilled in the art, in the case of deletion of essential regions from the adenovirus genome, the function encoded by these regions should preferably be provided trans by the producer cell, ie, E1, If some or all of the E2 and / or E4 regions are deleted from adenovirus, they are present in producer cells, eg, integrated into their genome, or in the form of so-called helper adenovirus or helper plasmids. There is a need. Adenovirus may also have a deletion in the E3 region that is not needed for replication, and therefore such deletion does not need to be complemented.

The usable producer cell (sometimes also referred to in the art or herein as "packaging cell" or "complementary cell") can be any producer cell to which the desired adenovirus can be transmitted. .. For example, transmission of recombinant adenovirus vectors takes place within producer cells that complement the deficiency in adenovirus. Such producer cells preferably have at least the adenovirus E1 sequence in their genome, thereby being capable of complementing recombinant adenovirus with a deletion in the E1 region. Producer cells that complement any E1, such as human retinal cells immortalized by E1, such as 911 or PER.C6 cells (US Pat. No. 5,994,128), E1 transformed sheep membrane cells (European Patent No. 1230354). ), E1 transformed A549 cells (see, eg, WO 98/39411, US Pat. No. 5,891,690), GH329: HeLa (Gao et al., 2000, Hum Gene Ther). 11: 213-19), 293, etc. can be used. In certain embodiments, the producer cells are, for example, HEK293 cells, or PER.C6 cells, or 911 cells, or IT293SF cells. The production of adenovirus vectors in producer cells has been reviewed in (Kovesdi et al., 2010, Viruses 2: 1681-703).

In certain embodiments, the E1-deficient adenovirus comprises the E4-orf6 coding sequence of a subgroup C adenovirus, such as Ad5. This allows transmission in well-known complementary cell lines expressing the Ad5 E1 gene of such adenovirus, such as 293 cells or PER.C6 cells (eg, Havenga et al., 2006, J Gen Virol 87: 2135-43; see International Publication No. 03/104467, which is incorporated herein by reference in its entirety).

The "heterologous nucleic acid" (also referred to herein as "transgene") in the vector of the invention is a nucleic acid that does not naturally exist in the vector, and according to the invention, the fusion polypeptide of the invention. The encoding nucleic acid is considered a heterologous nucleic acid if it is present in the vector. It is introduced into the vector, for example by standard molecular biology techniques. It can be cloned, for example, within the deleted E1 or E3 region of the adenovirus vector, or within the region between the E4 region and rITR. The transgene is generally operably linked to an expression control sequence. In a preferred embodiment, the transgene is cloned into the E1 region of the adenovirus vector.

The production of vectors such as DNA vectors, or recombinant adenovirus vectors, can be performed according to a variety of methods well known to those of skill in the art. In general, the production involves proliferation in cultured cells to make a substantial amount of vector material, subsequent collection of the vector from the cell culture medium, and typically subsequent removal of other substances to form a pharmaceutical composition. Further purification of the vector is required to obtain a purified vector that can be incorporated into the product (eg, Hoganson et al., 2002, BioProcessing J 1: 43-8; Evans et al., 2004, J Pharm Sci 93: 2458). -75). For example, methods for collecting adenovirus from producer cell cultures are widely described, for example, in WO 2005/080556. For example, WO 2010/060719 and Pamphlet 2011/098592 (both incorporated herein by reference) are suitable for obtaining and purifying large amounts of recombinant adenovirus. The method is described.

In certain aspects, the invention also provides a polypeptide encoded by a nucleic acid molecule according to the invention. Such a polypeptide comprises SEQ ID NO: 1. In certain embodiments, such polypeptides may comprise SEQ ID NO: 3 or SEQ ID NO: 5. The properties of such polypeptides are as described above. Such polypeptides can be used directly, for example, as a vaccine against HPV.

The present invention also provides a vaccine comprising a nucleic acid molecule, vector or polypeptide according to the present invention, and embodiments in each of these embodiments may include those described above. In a preferred embodiment, the vaccine according to the invention comprises a nucleic acid molecule according to the invention. In a further preferred embodiment, the vaccine comprises a vector according to the invention, preferably a DNA vector, an MVA vector, or a recombinant adenovirus vector.

In certain embodiments, the vaccine according to the invention comprises an additional active ingredient, eg, at least one HPV type different from HPV16, eg, a high-risk HPV type, eg HPV18, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, Includes nucleic acids encoding at least one epitope of the E6 and / or E7 protein of HPV56, HPV58, HPV59, HPV68, HPV73, or HPV82.

The term "vaccine" is a drug or composition that contains an active ingredient that is effective in inducing a prophylactic and / or therapeutic degree of immunity (in this case, therapeutic against HPV) in a subject against a particular pathogen or disease. Point to an object. Vaccines typically include nucleic acid molecules or vectors according to the invention, and pharmaceutically acceptable excipients. Upon administration to the subject, the polypeptide encoded by the nucleic acid molecule according to the invention will be expressed in the subject, thereby resulting in an immune response against the E6 and / or E7 antigen fragments present in the polypeptide. Become. The advantage of this molecule is that essentially all T cell epitopes of HPV16 E6 and E7 are present, so that a T cell response to any epitope present in wild-type E6 or E7 can be initiated in the vaccine. is there. In addition, the vaccine has all the safety and efficacy advantages as outlined above in nucleic acid molecules according to the invention.

For administration to humans, the present invention may use a pharmaceutical composition comprising a vector and a pharmaceutically acceptable carrier or excipient. In this case, the term "pharmaceutically acceptable" means that the carrier or excipient does not produce any unwanted or detrimental effects in the subject to which it is administered at the dose and concentration used. .. Such pharmaceutically acceptable excipients are well known in the art (Remington's Pharmaceutical Sciences, 18th edition, ARGennaro, Ed., Mack Publishing Company [1990]; Pharmaceutical Formulation Development of Peptides and Proteins, S. Frokjaer and L. Hovgaard, Eds., Taylor &amp; Francis [2000]; and Handbook of Pharmaceutical Excipients, 3rd edition, A.Kibbe, Ed., Pharmaceutical Press [2000]). Excipients are generally pharmacologically inert substances that are combined with the active ingredient of the drug. Excipients are commonly used to increase the amount of a formulation containing a potent active ingredient (hence the term "bulking agent", "filler" or "diluent"), thereby. Allows convenient and accurate dispensing of the drug substance in the production of dosage forms. Excipients can also contribute to various therapeutic enhancement purposes, such as promoting absorption or solubility of a drug, or other pharmacokinetic considerations. Excipients also contribute to in vitro stability, such as preventing denaturation over the expected shelf life, during the manufacturing process, as well as promoting powder fluidity or non-stickiness of the active substances involved. It can be useful in contributing to handling. The choice of suitable excipient also depends on the route of administration and dosage form, as well as the active ingredient and other factors.

The purified nucleic acid molecule, vector or polypeptide is preferably formulated and administered as a sterile solution, but lyophilized formulations are also available. The sterile solution is prepared by sterile filtration or essentially by other methods known in the art. The solution is then lyophilized or filled in a drug administration container. The pH of the solution is generally in the range of pH 3.0 to 9.5, for example pH 5.0 to 7.5. The nucleic acid molecule or vector or polypeptide is typically present in a solution having a suitable buffer, the solution of the vector may also contain salts. Optionally, a stabilizer, such as albumin, may be present. In certain embodiments, detergent is added. In certain embodiments, the vaccine may be formulated with an injectable formulation. These formulations contain effective amounts of nucleic acid molecules, vectors or polypeptides and are either sterile solutions, suspensions or lyophilized versions, optionally containing stabilizers or excipients.

For example, recombinant adenovirus vectors are stored in buffer (20 mM Tris, pH 8, 25 mM NaCl, 2.5% glycerol), which is also used in the Adenovirus World Standard (Hoganson et al., 2002, Bioprocessing J 1: 43-8). You may. Another useful preparation buffer suitable for administration to humans is 20 mM Tris, 2 mM MgCl.₂, 25 mM NaCl, sucrose 10% w / v, polysorbate 80 0.02% w / v. Another preparation buffer suitable for recombinant adenovirus is 10-25 mM citrate buffer (pH 5.9-6.2), 4-6% (w / w) hydroxypropyl-β-cyclodextrin (HBCD). ), 70-100 mM NaCl, 0.018-0.035% (w / w) polysorbate 80, and optionally 0.3-0.45% (w / w) ethanol. Obviously, many other buffers can be used and some examples of formulations suitable for storage or pharmaceutical administration of purified vectors are known.

In certain embodiments, the composition comprising the vector further comprises one or more adjuvants. An adjuvant is known in the art to further enhance the immune response to the antigenic determinant applied. The terms "adjudicul" and "immune stimulant" are used interchangeably herein and are defined as one or more substances that cause irritation of the immune system. In this regard, adjuvants are used to enhance the immune response to the polypeptide encoded by the nucleic acid molecule in the vector of the invention. As an example of a suitable adjuvant, aluminum hydroxide and / or aluminum phosphate and / or aluminum potassium phosphate (aluminium potassium) Aluminum salts such as phosphate); oil emulsion compositions (or oil-in-water compositions), such as squalene-water emulsions, such as MF59 (see, eg, WO 90/14837); saponin preparations, such as QS21 and immunity. Activated complex (ISCOMS), etc. (eg, US Pat. No. 5,057,540; International Publication No. 90/03184, International Publication No. 96/11711, International Publication No. 2004/004762, International Publication No. 2005 (See Pamphlet No. 002620); Bacterial or microbial derivatives include, examples include monophosphoryl lipid A (MPL), 3-O-deacylated MPL (3dMPL), oligonucleotides with CpG-motif, ADP- Examples include ribosylated bacterial toxins or variants thereof, such as E. coli thermolabile enterotoxin LT, cholera toxin CT, and the like. The vector-encoded adjuvant is, for example, by using a heterologous nucleic acid encoding a fusion of the C4 binding protein (C4bp) to the antigen of interest in the oligomerization domain (eg, Solabomi). et al., 2008, Infect Immun 76: 3817-23), or use vectors encoding both the transgene of interest and TLR-3 agonists, such as heterologous dsRNA (eg, WO 2007/100908). It is also possible to use it.

In other embodiments, the compositions of the invention do not contain an adjuvant.

The pharmaceutical composition may be administered to a subject, such as a human subject. The total dose of vaccine active ingredient provided to the subject during a single dose can vary as known to skilled practitioners, and for adenovirus is generally 1x10.⁷10 virus particles (vp) ~ 1 × 10¹²During vp, preferably 1x10⁸vp ~ 1 × 10¹¹During vp, for example 3x10⁸~5×10¹⁰During vp, for example 10⁹~3×10¹⁰Between vp. For DNA vaccines, the total amount of DNA per dose may be, for example, between 1 μg and 10 mg. When a gene gun is used for administration, a small amount, eg 10 μg, is typically used. For intramuscular injections, higher doses, such as up to 5 mg, are typically used.

Administration of the pharmaceutical composition can be performed using standard routes of administration. Non-limiting embodiments include, for example, parenteral administration by injection, such as intradermal, intramuscular, or subcutaneous or transdermal, or mucosal administration, such as intranasal, oral, vaginal, rectal, and the like. In one embodiment, the composition is administered by intramuscular injection, for example, to the deltoid muscles of the arm or the vastus lateralis muscle of the thigh. In certain embodiments, the vaccine is a DNA vaccine, which can be administered intradermally, for example by DNA tattooing (see, eg, Oosterhuis et al., 2012, Curr Top Microbiol Immunol 351: 221-50). This pathway can also be used for adenovirus vectors. In certain embodiments, the composition according to the invention comprises an adenoviral vector and is administered by intramuscular injection. One of ordinary skill in the art understands the various possibilities for administering a composition, eg, a vaccine, to induce an immune response against an antigen in a vaccine.

The subjects used herein are preferably mammals, such as rodents, such as mice, or non-human primates, or humans. Preferably, the subject is a human subject.

The vaccines of the invention are also known, for example, as (pre) cancerous lesions, and cervical intraepithelial neoplasia (CIN; cervical dysplasia and cervical interstitial tumors, potentially the uterus Precancerous transformation and overgrowth (dysplasia) of flat epithelial cells on the surface of the cervix) to cervical cancer (including (including) cervical flat epithelial cancer (SCC), etc.) Like before (for example, HPV It can be used to treat patients with one of the various stages of disease caused by HPV (particularly type 16) from accidental and persistent HPV infections (as detected by DNA testing). In addition, other HPV-induced tumors such as vulvar intraepithelial neoplasia (VIN), vaginal intraepithelial neoplasia (VaIN), penile intraepithelial neoplasia (PIN), anal intraepithelial neoplasia (AIN), and mesopharyngeal cancer (head and neck) More advanced stages of penis cancer, vaginal cancer, vulvar cancer and anal cancer can be targeted. Thus, the vaccines of the invention can target a wide range of HPV-induced lesions and also express precancerous stages of HPV-induced diseases such as (persistent) infection and / or tumor stages (E2, E6 and / or E7). Is most likely to be the most effective. Compounds that can interact with or overcome the anti-immunity evasion mechanism in advanced cancer cells, such as anti-PD1 / PD-L1 antibodies, anti-CTLA-4 antibodies such as ipilimumab, It can also be combined with anti-LAG-3 antibody, anti-CD25 antibody, IDO inhibitor, CD40 agonist antibody, CD137 agonist antibody, etc. (eg Hamid and Carvajal, 2013, Expert Opinion Biol Ther 13: 847-861; Mellman et. al., 2011, Nature Rev 480: 480-89). Therapeutic vaccination methods also, in principle, when the vaccine further comprises HPV type E6 and / or E7 (a sequence encoding) that causes external genital warts and is administered to subjects infected with such HPV type. , Can be used to treat external genital warts or their precursor lesions.

As used herein, "treating" refers to HPV16 in a patient. It means the administration of a vaccine to induce a therapeutic immune response against cells expressing E6 and / or E7 (epitope), which results in at least a reduction in the level of HPV16 infection and preferably complete elimination. As a result, the progression of HPV16-induced diseases such as tumors and / or their symptoms is at least slowed and preferably stopped. Preferably, vaccination also results in remission of more advanced stages of HPV-induced cancer. Since it is preferable to administer the vaccine to patients with a classified and established HPV infection, a vaccine encoding the corresponding HPV-type polypeptide can be administered. In the absence of screening, the vaccine can also be administered in a population likely to be infected with HPV, i.e. some of the sexually enthusiastic people. The vaccine of the present invention may also be administered to a subject not infected with HPV16, for example for prophylactic purposes, in combination with a vaccine against another HPV type, for example in a non-infected or otherwise uninfected subject. It is possible. Vaccines of the invention also include further treatment by other means, such as surgery (removal of lesions caused by HPV16 infection), or treatment with imiquimod (including TLR-7 / 8 agonists, eg, Dayaana et al., 2010). , Br J Cancer 102: 1129-36). The effectiveness of treatment can be measured by either cytology or HPV testing.

Vaccination comprises the step of administering the vaccine of the invention to a subject or patient at least once. It is also possible to provide one or more booster doses of one or more additional vaccines. When booster vaccination is performed, typically such booster vaccination corresponds to the first administration of an immunogenic composition with the same antigen to a subject (as referred to as "priming vaccination"). ) Later, it will be administered to the same subject for a period of 1 week to 1 year, preferably 2 weeks to 4 months. Other boost immune regimens also allow subjects to receive different vectors, such as one or more adenoviruses of different serotypes, or other vectors such as MVA, or DNA, or proteins as priming or booster vaccination. Is. In certain embodiments, the vaccine of the same form of the invention is administered to the same patient at least twice in a prime-boost regimen with, for example, the same recombinant adenovirus (such as Ad26) according to the invention. In certain embodiments, the vaccines of the invention are administered at least twice in a prime-boost regimen, but the vaccine vector is different, for example two different serotypes of adenoviral vector are used, eg, recombinant. Ad26 priming and recombinant Ad35 booster, or vice versa; or DNA priming and adenoviral vector booster, or vice versa; or adenoviral vector priming and MVA vector booster, or vice versa. Be done. In certain embodiments, the vaccine according to the invention is administered at least 3 times in a prime-boost-boost regimen. In some cases, additional booster administration may be added to the regimen.

Inducing a CTL response to HPV16 in a subject is also an aspect of the invention, comprising administering to the subject a vaccine according to the invention.

The present invention also describes the following non-limiting embodiments, i.e. 1) a nucleic acid encoding a polypeptide comprising SEQ ID NO: 1; 2) the polypeptide further comprises at least a portion of the HPV E2 protein. Nucleic acid; 3) HPV The nucleic acid according to embodiment 2, wherein at least a portion of the E2 protein is derived from the E2 protein of HPV16; 4) At least a portion of the E2 protein fused to the N-terminal side of the polypeptide having SEQ ID NO: 1. The nucleic acid according to embodiment 2; 5) the nucleic acid according to embodiment 2; 5) the nucleic acid comprising at least a part of the E2 protein fused to the C-terminal side of the polypeptide having SEQ ID NO: 1. The nucleic acid according to embodiment 3, wherein the peptide comprises at least a portion of the E2 protein fused to the N-terminal side of the polypeptide having SEQ ID NO: 1; 7) the C-terminal side of the polypeptide having SEQ ID NO: 1. The nucleic acid according to embodiment 3, which comprises at least a portion of the E2 protein fused to; 8) contains a variant of the E2 protein having a mutation at which at least a portion of the E2 protein suppresses DNA binding of E2. The nucleic acid according to embodiment 2; 9) The nucleic acid according to embodiment 3; 10) of the E2 protein, which comprises a variant of the E2 protein having a mutation in which at least a portion of the E2 protein suppresses DNA binding of E2. The nucleic acid according to embodiment 4, comprising a variant of the E2 protein having a mutation at least in part that suppresses DNA binding of E2; 11) Suddenly that at least part of the E2 protein suppresses DNA binding of E2. The nucleic acid according to embodiment 5, comprising a variant of the E2 protein having a mutation; 12) an embodiment comprising a variant of the E2 protein having a mutation at which at least a portion of the E2 protein suppresses DNA binding of E2. 6. The nucleic acid according to embodiment 7; 13) the nucleic acid according to embodiment 7; 14) encoding a polypeptide, comprising a variant of the E2 protein having a mutation at which at least a portion of the E2 protein suppresses DNA binding of E2. Vector containing the nucleic acid according to embodiment 1 in which the sequence is operably linked to the promoter; 15) Vector containing the nucleic acid according to embodiment 2 in which the sequence encoding the polypeptide is operably linked to the promoter. ;16) Vector containing the nucleic acid of embodiment 3, wherein the polypeptide-encoding sequence is operably linked to the promoter; 17) Polypeptide-encoding sequence is operably linked to the promoter, embodiment 4. 18) A vector containing a nucleic acid according to embodiment 5 in which a sequence encoding a polypeptide is operably linked to a promoter; 19) a vector containing a polypeptide according to 5 is operably linked to a promoter. Vector containing the nucleic acid according to embodiment 6; 20) Vector containing the nucleic acid according to embodiment 7 operably linked to a promoter with a sequence encoding the polypeptide; 21) Polypeptide. A vector comprising the nucleic acid according to embodiment 8 in which the encoding sequence is operably linked to the promoter; 22) The nucleic acid according to embodiment 9 in which the polypeptide-encoding sequence is operably linked to the promoter. 23) The vector encoding the polypeptide is operably linked to the promoter, the vector containing the nucleic acid according to embodiment 10; 24) the sequence encoding the polypeptide is operably linked to the promoter. Vector containing the nucleic acid according to embodiment 11; 25) Vector containing the nucleic acid according to embodiment 12 in which the sequence encoding the polypeptide is operably linked to the promoter; 26) The sequence encoding the polypeptide is the promoter. 27) The vector according to embodiment 13; the vector according to embodiment 14; 28) the vector is an adenovirus, according to embodiment 15. 29) The vector is an adenovirus, the vector according to embodiment 16; 30) the vector is an adenovirus, the vector according to embodiment 17; 31) the vector is an adenovirus, according to embodiment 18. 32) The vector according to embodiment 19 where the vector is an adenovirus; 33) The vector according to embodiment 20 where the vector is an adenovirus;34) The vector according to embodiment 21 where the vector is adenovirus; 35) the vector according to embodiment 22 where the vector is adenovirus; 36) the vector according to embodiment 23 where the vector is adenovirus. 37) The vector according to embodiment 24, the vector is adenovirus; 38) the vector according to embodiment 25, the vector is adenovirus; 39) the vector according to embodiment 26, wherein the vector is adenovirus. Vector; 40) The vector according to embodiment 27, wherein the adenovirus is a human adenovirus of serum type 26; 41) The vector according to embodiment 28, wherein the adenovirus is a human adenovirus of serum type 26; 42) The vector according to embodiment 29, wherein the adenovirus is a human adenovirus of serum type 26; 43) The vector according to embodiment 30, wherein the adenovirus is a human adenovirus of serum type 26; 44) The adenovirus is serum. Vector according to embodiment 31 which is a human adenovirus of type 26; 45) Vector according to embodiment 32 where adenovirus is a human adenovirus of serum type 26; 46) Human whose adenovirus is serum type 26. The vector according to embodiment 33, which is an adenovirus; 47) The vector according to embodiment 34, where the adenovirus is a human adenovirus of serum type 26; 48) The adenovirus is a human adenovirus of serum type 26. 49) The vector according to embodiment 35; 49) the adenovirus is a human adenovirus of serum type 26; 50) the vector according to embodiment 36; 50) the adenovirus is a human adenovirus of serum type 26, embodiment 37. 51) The vector according to embodiment 38, wherein the adenovirus is a human adenovirus of serum type 26; 52) The vector according to embodiment 39, wherein the adenovirus is a human adenovirus of serum type 26. 53) The vector according to embodiment 28, wherein the adenovirus is a human adenovirus of serum type 35;54) The vector according to embodiment 29, wherein the adenovirus is a human adenovirus of serum type 35; 55) The vector according to embodiment 30, wherein the adenovirus is a human adenovirus of serum type 35; 56) adenovirus. Is a human adenovirus of serum type 35, the vector according to embodiment 31; 57) the adenovirus is a human adenovirus of serum type 35, the vector according to embodiment 32; 58) the adenovirus is a serum type 35. The vector according to embodiment 33, which is a human adenovirus of the same; 59) the vector according to embodiment 34, where the adenovirus is a human adenovirus of serum type 35; 60) the human adenovirus whose adenovirus is of serum type 35. 61) The vector according to embodiment 35; 61) the adenovirus is a human adenovirus of serum type 35; 62) the adenovirus is a human adenovirus of serum type 35. The vector according to form 37; 63) the adenovirus is a human adenovirus of serum type 35, the vector according to embodiment 38; 64) the adenovirus is a human adenovirus of serum type 35, according to embodiment 39. 65) The vector according to embodiment 14 and a vaccine composition containing a pharmaceutically acceptable excipient; 66) The vector according to embodiment 15 and a vaccine containing a pharmaceutically acceptable excipient. Composition; 67) Vaccine composition comprising the vector according to embodiment 16 and a pharmaceutically acceptable excipient; 68) A vaccine comprising the vector according to embodiment 17 and a pharmaceutically acceptable excipient. Composition; 69) A vaccine composition comprising the vector according to embodiment 18 and a pharmaceutically acceptable excipient; 70) A vaccine comprising the vector according to embodiment 19 and a pharmaceutically acceptable excipient. Composition; 71) Vaccine composition comprising the vector according to embodiment 20 and a pharmaceutically acceptable excipient; 72) The vector according to embodiment 21 and a vaccine comprising a pharmaceutically acceptable excipient. Composition;73) The vector according to embodiment 22 and a vaccine composition comprising a pharmaceutically acceptable excipient; 74) the vector according to embodiment 23 and a vaccine composition comprising a pharmaceutically acceptable excipient; 75) The vector according to embodiment 24 and a vaccine composition comprising a pharmaceutically acceptable excipient; 76) the vector according to embodiment 25 and a vaccine composition comprising a pharmaceutically acceptable excipient; 77) The vector according to embodiment 26 and a vaccine composition comprising a pharmaceutically acceptable excipient; 78) the vector according to embodiment 27 and a vaccine composition comprising a pharmaceutically acceptable excipient; 79) The vector according to embodiment 28 and a vaccine composition comprising a pharmaceutically acceptable excipient; 80) the vector according to embodiment 29 and a vaccine composition comprising a pharmaceutically acceptable excipient; 81) The vector according to embodiment 30 and a vaccine composition comprising a pharmaceutically acceptable excipient; 82) The vector according to embodiment 31 and a vaccine composition comprising a pharmaceutically acceptable excipient; 83) The vector according to embodiment 32 and a vaccine composition comprising a pharmaceutically acceptable excipient; 84) The vector according to embodiment 33 and a vaccine composition comprising a pharmaceutically acceptable excipient; 85) The vector according to embodiment 34 and a vaccine composition comprising a pharmaceutically acceptable excipient; 86) The vector according to embodiment 35 and a vaccine composition comprising a pharmaceutically acceptable excipient; 87) The vector according to embodiment 36 and a vaccine composition comprising a pharmaceutically acceptable excipient; 88) the vector according to embodiment 37 and a vaccine composition comprising a pharmaceutically acceptable excipient; 89) The vector according to embodiment 38 and a vaccine composition comprising a pharmaceutically acceptable excipient; 90) the vector according to embodiment 39 and a vaccine composition comprising a pharmaceutically acceptable excipient; 91) A vaccine composition comprising the vector according to embodiment 40 and a pharmaceutically acceptable excipient;92) The vector according to embodiment 41 and a vaccine composition comprising a pharmaceutically acceptable excipient; 93) the vector according to embodiment 42 and a vaccine composition comprising a pharmaceutically acceptable excipient; 94) The vector according to embodiment 43 and a vaccine composition comprising a pharmaceutically acceptable excipient; 95) the vector according to embodiment 44 and a vaccine composition comprising a pharmaceutically acceptable excipient; 96) The vector according to embodiment 45 and a vaccine composition comprising a pharmaceutically acceptable excipient; 97) the vector according to embodiment 46 and a vaccine composition comprising a pharmaceutically acceptable excipient; 98) The vector according to embodiment 47 and a vaccine composition comprising a pharmaceutically acceptable excipient; 99) the vector according to embodiment 48 and a vaccine composition comprising a pharmaceutically acceptable excipient; 100) The vector according to embodiment 49 and a vaccine composition comprising a pharmaceutically acceptable excipient; 101) the vector according to embodiment 50 and a vaccine composition comprising a pharmaceutically acceptable excipient; 102) The vector according to embodiment 51 and a vaccine composition comprising a pharmaceutically acceptable excipient; 103) The vector according to embodiment 52 and a vaccine composition comprising a pharmaceutically acceptable excipient; 104) The vector according to embodiment 53 and a vaccine composition comprising a pharmaceutically acceptable excipient; 105) the vector according to embodiment 54 and a vaccine composition comprising a pharmaceutically acceptable excipient; 106) The vector according to embodiment 55 and a vaccine composition comprising a pharmaceutically acceptable excipient; 107) The vector according to embodiment 56 and a vaccine composition comprising a pharmaceutically acceptable excipient; 108) The vector according to embodiment 57 and a vaccine composition comprising a pharmaceutically acceptable excipient; 109) the vector according to embodiment 58 and a vaccine composition comprising a pharmaceutically acceptable excipient; 110) A vaccine composition comprising the vector according to embodiment 59, and a pharmaceutically acceptable excipient;111) Vaccine composition comprising the vector according to embodiment 60 and a pharmaceutically acceptable excipient; 112) Vaccine composition comprising the vector according to embodiment 61 and a pharmaceutically acceptable excipient; 113) Vaccine composition comprising the vector according to embodiment 62 and a pharmaceutically acceptable excipient; 114) Vaccine composition comprising the vector according to embodiment 63 and a pharmaceutically acceptable excipient; 115) Vaccine composition comprising the vector according to embodiment 64 and a pharmaceutically acceptable excipient; 116) a method for inducing an immune response against HPV in a subject, any of embodiments 65-115. A method comprising the step of administering the vaccine composition according to any one to a subject; 117) a method for treating a persistent HPV (type 16) infection, any one of embodiments 65-115. A method comprising the step of administering the vaccine described in 1 to a subject suffering from a persistent HPV infection; 118) a method for treating an intraepithelial tumor (VIN) (with an underlying HPV type 16 infection). , A method comprising the step of administering the vaccine according to any one of embodiments 65-115 to a subject suffering from VIN; 119) to treat genital cancer (with underlying HPV16 infection). 120) (with underlying HPV16 infection), comprising the step of administering the vaccine according to any one of embodiments 65-115 to a subject suffering from genital cancer. A method for treating a cervical intraepithelial tumor (CIN), comprising the step of administering the vaccine according to any one of embodiments 65-115 to a subject suffering from CIN; 121). A method for treating cervical cancer (with underlying HPV16 infection), wherein the vaccine according to any one of embodiments 65-115 is administered to a subject suffering from cervical cancer. Including steps, methods;122) A method for treating penile cancer (with underlying HPV16 infection), wherein the vaccine according to any one of embodiments 65 to 115 is applied to a subject suffering from penile cancer. A method comprising the step of administration; 123) a method for treating an intraepithelial neoplasia (PIN) (with an underlying HPV16 infection), according to any one of embodiments 65-115. A method comprising the step of administering a vaccine to a subject suffering from PIN; 124) a method for treating penile cancer (with underlying HPV16 infection), any one of embodiments 65-115. A method comprising the step of administering one of the vaccines to a subject suffering from penile cancer; 125) a method for treating a vaginal intraepithelial tumor (VaIN) (with underlying HPV16 infection). , A method comprising the step of administering the vaccine according to any one of embodiments 65-115 to a subject suffering from VaIN; 126) for treating vaginal cancer (with underlying HPV16 infection). A method comprising administering to a subject suffering from vaginal cancer the vaccine according to any one of embodiments 65-115; 127) anal epithelium (with underlying HPV16 infection). A method for treating an internal tumor (AIN), comprising the step of administering the vaccine according to any one of embodiments 65-115 to a subject suffering from AIN; 128) (underlying). A method for treating anal cancer (with HPV16 infection), comprising the step of administering the vaccine according to any one of embodiments 65-115 to a subject suffering from anal cancer; 129) Vaccine comprising SEQ ID NO: 1; 130) Vaccine according to embodiment 129, further comprising at least a portion of the HPV E2 protein; 131) At least a portion of the HPV E2 protein is derived from the E2 protein of HPV16. The polypeptide according to embodiment 130;132) The polypeptide according to embodiment 130, wherein at least a portion of the E2 protein is fused to the N-terminal side of the polypeptide having SEQ ID NO: 1; 133) A polypeptide having at least a portion of the E2 protein having SEQ ID NO: 1. The polypeptide according to embodiment 130, which is fused to the C-terminal side of the protein; 134) The poly according to embodiment 131, which is fused to the N-terminal side of a polypeptide having SEQ ID NO: 1. Peptide; 135) The polypeptide according to embodiment 131, wherein at least a portion of the E2 protein is fused to the C-terminal side of the polypeptide having SEQ ID NO: 1; 136) At least a portion of the E2 protein is DNA-binding to E2. The polypeptide according to embodiment 130, comprising a variant of the E2 protein having a mutation that suppresses the DNA binding of E2; 137) a variant of the E2 protein that has a mutation in which at least a portion of the E2 protein suppresses the DNA binding of E2. 138) The polypeptide according to embodiment 132, comprising a variant of the E2 protein having a mutation in which at least a portion of the E2 protein suppresses DNA binding of E2; 139) The polypeptide according to embodiment 133, comprising a variant of the E2 protein having a mutation in which at least part of the E2 protein suppresses DNA binding of E2; 140) DNA of at least part of the E2 protein. The polypeptide according to embodiment 134, comprising a variant of the E2 protein having a binding-suppressing mutation; 141) an E2 protein having a mutation in which at least a portion of the E2 protein has a mutation that suppresses the DNA binding of E2. The polypeptide of embodiment 135, including the variant; 142) encodes the polypeptide of SEQ ID NO: 3, the nucleic acid of embodiment 3; 143) encodes the polypeptide of SEQ ID NO: 5. The nucleic acid according to embodiment 3; 144) The vector encoding the nucleic acid according to embodiment 142, in which the sequence encoding the polypeptide is operably linked to the promoter;145) The vector encoding the nucleic acid of embodiment 143, in which the sequence encoding the polypeptide is operably linked to the promoter; 146) the vector of embodiment 144, the vector of embodiment 144; 147) vector. Is an adenovirus, the vector according to embodiment 145; 148) the adenovirus is a human adenovirus of serum type 26; 149) the vector according to embodiment 146; There, the vector according to embodiment 147; 150) the adenovirus is a human adenovirus of serum type 35, the vector according to embodiment 146; 151) the adenovirus is a human adenovirus of serum type 35, embodiment. 147 of the vector; 152) the vector of embodiment 144 and a vaccine composition comprising a pharmaceutically acceptable excipient; 153) the vector of embodiment 145 and a pharmaceutically acceptable excipient. Vaccine composition comprising; 154) the vector according to embodiment 146, and a vaccine composition comprising a pharmaceutically acceptable excipient; 155) the vector according to embodiment 147, and a pharmaceutically acceptable excipient. Vaccine composition comprising; 156) the vector according to embodiment 148, and a vaccine composition comprising a pharmaceutically acceptable excipient; 157) the vector according to embodiment 149, and a pharmaceutically acceptable excipient. Vaccine composition comprising; 158) the vector according to embodiment 150, and a vaccine composition comprising a pharmaceutically acceptable excipient; 159) the vector according to embodiment 151, and a pharmaceutically acceptable excipient. 160) A method for inducing an immune response against HPV in a subject, comprising the step of administering to the subject the vaccine composition according to any one of embodiments 152-159. 161) A method for treating an intraepithelial neoplasia (VIN), comprising the step of administering the vaccine according to any one of embodiments 152-159 to a subject suffering from VIN;162) A method for treating genital cancer, comprising the step of administering the vaccine according to any one of embodiments 152 to 159 to a subject suffering from genital cancer; 163). A method for treating a cervical intraepithelial tumor (CIN), comprising the step of administering the vaccine according to any one of embodiments 152-159 to a subject suffering from CIN; 164). A method for treating cervical cancer, comprising the step of administering the vaccine according to any one of embodiments 152-159 to a subject suffering from cervical cancer; 165) mesopharyngeal. A method for treating cancer, comprising the step of administering the vaccine according to any one of embodiments 152-159 to a subject suffering from cervical cancer; 166) Intraepithelial tumor of the penis A method for treating (PIN), comprising the step of administering the vaccine according to any one of embodiments 152-159 to a subject suffering from PIN; 167) treating penis cancer. 168) Treatment of a vaginal intraepithelial tumor (VaIN), comprising the step of administering the vaccine according to any one of embodiments 152-159 to a subject suffering from penile cancer. 169) A method for treating vaginal cancer, comprising the step of administering the vaccine according to any one of embodiments 152-159 to a subject suffering from VaIN. , A method comprising the step of administering the vaccine according to any one of embodiments 152 to 159 to a subject suffering from vaginal cancer; 170) a method for treating an intraepithelial tumor (AIN). , A method comprising the step of administering the vaccine according to any one of embodiments 152 to 159 to a subject suffering from AIN; 171) a method for treating anal cancer, embodiments 152 to 159. Provided is a method comprising the step of administering the vaccine according to any one of the above to a subject suffering from anal cancer.171) A method for treating anal cancer, comprising the step of administering the vaccine according to any one of embodiments 152 to 159 to a subject suffering from anal cancer.

In the practice of the present invention, conventional techniques of immunology, molecular biology, microbiology, cell biology, and recombinant DNA will be used within the scope of the techniques, unless otherwise indicated. For example, Sambrook, Fritzch and Maniatis, Molecular Cloning: A Laboratory Manual, 2nd edition, 1989; Current Protocols in Molecular Biology, Ausubel FM, et al., Eds, 1987; the series Methods in Enzymology (Academic Press, Inc.); See PCR2: A Practical Approach, MacPherson MJ, Hams BD, Taylor GR, eds, 1995; Antibodies: A Laboratory Manual, Harlow and Lane, eds, 1988.

The present invention will be further described in the following examples. The examples are by no means limiting to the present invention. The examples are only useful for clarifying the present invention.

<p>Example 1: Construction of a Designer Polypeptide Containing Essentially All HPV16 E6 and E7 CTL epitopes The inventor has essentially all CTL epitopes of HPV16 E6 and E7 proteins and is expected / predicted potent. We designed a novel non-tumorized polypeptide (and the nucleic acid encoding it) that has the least number of neoepitope, which means an epitope that is not present in wild HPV16 E6 and E7 proteins. The polypeptides of the invention (sometimes also referred to herein as "E6E7SH") include the sequences provided by SEQ ID NO: 1. The codon-optimized nucleic acid encoding this polypeptide is provided in SEQ ID NO: 2.</p><p> The molecules of the invention are single molecules that offer a fabrication advantage over methods where multiple molecules are used. In addition, the polypeptides of the invention contain essentially all putative CTL epitopes present in wild-type E6 and E7 of HPV16, while at the same time being expected / predicted to be potentially immune predominant. It has the least number of potent neoepitope and therefore diverts the immune response from the relevant wild-type CTL epitope. Therefore, the constructs of the present invention are immunologically advantageous over molecules described by others, lacking any of the possible CTL epitopes and / or having more or stronger neoepitope.</p><p> For example, the construct of SEQ ID NO: 1 can be found on the IEDB website (http://tools.immuneepitope.org/analyze/html/mhc_binding.html,version 2009-09-01B) as "Peptide to MHC class I molecule". ANN (Lundegaard et al., 2008, Nucl Acids Res 36: W509-12) and SMM method (Peters et al., 2003, 2003, 20 most common HLA-A, 20 most common judgments using the NetMHCpan method (Hoof et al., 2009, Immunogenetics 61: 1-13) for Bioinformatics 19: 1765-72) and HLA-C. HLA-B and 20 most common HLA-C alleles (HLA-A)^*01:01, HLA-A^*02:01, HLA-A^*02:03, HLA-A^*02:06, HLA-A^*02:07, HLA-A^*03:01, HLA-A^*11:01, HLA-A^*23:01, HLA-A^*24:02, HLA-A^*26:01, HLA-A^*29:02, HLA-A^*30:01, HLA-A^*30:02, HLA-A^*31:01, HLA-A^*32:01, HLA-A^*33:01, HLA-A^*33:03, HLA-A^*34:01, HLA-A^*68:01, HLA-A^*68:02, HLA-B^*07:02, HLA-B^*07:04, HLA-B^*08:01, HLA-B^*13:01, HLA-B^*15:01, HLA-B^*18:01, HLA-B^*35:01, HLA-B^*37:01, HLA-B^*39:01, HLA-B^*40:01, HLA-B^*40:02, HLA-B^*40:06, HLA-B^*44:02, HLA-B^*44:03, HL-B * 46: 01, HLA-B^*48:01, HLA-B^*51:01, HLA-B^*52:01, HLA-B^*53:01, HLA-B^*58:01, HLA-C^*07:02, HLA-C^*04:01, HLA-C^*03:04, HLA-C^*01:02, HLA-C^*07:01, HLA-C^*06:02, HLA-C^*03:03, HLA-C^*08:01, HLA-C^*15:02, HLA-C^*12:02, HLA-C^*02:02, HLA-C^*05:01, HLA-C^*14:02, HLA-C^*03:02, HLA-C^*16:01, HLA-C^*08:02, HLA-C^*12:03, HLA-C^*04:03, HLA-C^*17:01, HLA-C^*It has only one neoepitope of 9 amino acids in length with the predicted binding affinity &lt;50 nM) for 14:03).</p><p> Non-limiting examples using the SMM prediction tool on the IEDB website are described by Oosterhuis et al., 2011, Int J Cancer 129: 397-406 and Oehlschlaeger et al., 2006, Vaccine 24: 2880-93. Such shuffled E6 and E7 sequences, respectively, in the core portion, have nine potential potent unique neoepitope (ANN or SMM) for most of the 20 HLA-A and HLA-B. It has an IC50 &lt;50nM). This further excludes the appendices used in the technique (where the appendix further contributes to the additional neoepitope and is due to the limited length of "overlap". And may miss the opportunity for more natural MHCII epitopes). Unsurprisingly, the improved molecules with variants with reportedly shuffled E6 and E7 proteins, described in WO 2013/083287, are the 20 most common HLA-A, the 20 most common. 22 unique neoepitope with 9 amino acid lengths with predicted IC50 &lt;50 nM (ANN, SMM or NetMHCPan) for common HLA-B and 20 most common HLA-C alleles Have.</p><p> Thus, the designer molecules of the invention are apparent in that they have significantly fewer predicted neoepitope than other published methods when E6 and E7 are shuffled to eliminate functionality. It is convenient.</p><p> Nucleic acid encoding the HPV16 E6E7SH molecule thus designed by the inventor (ie, the polypeptide having the amino acid sequence provided by SEQ ID NO: 1), ie, the nucleic acid sequence containing SEQ ID NO: 2, was synthesized and on the 5'end. Adjacent by the HindIII and Kozak sequences as well as the XbaI site on the 3'site (Invitrogen Life technologies, custom synthesis and standard molecular cloning in Germany).</p><p> The synthesized fragment was cloned using HindIII and XbaI into a standard expression vector pCDNA2004.Neo with both a bacterial resistance marker (ampicillin) and a mammalian resistance marker (neomycin), eg, (transient) gene transfer. A plasmid vector encoding the molecule of the present invention was obtained for experiments based on.</p><p> In addition to this, these molecules could also be used as a basis for additional molecules with additional characteristics. As a non-limiting example, several additional variants were prepared as follows.</p><p> The HPV16 E6E7SH fusion protein sequence can be combined with the sequences of other HPV16 early proteins to target individuals with persistent infections and expand the immune repertoire in immunized individuals. The immune response to E2 has been suggested to play an important role in the clearance of HPV16 infection (de Jong et al., 2002, Cancer Res 62: 472-479). The fusion of E2 to E6E7SH will provide a vaccine component with antigens for various stages of HPV-related cancers, from persistent infections to invasive cancers after LEEP surgery or recurrent / refractory diseases. Therefore, as a non-limiting example of such an embodiment, the inventor prepared a sequence encoding a fusion protein of E6E7SH and E2 at its N-terminus. Within the E2 sequence, modifications can be made to suppress DNA binding activity that can affect gene expression in cells expressing the fusion protein. The inventor is the wild type HPV16 The E2 protein, glycine at position 293, lysine at position 299, and cysteine at position 300 were mutated to valine, methionine and arginine, respectively. Each of these spontaneous mutations already completely suppresses the binding of E2 to the DNA sequence having the E2 binding domain (Prakash et al., 1992, Genes Dev 6: 105-16).</p><p> The obtained polypeptide is referred to as HPV16 E2E6E7SH and contains SEQ ID NO: 3. A codon-optimized sequence encoding this polypeptide is prepared and provided in SEQ ID NO: 4.</p><p> The inventor also constructed a variant in which the same E2 mutant protein fused to the C-terminus of the HPV16 E6E7SH fusion polypeptide, which yielded a polypeptide called HPV16 E6E7E2SH, containing SEQ ID NO: 5. The sequence encoding this construct is provided as SEQ ID NO: 6.</p><p> For the purposes of control, the inventor also presents a polypeptide having a wild-type sequence in full-length HPV16 E6 and E7 as a fusion protein (E6 from aa1 to 158 fused directly to E7 from aa1 to 98). An array encoding (referred to as E6E7wt in the specification) was constructed.</p><p> The inventor also tested the effect of adding the leader sequence to the polypeptide. As a non-limiting example, a sequence encoding an IgE reader sequence (see, eg, US Pat. No. 6,733,994) [the sequence of the leader peptide is provided in SEQ ID NO: 7] is provided in a construct, eg, an E6E7wt construct giving LSE6E7wt. , Also fused to some N-terminus within the E2E6E7SH construct giving LSE2E6E7SH. The effect was significantly (p &lt;0.05) enhanced immunogenicity compared to the same antigen without the LS sequence, as measured by E7 tetramer analysis in immune mice (eg, as seen in FIG. 9). As you can see).</p><p> The sequences encoding the E6E7SH polypeptides of the invention can be expressed, for example, from DNA constructs, RNA, or viral vectors, with or without E2. FIG. 1 shows the expression of a DNA vector expressing the above-mentioned transgene in HEK-293T cells at the time of transient gene transfer. After gene transfer, cells were collected and cell extracts analyzed by SDS-PAGE and Western blotting using antibodies against HPV16 E7. This experiment shows the expected expression of a fusion protein of appropriate size upon gene transfer of the expression vector.</p><p> An adenoviral vector can be used to express E6E7 with or without E2 and with or without additional sequences to enhance the immunogenicity of the encoded fusion protein. ..</p><p> The gene encoding the HPV16 E6E7wt control or HPV designer sequence described above was a gene optimized and synthesized for human expression by Geneart. The Kozak sequence (5'GCCACC3') was included immediately before the ATG start codon, and two stop codons (5'TGA TAA3') were added at the end of each coding sequence. The gene was inserted into the pAdApt35BSU plasmid and the pAdApt26 plasmid via the HindIII and XbaI sites (Havenga et al., 2006, J Gen Virol 87, 2135-43).</p><p> All adenoviruses were produced by a single homologous recombination in PER.C6 cells and produced as described above (for rAd35: Havenga et al., 2006, J Gen Virol 87: 2135-43; for rAd26). : Abbink et al., 2007, J Virol 81: 4654-63). PER.C6 cells (Fallaux et al., 1998, Hum Gene Ther 9: 1909-17) were maintained in Dulbecco's Modified Eagle's Medium (DMEM) with 10% fetal bovine serum (FBS) supplemented with 10 mM MgCl2. ..</p><p> Briefly, the Ad vector plasmid was gene-transduced into PER.C6 cells using lipofectamine according to the instructions provided by the manufacturer (Life Technologies). Cells were collected 1 day after reaching full cytopathic effect (CPE), thawed, centrifuged at 3,000 rpm for 5 minutes and stored at -20 ° C. The virus was plaque-purified and amplified in PER.C6 cells cultured in a single well of a multi-well 24 tissue culture plate. Further amplification was performed in T25 tissue culture flasks and subsequently in PER.C6 cells cultured in T175 tissue culture flasks. 3-5 ml from crew drysate prepared from cells obtained after the T175 flask was used to inoculate a 24-x T1000 5-layer tissue culture flask with a 70% confluent layer of PER.C6 cells. The virus was purified using a two-step CsCl purification method. Finally, the virus was stored in an aliquot at -85 ° C.</p><p> Ad35.HPV16-E6E7wt, and Ad35.HPV16-E6E7SH are wild HPV16 E6 and E7 protein fusion proteins (E6E7wt), as well as designer fusion protein variants as described above (E6E7SH, amino acid sequences provided by SEQ ID NO: 1). A recombinant adenovirus serotype 35 (Ad35) vector containing a codon-optimized nucleotide sequence for each expression of). The combined E6 and E7 sequences were placed under the control of the CMV promoter within the E1 region of the adenovirus genome lacking E1, E3. Ad26.HPV16-E6E7wt and Ad26.HPV16-E6E7SH are equivalent vectors based on recombinant adenovirus serotype 26.</p><p> Similarly, we generated recombinant adenovirus vectors based on Ad26 and Ad35 encoding the HPV16 E2E6E7SH (SEQ ID NO: 3) mutant. Similarly, Ad26 and Ad35 encoding the HPV16 E6E7E2SH (SEQ ID NO: 5) mutant were generated. We also generated an Ad35 vector encoding an E2E6E7SH fusion protein with an IgE leader sequence at the N-terminus, and named it Ad35.HPV16-LSE2E6E7SH. We also generated a control adenovirus with E6E7wt fused to the IgE leader sequence at the N-terminus.</p><p> Recombinant adenovirus was generated in PER.C6 cells and purified by centrifugation on a cesium chloride gradient.</p><p> Further examples of the constructs of the invention coupled to the repressor system are provided in the examples described below.</p><p>Example 2. Deficiency of transforming activity in designer constructs Wild-type HPV16 E6 and E7 proteins have the apparent potential for tumorigenesis as transforming activity in a particular assay (eg, colonization in a soft agar assay) (Massimi and Banks, 2005, Methods Mol Med 119: 381-395). The E6E7SH polypeptide described in Example 1 comprises fragments of the E6 and E7 proteins in a rearranged fashion. This is expected to rule out the possibility of tumorigenesis, as can be measured, for example, by significantly reduced transformation activity compared to any of the wild-type E6 and E7 proteins in such assays.</p><p> Others have reported that genetically shuffled mutants of HPV 16 E6 and E7 naturally lose their carcinogenic potential (Oehlschlaeger et al., 2006, Vaccine 24: 2880-93; Henken et al., 2012, Vaccine 30: 4259-66), which indicates that gene shuffling disrupts the wild-type function of the E6 and E7 proteins.</p><p> To assess the lack of tumorigenicity, the inventor found that our E6E7SH construct (eg, as described in Massimi and Banks, 2005, Methods Mol Med 119: 381-395) in soft agar on NIH3T3 cells. The ability to impart proliferative capacity was evaluated. Transfection of a plasmid expressing wild-type HPV16 E7 into NIH3T3 cells resulted in consistent colonization. In these assays, expression of wild-type HPV16 E6 alone did not result in cross-background colonization. This is consistent with published observations that E7wt is much more efficient than E6wt in this assay (Sedman et al., 1991, J Virol 65: 4860-66). The gene transfer of our E6E7SH construct did not result in the proliferation of cell colonies in soft agar in four independent experiments (Fig. 2), which is a trait in which the nucleic acid encoding the polypeptide E6E7SH of the present invention is associated with E7. Showed that he has lost his ability to convert.</p><p> The potential for tumorigenesis of E6 and E7 is associated with their ability to reduce levels of cellular proteins p53 and pRb, respectively. P53 and pRb degradation assays were performed to show that the nucleic acid constructs encoding the polypeptides E6E7SH of the present invention do not have the biological activity associated with wild-type E6 and E7 at the molecular level. That is, HPV16 E6wt and our E6E7SH construct were expressed in NCI-H1299 cells lacking endogenous p53 in the p53 degradation assay. HPV16 in the pRb degradation assay E7wt and E6E7SH constructs were expressed in pRb null Saos-2 cells. As can be seen in Figure 3, co-expression of p53 with E6wt (as opposed to E6E7SH) results in reduced p53 levels (panels A and B). Similarly, panels 3C and 3D show that co-expression of pRb with E7wt (as opposed to E6E7SH) results in reduced pRB levels. These data show that the nucleic acids encoding the polypeptides of the invention have no colony forming ability in soft agar, and that the major biological activities of the wild-type E6 and E7 polypeptides, namely p53 and pRb, respectively. It shows that it does not have inactivation.</p><p> To further demonstrate the safety of the nucleic acid construct encoding the polypeptide of the invention, the inventor used primary human foreskin keratinocytes, which are natural target cells for HPV-mediated transformation. Immortalization of primary human keratinocytes requires the action of both E6 and E7 wild-type (Munger et al., 1989, J Virol 63: 4417-21). This assay is probably the most physiologically relevant in vitro assay to demonstrate the safety of our construct (Massimi and Banks, 2005, Methods Mol Med). 119: 381-395). Lentivirus-transduced cells expressing wild-type E6 and E7 (E6E7wt) derived from HPV16 have an extended lifespan compared to non-transduced control cells (Fig. 4) and hTERT, a catalytic subunit of telomerase. Induces immortality in primary keratinocytes, as indicated by activation of (data not shown). Expression of the polypeptide of the invention (E6E7SH) cannot prolong lifespan compared to GFP-transduced or non-transduced keratinocytes. Similar results were obtained with two additional independent donors (data not shown). Taken together, these data show that our constructs have lost the ability to induce immortality in primary human keratinocytes, which is considered a highly physiological model.</p><p> Another construct in which fragments of HPV16 E6 and E7 were recombined in a different order also failed to immortalize the primary human foreskin keratinocytes. However, an extension of life of up to about 120-150 days was observed in the construct. This has shown some unpredictability in the art and demonstrates the superiority of designer molecules according to the invention in this safety-related aspect.</p><p> The experiments in this example simultaneously provide strong evidence of the lack of transforming activity of the nucleic acid encoding the polypeptide according to the invention and significantly improved safety over the HPV16 E6 and E7 wt constructs.</p><p>Example 3. Immune Response to E6E7SH Designer Constructs The inventor has prepared a DNA vector and an adenovirus vector as described in Example 1.</p><p> The inventor found that mice were immunized with wild-type E2, or a DNA plasmid encoding E6 or E7, and were more broadly cell-mediated than C57BL / 6 or Balb / c mice by immunization with HPV16 E2, E6 and E7 antigens. Based on the initial experiment when the response was induced by CB6F1, a CB6F1 mouse strain was used to measure the immune response. In separate experiments, mice were immunized with a DNA vector encoding the molecule of the invention and the cellular immune response was measured. An immune response specific for HPV16 E7 could be measured in mice immunized with a DNA plasmid expressing E6E7SH (Fig. 5).</p><p> The following data shown in this example are obtained from mouse experiments performed using an adenovirus vector.</p><p> To assess vaccine-induced immunogenicity, CB6F1 mice were immunized with an adenovector expressing E6E7wt, LSE6E7wt, E6E7SH (Ad35) or an adenovector not encoding a transgene (Empty). 5 × 10 for administration to mice⁹Virus particles (vp) and 1x10¹⁰Two doses of vp were tested. Two and eight weeks after immunization, mice were sacrificed and isolated splenocytes were stimulated overnight with a 15 mer peptide pool of HPV 16 E7. E7-specific responses after 2 and 8 weeks were analyzed by IFNγ ELISPOT. The data is shown in Figure 6.</p><p> This indicates that mouse Ad35.HPV16-E6E7SH immunization induces an E7-specific immune response, as measured by ELISPOT analysis. Furthermore, the results in FIG. 6 indicate that the addition of the N-terminal leader sequence to the transgene may enhance the immune response to the transgene expressed by adenovirus.</p><p> Next, the effect of adding E2 to the E6E7SH polypeptide was tested for immunogenicity. The Ad35 vector encoded a polypeptide having E2 fused to either the N-terminus (E2E6E7SH) or the C-terminus (E6E7E2SH). CB6F1 mouse, 1x10¹⁰Immunized at a dose of vp. Figures 7 (E7 tetramer staining) and Figure 8 (Panel C, IFNγ ELISPOT) show immune responses to E7, with immune responses tending to be higher for designer constructs containing E2 than for constructs without E2. However, the difference was not statistically significant. The response to E2 was higher for the adenoviral vector encoding E2 only than for the adenoviral vector with E2 fused to the E6E7SH designer polypeptide (Figure 8B), with a difference of E2 vs. E2E6E7SH and E2. It was significant for both E6E7E2SH (P value: &lt;0.05).</p><p> It can be concluded that a designer construct that further contains E2 can provide an additional immune response to E7, and in addition an immune response to E2, thus increasing the range of immune responses compared to a construct that does not contain E2.</p><p> Addition of the leader sequence has been shown to result in a higher E7-specific response when fused to the N-terminus of the wild-type E6 and E7 fusion proteins (Fig. 6C). Similarly, the effect of the leader sequence on the immunogenicity of the E2E6E7SH fusion protein was determined. Therefore, the Ad35 vector encoding the designer polypeptide with or without N-terminal E2 and the Ad35 vector encoding LSE2E6E7SH were used for mouse immunization, and 2 blood samples were used to measure the E7-specific immune response. Collected at weekly intervals (Fig. 9). As shown in Figures 7 and 8, the presence of E2 at either the N-terminus or the C-terminus fused to E6E7SH was more likely to enhance the immune response. The addition of the IgE leader sequence further enhanced the E7-specific response (Fig. 9B). All three adenovirus vectors encoding designer molecules according to the invention showed a sustained immune response over time, and the highest post-immune response corresponded to the highest response over the duration of the experiment.</p><p> It is concluded that the response induced by the designer construct further containing the N-terminal E2 can be enhanced by the addition of specific sequences that target the encoded protein to specific cellular compartments, such as the IgE leader sequence.</p><p> The cell-mediated immune response to the peptides of the invention can be induced using different types of adenoviral vectors. In previous experiments, the inventor used the Ad35 vector, while in the experiment of FIG. 10, mice were immunized with the Ad26 adenovirus vector expressing E2E6E7SH. The data show that immunization with an Ad26-based vaccine also induced E7-specific T cells. Furthermore, the results show that the second immunization with the Ad35 adenovirus vector expressing E2E6E7SH further boosted the cellular immune response (Fig. 10).</p><p>Example 4. Immunogenicity of Designer Constructs in Rhesus macaques To assess the ability of an adenovirus vector expressing the designer sequence of the invention to induce an immune response in non-human primates, an adenovector expressing E2E6E7SH. (Ad26) or 1x10 adenovector (Empty) that does not encode a transgene¹¹Rhesus macaques were immunized by intramuscular injection at a dose of vp. Eight weeks after immunization, the immune response was boosted by immunization with an Ad26 vector expressing the same antigen. After 16 weeks, the animals received another injection of the Ad35 vector expressing the same antigen. Blood samples were taken at several time points and isolated leukocytes were stimulated overnight in a peptide pool corresponding to HPV16 E2, E6 or E7. The specific response was measured by IFNγ ELISPOT. The data is shown in Figure 11. In addition, 10 and 18 weeks after prime immunization, peptide-specific cellular immune responses across novel junctions in the present invention were evaluated. Induction of IFNγ response is &lt;50 SFU / 1 × 10 in all animals⁶It was below the detection limit of PBMC (data not shown).</p><p> The data show that immunization of non-human primates with Ad26.HPV16-E2E6E7SH resulted in a cellular immune response against all three HPV16 proteins present in the encoded transgene, not against novel conjugation. Is shown. Additional immunization with Ad26.HPV16-E2E6E7SH could boost the response, and additional boost after 16 weeks with the corresponding Ad35 vector further enhanced the immune response specific for HPV16 E2, E6 and E7.</p><p> The late booster administration of Ad26.HPV16-E2E6E7SH 72 weeks later caused an increase in the HPV16 cell immune response again, which decreased after a few weeks (not shown).</p><p> In another experiment (not described), rhesus macaques were immunized by vaginal administration of a combination of two adenoviral vectors, one expressing HPV16 E6E7SH and the other expressing HPV16 L1 protein. A low but measurable cellular response to both E6 and E7 was measured within peripheral mononuclear blood cells. In these experiments, a strong cellular immune response to L1 was detected.</p><p>Example 5. Therapeutic effect in a mouse tumor model The polypeptide of the present invention can induce an HPV16-specific cellular immune response in animals and has a therapeutic effect on cells expressing HPV16 E6 and / or E7. Can be demonstrated. Therapeutic immunity, i.e. immunity after tumor growth has begun, can be used to demonstrate the efficacy of therapeutic HPV vaccine candidates. The therapeutic effects of the Ad26 and Ad35 vectors were tested in mice injected with TC-1 cells (mouse cells expressing HPV16 E6 and E7) (Lin et al., 1996, Cancer Res 56: 21-6). TC-1 cells will form solid tumors within days to weeks after subcutaneous injection in mice. In the absence of the vaccine, the tumor grew rapidly and reached a predetermined size of 1000 mm3 within 30 days (panels D and E). When this size is reached, the mice are slaughtered for ethical reasons.</p><p> Adenovirus expressing SLP (used as a positive control; Kenter et al., 2009, N Engl J Med 361: 1838-47; Zwaveling et al., 2002, J Immunol 169: 350-8) or HPV16-E2E6E7SH A significant reduction in TC-1-induced tumor growth was observed with the vector-based prime-boosted immune scheme (Figure 12, Panels B and C). More detailed examination (panels F and G) on the first 30 days after prime immunization shows that immunization with an adenovector expressing E2E6E7SH has a substantially greater effect on tumor growth than immunization with SLP. Is shown. The initial growth rate was much slower and in most cases the tumor contracted. Tumors were completely eradicated in 3 of 11 mice immunized with the adenovirus vector, which is reflected in the survival plot (Panel H).</p><p> In conclusion, immunization with adenoviral vectors expressing the polypeptides of the invention significantly reduced tumor growth or completely eradicated colonized tumors in a well-established loading model for HPV16-induced cancers.</p><p>Example 6: Utilization of a repressor system to improve the productivity and genetic stability of an adenovirus vector expressing an HPV-derived antigen Inserted into an adenovirus vector under the control of a strong and constitutively active promoter It has been previously reported that transgenes can have a negative effect on vector production depending on the properties of the transgene product (Yoshida &amp; Yamada, 1997, Biochem Biophys Res Commun 230: 426-30; Rubinchik et. al., 2000, Gene Ther 7: 875-85; Matthews et al., 1999, J Gen Virol 80: 345-53; Edholm et al., 2001, J Virol 75: 9579-84; Gall et al., 2007 , Mol Biotechnol 35: 263-73). Examples of productivity challenges for transgene-dependent vectors are inefficient vector rescue and proliferation, low final vector yields, and, in severe cases, rapid rapid transgene with defective transgene cassettes. Proliferation. To solve these problems, multiple studies explored the possibility of suppressing vector / transgene expression during vector replication in producer cells (Matthews et al., 1999, J Gen Virol). 80: 345-53; Edholm et al., 2001, J Virol 75: 9579-84; Gall et al., 2007, Mol Biotechnol 35: 263-73; Cottingham et al., 2012, Biotechnol Bioeng 109: 719-28 Gilbert et al., 2014, J Virol Methods 208: 177-88). In this regard, different inhibitory systems are pre-equipped in the context of Ad vectors, which, of course, improve vector productivity and genetic stability in vectors encoding different types of (inhibitory) transgenes. It is shown that.</p><p> Some of the adenovirus vectors described herein, as well as some other adenovirus vectors encoding specific HPV antigen variants, present some of the productivity challenges of the transgene-dependent vectors described above. However, it was acknowledged that it could probably be further improved in that regard. Therefore, the inventor can improve the production characteristics of Ad vectors expressing HPV-derived antigens as described herein by using a system for suppressing the expression of a vector / transgene. I tried to consider. To this end, the inventor has two existing repressor-operator systems, namely TetR / TetO (Yao &amp; Eriksson, 1999, Hum Gene Ther 10: 419-22, European Patent No. 0990041B1) and CymR /. CuO (Mullick et al., 2006, BMC Biotechnol 6:43) was installed on our adenovirus vector platform. Both TetR / TetO and CymR / CuO strains have been used in advance by others to improve adenovirus vector productivity through vector-transgene silencing during vector replication (). Gall et al., 2007, Mol Biotechnol 35: 263-73; Cottingham et al., 2012, Biotechnol Bioeng 109: 719-28; Gilbert et al., 2014, J Virol Methods 208: 177-88). The equipment of these two systems involved the production of adenovirus vectors expressing the gene of interest under the control of the CMV promoter with either the TetO or CuO sequence. In addition, the equipment required the production of cell lines that stably express each cognate repressor protein (ie, TetR or CymR).</p><p> Several E1-deficient Ad26 and Ad35-based vectors were generated in which the sequence encoding the heterologous polypeptide was operably linked to the CMV promoter with either the TetO or CuO operator sequence. First, specific TetO or CuO-containing sequences (SEQ ID NO: 11 and SEQ ID NO: 12, respectively) were inserted near the transcription initiation site (TSS) of the CMV promoter (SEQ ID NO: 13) of the pAdapt26 and pAdapt35.Bsu plasmids. (Abbink et al., 2007, J Virol 81: 4654-63; Havenga et al., 2006, J Gen Virol 87: 2135-43). The operator-containing sequence was inserted at exactly the same position as above for the two systems of CMV promoters (Yao &amp; Eriksson, 1999, Human Gene Ther 10: 419-22; European Patent No. 0990041B1; Mullick et. al., 2006, BMC Biotechnol 6:43; European Patent No. 1385946B1). For more information, TSS (as originally assigned; Stenberg et For al., 1984, J. Virol. 49: 190-9), the TetO and CuO-containing sequences were inserted directly downstream of positions -20 and +7, respectively. In SEQ ID NO: 13, these two positions correspond to positions 716 and 742, respectively. The obtained operator-containing CMV promoters are referred to as CMVTetO and CMVCuO, respectively. Different transgenes were then inserted downstream of the (modified) CMV promoter of the resulting construct using HindIII and XbaI restriction sites. These transgenes are genes encoding a fusion protein of green fluorescent protein and luciferase (GFP-Luc), LSE2E6E7SH from the present invention, and another polypeptide having some similarities to LSE2E6E7SH (in this example, " A construct called "HPVAg") was included. The HPVAg contains the same reader sequences that are present in LSE2E6E7SH, as well as the E2, E6, and E7 sequences of HPV16. Using the methods described herein, the resulting modified pAdapt26 and pAdapt35.Bsu plasmids are adenoviral vectors that express the above reporter and HPV transgene under the control of either the CMVTetO or CMVCuO promoter. Was used for the production of.</p><p> Cell lines expressing either TetR or CymR using PER.C6® with derivatives of plasmid pcDNA 6 / TR (Life Technologies, V1025-20) and pcDNA 6 / TR, respectively. ) Created by stable gene transfer of cells, where the TetR coding sequence (SEQ ID NO: 14 encoding polypeptide SEQ ID NO: 15) is a codon-optimized CymR coding sequence (SEQ ID NO: 16 encoding plasmid SEQ ID NO: 17). ) Replaced by. The production of stable cell lines is predominantly pcDNA 6 / TR using a transient gene transfer-based assay to screen for cell clones capable of suppressing the expression of CMVTetO or CMVCuO driven genes. It was carried out according to the instructions given by the supplier. The resulting PER.C6 / TetR and PER.C6 / CymR cell lines were analyzed for their ability to suppress transgene expression during vector replication within these cells. Experiments performed with vectors expressing GFP-Luc under the control of an operator-containing CMV promoter show that throughout the complete viral replication cycle in cell lines expressing the repressor corresponding to each operator sequence. A at least 10-fold reduction in luciferase gene expression was shown (data not shown). This confirmed that the PER.C6 / TetR and PER.C6 / CymR cell lines have the ability to suppress the expression of the vector / transgene in relation to the replication of the adenovirus vector.</p><p> The effect of adenovirus / transgene expression on TetR and CymR-mediated inhibition on vector yield was investigated for Ad35-based vectors expressing HPVAg (Fig. 13A). Therefore, for PER.C6, PER.C6 / TetR, and PER.C6 / CymR cell lines seeded at 3 x 105 cells / well in a 24-well plate well, 1000 virus particles / cell lasted for 3 hours. Four infections were performed with a vector expressing HPVAg from either the CMVTetO or CMVCuO promoters over time. As a control, parallel infection was performed using the corresponding vector expressing GFP-Luc instead of HPVAg. Four days after infection, Crudevirus lysate was prepared by subjecting the contents of the wells (ie infected cells and medium) to two freeze-thaw cycles. The adenovector titer was then measured by a protocol based on quantitative PCR specific for the Ad35 hexone sequence using a purified Ad35 vector with known virus particle titers as a standard. The results show that the Ad35 vector encoding HPVAg containing both TetO and CuO shows a reduced vector yield in normal PER.C6 cells compared to the control vector expressing GFP-Luc. .. In contrast, these same vectors yield as high yields as those obtained with the control vector when produced in cells expressing their cognate repressors (ie, TetR and CymR, respectively). Was done. These data indicate that suppression of transgene expression during vector production in producer cells can be beneficial to the productivity of Ad35 vectors carrying HPVAg as the transgene.</p><p> The effect that suppression of adenovirus transgene expression may have on vector yield was also examined for vectors derived from adenovirus serotype 26 (Ad26) (Fig. 13B). In the assay performed essentially as described above for the Ad35 vector, the Ad26 vector carrying the transgene controlled by the CMVTetO promoter, which encodes either GFP-Luc, HPVAg, or LSE2E6E7SH, was PER.C6 and PER. .C6 / TetR cells were used to infect with 1500 virus particles / cells. After 3 days, the infectious agent was collected and the virus particle titers were measured by a method based on quantitative PCR specific for Ad26 hexone sequences. The results show that the yield in the vector encoding HPVAg and LSE2E6E7SH in PER.C6 cells is lower than that obtained with the control vector encoding GFP-Luc. In contrast, in PER.C6 / TetR cells, both of these vectors showed as high titers as those obtained with the control vector. Summarizing the above results (in the Ad35 vector), these data show that suppression of transgene expression during adenovector production increases the yield of vectors expressing HPVAg and LSE2E6E7SH.</p><p> The inventor acknowledges a major challenge regarding the genetic stability of adenovirus vectors carrying the CMV promoter-driven transgene for HPVAg. For example, after several passages of this vector on PER.C6, it was found that the majority of the vector population consisted of mutant vectors carrying large deletions in the HPVAg coding sequence (data not shown). ).</p><p> The inventor can utilize a transgene expression-suppressing system, eg, one of the above two, to prevent genetic stability issues associated with HPVAg, which is inhibitory to the growth of transgenes, eg, vectors. Judging that it could be done. To test this, an Ad35-based vector with expression of HPVAg driven by the CMVCuO promoter was evaluated for the stability of the transgene cassette during growth of the vector in PER.C6 or PER.C6 / CymR cells. (Fig. 14). That is, the vector DNA was transfected into two different cell lines and the resulting viral plaque was grown under the agarose layer. Five viral plaques were isolated from each of the two gene transfers and then passaged separately over 10 consecutive viral passages in the same cell line (ie, the one used for gene transfer). Transgene integrity was assessed by PCR amplification of the transgene cassette at viral passage number 10 (VPN10), followed by gel electrophoresis of the resulting PCR product and analysis by Sanger sequencing. In addition, VPN7 passaged virus clones were evaluated for their ability to express HPVAg. It uses a passaged virus isolate to infect A549 cells with 1000 viral particles / cells, lyses the cells 48 hours after infection, and then expresses HPVAg specific to HPV16 E7. It was performed by analysis by Western blotting (Santa-Cruz Biotechnology) using a monoclonal antibody. According to the results of gel electrophoresis and sequencing analysis, all five virus isolates, each of which had been passaged to PER.C6, had a slight frameshift deletion or premature arrest in the transgene cassette. It has been shown to carry any of the mutations. In contrast, such deletions or mutations could not be detected in any of the vector isolates that had been passaged on the CymR expressing cell line (PER.C6 / CymR). Matching these data, all PER. While C6 / CymR growth vector isolates were able to express HPVAg, all PER.C6 growth vectors completely lost this ability, suggesting defect transgene cassettes in these vectors. In conclusion, our data show that the use of repressor systems, such as the CymR / CuO system, to suppress the expression of vector transgenes during vector growth carries transgenes that express HPVAg, eg. 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Array<chemistry num="1"><img file="JP6606571B2_D0001.tif" /></chemistry><chemistry num="2"><img file="JP6606571B2_D0002.tif" /></chemistry><chemistry num="3"><img file="JP6606571B2_D0003.tif" /></chemistry><chemistry num="4"><img file="JP6606571B2_D0004.tif" /></chemistry><chemistry num="5"><img file="JP6606571B2_D0005.tif" /></chemistry><chemistry num="6"><img file="JP6606571B2_D0006.tif" /></chemistry>SEQ ID NO: 7 (IgE leader peptide amino acid sequence) MDWTWILFLVAAATRVHS SEQ ID NO: 8 (nucleotide sequence encoding IgE leader peptide) ATGGACTGGACCTGGATCCTGTTCCTGGTGGCTGCCGCAACCCGGGTGCACAGC SEQ ID NO: 9 (aa HAVT20 leader peptide amino acid sequence) MACPGFLWALVISTCLEFSMA<chemistry num="7"><img file="JP6606571B2_D0007.tif" /></chemistry>SEQ ID NO: 11 (2xTetO-containing sequence) GAGCTCTCCCTATCAGTGATAGAGATCTCCCTATCAGTGATAGAGATCGTCGAC SEQ ID NO: 12 (CuO-containing sequence) AACAACAGACAATCTGGTCTGTTTGTA<chemistry num="8"><img file="JP6606571B2_D0008.tif" /></chemistry><chemistry num="9"><img file="JP6606571B2_D0009.tif" /></chemistry><chemistry num="10"><img file="JP6606571B2_D0010.tif" /></chemistry><chemistry num="11"><img file="JP6606571B2_D0011.tif" /></chemistry><chemistry num="12"><img file="JP6606571B2_D0012.tif" /></chemistry>SEQ ID NO: 18 (HPV16 E6, aa41 ~ 65) KQQLLRREVYDFAFRDLCIVYRDGN SEQ ID NO: 19 (HPV16 E7 aa43 ~ 77) GQAEPDRAHYNIVTFCCKCDSTLRLCVQSTHVDIR</p>