Untitled record

Full description

Sister Ar'a's background

The invention relates to the field of medicine and more specifically to nucleic acid and polypeptide components that can be used in therapeutic vaccines against human papillomavirus type 16.

The human papillomaviruses (HPVs) family consists of more than 100 species (also referred to as HPVs).

<p dir="rtl">5 subtypes) that are capable of infecting keratinocytes of the skin or mucous membranes. Up to 40 types of HPV have typically been transmitted through sexual contact and anogenital forms of HPV are very common in both men and women. Some types of HPV transmitted can cause Persistent infections with “high-risk” HPV types (i.e., types 16, 18, 31, 45) can be exacerbated by sexual intercourse.</p>

<p dir="rtl">10 Different from those that cause skin warts - even sites of pre-cancerous and invasive cancer lesions, that is, the cervix, vulva, vagina, penis, oropharynx and anus. Most forms of HPV infection disappear on their own within a year or two after infection. In healthy individuals, the turnover of CD4+ Th1 and Th2 T cells specific for early viral proteins E6, E2 and E7 from HPV-16, as well as CD8+ T cells specific for E6, moving into the skin upon antigenic testing, indicates</p>

<p dir="rtl">15 Successful defense against HPV-16 infection is commonly associated with a systemic T cell response against these early viral antigens. In a small number (approximately 1%) of infected individuals, HPV infection persists, eventually resulting in new neoplastic lesions in the genital organs. Among the high-risk HPVs, HPV16 and HPV18 are the major cause of cervical cancer, and together they cause About 70% of these cases, these two types also play a major role</p>

<p dir="rtl">20 In other HPV-induced cancers such as anal and oropharyngeal cancer. Globally, HPV is one of the most important infectious agents that result in cancer.</p>

7690

-3-

Vaccination against HPV is a feasible strategy to reduce the incidence or effects of infection with HPV

(van der Burg and Melief, 2011, Curr Opinion Immunol 23:252–257).

Prophylactic HPV vaccines, based on virus-sized particles (VLPs) formed by the L1 (envelope) protein of HPV types 16 and 18, are highly effective in preventing persistent infection5 and related disease with HPV16 and HPV18. These vaccines are thought to provide sterile immunity

By inducing neutralizing antibodies against L1 proteins. The addition of L1-based VLPs from high-risk HPV types could also increase the breadth of protection conferred by these vaccines.

However, while these vaccines can protect against primary infection (i.e., are protective), 10 there is no evidence of a beneficial effect on persistent genital lesions caused by HPV16 and HPV18.

JAMA298:743-53 (Hildesheim et al., 2007, so that they cannot be considered therapeutic vaccines against HPV).

Despite the introduction of these prophylactic vaccines, a significant number of people have already acquired or are at risk of developing persistently high risk of HPV infection and, therefore, are at risk of developing cancer. Vaccines are therapeutic to get rid of forms of infection

Persistent and mother-related HPV is an urgent unmet medical need.

Some attempts have been described that address this need. For example, in vitro experiments have been performed with a variety of different vaccination strategies, such as a fusion protein consisting of a heat shock protein (Hsp) from Mycobacterium bovis and HPV-16 E7, or a fusion protein consisting of a heat shock protein (Hsp) from Mycobacterium bovis and HPV-16 E7.

<p dir="rtl">20 Fusion of E7, E6 and L2 of 16-HPV and 18-L1-E7VLPs, chimeric HPV, poxviruses resulting from genetic recombination, expressing either E6 and E7 of 16-HPV and 18-HPV, or bovine papillomavirus E2, DNA vaccines expressing epitopes A patch of CTLs of E6 and E7 for HPV types 16-18 and 18-HPV, attenuated Listeria monocytogenes (Lm) that secretes HPV-16 E7 antigen, and synthetic long peptides (SLPs) that include HPV-16 E6 and E7 peptides.</p>

<p dir="rtl">25 While some of these methods show some laboratory efficacy, it is limited, and most have failed, highlighting the need for improvement in current strategies.</p>

7690

-4-

Integration of HPV proteins from early E6 and E7 is a necessary step in the process from infection to cancer, and sustained expression of E6 and E7 is required to maintain the tumor phenotype.

New cervical cancer cells. Therefore, E6 and E7 are good targets for therapeutic vaccination. As mentioned, some studies have shown that therapeutic vaccination for women with HPV is high risk

<p dir="rtl">5 It would induce recurrence in existing lesions. Kenter et al demonstrated prolonged and complete relapse in 47% of patients with vulvar interepithelial neoplasia (VIN) using SLPs derived from HPV16 E6 and E7 proteins and an adjuvant as a therapeutic vaccine (Kenter et al., 47). (2009, N Engl J Med 361:1838). Likewise, in a study a protein-based vaccine was combined (TA-CIN), consisting of an HPV16 fusion protein of E6 and E7.</p>

<p dir="rtl">10 and L2) with local immunomodulation in VIN 2/3 patients, showed complete remission in 63% of</p>

Disadvantages include patients (Daayana et al., 2010,Br J Cancer 102:1129-36).

Potential for long synthetic peptides in vaccine form Manufacturability at high volume levels and associated costs, need for an adjuvant generating potential cross-reactivity and adverse effects associated with vaccination (particularly pain and swelling). Due to the high level of discomfort it is unlikely that

<p dir="rtl">15 SLPs at an early stage of the disease when the ER rate is still high. Likewise, due to the need for topical imiquimod treatment in the case of TA-CIN treatment, tolerability is a notable issue as most women experience local and systemic side effects that persist for the duration of imiquimod treatment, affecting daily activities.</p>

A potential alternative is to use a nucleic acid-based vaccination such as DNA vaccines or viral vaccines 20 encoding the HPV protein E6 and/or E7 for vaccination.

However, HPV proteins E6 and E7 have oncogenic potential and therefore vaccination with vaccines containing nucleic acids encoding these proteins imposes a step towards cellular transformation.

Result of extended expression potential of antigens.

Therefore, in the case of genetic vaccination, non-oncogenic/detoxified forms may be used

<p dir="rtl">25 From E6 and/or E7 in order to rule out any risks of cell transformation as a result of vaccination. Loss of the oncogenic potential of untreated E6 and E7 is most commonly achieved by deletion and/or deletion</p>

7690

-5-

Substitution of residues known to be important for the function of these proteins (i.e.,...Smahel et al 281:231-38 2001, Virology; 27:40–431 Yan et al., 2009, Vaccine;

Wieking et al., 2012, Cancer Gene Ther 19: 667-74; WO 2009/106362

However, a drawback of these approaches is that they carry the risk of removing T cell epitopes

<p dir="rtl">5 and/or the introduction of new unwanted T cell epitopes into proteins, which may not lead to the desired immune response.</p>

In an alternative strategy to eliminate the neo-oncogenic potential of HPV from E6 and E7, modified forms (i.e., polypeptides in which protein fragments of the unprocessed type are rearranged) have been constructed from E6 and E7 proteins (i.e., Öhlschläger 24:93–2880). et al., 2006,Vaccine;

Oosterhuis et al., ;Oosterhuis et al., 2011, Int J Cancer 129: 397-406 10

Hum Gen Ther 23:12-1301 2012. However, these approaches still require the manufacture, formulation and administration of multiple molecules to ensure inclusion of all epitope peaks from both the E6 and E7 proteins, resulting in suboptimal logistic requirements and much higher costs. Furthermore, strategies described for the introduction of strong anomalous adhesive peaks were not presented in E6 and E7.

<p dir="rtl">15 Since immune responses may be diverted from the relevant E6/E7 epitopes to abnormal epitopes, the described constructs may not have optimal immunogenic properties.</p>

The closest industrial technology cases related to the subject of invention are the documents: A- Patent application (international) No. 2013083287, dated 06/13/2013. B- Application for patent (international), No. 2009106362, dated 09/03/2009.

20 C- Patent application (America) No. 2007014810 dated 01/18/2007.

OHLSCHLAGER P ET AL, "An improved rearranged Human Papillomavirus Type 16 E7 DNA vaccine candidate (HPV-16 E7SH) induces an E7 wildtype-specific T cell response", VACCINE, ELSEVIER LTD, GB 04/05/2006.

KOEN OOSTERHUIS ET AL, “Preclinical development of highly effective and safe DNA vaccines directed against HPV 16 E6 and E7

7690

-6-

.15/07/2011

However, document (C) revealed a multi-epitope containing parts of HPV16, although it has some laboratory effectiveness, but it is limited.

While document (B) revealed the use of non-generating images of tumors that still contain the name

<p dir="rtl">5 E6 and/or E7 in order to rule out any risks of cellular transformation as a result of vaccination. Loss of the oncogenic potential of unprocessed E6 and E7 is commonly achieved by deletion and/or substitution of residues known to be important for the function of these proteins, but carries the risk of removing T cell epitopes or introducing unwanted T cell epitopes. New proteins, which may not lead to the desired immune response.</p>

<p dir="rtl">10 Reference D explores an alternative strategy to eliminate the oncogenic potential of HPV from E6.</p>

<p dir="rtl">and E7, but methods of manufacturing, formulating, and administering multiple molecules to ensure inclusion of all binding epitopes from both E6 and E7 proteins result in suboptimal logistic requirements and significantly higher costs.</p>

While Exhibit A disclosed early molecules of some potency in the form of HPV vaccines, each of these molecules had one or more shortcomings. The polypeptide molecules of the invention are characterized by having a desirable safety profile, as acid is eliminated Nuclear power is powerful, easy to manufacture and economically feasible.

Reference E revealed the preclinical development of highly effective E6 and E7 HPV vaccines, but the potential drawbacks of long synthetic peptides in vaccine form are the need for an adjuvant 20 generating potential cross-reactivity and adverse effects associated with immunization.

Thus, there remains a need in the field for therapeutic vaccines against HPV, preferably with fewer drawbacks than previously described approaches.

General description of the invention

The present invention provides nucleic acid molecules encoding polypeptides comprising substantially all 25 of the T cell epitopes available from HPV16 E6 and E7 cancer proviruses, but nevertheless

7690

-7-

It is strongly reduced (compared to wt E6 and E7), even to undetectable transforming activity, by including fragments of re-recorded E6 and E7 proteins, while at the same time containing a minimal number of new unwanted epitopes. Contrast with molecules previously shown by others.

<p dir="rtl">5 The invention provides a nucleic acid fragment encoding a polypeptide comprising a sequence as stated in Sequence ID No.: 1.</p>

The encoded polypeptide also includes a leader sequence.

In certain embodiments, the encoded polypeptide also includes at least one epitope from the human papillomavirus (HPV) E2 protein, for example the HPV16 E2 protein.

<p dir="rtl">10 E2 to reduce DNA binding, for example by deletion or mutagenesis in its DNA binding domain. In certain embodiments, the encoded polypeptide comprises a sequence as given in SEQ ID NO: 3 or SEQ ID NO: 5.</p>

In certain embodiments, the DNA sequence is codon optimized, for example for genetic expression in human cells.

<p dir="rtl">15 In certain embodiments, the DNA sequence comprises a sequence as given in SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6.</p>

The invention also provides a vector comprising a nucleic acid portion according to the invention, wherein the sequence encoding the polypeptide is operatively ligated to an enhancer.

In certain embodiments, the vector is a DNA vector such as a plasmid. In other incarnations it is

<p dir="rtl">20 A vector is a viral vector, such as an MVA vector or a genetically recombinant adenoviral vector.</p>

In certain preferred embodiments, the vector is a genetically recombinant adenovirus.

In certain embodiments, the enhancer in the vector is operatively coupled to a repressor operator sequence, to which a repressor protein can bind so that genetic expression of the enhancer is suppressed in the presence of said repressor protein. In certain embodiments, the repressor sequence is a TetO sequence or a CuO sequence.

<p dir="rtl">25 The invention also provides a vaccine composition comprising a vector according to the invention, and a pharmaceutically acceptable excipient.</p>

The invention also provides a method for inducing an immune response against HPV, specifically HPV16, in a subject,

7690

-8-

10

15

The method includes administering to the subject a vaccine composition according to the invention. The invention also provides a vaccine according to the invention for use in inducing an immune response against HPV, specifically HPV16. In certain embodiments, the vaccine is administered to the subject more than once.

The invention also provides a method for treating any of: persistent HPV infection (particularly persistent HPV16 infection), VIN, cervical dysplasia (CIN), vaginal intraepithelial neoplasia (VaIN), anal intraepithelial neoplasia (VaIN). (AIN), cervical cancer (such as cervical squamous cell carcinoma (SCC), oropharyngeal cancer, penile cancer, vaginal cancer or anal cancer in a subject, the method comprising administering a vaccine to the subject according to the invention. The invention also provides a vaccine according to the invention for use in the treatment of any of: persistent HPV infection (particularly persistent HPV16 infection), VIN, cervical dysplasia (CIN), vaginal intraepithelial neoplasia (VaIN), Anal intraepithelial neoplasia (AIN), cervical cancer (such as cervical squamous cell carcinoma (SCC), oropharyngeal cancer, penile cancer, vaginal cancer or anal cancer in a subject.

The invention also provides a polypeptide comprising a sequence as given in SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 5.

Brief explanation of the drawings

Figure 1. Genotypic expression of HPV16 E6 and E7 fusion proteins. HEK 293T cells were transiently infected with DNA vectors that genetically express the transgenes indicated above the figure. After 24 hours infection, cells were collected and cell extracts were analyzed by SDS

<p dir="rtl">20 PAGE and western staining using an antibody against HPV16 E7 (top panel). Comparison sample confirms load. NF-kB (bottom panel) shows load similar to cell lysates in all lanes. A molecular weight barcode is indicated on the left. The expected sizes of the fusion proteins are: E6E7SH approximately 38 kDa; E2E6E7SH and E6E7E2SH approximately 75 kDa, LSE2E6E7SH approximately 78 kDa.</p>

<p dir="rtl">25 Figure 2. Colony formation on soft agar. A) A schematic representation of the soft agar experiment setup. B) Pictures</p>

7690

-9-

Representative micrographs at 40× magnification of cells in agar six weeks after publication. White arrows identify colonies observed in cells infected with E7wt. c) Colony quantification after six weeks of spreading on agar using GelcountTM and accompanying software. *: p &gt; 0.05 (Poisson regression model); **: non-minimum (general linear model with 5% non-minimum margin).

5

Figure 3. E6E7SH lost E6 and E7 activities. A) Representative western blot showing the absence of p53 degradation by E6E7SH. Human p53 null NCI-H1299 cells were co-infected with a plasmid that genetically expresses p53 in combination with a plasmid that genetically expresses HPV16 E6 wild-type, E6E7SH, or the empty vector. Indicates Lack of TF to uninfected cells after 24

<p dir="rtl">10 One hour after infection, cell lysates were prepared and 30 μg of total protein was loaded onto the gel. Top panel – p53 staining, middle panel – E6 staining, bottom panel – NF-κB staining (carrier comparison sample). (b) Quantification of p53 levels in four independent experiments. p53 signal was normalized to NF-αB signal. c) Staining Western demonstrates the lack of pRb degradation by E6E7SH. pRb empty 2-Saos cells are infected with a plasmid that genetically expresses pRb in</p>

<p dir="rtl">15 Combinations with a plasmid genetically expressing HPV16 E7 wild type, E6E7SH or empty vector. The absence of TF indicates uninfected cells. Twenty-four hours after infection, cell lysates were prepared and 10 μg of total protein was loaded onto the gel. Top panel – pRb staining, middle panel – E7 staining, bottom panel – NF-κB staining (carrier comparison sample). D) Quantification</p>

NF-κB. *: p &lt; to pRb signal in four independent experiments. The pRb signal was normalized to levels

<p dir="rtl">20 0.05 (ANOVA models); **: non-inferior (test based on 95% CI's derived from models</p>

ANOVA. The non-minimum value margin is set at 75%.

Figure 4. E6E7SH does not immunize primary human epidermal keratinocytes. Primary human epidermal keratinocytes were transduced with lens viruses encoding either E6- or E7-wild-type open reading frames for HPV16 (E6E7wt), E6E7SH, or eGFP sequences.

<p dir="rtl">25 Non-induced donor cells as a comparison sample. Only genetic expression of E6E7wt is induced</p>

Primary keratinocyte immunization as indicated by extended lifespan and hTERT activation

7690

-10-

Around day 200 (not shown). The cross symbol indicates that the cells die of old age and cannot be cultured either. For details see Example 2. Similar results were obtained for two additional monomers (not shown).

Figure 5. Immune response induced by E6E7SH after DNA immunization-IFNγ analysis

<p dir="rtl">5 ELISPOT. a. Immunization scheme. CB6F1 mice were vaccinated using plasmid DNA that genetically expresses E6E7SH or a plasmid that does not genetically express a transgene (comparison sample). Two weeks after vaccination, mice were sacrificed and dissociated splenocytes were stimulated overnight with 15mer peptide assemblies corresponding to E7. b. E7-specific immune responses in independent mice as measured by IFNγ ELISPOT experiments are given as spot forming units (SFU) per</p>

<p dir="rtl">10 610 spleen cells.</p>

Figure 6. Immunogenicity of E6E7SH–IFNγ ELISPOT analysis. (A). Immunization scheme. Mice were immunized using adenoviral vectors with primers as indicated. E7-specific responses at two weeks (B) and at eight weeks (C) analyzed by IFNγ ELISPOT (represented as spot-forming units (SFU) (per 610 spleen cells). Closed circles represent immune groups

<p dir="rtl">15 Using a dose of 1*1010 vp, the open circles represent a class immunized with 5*910 vp. The black bar represents the geometric mean of the responses. The dotted line indicates the lower limit of detection in the ELISPOT experiment. Post hoc Bonferroni ANOVA statistical analysis was performed on log transformed data. *: 0.05 &gt; p. For details see Example 3.</p>

Figure 7. Immunoprecipitation of E2E6E7SH–E7-Tatarmer staining. (A). Immunization plan. Done

<p dir="rtl">20 Immunization of CB6F1 mice using 1*1010 vp of adenoviral vector that genetically expresses the transgene as indicated. Two weeks after immunization, mice were sacrificed and the dissociated spleen cells were analyzed to determine the presence of CD8+ cells capable of cross-talk with tatarmart E749-57-H2-Db (b). The percentage of CD8+ T cells positive for tatarmart E7 is indicated on the y axis. Statistical analysis Post-hoc Bonferroni ANOVA on log transformed data,</p>

<p dir="rtl">25 The differences between the different E6E7SH variants are not statistically significant.</p>

Figure 8. Immunogenicity of E2E6E7SH–IFNγ ELISPOT analysis. (a). Immunization plan.

7690

-11-

The CB6F1 strain was vaccinated using adenoviral vectors that genetically express the transgenes indicated at the bottom of panels B and C. Two weeks after immunization, mice were sacrificed and dissociated splenocytes were stimulated overnight with 15mer peptide assemblies corresponding to E2 (b), E6 (not shown) or E7 (c). Responses were given as SFU for every 610 splenocytes. Analysis was performed

<p dir="rtl">5 Post-hoc Bonferroni ANOVA on log transformed data. The E2 response induced by adenoviral vectors encoding single E2 is superior to the response induced by polypeptides of the invention comprising E6 and E7 fragments. The difference is significant for E2 vs. E2E6E7SH and E2 vs. E6E7E2SH &gt; 0.05 (*: p). Post-hoc Bonferroni ANOVA statistical analysis was performed on log transformed data.</p>

<p dir="rtl">10 Figure 9. Persistent responses in an immune group. (A) Immunization scheme. CB6F1 mice were vaccinated using 1*1010 vp of Ad35 vectors that genetically express LSE2E6E7SH, E6E7SH, and E2E6E7SH variants, or using an adenoviral vector that does not genetically express a transgene (empty). Blood samples were drawn every two weeks to determine Percentage of CD8+ T cells allocated to E7 by Tetramer staining (B) Immune responses two weeks after immunization. The vector containing...</p>

<p dir="rtl">15 A leading sequence has a higher response than vectors without a leading sequence; LSE2E6E7SH vs</p>

0.05 &gt; E2E6E7SH (*: p(. )c) Kinetics of responses. Post-hoc Bonferroni ANOVA statistical analysis was performed on log-transformed data in the week 2 dataset. The E7 response induced by molecules containing E2 tends to be higher compared to the molecule without E2, although Although the results are not statistically significant.

<p dir="rtl">20 Figure 10. Use of different adenoviral vectors to enhance immune responses. (A). Immunization scheme. CB6F1 mice were immunized with an Ad26 vector that genetically expresses HPV16 E2E6E7SH (HPV16-Tx) or with an Ad26 vector that does not genetically express a transgene (empty). Two weeks later, immunizations were repeated using Ad35-based vectors as is Indicated at the bottom of the figure. Four weeks after the second immunization, the Arn group was sacrificed and blood samples were used to determine the percentage</p>

<p dir="rtl">25 Percentage of CD8+ T cells assigned to E7 by TetArmer staining (b). * indicates comparison of Ad26.HPV16-Tx/Ad35.HPV16-Tx versus Ad26.HPV16-Tx/Ad35.blank, &gt; p</p>

7690

-12-

0.05 (Student t test on log transformed data, using alpha = 0.01 for multiple comparisons). Figure 11. Cellular immunogenicity of E2E6E7SH in rhesus macaques. (a) Immunization scheme. Rhesus macaques were immunized on day 0: eight animals received Ad26. HPV16 E2E6E7SH and two control animals received Ad26.blank by intramuscular (i.m.) immunization.

<p dir="rtl">5 A booster immunization (Ad26.HPV16-E2E6E7SH or Ad26.blank) was given at weeks 8.</p>

At 16 weeks, animals received a second booster immunization using Ad35 vectors genetically expressing the same E2E6E7SH, while control animals received empty Ad35. The dose of adenoviral vectors was 1*1110 vp per immunization. MART blood draws were performed at multiple time points. (B) Cellular immune responses in PBMCs were measured by IFNγ ELISPOT. PBMCs were stimulated

<p dir="rtl">10 Using peptide combinations corresponding to HPV16 E2, E7 or E6, spot-forming units (SFU) were imaged in 1*610 PBMCs. Blank control animals (n=2) showed no detectable response. For details see Example 4.</p>

Figure 12- Therapeutic effect of adenoviral vectors expressing HPV16-E2E6E7SH. (A) 1-TC injection and immunization scheme. CB6F1 mice were subcutaneously injected with 1*510 cells

<p dir="rtl">15 1-TC on day zero. Six days later, when the tumor was evident, the group was immunized with two of the following:</p>

SLPs covering the immunodominant HPV16 epitope of E6 and E7 (i.e., HPV16 E6, aa41-65 (KQQLLRREVYDFAFRDLCIVYRDGN; sequence ID number: 18)

aa 43-77 of HPV16 E7

GQAEPDRAHYNIVTFCCKCDSTLRLCVQSTHVDIR); Sequence ID: 20 19) at 150 μM in a final volume of 200 μL of 0.9% saline supplemented with

5 nmol ODN1826-CpG (b) or Ad26.HPV16-E2E6E7SH (c). Mice received

Comparison of either CpG alone (d) or Ad26 null (e). All mice received a booster immunization on day 20. Mice that received Ad26 vectors in the primary immunization were subsequently immunized with the corresponding Ad35 vectors. The other mice received SLP mixed with Adjuvant with CpG or CpG alone as in 25 primary immunization types (B-E) Tumor measurement in mice injected with 1-TC. Tumor volume was calculated

In the form of (width2*length)/2. Mice were sacrificed when tumor volumes exceeded 1000 mm3. It is necessary

7690

-13-

Two mice were sacrificed due to a weight loss of more than 20% (indicated by two crescents). (F-G) Close-up view of panels B and C for the first 35 days. (H) Survival after 1-TC injection. Survival of mice treated with 1-TC was significantly increased. Ad.HPV16-E2E6E7SH compared to mice vaccinated with SLP and CpG (long-ranked test p&lt;0.05). Three of the mice were vaccinated with Ad.HPV16-

<p dir="rtl">5 E2E6E7SH The tumors were free at the end of the experiment (on day 92).</p>

Figure 13 - Adenovirus vectors carrying transient antigens encoding either HPVAg or LSE2E6E7SH display viral products that are expressed on cells and are capable of repressing the heritable expression of a transgene. A) Viral output assay of Ad35 vectors. PER.C6, PER.C6/CymR, and PER.C6/TetR cells were transfected with Ad35 vectors carrying transient genes encoding for

<p dir="rtl">10 GFP-Luc or HPVAg. These transient genes were driven by CMV promoters containing either CuO or TetO. Viral products were determined four days postinfection by a method based on Ad35 hexon-specific qPCR. B) Viral output assay of Ad26 vectors. PER.C6 and PER.C6/ TetR cells were transfected with Ad26 vectors carrying transient genes encoding HPVAg, GFP-Luc, or LSE2E6E7SH, all of which are driven by the CMV promoter.</p>

<p dir="rtl">15 Contains TetO. Viral products were determined three days postinfection by a method based on Ad26 hexon-specific qPCR. For details see Example 6.</p>

Figure 14 - Using a repressor system to suppress the expression of a vector gene during production prevents instability of the vector gene sequence in an adenoviral vector carrying a transient gene encoding HPVAg. An Ad35 vector expressing HPVAg under CMVCuO control was edited by transfection with DNA either in

<p dir="rtl">20 PER.C6 or PER.C6/CymR cell lines. The resulting viral panels—five from each cell line—were selected and used for serial infections of the selected cell lines. A) Analysis of integration of the transgenic chain region of the vector by PCR after 10 viral passages. PCR products obtained from viral isolation products passaged on PER.C6 and PER.C6/CymR are shown in the middle and right panels, respectively. PCR products that</p>

<p dir="rtl">25 The full-length sequences obtained from viral isolates passing PER.C6, 1, 2, 4, and 5, and those revealed for isolates passing PER.C6/CymR 1 through 5, appear to be</p>

7690

-14-

DNA sequences according to Sanger. Analysis of trace products by chromatograms (not shown) showed that all isolates grown on PER.C6, but not grown on PER.C6/CymR, contained either small frameshift deletions or immature stop mutations within the HPVAg coding sequence. b) Analysis of the ability of vectors to genetically express HPVAg after seven viral passages.

<p dir="rtl">5 A549 cells were transduced with PER.C6 and PER.C6/CymR viral isolates, and HPVAg genotype expression was analyzed by Western blot using an anti-HPV16 E7-specific antibody. The predicted size of HPVAg was 83 kDa. For details see Example 6.</p>

Detailed description:

<p dir="rtl">10 The presenter provides a nucleic acid fragment encoding a polypeptide comprising sequence identity number: 1. The polypeptide is a fused polypeptide, sometimes referred to herein as a polypeptide of the presenter, or a fused polypeptide of the presenter. This polypeptide is used to generate an immune response against the E6 and E7 proteins of HPV16, so the DNA molecule can be used as a therapeutic vaccine to prevent persistent and associated HPV16 infection.</p>

<p dir="rtl">15 The polypeptide of the invention is a carefully tailored molecule containing substantially the complete E6 and E7 amino acid sequences of HPV16 (lacking only one C-terminal amino acid of the original HPV16 E6 protein) in the form of fragments that are rearranged and partially recombined such that (essentially (All HPV16 E6 and E7 protein T cell epitopes are present. Early molecules of some potency have been described in the form of HPV vaccines by others (e.g. Kenter et al.</p>

al., 2009, N Engl J Med 361: 1838-47; Daayana et al., 2010, Br J 20 Cancer 102: 1129-36; Smahel et al., 2001, Virology 281: 231-38; Yan et al., 2009, Vaccine 27: 431–40; Öhlschläger et al., 2006, Vaccine 24:

2880–93; Oosterhuis et al., 2011, Int J Cancer 129: 397-406;

2013/083287 EP1183368, WO), but each of those molecules has one or more of 25 deficiencies. The polypeptide molecules designed by the invention are distinctive in at least one and several

7690

-15-

Typical features of the methods described later. Specifically, features of the molecules and/or vectors of the present invention include: (i) having a desirable safety feature, whereby the DNA is strongly reduced (compared to native E6 and E7 proteins), up to undetectable transforming activity; (ii) They are single nucleic acid molecules, which are easy to manufacture on an industrial scale in an economical manner, and do not have logical challenges in contrast to multi-molecule methods (iii).

Polypeptides encoded on essentially all T cell epitopes of the native HPV16 E6 and E7 proteins; (iv) the design of the encoded polypeptides minimizes the introduction of potentially unwanted, potent novel epitopes (i.e., epitopes not present in the native E6 and E7 proteins); and (v) in certain embodiments, does not rely on highly reactive adjuvants to elevate the response 10 Immunogenicity is required, therefore, innovative molecules represent a major step towards combining different properties in...

One design, they are essentially an excellent choice for therapeutic vaccination against HPV16.

These molecules could also potentially serve as prophylactic vaccines against HPV16, meaning they could potentially prevent ongoing infection with HPV16 in vaccinated subjects.

In certain embodiments, by careful design a number of novel epitopes with a length of nine 15 amino acids with an expected binding affinity of &gt;50 nM for 20 of the most common HLA-A have been reduced from 20 to

The most common of HLA-B and 20 of the most common of HLA-C alleles to only 1. This is a significant improvement over the components described by others, which for a single modified E6 protein contain more than 30 neo-epitopes, and these components will likely include significantly more neo-epitopes in sequences appended to these components to prevent loss.

Therefore, these epitopes have 20 (Öhlschläger et al., 2006, Vaccine 24: 2880–93).

The components of the invention feature significantly improved immunogenicity since the chances of a substituted immune response are reduced by E6 and E7 in the molecules of the invention, compared to methods described by others.

Skilled individuals can, using routine techniques, create 25 non-affecting nucleotide substitutions in the polypeptide sequence encoded by the polynucleotides described to reverse codon usage.

From any particular host organism in which the polypeptides are expressed. Therefore, unless otherwise described

7690

-16-

Therefore, a “nucleotide sequence encoding an amino acid sequence” includes all nucleotide sequences that are variants of one another and encode the same amino acid sequence. Nucleotide sequences that encode proteins and RNA can include introns.

In a preferred embodiment, the nucleic acid encoding the polypeptide according to the first example is a codon

<p dir="rtl">5 Enhancer of genetic expression in mammalian cells, preferably human cells. Codon optimization methods are known and have been described previously (eg 96/09378 WO). A sequence was considered codon optimized if a codon that was unfavorable compared to a wild-type sequence was replaced by a codon that was more favorable. In this document, a non-favorable codon is a codon It is used less frequently in an organism than another codon that encodes the same amino acid, and is more preferably a codon</p>

<p dir="rtl">10 A codon that is used more frequently in an organism than a non-preferred codon. can get</p>

A codon for a particular object must be frequently used in codon frequency tables, such as in<a href="http://www.kazusa.or.jp/codon">http://www.kazusa.or.jp/codon</a>. Preferably more than one non-preferred codon, e.g. more than 10%, 40%, 60%, 80% of the non-preferred codons, the most preferred (e.g. at least 90%) or all of the non-preferred codons, are replaced by codons that are more

<p dir="rtl">15 In preference. Preferably the most frequently used codons in an organism are used in</p>

Codon optimization sequences. Substitution by a preferred codon generally results in higher genotypic expression.

DNA sequences can be cloned using molecular and routine biotechnology techniques, or generated from...

New methods are developed by DNA synthesis, which can be performed using routine procedures by service companies

<p dir="rtl">20 It has work in the field of DNA synthesis and/or molecular cloning (eg GeneArt,</p>

Eurofins, Invitrogen, GenScripts

A skilled person will also realize that changes can be made to a protein, for example by amino acid substitutions, deletions, additions, etc., for example using routine molecular biological procedures. In general, conserved amino acid substitutions can be applied without loss of function or

<p dir="rtl">25 Polypeptide immunogenicity. This can be checked according to routine procedures well known to the skilled person.</p>

7690

-17-

In certain embodiments, the polypeptide encoded according to the invention also includes a leader sequence, also referred to as a signal sequence or signal peptide. This is a short peptide (typically 5-30 amino acids long) found at the N terminus of most newly synthesized proteins directed toward the secretory pathway. The presence of this sequence can lead to increased genetic expression and immunogenicity. Examples are not limited to

5 That may be used is an IgE leader peptide (see for example US 6,733,994; For example it has the sequence MDWTWILFLVAAATRVHS (SEQ ID: 7) or a leader peptide

HAVT20 (for example having the sequence MACPGFLWALVISTCLEFSMA (Sequence ID No.: 9)). One of these may optionally be added to the N terminus of a polypeptide of the invention. In other embodiments, the polypeptide of the invention does not include a leader sequence.

<p dir="rtl">10 There are various types of HPV (over 120 types are defined and indicated by number), and generally for each type that requires coverage by a vaccine, there may be a need to include type-specific antigens in the vaccine, although for certain antigens there may be some Symptomatic reactivity Types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, 73, and 82 are sexually transmitted “high-risk” HPVs that are carcinogenic and can lead to</p>

<p dir="rtl">15 Development of cervical dysplasia (CIN), hyperplasia of intraepithelial neoplasia (VIN), vaginal intraepithelial neoplasia (VaIN), penile intraepithelial neoplasia (PIN), and/or anal intraepithelial neoplasia (AIN). HPV according to the invention (i.e. the HPV from which it is derived).</p>

The E6 and E7 fragments of the encoding polypeptide) are HPV16. It can be used in subjects infected with HPV16. In certain embodiments it may also be possible to combine them with vaccines

<p dir="rtl">20 Against other types of HPV. In certain embodiments, it is combined with a vaccine against HPV with a high-risk type as defined above, for example with a vaccine against HPV18. In other embodiments, the vaccine of the invention is combined with a vaccine against one or more of HPV 18-45, -39, -35, -33, -31, -51, 52-, 56-, 58-, 59-, 68 -, -73, or -82. These combinations may be used, for example, if the actual type of HPV infection is not specific, or if there is an immune response with</p>

<p dir="rtl">25 Prophylaxis is required against more than one HPV type. Combinations of the invention's vaccines with vaccines against the HPV types that cause genital warts, such as HPV6 and/or HPV11, have also been investigated. be sequences of</p>

7690

-18-

These types of HPV and the proteins subsequently encoded (e.g. E2, E7, E6) are available to the skilled person in public databases, such as the GenBank sequence database provided by

.)National Center for Biotechnology Information (NCBI).

The polypeptide according to the invention comprises sequence ID No.: 1, and in one embodiment comprises a molecule

<p dir="rtl">5 DNA according to the invention of sequence identity number: 2.</p>

Sequences are presented herein from the 5' to 3' direction or from the N to C terminus, as done

10

Customize it in the field.

The polypeptide according to the invention comprises epitopes from the HPV16 E6 and E7 proteins. In certain embodiments, the polypeptide according to the invention also includes (and thus the nucleic acid encoding the polypeptide also encodes) at least an additional antigen or epitope(s) of such additional antigen.

This additional antigen is preferably an HPV antigen, preferably with the same HPV type as the E6 and E7 proteins in the polypeptide, i.e. HPV16. This additional antigen can therefore be an HPV protein or an immunogenic fragment thereof, and in certain embodiments comprising an E2 protein or a fragment thereof comprising an epitope of at least E2 of HPV, preferably from

<p dir="rtl">15 HPV16. These additional antigens or epitopes may be located internally between two E6 and/or E7 fragments in the polypeptide containing sequence ID 1, but preferably fused to the N-terminus or C-terminus of an E6/E7 polypeptide containing sequence ID 1. 1. Alternatively or additionally, amino acid sequences may be found where they stimulate an immune response. Thus, in certain embodiments the sister provides a nucleic acid molecule according to the sister, encoding a polypeptide</p>

<p dir="rtl">20 comprising sequence identity number: 1, wherein the polypeptide also includes at least one other antigen, for example the HPV E2 protein, or at least one epitope, but preferably</p>

More epitopes, including. One advantage of adding E2 antigen to the present invention is that E2 is known to be expressed early during infection/in low-grade ulcers where genetic expression of E6 and E7 remains very low. During the development towards cervical cancer is only expressed

E7 (Yugawa and Kiyono, and E6 As a result, levels of hereditary E2 25 are increased from

Rev Med Virol 19:97–113 2009). It allows the assembly of E6, E2 and E7 epitopes into a vaccine.

7690

-19-

One treatment in a broad target patient population, ranging from persistent infection to invasive cervical cancer (or other HPV16-induced cancers). 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 in its DNA-binding domain (compared to the wild-type E2 protein).

5 The 041328.1_HPV16 E2 protein (NP) sequence can be detected in the NCBI protein database (<a href="http://www.ncbi.nlm.nih.gov/protein(">(www.ncbi.nlm.nih.gov/protein)</a> Under the number 041328.1_NP. Multiple single amino acid changes in E2 such as K299M, G293V, or C300R in the C-terminal portion of that protein are known to break DNA binding. The advantage of using a variant or fragment of E2 that lacks DNA binding ability is that unpredictable transcriptional changes can be prevented by

<p dir="rtl">10 Direct binding to host cell DNA in expressed cells. The E2 protein or a fragment or variant thereof may be internally added, but preferably at the N or C terminus</p>

The polypeptide of the invention having sequence identity number: 1. In one embodiment, the nucleic acid molecule of the invention encodes a polypeptide having sequence identity number: 3. In one embodiment, the nucleic acid molecule of the invention has sequence identity number: 4. In another embodiment, it encodes a molecule

<p dir="rtl">15 The nucleic acid molecule of the invention is a polypeptide comprising sequence identity number: 5. In one embodiment, the nucleic acid molecule of the invention includes sequence identity number: 6.</p>

It will also be possible to perform additional combinations of the polypeptides designed by the invention with

Additional proteins, for example so-called carrier proteins, such as the heat shock protein calreticulin, Mycobacterium tuberculosis-70, IP10, or tetanus toxin C fragment (see Oosterhuis et al.

<p dir="rtl">20 2012, al., Human Gene Ther, above, for additional examples), which could also be improved</p>

Immune response to HPV E6 and E7 (and optionally E2) epitopes. The invention therefore also provides these additional fusion proteins, and the nucleic acids that encode them.

In certain embodiments, a nucleic acid fragment according to the invention is included in the vector. A “vector” as used herein is typically a vector material that artificially carries the foreign genetic material 25 into another cell, where it can be replicated and/or expressed genetically, and according to the invention it can be

Any nucleic acid molecule including a nucleic acid moiety according to the invention. Can be prepared accordingly

7690

-20-

For routine molecular biotechniques such as cloning. These vectors can be disseminated into at least one type of suitable host such as bacteria, yeast, insect cells, mammalian cells, and the like. The four main types of vectors are plasmids, viral vectors, cosmids, and artificial chromosomes. The vector itself is generally a DNA sequence consisting of a ligand (transgene;

In the present invention the nucleic acid encoding the fused polypeptide of the invention) and a sequence acting as a “basic strand” of the vector. The purpose of the vector that transfers genetic information to another cell is typically to separate, replicate, or express the conjugate in the target cell Preferably, the sequence encoding the polypeptide is functionally linked to an enhancer in the vector. The expression “adequately linked” indicates that the nucleotide sequence of interest binds to the enhancer in a way that allows expression.

<p dir="rtl">10 Genetic transmission of the nucleotide sequence (for example, in a host cell when the vector is introduced into a cell</p>

(host). Sequences regulating genetic expression may be conveniently linked to a transgene. In certain embodiments, the vector is designed to genetically express the transgene in a target cell, generally having a promoter sequence that derives genetic expression of the transgene. In certain embodiments , one or more vector elements used routinely such as cloning stop sequences, tail sequences

<p dir="rtl">15 Polyadenylation, UTRs, Kozak sequences, origin of replication, multiple replication sites, inheritance, antibiotic resistance, and additional sequences may exist, and a skilled person can design</p>

A vector such that it has the desired properties, for example for transcriptional replication in specific cells to spread and replicate the vector, and for genetic expression of the transgene from the vector in the target cells into which the vector is introduced. They are vectors that contain the DNA that encodes the polypeptide

<p dir="rtl">20 The fusion according to the invention, preferably designed for genetic expression in mammalian cells, is suitable as vaccines according to the invention. In certain embodiments, the vector according to the invention is a plasmid, cosmid, yeast artificial chromosome, bacterial artificial chromosome, viral vector, or the like. A person skilled in the art will be aware that different enzymes can be used to elicit the heritable expression of a gene in host cells. It includes some of the well-known and most well-known Moez Azts</p>

<p dir="rtl">25 They are used for genetic expression in eukaryotic cells based on enzymes derived from viruses, such as viruses</p>

adenovirus, e.g. E1A enhancer, enzyme derived from cytomegalovirus (CMV), e.g.

7690

-21-

CMV immediate early enhancer (IE) (referred to in this document as the CMV enhancer) (e.g. obtainable from Invitrogen, pcDNA), 40 Simian Virus SV40-derived enhancers (e.g. obtainable from cat.no . 631605, BD, pIRES Sciences), etc. Suitable enzymes can also be derived from eukaryotic cells, e.g.

<p dir="rtl">5 Methalothionine (MT) booster, EF-11 booster, ubiquitin C or UB6 booster</p>

actin enhancer, immunoglobulin enhancer, heat shock enhancer, and the like (see for example WO 2006/048459). A non-exclusive example of a suitable enhancer for eliciting gene expression in eukaryotic cells would be a CMV (US) 5,385,839 enhancer, For example an immediate early CMV booster, for example including –735.nt to +95 of enhancer/booster.

<p dir="rtl">10 A CMV immediate early gene, eg a CMV promoter as introduced herein using a sequence as given in SEQ ID NO: 13. A polyadenylation signal, eg a bovine growth hormone polyA signal (US 5,122,458), may be located behind Modified gene(s). Additional regulatory sequences may also be added. The expression “regulated sequence” is used interchangeably with “regulatory element” in this document and refers to a nucleic acid fragment, typically but not bound to DNA,</p>

<p dir="rtl">15 It modifies the transcription of the DNA sequence to which it is functionally linked, and thus acts as a transcription modifier. A structured sequence often includes DNA sequences that are transcription-binding domains recognized by the DNA-binding domains of transcriptional proteins and/or transcription factors, enhancers or repressors, etc. For example, it is convenient to pair a repressor sequence with a promoter, where the repressor sequence can bind to a repressor protein that can</p>

<p dir="rtl">20 It reduces or prevents the genetic expression of the transgene in a production cell line that expresses that repressor protein. This can improve the genotypic stability and/or expression levels of the DNA molecule during passage and/or when introduced at high quantities into the production cell lineage. These systems are described in the field. For example, a regulatory sequence may include one or more sequences of the tetracyclin operon (tetO) operon sequences, such that gene expression is inhibited in the presence of the operon repressor protein</p>

<p dir="rtl">25 tetarcyclin (tetR). In the absence of tetRcyclin, the tetR protein is able to bind to tetO sites.</p>

The transcription of a gene functionally linked to the tetO sites was repressed. In the presence of Tatar cyclin, however, a conformational change is prevented

7690

-22-

The tetR protein can bind to operator sequences, allowing transcription of functionally related genes to occur. In certain embodiments, a nucleic acid portion, for example when contained in a recombinant adenovirus vector, of the present invention may optionally comprise a tetO operably linked to an enhancer, such that the genetic expression of one or more transgenes in viruses is stabilized.

<p dir="rtl">5 Adenoids are produced in the producer cell lineage in which the tetR protein is expressed. Subsequently, gene expression can be uninhibited if the genetically recombinant adenovirus is introduced into a subject or into cells that do not express the tetR protein (e.g., International Patent Application 073513/07 WO). In certain other embodiments, it is possible to Optionally including a nucleic acid portion of the present invention, for example when present in a genetically recombinant adenovirus.</p>

<p dir="rtl">10 On the COMAT gene conversion system, gene expression is modified by binding a repressor (CymR) to an operator site (CuO), the position after the promoter (e.g., Mullick et al 6:43 2006. BMC Biotechnol). In this document, the term “repressor” refers to entities (e.g., proteins or other molecules) that have the ability to inhibit, interfere with, delay and/or repress the production of the heterologous protein product of a recombinant genetic expression vector.</p>

<p dir="rtl">15 For example, by overlapping a binding site at an appropriate location along the genetic expression vector, such as in a genetic expression cassette. Examples of repressors include CymR, tetR, lac repressor, trp repressor, gal repressor, lambda repressor, and other appropriate repressors known in the art. Examples of the use of the tetO/tetR actuator/repressor system and the CuO/CymR actuator/repressor system are presented in this document. Repressing the hereditary expression of a vector transgene during vector propagation can prevent instability of the transgene.</p>

<p dir="rtl">20 Producing vectors containing a transgene can increase invention during production. Thus, in some embodiments, vectors of the invention that can be repressed by binding to a repressor protein are enhanced, for example by having an enhancer that is operably coupled to the repressor operator sequence (e.g. in non-exclusive embodiments, a sequence containing TetO, e.g. For example, those mentioned in Sequence ID No. 11, or a sequence containing CuO, for example, those mentioned in Sequence ID No.: 11, or a sequence containing CuO, for example, those mentioned in Sequence ID No. 11.</p>

<p dir="rtl">25 Sequence No.: 12), to which a repressor protein may bind (for example a TetR protein, for example having an amino acid sequence as given in SEQ ID No.: 15, or a protein</p>

7690

-23-

CymR, for example, has the amino acid sequence as given in SEQ ID No. 17). In certain embodiments, the vector is a plasmid DNA molecule, or a fragment thereof. It can be used to inoculate DNA. Other platforms are also available for use as vectors. , for example live attenuated double-deletion strains Listeria monocytogenes.

<p dir="rtl">5 In other embodiments, the vector is a genetically recombinant viral vector, which may be replication-specific or hypo-replicative. In certain embodiments, a viral vector includes a recombinant DNA genome. In certain embodiments, a vector according to the invention is for example a genetically recombinant adenovirus, a genetically recombinant retrovirus, a genetically recombinant pox virus such as a vaccine virus (e.g. Modified Vaccinia Ankara</p>

<p dir="rtl">10 MVA)((, genetically recombinant alphavirus such as semliki forest virus, mariavirus recombinant</p>

Genetic linkage, such as genetically linked measles virus, or another genetically linked virus. In certain embodiments, a vector according to the hereafter MVA vector.

In preferred embodiments, the vector according to the invention is a genetically recombinant adenovirus. Advantages of adenoviruses for use as vaccines include ease of processing, portability, and...

<p dir="rtl">15 Good manufacturing on a large scale, and an excellent safety record based on many years of experience in research, development, manufacturing and clinical trials using multiple registered adenoviral vectors.</p>

20

25

Adenoviral vectors that have been used as vaccines generally provide a good immune response to the protein encoded by the transgene, including a cell-mediated immune response. An adenoviral vector according to the invention can be based on any type of adenovirus, and in certain embodiments a human adenovirus, which can be of any serotype. In other embodiments, it is an adenovirus, such as a chimpanzee or gorilla adenovirus, which may be of any Egyptian species. In certain embodiments, the vector according to the invention is a human adenovirus of serotype 5, 26 or 35. The preparation of genetically recombinant adenoviral vectors is well known in the art. In certain embodiments, an adenoviral vector according to the invention is deficient in at least one essential gene function in the E1 region, for example the E1a region and/or E1b region, of the adenoviral genome that is required for viral replication. In certain embodiments, it is a vector

7690

-24-

Adenovirus according to the type is missing in at least part of the non-essential E3 region. In certain embodiments, the vector is deficient in at least one essential gene function of the E1 region and at least part of the non-essential E3 region.

Adenoviral vectors, methods of their construction and methods of dissemination, are well known in the art and have been described

<p dir="rtl">5 In, for example, US patent numbers 5,559,099, 5,837,511, 5,846,782, 5,851,806, 5,994,106, 5,994,128, 5,965,541, 5,981,225, 6,040,174, 6.02 0.191, 6,113,913, and Thomas Shenk, “Adenoviridae and</p>

On their Replication", MS Horwitz, "Adenoviruses", Chapters 67 and 68 Virology, BN Fields et al., eds., 3d ed., Raven Press, Ltd., respectively, in

<p dir="rtl">10 New York (1996), and other references cited therein. Typically, it involves the creation of viral vectors</p>

adenoids using standard molecular biotechniques, such as those described in, e.g.

Sambrook et al., Molecular Cloning, a Laboratory Manual, 2d ed., Cold Spring Harbor Press, Cold Spring Harbor, NY (1989), Watson et al.,

Ausubel and Recombinant DNA, 2d ed., Scientific American Books (1992).

et al., Current Protocols in Molecular Biology, Wiley Interscience 15

Publishers, NY (1995), and other references cited therein.

Particularly preferred serotypes for recombinant adenovirus are human serotype 35 or human serotype 26. Preparation of rAd26 vectors has been described, for example, in WO 2007/104792 and in Virology: 81 Abbink et al., 2007

20 4654-63. The typical genome sequences of Ad26 were discovered in GenBank Accession

153474 EF and in serial ID No.: 1 of 2007/104792 WO. The preparation of rAd35 vectors is described, for example, in US Patent No. 7,270,811, in WO 00/70071, and at 77:71-8263 Vogels et al., 2003, J Virol. Typical genomic sequences of Ad35 were detected in 000019_GenBank Accession AC and in Figure 6 of

.WO 00/70071 25

In certain embodiments, the adenovirus is replication-deficient, for example because it contains...

7690

-25-

Deletion in the E1 region of the genome. As is known to a skilled person, in the case of deletions of a core region of the adenovirus genome, the functions encoded by those regions are introduced into translocations, preferably by the producer cell, i.e. when parts of or all of the E2, E1 and/or E4 regions are deleted from Adenovirus, this must be displayed in the product cell, for example integrated into its genome, or 5 in the form of so-called adenovirus helper or helper plasmids. It could be a virus

The adenoid also has a deletion in the E3 region, where it is dispensable for replication, and thus the deletion does not have to be complementary.

A producer cell (sometimes also referred to in the art and herein as a “packaging cell” or “complementary cell”) that may be used may be any producer cell in which a desired adenovirus can be propagated. For example, 10 The propagation of a genetically recombinant adenovirus vector is performed in producer cells that are deficient in integration of the adenovirus. These preferentially producing cells have at least one E1 adenovirus sequence in their genome and are thus able to complement recombinant adenoviruses with deletions in the E1 region. Any E1 complement-producing cell, such as E1-immunized human retinal cells, can be used e.g.

<p dir="rtl">15 Example 911 or PER.C6 cells (see US Pat. No. 5,994,128), E1 converted amino cells (see EP 1230354), E1 converted A549 cells (see e.g. 98/39411 WO, US Patent No. 5,891,690) , GH329: HeLa</p>

(Gao et al., 2000, Hum Gene Ther, 19:213-293) and the like. In certain embodiments, the cells produced are for example HEK293 cells, or

<p dir="rtl">20 PER.C6, 911 cells, IT293SF cells, etc. The production of viral vectors has been restored</p>

(Kovesdi et al., 2010, Viruses 2: 1681-703).

In certain embodiments, an E1-deficient adenovirus comprises an E4-orf6 coding sequence from a subgroup C adenovirus such as Ad5. This allows these adenoviruses to be propagated in well-known complementary cell lines expressing the E1 genes of Ad5, such as for example 293 cells or

Havenga et al., 2006, J Gen Virol 87: see, for example, PER.C6 25 cells

03/104467 2135-43; WO, the contents of which are incorporated herein by reference.

7690

-26-

The “heterogeneous nucleic acid” (hereinafter referred to as “genome”) in the vectors of the invention is nucleic acid present in a form not naturally present in the vector, and according to the present invention the nucleic acid encoding the fused polypeptide of the invention is considered It is a heterogeneous nucleic acid when it is present in a vector and is introduced into the vector, for example, by techniques

<p dir="rtl">5 Standard molecular vitality. It can be cloned, for example, into a deleted E1 or E3 region of an adenoviral vector, or into the region between the E4 region and the rITR. A transgene is generally linked in practice to sequences with comparable genotypic expression. In preferred embodiments, the transgene is transcribed into the E1 region of an adenoviral vector.</p>

Vector production can be carried out as DNA vectors, or genetically recombinant adenovirus vectors, depending on...

<p dir="rtl">10 For various methods well known to the person skilled in the art. In general, production involves diffusion into cultured cells to generate a large amount of vector material, followed by collection of the vector from the cell culture medium, typically followed by further purification of the vector to remove other materials and obtain purified vectors that can be formulated into pharmaceutical formulations (e.g., 2002). Hoganson et al</p>

BioProcessing J 1: 43-8; Evans et al., 2004, J Pharm Sci 93:2458-75

15

20

For example, methods for assembling adenovirus from productive cell cultures are described extensively in WO 2005/080556. For example WO 2010/060719, and WO 2011/098592, the contents of which are incorporated herein by reference, describe suitable methods for obtaining and purifying large quantities of genetically recombinant adenoviruses. In certain features, the invention also provides a polypeptide encoded by a nucleic acid fragment according to the invention. This polypeptide includes sequence ID number: 1. In certain embodiments, the polypeptide may include sequence identity No.: 3 or sequence identity No.: 5. The attributes of such polypeptide are as described above. This polypeptide, for example, can be used directly as...

Vaccine against HPV.

The invention also provides vaccines comprising nucleic acid molecules, vectors or polypeptides accordingly

<p dir="rtl">25 of the invention, wherein embodiments of each of those features may include those as described above.</p>

In preferred embodiments, the vaccine according to the study comprises a nucleic acid portion according to the study. in

7690

-27-

In additional preferred embodiments, the vaccine includes a vector according to the invention, preferably a DNA vector, an MVA vector, or a genetically recombinant adenovirus vector.

In certain embodiments, the vaccine according to the invention comprises other active components, for example nucleic acid encoding an epitope of at least one HPV E6 and/or E7 protein on

<p dir="rtl">5 Least different from HPV16, for example HPV type with high risk such as HPV18-31, -33, 35-, 39-, 45-, 51-, 52-, 56-, 58-, 59-, 68-, 73-. , or -82. The term “drug” refers to an agent or formulation containing an active ingredient that is effective in inducing a protective and/or therapeutic degree of immunity in a subject against a specific pathogen or disease, in the present in the form of a therapeutic against HPV. The vaccine typically comprises a nucleic acid molecule, or vector, according to the invention, and an acceptable excipient</p>

<p dir="rtl">10 Pharmaceutical. Upon administration to the subject, the polypeptide encoded by the nucleic acid molecule according to the invention will be expressed in the subject, which will trigger an immune response to antigenic E6 and/or E7 fragments present in the polypeptide. An advantage of the current molecules is that all HPV16 T cell adhesive epitopes from E6 and E7 are present and thus the T cell response can be fitted to any adhesive epitope present at E6 and E7 of the untreated type in the vaccine. The vaccine also has advantages</p>

<p dir="rtl">15 Safety and effectiveness as stated above for DNA molecules according to the invention.</p>

For administration to humans, the invention may use pharmaceutical compositions comprising a carrier, a pharmaceutically acceptable carrier substance or a pharmaceutically acceptable excipient. In the current context, the term “pharmaceutical acceptable” means that the carrier or excipient, at the doses and concentrations used, will not cause any unwanted or harmful effects in subjects taking it. Excipients are pharmaceutically acceptable

Remington's Pharmaceutical Sciences, 18th 20 known in the field (see; edition, AR Gennaro, Ed., Mack Publishing Company [1990

Pharmaceutical Formulation Development of Peptides and Proteins, S.

Handbook of ; Frokjaer and L. Hovgaard, Eds., Taylor &amp; Francis [2000 Pharmaceutical Excipients, 3rd edition, A. Kibbe, Ed., Pharmaceutical

<p dir="rtl">25 Press[2000]. An excipient is generally an inactive substance formulated with the active ingredient of a drug.</p>

Excipients are generally used to bulk formulas containing the potent active ingredients (hence

7690

-28-

Commonly referred to as “bulking agents,” “fillers,” or “diluents”), to allow the distribution of a drug substance when producing a dosage form. They may also serve for purposes of therapeutic enhancement, such as facilitating the absorption or dissolution of a drug, or as a Other Pharmacokinetics Excipients can also be useful in the manufacturing process, to aid in the processing of the intended active ingredient i.e. by facilitating the tractability of the powder

<p dir="rtl">5 To flow or give it non-stick properties, in addition to helping with stability in the laboratory, that is, by preventing or changing the naturalness over the expected shelf life. The selection of appropriate excipients also depends on the route of administration and dosage form, as well as the active ingredient and other factors.</p>

The purified DNA molecule, carrier or polypeptide is preferably formulated and administered as a sterile solution although a lyophilized preparation may also be used. is being prepared

<p dir="rtl">10 Sterilize solutions by sterile filtration or by other methods known in themselves in the art. The solutions are then freeze-dried or filled into pharmaceutical dosage containers. The pH of the solution generally falls in the range of pH 3 to 9.5, i.e., pH 5 to 7.5. A nucleic acid molecule, vector or polypeptide is usually in solution with a suitable buffer, and the carrier solution may also contain a salt. It can be found optionally</p>

<p dir="rtl">15 A stabilizing agent, such as albumin. In certain embodiments, a detergent is added. In certain embodiments, the vaccine may be formulated into an injectable preparation. These formulations contain either sterile liquid solutions, liquid suspensions or freeze-dried versions and optionally contain stabilizers or excipients.</p>

For example, the adenoviral vector resulting from gene recombination could be stored in...

Hogansonet al., (A buffer solution is used according to the international standard for adenovirus

<p dir="rtl">20 Bioprocessing J1:20:43-8:20 mM Tris pH 5, 25 mM</p>

Molar of NaCl, 2.5% glycerol. A buffer solution of another useful formulation suitable for administration to humans consists of 20 mM Tris, 2 mM MgCl2, 25 mM NaCl, sucrose 10% w/v, polysorbate-80 0.02% w/v. Another formulation buffer solution suitable for adenovirus induced reassortment includes 10-25 mM buffer solution.

25 of stearate pH 5.9-6.2, 4-6% (w/w) of hydroxypropyl-beta-cyclodextrin (HBCD), 70-100 mM of NaCl, 0.018-0.035% (w/w)

7690

-29-

of polysorbate-80, and optionally 0.45-0.3% (w/w) ethanol. Obviously, many buffer solutions can be used, and many examples of suitable storage formulations are known for pharmaceutical administration of purified vectors.

In certain embodiments a composition comprising the carrier also includes one or more auxiliaries.

<p dir="rtl">5 Adjuvants are known in the art to also increase the immune response to an applicable antigen. The terms “adjuvant” and “immunostimulant” are used interchangeably herein, and are defined as one or more substances that cause stimulation of the immune system. In this context, an adjuvant is used to enhance an immune response to polypeptides encoded by nucleic acid molecules in vectors of the invention. Examples of suitable adjuvants include salts</p>

<p dir="rtl">10 Aluminum as aluminum hydroxide, aluminum phosphate and/or potassium aluminum phosphate; Oil-emulsion compositions (or in oil-in-water compositions), including squalene-water emulsions, such as MF59 (see for example 90/14837 WO); saponin formulations, such as for example QS21 and immune-stimulating complexes (ISCOMS) (see, For example 5,057,540 US; WO, 90/03184 WO, 2005/002620 WO, 2004/004762, WO, 96/11711);</p>

<p dir="rtl">15 Bacterial or microbial derivatives, examples of which are ADP-ribosylated monophosphoryl lipid (MPL) (3dMPL), oligonucleotides containing a CpG regulatory sequence, ADP-ribosylated bacterial toxins or mutants thereof, e.g. Heat-unstable enterotoxin Lt from E. coli, E. coli toxin CT, etc. A vector-encoded adjuvant can also be used, i.e., by the use of heterologous DNA encoding a transduction domain fusion.</p>

<p dir="rtl">20 Oligomerization of the C4 binding protein (C4bp) to the intended antigen (i.e., Solabomiet al 3817-23:76 2008, Infect Immun), or by use of a vector encoding both an intended transgene and a 3-TLR antigen such as a heterologous dsRNA (i.e., 2007/100908 WO), or similar.</p>

In other embodiments, the compositions of the invention do not include auxiliary materials.

<p dir="rtl">25 Pharmaceutical compositions may be administered to a subject, i.e., a human subject. The total dose of the vaccine active ingredient supplied to a subject may be varied during a single administration as is</p>

7690

-30-

Known to the practitioner of the art, adenoviruses generally range between 1 × 710 viral particles (vp) and 1 × 1210 vp, preferably between 1 × 810 vp and 1 × 1110 vp, for example between 3 × 810 and 5 × 1010 vp, For example between 910 and 3 x 1010 vp for a DNA vaccine, the total amounts of DNA per administration can range e.g

<p dir="rtl">5 Example between 1 microgram and 10 mg. If a gene gun is used for administration, low doses are usually used, i.e., 10 micrograms. For intramuscular administration, higher doses are usually used, i.e., up to 5 mg.</p>

Administration of pharmaceutical formulations may be performed using standard routes of administration. Unrestricted embodiments include non-enteral administration, such as by injection, i.e., intradermally, intramuscularly, and so on, or

<p dir="rtl">10 Subcutaneous or transdermal, or intramucosal administration, i.e., in the nose, in the vagina, rectum, and the like. In one embodiment the composition is administered by intramuscular injection, i.e., into the deltoid muscle of the arm, or the vastus lateralis muscle of the thigh. In certain embodiments the vaccine is a DNA vaccine, which may for example be administered intradermally, i.e., by DNA tagging (see, for example, Oosterhuiset al., 2012, Curr Top</p>

<p dir="rtl">15 MicrobiolImmunol: 351:221-50. This approach is feasible for adenoviral vectors. In</p>

Certain embodiments include a composition according to the invention comprising an adenoviral vector and are administered by intramuscular injection. A person skilled in the art recognizes the various possibilities of administering a formulation, such as a vaccine, in order to induce an immune response to the antigen(s) in a vaccine.

A subject herein is preferably a mammal, e.g., a rodent, i.e., a mouse, 20 non-human primate, or a human. Preferably the submissive is a human submissive.

The vaccines of the invention may be used to treat patients suffering from one of the various stages of disease caused by HPV (specifically type 16), both incident and persistent infection with HPV as (i.e., as detected by HPV DNA tests), thus prior to the formation of Cancerous (pre-cancerous) lesions, as well as the formation of new cervical intraepithelial neoplasia (CIN); Also known as 25 cervical dysplasia, the formation or formation of interstitial neoplasia in the neck, which is a pre-malignant transformation and non-invasive growth.

Normal (dysplasia) of squamous cells on the surface of the cervix) up to and including cervical cancer (e.g.

7690

-31-

Cervical squamous cell carcinoma (SCC). In addition, new HPV-induced neoplasms can be targeted, such as vulvar intraepithelial neoplasm (VIN), vaginal intraepithelial neoplasm (VaIN), Or new tumors between penile epithelial cells (PIN), formation of new tumors between epithelial cells in the anus (AIN), as in more advanced stages 5 Oropharyngeal cancer (also known as cervical cancer), bronchial cancer, carcinoma

Vaginal, vulvar and anal cancer. The vaccines of the invention can also target a wide range of HPV-induced lesions and are likely to be most effective at pre-cancerous stages of HPV-induced disease, i.e., at (persistent) infection and/or de novo tumorigenesis stages, where expression For E6, E2 and/or E7, treatment with the patented vaccine can also be combined with 10 compounds that neutralize or can bypass immune escape mechanisms in advanced cancer cells, i.e., antibodies.

Anti-PD1/PD-L1, anti-4-CTLA antibodies such as ipilumab, anti-3-LAG antibodies, anti-CD25 antibodies, IDO inhibitors, anti-CD40 antibodies, anti-CD137 antibodies, and so on (see e.g. , Hamid and

Mellmanet al., Carvajal, 2013, Expert Opinion BiolTher 13: 847-861

2011). The therapeutic method of vaccination can also be used, Nature Rev 480: 480-89 15

Primarily for the treatment of external genital warts or their precursors in the case of a vaccine that also contains (sequences encoding) the E6 and/or E7 type of HPV causing genital warts and is administered to a subject infected with this type of HPV.

As used herein, the term “treatment” refers to the administration of a vaccine to induce an immune response

<p dir="rtl">20 Therapeutic treatment against cells expressing HPV16 epitopes with E6 and/or E7 in the patient, resulting in at least a reduction in the level and preferably complete clearance of HPV16 infection, which results in at least a slowing and preferably an arrest of the progression of disease caused by HPV16. Such as the formation of new tumors and/or their symptoms, treatment with the vaccine also preferably causes relief from more advanced stages of cancers caused by HPV. It is preferable to give the vaccine to patients with infection</p>

<p dir="rtl">25 Static HPV has been genotyped, such that a vaccine encoding a polypeptide of the corresponding HPV type can be administered. In the absence of testing, the vaccine may be administered to a portion of infected individuals</p>

7690

-32-

Most likely to be infected with HPV, i.e., sexually active individuals. The vaccine may also be administered to subjects who are not infected with HPV16, i.e., for prophylactic use, potentially in combination with a vaccine against another HPV type to which the patient has been infected, or alternatively in uninfected subjects. The vaccine of the invention may also be administered to a subject being treated by other means, i.e., surgical treatment (removal of a lesion by means of infection).

<p dir="rtl">5 HPV16), or treatment with amiquimod (containing the TLR 7/8 antigen, see, at</p>

For example, Dayaanaet al., 2010, Br J Cancer 102: 1129 – 36 can be done.

Measure the effect of treatment either by cytology or HPV testing.

Vaccination includes administering the invented vaccine to the subject or patient at least once. One or more booster administrations of one or more other drugs may also be provided. If it is done

<p dir="rtl">10 Booster vaccination Typically, this booster vaccination will be administered to the same subject at a point between one week and one year, preferably between two weeks and four months, after an immunogenic combination with the same antigen has been given to the subject for the first time (which in these cases is referred to as “ In alternative booster regimens, different vectors may also be administered, i.e., one or more adenoviruses of different serotypes, or other vectors such as MVA, DNA, or</p>

<p dir="rtl">15 Protein, to the subject in the form of a primary or booster vaccination. In certain embodiments, the same form of the vaccine of the invention is administered at least twice to the same patient in a prime and booster dosing regimen, i.e., with adenovirus resulting from the same genetic reassortment (such as Ad26) according to the invention. In certain embodiments, the vaccine is administered The vaccine is tested at least twice in a primary and booster regimen, but the vaccine vector is different, i.e., two different serotypes of adenoviral vectors are used,</p>

<p dir="rtl">20 That is, initial administration with Ad26 results in rejoining and a booster with Ad35 results in rejoining, or vice versa; Or initial dosing with DNA and boosting with an adenoviral vector, or vice versa; Or administering initial doses with an adenoviral vector and boosting with an MVA vector, or vice versa. In certain embodiments, the vaccine is administered according to at least three steps, in a prime-booster-booster regimen. Another booster may be added to the dosing regimen.</p>

<p dir="rtl">25 A further feature of the invention is to induce a CTL response against HPV16 in a subject, comprising administering a vaccine according to the invention to the subject.</p>

7690

-33-

The invention also provides the following non-exclusive embodiments:

<p dir="rtl">1) A nucleic acid encoding a polypeptide containing sequence ID number: 1;</p>

<p dir="rtl">2) nucleic acid according to embodiment 1, wherein the polypeptide also includes at least a portion of the HPV E2 protein;</p>

<p dir="rtl">5 3) Nucleic acid according to embodiment 2, wherein at least a portion of the HPV E2 protein is a protein</p>

E2 of HPV16;

<p dir="rtl">4) a nucleic acid according to embodiment 2, wherein the polypeptide comprises at least a portion of an E2 protein fused to the N-terminal side of the polypeptide with sequence identity number: 1;</p>

<p dir="rtl">5) Nucleic acid according to embodiment 2, wherein the polypeptide comprises at least a portion of an E2 protein</p>

<p dir="rtl">10 fused to the C-terminal side of a polypeptide with sequence identity no: 1;</p>

<p dir="rtl">6) Nucleic acid according to embodiment 3, wherein the polypeptide comprises at least a portion of an E2 protein</p>

fused to the N-terminal side of a polypeptide with sequence identity number: 1;

<p dir="rtl">7) Nucleic acid according to embodiment 3, wherein the polypeptide comprises at least a portion of an E2 protein fused to the C-terminal side of the polypeptide with sequence identity number: 1;</p>

<p dir="rtl">15 8) Nucleic acid according to embodiment 2, wherein at least part of the E2 protein comprises a variant of</p>

E2 protein with a mutation that abolishes DNA binding of E2;

<p dir="rtl">9) Nucleic acid according to embodiment 3, wherein at least part of the E2 protein comprises a variant of</p>

E2 protein with a mutation that abolishes DNA binding of E2;

<p dir="rtl">10) Nucleic acid according to embodiment 4, wherein at least a portion of the E2 protein comprises a variant of</p>

20 E2 protein with a mutation that abolishes DNA binding of E2;

<p dir="rtl">11) Nucleic acid according to embodiment 5, wherein at least part of the E2 protein comprises a variant of</p>

E2 protein with a mutation that abolishes DNA binding of E2;

<p dir="rtl">12) Nucleic acid according to embodiment 6, wherein at least a portion of the E2 protein comprises a variant of the E2 protein with a mutation that abolishes DNA binding of E2;</p>

<p dir="rtl">25 13) Nucleic acid according to embodiment 7, wherein at least part of the E2 protein comprises a variant of</p>

E2 protein with a mutation that abolishes DNA binding of E2;

7690

-34-

14) A vector comprising nucleic acid according to embodiment 1, wherein the polypeptide coding sequence is operably linked

with reinforcement;

15) A vector comprising nucleic acid according to embodiment 2, wherein the polypeptide coding sequence is operably linked

with reinforcement;

16) A vector comprising nucleic acid according to embodiment 3, wherein the polypeptide coding sequence is operably linked

with reinforcement;

17) A vector comprising nucleic acid according to embodiment 4, wherein the polypeptide coding sequence is operably linked

with reinforcement;

18) A vector comprising nucleic acid according to embodiment 5, wherein the polypeptide coding sequence is operably linked

<p dir="rtl">10 with reinforcement;</p>

19) A vector comprising nucleic acid according to embodiment 6, wherein the polypeptide coding sequence is operably linked

with reinforcement;

20) A vector comprising nucleic acid according to embodiment 7, wherein the polypeptide coding sequence is operably linked

15

20

25

with reinforcement;

21) A vector comprising nucleic acid according to embodiment 8, wherein the polypeptide coding sequence is operably linked

with reinforcement;

22) A vector comprising nucleic acid according to embodiment 9, wherein the polypeptide coding sequence is operably linked

with reinforcement;

<p dir="rtl">23) A vector comprising nucleic acid according to embodiment 10, wherein the polypeptide coding sequence is operably linked to an enhancer;</p>

<p dir="rtl">24) A vector comprising nucleic acid according to embodiment 11, wherein the polypeptide coding sequence is operably linked to an enhancer;</p>

<p dir="rtl">25) A vector comprising nucleic acid according to embodiment 12, wherein the polypeptide coding sequence is operably linked to an enhancer;</p>

<p dir="rtl">26) A vector comprising nucleic acid according to embodiment 13, wherein the polypeptide coding sequence is operably linked to an enhancer;</p>

7690

-35-

<p dir="rtl">27) A vector according to Embodiment 14, wherein the vector is an adenovirus;</p>

<p dir="rtl">28) A vector according to Embodiment 15, wherein the vector is an adenovirus;</p>

<p dir="rtl">29) A vector according to Embodiment 16, wherein the vector is an adenovirus;</p>

<p dir="rtl">30) A vector according to Embodiment 17, wherein the vector is an adenovirus;</p>

<p dir="rtl">5 31) A vector according to Embodiment 18, wherein the vector is an adenovirus;</p>

<p dir="rtl">32) A vector according to Embodiment 19, wherein the vector is an adenovirus;</p>

<p dir="rtl">33) A vector according to Embodiment 20, wherein the vector is an adenovirus;</p>

<p dir="rtl">34) A vector according to Embodiment 21, wherein the vector is an adenovirus;</p>

<p dir="rtl">35) A vector according to Embodiment 22, wherein the vector is an adenovirus;</p>

<p dir="rtl">10 36) A vector according to Embodiment 23, wherein the vector is an adenovirus;</p>

<p dir="rtl">37) A vector according to embodiment 24, wherein the vector is an adenovirus;</p>

<p dir="rtl">38) A vector according to Embodiment 25, wherein the vector is an adenovirus;</p>

<p dir="rtl">39) A vector according to embodiment 26, wherein the vector is an adenovirus;</p>

<p dir="rtl">40) A vector according to embodiment 27, wherein the adenovirus is a human adenovirus from</p>

<p dir="rtl">15 serotype 26;</p>

<p dir="rtl">41) A vector according to embodiment 28, wherein the adenovirus is a human adenovirus from</p>

serotype 26;

<p dir="rtl">42) a vector according to embodiment 29, wherein the adenovirus is a human adenovirus of serotype 26;</p>

20 43) A vector according to embodiment 30, wherein the adenovirus is a human adenovirus from

serotype 26;

<p dir="rtl">44) A vector according to embodiment 31, wherein the adenovirus is a human adenovirus from</p>

serotype 26;

<p dir="rtl">45) A vector according to embodiment 32, wherein the adenovirus is a human adenovirus from</p>

<p dir="rtl">25 serotype 26;</p>

<p dir="rtl">46) A vector according to embodiment 33, wherein the adenovirus is a human adenovirus from</p>

7690

-36-

serotype 26;

<p dir="rtl">47) a vector according to embodiment 34, wherein the adenovirus is a human adenovirus of serotype 26;</p>

<p dir="rtl">48) A vector according to embodiment 35, wherein the adenovirus is a human adenovirus from</p>

<p dir="rtl">5 serotype 26;</p>

<p dir="rtl">49) A vector according to embodiment 36, wherein the adenovirus is a human adenovirus from</p>

serotype 26;

<p dir="rtl">50) a vector according to embodiment 37, wherein the adenovirus is a human adenovirus of serotype 26;</p>

<p dir="rtl">10 51) A vector according to embodiment 38, wherein the adenovirus is a human adenovirus from</p>

serotype 26;

<p dir="rtl">52) A vector according to embodiment 39, wherein the adenovirus is a human adenovirus from</p>

serotype 26;

<p dir="rtl">53) A vector according to embodiment 28, wherein the adenovirus is a human adenovirus from</p>

<p dir="rtl">15 serotype 35;</p>

<p dir="rtl">54) A vector according to embodiment 29, wherein the adenovirus is a human adenovirus from</p>

serotype 35;

<p dir="rtl">55) A vector according to embodiment 30, wherein the adenovirus is a human adenovirus of serotype 35;</p>

20 56) A vector according to embodiment 31, wherein the adenovirus is a human adenovirus from

serotype 35;

<p dir="rtl">57) A vector according to embodiment 32, wherein the adenovirus is a human adenovirus from</p>

serotype 35;

<p dir="rtl">58) A vector according to embodiment 33, wherein the adenovirus is a human adenovirus from</p>

<p dir="rtl">25 serotype 35;</p>

<p dir="rtl">59) A vector according to embodiment 34, wherein the adenovirus is a human adenovirus from</p>

7690

-37-

serotype 35;

60) A vector according to embodiment 35, wherein the adenovirus is a human adenovirus from

serotype 35;

61) A vector according to embodiment 36, wherein the adenovirus is a human adenovirus from

<p dir="rtl">5 serotype 35;</p>

<p dir="rtl">62) A vector according to embodiment 37, wherein the adenovirus is a human adenovirus from</p>

serotype 35;

<p dir="rtl">63) a vector according to embodiment 38, wherein the adenovirus is a human adenovirus of serotype 35;</p>

10 64) A vector according to embodiment 39, wherein the adenovirus is a human adenovirus from

serotype 35;

<p dir="rtl">65) A vaccine composition comprising a vector according to embodiment 14, and a pharmaceutically acceptable excipient;</p>

<p dir="rtl">66) A vaccine composition comprising a vector according to embodiment 15, and a pharmaceutically acceptable excipient;</p>

<p dir="rtl">67) A vaccine composition comprising a vector according to embodiment 16, and a pharmaceutically acceptable excipient;</p>

15 68) A vaccine composition comprising a vector according to embodiment 17, and a pharmaceutically acceptable excipient;

<p dir="rtl">69) A vaccine composition comprising a vector according to embodiment 18, and a pharmaceutically acceptable excipient;</p>

<p dir="rtl">70) A vaccine composition comprising a vector according to embodiment 19, and a pharmaceutically acceptable excipient;</p>

<p dir="rtl">71) A vaccine composition comprising a vector according to embodiment 20, and a pharmaceutically acceptable excipient;</p>

<p dir="rtl">72) A vaccine composition comprising a vector according to embodiment 21, and a pharmaceutically acceptable excipient;</p>

20 73) A vaccine composition comprising a vector according to embodiment 22, and a pharmaceutically acceptable excipient;

<p dir="rtl">74) A vaccine composition comprising a vector according to embodiment 23, and a pharmaceutically acceptable excipient;</p>

<p dir="rtl">75) A vaccine composition comprising a vector according to embodiment 24, and a pharmaceutically acceptable excipient;</p>

<p dir="rtl">76) A vaccine composition comprising a vector according to embodiment 25, and a pharmaceutically acceptable excipient;</p>

<p dir="rtl">77) A vaccine composition comprising a vector according to embodiment 26, and a pharmaceutically acceptable excipient;</p>

25 78) A vaccine composition comprising a vector according to embodiment 27, and a pharmaceutically acceptable excipient;

<p dir="rtl">79) A vaccine composition comprising a vector according to embodiment 28, and a pharmaceutically acceptable excipient;</p>

7690

-38-

10

15

20

25

<p dir="rtl">80) A vaccine composition comprising a vector according to embodiment 29, and a pharmaceutically acceptable excipient;</p>

<p dir="rtl">81) A vaccine composition comprising a vector according to embodiment 30 and a pharmaceutically acceptable excipient;</p>

<p dir="rtl">82) A vaccine composition comprising a vector according to embodiment 31, and a pharmaceutically acceptable excipient;</p>

<p dir="rtl">83) A vaccine composition comprising a vector according to embodiment 32, and a pharmaceutically acceptable excipient;</p>

<p dir="rtl">84) A vaccine composition comprising a vector according to embodiment 33, and a pharmaceutically acceptable excipient;</p>

<p dir="rtl">85) A vaccine composition comprising a vector according to embodiment 34, and a pharmaceutically acceptable excipient;</p>

<p dir="rtl">86) A vaccine composition comprising a vector according to embodiment 35, and a pharmaceutically acceptable excipient;</p>

<p dir="rtl">87) A vaccine composition comprising a vector according to embodiment 36, and a pharmaceutically acceptable excipient;</p>

<p dir="rtl">88) A vaccine composition comprising a vector according to embodiment 37, and a pharmaceutically acceptable excipient;</p>

<p dir="rtl">89) A vaccine composition comprising a vector according to embodiment 38, and a pharmaceutically acceptable excipient;</p>

<p dir="rtl">90) A vaccine composition comprising a vector according to embodiment 39, and a pharmaceutically acceptable excipient;</p>

<p dir="rtl">91) A vaccine composition comprising a vector according to embodiment 40, and a pharmaceutically acceptable excipient;</p>

<p dir="rtl">92) A vaccine composition comprising a vector according to embodiment 41, and a pharmaceutically acceptable excipient;</p>

<p dir="rtl">93) A vaccine composition comprising a vector according to embodiment 42, and a pharmaceutically acceptable excipient;</p>

<p dir="rtl">94) A vaccine composition comprising a vector according to embodiment 43, and a pharmaceutically acceptable excipient;</p>

<p dir="rtl">95) A vaccine composition comprising a vector according to embodiment 44, and a pharmaceutically acceptable excipient;</p>

<p dir="rtl">96) A vaccine composition comprising a vector according to embodiment 45, and a pharmaceutically acceptable excipient;</p>

<p dir="rtl">97) A vaccine composition comprising a vector according to embodiment 46, and a pharmaceutically acceptable excipient;</p>

<p dir="rtl">98) A vaccine composition comprising a vector according to embodiment 47, and a pharmaceutically acceptable excipient;</p>

<p dir="rtl">99) A vaccine composition comprising a vector according to embodiment 48, and a pharmaceutically acceptable excipient;</p>

100) A vaccine composition comprising a vector according to embodiment 49, and a pharmaceutically acceptable excipient;

101) A vaccine composition comprising a vector according to embodiment 50 and a pharmaceutically acceptable excipient;

102) A vaccine composition comprising a vector according to embodiment 51, and a pharmaceutically acceptable excipient;

103) A vaccine composition comprising a vector according to embodiment 52, and a pharmaceutically acceptable excipient;

104) A vaccine composition comprising a vector according to embodiment 53, and a pharmaceutically acceptable excipient;

105) A vaccine composition comprising a vector according to embodiment 54, and a pharmaceutically acceptable excipient;

7690

-39-

106) A vaccine composition comprising a vector according to embodiment 55, and a pharmaceutically acceptable excipient;

107) A vaccine composition comprising a vector according to embodiment 56, and a pharmaceutically acceptable excipient;

108) A vaccine composition comprising a vector according to embodiment 57, and a pharmaceutically acceptable excipient;

109) A vaccine composition comprising a vector according to embodiment 58, and a pharmaceutically acceptable excipient;

5 110) A vaccine composition comprising a vector according to embodiment 59, and a pharmaceutically acceptable excipient;

111) A vaccine composition comprising a vector according to embodiment 60 and a pharmaceutically acceptable excipient;

112) A vaccine composition comprising a vector according to embodiment 61, and a pharmaceutically acceptable excipient;

113) A vaccine composition comprising a vector according to embodiment 62, and a pharmaceutically acceptable excipient;

114) A vaccine composition comprising a vector according to embodiment 63, and a pharmaceutically acceptable excipient;

<p dir="rtl">10 115) A vaccine composition comprising a vector according to embodiment 64, and a pharmaceutically acceptable excipient;</p>

<p dir="rtl">116) A method of inducing an anti-HPV immune response in a subject, comprising administering to the subject a vaccine composition according to any of embodiments 65-115;</p>

<p dir="rtl">117) A method for treating persistent HPV infection (type 16), comprising administering a vaccine according to any of embodiments 65-115 to a subject with persistent HPV infection;</p>

<p dir="rtl">15 118) A method for treating VIN (with HPV type 16 infection)</p>

latent), comprising administering a vaccine according to any of embodiments 65-115 to a subject suffering from VIN; 119) a method for treating vulvar cancer (with latent HPV type 16 infection), comprising administering a vaccine according to any of embodiments 65-115 to a subject suffering from VIN vulva;

120) A method for treating cervical dysplasia (CIN) (with latent HPV type 16 infection),

<p dir="rtl">20 comprising administering a vaccine according to any of embodiments 65-115 to a subject suffering from CIN;</p>

121) A method for treating cervical cancer (with latent HPV type 16 infection), comprising administering a vaccine according to any of embodiments 65-115 to a subject suffering from cervical cancer;

122) A method for treating oropharyngeal cancer (with latent HPV type 16 infection), comprising administering a vaccine according to any of embodiments 65-115 to a subject suffering from oropharyngeal cancer;

<p dir="rtl">25 123) A method for treating penile intraepithelial neoplasia (PIN) (with HPV infection).</p>

<p dir="rtl">16 latent), comprising administering a vaccine according to any of embodiments 65-115 to a subject suffering from</p>

7690

-40-

PIN;

124) A method for treating penile cancer (with latent HPV type 16 infection), comprising administering a vaccine according to any of embodiments 65-115 to a subject suffering from penile cancer;

125) A method for treating the formation of vaginal intraepithelial neoplasia (VaIN) with HPV infection.

<p dir="rtl">5 16 latent), comprising administering a vaccine according to any of embodiments 65-115 to a subject suffering from</p>

VaIN;

126) A method for treating vaginal cancer (with latent HPV type 16 infection), comprising administering a vaccine according to any of embodiments 65-115 to a subject suffering from vaginal cancer;

127) A method for treating anal intraepithelial neoplasia (AIN) (with latent HPV type 10 16 infection), comprising administering a vaccine according to any of embodiments 65-115 to a subject with

AIN;

128) A method for treating anal cancer (with latent HPV type 16 infection), comprising administering a vaccine according to any of embodiments 65-115 to a subject suffering from anal cancer;

129) A polypeptide containing sequence identity number: 1;

<p dir="rtl">15 130) A polypeptide according to embodiment 129, wherein the polypeptide also includes at least a portion of</p>

HPV E2 protein;

131) A polypeptide according to embodiment 130, wherein at least a portion of the HPV E2 protein is

E2 protein of HPV16;

132) A polypeptide according to embodiment 130, wherein at least a portion of the E2 protein is fused to the

<p dir="rtl">20 N-terminal to the polypeptide with sequence identity number: 1;</p>

133) A polypeptide according to embodiment 130, wherein at least a portion of the E2 protein is fused to the

C-terminal polypeptide with sequence identity number: 1;

134) A polypeptide according to embodiment 131, wherein at least a portion of the E2 protein is fused to the

N-terminal to the polypeptide with sequence identity number: 1;

<p dir="rtl">25 135) A polypeptide according to embodiment 131, wherein at least a portion of the E2 protein is fused to the</p>

C-terminal polypeptide with sequence identity number: 1;

7690

-41-

10

15

136) A polypeptide according to embodiment 130, wherein at least a portion of the E2 protein comprises a variant of the E2 protein with a mutation that abolishes DNA binding of E2;

137) A polypeptide according to embodiment 131, wherein at least a portion of the E2 protein comprises a variant

of an E2 protein with a mutation that abolishes DNA binding of E2;

138) A polypeptide according to embodiment 132, wherein at least a portion of the E2 protein comprises a variant

of an E2 protein with a mutation that abolishes DNA binding of E2;

139) A polypeptide according to embodiment 133, wherein at least a portion of the E2 protein comprises a variant

of an E2 protein with a mutation that abolishes DNA binding of E2;

140) A polypeptide according to embodiment 134, wherein at least a portion of the E2 protein comprises a variant

of an E2 protein with a mutation that abolishes DNA binding of E2;

141) A polypeptide according to embodiment 135, wherein at least a portion of the E2 protein comprises a variant

of an E2 protein with a mutation that abolishes DNA binding of E2;

142) A nucleic acid according to embodiment 3, encoding a polypeptide according to sequence identity number: 3;

143) A nucleic acid according to embodiment 3, encoding a polypeptide according to sequence identity number: 5;

144) A vector encoding a nucleic acid according to embodiment 142, wherein the polypeptide coding sequence is operably linked

with reinforcement;

145) A vector encoding a nucleic acid according to embodiment 143, wherein the polypeptide coding sequence is operably linked

with reinforcement;

146) A vector according to embodiment 144, wherein the vector is an adenovirus;

20 147) A vector according to embodiment 145, wherein the vector is an adenovirus;

148) A vector according to embodiment 146, wherein the adenovirus is a human adenovirus from

serotype 26;

149) A vector according to embodiment 147, wherein the adenovirus is a human adenovirus of serotype 26;

25 150) A vector according to embodiment 146, wherein the adenovirus is a human adenovirus from

serotype 35;

7690

-42-

10

151) A vector according to embodiment 147, wherein the adenovirus is a human adenovirus of serotype 35;

152) A vaccine composition comprising a vector according to embodiment 144, and a pharmaceutically acceptable excipient;

153) A vaccine composition comprising a vector according to embodiment 145, and a pharmaceutically acceptable excipient;

154) A vaccine composition comprising a vector according to embodiment 146, and a pharmaceutically acceptable excipient;

155) A vaccine composition comprising a vector according to embodiment 147, and a pharmaceutically acceptable excipient;

156) A vaccine composition comprising a vector according to embodiment 148, and a pharmaceutically acceptable excipient;

157) A vaccine composition comprising a vector according to embodiment 149, and a pharmaceutically acceptable excipient;

158) A vaccine composition comprising a vector according to embodiment 150, and a pharmaceutically acceptable excipient;

159) A vaccine composition comprising a vector according to embodiment 151, and a pharmaceutically acceptable excipient;

160) A method of inducing an anti-HPV immune response in a subject, comprising administering to the subject a vaccine composition according to any of embodiments 152-159;

161) A method for treating VIN, comprising administering a vaccine in accordance with either

Incarnations 152-159 of a submissive suffering from VIN;

15 162) A method for treating vulvar cancer, comprising administering a vaccine according to any of embodiments 152

159 For a submissive suffering from vulvar cancer;

163) A method for treating cervical dysplasia (CIN), comprising administering a vaccine according to any of embodiments 152-159 to a subject suffering from CIN;

164) A method of treating cervical cancer, including administering a vaccine according to any of the embodiments

20 159-152 for a subject suffering from cervical cancer;

165) A method for treating oropharyngeal cancer, comprising administering a vaccine according to any of embodiments 152-159 to a subject suffering from oropharyngeal cancer;

166) A method for treating penile intraepithelial neoplasia (PIN), comprising administering a vaccine according to any of embodiments 152-159 to a subject suffering from PIN;

25 167) A method for treating penile cancer, comprising administering a vaccine according to any of embodiments 152

159 For a submissive suffering from penile cancer;

7690

-43-

168) A method for treating vaginal intraepithelial neoplasia (VaIN), comprising administering a vaccine according to any of embodiments 152-159 to a subject suffering from VaIN;

169) A method for treating vaginal cancer, comprising administering a vaccine according to any of embodiments 152159 to a subject suffering from vaginal cancer;

<p dir="rtl">5 170) A method for treating the formation of anal intraepithelial neoplasia (AIN), which includes administering a vaccine according to</p>

of any of embodiments 152-159 of a subject suffering from AIN;

<p dir="rtl">171) A method for treating anal cancer, comprising administering a vaccine according to any of embodiments 152159 to a subject suffering from anal cancer.</p>

Unless otherwise indicated, the practice of this invention will use traditional techniques of validation, science

<p dir="rtl">10 Molecular biology, microbiology, cell biology, and DNA replication, which</p>

Sambrook, Fritsch and Maniatis, are within the skill of the field. See for example 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.); PCR2: A Practical Approach, MacPherson MJ, Hams BD, Taylor GR, eds, 1995; Antibodies: 15

A Laboratory Manual, Harlow and Lane, eds, 1988

The invention is also explained in the following examples. The examples are not limited in any way. It clearly works to clarify the most important sister.

Examples

<p dir="rtl">20 Example 1: Structure of a designed polypeptide that contains substantially all of the HPV16 E6 and E7 CTL epitopes We designed a novel, non-oncogenic polypeptide (and the DNA that encodes it) that contains substantially all of the CTL epitopes of the HPV16 E6 and E7 CTL proteins, and has a minimal number of novel epitopes. Anticipated/anticipated (new epitopes meaning epitopes not found in wild-type HPV16 E6 and E7 proteins). The polypeptide of the invention (sometimes also referred to as “E6E7SH”) includes</p>

<p dir="rtl">25 In this document (a sequence introduced in SEQ ID No. 1) an enhanced DNA is presented</p>

7690

-44-

With a codon that encodes the polypeptide, sequence identity number: 2.

The molecules of the invention are single molecules, providing a manufacturing advantage over strategies that use multiple molecules. In addition, the polypeptide of the invention comprises essentially all of the putative CTL epitopes found in E6 and E7 of wild-type HPV16, and in the same

<p dir="rtl">5 At the same time, there is a minimal number of potential new potent epitopes that can potentially immunodominate and thus shift the immune response from the relevant wild-type CTL epitopes. Components of the present invention are therefore more immunologically preferable than molecules described in other documents that either lack available CTL epitopes and/or contain more or stronger novel epitopes.</p>

<p dir="rtl">10 For example, a component with SEQ ID: 1 contains only one novel epitope nine amino acids long with a predicted binding affinity of &gt;50 nM for 20 of the most common HLA-A, 20 of the most common HLA-B, and 20 of the most common HLA-B. HLA-C alleles (01:01*HLA-A,</p>

HLA-A*, 02:01*HLA-A*, HLA-A*02:07, HLA-A*02:06, HLA-A*03:01 HLA-A*1, 24:02, HLA-A*11:01, 23:01,

<p dir="rtl">15 HLA-A*, 29:02*, HLA-A*31:01, HLA-A*30:02, HLA-A*30:01</p>

HLA-A*68:01, HLA-A*68:01, HLA-A*33:01, HLA-A*33:03, HLA-B*08, 68:02 HLA-B*07:04, HLA-A:01, HLA-B*07:02

HLA-B*35:01, 13:01*HLA-B*37:01, HLA-B*18:01, 15:01, 39:01*HLA-B*40 HLA-B*40:02, HLA-B*40:06:01, HLA-B*40:01

20 HLA-B*44:03, HL-B*46:01, HL-B*46:01, HLA-B*51:01,

HLA-B*, 52:01, HLA-C*, 07:02*, HLA-B*, 53:01, 58:01

HLA-C*1, 06:02*HLA-C*01:02, HLA-C*01:02, 03:04*HLA-C*12, 03:03* HLA-C*15:02, HLA-C*15:02, HLA-C*08:01, 02:02*HLA-C*16:01, HLA-C*14:02, 03:02, HLA-C*05:01,C,

25 HLA-C*, 08:02*, HLA-C*17:01, HLA-C*17:03, 04:03

14:03*C), as determined using Lundegaard et al., 2008, Nucl Acids (ANN

7690

-45-

Peters et al., 2003, Bioinformatics 19: (SMM and Method) Res 36: W509-12

Hoof et al., 2009, (NetMHCpan and HLA-B and HLA-A 72-1765 method) for

MHC class I prediction tool for peptide binding to HLA-C molecules (Immunogenetics 61: 1-13

On site IEDB

<a href="http://tools.immuneepitope.org/analyze/html/mhc_binding.html">http://tools.immuneepitope.org/analyze/html/mhc_binding.html</a>, version (5

.)2009-09-01B

By way of example, but not limited to, using the IEDB site's SMM prediction tool, E6 sequences

Oosterhuis et al., 2011, Int J Cancer 129: modified as described by E7, 406–397 on all epitopes and Öhlschläger et al., 2006, Vaccine 24: 2880–93

<p dir="rtl">10 The nine potentially powerful novel epitopes (ANN or SMM &lt; 50 nM IC50 nM) for the 20 most HLA-A and -B, in the core. This also excludes the epitopes used in this method (in which the epitopes will also contribute additional novel epitopes, and can Their loss on innate MHC II epitopes is more likely due to the limited length of the T-complex. In fact, an enhancer molecule as registered containing a variant with modified E6 and E7 proteins described in WO 2013/083287 contains 22</p>

<p dir="rtl">15 A unique novel epitope nine amino acids long with a predicted IC50 &gt;50 nM (ANN, SMM or NetMHCPan) for 20 of the most common HLA-A, 20 of the most common HLA-B and 20 of the most common HLA-C alleles.</p>

Therefore, the designer portions of the invention are clearly preferable in that they have a much smaller number of new predicted epitopes compared to other published methods where E6 and E7 are modified to remove

<p dir="rtl">20 Functional group.</p>

The nucleic acid encoding the designed HPV16 E6E7SH molecule (i.e., a polypeptide having the amino acid sequence as given by SEQ ID: 1) was synthesized, the nucleic acid sequence comprising SEQ ID: 2, flanked by a HindIII site and a Kozak sequence on the 5' end. And the XbaI website

On site '3) Custom synthesis and cloning of a standard molecule at Invitrogen Life technologies

<p dir="rtl">25 Germany(.</p>

The synthetic fragments were cloned using HindIII and XbaI into a standard expression vector,

7690

-46-

pCDNA2004.Neo, concealing both a bacterial resistance encoder (ampicillin) and a mammalian resistance encoder (neomycin), to obtain plasmid vectors encoding the molecule of the invention, for example for (transient) transcription experiments.

These molecules can be used as is, but also as a basis for other molecules that have 5 additional attributes. For example, but not limited to, some additional variables were prepared as described below.

The HPV16 E6E7SH fusion protein sequence can be assembled with other early HPV16 protein sequences to target people with persistent infection and to expand the immune repertoire in an immune person. Immune responses against E2 have been suggested to play an important role in the clearance of de Jong et al. (HPV16) infections.

Fusion of E6E7SH with E2 will lead to the production of E6E7SH. (al., 2002, Cancer Res 62: 472-479).

Vaccine component that hides antigens HPV-related cancer has not progressed from persistent infection to invasive cancer or recurrent/resistant disease after LEEP surgery. Therefore, by way of example and not limited to these embodiments, we have prepared a sequence encoding a fusion protein of E6E7SH with E2 at its N terminus. In the E2 sequence modifications can be made to abrogate the DNA binding activity which can affect the heritable expression of the gene.

<p dir="rtl">15 In cells that genetically express the fusion protein. We mutated a glycine at position 293, a lysine at position 299 and a cysteine at position 300 of the HPV16 wt E2 protein into two essential amino acids, respectively, methionine and arginine. Each of these mutations on their own already completely abolishes the link</p>

Prakash et al., 1992, E2 Genes Dev masking DNA binding domains with E2 sequences

.)6: 105-16

<p dir="rtl">20 The resulting polypeptide is denoted HPV16 E2E6E7SH and has sequence ID #: 3.</p>

A codon optimization sequence encoding this polypeptide was prepared and presented as SEQ ID NO: 4.

We have also created a variant in which the same E2 mutant protein is fused C-terminally to an HPV16 E6E7SH polypeptide, giving an excess of the polypeptide designated HPV16 E6E7E2SH, which has SEQ ID #5. The sequence encoding that component is provided as SEQ ID: Number: 6.

<p dir="rtl">25 For comparison purposes, we also generated sequences encoding a polypeptide containing the sequences of the type</p>

Wild-type HPV16 E6 full-length and E7 as fusion protein (E6 from 1 aa to 158

7690

-47-

fused directly to E7 from 1 aa to 98, here called E6E7wt(.

We also tested the effect of adding leader sequences to the polypeptide. By way of example, but not limited to, a sequence coding for a leader IgE sequence (see e.g. US 6,733,994) [the leader peptide sequence is provided in SEQ ID NO: 7] is fused at the N terminus to some component, e.g.

<p dir="rtl">5 The example is in the E6E7wt component, which blocks LSE6E7wt, and in the E2E6E7SH component, which blocks LSE2E6E7SH. Its immunogenic effect is significantly enhanced (p &lt; 0.05) compared to the same antigen without the LS sequence as measured by E7 tetramer analysis in immunized mice (as shown for example in Figure 9).</p>

Sequences encoding E6E7SH polypeptides of the invention can be expressed, with or without E2,

<p dir="rtl">10 For example from components of DNA, RNA or viral vectors. Figure 1 shows transgene expression in HEK-293T cells during transient infection using DNA vectors that transgenicly express the transgene as described above. After infection, cells were collected and cell extracts were analyzed by SDS-PAGE and Western staining using an antibody against HPV16 E7. This experiment demonstrates transgenic expression of the predicted fusion proteins at an appropriate size during infection of the expression vectors</p>

<p dir="rtl">15 Hereditary.</p>

Adenoviral vectors can be used to express E6E7, either with or without E2, and with or without additional sequences to increase immunogenicity of the encoded fusion protein.

The genes encoding the HPV16 E6E7 wt comparison or HPV construct sequences described above are genetically expressed in humans and synthetic, by Geneart. is included

<p dir="rtl">20 The 5' GCCACC (3' Kozak) sequence is immediately upstream of the ATG start codon, and two stop codons (5' 3' TGA TAA) are added at the end of the respective coding sequence. The genes were inserted into</p>

Havenga et al., 2006, J Gen (pAdApt26 and in pAdApt35BSU plasmid

Virol 87, 43-2135) via HindIII and XbaI sites.

All adenoviruses were generated in PER.C6 cells by a single homologous gene reassortment

Havenga et al., 2006, J Gen Virol 87: rAd35 25 Preserve the product as previously described (for

43–2135; For Abbink et al., 2007, J Virol 81: 4654-63: rAd26). PER.C6 cells

7690

-48-

Modified with Eagle in medium (Fallaux et al., 1998, Hum Gene Ther 9: 1909-17)

Dulbecco's DMEM (DMEM) with 10% fetal bovine serum (FBS), supplemented with 10 mM

.MgCl2

Briefly, PER.C6 cells were infected with Ad vector plasmids, using...

<p dir="rtl">5 Lipofectamine according to instructions provided by the manufacturer (Life Technologies). Cells were collected 1 day after reaching complete cytopathic effect (CPE), thawed, centrifuged for 5 minutes at 3,000 rpm, and stored at −20°C. Viruses were plate-purified and amplified in PER.C6 cells cultured in one eye of a 24-well multiwell tissue culture dish. Additional amplification was performed in PER.C6 cells cultured in a culture flask</p>

<p dir="rtl">10 T25 tissue and then into a T175 tissue culture flask. From the crude lysate prepared from cells generated after the T175 beaker, 3 to 5 ml was used to inoculate 24× T1000 five-layer tissue culture media containing 70% combined layers of PER.C6 cells. The virus was purified using the two-step CsCl purification method. Finally, the virus was stored in the coupons at −85°C.</p>

Ad35.HPV16-E6E7wt and Ad35.HPV16-E6E7SH are viral vectors

<p dir="rtl">15 Adenovirus recombinant gene serotype 35 (Ad35) comprises codon-augmented nucleotide sequences for genotypic expression of, respectively, a fusion protein of the wild-type HPV16 E6 and E7 proteins (E6E7wt), and a fusion protein variant designed as described above (E6E7SH, whose sequence The amino acid provided in Sequence ID No.: 1). The assembled E6 and E7 sequences are placed under the control of the CMV promoter in the E1 region of E1, an E3-deleted adenovirus genome.</p>

<p dir="rtl">20 Ad26.HPV16-E6E7wt and Ad26.HPV16-E6E7SH are equivalent vectors based on adenovirus serotype 26.</p>

Similarly, adenoviral vectors were produced based on the Ad26 and Ad35 gene recombinants encoding the HPV16 E2E6E7SH variant (SEQ ID NO: 3). Likewise, Ad26 and Ad35 encoding the HPV16 E6E7E2SH variant (SEQ ID NO: 5) were produced. Ad35 who

25 It encodes an E2E6E7SH fusion protein with an IgE leader sequence at the N terminus, termed Ad35.HPV16-LSE2E6E7SH. An adenovirus with E6E7wt fused to the IgE leader sequence has also been produced

7690

-49-

At the N-terminus.

Recombinant adenoviruses were generated on PER.C6 cells and purified by centrifugation on cesium chloride gradients.

Other examples of components of the invention that are coupled with suppressive systems are provided in a subsequent example below.

<p dir="rtl">5 Example 2. Lack of conversion activity of designed components</p>

Wild-type HPV16 E6 and E7 proteins have oncogenic potential, as they appear to be active

Conversion in certain experiments, such as colony formation in a soft agar experiment (Massimi and Banks).

As described E6E7SH 2005). It includes a polypeptide, Methods Mol Med 119: 381-395

In Example 1 the E6 and E7 protein fragments are rearranged. It is expected that this removes power

<p dir="rtl">10 Oncogenicity, as can be measured for example by a significantly low transforming activity by comparison</p>

with either E6 or E7 wt proteins in a similar manner.

Others report that gene-edited variants of HPV16 E6 and E7 actually lose their constitutive potency.

Öhlschläger et al., 2006, Vaccine 24: 2880–93; Henken et al., 2012, (for tumor

Vaccine 30:66-4259), demonstrating that modifying the gene destroys the wild-type functions of the E6 and E7 proteins 15 .

In order to evaluate the loss of oncogenic properties, we evaluated the ability of our E6E7SH components to confer potency

Growth in soft agar during NIH 3T3 cells (as described by e.g. Massimi).

Involved in cell injury (and Banks, 2005, Methods Mol Med 119: 381-395).

NIH3T3 using a plasmid that genetically expresses wild-type HPV16 E7 in a consistent manner.

<p dir="rtl">20 Colony. In these experiments, genotypic expression of wild-type HPV16 E6 alone did not cause formation</p>

Colony above the pain I return. This is consistent with published observations that E7wt is much more effective than...

E6wt infection in that experiment did not cause E6wt infection (Sedman et al., 1991, J Virol 65: 4860-66).

Using our E6E7SH component to grow cell colonies on soft agar (Figure 2) in...

Four independent experiments, demonstrating that nucleic acids encoding a polypeptide, E6E7SH, lose the 25-switching ability that binds to E7.

The oncogenic potency of E6 and E7 correlates with their ability to reduce levels of cellular p53 proteins

7690

-50-

and pRb, respectively. p53 and pRb degradation experiments were performed to demonstrate that the DNA encoding the inventive polypeptide component, E6E7SH, does not have the bioactivity associated with wild-type E6 and E7 at the molecular level. Briefly, our HPV16 E6wt and E6E7SH components were expressed in NCI-H1299 cells lacking endogenous p53 to experiment with p53 degradation. For pRb degradation experiment 5, the HPV16 E7wt and E6E7SH components were expressed in pRb null Saos cells.

<p dir="rtl">2. As can be seen in Figure 3, co-expression of p53 with E6wt, but not with E6E7SH, leads to low p53 levels (panels A and B). Likewise, panels 3C and 3D show that co-expression of pRb with E7wt, but not with E6E7SH These data show that the DNA encoding the inventive polypeptide 10 has no colony-forming ability in soft agar and does not contaminate the biological activities of the wild type.</p>

of E6 and E7 polypeptides, inactivating p53 and pRb, respectively.

To further demonstrate the safety of the DNA components encoding the polypeptide of the invention, we used primary human lock keratinocytes that are the natural target cells for HPV-induced transformation. Immunization of primary human keratinocytes requires the action of both E6 and E7

This experiment is in the form of a wild type (Munger et al., 1989, J Virol 63: 4417-21) 15 Massimi and Banks, (2005). Probably the most physiologically relevant in vitro experiment to elucidate the safety of our components. Cells transformed using lens viruses, Methods Mol Med 119: 381-395

Cells expressing wild-type E6 and E7 of HPV16 (E6E7wt) immunized into primary keratinocytes as indicated by an extension of their productive life compared to untransformed control cells 20 (Figure 4) and activation of hTERT, the catalytic subunit of the telomeric telomerase (data not shown). He is

Genetic expression of the polypeptide of the invention (E6E7SH) was unable to extend productive lifespan compared to GFP-transformed or non-transformed keratinocytes. A similar result was obtained in additional independent donors (data not shown). Taken together, these data demonstrate that our components It loses the ability to induce alloimmunization in primary human erythroid cells and is considered a highly physiological model.

<p dir="rtl">25 Another component in which the HPV16 E6 and E7 fragments are recombined in another arrangement is unable to populate primary human keratinocytes. However, extended lifetime expansion has been observed up to approx</p>

7690

-51-

150-120 days for that structure. This indicates some unpredictability in that area, and demonstrates the superiority of molecules designed according to the invention in this safety feature.

All the experiments together in this example provide strong evidence for the lack of transforming activity of the nucleic acids encoding polypeptides of the invention and thus strongly improved safety over the HPV16 E6 and E7 wt components.

<p dir="rtl">5 Example 3. Immune responses to E6E7SH designer components</p>

<p dir="rtl">DNA vectors and adenoviral vectors were prepared, as described in Example 1.</p>

The CB6F1 mouse strain was used to measure immune responses, based on initial experiments in which mice were immunized with DNA plasmids encoding wild-type E2, E6 or E7, and immunization with E6, HPV16 E2 and E7 antigens induces a broader cellular immune response in

<p dir="rtl">10 CB6F1 than in 6/C57BL mice or Balb/c mice. In a separate experiment, mice were vaccinated with DNA vectors encoding the inventive molecules, and cellular immune responses were measured. Immune responses specific to HPV16 E7 can be measured in immunized mice using DNA plasmids that genetically express E6E7SH (Figure 5).</p>

The data shown below in this example are from mouse experiments performed using adenoviral vectors.

To evaluate vaccine-induced immunogenicity, CB6F1 mice were immunized with adenoviral vectors (Ad35) that genetically express E6E7wt, LSE6E7wt, LSE6E7SH, E6E7SH, or adenoviral vectors that do not encode a transgene (empty). Both doses were tested for administration to mice: 5*⁹10 viral particles (vp) and 1*¹⁰10 vp. Two and eight weeks after immunization, mice were sacrificed and the dissociated splenocytes were stimulated throughout

<p dir="rtl">20 Overnight using the HPV16 E7 15mer peptide bath. Tailored responses to E7 at two weeks and at eight weeks were analyzed by IFNγ ELISPOT. The data are presented in Figure 6. This shows that immunization of mice with Ad35.HPV16-E6E7SH induces E7-specific immune responses as measured by ELISPOT analysis. In addition, the results in Figure 6 demonstrate the possibility of improving the immune response against a transgene expressed by an adenovirus by...</p>

<p dir="rtl">25 Addition of an N-terminal leader sequence to the transgene.</p>

After addition of E2 to E6E7SH, the polypeptide was tested for immunogenicity. Vector encryption

7690

-52-

Ad35 polypeptides that have E2 either fused to the N terminus (E2E6E7SH) or to the C terminus (E6E7E2SH). CB6F1 mice were immunized with a dose of vp¹⁰10x1. Figure 7 (E7-Tatarmer staining) and Figure 8 (panel C, IFNγ ELISPOT) show that the immune responses against E7 to designer components including E2 tend to be elevated compared to the component without E2, although the differences are not quite large. Significant response against E2 to viral vectors

adenoviruses encode only E2 compared to the response to adenoviral vectors that have E2 fused to the designer E6E7SH polypeptide (Figure 8B), with significant differences for both E2 versus E2E6E7SH and E2 versus E6E7E2SH (p &lt; 0.05).

It is concluded that designed components that also include E2 can still provide an immune response against E7, and in addition also provide an immune response against E2, thus increasing the breadth of the immune response.

Above ingredients that do not include E2.

It was shown that addition of a leader sequence produces higher E7-specific responses when fused to the N terminus of a wild-type E6 and E7 fusion protein (Figure 6C). Likewise, the effect of the leader sequence on immunogenicity was determined for an E2E6E7SH fusion protein. Therefore, Ad35 vectors encoding the polypeptide were used

<p dir="rtl">15 designed, with or without N-terminal E2. The Ad35 vector encoding LSE2E6E7SH was used to vaccinate mice, and blood samples were drawn at two-week intervals to measure specific immune responses to E7 (Figure 9). As can be seen in Figures 7 and 8, the presence of E2 at any N-terminus tends to or C fused to E6E7SH increased immune responses. Addition of the IgE leader sequence also increased a specific response to E7 (Figure 9b). Over time sustained immune responses were observed for all vectors.</p>

<p dir="rtl">20 The three adenovirus encoding molecules designed according to the invention, the highest response yet</p>

Immunization corresponded to higher responses over the duration of the experiment.

It has been concluded that responses induced by a designed component that also includes an N-terminal E2 can be increased by the addition of specific sequences, for example, an IgE leader sequence, that target the encoded protein to specific cellular chambers.

<p dir="rtl">25 A cellular immune response against the peptide of the invention can be induced using various types of adenoviral vectors. In the previous experiment we used Ad35 vectors, while in the experiment of Figure 10,</p>

7690

-53-

Immunization of mice using an Ad26 adenoviral vector that genetically expresses E2E6E7SH. The data also show that immunization with an Ad26-based vaccine induces E7-specific T cells. In addition, the results show that a second immunization with an Ad35 adenoviral vector that genetically expresses E2E6E7SH also enhances cellular immune responses (Figure 10).

<p dir="rtl">5 Example 4. Immunogenicity of designed components in rhesus macaque.</p>

To evaluate the ability of adenoviral vectors genetically expressing the designer sequence of the invention to induce immune responses in non-human protozoa, rhesus macaques were immunized by intracellular injection with adenoviral vectors (Ad26) genetically expressing E2E6E7SH or adenoviral vectors not encoding a transgene (Ad26). empty), using a dose of 1*¹¹10 vp. Eight weeks later responses were enhanced

<p dir="rtl">10 Immunity to immunization by immunization using Ad26 vectors that genetically express the same antigen. At week 16, the animals received additional injections using Ad35 vectors that genetically express the same antigen. Blood samples were drawn at multiple time points and separated white blood cells were stimulated overnight with peptide pools corresponding to E6, HPV16 E2 or E7. Specific responses were measured by IFNγ ELISPOT. The data are shown in Figure 11. In addition, at week 10</p>

<p dir="rtl">15 At week 18 after primary immunization, the cellular immune response specific to the peptides was assessed</p>

New intersections are provided in the sister district. An IFNγ response is induced in all animals below a detection limit of &gt;50 SFU per 1*⁶10 PBMC (data not shown).

Alpesanat demonstrates that immunization of non-human protozoa with Ad26.HPV16-E2E6E7SH

It elicits cellular immune responses against all three HPV16 proteins found in the 20-encoded transgene, but not against novel junctions. Responses may be enhanced by additional immunization

Using Ad26.HPV16-E2E6E7SH and an additional boost at week 16 with the corresponding Ad35 vector also increased specific immune responses to E6, HPV16 E2 and E7.

A subsequent booster at week 72 with Ad26.HPV16-E2E6E7SH again increases the HPV16 cell-mediated immune response, which after a few weeks is rejected (not shown).

<p dir="rtl">25 In a separate trial (not shown), rhesus macaques were vaccinated by intravaginal administration with...</p>

A combination of two adenoviral vectors, one genetically expressing HPV16 E6E7SH and the other

7690

-54-

HPV16 L1 protein. Low but measurable cytokine responses were measured in peripheral blood mononuclear cells versus both E6 and E7. In those trials, strong cellular immune responses against L1 were detected.

Example 5. Therapeutic efficacy in a murine tumor model

<p dir="rtl">5 The polypeptide of the invention is capable of inducing an HPV16-specific cellular immune response in animals, which can exert a therapeutic effect on cells that genetically express HPV16 E6 and/or E7. Therapeutic immunization, i.e. immunization after the onset of tumor growth, can be used to demonstrate the effectiveness of selecting a therapeutic HPV vaccine. The therapeutic effect of Ad26 and Ad35 vectors was tested in a group of mice treated with...</p>

Lin et al., 1996, Cancer (E7) and HPV16 (E6) class cells that genetically express TC-1 cells.

<p dir="rtl">10 Res 6:21-21 TC cells will form a solid tumor within a few days to weeks after injection</p>

Subcutaneous in Ern category. Without vaccine, the blastula grows rapidly and reaches a predetermined size of 1000 mm³ within 30 days (panels D and E). While reaching this size the mice are sacrificed for ethical reasons. Using the primary booster immunization scheme using SLPs (used as a positive comparison sample;

Kenter et al., 2009, N Engl J Med 361:1838-47; Zwaveling et al., 2002, J

<p dir="rtl">15 Immunol 169:350-8) or adenoviral vectors genetically expressing HPV16-E2E6E7SH,</p>

A reduction in the growth of 1-TC-induced tumors was observed (Figure 12, panels b and c). Closer examination of the first 30 days after primary immunizations (panels f and g) shows that immunization with adenoviral vectors that genetically express E2E6E7SH has a greater effect on Vaccination with SLPs significantly reduces tumor growth. The initial growth rate is much lower and in most cases the tumors shrink.

<p dir="rtl">20 In 3 of 11 mice immunized with adenoviral vectors, the tumors were completely eliminated, which is reflected in the survival plot (panel H).</p>

In conclusion, immunization with adenoviral vectors that genetically express a polypeptide of the invention significantly reduces tumor growth or completely eliminates tumor formation in a well-performed knockout model of HPV16-induced cancer.

<p dir="rtl">25 Example 6: Using repressor systems to improve the productivity and genetic stability of adenoviral vectors that genetically express HPV-derived antigens</p>

7690

-55-

It has been reported that transgenes introduced into adenoviral vectors under the control of strong conformationally active enzymes can, depending on the properties of the transgene product, negatively affect the production of

Yoshida &amp; Yamada, 1997, Biochem Biophys Res Commun 230:426-(The transporter

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., 5

Mol Biotechnol 35:263-73 2007. Examples of problems with transgene-based vector production include inefficient vector rescue and growth, low final vector yields, and, in severe cases, rapid growth of viral mutants using defective transgene cassettes. To solve these problems, several studies have discovered the ability to genetically mute the expression of a transgene for a reproductive vector in productive cells.

Matthews et al., 1999, J Gen Virol 80:345-53; Edholm et al., 2001, J (10

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, various suppression systems have been previously implemented in the context of vectors.

Ad It actually shows improved vector productivity and genetic stability of vectors encoding 15 different types of transgenes (inhibitors).

It has been noted that some of the adenoviral vectors described herein, as well as some of the other adenoviral vectors encoding specific HPV antigenic variants, exhibit some of the transgene-based vector production problems described above, and therefore could potentially be improved upon. connection. We therefore propose to examine whether systems can be used to suppress genetic expression

<p dir="rtl">20 The vector transgene can improve the production characteristics of Ad vectors that genetically express HPV-derived antigens as described herein. For this purpose, we have implemented two Kazem-Operation systems</p>

Yao &amp; Eriksson, 1999, Hum Gene Ther 10:419-22, CymR/CuO and EP0990041B1 (current TetO/TetR, i.e., 2006, BMC Biotechnol 6:43).

In our adenoviral vector platform. Both the TetO/TetR 25 and CymR/CuO systems have been used previously by others to improve adenoviral vector production through gene suppression.

Gall et al., 2007, Mol Biotechnol 35:263-73; (The axis of the conveyor while the conveyor is repeating

7690

-56-

Cottingham et al., 2012, Biotechnol Bioeng 109:719-28; Gilbert et al.,

J Virol Methods 208:177-88 2014. Implementation of these two systems involves generating adenoviral vectors that genetically express genes of interest under the control of either a CMV promoter containing a TetO or CuO sequence. Furthermore, the implementation is called generation of cell lines. Which expresses and inheritance in a way

<p dir="rtl">5 Stable from similar special repressor proteins (i.e. TetR or CymR).</p>

Ad26- and Ad35-based, multiple E1-deleted vectors have been generated in which sequences encoding heterologous polypeptides are functionally ligated to a CMV promoter containing either TetO or CuO driver sequences. First, a sequence containing specific TetO- or CuOs (SEQ ID: 11 and SEQ ID: 12, respectively) was inserted near the transcription start site (TSS).

<p dir="rtl">10 With the CMV promoter (SEQ ID: 13) from pAdapt26 and pAdapt35.Bsu plasmids</p>

Abbink et al., 2007, J Virol 81:4654-63; Havenga et al., 2006, J Gen (

Virol 87:2135-43). Sequences containing the operator at the same positions were inserted

Yao &amp; Eriksson, 1999, Human Gene (as previously described for the CMV promoter microsystems Ther 10:419-22; EP0990041B1; Mullick et al., 2006, BMC Biotechnol

<p dir="rtl">15 6:43; EP1385946B1). In particular, for TSS (as originally characterized; Stenberg</p>

In the form of CuOs and -TetO, a sequence containing (et al., 1984, J. Virol) was introduced. 49: 190-9

Direct after positions -20 and +7, respectively. In sequence identity number: 13, these two positions correspond to positions 716 and 742, respectively. CMV primers containing the resulting operator are named, respectively, CMVTetO and CMVCuO. Subsequently, various transgenes were introduced after Moazazat

<p dir="rtl">20 (Edited) CMV components generated using HindIII and XbaI restriction sites. These include genes</p>

The transgene encodes a fusion protein of rice green fluorescent protein (GFP-Luc), LSE2E6E7SH of the present invention, and another polypeptide with some homology to LSE2E6E7SH (a component referred to in this example as “HPVAg”). HPVAg comprises the same leader sequence as Illustrated in LSE2E6E7SH, as well as the E6, E2, and E7 sequences of HPV16 using methods as

<p dir="rtl">25 As described herein, the resulting modified pAdapt26 and pAdapt35.Bsu plasmids were used to generate adenoviral vectors that genetically express the above-mentioned transmitter and the HPV transgene below.</p>

7690

-57-

Control either CMVTetO or CMVCuO booster.

Either TetR or CymR cell lines were generated by stable infection of PER.C6® cells using, respectively, the LifeTechnologies plasmid, V1025-20 (pcDNA).^TM6/TR) and is derived from pcDNA^TM6/TR where the TetR coding sequence is replaced (SEQ ID: 14, which encodes

<p dir="rtl">5 Polypeptide SEQ ID #: 15) with the CymR coding sequence with an optimized codon (SEQ ID #: 16, which encodes polypeptide SEQ ID #: 17). Generation of a highly stable cell line was performed as described by the pcDNA supplier.^TM6/TR used a transient infection-based experiment to screen cell cultures capable of repressing the genetic expression of genes derived from CMVTetO or CMVCuO. The resulting PER.C6/TetR and PER.C6/CymR cell lines were analyzed for their susceptibility10 to suppressing transgene expression during vector replication in those cells. Experiments performed using vectors that genetically express GFP-Luc under the control of a CMV primer containing at least the operon show a 10-fold reduction in genetic expression of the rice lucifer gene over the entire viral replication cycle in cell lines that genetically express the repressor corresponding to the operon sequences. (data not shown). This confirms that the PER.C6/TetR and PER.C6/CymR cell lines were capable of</p>

<p dir="rtl">15 Repression of hereditary expression of the vector transgene in the context of duplication of adenoviral gene vectors.</p>

The effect of TetR- and CymR-induced suppression of genetic expression of the adenoviral vector transgene on vector yields was examined to select Ad35-based vectors that genetically express HPVAg (Figure 13a). To achieve this, PER.C6/TetR cell lines were exposed to .C6, and PER.C6/CymR, published at 3*⁵10 A cell for each eye in 24-eye dishes, to quadrilateral lesions - at

<p dir="rtl">20 1000 virus particles per cell for three hours - using vectors that genetically express HPVAg from</p>

Either Moaz Azat CMVTetO or CMVCuO. For comparison, parallel infections were performed using corresponding vectors that genetically express GFP-Luc instead of HPVAg. Four days after infection, crude viral lysates were prepared by subjecting the ophthalmic contents (i.e., infected cells and medium) to two freeze-thaw cycles. Adenoviral vector titers were then determined by a quantitative PCR-based protocol.

<p dir="rtl">25 Custom Ad35 Hexon Sequence Purified Ad35 vector is used using a known virus particle titer as the standard. The results demonstrate that both Ad35 vectors encoding HPVAg containing TetO-</p>

7690

-58-

and CuO-, compared to comparator vectors that transgenicly express GFP-Luc, show lower vector yields on normal PER.C6 cells. In contrast, when produced in cells that genetically express their cognate repressors (i.e., TetR and CymR, respectively), the same vectors give yields as high as those generated using the control vectors. These data indicate that genetically suppressing the expression of the gene

<p dir="rtl">5 The transgene during vector production in productive cells could be beneficial for the production of Ad35 vectors carrying HPVAg as a transgene.</p>

The effect of suppression of genetic expression of the transgene of an adenovirus vector may be found in vector cultures that were also screened for vectors derived from adenovirus serotype 26 (Ad26) (Figure 13b). In an experiment carried out essentially as described above for Ad35 vectors, It was completed

<p dir="rtl">10 Use Ad26 vectors carrying CMVTetO promoter-controlled transgenes encoding either HPVAg, GFP-Luc, or LSE2E6E7SH to infect PER.C6 and PER.C6/TetR cells at 1,500 virus particles per cell. Three days later, infections were collected and the identified virus particle was assayed by a quantitative PCR-based method specific to the Ad25 hexon sequence. The results show that in PER.C6 cells the yields of vectors encoding HPVAg and LSE2E6E7SH are lower than those</p>

<p dir="rtl">15 Using the comparator vector encoding GFP-Luc. In contrast, on PER.C6/TetR cells, both vectors show titers that are higher than the output for the comparator vector. In combination with the results above (for Ad35 vectors), these data indicate that suppression of transgene expression during adenoviral vector production increases the yields of vectors that genetically express HPVAg and LSE2E6E7SH.</p>

We have observed fundamental problems with the genetic stability of an adenovirus vector carrying a 20 transgene derived from the CMV promoter for HPVAg. For example, it was observed that after several traffic cycles

Therefore the vector on PER.C6 most of the vector assembly consists of a mutant vector carrying a large deletion in the HPVAg coding sequence (data not shown).

We attribute this to the fact that the use of a genotypic expression suppressor system for the transformant, such as one of the two described above, can prevent genetic invariance problems associated with transformants, such as HPVAg, which are

<p dir="rtl">25 Inhibiting the growth of the carrier. To test this, an Ad35-based vector is evaluated using genetic expression of Ad35</p>

CMVCuO promoter-derived HPVAg to determine conversion cassette stability during vector growth on any

7690

-59-

of PER.C6 or PER.C6/CymR cells (Figure 14). Briefly, vector DNA was transformed in two different cell lines and the resulting viral plaques were left to grow under an agarose layer. From each of the two infections, five viral plaques were isolated and passaged separately Also on the same cell lineage (i.e. the one that was used for infection), for ten consecutive viral passages. The integrity of the transformation is evaluated.

<p dir="rtl">5 By PCR amplification of the conversion cassette at viral passage number ten (VPN10), and subsequent analysis of the resulting PCR products by gel electrophoresis and Sanger sequencing. In addition, at VPN7, passaged viral colonies were assayed to determine their ability to express HPVAg. This was performed using nucleases Isolation of viral bystanders to infect A549 cells at 1,000 virus particles per cell, lysing cells at 48 h postinfection, and then analyzing genotypic expression of HPVAg by</p>

<p dir="rtl">10 Western staining using a monoclonal antibody directed against Santa-Cruz (HPV16 E7 Biotechnology). Results of gel electrophoresis and sequencing analyzes show that all five viral segregation products passed PER.C6, each carrying either small frameshift deletions or mutations. Premature stops within the transposition cassette In contrast, such deletions or mutations may not be detected in any vector segregation products that have passed into the CymR-expressing cell lineage.</p>

<p dir="rtl">15 (PER.C6/CymR). In agreement with these data, all vector shedding products that propagate PER.C6/CymR express HPVAg, while all PER.C6 growth vectors completely lose that ability, suggesting defective conversion cassettes for those Vectors In conclusion, our data demonstrate that using a repression system, for example such as the CymR/CuO system, to repress genetic expression of a transforming vector during vector propagation is an effective way to prevent severe transforming cassette instability, such as is evident for vectors.</p>

<p dir="rtl">20 Carry genetic expression of the HPVAg conversion.</p>

Didn't I come back?

Abbink P, Lemckert AA, Ewald BA, Lynch DM, Denholtz M, Smits S, Holterman L, Damen I, Vogels R, Thorner AR, O'Brien KL, Carville A, Mansfield KG, Goudsmit J, Havenga MJ, Barouch DH ( 2007) Comparative seroprevalence and immunogenicity of six rare serotype recombinant 25 adenovirus vaccine vectors from subgroups B and D. J Virol 81:4654-4663

7690

-60-

Ausubel FM (1995) Short protocols in molecular biology: a compendium of methods from current protocols in molecular biology. Wiley, [Chichester] Cottingham MG, Carroll F, Morris SJ, Turner AV, Vaughan AM, Kapulu MC, Colloca S, Siani L, Gilbert SC, Hill AV (2012) Preventing spontaneous genetic rearrangements in the transgene cassettes of adenovirus vectors. 5 Biotechnol Bioeng 109:719-728

Daayana S, Elkord E, Winters U, Pawlita M, Roden R, Stern PL, Kitchener HC (2010) Phase II trial of imiquimod and HPV therapeutic vaccination in patients with vulval intraepithelial neoplasia. Br J Cancer 102:1129-1136

de Jong A, van der Burg SH, Kwappenberg KM, van der Hulst JM, Franken 10 KL, Geluk A, van Meijgaarden KE, Drijfhout JW, Kenter G, Vermeij P, Melief CJ, Offringa R (2002) Frequent detection of human papillomavirus 16 E2-specific T-helper immunity in healthy subjects. Cancer Res 62:472-479

Edholm D, Molin M, Bajak E, Akusjarvi G (2001) Adenovirus vector designed for expression of toxic proteins. J Virol 75:9579-9584 15

Evans RK, Nawrocki DK, Isopi LA, Williams DM, Casimiro DR, Chin S, Chen M, Zhu DM, Shiver JW, Volkin DB (2004) Development of stable liquid formulations for adenovirus-based vaccines. J Pharm Sci 93:2458-2475

Fallaux FJ, Bout A, van der Velde I, van den Wollenberg DJ, Hehir KM, Keegan J, Auger C, Cramer SJ, van Ormondt H, van der Eb AJ, Valerio D, 20 Hoeben RC (1998) New helper cells and Matched early region 1-deleted adenovirus vectors prevent generation of replication-competent adenoviruses. Hum Gene Ther 9:1909-1917

Frøkjær S, Hovgaard L (2000) Pharmaceutical formulation development of peptides and proteins. Taylor &amp; Francis, London 25

Gall JG, Lizonova A, EttyReddy D, McVey D, Zuber M, Kovesdi I, Aughtman

7690

-61-

<p>B, King CR, Brough DE (2007) Rescue and production of vaccine and therapeutic adenovirus vectors expressing inhibitory transgenes. Mol</p>

Biotechnol 35:263-273

Gao GP, Engdahl RK, Wilson JM (2000) A cell line for high-yield production of E1-deleted adenovirus vectors without the emergence of replication-5 competent viruses. Hum Gene Ther 11:213-219

Gennaro AR (1990) Remington's pharmaceutical sciences. Mack Gilbert R, Guilbault C, Gagnon D, Bernier A, Bourget L, Elahi SM, Kamen A, Massie B (2014) Establishment and validation of new complementing cells for production of E1-deleted adenovirus vectors in serum-free 10 suspension culture. J Virol Methods 208:177-188

Hamid O, Carvajal RD (2013) Anti-programmed death-1 and anti-programmed death-ligand 1 antibodies in cancer therapy. Expert Opin Biol

Ther 13:847-861

Harlow E, Lane D (1988) Antibodies: a laboratory manual. Cold Spring 15 Harbor Laboratory, New York

Havenga M, Vogels R, Zuijdgeest D, Radosevic K, Mueller S, Sieuwerts M, Weichold F, Damen I, Kaspers J, Lemckert A, van Meerendonk M, van der Vlugt R, Holterman L, Hone D, Skeiky Y, Mintardjo R , Gillissen G, Barouch D, Sadoff J, Goudsmit J (2006) Novel replication-incompetent adenoviral 20 B-group vectors: high vector stability and yield in PER.C6 cells. J Gen Virol 87:2135-2143

Henken FE, Oosterhuis K, Ohlschlager P, Bosch L, Hooijberg E, Haanen JB, Steenbergen RD (2012) Preclinical safety evaluation of DNA vaccines encoding modified HPV16 E6 and E7. Vaccine 30:4259-4266 25

Hildesheim A, Herrero R, Wacholder S, Rodriguez AC, Solomon D, Bratti

7690

-62-

MC, Schiller JT, Gonzalez P, Dubin G, Porras C, Jimenez SE, Lowy DR (2007) Effect of human papillomavirus 16/18 L1 viruslike particle vaccine among young women with preexisting infection: a randomized trial. JAMA 298:743-753

Hoganson DK, Ma JC, Asato L, Ong M, Printz MA, Huyghe BG, Sosnowshi 5 BA, D'Andrea MJ (2002) Development of a stable adenoviral vector formulation. Bioprocess J 1:43-48

Hoof I, Peters B, Sidney J, Pedersen LE, Sette A, Lund O, Buus S, Nielsen M (2009) NetMHCpan, a method for MHC class I binding prediction beyond humans. Immunogenetics 61:1-13 10

Horwitz MS (1996) Adenoviruses. In: Fields BN, Knipe DM, Baines JD (eds) Virology. Raven Press Ltd, New York

Kenter GG, Welters MJ, Valentijn AR, Lowik MJ, Berends-van der Meer DM, Vloon AP, Essahsah F, Fathers LM, Offringa R, Drijfhout JW, Wafelman AR, Oostendorp J, Fleuren GJ, van der Burg SH, Melief CJ 15 (2009) Vaccination against HPV-16 oncoproteins for vulvar intraepithelial neoplasia. N Engl J Med 361:1838-1847

Kibbe AH (2000) Handbook of pharmaceutical excipients. Pharmaceutical Press, London

Kovesdi I, Hedley SJ (2010) Adenoviral producer cells. Viruses 2:1681-20

1703

Lin KY, Guarnieri FG, Staveley-O'Carroll KF, Levitsky HI, August JT, Pardoll DM, Wu TC (1996) Treatment of established tumors with a novel vaccine that enhances major histocompatibility class II presentation of tumor antigen. Cancer Res 56:21-26 25

Lundegaard C, Lamberth K, Harndahl M, Buus S, Lund O, Nielsen M (2008)

7690

-63-

NetMHC-3.0: accurate web accessible predictions of human, mouse and monkey MHC class I affinities for peptides of length 8-11. Nucleic Acids Res 36:W509-512

Massimi P, Banks L (2005) Transformation Assays for HPV Oncoproteins.

In: Davy C, Doorbar J (eds) Human papillomaviruses: methods and 5 protocols. Vol 119: Methods in Molecular Medicine Springer, Berlin, pp 381-395

Matthews DA, Cummings D, Evelegh C, Graham FL, Prevec L (1999) Development and use of a 293 cell line expressing lac repressor for the rescue of recombinant adenoviruses expressing high levels of rabbit virus 10 glycoprotein. J Gen Virol 80(Pt 2):345-353

McPherson MJ, Hames BD, Taylor GR (1995) PCR 2: a practical

approach. IRL Press at Oxford University Press, Oxford

Mellman I, Coukos G, Dranoff G (2011) Cancer immunotherapy comes of age. Nature 480:480-489 15

Mullick A, regulated expression in mammalian cells. BMC Biotechnol 6:43

Munger K, Phelps WC, Bubb V, Howley PM, Schlegel R (1989) The E6 20 and E7 genes of the human papillomavirus type 16 together are necessary and sufficient for transformation of primary human keratinocytes. J Virol 63:4417-4421

Ogun SA, Dumon-Seignovert L, Marchand JB, Holder AA, Hill F (2008)

The oligomerization domain of C4-binding protein (C4bp) acts as an 25 adjuvant, and the fusion protein comprised of the 19-kilodalton merozoite

7690

-64-

Surface protein 1 fused with the murine C4bp domain protects mice against malaria. Infect Immun 76:3817-3823

Oosterhuis K, Aleyd E, Vrijland K, Schumacher TN, Haanen JB (2012a) Rational Design of DNA Vaccines for the Induction of Human Papillomavirus

Type 16 E6- and E7-Specific Cytotoxic T-Cell Responses. Hum Gene 5

Ther 23:1301-1312

Oosterhuis K, Ohlschlager P, van den Berg JH, Toebes M, Gomez R, Schumacher TN, Haanen JB (2011) Preclinical development of highly effective and safe DNA vaccines directed against HPV 16 E6 and E7. Int J

Cancer 129:397-406 10

Oosterhuis K, van den Berg JH, Schumacher TN, Haanen JB (2012b) DNA vaccines and intradermal vaccination by DNA tattooing. Curr Top Microbiol

Immunol 351:221-250

Peters B, Tong W, Sidney J, Sette A, Weng Z (2003) Examining the independent binding assumption for binding of peptide epitopes to MHC-I 15

molecules. Bioinformatics 19:1765-1772

Prakash SS, Grossman SR, Pepinsky RB, Laimins LA, Androphy EJ (1992) Amino acids necessary for DNA contact and dimerization imply novel motifs

in the papillomavirus E2 trans-activator. Genes Dev 6:105-116

Rubinchik S, Ding R, Qiu AJ, Zhang F, Dong J (2000) Adenoviral vector 20 which delivers FasL-GFP fusion protein regulated by the tet-inducible expression system. Gene Ther 7:875-885

Sambrook JFEFMT (1989) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY

Sedman SA, Barbosa MS, Vass WC, Hubbert NL, Haas JA, Lowy DR, 25 Schiller JT (1991) The full-length E6 protein of human papillomavirus type

7690

-65-

16 has transforming and trans-activating activities and cooperates with E7 to immortalize keratinocytes in culture. J Virol 65:4860-4866

Shenk T (1996) Adenoviridae and their replication. In: Fields BN, Knipe DM, Baines JD (eds) Virology. Raven Press Ltd, New York

Smahel M, Sima P, Ludvikova V, Vonka V (2001) Modified HPV16 E7 5 Genes as DNA Vaccine against E7-Containing Oncogenic Cells. Virology 281:231-238

van der Burg SH, Melief CJ (2011) Therapeutic vaccination against human papilloma virus induced malignancies. Curr Opin Immunol 23:252-257

Watson JD (1992) Recombinant DNA. Scientific American Books, New York 10 Wieking BG, Vermeer DW, Spanos WC, Lee KM, Vermeer P, Lee WT, Xu

<p>Y, Gabitzsch ES, Balcaitis S, Balint JP, Jr., Jones FR, Lee JH (2012) A non-oncogenic HPV 16 E6/E7 vaccine enhances treatment of HPV</p>

expressing tumors. Cancer Gene Ther 19:667-674

Yan J, Reichenbach DK, Corbitt N, Hokey DA, Ramanathan MP, McKinney 15 KA, Weiner DB, Sewell D (2009) Induction of antitumor immunity in vivo following delivery of a novel HPV-16 DNA vaccine encoding an E6/E7 fusion antigen . Vaccine 27:431-440

Yao F, Eriksson E (1999) A novel tetracycline-inducible viral replication switch. Hum Gene Ther 10:419-427 20

Yoshida Y, Hamada H (1997) Adenovirus-mediated inducible gene expression through tetracycline-controlled transactivator with nuclear localization signal. Biochem Biophys Res Commun 230:426-430

Yugawa T, Kiyono T (2009) Molecular mechanisms of cervical carcinogenesis by high-risk human papillomaviruses: novel functions of E6 25 and E7 oncoproteins. Rev Med Virol 19:97-113

7690

-66-

Zwaveling S, Ferreira Mota SC, Nouta J, Johnson M, Lipford GB, Offringa R, van der Burg SH, Melief CJ (2002) Established human papillomavirus type 16-expressing tumors are effectively eradicated following vaccination with long peptides. J Immunol 169:350-358

<p dir="rtl">5 Table I sequences</p>

SEQ ID NO: 1 (HPV16-E6E7SH, amino acid sequence from designer HPV16 E6/E7 polypeptide)

MHQKRTAMFQ DPQERPRKLP QLCTELQTTI HDIILECVYC

KQQLEDEIDG PAGQAEPDRA HYNIVTFCCK CDSTLRLCVQ

STHVDIRTLE DLLMGTLGIV CPICSQKPGT TLEQQYNKPL CDLLIRCINC 10

QKPLCPEEKQ RHLDKKQRFH NIRGRWTGRC MSCCRSSRTR

RETQMHGDTP TLHEYMLDLQ PETTDLYCYE QLNDSSEEED

EIDGPAGQAE PDRAHYNIVT FCCQLCTELQ TTIHDIILEC VYCKQQLLRR

EVYDFAFRDL CIVYRDGNPY AVCDKCLKFY SKISEYRHYC

YSLYGTTLEQ QYNKPLCDLL IRCINCQK 15

SEQ ID NO: 2 (HPV16-E6E7SH, a nucleotide sequence encoding an amino acid sequence from the designer HPV16 E6/E7 polypeptide)

<tr><td><p>ATGCACCAGA</p></td><td><p>AACGGACCGC</p></td><td><p>CATGTTCCAG</p></td><td colspan="2"><p>GACCCCCAGG</p></td></tr><tr><td><p>AACGGCCCAG</p></td><td><p>AAAGCTGCCC</p></td><td><p>CAGCTGTGCA</p></td><td><p>CCGAGCTGCA</p></td><td></td></tr><tr><td><p>GACCACCATC</p></td><td><p>CACGACATCA</p></td><td><p>TCCTGGAATG</p></td><td><p>CGTGTACTGC</p></td><td><p>20</p></td></tr><tr><td><p>AAGCAGCAGC</p></td><td><p>TGGAAGATGA</p></td><td><p>GATCGACGGC</p></td><td><p>CCTGCTGGCC</p></td><td></td></tr><tr><td><p>AGGCCGAACC</p></td><td><p>CGACAGAGCC</p></td><td><p>CACTACAATA</p></td><td><p>TCGTGACCTT</p></td><td></td></tr><tr><td><p>CTGCTGCAAG</p></td><td><p>TGCGACAGCA</p></td><td><p>CCCTGCGGCT</p></td><td><p>GTGCGTGCAG</p></td><td></td></tr><tr><td><p>AGCACCCACG</p></td><td><p>TGGACATCCG</p></td><td><p>GACCCTGGAA</p></td><td><p>GATCTGCTGA</p></td><td></td></tr><tr><td><p>TGGGCACCCT</p></td><td><p>GGGCATCGTG</p></td><td><p>TGCCCCATCT</p></td><td><p>GCAGCCAGAA</p></td><td><p>25</p></td></tr><tr><td><p>GCCCGGCACC</p></td><td><p>ACCCTGGAAC</p></td><td><p>AGCAGTACAA</p></td><td><p>CAAGCCCCTG</p></td><td></td></tr>

7690

-67-

<tr><td><p>TGCGACCTGC</p></td><td><p>TGATCCGGTG</p></td><td><p>CATCAACTGC</p></td><td colspan="2"><p>CAGAAACCCC</p></td></tr><tr><td><p>TGTGCCCCGA</p></td><td><p>GGAAAAGCAG</p></td><td><p>CGGCACCTGG</p></td><td><p>ACAAGAAGCA</p></td><td></td></tr><tr><td><p>GCGGTTCCAC</p></td><td><p>AACATCCGGG</p></td><td><p>GCAGATGGAC</p></td><td><p>AGGCAGATGC</p></td><td></td></tr><tr><td><p>ATGAGCTGCT</p></td><td><p>GCAGAAGCAG</p></td><td><p>CCGGACCAGA</p></td><td><p>CGGGAAAACCC</p></td><td></td></tr><tr><td><p>AGATGCACGG</p></td><td><p>CGACACCCCC</p></td><td><p>ACCCTGCACG</p></td><td><p>AGTACATGCT</p></td><td><p>5</p></td></tr><tr><td><p>GGACCTGCAG</p></td><td><p>CCCGAGACAA</p></td><td><p>CCGACCTGTA</p></td><td><p>CTGCTACGAG</p></td><td></td></tr><tr><td><p>CAGCTGAACG</p></td><td><p>ACAGCAGCGA</p></td><td><p>GGAAGAGGAC</p></td><td><p>GAGATTGACG</p></td><td></td></tr><tr><td><p>GACCCGCTGG</p></td><td><p>ACAGGCCGAG</p></td><td><p>CCTGACCGGG</p></td><td><p>CTCACTATAA</p></td><td></td></tr><tr><td><p>CATCGTGACA</p></td><td><p>TTTTGCTGTC</p></td><td><p>AGCTCTGTAC</p></td><td><p>TGAACTCCAG</p></td><td></td></tr><tr><td><p>ACAACAATTC</p></td><td><p>ACGATATTAT</p></td><td><p>TCTCGAATGT</p></td><td><p>GTGTATTGTA</p></td><td><p>10</p></td></tr><tr><td><p>AACAGCAGCT</p></td><td><p>CCTGCGGAGA</p></td><td><p>GAGGTGTACG</p></td><td><p>ACTTCGCCTT</p></td><td></td></tr><tr><td><p>CCGGGACCTC</p></td><td><p>TGCATCGGTGT</p></td><td><p>ATCGGGACGG</p></td><td><p>CAACCCCTAC</p></td><td></td></tr><tr><td><p>GCCGTGTGCG</p></td><td><p>ACAAGTGCCT</p></td><td><p>GAAGTTCTAC</p></td><td><p>AGCAAGATCA</p></td><td></td></tr><tr><td><p>GCGAGTACCG</p></td><td><p>GCACTACTGC</p></td><td><p>TACAGCCTGT</p></td><td><p>ACGGAACAAC</p></td><td></td></tr><tr><td><p>ACTCGAACAG</p></td><td><p>CAGTATAACA</p></td><td><p>AACCACTCTG</p></td><td><p>TGATCTGCTG</p></td><td><p>15</p></td></tr>

ATTCGCTGTA TCAATTGTCA GAAGTGATAA

SEQ ID NO: 3 (HPV16 E2E6E7SH, amino acid sequence from HPV16 designer)

E2/E6/E7 polypeptide (

METLCQRLNVCQDKILTHYENDSTDLRDHIDYWKHMRLECAIYYKAREMG

FKHINHQVVPTLAVSKNKALQAIELQLTLETIYNSQYSNEKWTLQDVSLEVY 20

LTAPTGCIKKHGYTVEVQFDGDICNTMHYTNWTHIYICEEASVTVVEGQVD

YYGLYYVHEGIRTYFVQFKDDAEKYSKNKVWEVHAGGQVILCPTSVFSSN

EVSSPEIIRQHLANHPAATHTKAVALGTEETQTTIQRPRSEPDTGNPCHTT

KLLHRDSVDSAPILTAFNSSHKGRINCNSNTTPIVHLKVDANTLMRLRYRFK

KHCTLYTAVSSTWHWTGHNVKHKSAIVTLTYDSEWQRDQFLSQVKIPKTI 25

TVSTGFMSIMHQKRTAMFQDPQERPRKLPQLCTELQTTIHDIILECVYCKQ

7690

-68-

QLEDEIDGPAGQAEPDRAHYNIVTFCCKCDSTLRLCVQSTHVDIRTLEDLL

MGTLGIVCPICSQKPGTTLEQQYNKPLCDLLIRCINCQKPLCPEEKQRHLD

KKQRFHNIRGRWTGRCMSCCRSSRTRRETQMHGDTPTLHEYMLDLQPE

TTDLYCYEQLNDSSEEEDEIDGPAGQAEPDRAHYNIVTFCCQLCTELQTTI

HDIILECVYCKQQLLRREVYDFAFRDLCIVYRDGNPYAVCDKCLKFYSKISE 5

YRHYCYSLYGTTLEQQYNKPLCDLLIRCINCQK

Sequence ID No: 4 (HPV16 E2E6E7SH, nucleotide sequence encoding the designer HPV16 polypeptide E6/E7/E2).

ATGGAAACCCTGTGCCAGCGGCTGAACGTGTGCCAGGACAAGATCCT

GACCCACTACGAGAACGACAGCACGACCTGCGGGACCACATCGACT 10

ACTGGAAGCACATGCGGCTGGAATGCGCCATCTACTACAAGGCCAGA

GAGATGGGCTTCAAGCACATCAACCACCAGGTGGTGCCCACCCTGGC

CGTGTCCAAGAACAAGGCCCTGCAGGCCATCGAGCTGCAGCTGACCC

TGGAAACCATCTACAACAGCCAGTACAGCAACGAGAAGTGGACCCTGC

AGGACGTGTCCCTGGAAGTGTACCTGACCGCTCCCACCGGCTGCATC 15

AAGAAACACGGCTACACCGTGGAAGTGCAGTTCGACGGCGACATCTG

CAACACCATGCACTACACCAACTGGACCCACATCTACATCTGCGAAGA

GGCCAGCGTGACCGTGGTGGAAAGGCCAGGTGGACTACTACGGCCCTGT

ACTACGTGCACGAGGGCATCCGGACCTACTTCGTGCAGTTCAAGGAC

GACGCCGAGAAGTACAGCAAGAACAAAGTGTGGGAGGTGCACGCTGG 20

CGGCCAGGTCATCCTGTGCCCCACCAGCGTTGTTCAGCAGCAACGAGG

TGTCCAGCCCCGAGATCATCCGGCAGCACCTGGCCAATCACCCTGCC

GCCACCCACACAAAGGCCGTGGCCCTGGGGCACCGAGGAAACCCAGAC

CACCATCCAGCGGCCCAGAAGCGAGCCCGACACCGGCAATCCCTGCC

ACACCACCAAGCTGCTGCACCGGGACAGCGTGGACAGCGCCCCTATC 25

CTGACCCGCCTTCAACAGCAGCCACAAGGGCCGGATCAACTGCAACAG

7690

-69-

CAACACCACCCCCATCGTGCACCTGAAGGTGGACGCCAACACCCTGA TGCGGCTGCGGTACAGATTCAAGAAGCACTGCACCCTGTACACCGCC

GTGTCCTCCACCTGGCACTGGACCGGCCACAACGTGAAGCACAAGAG

CGCCATCGTGACCCTGACCTACGACAGCGAGTGGCAGCGGGACCAGT

TCCTGAGCCAGGTCAAAATCCCCAAGACCATCACCGTGTCCACCGGCT 5

TCATGAGCATCATGCACCAGAAACGGACCGCCATGTTCCAGGACCCCC AGGAACGGCCCAGAAAGCTGCCCCAGCTGTGCACCGAGCTGCAGACC

ACCATCCACGACATCATCCTGGAATGCGTGTACTGCAAGCAGCAGCTG

GAAGATGAGATCGACGCCCTGCTGGCCAGGCCGAACCCGACAGAGC CCACTACAATATCGTGACCTTCTGCTGCAAGTGCGACAGCACCCTGCG 10

GCTGTGCGTGCAGAGCACCCACGTGGACATCCGGACCCTGGAAGATC TGCTGATGGGCACCCTGGGCATCGGTGTGCCCCATCTGCAGCCAGAAG

CCCGGCACCACCCTGGAACAGCAGTACAACAAGCCCCTGTGCGACCT

GCTGATCGGTGCATCAACTGCCAGAAACCCCTGTGCCCCGAGGAAA

AGCAGCGGCACCTGGACAAGAAGCAGCGGTTCCACAACATCCGGGGC 15

AGATGGACAGGCAGATGCATGAGCTGCTGCAGAAGCAGCCGGACCAG ACGGGAAACCCAGATGCACGGCGACACCCCCACCCTGCACGAGTACA

TGCTGGACCTGCAGCCCGAGACAACCGACCTGTACTGCTACGAGCAG

CTGAACGACAGCAGCGAGGAAAGAGGACGAGATTGACGGACCCGCTGG ACAGGCCGAGCCTGACCGGGCTCACTATAACATCGTGACATTTTGCTG 20

TCAGCTCTGTACTGAACTCCAGACAACAATTCACGATATTATTCTCGAA

TGTGTGTATTGTAAACAGCAGCTCCTGCGGAGAGAGGTGTACGACTTC

GCCTTCCGGGACCTCTGCATCGTGTATCGGGACGGCAACCCCTACGC

CGTGTGCGACAAGTGCCCTGAAGTTCTACAGCAAGATCAGCGAGTACCG GCACTACTGCTACAGCCTGTACGGAACAACACTCGAACAGCAGTATAA 25

CAAACCACTCTGTGATCTGCTGATTCGCTGTATCAATTGTCAGAAGTGA

7690

-70-

TAA

SEQ ID NO: 5 (HPV16 E6E7E2SH, amino acid sequence encoding the HPV16 designer)

E6/E7/E2 polypeptide

MHQKRTAMFQDPQERPRKLPQLCTELQTTIHDIILECVYCKQQLEDEIDGP

AGQAEPDRAHYNIVTFCCKCDSTLRLCVQSTHVDIRTLEDLLMGTLGIVCPI 5

CSQKPGTTLEQQYNKPLCDLLIRCINCQKPLCPEEKQRHLDKKQRFHNIR

GRWTGRCMSCCRSSRTRRETQMHGDTPTLHEYMLDLQPETTDLYCYEQ

LNDSSEEEDEIDGPAGQAEPDRAHYNIVTFCCQLCTELQTTIHDIILECVYC

KQQLLRREVYDFAFRDLCIVYRDGNPYAVCDKCLKFYSKISEYRHYCYSLY

GTTLEQQYNKPLCDLLIRCINCQKMETLCQRLNVCQDKILTHYENDSTDLR 10

DHIDYWKHMRLECAIYYKAREMGFKHINHQVVPTLAVSKNKALQAIELQLT

LETIYNSQYSNEKWTLQDVSLEVYLTAPTGCIKKHGYTVEVQFDGDICNTM

HYTNWTHIYICEEASVTVVEGQVDYYGLYYVHEGIRTYFVQFKDDAEKYSK

NKVWEVHAGGQVILCPTSVFSSNEVSSPEIIRQHLANHPAATHTKAVALGT

EETQTTIQRPRSEPDTGNPCHTTKLLHRDSVDSAPILTAFNSSHKGRINCN 15

SNTTPIVHLKVDANTLMRLRYRFKKHCTLYTAVSSTWHWTGHNVKHKSAI

VTLTYDSEWQRDQFLSQVKIPKTITVSTGFMSI

Sequence ID No: 6 (HPV16 E6E7E2SH), nucleotide sequence encoding the HPV16 designer.

E6/E7/E2 polypeptide

ATGCACCAGAAACGGACCGCCATGTTCCAGGACCCCCAGGAACGGCC 20

CAGAAAGCTGCCCCAGCTTGTGCACCGAGCTGCAGACCACCATCCACG

ACATCATCCTGGAATGCGTGTACTGCAAGCAGCAGCTGGAAGATGAGA

TCGACGGCCCTGCTGGCCAGGCCGAACCCGACAGAGCCCACTACAAT

ATCGTGACCTTCTGCTGCAAGTGCGACAGCACCCTGCGGCTGTGCGT

GCAGAGCACCCACGTGGACATCCGGACCCTGGAAGATCTGCTGATGG 25

GCACCCTGGGCATCGGTGTGCCCCATCTGCAGCCAGAAGCCCGGCACC

7690

-71-

ACCCTGGAACAGCAGTACAACAAGCCCCTGTGCGACCTGCTGATCCG

GTGCATCAACTGCCAGAAAACCCCTGTGCCCCGAGGAAAAGCAGCGGC

ACCTGGACAAGAACAGCGGTTCCACAACATCCGGGGCAGATGGACA

GGCAGATGCATGAGCTGCTGCAGAAGCAGCCGGACCAGACGGGAAAC

CCAGATGCACGGCGACACCCCACCTGCACGAGTACATGCTGGACC 5

TGCAGCCCGAGACAACCGACCTGTACTGCTACGAGCAGCTGAACGAC

AGCAGCGAGGAAGAGGACGAGATTGACGGACCCGCTGGACAGGCCG

AGCCTGACCGGGCTCACTATAACATCGTGACATTTTGCTTGTCAGCTCT

GTACTGAACTCCAGACAACAATTCACGATATTATTCTCGAATGTGTGTA

TTGTAAACAGCAGCTCCTGCGGAGAGAGGTGTACGACTTCGCCTTCCG 10

GGACCTCTGCATCGTGTATCGGGACGGCAACCCCTACGCCGTTGTGCG

ACAAGTGCCTGAAGTTCTACAGCAAGATCAGCGAGTACCGGCACTACT

GCTACAGCCTGTACGGAACAACACTCGAACAGCAGTATAACAAACCAC

TCTGTGATCTGCTGATTCGCTGTATCAATTGTCAGAAGATTGGAAACCCT

GTGCCAGCGGCTGAACGTTGCCAGGACAAGATCCTGACCCACTACG 15

AGAACGACAGCACGACCTGCGGGACCACATCGACTACTGGAAGCAC

ATGCGGCTGGAAATGCGCCATCTACTACAAGGCCAGAGAGATGGGCTT

CAAGCACATCAACCACCAGGTGGTGCCCACCCTGGCCGTGTCCAAGA

ACAAGGCCCTGCAGGCCATCGAGCTGCAGCTGACCCTGGAAACCATC

TACAACAGCCAGTACAGCAACGAGAAGTGGACCCTGCAGGACGTGTC 20

CCTGGAAGTGTAACCTGACCGCTCCCACCGGCTGCATCAAGAAACACG

GCTACACCGTGGAAGTGCAGTTCGACGGCGACATCTGCAACACCATG

CACTACACCAACTGGACCCACATCTACATCTGCGAAAGAGGCCAGCGTG

ACCGTGGTGGAAGGCCAGTGGACTACTACGGCCCTGTACTACGTGCA

CGAGGGCATCCGGACCTACTTCGTGCAGTTCAAGGACGACGCCGAGA 25

AGTACAGCAAGAACAAAGTGTGGGAGGTGCACGCTGGCGGCCAGGTC

7690

-72-

ATCCTGTGCCCCACCAGCGTGTTCAGCAGCAACGAGGTGTCCAGCCC

CGAGATCATCCGGCAGCACCTGGCCAATCACCCTGCCGCCACCCACA

CAAAGGCCGTGGCCCTGGGCACCGAGGAAACCCAGACCACCATCCAG

CGGCCCAGAAGCGAGCCCGACACCGGCAATCCCTGCCACACCACCAA

GCTGCTGCACCGGGACAGCGTGGACAGCGCCCCTATCCTGACCGCCT 5

TCAACAGCAGCCACAAGGGCCGGATCAACTGCAACAGCAACACCACC

CCCATCGTGCACCTGAAGGTGGACGCCAACACCCTGATGCGGCTGCG

GTACAGATTCAAGAAGCACTGCACCCTGTACACCGCCGTGTCCTCCAC

CTGGCACTGGACCGGCCACAACGTGAAGCACAAAGAGCGCCATCGTGA

CCCTGACCTACGACAGCGAGTGGCAGCGGGACCAGTTCCTGAGCCAG 10

GTCAAAATCCCCAAGACCATCACCGTGTCCACCGGCTTCATGAGCATC

TGATAA

SEQUENCE ID NO: 7 (amino acid sequence of IgE leader peptide)

MDWTWILFLVAAATRVHS

15 Sequence ID: 8 (nucleotide sequence encoding a leader IgE peptide)

ATGGACTGGACCTGGATCCTGTTCCTGGTGGCTGCCGCAACCCGGGT

GCACAGC

Sequence ID No.: 9 (amino acid sequence of the leader peptide aa HAVT20)

MACPGFLWALVISTCLEFSMA

20 SEQ ID NO: 10 (nucleotide sequence encoding HAVT20 leader peptide)

ATGGCCTGCCCCGGCTTTCTGTGGGCCCTGGTCATCAGCACCTGTCT

GGAATTCAGCATGGCC

Sequence ID: 11 (sequence containing 2xTetO)

GAGCTCTCCCTATCAGTGATAGAGATCTCCCTATCAGTGATAGAGATC

GTCGAC 25

Sequence ID No.: 12 (sequence containing CuO)

7690

-73-

AACAAACAGACAATCTGGTCTGTTTGTA

SEQ ID: 13 (CMV promoter present in pAdapt26 and pAdapt35 plasmids)

TCAATATTGGCCATTAGCCATATTATTCATTGGTTATATAGCATAAATCA

ATATTGGCTATTGGCCATTGCATACGTTGTACCATATCATAATATGTAC

ATTTATATTGGCTCATGTCCAACATTACCGCCATGTTGACATTGATTATT 5

GACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCA

TATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGC

TGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTT

CCCATAGTAACGCCAATAAGGGACTTTCCATTGACGTCAATGGGTGGAG

TATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGC 10

CAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGC

ATTATGCCCAGTACATGACCTTATGGGGACTTTCCTACTTGGCAGTACAT

CTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTAC

ATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTC

CACCCCATTGACGTCAATGGGAGTTTTGTTTTGGCACCAAAATCAACGG 15

GACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGGC

GGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCGTTTAGTG

AACCGTCAGATCGCCTGGAGACGCCATCCACGCTGTTTTGACCTCCAT

AGAAAGACACCGGGACCGATCCAGCCTCCGCGGCCGGGAACGGTGCA

TTGGA 20

SEQ ID NO: 14 (TetR, nucleotide sequence encoding the amino acid sequence of TetR polypeptide expressed by pcDNA^TM6/TR(

ATGTCTAGATTAGATAAAAGTAAAGTGATTAAACAGCGCATTAGAGCTGC

TTAATGAGGTCGGAATCGAAGGTTTAACAACCCGTAAACTCGCCCAGA

AGCTAGGTGTAGAGCAGCCTACATTGTATTGGCATGTAAAAAATAAGC 25

GGGCTTTGCTCGACGCCTTAGCCATTGGAGATGTTAGATAGGCACCATA

7690

-74-

CTCACTTTTGCCCTTTAGAAGGGGAAAGCTGGCAAGATTTTTTACGTAA

TAACGCTAAAAGTTTTAGATGTGCTTTACTAAGTCATCGCGATGGAGCA

AAAGTACATTTAGGTACACGGCCTACAGAAAAACAGTATGAAACTCTCG

AAAATCAATTAGCCTTTTTATGCCAACAAGGTTTTTCACTAGAGAATGCA

TTATATGCACTCAGCCGCTGTGGGGCATTTTACTTTAGGTTGCGTATTGG 5

AAGATCAAGAGCATCAAGTCGCTAAAGAAGAAAGGGAAACACCTACTA

CTGATAGTATGCCGCCATTATTACGACAAGCTATCGAATTATTTGATCA

CCAAGGTGCAGAGCCAGCCTTCTTATTCGGCCTTGAATTGATCATATG

CGGATTAGAAAAACAACTTAAATGTGAAAGTGGGGTCCGCGTACAGCGG

ATCCCGGGAATTCAGATCTTATTAA 10

SEQ ID NO: 15 (TetR, amino acid sequence of TetR polypeptide expressed by

)pcDNA^TM6/TR

MSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWHVKNKRAL

LDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHL

GTRPTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEH 15

QVAKEERETPTTDSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKC

ESGSAYSGSREFRSY

SEQ ID NO: 16 (CymR, a nucleotide sequence encoding an amino acid sequence of the CymR polypeptide)

ATGTCTCCCAAACGACGGACTCAAGCGGAAAGGGCAATGGAAACTCA 20

GGGTAAGCTGATTGCCGCGCTTCTGGGAGTGCTGCGAGAGAAAGGGT

ATGCCGGGTTTCGCATAGCCGACGTTCCTGGAGCTGCAGGCGTAAGC

AGAGGAGCCCAATCTCATCACTTTCCGACCAAGCTGGAGCTTTTGCTG

GCTACCTTCGAATGGCTGTACGAGCAGATCACGGAAAGGAGTCGTGCT

AGGCTGGCCAAGCTGAAACCCGAGGATGATGTCATTCAGCAGATGCT 25

GGACGATGCAGCCGAGTTCTTCCTGGACGACGACTTCAGCATCAGTCT

7690

-75-

CGACCTCATCGTAGCCGCAGATCGCGATCCAGCTTTGCGCGAGGGCA

TACAGAGAACAGTCGAGCGGAATCGGTTTGTGGTGGAGGACATGTGG

CTTGGTGTTCTGGTGAGCAGAGGCCTCTCACGGGATGATGCCGAGGA

CATCCTGTGGCTGATCTTTAACTCCGTCAGAGGTTGGCAGTGAGGTC

CCTTTGGCAGAAGGACAAAGAACGGTTTGAACGTGTGCGAAACTCAAC 5

ACCGAGATTGCTAGGGAACGCTACGCCAAGTTCAAGAGATGA

SEQ ID NO: 17 (CymR, amino acid sequence of CymR polypeptide)

MSPKRRTQAERAMETQGKLIAAALGVLREKGYAGFRIADVPGAAGVSRG

AQSHHFPTKLELLLATFEWLYEQITERSRARLAKLKPEDDVIQQMLDDAAE

FFLDDDFSISLDLIVAAADRDPALREGIQRTVERNRFVVEDMWLGVLVSRGL 10

SRDDAEDILWLIFNSVRGLAVRSLWQKDKERFERVRNSTLEIARERYAKFK

R

SEQ ID NO: 18 (HPV16 E6, aa41-65)

KQQLLRREVYDFAFRDLCIVYRDGN

<p dir="rtl">15 Sequence ID No: 19 (77-43 HPV16 E7 aa)</p>

GQAEPDRAHYNIVTFCCKCDSTLRLCVQSTHVDIR

7690

-76-