EA049710B1 - VACCINES AGAINST RECURRENT RESPIRATORY PAPILLOMATOSIS AND METHODS OF THEIR USE - Google Patents

Abstract

The present invention provides nucleic acid molecules encoding an HPV antigen. Also provided are vaccines against human papillomavirus (HPV) comprising the nucleic acids, methods for inducing immune responses, and methods for prophylactic and/or therapeutic immunization of individuals against recurrent respiratory papillomatosis (RRP). Pharmaceutical compositions, recombinant vaccines comprising a DNA plasmid, and live attenuated vaccines are described, as well as methods for inducing an immune response for the treatment or prevention of RRP.

Description

Cross-reference to related applications

The present invention claims priority to U.S. Provisional Patent Application No. 63/024,912, filed May 14, 2020, the contents of which are incorporated herein in their entirety.

List of sequences

This invention contains a sequence listing provided in electronic form in ASCII format and hereby incorporated by reference in its entirety. A copy of said ASCII file created on May 14, 2021 is named 104409_000619_US_SL.txt and is 37913 bytes in size.

Field of technology

The present invention relates to vaccines against human papillomavirus (HPV), methods for inducing immune responses and methods for prophylactic and/or therapeutic immunization of individuals against HPV6 and/or HPV11, as well as methods for preventing or treating recurrent respiratory papillomatosis (RRP).

State of the art

Human papillomavirus-associated (HPV+) malignancies are an emerging global epidemic (Gradishar et al., JNCCN 2014;12(4):542-90). HPV-associated aerodigestive precancerous lesions and malignancies can occur in the oropharynx, larynx, and upper respiratory tract. Although the roles of HPV6 and HPV11 in the etiology of most aerodigestive malignancies remain unclear, their causal association with recurrent respiratory papillomatosis (RRP) (Mounts et al., PNAS USA 1982;79(17):5425-9; Gissmann et al., PNAS USA 1983;80(2):560-3; Bonagura et al., APMIS. 2010;118(6-7):455-70), the most common benign tumor of the laryngeal epithelium, is widely accepted. RRP is rare, with an estimated incidence of 1.8 cases per 100,000 adults in the United States (Winton et al., NEJM 2005;352(25):2589-97). Although most lesions are benign, some undergo malignant transformation, and patients with RRP have an increased risk of developing laryngeal neoplasia and carcinoma (Omland et al., PloS One. 2014;9(6):e99114).

Recurrent respiratory papillomatosis remains a challenging disease affecting children and adults, resulting in annual health care costs in the United States estimated at $120 million, with annual costs per patient approaching $60,000. Although the prevalence of RRP has decreased, RRP remains the most common benign laryngeal neoplasm in children. RRP is unique in its high rate of multifocal recurrence, high burden on patient quality of life, and high associated health care costs. Thus, there is a need for improved compositions and methods for treating or preventing RRP. The present invention addresses this unmet need.

Brief description of the essence of the invention

Disclosed herein are nucleic acid molecules encoding a human papillomavirus (HPV) antigen, wherein the HPV antigen comprises an HPV6 antigenic domain and an HPV11 antigenic domain. In some embodiments, the HPV6 antigenic domain comprises an HPV6 E6 antigenic domain and an HPV6 E7 antigenic domain. In some embodiments, the HPV11 antigenic domain comprises an HPV11 E6 antigenic domain and an HPV11 E7 antigenic domain. The nucleic acid molecules provided herein may encode an HPV antigen comprising the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 11; an amino acid sequence that is at least 95% homologous to SEQ ID NO: 1 or SEQ ID NO: 11, or a fragment of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 11, or an amino acid sequence that is at least 95% homologous to a fragment of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 11. In some aspects, the nucleic acid molecules comprise a nucleotide sequence that is at least 95% homologous to SEQ ID NO: 2 or SEQ ID NO: 12; the nucleotide sequence of SEQ ID NO: 2, or the nucleotide sequence of SEQ ID NO 12.

In some embodiments of the invention, the nucleic acid sequence encoding the antigenic domain of HPV11 is located 5' with respect to the nucleic acid sequence encoding the antigenic domain of HPV6. In alternative embodiments of the invention, the nucleic acid sequence encoding the antigenic domain of HPV6 is located 5' with respect to the nucleic acid sequence encoding the antigenic domain of HPV11.

The nucleic acid molecules may comprise a nucleic acid sequence encoding one or more post-translational cleavage sites, one or more translational skip sites, or both. Such sequence(s) may be interposed between the nucleic acid sequence encoding the HPV6 antigenic domain and the nucleic acid sequence encoding the antigenic domain

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HPV11, between a nucleic acid sequence encoding the HPV6 E6 antigenic domain and a nucleic acid sequence encoding the HPV6 E7 antigenic domain, between a nucleic acid sequence encoding the HPV11 E6 antigenic domain and a nucleic acid sequence encoding the HPV11 E7 antigenic domain, or any combination thereof.

Also provided herein are expression vectors comprising any of the disclosed nucleic acid molecules. In some embodiments, the expression vector is a DNA plasmid. As an example, the expression vector may comprise the nucleotide sequence of SEQ ID NO: 3.

Also disclosed herein are immunogenic proteins comprising an antigenic domain of human papillomavirus (HPV) 6 fused to an antigenic domain of HPV11. In some embodiments, the antigenic domain of HPV6 comprises HPV6 E6 and HPV6 E7; the antigenic domain of HPV11 comprises HPV11 E6 and HPV11 E7, or both. The antigenic domain of HPV11 may be N-terminal or C-terminal to the antigenic domain of HPV6. The immunogenic proteins may comprise one or more post-translational cleavage sites, one or more translational skip sites, or both. Such sites may be located between the antigenic domains of HPV6 and HPV11, between the antigenic domains of HPV6 E6 and HPV6 E7, between the antigenic domains of HPV11 E6 and HPV11 E7, or any combination thereof.

According to some embodiments of the invention, the immunogenic protein comprises the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 11, or an amino acid sequence that is at least 95% homologous to the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 11, or a fragment of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 11, or an amino acid sequence that is at least 95% homologous to a fragment of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 11.

Also provided herein are compositions comprising nucleic acid molecules, expression vectors, immunogenic proteins, or any combination thereof, and a pharmaceutically acceptable carrier. In some aspects, vaccines comprising nucleic acid molecules, expression vectors, immunogenic proteins, or any combination thereof are provided. In some embodiments, pharmaceutical compositions comprising nucleic acid molecules, expression vectors, immunogenic proteins, or any combination thereof, and an adjuvant are provided. An adjuvant can be, for example, interleukin 12 (IL-12). In some embodiments, IL-12 is encoded by a nucleic acid molecule, such as an expression vector. In some embodiments, the adjuvant comprises a nucleic acid molecule comprising a nucleotide sequence encoding the p35 subunit of IL-12, the p40 subunit of IL-12, or both. For example, a nucleotide sequence encoding a p35 subunit of IL-12 may comprise a nucleotide sequence selected from the group consisting of a nucleotide sequence that encodes SEQ ID NO: 6; a nucleotide sequence that is at least 95% homologous to a nucleotide sequence that encodes SEQ ID NO: 6; a fragment of a nucleotide sequence that encodes SEQ ID NO: 6; and a nucleotide sequence that is at least 95% homologous to a fragment of a nucleotide sequence that encodes SEQ ID NO: 6. In some aspects, a nucleotide sequence encoding a p40 subunit of IL-12 comprises a nucleotide sequence selected from the group consisting of a nucleotide sequence that encodes SEQ ID NO: 8; a nucleotide sequence that is at least 95% homologous to a nucleotide sequence that encodes SEQ ID NO: 8; a fragment of a nucleotide sequence that encodes SEQ ID NO: 8; and a nucleotide sequence that is at least 95% homologous to a fragment of a nucleotide sequence that encodes SEQ ID NO: 8. According to some embodiments of the invention, a nucleotide sequence encoding IL-12 comprises a nucleotide sequence selected from the group consisting of the nucleotide sequence of SEQ ID NO: 4; a nucleotide sequence that is at least 95% homologous to the nucleotide sequence of SEQ ID NO: 4; a fragment of the nucleotide sequence of SEQ ID NO: 4, and a nucleotide sequence that is at least 95% homologous to a fragment of the nucleotide sequence of SEQ ID NO: 4.

Additionally, described herein are methods for inducing an immune response in a subject by administering to the subject an effective amount of any of the disclosed nucleic acid molecules, expression vectors, immunogenic proteins, pharmaceutical compositions, or vaccines, thereby inducing an immune response.

Also provided in this document are methods for prophylactically or therapeutically immunizing a subject against HPV6 and/or HPV11 comprising administering to the subject an effective amount of any of the disclosed nucleic acid molecules, expression vectors, immunogenic proteins, pharmaceutical compositions or vaccines, thereby inducing an immune response.

- 2 049710 against HPV6, HPV11 or both.

Also provided herein are methods of treating or preventing recurrent respiratory papillomatosis (RRP) in a subject comprising administering to the subject an effective amount of any of the disclosed nucleic acid molecules, expression vectors, immunogenic proteins, pharmaceutical compositions, or vaccines, thereby treating or preventing RRP. RRP may be juvenile RRP or adult RRP.

According to some embodiments of the disclosed methods, a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 12 is administered to a subject.

In some aspects of the disclosed methods, the subject is further administered an adjuvant. The adjuvant can be interleukin-12 (IL-12). IL-12 can be encoded by a nucleic acid molecule, such as, for example, an expression vector or a plasmid. In some embodiments, the adjuvant comprises a nucleic acid molecule comprising a nucleotide sequence encoding the p35 subunit of IL-12, the p40 subunit of IL-12, or both. The nucleotide sequence encoding p35 can comprise a nucleotide sequence selected from the group consisting of a nucleotide sequence that encodes SEQ ID NO: 6; a nucleotide sequence that is at least 95% homologous to a nucleotide sequence that encodes SEQ ID NO: 6; a fragment of a nucleotide sequence that encodes SEQ ID NO: 6; and a nucleotide sequence that is at least 95% homologous to a fragment of a nucleotide sequence that encodes SEQ ID NO: 6. The nucleotide sequence encoding p40 may include a nucleotide sequence selected from the group consisting of a nucleotide sequence that encodes SEQ ID NO: 8; a nucleotide sequence that is at least 95% homologous to a nucleotide sequence that encodes SEQ ID NO: 8; a fragment of a nucleotide sequence that encodes SEQ ID NO: 8; and a nucleotide sequence that is at least 95% homologous to a fragment of a nucleotide sequence that encodes SEQ ID NO: 8. In some aspects, the nucleotide sequence encoding IL-12 may include a nucleotide sequence selected from the group consisting of the nucleotide sequence of SEQ ID NO: 4; a nucleotide sequence that is at least 95% homologous to the nucleotide sequence of SEQ ID NO: 4; a fragment of the nucleotide sequence of SEQ ID NO: 4 and a nucleotide sequence that is at least 95% homologous to a fragment of the nucleotide sequence of SEQ ID NO: 4. According to some embodiments of the invention, a nucleic acid molecule comprising a nucleotide sequence encoding IL-12 is pGX6010.

In some embodiments of the invention, the subject is a human.

According to some embodiments of the methods provided herein, the methods comprise administering pGX3024 and pGX6010 to a subject. In some aspects of the methods, pGX3024 and pGX6010 are administered as a composition, such as INO-3107. Some embodiments of the methods comprise administering 6 milligrams of pGX3024 and 0.25 milligrams of pGX6010 to a subject. In some aspects of the disclosed methods, the administration comprises intradermal or intramuscular injection. The administration may further comprise electroporation.

Brief description of graphic materials

The brief description, as well as the following detailed description, become more understandable when read in conjunction with the accompanying graphic materials. For the purpose of illustrating the disclosed methods, illustrative embodiments of the invention are shown in the graphic materials; however, the methods are not limited to the specific disclosed embodiments of the invention. In the graphic materials:

Figure 1A shows a schematic of the antigens encoded in different HPV6 and/or HPV11 plasmids. The individual antigens are separated by a P2A cleavage site for translational skipping and a furin cleavage site for post-translational cleavage of the P2A sequence. The DNA plasmid pGX3024 encodes the E6 and E7 SynCon® consensus antigens of both HPV6 and HPV11. Figure 1B shows a map of plasmid pGX3024.

Figure 2 shows the expression of pGX3024 E6 and E7 protein antigens in vitro. HEK-293T cells were transfected with either pGX3024, positive control pGX3021 or pGX3022, or empty negative control pGX0001 using Lipofectamine 3000 transfection reagent. Cells were harvested 48 h post-transfection, and cell lysates were analyzed with anti-HPV11 E7 antibody (left panel) or anti-2A antibody (middle panel) by Western blotting. Blots were stripped and reprobed with anti-βactin antibody (right panel) to confirm equal protein loading. E6 and E7 proteins were detected in cells transfected with pGX3024 and the control plasmids pGX3021 and pGX3022, but not with the negative control plasmid pGX0001.

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Figure 3 shows HPV6- and HPV11-specific cellular responses following immunization of C57BL/6 mice with pGX3024. C57BL/6 mouse splenocytes were harvested one week after immunization with either pGX3024, control plasmids encoding HPV6 (pGX3021) or HPV11 (pGX3022) antigens, or a negative control plasmid (pGX0001). Specific cellular responses to HPV6 and HPV11 E6 and E7 peptides were measured by IFN-γ ELISpot assay. Asterisk indicates significant difference in overall cellular response compared to pGX0001 control by one-way ANOVA with Dunnett's post hoc test.

Figure 4 shows humoral responses to HPV6 and HPV11 following immunization of C57BL/6 mice with pGX3024. C57BL/6 mouse serum samples were collected before immunization (week 0) and after the first (week 2) and second (week 3) immunizations with either pGX3024 or a negative control plasmid (pGX0001). Specific IgG-binding antibodies against HPV6 E7 (left panel) or HPV11 E7 (right panel) antigens were measured by ELISA.

Figure 5 shows HPV6- and HPV11-specific cellular responses following immunization of BALB/6 mice with pGX3024. BALB/c mouse splenocytes were collected one week after immunization with the indicated dose of either pGX3024 alone or in combination with the indicated dose of mouse IL-12 plasmid (pGX6012) or negative control plasmid (pGX0001). Specific cellular responses to HPV6 and HPV11 E6 and E7 peptides were measured by IFN-γ ELISpot assay. Asterisk indicates significant difference in overall cellular response compared to pGX0001 control by one-way ANOVA with Dunnett's test.

Figure 6 shows humoral responses to HPV6 and HPV11 following immunization of BALB/c mice with pGX3024. BALB/c mouse serum samples were collected before immunization (week 0) and after the first (week 2) and second (week 3) immunizations with the indicated dose of either pGX3024 alone or in combination with the indicated dose of mouse IL-12 plasmid (pGX6012) or negative control plasmid (pGX0001). Specific IgG-binding antibodies against HPV6 E7 (left panel) or HPV11 E7 (right panel) antigens were measured by ELISA. Asterisk indicates significant difference and ns indicates no significant difference compared to week 0 by two-way ANOVA.

Figure 7 shows the time course of HPV6- and HPV11-specific cellular responses following immunization of NZW rabbits with INO-3107. Peripheral blood mononuclear cells (PBMCs) from NZW rabbits were collected at the indicated time points following immunization with either INO-3107 or 1X sodium citrate-buffered saline (SSC). Specific cellular responses to HPV6 E6 and E7 peptides and HPV11 E6 and E7 peptides were measured by IFN-γ ELISpot assay. Data are presented as the sum of responses to HPV6 E6 and E7 (left panel) or HPV11 E6 and E7 (right panel) for individual animals.

Figure 8 shows HPV6- and HPV11-specific cellular responses following immunization of NZW rabbits with INO-3107. Week 11 (two weeks after the fourth immunization), T cell responses against HPV6 E6, HPV6 E7, HPV11 E6, or HPV11 E7 antigens in NZW rabbits at week 11 (two weeks after the fourth immunization) using INO-3107 or 1X SSC as described in Figure 7. Data are presented for individual rabbits (left panel) or the mean ± SEM for each treatment group (right panel). Asterisk indicates a significant difference (p < 0.05) as determined by the Mann-Whitney U test.

Figure 9 shows the time course of antibody responses to HPV6 and HPV11 following immunization of NZW rabbits with INO-3107. Serum samples from NZW rabbits were collected at the indicated time points following immunization with INO-3107 or 1X SSC. Specific antibody responses to HPV6 E7 (left panel) and HPV11 E7 (right panel) antigens were measured by IgG binding ELISA. Data are presented for individual animals.

Figure 10 shows the time course of body weight measurements in NZW rabbits treated with INO-3107 (left panel) or 1X SSC (right panel).

Figure 11 shows the time course of HPV6- and HPV11-specific cellular responses following immunization of Hartley guinea pigs with pGX3024. Guinea pigs (n of 5) were immunized at weeks 0, 2, and 4 with 100 μg of pGX3024 administered by intradermal CELLECTRA electroporation. Untreated guinea pigs (n of 2) served as negative controls. Guinea pig peripheral blood mononuclear cells (PBMCs) were collected at the indicated time points following immunization with pGX3024 or from untreated guinea pigs. Specific cellular responses to HPV6 E6 and E7 peptides and HPV11 E6 and E7 peptides were measured by IFN-γ ELISpot assay. Data are presented as mean ± SEM for each HPV6 E6 and E7 treatment group.

Figure 12 shows the time course of HPV6- and HPV11-specific antibody responses following immunization of Hartley guinea pigs with pGX3024. Hartley guinea pigs (n of 5) were immunized at weeks 0, 2, and 4 with 100 μg of pGX3024 administered by intradermal CELLECTRA electroporation. At the indicated time points following immunization with pGX3024, guinea pig serum samples were collected for measurement of specific IgG-binding antibodies.

- 4 049710 against HPV6 E7 (left) or HPV11 E7 (right) antigens by ELISA. Data are presented as mean ± SEM of five animals.

Figure 13 shows the immune responses induced following intradermal delivery of INO-3107 to NZW rabbits. Cellular immune responses were assessed by IFN-γ ELISpot before immunization (week 0) and two weeks after each immunization (weeks 2, 5, and 8). The combined immune response to both HPV6 and HPV11 antigens was increased after each immunization, with T cell responses being more specific for HPV6 E6 and HPV11 E6.

Detailed description of illustrative embodiments of the invention

The disclosed nucleic acid molecules, proteins, vaccines and methods may be better understood from the following detailed description together with the accompanying drawings, which form a part of the present invention. It should be understood that the disclosed nucleic acid molecules, proteins, vaccines and methods are not limited to the specific nucleic acid molecules, proteins, vaccines and methods described and/or shown herein, and that the terminology used herein is intended to describe particular embodiments of the invention by way of example only and is not intended to limit the claimed nucleic acid molecules, proteins, vaccines and methods.

Unless specifically stated otherwise, any description of a possible mechanism or mode of action or cause of improvement is intended for illustrative purposes only, and the disclosed nucleic acid molecules, proteins, vaccines and methods shall not be limited by the correctness or incorrectness of any such proposed mechanism or mode of action or cause of improvement.

Throughout the text, the descriptions refer to compositions and methods of using said compositions. If the present invention describes or claims a feature or embodiment of the invention related to a composition, such feature or embodiment of the invention applies equally to methods of using said composition. Similarly, if the present invention describes or claims a feature or embodiment of the invention related to a method of using a composition, such feature or embodiment of the invention applies equally to the composition.

It should be appreciated that certain features of the disclosed nucleic acid molecules, proteins, vaccines and methods, which are described herein in the context of separate embodiments for clarity, may also be provided in combination in a single embodiment. Conversely, various features of the disclosed nucleic acid molecules, proteins, vaccines and methods, which are described in the context of a single embodiment for brevity, may also be provided separately or in any subcombination.

Unless otherwise defined, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art. In case of conflict, the present document, including definitions, controls. Preferred methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All publications, patent applications, patents, and other literature references mentioned herein are hereby incorporated by reference in their entirety. The materials, methods, and examples described herein are for illustrative purposes only and are not intended to be limiting. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

As used herein, the terms comprise, include, having, may, include, and variations thereof are open-ended transitional expressions, terms, or words that do not exclude the possibility of additional acts or structures. The term including is intended to include examples encompassed by the terms consisting essentially of and consisting of; similarly, the term consisting essentially of is intended to include examples encompassed by the term consisting of. The present invention also contemplates other embodiments comprising, consisting of, and consisting essentially of embodiments or elements disclosed herein, whether or not expressly stated.

The singular forms include plural references unless the context clearly indicates otherwise.

When numerical ranges with the same degree of precision are listed in this document, each intermediate numerical value is explicitly provided. For example, in the case of the range 6-9, the numbers 7 and 8 are provided in addition to 6 and 9, and in the case of the range 6.0-7.0, the numbers 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly provided.

Certain quantitative expressions in this document are not accompanied by the term approximately. It is understood that, whether or not the term approximately is explicitly used, each quantity given is intended to represent

- 5 049710 actual specified value, and is also intended to denote an approximation to such specified value that can be reasonably deduced from the state of the art, including approximations due to experimental and/or measurement conditions for such value.

An adjuvant, as used herein, means any molecule added to the immunogenic compositions described herein to enhance the immunogenicity of the antigens and antigen-encoding nucleic acid molecules and sequences described below.

The antigen refers to proteins having an HPV6 E6 domain, an HPV6 E7 domain, an HPV11 E6 domain, an HPV11 E7 domain, or any combination thereof, and preferably to a fusion protein of an HPV6 E6 domain, an HPV6 E7 domain, an HPV11 E6 domain, and an HPV11 E7 domain with an endeoproteolytic cleavage site between each domain. The antigens include SEQ ID NO: 1; fragments thereof of the length specified herein, variants, i.e. proteins with sequences homologous to SEQ ID NO: 1 as specified herein, fragments of variants having the length specified herein, and combinations thereof. The antigens may have the IgE leader sequence of SEQ ID NO: 10 or, alternatively, may have such a sequence deleted from the N-terminal end. For example, an HPV antigen comprising an HPV6 E6 domain, an HPV6 E7 domain, an HPV11 E6 domain, and an HPV11 E7 domain, with or without an endoproteolytic cleavage site between each domain, may have an IgE leader sequence located N-terminal to the N-terminus of the HPV domain of the HPV antigen. Antigens may optionally include signal peptides, such as peptides from other proteins.

The term biosimilar (of an approved reference product/biological medicinal product, i.e. a medicinal product included in the reference list) refers to a biological product that is highly similar to the reference product, despite minor differences in clinically inactive components, with no clinically meaningful differences between the biosimilar and the reference product in terms of safety, purity and potency, based on data obtained from (a) analytical studies that demonstrate that the biological product is highly similar to the reference product, despite minor differences in clinically inactive components; (b) animal studies (including toxicity assessment); and/or (c) a clinical study or studies (including immunogenicity and pharmacokinetic assessment or pharmacodynamic assessment) sufficient to demonstrate safety, purity and potency in one or more relevant conditions of use for which the reference product is licensed and intended to be used and for which the biosimilar is being licensed. A biosimilar may be an interchangeable product that can be substituted for the reference product in the pharmacy without intervention by the treating physician. To meet the additional standard of interchangeability, the biosimilar must be expected to produce the same clinical outcome as the reference product in any given patient and, if the biosimilar is administered to an individual more than once, the risk in terms of safety or loss of efficacy of alternating or switching from the biosimilar to the reference product is no greater than the risk of using the reference product without such alternation or switching. The biosimilar uses the same mechanisms of action for the intended conditions of use to the extent that these mechanisms are known for the reference product. The condition or conditions of use prescribed, recommended, or suggested in the labeling proposed for the biosimilar have been previously approved for the reference product. The route of administration, dosage form, and/or strength of the biosimilar are the same as the reference product, and the biosimilar is manufactured, processed, packaged, or stored in a facility that meets standards designed to ensure the safety, purity, and potency of the biosimilar. The biosimilar may include minor modifications in the amino acid sequence compared to the reference product, such as N- or C-terminal truncations, that are not expected to alter the potency of the biosimilar.

In the context of this document, a coding sequence or coding nucleic acid may mean nucleic acids (RNA or DNA molecule) that contain a nucleotide sequence that codes for a protein. The coding sequence may further contain initiation and termination signals operably linked to regulatory elements, including a promoter and a polyadenylation signal, capable of directing expression in cells of an individual or mammal to which the nucleic acid is administered.

In the context of this document, a complementary sequence or complementarity refers to a nucleic acid molecule that has Watson-Crick (e.g., AT/U and C-G) or Hoogsteen base pairing between nucleotides or nucleotide analogs with a reference nucleic acid molecule.

Consensus or consensus sequence, as used herein, means a polypeptide sequence based on an alignment analysis of multiple sequences for the same gene from different organisms.

- 6 049710 nucleic acid sequences that encode a consensus polypeptide sequence. Immunogenic compositions containing proteins comprising consensus sequences and/or nucleic acid molecules encoding such proteins can be used to induce broad immunity against an antigen.

In the context of this document, the terms electroporation, electropermeabilization, or electrokinetic enhancement (EK) are used interchangeably and may refer to the use of a transmembrane electric field pulse to induce microscopic pathways (pores) in a biomembrane; their presence allows biomolecules such as plasmids, oligonucleotides, siRNA, drugs, ions, and water to cross from one side of the cell membrane to the other.

A fragment, as used herein in relation to nucleic acid sequences, means a nucleic acid sequence or a portion thereof that encodes a polypeptide capable of eliciting an immune response in a mammal that cross-reacts with an antigen disclosed herein. Fragments may be DNA fragments selected from at least one of the various nucleotide sequences that encode protein fragments set forth below. Fragments may comprise at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of one or more of the nucleic acid sequences set forth below. In some embodiments, the fragments may comprise at least 20 nucleotides or more, at least 30 nucleotides or more, at least 40 nucleotides or more, at least 50 nucleotides or more, at least 60 nucleotides or more, at least 70 nucleotides or more, at least 80 nucleotides or more, at least 90 nucleotides or more, at least 100 nucleotides or more, at least 150 nucleotides or more, at least 200 nucleotides or more, at least 250 nucleotides or more, at least 300 nucleotides or more, at least 350 nucleotides or more, at least 400 nucleotides or more, at least 450 nucleotides or more, at least 500 nucleotides or more, at least 550 nucleotides or more, at least 600 nucleotides or more than at least 650 nucleotides or more, at least 700 nucleotides or more, at least 750 nucleotides or more, at least 800 nucleotides or more, at least 850 nucleotides or more, at least 900 nucleotides or more, at least 950 nucleotides or more, or at least 1000 nucleotides or more of at least one of the nucleic acid sequences set forth below.

A fragment or immunogenic fragment, in relation to a polypeptide sequence, means a polypeptide capable of eliciting an immune response in a mammal that cross-reacts with an antigen disclosed herein. Fragments may be polypeptide fragments selected from at least one of the various amino acid sequences set forth below. A fragment of consensus proteins may comprise at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of a consensus protein. In some embodiments, fragments of consensus proteins may comprise at least 20 amino acids or more, at least 30 amino acids or more, at least 40 amino acids or more, at least 50 amino acids or more, at least 60 amino acids or more, at least 70 amino acids or more, at least 80 amino acids or more, at least 90 amino acids or more, at least 100 amino acids or more, at least 110 amino acids or more, at least 120 amino acids or more, at least 130 amino acids or more, at least 140 amino acids or more, at least 150 amino acids or more, at least 160 amino acids or more, at least 170 amino acids or more, at least 180 amino acids or more of a protein sequence disclosed herein.

As used herein, the term genetic construct refers to DNA or RNA molecules that contain a nucleotide sequence that encodes a protein. The coding sequence contains initiation and termination signals operably linked to regulatory elements, including a promoter and a polyadenylation signal, capable of directing expression in cells of an individual to which the nucleic acid molecule is administered. As used herein, the term expressible form refers to genetic constructs that contain the necessary regulatory elements operably linked to a coding sequence that encodes a protein such that, when present in a cell of an individual, the coding sequence will be expressed.

The term homology, as used in this document, refers to the degree

- 7 049710 complementarity. This may be partial homology or complete homology (i.e., identity). A partially complementary sequence that at least partially inhibits hybridization of a fully complementary sequence to a target nucleic acid is referred to using the functional term substantially homologous. When used in relation to a double-stranded nucleic acid sequence, such as a cDNA or genomic clone, the term substantially homologous, as used herein, refers to a probe that can hybridize to a strand of the double-stranded nucleic acid sequence under low stringency conditions. When used in relation to a single-stranded nucleic acid sequence, the term substantially homologous, as used herein, refers to a probe that can hybridize to (i.e., is complementary to) a template sequence of a single-stranded nucleic acid under low stringency conditions.

As used herein, identical or identity with respect to two or more nucleic acid or polypeptide sequences means that the sequences have a specified percentage of residues that are the same over a specified region. The percentage can be calculated by optimally aligning the two sequences, comparing the two sequences over a specified region, determining the number of positions at which identical residues occur in both sequences to obtain the number of matching positions, dividing the number of matching positions by the total number of positions in the specified region, and multiplying the result by 100 to obtain the percent sequence identity. In cases where the two sequences are of different lengths or the alignment results in one or more staggered ends and the specified region of comparison includes only one sequence, the residues of one sequence are included in the denominator but not the numerator in the calculation. When comparing DNA and RNA, thymine (T) and uracil (U) can be considered equivalent. Identity can be calculated manually or using a computer algorithm for sequences such as BLAST or BLAST 2.0.

As used herein, INO-3107 refers to an immunogenic composition of two DNA plasmids: DNA plasmid pGX3024 encoding an HPV antigen comprising the SynCon® E6 and E7 antigens of both HPV6 and HPV11, in combination with DNA plasmid pGX6010 encoding human IL-12. The amino acid sequence of the HPV antigen comprising the E6 and E7 SynCon® antigens of both HPV6 and HPV11 is set forth in SEQ ID NO: 1. The nucleotide sequence encoding the HPV antigen comprising the E6 and E7 SynCon® antigens of both HPV6 and HPV11 is set forth in SEQ ID NO: 2. The nucleic acid sequence of the DNA plasmid pGX3024 is set forth in SEQ ID NO: 3. The sequence of the DNA plasmid pGX6010 is set forth in SEQ ID NO: 4. INO-3107 may further comprise a sodium citrate buffered saline. The medicinal product INO-3107 refers to an immunogenic composition containing 6.25 mg total plasmid/ml (6 mg/ml pGX3024, 0.25 mg/ml pGX6010) in 150 mM sodium chloride and 15 mM sodium citrate, pH 7.

In the context of this document, an immune response means the activation of the immune system of a host, such as a mammal, in response to the introduction of an antigen. An immune response may take the form of a cellular or humoral response, or both.

In the context of this document, a nucleic acid or an oligonucleotide or a polynucleotide means at least two nucleotides covalently linked together. The illustration of one strand also determines the sequence of the complementary strand. Thus, a nucleic acid also encompasses the complementary strand of the illustrated single strand. Many variants of a nucleic acid can be used for the same purpose as a given nucleic acid. Thus, a nucleic acid also encompasses substantially identical nucleic acids and their complementary sequences. One strand provides a probe that can hybridize to a target sequence under stringent hybridization conditions. Thus, a nucleic acid also encompasses a probe that hybridizes under stringent hybridization conditions.

Nucleic acids may be single-stranded or double-stranded, or may contain portions of both double-stranded and single-stranded sequences. A nucleic acid may be DNA, either genomic or cDNA, RNA, or a hybrid, in which the nucleic acid may contain combinations of deoxyribo- and ribonucleotides, and combinations of bases including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine, hypoxanthine, isocytosine, and isoguanine. Nucleic acids may be produced by chemical synthesis or recombinant methods.

In the context of this document, operably linked means that the expression of a gene is under the control of a promoter to which it is spatially linked. A promoter may be located 5' (upstream) or 3' (downstream) of the gene under its control. The distance between the promoter and the gene may be approximately the same as the distance between the promoter and the gene it controls in the gene from which the promoter is derived. As is known in the art, this distance may be varied without loss of promoter function.

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In the context of this document, a peptide, protein or polypeptide may mean a linked sequence of amino acids and may be natural, synthetic or a modification or a combination of natural and synthetic.

As used herein, a promoter means a synthetic or naturally occurring molecule that is capable of providing, activating or enhancing the expression of a nucleic acid in a cell. A promoter may contain one or more specific transcriptional regulatory sequences to further enhance expression and/or to alter the spatial expression and/or temporal expression thereof. A promoter may also contain distal enhancer or repressor elements that may be located even several thousand base pairs from the transcription start site. A promoter may be obtained from sources including viruses, bacteria, fungi, plants, insects and animals. A promoter may regulate the expression of a gene component constitutively or differentially with respect to the cell, tissue or organ in which expression occurs, or with respect to the developmental stage at which expression occurs, or in response to external stimuli such as physiological stresses, pathogens, metal ions or inducing agents. Representative examples of promoters include the bacteriophage T7 promoter, the bacteriophage T3 promoter, the SP6 promoter, the lac operator promoter, the tac promoter, the BO40 late promoter, the BO40 early promoter, the BCP-LTR promoter, the CMV IE promoter, the BO40 early promoter or the SV 40 late promoter, and the CMV IE promoter.

In the context of this document, signal peptide and leader sequence are used interchangeably and refer to an amino acid sequence that may be attached to the amino terminus of a protein described herein. Signal peptides/leader sequences typically direct the localization of a protein. As used herein, signal peptides/leader sequences may facilitate secretion of a protein from the cell in which it is produced. Following secretion from the cell, signal peptides/leader sequences are often cleaved from the remainder of the protein, often referred to as the mature protein. Signal peptides/leader sequences are attached to the amino terminus (i.e., N-terminus) of the protein.

In the context of this document, substantially complementary means that the first sequence is at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the complementary sequence of the second sequence over a region of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 180, 270, 360, 450, 540 or more nucleotides or amino acids, or that the two sequences hybridize under stringent hybridization conditions.

In the context of this document, substantially identical means that the first and second sequences are at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical over a region of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 180, 270, 360, 450, 540 or more nucleotides or amino acids, or, in relation to nucleic acids, if the first sequence is substantially complementary to the complementary sequence of the second sequence.

As used herein, the term "subject in need thereof" means a human or non-human mammal that exhibits one or more symptoms or indicators of recurrent respiratory papillomatosis (RRP) and/or has been diagnosed with RRP and is in need of treatment therefor. In many embodiments, the term "subject" may be used interchangeably with the term "patient." For example, a human subject may be diagnosed with RRP and/or one or more symptoms or indicators including, but not limited to, hoarseness, weak cry, chronic cough, breathing difficulties, shortness of breath, recurrent upper respiratory tract infections, pneumonia, dysphagia, stridor, failure to thrive, and/or airway tumors. For example, the term includes subjects that have been newly diagnosed. In some embodiments, the term includes subjects for whom treatment according to the disclosed methods is an initial treatment (e.g., first-line treatment where the patient has not received prior systemic treatment for RRP). In some embodiments of the invention, the expression includes subjects for whom treatment according to the disclosed methods is a second-line treatment, wherein the patient has previously received treatment with standard of clinical practice therapy, including, but not limited to, surgery, antiviral therapy, and tracheostomy.

As used herein, the terms "treat," "treat," or "treatment" mean alleviating symptoms, eliminating the cause of symptoms on a temporary or permanent basis, delaying or suppressing tumor growth, reducing tumor cell load or tumor burden, causing tumor regression, causing tumor shrinkage, necrosis, and/or disappearance, preventing tumor recurrence, preventing or suppressing malignant transformation, and/or increasing the survival of a subject.

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As used in this document, unless otherwise specified, the term clinically proven (whether used independently or to modify the terms safe and/or effective) means demonstrated by a clinical trial, where the clinical trial met the approval standards of the US Food and Drug Administration, EMA or the relevant national regulatory authority. For example, the clinical trial(s) described in the examples provided in this document may serve as evidence.

The term clinically demonstrated safety for a human papillomavirus (HPV) antigen dose, dosing regimen, treatment, or route (e.g., HPV antigen administered as the drug product pGX3024 or INO-3107 or a biosimilar thereof) refers to a favorable risk-benefit ratio with an acceptable incidence and/or acceptable severity of treatment-emergent adverse events (called TEAEs), compared with the standard of clinical practice or another comparator. An TEAE is an unfavorable medical occurrence in a patient administered a medicinal product. One measure of safety is the incidence of TEAEs classified by the National Cancer Institute (NCI) in accordance with the CTCAE Common Toxicity Criteria for Adverse Events v5.0.

As used herein, the terms clinically proven effective and clinically proven effective in the context of a dose, dosing regimen, treatment, or method refer to the effectiveness of a particular dose, dosing regimen, or treatment. Efficacy can be measured based on a change in the course of a disease in response to an agent of the present invention. For example, a human papillomavirus (HPV) antigen (e.g., an HPV antigen administered as a pGX3024 or INO-3107 drug product or a biosimilar thereof) is administered to a patient in an amount and for a time sufficient to cause an improvement, preferably a sustained improvement, in at least one indicator reflecting the severity of the disorder being treated. Various indicators reflecting the extent of the disease, illness, or condition of the subject can be assessed to determine the adequacy of the amount and time of treatment. Such indicators include, for example, clinically recognized indicators of disease severity, symptoms, or manifestations of the disorder of interest. The degree of improvement is usually determined by the physician, who may do so on the basis of signs, symptoms, biopsies, or other test results, as well as questionnaires given to the subject, such as disease-specific quality of life questionnaires. Improvement may be evidenced by an improvement in the disease activity index, relief of clinical symptoms, or any other measure of disease activity. For example, human papillomavirus (HPV) antigen (eg, HPV antigen administered as the drug product pGX3024 or INO-3107 or their biosimilar) may be administered to achieve an improvement in the patient's condition associated with a decrease in the frequency of surgery for RRP, a change in the RRP staging score, an increase in the interval between surgeries, clearance of HPV, or a decrease in the burden of disease.

In the context of this document, a variant with respect to a nucleic acid means (i) a part or fragment of a specified nucleotide sequence; (ii) a complementary sequence of a specified nucleotide sequence or a part thereof; (iii) a nucleic acid that is substantially identical to a specified nucleic acid or its complementary sequence; or (iv) a nucleic acid that hybridizes under stringent conditions to a specified nucleic acid, its complementary sequence or sequences substantially identical thereto. A variant may be a nucleic acid sequence that is substantially identical over the entire length of a complete gene sequence or a fragment thereof. A nucleic acid sequence may be 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical over the full length of the gene sequence or a fragment thereof.

A variant of a polypeptide is a polypeptide that differs in amino acid sequence by insertion, deletion or conservative substitution of amino acids, but retains at least one biological activity of said polypeptide. A variant may also mean a protein with an amino acid sequence that is substantially identical to said protein with an amino acid sequence that retains at least one biological activity. A variant may be an amino acid sequence that is substantially identical over the full length of the amino acid sequence or a fragment thereof. The amino acid sequence may be 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical over the full length of the amino acid sequence or a fragment thereof.

In the context of this document, a vector means a nucleic acid sequence containing an origin of replication. A vector may be a viral vector,

- 10 049710 bacteriophage, bacterial artificial chromosome or yeast artificial chromosome. The vector may be a DNA or RNA vector. The vector may be a self-replicating extrachromosomal vector, and in one embodiment of the invention is an expression plasmid. The vector may contain or include one or more heterologous nucleic acid sequences.

As used herein, the phrase in combination with means that the HPV6 E6 and E7 antigens and the HPV11 E6 and E7 antigens are administered to the subject simultaneously with, immediately before, or immediately after the administration of the adjuvant. In some embodiments of the invention, the HPV6 E6 and E7 antigens and the HPV11 E6 and E7 antigens are administered as a co-formulation with the adjuvant.

As used in this document, unless otherwise specified, the term clinically proven (whether used independently or to modify the terms safe and/or effective) means proven by a clinical trial, where the clinical trial met the approval standards of the US Food and Drug Administration, EMA or the relevant national regulatory authority. For example, the clinical trial described in the examples provided in this document could serve as evidence.

The term clinically proven safety, as applied to a dose, dosing regimen, treatment, or method with HPV6 E6 and E7 antigens and HPV11 E6 and E7 antigens (e.g., administered as pGX3024) in combination with an adjuvant such as IL-12 (e.g., administered as pGX6010), refers to a favorable risk-benefit ratio with an acceptable incidence and/or acceptable severity of treatment-emergent adverse events (called AEs or TEAEs), compared to the standard of clinical practice or another comparator. An adverse event is an unfavorable medical occurrence in a subject administered a medical product.

As used herein, the terms clinically proven effective and clinically proven effective in the context of a dose, dosing regimen, treatment, or method refer to the effectiveness of a particular dose, dosing regimen, or treatment. Efficacy can be measured based on a change in the course of a disease in response to an agent of the present invention. For example, a combination of HPV6 E6 and E7 antigens and HPV11 E6 and E7 antigens (e.g., administered as pGX3024) with an adjuvant such as IL-12 (e.g., administered as pGX6010) is administered to a subject in an amount and for a time sufficient to cause an improvement, preferably a sustained improvement, in at least one measure reflecting the severity of the disorder being treated. Various measures reflecting the extent of the disease, illness, or condition of the subject can be assessed to determine the adequacy of the amount and duration of treatment. Such measures include, for example, clinically recognized measures of disease severity, symptoms, or manifestations of the disorder of interest. The degree of improvement is usually determined by the physician, who may do so on the basis of signs, symptoms, biopsies, or other test results, as well as questionnaires given to the subject, such as disease-specific quality of life questionnaires. For example, a combination of HPV6 E6 and E7 antigens and HPV11 E6 and E7 antigens (e.g., administered as pGX3024) with an adjuvant such as IL-12 (e.g., administered as pGX6010) may be administered to achieve improvement in a patient's condition associated with RRP. Improvement may be evidenced by improvement in the disease activity index, relief of clinical symptoms, or any other measure of disease activity.

Provided herein are nucleic acid molecules, proteins, immunogenic compositions, including vaccines, and methods of using them to induce an immune response and/or prevent or treat RRP. The immunogenic compositions preferably comprise a human papillomavirus (HPV) antigen comprising an HPV6 E6 antigenic domain, an HPV6 E7 antigenic domain, an HPV11 E6 antigenic domain, and an HPV11 E7 antigenic domain. The disclosed immunogenic compositions are the result of a multi-phase strategy in which modified consensus sequences were created and genetic modifications were made, including codon optimization, RNA optimization, and the addition of a highly effective immunoglobin leader sequence. The immunogenic compositions can be used to protect against multiple HPV strains, thereby treating, preventing, and/or protecting against HPV-based pathologies. In particular, the immunogenic compositions can be used to prevent or treat HPV6 and/or HPV11-based pathologies. Immunogenic compositions can significantly induce an immune response in a subject to whom the immunogenic composition is administered, thereby protecting against and treating HPV6 infection, HPV11 infection, or both.

The vaccine may be a DNA vaccine, a peptide vaccine, or a combination of a DNA vaccine and a peptide vaccine. The DNA vaccine may include a nucleic acid sequence encoding an HPV antigen. The nucleic acid sequence may be DNA, RNA, cDNA, a variant thereof, a fragment thereof, or a combination thereof. The nucleic acid sequence may also include additional sequences encoding linker, leader, or marker sequences that are linked to the HPV antigen by a peptide bond.

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A peptide vaccine may include an HPV antigenic peptide, an HPV antigenic protein, a variant thereof, a fragment thereof, or a combination thereof. A combined DNA and peptide vaccine may include the nucleic acid sequence described above encoding an HPV antigen and an HPV antigenic peptide or protein, wherein the HPV antigenic peptide or protein and the encoded HPV antigen have the same amino acid sequence.

The vaccine may elicit a humoral immune response in a subject administered the vaccine. The humoral immune response induced may be specific to the HPV6 E6 antigenic domain, the HPV6 E7 antigenic domain, the HPV11 E6 antigenic domain, the HPV11 E7 antigenic domain, or any combination thereof. The humoral immune response induced may react with the HPV6 E6 antigen, the HPV6 E7 antigen, the HPV11 E6 antigen, the HPV11 E7 antigen, or any combination thereof. The humoral immune response may be induced in a subject administered the vaccine by about 1.5 times to about 16 times, about 2 times to about 12 times, or about 3 times to about 10 times. The humoral immune response in a subject administered the vaccine can be induced to be at least about 1.5-fold, at least about 2.0-fold, at least about 2.5-fold, at least about 3.0-fold, at least about 3.5-fold, at least about 4.0-fold, at least about 4.5-fold, at least about 5.0-fold, at least about 5.5-fold, at least about 6.0-fold, at least about 6.5-fold, at least about 7.0-fold, at least about 7.5-fold, at least about 8.0-fold, at least about 8.5-fold, at least about 9.0-fold, at least about 9.5-fold, at least about 10.0-fold, at least about 10.5-fold, at least about 11.0-fold, at least about 11.5-fold, at least about 12.0 times, at least about 12.5 times, at least about 13.0 times, at least about 13.5 times, at least about 14.0 times, at least about 14.5 times, at least about 15.0 times, at least about 15.5 times, or at least about 16.0 times.

The humoral immune response induced by the vaccine may include an increased level of IgG antibodies associated with a subject who has been administered the vaccine compared to a subject who has not been administered the vaccine. Such IgG antibodies may be specific for HPV6 E6 antigen, HPV6 E7 antigen, HPV11 E6 antigen, HPV11 E7 antigen, or any combination thereof. Such IgG antibodies may react with HPV6 E6 antigen, HPV6 E7 antigen, HPV11 E6 antigen, HPV11 E7 antigen, or any combination thereof. The level of IgG antibodies associated with a subject who has been administered the vaccine may be increased by about 1.5-fold to about 16-fold, about 2-fold to about 12-fold, or about 3-fold to about 10-fold compared to a subject who has not been administered the vaccine. The level of IgG antibodies associated with a subject who has been administered the vaccine may be increased by at least about 1.5-fold, at least about 2.0-fold, at least about 2.5-fold, at least about 3.0-fold, at least about 3.5-fold, at least about 4.0-fold, at least about 4.5-fold, at least about 5.0-fold, at least about 5.5-fold, at least about 6.0-fold, at least about 6.5-fold, at least about 7.0-fold, at least about 7.5-fold, at least about 8.0-fold, at least about 8.5-fold, at least about 9.0-fold, at least about 9.5-fold, at least about 10.0-fold, at least about 10.5-fold, at least about 11.0-fold, at least about 11.5-fold, at least about 12.0 times, at least about 12.5 times, at least about 13.0 times, at least about 13.5 times, at least about 14.0 times, at least about 14.5 times, at least about 15.0 times, at least about 15.5 times, or at least about 16.0 times compared to a subject who was not administered the vaccine.

A vaccine may elicit a cellular immune response in a subject to which the vaccine has been administered. The induced cellular immune response may be specific for HPV6 E6 antigen, HPV6 E7 antigen, HPV11 E6 antigen, HPV11 E7 antigen, or any combination thereof. The induced cellular immune response may be reactive for HPV6 E6 antigen, HPV6 E7 antigen, HPV11 E6 antigen, HPV11 E7 antigen, or any combination thereof. The induced cellular immune response may involve the induction of a T cell response. The induced T cell response may be reactive for HPV6 E6 antigen, HPV6 E7 antigen, HPV11 E6 antigen, HPV11 E7 antigen, or any combination thereof. The induced T cell response may be polyfunctional. An induced cellular immune response may include the induction of a T cell response in which T cells produce interferon gamma (IFN-γ). An induced cellular immune response may include increased T cell

- 12 049710 response associated with a subject who has been administered the vaccine compared to a subject who has not been administered the vaccine. The T cell response associated with a subject who has been administered the vaccine may be increased by about 2-fold to about 30-fold, about 3-fold to about 25-fold, or about 4-fold to about 20-fold compared to a subject who has not been administered the vaccine. The T cell response associated with a subject administered the vaccine can be increased by at least about 1.5-fold, at least about 2.0-fold, at least about 3.0-fold, at least about 4.0-fold, at least about 5.0-fold, at least about 6.0-fold, at least about 6.5-fold, at least about 7.0-fold, at least about 7.5-fold, at least about 8.0-fold, at least about 8.5-fold, at least about 9.0-fold, at least about 9.5-fold, at least about 10.0-fold, at least about 10.5-fold, at least about 11.0-fold, at least about 11.5-fold, at least about 12.0-fold, at least about 12.5-fold, at least about 13.0-fold, at least about 13.5-fold, at least about 14.0 times, at least about 14.5 times, at least about 15.0 times, at least about 16.0 times, at least about 17.0 times, at least about 18.0 times, at least about 19.0 times, at least about 20.0 times, at least about 21.0 times, at least about 22.0 times, at least about 23.0 times, at least about 24.0 times, at least about 25.0 times, at least about 26.0 times, at least about 27.0 times, at least about 28.0 times, at least about 29.0 times, or at least about 30.0 times compared to a subject who was not administered the vaccine.

The vaccine of the present invention may have properties necessary for effective vaccines, such as safety, such that the vaccine itself does not cause disease or death; protection against disease resulting from exposure to live pathogens such as viruses or bacteria; inducing antibodies to prevent the creation of cells; inducing protective T cells against intracellular pathogens; and providing ease of administration, few side effects, biological stability, and low cost per dose.

The vaccine may further induce an immune response when administered into various tissues, such as muscle or skin. The vaccine may also induce an immune response when administered by electroporation or injection, subcutaneously or intramuscularly.

An HPV antigen is capable of inducing an immune response in a mammal against one or more HPV strains. Thus, HPV antigens are disclosed herein that include an HPV6 antigenic domain and an HPV11 antigenic domain. The HPV6 antigenic domain may be located N-terminal or C-terminal to the HPV11 antigenic domain.

In some embodiments, the HPV6 antigenic domain comprises the HPV6 E6 antigenic domain, a fragment thereof, or a variant thereof and the HPV6 E7 antigenic domain, a fragment thereof, a variant thereof, or a combination thereof. The HPV6 E6 antigenic domain may be located N-terminal or C-terminal to the HPV6 E7 antigenic domain. In some embodiments, the HPV6 E6 antigenic domain may comprise epitope(s) that make it particularly effective as an immunogen against which an immune response can be elicited. The HPV6 E6 antigenic domain may be a consensus sequence derived from two or more HPV6 strains. The HPV6 E6 antigenic domain may comprise the consensus sequence and/or modification(s) to improve expression. The modification may include codon optimization, RNA optimization, addition of a Kozak sequence to enhance translation initiation, and/or addition of an immunoglobulin leader sequence to enhance the immunogenicity of the HPV6 E6 antigen domain. The HPV6 E6 consensus antigen domain may include a signal peptide, such as an immunoglobulin signal peptide, such as, but not limited to, an immunoglobulin E (IgE) or immunoglobulin (IgG) signal peptide. In some embodiments, the HPV6 E6 consensus antigen domain may include a hemagglutinin (HA) tag. The HPV6 E6 consensus antigen domain may be engineered to induce stronger and broader cellular and/or humoral immune responses compared to a corresponding codon-optimized HPV6 E6 antigen domain. In some embodiments, the HPV6 E7 antigenic domain may comprise epitope(s) that make it particularly useful as an immunogen against which an immune response can be elicited. The HPV6 E7 antigenic domain may be a consensus sequence derived from two or more HPV6 strains. The HPV6 E7 antigenic domain may comprise the consensus sequence and/or modification(s) to improve expression. The modification may comprise codon optimization, RNA optimization, addition of a Kozak sequence to enhance translation initiation, and/or addition of an immunoglobulin leader sequence to enhance the immunogenicity of the HPV6 E7 antigenic domain.

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The HPV6 E7 consensus antigen domain may include a signal peptide, such as an immunoglobulin signal peptide, such as, but not limited to, an immunoglobulin E (IgE) or immunoglobulin (IgG) signal peptide. In some embodiments, the HPV6 E7 consensus antigen domain may include a hemagglutinin (HA) tag. The HPV6 E7 consensus antigen domain may be engineered to induce stronger and broader cellular and/or humoral immune responses compared to a corresponding codon-optimized HPV6 E7 antigen domain.

In some embodiments, the HPV1 antigenic domain comprises the HPV11 E6 antigenic domain, a fragment thereof, or a variant thereof and the HPV11 E7 antigenic domain, a fragment thereof, a variant thereof, or a combination thereof. The HPV11 E6 antigenic domain may be located N-terminal or C-terminal to the HPV11 E7 antigenic domain. In some embodiments, the HPV11 E6 antigenic domain may comprise epitope(s) that make it particularly effective as an immunogen against which an immune response can be elicited. The HPV11 E6 antigenic domain may be a consensus sequence derived from two or more HPV11 strains. The HPV11 E6 antigenic domain may comprise the consensus sequence and/or modification(s) to improve expression. The modification may include codon optimization, RNA optimization, addition of a Kozak sequence to enhance translation initiation, and/or addition of an immunoglobulin leader sequence to enhance the immunogenicity of the HPV11 E6 antigen domain. The HPV11 E6 consensus antigen domain may include a signal peptide, such as an immunoglobulin signal peptide, such as, but not limited to, an immunoglobulin E (IgE) or immunoglobulin (IgG) signal peptide. In some embodiments, the HPV11 E6 consensus antigen domain may include a hemagglutinin (HA) tag. The HPV11 E6 consensus antigen domain may be engineered to induce stronger and broader cellular and/or humoral immune responses compared to a corresponding codon-optimized HPV11 E6 antigen domain. In some embodiments, the HPV11 E7 antigenic domain may include epitope(s) that make it particularly effective as an immunogen against which an immune response can be elicited. The HPV11 E7 antigenic domain may be a consensus sequence derived from two or more HPV11 strains. The HPV11 E7 antigenic domain may include the consensus sequence and/or modification(s) to improve expression. The modification may include codon optimization, RNA optimization, addition of a Kozak sequence to enhance translation initiation, and/or addition of an immunoglobulin leader sequence to enhance the immunogenicity of the HPV11 E7 antigenic domain. The HPV11 E7 consensus antigenic domain may include a signal peptide, such as an immunoglobulin signal peptide, such as, but not limited to, an immunoglobulin E (IgE) or an immunoglobulin (IgG) signal peptide. In some embodiments of the invention, the HPV11 E7 consensus antigen domain may include a hemagglutinin (HA) tag. The HPV11 E7 consensus antigen domain may be engineered to induce stronger and broader cellular and/or humoral immune responses compared to the corresponding codon-optimized HPV11 E7 antigen domain.

In some aspects, the HPV antigen comprises the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 11; an amino acid sequence that is at least about 95%, about 96%, about 97%, about 98%, or about 99% homologous to the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 11; an immunogenic fragment of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 11; or an amino acid sequence that is at least about 95%, about 96%, about 97%, about 98%, or about 99% homologous to an immunogenic fragment of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 11.

Fragments of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 11 may constitute 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more percent of the length of the full-length amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 11. Fragments of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 11 may constitute 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more percent of the length of the full-length amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 11. Fragments of SEQ ID NO: 1 or SEQ ID NO: 11 may be 100% identical to the full-length reference sequence, except for the absence of at least one amino acid at the N- and/or C-terminus, in each case with or without signal peptides and/or methionine at position 1. Fragments of SEQ ID NO: 1 or SEQ ID NO: 11 may be 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more,

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91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more percent of the length of the full-length SEQ ID NO: 1 or SEQ ID NO: 11, excluding any added heterologous signal peptide. The fragment may preferably include a fragment of SEQ ID NO: 1 or SEQ ID NO: 11 that is 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more homologous to SEQ ID NO: 1 or SEQ ID NO: 11, and further includes an N-terminal methionine or a heterologous signal peptide that is not taken into account when calculating the percent homology. The fragments may further comprise an N-terminal methionine and/or a signal peptide, such as an immunoglobulin signal peptide, for example an IgE or IgG signal peptide. The N-terminal methionine and/or signal peptide may be linked to the fragment.

In some embodiments, fragments of SEQ ID NO: 1 or SEQ ID NO: 11 may comprise 100 or more residues; in some embodiments, 200 or more residues; in some embodiments, 300 or more residues; in some embodiments, 400 or more residues; and in some embodiments, 500 or more residues.

In some aspects, the HPV antigen may comprise HPV6 and HPV11 antigenic domains separated by one or more post-translational cleavage sites, one or more translational skip-over sites, or both. In some embodiments, the post-translational cleavage site is located between the HPV6 and HPV11 antigenic domains, between the HPV6 E6 and E7 antigenic domains, and/or between the HPV 11 E6 and E7 antigenic domains. In some embodiments, the translational skip-over site is located between the HPV6 and HPV11 antigenic domains, between the HPV6 E6 and E7 antigenic domains, and/or between the HPV11 E6 and E7 antigenic domains. In some embodiments, the post-translational cleavage site and the translational skip site are located between the HPV6 and HPV11 antigenic domains, between the HPV6 E6 and E7 antigenic domains, and/or between the HPV11 E6 and E7 antigenic domains. In some embodiments, the post-translational cleavage site is a furin cleavage site. In some embodiments, the translational skip site is a P2A site. In some aspects, the immunogenic HPV protein comprises the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 11.

In some aspects, the HPV antigen comprises an amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 11; an amino acid sequence that is at least about 95%, about 96%, about 97%, about 98%, or about 99% homologous to SEQ ID NO: 1 or SEQ ID NO: 11; an immunogenic fragment of SEQ ID NO: 1 or SEQ ID NO: 11; or an amino acid sequence that is at least about 95%, about 96%, about 97%, about 98%, or about 99% homologous to an immunogenic fragment of SEQ ID NO: 1 or SEQ ID NO: 11.

The proteins of the present invention can be created using well-known techniques. In some embodiments, for example, DNA molecules that encode a protein of the present invention can be inserted into a commercially available expression vector for use in an expression system. The resulting protein is recovered from the culture either by lysis of the cells or from the culture medium, as appropriate and known to those skilled in the art. One skilled in the art can, using well-known techniques, isolate the protein obtained using such expression systems. The methods for purifying protein from natural sources using antibodies that specifically bind to a particular protein, described above, can equally be used to purify protein produced by recombinant DNA technology. In addition to producing proteins by recombinant techniques, automated peptide synthesizers can also be used to produce isolated, substantially pure protein.

The HPV antigen may be a nucleic acid molecule that encodes the HPV6-HPV11 fusion antigen disclosed herein. The nucleotide sequence encoding the HPV11 antigenic domain may be located 5' or 3' relative to the nucleotide sequence encoding the HPV6 antigenic domain.

In some embodiments of the invention, the nucleic acid sequence encoding the HPV6 antigenic domain comprises a nucleic acid sequence encoding the HPV6 E6 antigenic domain and the HPV6 E7 antigenic domain. The nucleic acid sequence encoding the HPV6 E6 antigenic domain may be located 5' or 3' relative to the nucleic acid sequence encoding the HPV6 E7 antigenic domain.

In some embodiments of the invention, the nucleic acid sequence encoding the HPV11 antigenic domain comprises a nucleic acid sequence encoding the HPV11 E6 antigenic domain and the HPV11 E7 antigenic domain. The nucleic acid sequence encoding the HPV11 E6 antigenic domain may be located 5' or 3' relative to the nucleic acid sequence encoding the HPV11 E7 antigenic domain.

- 15 049710

In some aspects, nucleotide sequences encoding antigenic domains of an HPV antigen may be separated by nucleotide sequences encoding one or more post-translational cleavage sites, one or more translational skip sites, or both. In some embodiments, a nucleotide sequence encoding a post-translational cleavage site is located between nucleotide sequences encoding the antigenic domains of HPV6 and HPV11, between nucleotide sequences encoding the antigenic domains of HPV6 E6 and E7, between nucleotide sequences encoding the antigenic domains of HPV11 E6 and E7, or any combination thereof. In some embodiments of the invention, the nucleotide sequences encoding the translational skip site are located between the nucleotide sequences encoding the HPV6 and HPV11 antigenic domains, between the nucleotide sequences encoding the HPV6 E6 and E7 antigenic domains, between the nucleotide sequences encoding the HPV11 E6 and E7 antigenic domains, or any combination thereof. In some embodiments of the invention, the nucleotide sequences encoding the post-translational cleavage site and the translational skip site are located between the nucleotide sequences encoding the HPV6 and HPV11 antigenic domains, between the nucleotide sequences encoding the HPV6 E6 and E7 antigenic domains, between the nucleotide sequences encoding the HPV11 E6 and E7 antigenic domains, or any combination thereof. In some embodiments, the post-translational cleavage site is a furin cleavage site. In some embodiments, the translational skip site is a P2A site.

In some embodiments of the invention, a nucleic acid sequence encoding an HPV antigen comprises the nucleotide sequence of SEQ ID NO: 2 or SEQ ID NO: 12; a nucleotide sequence that is at least about 95%, about 96%, about 97%, about 98%, or about 99% homologous to the nucleotide sequence of SEQ ID NO: 2 or SEQ ID NO: 12; a fragment of the nucleotide sequence of SEQ ID NO: 2 or SEQ ID NO: 12; or a nucleotide sequence that is at least about 95%, about 96%, about 97%, about 98%, or about 99% homologous to a fragment of the nucleotide sequence of SEQ ID NO: 2 or SEQ ID NO: 12. The fragments may further comprise coding sequences for an N-terminal methionine and/or a signal peptide, such as an immunoglobulin signal peptide, for example, an IgE or IgG signal peptide. A coding sequence encoding an N-terminal methionine and/or a signal peptide may be linked to a fragment.

Nucleic acid molecules that include a nucleotide sequence encoding an immunogen(s) may be operably linked to regulatory elements. The nucleic acid molecule may be DNA, RNA, cDNA, a variant thereof, a fragment thereof, or a combination thereof. The nucleic acid sequence may also include additional sequences encoding linker or marker sequences that are linked to the antigen by a peptide bond.

In some aspects, a nucleic acid molecule encoding an HPV antigen is an expression vector. The expression vector may be a circular plasmid or a linear nucleic acid. The expression vector is capable of directing the expression of a particular nucleotide sequence in a corresponding subject cell. The expression vector may have a promoter operably linked to the antigen-encoding nucleotide sequence, which may be operably linked to termination signals. The expression vector may also contain sequences necessary for the proper translation of the nucleotide sequence. The expression vector containing the nucleotide sequence of interest may be chimeric, meaning that at least one of its components is heterologous with respect to at least one of its other components. Expression of the nucleotide sequence in the expression cassette may occur under the control of a constitutive promoter or an inducible promoter that initiates transcription only when the host cell is exposed to a certain external stimulus. In the case of a multicellular organism, a promoter may also be specific to a particular tissue, organ, or developmental stage.

In one embodiment, the nucleic acid is an RNA molecule. Accordingly, in one embodiment, the invention provides an RNA molecule encoding one or more polypeptides of interest. The RNA may be a plus strand. Accordingly, in some embodiments, the RNA molecule may be translated by cells without the need for any intermediate replication steps, such as reverse transcription. An RNA molecule useful in the present invention may have a 5' cap (e.g., 7-methylguanosine). This cap may enhance in vivo translation of the RNA. The 5' nucleotide of an RNA molecule useful in the present invention may have a 5' triphosphate group.

- 16 049710 capped RNA, it may be linked to 7-methylguanosine via a 5'-to-5' bridge. The RNA molecule may have a 3' polyA tail. It may also include a polyA polymerase recognition sequence (e.g., AAUAAA) near the 3' end. The RNA molecule useful in the present invention may be single-stranded. In some embodiments, the RNA molecule may be a naked RNA molecule. In one embodiment, the RNA molecule is contained in a vector.

In one embodiment, the RNA has a 5' and 3' UTR. In one embodiment, the 5' UTR is between zero and 3000 nucleotides in length. The length of the 5' and 3' UTR sequences added to the coding region can be varied in a variety of ways, including, but not limited to, designing PCR primers that anneal to different regions of the UTR. Using this approach, one skilled in the art can modify the length of the 5' and 3' UTR as needed to achieve optimal translation efficiency after transfection of the transcribed RNA.

The 5' and 3' UTRs may be naturally occurring, endogenous 5' and 3' UTRs for the gene of interest. Alternatively, UTR sequences that are not endogenous to the gene of interest may be added by incorporating UTR sequences into the forward and reverse primers or by any other modifications to the template. The use of UTR sequences that are not endogenous to the gene of interest may be used to modify the stability and/or translation efficiency of the RNA. For example, it is known that AU-rich elements in 3' UTR sequences may decrease RNA stability. Therefore, the 3' UTR may be selected or designed to enhance the stability of the transcribed RNA based on properties of the UTR that are well known in the art.

In one embodiment, the 5' UTR may comprise a Kozak sequence from an endogenous gene. Alternatively, when a 5' UTR that is not endogenous to the gene of interest is added by PCR as described above, the Kozak consensus sequence can be redesigned by adding the 5' UTR sequence. Kozak sequences can improve the translation efficiency of some RNA transcripts, but are not required for all RNAs to ensure efficient translation. The requirement for Kozak sequences for many RNAs is known in the art. In other embodiments, the 5' UTR can be derived from an RNA virus whose RNA genome is stable in cells. In other embodiments, various nucleotide analogs can be used in the 3' or 5' UTR to interfere with exonuclease degradation of the RNA.

In one embodiment of the invention, the RNA has both a cap at the 5' end and a 3' poly(A) tail, which determine ribosome binding, translation initiation, and RNA stability in the cell.

In one embodiment of the invention, the RNA is RNA with modified nucleosides. RNA with modified nucleosides has certain advantages over unmodified RNA, including, for example, increased stability, low or no natural immunogenicity, and increased translation.

The expression vector may be a circular plasmid that can transform the target cell by integrating into the cellular genome or exist extrachromosomally (e.g., an autonomously replicating plasmid with an origin of replication). The vector may be pVAX, pcDNA3.0, provax, or any other expression vector capable of expressing DNA encoding the antigen and allowing the cell to translate the sequence into an antigen recognized by the immune system.

Also provided herein is an immunogenic composition of a linear nucleic acid or a linear expression cassette (LEC) that is capable of being efficiently delivered to a subject by electroporation and expressing one or more desired antigens. The LEC may be any linear DNA in which the phosphate backbone has been removed. The DNA may encode one or more antigens. The LEC may include a promoter, an intron, a stop codon, and/or a polyadenylation signal. Expression of the antigen may be controlled by the promoter. The LEC may be free of any antibiotic resistance genes and/or a phosphate backbone. The LEC may be free of other nucleotide sequences unrelated to expression of the desired antigen gene. The LEC may be derived from any plasmid that is capable of linearization. The plasmid may be capable of expressing the antigen. The plasmid may be pNP (Puerto Rico/34) or pM2 (New Caledonia/99). The plasmid may be WLV009, pVAX, pcDNA3.0, or provax, or any other expression vector capable of expressing DNA encoding the antigen and allowing the cell to translate the sequence into an antigen recognized by the immune system. The LEC may be pcrM2. The LEC may be pcrNP. pcrNP and pcrMR may be derived from pNP (Puerto Rico/34) and pM2 (New Caledonia/99), respectively.

The vector may comprise a heterologous nucleic acid encoding the antigens described above and may further comprise an initiation codon, which may be located upstream of the sequence(s) encoding one or more cancer antigens, and a stop codon, which

- 17 049710 may be located downstream of the sequence(s) encoding the antigen(s) described above.

The vector may have a promoter. The promoter may be any promoter that is capable of directing gene expression and regulating expression of the isolated nucleic acid. Such a promoter is a cis-acting sequence element that is necessary for transcription by DNA-dependent RNA polymerase that transcribes the antigen sequence described herein. The choice of promoter used to direct expression of a heterologous nucleic acid depends on the particular application. The promoter may be located approximately the same distance from the transcription start site in the vector as it is from the transcription start site in its natural environment. However, this distance may be varied without loss of promoter function.

The initiation codon and the termination codon may be in frame with the coding sequence(s) of the antigens described above. The vector may also include a promoter that is operably linked to the coding sequence(s) of the antigens described above. The promoter operably linked to the coding sequence(s) may be a promoter from simian virus 40 (BO40), a murine mammary tumor virus (MMT) promoter, a human immunodeficiency virus (HIV) promoter such as the bovine immunodeficiency virus (BIV) long terminal repeat (LTR) promoter, a Moloney virus promoter, an avian leukemia virus (ALV) promoter, a cytomegalovirus (CMV) promoter such as the CMV immediate early promoter, an Epstein-Barr virus (EBV) promoter, or a Rous sarcoma virus (RSV) promoter. The promoter may also be a promoter from a human gene, such as human actin, human myosin, human hemoglobin, human muscle creatinine, or human metallothionein. The promoter may also be a tissue-specific promoter, such as a muscle- or skin-specific promoter, natural or synthetic. Examples of such promoters are described in U.S. Patent Application Publication No. US 20040175727, the entire contents of which are incorporated herein by reference.

The vector may also include a polyadenylation signal, which may be downstream of the sequence(s) encoding the antigens and/or antibodies described above. The polyadenylation signal may be a BO40 polyadenylation signal, a LTR polyadenylation signal, a bovine growth hormone (bGH) polyadenylation signal, a human growth hormone (hGH) polyadenylation signal, or a human β-globin polyadenylation signal. The BO40 polyadenylation signal may be the polyadenylation signal from the pCEP4 vector (Invitrogen, San Diego, CA).

The vector may also include an enhancer of the antigens described above. The enhancer may be necessary for expression. The enhancer may be a human actin enhancer, a human myosin enhancer, a human hemoglobin enhancer, a human muscle creatinine enhancer, or a viral enhancer such as one of CMV, FMDV, HRV, or EBV.

The vector may include an enhancer and an intron with functional splice donor and acceptor sites. The vector may contain a transcription termination region downstream of the structural gene to ensure efficient termination. The termination region may be derived from the same gene as the promoter sequence or may be derived from different genes.

The vector may also include elements or reagents that interfere with its integration into the chromosome. The vector may also contain a mammalian origin of replication for extrachromosomal maintenance of the vector and production of multiple copies of the vector in the cell. The vector may be pVAX1, pCEP4, or pREP4 from Invitrogen (San Diego, CA), which may contain an Epstein-Barr virus origin of replication and the coding region of the EBNA-1 nuclear antigen, which may provide for high copy number episomal replication without integration. The vector may be pVAX1 or a pVAX1 variant with alterations, such as the plasmid variant described herein. The pVax1 variant plasmid is a plasmid variant on the 2998 base pair backbone of the pVAX1 vector (Invitrogen, Carlsbad, CA). The CMV promoter is located at bases 137-724. The T7 promoter/priming site is located at bases 664-683. The multiple cloning sites are located at bases 696-811. The bovine GR polyadenylation signal is located at bases 829-1053. The kanamycin resistance gene is located at bases 1226-2020. The pUC start is located at bases 2320-2993.

Based on the pVAXl sequence available from Invitrogen, the following mutations were found in the pVAXl sequence:

C>G241 in the CMV promoter;

C>T 1942 backbone, downstream of the bovine growth hormone polyadenylation signal (bGH polyA);

A> - 2876 backbone, downstream of the kanamycin resistance gene;

C>T 3277 at the pUC origin of replication (Ori), a high-copy mutation (see Nucleic. Acid. Research 1985);

- 18 049710

G>C 3753 at the very end of Ori pUC upstream of the RNASeH site.

Base pairs 2, 3, and 4 in the backbone upstream of the CMV promoter are changed from ACT to CTG.

The vector backbone may be pAV0242. The vector may be a replication-deficient adenovirus type 5 (Ad5) vector.

The vector may also include a regulatory sequence that may be well suited for gene expression in a mammalian or human cell into which the vector is introduced. The antigen sequences disclosed herein may include a codon that may provide for more efficient transcription of the coding sequence in the host cell.

The vector may be pSE420 (Invitrogen, San Diego, CA), which can be used to produce protein in Escherichia coli (E. coli). The vector may also be pYES2 (Invitrogen, San Diego, CA), which can be used to produce protein in Saccharomyces cerevisiae yeast strains. The vector may also be the MAXBAC™ Complete Baculovirus Expression System (Invitrogen, San Diego, CA), which can be used to produce protein in insect cells. The vector may also be pcDNA I or pcDNA3 (Invitrogen, San Diego, CA), which can be used to produce protein in mammalian cells, such as Chinese hamster ovary (CHO) cells. Vector may refer to vectors or expression systems for producing a protein using conventional techniques and readily available starting materials, including those mentioned in Sambrook et al., Molecular Cloning and Laboratory Manual, Second Ed., Cold Spring Harbor (1989), incorporated herein by reference in its entirety.

A typical DNA plasmid comprises SEQ ID NO: 3.

The immunogenic compositions of the present invention may comprise an HPV antigen of the present invention, a recombinant vaccine comprising a nucleotide sequence that encodes an HPV antigen of the present invention, a live attenuated pathogen that encodes an HPV antigen of the present invention and/or comprises an HPV antigen of the present invention; a killed pathogen comprising an HPV antigen of the present invention; or a composition, such as a liposome or a subunit vaccine, that comprises an HPV antigen of the present invention. The present invention further relates to pharmaceutical compositions, such as, but not limited to, injectable pharmaceutical compositions, that comprise the disclosed immunogenic compositions.

The immunogenic compositions of the present invention can be formulated with suitable pharmaceutically acceptable carriers, excipients and other means that provide suitable transport, delivery, tolerability, etc. The pharmaceutically acceptable excipient can be functional molecules such as carrier media, vehicles or diluents.

The pharmaceutically acceptable excipient may be a transfection facilitating agent, which may include surface-active agents such as immunostimulatory complexes (ISCOMS), incomplete Freund's adjuvant, an LPS analog including monophosphoryl lipid A, muramyl peptides, quinone analogs, vesicles such as squalene, hyaluronic acid, lipids, liposomes, calcium ions, viral proteins, polyanions, polycations or nanoparticles, or other known transfection facilitating agents.

The pharmaceutically acceptable excipient may be an adjuvant. In some aspects, compositions and vaccines are provided that include an HPV antigen of the present invention in combination with an adjuvant. The adjuvant may be other genes that are expressed on an alternative plasmid or that are delivered as proteins in combination with the HPV antigen of the present invention. The adjuvant may be selected from the group consisting of α-interferon (IFN-α), β-interferon (IFN-β), γ-interferon, platelet-derived growth factor (PDGF), TNFa, TNF[1. GM-CSF, epidermal growth factor (EGF), cutaneous T-cell-attracting chemokine (CTACK), thymus-expressed epithelial chemokine (TECK), mucosal epithelial chemokine (MEC), IL-12, IL-15, MHC, CD80, CD86, including IL-15 with deleted signal sequence and optionally containing signal peptide from IgE. The adjuvant can be IL-12, IL-15, IL-28, CTACK, TECK, platelet-derived growth factor (PDGF), TNF, TNF, GM-CSF, epidermal growth factor (EGF), IL-1, IL-2, IL-4, IL-5, PD-1, IL10, IL-12, IL-18 or a combination thereof.

Other genes that may be useful as adjuvants include those encoding MCP-1, MIP-la, MIP-1p, IL-8, RANTES, L-selectin, P-selectin, E-selectin, CD34, GlyCAM-1, MadCAM-1, LFA-1, VLA-1, Mac-1, p150.95, PECAM, ICAM-1, ICAM-2, ICAM-3, CD2, LFA-3, M-CSF, G-CSF, IL-4, mutant forms of IL-18, CD40, CD40L, vascular growth factor, fibroblast growth factor, IL-7, IL-22, nerve growth factor, vascular endothelial growth factor, Fas, TNF receptor, Flt, Apo-1, p55, WSL-1, DR3, TRAMP, Apo-3, AIR, LARD, NGRF, DR4, DR5, KILLER, TRAIL-R2, TRICK2, DR6, caspase ICE, Fos, c-jun, Sp-1, Ap-1, Ap-2, p38, p65Rel, MyD88, IRAK, TRAF6, IkB,

- 19 049710 inactive NIK, SAP K, SAP-1, JNK, interferon response genes, NFkB, Bax, TRAIL, TRAILrec, TRAILrecDRC5, TRAIL-R3, TRAIL-R4, RANK, RANK LIGAND, Ox40, Ox40 LIGAND, NKG2D, MICA, MICB, NKG2A, NKG2B, NKG2C, NKG2E, NKG2F, TAP1, TAP2 and their functional fragments.

In some embodiments, the adjuvant is interleukin-12 (IL12). IL-12 may be included in the vaccine in the form of its p35 and p40 subunits. The IL-12 adjuvant may be administered to a subject in the form of its p35 and p40 subunits. The p35 and p40 subunits of IL-12 may be encoded by the same expression vector or by separate expression vectors. The nucleic acid molecule encoding IL-12 may be the same as or different from the nucleic acid molecule encoding the HPV6 antigen fused to the HPV11 antigen. In some aspects, the nucleic acid molecule encoding IL-12 comprises a nucleotide sequence encoding the p35 subunit of IL-12, the p40 subunit of IL-12, or both. A nucleic acid molecule encoding the p35 subunit of IL-12 may comprise a nucleotide sequence that encodes SEQ ID NO: 6; a nucleotide sequence that is at least about 95%, about 96%, about 97%, about 98%, or about 99% homologous to the nucleotide sequence that encodes SEQ ID NO: 6; a fragment of the nucleotide sequence that encodes SEQ ID NO: 6; or a nucleotide sequence that is at least about 95%, about 96%, about 97%, about 98%, or about 99% homologous to a fragment of the nucleotide sequence that encodes SEQ ID NO: 6. A nucleic acid molecule encoding the p40 subunit of IL-12 may comprise a nucleotide sequence that encodes SEQ ID NO: 8; a nucleotide sequence that is at least about 95%, about 96%, about 97%, about 98%, or about 99% homologous to the nucleotide sequence that encodes SEQ ID NO: 8; a fragment of the nucleotide sequence that encodes SEQ ID NO: 8; or a nucleotide sequence that is at least 95% homologous to a fragment of the nucleotide sequence that encodes SEQ ID NO: 8. A nucleic acid molecule encoding the p35 subunit of IL-12 can include the nucleotide sequence of SEQ ID NO: 5; a nucleotide sequence that is at least about 95%, about 96%, about 97%, about 98%, or about 99% identical to the nucleotide sequence of SEQ ID NO: 5; a fragment of the nucleotide sequence of SEQ ID NO: 5; or a nucleotide sequence that is at least about 95%, about 96%, about 97%, about 98%, or about 99% identical to a fragment of the nucleotide sequence of SEQ ID NO: 5. A nucleic acid molecule encoding the p40 subunit of IL-12 may include the nucleotide sequence of SEQ ID NO: 7; a nucleotide sequence that is at least about 95%, about 96%, about 97%, about 98%, or about 99% identical to the nucleotide sequence of SEQ ID NO: 7; a fragment of the nucleotide sequence of SEQ ID NO: 7; or a nucleotide sequence that is at least about 95%, about 96%, about 97%, about 98%, or about 99% identical to a fragment of the nucleotide sequence of SEQ ID NO: 7.

In some embodiments of the described immunogenic compositions, the composition comprises pGX3024 and pGX6010. In some embodiments of the invention, the composition is INO-3107.

As used herein, a buffer refers to a buffered solution that is resistant to changes in pH due to the action of its acid-base conjugate components. The buffer typically has a pH of about 4.0 to about 8.0, such as about 5.0 to about 7.0. In some embodiments, the buffer is a sodium citrate buffered saline (SSC). In some embodiments in which the immunogenic composition comprises a vector comprising a nucleic acid molecule encoding an HPV antigen as described above, the immunogenic composition comprises 6 mg/mL of the vector in a buffer, such as, but not limited to, SSC buffer. In some embodiments, the immunogenic composition comprises 6 mg/mL of the pGX3024 DNA plasmid in a buffer. In some embodiments wherein the immunogenic composition further comprises a separate vector comprising a nucleic acid molecule encoding the p35 subunit of IL-12, the p40 subunit of IL-12, or both, the immunogenic composition comprises 0.25 mg/mL of the separate vector in buffer. In some embodiments, the immunogenic composition comprises 0.25 mg/mL of the pGX6010 DNA plasmid in buffer in addition to the vector comprising a nucleic acid molecule encoding an HPV antigen (e.g., pGX3024).

The pharmaceutical compositions of the present invention are formulated according to the route of administration used. In cases where the pharmaceutical compositions are injectable pharmaceutical compositions, they are sterile, pyrogen-free and particle-free. Preferably, an isotonic formulation is used. In general, additives for providing isotonicity may include sodium chloride, dextrose, mannitol, sorbitol and lactose. In some cases,

- 20 049710 isotonic solutions, such as phosphate-buffered saline. Stabilizers include gelatin and albumin. In some embodiments of the invention, a vasoconstrictor is added to the composition.

Also provided herein are methods for treating, protecting against and/or preventing a disease in a subject in need thereof by administering to the subject a vaccine of the present invention. Administration of the vaccine to the subject may cause or induce an immune response in the subject. Thus, provided are methods for inducing an immune response in a subject comprising administering to the subject an effective amount of an HPV antigen of the present invention, thereby inducing an immune response.

Also provided are methods of prophylactically or therapeutically immunizing a subject against HPV6, HPV11, or both, comprising administering to the subject an effective amount of an HPV antigen of the present invention, thereby inducing an immune response against HPV6, HPV11, or both. Also provided are methods of treating or preventing recurrent respiratory papillomatosis (RRP) in a subject, comprising administering to the subject an effective amount of an HPV antigen of the present invention, thereby treating or preventing RRP.

The induced immune response can be used to treat, prevent and/or protect against a disease, such as pathologies associated with HPV infection. In some embodiments of the invention, methods are provided for treating, protecting against and/or preventing RRP in a subject in need thereof by administering an HPV antigen of the present invention to the subject. The induced immune response provides the subject to which the vaccine is administered with resistance to one or more HPV strains. The induced immune response can include an induced humoral immune response and/or an induced cellular immune response.

The subject may be a mammal such as a human, horse, cow, pig, sheep, cat, dog, rabbit, guinea pig, rat, or mouse.

The vaccine dose may be from 1 μg to 10 mg of total plasmid per injection, preferably 6.25 mg of total plasmid per injection. The vaccine may be administered every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 days. The number of vaccine doses for effective treatment may be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

A vaccine may be administered prophylactically or therapeutically. In prophylactic administration, vaccines may be administered in an amount sufficient to induce an immune response. In therapeutic applications, vaccines are administered to a subject in need thereof in an amount sufficient to induce a therapeutic effect. An amount sufficient to achieve this purpose is defined as a therapeutically effective dose. An amount effective for such an application will depend, for example, on the particular composition of the vaccine regimen administered, the route of administration, the stage and severity of the disease, the general health of the patient, and the judgment of the physician prescribing the treatment.

The vaccine can be administered by methods well known in the art, as described by Donnelly et al. (Ann. Rev. Immunol. 15:617-648 (1997)); Felgner et al. (U.S. Patent No. 5,580,859, issued December 3, 1996); Felgner (U.S. Patent No. 5,703,055, issued December 30, 1997); and Carson et al. (U.S. Patent No. 5,679,647, issued October 21, 1997), all of which are incorporated herein by reference in their entireties. The DNA in the vaccine can be complexed with particles or beads that can be administered to an individual, for example, using a vaccine gun. A person skilled in the art knows that the choice of a pharmaceutically acceptable carrier, including a physiologically acceptable compound, depends, for example, on the route of administration of the expression vector.

In some embodiments of the methods described, pGX3024 and pGX6010 are administered to the subject. In some embodiments, pGX3024 and pGX6010 are administered to the subject as INO-3107. In some embodiments, pGX3024 and pGX6010 are administered to the subject as the drug product INO-3107, comprising 6.25 mg total plasmid/mL (6 mg/mL pGX3024, 0.25 mg/mL pGX6010) in 150 mM sodium chloride and 15 mM sodium citrate, pH 7.

The vaccine can be delivered by a variety of routes. Typical delivery routes include parenteral administration, such as intradermal, intramuscular, or subcutaneous delivery. Other routes include oral administration, intranasal, and intravaginal routes. With respect to DNA in the vaccine, in particular, the vaccine can be delivered to the interstitial spaces of human tissues (Felgner et al., U.S. Patent Nos. 5,580,859 and 5,703,055, the entire contents of which are incorporated herein by reference in their entirety). The vaccine can also be injected into muscle, or can be administered by intradermal or subcutaneous injection, or transdermally, such as by iontophoresis. Epidermal administration of the vaccine can also be used. Epidermal administration may involve mechanical or chemical irritation of the outer layer of the epidermis to stimulate an immune response to the irritant (Carson et al., U.S. Patent No. 5,679,647, the contents of which are incorporated herein by reference in their entirety.

- 21 049710 in its volume).

According to some embodiments of the invention, the vaccine is delivered to an individual to modulate the activity of the individual's immune system and thereby enhance the immune response against HPV to treat RRP. When a nucleic acid molecule encoding an HPV antigen of the present invention is taken up by the cells of an individual, the nucleotide sequence is expressed in the cells and thus the protein is delivered to the individual. Methods are provided for delivering protein coding sequences on a nucleic acid molecule, such as a plasmid, as part of recombinant vaccines and as part of attenuated vaccines, as isolated proteins or proteins included in a vector.

The methods comprise administering to a subject an HPV antigen of the present invention. In some aspects, the methods comprise the step of administering the present nucleic acid molecules followed by electroporation.

The disclosed methods may include introducing multiple copies of a single nucleic acid molecule, such as a single plasmid, or multiple copies of two or more different nucleic acid molecules, such as two or more different plasmids. For example, the methods may include introducing two, three, four, five, six, seven, eight, nine, or ten or more different nucleic acid molecules.

In some embodiments, the disclosed methods for inducing an immune response or methods for preventing or treating RRP further comprise administering an adjuvant to the subject. In some embodiments, the adjuvant is IL-12. IL-12 may be included in the vaccine in the form of its p35 and p40 subunits. The IL-12 adjuvant may be administered to the subject in the form of its p35 and p40 subunits. The p35 and p40 subunits of IL-12 may be encoded by the same expression vector or by separate expression vectors. In one embodiment, the coding sequence of p35 IL-12 is shown in SEQ ID NO: 5. In one embodiment, the p35 subunit of IL-12 has the amino acid sequence shown in SEQ ID NO: 6. In one embodiment, the coding sequence of p40 IL-12 is shown in SEQ ID NO: 7. In one embodiment, the p40 subunit of IL-12 has the amino acid sequence shown in SEQ ID NO: 8. In some embodiments, the expression vector is pGX6012 or pGX6010. In some embodiments, the methods have clinically demonstrated safety, clinically demonstrated efficacy, or both.

In some embodiments, the method comprises simultaneously administering (a) an immunogenic composition comprising an HPV antigen as disclosed herein and (b) a composition comprising a nucleic acid molecule encoding one or more IL12 subunits (e.g., p35 and/or p40) as disclosed herein. In some embodiments, the method comprises administering a composition comprising a nucleic acid molecule encoding one or more IL-12 subunits (e.g., p35 and/or p40) as disclosed herein after prior administration of a composition comprising a nucleic acid molecule encoding an HPV antigen as disclosed herein. In some embodiments of the invention, the method comprises administering a composition comprising a nucleic acid molecule encoding an HPV antigen disclosed herein after pre-administration of a composition comprising a nucleic acid molecule encoding one or more IL12 subunits (e.g., p35 and/or p40) disclosed herein.

Routes of administration include, but are not limited to, intramuscular, intranasal, intraperitoneal, intradermal, subcutaneous, intravenous, intraarterial, intraocular, and oral, as well as topical, transdermal, inhalation, or by suppository or into mucosal tissue, such as by lavage of vaginal, rectal, urethral, buccal, and sublingual tissue. Preferred routes of administration include intramuscular, intraperitoneal, intradermal, and subcutaneous injection. Genetic constructs may be administered by means including, but not limited to, electroporation methods and devices, conventional syringes, needle-free injection devices, or microparticle bombardment devices.

The vaccine can be administered by electroporation, such as by the method described in U.S. Patent No. 7,664,545, the contents of which are incorporated herein by reference. Electroporation can be accomplished by the method and/or apparatus described in U.S. Patent Nos. 6,302,874; 5,676,646; 6,241,701; 6,233,482; 6,216,034; 6,208,893; 6,192,270; 6,181,964; 6,150,148; 6,120,493; 6,096,020; 6,068,650; and 5,702,359, the contents of which are incorporated herein by reference in their entireties. Electroporation can be accomplished using a minimally invasive device.

The minimally invasive electroporation device (MID) may be an apparatus for injecting the vaccine and associated fluid described above into body tissue. The device may include a hollow needle, a DNA cassette, and fluid delivery means, and the device is adapted to actuate the fluid delivery means upon use in such a manner as to simultaneously (e.g., automatically) inject the DNA into body tissue during

- 22 049710 insertion of a needle into the specified tissue of the body. The advantage of this method is that the possibility of gradual injection of DNA and associated fluid during the insertion of the needle leads to a more uniform distribution of the fluid throughout the tissue of the body. The pain experienced during the injection can be reduced due to the distribution of the DNA injected over a larger area.

The MID may inject the vaccine into tissue without the use of a needle. The MID may inject the vaccine as a small stream or jet with such force that the vaccine pierces the surface of the tissue and penetrates into the underlying tissue and/or muscle. The force creating the small stream or jet may be provided by expanding a compressed gas, such as carbon dioxide, through a microorifice for a fraction of a second. Examples of minimally invasive electroporation devices and methods of using them are described in U.S. Patent Application Publication No. 20080234655; U.S. Patent Nos. 6,520,950; 7,171,264; 6,208,893; 6,009,347; 6,120,493; 7,245,963; 7,328,064; and 6,763,264, the contents of each of which are incorporated herein by reference.

The IUI may include an injector that creates a high velocity fluid jet that painlessly pierces tissue. Such needle-free injectors are commercially available. Examples of needle-free injectors that may be used herein include those described in U.S. Patent Nos. 3,805,783; 4,447,223; 5,505,697 and 4,342,310, the contents of each of which are incorporated herein by reference.

The desired vaccine in a form suitable for direct or indirect electrotransfer may be administered (e.g., injected) using a needle-free injector into the tissue to be treated, typically by contacting the tissue surface with the injector in such a way as to initiate the delivery of a jet of the agent with a force sufficient to penetrate the tissue. For example, if the tissue to be treated is mucosa, skin or muscle, the agent is directed onto the mucosal or skin surface with a force sufficient to penetrate the stratum corneum and into the dermal layers or into the underlying tissue and muscle, respectively.

Needle-free injectors are well suited for delivering vaccines to all types of tissues, especially to the skin and mucosa. In some embodiments of the invention, a needle-free injector can be used to deliver a liquid containing a vaccine to the surface and into the skin or mucosa of a subject. Representative examples of various types of tissues that can be treated using the methods of the present invention include the pancreas, larynx, nasopharynx, hypopharynx, oropharynx, lip, throat, lung, heart, kidney, muscle, breast, colon, prostate, thymus, testicle, skin, mucosal tissue, ovary, blood vessels, or any combination thereof.

The MDI may have needle electrodes that electroporate tissue. Generating pulses between multiple pairs of electrodes in an electrode array, such as those arranged in rectangular or square configurations, provides improved results compared to generating pulses between a pair of electrodes. For example, U.S. Patent No. 5,702,359, titled Needle Electrodes for Indirect Drug and Gene Delivery, provides an array of needles in which multiple pairs of needles can be pulsed during a therapeutic treatment. In this invention, which is incorporated herein by reference as set forth in its entirety, the needles are arranged in a circular array but have connectors and a switching device that allows pulses to be generated between opposing pairs of needle electrodes. A pair of needle electrodes can be used to deliver recombinant expression vectors to cells. Such a device and system are described in U.S. Patent No. 6,763,264, the contents of which are incorporated herein by reference. Alternatively, a single-needle device may be used that provides DNA injection and electroporation using a single needle resembling a conventional injection needle and delivers pulses of lower voltage than those delivered by currently used devices, thereby reducing the electrical sensation experienced by the patient.

The MDI may include one or more electrode arrays. The arrays may include two or more needles of the same or different diameters. The needles may be spaced evenly or unevenly apart. The needles may be from 0.005 inches to 0.03 inches, from 0.01 inches to 0.025 inches, or from 0.015 inches to 0.020 inches. The needle diameter may be 0.0175 inches. The needles may be spaced at a distance of 0.5 mm, 1.0 mm, 1.5 mm, 2.0 mm, 2.5 mm, 3.0 mm, 3.5 mm, 4.0 mm, or more.

The IED may consist of a pulse generator and two or more vaccine needle injectors that deliver vaccine and electroporation pulses in a single step. The pulse generator may provide flexible programming of pulse and injection parameters using a personal computer with a flash card, as well as complete recording and storage of electroporation and patient data. The pulse generator may deliver different volt pulses for short periods of time. For example, the pulse generator may deliver three 15 volt pulses of 100 ms duration. An example of such an IED is the Elgen 1000 system of Inovio Biomedical Corporation, which is described in U.S. Patent No. 7,328,064, the contents of which are incorporated herein by

- 23 049710 links.

The MDI may be a CELLECTRA® device and system (Inovio Pharmaceuticals, BlueBell, PA), which is a modular electrode system that facilitates the introduction of a macromolecule, such as DNA, into cells of a selected tissue of an organism or plant. The modular electrode system may comprise a plurality of needle electrodes; a hypodermic needle; an electrical connector that provides a conductive connection from a programmable direct current pulse controller to the plurality of needle electrodes; and a power source. An operator may take a plurality of needle electrodes that are supported on a support structure and firmly insert them into a selected tissue in an organism or plant. The macromolecule is then delivered into the selected tissue through the hypodermic needle. The programmable direct current pulse controller is activated and a direct current electrical pulse is applied to the plurality of needle electrodes. The applied direct current electrical pulse facilitates the introduction of the macromolecule into the cell between the plurality of electrodes. Cell death due to cell overheating is minimized by limiting the power dissipation in the tissue using direct current pulses. The CELLECTRA device and system are described in U.S. Patent No. 7,245,963, the contents of which are incorporated herein by reference. The CELLECTRA® device may be a CELLECTRA 2000® device or a CELLECTRA® 3PSP device. The CELLECTRA® 2000 device is configured by the manufacturer to support either ID (intradermal) or IM (intramuscular) administration. The CELLECTRA™ 2000 includes a CELLECTRA™ pulse generator, an appropriate applicator, a disposable sterile matrix, and a disposable sheath (for ID only). The DNA plasmid is delivered separately by injection with a needle and syringe into the area defined by the electrodes immediately prior to the electroporation procedure.

The IUD may be the Elgen 1000 system (Inovio Pharmaceuticals). The Elgen 1000 system may include a device providing a hollow needle; and a means for delivering a fluid, wherein the apparatus is adapted to actuate the means for delivering the fluid when used in a manner that simultaneously (e.g., automatically) injects a fluid, a vaccine described herein, into a body tissue during insertion of the needle into said body tissue. The advantage is that the fluid may be injected gradually during insertion of the needle, resulting in a more uniform distribution of the fluid throughout the body tissue. It is also believed that pain experienced during injection is reduced due to the distribution of the volume of injected fluid over a larger area.

In addition, automatic fluid injection facilitates automatic monitoring and recording of the actual dose of fluid injected. If desired, this data can be stored by the control unit for documentation purposes.

It should be understood that the injection rate may be either linear or non-linear, and that the injection may be performed after the needles have been inserted through the skin of the subject to be treated and during their further insertion into the body tissue. Suitable tissues into which the liquid may be injected by the apparatus of the present invention include tumor tissue, skin or muscle tissue.

The apparatus also includes needle insertion means for guiding the insertion of the needle into body tissue. The rate of injection of the fluid is controlled by the rate of insertion of the needle. The advantage of this is that both the insertion of the needle and the injection of the fluid can be controlled so that the insertion rate matches the injection rate if necessary. This also facilitates the user's operation of the apparatus. If necessary, means can be provided for automatically inserting the needle into body tissue.

The user can select the moment at which the injection of the fluid begins. Ideally, however, the injection begins when the tip of the needle reaches the muscle tissue, and the device may include means for determining when the needle has been inserted to a sufficient depth to begin the injection of the fluid. This means that the injection of the fluid can be programmed to begin automatically when the needle reaches the desired depth (usually the depth at which the muscle tissue begins). The depth at which the muscle tissue begins can, for example, be taken as a predetermined depth of insertion of the needle, such as a value of 4 mm, which is considered sufficient for the needle to pass through the skin layer.

The detection means may include an ultrasound probe. The detection means may include means for detecting a change in impedance or resistance. In this case, the means may not record the depth of insertion of the needle into body tissue, but may be adapted to detect a change in impedance or resistance as the needle advances from another type of body tissue into muscle. Either of these alternatives provides a relatively accurate and easy-to-use means of determining that an injection can begin. If desired, the depth of insertion of the needle may be recorded and may be used to monitor the injection of fluid, for example, the volume of fluid injected is determined by recording the depth of insertion of the needle.

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The device may further comprise a base for supporting the needle and a housing for accommodating the base therein, wherein the base is movable relative to the housing, so that the needle is drawn into the housing when the base is in the first rear position relative to the housing, and the needle exits the housing when the base is in the second front position inside the housing. This is advantageous for the user, since the housing may be positioned on the patient's skin, and then the needles may be inserted into the patient's skin by moving the housing relative to the base.

As stated above, it is desirable to achieve a controlled injection rate of the fluid so that the fluid is uniformly distributed along the length of the needle when it is inserted into the skin. The fluid delivery means may include piston drive means adapted to inject the fluid at a controlled rate. The piston drive means may, for example, be driven by a servo drive. However, the piston drive means may be driven by moving the base in an axial direction relative to the housing. It should be understood that alternative means may be provided for delivering the fluid. Thus, for example, instead of a syringe and piston system, a closed container may be provided that can be compressed to deliver the fluid at a controlled or uncontrolled rate.

The apparatus described above can be used for any type of injection. However, it is expected to be particularly applicable in the field of electroporation, so it may additionally include means for applying voltage to the needle. This allows the needle to be used not only for injections, but also as an electrode during electroporation. This is particularly advantageous, as it means that the electric field is applied to the same area as the liquid being injected. Traditionally, electroporation has had the problem of being very difficult to accurately align the electrode with the previously injected liquid, so users tend to inject a larger volume of liquid than required over a larger area and apply the electric field over a larger area to try to ensure overlap between the injected substance and the electric field. Using the present invention, both the volume of liquid being injected and the magnitude of the applied electric field can be reduced while achieving good alignment between the electric field and the liquid.

Further provided herein are kits that can be used to treat a subject using the above-described vaccination methods. The kits can include a vaccine. The kits can also include instructions for performing the above-described vaccination method and/or for using the kit. The instructions included in the kits can be attached to the packaging material or can be included as a package insert. Although the instructions are typically written or printed materials, they are not limited to them. The present invention contemplates any medium capable of storing and transmitting them to an end user. Such media include, but are not limited to, electronic storage media (e.g., magnetic disks, tapes, cartridges), optical media (e.g., CD-ROM), and the like. As used herein, the term instructions can include an Internet site address where the instructions are provided.

Illustrative embodiments of the invention

Embodiment of the invention 1. A nucleic acid molecule encoding a human papillomavirus (HPV) antigen, wherein the HPV antigen comprises an HPV6 antigenic domain and an HPV11 antigenic domain.

Embodiment of the invention 2. A nucleic acid molecule according to embodiment of the invention 1, wherein the HPV6 antigenic domain is the HPV6 E6-E7 fusion antigen.

Embodiment 3. A nucleic acid molecule according to embodiment 1 or 2 of the invention, wherein the HPV11 antigenic domain is the HPV11 E6-E7 fusion antigen.

Embodiment 4. A nucleic acid molecule according to any of the previous embodiments of the invention, wherein the HPV antigen comprises the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 11; or an amino acid sequence that is at least 95% homologous to SEQ ID NO: 1 or SEQ ID NO: 11.

Embodiment 5. A nucleic acid molecule according to any of the previous embodiments of the invention, comprising a nucleotide sequence that is at least 95% homologous to SEQ ID NO: 2 or SEQ ID NO: 12;

the nucleotide sequence of SEQ ID NO: 2; or the nucleotide sequence of SEQ ID NO: 12.

Embodiment of the invention 6. A nucleic acid molecule according to any of the previous embodiments of the invention, wherein the nucleic acid sequence encoding the antigenic domain of HPV11 is located 5' with respect to the nucleic acid sequence encoding the antigenic domain of HPV6.

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Embodiment 7. A nucleic acid molecule according to any of the previous embodiments of the invention, wherein the HPV6 antigenic domain and the HPV11 antigenic domain are separated by one or more post-translational cleavage sites, one or more translational skip sites, or both.

Embodiment 8. A nucleic acid molecule according to embodiment 2, wherein the HPV6 E6 antigenic domain and the HPV6 E7 antigenic domain are separated by one or more post-translational cleavage sites, one or more translational skip sites, or both.

Embodiment 9. A nucleic acid molecule according to embodiment 4, wherein the HPV11 E6 antigenic domain and the HPV11 E7 antigenic domain are separated by one or more post-translational cleavage sites, one or more translational skip sites, or both.

Embodiment of the invention 10. An expression vector comprising a nucleic acid molecule according to any of the previous embodiments of the invention.

Embodiment of the invention 11. An expression vector according to embodiment of the invention 10, comprising a DNA plasmid.

Embodiment of the invention 12. An expression vector according to embodiment of the invention 10, comprising the nucleotide sequence SEQ ID NO: 3.

Embodiment of the invention 13. An immunogenic protein comprising the antigenic domain of human papillomavirus (HPV) 6 and the antigenic domain of HPV11.

Embodiment of the invention 14. An immunogenic protein according to embodiment of the invention 14, wherein the HPV6 antigenic domain comprises the HPV6 E6 antigenic domain and the HPV6 E7 antigenic domain.

Embodiment 15. An immunogenic protein according to embodiment 13 or 14, wherein the HPV11 antigenic domain comprises the HPV11 E6 antigenic domain and the HPV11 E7 antigenic domain.

Embodiment 16. An immunogenic protein according to any of embodiments 13-15, comprising the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 11; or an amino acid sequence that is at least 95% homologous to the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 11.

Embodiment of the invention 17. An immunogenic protein according to any of embodiments of the invention 13-16, wherein the HPV11 antigenic domain is located N-terminally with respect to the HPV6 antigen.

Embodiment 18. An immunogenic protein according to any one of embodiments 13-17, wherein the HPV6 antigenic domain and the HPV11 antigenic domain are separated by one or more post-translational cleavage sites, one or more translational skip sites, or both.

Embodiment 19. An immunogenic protein according to embodiment 14, wherein the HPV6 E6 antigenic domain and the HPV6 E7 antigenic domain are separated by one or more post-translational cleavage sites, one or more translational skip sites, or both.

Embodiment 20. An immunogenic protein according to embodiment 15, wherein the HPV11 E6 antigenic domain and the HPV11 E7 antigenic domain are separated by one or more post-translational cleavage sites, one or more translational skip sites, or both.

Embodiment of the invention 21. A vaccine comprising a nucleic acid molecule according to any of embodiments of the invention 1-9 or an expression vector according to any of embodiments of the invention 10-12 and a pharmaceutically acceptable excipient.

Embodiment of the invention 22. A pharmaceutical composition comprising a nucleic acid molecule according to any of embodiments of the invention 1-9 or an expression vector according to any of embodiments of the invention 10-12 and a pharmaceutically acceptable excipient.

Embodiment of the invention 23. A pharmaceutical composition according to embodiment of the invention 22, including an adjuvant.

Embodiment of the invention 24. A pharmaceutical composition according to embodiment of the invention 23, wherein the adjuvant comprises interleukin-12 (IL-12).

Embodiment of the invention 25. A pharmaceutical composition according to embodiment of the invention 24, wherein IL-12 is encoded by a nucleic acid molecule.

Embodiment of the invention 26. A pharmaceutical composition according to embodiment of the invention 25, wherein the nucleic acid molecule encoding IL-12 is an expression vector.

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Embodiment of the invention 27. A vaccine comprising an immunogenic protein according to any of embodiments of the invention 13-20.

Embodiment of the invention 28. A pharmaceutical composition comprising an immunogenic protein according to any of embodiments of the invention 13-20 and a pharmaceutically acceptable excipient.

Embodiment of the invention 29. A pharmaceutical composition according to embodiment of the invention 28, including an adjuvant.

Embodiment of the invention 30. A pharmaceutical composition according to embodiment of the invention 29, wherein the adjuvant comprises interleukin-12 (IL-12).

Embodiment 31. A pharmaceutical composition according to embodiment 23 or 29, wherein the adjuvant comprises a nucleic acid molecule comprising a nucleotide sequence encoding the p35 subunit of IL-12, the p40 subunit of IL-12, or both.

Embodiment 32. A pharmaceutical composition according to embodiment 31, wherein the nucleotide sequence encoding the p35 IL-12 subunit comprises a nucleotide sequence selected from the group consisting of a nucleotide sequence encoding SEQ ID NO: 6; or a nucleotide sequence that is at least 95% homologous to the nucleotide sequence encoding SEQ ID NO: 6.

Embodiment 33. A pharmaceutical composition according to embodiment 31 or 32, wherein the nucleotide sequence encoding the p40 subunit of IL-12 comprises a nucleotide sequence selected from the group consisting of a nucleotide sequence encoding SEQ ID NO: 8; or a nucleotide sequence that is at least 95% homologous to the nucleotide sequence encoding SEQ ID NO: 8.

Embodiment 34. A pharmaceutical composition according to any of embodiments 31, 32 or 33, wherein the nucleotide sequence encoding IL-12 comprises a nucleotide sequence selected from the group consisting of the nucleotide sequence of SEQ ID NO: 4; or a nucleotide sequence that is at least 95% homologous to SEQ ID NO: 4.

Embodiment 35. A pharmaceutical composition according to any one of embodiments 31-34, wherein the nucleic acid molecule comprising a nucleotide sequence encoding the p35 IL-12 subunit, the p40 IL-12 subunit, or both, is an expression vector.

Embodiment 36. A pharmaceutical composition according to embodiment 35, wherein the expression vector comprising a nucleic acid molecule encoding the p35 subunit of IL-12, the p40 subunit of IL-12, or both, is the same expression vector or an expression vector different from the expression vector comprising a nucleic acid molecule encoding an HPV antigen.

Embodiment 37. A pharmaceutical composition according to any of embodiments 22-26 or 28-36, wherein the pharmaceutically acceptable excipient comprises a buffer, optionally a saline buffer with sodium citrate, optionally a buffer comprising 150 mM sodium chloride and 15 mM sodium citrate, pH 7.

Embodiment 38. A pharmaceutical composition according to embodiment 37, wherein the composition comprises 6 mg of a vector encoding an HPV antigen per milliliter of sodium citrate saline buffer and 0.25 mg of a vector encoding the p35 IL-12 subunit, the p40 IL-1 subunit, or both, per milliliter of buffer.

Embodiment 39. A pharmaceutical composition according to embodiment 38, wherein the composition comprises 6 mg pGX3024 per milliliter of sodium citrate buffered saline and 0.25 mg pGX6010 per milliliter of buffer.

Embodiment 40. A method for inducing an immune response in a subject, comprising administering to the subject an effective amount of a nucleic acid molecule according to any one of embodiments 1-9, an expression vector according to any one of embodiments 10-12, an immunogenic protein according to any one of embodiments 13-20, a vaccine according to embodiment 21 or embodiment 27, or a pharmaceutical composition according to any one of embodiments 22-26 or 28-39, thereby inducing an immune response.

Embodiment 41. A method for prophylactic or therapeutic immunization of a subject against HPV6 and/or HPV11, comprising administering to the subject an effective amount of a nucleic acid molecule according to any one of embodiments 1-9, an expression vector according to any one of embodiments 10-12, an immunogenic protein according to any one of embodiments 13-20, a vaccine according to embodiment

- 27 049710 of invention 21 or embodiment of invention 27 or a pharmaceutical composition according to any of embodiments of invention 22-26 or 28-39, thereby inducing an immune response against HPV6, HPV11 or both.

Embodiment 42. A method for treating or preventing recurrent respiratory papillomatosis (RRP) in a subject, comprising administering to the subject an effective amount of a nucleic acid molecule according to any one of embodiments 1-9, an expression vector according to any one of embodiments 10-12, an immunogenic protein according to any one of embodiments 13-20, a vaccine according to embodiment 21 or embodiment 27, or a pharmaceutical composition according to any one of embodiments 22-26 or 28-39, thereby treating or preventing RRP.

Embodiment of the invention 43. The method according to embodiment of the invention 42, wherein the RRP is juvenile RRP or adult RRP.

Embodiment 44. The method according to any of embodiments 40-43, wherein the nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 12.

Embodiment 45. The method according to any of embodiments 40-44, further comprising administering an adjuvant to the subject.

Embodiment 46. The method according to embodiment 45, wherein the adjuvant is interleukin-12 (IL12).

Embodiment 47. The method according to embodiment 46, wherein IL12 is encoded by a nucleic acid molecule.

Embodiment 48. The method according to embodiment 45, wherein the adjuvant comprises a nucleic acid molecule comprising a nucleotide sequence encoding the p35 subunit of IL-12, the p40 subunit of IL-12, or both.

Embodiment 49. The method according to embodiment 48, wherein the nucleotide sequence encoding the p35 subunit of IL-12 comprises a nucleotide sequence selected from the group consisting of a nucleotide sequence encoding SEQ ID NO: 6; or a nucleotide sequence that is at least 95% homologous to the nucleotide sequence encoding SEQ ID NO: 6.

Embodiment 50. The method according to embodiment 48 or 49, wherein the nucleotide sequence encoding the p40 subunit of IL-12 comprises a nucleotide sequence selected from the group consisting of a nucleotide sequence encoding SEQ ID NO: 8; or a nucleotide sequence that is at least 95% homologous to the nucleotide sequence encoding SEQ ID NO: 8.

Embodiment 51. The method according to embodiment 47, wherein the nucleic acid molecule encoding IL12 comprises a nucleotide sequence selected from the group consisting of the nucleotide sequence of SEQ ID NO: 4; or a nucleotide sequence that is at least 95% homologous to SEQ ID NO: 4.

Embodiment 52. The method according to embodiment 47, wherein the nucleic acid molecule encoding IL-12 is an expression vector, optionally a plasmid.

Embodiment 53. The method according to embodiment 52, wherein the plasmid is pGX6010.

Embodiment of the invention 54. The method according to any of the embodiments of the invention 40-53, wherein the subject is a human.

Embodiment 55. The method according to any of embodiments 40-54, wherein the administration comprises intradermal or intramuscular injection.

Embodiment of the invention 56. The method according to embodiment of the invention 55, wherein the administration additionally includes electroporation.

Embodiment of the invention 57. Use of an effective amount of a nucleic acid molecule according to any of embodiments of the invention 1-9, an expression vector according to any of embodiments of the invention 10-12, or an immunogenic protein according to any of embodiments of the invention 13-20 in the manufacture of a prophylactic or medicinal product.

Embodiment 58. Use of an effective amount of a nucleic acid molecule according to any of embodiments 1-9, an expression vector according to any of embodiments 10-12, or an immunogenic protein according to any of embodiments 13-20 in the manufacture of a prophylactic or medicinal product

- 28 049710 of a drug for the prevention or treatment of human papillomavirus (HPV) 6 or HPV11 infection.

Embodiment 59. Use of an effective amount of a nucleic acid molecule according to any of embodiments 1-9, an expression vector according to any of embodiments 10-12, an immunogenic protein according to any of embodiments 13-20, a vaccine according to embodiment 21 or embodiment 27, or a pharmaceutical composition according to any of embodiments 22-26 or 28-39 for the prevention or treatment of human papillomavirus (HPV) 6 or HPV11 infection.

Embodiment 60. Use of an effective amount of a nucleic acid molecule according to any of embodiments 1-9, an expression vector according to any of embodiments 10-12, an immunogenic protein according to any of embodiments 13-20, a vaccine according to embodiment 21 or embodiment 27, or a pharmaceutical composition according to any of embodiments 22-26 or 28-39 for the prevention or treatment of recurrent respiratory papillomatosis (RRP).

Embodiment of the invention 61. Use according to embodiment of the invention 60, wherein the RRP is juvenile RRP or adult RRP.

Embodiment of the invention 62. Use according to any of embodiments of the invention 57-61, wherein the nucleic acid molecule comprises the nucleotide sequence SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 12.

Embodiment of the invention 63. Use according to any of the embodiments of the invention 57-62 in combination with an adjuvant.

Embodiment of the invention 64. Use according to embodiment of the invention 63, wherein the adjuvant is interleukin-12 (IL-12).

Embodiment 65. Use according to embodiment 64, wherein IL-12 is encoded by a nucleic acid molecule.

Embodiment 66. Use according to embodiment 65, wherein the adjuvant comprises a nucleic acid molecule comprising a nucleotide sequence encoding the p35 subunit of IL-12, the p40 subunit of IL-12, or both.

Embodiment 67. Use according to embodiment 66, wherein the nucleotide sequence encoding the p35 IL-12 subunit comprises a nucleotide sequence selected from the group consisting of a nucleotide sequence encoding SEQ ID NO: 6; or a nucleotide sequence that is at least 95% homologous to the nucleotide sequence encoding SEQ ID NO: 6.

Embodiment 68. Use according to embodiment 66 or 67, wherein the nucleotide sequence encoding the p40 subunit of IL-12 comprises a nucleotide sequence selected from the group consisting of a nucleotide sequence encoding SEQ ID NO: 8; or a nucleotide sequence that is at least 95% homologous to the nucleotide sequence encoding SEQ ID NO: 8.

Embodiment 69. Use according to embodiment 65, wherein the nucleic acid molecule encoding IL-12 comprises a nucleotide sequence selected from the group consisting of the nucleotide sequence of SEQ ID NO: 4; or a nucleotide sequence that is at least 95% homologous to SEQ ID NO: 4.

Embodiment of the invention 70. Use according to embodiment of the invention 65, wherein the nucleic acid molecule encoding IL-12 is an expression vector, optionally a plasmid.

Embodiment of the invention 71. Use according to embodiment of the invention 70, wherein the plasmid is pGX6010.

The present invention is further illustrated in the following examples. It should be understood that these examples, although indicating embodiments of the invention, are provided for illustrative purposes only. From the above discussion and these examples, one skilled in the art can ascertain the important characteristics of the present invention and, without departing from the spirit and scope thereof, can make various changes and modifications of the present invention to adapt it to various needs and conditions. Thus, various modifications of the present invention in addition to those illustrated and described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are also within the scope of the appended claims.

Each of the U.S. patents, U.S. patent applications, and references cited in this disclosure are hereby incorporated by reference in their entirety.

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Examples

Generation of fusion immunogen DNA constructs based on HPV6 and HPV11 E6/E7 consensus sequences.

HPV6 E6/E7 and HPV11 E6/E7 consensus sequences were generated using sequences obtained from GenBank. Two point mutations were introduced into the HPV6 E6 and HPV11 E6 proteins to inhibit the ability to bind and degrade p53. One point mutation was introduced into the HPV6 E7 and HPV11 E7 protein to abolish p130 binding. The sequences were optimized using Inovio's proprietary gene optimization algorithm. Figure 1A shows a schematic of the antigens encoded in the various HPV6 and/or HPV11 plasmids. Individual antigens are separated by a P2A cleavage site for translational skipping and a furin cleavage site for post-translational cleavage of the P2A sequence. The DNA plasmid pGX3024 encodes the SynCon® consensus antigens E6 and E7 of both HPV6 and HPV11. Figure 1B shows a map of plasmid pGX3024.

In vitro transfection and Western blot analyses.

In vitro expression of pGX3024. pGX3024-mediated expression of HPV6 and HPV11 E6 and E7 antigens was confirmed by Western blot analysis after in vitro transfection of HEK-293T cells with pGX3024. Cells transfected with plasmids encoding HPV6 or HPV11 E6 and E7 antigens (pGX3021 and pGX3022, respectively) served as positive controls, and cells transfected with the empty plasmid backbone (pGX0001) served as negative controls.

The day before transfection, HEK-293T cells were seeded at 80% confluence in 6-well tissue culture dishes. The following day, cells were transfected according to the manufacturer's recommendations with 3 μg of plasmid DNA using Lipofectamine 3000 Transfection Reagent (Thermo Scientific). Forty-eight hours after transfection, cell lysates were collected and protein concentrations were determined using the BCA assay (Quick Start™ Bradford Protein Assay, BioRad). Thirty micrograms (30 μg) of cell lysates were loaded onto a 12% Bis-Tris acrylamide gel (Thermo Scientific) and transferred to a PVDF membrane. Precision Plus Protein™ Dual Xtra Prestained Protein Standards (BioRad, Cat. #1610377) were loaded as a molecular weight standard. After transfer, blots were washed in 1x PBS/0.05% Tween-20 and blocked for 1 h at room temperature in 1x PBS/0.05% Tween-20/5% Milk, then probed overnight with diluted HPV11 E7 antibody (Genetex) at room temperature overnight. The next day, blots were washed, then incubated with anti-mouse IgG with HRP (Bethyl Laboratories) 1:10,000 for 1 h at room temperature, then washed again. For detection, blots were incubated for 5 min with ECL Prime Western Blotting Substrate (GE Lifesciences/Amersham, cat. #RPN2232/89168-782). Blots were visualized on Protein Simple FluorChem® by chemiluminescence. After detection, blots were stripped by adding Restore reagent (Thermofisher, cat #21059) for 15 min, then washed and probed with anti-2A antibody (EMD Millipore) or anti-β-actin antibody (Santa Cruz Biotech) and developed as before.

As shown in Fig. 2, E6 and E7 proteins were detected in cells transfected with pGX3024 and the control plasmids pGX3021 and pGX3022, but not with the negative control plasmid pGX0001. E7 protein expression was detected using a commercially available anti-HPV11 E7 antibody (Fig. 2, left panel). E6 proteins were detected in cells transfected with pGX3024 but not with the negative control plasmid pGX0001. E7 antigen was detected in cells transfected with the HPV6 antigen-only plasmid (pGX3021) as well as the HPV11 antigen-only plasmid (pGX3022), indicating cross-reactivity of the HPV11 E7 antibody to both HPV6 and HPV11 E7 antigens and suggesting that the bands detected in pGX3024-transfected cells represent expression of both HPV6 and HPV11 E7 proteins.

After evaluation of several commercially available reagents, no antibody specifically recognizing HPV6 or HPV11 E6 proteins could be identified. However, using an anti-HPV11 E7 probe, two specific bands with predicted molecular masses of ~10.7 kDa and ~13 kDa were detected for E7 and E7-Furinl/P2A, respectively, indicating partial furin cleavage of the P2A sequence in this in vitro system (Fig. 2, left panel). Therefore, expression of E6 and E7 antigens could be detected using an anti-2A antibody probe against the partially cleaved protein products (Fig. 2, middle panel). A band representing ~20 kDa E6-Furin/P2A was detected in pGX3024 but not in pGX0001 control-transfected cells after probing with the anti-2A antibody. In addition, an E7-Furin/P2A band of ~15 kDa was detected in cells transfected with pGX3024 but not the control pGX0001, consistent with that detected using the anti-HPV11 E7 antibody probe.

Mouse IFN-γ ELISpot.

pGX3024 was evaluated for immunogenicity in two mouse models (Figs. 3–6). The cellular and humoral immune responses induced by the pGX3024 DNA vaccine were characterized in the C57BL/6 mouse model in two independent studies. Female C57BL/6 mice (n = 5 or 10 per group in

- 30 049710 study 1 or study 2, respectively) received two immunizations two weeks apart by intramuscular electroporation (IM-EP) with 20 μg of pGX3024 vaccine or control DNA vaccine. T cell responses were measured one week after the second immunization by IFN-γ ELISpot after stimulation of splenocytes with HPV6/HPV11 E6 or E7 peptides. After euthanasia, mouse spleens were isolated and placed in a tube containing 5 ml of R10 medium (RPMI 1640 supplemented with 10% fetal bovine serum, 1% penicillin-streptomycin, and 0.001% 2-mercaptoethanol). Splenocytes were isolated by mechanical disruption of the spleen using a GentleMACS Dissociator (Miltenyi Biotech) then filtered using a 40 μm cell strainer (BD Falcon). After centrifugation, the resuspended cell pellet was treated with ACK Lysis Buffer (Lonza) for 5 min to lyse red blood cells. Splenocytes were washed in PBS, centrifuged, resuspended in R10 medium and counted using Vi-cell (Beckman Coulter). To assess antigen-specific responses, IFN-γ ELISpot kits were purchased from MabTech (MabTech #3321-4APW-10). Precoated 96-well plates were washed in PBS according to the manufacturer's protocol and blocked with R10 medium for 2 h at room temperature. Isolated splenocytes were resuspended in R10 medium and seeded in triplicate at 2* ¹⁰⁵ cells per well. Overlapping 15-mer peptides for HPV6 E6 and E7 proteins and HPV11 E6 and E7 proteins were synthesized. These peptides were resuspended in DMSO (Sigma) and combined at a concentration of ~2 μg/ml per peptide into two peptide pools for each antigen (HPV6 E6, HPV6 E7, HPV11 E6, and HPV11 E7) for cell stimulation. Concavalin A (Sigma) at 5 μg/ml was used as a positive control and complete medium with DMSO was used as a negative control. Plates were incubated for a minimum of 18 h at 37°C with 5% CO2. For development, the plates were first washed in PBS and then incubated with biotinylated anti-mouse IFN-γ detection antibody (R4-6A2-biotin) for 2 h at room temperature. After washing, the plates were then incubated with CΊreptavidin-ALP (MabTech, #3321-4APW-10) for 1 h at room temperature. Spots were detected using filtered substrate solution (BCIP/NBT-plus) according to the manufacturer's instructions (MabTech). After drying the plates, spots were counted using an automated ELISpot reader (Cellular Technology). The mean spot formation unit (MSU) was corrected to 1* ¹⁰⁶ splenocytes, and antigen-specific responses were presented as the number of IFN-γ MSU per 1*106 splenocytes in excess of the DMSO control.

HPV6- and HPV11-specific T cell responses were detected in mice following immunization with pGX3024 but not the negative control pGX0001 (Fig. 3). No significant differences in T cell responses were observed in mice immunized with pGX3024 compared with mice immunized with pGX3021 and/or pGX3022 as determined by one-way ANOVA, Tukey's post hoc test (Fig. 3). Immunization with pGX3024 induced T cell responses against HPV6 E7, HPV11 E6, and HPV11 E7 antigens, but not against HPV6 E6 antigen in this model. Inbred C57BL/6 mice have a single MHC haplotype (H2b), which may explain the lack of cellular responses to HPV6 E6 in this model. T cell responses were highly reproducible in two independent studies.

Antibodies against HPV6 and HPV11 E7 antigens were measured by IgG binding ELISA in serum samples collected before immunization (week 0) and after the first (week 2) and second (week 3) immunizations with either pGX3024 or the negative control plasmid (pGX0001).

IgG antigen binding ELISA

Nunc™ high-binding 96-well plates (Thermo Scientific) were coated with 0.5 μg/ml of each protein (HPV6 E7 and HPV11 E7 - Tulip BioLabs) in 1x PBS (Thermo Scientific) overnight at 4°C. The next day, the plates were washed with 1x PBS + 0.05% Tween-20 and blocked with 3% BSA in PBS + Tween-20 for 2 h at 37°C. The plates were then washed as before, serially diluted serum samples were added, and the plates were incubated for 2 h at 37°C. The plates were washed and incubated with a 1:10,000 dilution of anti-mouse or anti-rabbit IgG secondary antibody with HRP (Bethyl Laboratories, Inc) for 1 h at room temperature. The plates were washed and 100 µl/well SureBlue TMP Substrate (KPL 5120-0077) was added. The reaction was stopped by adding 100 µl/well TMB Stop Solution (KPL 5150-0021) after 6 min of incubation and the plates were read on a Biotek Synergy plate reader at 450 nm.

HPV6 E7-binding antibodies were detected in mice immunized with pGX3024, but not pGX0001, after the first and second immunizations (Fig. 4, left panel). Overall, HPV11 E7-binding antibodies were reduced compared with HPV6 E7-binding antibodies in immunized mice; however, HPV11 E7-binding antibodies were higher after two immunizations (week 3) in mice immunized with pGX3024 compared with pGX0001 (Fig. 4, right panel).

Immunogenicity of pGX3024 in BALB/c mice. As mentioned previously, C57BL/6 mice have a single MHC haplotype (H2b), which may explain the lack of cellular response to HPV6 E6 in this model.

- 31 049710

Therefore, the cellular responses induced by the pGX3024 vaccine were investigated in a BALB/c mouse model with a different MHC haplotype (H2d). The effect of pGX3024 dose and combination with a plasmid encoding murine IL-12 adjuvant (pGX6012) was also investigated. Female BALB/c mice (n = 6 per group) received two immunizations two weeks apart by intramuscular electroporation (IM-EP) delivering 1 μg, 5 μg, 10 μg, or 20 μg of pGX3024 DNA vaccine alone or 5 μg of pGX3024 adjuvanted with 2 μg or 11 μg of pGX6012 murine IL-12 plasmid. Mice immunized with 40 μg of empty pGX0001 served as negative controls. T cell responses were measured one week after the second immunization by IFN-γ ELISpot after stimulation of splenocytes with HPV6/HPV11 E6 or E7 peptides as shown in Fig. 5. HPV6- and HPV11-specific T cell responses were detected in mice after immunization with all dose levels of pGX3024, but not pGX0001, in a dose-dependent manner. Total cellular responses to HPV6 and HPV11 increased with increasing pGX3024 dose. In contrast to the C57BL/6 model, pGX3024-immunized BALB/c mice elicited T cell responses against HPV6 E6 antigen, as well as HPV6 E7, HPV11 E6, and HPV11 E7 antigens. No significant differences in T cell responses were observed in mice immunized with 5 μg pGX3024 with or without any dose level of pGX6012, as determined by one-way ANOVA, Tukey's post hoc test.

Antibodies against HPV6 and HPV11 E7 antigens were measured by IgG binding ELISA in serum samples collected before immunization (week 0) and after the first (week 2) and second (week 3) immunizations with either 5 μg, 10 μg, or 20 μg of pGX3024 DNA vaccine alone or 5 μg of pGX3024 adjuvanted with 2 μg of murine IL-12 plasmid (pGX6012). Sera from mice immunized with 40 μg of empty pGX0001 plasmid served as negative controls. Levels of antibodies binding to HPV6 E7 and HPV11 E7 were significantly increased at week 3 compared with week 0 in BALB/c mice immunized with pGX3024, regardless of dose or the presence of IL-12 plasmid, but not in pGX0001-treated mice (Fig. 6). Antibody levels were also significantly increased following a single immunization (week 2) with 20 μg pGX3024 or 5 μg pGX3024 adjuvanted with 2 μg murine IL-12 plasmid. A trend toward increased antibody binding against both antigens was observed with the addition of murine IL-12 to 5 μg pGX3024, particularly at the week 2 time point.

Immunogenicity study in rabbits.

INO-3107 (pGX3024 with pGX6010) was evaluated for immunogenicity and safety in a rabbit model.

New Zealand White (NZW) rabbits (n = 5 per group) received four immunizations at three-week intervals with INO-3107 (6 mg pGX3024 and 0.25 mg pGX6010 co-formulated in 1 ml 1x SSC) or 1 ml 1x SSC (negative control) by intramuscular (IM) injection into the quadriceps muscle followed by electroporation (EP) using a CELLECTRA® 2000 Electroporation Device. Cellular and humoral immune responses were assessed by IFN-γ ELISpot and IgG binding ELISA, respectively, prior to immunization (week 0) and two weeks after each immunization (weeks 2, 5, 8 and 11). Physiological parameters including body weights, hematology, serum chemistry, and general appearance were monitored throughout the study as indicators of vaccine safety and animal health.

Cellular responses in rabbits were assessed using an IFN-γ ELISpot assay following stimulation with PBMCs with HPV6/HPV11 E6 and E7 peptides. SepMate 15 mL tubes were filled with 3.5 mL Ficoll-Paque gradient and allowed to equilibrate at room temperature. Whole blood was collected into K2EDTA tubes and inverted several times to mix. Blood was diluted with Hanks' balanced salt solution (HBSS) and slowly layered on top of the Ficoll-Paque gradient in SepMate tubes. The tubes were centrifuged, and the buffy coats were collected and placed in a fresh 15 mL tube. Cells were washed by diluting with R10 medium (RPMI 1640 supplemented with 10% fetal bovine serum, 1% penicillin-streptomycin, and 0.001% 2-mercaptoethanol). The cell pellet was resuspended in ACK Lysis Buffer (Lonza) for red blood cell lysis and incubated at room temperature for 4 min. PBMCs were washed, centrifuged, and resuspended in R10 medium and counted using Vi-cell (Beckman Coulter). Rabbit IFN-γ ELISpot kits (MabTech (MabTech, #3110-4HPW-10)) were used to assess antigen-specific responses. Plates were prepared according to the manufacturer's protocol and rabbit PBMCs were seeded in triplicate at 2 ^χ 10 ⁵ cells per well. Overlapping 15-mer peptides for the E6 and E7 proteins from HPV6 and HPV11 were used to stimulate cells. Phorbol 12-myristate 13-acetate and ionomycin (PMA/I) (Sigma) and DMSO-containing medium served as positive and negative controls, respectively. Plates were incubated for a minimum of 18 h at 37°C with 5% CO2. The following day, plates were developed according to the manufacturer's protocol and, after the plates had dried, spots were counted using an automated ELISpot reader (Cellular Technology). The mean spot forming unit (MSU) was corrected to 1 ^χ 10 ⁶ splenocytes, and antigen-specific responses were expressed as the number of IFN-γ MSU per 1 ^χ 10 ⁶ splenocytes in excess of the DMSO control.

HPV6- and HPV11-specific T-cell responses above baseline were detected in all

- 32,049,710 rabbits after immunization with INO-3107, but not in rabbits treated with 1x SSC. T cell responses to HPV6 and HPV11 were enhanced, as they increased after each subsequent immunization (Fig. 7). INO-3107 immunization induced T cell responses against HPV6 and HPV11 E6 antigens, but not against HPV6 or HPV11 E7 antigens in this model (Fig. 8).

Humoral responses against HPV6 and HPV11 E7 antigens were measured by IgG binding ELISA in serum samples collected before and two weeks after each immunization. The time course of antibody levels is shown in Fig. 9. HPV6 or HPV11 E7 binding antibodies were detected in 4 of 5 rabbits immunized with INO-3107 but not in rabbits treated with 1x SSC. Overall, HPV6 E7 binding antibodies were reduced compared to HPV11 E7 binding antibodies in immunized rabbits. Together, the ELISpot and ELISA data support the immunogenicity of all INO-3107-encoded antigens in the rabbit model.

In addition to INO-3107-mediated immune responses, physiological parameters including body weights, hematology, serum chemistry, and general appearance were monitored throughout the study as indicators of vaccine safety and animal health. No significant differences in body weights or changes in body weight over time were observed in INO-3107-treated rabbits (Fig. 10). No changes were observed in monitoring general appearance (nose, eyes, coat, movement) in any treatment group during the study. Serum hematology and clinical chemistry samples were collected prior to immunization and at week 5, week 8, and week 11 post-immunization, and results were submitted to an independent veterinary clinical pathologist (IDEXX BioAnalytics). Compared with baseline values, INO-3107 administration resulted in a modest increase in lymphocyte counts at week 5 (but not at week 8 or week 11) in NZW rabbits. No other hematological or clinical chemistry parameters in serum were observed following INO-3107 administration that would indicate a biologically significant effect.

Evaluation of intradermal (ID) delivery in the rabbit.

To evaluate cellular immune responses, an intradermal (ID) injection study was performed in NZW rabbits. NZW rabbits (n = 5) were immunized three times at three-week intervals with INO-3107 formulated with 1 mg pGX3024 in 0.1 ml 1x SSC via ID delivery. Rabbit IFN-γ ELISpots, as described previously, were performed prior to the first vaccination and at weeks 2, 5, and 8. The combined immune response to both HPV6 and HPV11 antigens increased after each immunization, with T cell responses being more specific for HPV6 E6 and HPV11 E6 (Fig. 13).

Evaluation of intradermal (ID) delivery in a guinea pig model.

Hartley guinea pigs (n = 5) received three immunizations with pGX3024 (0.1 mg formulated in 0.1 ml 1x SSC) by intradermal (ID) injection followed by electroporation (EP) using a CELLECTRA® 2000 Electroporation Device at two-week intervals. Untreated guinea pigs served as negative controls (n = 2). Cellular and humoral immune responses were assessed by IFN-γ ELISpot and IgG binding ELISA, respectively, prior to immunization (week 0) and two weeks after each immunization (weeks 2, 4 and 6).

Animal housing, immunization, and sample collection were performed at Acculab Life Sciences (San Diego, CA) under an IACUC-approved protocol in compliance with the Animal Welfare Act, PHS policy, AAALACi guidelines, USDA, and other federal laws and regulations pertaining to animals and animal experiments. All sample analyses were performed at Inovio (San Diego, CA). pGX3024 was formulated in 1x SSC for a final amount of 0.1 mg in 0.1 mL dosing solution and stored at 2-8°C until use. Female Hartley guinea pigs, 8 weeks of age, were randomized into 2 groups of 5 or 2 animals each according to the table. Each treatment procedure was performed by intradermal (ID) injection of 100 μl of Mantoux dosing solution into the skin followed by electroporation using a CELLECTRA 2000® Adaptive Constant Current Electroporation device with a 3P grid (Inovio Pharmaceuticals) according to the manufacturer's protocol.

Research plan

Group number (n) Plasmid Number of injection sites ^/location/ Tx Device for EP and injection method Injection volume (µl) DNA/plasmid dose (µg) 1 (5) pGX3024 1/ID CELLECTRA 3P 100 100 2 (2) Previously untreated - - - -

Animals in Group 1 received a total of three immunizations at two-week intervals.

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Serum samples were collected from all animals for humoral immunogenicity assessments at week 0, week 2, week 4, and week 6. Whole blood samples were collected from all animals for cellular immunogenicity assessments at week 2, week 4, and week 6.

Guinea pig IFN-γ ELISpot. Guinea pig IFN-γ ELISpot was performed according to the methods described in Schultheis, et al., J Vis Exp. 2019;(143):10.3791/58595, published January 20, 2019, doi:10.3791/58595. Peripheral blood was collected from the jugular vein of each anesthetized animal and immediately transferred to EDTA blood collection tubes. Blood was diluted 1:1 with phosphate-buffered saline. Diluted blood was layered on FicollPaque Plus (GE Healthcare Life Sciences) in SepMate™ tubes (Stemcell) and centrifuged (1200 g, 10 minutes, 24°C). PBMCs were resuspended in R10 medium at a P10 of ⁶ cells/ml and plated at 100 μl/well in 96-well Millipore IP plates (Millipore) precoated with 5 μg/ml of the anti-IFN-γ primary antibody V-E4 (a gift from Dr. Schafer, Robert Koch Institute, Berlin, Germany) blocked with R10 medium. Cells were supplemented with 100 μl of HPV6 E6, HPV6 E7, HPV11 E6, or HPV11 E7 peptide pools or phorbol 12-myristate 13-acetate (PMA)/lonomycin stimulants. Samples were analyzed in triplicate. After incubation in humidified 5% CO2 at 37°C for 18 h, cells were removed by washing and 100 μl/well of 2 μg/ml biotinylated anti-IFN-γ secondary antibody N-G3 diluted in blocking buffer were added. After 2 h incubation and washing, alkaline phosphatase-conjugated streptavidin (MabTech) was added at 100 μl/well for 1 h at room temperature. After washes, wells were incubated for 6-12 min at room temperature with 100 μl/well of nitroblue tetrazolium/5-bromo-4-chloro-3'-indolyphosphate (BHIF/HCT) detection reagent substrate (MabTech). Positive interferon-gamma spots were visualized, analyzed and counted using a CTL-Immunospot® S6 ELISPOT plate reader and CTL-Immunospot® software. Antigen-specific responses were determined by subtracting the number of spots in DMSO-treated wells from those treated with peptides.

Results are shown as spot forming units (SFU)/ ¹⁰⁶ PBMCs of individual animals obtained from triplicate wells. HPV6- and HPV11-specific T cell responses above baseline were detected in guinea pigs immunized with pGX3024 but not in naive guinea pigs (Fig. 11). Immunization with pGX3024 induced T cell responses against HPV6 and HPV11 E6 and E7 antigens in this model (Fig. 11).

Antigen binding ELISA. Nunc™ high-binding 96-well plates (Thermo Scientific) were coated with 1 μg/ml recombinant HPV6 E7 or HPV11 E7 proteins in 1x Dulbecco's phosphate-buffered saline (PBS) (Thermo Scientific) overnight at 4°C. The following day, the plates were washed with 1x PBS + 0.05% Tween®-20 and blocked with 3% BSA in PBS + 0.05% Tween®-20 for 2 h at room temperature. The plates were then washed as before, serially diluted serum samples were added and the plates were incubated for 2 h at room temperature. Plates were washed and incubated with horseradish peroxidase (HRP)-conjugated anti-guinea pig IgG secondary antibody (Sigma) at a dilution of 1:10,000 for 1 h at room temperature. Plates were washed and 100 µl/well SureBlue™ TMB Substrate (KPL 5120-0077) was added. The reaction was stopped by adding 100 µl/well TMB Stop Solution (KPL 5150-0021) after 6 incubations and plates were read on a Biotek Synergy plate reader at 450 nm.

Humoral responses against HPV6 E7 and HPV11 E7 antigens were measured by IgG binding ELISA in serum samples collected before immunization and two weeks after each immunization. The time course of antibody levels is shown in Fig. 12. HPV6 E7- and HPV11 E7-binding antibodies were detected in guinea pigs after immunization with pGX3024.

Phase 1/2 clinical trial of INO-3107 with electroporation (EP) in subjects with HPV6- and/or HPV11-associated recurrent respiratory papillomatosis (RRP) [ClinicalTrials.gov identifier: NCT04398433]

This is a phase 1/2, open-label, multicenter trial to evaluate the safety, tolerability, immunogenicity, and efficacy of INO-3107 in subjects with HPV6- and/or HPV11-associated recurrent respiratory papillomatosis (RRP). INO-3107 will be administered IM followed by EP to subjects on day 0, weeks 3, 6, and 9.

The study will include approximately 20 adults (>18 years) diagnosed with either juvenile-onset RAD (J-O RAD), defined by age at first diagnosis <12 years, or adult-onset RAD (A-O RAD), defined by age at first diagnosis >12 years. The study population is divided into two cohorts. Cohort A: participants diagnosed with juvenile-onset RAD, defined by age at first RAD diagnosis <12 years. Cohort B: participants diagnosed with adult-onset RAD, defined by age at first RAD diagnosis >12 years.

This study will have a safety run-in period of up to six subjects with a one-week waiting period between each participant enrolled. Safety and tolerability will be assessed throughout the study once

- 34 049710 tolerability. Tolerability will be determined by the incidence of dose-limiting toxicity (DLT), which is defined as:

treatment-related nonhematologic toxicity > grade 3 according to NCI Common Terminology Criteria for Adverse Events (CTCAE, version 5.0) that does not respond to supportive care and lasts for more than 48 hours, or treatment-related hematologic toxicity > grade 3 according to NCI CTCAE v5.0 that does not respond to supportive care and lasts for more than 48 hours.

Subjects will undergo routine surgical removal of papilloma(s) during the screening period for 14 days prior to the Day 0 dose (papilloma removal and Day 0 dose may be performed on the same day if other eligibility criteria are met). Biopsy tissue will be collected and evaluated for secondary and exploratory endpoints. Disease status will be monitored throughout the trial.

Cohort A (participants with juvenile RRP) will receive a single intramuscular (IM) injection of 6.25 mg INO-3107 drug product followed by electroporation (EP) using CELLECTRA® 2000 on Day 0, Week 3, Week 6, and Week 9. Cohort B (participants with adult RRP) will receive a single intramuscular (IM) injection of 6.25 mg INO-3107 drug product followed by EP using CELLECTRA® 2000 on Day 0, Week 3, Week 6, and Week 9.

The primary objective of the study is to evaluate the safety and tolerability of INO-3107 in subjects with HPV6 and/or HPV11-associated RRP. The primary endpoint is safety and tolerability, assessed by reporting adverse events (AEs) and serious adverse events (SAEs). The primary outcome measure is the percentage of subjects with adverse events (AEs) and serious adverse events (SAEs) [time frame: screening up to week 52 (up to approximately 1 year)]. An adverse event (AE) is any undesirable medical occurrence in a subject or clinical trial participant who is administered a pharmaceutical product and that does not necessarily have a causal relationship with the treatment. An AE may include any unfavorable or undesirable sign (including abnormal laboratory results), symptom, or disease temporally associated with the use of a medicinal (investigational) product, whether or not related to the medicinal (investigational) product. A serious adverse event (SAE) is any undesirable medical occurrence that, at any dose: 1. results in death; 2. is a life-threatening event; but does not include an event that, if it occurred in a more severe form, could result in death; 3. requires hospitalization or prolongation of current hospitalization; 4. results in permanent or significant disability/incapacity; 5. results in a congenital anomaly or developmental defect.

The secondary objectives of the study are:

1) to evaluate the efficacy of the medicinal product INO-3107, as determined by the incidence of surgical interventions for RRP during the year after the first dose of the study product compared with the incidence during the year prior to day 0;

2) evaluation of the efficacy of the drug INO-3107 based on changes in the staging assessment of RRP over time;

3) evaluation of the cellular immune response to the medicinal product INO-3107 when administered IM followed by EP;

4) evaluation of the immunogenicity of the medicinal product INO-3107 based on proinflammatory and immunosuppressive elements in the resected tumor tissue at the start of the study and, if present, during subsequent tissue resections;

5) assess any potential association of microRNA (miRNA) profile with reduced incidence of surgical intervention for RRP.

Secondary endpoints are:

1) the number of surgeries for RRP in the 52 weeks after day 0 compared with the number of surgeries for RRP in the year before the day 0 dose [time frame: screening before week 52 (up to approximately 1 year);

2) change in RRP staging score over time [time frame: screening, day 0, weeks 6, 11, 26, 52 (up to approximately 1 year)]; the RRP staging score will be determined using a modified Derkay staging tool; it includes both a subjective functional assessment of clinical parameters and an anatomical assessment of disease extent; the anatomical assessment can then be used in combination with the functional assessment to measure the clinical course and response to therapy of an individual patient over time;

3) change from baseline in the magnitude of response in the IFN-γ enzyme-linked immunosorbent assay (ELISpot IFN-γ) for IFN-γ-secreting cells in peripheral blood mononuclear cells (PBMCs) [time frame: baseline, weeks

- 35 049710

6, 9, 11, 26, 52];

4) change from baseline in flow cytometry response magnitude for T cell phenotype and lytic potential in PBMCs [time frame: baseline, weeks 6, 9, 11, 26, 52];

5) change from baseline in the magnitude of the response of resected tumor tissue to proinflammatory and immunosuppressive elements [time frame: baseline and subsequent tissue resections, up to week 52 (up to approximately 1 year)];

6) change from baseline in microRNA (miRNA) expression levels associated with a reduction in the incidence of surgery for RRP [time frame: baseline and week 6].

The research objectives of the trial are:

1) description of virological clearance of HPV6 and/or 11 in resected tissue, if available;

2) evaluation of the humoral immune response to the medicinal product INO-3107 when administered IM followed by EP;

3) evaluation of the immunogenicity of the medicinal product INO-3107 based on proinflammatory and immunosuppressive elements in peripheral blood;

4) to evaluate circulating free HPV DNA (csDNA HPV) 6/11 as a correlate of disease burden and clinical outcomes in patients with RRP treated with INO-3107.

The exploratory endpoints are:

1) elimination of HPV6/11 in resected tissue compared to baseline;

2) antigen-specific humoral immune responses assessed using ELISA;

3) assessment of proinflammatory and immunosuppressive elements in peripheral blood and

4) the amount of HPV 6/11 ctDNA before and after administration of the drug product INO-3107 as a correlate of disease burden and clinical outcomes in patients with RRP treated with the drug product INO-3107.

Efficacy assessment. A detailed history will be obtained for each subject, including documentation of HPV-6 and/or HPV-11-associated RRP, a list of surgeries and therapies for RRP received within 3 years prior to screening, and any periods of remission. To be eligible for the study, subjects must have had at least two surgeries (including laser) for RRP in the year prior to Day 0. Subjects must be in need of surgery for RRP at study entry and have had surgical removal of their papilloma(s) at screening within 14 days prior to the Day 0 dose to maximize standardization of baseline staging across subjects. Efficacy assessment will be based on the number of surgeries for RRP within 52 weeks after Day 0 compared with the number of surgeries for RRP in the year prior to the Day 0 dose. Surgeries for RRP include laser therapy. As a secondary endpoint, the trial will also assess changes in PPR staging over time.

Safety assessment. Subjects will be monitored from the time of informed consent until week 52 or the last subject visit. The safety of the INO-3107 medicinal product will be measured and assessed in accordance with CTCAE v5.0. Clinically significant changes in laboratory parameters and vital signs compared to baseline assessments will be assessed.

An adverse event is any undesirable medical occurrence in a patient or clinical trial subject who is administered a pharmaceutical product and which does not necessarily have a causal relationship with the treatment. Therefore, an AE may be any unfavourable and undesirable sign (including, for example, an abnormal laboratory result), symptom or disease temporally associated with the use of a medicinal product, whether or not considered related to the medicinal product. Adverse events (AEs) include the following: complications before or after treatment arising from a protocol procedure during or after screening (before administration of the clinical trial medicinal product); any pre-existing condition, other than the condition being investigated in the study, that increases in severity or changes in nature during or as a result of the medicinal product administration phase of the clinical trial; complications of pregnancy. Adverse events (AEs) do not include the following: medical or surgical procedures performed (e.g., surgery, endoscopy, tooth extraction, blood transfusion), but the condition that led to the procedure is an AE; pre-existing diseases or conditions or laboratory abnormalities present or identified before the screening visit that have not worsened; recurrence of RRP; situations in which an adverse medical event has not occurred (e.g., hospitalization for elective surgery, community hospitalization and/or

- 36 049710 hospitalization for convenience); overdose without clinical consequences; any medical condition or clinically significant laboratory abnormality with an onset date before informed consent is not an AE; uncomplicated pregnancy; induced elective abortion to terminate pregnancy without medical indications.

Adverse drug reactions (ADRs) include all harmful and undesirable responses to a medicinal product, associated with any dose. This means that a causal relationship between the medicinal product and the adverse event is at least a reasonable possibility (i.e., an association cannot be excluded).

A serious adverse event (SAE) is any undesirable medical event that, at any dose, results in death; is life-threatening; requires hospitalization or prolongation of current hospitalization; results in permanent or significant disability/incapacity; results in a congenital anomaly or developmental defect and/or is a major medical event.

An unexpected adverse drug reaction is an adverse reaction the nature or severity of which is not consistent with the information about the product being used. An unexpected (severe) adverse device effect (UADE) is any serious adverse effect on health or safety, or any life-threatening problem or death, caused by or associated with a device, if the effect, problem or death was not previously defined by nature, severity or incidence in the study plan or application (including supplemental plans or applications), or any other unexpected serious problem associated with a device that relates to the rights, safety or welfare of subjects.

Immunogenicity assessment. The study will evaluate humoral and cell-mediated immune responses in blood samples collected at the baseline visit (i.e., Screening and Day 0 pre-dose) and at Weeks 6, 9, 11, 26, and 52. Tissue samples will be collected at baseline and as clinically indicated during the study. Testing may include, but is not limited to, ELISA, ELISpot, flow cytometry, immunohistochemistry (IHC), Nanostring on peripheral blood samples and/or resected tissue (baseline and relapse, if any).

miRNA profiling can be performed using tissue obtained at screening, day 0, and relapse. Evaluation of day 0 and screening samples will allow the study of algorithms for predicting response to treatment with INO-3107. Samples evaluated at relapse will allow the characterization of how changes in miRNA profiles may be associated with the likelihood of relapse.

Virologic assessment: The trial will assess the presence of HPV6/11 DNA in tissue and peripheral blood samples before and after study treatment as described.

Main inclusion criteria.

Histologically confirmed HPV6- or HPV11-positive respiratory papilloma or confirmation of low-risk HPV positivity using a sponsor-approved HPV6/11 type-specific assay.

Requirement for frequent intervention for RRP to remove or resect respiratory papilloma, defined as at least 2 surgical (including laser) interventions for RRP within the year up to and including day 0.

Must be an appropriate candidate for the upcoming surgical intervention in the opinion of the investigator and according to the RRP staging scale.

Adequate bone marrow, liver, and kidney function as defined by absolute neutrophil count (ANC) > 1,000 cells/ ^mm3 , platelets > 50,000/ ^mm3 , hemoglobin > 9 g/dL; serum total bilirubin concentrations within 1.5 x upper limit of normal (ULN), AST and ALT within 1.5 x ULN, and serum creatinine < 1.5 x ULN.

Participants must meet one of the following requirements: not be of childbearing potential (> 12 months of uninduced amenorrhea confirmed by follicle-stimulating hormone [FSH] unless on hormone replacement therapy);

be surgically sterile (vasectomy in men or absence of ovaries and/or uterus in women);

agree to use one highly effective or combined method of contraception with a failure rate of < 1% per year during the treatment period and until at least week 12 after the last dose; or agree to abstain from sexual intercourse.

Main exclusion criteria.

Recipient of therapy aimed at treating RRP disease (other than surgery or ablation), including but not limited to antiviral drugs (including cidofovir), radiation, chemotherapy, antiangiogenic therapy (including bevacizumab),

- 37 049710 prophylactic HPV vaccination (including Gardasil) as a therapeutic intervention or treatment with an experimental agent within 3 months prior to day 0.

Current or recent (within 1 year) evidence of an autoimmune disease that has required treatment with systemic immunosuppressive agents, with the exception of vitiligo, childhood asthma that has resolved, type 1 diabetes, residual hypothyroidism that requires only hormone replacement therapy, or psoriasis that does not require systemic treatment.

Diagnosis of immunodeficiency or treatment with systemic immunosuppressive therapy within 28 days prior to the first dose of study treatment, including systemic corticosteroids.

High risk of bleeding or need for anticoagulants to control known bleeding diathesis.

Recipient of any live virus-containing vaccine within 4 weeks prior to the first dose of study treatment or any non-live virus-containing vaccine within two weeks prior to the first dose of study treatment.

History of clinically significant, medically unstable illness that, in the opinion of the investigator, may compromise the safety of the participant, interfere with trial assessment or endpoint evaluation, or otherwise affect the validity of the trial results (this may include chronic renal failure; myocardial ischemia or infarction; New York Heart Association (NYHA) class III/IV heart disease); any cardiac pre-excitation syndromes (such as Wolff-Parkinson-White syndrome; cardiomyopathy or clinically significant arrhythmias); current malignancy other than treated basal and squamous cell skin cancer, prostate cancer, or cervical carcinoma in situ; HIV that may interfere with the ability to mount an immune response to study therapy; drug or alcohol dependence).

Less than two acceptable sites for IM injection, including the deltoid and anterolateral quadriceps muscles [unacceptable sites include: tattoos, keloids, or hypertrophic scars within 2 cm of the intended treatment site; a cardioverter-defibrillator or pacemaker (to prevent life-threatening arrhythmia) located ipsilateral to the deltoid injection site (unless deemed acceptable by a cardiologist); metallic implants or an implantable medical device at the intended treatment site].

Pregnancy or current breastfeeding.

Treatment in a clinical trial. The drug product INO-3107 is the investigational product to be used in this study. The drug product INO-3107 contains a DNA plasmid for the expression of the E6 and E7 proteins of the HPV11 and HPV6 genes (pGX3024) and an expression plasmid expressing the subunits of human IL-12 (pGX6010). The drug product INO-3107 is a clear, colorless solution containing 6.25 mg total plasmid/mL (6 mg/mL pGX3024, 0.25 mg/mL pGX6010) in 150 mM sodium chloride and 15 mM sodium citrate, pH 7. A minimum volume of 1 mL will be filled into 2-mL clear glass vials for intramuscular injection.

Subjects will receive a single injection of 6.25 mg of INO-3107 drug product intramuscularly followed by EN using the CELLECTRA® 2000 EN device on Day 0, Week 3, Week 6, and Week 9.

The populations to be analyzed will be the following: the intent-to-treat (IT) population includes all subjects who meet the inclusion criteria;

the modified intent-to-treat (mITT) population includes all subjects receiving at least one dose of the INO-3107 medicinal product;

The per-protocol (PP) population includes subjects receiving all doses of INO-3107 and having no protocol violations; subjects excluded from the PP population will be identified and documented prior to trial database lock;

The safety analysis set includes all subjects receiving at least one dose of the INO-3107 drug product.

Peripheral Blood Immunogenicity Assessments. Whole blood and serum samples will be obtained at the Baseline visit (Screening and Day 0 pre-dose) and at Weeks 6, 9, 11, 26, and 52. Peripheral blood mononuclear cells (PBMCs) will be isolated from whole blood samples. Cellular immune activity may be assessed using gene expression assays, IFN-γ enzyme-linked immunosorbent assay (ELISpot IFN-γ), and flow cytometry. Cellular immune activity may be further assessed using flow cytometry to perform a lytic granule loading assay. The following extrinsic cellular markers can be examined in the lytic granule loading assay: CD3, CD4, CD8 (T cell identification), Ki67, CD137, CD38 and CD69 (T cell activation markers), as well as PD-1 (exhaustion/activation marker), Tim-3 and Lag-3. The lytic granule loading assay can

- 38 049710 additionally analyze the following intracellular markers: granzyme A, granzyme B, granulysin and perforin (proteins involved in lytic degranulation and cytotoxic potential).

miRNA profiling will be performed using plasma collected at Screening, Day 0, and Week 6. Evaluation of Day 0 and Screening samples will enable the study of algorithms for predicting treatment response to INO-3107. Samples evaluated at Week 6 will enable the characterization of how changes in miRNA profiles may be associated with ultimate treatment success or failure.

A standard binding ELISA can be performed to measure the anti-HPV6/11 antibody response induced by the INO-3107 drug product.

HPV6/11 testing: Whole blood will be collected pre-dose on day 0 and at weeks 6, 11, 26, and 52 to measure HPV6/11 ctDNA.

Description of statistical methods.

Primary analyses: The primary analyses for this trial are the safety analysis, including analysis of treatment-emergent adverse events (TEAEs) and clinically important changes from baseline in laboratory safety parameters. In this trial, TEAEs are defined as any AE occurring from day 0 post-dose of study drug (IM+EP) through 30 days post-last dose. All TEAEs will be summarized by frequency in the safety analysis population. These frequencies will be presented overall, by system organ class, and preferably by percentage of subjects affected. Additional frequencies will be presented taking into account the greatest severity and the closest relationship to study treatment. Multiple occurrences of the same AE will be counted only once, in accordance with a worst-case approach for severity and relationship to study treatment. The main safety summary will be based on TEAEs. For this summary, Preferred Terminology event rates will be calculated along with 95% confidence intervals using the Clopper-Pearson exact method. Individual summaries will be based on events occurring within 7 days of any dose and regardless of when they occurred. AEs and SAEs that are not TEAEs or serious TEAEs will be are presented in the form of lists.

For AE data, partial start dates will be referenced to the treatment start date to conservatively represent the event as treatment-induced if the partial date corresponds to the study treatment start date. Otherwise, it will be referenced to the earliest date corresponding to the partial date. A completely missing start date will be considered the day of treatment start. Partial stop dates will be taken as the latest possible day corresponding to the partial date.

The duration of the adverse event will be calculated as (cessation date - start date) + 1.

Laboratory response variables will be summarized descriptively by time point and as changes from baseline, including 95% confidence intervals. Shifts from baseline according to CTCAE will also be presented. Laboratory values considered clinically significant will be presented as lists.

All safety analyses will be conducted in subjects in the safety analysis set.

The analyses will be summarized and presented by the number of previous surgeries (< 2, 3-5 and > 6) and overall.

Secondary analyses.

Efficacy. The incidence of surgery for RRP in the year following the first dose of INO-3107 compared with the incidence in the year prior to the day 0 dose will be descriptively summarized using mean fold change and 95% CI based on the t-distribution. Changes in RRP staging scores from baseline before dosing to each post-dose assessment will be analyzed. Median changes and corresponding 95% CIs will be calculated.

The relationship between the efficacy endpoint and siRNA results will be examined. The relationship will be examined using regression models that model the efficacy endpoint versus siRNA results as covariates.

Intervals between surgeries will also be summarized. Analyses will be summarized and presented by number of prior surgeries (<2, 3-5, and >6) and overall. Efficacy analyses will be performed using the mNL population. The per-protocol population will also be used for secondary analyses.

Immunogenicity. Increases from baseline in interferon-γ ELISpot and flow cytometry response values will be summarized. Median increases and corresponding 95% confidence intervals will be calculated. Changes from baseline in tumor tissue response values will be summarized. Mean increases and corresponding 95% confidence intervals based on the t-distribution will be calculated. Valid samples for statistical analysis will be those collected in

- 39 049710 for 7 days after the specified visit. Baseline is defined as the last measurement before the first administration of treatment. The mNL population will be used for immunogenicity analysis. Analyses will be summarized and presented by number of prior surgeries (< 2, 3-5, and > 6) and overall.

Research analyses.

Efficacy. HPV clearance will be summarized; the percentage of subjects with HPV6/11 clearance in resected tumor tissue compared to baseline will be calculated. The relationship between HPV6/11 ccDNA before and after INO-3107 administration as a correlate of disease burden and clinical outcomes in patients with RPC will be examined using regression models. Analyses will be summarized and presented by number of prior surgeries (<2, 3-5, and >6) and overall.

Immunogenicity. Post-baseline ELISA titers will be analyzed with geometric means. Peripheral blood gene expression changes from baseline will be summarized. Analysis will be summarized and presented by number of prior surgeries (<2, 3-5, and >6) and overall.

Sequences and sequence identifiers.

>pGX3024 insertion of amino acid sequence only without IgE leader sequence <SEQ ID NO: 1>

ESKDASTSATSIDQLCKTFNLSLHTLQIQCVFCRNALTTAEIYAYAYKNLKVVWRDNFPFAACACC LELQGKINQYRHFNYAAYAPTVEEETNEDILKVLIRCYLCHKPQCEIEKLKHILGKARFIKLNNQRKGRC LHCWTTCMEDLLPRGRKRRSGSGATNFSLLKQAGDVEENPGPHGRLVTLKDIVLDLQPPDPVGLHAYE QLEDSSEDEVDKVDKQDSQPLTQHYQILTCCCGCDSNVRLVVECTDGDIRQLQDLLLGTLNIVCPICAP KPRGRKRRSGSGATNFSLLKQAGDVEENPGPESANASTSATTIDQLCKTFNLSMHTLQINCVFCKNALT TAEIYSYAYKQLKVLFRGGYPYAACACCLEFHGKINQYRHFDYAGYATTVEEETKQDILDVLIRCYLCH KPQCEVEKVKHILTKARFIKLNCTRKGRCLHCWTTCMEDMLPRGRKRRSGSGATNFSLLKQAGDVEEN PGPHGRHVTLKDIVLDLQPPDPVGLHAYEQLVDSSEDEVDEVDGQDSQPLKQHYQIVTCCCGCDSNVR LVVQCTETDIREVQQLLLGTLNIVCPICAPKT >pGX3024_insert only without IgE leader sequence <SEQ ID NO: 2>

GAGAGCAAGGATGCCAGCACAAGCGCCACCAGCATCGACCAGCTTTGCAAGACCTTTAACCT GAGCCTGCACACACTTCAGATCCAGTGTGTCTTCTGCCGAAATGCTCTGACAACAGCAGAAATCTA CGCCTACGCCTACAAACCTGAAGGTGGTGTGGAGAGACAACTTTCCTTTCGCTGCCTGCGCTTG CTGCCTGGAGCTGCAGGGCAAGATCAATCAGTACCGGCACTTCAACTACGCTGCCTACGCCCCTAC AGTGGAGGAGGAAACAAACGAAGACATCCTGAAGGTGCTGATCAGATGCTACCTCTGCCACAAGC CACAGTGTGAAATCGAGAAGCTGAAGCACATTCTGGGCAAGGCCAGATTTATCAAGCTGAACAAC CAGAGAAAGGGAAGATGTCTGCACTGTTGGACAACCTGCATGGAGGACCTGCTGCCCAGAGGCAG AAAGAGAAGATCTGGCAGCGGAGCTACCAACTTCTCTCTGCTGAAGCAGGCTGGAGATGTTGAGG AGAACCCAGGCCCTCACGGCCGGCTGGTCACCCTGAAGGATATCGTGCTGGATCTGCAGCCCCCTG ATCCTGTGGGCCTTCACGCCTACGAACAGCTGGAGGACAGCTCTGAAGACGAAGTGGACAAGGTG GACAAGCAGGACTCTCAGCCTCTGACACAGCACTATCAGATCCTGACCTGCTGCTGCGGCTGTGAC TCTAACGTGAGACTGGTGGTGGAGTGCACCGATGGAGACATCAGACAGCTGCAGGACCTGCTGCT GGGTACCCTGAACATTGTGTGTCCTATCTGTGCTCCAAAGCCAAGAGGCAGGAAAAGAAGATCCG GCAGCGGAGCCACCAATTTCTCCCTGCTGAAGCAAGCTGGAGATGTGGAGGAGAACCCTGGCCCT GAGAGCGCCAACGCCAGCACATCCGCCACCACCATCGACCAGCTGTGCAAGACCTTCAACCTGAG CATGCACACACTGCAGATCAACTGTGTCTTCTGCAAGAATGCCCTGACCACAGCAGAGATCTACAG CTACGCCTACAAGCAGCTGAAGGTGCTGTTCAGAGGCGGCTACCCTTATGCTGCCTGTGCCTGCTG CCTGGAGTTCCACGGCAAGATCAACCAGTACAGACACTTCGACTACGCTGGCTACGCCACCACAGT GGAAGAGGAAACAAAGCAGGACATCCTGGACGTGCTGATCCGATGCTACCTGTGCCACAAGCCTC AGTGTGAAGTGGAAAAAGTGAAGCACATCCTGACCAAGGCCAGATTCATCAAGCTGAACTGCACC AGAAAAGGCAGATGCCTGCACTGCTGGACCACCTGCATGGAAGACATGCTGCCTAGAGGCAGAAA AAGAAGAAGCGGCTCTGGAGCCACCAACTTTTCCCTGCTGAAACAAGCTGGAGACGTGGAGGAAA ACCCTGGCCCTCACGGCAGACACGTGACACTGAAGGACATCGTGCTGGACCTGCAGCCTCCTGACC CTGTGGGCCTGCACGCCTACGAGCAGCTGGTGGACAGCAGCGAGGACGAAGTGGACGAAGTGGAT GGCCAGGACAGCCAGCCTCTGAAGCAGCACTACCAGATCGTCACCTGCTGCTGTGGCTGTGATAGC AATGTGAGGGCTGGTGGTGCAGTGCACAGAAACAGACATCAGAGAAGTGCAGCAACTGCTGCTGGG CACCCTGAACATCGTGTGTCCCATCTGTGCTCCCAAGACATGATAA >pGX3024_full sequence <SEQ ID NO: 3>

gctgcttegcgatgtacgggccagatacgcgttgacattgattattgactagttattaatagtaatcaattacggggteattagttcatagcccatatatggag ttccgcgttacataacttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagtaacgccaatagg gactttccattgacgtcaatgggtggagtatttacggtaaactgcccacttggcagtacateaagtgtateatatgccaagtacgccccctattgacgtcaatgacggta aatggcccgcctggcattatgcccagtacatgaccttatgggactttectacttggcagtacatetacgtattagtcategctattaccatggtgatgcggttttggcagt acatcaatgggcgtggatagcggtttgactcacggggatttccaagtctccaccccattgacgtcaatgggagtttgttttggcaccaaaatcaacgggactttccaaa atgtcgtaacaactccgccccattgacgcaaatgggcggtaggcgtgtacggtgggaggtctatataagcagagctctctggctaactagagaacccactgcttact

- 40 049710 ggcttatcgaaattaatacgactcactatagggagacccaagctggctagcgtttaaacttaagcttggtaccgagctcggatecgccaccatggattggacctggat tctetttetegttgccgctgctactcgcgttcatagtgagagcaaggatgccagcacaagcgccaccagcategaccagctttgcaagacctttaacctgagcctgca cacactteagatccagtgtgtettetgccgaaatgctctgacaacagcagaaatetacgcctacgcctacaaaaacctgaaggtggtgtggagagacaactttectttc gctgcctgcgcttgctgcctggagctgcagggcaagatcaatcagtaccggcacttcaactacgctgcctacgcccctacagtggagggaggaaacaaacgaaga catcctgaaggtgctgatcagatgctacctctgccacaagccacagtgtgaaatcgagaagctgaagcacattctgggcaaggccagatttatcaagctgaacaac cagagaaagggaagatgtctgcactgttggacaacctgcatggaggacctgctgcccagaggcagaaagagaagatctggcagcggagctaccaacttctctct gctgaagcaggctggagatgttgaggagaacccaggccctcacggccggctggtcaccctgaaggatatcgtgctggatctgcagccccctgatcctgtgggcct tcacgcctacgaacagctggaggacagctctgaagacgaagtggacaaggtggacaagcaggactctcagcctctgacacagcactatcagatcctgacctgct gctgcggctgtgactctaacgtgagactggtggtggagtgcaccgatggagacatcagacagctgcaggacctgctgctgggtaccctgaacattgtgtgtcctatc tgtgctccaaagccaagaggcaggaaaagaagatccggcagcggagccaccaatttctccctgctgaagcaagctggagatgtggaggagaaccctggccctg agagcgccaacgccagcacatccgccaccaccatcgaccagctgtgcaagaccttcaacctgagcatgcacacactgcagatcaactgtgtcttctgcaagaatg ccctgaccacagcagagatctacagctacgcctacaagcagctgaaggtgctgttcagaggcggctacccttatgctgcctgtgcctgctgcctggagttccacgg caagatcaaccagtacagacacttcgactacgctggctacgccaccacagtggaagaggaaacaaagcaggacatcctggacgtgctgatccgatgctacctgtg ccacaagcctcagtgtgaagtggaaaaagtgaagcacatcctgaccaaggccagattcatcaagctgaactgcaccagaaaaggcagatgcctgcactgctgga ccacctgcatggaagacatgctgcctagaggcagaaaaagaagaagcggctctggagccaccaacttttccctgctgaaacaagctggagacgtggaggaaaac cctggccctcacggcagacacgtgacactgaaggacatcgtgctggacctgcagcctcctgaccctgtgggcctgcacgcctacgagcagctggtggacagcag cgaggacgaagtggacgaagtggatggccaggacagccagcctctgaagcagcactaccagatcgtcacctgctgctgtggctgtgatagcaatgtgaggctgg tggtgcagtgcacagaaacagacatcagagaagtgcagcaactgctgctgggcaccctgaacatcgtgtgtcccatctgtgctcccaagacatgataactcgagtc tagagggcccgtttaaacccgctgatcagcctcgactgtgccttctagttgccagccatctctgttgtttgcccctcccccgtgccttccttgaccctggaaggtgccactc ccactgtcctttcctaataaaatgaggaaattgcatcgcattgtctgagtaggtgtcattctattctggggggtggggtggggcaggacagcaagggggaggattgg gaagacaatagcaggcatgctggggatgcggtgggctctatggcttctactgggcggttttatggacagcaagcgaaccggaattgccagctggggcgccctctg gtaaggttgggaagccctgcaaagtaaactggatggctttcttgccgccaaggatctgatggcgcaggggatcaagctctgatcaagagacaggatgaggatcgtt tcgcatgattgaacaagatggattgcacgcaggttctccggccgcttgggtggagaggctattcggctatgactgggcacaacagacaatcggctgctctgatgcc gccgtgttccggctgtcagcgcaggggcgcccggttctttttgtcaagaccgacctgtccggtgccctgaatgaactgcaagacgaggcagcgcggctatcgtgg ctggccacgacgggcgttccttgcgcagctgtgctcgacgttgtcactgaagcgggaagggactggctgctattgggcgaagtgccggggcaggatctcctgtca tctcaccttgctcctgccgagaaagtatccatcatggctgatgcaatgcggcggctgcatacgcttgatccggctacctgcccattcgaccaccaagcgaaacatcg catcgagcgagcacgtactcggatggaagccggtcttgtcgatcaggatgatctggacgaagagcatcaggggctcgcgccagccgaactgttcgccaggctca aggcgagcatgcccgacggcgaggatctcgtcgtgacccatggcgatgcctgcttgccgaatatcatggtggaaaatggccgcttttctggattcatcgactgtggc cggctgggtgtggcggaccgctatcaggacatagcgttggctacccgtgatattgctgaagagcttggcggcgaatgggctgaccgcttcctcgtgctttacggtat cgccgctcccgattcgcagcgcatcgccttctatcgccttcttgacgagttcttctgaattattaacgcttacaatttcctgatgcggtattttctccttacgcatctgtgcg gtatttcacaccgcatcaggtggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaatacattcaaatatgtatccgctcatgagacaataaccct gataaatgcttcaataatagcacgtgctaaaacttcatttttaatttaaaaggatctaggtgaagatcctttttgataatctcatgaccaaaatcccttaacgtgagttttcgtt ccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaccaccgctaccagcggtg gtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgttcttctagtgtagccgtagttaggccacca cttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacga tagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtg agctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagg gggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccag caacgcggcctttttacggttcctggccttttgctggccttttgctcacatgttctt

SEQ ID NO: 4 - Full-length pGX6010 sequence and note:

Elements:

Base pairs:

Kanamycin resistance: pUC Ori: bGR polyA: XhoI site:

Coding sequence of huIL-12 p35:

298-1059

1227-1900 2290-2528 2551-2556

2557-3216 (Note: the p35 subunit is encoded on the opposite strand) 3217-3224 3209-3711 4200-5024 5030-5035 5036-6022 6023-6028 6024-6229

PmeI website:

oCMV promoter:

hCMV promoter:

SalI website:

huIL-12 p40:

MluI website:

BO40 polyA:

aaatgggggc gctgaggtct gcctcgtgaa gaaggtgttg ctgactcata ccaggcctga 60 atcgccccat catccagcca gaaagtgagg gagccacggt tgatgagagc tttgttgtag 120 gtggaccagt tggtgatttt gaacttttgc tttgccacgg aacggtctgc gttgtcggga 180 agatgcgtga tctgatcctt caactcagca aaagttcgat ttattcaaca aagccgccgt 240 cccgtcaagt cagcgtaatg ctctgccagt gttacaacca attaaccaat tctgcgttca 300 aaatggtatg cgttttgaca catccactat atatccgtgt cgttctgtcc actcctgaat 360 cccattccag aaattctcta gcgattccag aagtttctca gagtcggaaa gttgaccaga 420

- 41 049710 cattacgaac tggcacagat ggtcataacc tgaaggaaga tctgattgct taactgcttc 480 agttaagacc gacgcgctcg tcgtataaca gatgcgatga tgcagaccaa tcaacatggc 540 acctgccatt gctacctgta cagtcaagga tggtagaaat gttgtcggtc cttgcacacg 600 aatattacgc catttgcctg catattcaaa cagctcttct acgataaggg cacaaatcgc 660 atcgtggaac gtttgggctt ctaccgattt agcagtttga tacactttct ctaagtatcc 720 acctgaatca taaatcggca aaatagagaa aaattgacca tgtgtaagcg gccaatctga 780 ttccacctga gatgcataat ctagtagaat ctcttcgcta tcaaaattca cttccacctt 840 ccactcaccg gttgtccatt catggctgaa ctctgcttcc tctgttgaca tgacacacat 900 catctcaata tccgaatacg gaccatcagt ctgacgacca agagagccat aaacaccaat 960 agccttaaca tcatccccat atttatccaa tattcgttcc ttaatttcat gaacaatctt 1020 cattctttct tctctagtca ttattattgg tccgttcata acaccccttg tattactgtt 1080 tatgtaagca gacagtttta ttgttcatga tgatatattt ttatcttgtg caatgtaaca 1140 tcagagattt tgagacacaa cgtggctttc cccggcccat gaccaaaatc ccttaacgtg 1200 agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc 1260 ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg 1320 tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag 1380 cgcagatacc aaatactgtt cttctagtgt agccgtagtt aggccaccac ttcaagaact 1440 ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg 1500 gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc 1560 ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg 1620 aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg 1680 cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag 1740 ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc 1800 gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct 1860 ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc 1920 ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc 1980 gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga agagcgcctg atgcggtatt 2040 ttctccttac gcatctgtgc ggtatttcac accgcatatg gtgcactctc agtacaatct 2100 gctctgatgc cgcatagtta agccagtatc tgctccctgc ttgtgtgttg gaggtcgctg 2160 agtagtgcgc gagcaaaatt taagctacaa caaggcaagg cttgaccgac aattgcatga 2220 agaatctgct tagggttagg cgttttgcgc tgcttcgcga tgtacgggcc agatatagcc 2280 gcggcatcga tgataattcg gcttatttaa attccccagc atgcctgcta ttgtcttccc 2340 aatcctcccc cttgctgtcc tgccccaccc caccccccag aatagaatga cacctactca 2400 gacaatgcga tgcaatttcc tcattttatt aggaaaggac agtgggagtg gcaccttcca 2460 gggtcaagga aggcacgggg gaggggcaaa caacagatgg ctggcaacta gaaggcacag 2520 tcgaggctga tcagcgagct cggcgcgcct ctcgagttag gaagcgttca ggtatgacat 2580 gacccgatca atagtgacag cccgaatccg aaaggcatgc agcagaatgc acagcttgat 2640 ttttgtctta taaaagtcgg gttcctccag actagacttc tgtgggacgg tttcgctatt 2700 gaagttcagg gcctgcatca gctcgtcaat cactgccagc atattctgat ccagaaagat 2760 ctgtcgttta gggtccatca gcagcttagc gttcatggtt ttgaattcca cctgatacat 2820 cttcagatcc tcgtagatgg agctcaggca cagtgccatc atgaagctgg tcttgcgact 2880 agccaggcaa gacccgttgg tgatgaagga agtctccctg ctattcagac atgattcgtt 2940 cttggtcagc tccagtggca ggcaggcttc gactgtggag gttttgtcct tagtaatatc 3000 ctcgtggtcg atttcctcag aagtacaagg gtaaaactcc agtgtctgtc tagctttctg 3060 cagcatattg gacacggccc gcagcaggtt ctggctatgg tgcaggcagg ggaacatgcc 3120 aggatcgggg gtagcgacag gcagatttcg agccagtgac aggtgatcca gcaggaccag 3180 ggtagcgacc agcagcaggg accgagcggg gcacatgttt aaacgctcct ccgacgtccc 3240 caggcagaat ggcggttccc taaacgagca ttgcttatat agacctccca ttaggcacgc 3300 ctaccgccca tttacgtcaa tggaacgccc atttgcgtca ttgcccctcc ccattgacgt 3360 caatggggat gtacttggca gccatcgcgg gccatttacc gccattgacg tcaatgggag 3420 tactgccaat gtaccctggc gtacttccaa tagtaatgta cttgccaagt tactattaat 3480 agatattgat gtactgccaa gtgggccatt taccgtcatt gacgtcaata gggggcgtga 3540 gaacggatat gaatgggcaa tgagccatcc cattgacgtc aatggtgggt ggtcctattg 3600 acgtcaatgg gcattgagcc aggcgggcca tttaccgtaa ttgacgtcaa tgggggaggc 3660 gccatatacg tcaataggac cgcccatatg acgtcaatag gaaagaccat gctaagccga 3720 attatcgcgg ctatctgagg ggactagggt gtgtttaggc gaaaagcggg gcttcggttg 3780 tacgcggtta ggagtcccct caggatatag tagtttcgct tttgcatagg gagggggaaa 3840 tgtagtctta tgcaatactc ttgtagtctt gcaacatggt aacgatgagt tagcaacatg 3900 ccttacaagg agagaaaaag caccgtgcat gccgattggt ggaagtaagg tggtacgatc 3960 gtgccttatt aggaaggcaa cagacgggtc tgacatggat tggacgaacc actgaattcc 4020 gcattgcaga gatattgtat ttaagtgcct agctcgatac aataaacgcc atttgaccat 4080 tcaccacatt ggtgtgcacc tccaagcttc gaccaattct catgtttgac agcttatcat 4140

- 42 049710 cgcagatccg ggcaacgttg ttgccattgc tgcaggcgca gaactggtag gtatggaaga 4200 tctatacatt gaatcaatat tggcaattag ccatattagt cattggttat atagcataaa 4260 tcaatattgg ctattggcca ttgcatacgt tgtatctata tcataatatg tacatttata 4320 ttggctcatg tccaatatga ccgccatgtt gacattgatt attgactagt tattaatagt 4380 aatcaattac ggggtcatta gttcatagcc catatatgga gttccgcgtt acataactta 4440 cggtaaatgg cccgcctggc tgaccgccca acgacccccg cccattgacg tcaataatga 4500 cgtatgttcc catagtaacg ccaataggga ctttccattg acgtcaatgg gtggagtatt 4560 tacggtaaac tgcccacttg gcagtacatc aagtgtatca tatgccaagt ccgcccccta 4620 ttgacgtcaa tgacggtaaa tggcccgcct ggcattatgc ccagtacatg accttacggg 4680 actttcctac ttggcagtac atctacgtat tagtcatcgc tattaccatg gtgatgcggt 4740 tttggcagta catcaatggg cgtggatagc ggtttgactc acggggattt ccaagtctcc 4800 accccattga cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac tttccaaaat 4860 gtcgtaacaa ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg tgggaggtct 4920 atataagcag agctcgttta gtgaaccgtc agatcgcctg gagacgccat ccacgctgtt 4980 ttgacctcca tagaagacac cgggaccgat ccagcctccg cgggcgcgcg tcgacatgtg 5040 ccatcagcag ctggtcatct cttggtttag tctggtgttt ctggcttctc cactggtcgc 5100 tatctgggaa ctgaaaaagg atgtgtacgt ggtcgagctg gactggtatc cagatgcacc 5160 cggagaaatg gtggtcctga cctgcgacac acccgaggaa gatggcatca cttggaccct 5220 ggaccagagc tccgaggtgc tgggatctgg caagacactg actattcagg tcaaagaatt 5280 cggggatgcc ggacagtaca catgtcacaa gggcggggag gtgctgagtc actcactgct 5340 gctgctgcat aagaaagaag acggcatctg gtctactgac attctgaagg atcagaaaga 5400 gcctaagaac aaaaccttcc tgagatgcga agctaagaat tatagtggga ggtttacctg 5460 ttggtggctg accacaatct caactgacct gacctttagc gtgaaatcta gtagggggtc 5520 aagcgatcca cagggagtga cctgcggagc agctacactg agcgccgagc gggtgagagg 5580 agacaacaag gagtacgaat atagtgtcga gtgccaggaa gattcagcct gtcccgcagc 5640 cgaggaatcc ctgcctatcg aagtgatggt ggacgctgtg cacaagctga aatacgaaaa 5700 ctacacatcc tctttcttta ttcgcgacat cattaagcca gatcccccta aaaacctgca 5760 gctgaagccc ctgaaaaatt cccgacaggt ggaggtctct tgggaatacc ctgatacatg 5820 gagcactcca cattcttatt tcagtctgac tttttgcgtg caggtccagg gcaagagcaa 5880 aagggagaag aaagaccgcg tgttcaccga taagacatcc gctactgtca tctgtcgaaa 5940 aaacgcaagc atttccgtgc gggcacagga taggtattat tccagcagtt ggtctgagtg 6000 ggcttccgtc ccttgtagtt gaacgcgtaa aaagatccag acatgataag atacattgat 6060 gagtttggac aaaccacaac tagaatgcag tgaaaaaaat gctttatttg tgaaatttgt 6120 gatgctattg ctttatttgt aaccattata agctgcaata aacaagttaa caacaacaat 6180 tgcattcatt ttatgtttca ggttcagggg gaggtgtggg aggtttttt 6229

SEQ ID NO: 5 - DNA sequence of p35:

ATGTGTCCAGCGCGCAGCCTCCTCCTTGTGGCTACCCTGGTCCTCCTGGACCACCTCAGTTTGG CCAGAAACCTCCCCGTGGCCACTCCAGACCCAGGAATGTTCCCATGCCTTCACCACTCCCAAAACC TGCTGAGGGCCGTCAGCAACATGCTCCAGAAGGCCAGACAAACTCTAGAATTTTACCCTTGCACTT CTGAAGAGATTGATCATGAAGATATCACAAAAGATAAAACCAGCACAGTGGAGGCCTGTTTACCA TTGGAATTAACCAAGAATGAGAGTTGCCTAAATTCCAGAGAGACCTCTTTCATAACTAATGGGAGT TGCCTGGCCTCCAGAAAGACCTCTTTTATGATGGCCCTGTGCCTTAGTAGTATTTATGAAGACTTGA AGATGTACCAGGTGGAGTTCAAGACCATGAATGCAAAGCTTCTGATGGATCCTAAGAGGCAGATCT TTCTAGATCAAAACATGCTGGCAGTTATTGATGAGCTGATGCAGGCCCTGAATTTCAACAGTGAGA CTGTGCCACAAAAATCCTCCCTTGAAGAACCGGATTTTTATAAAACTAAAATCAAGCTCTGCATAC TTCTTCATGCTTTCAGAATTCGGGCAGTGACTATTGATAGAGTGATGAGCTATCTGAATGCTTCCTAA

SEQ ID NO: 6 - Amino acid sequence of p35:

MCPARSLLLLVATLVLLDHLSLARNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCT SEEIDHEDITKDKTSTVEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQV EFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEPDFYKTKIKLCILLHAFRIRA VTIDRVMSYLNAS*

SEQ ID NO: 7 - DNA sequence of p40:

ATGTGTCACCAGCAGTTGGTCATCTCTTGGTTTTCCCTGGTTTTTCTGGCATCTCCCCTCGTGG CCATATGGGAACTGAAGAAAGATGTTTATGTCGTAGAATTGGATTGGTATCCGGATGCCCCTGGAG AAATGGTGGTCCTCACCTGTGACACCCCTGAAGAAGATGGTATCACCTGGACCTTGGACCAGAGCA GTGAGGTCTTAGGCTCTGGCAAAACCCTGACCATCCAAGTCAAAGAGTTTGGAGATGCTGGCCAGT ACACCTGTCACAAAGGAGGCGAGGTTCTAAGCCATTCGCTCCTGCTGCTTCACAAAAAGGAAGATG GAATTTGGTCCACTGATATTTTAAAGGACCAGAAAGAACCCAAAAATAAGACCTTTCTAAGATGCG AGGCCAAGAATTATTCTGGACGTTTCACCTGCTGGTGGCTGACGACAATCAGTACTGATTTGACAT TCAGTGTCAAAAGCAGCAGAGGCTCTTCTGACCCCCAAGGGGTGACGTGCGGAGCTGCTACACTCT CTGCAGAGAGAGTCAGAGGGGACAACAAGGAGTATGAGTACTCAGTGGAGTGCCAGGAGGACAG TGCCTGCCCAGCTGCTGAGGAGAGTCTGCCCATTGAGGTCATGGTGGATGCCGTTCACAAGCTCAA

- 43 049710

GTATGAAAACTACACCAGCAGCTTCTTCATCAGGGACATCATCAAACCTGACCCACCCAAGAACTT GCAGCTGAAGCCATTAAAGAATTCTCGGCAGGTGGAGGTCAGCTGGGAGTACCCTGACACCTGGA GTACTCCACATTCCTACTTCTCCCTGACATTCTGCGTTCAGGTCCAGGGCAAGAGCAAGAGAGAAA TAG

SEQ ID NO: 8 Amino acid sequence of p40:

MCHQQLVISWFSLVFLASPLVAIWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQ SSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEA KNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPA AEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSL TFCVQVQGKSKREKKDRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCS*

SEQ ID NO:9 IgE DNA leader sequence atggactgga cctggatcct gttcctggtg gccgctgcca cacgggtgca cagc

SEQ ID NO: 10 Leader sequence of IgE protein

Met Asp Trp Thr Trp Ile Leu Phe Leu Val Ala Ala Ala Thr Arg

Val His Ser >pGX3024 insertion of amino acid sequence with IgE leader sequence <SEQ ID NO: 11>

MDWTWILFLVAAATRVHSESKDASTSATSIDQLCKTFNLSLHTLQIQCVFCRNALTTAEIYAYAYK NLKVVWRDNFPFAACACCLELQGKINQYRHFNYAAYAPTVEEETNEDILKVLIRCYLCHKPQCEIEKLK HILGKARFIKLNNQRKGRCLHCWTTCMEDLLPRGRKRRSGSGATNFSLLKQAGDVEENPGPHGRLVTL KDIVLDLQPPDPVGLHAYEQLEDSSEDEVDKVDKQDSQPLTQHYQILTCCCGCDSNVRLVVECTDGDIR QLQDLLLGTLNIVCPICAPKPRGRKRRSGSGATNFSLLKQAGDVEENPGPESANASTSATTIDQLCKTFN LSMHTLQINCVFCKNALTTAEIYSYAYKQLKVLFRGGYPYAACACCLEFHGKINQYRHFDYAGYATTV EEETKQDILDVLIRCYLCHKPQCEVEKVKHILTKARFIKLNCTRKGRCLHCWTTCMEDMLPRGRKRRSG SGATNFSLLKQAGDVEENPGPHGRHVTLKDIVLDLQPPDPVGLHAYEQLVDSSEDEVDEVDGQDSQPL KQHYQIVTCCCGCDSNVRLVVQCTETDIREVQQLLLGTLNIVCPICAPKT >pGX3024_insert with only IgE leader sequence <SEQ ID NO: 12>

ATGGATTGGACCTGGATTCTCTTTCTCGTTGCCGCTGCTACTCGCGTTCATAGTGAGAGCAAG GATGCCAGCACAAGCGCCACCAGCATCGACCAGCTTTGCAAGACCTTTAACCTGAGCCTGCACACA CTTCAGATCCAGTGTGTCTTCTGCCGAAATGCTCTGACAACAGCAGAAATCTACGCCTACGCCTAC AAAAACCTGAAGGTGGTGTGGAGAGACAACTTTCCTTTCGCTGCCTGCGCTTGCTGCCTGGAGCTG CAGGGCAAGATCAATCAGTACCGGCACTTCAACTACGCTGCCTACGCCCCTACAGTGGAGGAGGA AACAAACGAAGACATCCTGAAGGTGCTGATCAGATGCTACCTCTGCCACAAGCCACAGTGTGAAA TCGAGAAGCTGAAGCACATTCTGGGCAAGGCCAGATTTATCAAGCTGAACAACCAGAGAAAGGGA AGATGTCTGCACTGTTGGACAACCTGCATGGAGGACCTGCTGCCCAGAGGCAGAAAGAGAAGATC TGGCAGCGGAGCTACCAACTTCTCTCTGCTGAAGCAGGCTGGAGATGTTGAGGAGAACCCAGGCCC TCACGGCCGGCTGGTCACCCTGAAGGATATCGTGCTGGATCTGCAGCCCCCTGATCCTGTGGGCCT TCACGCCTACGAACAGCTGGAGGACAGCTCTGAAGACGAAGTGGACAAGGTGGACAAGCAGGACT CTCAGCCTCTGACACAGCACTATCAGATCCTGACCTGCTGCTGCGGCTGTGACTCTAACGTGAGAC TGGTGGTGGAGTGCACCGATGGAGACATCAGACAGCTGCAGGACCTGCTGCTGGGTACCCTGAAC ATTGTGTGTCCTATCTGTGCTCCAAAGCCAAGAGGCAGGAAAAGAAGATCCGGCAGCGGAGCCAC CAATTTCTCCCTGCTGAAGCAAGCTGGAGATGTGGAGGAGAACCCTGGCCCTGAGAGCGCCAACG CCAGCACATCCGCCACCACCATCGACCAGCTGTGCAAGACCTTCAACCTGAGCATGCACACACTGC AGATCAACTGTGTCTTCTGCAAGAATGCCCTGACCACAGCAGAGATCTACAGCTACGCCTACAAGC AGCTGAAGGTGCTGTTCAGAGGCGGCTACCCTTATGCTGCCTGTGCCTGCTGCCTGGAGTTCCACG GCAAGATCAACCAGTACAGACACTTCGACTACGCTGGCTACGCCACCACAGTGGAAGAGGAAACA AAGCAGGACATCCTGGACGTGCTGATCCGATGCTACCTGTGCCACAAGCCTCAGTGTGAAGTGGAA AAAGTGAAGCACATCCTGACCAAGGCCAGATTCATCAAGCTGAACTGCACCAGAAAAGGCAGATG CCTGCACTGCTGGACCACCTGCATGGAAGACATGCTGCCTAGAGGCAGAAAAAGAAGAAGCGGCT CTGGAGCCACCAACTTTTCCCTGCTGAAACAAGCTGGAGACGTGGAGGAAAACCCTGGCCCTCACG GCAGACACGTGACACTGAAGGACATCGTGCTGGACCTGCAGCCTCCTGACCCTGTGGGCCTGCACG CCTACGAGCAGCTGGTGGACAGCAGCGAGGACGAAGTGGACGAAGTGGATGGCCAGGACAGCCA GCCTCTGAAGCAGCACTACCAGATCGTCACCTGCTGCTGTGGCTGTGATAGCAATGTGAGGCTGGT GGTGCAGTGCACAGAAACAGACATCAGAGAAGTGCAGCAACTGCTGCTGGGCACCCTGAACATCG TGTGTCCCATCTGTGCTCCCAAGACATGATAA

-

Claims (52)

CLAUSE OF INVENTION 1. A nucleic acid molecule encoding a human papillomavirus (HPV) antigen, wherein the HPV antigen comprises the amino acid sequence of SEQ ID NO: 1; amino acid sequence SEQ ID NO: 11; an amino acid sequence that is at least 95% identical to SEQ ID NO: 1; or an amino acid sequence that is at least 95% identical to SEQ ID NO: 11. 2. A nucleic acid molecule according to claim 1, comprising a nucleotide sequence that is at least 95% identical to SEQ ID NO: 2; a nucleotide sequence that is at least 95% identical to SEQ ID NO: 12; the nucleotide sequence of SEQ ID NO: 2 or the nucleotide sequence of SEQ ID NO: 12. 3. A nucleic acid molecule according to claim 1, containing a nucleotide sequence encoding an IgE leader sequence. 4. A nucleic acid molecule according to claim 3, wherein the IgE leader sequence comprises the amino acid sequence of SEQ ID NO: 10. 5. A nucleic acid molecule according to claim 3, wherein the nucleotide sequence encoding the IgE leader sequence comprises SEQ ID NO: 9. 6. An expression vector containing a nucleic acid molecule according to claim 1. 7. An expression vector according to claim 6, containing a DNA plasmid. 8. An expression vector according to claim 6, containing the nucleotide sequence SEQ ID NO: 3. 9. An immunogenic protein comprising the amino acid sequence of SEQ ID NO: 1; amino acid sequence SEQ ID NO: 11; an amino acid sequence that is at least 95% identical to SEQ ID NO: 1; or an amino acid sequence that is at least 95% identical to SEQ ID NO: 11. 10. A vaccine against human papillomavirus infection, comprising an expression vector according to claim 6 and a pharmaceutically acceptable excipient. 11. A pharmaceutical composition containing an expression vector according to claim 6 and a pharmaceutically acceptable excipient. 12. A pharmaceutical composition according to claim 11, containing an adjuvant. 13. The pharmaceutical composition according to claim 12, characterized in that the adjuvant contains interleukin-12 (IL-12), the p35 subunit of IL12, the p40 subunit of IL12, or the p35 subunit of IL12 and the p40 subunit of IL12. 14. The pharmaceutical composition according to claim 12, characterized in that the adjuvant contains a nucleic acid molecule containing a nucleotide sequence encoding interleukin-12 (IL12), a nucleic acid molecule containing a nucleotide sequence encoding the p35 subunit of IL12, a nucleotide sequence encoding the p40 subunit of IL12, a nucleotide sequence encoding the p35 subunit of IL12 and a nucleotide sequence encoding the p40 subunit of IL12, or a nucleotide sequence encoding the p35 subunit of IL12 and a nucleotide sequence encoding the p40 subunit of IL12. 15. The pharmaceutical composition according to claim 11, characterized in that the nucleic acid molecule containing a nucleotide sequence encoding IL-12, a nucleic acid molecule containing a nucleotide sequence encoding the p35 subunit of IL12, a nucleic acid molecule containing a nucleotide sequence encoding the p40 subunit of IL12, a nucleic acid molecule containing a nucleotide sequence encoding the p35 subunit of IL12 and a nucleotide sequence encoding the p40 subunit of IL12, or a nucleic acid molecule containing a nucleotide sequence encoding the p35 subunit of IL12 and a nucleic acid molecule containing a nucleotide sequence encoding the p40 subunit of IL12, is an expression vector. 16. A vaccine against human papillomavirus infection, comprising an immunogenic protein according to claim 9 and a pharmaceutically acceptable excipient. 17. A pharmaceutical composition containing an immunogenic protein according to claim 9 and a pharmaceutically acceptable excipient. 18. A pharmaceutical composition according to claim 17, containing an adjuvant. 19. The pharmaceutical composition according to claim 18, characterized in that the adjuvant contains interleukin-12 (IL-12), the p35 subunit of IL12, the p40 subunit of IL12, or the p35 subunit of IL12 and the p40 subunit of IL12. 20. The pharmaceutical composition according to claim 14, characterized in that the nucleotide sequence encoding the p35 subunit of IL-12 contains a nucleotide sequence selected from - 45 049710 a group consisting of a nucleotide sequence that encodes SEQ ID NO: 6; a nucleotide sequence that is at least 95% identical to the nucleotide sequence that encodes SEQ ID NO: 6; the nucleotide sequence of SEQ ID NO: 5; or a nucleotide sequence that is at least 95% identical to SEQ ID NO: 5. 21. The pharmaceutical composition according to claim 14, characterized in that the nucleotide sequence encoding the p40 subunit of IL-12 contains a nucleotide sequence selected from the group consisting of a nucleotide sequence that encodes SEQ ID NO: 8; a nucleotide sequence that is at least 95% identical to the nucleotide sequence that encodes SEQ ID NO: 8; the nucleotide sequence of SEQ ID NO: 7; or a nucleotide sequence that is at least 95% identical to SEQ ID NO: 7. 22. The pharmaceutical composition according to claim 14, characterized in that the nucleotide sequence encoding IL-12 comprises a nucleotide sequence selected from the group consisting of the nucleotide sequence of SEQ ID NO: 4; or a nucleotide sequence that is at least 95% identical to SEQ ID NO: 4. 23. The pharmaceutical composition according to claim 15, characterized in that the expression vector containing a nucleotide sequence encoding the p35 subunit of IL12, a nucleotide sequence encoding the p35 subunit of IL12, a nucleotide sequence encoding the p40 subunit of IL12, or a nucleotide sequence encoding the p35 subunit of IL12 and a nucleotide sequence encoding the p40 subunit of IL12, is the same expression vector or an expression vector different from the expression vector containing a nucleic acid molecule encoding an HPV antigen. 24. The pharmaceutical composition according to claim 11, characterized in that the pharmaceutically acceptable excipient contains a buffer, optionally a saline buffer with sodium citrate, optionally a buffer containing 150 mM sodium chloride and 15 mM sodium citrate, pH level 7. 25. The pharmaceutical composition according to claim 24, characterized in that the composition contains 6 mg of an expression vector encoding an HPV antigen per milliliter of buffer and 0.25 mg of an expression vector containing a nucleotide sequence encoding interleukin-12 (IL12), a nucleotide sequence encoding the p35 subunit of IL12, a nucleotide sequence encoding the p40 subunit of IL12, or a nucleotide sequence encoding the p35 subunit of IL12 and a nucleotide sequence encoding the p40 subunit of IL12, per milliliter of buffer. 26. The pharmaceutical composition according to claim 25, characterized in that the composition contains 6 mg of DNA plasmid containing the nucleotide sequence of SEQ ID NO: 3 per milliliter of buffer and 0.25 mg of DNA plasmid containing the nucleotide sequence of SEQ ID NO: 4 per milliliter of buffer. 27. A method for inducing an immune response in a subject, comprising administering to the subject an effective amount of the pharmaceutical composition of claim 11, thereby inducing an immune response. 28. A method for prophylactic or therapeutic immunization of a subject against HPV6 and/or HPV11, comprising administering to the subject an effective amount of the pharmaceutical composition of claim 11, thereby inducing an immune response against HPV6, HPV11, or both. 29. A method of treating or preventing recurrent respiratory papillomatosis (RRP) in a subject, comprising administering to the subject an effective amount of a pharmaceutical composition according to claim 11, thereby treating or preventing RRP. 30. The method according to paragraph 29, characterized in that the RRP is juvenile RRP or adult RRP. 31. The method according to any one of claims 27-29, characterized in that the nucleic acid molecule comprises the nucleotide sequence SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 12. 32. The method according to any one of claims 27-29, further comprising administering an adjuvant to the subject. 33. The method according to claim 32, characterized in that the adjuvant contains interleukin-12 (IL-12), the p35 subunit of IL12, the p40 subunit of IL12, or the p35 subunit of IL12 and the p40 subunit of IL12. 34. The method according to claim 32, characterized in that the adjuvant contains a nucleic acid molecule containing a nucleotide sequence encoding interleukin-12 (IL12), a nucleic acid molecule containing a nucleotide sequence encoding the p35 subunit of IL12, a nucleotide sequence encoding the p40 subunit of IL12, a nucleotide sequence encoding the p35 subunit of IL12, and a nucleotide sequence encoding the p40 subunit of IL12, or a nucleotide sequence encoding the p35 subunit of IL12 and a nucleotide sequence encoding the p40 subunit of IL12. 35. The method according to claim 34, characterized in that the nucleotide sequence encoding the subject - 46 049710 unit p35 IL-12, contains a nucleotide sequence selected from the group consisting of a nucleotide sequence that encodes SEQ ID NO: 6; a nucleotide sequence that is at least 95% identical to the nucleotide sequence that encodes SEQ ID NO: 6; the nucleotide sequence of SEQ ID NO: 5; or a nucleotide sequence that is at least 95% identical to SEQ ID NO: 5. 36. The method according to claim 34, characterized in that the nucleotide sequence encoding the p40 subunit of IL-12 contains a nucleotide sequence selected from the group consisting of a nucleotide sequence that encodes SEQ ID NO: 8; a nucleotide sequence that is at least 95% identical to the nucleotide sequence that encodes SEQ ID NO: 8; the nucleotide sequence of SEQ ID NO: 7; or a nucleotide sequence that is at least 95% identical to SEQ ID NO: 7. 37. The method according to claim 34, characterized in that the nucleic acid molecule encoding IL-12 comprises a nucleotide sequence selected from the group consisting of the nucleotide sequence of SEQ ID NO: 4; or a nucleotide sequence that is at least 95% identical to SEQ ID NO: 4. 38. The method according to claim 34, characterized in that a nucleic acid molecule comprising a nucleotide sequence encoding IL-12, a nucleic acid molecule comprising a nucleotide sequence encoding the p35 subunit of IL12, a nucleic acid molecule comprising a nucleotide sequence encoding the p40 subunit of IL12, a nucleic acid molecule comprising a nucleotide sequence encoding the p35 subunit of IL12 and a nucleotide sequence encoding the p40 subunit of IL12, or a nucleic acid molecule comprising a nucleotide sequence encoding the p35 subunit of IL12 and a nucleic acid molecule comprising a nucleotide sequence encoding the p40 subunit of IL12, is an expression vector, optionally a plasmid. 39. The method according to any of paragraphs 27-29, characterized in that the subject is a human being. 40. The method according to any one of paragraphs 27-29, characterized in that the administration includes intradermal or intramuscular injection. 41. The method according to claim 40, characterized in that the introduction additionally includes electroporation. 42. Use of an effective amount of the expression vector according to claim 6 in the manufacture of a prophylactic or medicinal product for the prevention or treatment of recurrent respiratory papillomatosis (RRP). 43. Use of an effective amount of the expression vector according to claim 6 in the manufacture of a prophylactic or medicinal product for the prevention or treatment of human papillomavirus (HPV) 6 or HPV 11 infection. 44. Use of an effective amount of the pharmaceutical composition according to claim 11 for the prevention or treatment of human papillomavirus (HPV) 6 or HPV11 infection. 45. Use of an effective amount of a pharmaceutical composition according to claim 11 for the prevention or treatment of recurrent respiratory papillomatosis (RRP). 46. Use according to paragraph 45, characterized in that the RRP is juvenile RRP or adult RRP. 47. The use according to any one of claims 42-44, characterized in that the nucleic acid molecule comprises the nucleotide sequence SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 12. 48. Use according to any of paragraphs 42-47 in combination with an adjuvant. 49. The use according to claim 48, characterized in that the adjuvant contains interleukin-12 (IL-12), the p35 subunit of IL12, the p40 subunit of IL12, or the p35 subunit of IL12 and the p40 subunit of IL12. 50. The use according to claim 48, characterized in that the adjuvant comprises a nucleic acid molecule comprising a nucleotide sequence encoding interleukin-12 (IL12), a nucleic acid molecule comprising a nucleotide sequence encoding the p35 subunit of IL12, a nucleotide sequence encoding the p40 subunit of IL12, a nucleotide sequence encoding the p35 subunit of IL12 and a nucleotide sequence encoding the p40 subunit of IL12, or a nucleotide sequence encoding the p35 subunit of IL12 and a nucleotide sequence encoding the p40 subunit of IL12. 51. The use according to claim 50, characterized in that the nucleotide sequence encoding the p35 subunit of IL-12 comprises a nucleotide sequence selected from the group consisting of a nucleotide sequence that encodes SEQ ID NO: 6; a nucleotide sequence that is at least 95% identical to the nucleotide sequence that encodes SEQ ID NO: 6; a nucleotide sequence that encodes SEQ ID NO: 5; or a nucleotide sequence that is at least 95% identical to SEQ ID NO: 5. 52. The use according to claim 50, characterized in that the nucleotide sequence encoding -