Vaccines for recurrent respiratory papillomatosis and methods for using the same

Abstract

The use of anti-HPV immunogens and nucleic acid molecules encoding them for the treatment and prevention of RRP is disclosed.SOLUTION: Pharmaceutical compositions, recombinant vaccines comprising the DNA plasmids, and live attenuated vaccines are disclosed, as well as methods of inducing an immune response to treat or prevent RRP.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to and the benefit of U.S. Provisional Patent Application No. 62 / 925,283, filed October 24, 2019, which is incorporated herein by reference in its entirety.

[0002] The present invention relates to improved human papillomavirus (HPV) vaccines, improved methods for inducing an immune response, and for prophylactically and / or therapeutically immunizing individuals against recurrent respiratory papillomatosis (RRP). [Background technology]

[0003] Human papillomavirus-associated (HPV+) malignancies are an emerging global epidemic (Gradishar et al., Journal of the National Comprehensive Cancer Network: 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 role of HPV6 in the pathogenesis of most aerodigestive malignancies remains unclear, its role is widely accepted as causally involved in recurrent respiratory papillomatosis (RRP), the most common benign tumor of the laryngeal epithelium (Mounts et al., Proc Natl Acad Sci US A. 1982;79(17):5425-9; Gissmann et al., Proc Natl Acad Sci US A. 1983;80(2):560-3; Bonagura et al., APMIS. 2010;118(6-7):455-70). RRP is rare, with an estimated incidence of 1.8 per 100,000 adults in the United States (Winton et al., The New England Journal of Medicine. 2005;352(25):2589-97). Although most lesions are benign, some undergo malignant transformation, and patients with RRP have a higher risk of developing laryngeal tumors and carcinomas (Omland et al., PloS one. 2014;9(6):e99114).

[0004] The clinical course of RRP can vary greatly among affected individuals. Treatment options, including active monitoring without treatment, surgery, radiation therapy, or a combination, depend on several factors. Repeated surgical removal of papillomas for symptomatic management typically remains the mainstay of treatment (Derkay et al., Otolaryngol Clin North Am. 2019;52(4):669-79). In some cases, malignant transformation can occur, which is usually associated with a poor prognosis. Some such patients with malignant disease may be candidates for salvage therapy, including potentially curative surgery (Richey et al., Otolaryngology--Head and Neck Surgery: Official Journal of the American Academy of Otolaryngology-Head and Neck Surgery. 2007;136(1):98-103). Although selected patients in this setting may benefit from radiation, the morbidity of this approach is substantial (Mendenhall et al., American Journal of Clinical Oncology. 2008;31(4):393-8). Recently, a phase II trial of pembrolizumab in patients with RRP demonstrated a 43% response rate, supporting the rationale for immunotherapeutic management of RRP (Pai et al., Journal of Clinical Oncology. 2019;37(15_suppl):2502).

[0005] Thus, there is a need in the art for improved compositions and methods for the treatment or prevention of RRP. The present invention fulfills this unmet need. Summary of the Invention

[0006] Aspects of the present invention provide compositions comprising at least one nucleotide sequence comprising an HPV6 E6-E7 fusion antigen, and their use for the treatment or prevention of RRP.

[0007] Another aspect provides compositions comprising one or more nucleotide sequences encoding an HPV6 E6-E7 fusion antigen selected from the group consisting of a nucleotide sequence encoding SEQ ID NO: 2, a nucleotide sequence that is at least 95% homologous to a nucleotide sequence encoding SEQ ID NO: 2, and a nucleotide sequence that is at least 95% homologous to a fragment of a nucleotide sequence encoding SEQ ID NO: 2. In some embodiments, the nucleotide sequence encoding the HPV6 E6-E7 fusion antigen does not have a 5' leader sequence that is a nucleotide sequence encoding SEQ ID NO: 4.

[0008] In another aspect of the invention, compositions are provided comprising one or more nucleotide sequences encoding an HPV6 E6-E7 fusion antigen selected from the group consisting of: SEQ ID NO: 1, a nucleotide sequence that is at least 95% homologous to SEQ ID NO: 1, a fragment of SEQ ID NO: 1, a nucleotide sequence that is at least 95% homologous to a fragment of SEQ ID NO: 1. In some embodiments, the nucleotide sequence encoding the HPV6 E6-E7 fusion antigen does not have a leader sequence at the 5' end having the nucleotide sequence SEQ ID NO: 3.

[0009] The nucleotide sequence provided may be a plasmid.

[0010] In an additional aspect, pharmaceutical compositions comprising the disclosed nucleotide sequences are provided.

[0011] In some embodiments, there are methods for treating or preventing RRP in an individual by inducing an effective immune response in the individual, comprising administering to the individual a composition comprising one or more of the provided nucleotide sequences, preferably comprising introducing the provided nucleotide sequences into the individual by electroporation.

[0012] In some aspects, the method further comprises administering to the individual a composition comprising an adjuvant. In one embodiment, the method further comprises administering to the individual a composition comprising a nucleic acid molecule encoding IL-12. For example, in certain embodiments, the method further comprises administering to the individual a composition comprising a nucleic acid molecule encoding one or more of the p35 and p40 subunits of IL-12.

[0013] In some aspects, the method comprises administering to the individual a nucleic acid molecule comprising a nucleotide sequence encoding one or more of the p35 and p40 subunits of IL-12. In one embodiment, the nucleotide sequence encoding p35 comprises a nucleotide sequence selected from the group consisting of a nucleotide sequence encoding SEQ ID NO:6, a nucleotide sequence that is at least 95% homologous to a nucleotide sequence encoding SEQ ID NO:6, a fragment of a nucleotide sequence encoding SEQ ID NO:6, and a nucleotide sequence that is at least 95% homologous to a fragment of a nucleotide sequence encoding SEQ ID NO:6. In one embodiment, the nucleotide sequence encoding p40 comprises a nucleotide sequence selected from the group consisting of a nucleotide sequence encoding SEQ ID NO:8, a nucleotide sequence that is at least 95% homologous to a nucleotide sequence encoding SEQ ID NO:8, a fragment of a nucleotide sequence encoding SEQ ID NO:8, and a nucleotide sequence that is at least 95% homologous to a fragment of a nucleotide sequence encoding SEQ ID NO:8. [Brief explanation of the drawings]

[0014] [Figure 1] Comparative 3D models of the HPV6 E6 and HPV6 E7 SynCon antigens. E6 is modeled as a monomer, and the ordered C-terminal region of E7 is modeled as a homodimer. The disordered N-terminus is shown in the diagram. Both are visualized in ribbon format with side chains and transparent solvent-exposed surfaces. The zinc finger motifs on both models are annotated. [Figure 2]Interferon gamma is produced by HPV6 E6- and HPV6 E7-specific T cells in RRP patients. Subjects 603 (top panel) and 604 (bottom panel) were followed for their ability to produce interferon gamma in an ELISpot assay longitudinally throughout the study. E6-specific activity is represented by the dashed blue line, E7-specific activity is represented by the solid blue line, and the sum of both antigens is represented by the solid black line. Long-term follow-up (LTFU) time points are noted and illustrated in terms of time after completion of dose 4. [Figure 3] INO-3106 activates HPV6-specific cytotoxic lymphocytes in RRP patients. Flow cytometry was performed to evaluate activation marker expression on HPV6-specific CD8+ T cells collected from subjects before and after immunotherapy. The expression of CD137 and CD38 in patient 604 before (upper panel) and after (lower panel) treatment with INO-3106 is shown in the left column. The expression of Ki67 and CD69 in patient 604 before (upper panel) and after (lower panel) treatment with INO-3106 is shown in the right column. [Figure 4A] Immune gene transcripts are differentially regulated in an HPV6-specific manner following treatment with INO-3106. Heatmaps showing the fold difference of differentially expressed genes in stimulated versus unstimulated cells before and after vaccination. (Figure 4A) Fold change (≧2-fold) in gene expression in cells stimulated with peptide pool versus medium alone over 24 hours. (Figure 4B) Fold change (≧2-fold) in gene expression after 11 days of T cell expansion followed by restimulation of cells with peptide pool versus medium alone for 24 hours. Data were transformed to log2 fold change, with red indicating upregulation and green indicating downregulation. [Figure 4B] Same as above. [Figure 5]Treatment of RRP patients with INO-3106 confers clinical benefit in the form of avoidance of surgery. Top panel—Swimmer plot showing the length of time in days that subjects 604 and 603 were surgery-free. The dotted red line indicates the time point at which surgery would be expected based on their previous surgery frequency before intervention with INO-3106. Φ indicates the time point at which subject 604 required surgery. λ indicates that subject 603 remains surgery-free at the indicated time point. Bottom left panel—Green bars, tracing to the left y-axis, show the magnitude of HPV6-specific CD8+ T cells expressing CD38, Ki67, granzyme A, granzyme B, and perforin. Blue bars, tracing to the right y-axis, show the fold change in surgery-free time relative to the expected surgery frequency for these subjects. Bottom right—Chart shows patient ID, fold increase in surgery-free time experienced by these subjects after treatment with INO-3106, total surgery-free time, and increase in surgery-free time. [Figure 6] 6 shows the results of an experiment assessing HPV6 E6 and E7 cellular immune responses for subject 601.

[0015] Detailed Description of the Preferred Embodiments Definition. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise.

[0016] For the recitation of numerical ranges herein, each intervening number is expressly contemplated to the same degree of precision. For example, for the range 6 to 9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0 to 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 expressly contemplated.

[0017] a. adjuvant As used herein, "adjuvant" can mean any molecule that encodes a nucleic acid sequence as described below, added to the DNA plasmid vaccines described herein to enhance the antigenicity of one or more antigens encoded by the DNA plasmid.

[0018] b.Antibodies "Antibody" may refer to antibodies of the classes IgG, IgM, IgA, IgD, or IgE, or fragments, fragments, or derivatives thereof, including Fab, F(ab'), Fd, and single-chain antibodies, diabodies, bispecific antibodies, bifunctional antibodies, and derivatives thereof. The antibody may be an antibody isolated from a mammalian serum sample, a polyclonal antibody, an affinity-purified antibody, or a mixture thereof, which exhibits sufficient binding specificity for a desired epitope or a sequence derived therefrom.

[0019] c.Antigen "Antigen" refers to a protein having an HPV E6 or HPV E7 domain, and preferably an E6 and E7 fusion with an endoprotease cleavage site therebetween. Antigens include SEQ ID NO: 2 (subtype 6), fragments thereof of lengths described herein, variants, i.e., proteins having a sequence homologous to SEQ ID NO: 2 described herein, fragments of variants of lengths described herein, and combinations thereof. Antigens may have the IgE leader sequence of SEQ ID NO: 4, or may have such sequence removed from the N-terminus. Antigens may optionally include a signal peptide, such as one from another protein.

[0020] d. coding sequence As used herein, "coding sequence" or "encoding nucleic acid" may be meant to refer to a nucleic acid (RNA or DNA molecule) comprising a nucleotide sequence encoding an antigen as described in section c above. The coding sequence may further comprise initiation and termination signals operably linked to regulatory elements, including a promoter and polyadenylation signal, capable of directing expression in the cells of an individual or mammal to which the nucleic acid is administered. The coding sequence may further comprise a sequence encoding a signal peptide, e.g., an IgE leader sequence such as SEQ ID NO:3.

[0021] e. complement As used herein, "complement" or "complementary" can refer to nucleic acids and can refer to Watson-Crick (e.g., AT / U and CG) or Hoogsteen base pairing between nucleotides or nucleotide analogs of a nucleic acid molecule.

[0022] f. Fragment "Fragment" may refer to a polypeptide fragment of an antigen that is capable of eliciting an immune response in a mammal against the antigen. A fragment of an antigen may be 100% identical to the full length, except for the deletion of at least one amino acid from the N- and / or C-terminus, in each case with or without a signal peptide and / or methionine at position 1. Fragments may comprise 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, or 99% or more of the length of a particular full-length antigen, excluding any added heterologous signal peptide. Fragments preferably include fragments of polypeptides that are 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more homologous to the antigen, and may additionally include an N-terminal methionine or heterologous signal peptide, which are not included in calculating the percent homology. The fragment may further comprise a signal peptide, such as an N-terminal methionine and / or an immunoglobulin signal peptide, e.g., an IgE or IgG signal peptide. The N-terminal methionine and / or signal peptide may be linked to the fragment of the antigen.

[0023] Fragments of nucleic acid sequences encoding antigens can be 100% identical to the full length, except for lacking at least one nucleotide from the 5' and / or 3' end, in each case with or without sequences encoding a signal peptide and / or methionine at position 1. Fragments can comprise 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, or 99% or more percent of the length of the specified full-length coding sequence, excluding any added heterologous signal peptide. Fragments preferably include fragments encoding polypeptides that are 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more homologous to the antigen, and may optionally include sequences encoding an N-terminal methionine or heterologous signal peptide, which are not included in calculating percent homology. The fragment may further comprise a coding sequence for a signal peptide, such as an N-terminal methionine and / or an immunoglobulin signal peptide, e.g., an IgE or IgG signal peptide. The coding sequence encoding the N-terminal methionine and / or signal peptide may be linked to the fragment of the coding sequence.

[0024] g.Identical As used herein, "identical" or "identity" in the context of two or more nucleic acid or polypeptide sequences can mean that the sequences have a specific percentage of residues that are the same across a specific region. The percentage can be calculated by optimally aligning the two sequences, comparing the two sequences across a specific region, determining the number of positions where 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 specific region, and raising the result to the power of 100 to obtain the percentage of sequence identity. If the two sequences have different lengths or the alignment results in one or more skewed ends, and a specific comparison region contains only a single sequence, the residues of the single sequence are included in the denominator rather than the numerator of the calculation. When comparing DNA and RNA, thymine (T) and uracil (U) can be considered equivalent. Identity can be determined manually or by using a computer sequence algorithm such as BLAST or BLAST 2.0.

[0025] h. immune response As used herein, "immune response" can refer to the activation of a host's immune system, e.g., a mammal's immune system, in response to the introduction of one or more antigens via the provided DNA plasmid vaccines. The immune response can be in the form of a cellular or humoral response, or both.

[0026] i.Nucleic acid As used herein, "nucleic acid" or "oligonucleotide" or "polynucleotide" can refer to at least two nucleotides covalently linked together. A depiction of a single strand also defines the sequence of the complementary strand. Thus, a nucleic acid also encompasses the complementary strand of the depicted 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 complements. A single 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.

[0027] Nucleic acids can be single-stranded or double-stranded, or can contain portions of both double-stranded and single-stranded sequences. Nucleic acids can be DNA, both genomic and cDNA, RNA, or hybrids, and can contain combinations of deoxyribonucleotides and ribonucleotides, as well as combinations of bases including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine, hypoxanthine, isocytosine, and isoguanine. Nucleic acids can be obtained by chemical synthesis or by recombinant methods.

[0028] j. operably linked As used herein, "operably linked" may mean that the expression of a gene is under the control of a promoter to which it is spatially connected. The 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, variations in this distance may be accommodated without loss of promoter function.

[0029] k. Promoter As used herein, a "promoter" can refer to a synthetic or naturally occurring molecule that can confer, activate, or enhance expression of a nucleic acid in a cell. A promoter can contain one or more specific transcriptional regulatory sequences to further enhance expression and / or modify its spatial and / or temporal expression. A promoter can also contain distal enhancer or repressor elements, which can be located as much as several thousand base pairs from the start site of transcription. Promoters can be derived from sources including viruses, bacteria, fungi, plants, insects, and animals. A promoter can regulate the expression of genetic components constitutively or differentially depending on the cell, tissue, or organ in which expression occurs, or on the developmental stage in which expression occurs, or in response to external stimuli such as physiological stress, pathogens, metal ions, or inducers. Representative examples of promoters include the bacteriophage T7 promoter, bacteriophage T3 promoter, SP6 promoter, lac operator-promoter, tac promoter, SV40 late promoter, SV40 early promoter, RSV-LTR promoter, CMV IE promoter, SV40 early promoter or SV40 late promoter, and CMV IE promoter.

[0030] l. Stringent hybridization conditions As used herein, "stringent hybridization conditions" refers to conditions under which a first nucleic acid sequence (e.g., a probe) hybridizes to a second nucleic acid sequence (e.g., a target) as in a complex mixture of nucleic acids. Stringent conditions are sequence-dependent and will vary in different circumstances. Stringent conditions may be selected to be approximately 5-10°C lower than the thermal melting point (Tm) of a particular sequence at a defined ionic strength and pH. The Tm may be the temperature (under defined ionic strength, pH, and nucleic acid concentration) at which 50% of the probes complementary to the target hybridize to the target sequence at equilibrium (because the target sequence is present in excess, at Tm, 50% of the probes are occupied at equilibrium). Stringent conditions can be those in which the salt concentration is less than about 1.0 M sodium ion, e.g., about 0.01 to 1.0 M sodium ion (or other salt), at pH 7.0 to 8.3, and the temperature is at least about 30°C for short probes (e.g., about 10 to 50 nucleotides) and at least about 60°C for long probes (e.g., more than about 50 nucleotides). Stringent conditions can also be achieved with the addition of destabilizing agents such as formamide. For selective or specific hybridization, a positive signal can be at least 2 to 10 times background hybridization. Exemplary stringent hybridization conditions include: 50% formamide, 5x SSC, and 1% SDS, incubated at 42°C, or 5x SSC, 1% SDS, incubated at 65°C, washed in 0.2x SSC, and 0.1% SDS at 65°C.

[0031] m. Substantially complementary As used herein, "substantially complementary" can mean that a first sequence is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to the complement of a 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 or more nucleotides or amino acids, or that the two sequences hybridize under stringent hybridization conditions.

[0032] n. Substantially identical As used herein, "substantially identical" can mean that a first and second sequence are at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 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 or more nucleotides or amino acids, or to nucleic acids, where the first sequence is substantially complementary to the complement of the second sequence.

[0033] o.Variant "Variant," as used herein with respect to a nucleic acid, can mean (i) a portion or fragment of a referenced nucleotide sequence, (ii) the complement of a referenced nucleotide sequence or a portion thereof, (iii) a nucleic acid that is substantially identical to a referenced nucleic acid or its complement, or (iv) a nucleic acid that hybridizes under stringent conditions to a referenced nucleic acid, its complement, or a sequence substantially identical thereto.

[0034] "Variant" refers to a peptide or polypeptide that differs in amino acid sequence by amino acid insertion, deletion, or conservative substitution but retains at least one biological activity. A variant can also refer to a protein having an amino acid sequence substantially identical to a reference protein having an amino acid sequence that retains at least one biological activity. Conservative amino acid substitutions, i.e., replacing an amino acid with a different amino acid having similar properties (e.g., hydrophilicity, degree, and distribution of charged regions), are recognized in the art as typically resulting in minor changes. These minor changes can be identified, in part, by considering the hydrophilicity index of the amino acid, as understood in the art. Kyte et al., J. Mol. Biol. 157:105-132 (1982). The hydropathic index of an amino acid is based on consideration of its hydrophobicity and charge. It is known in the art that amino acids with similar hydropathic indexes can be substituted and still retain protein function. In one embodiment, amino acids with hydropathic indexes of ±2 are substituted. The hydrophilicity of amino acids can also be used to identify substitutions that result in proteins that retain biological function. Consideration of the hydrophilicity of amino acids in the context of a peptide allows for calculation of the peptide's greatest local average hydrophilicity, a useful measure that has been reported to correlate well with antigenicity and immunogenicity. U.S. Pat. No. 4,554,101, incorporated herein by reference in its entirety, discloses that substitution of amino acids with similar hydrophilicity values ​​can result in peptides that retain biological activity, e.g., immunogenicity, as understood in the art. Substitutions can be made with amino acids with hydrophilicity values ​​within ±2 of each other. Both the hydrophobicity index and hydrophilicity value of an amino acid are influenced by the specific side chain of that amino acid. Consistent with this observation, it is understood that amino acid substitutions that are compatible with biological function depend on the relative similarity of amino acids, particularly their side chains, as revealed by hydrophobicity, hydrophilicity, charge, size, and other properties.

[0035] p. Vector As used herein, "vector" can refer to a nucleic acid sequence that contains a replication origin.Vector can be a plasmid, bacteriophage, bacterial artificial chromosome, or yeast artificial chromosome.Vector can be a DNA or RNA vector.Vector can be either a self-replicating extrachromosomal vector or a vector that is integrated into host genome.

[0036] Improved vaccines are disclosed that result from a multiphase strategy to enhance the cellular immune response induced by immunogens. Modified consensus sequences have been generated. Genetic modifications, including codon optimization, RNA optimization, and the addition of highly efficient immunoglobulin leader sequences, are also disclosed. The novel constructs are designed to induce stronger and broader cellular immune responses than the corresponding codon-optimized immunogens.

[0037] Improved HPV vaccines are based on proteins and gene constructs encoding proteins with epitopes that are particularly effective as immunogens to mediate preventive or therapeutic strategies against RRP. Thus, the vaccine can induce a therapeutic or preventive immune response. In some embodiments, the means for delivering the immunogen is a DNA vaccine, a recombinant vaccine, a protein subunit vaccine, a composition containing an immunogen, an attenuated vaccine, or a killed vaccine. In some embodiments, the vaccine comprises a combination selected from the group consisting of one or more DNA vaccines, one or more recombinant vaccines, one or more protein subunit vaccines, one or more compositions containing an immunogen, one or more attenuated vaccines, and one or more killed vaccines.

[0038] According to some embodiments, the vaccine is delivered to an individual to regulate the activity of the individual's immune system, thereby enhancing the immune response to HPV, which treats RRP.When the nucleic acid molecule encoding the protein is taken up by the individual's cells, the nucleotide sequence is expressed in the cells, and the protein is thereby delivered to the individual.Methods are provided for delivering the coding sequence of the protein on a nucleic acid molecule such as a plasmid as part of a recombinant vaccine, as part of an attenuated vaccine, as an isolated protein, or as part of a vector.

[0039] Compositions and methods are provided that provide prophylactic and / or therapeutic treatment for RRP in an individual.

[0040] The composition for delivering a nucleic acid molecule comprising a nucleotide sequence encoding an immunogen is operably linked to a regulatory element. The composition may comprise a plasmid encoding the immunogen, a recombinant vaccine comprising a nucleotide sequence encoding the immunogen, a live attenuated pathogen encoding and / or comprising a protein of the present invention, a killed pathogen comprising a protein of the present invention, or a liposome or subunit vaccine comprising a protein of the present invention. The present invention further relates to an injectable pharmaceutical composition comprising the composition.

[0041] An embodiment of the present invention provides a composition comprising at least one nucleotide sequence comprising an HPV6 E6-E7 fusion antigen.

[0042] Another aspect provides a composition comprising one or more nucleotide sequences encoding an HPV6 E6-E7 fusion antigen selected from the group consisting of a nucleotide sequence encoding SEQ ID NO:2, a nucleotide sequence that is at least 95% homologous to a nucleotide sequence encoding SEQ ID NO:2, a fragment of a nucleotide sequence encoding SEQ ID NO:2, and a nucleotide sequence that is at least 95% homologous to a fragment of a nucleotide sequence encoding SEQ ID NO:2.

[0043] In some embodiments, the composition comprises an HPV6 E6-E7 fusion antigen selected from the group consisting of a nucleotide sequence encoding SEQ ID NO:2, a nucleotide sequence that is at least 95% homologous to a nucleotide sequence encoding SEQ ID NO:2, a fragment of a nucleotide sequence encoding SEQ ID NO:2, and a nucleotide sequence that is at least 95% homologous to a fragment of a nucleotide sequence encoding SEQ ID NO:2.

[0044] In another aspect of the present invention, there is provided a composition comprising one or more nucleotide sequences encoding an HPV6 E6-E7 fusion antigen selected from the group consisting of: SEQ ID NO:1, a nucleotide sequence that is at least 95% homologous to SEQ ID NO:1, a fragment of SEQ ID NO:1, a nucleotide sequence that is at least 95% homologous to a fragment of SEQ ID NO:1.

[0045] In some embodiments, the nucleotide sequences described herein lack a leader sequence. In one embodiment, the nucleotide sequence comprising the HPV6 E6-E7 fusion antigen lacks a leader sequence. In particular, an HPV6 E6-E7 fusion antigen comprising a nucleotide sequence encoding SEQ ID NO:2 lacks a 5'-terminal leader sequence, for example, a nucleotide sequence encoding SEQ ID NO:4. In particular, an HPV6 E6-E7 fusion antigen comprising the nucleotide sequence SEQ ID NO:1 lacks a 5'-terminal leader sequence, for example, a nucleotide sequence encoding SEQ ID NO:3.

[0046] In some embodiments, the nucleotide sequences of the invention may be 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, preferably 95%, 96%, 97%, 98%, or 99%, or 98% or 99% homologous to the nucleotide sequences provided.

[0047] The provided nucleotide sequences can be contained in one of a variety of known vectors or delivery systems, including plasmids, viral vectors, lipid vectors, nanoparticles, preferably plasmids.

[0048] In an additional aspect, pharmaceutical compositions comprising the disclosed nucleotide sequences are provided.

[0049] In some embodiments, there are methods for inducing an effective immune response in an individual against two or more subtypes of HPV, thereby providing prophylactic or therapeutic treatment for RRP, comprising administering to the individual a composition comprising one or more of the provided nucleotide sequences, preferably the composition having two or more antigens. The method preferably comprises introducing the provided nucleotide sequences into the individual by electroporation.

[0050] SEQ ID NO:1 comprises a nucleotide sequence encoding a consensus immunogen of HPV6 E6 and E7 proteins. SEQ ID NO:1 comprises an IgE leader sequence SEQ ID NO:3 linked to the nucleotide sequence at the 5' end of SEQ ID NO:1. SEQ ID NO:2 comprises the amino acid sequence of a consensus immunogen of HPV6 E6 and E7 proteins. SEQ ID NO:2 comprises an IgE leader sequence SEQ ID NO:4 at the N-terminus of the consensus immunogen sequence. The IgE leader sequence is SEQ ID NO:4 and can be encoded by SEQ ID NO:3. Further information regarding HPV6 E6-E7 fusion antigens can be found in at least U.S. Patent No. 9,050,287, which is incorporated by reference in its entirety.

[0051] In some embodiments, the vaccine comprises SEQ ID NO:2, or a nucleic acid molecule encoding SEQ ID NO:2.

[0052] A fragment of SEQ ID NO:2 can be 100% identical to the full length except for lacking at least one amino acid from the N- and / or C-terminus, in each case with or without a signal peptide and / or methionine at position 1. A fragment of SEQ ID NO:2 can comprise 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 full-length SEQ ID NO:2, excluding any added heterologous signal peptide. Fragments preferably include fragments of SEQ ID NO:2 that are 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more homologous to SEQ ID NO:2, and additionally include an N-terminal methionine or heterologous signal peptide, which are not included in calculating percent homology. The fragment can further comprise a signal peptide, such as an N-terminal methionine and / or an immunoglobulin signal peptide, for example, an IgE or IgG signal peptide. The N-terminal methionine and / or signal peptide can be linked to the fragment.

[0053] Fragments of nucleic acid sequence SEQ ID NO: 1 can be 100% identical to the full length, except for the deletion of at least one nucleotide from the 5' and / or 3' end, in each case with or without sequences encoding a signal peptide and / or methionine at position 1. Fragments can comprise 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, or 99% or more of the length of the full-length coding sequence SEQ ID NO: 1, excluding any added heterologous signal peptide. Fragments preferably include fragments encoding polypeptides that are 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more homologous to antigen SEQ ID NO: 2, and optionally, can include sequences encoding an N-terminal methionine or heterologous signal peptide, which are not included in calculating percent homology. The fragment may further comprise a coding sequence for a signal peptide, such as an N-terminal methionine and / or an immunoglobulin signal peptide, e.g., an IgE or IgG signal peptide. A coding sequence encoding an N-terminal methionine and / or a signal peptide may be linked to the fragment.

[0054] In some embodiments, a fragment of SEQ ID NO: 1 may comprise 786 or more nucleotides, in some embodiments, 830 or more nucleotides, in some embodiments, 856 or more nucleotides, and in some embodiments, 865 or more nucleotides. In some embodiments, a fragment of SEQ ID NO: 1, such as those described herein, may further comprise a coding sequence for an IgE leader sequence. In some embodiments, a fragment of SEQ ID NO: 1 does not comprise a coding sequence for an IgE leader sequence.

[0055] In some embodiments, a fragment of SEQ ID NO:2 may comprise 252 or more amino acids, in some embodiments, 266 or more amino acids, in some embodiments, 275 or more amino acids, and in some embodiments, 278 or more amino acids.

[0056] In one embodiment, the HPV6 E6-E7 immunogen or a nucleic acid molecule encoding the HPV6 E6-E7 immunogen is administered in combination with IL- 12. In one embodiment, the IL-12 is encoded from a synthetic DNA plasmid.

[0057] In some embodiments, the methods involve administering a composition comprising a nucleic acid molecule encoding the p35 and / or p40 subunit of IL-12.

[0058] SEQ ID NO: 5 comprises the nucleotide sequence encoding the p35 subunit of IL-12. SEQ ID NO: 6 comprises the amino acid sequence of the p35 subunit of IL-12.

[0059] In some embodiments, the vaccine comprises SEQ ID NO:6, or a nucleic acid molecule encoding SEQ ID NO:6.

[0060] A fragment of SEQ ID NO:6 can be 100% identical to the full length except for lacking at least one amino acid from the N- and / or C-terminus, in each case with or without a signal peptide and / or methionine at position 1. A fragment of SEQ ID NO:6 can comprise 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 full-length SEQ ID NO:6, excluding any added heterologous signal peptide. Fragments preferably include fragments of SEQ ID NO:6 that are 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more homologous to SEQ ID NO:6, and may additionally include an N-terminal methionine or heterologous signal peptide, which are not included in calculating percent homology. The fragment can further comprise a signal peptide, such as an N-terminal methionine and / or an immunoglobulin signal peptide, for example, an IgE or IgG signal peptide. The N-terminal methionine and / or signal peptide can be linked to the fragment.

[0061] Fragments of nucleic acid sequence SEQ ID NO: 5 can be 100% identical to the full length, except for the deletion of at least one nucleotide from the 5' and / or 3' end, in each case with or without sequences encoding a signal peptide and / or methionine at position 1. Fragments can comprise 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, or 99% or more of the length of the full-length coding sequence SEQ ID NO: 5, excluding any added heterologous signal peptide. Fragments preferably include fragments encoding polypeptides that are 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more homologous to the antigen SEQ ID NO: 6, and can optionally include sequences encoding an N-terminal methionine or heterologous signal peptide, which are not included in calculating the percent homology. The fragment may further comprise a coding sequence for a signal peptide, such as an N-terminal methionine and / or an immunoglobulin signal peptide, e.g., an IgE or IgG signal peptide. A coding sequence encoding an N-terminal methionine and / or a signal peptide may be linked to the fragment.

[0062] In some embodiments, a fragment of SEQ ID NO:5 may comprise 500 or more nucleotides, in some embodiments 550 or more nucleotides, in some embodiments 600 or more nucleotides, and in some embodiments 630 or more nucleotides. In some embodiments, a fragment of SEQ ID NO:5, such as those described herein, may further comprise a coding sequence for an IgE leader sequence. In some embodiments, a fragment of SEQ ID NO:5 does not comprise a coding sequence for an IgE leader sequence.

[0063] In some embodiments, a fragment of SEQ ID NO: 6 may comprise 150 or more amino acids, in some embodiments, 175 or more amino acids, in some embodiments, 200 or more amino acids, and in some embodiments, 210 or more amino acids.

[0064] SEQ ID NO: 7 contains the nucleotide sequence encoding the p40 subunit of IL-12. SEQ ID NO: 8 contains the amino acid sequence of the p35 subunit of IL-12.

[0065] In some embodiments, the vaccine comprises SEQ ID NO:8, or a nucleic acid molecule encoding SEQ ID NO:8.

[0066] A fragment of SEQ ID NO:8 can be 100% identical to the full length except for lacking at least one amino acid from the N- and / or C-terminus, in each case with or without a signal peptide and / or methionine at position 1. A fragment of SEQ ID NO:8 can comprise 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 full-length SEQ ID NO:8, excluding any added heterologous signal peptide. Fragments preferably include fragments of SEQ ID NO:8 that are 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more homologous to SEQ ID NO:8, and may additionally include an N-terminal methionine or heterologous signal peptide, which are not included in calculating percent homology. The fragment can further comprise a signal peptide, such as an N-terminal methionine and / or an immunoglobulin signal peptide, for example, an IgE or IgG signal peptide. The N-terminal methionine and / or signal peptide can be linked to the fragment.

[0067] Fragments of nucleic acid sequence SEQ ID NO:7 can be 100% identical to the full length, except for the deletion of at least one nucleotide from the 5' and / or 3' end, in each case with or without sequences encoding a signal peptide and / or methionine at position 1. Fragments can comprise 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, or 99% or more of the length of the full-length coding sequence SEQ ID NO:7, excluding any added heterologous signal peptide. Fragments preferably include fragments encoding polypeptides that are 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more homologous to the antigen SEQ ID NO:8, and can optionally include sequences encoding an N-terminal methionine or heterologous signal peptide, which are not included in calculating the percent homology. The fragment may further comprise a coding sequence for a signal peptide, such as an N-terminal methionine and / or an immunoglobulin signal peptide, e.g., an IgE or IgG signal peptide. A coding sequence encoding an N-terminal methionine and / or a signal peptide may be linked to the fragment.

[0068] In some embodiments, a fragment of SEQ ID NO:7 may comprise 850 or more nucleotides, in some embodiments, 900 or more nucleotides, in some embodiments, 930 or more nucleotides, and in some embodiments, 960 or more nucleotides. In some embodiments, a fragment of SEQ ID NO:7, such as those described herein, may further comprise a coding sequence for an IgE leader sequence. In some embodiments, a fragment of SEQ ID NO:7 does not comprise a coding sequence for an IgE leader sequence.

[0069] In some embodiments, a fragment of SEQ ID NO: 8 may comprise 250 or more amino acids, in some embodiments, 275 or more amino acids, in some embodiments, 300 or more amino acids, and in some embodiments, 315 or more amino acids.

[0070] In some embodiments, the method comprises simultaneous administration of (a) a composition comprising a nucleic acid molecule encoding an HPV6 antigen (e.g., HPV6 E6-E7 fusion antigen) disclosed herein and (b) a composition comprising a nucleic acid molecule encoding one or more IL-12 subunits (e.g., p35 and / or p40) disclosed herein. In some embodiments, the method comprises prior administration of a composition comprising a nucleic acid molecule encoding an HPV6 antigen (e.g., HPV6 E6-E7 fusion antigen) disclosed herein followed by administration of a composition comprising a nucleic acid molecule encoding one or more IL-12 subunits (e.g., p35 and / or p40) disclosed herein. In some embodiments, the method comprises prior administration of a composition comprising a nucleic acid molecule encoding one or more IL-12 subunits (e.g., p35 and / or p40) disclosed herein followed by administration of a composition comprising a nucleic acid molecule encoding an HPV6 antigen (e.g., HPV6 E6-E7 fusion antigen) disclosed herein.

[0071] Provided herein are methods for treating or preventing RRP in a subject by inducing an immune response in the individual against HPV, comprising administering to the individual a composition comprising a nucleic acid sequence provided herein. In some embodiments, the method also comprises introducing the nucleic acid sequence into the individual by electroporation.

[0072] In some aspects, there are methods for treating or preventing RRP in a subject by inducing an immune response in the individual against HPV, the method comprising administering to the individual a composition comprising an amino acid sequence provided herein. In some embodiments, the method also comprises introducing the amino acid sequence into the individual by electroporation.

[0073] Improved vaccines include proteins and gene constructs that encode proteins with epitopes that are particularly effective as immunogens that can induce anti-HPV immune responses. Thus, vaccines can be provided to induce therapeutic or prophylactic immune responses. In some embodiments, the means for delivering the immunogen is a DNA vaccine, a recombinant vaccine, a protein subunit vaccine, a composition containing an immunogen, an attenuated vaccine, or a killed vaccine. In some embodiments, the vaccine comprises a combination selected from the group consisting of one or more DNA vaccines, one or more recombinant vaccines, one or more protein subunit vaccines, one or more compositions containing an immunogen, one or more attenuated vaccines, and one or more killed vaccines.

[0074] Aspects of the invention provide methods for delivering coding sequences for proteins on nucleic acid molecules such as plasmids, as isolated proteins or as part of a vector, as part of a recombinant vaccine and as part of an attenuated vaccine.

[0075] According to some aspects of the present invention, compositions and methods for prophylactically and / or therapeutically immunizing an individual are provided.

[0076] DNA vaccines are described in U.S. Patent Nos. 5,593,972, 5,739,118, 5,817,637, 5,830,876, 5,962,428, 5,981,505, 5,580,859, 5,703,055, 5,676,594, and the priority applications cited therein, each of which is incorporated herein by reference.In addition to the delivery protocols described in these applications, alternative methods of delivering DNA are described in U.S. Patent Nos. 4,945,050 and 5,036,006, both of which are incorporated herein by reference.

[0077] The present invention relates to improved live attenuated vaccines, improved killed vaccines, and improved vaccines that use recombinant vectors to deliver foreign genes encoding antigens, as well as subunit and glycoprotein vaccines. Examples of live attenuated vaccines, those that use recombinant vectors to deliver foreign antigens, subunit vaccines, and glycoprotein vaccines are described in U.S. Patent Nos. 4,510,245, 4,797,368, 4,722,848, 4,790,987, 4,920,209, 5,017,487, 5,077,044, 5,110,587, 5,112,749, 5,174,993, 5,223,424, 5,225,336, 5,240,703, 5,242,829, 5,294,441, and 5,294,548. Nos. 5,310,668, 5,387,744, 5,389,368, 5,424,065, 5,451,499, 5,453,364, 5,462,734, 5,470,734, 5,474,935, 5,482,713, 5,591,439, 5,643,579, 5,650,309, 5,698,202, 5,955,088, 6,034,298, 6,042,836, 6,156,319, and 6,589,529, each of which is incorporated herein by reference.

[0078] When taken up by a cell, the genetic construct(s) may remain present in the cell as a functional extrachromosomal molecule and / or be integrated into the cell's chromosomal DNA. DNA may be introduced into the cell, where it remains as separate genetic material in the form of a plasmid(s). Alternatively, linear DNA capable of chromosomal integration may be introduced into the cell. When introducing DNA into the cell, reagents may be added to promote DNA integration into the chromosome. DNA sequences useful for promoting integration may also be included in the DNA molecule. Alternatively, RNA may be administered to the cell. It is also contemplated to provide the genetic construct as a linear microchromosome containing a centromere, telomeres, and an origin of replication. The genetic construct may remain part of the genetic material in an attenuated live microorganism or recombinant microbial vector that survives in the cell. The genetic construct may be part of the genome of a recombinant viral vaccine, where the genetic material is either integrated into the cell's chromosome or remains extrachromosomal. The genetic construct contains the regulatory elements necessary for gene expression of the nucleic acid molecule. These elements include a promoter, start codon, stop codon, and polyadenylation signal. In addition, enhancers are often required for gene expression of sequences encoding target proteins or immunomodulatory proteins. These elements must be operably linked to the sequence encoding the desired protein, and the regulatory elements must be operative in the individual to whom they are administered.

[0079] The start codon and stop codon are generally considered to be part of the nucleotide sequence encoding the desired protein. However, these elements must be functional in the individual to whom the gene construct is administered. The start codon and stop codon must be in frame with the coding sequence.

[0080] The promoter and polyadenylation signal used must be functional in the cells of the individual.

[0081] Examples of promoters useful in practicing the present invention, particularly in the production of genetic vaccines for humans, include, but are not limited to, promoters from simian virus 40 (SV40), mouse mammary tumor virus (MMTV) promoter, human immunodeficiency virus (MV) such as the BIV long terminal repeat (LTR) promoter, Moloney virus, ALV, cytomegalovirus (CMV) such as the CMV immediate-early promoter, Epstein-Barr virus (EBV), Rous sarcoma virus (RSV), and human genes such as human actin, human myosin, human hemoglobin, human muscle creatine, and human metallothionein.

[0082] Examples of polyadenylation signals useful for carrying out the present invention, particularly in the production of human genetic vaccines, include, but are not limited to, SV40 polyadenylation signals and LTR polyadenylation signals. In particular, the SV40 polyadenylation signal found in pCEP4 plasmid (Invitrogen, San Diego CA), known as the SV40 polyadenylation signal, is used.

[0083] In addition to the regulatory elements required for DNA expression, other elements can also be included in DNA molecule.Such additional elements include enhancer.Enhancer can be selected from the group including but not limited to human actin, human myosin, human hemoglobin, human muscle creatine, and viral enhancer such as those derived from CMV, RSV and EBV.

[0084] The gene construct can be provided with a mammalian origin of replication to maintain the construct extrachromosomally and produce multiple copies of the construct in cells. Plasmids pVAX 1, pCEP4, and pREP4 from Invitrogen (San Diego, CA) contain the Epstein-Barr virus origin of replication and the nuclear antigen EBNA-1 coding region, which provides high-copy episomal replication without integration.

[0085] In some preferred embodiments related to immunization applications, nucleic acid molecule(s) are delivered that contain a nucleotide sequence encoding a protein of the invention and, in addition, a gene for a protein that further enhances the immune response to such target protein. Examples of such genes are those encoding other cytokines and lymphokines such as alpha-interferon, gamma-interferon, platelet-derived growth factor (PDGF), TNFα, TNFβ, GM-CSF, epidermal growth factor (EGF), IL-1, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12, IL-18, MHC, CD80, CD86, and IL-15, including IL-15 with the signal sequence deleted and optionally including a signal peptide from IgE. Other genes that may be useful include those encoding: MCP-1, MIP-1α, MIP-1p, IL-8, RANTES, L-selectin, P-selectin, E-selectin, CD34, GlyCAM-1, MadCAM-1, LFA-1, VLA-1, Mac-1, pl50.95, PECAM, ICAM-1, ICAM-2, ICAM-3, CD2, LFA-3, M-CSF, G-CSF, IL-4, mutant forms of IL-18, CD4 0, CD40L, vascular growth factor, IL-7, 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, 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

[0086] If, for any reason, it is desirable to eliminate cells that receive the gene construct, additional elements can be added that target cells for destruction. An expressible form of the herpes thymidine kinase (tk) gene can be included in the gene construct. The drug ganciclovir can be administered to an individual, causing the selective killing of any cells that produce tk, thus providing a means for the selective destruction of cells that carry the gene construct.

[0087] To maximize protein production, regulatory sequences can be selected that are adequate for gene expression in the cells to which the construct is administered.Furthermore, codons that are most efficiently transcribed in cells can be selected.Those skilled in the art can generate DNA constructs that are functional in cells.

[0088] In some embodiments, genetic constructs can be provided in which the coding sequences for the proteins described herein are linked to an IgE signal peptide. In some embodiments, the proteins described herein are linked to an IgE signal peptide.

[0089] In some embodiments in which proteins are used, for example, one skilled in the art can use well-known techniques to produce and isolate proteins of the invention. In some embodiments in which proteins are used, for example, one skilled in the art can use well-known techniques to insert a DNA molecule encoding a protein of the invention into a commercially available expression vector for use in a well-known expression system. For example, the commercially available plasmid pSE420 (Invitrogen, San Diego, Calif.) can be used for protein production in E. coli. The commercially available plasmid pYES2 (Invitrogen, San Diego, Calif.) can be used for production in, for example, S. cerevisiae strains of yeast. The commercially available MAXBAC™ complete baculovirus expression system (Invitrogen, San Diego, Calif.) can be used for production in, for example, insect cells. The commercially available plasmids pcDNA I or pcDNA3 (Invitrogen, San Diego, Calif.) can be used for production in mammalian cells, such as Chinese hamster ovary cells. Those skilled in the art can use these commercial expression vectors and expression systems, or others, to produce proteins using routine techniques and readily available starting materials. (See, for example, Sambrook et al., Molecular Cloning a Laboratory Manual, Second Ed. Cold Spring Harbor Press (1989), incorporated herein by reference.) Thus, desired proteins can be prepared in both prokaryotic and eukaryotic systems, resulting in a spectrum of processed forms of the protein.

[0090] Those skilled in the art may use other commercially available expression vectors and expression systems or produce vectors using well-known methods and readily available starting materials. Expression systems containing the necessary control sequences, such as promoters and polyadenylation signals, and preferably enhancers, are readily available for a variety of hosts and are known in the art. See, for example, Sambrook et al., Molecular Cloning a Laboratory Manual, Second Ed. Cold Spring Harbor Press (1989). The gene construct contains a protein-coding sequence operably linked to a promoter that is functional in the cell line into which the construct will be transfected. Examples of constitutive promoters include promoters from cytomegalovirus or SV40. Examples of inducible promoters include the mouse mammary leukemia virus or metallothionein promoter. Those skilled in the art can easily produce gene constructs useful for transfecting cells with DNA encoding the protein of the present invention from readily available starting materials. An expression vector containing DNA encoding the protein is used to transform a compatible host, which is then cultured and maintained under conditions that allow expression of the foreign DNA.

[0091] The produced protein is recovered by lysing cells or from the culture medium as needed, as known to those skilled in the art.Those skilled in the art can use well-known techniques to isolate the protein produced by using such expression systems.As described above, the method of purifying protein from natural sources using antibodies that specifically bind to specific proteins can be equally applied to the purification of proteins produced by recombinant DNA methodology.

[0092] In addition to producing proteins by recombinant techniques, automated peptide synthesizers can also be used to produce isolated, essentially pure proteins. Such techniques are well known to those skilled in the art and are useful for derivatives having substitutions not provided for in DNA-encoded protein production.

[0093] Nucleic acid molecules can be delivered using any of several well-known techniques, including DNA injection (also called DNA vaccination), recombinant vectors such as recombinant adenovirus, recombinant adenovirus-associated virus, and recombinant vaccinia.

[0094] 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 suppository, or by irrigation into mucosal tissues, such as vaginal, rectal, urethral, ​​buccal, and sublingual tissues. Preferred routes of administration include intramuscular, intraperitoneal, intradermal, and subcutaneous injection. Gene constructs may be administered by means including, but not limited to, electroporation methods and devices, conventional syringes, needleless injection devices, or "microprojectile bombardment gene guns."

[0095] Examples of preferred electroporation devices and methods for enhancing delivery of DNA vaccines include those described in U.S. Patent No. 7,245,963 by Draghia-Akli et al., and U.S. Patent Publication No. 2005 / 0052630 filed by Smith et al., the contents of which are incorporated herein by reference in their entireties. Also preferred are electroporation devices and methods for enhancing delivery of DNA vaccines provided in co-pending, commonly owned U.S. patent application Ser. No. 11 / 874,072, filed October 17, 2007, which claims benefit under 35 U.S.C. 119(e) to U.S. Provisional Application No. 60 / 852,149, filed October 17, 2006, and U.S. Provisional Application No. 60 / 978,982, filed October 10, 2007, all of which are incorporated herein by reference in their entireties.

[0096] The following are examples of embodiments using electroporation techniques, discussed in more detail in the patent references mentioned above: The electroporation device can be configured to deliver pulses of energy to a desired tissue in a mammal, generating a constant current similar to a current input preset by a user. The electroporation device includes an electroporation component and an electrode assembly or handle assembly. The electroporation component can include or incorporate one or more of various elements of an electroporation device, including a controller, a current waveform generator, an impedance tester, a waveform logger, an input element, a status reporting element, a communication port, a memory component, a power supply, and a power switch. The electroporation component can function as one element of the electroporation device, with other elements being separate elements (or components) that communicate with the electroporation component. In some embodiments, the electroporation component can function as more than one element of the electroporation device, which may communicate with yet other elements of the electroporation device that are separate from the electroporation component. The use of electroporation technology to deliver improved HPV vaccines is not limited by the elements of the electroporation device being part of a single electromechanical or mechanical device, as the elements can function as a single device or as separate elements that communicate with each other. The electroporation component can deliver pulses of energy that generate a constant current in the desired tissue and includes a feedback mechanism. The electrode assembly includes an electrode array having multiple electrodes in a spatial arrangement, and the electrode assembly receives the pulses of energy from the electroporation component and delivers them to the desired tissue through the electrodes. At least one of the multiple electrodes is neutral during delivery of the pulses of energy, measures the impedance at the desired tissue, and communicates the impedance to the electroporation component.A feedback mechanism can receive the measured impedance and can adjust the pulses of energy delivered by the electroporation component to maintain a constant current.

[0097] In some embodiments, the multiple electrodes can deliver pulses of energy in a decentralized pattern. In some embodiments, the multiple electrodes can deliver pulses of energy in a decentralized pattern through control of the electrodes under a programmed sequence, the programmed sequence being input into the electroporation component by a user. In some embodiments, the programmed sequence includes multiple pulses delivered sequentially, each pulse of the multiple pulses being delivered by at least two active electrodes with one indifferent electrode measuring impedance, and each subsequent pulse of the multiple pulses being delivered by a different one of the at least two active electrodes with one indifferent electrode measuring impedance.

[0098] In some embodiments, the feedback mechanism is implemented by either hardware or software. Preferably, the feedback mechanism is implemented by an analog closed-loop circuit. Preferably, this feedback occurs every 50 μs, 20 μs, 10 μs, or 1 μs, but is preferably real-time feedback or instantaneous (i.e., substantially instantaneous, as determined by available techniques for determining response time). In some embodiments, an indifferent electrode measures the impedance in the desired tissue and communicates the impedance to the feedback mechanism, which responds to the impedance and adjusts the pulse of energy to maintain a constant current at a value similar to the preset current. In some embodiments, the feedback mechanism maintains a constant current continuously and instantaneously during delivery of the pulse of energy.

[0099] In some embodiments, the nucleic acid molecule is delivered to cells in conjunction with the administration of a polynucleotide function enhancer or genetic vaccine facilitator. Polynucleotide function enhancers are described in U.S. Patent Nos. 5,593,972, 5,962,428, and International Patent Application No. PCT / US94 / 00899, filed January 26, 1994, each of which is incorporated herein by reference. Genetic vaccine facilitator agents are described in U.S. Application No. 021,579, filed April 1, 1994, each of which is incorporated herein by reference. The adjuvant administered in conjunction with the nucleic acid molecule may be administered as a mixture with the nucleic acid molecule, or may be administered separately and simultaneously, before or after administration of the nucleic acid molecule. In addition, other agents that may function as transfection and / or replication and / or inflammatory agents and may be co-administered with GVFs include growth factors, cytokines, and lymphocytes, such as α-interferon, γ-interferon, GM-CSF, platelet-derived growth factor (PDGF), TNF, epidermal growth factor (EGF), IL-1, IL-2, IL-4, IL-6, IL-10, IL-12, and IL-15, as well as fibroblast growth factors, surfactants, such as immune-stimulating complexes (ISCOMS), Freund's incomplete adjuvant, LPS analogs including monophosphoryl lipid A (WL), muramyl peptides, quinone analogs, and vesicles such as squalene and squalene; hyaluronic acid may also be used and administered in conjunction with gene constructs. In some embodiments, immunomodulatory proteins may be used as GVFs. In some embodiments, nucleic acid molecules are provided in association with PLG to enhance delivery / uptake.

[0100] Pharmaceutical compositions according to the present invention contain about 1 nanogram to about 2000 micrograms of DNA. In some preferred embodiments, pharmaceutical compositions according to the present invention contain about 5 nanograms to about 1000 micrograms of DNA. In some preferred embodiments, pharmaceutical compositions contain about 10 to about 800 micrograms of DNA. In some preferred embodiments, pharmaceutical compositions contain about 0.1 to about 500 micrograms of DNA. In some preferred embodiments, pharmaceutical compositions contain about 1 to about 350 micrograms of DNA. In some preferred embodiments, pharmaceutical compositions contain about 25 to about 250 micrograms of DNA. In some preferred embodiments, pharmaceutical compositions contain about 100 to about 200 micrograms of DNA.

[0101] The pharmaceutical compositions according to the present invention are formulated according to the mode of administration to be used. When the pharmaceutical compositions are injectable pharmaceutical compositions, they are sterile, pyrogen-free, and particulate-free. An isotonic formulation is preferably used. Common additives for isotonicity include sodium chloride, dextrose, mannitol, sorbitol, and lactose. In some cases, an isotonic solution such as phosphate-buffered saline is preferred. Stabilizers include gelatin and albumin. In some embodiments, a vasoconstrictor is added to the formulation.

[0102] According to some embodiments of the present invention, a method for inducing an immune response is provided. The vaccine can be a protein-based, live attenuated vaccine, a cellular vaccine, a recombinant vaccine, or a nucleic acid or DNA vaccine. In some embodiments, a method for inducing an immune response in an individual to an immunogen, including a method for inducing a mucosal immune response, comprises administering to the individual one or more of a CTACK protein, a TECK protein, an MEC protein, and functional fragments thereof, or expressible coding sequences thereof, in combination with an isolated nucleic acid molecule encoding a protein of the invention and / or a recombinant vaccine encoding a protein of the invention and / or a subunit vaccine encoding a protein of the invention and / or a live attenuated vaccine and / or a killed vaccine. One or more of a CTACK protein, a TECK protein, an MEC protein, and functional fragments thereof can be administered before, simultaneously with, or after administration of an isolated nucleic acid molecule encoding an immunogen and / or a recombinant vaccine encoding an immunogen and / or a subunit vaccine comprising an immunogen and / or a live attenuated vaccine and / or a killed vaccine. In some embodiments, an isolated nucleic acid molecule encoding one or more proteins selected from the group consisting of CTACK, TECK, MEC, and functional fragments thereof is administered to the individual.

[0103] The present invention is further illustrated in the following examples. It should be understood that these examples, while illustrating embodiments of the present invention, are provided by way of illustration only. From the above discussion and these examples, those skilled in the art can ascertain the essential features of the present invention, and can make various changes and modifications to the present invention to adapt it to various uses and conditions without departing from its spirit and scope. Thus, various modifications of the present invention, in addition to those shown and described herein, will be apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims.

[0104] Each of the U.S. patents, U.S. applications, and references cited throughout this disclosure is incorporated herein by reference in its entirety. [Example]

[0105] The present invention is further defined in the following examples. It should be understood that these examples, while showing preferred embodiments of the present invention, are provided by way of illustration only. From the above discussion and these examples, those skilled in the art can ascertain the essential features of the present invention and can make various changes and modifications to the present invention to adapt it to various uses and conditions without departing from the spirit and scope thereof. Thus, various modifications of the present invention, in addition to those shown and described herein, will be apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims.

[0106] Example 1 Recurrent respiratory papillomatosis (RRP) is a rare disorder characterized by the formation of papillomas in the aerodigestive tract, usually associated with human papillomavirus (HPV) subtypes 6 and 11. Current treatment of HPV6-associated RRP and invasive malignancies could potentially be improved by the addition of HPV-specific immunotherapy. While available prophylactic HPV vaccines can generate neutralizing antibodies against the HPV major capsid protein L1, they have not demonstrated therapeutic efficacy against HPV infection or existing lesions and are unlikely to generate cytolytic T cell responses (Lin et al., Immunologic research. 2010;47(1-3):86-112). On the other hand, HPV-specific immunotherapy may have therapeutic potential to eliminate existing lesions and infections by generating immunity against the HPV virus itself and HPV-infected cells. The HPV E6 and E7 oncoproteins represent ideal targets for this type of therapeutic intervention due to their constitutive expression in HPV-associated tumors and their important role in the induction and maintenance of HPV-associated disease (Lin et al., Immunologic research. 2010;47(1-3):86-112).

[0107] The experiments presented herein investigate the efficacy of INO-3106, a DNA plasmid-based immunotherapy targeting the E6 and E7 proteins of HPV6, to form a robust immune T cell response to treat RRP.

[0108] In this study, experiments were conducted to evaluate the efficacy of INO-3106, a novel HPV6-specific immunotherapy consisting of a synthetic consensus DNA sequence encoding HPV6 E6 and E7 (Figure 1), proteins required for HPV6-induced transformation of cancer and tumor maintenance. Synthetic DNA plasmids offer several potential advantages as an immunotherapy platform, including the ability to elicit a strong immune response without evidence of genomic integration, a favorable safety profile, stability, and relative ease of manufacturing (Saha et al., Recent Pat DNA Gene Seq. 2011;5(2):92-6). Preclinical studies of INO-3106 have demonstrated a strong and specific immune response against HPV6 in animal models (Shin et al., Human vaccines & immunotherapeutics. 2012;8(4):470-8). An HPV16 / 18-specific therapy (VGX-3100, Inovio Pharmaceuticals, Inc.), designed and evaluated based on the same synthetic consensus platform, demonstrated cellular immune responses that correlated with clinical benefit in the form of dysplastic lesion regression and clearance of HPV16 / 18 infection, supporting late-stage clinical trials targeting HPV16- and 18-associated disease (Bagarazzi et al., Sci Transl Med. 2012;4(155):155ra38).

[0109] Preclinical studies have shown that the immunogenicity of DNA vaccines can be substantially increased by the use of cytokine adjuvants (Chattergoon et al., Vaccine. 2004;22(13-14):1744-50; Hanlon et al., JVirol. 2001;75(18):8424-33; Kim et al., JInterferon Cytokine Res. 1998;18(7):537-47; Kim et al., Eur JImmunol. 1998;28(3):1089-103; Operschall et al., JClinVirol. 1999;13(1-2):17-27). Importantly, engineered plasmid IL-12 gene adjuvants have been shown to enhance immunogenicity in humans when delivered using the CELLECTRA® device (Kalams et al., Journal of Infectious Diseases. 2013; Tebas et al., The Journal of infectious diseases. 2019).In multiple clinical trials using both cutaneous delivery and local muscle targeting, optimized DNA delivery via the CELLECTRA® device has been established as a highly reproducible method for rapidly generating immunity in humans for a variety of purposes, ranging from inductive prophylactic settings to therapeutic approaches (Kalams et al., Journal of Infectious Diseases. 2013; Tebas et al., The Journal of infectious diseases. 2019; Tebas et al., The New England journal of medicine. 2017; Bagarazzi et al., SciTranslMed. 2012;4(155):155ra38; Morrow et al., Molecular therapy oncolytics. 2016;3(16025); Trimble et al., Lancet. 2015;386(10008):2078-88; Morrow et al., Clinical cancer research: an official journal of the American Association for Cancer Research Research.2018;24(2):276-94, Aggarwal et al., Clinical cancer research: an official journal of the American Association for Cancer Research.2019;25(1):110-24, Morrow et al., Molecular therapy: the journal of the American Society of Gene Therapy.2015;23(3):591-601).

[0110] Here, this study demonstrates the safety and immunogenicity of a pilot study of INO-3106, with or without INO-9012 (IL-12 adjuvant), delivered intramuscularly (IM) via the EP and CELLECTRA® devices in patients with HPV6-associated RRP or malignancies. Data from this study indicate that immunotherapy with INO-3106 and IL-12 adjuvant may be a non-invasive, immune-mediated treatment option for RRP.

[0111] In this single-center, open-label, Phase 1 study, subjects with HPV6-positive RRP and malignant tumors were administered INO-3106, a DNA plasmid immunotherapy targeting the HPV6 E6 and E7 proteins, with or without INO-9012. The DNA plasmid immunotherapy encodes IL-12 and was delivered intramuscularly (IM) in combination with electroporation (EP) using the CELLECTRA® device. Patients received escalating doses of INO-3106: 3 mg once, followed by three additional doses of 6 mg, separated by three weeks. The third and fourth doses were co-administered with INO-9012. The primary objective of this study was to evaluate the safety and tolerability of INO-3106 with and without INO-9012. Secondary objectives included determining the cellular immune response to INO-3106 with and without INO-9012. Exploratory objectives included preliminary clinical efficacy of the therapy.

[0112] Four patients consented, and three met all inclusion and exclusion criteria and were enrolled in the study. The study therapy was well tolerated, with no related serious adverse events (AEs), and all related adverse events (AEs) were low-grade. Injection site pain was the most common related AE reported in all patients. Immunogenicity was demonstrated by multiple immunoassays, demonstrating the engagement and expansion of HPV6-specific cellular responses, including those characteristic of cytotoxic T cells. Preliminary efficacy was demonstrated in these patients in the form of a change in the frequency of surgery for growth resection. Prior to intervention, both patients required surgery approximately every 180 days. One patient demonstrated a more than three-fold increase in freedom from surgery (584 days), and another patient remained completely surgery-free at the time of last contact at 915 days, a more than five-fold increase in the interval between surgeries.

[0113] The experiments presented herein demonstrate that INO-3106, with and without INO-9012, was well tolerated, immunogenic, and demonstrated preliminary efficacy in patients with HPV6-associated RRP aerodigestive lesions.

[0114] The materials and methods used in these experiments are described here.

[0115] Study population This was a prospective, open-label, phase 1 trial. Male and female patients aged 18 years or older were eligible for enrollment. To be eligible, patients must have histologically documented HPV6-associated aerodigestive papilloma, premalignant lesions, or invasive aerodigestive malignancies for which recent therapy (e.g., radiation, chemotherapy) was completed at least 2 months before the first dose of study treatment. Patients must have an ECOG score of 0-1, with hepatic, renal, liver, and bone marrow function within normal ranges. Patients were excluded if they had anticipated use of immunosuppressants, required use of systemic steroids, the presence of cardiac preexcitation syndrome, or evidence of pregnancy or breastfeeding. Written informed consent was obtained from each patient before any evaluations were performed. The clinical trial was conducted in accordance with the ethical guidelines of the Declaration of Helsinki.

[0116] Immunotherapy and Electroporation Using the CELLECTRA® Device INO-3106 is a DNA plasmid encoding the E6 and E7 proteins of HPV type 6 formulated in sterile water for injection. INO-3106 contains the nucleotide sequence of SEQ ID NO: 1, which encodes the amino acid sequence of SEQ ID NO: 2. INO-9012 consists of a DNA plasmid encoding synthetic human IL-12 (p35 and p40 subunits), similarly formulated in sterile water for injection. INO-9012 contains the nucleotide sequence of SEQ ID NO: 5, which encodes the amino acid sequence of SEQ ID NO: 6 (the p35 subunit of IL-12), and the nucleotide sequence of SEQ ID NO: 7, which encodes the amino acid sequence of SEQ ID NO: 8 (the p40 subunit of IL-12). Both INO-3106 and INO-9012 were engineered using proprietary technology (Inovio Pharmaceuticals, Inc.) as previously described (Yan et al., Vaccine. 2008;26(40):5210-5, Yan et al., Vaccine. 2009;27(3):431-40). The CELLECTRA® 2000 adaptive constant-current electroporation device (Inovio Pharmaceuticals, Inc.) delivers three 52-ms controlled electrical pulses, spaced 1 second apart, to the injection site through a sterile, disposable array. When inserted into tissue, the needle array centers around the site of immunotherapy injection, creating transient pores in cell membranes to enhance cell transfection. INO-3106, with or without INO-9012, was delivered intramuscularly in a 1 mL volume, followed immediately by electroporation with the CELLECTRA® device. Treatment or dose is defined as the injection of DNA plasmid followed by EP.

[0117] Test Design After informed consent, each patient was assigned a unique patient identification code. Screening procedures to determine eligibility and collect baseline characteristics were completed within 28 days prior to the first dose. Patients received escalating doses of INO-3106, with the first dose (Day 0) delivering 3 mg of INO-3106, the second dose (Week 3) delivering 6 mg of INO-3106, and the third dose (Week 6) and fourth dose (Week 9) delivering 6 mg of INO-3106 with 1 mg of INO-9012. Each dose was delivered 3 weeks apart to allow for observation of the occurrence of any Grade 2 or higher related systemic adverse events (AEs). In total, all patient participation included a 9-week treatment period, followed by a 6-month long-term follow-up period from the last dose.

[0118] The primary objective of this study was to evaluate the safety and tolerability of INO-3106 with and without INO-9012. Secondary objectives were to determine humoral and cellular immune responses to INO-3106 with and without INO-9012, and exploratory objectives were to evaluate preliminary clinical efficacy of the treatment and, if possible, correlate efficacy with immune cell infiltration in tissues after dosing.

[0119] The trial was registered with ClinicalTrials.gov under the identifier NCT02241369. The study protocol conformed to the ethical guidelines of the 1975 Declaration of Helsinki and was reviewed and approved by the center's Institutional Review Board.

[0120] Safety evaluation The occurrence of local and systemic adverse events (AEs), vital signs, 12-lead electrocardiograms (ECGs), and laboratory abnormalities were monitored from the date of informed consent until the last follow-up visit. In particular, injection site reactions, including pain, pruritus, erythema, induration, and bruising, were assessed on the day of each treatment and for 7 consecutive days after treatment. Patients were asked about the occurrence of new AEs or illnesses and the use of concomitant medications at each visit. All events were graded according to the Common Terminology Criteria for Adverse Events (CTCAE), version 4.03, and coded using MedDRA, version 21.

[0121] Further enrollment and treatment were immediately discontinued if one-third or more patients experienced related events requiring expedited reporting, if any patient experienced a serious adverse event (SAE), unexpected grade 4 toxicity, a potentially life-threatening AE assessed as related to study treatment, or death, if three or more patients experienced the same related grade 3 or 4 AE, or if any patient reported grade 3 anaphylaxis.

[0122] Women of reproductive potential were required to complete a pregnancy test at screening and within 3 days before each dose. Treatment was discontinued in women if the pregnancy test result was positive. Laboratory parameters, including hematology, coagulation, serum chemistry (including liver function), and creatine phosphokinase (CPK), were monitored throughout the study and evaluated locally at the center.

[0123] 3D antigen modeling Comparative models were constructed using Bioluminate (2019-2 release, Schrodinger, New York, NY) and visualized with Discovery Studio Visualizer (Dassault Systèmes BIOVIA, San Diego, CA).

[0124] Interferon gamma ELISpot Whole blood was collected into ACD-A tubes, and peripheral blood mononuclear cells (PBMCs) were isolated within 24 hours of collection. Samples were collected at baseline, at the time of immunotherapy administration, and at each follow-up visit, and PBMCs were cryopreserved in batches for immune analysis. T cell and antibody responses to HPV6 E6 and E7 antigens were determined by interferon-γ ELISpot and ELISA, respectively, as previously described (Bagarazzi et al., SciTranslMed. 2012;4(155):155ra38).

[0125] Flow cytometry PBMCs were collected after overnight cryopreservation in cell culture medium, spun, washed, and resuspended the following day. After counting, 1 × 10 6 PBMCs from patients with sufficient samples were seeded into 96-well plates in R10 medium. For antigen-specific responses, cells were stimulated for 5 days with a combination of pooled HPV6 E6 and E7 peptides at a concentration of 2 μg / ml. An irrelevant peptide was used as a negative control (OVA), and concanavalin A (Sigma-Aldrich) was used as a positive control. No costimulatory antibodies or cytokines were added to the cell cultures at any time. At the end of the 5-day incubation period, cells were stained for CD3-BUV737, CD4-APC-Cy7, CD14-BUV395, CD-16-BUV395, CD137-APC, granulysin-AF488 CD-19-BUV395, CD38-BV786, CD8-BV650, granzyme B-AF700 (BD Biosciences), granzyme A-PECy7 (ThermoFisher), PD-1-PEDazzle, perforin-BV421, Ki67-BV605, and CD69-BV711 (BioLegend). Staining for extracellular markers (CD4, CD8, CD137, CD69, CD38, and PD-1) was performed first, followed by permeabilization for staining for the remaining markers. CD3 was stained intracellularly to account for the downregulation of this marker after cell activation. The acquired data were analyzed using FlowJo software version X.0.7 or later (Tree Star).

[0126] Antigen-specific PBMC stimulation for gene expression analysis For short-term stimulation—cryopreserved PBMCs were thawed, rested overnight, and stimulated with either DMSO (negative control) or HPV6 E6 and E7 overlapping peptide pool (OLP) for 22 hours at 37°C, 5% CO2, and 95% humidity. After stimulation, culture supernatants were collected and stored at -20°C. Cells were then lysed using buffer RLT (Qiagen) and stored at -80°C.

[0127] For long-term stimulation, cryopreserved PBMCs were thawed, rested overnight, and stimulated with HPV6 E6 and E7 OLPs at 37°C, 5% CO2, and 95% humidity for 11 days. Fresh medium containing IL-2 and IL-7 was added at 10 U / mL and 10 ng / mL, respectively, on days 1, 4, 6, and 8. On day 11, PBMCs were washed and rested overnight at 37°C, 5% CO2, and 95% humidity. After overnight rest, PBMCs were restimulated with either DMSO (negative control) or HPV6 E6 and E7 OLPs for 22 hours. At the end of the 22-hour stimulation, cell supernatants were collected and stored at -20°C. Cells were then lysed and stored at -80°C.

[0128] Multiplex gene expression analysis Cell lysates were thawed in batches of 12 according to the manufacturer's instructions and hybridized to capture probes and fluorophore barcode reporter probes using the nCounter (NanoString) GX Human Immunology V2 panel, consisting of 594 genes and 15 internal reference controls. Samples were then loaded into an automated nCounter Prep Station (Nanostring) for hybridization of capture probes to translucent cartridges. Gene expression was then measured by direct counting of reporter probes in each sample lane using an nCounter Digital Analyzer (Nanostring).

[0129] statistical methods Subjects who received at least one dose of treatment were included in safety analyses. The incidence of AEs, including SAEs and injection site reactions, was estimated with exact 95% confidence intervals. Analyses related to secondary and exploratory endpoints utilized subjects who received the assigned number of doses. Secondary analyses estimated immune response parameters. Exploratory analyses estimated clinical response and histopathological assessment parameters. For continuous outcomes, mean / median and 95% confidence intervals were calculated, and for binary outcomes, proportions and exact 95% confidence intervals were calculated using the Clopper-Pearson method.

[0130] The results of the experiment are now described.

[0131] Patient characteristics and disposition In total, four patients consented and were screened for eligibility. Three patients met all inclusion and exclusion criteria and were enrolled between October 2014 and September 2017. Demographic and baseline characteristics are summarized in Table 1. Of these three, two patients presented with HPV6-associated RRP (both with vocal cord disease), and one patient had invasive malignancy (initial disease located in the trachea, with squamous cell carcinoma in the pharynx presenting at study enrollment). All three patients completed all four doses, receiving 3 mg of INO-3106 on day 0, 6 mg of INO-3106 at week 3, and 6 mg of INO-3106 with 1 mg of INO-9012 at weeks 6 and 9, all delivered intramuscularly via the CELLECTRA® device. Two patients completed the 6-month long-term follow-up period after their last dose of treatment. One patient did not complete the long-term follow-up and reported a non-study-related conflict as the primary reason for withdrawal from the study. All three patients are included in the safety analysis set. [Table 1]

[0132] Safety and tolerability of INO-3106 and INO-9012 by EP INO-3106 and INO-9012 delivered via EP were well tolerated. Treatment-emergent AEs included injection site pain (three related Grade 1 events), fever (one unrelated Grade 1 event), and urinary tract infection (one unrelated Grade 2 event). All patients reported injection site pain, which was treated with medication in most cases and led to recovery. One treatment-emergent SAE, a Grade 3 monoplegia requiring hospitalization, was reported in the study but was assessed as unrelated to study treatment. No patients discontinued continued study treatment or continued study participation due to either an AE or tolerability of the EP. No Grade 4 events or deaths were reported during the course of the study. All patients experienced changes in laboratory parameters, the majority of which included minor fluctuations in hematology values; however, all abnormal laboratory values ​​were determined to be clinically insignificant.

[0133] INO-3106 induces IFNγ production and expression of activation markers and lytic proteins in T cells from treated RRP patients Assessment of HPV6 E6 and E7 cellular immune responses was performed for all three patients enrolled in the study (Figures 2 and 6). Because subject 601 was not an RRP patient and has limited data due to death related to non-treatment events, immunological information relevant to this subject can be found in Figure 6. Cellular immune responses were first addressed by performing overnight IFNγ ELISpot without the addition of supplemental cytokines on isolated peripheral blood mononuclear cells (PBMCs) obtained before and after INO-3106 dosing. The results of this assessment indicate that patient 603 demonstrated very low baseline activity to HPV6 E6 and E7 antigens in the form of antigen-specific IFNγ secretion (Figure 2). Specifically, 10% of patients with E6 or E7 antigens were resistant to IFNγ. 6 Although fewer than 20 spots per PBMC were observed, patient 604 demonstrated a reasonably robust cellular response to these antigens at study entry, with 10 6 E6 spot-forming units per PBMC approached 150 spots, and E7 exceeded 50 spots (Figure 2).

[0134] Treatment with INO-3106 increased HPV6 E6- and E7-specific cellular responses above baseline in patient 603, including a total response to more than 50 HPV6 antigen spots. Patient 604 did not demonstrate an increase in IFNγ spots in response to treatment (Figure 2). However, among responding patients, the time to peak response varied and was difficult to accurately assess. Specifically, death from a non-treatment-related event occurred in patient 601 after the fourth dose of INO-3106; therefore, post-treatment follow-up was unavailable; peak response was observed after the third dose. Patient 603 demonstrated a peak response 6 months after the final dose of INO-3106, which may be related to changes in viral activation / antigen target expression during that period or may reflect the dynamics of the patient's immune system, which continues to build and support a large pool of HPV6-specific T cells.

[0135] It has previously been shown that IFNγ production, while indicative of a Th1 immune response, does not correlate 1:1 with lytic activity (Morrow et al., Molecular therapy: the journal of the American Society of Gene Therapy. 2015;23(3):591-601; Morrow et al., Clinical cancer research: an official journal of the American Association for Cancer Research. 2017;DOI:10.1158 / 1078-0432.CCR-17-2335; Trimble et al., Lancet. 2015;386(10008):2078-88; Migueles et al., PLoS pathogens. 2011;7(2):e1002002; Varadarajan et al., The Journal of clinical investigation. 2011;121(11):4322-31). The cytolytic response by CD8+ T cells is understood to be a critical component of the immune response, controlling and eliminating virally infected cells. Therefore, flow cytometry was performed on PBMCs from patients 603 and 604 with sufficient samples isolated before and after dosing with INO-3106 to assess the ability of HPV6-specific CD8+ T cells to load granzymes and perforin in response to treatment. To do so, the CD8+ T cell compartment was analyzed for immune activation via antigen-specific expression of cell surface markers such as CD38, CD69, CD137, and Ki67 (Figure 3), as well as for lytic potential as determined by the presence of granulysin (Gnly), granzyme A (GrzA), granzyme B (GrzB), and perforin (Prf) after in vitro stimulation with cognate antigen. Table 2 shows the antigen-specific modulation of these markers before and after treatment with INO-3106. Consistent with the ELISpot response, patient 603 demonstrated a robust increase in various CD8+ T cells expressing activation markers as well as lytic proteins.Most notably, expression of CD38 and / or Ki67, combined with markers of lytic potential such as granzyme A, granzyme B, and perforin, dramatically increased after treatment with INO-3106, reaching values ​​exceeding 3% of total CD8+ T cells specific for HPV6 E6 and E7 antigens (Table 2A, Figure 3). Conversely, consistent with the ELISpot results showing a lack of robust T cell proliferation, patient 604 (Table 2B, Figure 3) showed a smaller increase in CD8+ T cell responses compared to patient 603. Interestingly, although smaller in magnitude than patient 603, the phenotype of putative CTLs induced in patient 604 indicates the possibility of more highly activated CD8+ T cells, as the population most likely to expand after treatment consisted of co-expression of three (CD38, CD137, Ki67) activation markers or all four (the previous three plus CD69).Co-expression of this number of activation markers at the same time is much rarer (Tebas et al., The New England Journal of Medicine. 2017; Bagarazzi et al. SciTranslMed. 2012;4(155):155 ra 38; Morrow et al. Molecular therapy oncolytics. 2016;3(16025; Trimble et al., Lancet. 2015;386(10008):2078-88; Morrow et al., Clinical cancer research: an official journal of the American Association for Cancer Research. 2018;24(2):276-94; Aggarwal et al., Clinical cancer research: an official journal of the American Association for Cancer Research. Research. 2019;25(1):110-24), previous investigations of CD8+ T cells expressing multiple activation markers have shown that these cells express granzymes and can effectively induce apoptosis in targets expressing cognate antigens (Duhen et al., Nat Commun. 2018 Jul 13;9(1):2724. doi:10.1038 / s41467-018-05072-0). Indeed, this last notion is further supported when noting that patient 604 showed increased expression not only of granzymes and perforin, but also of granulysin. While the limitations of this small sample size need to be considered, the results of this evaluation demonstrate that INO-3106 potentially leads to the induction of CD8+ T cells capable of activation in the context of antigen exposure and that these cells are capable of synthesizing granzymes, perforin, and granulysin, thus exhibiting a distinct HPV6-specific CTL phenotype. [Table 2A] [Table 2B]

[0136] INO-3106 alters the immune transcriptional profile of T cells in RRP patients. Short-term stimulation (24 h) of patient PBMCs was performed, followed by analysis of immune gene transcripts found to be differentially regulated in response to stimulation with HPV6 E6 and E7-derived peptide pools. Data from patient 601 can be seen in Figure 6. For patients 603 and 604, gene transcription was mostly associated with upregulation of proinflammatory signatures after immunotherapy. Patient 603 showed minimal differential gene expression at dose 2 compared to baseline. However, at the 2-week follow-up visit, significant upregulation of genes related to innate immune responses (CXCL10, CXCL9, CCL7, CCL8), IFNγ pathways (GBP1, GBP5), cell-cell interactions (CD209, MRC1), and B cell help (CXCL13) was observed in cells stimulated with the peptide pool compared to unstimulated cells. Patient 604 also showed gene upregulation at the 2-week follow-up visit, with a signature similar to that observed in patient 603 (CXCL10, CXCL9, Stat1, GBP1, GBP5, CCL8). Additionally, for patient 604, upregulation of markers indicative of adaptive cell activation (CD274, TNFSF13B) was observed. While gene upregulation in patient 603 was transient, patient 604 largely maintained this signature at later follow-up visits 3 and 6 months after dose 4 (Figure 4A, Table 3). Furthermore, specifically for patient 604, expression of IDO1, a molecule expressed by antigen-presenting cells, rapidly increased over time, showing an approximately 4-fold difference at baseline, which increased 8-fold at dose 2, 10-fold at the 2-week follow-up, nearly 13-fold at the 3-month follow-up, and peaked with a 65-fold increase at the 6-month follow-up (Figure 4A, Table 3).

[0137] Eleven days of in vitro culture, followed by 24 hours of antigen restimulation, allowed us to visualize antigen-specific T cells. The stimulation conditions favored the proliferation of T cells over all other cell types, including B cells and other APCs. Analysis revealed reduced levels of differential gene expression compared to ex vivo stimulation. Overall, both RRP patients showed similar patterns of gene upregulation, with few upregulated genes at baseline (patient 603 - 0 genes and patient 604 - 7 genes), but increased differential expression at post-immunotherapy time points (patient 603 - 4 genes at dose 4, 7 genes at 2-week follow-up, 3 genes at 3-month and 6-month follow-up, respectively; patient 604 - 12 genes at dose 2, 9 genes at 2-week follow-up, 10 genes at 3-month follow-up, and 22 genes at 6-month follow-up) (Figure 4B). For both RRP patients, the upregulated gene expression profiles in stimulator cells from post-immunotherapy samples were primarily associated with T cell activation and functionality (CD276, TNFRSF8, TNFRSF9, GZMB) and B cell help (IL-21, CXCL13). See Table 4 for a complete list of differentially expressed genes for each subject enrolled in the study. Increased expression of CXCL10 and CXCL9 was also observed post-immunotherapy for all subjects, although in patient 604, these markers were already elevated at baseline.

[0138] INO-3106 reduces the need for surgical intervention for the treatment of RRP Prior to enrollment in the study, subjects 603 and 604 required surgical intervention to remove respiratory papillomas approximately every 180 days. Assuming this pattern continued, the expected number of required surgical interventions over the course of the study would be four for subject 603 and two for subject 604. However, throughout the study, neither subject required surgical intervention for removal of airway papillomas, constituting a clinical change in the need for intervention in the treatment of this disease. Post-study follow-up of these subjects reveals that subject 603 has not required surgical intervention in the treatment of his disease as of this publication, remaining surgery-free for a total of more than 915 days. 584 days later, subject 604 had a disease recurrence that required surgical intervention to adequately treat, representing an overall reduction in surgical frequency of more than three-fold (Figure 5). The difference in outcomes for these patients prompted investigation of whether any of the immunological data generated during the study indicated a differential clinical response to treatment. Flow cytometry evaluation showed that subject 604 had more robust immune activity in the form of HPV6-specific CTLs than subject 603 (Figure 5). Without wishing to be bound by any particular theory, it is therefore believed that differences in both the magnitude of the response and the pattern of activation marker expression on subject CTLs may be associated with the durability of clinical impact.

[0139] Results of a Phase I safety and immunological clinical trial of HPV6 E6 / E7-specific targeted DNA immunotherapy, administered intramuscularly and delivered via electroporation with the CELLECTRA® device, with and without an IL-12 DNA adjuvant in three patients with HPV6-associated aerodigestive precancerous lesions and malignancies are reported herein. The immunotherapy was well tolerated. There were no treatment-related serious adverse events (SAEs), and the most frequent treatment-emergent AE was injection site reaction. All patients demonstrated induction of cellular responses to HPV6 E6 and E7 antigens, as demonstrated by at least one immunological assessment. Notably, both evaluable RRP patients achieved clinical benefit from treatment with INO-3106, primarily in the form of delayed intervention (e.g., surgery) compared with their prestudy surgical frequency. Furthermore, the fact that patients who demonstrated more robust cellular activity after INO-3106 treatment remained surgery-free, while patients with less robust cellular activity delayed but did not completely avoid surgery, suggests a possible causal relationship between the induction of HPV6-specific cellular responses and the type and duration of clinical benefit. These results are encouraging and raise the idea that, in certain cases, additional dosing to continue enhancing the cellular response may be preferable in this treatment setting. INO-3106 treatment resulted in the induction of HPV6-specific cellular responses across various immunoassays. Confirmation of IFNγ production using ELISpot and the expression of activation markers concomitant with granzyme and perforin synthesis on CD8+ T cells via flow cytometry indicate that INO-3106 drove the induction of a proinflammatory immune response that included T cells with characteristics of highly activated cytotoxic lymphocytes. These results are further highlighted by the observation of dynamic regulation of proinflammatory and regulatory gene transcripts in PBMCs after completion of treatment. Specifically, genes associated with the IFNγ pathway, such as CXCL10 and GBP1, were upregulated after both short- and long-term stimulation.A recent study in head and neck squamous cell carcinoma reported that a composite score based on IFNγ, CXCL9, CXCL10, IDO1, HLA-DRA, and STAT1 significantly correlated with treatment response rates, indicating that an IFNγ-based signature is associated with therapeutic benefit (Ahn et al., Laryngoscope. 2018;128(1):E27-E32). Furthermore, increased expression of granzyme B and TNFRSF9 was observed, confirming cytotoxic lymphocyte activity at the transcriptome level. The necessity of this nature of T cell responses in combating HPV-driven disease has been exemplified in two previous clinical trials of DNA-based immunotherapy, both of which were delivered using the CELLECTRA® device. In the context of HPV-associated cervical dysplasia, clinical response to treatment with VGX-3100 (a DNA immunotherapy for HPV16 / 18) in the form of lesion regression accompanied by clearance of HPV infection was statistically associated with the presence of robust cellular responses, including IFNγ and CD8+ T cells displaying cytotoxic phenotypic markers (Trimble et al., Lancet. 2015;386(10008):2078-88). Additionally, in another study investigating the treatment of HPV-associated squamous cell carcinoma of the oropharynx, patients with metastatic cancer who achieved a complete response to nivolumab treatment after treatment with MEDI0457 (a DNA immunotherapy for HPV16 / 18 with a plasmid-encoded IL-12 adjuvant) were found to have a robust therapy-driven expansion of PD1+ cytotoxic T cells (Aggarwal et al., Clinical cancer research: an official journal of the American Association for Cancer Research. 2019;25(1):110-24). Thus, this study provides further evidence that HPV-specific immunotherapy delivered by the CELLECTRA® device induces the generation of potent T cell responses that have the potential to clinically impact HPV-associated tumorigenesis.

[0140] The data collected from this study are the first to demonstrate that HPV-specific immunotherapy may impact the clinical status of patients with HPV6-associated recurrent respiratory papillomatosis and may be able to act as an additional or alternative adjuvant therapy. These findings complement data presented earlier this year showing that administration of pembrolizumab was associated with a reduced need for routine surgical intervention (Pai et al., Journal of Clinical Oncology. 37.2502-2502.10.1200 / JCO.2019.37.15_suppl.2502). Together, these findings provide early support for the use of immunotherapeutic approaches in the management of these patients. The standard of care for the treatment of this disease is repeated surgical intervention, which presents several complications and is unlikely to completely eradicate recurrent lesions because latent virus may reside in adjacent tissues (Chow et al., APMIS. 2010;118(6-7):422-49). Other non-surgical adjuvant interventions are indicated in patients with rapid regrowth of lesions or invasive disease, but such therapies also have inherent risks and require further evaluation to determine the optimal treatment regimen (Derkay et al., Otolaryngol Clin North Am. 2019;52(4):669-79). The limitations of current treatments highlight the need to identify non-invasive immune-mediated approaches to treat patients with HPV-positive aerodigestive disease. Indeed, prophylactic HPV vaccines have been reported to reduce papilloma growth and extend the time between interventions, but determining therapeutic efficacy requires ongoing evaluation (Makiyama et al., J Voice. 2017;31(1):104-6). Similarly, PD-1 / PD-L1 blockade represents a rational approach to treat RRP, but its impact on onset and clinical outcomes is poorly characterized (Ahn et al., Laryngoscope. 2018;128(1):E27-E32).

[0141] Data generated from this study indicate that immunotherapy using INO-3106 and an IL-12 adjuvant as a non-invasive immune-mediated approach may offer an option to address existing treatment deficiencies in RRP.

Claims

1. 1. A method for treating or preventing recurrent respiratory papillomatosis (RRP) in an individual, comprising administering to said individual a composition comprising a nucleic acid molecule encoding an HPV6 antigen.

2. The method of claim 1, wherein the HPV6 antigen is an HPV6 E6-E7 fusion antigen.

3. the nucleic acid molecule a nucleotide sequence encoding SEQ ID NO:2; a nucleotide sequence that is at least 95% homologous to the nucleotide sequence encoding SEQ ID NO:2; a fragment of a nucleotide sequence encoding SEQ ID NO:2; 3. The method of claim 2, comprising one or more nucleotide sequences selected from the group consisting of: a nucleotide sequence that is at least 95% homologous to a fragment of the nucleotide sequence encoding SEQ ID NO:2;

4. 4. The method of claim 3, wherein the nucleic acid molecule comprises a nucleotide sequence that is at least 98% homologous to a nucleotide sequence encoding SEQ ID NO:

2.

5. 4. The method of claim 3, wherein the nucleic acid molecule comprises a nucleotide sequence that is at least 99% homologous to a nucleotide sequence encoding SEQ ID NO:

2.

6. 4. The method of claim 3, wherein the nucleotide sequence encoding the HPV6 E6-E7 fusion antigen does not have a 5'-terminal leader sequence that is a nucleotide sequence encoding SEQ ID NO:

4.

7. the nucleic acid molecule a nucleotide sequence comprising SEQ ID NO: 1; a nucleotide sequence that is at least 95% homologous to SEQ ID NO: 1; a fragment of SEQ ID NO: 1; 4. The method of claim 3, comprising one or more nucleotide sequences selected from the group consisting of: a nucleotide sequence that is at least 95% homologous to a fragment of SEQ ID NO:1;

8. 4. The method of claim 3, wherein the nucleic acid molecule comprises a nucleotide sequence that is at least 98% homologous to SEQ ID NO:

1.

9. 4. The method of claim 3, wherein the nucleic acid molecule comprises a nucleotide sequence that is at least 99% homologous to SEQ ID NO:

1.

10. The method of claim 3 , wherein the nucleic acid molecule is a plasmid.

11. The method of claim 3 , wherein the composition is a pharmaceutical composition.

12. 10. The method of claim 1, further comprising administering to the individual a composition comprising an adjuvant.

13. The method of claim 1, further comprising administering to the individual a nucleic acid molecule comprising a nucleotide sequence encoding one or more of the p35 and p40 subunits of IL-12.

14. the nucleotide sequence encoding p35 is a nucleotide sequence encoding SEQ ID NO:6; a nucleotide sequence that is at least 95% homologous to the nucleotide sequence encoding SEQ ID NO:6; a fragment of a nucleotide sequence encoding SEQ ID NO:6; 14. The method of claim 13, wherein the nucleotide sequence comprises a nucleotide sequence selected from the group consisting of: a nucleotide sequence that is at least 95% homologous to a fragment of the nucleotide sequence encoding SEQ ID NO:6;

15. the nucleotide sequence encoding p40 is a nucleotide sequence encoding SEQ ID NO:8; a nucleotide sequence that is at least 95% homologous to the nucleotide sequence encoding SEQ ID NO:8; and a fragment of the nucleotide sequence encoding SEQ ID NO:

8.

16. A nucleotide sequence that is at least 95% homologous to a fragment of the nucleotide sequence encoding SEQ ID NO:

8.

17. The method of claim 3 , wherein administering the nucleic acid molecule to the individual comprises electroporation.

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