Improved vaccine for recurrent respiratory papillomatosis and method of using it

Abstract

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

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims priority and benefit to U.S. Provisional Patent Application No. 62 / 925,283, filed October 24, 2019, which is hereby incorporated by reference in its entirety.

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

Background Art

[0003] Human papillomavirus (HPV)-associated malignancies are a newly emerging global epidemic (Gradishar et al., Journal of the National Comprehensive Cancer Network: JNCCN. 2014; 12(4): 542-90). HPV-associated precancerous lesions and malignancies of the airway and gastrointestinal tract can occur in the oropharynx, larynx, and upper respiratory tract. The role of HPV6 in the pathogenesis of most malignancies of the respiratory and gastrointestinal tract remains unclear, but its role is widely accepted as causal 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). Most lesions are benign, but 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 considerably among affected individuals. Treatment options, including active monitoring without treatment, surgery, radiotherapy, or a combination of these, depend on several factors. Repeated surgical removal of papillomas for symptomatic management usually remains the cornerstone 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 American Academy of Otolaryngology-Head and Neck Surgery. 2007;136(1):98-103). While selected patients in this environment may benefit from radiation therapy, the incidence 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 showed a 43% response rate, supporting the rationale for immunotherapy management of RRP (Pai et al., Journal of Clinical Oncology. 2019;37(15_suppl):2502).

[0005] Therefore, there is a need in the art for improved compositions and methods for the treatment or prevention of RRP. The present invention satisfies this unmet need. [Overview of the project]

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

[0007] Another embodiment 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 the nucleotide sequence encoding SEQ ID NO: 2, and a nucleotide sequence that is at least 95% homologous to a fragment of the 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' terminal leader sequence which is the nucleotide sequence encoding SEQ ID NO: 4.

[0008] In another aspect of the present invention, a composition is 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 at least 95% homologous to SEQ ID NO: 1, a fragment of SEQ ID NO: 1, and a nucleotide sequence 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 nucleotide sequence SEQ ID NO: 3.

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

[0010] In additional embodiments, a pharmaceutical composition comprising the disclosed nucleotide sequence is 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, the methods comprising administering to the individual a composition comprising one or more of the provided nucleotide sequences. The methods preferably include the step of introducing the provided nucleotide sequences into the individual by electroporation.

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

[0013] In some embodiments, the method involves administering to an individual a nucleic acid molecule containing a nucleotide sequence encoding one or more of the p35 and p40 subunits of IL-12. In one embodiment, the nucleotide sequence encoding p35 includes 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 the nucleotide sequence encoding SEQ ID NO: 6, a fragment of the nucleotide sequence encoding SEQ ID NO: 6, and a nucleotide sequence that is at least 95% homologous to the fragment of the nucleotide sequence encoding SEQ ID NO: 6. In one embodiment, the nucleotide sequence encoding p40 includes 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 the nucleotide sequence encoding SEQ ID NO: 8, a fragment of the nucleotide sequence encoding SEQ ID NO: 8, and a nucleotide sequence that is at least 95% homologous to the fragment of the nucleotide sequence encoding SEQ ID NO: 8. [Brief explanation of the drawing]

[0014] [Figure 1] This is a comparative 3D model 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 figure. Both are visualized in ribbon format with side chains and a transparent solvent-exposed surface. 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 (upper panel) and 604 (lower panel) were longitudinally tracked throughout the study for their ability to produce interferon-gamma in the ELISpot assay. E6-specific activity is shown by a blue dashed line, E7-specific activity by a blue solid line, and the sum of both antigens by a black solid line. Long-term follow-up (LTFU) time points are described and explained in relation to the time after completion of dose 4. [Figure 3] INO-3106 activates HPV6-specific cytotoxic lymphocytes in RRP patients. Flow cytometry was performed to evaluate the expression of activation markers on HPV6-specific CD8+ T cells collected from subjects before and after immunotherapy. The expression of CD137 and CD38 before (upper panel) and after (lower panel) treatment with INO-3106 in patient 604 is shown in the left column. The expression of Ki67 and CD69 before (upper panel) and after (lower panel) treatment with INO-3106 in patient 604 is shown in the right column. [Figure 4A] Immunogen transcripts are differentially regulated in an HPV6-specific manner after treatment with INO-3106. Heatmaps showing the differential gene expression ratio differences between stimulated and unstimulated cells before and after vaccination. (Figure 4A) Changes in gene expression ratio (≥2x) in cells stimulated with peptide pool versus medium alone over 24 hours. (Figure 4B) Changes in gene expression ratio (≥2x) after 11 days of T cell proliferation, followed by 24 hours of restimulation of cells with peptide pool versus medium alone. Data are converted to log2 ratio changes, with red indicating upregulation and green indicating downregulation. [Figure 4B] Same as above. [Figure 5]Treatment of RRP patients with INO-3106 provides clinical benefit in the form of surgery avoidance. Top panel - Swimmer plot showing the length of time in days that subjects 604 and 603 did not require surgery. The red dotted line indicates the point at which surgery would have been expected based on the previous surgery frequency before intervention with INO-3106. Φ indicates the point at which subject 604 required surgery. λ indicates that subject 603 remained surgery-free at the indicated point. Bottom left panel - Green bars track to the left y-axis and show the size of HPV6-specific CD8+ T cells expressing CD38, Ki67, granzyme A, granzyme B, and perforin. Blue bars track to the right y-axis and show the multiplier change in surgery-free time relative to the expected surgery frequency for these subjects. Bottom right - Chart shows patient ID, multiplier 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] The results of experiments evaluating HPV6 E6 and E7 cellular immune responses in subject 601 are shown.

[0015] Detailed description of preferred embodiments Definition. The terms used herein are for the purpose of describing specific embodiments and are not intended to limit them. Where used herein and in the appended claims, the singular forms "a," "an," and "the" include plural references unless the context expressly indicates otherwise.

[0016] In the enumeration of numerical ranges as described herein, each number intervening between them is explicitly intended with the same degree of precision. For example, in the range 6–9, the numbers 7 and 8 are intended in addition to 6 and 9, and in the range 6.0–7.0, the numbers 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly intended.

[0017] a. Adjuvant As used herein, "adjuvant" refers to any molecule that is added to a DNA plasmid vaccine described herein to enhance the antigenicity of one or more antigens encoded by the DNA plasmid and that encodes a nucleic acid sequence described below.

[0018] b. Antibody "Antibody" can mean an antibody of class IgG, IgM, IgA, IgD, or IgE, or fragments, fragments, or derivatives thereof, including Fab, F(ab’)2, Fd, and single-chain antibodies, diabodies, bispecific antibodies, bifunctional antibodies, and derivatives thereof. The antibody can 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 the 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 of that length described herein, variants, i.e., proteins having a sequence homologous to SEQ ID NO: 2 described herein, fragments of variants having the lengths described herein, and combinations thereof. The antigen may have the IgE leader sequence of SEQ ID NO: 4, or such a sequence may be removed from the N-terminus. The antigen may optionally include a signal peptide, such as one from another protein.

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

[0021] e. Complement As used herein, "complement" or "complementary" may refer to a nucleic acid and may refer to Watson-Crick (e.g., A-T / U and C-G) or Hoogsteen base pairing between nucleotides or nucleotide analogs of a nucleic acid molecule.

[0022] f. Fragment "Fragment" may mean a polypeptide fragment of an antigen capable of inducing an immune response in a mammal against the antigen. The fragment of the antigen can be 100% identical to the full length, except that in each case it lacks at least one amino acid from the N and / or C terminus, with or without a signal peptide at position 1. The fragment 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, 99% or more of the length of a particular full-length antigen, excluding any heterologous signal peptide added. The fragment preferably comprises a fragment of a polypeptide that is 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more homologous to the antigen, and may further include an N-terminal methionine or heterologous signal peptide not included when calculating the percent homology. The fragment may further include an N-terminal methionine and / or an immunoglobulin signal peptide, such as 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 an antigen may be 100% identical to the full-length sequence, except that, in each case, they may or may not contain a sequence encoding a signal peptide and / or methionine at position 1, and that they are missing at least one nucleotide from the 5' and / or 3' ends. Fragments may contain 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more of the length of a particular full-length coding sequence, excluding any added heterologous signal peptides. Preferably, fragments may include a polypeptide that is 95%, 96%, 97%, 98%, or 99% homologous to the antigen, and may also optionally include a sequence encoding an N-terminal methionine or heterologous signal peptide that is not included when calculating the homology percentage. The fragment may further contain a coding sequence for an N-terminal methionine and / or immunoglobulin signal peptide, such as an IgE or IgG signal peptide. The coding sequence encoding the N-terminal methionine and / or signal peptide may be ligated to the coding sequence fragment.

[0024] g. Same As used herein in the context of two or more nucleic acid or polypeptide sequences, “identical” or “sameness” may mean that the sequences have a certain percentage of residues that are the same across a particular region. The percentage may be calculated by optimally aligning the two sequences, comparing them across a particular 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 particular 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 mismatched ends and the particular comparison region contains only a single sequence, the residues of the single sequence are included in the denominator, not the numerator, of the calculation. When comparing DNA and RNA, thymine (T) and uracil (U) may be considered equivalent. Identity may be performed manually or by using computer sequencing algorithms such as BLAST or BLAST 2.0.

[0025] h. Immune response As used herein, “immune response” may mean the activation of the host’s immune system, e.g., the mammalian immune system, in response to the introduction of one or more antigens via the provided DNA plasmid vaccine. The immune response may take the form of a cellular or humoral response, or both.

[0026] i.Nucleic acid As used herein, “nucleic acid,” “oligonucleotide,” or “polynucleotide” may mean at least two nucleotides covalently bonded together. A single-stranded description also defines the sequence of the complementary strand. Thus, nucleic acid also encompasses the complementary strand of the single-stranded molecule being described. Many variants of nucleic acid may be used for the same purposes as a given nucleic acid. Thus, 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, nucleic acid also encompasses probes that hybridize under stringent hybridization conditions.

[0027] Nucleic acids can be single-stranded or double-stranded, or may contain portions of both double-stranded and single-stranded sequences. Nucleic acids can be DNA, both genomic and cDNA, RNA, or hybrids, and may 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. Connected in an operable manner 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 originates. As is known in the art, variations in this distance can be adapted without loss of promoter function.

[0029] k. Promoter As used herein, “promoter” may mean a synthetic or naturally occurring molecule that can confer, activate, or enhance the expression of nucleic acids in cells. A promoter may include one or more specific transcriptional regulatory sequences to further enhance expression and / or modify its spatial and / or temporal expression. A promoter may also include distal enhancer or repressor elements, which may be located several thousand base pairs from the transcription start site. Promoters may be derived from sources including viruses, bacteria, fungi, plants, insects, and animals. Promoters may constitutively or differentially regulate the expression of gene components in relation to the cell, tissue, or organ in which expression occurs, or to 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” may mean conditions under which a first nucleic acid sequence (e.g., a probe) hybridizes to a second nucleic acid sequence (e.g., a target) like a complex mixture of nucleic acids. Stringent conditions are sequence-dependent and will vary in different situations. 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. Tm may be the temperature (under defined ionic strength, pH, and nucleic acid concentration) at which 50% of the probe complementary to the target hybridizes to the target sequence at equilibrium (at Tm, 50% of the probe is occupied at equilibrium due to the excess presence of the target sequence). Stringent conditions may be those where the salt concentration is less than about 1.0 M sodium ions at pH 7.0–8.3, e.g., a sodium ion concentration of about 0.01–1.0 M (or other salts), and the temperature is at least about 30°C for short probes (e.g., about 10–50 nucleotides) and at least about 60°C for long probes (e.g., more than about 50 nucleotides). Stringent conditions can also be achieved by adding an stabilizer such as formamide. For selective or specific hybridization, the positive signal may be at least 2–10 times the 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” may mean that the first sequence is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to the complement of the second sequence over a region of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 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” may mean that the first and second sequences are substantially complementary to the complement of the second sequence, and are identical by at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% across regions of 8, 9, 10, 100 or more nucleotides or amino acids, or with respect to the nucleic acid.

[0033] o. variant As used herein with respect to nucleic acids, “variant” may mean (i) a portion or fragment of a referenced nucleotide sequence, (ii) a complement of a referenced nucleotide sequence or a portion thereof, (iii) a nucleic acid substantially identical to a referenced nucleic acid or its complement, or (iv) a nucleic acid that hybridizes under stringent conditions with a referenced nucleic acid, its complement, or a sequence substantially identical thereto.

[0034] A “variant” refers to a peptide or polypeptide that differs in its amino acid sequence due to an amino acid insertion, deletion, or conservative substitution, but retains at least one biological activity. A variant may also mean a protein having an amino acid sequence that is substantially identical to a reference protein having an amino acid sequence that retains at least one biological activity. Conservative substitutions of amino acids, i.e., replacing an amino acid with a different amino acid having similar properties (e.g., hydrophilicity, degree, and distribution of charged regions), are typically recognized in the art as resulting in only slight changes. These slight changes can be partially identified by considering the hydrophilicity index of amino acids, as understood in the art. Kyte et al., J.Mol.Biol.157:105-132 (1982). The hydroxyl index of amino acids is based on consideration of their hydrophobicity and charge. It is known in the art that amino acids with similar hydroxyl indices can be substituted and still retain protein function. In one embodiment, amino acids with hydroxyl indices of ±2 are substituted. The hydrophilicity of amino acids can also be used to reveal substitutions that result in proteins that retain biological function. Considering the hydrophilicity of amino acids in the context of peptides allows for the calculation of the peptide's maximum local mean hydrophilicity, a useful measure that has been reported to correlate well with antigenicity and immunogenicity. U.S. Patent No. 4,554,101, fully incorporated herein by reference, is used. Substitutions of amino acids with similar hydrophilicity values ​​can result in peptides that retain biological activity, e.g., immunogenicity, as understood in the art. Substitutions may be made with amino acids having 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 its observations, amino acid substitutions that are compatible with biological function are understood to depend on the relative similarity of amino acids, particularly their side chains, as revealed by their hydrophobicity, hydrophilicity, charge, size, and other properties.

[0035] p. Vector As used herein, “vector” may mean a nucleic acid sequence containing an origin of replication. A vector may be a plasmid, bacteriophage, bacterial artificial chromosome, or yeast artificial chromosome. A vector may be a DNA or RNA vector. A vector may be either a self-replicating extrachromosomal vector or a vector integrated into the host genome.

[0036] Improved vaccines resulting from multiphase strategies for enhancing immunogen-induced cellular immune responses are disclosed. Modified consensus sequences were generated. Genetic modifications, including codon optimization, RNA optimization, and the addition of highly efficient immunoglobulin reader sequences, are also disclosed. Novel constructs are designed to induce more potent and broader cellular immune responses than corresponding codon-optimized immunogens.

[0037] Improved HPV vaccines are based on proteins and gene constructs encoding proteins having epitopes that are particularly effective as immunogens to mediate prophylactic or therapeutic strategies against RRPs. Thus, the vaccines can induce therapeutic or prophylactic immune responses. In some embodiments, the means of delivering the immunogen are DNA vaccines, recombinant vaccines, protein subunit vaccines, compositions containing immunogens, attenuated vaccines, or dead bacterial vaccines. 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 immunogens, one or more attenuated vaccines, and one or more dead bacterial vaccines.

[0038] According to some embodiments, vaccines are delivered to an individual to modulate the activity of the individual's immune system and thereby enhance the immune response to HPV that treats RRP. When a protein-coding nucleic acid molecule 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 protein-coding sequences on nucleic acid molecules such as plasmids as part of a recombinant vaccine and as part of an attenuated vaccine, as part of an isolated protein or vector.

[0039] Compositions and methods are provided that offer prophylactic and / or therapeutic treatment for RRP in individuals.

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

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

[0042] Another embodiment 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 the nucleotide sequence encoding SEQ ID NO: 2, a fragment of the nucleotide sequence encoding SEQ ID NO: 2, and a nucleotide sequence that is at least 95% homologous to the fragment of the 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 the nucleotide sequence encoding SEQ ID NO: 2, a fragment of the nucleotide sequence encoding SEQ ID NO: 2, and a nucleotide sequence that is at least 95% homologous to the fragment of the nucleotide sequence encoding SEQ ID NO: 2.

[0044] In another aspect of the present invention, a composition is 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 at least 95% homologous to SEQ ID NO: 1, a fragment of SEQ ID NO: 1, and a nucleotide sequence 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, a nucleotide sequence comprising an 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' leader sequence, for example, a nucleotide sequence encoding SEQ ID NO: 4. In particular, an HPV6 E6-E7 fusion antigen comprising nucleotide sequence SEQ ID NO: 1 lacks a 5' leader sequence, for example, nucleotide sequence SEQ ID NO: 3.

[0046] In some embodiments, the nucleotide sequences of the present 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 provided nucleotide sequences.

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

[0048] In additional embodiments, a pharmaceutical composition comprising the disclosed nucleotide sequence is provided.

[0049] In some embodiments, there are methods for inducing an effective immune response in an individual to 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 comprising two or more antigens. The method preferably comprises the step of introducing the provided nucleotide sequences into the individual by electroporation.

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

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

[0052] Fragments of SEQ ID NO: 2 may be 100% identical to the full-length SEQ ID NO: 2, except that, in each case, they lack at least one amino acid from the N and / or C-terminus, with or without the signal peptide and / or methionine at position 1. Fragments of SEQ ID NO: 2 may contain 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 SEQ ID NO: 2, excluding any added heterologous signal peptides. Preferably, the fragments include SEQ ID NO: 2 fragments 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 may also contain N-terminal methionine or heterologous signal peptides that are not included when calculating the homology percentage. The fragment may further contain an N-terminal methionine and / or an immunoglobulin signal peptide, such as an IgE or IgG signal peptide. The N-terminal methionine and / or signal peptide may be ligated to the fragment.

[0053] A fragment of nucleic acid sequence SEQ ID NO: 1 may be 100% identical to the full-length sequence, except that, in each case, it may or may not contain a sequence encoding a signal peptide and / or methionine at position 1, and may be missing at least one nucleotide from the 5' and / or 3' ends. The fragment may contain 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more of the length of the full-length coding sequence SEQ ID NO: 1, excluding any added heterologous signal peptides. Preferably, the fragment contains a polypeptide that is 95%, 96%, 97%, 98%, or 99% homologous to antigen SEQ ID NO: 2, and may also optionally contain a sequence encoding an N-terminal methionine or heterologous signal peptide that is not included when calculating the homology percentage. The fragment may further contain a coding sequence for an N-terminal methionine and / or immunoglobulin signal peptide, such as an IgE or IgG signal peptide. The coding sequence encoding the N-terminal methionine and / or signal peptide may be ligated to the fragment.

[0054] In some embodiments, the SEQ ID NO: 1 fragment may contain 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, the SEQ ID NO: 1 fragment, such as those described herein, may further contain the coding sequence of an IgE reader sequence. In some embodiments, the SEQ ID NO: 1 fragment does not contain the coding sequence of an IgE reader sequence.

[0055] In some embodiments, the SEQ ID NO: 2 fragment may contain 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, an HPV6 E6-E7 immunogen or a nucleic acid molecule encoding an HPV6 E6-E7 immunogen is administered in combination with IL-12. In one embodiment, IL-12 is encoded from a synthetic DNA plasmid.

[0057] In some embodiments, the method involves administering a composition comprising nucleic acid molecules encoding the p35 and / or p40 subunits of IL-12.

[0058] Sequence ID 5 contains the nucleotide sequence encoding the p35 subunit of IL-12. Sequence ID 6 contains the amino acid sequence of the p35 subunit of IL-12.

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

[0060] Fragments of SEQ ID NO: 6 may be 100% identical to the full-length SEQ ID NO: 6, except that, in each case, they lack at least one amino acid from the N and / or C-terminus, with or without the signal peptide and / or methionine at position 1. Fragments of SEQ ID NO: 6 may contain 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 SEQ ID NO: 6, excluding any added heterologous signal peptides. Preferably, the fragment contains a fragment of SEQ ID NO: 6 that is 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more homologous to SEQ ID NO: 6, and may also contain an N-terminal methionine or heterologous signal peptide that is not included when calculating the homology percentage. The fragment may further contain an N-terminal methionine and / or an immunoglobulin signal peptide, such as an IgE or IgG signal peptide. The N-terminal methionine and / or signal peptide may be ligated to the fragment.

[0061] Fragments of nucleic acid sequence SEQ ID NO: 5 may be 100% identical to the full-length sequence, except that, in each case, they lack at least one nucleotide from the 5' and / or 3' ends, with or without the sequence encoding the signal peptide and / or methionine at position 1. Fragments may contain 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more of the length of the full-length coding sequence SEQ ID NO: 5, excluding any added heterologous signal peptides. Preferably, fragments may include a polypeptide that is 95%, 96%, 97%, 98%, or 99% homologous to antigen SEQ ID NO: 6, and may also optionally include a sequence encoding the N-terminal methionine or heterologous signal peptide that is not included when calculating the homology percentage. The fragment may further contain a coding sequence for an N-terminal methionine and / or immunoglobulin signal peptide, such as an IgE or IgG signal peptide. The coding sequence encoding the N-terminal methionine and / or signal peptide may be ligated to the fragment.

[0062] In some embodiments, the SEQ ID NO: 5 fragment may contain 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, the SEQ ID NO: 5 fragment, such as those described herein, may further contain the coding sequence of an IgE reader sequence. In some embodiments, the SEQ ID NO: 5 fragment does not contain the coding sequence of an IgE reader sequence.

[0063] In some embodiments, the SEQ ID NO: 6 fragment may contain 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] Sequence ID 7 contains the nucleotide sequence encoding the p40 subunit of IL-12. Sequence ID 8 contains the amino acid sequence of the p35 subunit of IL-12.

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

[0066] Fragments of SEQ ID NO: 8 may be 100% identical to the full-length SEQ ID NO: 8, except that, in each case, they lack at least one amino acid from the N and / or C-terminus, with or without the signal peptide and / or methionine at position 1. Fragments of SEQ ID NO: 8 may contain 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 SEQ ID NO: 8, excluding any added heterologous signal peptides. Preferably, the fragment contains a fragment of SEQ ID NO: 8 that is 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more homologous to SEQ ID NO: 8, and may also contain an N-terminal methionine or heterologous signal peptide that is not included when calculating the homology percentage. The fragment may further contain an N-terminal methionine and / or an immunoglobulin signal peptide, such as an IgE or IgG signal peptide. The N-terminal methionine and / or signal peptide may be ligated to the fragment.

[0067] Fragments of nucleic acid sequence SEQ ID NO: 7 may be 100% identical to the full-length sequence, except that, in each case, they lack at least one nucleotide from the 5' and / or 3' ends, with or without the sequence encoding the signal peptide and / or methionine at position 1. Fragments may contain 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the length of the full-length coding sequence SEQ ID NO: 7, excluding any added heterologous signal peptides. Preferably, fragments include a polypeptide that is 95%, 96%, 97%, 98%, or 99% homologous to antigen SEQ ID NO: 8, and may also optionally include a sequence encoding the N-terminal methionine or heterologous signal peptide that is not included when calculating the homology percentage. The fragment may further contain a coding sequence for an N-terminal methionine and / or immunoglobulin signal peptide, such as an IgE or IgG signal peptide. The coding sequence encoding the N-terminal methionine and / or signal peptide may be ligated to the fragment.

[0068] In some embodiments, the SEQ ID NO: 7 fragment may contain 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, the SEQ ID NO: 7 fragment, such as those described herein, may further contain the coding sequence of an IgE reader sequence. In some embodiments, the SEQ ID NO: 7 fragment does not contain the coding sequence of an IgE reader sequence.

[0069] In some embodiments, the SEQ ID NO: 8 fragment may contain 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 includes the simultaneous administration of (a) a composition comprising a nucleic acid molecule encoding an HPV6 antigen (e.g., HPV6 E6-E7 fusion antigen) as disclosed herein, and (b) a composition comprising a nucleic acid molecule encoding one or more IL-12 subunits (e.g., p35 and / or p40) as disclosed herein. In some embodiments, the method includes administering a composition comprising a nucleic acid molecule encoding one or more IL-12 subunits (e.g., p35 and / or p40) as disclosed herein, after pre-administration of a composition comprising a nucleic acid molecule encoding an HPV6 antigen (e.g., HPV6 E6-E7 fusion antigen) as disclosed herein. In some embodiments, the method includes administering a composition comprising a nucleic acid molecule encoding an HPV6 antigen (e.g., HPV6 E6-E7 fusion antigen) as disclosed herein, after pre-administration of a composition comprising a nucleic acid molecule encoding one or more IL-12 subunits (e.g., p35 and / or p40) as disclosed herein.

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

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

[0073] The improved vaccine comprises proteins and gene constructs encoding proteins having epitopes that are particularly effective as immunogens in which an anti-HPV immune response can be induced. Thus, the vaccine can be provided to induce a therapeutic or prophylactic immune response. In some embodiments, the means for delivering the immunogen are DNA vaccines, recombinant vaccines, protein subunit vaccines, compositions containing immunogens, attenuated vaccines, or dead bacterial vaccines. 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 immunogens, one or more attenuated vaccines, and one or more dead bacterial vaccines.

[0074] Aspects of the present invention provide a method for delivering a protein coding sequence on a nucleic acid molecule, such as a plasmid, as part of an isolated protein or vector, both 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 immunizing individuals prophylactically and / or therapeutically are provided.

[0076] DNA vaccines are described in U.S. Patents No. 5,593,972, No. 5,739,118, No. 5,817,637, No. 5,830,876, No. 5,962,428, No. 5,981,505, No. 5,580,859, No. 5,703,055, No. 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 those applications, alternative methods for delivering DNA are described in U.S. Patents No. 4,945,050 and No. 5,036,006, both of which are incorporated herein by reference.

[0077] The present invention relates to improved live attenuated vaccines, improved dead bacterial vaccines, and improved vaccines that use recombinant vectors to deliver exogenous genes encoding antigens, as well as subunit and glycoprotein vaccines. Examples of live attenuated vaccines, those using recombinant vectors to deliver exogenous antigens, subunit vaccines, and glycoprotein vaccines are U.S. Patents No. 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. These are listed in publications No. 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, which are incorporated herein by reference, respectively.

[0078] When taken up by cells, gene constructs may remain in the cell as functional extrachromosomal molecules and / or be incorporated into the cell's chromosomal DNA. DNA may be introduced into cells and remain as separate genetic material in the form of plasmids. Alternatively, linear DNA that can be incorporated into chromosomes may be introduced into cells. When DNA is introduced into cells, reagents that promote DNA integration into chromosomes may be added. DNA sequences useful for promoting integration may also be included in the DNA molecule. Alternatively, RNA may be administered to cells. It is also intended to provide gene constructs as linear microchromosomes containing centromeres, telomeres, and origins of replication. Gene constructs may remain as part of the genetic material in attenuated live microorganisms or recombinant microbial vectors that survive in cells. Gene constructs may be part of the genome of recombinant viral vaccines, where the genetic material is either incorporated into the cell's chromosomes or remains extrachromosomal. Gene constructs contain regulatory elements necessary for gene expression in nucleic acid molecules. These elements include promoters, start codons, stop codons, and polyadenylation signals. In addition, enhancers are often required for the gene expression of sequences encoding target proteins or immunomodulatory proteins. These elements must be operably ligated to the sequences encoding the desired proteins, and the regulatory elements must be operable in the individual to whom they are administered.

[0079] The start and stop codons are generally considered to be part of the nucleotide sequence that codes for the desired protein. However, these elements must be functional in the individual to which the gene construct is administered. The start and stop codons must be in-frame with the coding sequence.

[0080] The promoters and polyadenylation signals used must be functional within the cells of the organism.

[0081] Examples of promoters useful for carrying out the present invention, particularly in the production of gene vaccines for humans, include, but are not limited to, promoters from human immunodeficiency viruses (MV) such as Simian virus 40 (SV40), mouse mammary tumor virus (MMTV) promoter, and BIV long-terminal repeat (LTR) promoter; promoters from cytomegaloviruses (CMV) such as Moloney virus, ALV, and CMV initial promoter; Epstein-Barr virus (EBV); Roussarcoma virus (RSV); and promoters from human genes such as human actin, human myosin, human hemoglobin, human muscle creatine, and human metallothionein.

[0082] In particular, in the production of gene vaccines for humans, examples of polyadenylation signals useful for carrying out the present invention include, but are not limited to, the SV40 polyadenylation signal and the LTR polyadenylation signal. In particular, the SV40 polyadenylation signal, located in the pCEP4 plasmid (Invitrogen, San Diego CA), is used.

[0083] In addition to the regulatory elements necessary for DNA expression, other elements may also be included in the DNA molecule. Such additional elements include enhancers. Enhancers may be selected from a group that includes, but is not limited to, human actin, human myosin, human hemoglobin, human muscle creatine, and viral enhancers such as those derived from CMV, RSV, and EBV.

[0084] Genetic constructs can incorporate mammalian origins of replication to maintain the construct outside the chromosome and produce multiple copies of the construct in the cell. Plasmids pVAX 1, pCEP4, and pREP4 from Invitrogen (San Diego, CA) contain an Epstein-Barr virus origin of replication and a nuclear antigen EBNA-1 coding region that results in high-copy episomal replication without incorporation.

[0085] In some preferred embodiments relating to immunization applications, nucleic acid molecules (may include) containing nucleotide sequences encoding the proteins of the present invention, and, in addition, genes for proteins that further enhance the immune response to such target proteins. Examples of such genes include 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 other cytokines and lymphokines such as IL-15, including IL-15 with a deleted signal sequence and optionally containing a signal peptide from IgE. Other potentially useful genes 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, variant forms of IL-18, and 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 it is desirable for any reason to eliminate cells that accept the gene construct, additional elements that target cell destruction can be added. An expressible form of the herpesthymidine kinase (TK) gene can be included in the gene construct. The drug ganciclovir can be administered to an individual, and the drug causes selective killing of any cell-producing TK, thus providing a means for the selective destruction of cells having the gene construct.

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

[0088] In some embodiments, gene constructs may be provided in which the coding sequence of the protein described herein is linked to an IgE signal peptide.

[0089] In some embodiments in which the protein is used, for example, those skilled in the art can generate and isolate the protein of the present invention using well-known techniques. In some embodiments in which the protein is used, for example, those skilled in the art can insert the DNA molecule encoding the protein of the present invention into an expression vector commercially available for use in well-known expression systems using well-known techniques. For example, the commercially available plasmid pSE420 (Invitrogen, San Diego, Calif.) can be used for the production of the protein in E. coli. The commercially available plasmid pYES2 (Invitrogen, San Diego, Calif.) can be used, for example, for production in the yeast strain S. cerevisiae. The commercially available MAXBAC® complete baculovirus expression system (Invitrogen, San Diego, Calif.) can be used, for example, for production in insect cells. The commercially available plasmid pcDNA I or pcDNA3 (Invitrogen, San Diego, Calif.) can be used, for example, 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, the desired protein can be prepared in both prokaryotic and eukaryotic systems, resulting in a spectrum of processed protein forms.

[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 necessary regulatory sequences such as promoters and polyadenylation signals, and preferably enhancers, are readily available and known in the art for various hosts. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Ed., Cold Spring Harbor Press (1989). The gene construct comprises a protein-coding sequence operably linked to a promoter that is functional in the cell line to which the construct is transfected. Examples of constitutive promoters include promoters from cytomegalovirus or SV40. Examples of inductive promoters include mouse mammary leukemia virus or metallothionein promoters. Those skilled in the art can readily produce gene constructs useful for transfecting cells with DNA encoding the protein of the present invention from readily available starting materials. Using an expression vector containing the protein-coding DNA, a suitable host is transformed, which is then cultured and maintained under conditions in which the foreign DNA expression occurs.

[0091] The produced proteins are recovered by lysing the cells or, if necessary, from culture media known to those skilled in the art. Those skilled in the art can isolate the proteins produced using such expression systems using well-known techniques. The method of purifying proteins from natural sources using antibodies that specifically bind to particular proteins, as described above, can be equally applied to the purification of proteins produced by recombinant DNA methodologies.

[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 when the derivatives have substitutions not provided in DNA-coding protein production.

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

[0094] Routes of administration include, but are not limited to, intramuscular, intranasal, intraperitoneal, intradermal, subcutaneous, intravenous, intra-arterial, intraocular, and oral, as well as topical, transdermal, inhalation, or suppository administration, or by lavage into mucosal tissues, such as the vagina, rectum, urethra, 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 apparatus, conventional syringes, needle-free injectors, or "microprojectile impulse gene guns."

[0095] Examples of preferred electroporation apparatuses and methods for facilitating the 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 2005 / 0052630 filed by Smith, et al., the contents of which are incorporated herein by reference in their entirety. Also preferred are the electroporation apparatuses and methods for facilitating the delivery of DNA vaccines provided in U.S. Provisional Application No. 60 / 852,149 filed October 17, 2006, and the concurrently pending shared U.S. Patent Application No. 11 / 874072 filed October 10, 2007, all of which are incorporated herein by reference in their entirety.

[0096] The following is an example of an embodiment using electroporation technology, which is discussed in more detail in the patent references mentioned above: An electroporation apparatus can be configured to deliver pulses of energy to a desired tissue of a mammal, generating a constant current similar to a current input preset by the user. The electroporation apparatus includes an electroporation component and an electrode assembly or handle assembly. The electroporation component may include and incorporate one or more of the various elements of the electroporation apparatus, 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 may function as one element of the electroporation apparatus, and the other elements may be separate elements (or components) that communicate with the electroporation component. In some embodiments, the electroporation component may function as two or more elements of the electroporation apparatus, which may communicate with yet another element of the electroporation apparatus separate from the electroporation component. The use of electroporation technology for delivering improved HPV vaccines is not limited by the elements of an electroporation device existing as part of a single electromechanical or mechanical device, since the elements can function as a single device or as separate elements communicating with one another. The electroporation component can deliver pulses of energy that generate a constant current in the desired tissue and includes a feedback mechanism. An electrode assembly includes an electrode array having multiple electrodes in a spatial arrangement, the electrode assembly receives 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 the delivery of the pulses of energy, measures the impedance in the desired tissue, and communicates the impedance to the electroporation component.The feedback mechanism can receive the measured impedance and adjust the pulse of energy delivered by the electroporation component to maintain a constant current.

[0097] In some embodiments, multiple electrodes can deliver pulses of energy in a dispersed pattern. In some embodiments, multiple electrodes can deliver pulses of energy in a dispersed pattern through control of the electrodes under a programmed sequence, the programmed sequence being input to the electroporation components 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 having one neutral electrode measuring impedance, and subsequent pulses of the multiple pulses being delivered by one different of the at least two active electrodes having one neutral electrode measuring impedance.

[0098] In some embodiments, the feedback mechanism is implemented either in 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 preferably in real time or instantaneous (i.e., substantially instantaneous as determined by the techniques available to determine the response time). In some embodiments, a neutral electrode measures the impedance in the desired tissue and communicates the impedance to the feedback mechanism, which responds to the impedance and adjusts the energy pulse to maintain a constant current at a value similar to a preset current. In some embodiments, the feedback mechanism maintains a constant current continuously and instantaneously during the delivery of the energy pulse.

[0099] In some embodiments, nucleic acid molecules are delivered to cells in conjunction with the administration of polynucleotide function enhancers or gene vaccine enhancers. Polynucleotide function enhancers are described in U.S. Patent No. 5,593,972, U.S. Patent No. 5,962,428, and International Patent Application PCT / US94 / 00899, filed on January 26, 1994, respectively, which are incorporated herein by reference. Gene vaccine enhancers are described in U.S. Application No. 021,579, filed on April 1, 1994, which are incorporated herein by reference. The adjuvants administered in conjunction with the nucleic acid molecules may be administered as a mixture with the nucleic acid molecules, or they may be administered separately and simultaneously before or after the administration of the nucleic acid molecules. In addition, other agents that may function as transfection agents and / or replication agents and / or inflammatory agents and may be co-administered with GVF include growth factors, cytokines, and lymphocytes, such as α-interferon, gamma-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 factor, surfactants, such as immunostimulatory complexes (ISCOMS), Freund's incomplete adjuvants, LPS analogs including monophosphoryl lipid A (WL), muramil 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 GVF. In some embodiments, nucleic acid molecules are provided in association with PLG to enhance delivery / uptake.

[0100] The pharmaceutical composition according to the present invention contains about 1 nanogram to about 2000 micrograms of DNA. In some preferred embodiments, the pharmaceutical composition according to the present invention contains about 5 nanograms to about 1000 micrograms of DNA. In some preferred embodiments, the pharmaceutical composition contains about 10 to about 800 micrograms of DNA. In some preferred embodiments, the pharmaceutical composition contains about 0.1 to about 500 micrograms of DNA. In some preferred embodiments, the pharmaceutical composition contains about 1 to about 350 micrograms of DNA. In some preferred embodiments, the pharmaceutical composition contains about 25 to about 250 micrograms of DNA. In some preferred embodiments, the pharmaceutical composition contains 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. If the pharmaceutical compositions are injectable, they are sterile, pyrogen-free, and particulate-free. Isotonic formulations are preferably used. Generally, additives for isotonicity include sodium chloride, dextrose, mannitol, sorbitol, and lactose. In some cases, isotonic solutions such as phosphate-buffered saline are preferred. Stabilizers include gelatin and albumin. In some embodiments, vasoconstrictors are added to the formulation.

[0102] According to several embodiments of the present invention, methods for inducing an immune response are provided. The vaccine may be protein-based, a live attenuated vaccine, a cell 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 an individual one or more of the CTACK protein, TECK protein, MEC protein, and their functional fragments, or their expressible coding sequences, in combination with an isolated nucleic acid molecule encoding the protein of the present invention and / or a recombinant vaccine encoding the protein of the present invention and / or a subunit vaccine encoding the protein of the present invention and / or a live attenuated vaccine and / or a dead bacterial vaccine. One or more of the CTACK protein, TECK protein, MEC protein, and their functional fragments may be administered before, simultaneously with, or after the administration of an isolated nucleic acid molecule encoding the immunogen and / or a recombinant vaccine encoding the immunogen and / or a subunit vaccine containing the immunogen and / or a live attenuated vaccine and / or a dead bacterial vaccine. In some embodiments, isolated nucleic acid molecules encoding one or more proteins selected from the group consisting of CTACK, TECK, MEC, and their functional fragments are administered to an individual.

[0103] The present invention is further illustrated in the following embodiments. These embodiments illustrate the present invention, but should be understood to be provided for illustrative purposes only. From the above considerations and these embodiments, those skilled in the art can identify the essential features of the present invention and make various changes and modifications to the invention to adapt it to various uses and conditions without departing from its spirit and scope. Thus, in addition to those shown and described herein, various modifications of the invention 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 are incorporated herein by reference in their entirety. [Examples]

[0105] The present invention is further defined in the following embodiments. These embodiments illustrate preferred embodiments of the invention, but should be understood to be provided for illustrative purposes only. From the above considerations and these embodiments, those skilled in the art will be able to identify the essential features of the invention and make various changes and modifications to the invention to adapt it to various uses and conditions without departing from its spirit and scope. Thus, in addition to those shown and described herein, various modifications of the invention 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 development of papillomas in the airways and gastrointestinal tract, usually associated with human papillomavirus (HPV) subtypes 6 and 11. Current treatments for HPV6-associated RRP and invasive malignancies may be potentially improved by the addition of HPV-specific immunotherapy. While available prophylactic HPV vaccines can generate neutralizing antibodies against HPV main capsid protein L1, they have not demonstrated therapeutic efficacy against HPV infection or pre-existing lesions and are unlikely to induce a cytolytic T-cell response (Lin et al., Immunologic research. 2010;47(1-3):86-112). HPV-specific immunotherapy, on the other hand, may have therapeutic potential to eliminate pre-existing lesions and infections by generating immunity against the HPV virus itself and HPV-infected cells. HPV E6 and E7 oncoplastic proteins represent ideal targets for this type of therapeutic intervention due to their constitutive expression in HPV-associated tumors and their crucial roles in the induction and maintenance of HPV-associated diseases (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 HPV6 E6 and E7 proteins, in forming a robust immune T-cell response to treat RRP.

[0108] This study experimentally evaluated the efficacy of INO-3106, a novel HPV6-specific immunotherapy consisting of synthetic consensus DNA sequences encoding HPV6 E6 and E7 (Figure 1), proteins essential for HPV6-induced transformation and tumor maintenance in cancer. Synthetic DNA plasmids offer several potential advantages as an immunotherapy platform, including the ability to induce a potent immune response without evidence of genomic integration, a favorable safety profile, stability, and relative ease of manufacture (Saha et al., Recent Pat DNA Gene Seq. 2011;5(2):92-6). Preclinical studies of INO-3106 have demonstrated a potent and specific immune response to HPV6 in animal models (Shin et al., Human vaccines & immunotherapeutics. 2012;8(4):470-8). HPV16 / 18-specific therapies (VGX-3100, Inovio Pharmaceuticals, Inc.) designed and evaluated based on the same synthetic consensus platform demonstrated cellular immune responses correlated with clinical benefits in the form of regression of dysplastic lesions and elimination of HPV16 / 18 infection, supporting late-stage clinical trials targeting HPV16 and 18-related diseases (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., EurJImmunol. 1998; 28(3): 1089-103, Operschall et al., JClinVirol. 1999; 13(1-2): 17-27). Importantly, the engineered plasmid IL-12 gene adjuvant has 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 targeting both skin delivery and local muscle, 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, from induced prophylactic environments 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.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] This study demonstrates the safety and immunogenicity of a pilot trial of INO-3106 with or without INO-9012 (IL-12 adjuvant), delivered intramuscularly (IM) via EP and CELLECTRA® devices, in patients with HPV6-associated RRP or malignancies. The data from this trial suggest 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 trial, subjects with HPV6-positive RRPs and malignancies were administered INO-3106, a DNA plasmid immunotherapy targeting HPV6 E6 and E7 proteins, with or without INO-9012, and IL-12-encoding DNA plasmid immunotherapy delivered intramuscularly (IM) in combination with electroporation (EP) using a CELLECTRA® device. Patients received escalating doses of INO-3106, 3 mg once, followed by three additional doses of 6 mg, with each dose spaced 3 weeks apart, and the third and fourth doses were co-administered with INO-9012. The primary objective of this trial was to evaluate the safety and tolerability of INO-3106 with and without INO-9012. A secondary objective was to determine 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 patients met all inclusion and exclusion criteria and were enrolled in the trial. The study therapy was well-tolerated, with no associated serious adverse events, and all associated adverse events (AEs) were low-grade. Injection site pain was the most common associated AE reported in all patients. Immunogenicity was demonstrated by multiple immunoassays, showing involvement and amplification of HPV6-specific cellular responses, including cytotoxic T cell features. In these patients, preliminary efficacy was demonstrated in the form of a change in the frequency of surgery for growth resection. Prior to the intervention, both patients required surgery approximately every 180 days. One patient showed a more than 3-fold increase in surgery avoidance (584 days), and the other patient remained completely surgery-free at the last contact at 915 days, a more than 5-fold increase in the interval between surgeries.

[0113] The experiments presented herein demonstrate that INO-3106 is well-tolerated, immunogenic, and demonstrates preliminary efficacy in patients with HPV6-associated RRP airway and gastrointestinal lesions, both with and without INO-9012.

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

[0115] Test group This was a prospective, open-label, phase 1 trial. Male and female patients aged 18 years or older were enrolled. To be eligible, patients had to have histologically documented HPV6-related airway / gastrointestinal papillomas, pre-malignant lesions, or invasive airway / gastrointestinal malignancies in which recent therapy (e.g., radiotherapy, chemotherapy) had been completed at least two months prior to the first dose of the study treatment. Patients had to have ECOG 0-1 with normal liver, kidney, and bone marrow function. Patients were excluded if there was evidence of immunosuppression or anticipated use of immunosuppressants, required use of systemic steroids, presence of pre-excitation cardiac syndrome, or pregnancy or lactation. Written informed consent was obtained from each patient before any evaluation was 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 encoded with HPV6-type E6 and E7 proteins, 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 encoded with 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 designed 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 controlled 52 ms electrical pulses at 1-second intervals to the injection site via a sterile, disposable array. When inserted into tissue, the needle array centers around the immunotherapy injection site, forming transient pores within the cell membrane to enhance cell transfection. INO-3106 was delivered intramuscularly in 1 mL volume with or without INO-9012, followed immediately by EP using the CELLECTRA® device. The treatment or dosage is defined as injection of a DNA plasmid, followed by EP.

[0117] Test design Following 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) being 3 mg of INO-3106, the second dose (week 3) being 6 mg of INO-3106, and the third (week 6) and fourth (week 9) doses being 6 mg of INO-3106 with 1 mg of INO-9012. Each dose was delivered with a 3-week interval to allow observation for any grade 2 or higher related systemic adverse events (AEs). In total, all patients' 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. A secondary objective was to determine the humoral and cellular immune responses to INO-3106 with and without INO-9012, and an exploratory objective was to evaluate the preliminary clinical efficacy of the treatment and, if possible, correlate efficacy with post-dose immune cell infiltration in tissues.

[0119] The trial was registered on ClinicalTrials.gov with identifier NCT02241369. This trial protocol complies with the ethical guidelines of the 1975 Declaration of Helsinki and was reviewed and approved by the center's institutional review board.

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

[0121] Further enrollment and treatment were immediately discontinued if more than one-third of patients experienced an event requiring emergency reporting, if any patient experienced a serious adverse event (SAE), an unexpected grade 4 toxicity, a potentially life-threatening AE assessed as related to the 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 age were required to complete pregnancy tests at the time of screening and within three days prior to each dose. Treatment was discontinued in women who tested positive for pregnancy. Laboratory parameters, including hematology, coagulation, serological chemistry (including liver function), and creatine phosphokinase (CPK), were monitored throughout the study and locally evaluated at the center.

[0123] Antigen 3D Modeling The comparative model was built using Bioluminate (2019-2 release, Schrodinger, New York, NY) and visualized using Discovery Studio Visualizer (Dassault Systèmes BIOVIA, San Diego, CA).

[0124] Interferon-gamma ELISpot Whole blood was collected in ACD-A tubes, and peripheral blood mononuclear cells (PBMCs) were isolated within 24 hours of collection. Samples were collected at baseline, at immunotherapy administration, and at each follow-up visit, and PBMCs were cryopreserved in batches for immunoassay. 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 frozen overnight in cell culture medium, then harvested, rotated, washed, and resuspended the following day. After counting, 1 × 10⁻⁶ PBMCs were obtained. 6 Individual PBMCs were seeded into 96-well plates in R10 medium from patients with sufficient sample volume. For antigen-specific response, cells were stimulated for 5 days with a pooled combination of peptides corresponding to HPV6 E6 and E7 at a concentration of 2 μg / ml, with an unrelated peptide used as a negative control (OVA) and concanavalin A used as a positive control (Sigma-Aldrich). No co-stimulatory antibodies or cytokines were added to the cell cultures at any point. 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, granulosin-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, PD-1) was performed first, followed by permeabilization for staining the remaining markers. To account for the downregulation of markers after cell activation, CD3 was stained intracellularly. 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 Short-term stimulation: Frozen PBMCs were thawed, rested overnight, and stimulated for 22 hours at 37°C, 5% CO2, and 95% humidity with either DMSO (negative control) or HPV6 E6 and E7 duplicate peptide pool (OLP). After stimulation, the culture supernatant was collected and stored at -20°C. The cells were then lysed using buffer RLT (Qiagen) and stored at -80°C.

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

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

[0129] statistical methods Participants who received at least one dose of treatment were included in the safety analysis. The incidence of adverse events (AEs), including SAEs and injection site reactions, was estimated along with accurate 95% confidence intervals. Analyses related to secondary and exploratory endpoints utilized participants who received an assigned number of doses. Secondary analyses estimated immune response parameters. Exploratory analyses estimated clinical response and histopathological evaluation parameters. For continuous results, mean / median values ​​and 95% confidence intervals were calculated, and for binary results, percentages and accurate 95% confidence intervals were calculated using the Cropper-Pearson method.

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

[0131] Patient characteristics and properties In total, four patients consented and were screened for eligibility. Three patients met all inclusion and exclusion criteria and were enrolled from October 2014 to September 2017. Demographics and baseline characteristics are summarized in Table 1. Of these three patients, two presented with HPV6-related RRPs (both with vocal cord disease), and one had an invasive malignancy (initial disease was located in the trachea, with squamous cell carcinoma in the pharynx present at trial 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 a 6-month long-term follow-up period after the final dose of treatment. One patient did not complete the long-term follow-up and reported non-trial-related conflict as the primary reason for dropping out of the trial. All three patients were included in the safety analysis set. [Table 1]

[0132] Safety and tolerability of INO-3106 and INO-9012 according to EP INO-3106 and INO-9012, delivered via EP, were well-tolerated. AEs that occurred during treatment included injection site pain (3 related grade 1 events), fever (1 unrelated grade 1 event), and urinary tract infection (1 unrelated grade 2 event). All patients reported injection site pain, which in most cases was treated with the medication and led to recovery. One SAE occurring during treatment of grade 3 monoplegia requiring hospitalization was reported in the study but was assessed as unrelated to the study treatment. No patients discontinued continued study treatment or continued participation in the study due to AEs 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, most of which included minor fluctuations in hematological values; however, all abnormal clinical laboratory values ​​were determined to be clinically insignificant.

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

[0134] Treatment with INO-3106 included a total response to over 50 HPV6 antigen spots and increased HPV6 E6 and E7 specific cellular responses above baseline in patient 603. Patient 604 did not show an increase in IFNγ spots in response to treatment (Figure 2), but the time to peak response varied among responding patients and was difficult to accurately assess. Specifically, a non-treatment-related death occurred in patient 601 after the fourth dose of INO-3106, making post-treatment follow-up unavailable, and the peak response was observed after the third dose. Patient 603 showed 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, building and supporting a large pool of HPV6-specific T cells.

[0135] Previous studies have shown that IFNγ production indicates a Th1 immune response but 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 crucial component of the immune response that controls and eliminates virus-infected cells. Therefore, flow cytometry was performed on PBMCs from patients 603 and 604 with sufficient samples isolated before and after administration of INO-3106 to evaluate the ability of HPV6-specific CD8+ T cells to load granzymes and perforins in response to treatment. For this purpose, 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 determined by the presence of granulysin (Gnly), granzyme A (GrzA), granzyme B (GrzB), and perforin (Prf) after in vitro stimulation with allogeneic antigens. Table 2 shows the antigen-specific modulation of these markers before and after treatment with INO-3106. Consistent with the ELISpot response, patient 603 showed a robust elevation of various CD8+ T cells expressing soluble proteins and activation markers simultaneously.Most notably, the expression of CD38 and / or Ki67 combined with lytic markers 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 to HPV6 E6 and E7 antigens (Table 2A, Figure 3). Conversely, consistent with ELISpot results showing a lack of robust T cell proliferation, patient 604 (Table 2B, Figure 3) showed a smaller increase in CD8+ T cell response in terms of size when compared to patient 603. Interestingly, although smaller in size than patient 603, the phenotype of putative CTLs induced in patient 604 suggests the possibility of more highly active CD8+ T cells, as the population most likely to increase after treatment consisted of co-expression of three (CD38, CD137, Ki67) activation markers or all four (CD69 in addition to the previous three).At the same time, the co-expression of this number of activation markers is far 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, vMorrow 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. Previous studies of CD8+ T cells expressing multiple activation markers (Research. 2019;25(1):110-24) have shown that these cells express granzymes and can effectively induce apoptosis at targets expressing alloantigens (Duhen et al., Nat Commun. 2018 Jul 13;9(1):2724. doi:10.1038 / s41467-018-05072-0). Indeed, this last idea is further supported when we note that patient 604 showed increased expression not only of granzymes and perforin but also of granulysin. While the limitations of this small sample size must be considered, the results of this evaluation suggest that INO-3106 potentially leads to the induction of CD8+ T cells that can be activated in the context of antigen exposure, and that these cells are capable of synthesizing granzymes, perforin, and granulysin, and thus exhibit a distinct HPV6-specific CTL phenotype. [Table 2A] [Table 2B]

[0136] INO-3106 alters the immunotranscriptional profile of T cells in RRP patients. Patient PBMCs were subjected to short-term stimulation (24 hours), followed by analysis of immunogene transcripts found to be specifically regulated in response to stimulation with HPV6 E6 and E7-derived peptide pools. Data from patient 601 can be found in Figure 6. For patients 603 and 604, gene transcription was largely associated with upregulation of pro-inflammatory signatures after immunotherapy. Patient 603 showed only slight 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), genes related to the IFNγ pathway (GBP1, GBP5), intercellular interactions (CD209, MRC1), and B-cell help (CXCL13) was observed in peptide pool-stimulated cells compared to unstimulated cells. Patient 604 also showed gene upregulation at the 2-week follow-up visit, possessing a similar signature (CXCL10, CXCL9, Stat1, GBP1, GBP5, CCL8) to that observed in patient 603. In addition, upregulation of markers indicating adaptive cell activation (CD274, TNFSF13B) was observed in patient 604. While the gene upregulation in patient 603 was transient, patient 604 largely maintained this signature at the 3-month and 6-month follow-up visits after dose 4 (Figure 4A, Table 3). Furthermore, particularly in patient 604, the expression of IDO1, a molecule expressed by antigen-presenting cells, increased rapidly over time, showing an approximately 4-fold difference at baseline, increasing to an 8-fold increase at dose 2, a 10-fold increase at the 2-week follow-up, and nearly a 13-fold increase at the 3-month follow-up, peaking at 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 for the observation of antigen-specific T cells. The stimulation conditions favored T cell proliferation over all other cell types, including B cells and other APCs. Analysis revealed reduced levels of differential gene expression compared to after 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 the time point after immunotherapy (Patient 603 - 4 genes at dose 4, 7 genes at 2-week follow-up, and 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). In both RRP patients, upregulated gene expression profiles in stimulated cells from 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 in all subjects after immunotherapy, although in patient 604, these markers were already elevated at baseline.

[0138] INO-3106 reduces the need for surgical intervention in the treatment of RRP. Prior to enrollment in the trial, subjects 603 and 604 required surgical intervention to remove respiratory papillomas approximately every 180 days. Assuming this pattern continued, the expected number of surgical interventions required over the course of the trial would be four for subject 603 and two for subject 604. However, throughout the trial, neither subject required surgical intervention to remove airway papillomas, constituting a clinical change in the need for intervention in the treatment of this disease. Post-trial follow-up of these subjects revealed that subject 603 had not required surgical intervention in the treatment of the disease at the time of this publication and had not undergone surgery for a total of more than 915 days. After 584 days, subject 604 had a recurrence of the disease requiring surgical intervention to be adequately treated, and the overall reduction in surgical frequency was more than threefold (Figure 5). The differences in outcomes in these patients prompted an investigation into whether any of the immunological data generated during the trial indicated a differential clinical response to treatment. Flow cytometry analysis showed that subject 604 exhibited more robust immunoactivity in the form of HPV6-specific CTLs than subject 603 (Figure 5). While we do not wish to be constrained by any particular theory, it is therefore conceivable that differences in both the magnitude of the response and the pattern of activation marker expression on the target CTLs may be related to the persistence of clinical effects.

[0139] This document reports the results of a Phase I safety and immunological clinical trial of HPV6 E6 / E7-specific targeted DNA immunotherapy with and without IL-12 DNA adjuvant, delivered intramuscularly and via electroporation using a CELLECTRA® device, in three patients with HPV6-related precancerous lesions and malignancies of the airway and gastrointestinal tract. Administration of the immunotherapy was well-tolerated. There were no treatment-related SAEs, and the most frequent AE occurring during treatment was injection site reactions. All patients showed induction of cellular responses to HPV6 E6 and E7 antigens, as demonstrated by at least one immunological assessment. In particular, both evaluable RRP patients derive clinical benefit from treatment with INO-3106, primarily in the form of delayed therapeutic interventions (e.g., surgery) compared to their pre-trial surgical frequency. Furthermore, the fact that patients who showed more robust cellular activity after INO-3106 treatment remained surgery-free, while those with less robust cellular activity, though delayed, did not completely avoid surgery, suggests a possible causal relationship between the induction of HPV6-specific cellular responses and the type / duration of clinical benefit. These results are promising and suggest that, in certain cases, additional medication to continue enhancing cellular responses may be preferable in this therapeutic environment. Treatment with INO-3106 resulted in the induction of HPV6-specific cellular responses across various immunoassays. Confirmation of IFNγ production using ELISpot, as well as confirmation of the expression of activation markers associated with granzyme and perforin synthesis on CD8+ T cells via flow cytometry, indicates that INO-3106 drove the induction of a pro-inflammatory immune response involving T cells with highly activated cytotoxic lymphocyte characteristics. These results are further highlighted by the observation of dynamic regulation of pro-inflammatory 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-term and long-term stimulation.A recent trial 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 rate, demonstrating that an IFNγ-based signature is associated with treatment benefit (Ahn et al., Laryngoscope. 2018;128(1):E27-E32). Furthermore, increased expression of granzyme B and TNFRSF9 was observed, confirming the activity of cytotoxic lymphocytes at transcriptome levels. The need for a T-cell response of this nature in the fight against HPV-driven disease has been exemplified in two previous clinical trials of DNA-based immunotherapy, both delivered using the CELLECTRA® device. In the context of HPV-associated cervical dysplasia, clinical responses to treatment with VGX-3100 (DNA immunotherapy for HPV16 / 18) in the form of lesion regression associated with elimination of HPV infection were statistically associated with the presence of a robust cellular response, including IFNγ and CD8+ T cells exhibiting cytotoxic phenotypic markers (Trimble et al., Lancet. 2015;386(10008):2078-88). In addition, another trial investigating the treatment of HPV-associated squamous cell carcinoma of the oropharynx found that patients with metastatic cancer who achieved a complete response to nivolumab treatment after treatment with MEDI0457 (DNA immunotherapy for HPV16 / 18 with plasmid-coded IL-12 adjuvant) had a therapy-driven robust proliferation 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). Therefore, this study provides further evidence that HPV-specific immunotherapy delivered by the CELLECTRA® device induces the generation of a potent T-cell response that may clinically impact HPV-related tumorigenesis.

[0140] The data collected from this trial are the first to demonstrate that HPV-specific immunotherapy may impact the clinical status of patients with HPV6-associated recurrent respiratory papillomatosis and may act as an add-on or alternative adjuvant therapy. These findings complement data presented to date this year that pembrolizumab administration 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 data supporting the use of immunotherapy approaches in the management of these patients. The standard treatment for this disease is repeated surgical intervention, which presents several complications and has a low probability of completely eradicating lesion recurrence due to the possibility of latent virus presence in adjacent tissues (Chow et al., APMIS. 2010; 118(6-7): 422-49). Other non-surgical adjuvant interventions are indicated in patients with rapid lesion regrowth or invasive disease, but such therapies also carry 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 immunomediated approaches for treating patients with HPV-positive respiratory and gastrointestinal disease. Indeed, prophylactic HPV vaccination has been reported to reduce papilloma growth and extend inter-intervention time, but continuous evaluation is needed to determine treatment efficacy (Makiyama et al., J Voice. 2017;31(1):104-6). Similarly, PD-1 / PD-L1 inhibition represents a rational approach to treating RRP, but its impact on expression and clinical outcomes is not well characterized (Ahn et al., Laryngoscope. 2018;128(1):E27-E32).

[0141] The data generated from this study suggest that immunotherapy using INO-3106 and IL-12 adjuvants may offer an option to address existing treatment shortcomings in RRP as a non-invasive immune-mediated approach.

Claims

1. A composition for treating or preventing recurrent respiratory papillomatosis (RRP) in an individual, comprising a nucleic acid molecule encoding the HPV6 antigen and a nucleic acid molecule containing a nucleotide sequence encoding one or more of the p35 and p40 subunits of IL-12, The aforementioned HPV6 antigen is an HPV6 E6-E7 fusion antigen. The nucleic acid molecule encoding the HPV6 antigen, The nucleotide sequence encoding Sequence ID No. 2, The nucleotide sequence includes a nucleotide sequence selected from the group consisting of nucleotide sequences that are at least 95% homologous to the nucleotide sequence encoding Sequence ID No. 2, The nucleotide sequence encoding p35 of IL-12 is The nucleotide sequence encoding sequence number 6, The nucleotide sequence includes a nucleotide sequence selected from the group consisting of nucleotide sequences that are at least 95% homologous to the nucleotide sequence encoding SEQ ID NO: 6, The nucleotide sequence encoding IL-12 p40 is The nucleotide sequence encoding Sequence ID No. 8, A composition comprising a nucleotide sequence selected from the group consisting of nucleotide sequences that are at least 95% homologous to the nucleotide sequence encoding Sequence ID No.

8.

2. The composition according to claim 1, wherein the nucleic acid molecule comprises a nucleotide sequence that is at least 98% homologous to the nucleotide sequence encoding Sequence ID No.

2.

3. The composition according to claim 1, wherein the nucleic acid molecule comprises a nucleotide sequence that is at least 99% homologous to the nucleotide sequence encoding Sequence ID No.

2.

4. The composition according to claim 1, wherein the nucleotide sequence encoding the HPV6 E6-E7 fusion antigen does not have a 5' terminal leader sequence which is the nucleotide sequence encoding SEQ ID NO:

4.

5. The nucleic acid molecule The nucleotide sequence containing sequence number 1, A nucleotide sequence that is at least 95% homologous to Sequence ID No. 1, Fragment of sequence number 1, The composition according to claim 1, comprising one or more nucleotide sequences selected from the group consisting of a nucleotide sequence that is at least 95% homologous to the fragment of sequence number 1.

6. The composition according to claim 1, wherein the nucleic acid molecule comprises a nucleotide sequence that is at least 98% homologous to SEQ ID NO:

1.

7. The composition according to claim 1, wherein the nucleic acid molecule comprises a nucleotide sequence that is at least 99% homologous to SEQ ID NO:

1.

8. The composition according to claim 1, wherein the nucleic acid molecule is a plasmid.

9. The composition according to claim 1, wherein the composition is a pharmaceutical composition.

10. The composition according to claim 1, further comprising administering a composition containing an adjuvant to the individual.

11. The composition according to claim 1, wherein administering the nucleic acid molecule to the individual includes electroporation.

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