Vaccines for recurrent respiratory papillomatosis and methods of use thereof

Abstract

Provided herein are nucleic acid molecules encoding HPV antigens. Also provided are vaccines against human papillomavirus (HPV), methods of inducing an immune response, and methods of prophylactically and / or therapeutically immunizing individuals against recurrent respiratory papillomatosis (RRP) comprising the nucleic acid. 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 the benefit of U.S. Provisional Application No. 63 / 379,415, filed October 13, 2022, U.S. Provisional Application No. 63 / 476,049, filed December 19, 2022, U.S. Provisional Application No. 63 / 485,219, filed February 15, 2023, U.S. Provisional Application No. 63 / 487,488, filed February 28, 2023, U.S. Provisional Application No. 63 / 488,352, filed March 3, 2023, and U.S. Provisional Application No. 63 / 499,002, filed April 28, 2023. Each of these applications is incorporated herein by reference in its entirety.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in .XML format and is incorporated herein by reference in its entirety. The .XML copy, created on October 13, 2023, is named "104409000897_Sequence Listing.xml" and is 29445 bytes in size. The Sequence Listing contained in this .XML file is a part of the present specification and is incorporated herein by reference in its entirety.

[0003] The present invention relates to human papillomavirus (HPV) vaccines, methods for inducing an immune response, methods for prophylactically and / or therapeutically immunizing individuals against HPV6 and / or HPV11, and methods for preventing or treating recurrent respiratory papillomatosis (RRP). [Background technology]

[0004] Human papillomavirus-associated (HPV+) malignancies are an emerging global epidemic (Gradishar et al., JNCCN 2014;12(4):542-90). HPV-associated aerodigestive precancerous lesions and malignancies can occur in the oropharynx, larynx, and upper respiratory tract. Although the role of HPV6 and HPV11 in the pathogenesis of most aerodigestive malignancies remains unclear, they are widely accepted as causally involved in recurrent respiratory papillomatosis (RRP) (Mounts et al., PNAS USA 1982;79(17):5425-9; Gissmann et al., PNAS USA 1983;80(2):560-3; Bonagura et al., APMIS. 2010;118(6-7):455-70), the most common benign tumor of the laryngeal epithelium. RRP is rare, with an estimated incidence of 1.8 per 100,000 adults in the United States (Winton et al., NEJM 2005;352(25):2589-97). While most lesions are benign, some undergo malignant transformation, and patients with RRP have a higher risk of developing laryngeal tumors and carcinomas (Omland et al., PloS One. 2014;9.6):e99114).

[0005] Recurrent respiratory papillomatosis remains a devastating disease affecting children and adults, resulting in an estimated annual healthcare-related cost of $120 million in the United States, with annual costs approaching $60,000 per patient. While the prevalence of RRP has declined, it remains the most common benign laryngeal neoplasm in children. RRP is unique in its high rate of multisite recurrence, its significant burden on patients' quality of life, and its associated high healthcare costs. Thus, there is a need for improved compositions and methods for the treatment or prevention of RRP. The present invention fulfills this unmet need. Summary of the Invention

[0006] Provided herein is a method for inducing an immune response in a subject by administering to the subject an effective amount of a nucleic acid molecule encoding a human papillomavirus (HPV) antigen comprising an HPV6 antigenic domain and an HPV11 antigenic domain, where the HPV6 antigenic domain is an HPV6 E6-E7 fusion antigen and the HPV11 antigenic domain is an HPV11 E6-E7 fusion antigen, thereby inducing an immune response. Also provided is a method for prophylactically or therapeutically immunizing a subject against HPV6 and / or HPV11, the method comprising administering to the subject an effective amount of a nucleic acid molecule encoding a human papillomavirus (HPV) antigen comprising an HPV6 antigenic domain and an HPV11 antigenic domain, where the HPV6 antigenic domain is an HPV6 E6-E7 fusion antigen and the HPV11 antigenic domain is an HPV11 E6-E7 fusion antigen, thereby inducing an immune response against HPV6, HPV11, or both. Further provided is a method for treating or preventing recurrent respiratory papillomatosis (RRP) in a subject, the method comprising administering to the subject an effective amount of a nucleic acid molecule encoding a human papillomavirus (HPV) antigen comprising an HPV6 antigenic domain and an HPV11 antigenic domain, wherein the HPV6 antigenic domain is an HPV6 E6-E7 fusion antigen and the HPV11 antigenic domain is an HPV11 E6-E7 fusion antigen, thereby treating or preventing RRP.

[0007] Administration can be intradermal or intramuscular. According to some embodiments, administration is by injection through a standard needle or by injection with a side port needle. Administration can further include electroporation.

[0008] According to some embodiments of the method, the number of RRP surgical interventions in a population of human subjects diagnosed with RRP is reduced in the 52 weeks following administration of the pharmaceutical composition, compared to the number of RRP surgical interventions in the 52 weeks prior to administration of the pharmaceutical composition to the population of human subjects diagnosed with RRP. For example, the method may reduce the number of RRP surgical interventions in at least about 76% of the population of human subjects diagnosed with RRP in the 52 weeks following administration of the pharmaceutical composition, compared to the number of RRP surgical interventions in the 52 weeks prior to administration of the pharmaceutical composition to the population of human subjects diagnosed with RRP. The method may reduce the number of RRP surgical interventions in about 70% to about 95%, about 75% to about 85%, about 75% to about 80%, about 75%, about 76%, about 77%, about 78%, about 79%, or about 80% of the population of human subjects diagnosed with RRP in the 52 weeks following administration of the pharmaceutical composition, compared to the number of RRP surgical interventions in the 52 weeks prior to administration of the pharmaceutical composition to the population of human subjects diagnosed with RRP. According to some embodiments, in the 52 weeks following administration of the pharmaceutical composition, the median reduction in the number of RRP surgical interventions is about 3. According to some embodiments of the disclosed methods, in the 52 weeks following administration of the pharmaceutical composition, the number of RRP surgical interventions may be reduced in about 90% of a population of human subjects diagnosed with RRP, compared to the number of RRP surgical interventions in the 52 weeks prior to administration of the pharmaceutical composition to the population of human subjects diagnosed with RRP.

[0009] According to some embodiments, the method increases the number of HPV6-specific activated CD4 and HPV11-specific activated CD4 and / or activated lytic CD8 T cells.

[0010] In some embodiments, the method increases the number of HPV6- and HPV11-specific CD4 cells expressing CD38 52 weeks after administration of the pharmaceutical composition in about 85% of a population of human subjects diagnosed with RRP compared to the number of HPV6- and HPV11-specific CD4 cells expressing CD38 52 weeks before administration of the pharmaceutical composition to the population of human subjects diagnosed with RRP. The method may increase the number of HPV6- and HPV11-specific CD4 cells expressing CD38 52 weeks after administration of the pharmaceutical composition in about 70% to about 95%, about 75% to about 90%, about 85% to about 90%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, or about 89% of a population of human subjects diagnosed with RRP compared to the number of HPV6- and HPV11-specific CD4 cells expressing CD38 52 weeks before administration of the pharmaceutical composition to the population of human subjects diagnosed with RRP.

[0011] In some embodiments, the method increases the number of HPV6- and HPV11 E6-specific CD8 cells expressing CD38, granzyme A, granzyme B, and perforin 52 weeks after administration of the pharmaceutical composition compared to the number of HPV6- and HPV11 E6-specific CD8 cells expressing CD38, granzyme A, granzyme B, and perforin 52 weeks before administration of the pharmaceutical composition to the population of human subjects diagnosed with RRP. For example, the method may increase the number of HPV6- and HPV11 E6-specific CD8 cells expressing CD38, granzyme A, granzyme B, and perforin 52 weeks after administration of the pharmaceutical composition in about 85% of a population of human subjects diagnosed with RRP compared to the number of HPV6- and HPV11 E6-specific CD8 cells expressing CD38, granzyme A, granzyme B, and perforin 52 weeks before administration of the pharmaceutical composition to the population of human subjects diagnosed with RRP. The method may increase the number of HPV6- and HPV11 E6-specific CD8 cells expressing CD38, granzyme A, granzyme B, and perforin during the 52 weeks following administration of the pharmaceutical composition in about 70% to about 95%, about 75% to about 90%, about 85% to about 90%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, or about 89% of a population of human subjects diagnosed with RRP, compared to the number of HPV6- and HPV11 E6-specific CD8 cells expressing CD38, granzyme A, granzyme B, and perforin during the 52 weeks prior to administration of the pharmaceutical composition to the population of human subjects diagnosed with RRP.

[0012] Further provided herein is the use of an effective amount of a nucleic acid molecule encoding a human papillomavirus (HPV) antigen comprising an HPV6 antigenic domain and an HPV11 antigenic domain, wherein the HPV6 antigenic domain is an HPV6 E6-E7 fusion antigen and the HPV11 antigenic domain is an HPV11 E6-E7 fusion antigen, for inducing an immune response in a subject. Also provided is the use of an effective amount of a nucleic acid molecule encoding a human papillomavirus (HPV) antigen comprising an HPV6 antigenic domain and an HPV11 antigenic domain, wherein the HPV6 antigenic domain is an HPV6 E6-E7 fusion antigen and the HPV11 antigenic domain is an HPV11 E6-E7 fusion antigen, for prophylactically or therapeutically immunizing a subject against HPV6 and / or HPV11. Further disclosed is the use of an effective amount of a nucleic acid molecule encoding a human papillomavirus (HPV) antigen comprising an HPV6 antigenic domain and an HPV11 antigenic domain in a method for treating or preventing recurrent respiratory papillomatosis (RRP) in a subject, wherein the HPV6 antigenic domain is an HPV6 E6-E7 fusion antigen and the HPV11 antigenic domain is an HPV11 E6-E7 fusion antigen.

[0013] Further provided is use of a nucleic acid molecule encoding a human papillomavirus (HPV) antigen comprising an HPV6 antigenic domain and an HPV11 antigenic domain in the preparation of a medicament for inducing an immune response in a subject, for prophylactically or therapeutically immunizing a subject against HPV6 and / or HPV11, or for treating or preventing recurrent respiratory papillomatosis (RRP) in a subject, wherein the HPV6 antigenic domain is an HPV6 E6-E7 fusion antigen and the HPV11 antigenic domain is an HPV11 E6-E7 fusion antigen.

[0014] The nucleic acid molecules for use as disclosed herein can be formulated with pharmaceutically acceptable excipients in pharmaceutical compositions. The nucleic acid molecules can be formulated for intradermal or intramuscular injection, and optionally for electroporation. According to some embodiments, the nucleic acid molecules can be formulated for intramuscular administration using a standard needle or a side port needle.

[0015] According to some embodiments of the disclosed uses, the use reduces the number of RRP surgical interventions in a population of human subjects diagnosed with RRP in the 52 weeks after administration of the pharmaceutical composition compared to the number of RRP surgical interventions in the 52 weeks prior to administration of the pharmaceutical composition to the population of human subjects diagnosed with RRP. For example, the use may reduce the number of RRP surgical interventions in at least about 76% of the population of human subjects diagnosed with RRP in the 52 weeks after administration of the pharmaceutical composition compared to the number of RRP surgical interventions in the 52 weeks prior to administration of the pharmaceutical composition to the population of human subjects diagnosed with RRP. This use may reduce the number of RRP surgical interventions in about 70% to about 95%, about 75% to about 85%, about 75% to about 80%, about 75%, about 76%, about 77%, about 78%, about 79%, or about 80% of a population of human subjects diagnosed with RRP in the 52 weeks following administration of the pharmaceutical composition, compared to the number of RRP surgical interventions in the 52 weeks prior to administration of the pharmaceutical composition to the population of human subjects diagnosed with RRP. In some embodiments, the median reduction in the number of RRP surgical interventions in the 52 weeks following administration of the pharmaceutical composition is about 3.

[0016] According to some embodiments, the use increases the number of HPV6- and HPV11-specific CD4 cells expressing CD38 in at least about 85% of a population of human subjects diagnosed with RRP 52 weeks after administration of the pharmaceutical composition compared to the number of INO-3107-specific CD4 cells expressing CD38 in the 52 weeks prior to administration of the pharmaceutical composition to the population of human subjects diagnosed with RRP. This use may increase the number of HPV6- and HPV11-specific CD4 cells expressing CD38 in about 70% to about 95%, about 75% to about 90%, about 85% to about 90%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, or about 89% of a population of human subjects diagnosed with RRP, compared to the number of HPV6- and HPV11-specific CD4 cells expressing CD38 in the 52 weeks prior to administration of the pharmaceutical composition to the population of human subjects diagnosed with RRP.

[0017] In some embodiments, the use increases the number of HPV6- and HPV11 E6-specific CD8 cells that express CD38, granzyme A, granzyme B, and perforin in at least 85% of a population of human subjects diagnosed with RRP 52 weeks after administration of the pharmaceutical composition compared to the number of HPV6- and HPV11 E6-specific CD8 cells that express CD38, granzyme A, granzyme B, and perforin in the 52 weeks prior to administration of the pharmaceutical composition to the population of human subjects diagnosed with RRP. This use increases the number of HPV6- and HPV11 E6-specific CD8 cells expressing CD38, granzyme A, granzyme B, and perforin in about 70% to about 95%, about 75% to about 90%, about 85% to about 90%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, or about 89% of a population of human subjects diagnosed with RRP during the 52 weeks following administration of the pharmaceutical composition, compared to the number of HPV6- and HPV11 E6-specific CD8 cells expressing CD38, granzyme A, granzyme B, and perforin during the 52 weeks prior to administration of the pharmaceutical composition to the population of human subjects diagnosed with RRP.

[0018] According to some embodiments, the number of RRP surgical interventions may be reduced in at least about 90% of a population of human subjects diagnosed with RRP in the 52 weeks following administration of the pharmaceutical composition, compared to the number of RRP surgical interventions in the 52 weeks prior to administration of the pharmaceutical composition to the population of human subjects diagnosed with RRP. According to some embodiments, the number of RRP surgical interventions may be reduced in about 70% to about 95%, about 75% to about 90%, about 85% to about 90%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, or about 95% of a population of human subjects diagnosed with RRP in the 52 weeks following administration of the pharmaceutical composition, compared to the number of RRP surgical interventions in the 52 weeks prior to administration of the pharmaceutical composition to the population of human subjects diagnosed with RRP.

[0019] According to some embodiments, the use increases the number of HPV6-specific activated CD4 and HPV11-specific activated CD4 and / or activated lytic CD8 T cells.

[0020] RRP can be juvenile-onset RRP or adult-onset RRP.

[0021] According to some embodiments of the disclosed methods and uses, the HPV antigen comprises the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:11, or an amino acid sequence at least 95% homologous to SEQ ID NO:1 or SEQ ID NO:11. The nucleic acid molecule comprises a nucleotide sequence at least 95% homologous to SEQ ID NO:2 or SEQ ID NO:12, the nucleotide sequence of SEQ ID NO:2, or the nucleotide sequence of SEQ ID NO:12. The nucleic acid sequence encoding the HPV11 antigenic domain may be located relative to or 5' to the nucleic acid sequence encoding the HPV6 antigenic domain. In some embodiments, the HPV6 antigenic domain and the HPV11 antigenic domain are separated by one or more post-translational cleavage sites, one or more translational skip sites, or both. The HPV6 E6 antigenic domain and the HPV6 E7 antigenic domain may be separated by one or more post-translational cleavage sites, one or more translational skip sites, or both. Similarly, the HPV11 E6 antigenic domain and the HPV11 E7 antigenic domain may be separated by one or more post-translational cleavage sites, one or more translational skip sites, or both.

[0022] The nucleic acid molecule of the disclosed methods and uses can be an expression vector, optionally a DNA plasmid. In some embodiments, the expression vector comprises the nucleotide sequence of SEQ ID NO:3.

[0023] The nucleic acid molecule may be a component of a pharmaceutical composition further comprising a pharmaceutically acceptable excipient. The pharmaceutically acceptable excipient may include a buffer, optionally a saline-sodium citrate buffer. In some embodiments, the pharmaceutical composition comprises 6 mg of a vector encoding an HPV antigen per milliliter of buffer and 0.25 mg of a vector encoding the p35 subunit of IL-12, the p40 subunit of IL-1, or both per milliliter of buffer. For example, the pharmaceutical composition may comprise 6 mg of pGX3024 per milliliter of buffer and 0.25 mg of pGX6010 per milliliter of buffer.

[0024] The summary and the following detailed description will be better understood when read in conjunction with the appended drawings. For the purpose of illustrating the disclosed invention, there are shown in the drawings exemplary embodiments thereof; however, the invention is not limited to the particular embodiments disclosed. The drawings are as follows: [Brief explanation of the drawings]

[0025] [Figure 1A] Schematic diagram of the antigens encoded by different HPV6 and / or HPV11 plasmids. Individual antigens are separated by a P2A cleavage site for translational skipping and a furin cleavage site for post-translational cleavage of the P2A sequence. DNA plasmid pGX3024 encodes the consensus SynCon® E6 and E7 antigens for both HPV6 and HPV11. [Figure 1B] A plasmid map of pGX3024 is provided. [Figure 2]Figure 1 shows in vitro expression of pGX3024 E6 and E7 protein antigens. HEK-293T cells were transfected with either the pGX3024 plasmid, the positive control pGX3021 or pGX3022 plasmid, or the negative control empty pGX0001 plasmid using Lipofectamine 3000 transfection reagent. Cells were harvested 48 hours posttransfection, and cell lysates were then probed by Western blot with anti-HPV11 E7 (left panel) or anti-2A (center panel) antibodies. Blots were stripped and reprobed with anti-β-actin antibodies (right panel) to confirm equal protein loading. E6 and E7 proteins were detected in cells transfected with pGX3024 and the control pGX3021 and pGX3022 plasmids, but not with the negative control pGX0001 plasmid. [Figure 3] Figure 1 shows HPV6- and HPV11-specific cellular responses after pGX3024 immunization of C57BL / 6 mice. C57BL / 6 mouse splenocytes were collected one week after immunization with either pGX3024, control plasmids encoding HPV6 (pGX3021) or HPV11 (pGX3022) antigens, or a negative control plasmid (pGX0001). Specific cellular responses to HPV6 and HPV11 E6 and E7 peptides were measured by IFNγ ELISpot assay. Asterisks indicate significant differences in total cellular responses compared to the pGX0001 control by one-way ANOVA with Dunnett's post-hoc test. [Figure 4]Figure 1 shows the effect of a plasmid encoding mouse IL-12 (pGX6012) on cellular responses to HPV6 and HPV11 E6 and E7 peptides after immunization with pGX3024 in C57BL / 6 mice. C57BL / 6 mouse splenocytes were collected 14 days after a single immunization with pGX3024 alone, or increasing doses of plasmid encoding mouse IL-12 (pGX6012) or a control plasmid (pGX0001). Specific cellular responses to HPV6 and HPV11 E6 and E7 peptides were measured by IFNγ ELISpot assay. Error bars are SEM. Asterisks indicate significant differences in total cellular responses compared to pGX3024 alone by one-way ANOVA with Dunnett's post-hoc test. [Figure 5A] Figure 1 shows the effect of a plasmid encoding murine IL-12 (pGX6012) on humoral responses to HPV6, 11 E6, and E7 peptides after immunization with pGX3024 in C57BL / 6 mice. C57BL / 6 mouse serum samples were collected before (week 0) immunization with either pGX3024 alone or increasing doses of pIL-12 (pGX6012) or control plasmid (pGX0001). Specific IgG-binding antibodies against HPV6 E6, HPV6 E7, HPV11 E6, or HPV11 E7 antigens were measured by ELISA. Error bars are SEM. [Figure 5B] Figure 1 shows the effect of a plasmid encoding murine IL-12 (pGX6012) on humoral responses to HPV6, 11 E6, and E7 peptides after immunization with pGX3024 in C57BL / 6 mice. C57BL / 6 mouse serum samples were collected (week 2) after a single immunization with either pGX3024 alone or increasing doses of pIL-12 (pGX6012) or a control plasmid (pGX0001). Specific IgG-binding antibodies against HPV6 E6, HPV6 E7, HPV11 E6, or HPV11 E7 antigens were measured by ELISA. Error bars are SEM. Asterisks indicate significant differences in total cellular responses compared to pGX3024 alone by one-way ANOVA with Dunnett's post-hoc test. [Figure 6]Figure 1 shows HPV6 and HPV11 humoral responses after pGX3024 immunization of C57BL / 6 mice. C57BL / 6 mouse serum samples were collected before immunization (week 0) and after the first (week 2) and second (week 3) immunizations with either pGX3024 or a negative control plasmid (pGX0001). Specific IgG-binding antibodies against the HPV6 E7 (left panel) or HPV11 E7 (right panel) antigen were measured by ELISA. [Figure 7] Figure 1 shows HPV6- and HPV11-specific cellular responses after pGX3024 immunization of BALB / c mice. Splenocytes from BALB / c mice were collected one week after immunization with the indicated doses of pGX3024 alone or in combination with the indicated doses of plasmid mouse IL-12 (pGX6012) or negative control plasmid (pGX0001). Specific cellular responses to HPV6 and HPV11 E6 and E7 peptides were measured by IFNγ ELISpot assay. Asterisks indicate significant differences in total cellular responses compared to the pGX0001 control by one-way ANOVA with Dunnett's post-hoc test. [Figure 8] Figure 1 shows HPV6 and HPV11 humoral responses after pGX3024 immunization of BALB / c mice. BALB / c mouse serum samples were collected before immunization (week 0) and after the first (week 2) and second (week 3) immunizations with the indicated doses of pGX3024 alone or in combination with the indicated doses of plasmid mouse IL-12 (pGX6012) or negative control plasmid (pGX0001). Specific IgG-binding antibodies against the HPV6 E7 (left panel) or HPV11 E7 (right panel) antigens were measured by ELISA. Asterisks indicate significance compared to week 0 by two-way ANOVA, and "ns" indicates no significant difference. [Figure 9]Figure 1 shows the time course of HPV6-specific and HPV11-specific cellular responses after INO-3107 immunization of NZW rabbits. NZW rabbit peripheral blood mononuclear cells (PBMCs) were collected at the indicated time points after immunization with either INO-3107 or 1x saline-sodium citrate buffer (SSC). Specific cellular responses to HPV6 E6 and E7 peptides and HPV11 E6 and E7 peptides were measured by IFNγ ELISpot assay. Data are shown as the sum of HPV6 E6 and E7 (left panel) or HPV11 E6 and E7 (right panel) responses for individual animals. [Figure 10] Figure 9 shows HPV6- and HPV11-specific cellular responses following INO-3107 immunization of NZW rabbits. At week 11 (2 weeks after the fourth immunization), T cell responses to HPV6 E6, HPV6 E7, HPV11 E6, or HPV11 E7 antigens for NZW rabbits were measured with either INO-3107 or 1x SSC as shown in Figure 9. Data are shown for individual rabbits (left panel) or mean ± SEM for each treatment group (right panel). Asterisks indicate significant differences (p<0.05) as determined by the Mann-Whitney U test. [Figure 11]

[0033] Figure 1 shows the time course of HPV6 and HPV11 humoral responses after INO-3107 immunization of NZW rabbits. NZW rabbit serum samples were collected at the indicated time points after immunization with either INO-3107 or 1xSSC. Specific humoral responses to HPV6 E7 (left panel) and HPV11 E7 (right panel) antigens were measured by IgG-binding ELISA. Data are shown for individual animals. [Figure 12] Time course of body weight measurements of NZW rabbits administered INO-3107 (left panel) or 1×SSC (right panel) is shown. [Figure 13]Figure 1 shows the time course of HPV6-specific and HPV11-specific cellular responses after pGX3024 immunization in Hartley guinea pigs. Guinea pigs (n=5) were immunized with 100 μg of pGX3024 administered by CELLECTRA intradermal electroporation at weeks 0, 2, and 4. Naive guinea pigs (n=2) served as negative controls. Guinea pig peripheral blood mononuclear cells (PBMCs) were collected at the indicated time points after immunization with pGX3024 or from naive guinea pigs. Specific cellular responses to HPV6 E6 and E7 peptides and HPV11 E6 and E7 peptides were measured by IFNγ ELISpot assay. Data are presented as the mean ± SEM for each HPV6 E6 and E7 treatment group. [Figure 14] Figure 1 shows the time course of HPV6-specific and HPV11-specific humoral responses following pGX3024 immunization of Hartley guinea pigs. Hartley guinea pigs (n=5) were immunized at weeks 0, 2, and 4 with 100 μg of pGX3024 administered by CELLECTRA intradermal electroporation. Guinea pig serum samples were collected at the indicated time points after immunization with pGX3024 for measurement of specific IgG-binding antibodies against the HPV6 E7 (left) or HPV11 E7 (right) antigens by ELISA. Data are shown as the mean ± SEM of 5 animals. [Figure 15] Figure 1 shows the immune response induced after intradermal delivery of INO-3107 to NZW rabbits. Cellular immune responses were assessed by IFNγ ELISpot before immunization (week 0) and two weeks after each immunization (weeks 2, 5, and 8). The combined immune response to both antigens, HPV6 and HPV11, increased after each immunization, and T cell responses were more HPV6 E6- and HPV11 E6-specific. [Figure 16]Figure 1 shows cellular IFN-γ responses in NZW rabbit PBMCs. Total T cell responses to HPV6 E6, HPV11 E6, HPV6 E7, and HPV11 E7 antigens were measured by IFNγ ELISpot assay in NZW rabbits at prebleed, D14, D35, D56, and D77. Error bars are SEM. Asterisks indicate significant differences between groups by one-way ANOVA, multiple comparison test. [Figure 17A] Figure 1 shows humoral responses in NZW rabbit serum. NZW rabbit serum samples were collected before (day 0) and after four immunizations (day 77) with pGX3024 and human pIL-12 delivered ID or IM. Specific IgG binding antibodies against HPV6 E6 antigen were measured by ELISA. Serum dilutions started at 1:800 for HPV6 E6. Error bars are SEM. [Figure 17B] Humoral responses in NZW rabbit serum are shown. NZW rabbit serum samples were collected before (day 0) and after four immunizations (day 77) with pGX3024 and human pIL-12 delivery ID or IM. Specific IgG binding antibodies against HPV6 E7 antigen were measured by ELISA. Serum dilutions started at 1:200 for HPV6 E7. Error bars are SEM. [Figure 17C] Figure 1 shows humoral responses in NZW rabbit serum. NZW rabbit serum samples were collected before (day 0) and after four immunizations (day 77) with pGX3024 and human pIL-12 delivered ID or IM. Specific IgG binding antibodies against HPV11 E6 antigen were measured by ELISA. Serum dilutions started at 1:800 for HPV11 E6. Error bars are SEM. [Figure 17D] Figure 1 shows humoral responses in NZW rabbit serum. NZW rabbit serum samples were collected before (day 0) and after four immunizations (day 77) with pGX3024 and human pIL-12 delivery ID or IM. Specific IgG binding antibodies against HPV11 E7 antigen were measured by ELISA. Serum dilutions started at 1:200 for HPV11 E7. Error bars are SEM. [Figure 18] 1 shows the study design of a Phase 1 / 2 clinical trial (INO-3107 by electroporation (EP) in subjects with HPV-6-associated recurrent respiratory papillomatosis (RRP) and / or HPV-11-associated RRP [ClinicalTrials.gov Identifier: NCT04398433]). [Figure 19] Interim data are presented for TEAEs occurring in two or more patients in the clinical trial (ClinicalTrials.gov Identifier: NCT04398433) (n=32). The most frequently reported TEAEs were related to administration (injection site or procedural pain). 17 / 20 TEAEs (85.0%) occurred within 7 days of administration. [Figure 20] Interim data are presented for the number of RRP procedures (operating room or office-based) in the year after INO-3107 administration versus the previous year for individual patients (n=32) in the clinical trial (ClinicalTrials.gov Identifier: NCT04398433). Overall, 26 patients (81.3%) had reduced surgical interventions in the year after INO-3107 administration versus the previous year, of which 9 (28.1%) did not require surgical intervention. [Figure 21] Interim data for RRP staging scores in a clinical trial (ClinicalTrials.gov Identifier: NCT04398433) (n=32) are shown. Improvement was seen in the mean total site score at week 52 (10.7 points lower than baseline), while mean symptom scores were similar at week 52 compared to baseline. [Figure 22]

[0023] Figure 1 shows interim data on the number of surgeries (operating room or office-based) in the year after administration of INO-3107 via standard needle injection versus the previous year for individual patients (mITT population, n=21) in a clinical trial (ClinicalTrials.gov Identifier: NCT04398433). The graph is organized to show the data as a function of the number of procedures in the previous year. Numbering does not reflect the order of enrollment. [Figure 23A]Figure 23A shows the frequencies of HPV-6-specific activated CD4 and HPV-11-specific activated CD4 T cells and cytolytic CD8 T cells (mITT population, n=21) assessed in a clinical trial (ClinicalTrials.gov Identifier: NCT04398433). Figure 23B shows the total CD4 response over time. Figure 23A is a box plot; the boxes represent the 25th and 75th percentiles, the horizontal line dividing the box in half is the median, and the + is the mean. [Figure 23B] Figure 23B shows the frequencies of HPV-6-specific activated CD4 and HPV-11-specific activated CD4 T cells and cytolytic CD8 T cells (mITT population, n=21) assessed in a clinical trial (ClinicalTrials.gov Identifier: NCT04398433). Figure 23B shows the contribution of HPV-6-specific CD4 and HPV-11-specific CD4 T cells to the peak response. [Figure 23C] Figure 23C shows the frequencies of HPV-6-specific activated CD4 and HPV-11-specific activated CD4 T cells and cytolytic CD8 T cells (mITT population, n=21) assessed in a clinical trial (ClinicalTrials.gov Identifier: NCT04398433). Figure 23C shows the total CD8 response over time. Figure 23C is a box plot; the boxes represent the 25th and 75th percentiles, the horizontal line dividing the box in half is the median, and the + is the mean. [Figure 23D]Figure 23D shows the frequencies of HPV-6-specific activated CD4 and HPV-11-specific activated CD4 T cells and cytolytic CD8 T cells (mITT population, n=21) assessed in a clinical trial (ClinicalTrials.gov Identifier: NCT04398433). Figure 23D shows the contribution of HPV-6-specific CD8 and HPV-11-specific CD8 T cells to the peak response. The peak response was the highest response observed during the study, excluding pre-dose and week 52 time points. The memory response was measured at week 52. Grz = granzyme A, GrzB = granzyme b, HPV = human papillomavirus, Prf = perforin, mITT = modified intent to treat. Increases in INO-3107-specific CD4 cells expressing activation markers (CD38) and HPV-6 and HPV-11 E6-specific CD8 cells expressing a combination of activation markers (CD38) and lytic capacity (granzyme A, granzyme B, perforin) were observed after vaccination. Regarding CD8 T cell activity, 18 / 21 (85.7%) patients demonstrated antigen-specific cell activity above pre-dose levels, as demonstrated by flow cytometry. This was similar for CD4 T cells in 18 / 21 (85.7%) patients. The proportions of antigen-specific CD8 and CD4 cells expressing activation markers and CD8 cells expressing lytic capacity markers were similar for HPV-6 and HPV-11. Increased frequencies of activated INO-3107-specific CD4 and CD8 cells were observed in week 52 samples compared to baseline. [Figure 24A] 1 shows exemplary images of responders at baseline. [Figure 24B] 1 shows exemplary images of responders after treatment with INO-3107. [Figure 24C] 1 shows exemplary images of non-responders at baseline. [Figure 24D] 1 shows exemplary images of a non-responder after treatment with INO-3107. DETAILED DESCRIPTION OF THE INVENTION

[0026] The disclosed nucleic acid molecules, proteins, vaccines, and methods may be more readily understood by reference to the following detailed description, which is set forth in connection with the accompanying drawings, which form a part of this disclosure: It is to be understood that the disclosed nucleic acid molecules, proteins, vaccines, and methods are not limited to the specific nucleic acid molecules, proteins, vaccines, and methods described and / or illustrated herein, and that the terminology used herein is for the purpose of describing particular embodiments by way of example only and is not intended to limit the claimed nucleic acid molecules, proteins, vaccines, and methods.

[0027] Unless specifically stated otherwise, any explanation of a possible mechanism or mode of action, or reason for improvement, is intended to be exemplary only, and the disclosed nucleic acid molecules, proteins, vaccines, and methods should not be limited by the accuracy or inaccuracy of any such proposed mechanism or mode of action, or reason for improvement.

[0028] Throughout this specification, descriptions refer to compositions and methods of using the compositions. Where this disclosure describes or claims features or embodiments related to compositions, such features or embodiments are equally applicable to methods of using the compositions. Similarly, where this disclosure describes or claims features or embodiments related to methods of using a composition, such features or embodiments are equally applicable to the compositions.

[0029] It should be understood that certain features of the disclosed nucleic acid molecules, proteins, vaccines, and methods that are described herein in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the disclosed nucleic acid molecules, proteins, vaccines, and methods that are described for clarity in the context of a single embodiment may also be provided separately or in any subcombination.

[0030] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In case of conflict, the present specification, including definitions, will control. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, exemplary methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and are not intended to be limiting. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0031] The terms "comprise(s)," "include(s)," "having," "having," "can," "containing," and variations thereof, as used herein, are intended to be open-ended transitional phrases, terms, or words that do not preclude the possibility of additional acts or structures. The term "comprising" is intended to include examples encompassed by the terms "consisting essentially of" and "consisting of," and similarly, the term "consisting essentially of" is intended to include examples encompassed by the term "consisting of." The present disclosure also contemplates other embodiments that "comprise," "consist of," and "consist essentially of" the embodiments or elements set forth herein, whether or not explicitly stated.

[0032] The singular forms "a," "and," and "the" include plural references unless the context clearly dictates otherwise.

[0033] For the recitation of numerical ranges herein, each intervening number is expressly contemplated to the same degree of precision. For example, for the range of 6 to 9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range of 6.0 to 7.0, the numbers 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are expressly contemplated.

[0034] Some of the quantitative expressions set forth herein are not qualified by the term "about." It is understood that, whether or not the term "about" is explicitly used, all given amounts are intended to refer to the actual given value, and also to refer to approximations about such given value that would be reasonably estimated based on a person skilled in the art, including approximations resulting from experimental and / or measurement conditions for such value.

[0035] As used herein, "adjuvant" means any molecule added to the immunogenic compositions described herein to enhance the immunogenicity of the antigens and nucleic acid molecules encoding the antigens and sequences described below.

[0036] "Antigen" refers to a protein having the HPV6 E6 domain, the HPV6 E7 domain, the HPV11 E6 domain, the HPV11 E7 domain, or any combination thereof, preferably a fusion protein of the HPV6 E6 domain, the HPV6 E7 domain, the HPV11 E6 domain, and the HPV11 E7 domain with an endoproteolytic cleavage site between each domain. Antigens include SEQ ID NO: 1, fragments thereof of lengths described herein, variants thereof, i.e., proteins having sequence identity to SEQ ID NO: 1 described herein, fragments of the variants of lengths described herein, and combinations thereof. The antigen may have the IgE leader sequence of SEQ ID NO: 10, or such sequence may be removed from the N-terminus. For example, an HPV antigen comprising the HPV6 E6 domain, the HPV6 E7 domain, the HPV11 E6 domain, and the HPV11 E7 domain may have an IgE leader sequence located N-terminal to the N-terminal HPV domain of the HPV antigen, with or without an endoproteolytic cleavage site between each domain. The antigen may optionally include a signal peptide, such as one from another protein.

[0037] The term "biosimilar" (of an approved reference product / biological drug, i.e., a reference-listed drug) refers to a biological product that is highly similar to the reference product, despite minor differences in clinically inactive components, in terms of safety, purity, and efficacy, with no clinically meaningful differences between the biosimilar and the reference product, based on data obtained from: (a) analytical studies showing that the biological product is highly similar to the reference product, despite minor differences in clinically inactive components; (b) animal studies (including evaluation of toxicity); and / or (c) clinical studies (including evaluation of immunogenicity and pharmacokinetics or pharmacodynamics) sufficient to demonstrate safety, purity, and efficacy under one or more appropriate conditions of use for which the reference product is licensed and intended to be used, and for which a license for the biosimilar is sought. A biosimilar may be an interchangeable product that can be substituted for the reference product in pharmacies without the intervention of a prescribing healthcare professional. To meet the additional criteria of "interchangeability," a biosimilar is expected to produce the same clinical results as the reference product in any given patient, and when the biosimilar is administered multiple times to an individual, the risks in terms of safety or reduced efficacy of substituting or switching between use of the biosimilar and use of the reference product are no greater than the risks of using the reference product without such substitution or switching. The biosimilar utilizes the same mechanism of action for the proposed conditions of use, to the extent that the mechanism is known for the reference product. The conditions of use specified, recommended, or proposed in the proposed labeling for the biosimilar have previously been approved for the reference product. The route of administration, dosage form, and / or strength of the biosimilar are the same as those of the reference product, and the biosimilar is manufactured, processed, packaged, or held in facilities that meet standards designed to ensure that the biosimilar remains safe, pure, and potent. A biosimilar may contain minor modifications in amino acid sequence, such as N- or C-terminal truncations, that are not expected to alter biosimilar performance when compared to the reference product.

[0038] As used herein, "coding sequence" or "encoding nucleic acid" refers to a nucleic acid (RNA or DNA molecule) comprising a nucleotide sequence that encodes a protein. The coding sequence may further comprise initiation and termination signals operably linked to control elements comprising a promoter and polyadenylation signal capable of directing expression in the cells of an individual or mammal to which the nucleic acid is administered.

[0039] As used herein, "complement" or "complementary" refers to a nucleic acid molecule having Watson-Crick (e.g., AT / U and CG) or Hoogsteen base pairing between nucleotides or nucleotide analogs and a reference nucleic acid molecule.

[0040] As used herein, "consensus" or "consensus sequence" refers to a polypeptide sequence based on an analysis of the alignment of multiple sequences for the same gene from different organisms. Nucleic acid sequences encoding consensus polypeptide sequences can be prepared. Immunogenic compositions comprising proteins containing consensus sequences and / or nucleic acid molecules encoding such proteins can be used to induce broad immunity against an antigen.

[0041] "Electroporation," "electropermeabilization," or "electrokinetic enhancement" ("EP"), as used interchangeably herein, refers to the use of transmembrane electric field pulses to induce microscopic pathways (pores) in biological membranes, the presence of which allows the passage of biological molecules, such as plasmids and vectors, oligonucleotides, siRNA, drugs, ions, and water, from one side of the cell membrane to the other.

[0042] As used herein, a "fragment" with respect to a nucleic acid sequence refers to a nucleic acid sequence, or a portion thereof, that encodes a polypeptide capable of eliciting an immune response in a mammal that cross-reacts with an antigen disclosed herein. The fragment may be a DNA fragment selected from at least one of the various nucleotide sequences encoding protein fragments described below. The fragment may comprise at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of one or more of the nucleic acid sequences described below. In some embodiments, the fragment comprises at least 20 or more nucleotides, at least 30 or more nucleotides, at least 40 or more nucleotides, at least 50 or more nucleotides, at least 60 or more nucleotides, at least 70 or more nucleotides, at least 80 or more nucleotides, at least 90 or more nucleotides, at least 100 or more nucleotides, at least 150 or more nucleotides, at least 200 or more nucleotides, at least 250 or more nucleotides, at least 300 or more nucleotides, at least 350 or more nucleotides, at least 400 or more nucleotides, at least 450 or more nucleotides, at least 500 or more nucleotides, at least 550 or more nucleotides, at least 600 or more nucleotides, at least 650 or more nucleotides, at least 700 or more nucleotides, at least 750 or more nucleotides, at least 800 or more nucleotides, at least 850 or more nucleotides, at least 900 or more nucleotides, at least 950 or more nucleotides, or at least 1000 or more nucleotides of at least one of the nucleic acid sequences set forth below.

[0043] "Fragment" or "immunogenic fragment," with respect to a polypeptide sequence, means a polypeptide capable of eliciting an immune response in a mammal that cross-reacts with an antigen disclosed herein. The fragment can be a polypeptide fragment selected from at least one of the following various amino acid sequences: A fragment of a consensus protein can comprise at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of the consensus protein. In some embodiments, a fragment of a consensus protein can comprise at least 20 or more amino acids, at least 30 or more amino acids, at least 40 or more amino acids, at least 50 or more amino acids, at least 60 or more amino acids, at least 70 or more amino acids, at least 80 or more amino acids, at least 90 or more amino acids, at least 100 or more amino acids, at least 110 or more amino acids, at least 120 or more amino acids, at least 130 or more amino acids, at least 140 or more amino acids, at least 150 or more amino acids, at least 160 or more amino acids, at least 170 or more amino acids, or at least 180 or more amino acids of a protein sequence disclosed herein.

[0044] As used herein, the term "genetic construct" refers to a DNA or RNA molecule comprising a nucleotide sequence that encodes a protein. The coding sequence includes initiation and termination signals operably linked to control elements, including a promoter, and a polyadenylation signal that are capable of directing expression in the cells of an individual to whom the nucleic acid molecule is administered. As used herein, the term "expressible form" refers to a genetic construct that contains the necessary control elements operably linked to a coding sequence that encodes a protein such that the coding sequence is expressed when present in the cells of an individual.

[0045] The term "homology" as used herein refers to the degree of complementarity. There can be partial or complete homology (i.e., identity). Partially complementary sequences that at least partially inhibit a fully complementary sequence from hybridizing to a target nucleic acid are referred to using the functional term "substantially homologous." When used in reference to a double-stranded nucleic acid sequence, such as a cDNA or genomic clone, the term "substantially homologous" as used herein refers to a probe that can hybridize to a strand of the double-stranded nucleic acid sequence under low stringency conditions. When used in reference to a single-stranded nucleic acid sequence, the term "substantially homologous" as used herein refers to a probe that can hybridize to (i.e., is the complement of) a single-stranded nucleic acid template sequence under low stringency conditions.

[0046] As used herein, "identical" or "identity" in the context of two or more nucleic acid or polypeptide sequences means that the sequences have a specified percentage of residues that are the same over a specified region. The percentage can be calculated by optimally aligning the two sequences, comparing the two sequences over a specified region, determining the number of positions 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 specified region, and multiplying the result by 100 to obtain the percentage of sequence identity. If the two sequences are of different lengths, or the alignment results in one or more skewed ends, and a particular comparison region contains only a single sequence, the residues of the single sequence are included in the denominator rather than the numerator of the calculation. When comparing DNA and RNA, thymine (T) and uracil (U) can be considered equivalent. Identity can be performed manually or using a computer sequence algorithm such as BLAST or BLAST 2.0.

[0047] As used herein, "INO-3107" refers to an immunogenic composition of two DNA plasmids: DNA plasmid pGX3024 encoding HPV antigens, including SynCon® E6 and E7 antigens, for both HPV6 and HPV11, in combination with DNA plasmid pGX6010, encoding human IL-12. The amino acid sequences of HPV antigens, including SynCon® E6 and E7 antigens, for both HPV6 and HPV11, are provided in SEQ ID NO: 1. The nucleotide sequence encoding HPV antigens, including SynCon® E6 and E7 antigens, for both HPV6 and HPV11, is provided in SEQ ID NO: 2. The nucleic acid sequence of DNA plasmid pGX3024 is set forth in SEQ ID NO: 3. The sequence of DNA plasmid pGX6010 is set forth in SEQ ID NO: 4. "INO-3107" may further comprise a saline-sodium citrate buffer. "INO-3107 drug product" refers to an immunogenic composition containing 6.25 mg of total plasmid / mL (6 mg / mL pGX3024, 0.25 mg / mL pGX6010) in 150 mM sodium chloride and 15 mM sodium citrate, pH 7.

[0048] As used herein, "immune response" refers to the activation of a host's immune system, e.g., a mammal's immune system, in response to the introduction of an antigen. The immune response can be in the form of a cellular or humoral response, or both.

[0049] As used herein, "nucleic acid" or "oligonucleotide" or "polynucleotide" refers to at least two nucleotides covalently linked to each other. A depiction of a single strand also defines the sequence of the complementary strand. Thus, a nucleic acid also encompasses the complementary strand of a depicted single strand. Many variants of a nucleic acid can be used for the same purpose as a given nucleic acid. Thus, a nucleic acid also encompasses substantially identical nucleic acids and their complements. A single strand provides a probe that can hybridize to a target sequence under stringent hybridization conditions. Thus, a nucleic acid also encompasses a probe that hybridizes under stringent hybridization conditions.

[0050] Nucleic acids can be single-stranded or double-stranded, or can contain portions of both double-stranded and single-stranded sequence. Nucleic acids can be DNA, both genomic and cDNA, RNA, or hybrids, and can contain combinations of deoxyribonucleotides and ribonucleotides, with base combinations including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine, hypoxanthine, isocytosine, and isoguanine. Nucleic acids can be obtained by chemical synthesis or recombinant methods.

[0051] As used herein, "operably linked" means 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 it is derived. As is known in the art, variations in this distance can be accommodated without loss of promoter function.

[0052] As used herein, "peptide," "protein," or "polypeptide" can refer to a linked sequence of amino acids, which can be natural, synthetic, or a modified or combination of natural and synthetic.

[0053] As used herein, a "promoter" refers to a synthetic or naturally occurring molecule capable of conferring, activating, or enhancing expression of a nucleic acid in a cell. A promoter may contain one or more specific transcriptional regulatory sequences to further enhance expression and / or modify its spatial and / or temporal expression. A promoter may also contain distal enhancer or repressor elements, which may be located as far as several thousand base pairs from the start site of transcription. Promoters may be derived from sources including viruses, bacteria, fungi, plants, insects, and animals. A promoter may constitutively or differentially regulate expression of a genetic component with respect to the cell, tissue, or organ in which expression occurs, or with respect 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.

[0054] "Signal peptide" and "leader sequence" are used interchangeably herein and refer to an amino acid sequence that can be linked at the amino terminus of a protein described herein. A signal peptide / leader sequence typically directs the localization of a protein. As used herein, a signal peptide / leader sequence can facilitate secretion of a protein from the cell in which it is produced. A signal peptide / leader sequence is often cleaved from the remainder of the protein, often referred to as the mature protein, upon secretion from the cell. A signal peptide / leader sequence is linked at the amino terminus (i.e., N-terminus) of a protein.

[0055] As used herein, "substantially complementary" means that a first sequence is substantially complementary to a second sequence over a region of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 180, 270, 360, 450, 540, or more nucleotides or amino acids. It means that a sequence is at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the complement of a second sequence, or that the two sequences hybridize under stringent hybridization conditions.

[0056] As used herein, "substantially identical" means that the first and second sequences are at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 180, 270, 360, 450, 540 or more nucleotides or amino acids apart. "Identity" means being at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical over the entire nucleic acid region, or, with respect to nucleic acids, that a first sequence is substantially complementary to the complement of a second sequence.

[0057] As used herein, the phrase "subject in need thereof" refers to a human or non-human mammal exhibiting one or more symptoms or indications of recurrent respiratory papillomatosis (RRP) and / or diagnosed with RRP and in need of treatment thereof. In many embodiments, the term "subject" can be used interchangeably with the term "patient." For example, a human subject can be diagnosed with RRP and / or one or more symptoms or indications, including, but not limited to, hoarseness, weak crying, chronic cough, breathing problems, difficulty breathing, recurrent upper respiratory tract infections, pneumonia, dysphagia, wheezing, growth failure, and / or respiratory tumors. For example, the phrase includes newly diagnosed subjects. In some embodiments, the phrase includes subjects for whom treatment with the disclosed methods is an initial treatment (e.g., a "first-line" treatment in which the patient has not received prior systemic treatment for RRP). In certain embodiments, this manifestation includes subjects for whom treatment with the disclosed methods is a "second line" treatment, where the patient has previously been treated with "standard of care" therapies, including but not limited to surgery, antiviral therapy, and tracheostomy.

[0058] As used herein, the terms "treat", "treating" and the like mean alleviating symptoms, temporarily or permanently eliminating the cause of symptoms, slowing or inhibiting tumor growth, reducing tumor cell or tumor burden, promoting tumor regression, causing tumor shrinkage, necrosis and / or disappearance, preventing tumor recurrence, preventing or inhibiting malignant transformation, and / or increasing the survival of a subject.

[0059] As used herein, unless otherwise specified, the term "clinically proven" (whether used independently or to modify the terms "safe" and / or "effective") shall mean that it has been proven by a clinical trial that has met the approval standards of the US Food and Drug Administration, the EMA, or a corresponding national regulatory authority. For example, the proof may be provided by the clinical trial described in the examples provided herein.

[0060] The term "clinically proven safety," when associated with a dose, dosage regimen, treatment, or method using a human papillomavirus (HPV) antigen (e.g., an HPV antigen administered as a pGX3024 or INO-3107 drug or a biosimilar thereof), refers to a favorable risk:benefit ratio with an acceptable frequency and / or acceptable severity of treatment-emergent adverse events (termed TEAEs) compared to standard of care or another comparator. An adverse event is an untoward medical occurrence in a patient administered the drug. One measure of safety is the incidence of National Cancer Institute (NCI) adverse events (AEs) graded according to the Common Toxicity Criteria for Adverse Events CTCAE v5.0.

[0061] The terms "clinically proven effective" and "clinically proven effective" as used herein in the context of a dose, dosage regimen, treatment, or method refer to the effectiveness of a particular dose, dosage, or treatment regimen. Efficacy can be measured based on changes in the course of a disease in response to the agent of the present invention. For example, a human papillomavirus (HPV) antigen (e.g., an HPV antigen administered as a pGX3024 or INO-3107 pharmaceutical or a biosimilar thereof) is administered to a patient in an amount and for a time sufficient to induce an improvement, preferably a sustained improvement, in at least one indicator reflecting the severity of the disorder being treated. To determine whether the amount and duration of treatment are sufficient, various indicators reflecting the extent of the subject's illness, disease, or condition can be evaluated. Such indicators include, for example, clinically recognized indicators of disease severity, symptoms, or signs of the disorder in question. The degree of improvement is generally determined by a physician, who can make this determination based on signs, symptoms, biopsy, or other test results, and can also use questionnaires administered to the subject, such as quality of life questionnaires developed for a given disease. Improvement can be indicated by an improvement in indicators of disease activity, an improvement in clinical symptoms, or any other measure of disease activity. For example, human papillomavirus (HPV) antigens (e.g., HPV antigens administered as pGX3024 or INO-3107 pharmaceuticals or their biosimilars) can be administered to achieve a reduction in the frequency of RRP surgical intervention, a change in RRP stage evaluation score, an increase in the interval between surgeries, or an improvement in patient condition related to HPV clearance or a decrease in disease burden.

[0062] "Variant," as used herein with respect to nucleic acids, can refer to (i) a portion or fragment of a referenced nucleotide sequence, (ii) the complement of a referenced nucleotide sequence or a portion thereof, (iii) a nucleic acid that is substantially identical to a referenced nucleic acid or its complement, or (iv) a nucleic acid that hybridizes under stringent conditions to a referenced nucleic acid, its complement, or a sequence substantially identical thereto. A variant can be a nucleotide sequence that is substantially identical over the full length of a complete gene sequence or a fragment thereof. The nucleotide sequence can be 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical over the full length of a gene sequence or a fragment thereof.

[0063] A "variant," with respect to a polypeptide, is a polypeptide that differs in amino acid sequence by amino acid insertions, deletions, or conservative substitutions, but retains at least one biological activity of the reference polypeptide. A variant can also refer to 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. A variant can be an amino acid sequence that is substantially identical over the full length of the amino acid sequence or a fragment thereof. The amino acid sequence can be 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical over the full length of the amino acid sequence or a fragment thereof.

[0064] As used herein, "vector" refers to a nucleic acid sequence containing an origin of replication. A vector can be a viral vector, a bacteriophage, a bacterial artificial chromosome, or a yeast artificial chromosome. A vector can be a DNA or RNA vector. A vector can be a self-replicating extrachromosomal vector, and in one embodiment, is an expression plasmid. A vector can contain or include one or more heterologous nucleic acid sequences.

[0065] As used herein, the phrase "in combination with" means that the HPV6 E6 and E7 antigens and HPV11 E6 and E7 antigens are administered to a subject simultaneously with, immediately before, or immediately after administration of an adjuvant. In certain embodiments, the HPV6 E6 and E7 antigens and HPV11 E6 and E7 antigens are administered as a co-formulation with an adjuvant.

[0066] As used herein, unless otherwise specified, the term "clinically proven" (whether used independently or to modify the terms "safe" and / or "effective") shall mean that it has been proven by a clinical trial that has met the approval standards of the US Food and Drug Administration, the EMA, or a corresponding national regulatory authority. For example, the proof may be provided by the clinical trial described in the examples provided herein.

[0067] The term "clinically proven safety," when associated with a dose, dosage regimen, treatment, or method using HPV6 E6 and E7 antigens and HPV11 E6 and E7 antigens (e.g., administered as pGX3024) in combination with an adjuvant such as IL-12 (e.g., administered as pGX6010), refers to a favorable risk:benefit ratio with an acceptable frequency and / or severity of treatment-emergent adverse events (also called AEs or TEAEs) compared to a standard of care or another comparator. An adverse event is an untoward medical occurrence in a subject administered a medicinal product.

[0068] The terms "clinically proven effective" and "clinically proven effective" as used herein in the context of a dose, dosage regimen, treatment, or method refer to the effectiveness of a particular dose, dosage, or treatment regimen. Efficacy can be measured based on changes in the course of a disease in response to the agent of the present invention. For example, a combination of HPV6 E6 and E7 antigens and HPV11 E6 and E7 antigens (e.g., administered as pGX3024) and an adjuvant such as IL-12 (e.g., administered as pGX6010) is administered to a subject in an amount and for a time sufficient to induce an improvement, preferably a sustained improvement, in at least one indicator reflecting the severity of the disorder being treated. To determine whether the amount and duration of treatment are sufficient, various indicators reflecting the extent of the subject's illness, disease, or condition can be evaluated. Such indicators include, for example, clinically recognized indicators of disease severity, symptoms, or signs of the disorder in question. The degree of improvement is generally determined by a physician, who can make this determination based on signs, symptoms, biopsy, or other test results, and can also use questionnaires administered to the subject, such as quality of life questionnaires developed for a given disease. For example, a combination of HPV6 E6 and E7 antigens and HPV11 E6 and E7 antigens (e.g., administered as pGX3024) and an adjuvant such as IL-12 (e.g., administered as pGX6010) can be administered to achieve an improvement in the patient's condition associated with RRP. Improvement can be indicated by an improvement in indicators of disease activity, an improvement in clinical symptoms, or any other measure of disease activity.

[0069] Provided herein are pharmaceutical compositions, including nucleic acid molecules, proteins, immunogenic compositions, and vaccines, as well as methods of using them to induce an immune response and / or prevent or treat RRP. The immunogenic compositions preferably contain human papillomavirus (HPV) antigens, including the HPV6 E6 antigenic domain, the HPV6 E7 antigenic domain, the HPV11 E6 antigenic domain, and the HPV11 E7 antigenic domain. The disclosed immunogenic compositions result from a multiphase strategy in which modified consensus sequences are generated and genetic modifications, including codon optimization, RNA optimization, and the addition of highly efficient immunoglobulin leader sequences, are performed. The immunogenic compositions can be used to protect against multiple strains of HPV, thereby treating, preventing, and / or protecting against HPV-based pathologies. In particular, the immunogenic compositions can be used to prevent or treat HPV6- and / or HPV11-based pathologies. The immunogenic composition can significantly induce an immune response in a subject to which the immunogenic composition is administered, thereby protecting against and treating HPV6 infection, HPV11 infection, or both.

[0070] The vaccine may be a DNA vaccine, a peptide vaccine, or a combination vaccine of DNA and peptide. The DNA vaccine may contain a nucleic acid sequence encoding an HPV antigen. The nucleic acid sequence may be DNA, RNA, cDNA, a variant thereof, a fragment thereof, or a combination thereof. The nucleic acid sequence may also contain an additional sequence encoding a linker, leader, or tag sequence linked to the HPV antigen by a peptide bond. The peptide vaccine may contain an HPV antigen peptide, an HPV antigen protein, a variant thereof, a fragment thereof, or a combination thereof. The DNA and peptide combination vaccine may contain the above-mentioned nucleic acid sequence encoding the HPV antigen and an HPV antigen peptide or protein, wherein the HPV antigen peptide or protein and the encoded HPV antigen have the same amino acid sequence.

[0071] The vaccine can induce a humoral immune response in a subject administered the vaccine. The induced humoral immune response can be specific to the HPV6 E6 antigenic domain, the HPV6 E7 antigenic domain, the HPV11 E6 antigenic domain, the HPV11 E7 antigenic domain, or any combination thereof. The induced humoral immune response can be reactive with the HPV6 E6 antigen, the HPV6 E7 antigen, the HPV11 E6 antigen, the HPV11 E7 antigen, or any combination thereof. The humoral immune response can be induced about 1.5-fold to about 16-fold, about 2-fold to about 12-fold, or about 3-fold to about 10-fold in a subject administered the vaccine. A humoral immune response can be induced in a subject receiving the vaccine by at least about 1.5-fold, at least about 2.0-fold, at least about 2.5-fold, at least about 3.0-fold, at least about 3.5-fold, at least about 4.0-fold, at least about 4.5-fold, at least about 5.0-fold, at least about 5.5-fold, at least about 6.0-fold, at least about 6.5-fold, at least about 7.0-fold, at least about 7.5-fold, at least about 8.0-fold, at least about 8.5-fold, at least about 9.0-fold, at least about 9.5-fold, at least about 10.0-fold, at least about 10.5-fold, at least about 11.0-fold, at least about 11.5-fold, at least about 12.0-fold, at least about 12.5-fold, at least about 13.0-fold, at least about 13.5-fold, at least about 14.0-fold, at least about 14.5-fold, at least about 15.0-fold, at least about 15.5-fold, or at least about 16.0-fold.

[0072] The humoral immune response induced by the vaccine can include an increase in the level of IgG antibodies associated with vaccinated subjects compared to unvaccinated subjects. These IgG antibodies can be specific to HPV6 E6 antigen, HPV6 E7 antigen, HPV11 E6 antigen, HPV11 E7 antigen, or any combination thereof. These IgG antibodies can be reactive with HPV6 E6 antigen, HPV6 E7 antigen, HPV11 E6 antigen, HPV11 E7 antigen, or any combination thereof. The level of IgG antibodies associated with vaccinated subjects can be increased by about 1.5-fold to about 16-fold, about 2-fold to about 12-fold, or about 3-fold to about 10-fold compared to unvaccinated subjects. The level of IgG antibodies associated with a subject receiving the vaccine may be increased by at least about 1.5-fold, at least about 2.0-fold, at least about 2.5-fold, at least about 3.0-fold, at least about 3.5-fold, at least about 4.0-fold, at least about 4.5-fold, at least about 5.0-fold, at least about 5.5-fold, at least about 6.0-fold, at least about 6.5-fold, at least about 7.0-fold, at least about 7.5-fold, at least about 8.0-fold, at least about 8.5-fold, at least about 9.0-fold, at least about 9.5-fold, at least about 10.0-fold, at least about 10.5-fold, at least about 11.0-fold, at least about 11.5-fold, at least about 12.0-fold, at least about 12.5-fold, at least about 13.0-fold, at least about 13.5-fold, at least about 14.0-fold, at least about 14.5-fold, at least about 15.0-fold, at least about 15.5-fold, or at least about 16.0-fold compared to a subject not receiving the vaccine.

[0073] The vaccine may induce a cellular immune response in a subject administered the vaccine. The induced cellular immune response may be specific to HPV6 E6 antigen, HPV6 E7 antigen, HPV11 E6 antigen, HPV11 E7 antigen, or any combination thereof. The induced cellular immune response may be reactive with HPV6 E6 antigen, HPV6 E7 antigen, HPV11 E6 antigen, HPV11 E7 antigen, or any combination thereof. The induced cellular immune response may include eliciting a T cell response. The induced T cell response may be reactive with HPV6 E6 antigen, HPV6 E7 antigen, HPV11 E6 antigen, HPV11 E7 antigen, or any combination thereof. The induced T cell response may be polyfunctional. The induced cellular immune response may include eliciting a T cell response in which the T cells produce interferon-gamma (IFN-γ). The induced cellular immune response can include an increase in T cell responses associated with the vaccinated subject compared to unvaccinated subjects, which can be about 2-fold to about 30-fold, about 3-fold to about 25-fold, or about 4-fold to about 20-fold increased T cell responses associated with the vaccinated subject compared to unvaccinated subjects.The T cell response associated with a vaccinated subject may be at least about 1.5 fold, at least about 2.0 fold, at least about 3.0 fold, at least about 4.0 fold, at least about 5.0 fold, at least about 6.0 fold, at least about 6.5 fold, at least about 7.0 fold, at least about 7.5 fold, at least about 8.0 fold, at least about 8.5 fold, at least about 9.0 fold, at least about 9.5 fold, at least about 10.0 fold, at least about 10.5 fold, at least about 11.0 fold, at least about 11.5 fold, at least about 12.0 fold, at least about 13.0 fold, at least about 14.0 fold, at least about 15.0 fold, at least about 16.0 fold, at least about 17.0 fold, at least about 18.0 fold, at least about 19.0 fold, at least about 20.0 fold, at least about 21.0 fold, at least about 22.0 fold, at least about 23.0 fold, at least about 24.0 fold, at least about 25.0 fold, at least about 26.0 fold, at least about 27.0 fold, at least about 28.0 fold, at least about 29.0 fold, at least about 30.0 fold, at least about 31.0 fold, at least about 32.0 fold, at least about 33.0 fold, at least about 34.0 fold, at least about 35.0 fold, at least about 36.0 fold, at least about 37.0 fold, at least about 38.0 fold, at least about 39.0 fold, at least about 40.0 fold, at least about The increase may be about 12.5-fold, at least about 13.0-fold, at least about 13.5-fold, at least about 14.0-fold, at least about 14.5-fold, at least about 15.0-fold, at least about 16.0-fold, at least about 17.0-fold, at least about 18.0-fold, at least about 19.0-fold, at least about 20.0-fold, at least about 21.0-fold, at least about 22.0-fold, at least about 23.0-fold, at least about 24.0-fold, at least about 25.0-fold, at least about 26.0-fold, at least about 27.0-fold, at least about 28.0-fold, at least about 29.0-fold, or at least about 30.0-fold.

[0074] The vaccines of the present invention may possess the characteristics required for an effective vaccine, such as being safe so that the vaccine itself does not cause illness or death, protecting against illness resulting from exposure to live pathogens such as viruses or bacteria, inducing antibodies to prevent cellular proliferation, inducing protective T cells against intracellular pathogens, and providing ease of administration, few side effects, biological stability, and low cost per dose.

[0075] The vaccine may further induce an immune response when administered into various tissues, such as muscle or skin. The vaccine may further induce an immune response when administered via electroporation, injection, or subcutaneously or intramuscularly.

[0076] HPV antigens can induce an immune response in a mammal against one or more HPV strains. Thus, disclosed herein are HPV antigens comprising an HPV6 antigenic domain and an HPV11 antigenic domain. The HPV6 antigenic domain can be located at the N-terminus or C-terminus of the HPV11 antigenic domain.

[0077] In some embodiments, the HPV6 antigenic domain comprises an HPV6 E6 antigenic domain, a fragment thereof, or a variant thereof, and an HPV6 E7 antigenic domain, a fragment thereof, a variant thereof, or a combination thereof. The HPV6 E6 antigenic domain may be located at the N-terminus or C-terminus of the HPV6 E7 antigenic domain. In some embodiments, the HPV6 E6 antigenic domain may contain an epitope that makes it particularly effective as an immunogen to which an immune response can be elicited. The HPV6 E6 antigenic domain may be a consensus sequence derived from two or more strains of HPV6. The HPV6 E6 antigen may include consensus sequences and / or modifications to improve expression. Modifications may include codon optimization, RNA optimization, addition of a Kozak sequence to increase translation initiation, and / or addition of an immunoglobulin leader sequence to increase the immunogenicity of the HPV6 E6 antigenic domain. The HPV6 E6 consensus antigenic domain may include a signal peptide, such as, but not limited to, an immunoglobulin signal peptide, such as an immunoglobulin E (IgE) or immunoglobulin (IgG) signal peptide. In some embodiments, the HPV6 E6 consensus antigenic domain may include a hemagglutinin (HA) tag. The HPV6 E6 consensus antigenic domain may be designed to elicit a stronger and broader cellular and / or humoral immune response than the corresponding codon-optimized HPV6 E6 antigenic domain. In some embodiments, the HPV6 E7 antigenic domain may include an epitope that makes it particularly effective as an immunogen against which an immune response can be induced. The HPV6 E7 antigenic domain may be a consensus sequence derived from two or more strains of HPV6. The HPV6 E7 antigen may include consensus sequences and / or modifications to improve expression. Modifications may include codon optimization, RNA optimization, addition of a Kozak sequence to increase translation initiation, and / or addition of an immunoglobulin leader sequence to increase the immunogenicity of the HPV6 E7 antigenic domain. The HPV6 E7 consensus antigenic domain can include a signal peptide, such as an immunoglobulin signal peptide, for example, but not limited to, an immunoglobulin E (IgE) or immunoglobulin (IgG) signal peptide.In some embodiments, the HPV6 E7 consensus antigenic domain may comprise a hemagglutinin (HA) tag. The HPV6 E7 consensus antigenic domain may be designed to elicit a stronger and broader cellular and / or humoral immune response than the corresponding codon-optimized HPV6 E7 antigenic domain.

[0078] In some embodiments, the HPV11 antigenic domain comprises an HPV11 E6 antigenic domain, a fragment thereof, or a variant thereof, and an HPV11 E7 antigenic domain, a fragment thereof, a variant thereof, or a combination thereof. The HPV11 E6 antigenic domain may be located N-terminal or C-terminal to the HPV11 E7 antigenic domain. In some embodiments, the HPV11 E6 antigenic domain may contain an epitope that makes it particularly effective as an immunogen to elicit an immune response. The HPV11 E6 antigenic domain may be a consensus sequence derived from two or more strains of HPV11. The HPV11 E6 antigen may include consensus sequences and / or modifications to improve expression. Modifications may include codon optimization, RNA optimization, addition of a Kozak sequence to increase translation initiation, and / or addition of an immunoglobulin leader sequence to increase the immunogenicity of the HPV11 E6 antigenic domain. The HPV11 E6 consensus antigenic domain may include a signal peptide, such as, but not limited to, an immunoglobulin signal peptide, such as an immunoglobulin E (IgE) or immunoglobulin (IgG) signal peptide. In some embodiments, the HPV11 E6 consensus antigenic domain may include a hemagglutinin (HA) tag. The HPV11 E6 consensus antigenic domain may be designed to elicit a stronger and broader cellular and / or humoral immune response than the corresponding codon-optimized HPV11 E6 antigenic domain. In some embodiments, the HPV11 E7 antigenic domain may include an epitope that makes it particularly effective as an immunogen against which an immune response can be induced. The HPV11 E7 antigenic domain may be a consensus sequence derived from two or more strains of HPV11. The HPV11 E7 antigen may include consensus sequences and / or modifications to improve expression. Modifications may include codon optimization, RNA optimization, the addition of a Kozak sequence to increase translation initiation, and / or the addition of an immunoglobulin leader sequence to increase the immunogenicity of the HPV11 E7 antigenic domain.The HPV11 E7 consensus antigenic domain may include a signal peptide, such as an immunoglobulin signal peptide, for example, but not limited to, an immunoglobulin E (IgE) or immunoglobulin (IgG) signal peptide. In some embodiments, the HPV11 E7 consensus antigenic domain may include a hemagglutinin (HA) tag. The HPV11 E7 consensus antigenic domain may be designed to elicit a stronger and broader cellular and / or humoral immune response than the corresponding codon-optimized HPV11 E7 antigenic domain.

[0079] In some embodiments, the HPV antigen comprises the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:11, an amino acid sequence that is at least about 95%, about 96%, about 97%, about 98%, or about 99% identical to the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:11, an immunogenic fragment of the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:11, or an amino acid sequence that is at least about 95%, about 96%, about 97%, about 98%, or about 99% homologous to an immunogenic fragment of the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:11.

[0080] A fragment of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 11 can comprise 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more of the full length of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 11. ... A fragment of SEQ ID NO:1 or SEQ ID NO:11 can be 100% identical to the full-length reference sequence except for lacking at least one amino acid from the N- and / or C-terminus, in each case with or without a signal peptide and / or methionine at position 1. A fragment of SEQ ID NO:1 or SEQ ID NO:11 can comprise a percent of 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 full-length SEQ ID NO:1 or SEQ ID NO:11, excluding any added heterologous signal peptide. Fragments preferably include fragments of SEQ ID NO:1 or SEQ ID NO:11 that are 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more homologous to SEQ ID NO:1 or SEQ ID NO:11, and additionally include an N-terminal methionine or heterologous signal peptide, which are not included when calculating percent homology. The fragment can further comprise an N-terminal methionine and / or a signal peptide, such as an immunoglobulin signal peptide, e.g., an IgE or IgG signal peptide. The N-terminal methionine and / or signal peptide can be linked to the fragment.

[0081] In some embodiments, a fragment of SEQ ID NO:1 or SEQ ID NO:11 may contain 100 or more residues, in some embodiments, 200 or more residues, in some embodiments, 300 or more residues, in some embodiments, 400 or more residues, and in some embodiments, 500 or more residues.

[0082] In some aspects, the HPV antigens may comprise HPV6 and HPV11 antigenic domains separated by one or more post-translational cleavage sites, one or more translational skip sites, or both. In some embodiments, the post-translational cleavage site is located between the HPV6 and HPV11 antigenic domains, between the HPV6 E6 and HPV6 E7 antigenic domains, and / or between the HPV11 E6 and HPV11 E7 antigenic domains. In some embodiments, the translational skip site is located between the HPV6 and HPV11 antigenic domains, between the HPV6 E6 and HPV6 E7 antigenic domains, and / or between the HPV11 E6 and HPV11 E7 antigenic domains. In some embodiments, the post-translational cleavage site and the translation skip site are located between the HPV6 antigenic domain and the HPV11 antigenic domain, between the HPV6 E6 antigenic domain and the HPV6 E7 antigenic domain, and / or between the HPV11 E6 antigenic domain and the HPV11 E7 antigenic domain. In some embodiments, the post-translational cleavage site is a furin cleavage site. In some embodiments, the translation skip site is a P2A site. In some aspects, the HPV immunogenic protein comprises the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 11.

[0083] In some embodiments, the HPV antigen comprises the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:11, an amino acid sequence that is at least about 95%, about 96%, about 97%, about 98%, or about 99% homologous to SEQ ID NO:1 or SEQ ID NO:11, an immunogenic fragment of SEQ ID NO:1 or SEQ ID NO:11, or an amino acid sequence that is at least about 95%, about 96%, about 97%, about 98%, or about 99% homologous to an immunogenic fragment of SEQ ID NO:1 or SEQ ID NO:11.

[0084] The proteins of the present invention can be produced using well-known techniques. In some embodiments, for example, a DNA molecule encoding a protein of the present invention can be inserted into a commercially available expression vector for use in an expression system. The produced protein is recovered by lysing the cells or, if necessary, from the culture medium as known to those of skill in the art. Those skilled in the art can isolate proteins produced using such expression systems using well-known techniques. The methods for purifying proteins from natural sources using antibodies that specifically bind to a particular protein, as described above, can be equally applied to purifying proteins produced by recombinant DNA methodology. In addition to producing proteins by recombinant techniques, automated peptide synthesizers can also be used to produce isolated, essentially pure proteins.

[0085] The HPV antigen can be a nucleic acid molecule encoding an HPV6-HPV11 fusion antigen disclosed herein. The nucleotide sequence encoding the HPV11 antigenic domain can be located 5' or 3' to the nucleotide sequence encoding the HPV6 antigenic domain.

[0086] In some embodiments, the nucleic acid sequence encoding the HPV6 antigenic domain comprises a nucleic acid sequence encoding the HPV6 E6 antigenic domain and an HPV6 E7 antigenic domain. The nucleic acid sequence encoding the HPV6 E6 antigenic domain can be located 5' or 3' to the nucleic acid sequence encoding the HPV6 E7 antigenic domain.

[0087] In some embodiments, the nucleic acid sequence encoding the HPV11 antigenic domain comprises a nucleic acid sequence encoding the HPV11 E6 antigenic domain and an HPV11 E7 antigenic domain. The nucleic acid sequence encoding the HPV11 E6 antigenic domain can be located 5' or 3' to the nucleic acid sequence encoding the HPV11 E7 antigenic domain.

[0088] In some aspects, nucleotide sequences encoding antigenic domains of HPV antigens may be separated by nucleotide sequences encoding one or more post-translational cleavage sites, one or more translational skip sites, or both. In some embodiments, the nucleotide sequence encoding the post-translational cleavage site is located between the nucleotide sequence encoding the HPV6 antigenic domain and the nucleotide sequence encoding the HPV11 antigenic domain, between the nucleotide sequence encoding the HPV6 E6 antigenic domain and the nucleotide sequence encoding the HPV6 E7 antigenic domain, between the nucleotide sequence encoding the HPV11 E6 antigenic domain and the nucleotide sequence encoding the HPV11 E7 antigenic domain, or any combination thereof. In some embodiments, the nucleotide sequence encoding the translational skip site is located between the nucleotide sequence encoding the HPV6 antigenic domain and the nucleotide sequence encoding the HPV11 antigenic domain, between the nucleotide sequence encoding the HPV6 E6 antigenic domain and the nucleotide sequence encoding the HPV6 E7 antigenic domain, between the nucleotide sequence encoding the HPV11 E6 antigenic domain and the nucleotide sequence encoding the HPV11 E7 antigenic domain, or any combination thereof. In some embodiments, the nucleotide sequence encoding the post-translational cleavage site and the translation skip site is located between the nucleotide sequence encoding the HPV6 antigenic domain and the nucleotide sequence encoding the HPV11 antigenic domain, between the nucleotide sequence encoding the HPV6 E6 antigenic domain and the nucleotide sequence encoding the HPV6 E7 antigenic domain, between the nucleotide sequence encoding the HPV11 E6 antigenic domain and the nucleotide sequence encoding the HPV11 E7 antigenic domain, or any combination thereof. In some embodiments, the post-translational cleavage site is a furin cleavage site. In some embodiments, the translation skip site is a P2A site.

[0089] In some embodiments, the nucleic acid sequence encoding the HPV antigen comprises the nucleotide sequence of SEQ ID NO:2 or SEQ ID NO:12, a nucleotide sequence at least about 95%, about 96%, about 97%, about 98%, or about 99% homologous to the nucleotide sequence of SEQ ID NO:2 or SEQ ID NO:12, a fragment of the nucleotide sequence of SEQ ID NO:2 or SEQ ID NO:12, or a nucleotide sequence at least about 95%, about 96%, about 97%, about 98%, or about 99% homologous to a fragment of the nucleotide sequence of SEQ ID NO:2 or SEQ ID NO:12. The fragment can further comprise a coding sequence for a signal peptide, such as an N-terminal methionine and / or an immunoglobulin signal peptide, e.g., an IgE or IgG signal peptide. A coding sequence encoding an N-terminal methionine and / or a signal peptide can be linked to the fragment.

[0090] The nucleic acid molecule comprising the nucleotide sequence encoding the immunogen can be operably linked to a regulatory element. The nucleic acid molecule can be DNA, RNA, cDNA, a variant thereof, a fragment thereof, or a combination thereof. The nucleic acid sequence can also include an additional sequence encoding a linker or tag sequence linked to the antigen by a peptide bond.

[0091] In some embodiments, the nucleic acid molecule encoding an HPV antigen is an expression vector. The expression vector can be a circular plasmid or a linear nucleic acid. The expression vector can direct the expression of a specific nucleotide sequence in an appropriate target cell. The expression vector can have a promoter operably linked to the nucleotide sequence encoding the antigen, which can be operably linked to a termination signal. The expression vector can also contain sequences necessary for proper translation of the nucleotide sequence. The expression vector containing the nucleotide sequence of interest can be chimeric, meaning that at least one of its components is heterologous with respect to at least one of the other components. The expression of the nucleotide sequence in the expression cassette can be under the control of a constitutive or inducible promoter, which initiates transcription only when the host cell is exposed to some specific external stimulus. In the case of a multicellular organism, the promoter can also be specific to a particular tissue, organ, or developmental stage.

[0092] In one embodiment, the nucleic acid is an RNA molecule. Thus, in one embodiment, the present invention provides an RNA molecule encoding one or more polypeptides of interest. The RNA can be positive-stranded. Thus, in some embodiments, the RNA molecule can be translated by cells without the need for any intervening replication step, such as reverse transcription. RNA molecules useful in the present invention can have a 5' cap (e.g., 7-methylguanosine). This cap can improve in vivo translation of the RNA. The 5' nucleotide of an RNA molecule useful in the present invention can have a 5' triphosphate group. In capped RNA, this can be linked to the 7-methylguanosine via a 5'-5' bridge. The RNA molecule can have a 3' poly-A tail. It can also include a poly-A polymerase recognition sequence (e.g., AAUAAA) near its 3' end. RNA molecules useful in the present invention can be single-stranded. In some embodiments, the RNA molecule is a naked RNA molecule. In one embodiment, the RNA molecule is contained within a vector.

[0093] In one embodiment, the RNA has 5' and 3' UTRs. In one embodiment, the 5' UTR is 0 to 3,000 nucleotides long. The length of the 5' and 3' UTR sequences added to the coding region can be varied by various methods, including, but not limited to, designing PCR primers that anneal to different regions of the UTR. Using this approach, one skilled in the art can modify the 5' and 3' UTR lengths necessary to achieve optimal translation efficiency after transfection of the transcribed RNA.

[0094] The 5' and 3' UTRs can be naturally occurring endogenous 5' and 3' UTRs for the gene of interest. Alternatively, UTR sequences that are not endogenous to the gene of interest can be added by incorporating UTR sequences into the forward and reverse primers or by any other modification of the template. The use of UTR sequences that are not endogenous to the gene of interest can be useful for modifying RNA stability and / or translation efficiency. For example, it is known that AU-rich elements in 3' UTR sequences can increase RNA stability. Therefore, 3' UTRs can be selected or designed to increase the stability of transcribed RNA based on the properties of UTRs that are well known in the art.

[0095] In one embodiment, the 5' UTR may contain the Kozak sequence of the endogenous gene. Alternatively, if a 5' UTR that is not endogenous to the gene of interest is added by PCR as described above, the consensus Kozak sequence can be redesigned by adding the 5' UTR sequence. While the Kozak sequence can increase the efficiency of translation of some RNA transcripts, it does not appear to be required for efficient transcription of all RNAs. The requirement for a Kozak sequence for many RNAs is known in the art. In another embodiment, the 5' UTR may be derived from an RNA virus, whose RNA genome is stable in cells. In another embodiment, various nucleotide analogs can be used in the 3' or 5' UTR to prevent exonuclease degradation of the RNA.

[0096] In one embodiment, the RNA has a cap at both the 5' end and a 3' poly(A) tail, which determines ribosome binding, translation initiation, and RNA stability in the cell.

[0097] In one embodiment, the RNA is nucleoside-modified RNA. Nucleoside-modified RNA has certain advantages over unmodified RNA, including, for example, increased stability, low or no natural immunogenicity, and improved translation.

[0098] The expression vector may be a circular plasmid that can transform the target cell by integrating into the cell genome, or may exist extrachromosomally (e.g., a self-replicating plasmid with an origin of replication). The vector may be pVAX, pcDNA3.0, or provax, or any other expression vector that can express DNA encoding an antigen and allow the cell to translate the sequence into an antigen that is recognized by the immune system.

[0099] Also provided herein are linear nucleic acid immunogenic compositions or linear expression cassettes ("LECs") that can be efficiently delivered to a subject by electroporation and express one or more desired antigens. LECs can be any linear DNA that lacks a phosphate backbone. The DNA can encode one or more antigens. LECs can contain a promoter, introns, stop codons, and / or polyadenylation signals. Expression of the antigen can be controlled by a promoter. LECs may not contain any antibiotic resistance genes and / or a phosphate backbone. LECs may be free of other nucleotide sequences unrelated to desired antigen gene expression. LECs can be derived from any plasmid that can be linearized. The plasmid can be capable of expressing the antigen. The plasmid can be pNP (Puerto Rico / 34) or pM2 (New Caledonia / 99). The plasmid can be WLV009, pVAX, pcDNA3.0, or provax, or any other expression vector capable of expressing DNA encoding an antigen and allowing the cell to translate the sequence into an antigen recognized by the immune system. The LEC can be pcrM2. The LEC can be pcrNP. pcrNP and pcrMR can be derived from pNP (Puerto Rico / 34) and pM2 (New Caledonia / 99), respectively.

[0100] The vector may comprise a heterologous nucleic acid encoding the above antigens, and may further comprise an initiation codon that may be upstream of one or more cancer antigen coding sequence(s) and a termination codon that may be downstream of the coding sequence(s) of the above antigens.

[0101] The vector may have a promoter. The promoter may be any promoter capable of driving gene expression and controlling the expression of the isolated nucleic acid. Such a promoter is a cis-acting sequence element required for transcription via DNA-dependent RNA polymerase, which transcribes the antigen sequence described herein. The choice of promoter used to direct the expression of heterologous nucleic acid depends on the specific application. The promoter may be located at approximately the same distance from the transcription start in the vector as it is from the transcription start site in its natural setting. However, variations in this distance can be accommodated without loss of promoter function.

[0102] The initiation and termination codons may be in frame with the coding sequence(s) of the antigen. The vector may also include a promoter operably linked to the coding sequence(s) of the antigen. The promoter operably linked to the coding sequence(s) of the antigen may be a promoter derived from simian virus 40 (SV40), a mouse mammary tumor virus (MMTV) promoter, a human immunodeficiency virus (HIV) promoter such as the bovine immunodeficiency virus (BIV) long terminal repeat (LTR) promoter, a Moloney virus promoter, an avian leukosis virus (ALV) promoter, a cytomegalovirus (CMV) promoter such as the CMV immediate early promoter, an Epstein-Barr virus (EBV) promoter, or a Rous sarcoma virus (RSV) promoter. The promoter may also be a promoter derived from a human gene such as human actin, human myosin, human hemoglobin, human muscle creatine, or human metallothionein. The promoter may also be a tissue-specific promoter, such as a natural or synthetic muscle- or skin-specific promoter. Examples of such promoters are described in US Patent Application Publication No. 2004 / 0175727, the contents of which are incorporated herein in their entirety.

[0103] The vector may also contain a polyadenylation signal downstream of the coding sequence(s) of the antigen and / or antibody. The polyadenylation signal may be an SV40 polyadenylation signal, an LTR polyadenylation signal, a bovine growth hormone (bGH) polyadenylation signal, a human growth hormone (hGH) polyadenylation signal, or a human β-globin polyadenylation signal. The SV40 polyadenylation signal may be a polyadenylation signal derived from the pCEP4 vector (Invitrogen, San Diego, CA).

[0104] The vector may also contain an enhancer upstream of the antigen. The enhancer may be necessary for expression. The enhancer may be human actin, human myosin, human hemoglobin, human muscle creatine, or a viral enhancer such as those derived from CMV, HA, RSV, or EBV.

[0105] The vector may contain an enhancer and intron with functional splice donor and acceptor sites. The vector may contain a transcription termination region downstream of the structural gene to provide for efficient termination. The termination region may be obtained from the same gene as the promoter sequence or may be obtained from a different gene.

[0106] The vector may further contain elements or reagents that inhibit its integration into a chromosome. The vector may contain a mammalian origin of replication to maintain the vector extrachromosomally and produce multiple copies of the vector within a cell. The vector may be pVAX1, pCEP4, or pREP4 from Invitrogen (San Diego, CA), which may contain the Epstein-Barr virus origin of replication and the nuclear antigen EBNA-1 coding region, which may produce high-copy episomal replication without integration. The vector may be pVAX1 or a pVAX1 variant with alterations, such as the variant plasmids described herein. The variant pVax1 plasmid is a 2998 base pair variant of the backbone vector plasmid pVAX1 (Invitrogen, Carlsbad, CA). The CMV promoter is located at bases 137-724. The T7 promoter / priming site is at bases 664-683. The multiple cloning site is at bases 696-811. The bovine GH polyadenylation signal is at bases 829 to 1053. The kanamycin resistance gene is at bases 1226 to 2020. The pUC origin is at bases 2320 to 2993.

[0107] Based on the sequence of pVAX1 available from Invitrogen, the following mutations were found in the sequence of pVAX1: C>G241 in CMV promoter C>T 1942 backbone, downstream of the bovine growth hormone polyadenylation signal (bGH polyA) A>- 2876 backbone, downstream of the kanamycin gene C>T 3277 High copy number mutation in the pUC origin of replication (Ori) (see Nucleic Acid Research 1985) G>C 3753 Extreme end of pUC Ori upstream of RNASeH site Base pairs 2, 3, and 4 are changed from ACT to CTG in the backbone upstream of the CMV promoter.

[0108] The backbone of the vector may be pAV0242. The vector may be a replication-deficient adenovirus type 5 (Ad5) vector.

[0109] The vector may also include regulatory sequences that may be well suited for gene expression in mammalian or human cells into which the vector is administered. The antigen sequences disclosed herein may include codons that may allow for more efficient transcription of the coding sequence in the host cell.

[0110] The vector may be pSE420 (Invitrogen, San Diego, Calif.), which can be used for protein production in Escherichia coli (E. coli). The vector may also be pYES2 (Invitrogen, San Diego, Calif.), which can be used for protein production in Saccharomyces cerevisiae strains of yeast. The vector may also be the MAXBAC™ complete baculovirus expression system (Invitrogen, San Diego, Calif.), which can be used for protein production in insect cells. The vector may also be pcDNA I or pcDNA3 (Invitrogen, San Diego, Calif.), which can be used for protein production in mammalian cells, such as Chinese hamster ovary (CHO) cells. The vector may be an expression vector or system for producing proteins by routine techniques and readily available starting materials, including Sambrook et al., Molecular Cloning and Laboratory Manual, Second Ed., Cold Spring Harbor (1989), which is incorporated by reference in its entirety.

[0111] An exemplary DNA plasmid comprises SEQ ID NO:3.

[0112] The immunogenic compositions of the present invention may comprise compositions such as HPV antigens of the present invention, recombinant vaccines comprising nucleotide sequences encoding HPV antigens of the present invention, live attenuated pathogens encoding and / or comprising HPV antigens of the present invention, killed pathogens comprising HPV antigens of the present invention, or liposomal or subunit vaccines comprising HPV antigens of the present invention. The present invention further relates to pharmaceutical compositions, including, but not limited to, injectable pharmaceutical compositions comprising the disclosed immunogenic compositions.

[0113] The immunogenic compositions of the present invention may be formulated with suitable pharmaceutically acceptable carriers, excipients, and other agents that provide suitable transport, delivery, tolerability, etc. A pharmaceutically acceptable excipient may be a functional molecule such as a vehicle, carrier, or diluent.

[0114] The pharmaceutically acceptable excipient may be a transfection-facilitating agent, which may include surfactants such as immune stimulating complexes (ISCOMS), Freund's incomplete adjuvant, LPS analogs including monophosphoryl lipid A, muramyl peptides, quinone analogs, vesicles such as squalene and squalene, hyaluronic acid, lipids, liposomes, calcium ions, viral proteins, polyanions, polycations, or nanoparticles, or other known transfection-facilitating agents.

[0115] The pharmaceutically acceptable excipient may be an adjuvant. In some embodiments, compositions and vaccines are provided that include the HPV antigens of the present invention in combination with an adjuvant. The adjuvant may be another gene expressed in an alternative plasmid or delivered as a protein in combination with the HPV antigens of the present invention. The adjuvant may be selected from the group consisting of α-interferon (IFN-α), β-interferon (IFN-β), γ-interferon, platelet-derived growth factor (PDGF), TNFα, TNFβ, GM-CSF, epidermal growth factor (EGF), cutaneous T cell-attracting chemokine (CTACK), epithelial thymus-expressed chemokine (TECK), mucosal-associated epithelial chemokine (MEC), IL-12, IL-15, MHC, CD80, CD86, including IL-15 with a deleted signal sequence and optionally including a signal peptide from IgE. The adjuvant can be IL-12, IL-15, IL-28, CTACK, TECK, 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, or a combination thereof.

[0116] Other genes that may be useful as adjuvants include MCP-1, MIP-1a, MIP-1p, IL-8, RANTES, L-selectin, P-selectin, E-selectin, CD34, GlyCAM-1, MadCAM-1, LFA-1, VLA-1, Mac-1, p150.95, PECAM, ICAM-1, ICAM-2, ICAM-3, CD2, LFA-3, M-CSF, G-CSF, IL-4, variants of IL-18, CD40, CD40L, and vascular growth factor. , fibroblast growth factor, IL-7, IL-22, nerve growth factor, vascular endothelial growth factor, Fas, TNF receptor, Flt, Apo-1, p55, WSL-1, DR3, TRAMP, Apo-3, AIR, LARD, NGRF, DR4, DR 5, 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 These include those encoding 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 functional fragments thereof.

[0117] In certain embodiments, the adjuvant is interleukin-12 (IL12). IL12 can be included in the vaccine in the form of its p35 and p40 subunits. The adjuvant IL-12 can be administered to a subject as its p35 and p40 subunits. The IL12 p35 and p40 subunits can be encoded by the same expression vector or by separate expression vectors. The nucleic acid molecule encoding IL12 can be the same as or different from the nucleic acid molecule encoding the HPV6 antigen fused to the HPV11 antigen. In some aspects, the nucleic acid molecule encoding IL12 comprises a nucleotide sequence encoding the p35 subunit of IL-12, the p40 subunit of IL-12, or both. A nucleic acid molecule encoding the p35 subunit of IL12 may comprise a nucleotide sequence encoding SEQ ID NO:6, a nucleotide sequence that is at least about 95%, about 96%, about 97%, about 98%, or about 99% homologous to the nucleotide sequence encoding SEQ ID NO:6, a fragment of the nucleotide sequence encoding SEQ ID NO:6, or a nucleotide sequence that is at least about 95%, about 96%, about 97%, about 98%, or about 99% homologous to a fragment of the nucleotide sequence encoding SEQ ID NO:6. A nucleic acid molecule encoding the p40 subunit of IL12 may comprise a nucleotide sequence encoding SEQ ID NO:8, a nucleotide sequence that is at least about 95%, about 96%, about 97%, about 98%, or about 99% homologous to the nucleotide sequence encoding SEQ ID NO:8, a fragment of the nucleotide sequence encoding SEQ ID NO:8, or a nucleotide sequence that is at least about 95% homologous to a fragment of the nucleotide sequence encoding SEQ ID NO:8. A nucleic acid molecule encoding the p35 subunit of IL12 may comprise the nucleotide sequence of SEQ ID NO:5, a nucleotide sequence that is at least about 95%, about 96%, about 97%, about 98%, or about 99% identical to the nucleotide sequence of SEQ ID NO:5, a fragment of the nucleotide sequence of SEQ ID NO:5, or a nucleotide sequence that is at least about 95%, about 96%, about 97%, about 98%, or about 99% identical to a fragment of the nucleotide sequence of SEQ ID NO:5.A nucleic acid molecule encoding the p40 subunit of IL12 may comprise the nucleotide sequence of SEQ ID NO:7, a nucleotide sequence that is at least about 95%, about 96%, about 97%, about 98%, or about 99% identical to the nucleotide sequence of SEQ ID NO:7, a fragment of the nucleotide sequence of SEQ ID NO:7, or a nucleotide sequence that is at least about 95%, about 96%, about 97%, about 98%, or about 99% identical to a fragment of the nucleotide sequence of SEQ ID NO:7.

[0118] In some embodiments of the described immunogenic compositions, the composition comprises pGX3024 and pGX6010. In some embodiments, the composition is INO-3107.

[0119] As used herein, "buffer" refers to a buffered solution that resists changes in pH due to the action of its acid-base conjugate components. A buffer generally has a pH of about 4.0 to about 8.0, e.g., about 5.0 to about 7.0. In some embodiments, the buffer is saline-sodium citrate (SSC) buffer. In some embodiments, where the immunogenic composition comprises a vector containing a nucleic acid molecule encoding an HPV antigen described above, the immunogenic composition comprises 6 mg / ml of the vector in a buffer, such as, but not limited to, an SSC buffer. In some embodiments, the immunogenic composition comprises 6 mg / ml of the DNA plasmid pGX3024 in a buffer. In some embodiments, where the immunogenic composition further comprises a separate vector containing a nucleic acid molecule encoding the p35 subunit of IL-12, the p40 subunit of IL-12, or both, the immunogenic composition comprises 0.25 mg / ml of the separate vector in a buffer. In some embodiments, the immunogenic composition comprises the DNA plasmid pGX6010 at 0.25 mg / mL in a buffer in addition to a vector (eg, pGX3024) comprising a nucleic acid molecule encoding an HPV antigen.

[0120] The pharmaceutical composition according to the present invention is formulated according to the mode of administration used.When the pharmaceutical composition is an injectable pharmaceutical composition, it is sterile, pyrogen-free, and particulate-free.An isotonic formulation is preferably used.Generally, additives for isotonicity can include sodium chloride, dextrose, mannitol, sorbitol, and lactose.In some cases, an isotonic solution such as phosphate buffered saline is preferred.Stabilizers include gelatin and albumin.In some embodiments, a vasoconstrictor is added to the formulation.

[0121] Also provided herein are methods for treating, protecting against, and / or preventing disease in a subject in need thereof by administering a vaccine of the present invention to the subject. Administering the vaccine to a subject can induce or elicit an immune response in the subject. Thus, provided are methods for inducing an immune response in a subject, comprising administering to the subject an effective amount of an HPV antigen of the present invention, thereby inducing an immune response.

[0122] Further provided is a method for prophylactically or therapeutically immunizing a subject against HPV6, HPV11, or both, comprising administering to the subject an effective amount of an HPV antigen of the present invention, thereby inducing an immune response against HPV6, HPV11, or both. Also provided is a method for treating or preventing recurrent respiratory papillomatosis (RRP) in a subject, comprising administering to the subject an effective amount of an HPV antigen of the present invention, thereby treating or preventing RRP.

[0123] The induced immune response can be used to treat, prevent, and / or protect against diseases, e.g., conditions associated with HPV infection. In some embodiments, methods are provided for treating, protecting against, and / or preventing RRP in a subject in need thereof by administering an HPV antigen of the present invention to the subject. The induced immune response provides the vaccinated subject with resistance to one or more HPV strains. The induced immune response can include an induced humoral immune response and / or an induced cellular immune response.

[0124] Administration of the HPV antigen reduces the number of RRP surgical interventions. In some embodiments, the method reduces the number of RRP surgical interventions in a 52-week period after administration of a pharmaceutical composition comprising an HPV antigen to a population of human subjects diagnosed with RRP, compared to the number of RRP surgical interventions in a 52-week period prior to administration of the pharmaceutical composition to the population of human subjects diagnosed with RRP. The method may reduce the number of RRP surgical interventions in about 70% to about 95%, about 75% to about 85%, about 75% to about 80%, about 75%, about 76%, about 77%, about 78%, about 79%, or about 80% of the population of human subjects diagnosed with RRP, compared to the number of RRP surgical interventions in a 52-week period prior to administration of the pharmaceutical composition to the population of human subjects diagnosed with RRP. For example, the method may reduce the number of RRP surgical interventions in at least about 76% of a population of human subjects diagnosed with RRP in the 52 weeks following administration of the pharmaceutical composition, compared to the number of RRP surgical interventions in the 52 weeks preceding administration of the pharmaceutical composition to the population of human subjects diagnosed with RRP. According to some embodiments of the disclosed method, the number of RRP surgical interventions may be reduced in about 90% of a population of human subjects diagnosed with RRP in the 52 weeks following administration of the pharmaceutical composition, compared to the number of RRP surgical interventions in the 52 weeks preceding administration of the pharmaceutical composition to the population of human subjects diagnosed with RRP. According to some embodiments, the median reduction in the number of RRP surgical interventions in the 52 weeks following administration of the pharmaceutical composition is about 3.

[0125] According to some embodiments, the method increases the number of HPV6-specific activated CD4 and HPV11-specific activated CD4 and / or activated lytic CD8 T cells.

[0126] The method may increase the number of HPV6- and HPV11-specific CD4 cells expressing CD38 52 weeks after administration of the pharmaceutical composition in about 70% to about 95%, about 75% to about 90%, about 85% to about 90%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, or about 89% of a population of human subjects diagnosed with RRP compared to the number of HPV6- and HPV11-specific CD4 cells expressing CD38 52 weeks before administration of the pharmaceutical composition to the population of human subjects diagnosed with RRP. In some embodiments, the method increases the number of HPV6- and HPV11-specific CD4 cells expressing CD38 52 weeks after administration of the pharmaceutical composition in about 85% of a population of human subjects diagnosed with RRP compared to the number of HPV6- and HPV11-specific CD4 cells expressing CD38 52 weeks before administration of the pharmaceutical composition to the population of human subjects diagnosed with RRP.

[0127] In some embodiments, the method increases the number of HPV6- and HPV11 E6-specific CD8 cells that express CD38, granzyme A, granzyme B, and perforin 52 weeks after administration of the pharmaceutical composition compared to the number of HPV6- and HPV11 E6-specific CD8 cells that express CD38, granzyme A, granzyme B, and perforin 52 weeks before administration of the pharmaceutical composition to a population of human subjects diagnosed with RRP. The method may increase the number of HPV6- and HPV11 E6-specific CD8 cells expressing CD38, granzyme A, granzyme B, and perforin during the 52 weeks following administration of the pharmaceutical composition in about 70% to about 95%, about 75% to about 90%, about 85% to about 90%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, or about 89% of a population of human subjects diagnosed with RRP, compared to the number of HPV6- and HPV11 E6-specific CD8 cells expressing CD38, granzyme A, granzyme B, and perforin during the 52 weeks prior to administration of the pharmaceutical composition to the population of human subjects diagnosed with RRP. For example, the method may increase the number of HPV6- and HPV11 E6-specific CD8 cells expressing CD38, granzyme A, granzyme B, and perforin in at least about 85% of a population of human subjects diagnosed with RRP 52 weeks after administration of the pharmaceutical composition compared to the number of HPV6- and HPV11 E6-specific CD8 cells expressing CD38, granzyme A, granzyme B, and perforin in the 52 weeks prior to administration of the pharmaceutical composition to the population of human subjects diagnosed with RRP.

[0128] The subject can be a mammal, such as a human, horse, cow, pig, sheep, cat, dog, rabbit, guinea pig, rat, or mouse.

[0129] The vaccine dose can be 1 μg to 10 mg of total plasmid per injection, preferably 6.25 mg of total plasmid per injection. The vaccine can be administered every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 days. The number of vaccines for effective treatment can be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0130] Vaccines can be administered prophylactically or therapeutically. In prophylactic administration, vaccines can be administered in an amount sufficient to induce an immune response. In therapeutic applications, vaccines are administered to a subject in need thereof in an amount sufficient to induce a therapeutic effect. An amount adequate to accomplish this is defined as a "therapeutically effective dose." Amounts effective for this use will depend, for example, on the particular composition of the vaccine regimen administered, the method of administration, the stage and severity of the disease, the patient's general health, and the judgment of the prescribing physician.

[0131] Vaccines can be administered by methods known in the art, such as those described in Donnelly et al. (Ann. Rev. Immunol. 15:617-648 (1997)), Felgner et al. (U.S. Patent No. 5,580,859, issued December 3, 1996), Felgner (U.S. Patent No. 5,703,055, issued December 30, 1997), and Carson et al. (U.S. Patent No. 5,679,647, issued October 21, 1997), the contents of all of which are incorporated herein by reference in their entireties. The vaccine DNA can be complexed to particles or beads that can be administered to an individual using, for example, a vaccine gun. One of skill in the art will know that the choice of a pharmaceutically acceptable carrier, including a physiologically acceptable compound, will depend, for example, on the route of administration of the expression vector.

[0132] In some embodiments of the described methods, the subject is administered pGX3024 and pGX6010. In some embodiments, pGX3024 and pGX6010 are administered to the subject as INO-3107. In some embodiments, pGX3024 and pGX6010 are administered to the subject as an INO-3107 formulation containing 6.25 mg total plasmid / mL (6 mg / mL pGX3024, 0.25 mg / mL pGX6010) in 150 mM sodium chloride and 15 mM sodium citrate, pH 7.

[0133] Vaccines can be delivered via various routes. Typical delivery routes include parenteral administration, such as intradermal, intramuscular, or subcutaneous delivery. Other routes include oral, intranasal, and intravaginal administration. Vaccines can be delivered to the interstitial space of an individual's tissues, particularly for vaccine DNA (Felgner et al., U.S. Pat. Nos. 5,580,859 and 5,703,055, the contents of all of which are incorporated herein by reference in their entirety). Vaccines can also be administered into the muscle or transdermally, such as via intradermal or subcutaneous injection or by iontophoresis. Epidermal administration of vaccines can also be used. Epidermal administration can involve mechanically or chemically irritating the outermost layer of the epidermis to stimulate an immune response to the irritant (Carson et al., U.S. Pat. No. 5,679,647, the contents of which are incorporated herein by reference in their entirety).

[0134] According to some embodiments, vaccines are delivered to individuals to modulate the activity of the individual's immune system, thereby enhancing the immune response to HPV to treat RRP. When a nucleic acid molecule encoding an HPV antigen of the present invention is taken up by the individual's cells, the nucleotide sequence is expressed in the cells, thereby delivering the protein to the individual. Methods are provided for delivering the coding sequence for the protein on a nucleic acid molecule, such as a plasmid, as part of a recombinant vaccine, as part of an attenuated vaccine, as an isolated protein, or as part of a vector.

[0135] The methods include administering to the subject an HPV antigen of the present invention. In some embodiments, the methods include introducing a provided nucleic acid molecule followed by electroporation.

[0136] The disclosed methods can involve the administration of multiple copies of a single nucleic acid molecule, such as a single plasmid, or multiple copies of two or more different nucleic acid molecules, such as two or more different plasmids. For example, the methods can involve the administration of 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more different nucleic acid molecules.

[0137] In certain embodiments, the disclosed methods of inducing an immune response or preventing or treating RRP further comprise administering an adjuvant to a subject. In certain embodiments, the adjuvant is IL12. IL12 can be included in the vaccine in the form of its p35 and p40 subunits. The adjuvant IL-12 can be administered to a subject as its p35 and p40 subunits. The IL12p35 and p40 subunits can be encoded by the same expression vector or separate expression vectors. In one embodiment, the sequence encoding IL12p35 is as set forth in SEQ ID NO:5. In one embodiment, the IL12p35 subunit has the amino acid sequence set forth in SEQ ID NO:6. In one embodiment, the sequence encoding IL12p40 is as set forth in SEQ ID NO:7. In one embodiment, the IL12p40 subunit has the amino acid sequence set forth in SEQ ID NO:8. In some embodiments, the expression vector is pGX6012 or pGX6010. In certain embodiments, the method is clinically proven safe, clinically proven effective, or both.

[0138] In some embodiments, the method comprises simultaneous administration of (a) an immunogenic composition comprising an HPV antigen disclosed herein, and (b) a composition comprising a nucleic acid molecule encoding one or more IL-12 subunits (e.g., p35 and / or p40) disclosed herein. In some embodiments, the method comprises prior administration of a composition comprising a nucleic acid molecule encoding an HPV antigen disclosed herein, followed by administration of a composition comprising a nucleic acid molecule encoding one or more IL-12 subunits (e.g., p35 and / or p40) disclosed herein. In some embodiments, the method comprises prior administration of a composition comprising a nucleic acid molecule encoding one or more IL-12 subunits (e.g., p35 and / or p40) disclosed herein, followed by administration of a composition comprising a nucleic acid molecule encoding an HPV antigen disclosed herein.

[0139] Routes of administration include, but are not limited to, intramuscular, intranasal, intraperitoneal, intradermal, subcutaneous, intravenous, intraarterial, intraocular, and oral, as well as topical, transdermal, inhalation, or suppository administration, or into mucosal tissues, such as by irrigation into vaginal, rectal, urethral, ​​buccal, and sublingual tissues. Preferred routes of administration include intramuscular, intraperitoneal, intradermal, and subcutaneous injection. Gene constructs may be administered by means including, but not limited to, electroporation methods and devices, conventional syringes, standard needles, side-port needles (see U.S. Patent Application Publication No. 2023 / 0017972, incorporated herein by reference), needleless injection devices, or "microprojectile bombardment."

[0140] The vaccine can be administered via electroporation, such as by the method described in U.S. Patent No. 7,664,545, the contents of which are incorporated herein by reference. Electroporation can be by the methods and / or devices described in U.S. Patent Nos. 6,302,874, 5,676,646, 6,241,701, 6,233,482, 6,216,034, 6,208,893, 6,192,270, 6,181,964, 6,150,148, 6,120,493, 6,096,020, 6,068,650, and 5,702,359, the contents of which are incorporated herein by reference in their entireties. Electroporation can be performed via minimally invasive devices.

[0141] A minimally invasive electroporation device ("MID") may be a device for injecting the above-mentioned vaccines and associated fluids into body tissue. The device may comprise a hollow needle, a DNA cassette, and a fluid delivery means, and the device is adapted to activate the fluid delivery means during use so as to simultaneously (e.g., automatically) inject the DNA into the body tissue during insertion of the needle into said body tissue. This has the advantage that the ability to gradually inject the DNA and associated fluids while the needle is being inserted results in a more uniform distribution of the fluids through the body tissue. Pain experienced during injection may be reduced due to the distribution of the injected DNA over a larger area.

[0142] MIDs can inject vaccines into tissue without the use of needles. MIDs can inject vaccines as a small stream or burst with such force that the vaccine penetrates the surface of the tissue and enters the underlying tissue and / or muscle. The force behind the small stream or burst can be provided by the expansion of a compressed gas, such as carbon dioxide, through a tiny orifice within a fraction of a second. Examples of minimally invasive electroporation devices and methods for using them are described as published in U.S. Patent Application No. 20080234655, U.S. Patent Nos. 6,520,950, 7,171,264, 6,208,893, 6,009,347, 6,120,493, 7,245,963, 7,328,064, and 6,763,264, the contents of which are incorporated herein by reference.

[0143] MID can be equipped with a syringe that produces a high-speed jet of liquid that penetrates tissue painlessly.Such needleless syringes are commercially available.The needleless syringe that can be used herein includes, for example, those described in U.S. Patent No. 3,805,783, U.S. Patent No. 4,447,223, U.S. Patent No. 5,505,697 and U.S. Patent No. 4,342,310, the contents of which are incorporated herein by reference.

[0144] The desired vaccine, in a form suitable for direct or indirect electrotransport, can be introduced (e.g., injected) into the tissue to be treated using a needleless syringe, typically by contacting the tissue surface with the syringe to actuate delivery of a burst of agent with sufficient force to cause penetration of the vaccine into the tissue. For example, if the tissue to be treated is mucosa, skin, or muscle, the agent is propelled toward the mucosa or skin surface with sufficient force to penetrate the stratum corneum into the skin layers, or into underlying tissue and muscle, respectively.

[0145] Needle-free injectors are well suited for delivering vaccines to all types of tissue, particularly skin and mucous membranes. In some embodiments, needle-free injectors can be used to propel a liquid containing a vaccine onto a surface and into the skin or mucous membrane of a subject. Representative examples of various types of tissue that can be treated using the methods of the present invention include the pancreas, larynx, nasopharynx, hypopharynx, oropharynx, lips, throat, lung, heart, kidney, muscle, breast, colon, prostate, thymus, testis, skin, mucosal tissue, ovary, blood vessels, or any combination thereof.

[0146] The MID may have needle electrodes for electroporating tissue. For example, pulsing between multiple pairs of electrodes in a multi-electrode array configured in a rectangular or square pattern provides improved results compared to pulsing between a pair of electrodes. For example, U.S. Patent No. 5,702,359, entitled "Needle Electrodes for Mediated Delivery of Drugs and Genes," discloses a needle array in which multiple pairs of needles can be pulsed during a therapeutic procedure. In that application, which is incorporated herein by reference as if fully set forth, the needles are arranged in a circular array, but have connectors and switching devices that allow pulsing between opposing pairs of needle electrodes. Paired needle electrodes can be used to deliver recombinant expression vectors to cells. Such devices and systems are described in U.S. Patent No. 6,763,264, the contents of which are incorporated herein by reference. Alternatively, a single-needle device can be used that allows DNA injection and electroporation through a single needle similar to a conventional injection needle, but applies lower-voltage pulses than those delivered by currently used devices, thus reducing the electrical sensation experienced by the patient.

[0147] The MID may comprise one or more electrode arrays. The array may comprise two or more needles of the same or different diameters. The needles may be evenly or unevenly spaced. The needles may be 0.005 inches to 0.03 inches, 0.01 inches to 0.025 inches, or 0.015 inches to 0.020 inches. The needles may be 0.0175 inches in diameter. The needles may be spaced 0.5 mm, 1.0 mm, 1.5 mm, 2.0 mm, 2.5 mm, 3.0 mm, 3.5 mm, 4.0 mm, or more apart.

[0148] The MID may consist of a pulse generator and two or more needle vaccine syringes that deliver vaccine and electroporation pulses in a single step. The pulse generator may allow flexible programming of pulse and injection parameters via a flash card-operated personal computer, as well as comprehensive recording and storage of electroporation and patient data. The pulse generator may deliver pulses of various voltages over short periods of time. For example, the pulse generator may deliver three 15-volt pulses of 100 ms duration. An example of such an MID is the Elgen 1000 system by Inovio Biomedical Corporation, which is described in U.S. Patent No. 7,328,064, the contents of which are incorporated herein by reference.

[0149] The MID can be the CELLECTRA® (Inovio Pharmaceuticals, Blue Bell Pa.) device and system, a controlled electrode system that facilitates the introduction of macromolecules, such as DNA, into cells of selected tissues in the body or plant. The controlled electrode system can include multiple needle electrodes, a hypodermic needle, an electrical connector providing conductive connection from a programmable constant current pulse controller to the multiple needle electrodes, and a power source. An operator can grasp the multiple needle electrodes mounted on a support structure and securely insert them into selected tissues in the body or plant. The macromolecule is then delivered to the selected tissue through the hypodermic needle. A programmable constant current pulse controller is activated, applying constant current electrical pulses to the multiple needle electrodes. The applied constant current electrical pulses facilitate the introduction of the macromolecule into cells between the multiple electrodes. Cell death due to overheating of cells is minimized by limiting power consumption in the tissue through the effectiveness of the constant current pulses. The CELLECTRA device and system are described in U.S. Patent No. 7,245,963, the contents of which are incorporated herein by reference. The CELLECTRA® device can be a CELLECTRA 2000® device or a CELLECTRA® 3PSP device. The CELLECTRA® 2000 device is configured by the manufacturer to support either ID (intradermal) or IM (intramuscular) administration. The CELLECTRA™ 2000 includes a CELLECTRA™ pulse generator, a suitable applicator, a disposable sterile array, and a disposable sheath (ID only). The DNA plasmid is delivered separately via needle and syringe injection in the area defined by the electrodes immediately prior to the electroporation procedure.

[0150] The MID may be the Elgen1000 system (Inovio Pharmaceuticals). The Elgen1000 system may comprise a device providing a hollow needle and a fluid delivery means, the device adapted to activate the fluid delivery means during use so as to simultaneously (e.g., automatically) inject a liquid vaccine described herein into a body tissue during insertion of the needle into said body tissue. An advantage is that the fluid can be injected gradually while the needle is being inserted, resulting in a more uniform distribution of the fluid through the body tissue. It is also believed that the pain experienced during injection is reduced due to the distribution of the volume of fluid injected over a larger area.

[0151] Additionally, automatic injection of liquid facilitates automatic monitoring and registration of the actual dose of liquid injected, and this data can be stored by the control unit for documentation, if desired.

[0152] It will be understood that the rate of injection may be linear or non-linear, and that injection may be performed after the needles have been inserted through the skin of the subject to be treated and while they are further inserted into the body tissue. Suitable tissues into which fluid may be injected by the device of the present invention include tumor tissue, skin, or muscle tissue.

[0153] The device further comprises needle insertion means for guiding the insertion of the needle into the body tissue. The rate of fluid injection is controlled by the rate of needle insertion. This has the advantage that both needle insertion and fluid injection can be controlled so that the rate of insertion matches the rate of injection as desired. It also makes the device easier for the user to operate. If desired, means can be provided for automatically inserting the needle into the body tissue.

[0154] The user can select when to begin the injection of the liquid. Ideally, however, the injection begins when the tip of the needle reaches the muscle tissue, and the device may include a means for sensing when the needle has been inserted deep enough to begin the fluid injection. This means that the device can be prompted to automatically begin the fluid injection when the needle reaches a desired depth (usually the depth at which the muscle tissue begins). The depth at which the muscle tissue begins can be a preset needle insertion depth, such as 4 mm, which is considered sufficient for the needle to penetrate the skin layer.

[0155] The sensing means may comprise an ultrasound probe. The sensing means may include means for sensing changes in impedance or resistance. In this case, the means may not be adapted to record the depth of the needle within the body tissue, but rather to sense changes in impedance or resistance as the needle moves through different types of body tissue and into muscle. Either of these alternatives provides a relatively accurate and easy to operate sensing means by which injection may be initiated. If desired, the needle insertion depth may further be recorded and used to control the injection of fluid, such that the volume of fluid injected is determined as the needle insertion depth is recorded.

[0156] The device may further comprise a base for supporting the needle and a housing for receiving the base therein, the base being movable relative to the housing such that the needle is retracted within the housing when the needle is in a first rearward position relative to the housing and extends out of the housing when the base is in a second forward position within the housing. This is advantageous for a user because the housing can be aligned over a patient's skin and then the needle can be inserted into the patient's skin by moving the housing relative to the base.

[0157] As mentioned above, it is desirable to achieve a controlled rate of fluid injection so that the fluid is evenly distributed along the length of the needle as it is inserted into the skin. The fluid delivery means may comprise a piston drive means adapted to inject the fluid at a controlled rate. The piston drive means may be actuated, for example, by a servomotor. However, the piston drive means may be actuated by axial movement of the base relative to the housing. It will be understood that alternative means for fluid delivery may be provided. Thus, for example, a closed container that can be compressed for fluid delivery at a controlled or uncontrolled rate may be provided in place of a syringe and piston system.

[0158] The above-described device can be used for any type of injection. However, it is envisioned to be particularly useful in the field of electroporation, and therefore may further include a means for applying a voltage to the needle. This allows the needle to be used not only for injection but also as an electrode during electroporation. This is particularly advantageous because it means that the electric field is applied to the same area as the injected fluid. A problem with electroporation in the past was that it was very difficult to accurately align the electrode with the previously injected fluid, so users tended to inject a larger amount of fluid than necessary over a larger area and apply the electric field over a wider area to ensure overlap between the injected material and the electric field. Using the present invention, both the volume of the injected fluid and the size of the applied electric field can be reduced while achieving good matching between the electric field and the fluid.

[0159] Further provided herein are kits that can be used to treat a subject using the above-described vaccination methods. The kits may include a vaccine. The kits may also include instructions for carrying out the above-described vaccination methods and / or methods of using the kit. The instructions included in the kits may be affixed to packaging or included as a package insert. The instructions are typically written or printed, but are not limited to such. Any medium capable of storing the instructions and transmitting them to an end user is contemplated by the present disclosure. Such media include, but are not limited to, electronic storage media (e.g., magnetic disks, tapes, cartridges), optical media (e.g., CD ROMs), and the like. As used herein, the term "instructions" may include the address of an internet site that provides the instructions. Illustrative Embodiments

[0160] Embodiment 1. A nucleic acid molecule encoding a human papillomavirus (HPV) antigen, wherein the HPV antigen comprises an HPV6 antigenic domain and an HPV11 antigenic domain.

[0161] Embodiment 2. The nucleic acid molecule of embodiment 1, wherein the HPV6 antigenic domain is an HPV6 E6-E7 fusion antigen.

[0162] Embodiment 3. The nucleic acid molecule of embodiment 1 or 2, wherein the HPV11 antigenic domain is an HPV11 E6-E7 fusion antigen.

[0163] Embodiment 4. The HPV antigen is: the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 11, or 10. The nucleic acid molecule of any one of the preceding embodiments, comprising an amino acid sequence that is at least 95% homologous to SEQ ID NO:1 or SEQ ID NO:11.

[0164] Embodiment 5. A nucleotide sequence that is at least 95% homologous to SEQ ID NO: 2 or SEQ ID NO: 12. the nucleotide sequence of SEQ ID NO: 2, or 12. The nucleic acid molecule of any one of the preceding embodiments, comprising the nucleotide sequence of SEQ ID NO: 12.

[0165] Embodiment 6. The nucleic acid molecule of any one of the preceding embodiments, wherein the nucleic acid sequence encoding the HPV11 antigenic domain is located 5' to the nucleic acid sequence encoding the HPV6 antigenic domain.

[0166] Embodiment 7. The nucleic acid molecule of any one of the preceding embodiments, wherein the HPV6 antigenic domain and the HPV11 antigenic domain are separated by one or more post-translational cleavage sites, one or more translational skip sites, or both.

[0167] Embodiment 8. The nucleic acid molecule of embodiment 2, wherein the HPV6 E6 antigenic domain and the HPV6 E7 antigenic domain are separated by one or more post-translational cleavage sites, one or more translational skip sites, or both.

[0168] Embodiment 9. The nucleic acid molecule of embodiment 4, wherein the HPV11 E6 antigenic domain and the HPV11 E7 antigenic domain are separated by one or more post-translational cleavage sites, one or more translational skip sites, or both.

[0169] Embodiment 10. An expression vector comprising a nucleic acid molecule according to any one of the preceding embodiments.

[0170] Embodiment 11. The expression vector of embodiment 10, comprising a DNA plasmid.

[0171] Embodiment 12. The expression vector of embodiment 10, comprising the nucleotide sequence of SEQ ID NO:3.

[0172] Embodiment 13. An immunogenic protein comprising a human papillomavirus (HPV) 6 antigenic domain and an HPV11 antigenic domain.

[0173] Embodiment 14. The immunogenic protein of embodiment 14, wherein the HPV6 antigenic domain comprises an HPV6 E6 antigenic domain and an HPV6 E7 antigenic domain.

[0174] Embodiment 15. The immunogenic protein of embodiment 13 or 14, wherein the HPV11 antigenic domain comprises an HPV11 E6 antigenic domain and an HPV11 E7 antigenic domain.

[0175] Embodiment 16. The amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 11, or 16. The immunogenic protein according to any one of embodiments 13 to 15, comprising an amino acid sequence that is at least 95% homologous to the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 11.

[0176] Embodiment 17. The immunogenic protein of any one of embodiments 13 to 16, wherein the HPV11 antigenic domain is located at the N-terminus of the HPV6 antigen.

[0177] Embodiment 18. The immunogenic protein of any one of embodiments 13 to 17, wherein the HPV6 antigenic domain and the HPV11 antigenic domain are separated by one or more post-translational cleavage sites, one or more translational skip sites, or both.

[0178] Embodiment 19. The immunogenic protein of embodiment 14, wherein the HPV6 E6 antigenic domain and the HPV6 E7 antigenic domain are separated by one or more post-translational cleavage sites, one or more translational skip sites, or both.

[0179] Embodiment 20. The immunogenic protein of embodiment 15, wherein the HPV11 E6 antigenic domain and the HPV11 E7 antigenic domain are separated by one or more post-translational cleavage sites, one or more translational skip sites, or both.

[0180] Embodiment 21. A vaccine comprising the nucleic acid molecule according to any one of embodiments 1 to 9 or the expression vector according to any one of embodiments 10 to 12, and a pharmaceutically acceptable excipient.

[0181] Embodiment 22. A pharmaceutical composition comprising the nucleic acid molecule according to any one of embodiments 1 to 9 or the expression vector according to any one of embodiments 10 to 12, and a pharmaceutically acceptable excipient.

[0182] Embodiment 23. The pharmaceutical composition of embodiment 22, comprising an adjuvant.

[0183] Embodiment 24. The pharmaceutical composition of embodiment 23, wherein the adjuvant comprises interleukin-12 (IL12).

[0184] Embodiment 25. The pharmaceutical composition of embodiment 24, wherein IL12 is encoded by a nucleic acid molecule.

[0185] Embodiment 26. The pharmaceutical composition of embodiment 25, wherein the nucleic acid molecule encoding IL12 is an expression vector.

[0186] Embodiment 27. A vaccine comprising an immunogenic protein according to any one of embodiments 13 to 20.

[0187] Embodiment 28. A pharmaceutical composition comprising an immunogenic protein according to any one of embodiments 13 to 20 and a pharmaceutically acceptable excipient.

[0188] Embodiment 29. The pharmaceutical composition of embodiment 28, comprising an adjuvant.

[0189] Embodiment 30. The pharmaceutical composition of embodiment 29, wherein the adjuvant comprises interleukin-12 (IL12).

[0190] Embodiment 31 The pharmaceutical composition of embodiment 23 or 29, wherein the adjuvant comprises a nucleic acid molecule comprising a nucleotide sequence encoding the p35 subunit of IL-12, the p40 subunit of IL-12, or both.

[0191] Embodiment 32. The nucleotide sequence encoding the p35 subunit of IL12 is a nucleotide sequence encoding SEQ ID NO:6, or 32. The pharmaceutical composition of embodiment 31, comprising a nucleotide sequence selected from the group consisting of nucleotide sequences that are at least 95% homologous to a nucleotide sequence encoding SEQ ID NO:6.

[0192] Embodiment 33. The nucleotide sequence encoding the p40 subunit of IL12 is a nucleotide sequence encoding SEQ ID NO:8, or 33. The pharmaceutical composition of embodiment 31 or 32, comprising a nucleotide sequence selected from the group consisting of nucleotide sequences that are at least 95% homologous to a nucleotide sequence encoding SEQ ID NO:8.

[0193] Embodiment 34. The nucleotide sequence encoding IL12 is the nucleotide sequence of SEQ ID NO: 4, or 34. The pharmaceutical composition of embodiment 31, 32 or 33, comprising a nucleotide sequence selected from the group consisting of nucleotide sequences that are at least 95% homologous to the nucleotide sequence of SEQ ID NO:4.

[0194] Embodiment 35. The pharmaceutical composition of any one of embodiments 31 to 34, wherein the nucleic acid molecule comprising a nucleotide sequence encoding the p35 subunit of IL-12, the p40 subunit of IL-12, or both, is an expression vector.

[0195] Embodiment 36. The pharmaceutical composition of embodiment 35, wherein the expression vector comprising the nucleic acid molecule encoding the p35 subunit of IL-12, the p40 subunit of IL-12, or both thereof is the same expression vector or a different expression vector as the expression vector comprising the nucleic acid molecule encoding the HPV antigen.

[0196] Embodiment 37. The pharmaceutical composition of any one of embodiments 22-26 or 28-36, wherein the pharmaceutically acceptable excipient comprises a buffer, optionally a saline-sodium citrate buffer, optionally a buffer comprising 150 mM sodium chloride and 15 mM sodium citrate (pH 7).

[0197] Embodiment 38. The pharmaceutical composition of embodiment 37, wherein the composition comprises 6 mg of vector encoding an HPV antigen per milliliter of saline-sodium citrate buffer and 0.25 mg of vector encoding the p35 subunit of IL-12, the p40 subunit of IL-1, or both per milliliter of buffer.

[0198] Embodiment 39. The pharmaceutical composition of embodiment 38, wherein the composition comprises 6 mg of pGX3024 per milliliter of saline-sodium citrate buffer and 0.25 mg of pGX6010 per milliliter of buffer.

[0199] Embodiment 40. A method for inducing an immune response in a subject, comprising administering to the subject an effective amount of a nucleic acid molecule described in any one of embodiments 1 to 9, an expression vector described in any one of embodiments 10 to 12, an immunogenic protein described in any one of embodiments 13 to 20, a vaccine described in embodiments 21 to 27, or a pharmaceutical composition described in any one of embodiments 22 to 26 or 28 to 39, thereby inducing an immune response.

[0200] Embodiment 41. A method for prophylactically or therapeutically immunizing a subject against HPV6 and / or HPV11, comprising administering to the subject an effective amount of a nucleic acid molecule described in any one of embodiments 1 to 9, an expression vector described in any one of embodiments 10 to 12, an immunogenic protein described in any one of embodiments 13 to 20, a vaccine described in embodiments 21 to 27, or a pharmaceutical composition described in any one of embodiments 22 to 26 or 28 to 39, thereby inducing an immune response against HPV6, HPV11, or both.

[0201] Embodiment 42. A method for treating or preventing recurrent respiratory papillomatosis (RRP) in a subject, comprising administering to the subject an effective amount of a nucleic acid molecule described in any one of embodiments 1 to 9, an expression vector described in any one of embodiments 10 to 12, an immunogenic protein described in any one of embodiments 13 to 20, a vaccine described in embodiments 21 to 27, or a pharmaceutical composition described in any one of embodiments 22 to 26 or 28 to 39, thereby treating or preventing RRP.

[0202] Embodiment 43. The method of embodiment 42, wherein the RRP is juvenile-onset RRP or adult-onset RRP.

[0203] Embodiment 44. The method of any one of embodiments 40 to 43, wherein the nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 12.

[0204] Embodiment 45. The method of any one of embodiments 40 to 44, further comprising administering an adjuvant to the subject.

[0205] Embodiment 46. The method of embodiment 45, wherein the adjuvant is interleukin-12 (IL12).

[0206] Embodiment 47. The method of embodiment 46, wherein IL12 is encoded by a nucleic acid molecule.

[0207] Embodiment 48 The method of embodiment 45, wherein the adjuvant comprises a nucleic acid molecule comprising a nucleotide sequence encoding the p35 subunit of IL-12, the p40 subunit of IL-12, or both.

[0208] Embodiment 49. The nucleotide sequence encoding the p35 subunit of IL-12 is a nucleotide sequence encoding SEQ ID NO:6, or 49. The method of embodiment 48, comprising a nucleotide sequence selected from the group consisting of nucleotide sequences that are at least 95% homologous to a nucleotide sequence encoding SEQ ID NO:6.

[0209] Embodiment 50. The nucleotide sequence encoding the p40 subunit of IL-12 is a nucleotide sequence encoding SEQ ID NO:8, or 50. The method of embodiment 48 or 49, comprising a nucleotide sequence selected from the group consisting of nucleotide sequences that are at least 95% homologous to a nucleotide sequence encoding SEQ ID NO:8.

[0210] Embodiment 51. The nucleic acid molecule encoding IL12 comprises: the nucleotide sequence of SEQ ID NO: 4, or 48. The method of embodiment 47, comprising a nucleotide sequence selected from the group consisting of nucleotide sequences that are at least 95% homologous to the nucleotide sequence of SEQ ID NO:4.

[0211] Embodiment 52 The method of embodiment 47, wherein the nucleic acid molecule encoding IL12 is an expression vector, optionally a plasmid.

[0212] Embodiment 53. The method of embodiment 52, wherein the plasmid is pGX6010.

[0213] Embodiment 54. The method of any one of embodiments 40 to 53, wherein the subject is a human.

[0214] Embodiment 55. The method of any one of embodiments 40 to 54, wherein administering comprises intradermal or intramuscular injection.

[0215] Embodiment 56. The method of embodiment 55, wherein administering further comprises electroporation.

[0216] Embodiment 57. Use of an effective amount of a nucleic acid molecule described in any one of embodiments 1 to 9, an expression vector described in any one of embodiments 10 to 12, or an immunogenic protein described in any one of embodiments 13 to 20 in the manufacture of a prophylactic or pharmaceutical agent.

[0217] Embodiment 58. Use of an effective amount of a nucleic acid molecule according to any one of embodiments 1 to 9, an expression vector according to any one of embodiments 10 to 12, or an immunogenic protein according to any one of embodiments 13 to 20 in the manufacture of a prophylactic or medicinal product for preventing or treating human papillomavirus (HPV) 6 or HPV11 infection.

[0218] Embodiment 59. Use of an effective amount of a nucleic acid molecule according to any one of embodiments 1 to 9, an expression vector according to any one of embodiments 10 to 12, an immunogenic protein according to any one of embodiments 13 to 20, a vaccine according to embodiments 21 to 27, or a pharmaceutical composition according to any one of embodiments 22 to 26 or 28 to 39 for preventing or treating human papillomavirus (HPV) 6 or HPV11 infection.

[0219] Embodiment 60. Use of an effective amount of a nucleic acid molecule according to any one of embodiments 1 to 9, an expression vector according to any one of embodiments 10 to 12, an immunogenic protein according to any one of embodiments 13 to 20, a vaccine according to embodiments 21 to 27, or a pharmaceutical composition according to any one of embodiments 22 to 26 or 28 to 39 for preventing or treating recurrent respiratory papillomatosis (RRP).

[0220] Embodiment 61. The use according to embodiment 60, wherein the RRP is juvenile-onset RRP or adult-onset RRP.

[0221] Embodiment 62. The use according to any one of embodiments 57 to 61, wherein the nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 12.

[0222] Embodiment 63. Use according to any one of embodiments 57 to 62 in combination with an adjuvant.

[0223] Embodiment 64. The use according to embodiment 63, wherein the adjuvant is interleukin-12 (IL12).

[0224] Embodiment 65. The use according to embodiment 64, wherein IL12 is encoded by a nucleic acid molecule.

[0225] Embodiment 66. The use of embodiment 65, wherein the adjuvant comprises a nucleic acid molecule comprising a nucleotide sequence encoding the p35 subunit of IL-12, the p40 subunit of IL-12, or both.

[0226] Embodiment 67. The nucleotide sequence encoding the p35 subunit of IL-12 is a nucleotide sequence encoding SEQ ID NO:6, or 67. The use of embodiment 66, comprising a nucleotide sequence selected from the group consisting of nucleotide sequences that are at least 95% homologous to a nucleotide sequence encoding SEQ ID NO:6.

[0227] Embodiment 68. The nucleotide sequence encoding the p40 subunit of IL-12 is a nucleotide sequence encoding SEQ ID NO:8, or 68. The use according to embodiment 66 or 67, comprising a nucleotide sequence selected from the group consisting of nucleotide sequences that are at least 95% homologous to a nucleotide sequence encoding SEQ ID NO:8.

[0228] Embodiment 69. The nucleotide sequence encoding IL12 is the nucleotide sequence of SEQ ID NO: 4, or 66. The use of embodiment 65, comprising a nucleotide sequence selected from the group consisting of nucleotide sequences that are at least 95% homologous to the nucleotide sequence of SEQ ID NO:4.

[0229] Embodiment 70. The use according to embodiment 65, wherein the nucleic acid molecule encoding IL12 is an expression vector, optionally a plasmid.

[0230] Embodiment 71. The use according to embodiment 70, wherein the plasmid is pGX6010.

[0231] The present invention is further illustrated in the following examples. It should be understood that these examples, while showing embodiments of the present invention, are provided by way of illustration only. From the above discussion and these examples, those skilled in the art can ascertain the essential features of the present invention and can make various changes and modifications to the present invention to adapt it to various uses and conditions without departing from the spirit and scope thereof. Thus, various modifications of the present invention, in addition to those shown and described herein, will be apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims.

[0232] Each of the U.S. patents, U.S. applications, and references cited throughout this disclosure is incorporated herein by reference in its entirety. [Example]

[0233] Generation of HPV6 and HPV11 E6 / E7 consensus-based fusion immunogen DNA constructs

[0234] HPV6 E6 / E7 and HPV11 E6 / E7 consensus sequences were generated using sequences obtained from GenBank. Two point mutations were introduced into the HPV6 E6 and HPV11 E6 proteins to inhibit their ability to bind and degrade p53. One point mutation was introduced into the HPV6 E7 and HPV11 E7 proteins to abolish p130 binding. The sequences were optimized using Inovio's proprietary gene optimization algorithm. Figure 1A shows a schematic diagram of the antigens encoded in different HPV6 and / or HPV11 plasmids. Individual antigens are separated by a P2A cleavage site for translational skipping and a furin cleavage site for post-translational cleavage of the P2A sequence. DNA plasmid pGX3024 encodes consensus SynCon® E6 and E7 antigens for both HPV6 and HPV11. Figure 1B provides a plasmid map of pGX3024.

[0235] In vitro transfection and Western blot analysis

[0236] Expression of pGX3024 in vitro. pGX3024-mediated HPV6 and HPV11 E6 and E7 antigen expression was confirmed by Western blot analysis after in vitro transfection of HEK-293T cells with the pGX3024 plasmid. Cells transfected with plasmids encoding either HPV6 or HPV11 E6 and E7 antigens (pGX3021 and pGX3022, respectively) served as positive controls, and cells transfected with the empty plasmid backbone (pGX0001) served as a negative control.

[0237] HEK-293T cells were seeded at 80% confluence in 6-well tissue culture dishes the day before transfection. The following day, cells were transfected with 3 μg of plasmid DNA using Lipofectamine 3000 transfection reagent (Thermo Scientific) according to the manufacturer's recommendations. 48 hours after transfection, cell lysates were harvested, and protein concentrations were determined by BCA assay (Quick Start™ Bradford Protein Assay, BioRad). 30 μg of cell lysate was loaded onto a 12% Bis-Tris acrylamide gel (Thermo Scientific) and transferred to a PVDF membrane. Precision Plus Protein™ Dual Xtra Prestained Protein Standards (BioRad, catalog number 1610377) were loaded as molecular weight references. After transfer, the blots were washed with 1x PBS / 0.05% Tween-20 and blocked in 1x PBS / 0.05% Tween-20 / 5% Milk for 1 hour at room temperature before being probed with diluted anti-HPV11 E7 (Genetex) overnight at room temperature. The next day, the blots were washed and then incubated with anti-mouse IgG HRP (Bethyl Laboratories) 1:10,000 for 1 hour at room temperature, then washed again. For detection, the blots were incubated with ECL Prime Western blotting substrate (GE Lifesciences / Amersham catalog number RPN2232 / 89168-782) for 5 minutes. The blots were imaged by chemiluminescence with Protein Simple FluorChem®. After detection, blots were stripped by adding Restore reagent (Thermofisher, cat#21059) for 15 minutes, then washed, probed with anti-2A (EMD Millipore) or anti-β-actin (Santa Cruz Biotech), and developed as before.

[0238] As shown in Figure 2, E6 and E7 proteins were detected in cells transfected with pGX3024 and the control pGX3021 and pGX3022 plasmids, but not in the negative control pGX0001 plasmid. E7 protein expression was detected using a commercially available anti-HPV11 E7 antibody (Figure 2, left panel). E7 protein was detected in cells transfected with pGX3024, but not in the negative control pGX0001 plasmid. E7 antigen was detected in cells transfected with the HPV6 antigen-only plasmid (pGX3021) and the HPV11 antigen-only plasmid (pGX3022), demonstrating that the anti-HPV11 E7 antibody is cross-reactive with both HPV6 and HPV11 E7 antigens, suggesting that the bands detected in pGX3024-transfected cells represent both HPV6 and HPV11 E7 protein expression.

[0239] After evaluating multiple commercially available reagents, we were unable to identify any antibodies that specifically recognized the HPV6 or HPV11 E6 protein. However, using an anti-HPV11 E7 probe, two specific bands were detected at the predicted molecular weights of approximately 10.7 kDa and 13 kDa for E7 and E7-Furin / P2A, respectively, indicating partial furin cleavage of the P2A sequence in this in vitro system (Figure 2, left panel). Therefore, E6 and E7 antigen expression could be detected using an anti-2A antibody probe against the partially cleaved protein products (Figure 2, center panel). After probing with anti-2A antibody, a band representing E6-Furin / P2A at approximately 20 kDa was detected in cells transfected with pGX3024 but not in cells transfected with the control pGX0001. We also detected an approximately 15-kDa E7-furin / P2A band in cells transfected with pGX3024 but not in cells transfected with the control pGX0001, consistent with that detected using an anti-HPV11 E7 probe.

[0240] Mouse IFN-γ ELISpot

[0241] pGX3024 was evaluated for immunogenicity in two mouse models (Figures 3-8). Cellular and humoral immune responses induced by the DNA vaccine pGX3024 were characterized in a C57BL / 6 mouse model in two independent studies. Female C57BL / 6 mice (n = 5 or 10 per group in Study 1 or Study 2) received two immunizations, 2 weeks apart, with 20 μg of pGX3024 or a control DNA vaccine delivered by intramuscular electroporation (IM-EP). Following splenocyte stimulation with HPV6 / HPV11 E6 or E7 peptides, T cell responses were measured 1 week after the second immunization by IFNγ ELISpot. After euthanasia, mouse spleens were isolated and placed in tubes containing 5 ml of R10 medium (RPMI 1640 supplemented with 10% fetal bovine serum, 1% penicillin-streptomycin, and 0.001% 2-mercaptoethanol). Splenocytes were isolated by mechanically disrupting the spleens using a GentleMACS Dissociator (Miltenyi Biotech) and then filtered through a 40 μm cell strainer (BD Falcon). After centrifugation, the resuspended cell pellet was treated with ACK lysis buffer (Lonza) for 5 minutes to lyse red blood cells. Splenocytes were washed with PBS, centrifuged, resuspended in R10 medium, and counted using a Vi-cell (Beckman Coulter) kit. A mouse IFN-γ ELISpot kit was purchased from MabTech (MabTech No. 3321-4APW-10) to assess antigen-specific responses. Pre-coated 96-well plates were washed with PBS and blocked with R10 medium for 2 hours at room temperature according to the manufacturer's protocol. Isolated splenocytes were resuspended in R10 medium and counted at 2 × 10 per well. 5Cells were seeded in triplicate. Overlapping 15-mer peptides of HPV6 E6 and E7 proteins and HPV11 E6 and E7 proteins were synthesized. The peptides were resuspended in DMSO (Sigma) and pooled into two peptide pools per antigen (HPV6 E6, HPV6 E7, HPV11 E6, and HPV11 E7) at a concentration of approximately 2 μg / ml per peptide for cell stimulation. Concanavalin A (Sigma) was used at 5 μg / ml as a positive control, and complete medium with DMSO was used as a negative control. Plates were incubated at 37°C and 5% CO2 for a minimum of 18 hours. For expression, plates were first washed with PBS and then incubated with a biotinylated anti-mouse IFN-γ detection antibody (R4-6A2-biotin) at room temperature for 2 hours. After washing, the plates were then incubated with streptavidin-ALP (MabTech No. 3321-4APW-10) for 1 hour at room temperature. Spots were detected using filtered substrate solution (BCIP / NBT-plus) according to the manufacturer's instructions (MabTech). Once the plates were dry, they were counted using an automated ELISpot reader (Cellular Technology). The average spot-forming units (SFU) was 1 x 10 6 Adjusted antigen-specific responses to 1 x 10 splenocytes greater than DMSO controls 6 The results were reported as the number of IFN-γ SFU per spleen cell.

[0242] HPV6- and HPV11-specific T cell responses were detected in mice immunized with pGX3024, but not with the negative control pGX0001 plasmid (Figure 3). As determined by one-way ANOVA and Tukey's post-hoc test, T cell responses in mice immunized with pGX3024 were not significantly different from those in mice immunized with pGX3021 and / or pGX3022 (Figure 3). Immunization with pGX3024 induced T cell responses to HPV6 E7, HPV11 E6, and HPV11 E7 antigens, but not HPV6 E6 antigen, in this model. Inbred C57BL / 6 mice have a single MHC haplotype (H2b), which may explain the lack of HPV6 E6 cellular responses in this model. T cell responses were highly reproducible between two independent studies.

[0243] Antibodies to HPV6 and HPV11 E7 antigens were measured by binding IgG ELISA in serum samples collected before immunization (week 0) and after the first (week 2) and second (week 3) immunizations with either pGX3024 or a negative control plasmid (pGX0001).

[0244] An immunogenicity study of pGX3024 with or without pIL-12 was conducted in C57BL / 6 mice. To determine whether the addition of a plasmid encoding IL-12 (pIL-12) augmented pGX3024 vaccine-induced cellular and humoral immune responses, a range of IL-12 doses was evaluated with a set pGX3024 dose after a single immunization. C57BL / 6 mice were administered pGX3024 with or without a range of doses of murine pIL-12. For species selectivity, pGX6012 (encoding murine IL-12) was used. Female C57BL / 6 mice (n = 8 per group) received a single immunization with 6 μg of control DNA or pGX3024 alone via electroporation (IM-EP), or with 0.031 μg, 0.063 μg, 0.125 μg, 0.25 μg, or 0.5 μg of pGX6012 delivered intramuscularly. Following splenocyte stimulation with HPV6 / HPV11 E6 or E7 peptides, T cell responses were measured by IFNγ ELISpot 14 days after immunization. HPV6- and HPV11-specific T cell responses were detected in mice after pGX3024 immunization, and a pIL-12 dose-dependent increase was observed (Figure 4). Addition of 0.25 μg or 0.50 μg of pIL-12 to the pGX3024 formulation resulted in a statistically significant increase in total HPV cellular responses.

[0245] Antibodies against HPV6 and HPV11 E6 and E7 antigens were measured by IgG ELISA in serum samples collected before (week 0) and after a single immunization (week 2) with either pGX3024 alone or increasing doses of murine pIL-12 (pGX6012) or control plasmid (pGX0001). HPV6 and HPV11 binding antibodies were low in mice immunized with pGX3024 alone (comparable to pGX0001) but significantly increased with 0.031 μg, 0.063 μg, and 0.125 μg of murine pIL-12 (Figures 5A and 5B). Notably, binding antibodies were detected for all four antigens. As expected, antibody titer levels were low because the animals received only a single immunization. Overall, we demonstrated enhanced cellular and humoral responses with plasmid IL-12 compared to pGX3024 alone in a C57BL / 6 mouse model, although the dose required to boost T cell responses compared to humoral responses varied. Significant enhancement of cellular responses was measured at ratios of 1:24 and 1:12 mouse pIL-12 to pGX3024. This ratio is consistent with the INO-3107 clinical dose, which includes a 1:24 ratio of human pIL-12 to pGX3024 (0.25 mg pIL-12 to 6 mg pGX3024).

[0246] IgG antigen binding ELISA

[0247] 96-well high-binding Nunc™ plates (Thermo Scientific) were coated overnight at 4°C with 0.5 μg / ml of each protein (HPV6 E7 and HPV11 E7-Tulip BioLabs) in 1× DPBS (Thermo Scientific). The next day, the plates were washed with 1× PBS + 0.05% Tween-20 and blocked with 3% BSA in PBS + Tween-20 for 2 hours at 37°C. The plates were then washed as before, serially diluted serum samples were added, and the plates were incubated for 2 hours at 37°C. The plates were washed and incubated with a 1:10,000 dilution of anti-mouse or rabbit IgG HRP secondary antibody (Bethyl Laboratories, Inc.) at room temperature for 1 hour. The plates were washed, and 100 μl / well of SureBlue TMP substrate (KPL5120-0077) was added to the plates. After 6 minutes of incubation, the reaction was stopped by adding 100 ul / well of TMB Stop Solution (KPL5150-0021) and the plate was read on a Biotek Synergy plate reader at a wavelength of 450 nm.

[0248] HPV6 E7-binding antibodies were detected in mice immunized with pGX3024 after the first and second immunizations, but not in mice immunized with pGX0001 (Figure 6, left panel). Generally, HPV11 E7-binding antibodies were reduced compared with HPV6 E7-binding antibodies in immunized mice; however, HPV11 E7-binding antibodies after two immunizations (week 3) were greater in mice immunized with pGX3024 compared with pGX0001 (Figure 6, right panel).

[0249] pGX3024 immunogenicity in BALB / c mice. As previously mentioned, C57BL / 6 mice have a single MHC haplotype (H2b), which may explain the lack of HPV6 E6 cellular responses in this model. Therefore, the cellular responses induced by the pGX3024 vaccine were investigated in a BALB / c mouse model with a different MHC haplotype (H2d). The effects of pGX3024 dose and its combination with a plasmid encoding a murine IL-12 adjuvant (pGX6012) were also investigated. Female BALB / c mice (n = 6 per group) received two immunizations, 2 weeks apart, via intramuscular electroporation (IM-EP) delivery of 5 μg pGX3024 adjuvanted with either 1 μg, 5 μg, 10 μg, or 20 μg of pGX3024 DNA vaccine alone or 2 μg or 11 μg of pGX6012 murine IL-12 plasmid. Mice immunized with 40 μg of empty pGX0001 plasmid served as a negative control. As shown in Figure 7, T cell responses were measured one week after the second immunization by IFNγ ELISpot after splenocyte stimulation with HPV6 / HPV11 E6 or E7 peptides. HPV6- and HPV11-specific T cell responses were detected in mice immunized with pGX3024 at all dose levels, but not with the pGX0001 plasmid in a dose-related manner. Total HPV6 and HPV11 cellular responses increased with increasing pGX3024 dose. Unlike the C57BL / 6 model, pGX3024-immunized BALB / c mice had T cell responses to the HPV6 E6 antigen as well as the HPV6 E7, HPV11 E6, and HPV11 E7 antigens. There were no significant differences in T cell responses between mice immunized with 5 μg of pGX3024 with or without any of the dose levels of pGX6012, as determined by one-way ANOVA, Tukey's post-hoc test.

[0250] Antibodies to HPV6 and HPV11 E7 antigens were measured by binding IgG ELISA in serum samples collected before immunization (week 0) and after the first (week 2) and second (week 3) immunizations with 5 μg, 10 μg, or 20 μg of pGX3024 DNA vaccine alone or 5 μg of pGX3024 adjuvanted with 2 μg of murine IL-12 plasmid (pGX6012). Serum from mice immunized with 40 μg of empty pGX0001 plasmid served as a negative control. HPV6 E7 and HPV11 E7 binding antibodies were significantly increased at week 3 compared to week 0 in BALB / c mice immunized with pGX3024, but not in pGX0001 mice, regardless of dose or the presence of IL-12 plasmid (Figure 8). Antibody levels were also significantly increased after a single immunization (week 2) with 20 μg of pGX3024 or 5 μg of pGX3024 adjuvanted with 2 μg of mouse IL-12 plasmid. Addition of mouse IL-12 to 5 μg of pGX3024 tended to increase antibody binding to both antigens, especially at week 2.

[0251] Rabbit immunogenicity study

[0252] INO-3107 (pGX3024, pGX6010) was evaluated for immunogenicity and safety in a rabbit model.

[0253] New Zealand White (NZW) rabbits (n = 5 per group) received four immunizations at 3-week intervals with INO-3107 (6 mg pGX3024 and 0.25 mg pGX6010 co-formulated in 1 mL 1x SSC) or 1 mL 1x SSC (negative control) via intramuscular (IM) injection into the quadriceps muscle followed by electroporation (EP) using a CELLECTRA® 2000 electroporation device. Cellular and humoral immune responses were assessed by IFNγ ELISpot and IgG-binding ELISA before immunization (week 0) and 2 weeks after each immunization (weeks 2, 5, 8, and 11), respectively. Physiological parameters, including body weight, hematology, serum chemistry, and general appearance, were monitored throughout the study as indicators of vaccine safety and animal health.

[0254] After stimulation of PBMCs with HPV6 / HPV11 E6 and E7 peptides, rabbit cell responses were evaluated by IFNγ ELISpot assay. A 15 ml SepMate tube was filled with 3.5 ml of a Ficoll-Paque gradient and equilibrated at room temperature. Whole blood was collected into a K2EDTA tube and then mixed by inversion several times. The blood was diluted with Hank's balanced salt solution (HBSS) and gently layered on top of the Ficoll-Paque gradient in the SepMate tube. The tube was spun, and the buffy coat was collected and placed in a fresh 15 ml tube. The cells were washed by dilution with R10 medium (RPMI 1640 supplemented with 10% fetal bovine serum, 1% penicillin-streptomycin, and 0.001% 2-mercaptoethanol). The cell pellet was resuspended in ACK lysis buffer (Lonza) to lyse red blood cells and incubated at room temperature for 4 minutes. PBMCs were washed, spun, resuspended in R10 medium, and counted using Vi-cell (Beckman Coulter). Antigen-specific responses were assessed using a rabbit IFN-γ ELISpot kit (MabTech (MabTech No. 3110-4HPW-10)). Plates were prepared according to the manufacturer's protocol, and rabbit PBMCs were added at 2 × 10 5Cells were seeded in triplicate at 10 ... 6 Adjusted antigen-specific responses to 1 x 10 splenocytes greater than DMSO controls 6 The results were reported as the number of IFN-γ SFU per spleen cell.

[0255] HPV6- and HPV11-specific T cell responses above baseline were detected in all rabbits after immunization with INO-3107, but not in rabbits treated with 1×SSC. HPV6 and HPV11 T cell responses increased following each successive immunization with INO-3107, and thus could be enhanced (FIG. 9). Immunization with INO-3107 induced T cell responses to HPV6 and HPV11 E6 antigens, but not HPV6 or HPV11 E7 antigens in this model (FIG. 10).

[0256] Humoral responses to HPV6 and HPV11 E7 antigens were measured by binding IgG ELISA in serum samples collected before and 2 weeks after each immunization. The time course of antibody levels is shown in Figure 11. HPV6 or HPV11 E7 binding antibodies were detected in four of five rabbits immunized with INO-3107, but not in rabbits dosed with 1xSSC. Overall, HPV6 E7 binding antibodies were reduced compared to HPV11 E7 binding antibodies in immunized rabbits. Taken together, the ELISpot and ELISA data confirmed the immunogenicity of all antigens encoded by INO-3107 in the rabbit model.

[0257] In addition to INO-3107-mediated immune responses, physiological parameters, including body weight, hematology, serum chemistry, and general appearance, were monitored throughout the study as indicators of vaccine safety and animal health. No significant differences in body weight or weight change over time were observed in rabbits administered INO-3107 (Figure 12). Additionally, no findings were observed during monitoring of general appearance (nose, eyes, fur, movement) for any treatment group during the study. Samples were collected for hematology and serum clinical chemistry analysis before immunization and at weeks 5, 8, and 11 post-immunization, and the results were submitted for independent review by a clinical veterinary pathologist (IDEXX BioAnalytics). Compared to baseline values, administration of INO-3107 resulted in a mild increase in lymphocyte counts at week 5 (but not at weeks 8 or 11) in NZW rabbits. There were no other hematological or serum clinical chemistry findings indicating biologically relevant effects from INO-3107 administration.

[0258] Evaluation of intradermal (ID) delivery in rabbits

[0259] To evaluate cellular immune responses, an intradermal (ID) injection study was performed in NZW rabbits. NZW rabbits (n=5) were immunized three times at 3-week intervals with INO-3107 formulated with 1 mg of pGX3024 in 0.1 mL of 1x SSC via ID delivery. Rabbit IFNγ ELISpot was performed before the first vaccination and at weeks 2, 5, and 8. The combined immune response to both antigens, HPV6 and HPV11, increased after each immunization, and T cell responses were more HPV6 E6 and HPV11 E6 specific (Figure 15).

[0260] Immunogenicity Study of INO-3107 Delivered by Intradermal Administration of CELLECTRA ID-EP in NZW Rabbits

[0261] The immunogenicity of INO-3107 (pGX3024 and pGX6010) was evaluated in 16-week-old female New Zealand White (NZW) rabbits after intradermal (ID) or intramuscular (IM) administration using the CELLECTRA EP device.

[0262] HPV6 and HPV11 antigen-specific cellular (FIG. 16) and humoral (FIGS. 17A-17D) responses were assessed pre-immunization (week 0) and 2 weeks after each immunization (weeks 2, 5, 8, and 11).

[0263] After stimulating PMBCs with HPV6 E6, HPV6 E7, HPV11 E6, and HPV11 E7 peptide pools, IFNγ cellular responses were assessed by IFNγ ELISpot assay before immunization (week 0) and 2 weeks after each immunization (weeks 2, 5, 8, and 11). HPV6 and HPV11 peptide pool-reactive cellular responses were detected in all groups after immunization with INO-3107. T cell responses above baseline were detected as early as week 2 and continued to increase after each successive immunization (Figure 16). HPV6 and HPV11 E7 cellular responses were lower than HPV6 and HPV11 E6 responses. Overall, T cell responses tended to be higher in the high-dose group (6 mg pGX3024 + 0.25 mg human pIL-12) compared with the low-dose group (0.8 mg pGX3024 + 0.2 mg human pIL-12). Statistically significant differences in total T cell responses between the high and low IM dose groups were detected at multiple time points.

[0264] INO-3107 can be delivered by electroporation (EP) at a range of doses to induce HPV6- and HPV11-specific T cell responses and boosted humoral binding antibodies after each administration. The highest immune responses were measured in the 6 mg INO-3107 IM delivery group. After immunization with a lower dose (1 mg), there was no significant difference in immune responses between ID or IM routes of administration, suggesting that ID delivery may be a viable administration route.

[0265] Evaluation of intradermal (ID) delivery in a guinea pig model

[0266] Hartley guinea pigs (n=5) received three immunizations at 2-week intervals with pGX3024 (0.1 mg formulated in a final 0.1 mL of 1×SSC) by intradermal (ID) injection followed by electroporation (EP) using a CELLECTRA® 2000 electroporation device. Naive guinea pigs served as negative controls (n=2). Cellular and humoral immune responses were assessed by IFNγ ELISpot and IgG-binding ELISA before immunization (week 0) and 2 weeks after each immunization (weeks 2, 4, and 6), respectively.

[0267] Animal care, immunizations, and sample collection were performed at Acculab Life Sciences (San Diego, CA) under IACUC-approved protocols in compliance with the Animal Welfare Act, PHS policy, AAALACi guidelines, USDA, and other federal laws and regulations related to animals and experiments involving animals. All sample analysis was performed at Inovio (San Diego, CA). pGX3024 was formulated in 1x SSC at a final concentration of 0.1 mg in 0.1 mL of dosing solution and stored at 2-8°C until use. Eight-week-old female Hartley guinea pigs were randomly divided into two groups of five or two animals each according to Table 1. Each treatment was delivered by Mantoux intradermal (ID) injection of 100 μL of dosing solution into the skin, followed by electroporation using a CELLECTRA2000® Adaptive Constant Current Electroporation Device (Inovio Pharmaceuticals) equipped with a 3P array, according to the manufacturer's protocol.

[0268] [Table 1]

[0269] Animals in Group 1 received a total of three immunizations at 2-week intervals. Serum samples were collected from all animals at weeks 0, 2, 4, and 6 for evaluation of humoral immunogenicity. Whole blood samples were collected from all animals at weeks 2, 4, and 6 for evaluation of cellular immunogenicity.

[0270] Guinea pig IFN-γ ELISpot. Guinea pig IFN-γ ELISpot was performed according to the method described by Schultheis, et al., J Vis Exp. 2019;(143):10.3791 / 58595. Published 2019 Jan 20. doi:10.3791 / 58595. Peripheral blood was collected from the jugular vein of each anesthetized animal and immediately transferred to an EDTA blood collection tube. The blood was diluted 1:1 with phosphate-buffered saline. The diluted blood was layered on Ficoll-Paque Plus (GE Healthcare Life Sciences) in a SepMate™ tube (Stemcell) and centrifuged (1200 g, 10 min, 24°C). PBMCs were collected at 1 × 10 in R10 medium. 6The cells were resuspended at 100 μl / well and plated onto 96-well Millipore IP plates (Millipore) previously coated with 5 μg / ml of primary anti-IFN-γ antibody V-E4 (provided by Prof. Schafer, Robert Koch Institute, Berlin, Germany) and blocked with R10 medium. 100 μl of HPV6 E6, HPV6 E7, HPV11 E6, or HPV11 E7 peptide pools or phorbol 12-myristate 13-acetate (PMA) / ionomycin stimuli were added to the cells. Samples were assayed in triplicate. After 18 hours of incubation at 37°C in a humidified atmosphere of 5% CO2, the cells were removed by washing, and 100 μl of 2 μg / ml of biotinylated secondary anti-IFN-γ antibody N-G3 diluted in blocking buffer was added per well. After a 2-hour incubation and wash, alkaline phosphatase-conjugated streptavidin (MabTech) was added at 100 μl per well for 1 hour at room temperature. After washing, the wells were incubated with nitro-blue tetrazolium / 5-bromo-4-chloro-3'-indolylphosphate (BCIP / NBT) detection reagent substrate (MabTech) at 100 μl per well for 6–12 minutes at room temperature. Interferon-gamma-positive spots were imaged, analyzed, and counted using a CTL-Immunospot® S6 ELISPOT plate reader and CTL-Immunospot® software. Antigen-specific responses were determined by subtracting the number of spots in DMSO-treated wells from those in peptide-treated wells.

[0271] Results are expressed as the spot-forming units (SFU) / 10 of individual animals obtained for triplicate wells. 6 Figure 13 shows HPV6- and HPV11-specific T cell responses above baseline were detected in guinea pigs after immunization with pGX3024, but not in naive guinea pigs (Figure 13). In this model, immunization with pGX3024 induced T cell responses against HPV6 and HPV11 E6 and E7 antigens (Figure 13).

[0272] Antigen-binding ELISA. 96-well high-binding Nunc™ plates (Thermo Scientific) were coated overnight at 4°C with 1 μg / ml recombinant HPV6 E7 or HPV11 E7 protein in 1 μg / ml Dulbecco's phosphate-buffered saline (DPBS) (Thermo Scientific). The next day, plates were washed with 1× PBS + 0.05% Tween®-20 and blocked with 3% BSA in PBS + 0.05% Tween®-20 for 2 hours at room temperature. Plates were then washed as before, serially diluted serum samples were added, and plates were incubated for 2 hours at room temperature. Plates were washed and incubated with a 1:10,000 dilution of anti-guinea pig IgG horseradish peroxidase (HRP) secondary antibody (Sigma) for 1 hour at room temperature. The plate was washed and 100 μl / well of SureBlue™ TMB Substrate (KPL5120-0077) was added to the plate. After a 6 minute incubation, the reaction was stopped by adding 100 μl / well of TMB Stop Solution (KPL5150-0021) and the plate was read on a Biotek Synergy plate reader at a wavelength of 450 nm.

[0273] Humoral responses to HPV6 E7 and HPV11 E7 antigens were measured by binding IgG ELISA in serum samples collected before and 2 weeks after each immunization. The time course of antibody levels is shown in Figure 14. After immunization with pGX3024, HPV6 E7 and HPV11 E7 binding antibodies were detected in guinea pigs.

[0274] Phase 1 / 2 Clinical Trial: INO-3107 by Electroporation (EP) in Controls with HPV-6 and / or HPV-11 Associated Recurrent Respiratory Papillomatosis (RRP) [ClinicalTrials.gov Identifier: NCT04398433]

[0275] This is a Phase 1 / 2, open-label, multicenter study to evaluate the safety, tolerability, immunogenicity, and efficacy of INO-3107 drug in subjects with HPV-6 and / or HPV-11 associated recurrent respiratory papillomatosis (RRP). INO-3107 drug will be administered IM to subjects on Day 0, Week 3, Week 6, and Week 9, followed by EP (Figure 18).

[0276] This study enrolled 32 adults (aged 18 years or older) diagnosed with either juvenile-onset RRP (JO RRP), defined by age <12 years at first diagnosis, or adult-onset RRP (AO RRP), defined by age ≥12 years at first diagnosis.

[0277] The study will have a safety run-in of up to six subjects with a one-week waiting period between each enrolled subject.

[0278] Safety and tolerability will be assessed throughout the study after tolerability has been established. Tolerability will be determined by the reported incidence of dose-limiting toxicities (DLTs), which are defined as follows: - Treatment-related NCI Common Terminology Criteria for Adverse Events (CTCAE, version 5.0) grade 3 or greater non-hematologic toxicity that is unresponsive to supportive care and lasts for more than 48 hours, or - Treatment-related NCI CTCAE v5.0 grade 3 or greater hematologic toxicity that is unresponsive to supportive care and lasts for more than 48 hours.

[0279] Subjects will undergo routine surgery to remove the papilloma during the screening period within 14 days prior to dosing on Day 0 (if other eligibility criteria are met, papilloma removal and Day 0 dose may be performed on the same day). Biopsy tissue will be collected and evaluated for secondary and exploratory endpoints. Disease status will be monitored throughout the study.

[0280] Subjects will receive a single 6.25 mg intramuscular (IM) injection of INO-3107 medication on Day 0, Week 3, Week 6, and Week 9, followed by electroporation (EP) using CELLECTRA® 2000. The primary objective of this study is to evaluate the safety and tolerability of INO-3107 medication in subjects with HPV-6 and / or HPV-11-associated RRP. The primary endpoints are safety and tolerability as assessed by reported adverse events (AEs) and serious adverse events (SAEs). The primary outcome measure is the proportion of participants with adverse events (AEs) and serious adverse events (SAEs) [time frame: up to week 52 screening (up to approximately 1 year)]. An adverse event (AE) is any untoward medical occurrence in a participant receiving the medication or in a clinical study participant that does not necessarily have a causal relationship to the treatment. AEs may include any untoward and unintended sign (including abnormal laboratory findings), symptom, or disease temporally associated with the use of a medicinal product (test), whether or not related to the medicinal product (test). A serious adverse event (SAE) is any untoward medical occurrence that is 1-5 of the following at any dose: 1. Results in death. 2. Is a life-threatening event, but does not include events that may have caused death if they occurred in a more severe form. 3. Requires inpatient hospitalization or prolongation of existing hospitalization. 4. Results in persistent or significant disability / incapacity. 5. Results in congenital abnormality / birth.

[0281] The secondary objectives of this study are 1 to 5 below. To evaluate the efficacy of INO-3107 drug product as determined by the frequency of RRP surgical interventions in the 1-year period after the first dose of test article compared with the 1-year frequency before Day 1.0. 2. To evaluate the efficacy of INO-3107 drug as assessed by changes in RRP stage assessment over time. 3. To evaluate the cellular immune response to INO-3107 drug when given IM followed by EP. 4. To evaluate the immunogenicity of the INO-3107 drug product as assessed by pro-inflammatory and immunosuppressive elements in resected tumor tissue at study entry and, if available, subsequent tissue resection. 5. To evaluate any potential association of microRNA (miRNA) profiles with the reduced frequency of RRP surgical interventions. The secondary endpoints are 1 to 5 below. Number of RRP surgical interventions in the year following Day 0 compared with the number of RRP surgical interventions in the year prior to Day 0 dosing [Timeframe: Screening up to Week 52 (up to approximately 1 year)] 2. Change in RRP staging score over time [Time frame: Screening, Day 0, Week 6, Week 11, Week 26, Week 52 (up to approximately 1 year)]. The RRP staging score is determined using a modified Derkay staging tool. It includes both a subjective functional assessment of clinical parameters and an anatomical assessment of disease distribution. The anatomical score can then be used in combination with the functional score to measure an individual patient's clinical course and response to therapy over time. 3. Antigen-specific cellular immune response assessed by: - Change from baseline in interferon-gamma enzyme-linked immunosorbent spot (IFN-γ ELISpot) response magnitude for IFN-γ secreting cells in peripheral blood mononuclear cells (PBMC) [Timeframe: Baseline, Week 6, Week 9, Week 11, Week 26, Week 52] - Change from baseline in flow cytometry response magnitude for T cell phenotype and lytic potential in PBMCs [Timeframe: Baseline, Week 6, Week 9, Week 11, Week 26, Week 52] 4. Change from baseline in magnitude of resected tumor tissue response for pro-inflammatory and immunosuppressive components [Time frame: baseline and subsequent tissue resections, up to week 52 (maximum of approximately 1 year)] 5. Change from baseline in microRNA (miRNA) expression associated with reduced frequency of RRP surgical intervention [time frame: baseline and week 6], as assessed by

[0282] The exploratory objectives of this study are 1 to 4 below. 1. To describe the virologic clearance of HPV-6 and / or 11 in excised tissue compared to baseline. 2. To evaluate the antigen-specific humoral immune response to INO-3107 drug as assessed by ELISA. 3. To evaluate the immunogenicity of INO-3107 drug as assessed by pro-inflammatory and immunosuppressive elements in peripheral blood. 4. To evaluate circulating free HPV DNA (cfHPV DNA) 6 / 11 as a correlate of disease burden and clinical outcomes in patients with RRP treated with INO-3107 drug. The exploratory endpoints are 1 to 5 below. 1. Clearance of HPV-6 / 11 in excised tissue compared to baseline, 2. Antigen-specific humoral immune responses assessed by ELISA, 3. Evaluation of pro-inflammatory and immunosuppressive factors in peripheral blood, and 4. cfHPV DNA6 / 11 load before and after INO-3107 drug as a correlate of disease burden and clinical outcome in RRP patients treated with INO-3107 drug. 5. Clinical and antigen-specific cellular and humoral immune responses assessed by the endpoints referenced above.

[0283] Efficacy Assessment: A detailed medical history for each subject will be conducted, including documentation of HPV-6 and / or HPV-11 RRP, a list of RRP surgeries and treatments that occurred within the three years prior to screening, and any periods of remission. To be eligible for the study, subjects must have undergone at least two surgical RRP interventions (including laser) in the year prior to and including Day 0. Subjects must have required an RRP intervention at the time of entry into the study and undergo surgical removal of a papilloma during screening within 14 days prior to Day 0 dosing to maximize standardization of baseline disease stage across subjects. Efficacy assessment will be based on the number of RRP surgical interventions in the 52 weeks after Day 0 compared to the number of RRP surgical interventions in the year prior to Day 0 dosing. RRP surgical interventions include laser therapy. The study will also assess change in RRP disease stage assessments over time.

[0284] Safety Assessment: Subjects will be followed for safety from the time they sign the informed consent through Week 52, or until the subject's last visit. Safety of INO-3107 drug will be measured and graded according to CTCAE v5.0. Clinically significant changes in laboratory parameters and vital signs from baseline assessment will be assessed.

[0285] An adverse event is any untoward medical occurrence in a patient or clinical investigational subject administered a medicinal product, and does not necessarily have a causal relationship to the treatment. Thus, an AE can be any untoward, unintended sign (including, for example, abnormal laboratory findings), symptom, or disease temporally associated with the use of a medicinal product, whether or not considered related to the medicinal product. Adverse events (AEs) include: pre- or post-treatment complications occurring as a result of protocol-mandated treatment during or after screening (prior to administration of clinical trial drug); any pre-existing condition that increases in severity or changes in nature during or as a result of the clinical trial drug administration phase, excluding conditions under investigation in this study; and pregnancy complications. Adverse events (AEs) do not include the following: a medical or surgical procedure (e.g., surgery, endoscopy, tooth extraction, blood transfusion) is performed but the condition leading to the procedure is an AE; a pre-existing disease or condition or laboratory abnormality present or detected before the screening visit; a recurrence of an RRP; a situation in which no adverse medical event has occurred (e.g., hospitalization for elective surgery, social and / or convenience hospitalization); an overdose without clinical sequelae; any medical condition or clinically significant laboratory abnormality with an onset date before informed consent was provided is not an AE; an uncomplicated pregnancy; an induced elective abortion to terminate a pregnancy without medical reasons.

[0286] Adverse drug reactions (ADRs) include all harmful and unintended responses to a medicinal product associated with any dose, meaning that a causal relationship between the medicinal product and the adverse event is at least reasonably possible (i.e., the relationship cannot be excluded).

[0287] A serious adverse event (SAE) is any unintended medical occurrence, at any dose, that results in death, is life-threatening, requires inpatient hospitalization or prolongs an existing hospitalization, results in persistent or significant disability / incapacity, results in a congenital anomaly or birth defect, and / or results in a significant medical event.

[0288] An unexpected adverse drug reaction is an adverse reaction whose nature or severity is inconsistent with the applicable product information. An unexpected (serious) adverse device effect (UADE) is any serious adverse effect to health or safety or any life-threatening problem or death caused by or associated with a device when the effect, problem, or death was not previously identified in its nature, severity, or degree of occurrence in the testing plan or use (including supplemental plans or uses), or when other unexpected serious problems related to the device that relate to the rights, safety, or welfare of the subject occur.

[0289] Immunogenicity Assessment: This study will explore humoral and cell-mediated immune responses in blood samples collected at baseline (i.e., pre-dose screening and day 0) and at weeks 6, 9, 11, 26, and 52. Tissue samples will be collected at baseline and as clinically indicated during the study. Tests may include, but are not limited to, ELISA, ELISpot, flow cytometry, immunohistochemistry (IHC), nanostring analysis on peripheral blood samples and / or excised tissues (at study entry and recurrence, if possible).

[0290] Virological Evaluation: The study will evaluate the presence of HPV-6 / 11 DNA in tissue samples and peripheral blood before and after study treatment as described.

[0291] MiRNA profiling can be performed using tissue obtained at screening, day 0, and recurrence. Evaluation of day 0 and screening samples will explore predictive algorithms for response to treatment with INO-3107. Samples evaluated from recurrence will illustrate how changes in miRNA profiles can be associated with the likelihood of recurrence.

[0292] Virological Evaluation: The study will assess the presence of HPV-6 / 11 DNA in tissue samples and peripheral blood before and after study treatment as described.

[0293] Important hiring criteria: · Histologically documented HPV-6 or HPV-11 positive respiratory papilloma or documentation of low-risk positive HPV. ·Requirement for frequent RRP interventions to remove or excise respiratory papillomas, defined as at least two RRP surgical (including laser) interventions in the year prior to and including day 0. Must be an appropriate candidate for future surgical intervention according to the investigator's judgment and RRP staging score. Adequate bone marrow, liver, and kidney function as defined by: ANC (absolute neutrophil count) ≥ 1000 cells / mm 3 , platelets ≥50,000 / mm 3 , hemoglobin ≥ 9 g / dL, total serum bilirubin concentration within 1.5 × upper limit of normal (ULN), AST and ALT within 1.5 × ULN, serum creatinine ≤ 1.5 × ULN. Male participants agree not to father a child and female participants, if of childbearing potential, agree not to become pregnant during the study.

[0294] Key exclusion criteria: Recipient of therapy (excluding surgery or ablation) for RRP disease, including but not limited to antiviral (including cidofovir), radiation, chemotherapy, antiangiogenic therapy (including bevacizumab), prophylactic HPV vaccination (including Gardasil), or therapy with an experimental agent within 3 months prior to Day 0. Ongoing or recent (within 1 year) evidence of an autoimmune disease requiring treatment with systemic immunosuppressive therapy, with the exception of vitiligo, resolved childhood asthma, type 1 diabetes, residual hypothyroidism requiring hormone replacement only, or psoriasis not requiring systemic treatment. Diagnosis of immunodeficiency or treatment with systemic immunosuppressive therapy, including systemic corticosteroids, within 28 days prior to the first dose of study treatment. · High risk of bleeding or requiring the use of anticoagulants to manage a known bleeding diathesis. Recipient of any live viral vaccine within 4 weeks prior to the first dose of study treatment or any non-live viral vaccine within 2 weeks prior to the first dose of study treatment. History of any clinically significant, medically unstable illness that, in the investigator's judgment, would jeopardize the participant's safety, interfere with study or endpoint assessment, or otherwise affect the validity of the study results (this may include chronic renal failure, myocardial ischemia or infarction, New York Heart Association (NYHA) Class III / IV heart disease, any cardiac pre-excitation syndrome (such as Wolff-Parkinson-White, cardiomyopathies, or clinically significant arrhythmias), current malignancy except for treated basal or squamous cell skin cancer, prostate cancer, or primary cervical cancer, HIV, or drug or alcohol dependence that may affect the ability to mount an immune response to the study therapy). Fewer than two acceptable sites are available for IM injection considering the deltoid and anterolateral quadriceps muscles [the following are unacceptable sites: tatoes, keloids, or hypertrophic scars located within 2 cm of the intended treatment site; cardioverter-defibrillators or pacemakers (to prevent life-threatening arrhythmias) located ipsilateral to the deltoid injection site (unless deemed acceptable by a cardiologist); metallic implants or implantable medical devices within the intended treatment site]. ·Pregnant or currently breastfeeding.

[0295] Clinical Trial Procedure: INO-3107 drug product is the test article used in this study. It contains a DNA plasmid (pGX3024) for the expression of the E6 and E7 proteins of the HPV11 and HPV6 genes and an expression plasmid (pGX6010) expressing the human IL-12 subunit. INO-3107 drug product is a clear, colorless solution containing 6.25 mg total plasmid / mL (6 mg / mL pGX3024, 0.25 mg / mL pGX6010) in 150 mM sodium chloride and 15 mM sodium citrate (pH 7). A minimum volume of 1 mL will be filled into a 2 mL clear glass vial for intramuscular injection.

[0296] Subjects will receive a single 6.25 mg injection of INO-3107 drug product intramuscularly on Day 0, Week 3, Week 6, and Week 9, followed by EP using a CELLECTRA® 2000 EP device.

[0297] The INO-3107 drug product is injected using a 2-inch needle and a CELLECTRA® 2000EP device. The 2-inch needles used in this study are one of two types: a standard 2-inch, 21-gauge injection needle or a 2-inch, 21-gauge injection side port needle (see U.S. Patent Application Publication No. 2023 / 0017972, incorporated herein by reference). The side port needle contains a sealed tip and a port along the side that allows for greater fluid distribution within the EP region of the intramuscular array. Twenty-one subjects will receive INO-3107 via a 2-inch, 21-gauge injection needle, and 11 subjects will receive INO-3107 via a 2-inch, 21-gauge side port needle.

[0298] The analysis populations were as follows: - The intention-to-treat (ITT) population includes all eligible subjects. - The modified intention-to-treat (mITT) population includes all subjects who receive at least one dose of INO-3107 medication. The per-protocol (PP) population will include subjects who receive all doses of INO-3107 drug and who have no protocol violations. Subjects excluded from the PP population will be identified and documented prior to study database lock. - The safety analysis set will include all subjects who received at least one dose of INO-3107 drug.

[0299] Peripheral Blood Immunogenicity Assessment: Whole blood and serum samples will be obtained at baseline (day 0, pre-screening and dosing), and at weeks 6, 9, 11, 26, and 52. Peripheral blood mononuclear cells (PBMCs) will be isolated from the whole blood samples. Assessment of cellular immune activity may be performed through gene expression, interferon-γ enzyme-linked immunosorbent spot (IFN-γ ELISpot), and flow cytometry assays. Additional assessment of cellular immune activity may be performed through flow cytometry to perform a lytic granule loading assay. The lytic granule loading assay may examine the following external cellular markers: CD3, CD4, CD8 (T cell identification), Ki67, CD137, CD38, and CD69 (T cell activation markers), as well as PD-1 (exhaustion / activation marker), Tim-3, and Lag-3. The lytic granule loading assay may further analyze the following intracellular markers: Granzyme A, Granzyme B, Granulysin and Perforin (proteins involved in lytic degranulation and cytotoxic potential).

[0300] MiRNA profiling will be performed using plasma obtained at screening, day 0, and week 6. Evaluation of day 0 and screening samples will explore predictive algorithms for response to treatment with INO-3107. Samples evaluated from week 6 will illustrate how changes in miRNA profiles may be associated with ultimate treatment success or failure.

[0301] A standard binding ELISA can be performed to measure the anti-HPV-6 / 11 antibody response induced by the INO-3107 drug product.

[0302] HPV-6 / 11 Testing: Whole blood will be collected on day 0 and before dosing at weeks 6, 11, 26 and 52 for measurement of cfHPV DNA-6 / 11.

[0303] Description of statistical methods:

[0304] Primary Analysis: The primary analysis of this study is a safety analysis of treatment-emergent adverse events (TEAEs) and clinically significant changes in safety laboratory parameters from baseline. TEAEs are defined for this study as any AE occurring after day 0 of study drug administration (IM+EP) through 30 days after the last dose. All TEAEs will be summarized in the safety population by frequency. These frequencies are presented overall, by system organ class, and by preferred term, and by the proportion of subjects affected. Additional frequencies are presented in terms of greatest severity and strongest relationship to study treatment. Multiple occurrences of the same AE will be counted only once, following a worst-case approach for severity and relationship to study treatment. The primary summary of safety data is based on TEAEs. In this summary, event frequencies for preferred time periods will be calculated with 95% confidence intervals using the Clopper-Pearson exact method. Individual summaries are based on events occurring within 7 days of any dose, regardless of when they occurred. AEs and SAEs that are not TEAEs or serious TEAEs are presented in the list.

[0305] For AE data, a partial start date is imputed to the date of treatment whenever a portion of the date matches that of the study treatment, in order to conservatively report the event as occurring during treatment. Otherwise, it is imputed to the earliest date that matches the partial date. Completely missing onset dates are imputed as the date of treatment. A partial stop date is considered the latest possible date that matches the partial date.

[0306] The duration of an adverse event will be calculated as (stop date - start date) + 1.

[0307] Laboratory response variables will be summarized descriptively by time point and as change from baseline with 95% confidence intervals. Shifts from baseline by CTCAE will also be shown. Laboratory values ​​considered clinically significant will be listed.

[0308] All safety analyses are performed on subjects in the safety analysis set.

[0309] The analysis is summarized and presented by number of previous surgical interventions (≦2, 3-5, and ≧6) and overall number.

[0310] Secondary analyses:

[0311] Efficacy: The frequency of RRP surgical interventions one year after the first dose of INO-3107 drug will be summarized descriptively using the mean fold change and 95% t-distribution-based CI compared to the one-year frequency before dosing on Day 0. Assessment of change in RRP staging score from pre-baseline dosing to post-each dose will be analyzed. Median changes and associated 95% confidence intervals will be calculated.

[0312] The relationship between efficacy endpoints versus miRNA outcomes is examined. The relationship is examined using a regression model that models the endpoint outcomes versus miRNA outcomes as regressor variables.

[0313] Inter-surgery intervals will also be summarized. Analyses will be summarized and presented by number of prior surgical interventions (≦2, 3-5, and ≧6) and overall number. Efficacy analyses using the mITT population will be performed. The per-protocol population will also be used for supportive analyses.

[0314] Immunogenicity: Interferon-γ ELISpot increase from baseline and magnitude of flow response will be summarized. Median increase and associated 95% confidence interval will be calculated. Change from baseline in magnitude of tumor tissue response will be summarized. Mean increase and associated 95% t-distribution-based confidence interval will be calculated. Valid samples for statistical analysis purposes will be collected within 7 days of the designated visit. Baseline is defined as the last measurement before the first treatment administration. The mITT population will be used for immunogenicity analysis. Analysis will be summarized and presented by number of prior surgical interventions (≦2, 3-5, and ≧6) and overall number.

[0315] For immunological evaluation, PBMCs were cryopreserved overnight in cell culture medium, then harvested, centrifuged, washed, and resuspended the following day. After counting, 1 × 106 PBMCs were seeded per well in R10 medium in a 96-well plate. Cells were stimulated for 5 days with a combination of 15-mer peptides overlapping by eight amino acid residues corresponding to the entire E6 and E7 antigens of HPV-6 and HPV-11. Assay background noise was addressed by using a control irrelevant peptide stimulation (OVA) during the incubation period, and its output was subtracted from the HPV-stimulated cells as a nonspecific signal. Concanavalin A (Sigma-Aldrich) was used as a positive control. All peptides were resuspended in dimethyl sulfoxide. No exogenous cytokines or costimulatory molecules were added during the culture period. At the end of the 5-day incubation period, plates were centrifuged to pellet the cells, samples were washed with phosphate-buffered saline, and cells were stained for intracellular and extracellular markers of T cell identity, activation, and lytic potential. Cell preparations were acquired using a Fortessa cytometer equipped with BD FACSDiva software (BD Biosciences).

[0316] RRP Severity Scoring Staging System

[0317] To standardize and improve the accuracy of grading papillomas identified during each flexible laryngoscopy, a modified stage score was created based on the RRP staging system originally published by Derkay et al., Laryngoscope, 1998, 108:935-937. Lesions were graded as superficial (<3 mm), protruding (4-8 mm), or bulky (>8 mm). The true vocal cord (using both the free margin and superior surface as distinct locations), ventricle, and false vocal cord were divided into three subsections from anterior to posterior. The epiglottis and posterior glottis were separated left and right. The epiglottic folds were clearly delineated, along with the left and right laryngeal tendons. Additionally, a definition and grading score was created for the widespread "papilloma carpet." When surgical endoscopy was performed, the same laryngeal assessment modality was utilized, along with evaluation of the subglottis (divided into left and right and anterior, lateral, and posterior segments) and trachea (divided into upper, middle, and distal segments). Staging assessment was further expanded to include a symptom score that identified the presence of voice changes, shortness of breath, dyspnea, and symptoms such as cough, throat clearing, burn, or pain. Laryngoscopy was performed, and severity scores were retrospectively verified by two independent otolaryngologists. Patients underwent office laryngoscopy and staging at screening and at weeks 6, 11, 26, and 52.

[0318] Exploratory analysis:

[0319] Efficacy: HPV clearance will be summarized and the proportion of subjects clearing HPV-6 / 11 compared to baseline in resected tumor tissue will be calculated. The relationship between cfHPV DNA 6 / 11 before and after INO-3107 treatment as a correlate of disease burden and clinical outcomes in RRP patients will be examined using regression models. Analyses will be summarized and presented by number of prior surgical interventions (≤2, 3-5, and ≥6) and overall number.

[0320] Immunogenicity: Post-baseline ELISA titers will be analyzed by geometric means. Changes in gene expression from baseline in peripheral blood will be summarized. Analyses will be summarized and presented by number of previous surgical interventions (≤2, 3-5, and ≥6) and overall number.

[0321] Interim Results

[0322] INO-3107 was evaluated in a Phase 1 / 2 open-label, multicenter study to assess its safety, tolerability, immunogenicity, and efficacy in 32 participants with HPV6- and / or HPV11-associated RRP (NCT:04398433).

[0323] Interim Results for Study Participants Receiving INO-3107 Medications

[0324] Demographic and baseline characteristics for the 32 study participants who received INO-3107 medication are summarized in Table 2.

[0325] [Table 2] HPV = human papillomavirus

[0326] Safety (primary endpoint). Safety data are shown in Table 3. Figure 19 shows TEAEs occurring in two or more patients. The most frequently reported TEAEs were related to administration (injection site or procedural pain). 17 / 20 TEAEs (85.0%) occurred within 7 days of administration.

[0327] [Table 3] AE = adverse event, SAE = serious adverse event, TEAE = treatment-emergent adverse event

[0328] Efficacy (Secondary Endpoint). Reduction in RRP Procedures. Overall, 26 patients (81.3%) had a reduction in surgical interventions in the year after INO-3107 administration compared to the previous year, of which 9 (28.1%) required no surgical intervention (Figure 20). The median change in number of procedures from the year before baseline to the subsequent year was -3 (95% CI: -3 to -2). The number of procedures in the previous year was 4 (95% CI: 3 to 5), and the number of procedures in the following year was 1 (95% CI: 1 to 3).

[0329] Efficacy (Secondary Endpoint): Improvement in RRP Staging Assessment Score. Improvement was seen in the mean total area score at week 52 (10.7 points lower than baseline), while mean symptom scores were similar at week 52 compared to baseline (Figure 21).

[0330] Interim results for study participants who received INO-3107 medication via standard needle injection

[0331] Demographic and baseline characteristics for the 21 study participants who received INO-3107 via standard needle injection are summarized in Table 4. In total, 16 (76.2%) patients had adult-onset RRP, and 5 (23.8%) had juvenile-onset RRP. The median (range) age was 48.0 (25-67) years, and 16 (76.2%) patients were male. The median number of surgical procedures in the year preceding Dose 1 (Day 0) was 4 (range, 2-7). All 21 patients received four doses of INO-3107, and 20 patients had complete 52-week follow-up data from Day 0 at the time of the data cutoff for the interim outcome. One patient was considered lost to follow-up but was included in the analysis because the patient subsequently had a follow-up visit after the 52-week visit. Non-surgical interventions were reported in one other patient who received bevacizumab every 3 weeks during the 52-week follow-up period.

[0332] [Table 4] HPV = human papillomavirus, mITT = modified intention to treat, RRP = recurrent respiratory papillomatosis * Of the 18 patients with available data, tissue samples from 19 patients were sent for HPV genotyping at screening, which was unsuccessful in one patient.

[0333] Safety (Primary Endpoint). Safety data are shown in Table 5. Overall, 15 (71.4%) patients had ≥1 TEAE during the study, of which 9 (42.9%) had a TEAE considered related to study treatment. Most TEAEs were Grade 1 in severity. The most frequently reported treatment-related TEAEs were injection site / procedural pain (n=8, 38.1%), fatigue (n=2, 9.5%), and injection site swelling (n=2, 9.5%). All treatment-related TEAEs were Grade 1 in severity.

[0334] Grade 3 TEAEs were reported in 3 (14.3%) patients, including one event each (4.8%) of aspartate aminotransferase increased, increased diastolic blood pressure, and syncope, none of which were considered treatment-related. There were no grade ≥4 TEAEs, TEAEs leading to treatment discontinuation or death, UADEs, or treatment-related SAEs.

[0335] [Table 5] AE = adverse event, MedDRA = ICH International Medical Dictionary, PT = preferred duration, SAE = serious adverse event, SOC = system organ class, TEAE = treatment-emergent adverse event, UADE = unexpected (serious) adverse device effect.

[0336] Efficacy (Secondary Endpoint). Reduction in RRP Surgical Procedures. Of 21 patients, 16 (76.2%) reduced surgical interventions in the year after INO-3107 administration compared to the previous treatment-free year, including 6 (37.5%) who did not require surgical intervention during the study period (Figure 22). The median change in surgical procedure number from the year before baseline (Day 0) to the year after Day 0 was -3 (less than 3), with a 95% CI of -3 to -1. The reduction in surgical procedure number was not significant in patients who had two surgeries in the year before Day 0 compared with patients who had more than two surgeries in the year before Day 0. Of 16 patients who had three or more surgeries in the previous year, 14 (87.5%) reduced the number of surgeries during the study period. Figures 24A-24D show exemplary images of responders (Figures 24A, 24B) and non-responders (Figures 24C, 24D) at baseline (Figures 24A, 24C) and after treatment with INO-3107 (Figures 24B, 24D).

[0337] Efficacy (Secondary Endpoint): Improvement in RRP Staging Assessment Score. There was an improvement in the total clinical score (total site score + total symptom score) from baseline to week 52 / end of study, which was 10.0 points lower than baseline (Table 6). The mean total site score was reduced by 9.7 points, while the mean symptom score was similar at week 52 / end of study compared to baseline (Table 6).

[0338] [Table 6] * The total clinical score is the sum of the total area and total symptom scores. CI = confidence interval, EOS = end of study, mITT = modified intention to treat, RRP = recurrent respiratory papillomatosis.

[0339] Immunology (Secondary Endpoint) Cellular Immune Responses. Increases in INO-3107-specific CD4 cells expressing activation markers (CD38) and HPV-6 and HPV-11 E6-specific CD8 cells expressing a combination of activation markers (CD38) and lytic potential (granzyme A, granzyme B, perforin) were observed after vaccination (Figures 19A-19D). "Peak" refers to the highest T cell response recorded during the study beyond day 0. For CD8 T cell activity, 18 / 21 (85.7%) patients demonstrated antigen-specific cellular activity above pre-dose levels, as demonstrated by flow cytometry. This was similar for CD4 T cells in 18 / 21 (85.7%) patients.

[0340] The proportions of antigen-specific CD8 and CD4 cells expressing activation markers and CD8 cells expressing lytic markers were similar for HPV-6 and HPV-11. Increased frequencies of activated INO-3107-specific CD4 and CD8 cells were observed in week 52 samples compared to baseline, indicating a sustained cellular "memory" response.

[0341] Interim results from this Phase 1 / 2 study suggest that INO-3107, administered by intramuscular injection via a standard needle followed by EP, is tolerable, immunogenic, and provides clinical benefit to adult patients with RRP. Treatment with INO-3107 resulted in the induction of HPV-6- and HPV-11-specific T cell responses. Flow cytometry confirmed the expression of activation markers, concomitant with the synthesis of granzymes and perforin, on CD8+ T cells, suggesting that INO-3107 induced a proinflammatory immune response involving T cells with characteristics of highly activated cytotoxic lymphocytes. Six of the 21 treated patients (28.6%) did not require surgical intervention during the 52-week study period.

[0342] Of the initial cohort of 21 patients, 15 (71.4%) patients experienced ≥1 TEAE, most of which were Grade 1 TEAEs, and 3 (14.3%) experienced Grade 3 TEAEs (non-treatment related). The most commonly reported TEAE was injection site or procedural pain (n=8, 38.1%). Sixteen patients underwent fewer surgical interventions in the year after INO-3107 administration, a median reduction of 3 interventions compared to the previous year. Modified Derkay severity scores showed improvement from baseline to Week 52. INO-3107 induced durable cellular responses to HPV-6 and HPV-11, with increased activated CD4 and CD8 T cells and CD8 cytolytic capacity.

[0343] Interim results for study participants who received INO-3107 medication via side-port needle injection

[0344] Safety (Primary Endpoint). INO-3107 was well tolerated, and all 11 patients completed study follow-up. Treatment-emergent adverse events (TEAEs) observed in the study were generally low-grade; 46% of patients experienced at least one TEAE, and 36% of patients reported at least one related TEAE, all of which were Grade 1 or 2. The only AEs reported by more than one patient were injection site pain (2 patients) and headache (2 patients).

[0345] Efficacy (Secondary Endpoint). The second cohort (study participants receiving INO-3107 via side-port needle injection) (n=11) demonstrated statistical significance based on the clinical endpoint of a reduction in the number of surgical interventions in the year after the first administration of INO-3107 compared with the year before treatment, with a median reduction of three surgical interventions (95% confidence interval 1, 4). In addition, 10 of 11 patients (90.9%) demonstrated a reduction in surgical interventions in the year after the first administration of INO-3107 compared with the number of surgeries in the year before study. Of these 10 patients, four did not require surgical intervention during the study period. In the year before treatment, the range of surgical interventions for these 11 patients was two to eight, with a median of five.

[0346] Immunology (Secondary Endpoint) Cellular immune response. Treatment with INO-3107 drug induced cellular responses to both HPV6 and HPV11, including both activated CD4 and activated lytic CD8 T cells. T cell responses were also observed at week 52, 43 weeks after the last treatment with INO-3107, indicating a sustained cellular memory response.

[0347] Interim results from this Phase 1 / 2 study suggest that the INO-3107 formulation administered by IM injection via a side-port needle, followed by EP, is well tolerated and immunogenic. Safety and efficacy results for study participants who received the INO-3107 drug product via side-port needle injection are consistent with those for study participants who received the INO-3107 formulation via standard needle injection.

[0348] Sequences and Sequence Identifiers >pGX3024 Insertion of only the amino acid sequence without the IgE leader sequence <SEQ ID NO: 1> ESKDASTSATSIDQLCKTFNLSLHTLQIQCVFCRNALTTAEIYAYAYKNLKVVWRDNFPFAACACCLELQGKINQYRHFNYAAYAPTVEEETNEDILKVLIRCYLCHKPQCEIEKLKHILGKARFIKLNNQRKGRCLHCWTTCM EDLLPRGRKRRSGSGATNFSLLKQAGDVEENPGPHGRLVTLKDIVLDLQPPDPVGLHAYEQLEDSSEDEVDKVDKQDSQPLTQHYQILTCCCGCDSNVRLVVECTDGDIRQLQDLLLGTLNIVCPICAPKPRGRKRRSGSGATNF SLLKQAGDVEENPGPESANASTSATTIDQLCKTFNLSMHTLQINCVFCKNALTTAEIYSYAYKQLKVLFRGGYPYAACACCLEFHGKINQYRHFDYAGYATTVEEETKQDILDVLIRCYLCHKPQCEVEKVKHILTKARFIKLNC TRKGRCLHCWTTCMEDMLPRGRKRRSGSGATNFSLLKQAGDVEENPGPHGRHVTLKDIVLDLQPPDPVGLHAYEQLVDSSEDEVDEVDGQDSQPLKQHYQIVTCCCGCDSNVRLVVQCTETDIREVQQLLLGTLNIVCPICAPKT >pGX3024_IgE insert only without leader sequence <SEQ ID NO: 2> >pGX3024_complete sequence<SEQ ID NO:3> Accession No. 4: pGX6010 Full-Length Sequence and Annotation:

Table 7

Claims

1. 1. A method for treating or preventing recurrent respiratory papillomatosis (RRP) in a subject in need thereof, the method comprising administering to the subject an effective amount of a pharmaceutical composition comprising a nucleic acid molecule encoding a human papillomavirus (HPV) antigen comprising an HPV6 antigenic domain and an HPV11 antigenic domain, thereby treating or preventing RRP, wherein the HPV6 antigenic domain is an HPV6 E6-E7 fusion antigen and the HPV11 antigenic domain is an HPV11 E6-E7 fusion antigen.

2. 10. The method of claim 1, wherein the administration comprises intradermal or intramuscular injection.

3. The method of claim 1 or 2, wherein the administering further comprises electroporation.

4. 10. The method of any one of claims 1 to 9, wherein the method reduces the number of RRP surgical interventions in the 52 weeks after the administration of the pharmaceutical composition compared to the number of RRP surgical interventions in the 52 weeks before the administration of the pharmaceutical composition.

5. 10. The method of claim 1, wherein administration of the pharmaceutical composition reduces RRP surgical interventions in at least about 80% of a population of human subjects diagnosed with RRP in the 52 weeks following administration compared to the number of RRP surgical interventions in the 52 weeks prior to administration of the pharmaceutical composition.

6. 10. The method of claim 1, wherein the administration comprises intramuscular injection with a standard needle.

7. 7. The method of claim 6, wherein administration of the pharmaceutical composition reduces RRP surgical interventions in the 52 weeks after administration of the pharmaceutical composition compared to the number of RRP surgical interventions in the 52 weeks prior to administration of the pharmaceutical composition in at least about 76% of a population of human subjects diagnosed with RRP.

8. 7. The method of claim 6, wherein administration of the pharmaceutical composition reduces the median number of RRP surgical interventions by about 3 in the 52 weeks after administration of the pharmaceutical composition compared to the number of RRP surgical interventions in the 52 weeks prior to administration of the pharmaceutical composition in a population of human subjects diagnosed with RRP.

9. 7. The method of claim 6, wherein the method increases the number of HPV-6-specific activated CD4 cells and HPV-11-specific activated CD4 cells that express CD38 52 weeks after said administration of the pharmaceutical composition compared to the number of HPV-6-specific activated CD4 cells and HPV-11-specific activated CD4 cells that express CD38 52 weeks before said administration of the pharmaceutical composition.

10. 7. The method of claim 6, wherein administration of the pharmaceutical composition increases the number of HPV-6-specific activated CD4 cells and HPV-11-specific activated CD4 cells that express CD38 52 weeks after administration of the pharmaceutical composition compared to the number of HPV-6-specific activated CD4 cells and HPV-11-specific activated CD4 cells that express CD38 52 weeks prior to administration of the pharmaceutical composition in at least about 85% of a population of human subjects diagnosed with RRP.

11. The method of claim 6, wherein the method increases the number of HPV6 and HPV11 E6-specific CD8 cells that express CD38, granzyme A, granzyme B and perforin 52 weeks after administration of the pharmaceutical composition compared to the number of HPV6 and HPV11 E6-specific CD8 cells that express CD38, granzyme A, granzyme B and perforin 52 weeks before administration of the pharmaceutical composition.

12. The method of claim 6, wherein administration of the pharmaceutical composition increases the number of HPV6 and HPV11 E6-specific CD8 cells that express CD38, granzyme A, granzyme B and perforin 52 weeks after administration of the pharmaceutical composition compared to the number of HPV6 and HPV11 E6-specific CD8 cells that express CD38, granzyme A, granzyme B and perforin 52 weeks before administration of the pharmaceutical composition in at least about 85% of a population of human subjects diagnosed with RRP.

13. The method of any one of claims 1 to 3, wherein the administration comprises intramuscular injection with a side port needle.

14. 14. The method of claim 13, wherein administration of the pharmaceutical composition reduces the number of RRP surgical interventions in the 52 weeks after administration compared to the number of RRP surgical interventions in the 52 weeks prior to administration in at least about 90% of a population of human subjects diagnosed with RRP.

15. 14. The method of claim 13, wherein the method increases the number of HPV6-specific activated CD4 and HPV11-specific activated CD4 and activated lytic CD8 T cells.

16. 7. The method of claim 6, wherein the method improves the RRP stage rating score compared to baseline.

17. 1. Use of a pharmaceutical composition comprising a nucleic acid molecule encoding a human papillomavirus (HPV) antigen comprising an HPV6 antigenic domain and an HPV11 antigenic domain in treating or preventing recurrent respiratory papillomatosis (RRP) in a subject in need thereof, wherein the HPV6 antigenic domain is an HPV6 E6-E7 fusion antigen and the HPV11 antigenic domain is an HPV11 E6-E7 fusion antigen.

18. 1. Use of a nucleic acid molecule encoding a human papillomavirus (HPV) antigen comprising an HPV6 antigenic domain and an HPV11 antigenic domain in the manufacture of a pharmaceutical composition for treating or preventing recurrent respiratory papillomatosis (RRP) in a subject in need thereof, wherein the HPV6 antigenic domain is an HPV6 E6-E7 fusion antigen and the HPV11 antigenic domain is an HPV11 E6-E7 fusion antigen.

19. 19. The use according to claim 17 or 18, wherein the pharmaceutical composition is formulated for administration by intradermal or intramuscular injection.

20. 19. The use according to claim 17 or 18, wherein the pharmaceutical composition is formulated for administration by intradermal or intramuscular injection using electroporation.

21. 21. The use of any one of claims 17 to 20, wherein the pharmaceutical composition reduces the number of RRP surgical interventions in a 52 week period after administration compared to the number of RRP surgical interventions in a 52 week period before administration.

22. 22. The use of any one of claims 17 to 21, wherein the pharmaceutical composition reduces RRP surgical interventions in a 52 week period after administration compared to the number of RRP surgical interventions in a 52 week period prior to administration in at least about 80% of a population of human subjects diagnosed with RRP.

23. The use according to any one of claims 17 to 22, wherein the pharmaceutical composition is administered by intramuscular injection with a standard needle.

24. 24. The use of claim 23, wherein the pharmaceutical composition reduces RRP surgical interventions in the 52 weeks following administration compared to the number of RRP surgical interventions in the 52 weeks prior to administration in at least about 76% of a population of human subjects diagnosed with RRP.

25. 25. The use of claim 24, wherein the pharmaceutical composition reduces the median number of RRP surgical interventions by about 3 in a 52-week period after administration compared to the number of RRP surgical interventions in a 52-week period prior to administration in a population of human subjects diagnosed with RRP.

26. 24. The use of claim 23, wherein the pharmaceutical composition increases the number of HPV-6-specific activated CD4 cells and HPV-11-specific activated CD4 cells that express CD38 52 weeks after administration compared to the number of HPV-6-specific activated CD4 cells and HPV-11-specific activated CD4 cells that express CD38 52 weeks before administration.

27. 24. The use of claim 23, wherein the pharmaceutical composition increases the number of HPV-6-specific activated CD4 cells and HPV-11-specific activated CD4 cells that express CD38 52 weeks after administration compared to the number of HPV-6-specific activated CD4 cells and HPV-11-specific activated CD4 cells that express CD38 52 weeks before administration in at least about 85% of a population of human subjects diagnosed with RRP.

28. The use of claim 23, wherein the pharmaceutical composition increases the number of HPV6 and HPV11 E6-specific CD8 cells that express CD38, granzyme A, granzyme B and perforin 52 weeks after administration compared to the number of HPV6 and HPV11 E6-specific CD8 cells that express CD38, granzyme A, granzyme B and perforin 52 weeks before administration.

29. The use of claim 23, wherein the pharmaceutical composition increases the number of HPV6 and HPV11 E6-specific CD8 cells that express CD38, granzyme A, granzyme B and perforin 52 weeks after administration compared to the number of HPV6 and HPV11 E6-specific CD8 cells that express CD38, granzyme A, granzyme B and perforin 52 weeks before administration in at least about 85% of a population of human subjects diagnosed with RRP.

30. The use according to any one of claims 17 to 20, wherein the pharmaceutical composition is administered by intramuscular injection with a side port needle.

31. 31. The use of claim 30, wherein the pharmaceutical composition reduces the number of RRP surgical interventions in a 52 week period after administration compared to the number of RRP surgical interventions in a 52 week period prior to administration in at least about 90% of a population of human subjects diagnosed with RRP.

32. The use according to claim 30, wherein the pharmaceutical composition increases the number of HPV6-specific activated CD4 and HPV11-specific activated CD4 and activated lytic CD8 T cells.

33. 24. The use of claim 23, wherein the pharmaceutical composition improves the RRP stage assessment score compared to baseline.

34. 10. The method or use according to any one of claims 1 to 9, wherein the RRP is juvenile onset RRP or adult onset RRP.

35. The HPV antigen is the amino acid sequence of SEQ ID NO: 1; the amino acid sequence of SEQ ID NO: 11; an amino acid sequence that is at least 95% homologous to SEQ ID NO: 1; or 10. The method or use of any one of claims 1 to 9, comprising an amino acid sequence that is at least 95% homologous to SEQ ID NO:

11.

36. The nucleic acid molecule encoding an HPV antigen is a nucleotide sequence that is at least 95% homologous to SEQ ID NO:2; a nucleotide sequence that is at least 95% homologous to SEQ ID NO: 12; the nucleotide sequence of SEQ ID NO: 2, or 10. The method or use according to any one of claims 1 to 9, comprising the nucleotide sequence of SEQ ID NO:

12.

37. 10. The method or use of claim 1, wherein the nucleic acid sequence encoding the HPV11 antigenic domain is located 5' to the nucleic acid sequence encoding the HPV6 antigenic domain.

38. 2. The method or use of claim 1, wherein the HPV6 antigenic domain and the HPV11 antigenic domain are separated by one or more post-translational cleavage sites, one or more translational skip sites, or both.

39. 2. The method or use of claim 1, wherein the HPV6 E6 antigenic domain and the HPV6 E7 antigenic domain are separated by one or more post-translational cleavage sites, one or more translational skip sites, or both.

40. 2. The method or use of claim 1, wherein the HPV11 E6 antigenic domain and the HPV11 E7 antigenic domain are separated by one or more post-translational cleavage sites, one or more translational skip sites, or both.

41. 10. The method or use according to any one of claims 1 to 9, wherein the nucleic acid molecule encoding an HPV antigen is an expression vector, optionally a plasmid.

42. 42. The method or use of claim 41 , wherein the expression vector comprises the nucleotide sequence of SEQ ID NO:

3.

43. 10. The method or use of claim 1, wherein the pharmaceutical composition further comprises an adjuvant.

44. 44. The method or use of claim 43, wherein the adjuvant comprises interleukin 12 (IL12), a nucleic acid molecule encoding IL12, a nucleic acid molecule comprising a nucleotide sequence encoding the p35 subunit of IL12, a nucleic acid molecule comprising a nucleotide sequence encoding the p40 subunit of IL12, a nucleic acid molecule comprising a nucleotide sequence encoding the p35 subunit of IL12, and a nucleic acid molecule comprising a nucleotide sequence encoding the p40 subunit of IL12, or a nucleic acid molecule comprising nucleotide sequences encoding the p35 subunit of IL12 and the p40 subunit of IL12.

45. 45. The method or use of claim 44, wherein the nucleic acid molecule encoding IL12, a p35 subunit, or a p40 subunit is an expression vector.

46. 46. ​​The method of claim 45, wherein the expression vector is pGX6010.

47. 10. The method or use of any one of claims 1 to 9, wherein the pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

48. 48. The method or use of claim 47, wherein the pharmaceutically acceptable excipient comprises a buffer.

49. 49. The method or use of claim 48, wherein the buffer is a saline-sodium citrate buffer.

50. 10. The method or use of any one of claims 1 to 9, wherein the pharmaceutical composition comprises 6 mg of an expression vector encoding an HPV antigen per milliliter of buffer solution.

51. 10. The method or use of claim 1, wherein the pharmaceutical composition comprises 0.25 mg of vector encoding the p35 subunit of IL12, the p40 subunit of IL12, or both, per milliliter of buffer solution.

52. 10. The method or use of claim 1, wherein the pharmaceutical composition comprises 6 mg of pGX3024 per milliliter of buffer and 0.25 mg of pGX6010 per milliliter of buffer.

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