Methods of treating high-grade squamous intraepithelial lesions (HSIL)

Abstract

The present invention describes a method of treating a high level of squamous intraepithelial lesion (HSIL) associated with human papilloma virus (HPV) type 16 or HPV type 18 of the cervix. The described methods include assessing the presence or absence of miRNA and isomiRs thereof in one or more biological samples.

Description

[0001] Cross-reference to related applications

[0002] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 380,861, filed Oct. 25, 2022, the disclosure of which is hereby incorporated by reference in its entirety.

[0003] Sequence listing

[0004] This application contains a Sequence Listing which is submitted electronically in XML format and is hereby incorporated by reference in its entirety. The XML copy, created on Oct. 19, 2023, is named 104409_000901_SequenceListing.xml and is 6,730 bytes in size. Technical Field

[0005] Methods of treating high-grade squamous intraepithelial lesions (HSIL) associated with human papillomavirus (HPV) type 16 or HPV type 18 of the cervix are provided. The methods provided include assessing the presence of miRNAs and their isomiRs in one or more biological samples. Background Art

[0006] MicroRNA (miRNA) is a class of endogenous small non-coding RNAs, typically ranging from 19-24 nucleotides. When loaded into micro-ribonucleoprotein (miRNP) or RNA-induced silencing complex (RISC), miRNA binds to the miRNA recognition element (MRE) on the mRNA target and exerts gene regulatory effects through post-transcriptional and / or translational mechanisms.

[0007] For the purpose of predicting the response to HPV type-specific treatment of HPV16- or HPV18-associated HSIL, miRNA profiles are needed. Summary of the Invention

[0008] The present invention provides a method for treating high-grade cervical intraepithelial neoplasia (HSIL) associated with human papillomavirus (HPV) type 16 or HPV type 18, the method comprising: (a) assessing the presence of a set of miRNAs and their isomiRs consisting of: hsa.miR.375.3p, hsa.miR.425.5p, hsa.miR.193a.5p, hsa.miR.199b.5p, hsa.miR.365a.3p, hsa.miR.223.5p, hsa.miR.148b.3p, hsa.miR.143.3p, hsa.miR.941, hsa.miR.340.5p, hsa.miR.151a.3p, hsa.miR.23a.3p in one or more biological samples from a subject having HSIL associated with HPV type 16 or HPV type 18; (b) calculating the normalized level of each of these miRNAs and their isomiRs in the set; (c) determining a miRNA signature based on the normalized levels of the miRNAs and their isomiRs in the set; (d) classifying the subject as biomarker positive or biomarker negative based on the miRNA signature; and (e) if the subject is classified as biomarker positive, administering to the subject a therapeutically effective amount of VGX-3100.

[0009] In certain embodiments, the presence of the miRNA is determined by RNA sequencing.

[0010] In further embodiments, the biological sample is a plasma sample.

[0011] In still further embodiments, the plasma sample is isolated from the subject prior to administration of VGX-3100.

[0012] In additional embodiments, the HPV type 16 or HPV type 18 associated HSIL of the cervix is determined by biopsy.

[0013] In some embodiments, VGX-3100 is administered to the subject by intramuscular injection followed by electroporation. In certain embodiments, VGX-3100 is administered to the subject at a dose of 6 mg. In further embodiments, VGX-3100 is administered to the subject three times over a 12-week period. In still further embodiments, VGX-3100 is formulated at a concentration of 6 mg / ml in 150 mM sodium chloride and 15 mM sodium citrate.

[0014] In certain embodiments, administration of VGX-3100 results in virological clearance of HPV-16 and / or HPV-18 and histopathological regression of cervical HSIL. In further embodiments, administration of VGX-3100 results in histopathological regression of cervical HSIL. In still further embodiments, administration of VGX-3100 results in virological clearance of HPV-16 and / or HPV-18. In certain embodiments, administration of VGX-3100 results in complete histopathological regression of cervical HSIL to normal. In further embodiments, administration of VGX-3100 results in complete histopathological regression of cervical HSIL to normal and virological clearance of HPV-16 and / or HPV-18. In still further embodiments, administration of VGX-3100 results in no histopathological progression. In certain embodiments, administration of VGX-3100 results in clearance of HPV-16 and / or HPV-18 infection from non-cervical anatomical sites. In further embodiments, administration of VGX-3100 results in improved humoral and cellular immune responses to VGX-3100 as assessed relative to baseline at 36 weeks after the third administration of VGX-3100 and at 36 weeks after administration of VGX-3100.

[0015] In certain embodiments, the results of VGX-3100 administration are evaluated at 36 weeks after administration of VGX-3100. Detailed Description

[0016] The disclosed method can be more readily understood by reference to the following detailed description, which forms a part of this disclosure. It should be understood that the disclosed method is not limited to the specific methods described and / or illustrated herein, and the terms used herein are for the purpose of describing particular embodiments by way of example only and are not intended to limit the claimed method.

[0017] Unless otherwise expressly stated, any description of a possible mechanism or mode of action or reason for improvement is intended to be illustrative only, and the disclosed method should not be limited by the correctness or incorrectness of any such proposed mechanism or mode of action or reason for improvement.

[0018] When expressing a series of numerical values, another embodiment includes from one specific value and / or to other specific values. Further, the values recited in a reference range include each value within that range. All ranges are inclusive and combinable. When a value is expressed as an approximation by use of the antecedent "about", it is to be understood that the specific value forms another embodiment. Unless the context clearly dictates otherwise, a reference to a specific numerical value includes at least that specific value.

[0019] It should be understood that, for clarity, certain features of the disclosed methods described herein in the context of separate embodiments may also be provided in combination in a single embodiment. Conversely, for brevity, the various features of the disclosed methods described in the context of a single embodiment may also be provided separately or in any sub-combination.

[0020] Throughout the specification and claims, various terms are used that are related to aspects of the specification. Unless otherwise indicated, such terms are to have their ordinary meaning in the art. Other specifically defined terms will be interpreted in a manner consistent with the definitions provided herein.

[0021] As used herein, the singular forms "a / an" and "the" include the plural.

[0022] Certain terms

[0023] As used herein, the term "about", when used in reference to a range of numbers, a critical value, or a specific value, is used to indicate that the recited value may vary by up to 10% from the listed value. Thus, the term "about" is used to encompass variations of ±10% or less, ±5% or less, ±1% or less, ±0.5% or less, or ±0.1% or less from the specified value.

[0024] As used herein, the term "at least one" means "one or more".

[0025] As used herein, the term "subject" refers to any animal, particularly a human. Thus, these methods are applicable to humans and non-human animals, but are most preferably used for humans. "Subject" and "patient" may be used interchangeably herein.

[0026] As used herein, the term "comprising" is intended to include instances covered by the terms "consisting essentially of" and "consisting of"; similarly, the term "consisting essentially of" is intended to include instances covered by the term "consisting of".

[0027] As used herein, "treatment" and like terms refer to, for example, reducing the symptoms and severity and / or frequency of high-grade cervical intraepithelial neoplasia (HSIL) associated with human papillomavirus (HPV) type 16 or HPV type 18 in the cervix and HSIL lesions; eliminating the symptoms of HPV type 16 or HPV type 18 infection, particularly HSIL lesions; and / or clearing HPV type 16 or HPV type 18 virus from the subject; and / or causing regression of low-grade squamous intraepithelial lesions (LSIL) or normal tissue in the cervix.

[0028] As used herein, the term "coding sequence" or "coding nucleic acid" can mean a nucleic acid (RNA or DNA molecule) that contains a nucleotide sequence encoding a polypeptide. The coding sequence can further include start and stop signals operably linked to regulatory elements, which include a promoter and a polyadenylation signal capable of directing expression in an individual or mammalian cell, and the nucleic acid is administered to an individual or a mammalian. The coding sequence can further include a sequence encoding a signal peptide, for example, an IgE leader sequence.

[0029] As used herein, the term "nucleic acid" or "oligonucleotide" or "polynucleotide" can mean at least two nucleotides covalently linked together. The description of a single strand also defines the sequence of the complementary strand. Thus, a nucleic acid also includes the complementary strand of the described 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 includes nucleic acids that are substantially the same and their complements. The single strand provides a probe that can hybridize to a target sequence under stringent hybridization conditions. Thus, a nucleic acid also includes a probe that hybridizes under stringent hybridization conditions. A nucleic acid can be single-stranded or double-stranded, or can contain portions of both double-stranded and single-stranded sequences. A nucleic acid can be DNA (both genomic and cDNA), RNA, or a hybrid, in which the nucleic acid can contain a combination of deoxyribonucleotides and ribonucleotides, as well as a combination of bases, including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine, hypoxanthine, isocytosine, and isoguanine. Nucleic acids can be obtained by chemical synthesis methods or recombinant methods.

[0030] As used herein, the term "operably linked" can mean that gene expression is under the control of a promoter that is spatially linked to it. The promoter can be located 5' (upstream) or 3' (downstream) of the gene it controls. The distance between the promoter and the gene can be approximately the same as the distance between the promoter and the gene it controls in the gene from which the promoter is derived. As is known in the art, changes in this distance can be accommodated without loss of promoter function.

[0031] As used herein, the term "promoter" can mean a synthetic or natural source molecule that is capable of conferring, activating, or enhancing the expression of a nucleic acid in a cell. The promoter can contain one or more specific transcriptional regulatory sequences to further enhance expression and / or alter its spatial and / or temporal expression. The promoter can also contain distal enhancer or repressor elements that can be located up to thousands of base pairs away from the transcription start site. Promoters can be derived from sources including viruses, bacteria, fungi, plants, insects, and animals. The promoter can regulate the expression of gene components constitutively or differentially 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.

[0032] As used herein, the term "vector" may refer to a nucleic acid sequence containing an origin of replication. The vector can be a plasmid, phage, bacterial artificial chromosome, or yeast artificial chromosome. The vector can be a DNA or RNA vector. The vector can be an extrachromosomal self-replicating vector or a vector integrated into the host genome.

[0033] As used herein, the term "adverse event" (AE) is defined according to the Common Terminology Criteria for Adverse Events (CTCAE) version 4.03 grading criteria. The investigator grades laboratory AEs and clinical AEs for the applicable subject population according to CTCAE version 4.03 at the following severity levels: grade 1 (mild), grade 2 (moderate), grade 3 (severe), grade 4 (life-threatening), and grade 5 (death).

[0034] As used herein, the term "isomiR" refers to an isoform of a canonical (mature) miRNA. IsomiRs may differ from classical miRNAs in the following ways: (1) addition and / or deletion of nucleotides at the 5' end of the classical miRNA; (2) addition and / or deletion of nucleotides at the 3' end of the classical miRNA; (3) addition and / or deletion of nucleotides at both the 5' and 3' ends of the classical miRNA; and (4) nucleotide substitutions within the sequence of the classical miRNA.

[0035] As used herein, the term "responder" refers to a study subject who achieves the primary endpoint of regression of HSIL lesions to LSIL or lower grade and elimination of HPV16 / HPV18 infection.

[0036] As used herein, the term "biomarker positive" refers to a subject who is predicted to be a "responder" as defined above according to biomarker characteristics (e.g., the RF_9_20 model) prior to administration of VGX-3100 or placebo.

[0037] As used herein, the term "PPV" refers to the number of biomarker-positive subjects who are responders / the number of biomarker-positive subjects.

[0038] As used herein, the term "NPV" refers to the number of biomarker-negative subjects who are responders / the number of biomarker-negative subjects.

[0039] As used herein, the term "accuracy" refers to the number of biomarker-positive subjects who are responders plus the number of biomarker-negative subjects who are non-responders / the number of subjects.

[0040] As used herein, the term "sensitivity" refers to the number of biomarker-positive subjects who are responders / the number of responders.

[0041] As used herein, the term "specificity" refers to the number of biomarker-negative subjects who are non-responders / the number of non-responders.

[0042] As used herein, the term "placebo" means the administration of a pharmaceutical composition that does not contain VGX-3100.

[0043] Methods of treatment and diagnosis

[0044] Provided herein are methods of treating high-grade cervical intraepithelial lesion (HSIL) associated with human papillomavirus (HPV) type 16 or HPV type 18, the methods comprising, consisting of, or consisting essentially of: (a) assessing the presence of a set of miRNAs and their isomiRs consisting of: hsa.miR.375.3p, hsa.miR.425.5p, hsa.miR.193a.5p, hsa.miR.199b.5p, hsa.miR.365a.3p, hsa.miR.223.5p, hsa.miR.148b.3p, hsa.miR.143.3p, hsa.miR.941, hsa.miR.340.5p, hsa.miR.151a.3p, hsa.miR.23a.3p in one or more biological samples from a subject having HSIL associated with HPV type 16 or HPV type 18; (b) calculating the normalized levels of each of these miRNAs and their isomiRs in the set; (c) determining a miRNA signature based on the normalized levels of the miRNAs and their isomiRs in the set; (d) classifying the subject as biomarker-positive or biomarker-negative based on the miRNA signature; and (e) if the subject is classified as biomarker-positive, administering to the subject a therapeutically effective amount of VGX-3100.

[0045] The present invention also provides VGX-3100 for use in a method of treating high-grade cervical intraepithelial neoplasia (HSIL) associated with human papillomavirus (HPV) type 16 or HPV type 18, the method comprising, consisting essentially of, or consisting of: (a) assessing the presence of a panel of miRNAs and their isomiRs consisting of: hsa.miR.375.3p, hsa.miR.425.5p, hsa.miR.193a.5p, hsa.miR.199b.5p, hsa.miR.365a.3p, hsa.miR.223.5p, hsa.miR.148b.3p, hsa.miR.143.3p, hsa.miR.941, hsa.miR.340.5p, hsa.miR.151a.3p, hsa.miR.23a.3p in one or more biological samples from a subject having HSIL associated with HPV type 16 or HPV type 18; (b) calculating the normalized level of each of these miRNAs and their isomiRs in the panel; (c) determining a miRNA signature based on the normalized levels of the miRNAs and their isomiRs in the panel; (d) classifying the subject as biomarker positive or biomarker negative based on the miRNA signature; and (e) administering to the subject a therapeutically effective amount of VGX-3100 if the subject is classified as biomarker positive.

[0046] The present invention also provides the use of VGX-3100 in the manufacture of a medicament for treating high-grade cervical intraepithelial neoplasia (HSIL) associated with human papillomavirus (HPV) type 16 or HPV type 18, said method comprising, consisting of, or consisting essentially of: (a) assessing the presence in one or more biological samples from a subject with HSIL associated with HPV type 16 or HPV type 18 of a set of miRNAs and their isomiRs consisting of: hsa.miR.375.3p, hsa.miR.425.5p, hsa.miR.193a.5p, hsa.miR.199b.5p, hsa.miR.365a.3p, hsa.miR.223.5p, hsa.miR.148b.3p, hsa.miR.143.3p, hsa.miR.941, hsa.miR.340.5p, hsa.miR.151a.3p, hsa.miR.23a.3p; (b) calculating the normalized level of each of these miRNAs and their isomiRs in the set; (c) determining a miRNA signature based on the normalized levels of the miRNAs and their isomiRs in the set; (d) classifying the subject as biomarker positive or biomarker negative based on the miRNA signature; and (e) administering to the subject a therapeutically effective amount of VGX-3100 if the subject is classified as biomarker positive.

[0047] The present invention provides a method for treating high-grade cervical intraepithelial neoplasia (HSIL) associated with human papillomavirus (HPV) type 16 or HPV type 18, the method comprising, consisting of, or consisting essentially of: (a) assessing the presence of a set of miRNAs and their isomiRs consisting of: hsa.miR.375.3p, hsa.miR.425.5p, hsa.miR.193a.5p, hsa.miR.199b.5p, hsa.miR.365a.3p, hsa.miR.223.5p, hsa.miR.148b.3p, hsa.miR.143.3p, hsa.miR.941, hsa.miR.340.5p, hsa.miR.151a.3p, hsa.miR.23a.3p in one or more biological samples from a subject having HSIL associated with HPV type 16 or HPV type 18; (b) calculating the normalized levels of each of these miRNAs and their isomiRs in the set; (c) determining a miRNA signature based on the normalized levels of the miRNAs and their isomiRs in the set; (d) classifying the subject as biomarker positive or biomarker negative based on the miRNA signature; and (e) if the subject is classified as biomarker negative, discontinuing administration of a therapeutically effective amount of VGX-3100 to the subject. In certain embodiments, the method further comprises treating the subject with loop electrosurgical excision procedure [LEEP] / large loop excision of the transformation zone [LLETZ], laser ablation, or conization.

[0048] In certain embodiments, a model having the ability to predict regression of cervical HSIL and clearance of HPV16 / 18 infection following treatment with VGX-3100 is used to determine the miRNA signature, and it has been found that the accuracy, PPV, NPV, sensitivity, and specificity are all superior when this model is applied to the treatment group as compared to placebo.

[0049] In certain embodiments, the presence of the miRNAs is determined by RNA sequencing.

[0050] In further embodiments, the biological sample is a plasma sample.

[0051] In still further embodiments, the plasma sample is isolated from the subject prior to administration of VGX-3100.

[0052] In additional embodiments, HPV type 16 or HPV type 18 associated HSIL of the cervix is determined by biopsy.

[0053] In some aspects of the present disclosure, the method includes evaluating one or more biological samples. In certain embodiments, the method includes evaluating one biological sample. In certain embodiments, the method includes evaluating two biological samples. In certain embodiments, the method includes evaluating three biological samples. In certain embodiments, the method includes evaluating four biological samples. In certain embodiments, the method includes evaluating five biological samples.

[0054] Also provided herein is a method of selecting a subject as a candidate for treatment with VGX-3100, the method comprising, consisting essentially of, or consisting of: (a) evaluating for the presence of a set of miRNAs and their isomiRs consisting of: hsa.miR.375.3p, hsa.miR.425.5p, hsa.miR.193a.5p, hsa.miR.199b.5p, hsa.miR.365a.3p, hsa.miR.223.5p, hsa.miR.148b.3p, hsa.miR.143.3p, hsa.miR.941, hsa.miR.340.5p, hsa.miR.151a.3p, hsa.miR.23a.3p in one or more biological samples from a subject having HPV type 16- or HPV type 18-associated HSIL; (b) calculating the normalized level of each of these miRNAs and their isomiRs in the set; (c) determining a miRNA signature based on the normalized levels of the miRNAs and their isomiRs in the set; (d) classifying the subject as a candidate for treatment with VGX-3100 based on the miRNA signature. In certain embodiments, the method further includes administering a therapeutically effective amount of VGX-3100 to the subject classified as a candidate for treatment with VGX-3100.

[0055] The present invention also provides a method for excluding a subject as a candidate for treatment with VGX-3100, the method comprising, consisting of, or consisting essentially of the following: (a) assessing the presence of a set of miRNAs and their isomiRs consisting of: hsa.miR.375.3p, hsa.miR.425.5p, hsa.miR.193a.5p, hsa.miR.199b.5p, hsa.miR.365a.3p, hsa.miR.223.5p, hsa.miR.148b.3p, hsa.miR.143.3p, hsa.miR.941, hsa.miR.340.5p, hsa.miR.151a.3p, hsa.miR.23a.3p in one or more biological samples from a subject with HPV type 16 or HPV type 18 associated HSIL; (b) calculating the normalized level of each of these miRNAs and their isomiRs in the set; (c) determining a miRNA signature based on the normalized levels of the miRNAs and their isomiRs in the set; (d) classifying the subject as not a candidate for treatment with VGX-3100 based on the miRNA signature. In certain embodiments, the method further comprises discontinuing the administration of a therapeutically effective amount of VGX-3100 to a subject not classified as a candidate for treatment with VGX-3100. In certain embodiments, the method further comprises treating the subject with loop electrosurgical excision procedure [LEEP] / large loop excision of the transformation zone [LLETZ], laser ablation, or conization.

[0056] In certain embodiments, administration of VGX-3100 results in virological clearance of HPV-16 and / or HPV-18 and histopathological regression of cervical HSIL. In further embodiments, administration of VGX-3100 results in histopathological regression of cervical HSIL. In still further embodiments, administration of VGX-3100 results in virological clearance of HPV-16 and / or HPV-18. In certain embodiments, administration of VGX-3100 results in complete histopathological regression of cervical HSIL to normal. In further embodiments, administration of VGX-3100 results in complete histopathological regression of cervical HSIL to normal and virological clearance of HPV-16 and / or HPV-18. In still further embodiments, administration of VGX-3100 results in no histopathological progression. In certain embodiments, administration of VGX-3100 results in clearance of HPV-16 and / or HPV-18 infection from non-cervical anatomical sites. In further embodiments, administration of VGX-3100 results in improved humoral and cellular immune responses to VGX-3100 as assessed relative to baseline at 36 weeks after the third administration of VGX-3100 and after administration of VGX-3100.

[0057] The present invention also provides methods for improving virological clearance of HPV-16 and / or HPV-18 and histopathological regression of cervical HSIL in a subject, the methods comprising, consisting of, or consisting essentially of: (a) assessing the presence of a set of miRNAs and their isomiRs consisting of: hsa.miR.375.3p, hsa.miR.425.5p, hsa.miR.193a.5p, hsa.miR.199b.5p, hsa.miR.365a.3p, hsa.miR.223.5p, hsa.miR.148b.3p, hsa.miR.143.3p, hsa.miR.941, hsa.miR.340.5p, hsa.miR.151a.3p, hsa.miR.23a.3p in one or more biological samples from a subject with HPV type 16- or HPV type 18-associated HSIL; (b) calculating the normalized level of each of these miRNAs and their isomiRs in the set; (c) determining a miRNA signature based on the normalized levels of the miRNAs and their isomiRs in the set; (d) classifying the subject as biomarker positive or biomarker negative based on the miRNA signature; and (e) administering to the subject a therapeutically effective amount of VGX-3100 if the subject is classified as biomarker positive. In certain embodiments, the improvement in virological clearance of HPV-16 and / or HPV-18 and histopathological regression of cervical HSIL is relative to administration of a placebo to the subject or group of subjects. In certain embodiments, the improvement in virological clearance of HPV-16 and / or HPV-18 and histopathological regression of cervical HSIL is relative to no treatment of the subject or group of subjects. In certain embodiments, the improvement in virological clearance of HPV-16 and / or HPV-18 and histopathological regression of cervical HSIL is relative to treatment of the subject or group of subjects with standard of care.

[0058] The present disclosure also provides methods for improving histopathological regression of cervical HSIL in a subject, the methods comprising, consisting of, or consisting essentially of: (a) assessing the presence of a set of miRNAs and their isomiRs consisting of: hsa.miR.375.3p, hsa.miR.425.5p, hsa.miR.193a.5p, hsa.miR.199b.5p, hsa.miR.365a.3p, hsa.miR.223.5p, hsa.miR.148b.3p, hsa.miR.143.3p, hsa.miR.941, hsa.miR.340.5p, hsa.miR.151a.3p, hsa.miR.23a.3p in one or more biological samples from a subject with HPV type 16- or HPV type 18-associated HSIL; (b) calculating the normalized levels of each of these miRNAs and their isomiRs in the set; (c) determining a miRNA signature based on the normalized levels of the miRNAs and their isomiRs in the set; (d) classifying the subject as biomarker positive or biomarker negative based on the miRNA signature; and (e) if the subject is classified as biomarker positive, administering to the subject a therapeutically effective amount of VGX-3100. In certain embodiments, improving histopathological regression of cervical HSIL is relative to administering a placebo to the subject or a group of subjects. In certain embodiments, improving histopathological regression of cervical HSIL is relative to not treating the subject or a group of subjects. In certain embodiments, improving histopathological regression of cervical HSIL is relative to treating the subject or a group of subjects with standard of care.

[0059] The present disclosure also provides methods for improving virological clearance of HPV-16 and / or HPV-18 in a subject, the methods comprising, consisting of, or consisting essentially of: (a) assessing the presence of a set of miRNAs and their isomiRs consisting of: hsa.miR.375.3p, hsa.miR.425.5p, hsa.miR.193a.5p, hsa.miR.199b.5p, hsa.miR.365a.3p, hsa.miR.223.5p, hsa.miR.148b.3p, hsa.miR.143.3p, hsa.miR.941, hsa.miR.340.5p, hsa.miR.151a.3p, hsa.miR.23a.3p in one or more biological samples from a subject with HPV type 16- or HPV type 18-associated HSIL; (b) calculating the normalized levels of each of these miRNAs and their isomiRs in the set; (c) determining a miRNA signature based on the normalized levels of the miRNAs and their isomiRs in the set; (d) classifying the subject as biomarker positive or biomarker negative based on the miRNA signature; and (e) administering to the subject a therapeutically effective amount of VGX-3100 if the subject is classified as biomarker positive. In certain embodiments, improving virological clearance of HPV-16 and / or HPV-18 is relative to administering a placebo to the subject or group of subjects. In certain embodiments, improving virological clearance of HPV-16 and / or HPV-18 is relative to not treating the subject or group of subjects. In certain embodiments, improving virological clearance of HPV-16 and / or HPV-18 is relative to treating the subject or group of subjects with standard of care.

[0060] The present disclosure also provides methods for achieving complete histopathological regression of cervical HSIL to normal in a subject, the methods comprising, consisting of, or consisting essentially of: (a) assessing the presence in one or more biological samples from a subject with HPV type 16 or HPV type 18 - associated HSIL of a set of miRNAs and their isomiRs consisting of: hsa.miR.375.3p, hsa.miR.425.5p, hsa.miR.193a.5p, hsa.miR.199b.5p, hsa.miR.365a.3p, hsa.miR.223.5p, hsa.miR.148b.3p, hsa.miR.143.3p, hsa.miR.941, hsa.miR.340.5p, hsa.miR.151a.3p, hsa.miR.23a.3p; (b) calculating the normalized level of each of these miRNAs and their isomiRs in the set; (c) determining a miRNA signature based on the normalized levels of the miRNAs and their isomiRs in the set; (d) classifying the subject as biomarker - positive or biomarker - negative based on the miRNA signature; and (e) if the subject is classified as biomarker - positive, administering to the subject a therapeutically effective amount of VGX - 3100. In certain embodiments, achieving complete histopathological regression of cervical HSIL to normal is relative to administering a placebo to the subject or a group of subjects. In certain embodiments, achieving complete histopathological regression of cervical HSIL to normal is relative to not treating the subject or a group of subjects. In certain embodiments, achieving complete histopathological regression of cervical HSIL to normal is relative to treating the subject or a group of subjects with standard of care.

[0061] The present disclosure also provides methods for achieving histopathologic complete regression of cervical HSIL to normal and virologic clearance of HPV-16 and / or HPV-18 in a subject, the methods comprising, consisting of, or consisting essentially of: (a) assessing the presence of a set of miRNAs and their isomiRs consisting of: hsa.miR.375.3p, hsa.miR.425.5p, hsa.miR.193a.5p, hsa.miR.199b.5p, hsa.miR.365a.3p, hsa.miR.223.5p, hsa.miR.148b.3p, hsa.miR.143.3p, hsa.miR.941, hsa.miR.340.5p, hsa.miR.151a.3p, hsa.miR.23a.3p in one or more biological samples from a subject having HPV type 16- or HPV type 18-associated HSIL; (b) calculating the normalized level of each of these miRNAs and their isomiRs in the set; (c) determining a miRNA signature based on the normalized levels of the miRNAs and their isomiRs in the set; (d) classifying the subject as biomarker positive or biomarker negative based on the miRNA signature; and (e) administering to the subject a therapeutically effective amount of VGX-3100 if the subject is classified as biomarker positive. In certain embodiments, achieving histopathologic complete regression of cervical HSIL to normal and virologic clearance of HPV-16 and / or HPV-18 is relative to administering a placebo to the subject or group of subjects. In certain embodiments, achieving histopathologic complete regression of cervical HSIL to normal and virologic clearance of HPV-16 and / or HPV-18 is relative to not treating the subject or group of subjects. In certain embodiments, achieving histopathologic complete regression of cervical HSIL to normal and virologic clearance of HPV-16 and / or HPV-18 is relative to treating the subject or group of subjects with standard of care.

[0062] The present invention also provides methods of improving histopathological progression-free in a subject, the methods comprising, consisting of, or consisting essentially of: (a) assessing the presence in one or more biological samples from a subject with HPV type 16 or HPV type 18 associated HSIL of a set of miRNAs and their isomiRs consisting of: hsa.miR.375.3p, hsa.miR.425.5p, hsa.miR.193a.5p, hsa.miR.199b.5p, hsa.miR.365a.3p, hsa.miR.223.5p, hsa.miR.148b.3p, hsa.miR.143.3p, hsa.miR.941, hsa.miR.340.5p, hsa.miR.151a.3p, hsa.miR.23a.3p; (b) calculating the normalized levels of each of these miRNAs and their isomiRs in the set; (c) determining a miRNA signature based on the normalized levels of the miRNAs and their isomiRs in the set; (d) classifying the subject as biomarker positive or biomarker negative based on the miRNA signature; and (e) if the subject is classified as biomarker positive, administering to the subject a therapeutically effective amount of VGX-3100. In certain embodiments, improving histopathological progression-free is relative to administering a placebo to the subject or a group of subjects. In certain embodiments, improving histopathological progression-free is relative to not treating the subject or a group of subjects. In certain embodiments, improving histopathological progression-free is relative to treating the subject or a group of subjects with standard of care.

[0063] The present disclosure also provides methods for improving clearance of HPV-16 and / or HPV-18 infection from non-cervical anatomical locations in a subject, the methods comprising, consisting essentially of, or consisting of: (a) assessing the presence of a set of miRNAs and their isomiRs consisting of: hsa.miR.375.3p, hsa.miR.425.5p, hsa.miR.193a.5p, hsa.miR.199b.5p, hsa.miR.365a.3p, hsa.miR.223.5p, hsa.miR.148b.3p, hsa.miR.143.3p, hsa.miR.941, hsa.miR.340.5p, hsa.miR.151a.3p, hsa.miR.23a.3p in one or more biological samples from a subject with HPV type 16 or HPV type 18 associated HSIL; (b) calculating the normalized level of each of these miRNAs and their isomiRs in the set; (c) determining a miRNA signature based on the normalized levels of the miRNAs and their isomiRs in the set; (d) classifying the subject as biomarker positive or biomarker negative based on the miRNA signature; and (e) administering to the subject a therapeutically effective amount of VGX-3100 if the subject is classified as biomarker positive. In certain embodiments, improving clearance of HPV-16 and / or HPV-18 infection from non-cervical anatomical locations is relative to administering a placebo to the subject or group of subjects. In certain embodiments, improving clearance of HPV-16 and / or HPV-18 infection from non-cervical anatomical locations is relative to not treating the subject or group of subjects. In certain embodiments, improving clearance of HPV-16 and / or HPV-18 infection from non-cervical anatomical locations is relative to treating the subject or group of subjects with standard of care.

[0064] The present invention also provides methods for improving humoral and cellular immune responses to VGX-3100 in a subject, as assessed relative to baseline, after the third administration of VGX-3100 and at 36 weeks after administration of VGX-3100, the methods comprising, consisting of, or consisting essentially of: (a) assessing the presence in one or more biological samples from a subject having HPV type 16- or HPV type 18-associated HSIL of a set of miRNAs and their isomiRs consisting of: hsa.miR.375.3p, hsa.miR.425.5p, hsa.miR.193a.5p, hsa.miR.199b.5p, hsa.miR.365a.3p, hsa.miR.223.5p, hsa.miR.148b.3p, hsa.miR.143.3p, hsa.miR.941, hsa.miR.340.5p, hsa.miR.151a.3p, hsa.miR.23a.3p; (b) calculating the normalized level of each of these miRNAs and their isomiRs in the set; (c) determining a miRNA signature based on the normalized levels of the miRNAs and their isomiRs in the set; (d) classifying the subject as biomarker positive or biomarker negative based on the miRNA signature; and (e) administering to the subject a therapeutically effective amount of VGX-3100 if the subject is classified as biomarker positive. In certain embodiments, improving clearance of HPV-16 and / or HPV-18 infection from non-cervical anatomical sites is relative to administration of a placebo to the subject or group of subjects. In certain embodiments, improving humoral and cellular immune responses to VGX-3100, as assessed relative to baseline, after the third administration of VGX-3100 and at 36 weeks after administration of VGX-3100 is relative to no treatment of the subject or group of subjects. In certain embodiments, improving humoral and cellular immune responses to VGX-3100, as assessed relative to baseline, after the third administration of VGX-3100 and at 36 weeks after administration of VGX-3100 is relative to treatment of the subject or group of subjects with standard of care.

[0065] In certain embodiments, the outcome of VGX-3100 administration or the improvement achieved by VGX-3100 administration is assessed at 36 weeks after administration of VGX-3100.

[0066] Route of administration and pharmaceutical composition

[0067] VGX-3100 can be delivered using any of several well-known techniques including DNA injection (also known as DNA vaccination), recombinant vectors such as recombinant adenovirus, recombinant adeno-associated virus, and recombinant vaccinia.

[0068] Routes of administration include, but are not limited to, intramuscular, intranasal, intraperitoneal, intradermal, subcutaneous, intravenous, intraarterial, intraocular, and oral, as well as topical, transdermal, by inhalation, or by suppository, or to mucosal tissues (such as by lavage to vaginal, rectal, urethral, oral, and sublingual tissues). Preferred routes of administration include intramuscular, intraperitoneal, intradermal, and subcutaneous injection. The genetic construct can be administered by means including, but not limited to, electroporation methods and devices, conventional syringes, needleless injection devices, or "gene guns." In some embodiments, VGX-3100 is administered to a subject by intramuscular injection. In certain embodiments, VGX-3100 is administered to a subject by intramuscular injection followed by electroporation.

[0069] Examples of electroporation devices and electroporation methods preferably used to facilitate DNA vaccine delivery include those described in U.S. Patent No. 7,245,963 to Draghia-Akli et al., and U.S. Patent Publication 2005 / 0052630 to Smith et al., the contents of which are hereby incorporated by reference in their entirety. Also preferred are electroporation devices and electroporation methods for facilitating DNA vaccine delivery provided in co-pending and co-owned U.S. Patent Application Serial No. 11 / 874,072, filed October 17, 2007, which claims the benefit of U.S. Provisional Application Serial No. 60 / 852,149, filed October 17, 2006, and U.S. Provisional Application Serial No. 60 / 978,982, filed October 10, 2007, all of which are hereby incorporated by reference in their entirety. In certain embodiments, the electroporation device is -5P device.

[0070] Examples of embodiments using electroporation technology are provided below and are discussed in more detail in the patent references discussed above: The electroporation device can be configured to deliver energy pulses to a desired tissue of a mammal, which pulses generate a constant current similar to a preset current input by the user. The electroporation device includes an electroporation component and an electrode assembly or a handle assembly. The electroporation component can include and incorporate one or more different elements of the electroporation device, including: a controller, a current waveform generator, an impedance tester, a waveform recorder, an input element, a status reporting element, a communication port, a memory component, a power source, and a power switch. The electroporation component can function as one element of the electroporation device, and the other elements are separate elements (or components) that communicate with the electroporation component. In some embodiments, the electroporation component can function as more than one element of the electroporation device, which can communicate with other elements of the electroporation device that are separate from the electroporation component. The delivery of a modified HPV vaccine using electroporation technology is not limited by the elements of the electroporation device that exist as part of an electromechanical or mechanical device, as these elements can function as one device or as separate elements that communicate with each other. The electroporation component is capable of delivering energy pulses that generate a constant current in the desired tissue and includes a feedback mechanism. The electrode assembly includes an electrode array having a plurality of electrodes in a spatial arrangement, wherein the electrode assembly receives an energy pulse from the electroporation component and delivers it through the electrodes to the desired tissue. At least one of the plurality of electrodes is neutral during the delivery of the energy pulse and measures the impedance in the desired tissue and transmits the impedance to the electroporation component. The feedback mechanism can receive the measured impedance and can adjust the energy pulse delivered by the electroporation component to maintain a constant current.

[0071] In some embodiments, the plurality of electrodes can deliver energy pulses in a dispersed mode. In some embodiments, the plurality of electrodes can deliver energy pulses in a dispersed mode under a programmed sequence through electrode control, and the programmed sequence is input by the user into the electroporation component. In some embodiments, the programmed sequence includes a plurality of pulses delivered in sequence, wherein each pulse of the plurality of pulses is delivered by at least two active electrodes with one neutral electrode having a measured impedance, and wherein subsequent pulses of the plurality of pulses are delivered by another of the at least two active electrodes with one neutral electrode having a measured impedance.

[0072] In some embodiments, the feedback mechanism is performed by hardware or software. Preferably, the feedback mechanism is performed by an analog closed-loop circuit. Preferably, this feedback occurs every 50 μs, 20 μs, 10 μs, or 1 μs, but preferably real-time feedback or instantaneous feedback (i.e., substantially instantaneous as determined by the available technology for determining response time). In some embodiments, the neutral electrode measures the impedance in the desired tissue and transmits the impedance to the feedback mechanism, and the feedback mechanism responds to the impedance and adjusts the energy pulse to maintain a constant current at a value similar to a preset current. In some embodiments, the feedback mechanism continuously and instantaneously maintains a constant current during the delivery of the energy pulse.

[0073] In some embodiments, a nucleic acid molecule is delivered to a cell in conjunction with the administration of a polynucleotide function enhancer or a gene vaccine promoter. Polynucleotide function enhancers are described in U.S. Sequence Nos. 5,593,972, 5,962,428, and International Application Sequence No. PCT / US94 / 00899, filed Jan. 26, 1994, each of which is incorporated herein by reference. An adjuvant co-administered with the nucleic acid molecule can be administered as a mixture with the nucleic acid molecule, or administered separately simultaneously with, before, or after the administration of the nucleic acid molecule. In addition, other reagents that can act as transfection agents and / or replication agents and / or inflammatory agents and can be co-administered with GVF include growth factors, cytokines, and lymphokines such as alpha-interferon, gamma-interferon, GM-CSF, platelet-derived growth factor (PDGF), TNF, epidermal growth factor (EGF), IL-1, IL-2, IL-4, IL-6, IL-10, IL-12, and IL-15, and fibroblast growth factor, surfactants such as immunostimulating complexes (ISCOMs), Freund's incomplete adjuvant, LPS analogs including monophosphoryl lipid A (MPL), muramyl peptides, quinone analogs, and vesicles (such as squalene and squalene), and hyaluronic acid can also be used in conjunction with the gene construct. In some embodiments, an immunomodulatory protein can be used as GVF. In some embodiments, a nucleic acid molecule bound to PLG is provided to enhance delivery / uptake.

[0074] The pharmaceutical composition according to the present invention comprises from about 1 nanogram to about 2000 micrograms of DNA. In some preferred embodiments, the pharmaceutical composition according to the present invention comprises from about 5 nanograms to about 1000 micrograms of DNA. In some preferred embodiments, the pharmaceutical composition contains from about 10 nanograms to about 800 micrograms of DNA. In some preferred embodiments, the pharmaceutical composition contains from about 0.1 to about 500 micrograms of DNA. In some preferred embodiments, the pharmaceutical composition contains from about 1 to about 350 micrograms of DNA. In some preferred embodiments, the pharmaceutical composition contains from about 25 to about 250 micrograms of DNA. In some preferred embodiments, the pharmaceutical composition contains from about 100 to about 200 micrograms of DNA. In certain embodiments, VGX-3100 is formulated at a concentration of 6 mg / ml.

[0075] The pharmaceutical compositions according to the present invention are formulated according to the mode of administration to be used. Where the pharmaceutical compositions are injectable pharmaceutical compositions, they are sterile, pyrogen-free and particulate-free. Isotonic formulations are preferably used. Generally, isotonic additives can include sodium chloride, glucose, mannitol, sorbitol and lactose. In some cases, isotonic solutions such as phosphate buffered saline are preferred. Stabilizers include gelatin and albumin. In some embodiments, vasoconstrictors are added to the formulation. In some preferred embodiments, the pharmaceutical composition contains from about 100 to about 200 micrograms of DNA. In certain embodiments, VGX-3100 is formulated at a concentration of 6 mg / ml in 150 mM sodium chloride and 15 mM sodium citrate.

[0076] In certain embodiments, VGX-3100 is administered to a subject at a dose of 6 mg. In further embodiments, VGX-3100 is administered to a subject three times over a 12-week period. In still further embodiments, the first dose of VGX-3100 is administered on day 0, the second dose of VGX-3100 is administered at week 4, and the third dose of VGX-3100 is administered at week 12.

[0077] Examples

[0078] The examples and embodiments described herein are for illustrative purposes only, and various modifications or changes proposed to those skilled in the art will be included within the spirit and scope of this application and the scope of the appended claims.

[0079] Example 1: Intramuscular delivery of VGX-3100 followed by CELLECTRA TMA Prospective, Randomized, Double-Blind, Placebo-Controlled Phase 3 Study of 5PSP Electroporation (EP) for the Treatment of HPV-16 and / or HPV-18-Related High-Grade Squamous Intraepithelial Lesions (HSIL) of the Cervix (REVEAL 1 Trial; Randomized Evaluation of VGX-3100 and Electroporation for the Treatment of Cervical HSIL) (Study HPV-301)

[0080] This document provides a non-limiting example of a prospective, randomized, double-blind, placebo-controlled study designed to demonstrate the safety and efficacy of VGX-3100 followed by EP in women with cervical HSIL associated with HPV-16 and / or HPV-18. The primary endpoints were histopathologic regression of cervical HSIL and virologic clearance of HPV-16 and / or HPV-18 at week 36. This clinical trial utilized a placebo-controlled design because this design provides scientific rigor to distinguish treatment effects, especially in cervical HSIL where spontaneous regression may occur.

[0081] Clinical trial objectives and endpoints:

[0082] Primary Objectives and Endpoints. The primary objective of this study was to determine the efficacy of VGX-3100 compared to placebo in histopathologic regression of cervical HSIL and virologic clearance of HPV-16 and / or HPV-18. The primary objective endpoint was evaluated by the proportion of subjects with no evidence of cervical HSIL on histological examination (i.e., biopsy or excisional treatment) and no evidence of HPV-16 and / or HPV-18 in cervical samples by type-specific HPV testing at the week 36 visit.

[0083] Secondary Objectives and Endpoints. The first secondary objective of this study was to evaluate the safety and tolerability of IM delivery of VGX-3100 followed by EP with CELLECTRA TM 5PSP. The endpoint of the first secondary objective was evaluated by: (a) the incidence and severity of local and systemic events at 7 days and 28 days after each study treatment and during the clinical trial (up to the week 88 visit); and (b) the incidence and severity of all AEs, including SAEs (e.g., Suspected Unexpected Serious Adverse Reactions [SUSAR], Unanticipated Adverse Device Events [UADE], and other unanticipated AEs) during the clinical trial (up to the week 88 visit).

[0084] The second secondary objective of this study was to determine the efficacy of VGX-3100 compared to placebo as measured by histopathologic regression of cervical HSIL. The endpoint of the second secondary objective was evaluated by the proportion of subjects with no evidence of cervical HSIL on histological examination (i.e., biopsy or excisional treatment) at the week 36 visit.

[0085] The third secondary objective of this study was to determine the efficacy of VGX-3100 compared to placebo, as measured by virological clearance of HPV-16 and / or HPV-18. The endpoint of the third secondary objective was evaluated by the proportion of subjects in whom no evidence of HPV-16 and / or HPV-18 was found in cervical samples at the Week 36 visit by type-specific HPV testing.

[0086] The fourth secondary objective of this study was to determine the efficacy of VGX-3100 compared to placebo, as measured by complete histopathological regression of cervical HSIL to normal. The endpoint of the fourth secondary objective was evaluated by the proportion of subjects in whom no evidence of low-grade squamous intraepithelial lesion (LSIL) or high-grade squamous intraepithelial lesion (HSIL) (i.e., no evidence of cervical intraepithelial neoplasia [CIN] 1, CIN2, or CIN3) was found by histological examination (i.e., biopsy or excision treatment) at the Week 36 visit.

[0087] The fifth secondary objective of this study was to determine the efficacy of VGX-3100 compared to placebo, as measured by both complete histopathological regression of cervical HSIL to normal and virological clearance of HPV-16 and / or HPV-18. The endpoint of the fifth secondary objective was evaluated by the proportion of subjects in whom no evidence of LSIL or HSIL (i.e., no evidence of CIN1, CIN2, or CIN3) was found by histological examination (i.e., biopsy or excision treatment) and no evidence of HPV-16 and / or HPV-18 was found by type-specific HPV testing at the Week 36 visit.

[0088] The sixth secondary objective of this study was to determine the efficacy of VGX-3100 compared to placebo, as measured by histopathological non-progression. The endpoint of the sixth secondary objective was evaluated by the proportion of subjects in whom cervical HSIL did not progress to cervical cancer from baseline by histological examination (i.e., biopsy or excision treatment) at the Week 36 visit.

[0089] The seventh secondary objective of this study was to describe the clearance of HPV-16 and / or HPV-18 infection from non-cervical anatomical sites. The endpoint of the seventh secondary objective was evaluated by the proportion of subjects in whom HPV-16 and / or HPV-18 was cleared from non-cervical anatomical sites (oropharynx, vagina, and intra-anal) in specimens at the Week 36 visit.

[0090] The eighth secondary objective of this study was to determine the humoral and cellular immune responses to VGX-3100 relative to placebo, as assessed from baseline, at the time of the third (3) dose and at the Week 36 visit. The endpoints for the eighth secondary objective were evaluated by: a) the levels of serum anti-HPV-16 and anti-HPV-18 antibody concentrations at the Week 15 and Week 36 visits; b) the magnitude of the IFN-γ ELISpot responses at baseline, and at the Week 15 and Week 36 visits; and c) the magnitude of the flow cytometry responses at baseline and at the Week 15 visit.

[0091] Exploratory objectives and endpoints. The first exploratory objective of this study was to evaluate the tissue immune response to VGX-3100 in cervical samples. The endpoint for the first exploratory objective was the assessment of markers, including but not limited to CD8+ and FoxP3+ infiltrating cells. Where samples permitted, additional assessments may have included visualization of granulysin, perforin, CD137, CD103, and PD-L1 in cervical tissue.

[0092] The second exploratory objective of this study was to describe the associations between the miRNA profile, DNA methylation profile, prior colposcopy, cytology, and HPV test results and histological regression at Week 36. The endpoints for the second exploratory objective were evaluated by combining colposcopy, cytology, HPV test results (at the Week 8, Week 15, and Week 28 visits), miRNA profile (at baseline and Week 8), and DNA methylation profile (at baseline and Week 15) with histological regression of cervical HSIL at the Week 36 visit.

[0093] The third exploratory objective of this study was to describe the durability of HPV-16 and / or HPV-18 virological clearance in subjects treated with VGX-3100 compared to subjects treated with placebo. The endpoint for the third exploratory objective was evaluated by the proportion of subjects without evidence of HPV-16 and / or HPV-18 infection at the Week 62 and Week 88 visits as determined by type-specific HPV testing.

[0094] The fourth exploratory objective of this study was to describe the patient-reported outcomes in subjects treated with VGX-3100. Enrolled subjects completed patient-reported outcome questionnaires at baseline, at Weeks 4 and 12, 8 - 14 days after each dose, and at Weeks 28, 36, 40, and 88.

[0095] The fifth exploratory objective of this study was to determine whether a tissue-based score (immune score) derived using immune markers at baseline could predict histological and virological responses to VGX-3100 at week 36. The endpoint of the fifth exploratory objective was evaluated by combining the immune score results of subjects treated with VGX-3100 with histological and virological outcomes at week 36.

[0096] Methods

[0097] This was a prospective, randomized, double-blind, placebo-controlled clinical trial designed to determine the efficacy, safety, and tolerability of intramuscular (IM) injection of VGX-3100 followed by electroporation (EP) delivery using the CELLECTRA TM 5PSP device in adult women histologically confirmed to have cervical HSIL (CIN2 and CIN3) associated with HPV-16 and / or HPV-18.

[0098] The clinical trial consisted of a screening period (up to 10 weeks), a treatment and follow-up period (36 weeks), and a long-term follow-up period (52 weeks). The total duration of participation in the clinical trial for each subject was up to 98 weeks.

[0099] Approximately 198 eligible subjects will be randomly assigned to receive 6 mg (in 1 mL) of VGX-3100 or placebo (ratio 2:1) by IM followed by EP. Subjects will be randomly assigned in a stratified manner according to the following: 1) the severity of CIN observed in the biopsy specimen at screening (CIN2 versus CIN3); 2) the body mass index (BMI) category on day 0 (≤25 kg / m 2 versus >25 kg / m 2 ), and 3) the age category on day 0 (<25 versus ≥25 years). To ensure that the proportion of subjects with CIN2 disease in the clinical trial is not too high, the percentage of enrolled subjects with CIN2 should not exceed 50% of the total number of enrolled subjects.

[0100] Screening period

[0101] All screening evaluations should be completed within 10 weeks after the first dose (day 0) of the clinical trial treatment, except for safety laboratory assessments, which should be performed within 45 days before day 0.

[0102] Diagnosis and main inclusion and exclusion criteria

[0103] To be eligible for the clinical trial, subjects must be at least 18 years old, give consent to participate, and undergo a cervical biopsy / cervical lesion biopsy at screening. The biopsy slides will be sent to a central pathology laboratory and reviewed blindly by the PAC to determine the presence of cervical HSIL during screening. To be eligible for randomization, the PAC should assign a histological diagnosis of cervical HSIL. At the same time, the subject's cervical ThinPrep TM specimen is tested by cobas TM HPV test and is positive for HPV-16 and / or HPV-18. The subject is required to provide informed consent for the use of any information collected before consent and before any additional clinical trial-specific procedures are performed.

[0104] Inclusion criteria

[0105] Subjects are eligible for inclusion in the clinical trial if they meet all of the following criteria: women who are at least 18 years old and have reached the minimum age of consent required by local regulations; subjects who have been diagnosed with cervical infection with HPV type 16 and / or 18 at the time of screening by cobas TM HPV test; subjects who have had a cervical tissue specimen / glass slide collected within 10 weeks before the anticipated date of the first administration of the clinical trial drug and submitted to the clinical trial PAC for diagnosis; subjects who have histological evidence of cervical HSIL confirmed by the PAC at screening; subjects who understand, consent to, and are able to comply with the requirements of the protocol and are willing and able to give voluntary consent to participate and sign the consent form before clinical trial-related activities; the investigator determines that the subject is an appropriate candidate for the protocol-specified surgery required at week 36 (i.e., excision, 4-quadrant biopsy with ECC, or 4-quadrant biopsy); subjects who have satisfactory colposcopy results at screening, defined as complete visualization of the squamous-columnar junction (type I or type II transformation zone) and complete visualization of the upper limit of acetowhite epithelium or suspected CIN disease; the subject's cervical lesion can be sampled with a biopsy instrument (e.g., Mini-Tischler device); the subject's cervical lesion is large enough to ensure that a visible lesion remains after screening biopsy; the subject must meet one of the following criteria regarding their reproductive capacity: a) postmenopausal, defined as spontaneous amenorrhea lasting more than 12 months; b) surgically sterilized due to absence of ovaries or bilateral tubal ligation / occlusion more than 12 months before screening; or c) women of childbearing potential (WOCBP) willing to use contraception from screening until week 36, with a failure rate of less than 1% per year if used correctly and consistently; and the investigator determines that the subject's screening ECG is normal or that there are no clinically significant findings on the screening ECG.

[0106] Exclusion criteria

[0107] Subjects will be excluded from this clinical trial if they meet any of the following criteria: the subject has microscopic or gross evidence of adenocarcinoma in situ (AIS), high-grade vulvar, vaginal (including cervical HPV-related lesions extending to the vaginal vault), or anal intraepithelial neoplasia or invasive cancer in any histopathological specimen at screening; the subject's cervical lesion cannot be fully visualized by colposcopy at screening due to extension to the upper end of the endocervical canal; the subject has a history of ECC that shows potential untreated cancer, untreated HSIL, indeterminate or inadequate diagnosis (ECC is not required as part of the clinical trial screening); the subject has received treatment for cervical HSIL within 4 weeks prior to screening; the subject is pregnant, lactating, or considering pregnancy as of the Week 36 visit; the subject has a history of prior therapeutic HPV vaccination (permission is given for licensed prophylactic HPV vaccines, e.g., Gardasil TM , Cervarix TM);Subjects had any unresolved abnormal clinical screening laboratory values within 45 days prior to Day 0 (Grade 1 or higher according to CTCAE version 4.03 and considered clinically significant by the investigator); Subjects had immunosuppression due to underlying diseases or treatments, including: a) a history of human immunodeficiency virus (HIV) or positive serological test at screening (within 45 days prior to Day 0); b) primary immunodeficiency; c) long-term use (≥7 days) of oral or parenteral glucocorticoids at a dose of ≥20 mg / day prednisone equivalent (inhaled, otic, and ophthalmic corticosteroids are permitted); d) currently using or expected to use disease-modifying doses of antirheumatic drugs (e.g., azathioprine, cyclophosphamide, cyclosporine, methotrexate) and biologic disease-modifying drugs such as tumor necrosis factor (TNF)-α inhibitors (e.g., infliximab, adalimumab, or etanercept); e) a history of solid organ or bone marrow transplantation; f) any other clinically significant immunosuppression or a history of clinically diagnosed autoimmune diseases that may endanger the safety of the subject or require treatment, may interfere with the assessment of the clinical trial or the evaluation of the endpoint, or otherwise affect the validity of the clinical trial results; g) malnutrition (e.g., medically significant unintentional weight loss, kwashiorkor, or marasmus) based on the investigator's clinical judgment, based on screening laboratory tests, medical history, and physical examination findings; Subjects had received any non-clinical trial-related inactivated vaccines within 2 weeks of dosing; Subjects had received any live vaccines not related to the clinical trial within 4 weeks of dosing (e.g., measles vaccine); Subjects currently had or had previously had clinically significant and medically unstable diseases that, in the investigator's judgment, could endanger the safety of the subject, interfere with the assessment of the clinical trial or the evaluation of the endpoint, or otherwise affect the validity of the clinical trial results (e.g., chronic renal failure; angina, myocardial ischemia, or infarction; congestive heart failure, cardiomyopathy, or clinically significant arrhythmia of Grade 3 or higher); Subjects had had a malignancy or received systemic treatment for malignancy within 2 years of screening (except for locally invasive anal and genital cancers and superficial skin cancers treated by curative therapy); Subjects had acute or chronic bleeding or coagulation disorders and were not suitable for IM injection, or had used blood thinners (e.g., anticoagulants or antiplatelet drugs) within 2 weeks of Day 0; Subjects had a history of seizures, unless they had not had a seizure for 5 years while using one (1) or fewer antiepileptic drugs; Subjects had a sustained, manually confirmed sitting systolic blood pressure >150 mmHg or <90 mmHg, or diastolic blood pressure >95 mmHg at screening or on Day 0; Subjects had a resting heart rate <50 beats per minute (unless attributed to exercise training) or >100 beats per minute at screening or on Day 0; Subjects had undergone major surgery within 4 weeks of Day 0;The subject has participated in an interventional clinical trial using a study compound or device within 30 days of signing the informed consent form; is permitted to participate in an observational study; has fewer than two (2) acceptable sites on the deltoid and anterolateral quadriceps muscles of the subject available for IM injection; has a tattoo, keloid, or hypertrophic scar within 2 cm of the intended treatment site; has an implantable cardioverter defibrillator or cardiac pacemaker at the ipsilateral deltoid injection site (to prevent life-threatening arrhythmias) (unless acceptable to the cardiologist); has a metal implant or implantable medical device within the EP area of the subject; is taking or dependent on drugs or alcohol, which the investigator believes will interfere with compliance with the clinical trial requirements; is a prisoner or is involuntarily incarcerated for the treatment of mental or physical (i.e., infectious) diseases; is an active-duty military personnel; is a clinical trial-related staff member or a family member of a clinical trial-related staff member; or has any disease or condition that the investigator believes may affect the safety of the subject or the assessment of any clinical trial endpoint.;

[0108] Treatment and long-term follow-up period

[0109] Eligible subjects receive three (3) doses of a 6-mg refrigerated formulation of VGX-3100 or placebo via IM (deltoid [preferred site] or anterolateral quadriceps [alternate site]), followed immediately by EP using the CELLECTRA TM 5PSP device. The first clinical trial treatment is conducted on Day 0, the second at Week 4, and the third (last) at Week 12. The first clinical trial treatment should be conducted as soon as possible after confirmation of the cervical HSIL diagnosis and HPV-16 and / or HPV-18 status, but no later than 10 weeks after the pathology adjudication committee (PAC) collects the biopsy specimens from the subject for diagnosis during screening, and the treatment is conducted concurrently with a positive test for HPV-16 and / or HPV 18.;

[0110] Clinical trial personnel assess the injection site before each clinical trial treatment, at least 30 minutes after treatment, and at 2 to 4 weeks after treatment. On the day of the clinical trial treatment, a participant diary card (PDC) is distributed to the subject. The subject is advised to record local and systemic adverse events (AEs) for 7 days in the PDC after each clinical trial treatment. At 8 to 14 days after the end of each clinical trial treatment, the subject is followed up by phone for PDC review of AEs and injection site reactions.;

[0111] Efficacy assessment includes histological examination (i.e., biopsy or excisional treatment), colposcopy, cytology, and HPV testing at screening and at specific visits on and after Day 0. Digital photographs of the cervix after application of acetic acid are used to record colposcopy findings. At Week 36, tissue for histopathological evidence of regression to be analyzed is obtained by excision (e.g., loop electrosurgical excision procedure [LEEP], large loop excision of the transformation zone [LLETZ], cold knife conization [CKC]) or by biopsy (4-quadrant biopsy or 4-quadrant biopsy combined with endocervical curettage [ECC]) based on the assessment of cytology results, high-risk HPV status, and colposcopy findings at Week 28.

[0112] All subjects are required to undergo long-term follow-up for safety, cytology, and HPV 16 and / or HPV-18 testing at 6 months and 1 year after histopathological assessment at Week 36.

[0113] Safety is assessed throughout the clinical trial, including monitoring local and systemic AEs within 7 days after each clinical trial treatment recorded on the PDC, and all adverse events (AEs) throughout the clinical trial, including serious AEs (SAEs), unanticipated device adverse events (UADEs), and other unanticipated AEs.

[0114] An independent Data and Safety Monitoring Board (DSMB) provides safety oversight. The DSMB is scheduled to meet quarterly. If a safety issue arises and if the proportion of subjects with regression in the VGX-3100 group is too low compared to the placebo group, the DSMB is responsible for notifying the sponsor; for this purpose, no formal interim analysis is conducted.

[0115] Immunogenicity assessment includes humoral and cell-mediated immune responses to VGX-3100 treatment in blood samples and evidence of elevated immune responses in cervical tissue samples.

[0116] Number of subjects (planned and analyzed)

[0117] A total of 198 subjects are planned to be randomly assigned at a ratio of 2:1 to receive 6 mg of VGX-3100 or placebo by IM, followed by EP.

[0118] A total of 201 subjects are randomly assigned to receive VGX-3100 + EP (138 subjects) or placebo + EP (63 subjects).

[0119] The percentage of subjects in each analysis set is summarized in Table 1.

[0120] Table 1: Analysis Sets

[0121]

[0122] Note: Two subjects were randomly assigned to the VGX-3100 + EP group but did not receive treatment.

[0123] Note: The denominator used in the percentage calculation is the subjects randomly assigned in each treatment.

[0124] Abbreviations: EP: Electroporation; ITT: Intention-to-treat analysis; N: Sample size within the group; n: Number of subjects.

[0125] Test product, dose and administration method, batch number

[0126] Study product

[0127] Generic Name: VGX-3100

[0128] Chemical Name: Circular double-stranded deoxyribonucleic acid, consisting of 3782 base pairs for the pGX3001 plasmid and 3824 base pairs for the pGX3002 plasmid.

[0129] Differential Name: Eukaryotic expression plasmid, containing the HPV 16 and 18 - E6 and E7 encoding transcription units controlled by a synthetic CMV promoter, as well as the elements required for replication and selection in Escherichia coli, namely the pUC replication origin (pUC Ori) and the kanamycin resistance gene (Kan R).

[0130] Detailed Description: VGX-3100 (HPV therapeutic vaccine) is a combination of two equal amounts of plasmids (i.e., a 6 mg dose will deliver 3 mg of each pGX3001 and pGX3002 plasmid): a) pGX3001: p16ConE6E7, a plasmid encoding the synthetic HPV16 consensus E6 and E7 fusion gene (“consensus HPV 16 - 6&7”) under the control of the cytomegalovirus immediate early (CMV) promoter into the pVAX1 backbone (Invitrogen, Carlsbad, CA); and b) pGX3002: p18ConE6E7, a plasmid encoding the synthetic HPV18 consensus E6 and E7 fusion gene (“consensus HPV 18 - 6&7”) under the control of the cytomegalovirus immediate early (CMV) promoter into the pVAX1 backbone (Invitrogen, Carlsbad, CA). VGX-3100 is described in WO2014 / 165291, which is incorporated herein by reference in its entirety. The nucleic acid and amino acid consensus sequences are shown in Tables 2 and 3, respectively.

[0131] Table 2. VGX-3100 Consensus Sequences (Nucleic Acid)

[0132]

[0133] Table 3. VGX-3100 Consensus Sequence (Amino Acids)

[0134]

[0135]

[0136] The test product VGX-3100 is provided as a solution containing 6 mg (a 1:1 mixture of SynCon TM HPV-16 E6 / E7 and HPV-18 E6 / E7 plasmids) in 150 mM sodium chloride and 15 mM sodium citrate. The VGX-3100 drug product is packaged in a clear glass cartridge and administered by IM injection (deltoid [preferred site] or anterolateral quadriceps [alternate site]), followed immediately by EP using the CELLECTRA TM 5PSP device.

[0137] The lot numbers of VGX-3100 used in the clinical trials were 1622-035 and 1816-045.

[0138] Control product, dose and administration method, batch number:

[0139] The control product (placebo) consists of a mixture of 150 mM sodium chloride and 15 mM sodium citrate. The placebo is packaged in a clear glass cartridge and administered by IM injection (deltoid [preferred site] or anterolateral quadriceps [alternate site]), followed immediately by EP using the CELLECTRA TM 5PSP device.

[0140] The lot number of the placebo used in the clinical trials was 1622-066.

[0141] Treatment duration:

[0142] Treatment was administered over 12 weeks. The first clinical trial treatment was given on Day 0, the second at Week 4, and the third (final) at Week 12.

[0143] Estimated objectives and concurrent events:

[0144] The estimation objectives and concurrent events were not defined in the clinical trial protocol or the Statistical Analysis Plan (SAP).

[0145] Statistical methods:

[0146] Sample size

[0147] A total of 198 subjects were planned to be randomly assigned at a ratio of 2:1 to receive 6 mg of VGX-3100 or placebo via IM, followed by EP. Assuming the true proportions of subjects achieving the primary endpoint are 35% and 14% for VGX-3100 and placebo, respectively, this sample size provides 90% power to show that VGX-3100 is superior to placebo. These proportions also include missing data (about 10%) classified as non-responders (failures). These assumptions are based on the results of the Phase 2 clinical trial.

[0148] Analysis set

[0149] The analysis sets are defined as follows:

[0150] Intention-to-Treat (ITT) set: The ITT set includes all randomly assigned subjects. Subjects in this sample were treated according to their random assignment. The primary efficacy analysis of this clinical trial used the Intention-to-Treat (ITT) set. Missing data were considered non-responders (failures) for the ITT efficacy analysis. If the CIN grade and HPV clearance at Week 36 could not be determined for a subject, their response outcome was missing. The ITT set was also used to summarize demographic information, baseline characteristics, dispositions, and protocol deviations.

[0151] Modified Intention-to-Treat (mITT) set: The mITT set includes all subjects who received at least one (1) dose of the clinical trial treatment and achieved the objective analysis endpoint. Subjects in this sample were treated according to their random assignment. The analysis of the mITT set was considered as support for the efficacy analysis of the corresponding ITT set and was also used as a sensitivity analysis regarding missing data.

[0152] Per-Protocol (PP) set: The PP set includes subjects who received all doses of the clinical trial treatment, did not violate the protocol, and achieved the objective analysis endpoint. Subjects in this sample were treated according to their random assignment. The analysis of the PP set was considered as support for the efficacy analysis of the corresponding ITT set. The additional efficacy analysis of the PP set utilized the Week 36 results and was independent of any procedures performed prior to Week 36, and thus was used as a sensitivity analysis regarding early intervention. Subjects excluded from the PP set were identified and recorded before the unblinding of the clinical trial database.

[0153] Safety set: The safety set includes all subjects who received at least one (1) dose of the clinical trial treatment. The treatment actually received by the subjects was analyzed.

[0154] Efficacy analysis

[0155] Primary analysis of the primary efficacy endpoint

[0156] The primary efficacy endpoint is the absence of evidence of cervical HSIL (i.e., no evidence of CIN2 and CIN3) on histological examination (i.e., biopsy or excision treatment) at week 36 and the absence of evidence of HPV-16 and / or HPV-18 in cervical samples by type-specific HPV testing.

[0157] The main hypothesis for superiority is: H0: δ ≤ 0 versus H1: δ > 0, where δ = P v - P p , and P v and P p represent the true population probabilities of the primary endpoint for VGX-3100 and placebo, respectively. The proportion in each treatment group is calculated as: the number of responders in the clinical trial population of the corresponding treatment group divided by the total number of responders and non-responders.

[0158] For the primary endpoint, histology at week 36 was assessed based on the first biopsy or surgical excision procedure on or after day 238; if multiple results occurred on the same day, the result with the lowest grade was used. The virological result for analysis should be the most recent result obtained on the same day as or before the histological result and on or after day 238. If a subject undergoes excision or cervical biopsy at any time on or after day 1 and before day 238, the subject is considered a non-responder.

[0159] Shows the number of responders and non-responders, the proportion of responders in each treatment group, and the difference in proportions between the two (2) treatment groups. Using the method of Miettinen and Nurminen, the superiority p-value based on the risk difference test and the corresponding 95% confidence interval (CI) were calculated. If the one-sided p-value < 0.025 and the lower limit of the corresponding 95% CI exceeds zero (0), a conclusion of superiority can be drawn.

[0160] The proportion of responders was also summarized separately based on stratification factors.

[0161] Analysis of the primary efficacy endpoint using the mITT set and the PP set was used as a sensitivity analysis and was considered supportive of the corresponding analysis using the ITT set.

[0162] Secondary efficacy endpoint analysis

[0163] The analysis method for the secondary efficacy endpoint is the same as that for the primary efficacy endpoint, including the sensitivity analysis, but the p-value for the secondary efficacy endpoint analysis was not calculated.

[0164] Immunogenicity analysis

[0165] Immunogenicity analysis was performed on the mITT set with at least one (1) immunogenicity measurement.

[0166] Serum anti-HPV-16 and anti-HPV-18 antibody concentration levels, as well as interferon (IFN)-γ enzyme-linked immunosorbent spot (ELISpot) response magnitudes, were assessed based on serum and peripheral blood mononuclear cells (PBMCs) isolated from whole blood collected at baseline, week 15, and week 36, respectively. Flow cytometry response magnitudes were assessed based on PBMCs isolated from whole blood collected at baseline and week 15.

[0167] At each visit, all endpoints were aggregated as continuous variables. The increases in ELISpot and flow cytometry relative to baseline at each post-baseline visit, as well as the titers in enzyme-linked immunosorbent assay (ELISA) at each post-baseline visit, were compared between treatment groups. These comparisons were analyzed using the median difference and the associated exact nonparametric 95% CI.

[0168] Exploratory endpoint analysis

[0169] Using a separate logistic regression model, the relationship between histological regression and virological clearance of cervical HSIL at the Week 36 visit (yes, no) was examined for each outcome, as well as a) miRNA results, b) DNA methylation results, c) colposcopy results, d) cytology results, e) HPV results, and f) baseline immune score results (tissue-based score derived using immune markers), where histological regression and virological clearance of cervical HSIL were used as the response variables, and each outcome and treatment group were used as regression variables. Odds ratios (ORs) and corresponding 95% CIs were provided for the regression variables. Durability (measured by clearance of HPV-16 and / or HPV-18 infection at Weeks 62 and 88) was summarized by treatment group as the number and percentage of subjects without evidence of HPV-16 and / or HPV-18 infection at each visit. Specifically, the virological outcome at Week 62 was the outcome between Day 420 and Day 448, and the virological outcome at Week 88 was the outcome between Day 602 and Day 630. Assessment of markers (including but not limited to CD8+ and FoxP3+ infiltrating cells) was performed, as well as visualization of granulysin, perforin, CD137, CD103, and PD-L1 in cervical tissue. Using the mean difference and the associated t-distribution-based 95% CI, the change in the magnitude of these tissue responses relative to baseline was compared between treatment groups at each post-baseline time point. Subjects enrolled in the United States, Canada, Mexico, Germany, and the United Kingdom were required to complete patient-reported outcome (PRO) questionnaires (36-item Short Form Survey [SF-36], EuroQol 5 Dimensions 5 Levels [EQ-5D-5L], and two [2] additional general PRO questions assessing quality of life [QoL] after resection or biopsy). For the SF-36, at each visit, the component scores for each of the eight (8) domains (physical function, role limitations due to physical problems, bodily pain, general health, vitality, social function, role limitations due to emotional problems, and mental health) were summarized by treatment group, as well as the change in score relative to baseline. The eight (8) SF-36 subscales were scored using QualityMetric Health Outcomes(tm) Scoring Software 5.0. For the EQ-5D-5L, at each visit, each of the five (5) domains (mobility, self-care, usual activities, pain / discomfort, anxiety / depression) and the overall health status score were summarized by treatment group and relative to baseline. For the two [2] additional general PRO questions assessing QoL after resection or biopsy, the time-to-outcome (median number of days to QoL deterioration) and the binary outcome of the yes / no question were summarized by treatment group. Using exact non-parametric 95% CIs, the difference in the median change in SF-36 scores relative to baseline was compared between treatment groups. EQ-5D-5L scores were analyzed in the same manner.The median difference in the number of days of QoL deterioration in the QoL questionnaire at Week 40 was analyzed using exact non-parametric 95% CIs. The proportional difference in the responses of yes / no QoL deterioration in the QoL questionnaire at Week 40 between treatment groups was analyzed using 95% Miettinen and Nurminen CIs. Additionally, PRO endpoints at each visit occurring after Week 36 were aggregated according to patients who received resection (excluding biopsies) and those who did not. Subjects in the mITT population with at least one (1) post-baseline corresponding measurement were used for PRO analysis.

[0170] Safety analysis

[0171] All safety analyses were conducted in the safety population.

[0172] Adverse events (AEs)

[0173] Adverse event verbatim reporting terms were coded using the latest version of the Medical Dictionary for Regulatory Activities (MedDRA) by System Organ Class (SOC) and Preferred Term (PT).

[0174] The adverse event summary tables included the number and percentage of subjects who experienced at least one (1) event by treatment group. The following AE summary tables were generated:

[0175] · An overview of the overall AEs, and those occurring within 28 days / 7 days after clinical trial treatment

[0176] · An overview of the treatment-emergent AEs (TEAEs) by SOC and PT, and those occurring within 28 days / 7 days after clinical trial treatment

[0177] · An overview of the TEAEs by dose number, SOC and PT, and those occurring within 28 days / 7 days after clinical trial treatment

[0178] · An overview of the TEAEs by dose number, Common Terminology Criteria for Adverse Events (CTCAE) grade, SOC and PT, and those occurring within 28 days / 7 days after clinical trial treatment

[0179] · An overview of the serious TEAEs by dose number, SOC and PT, and those occurring within 28 days / 7 days after clinical trial treatment

[0180] · An overview of the TEAEs by dose number, relationship to investigational product (IP) and EP, SOC and PT, and those occurring within 28 days / 7 days after clinical trial treatment

[0181] ·By dose number, relationship with IP and EP, overall incidence of serious TEAEs for SOC and PT, and occurring within 28 days / 7 days after clinical trial treatment

[0182] ·By dose number, overall incidence of ≥ grade 3 TEAEs for SOC and PT, and occurring within 28 days / 7 days after clinical trial treatment

[0183] ·By dose number, relationship with IP and EP, overall incidence of ≥ grade 3 TEAEs for SOC and PT, and occurring within 28 days / 7 days after clinical trial treatment

[0184] ·By dose number, overall incidence of AEs occurring during the treatment period of clinical trial treatment for SOC and PT, and occurring within 28 days / 7 days after clinical trial treatment

[0185] ·By dose number, overall incidence of AEs occurring during the treatment period of permanent discontinuation of clinical trial treatment for SOC and PT, and occurring within 28 days / 7 days after clinical trial treatment

[0186] ·By dose number, overall incidence of adverse events of special interest for SOC and PT, and occurring within 28 days / 7 days after clinical trial treatment

[0187] Subjects who experienced two (2) or more different AEs within the same MedDRA term and treatment regimen level are only counted once at that level, using the most extreme event (the most severe event in the intensity analysis and the relevant event in the analysis of the relationship with clinical trial treatment).

[0188] Data listings for AEs, SAEs, AEs leading to treatment discontinuation, AEs with CTCAE grade ≥ 3, AEs occurring within 28 days / 7 days after clinical trial treatment, and AEs leading to death are provided separately.

[0189] Incidence of adverse events

[0190] For AEs occurring within 28 days after any dose, the frequencies of SOC and PT events were compared between treatment groups with risk difference and 95% CI using the method of Miettinen and Nurminen. Since this analysis uses many event categories and generates many CIs, these CIs should be interpreted with caution.

[0191] Similar analyses were also performed for AEs within 7 days after any dose and for AEs during the clinical trial period after any dose.

[0192] Clinical laboratory evaluations

[0193] Safety laboratory tests (including hematology, serum chemistry, and urine analysis) are conducted during screening (within 30 days prior to Day 0), and a summary of the screening information is provided by treatment group and in total.

[0194] Vital signs, physical examinations, and other safety-related observations

[0195] At each visit, vital sign data are summarized by treatment group. Changes from baseline to each scheduled post-baseline visit are shown.

[0196] During the physical examination, body systems are evaluated as normal, abnormal, or not examined at each scheduled visit, and the percentage of subjects with abnormal physical examination findings is summarized by body system and treatment group at each time point.

[0197] ECG results are summarized by treatment group and in total for interpretive results, which include normal, clinically insignificant abnormalities, clinically significant abnormalities, and incomplete.

[0198] Definition of responders and non-responders for the primary endpoint

[0199] Responders and non-responders for the primary endpoint should be defined by considering both histopathological regression of cervical HSIL and virological (HPV-16 and / or HPV-18) clearance in cervical samples, as HPV persistence is an important factor in the clinical progression of HSIL. The responder definition also excludes subjects who underwent excision or cervical biopsy at any time between the initial dose and the 36-week endpoint tissue collection. This exclusion is intended to reduce the likelihood of an artificial increase in treatment effect due to the removal of HSIL tissue by unplanned intermittent biopsies and potential HPV-16 and / or HPV-18. Only subjects who underwent ECC prior to the 36-week endpoint collection will be considered for responder analysis.

[0200] To meet the criteria for responders, subjects must have the following:

[0201] 1. An acceptable histological specimen obtained at Week 36 that can be interpreted by an independent PAC; and

[0202] 2. An acceptable HPV ThinPrep TM sample obtained at Week 36 with associated valid HPV-test results. Responders are defined as:

[0203] 1. Subjects with no histological evidence of cervical HSIL at the Week 36 assessment

[0204] 2. Subjects with no evidence of HPV-16 and / or HPV-18 at the Week 36 assessment

[0205] 3. Subjects who did not receive an unplanned excision or biopsy sample between the initial dose and the Week 36 assessment.

[0206] Conversely, non - responders were defined as:

[0207] 1. Subjects with histological evidence of cervical HSIL, AIS, or cervical cancer at the Week 36 assessment, or

[0208] 2. Subjects with evidence of HPV - 16 or HPV - 18 at the Week 36 assessment, or

[0209] 3. Subjects who had an excision or biopsy sample obtained between the initial dose and the Week 36 assessment, or

[0210] 4. Subjects lacking an acceptable Week 36 histological specimen or HPV ThinPrep TM sample.

[0211] The absence of evidence of HSIL was histologically defined as negative, squamous atypia, or LSIL. The histopathological efficacy time limit was defined by patients who had a biopsy or surgical excision at any time starting 14 days before Week 36 from the protocol - specified target date. The histological endpoint was determined by the first tissue removal sample within this time limit. The most recent HPV clearance result (including results on the same date and within the same time limit) before tissue removal determined the HPV clearance endpoint.

[0212] Definition of responders and non-responders for the secondary endpoint

[0213] Responders and non - responders for the secondary endpoint of histopathological regression of cervical HSIL are defined in detail in Table 4.

[0214] Table 5: Definitions of Responders and Non - Responders for the Secondary Endpoint of Complete Histopathological Regression of Cervical HSIL

[0215]

[0216]

[0217] Abbreviations: AIS: Adenocarcinoma in situ; ECC: Endocervical curettage; HSIL: High - grade squamous intraepithelial lesion; LSIL: Low - grade squamous intraepithelial lesion.

[0218] a: The efficacy time limit was defined for those subjects who had a biopsy or surgical excision at any time starting 14 days before Week 36 from the protocol - specified target date. The histological endpoint was determined by the first tissue removal sample within the time limit.

[0219] b: Samples that received only ECC were excluded.

[0220] Responders and non-responders for the secondary endpoint of histopathological progression-free in cervical HSIL are defined in detail in Table 6.

[0221] Table 6: Definitions of Responders and Non-Responders for the Secondary Endpoint of Histopathological Progression-Free in Cervical HSIL

[0222]

[0223] Abbreviations: ECC: Endocervical Curettage; HSIL: High-Grade Squamous Intraepithelial Lesion.

[0224] a: Define the efficacy time limit for those subjects who have undergone biopsy or surgical resection at any time starting 14 days before week 36 from the protocol-specified target date. The histological endpoint is determined by the first tissue removal sample within the time limit.

[0225]

[0226]

[0227]

[0228] b: Exclude samples that only received ECC.

[0229] Responders and non-responders for the secondary endpoints of complete histopathological regression of cervical HSIL and virological clearance of HPV are defined in detail in Table 7.

[0229] Table 7: Definitions of Responders and Non-Responders for the Secondary Endpoints of Complete Histopathological Regression of Cervical HSIL and Virological Clearance of HPV

[0230]

[0231] Abbreviations: AIS: Adenocarcinoma in Situ; ECC: Endocervical Curettage; HPV: Human Papillomavirus; HSIL: High-Grade Squamous Intraepithelial Lesion; LSIL: Low-Grade Squamous Intraepithelial Lesion.

[0232] a: Absence of evidence of HSIL is histologically defined as negative, squamous atypia, or LSIL

[0233] b: Define the efficacy time limit for those subjects who have undergone biopsy or surgical resection at any time starting 14 days before week 36 from the protocol-specified target date. The histological endpoint is determined by the first tissue removal sample within the time limit. The most recent HPV clearance result before tissue removal (including results on the same date and within the same time limit) determines the HPV clearance endpoint.

[0234] c: Exclude samples that only received ECC.

[0235] Summary of results

[0236] Subject Disposition: Subject disposition is summarized in Table 8.

[0237] Table 8: Subject Disposition (ITT Population)

[0238]

[0239]

[0240]

[0241] Note: Two (2) subjects were randomized to the VGX-3100 + EP group but did not receive treatment.

[0242] Note: The denominator used in the percentage calculations was the subjects randomized in each treatment.

[0243] Abbreviations: EP: Electroporation; ITT: Intention-to-Treat; N: Sample size within group; n: Number of subjects.

[0244] Demographics and baseline characteristics

[0245] The mean age of the subjects was 31.5 years, and the age range was 20 to 55 years. Most subjects (77.1%) were white, non-Hispanic or Latino (82.6%). The mean BMI was 25.07 kg / m2, and the range was 16.5 to 56.5 kg / m2. The demographic characteristics of the two treatment groups were similar.

[0246] Randomization was stratified at baseline based on age, BMI, and CIN stage. The proportion of subjects between the treatment groups was similar in each individual stratum and combination of strata.

[0247] Sixteen subjects (8.0%) had received a prophylactic HPV vaccine prior to participating in the clinical trial: 14 subjects (10.1%) in the VGX-3100 + EP group and 2 subjects (3.2%) in the placebo + EP group. Most (76.6%) subjects had an abnormal result on their most recent Pap smear. The most recent Pap smear was performed between 21 and 735 days prior to baseline. Most subjects were diagnosed with CIN2 (43.9%) or CIN3 (39.8%). The median time from initial CIN diagnosis was 86.5 days, and the range was 21 to 4695 days. Fourteen subjects (7.0%) had received prior CIN treatment, most commonly LEEP (11 subjects [78.6%]). The median time from the last CIN treatment was 489.0 days, and the range was 250 to 4771 days.

[0248] The ITT set includes all randomly assigned subjects. The subjects in this sample were treated according to randomization. The primary efficacy analysis of this clinical trial used the intention-to-treat (ITT) set. Missing data were considered non-responders (failures) for the ITT efficacy analysis. If the CIN grade and HPV clearance at week 36 could not be determined for a subject, their response outcome was missing. The ITT set was also used to summarize demographic information, baseline characteristics, treatments, and protocol deviations.

[0249] The mITT set includes all subjects who received at least one (1) dose of the clinical trial treatment and achieved the objective analysis endpoint. The subjects in this sample were treated according to randomization. The analysis of the mITT set was considered to support the efficacy analysis of the corresponding ITT set and was also used as a sensitivity analysis regarding missing data.

[0250] The PP set includes subjects who received all doses of the clinical trial treatment, did not violate the protocol, and achieved the objective analysis endpoint. The subjects in this sample were treated according to randomization. The analysis of the PP set was considered to support the efficacy analysis of the corresponding ITT set. The additional efficacy analysis of the PP set utilized the results at week 36, independent of any procedures performed prior to week 36, and thus served as a sensitivity analysis regarding early intervention. Subjects excluded from the PP set were identified and recorded prior to unblinding the clinical trial database.

[0251] The safety set includes all subjects who received at least one (1) dose of the clinical trial treatment. The treatments actually received by the subjects were analyzed.

[0252] Table 9: Analysis Sets

[0253]

[0254] Note: The ITT set includes all randomly assigned subjects. The mITT set includes all subjects who received at least one (1) dose of the clinical trial treatment (based on subjects evaluated for any objective analysis endpoint). The per-protocol group includes subjects who have received all doses of the clinical trial treatment and have not deviated from the protocol (based on subjects evaluated for any objective analysis endpoint). The safety set includes subjects who received at least one (1) dose of the clinical trial treatment.

[0255] Please note that subjects who received a treatment protocol exactly opposite to the designated protocol and subjects who received a mixed treatment protocol are included in the column where the subjects were randomly assigned, but the safety outcome analysis for these subjects was based on the treatment they actually received.

[0256] Abbreviations: EP: Electroporation; N: Sample size within the group.

[0257] Efficacy results

[0258] Overall, for the ITT, mITT, and PP populations, for the primary endpoint of histopathologic regression of cervical HSIL and virologic clearance of HPV-16 and / or HPV-18 at Week 36, the percentage of responders was higher in the VGX-3100 + EP group compared to the placebo + EP group. In the mITT and PP populations, the percentage of responders for the primary endpoint was significantly higher in the VGX-3100 + EP group compared to the placebo + EP group (p-value < 0.025). Similar results were seen for the secondary endpoint of histopathologic regression of cervical HSIL at Week 36. For the other secondary endpoints (including virologic clearance of HPV16 and / or HPV-18 at Week 36; histopathologic regression of cervical HSIL to normal and virologic clearance of HPV-16 and / or HPV-18 at Week 36; histopathologic regression of cervical HSIL to normal at Week 36; and virologic clearance of HPV-16 and / or HPV-18 at non-cervical anatomic sites at Week 36), the percentage of responders was higher in the VGX 3100 + EP group compared to the placebo + EP group, and the lower limit of the 95% CI generally exceeded zero (0). The efficacy results are summarized below.

[0259] Primary Efficacy Endpoint

[0260] The primary endpoint was the absence of evidence of cervical HSIL on histological examination (i.e., biopsy or excision treatment) and the absence of evidence of HPV-16 and / or HPV-18 in cervical samples by type-specific HPV testing at the Week 36 visit.

[0261] In the ITT population, the percentage of responders (i.e., subjects with histopathologic regression of cervical HSIL and virologic clearance of HPV-16 and / or HPV-18 at Week 36) in the VGX-3100 + EP group was 22.5%, while the percentage of responders in the placebo + EP group was 11.1%. The difference between the two (2) groups of responders was not statistically significant (one-sided p-value = 0.029).

[0262] In the mITT population, 31 subjects (23.7%) in the VGX-3100 + EP group and 7 subjects (11.3%) in the placebo + EP group were considered responders. The difference between the two (2) groups of responders was statistically significant (one-sided p-value = 0.022), indicating that in the mITT population, VGX-3100 + EP was superior to placebo + EP in achieving histopathologic regression of cervical HSIL and virologic clearance of HPV-16 and / or HPV 18 at Week 36.

[0263] Among the PP population, 30 subjects (24.6%) in the VGX-3100 + EP group and 7 subjects (11.7%) in the placebo + EP group were considered responders. The difference between the two (2) groups of responders was statistically significant (one-sided p-value = 0.021), indicating that in the PP population, VGX 3100 + EP was superior to placebo + EP in achieving histopathological regression of cervical HSIL and virological clearance of HPV-16 and / or HPV 18 at week 36. The results of the PP population and the sensitivity analysis of the PP population supported the results of the mITT population.

[0264] Secondary efficacy endpoint

[0265] Histopathological regression of cervical HSIL at week 36: In the ITT population, the percentage of responders (i.e., subjects with histopathological regression of cervical HSIL at week 36) in the VGX-3100 + EP group was 31.9%, while the percentage of responders in the placebo + EP group was 19.0%. For the mITT population, the percentage of responders in the VGX-3100 + EP group was 33.6%, while the percentage of responders in the placebo + EP group was 19.4%, and the lower limit of the 95% CI of the difference in responder percentages exceeded zero (0), indicating that in the mITT population, VGX-3100 + EP was more efficacious in achieving histopathological regression of cervical HSIL at week 36. The results of the PP population and the sensitivity analysis of the PP population supported the results of the mITT population.

[0266] Virological clearance of HPV-16 and / or HPV-18 at week 36 : For the ITT population, the percentage of responders (34.1%) in the VGX-3100 + EP group was higher than the percentage of responders (15.9%) in the placebo + EP group. The lower limit of the 95% CI of the difference in responder percentages exceeded zero (0), indicating that VGX-3100 + EP was more efficacious in achieving virological clearance of HPV-16 and / or HPV-18 at week 36. The results of the mITT and PP populations and the sensitivity analysis of the PP population supported the results of the ITT population.

[0267] Histopathological regression of cervical HSIL to normal and virological clearance of HPV-16 and / or HPV-18 at week 36 : For the ITT population, the percentage of responders (18.1%) in the VGX-3100 + EP group was higher than the percentage of responders (6.3%) in the placebo + EP group. The lower limit of the 95% CI of the difference in responder percentages exceeded zero (0), indicating that VGX-3100 + EP was more efficacious in achieving histopathological regression of cervical HSIL to normal and virological clearance of HPV-16 and / or HPV-18 at week 36. The results of the mITT and PP populations and the sensitivity analysis of the PP population supported the results of the ITT population.

[0268] Histopathological regression of cervical HSIL to normal at week 36 : For the ITT population, the percentage of responders in the VGX-3100 + EP group (24.6%) was higher than the percentage of responders in the placebo + EP group (11.1%). The lower limit of the 95% CI for the difference in responder percentages exceeded zero (0), indicating that VGX-3100 + EP was superior in achieving histopathological regression of cervical HSIL to normal at week 36. The results for the mITT and PP populations and the sensitivity analysis for the PP population supported the ITT population results.

[0269] Cervical HSIL did not progress to cervical cancer at week 36 : For the ITT population, the percentage of responders in the VGX-3100 + EP group (84.1%) was similar to the percentage of responders in the placebo + EP group (85.7%). The 95% CI for the difference in responder percentages did not exclude zero (0), indicating that there was no difference in the efficacy of VGX-3100 + EP and placebo + EP in preventing the progression of cervical HSIL to cervical cancer at week 36. The results for the mITT and PP populations and the sensitivity analysis for the PP population supported the ITT population results.

[0270] Virological clearance of HPV-16 and / or HPV-18 at non-cervical anatomical sites at week 36 : In the ITT population, the percentage of responders (i.e., subjects with virological clearance of HPV-16 and / or HPV-18 in non-cervical anatomical locations at week 36) in the VGX-3100 + EP group was 20.3%, while the percentage of responders in the placebo + EP group was 9.5%. In the mITT population, the percentage of responders in the VGX-3100 + EP group was 24.8%, while the percentage of responders in the placebo + EP group was 10.7%. The lower limit of the 95% CI for the difference in responder percentages in the mITT population exceeded zero (0), indicating that in the mITT population, VGX-3100 + EP was superior in achieving virological clearance of HPV-16 and / or HPV-18 in non-cervical anatomical locations at week 36. The results for the PP population supported the mITT population results.

[0271] Subgroup analysis

[0272] Subgroup analyses of the primary and secondary efficacy endpoints were performed by prophylactic HPV vaccine exposure history (yes, no). The histopathological regression of cervical HSIL and the virological clearance of HPV-16 and / or HPV-18 at week 36 were also evaluated by stratification factors.

[0273] Among subjects without a history of prophylactic HPV vaccine exposure, VGX-3100 + EP was superior to placebo + EP in the following efficacy measures:

[0274] · Histopathological regression of cervical HSIL and virological clearance of HPV-16 and / or HPV-18 at week 36

[0275] · Virological clearance of HPV-16 and / or HPV-18 at week 36

[0276] ● Histopathological regression of cervical HSIL to normal and virological clearance of HPV-16 and / or HPV-18 at week 36

[0277] ● Histopathological regression of cervical HSIL to normal at week 36

[0278] · Virological clearance of HPV-16 and / or HPV-18 at non-cervical anatomical sites at week 36.

[0279] Regardless of prior prophylactic HPV vaccination, the percentage of responders in the VGX-3100+EP and placebo+EP groups was similar for the following efficacy measures:

[0280] ● Histopathological regression of cervical HSIL at week 36

[0281] · Non-progression of cervical HSIL to cervical cancer at week 36.

[0282] For all stratification combinations, the percentage of subjects with histopathological regression of cervical HSIL and virological clearance of HPV-16 and / or HPV-18 at week 36 was higher in the VGX-3100+EP group compared to the placebo+EP group. Among subjects aged ≥25 years, BMI ≤25 kg / m 2 and with CIN2, the difference in the percentage of responders was highest (5.8%).

[0283] Exploratory efficacy endpoints

[0284] Overall summary

[0285] The effects of prior miRNA and DNA methylation, colposcopy, cytology, and HPV results, baseline immune scores, and baseline biomarker status on histopathological regression of cervical HSIL and virological clearance of HPV-16 and / or HPV-18 at week 36 were evaluated. At the individual subject level, miRNA values at day 0 and week 8 and DNA methylation values at day 0 and week 15 did not significantly affect the odds of response, except for NKAIN2 methylated at day 0, where the odds of response decreased with increasing NKAIN2 at day 0. If the colposcopy result at week 15 showed improvement compared to no change, the odds of achieving a response at week 36 were 3.55-fold higher (95% CI: 1.69, 7.48), and if the colposcopy result at week 28 showed improvement compared to no change, the odds of achieving a response at week 36 were 2.93-fold higher (95% CI: 1.40, 6.13). If the cytology result at week 15 showed improvement compared to no change, the odds of achieving a response at week 36 were 2.24-fold higher (95% CI: 1.01, 4.99), and if the cytology result at week 28 showed improvement compared to possible progression, the odds of achieving a response at week 36 were 9.78-fold higher (95% CI: 1.23, 77.92). If HPV was cleared at weeks 8, 15, and 28, the odds of achieving a response at week 36 (95% CI) were 7.93-fold higher (2.92, 21.54), 10.65-fold higher (4.39, 25.86), and 27.83-fold higher (10.64, 72.76), respectively, compared to not being cleared at these time points, indicating that HPV clearance was associated with response and the response rate increased over time. The baseline immune score did not affect the odds of achieving a response (odds ratio [95% CI]: 0.99 [0.63, 1.56]). Compared to placebo + EP, VGX-3100 + EP showed superior efficacy in inducing histopathological regression of cervical HSIL and virological clearance of HPV-16 and / or HPV-18 at week 36 in subjects with a positive baseline biomarker status, but had no such effect on subjects with a negative baseline biomarker status.

[0286] Compared to the placebo + EP group, a higher percentage of subjects in the VGX-3100 + EP group had no evidence of cervical HPV-16 and / or HPV-18 at weeks 62 and 88; however, the difference between the groups narrowed at week 88.

[0287] Patient-reported outcome measures (SF-36, EQ-5D-5L, week 40 QoL response) were generally similar between the VGX-3100 + EP and placebo + EP groups.

[0288] Effect of baseline biomarker status on histopathological regression of cervical HSIL and virological clearance of HPV-16 and / or HPV-18 at week 36

[0289] Table 10 summarizes the impact of baseline biomarker status on histopathological regression of cervical HSIL and virological clearance of HPV-16 and / or HPV-18 at week 36 for the ITT population.

[0290] Biomarker status was determined based on a combination of the following 12 miRNAs: hsa.miR.375.3p, hsa.miR.425.5p, hsa.miR.193a.5p, hsa.miR.199b.5p, hsa.miR.365a.3p, hsa.miR.223.5p, hsa.miR.148b.3p, hsa.miR.143.3p, hsa.miR.941, hsa.miR.340.5p, hsa.miR.151a.3p, hsa.miR.23a.3p. These 12 miRNAs were then used to train a classification model that generated parameters to create a final model for defining individual subjects as "biomarker positive" or "biomarker negative".

[0291] Among subjects with a positive baseline biomarker status, 73.0% of subjects in the VGX-3100 + EP group and 6.3% of subjects in the placebo + EP group were considered responders. The difference in responder percentages (95% CI) was 66.7 (47.8, 88.6). The lower limit of the 95% CI exceeded zero (0), indicating that VGX-3100 + EP was more efficacious in inducing histopathological regression of cervical HSIL and virological clearance of HPV-16 and / or HPV-18 at week 36 among subjects with a positive baseline biomarker status.

[0292] Among subjects with a negative baseline biomarker status, two (2) subjects in the VGX-3100 + EP group and 11% of subjects in the placebo + EP group were considered responders. The difference in responder percentages (95% CI) was -8.9 (-19.1, 1.3). Table 10: Histopathological regression of cervical HSIL and virological clearance of HPV-16 and / or HPV-18 at week 36 by baseline biomarker status

[0293] (ITT population)

[0294]

[0295] Note: The denominator used in the calculation of the overall percentage was the number of subjects randomized. The denominator used in the calculation of the percentage for the baseline biomarker status was the number of subjects corresponding to biomarker positive, biomarker negative, and biomarker undetermined, respectively.

[0296] Abbreviations: CI: confidence interval; EP: electroporation; HPV: human papillomavirus; HSIL: high-grade squamous intraepithelial lesion; ITT: intention-to-treat; N: sample size within the group; n: number of subjects; NE: not estimated.

[0297] a: Responders were defined as: 1) subjects without histological evidence of cervical HSIL at the 36-week time point; and 2) subjects without evidence of HPV-16 and / or HPV-18. In addition, subjects who underwent resection or cervical biopsy at the initial dose or at any time after the initial dose and before the 36-week time point were considered non-responders. The histopathological efficacy time point was defined by biopsy or surgical resection at any time starting 14 days before the 36th week from the protocol-specified target date. The histological endpoint was determined by the first tissue removal sample within the time point. The most recent HPV clearance result (including results on the same date and within the same time point) before tissue removal determined the HPV clearance endpoint.

[0298] b: Biomarker status was determined based on a combination of the following 12 microRNAs: hsa.miR.375.3p, hsa.miR.425.5p, hsa.miR.193a.5p, hsa.miR.199b.5p, hsa.miR.365a.3p, hsa.miR.223.5p, hsa.miR.148b.3p, hsa.miR.143.3p, hsa.miR.941, hsa.miR.340.5p, hsa.miR.151a.3p, hsa.miR.23a.3p.

[0299] Immunogenicity results:

[0300] The immunogenicity results were as follows:

[0301] · As can be seen from the geometric mean of the reciprocal endpoint titers, VGX-3100 was immunogenic, and at weeks 8, 15, and 36, for both HPV-16E7 and HPV-18E7, the reciprocal endpoint titers in the VGX-3100+EP group were several-fold higher than those in the placebo+EP group. Anti-HPV-16E6 and anti-HPV-18E6 antibodies were not measured.

[0302] · At all post-baseline time points (Week 8, Week 15, and Week 36), compared with the placebo + EP group, the spot-forming units (SFU) per 10 6 PBMCs of HPV-16 E6, HPV-16 E7, HPV-18 E6, HPV-18 E7, and related combinations increased from baseline in the VGX-3100 + EP group.

[0303] · At Week 8, Week 15, and Week 36, the median increases in CEF and PMA levels in the two treatment groups seemed similar.

[0304] ● At Week 15, compared with the placebo + EP group, all parameters (including CD8+CD137+ perforin+, CD8+CD38+ perforin+, and CD8+CD69+ perforin+) showed increases in the VGX-3100 + EP group, indicating a stronger cellular immune response by flow cytometry (as measured by activated CD8+ T cells with lytic potential).

[0305] · The changes in CD8+, CD103+, FoxP3+, and perforin+ cells in normal cervical epithelium, normal stroma, CIN2 / 3 epithelium, and CIN2 / 3 stroma from baseline to Week 36 were small and were generally similar between the treatment groups.

[0306] Safety results:

[0307] Overall, for subjects with HPV-16 and / or HPV-18-related cervical HSIL, IM injection of VGX-3100 or placebo followed by EP was well tolerated. The safety results are summarized below.

[0308] ● By the end of the clinical trial, the incidence of TEAE was similar in the two treatment groups.

[0309] · A total of 96.3% of the subjects in the VGX-3100 + EP group and 98.4% of the subjects in the placebo + EP group reported at least one (1) TEAE.

[0310] ● AE (TEAE) occurring during treatment was considered severe in 9.6% of the subjects in the VGX-3100 + EP group and 9.7% of the subjects in the placebo + EP group. Most SAEs reported during treatment were in the SOC of benign, malignant, and tumors of unknown nature (including cysts and polyps). All SAEs were not related to IP or EP. One (1) subject died of a pulmonary embolism TEAE on Day 450 (i.e., 365 days after the 3rd dose of VGX-3100 + EP) during the clinical trial.

[0311] ·Overall, injection site pain, headache, fatigue, injection site erythema, injection site pruritus, myalgia, and injection site swelling were the most frequently reported TEAEs during the clinical trial, and the incidences of these TEAEs in the VGX-3100 + EP group and the placebo + EP group were as follows: 78.7% and 80.6% of the subjects reported injection site pain; 33.1% and 30.6% of the subjects reported headache; 28.7% and 27.4% of the subjects reported fatigue;

[0312] 25.0% and 22.6% of the subjects reported injection site erythema and injection site pruritus; 21.3% and 24.2% of the subjects reported myalgia; and 20.6% and 24.2% of the subjects reported injection site swelling.

[0313] ·The incidence of most TEAEs was not related to the number of doses received by the subjects. In the VGX-3100 + EP group and the placebo + EP group, a few TEAEs were reported more frequently after the first dose, and their incidences generally decreased after the second and third doses. These TEAEs included nausea, fatigue, injection site pain, injection site swelling, discomfort, myalgia, and headache. In the placebo + EP group, the incidences of injection-site erythema and injection site pruritus decreased after the first dose, but in the VGX-3100 + EP group, the incidences of injection site erythema and injection site pruritus increased slightly after the first dose. Most of these TEAEs were reported within 28 days after the clinical trial treatment.

[0314] ·Regardless of when they occurred after the administration of the clinical trial treatment, the intensity of most TEAEs was grade 1 or 2. 14.0% of the subjects in the VGX-3100 + EP group and 11.3% of the subjects in the placebo + EP group reported treatment-emergent TEAEs with CTCAE ≥ grade 3. The most common treatment-emergent TEAEs with CTCAE ≥ grade 3 included injection-site pain and headache.

[0315] ·Treatment-emergent AEs were related to IP or EP in 83.8% of the subjects in the VGX-3100 + EP group and 90.3% of the subjects in the placebo + EP group. Overall, by the end of the clinical trial, injection site pain, fatigue, and headache were the most frequently reported IP- or EP-related TEAEs. The investigators reported that by the end of the clinical trial, > 1% of the subjects had treatment-emergent AEs with CTCAE ≥ grade 3 severity related to IP, EP, or both, including injection site pain, myalgia, and headache. All of these TEAEs were reported within 7 days after the clinical trial treatment.

[0316] ●TEAEs that led to discontinuation of the clinical trial treatment included injection site pain and upper respiratory tract infection. VGX-3100 +

[0317] In the EP group, TEAE of injection site pain reported by another (1) subject within 7 days after the first dose of clinical trial treatment led to permanent discontinuation of clinical trial treatment.

[0318] ● Adverse pregnancy outcomes included spontaneous abortion (1 subject) and ectopic pregnancy (1 subject), both occurring in the placebo + EP group.

[0319] During the entire clinical trial, no TEAE related to abnormal laboratory results was reported. The vital signs and physical examination results of most subjects were normal, and few AEs were reported due to abnormal vital signs or physical examination results.

[0320] Conclusion

[0321] Compared with placebo + EP, VGX-3100 + EP showed superior efficacy in achieving histopathological regression of cervical HSIL and virological clearance of HPV-16 and / or HPV 18 at week 36, and was safe and well-tolerated in subjects with HPV-16 and / or HPV-18 related cervical HSIL.

[0322] Example 2: Establishing a predictive value for clinical response after VGX-3100 treatment of cervical HSIL using miRNA

[0323] In a phase 3 study of VGX-3100 for the treatment of HPV16 and / or HPV18 related HSIL (study number HPV-301 / REVEAL1; see Example 1), before administration of VGX-3100, miRNA was evaluated by RNA sequencing of plasma samples isolated from subjects to identify a miRNA signature that could predict clinical response, i.e., regression of cervical HSIL lesions and elimination of HPV16 and HPV18 infections at the efficacy assessment time point (week 36 of the study). The signature generated by the following method involved using a Next Seq555Dx instrument (Illumina, Inc.) and only using materials compliant with Good Manufacturing Practice (GMP).

[0324] To achieve this goal, the raw miRNA counts obtained from RNA sequencing were normalized relative to counts per million (CPM), and miRNAs showing near-zero variance in the VGX-3100 treatment group of all subjects (regardless of clinical outcome) were excluded as they do not convey differential information. The resulting values of miRNA reference sequences (defined as those that directly map to miRbase v22.1 <www_miRNA_org>) were used for classification and regression tree (CART) (Paradiso et al., BRCA germline mutation test for all woman with ovarian cancer? BMC Cancer (2019); Talhou et al., Confirmation of ProMisE: A simple, genomics-based clinical classifier for endometrial cancer. Cancer (2017)) and random forest analysis using the statistical computing language R (v3.6.2) (Toth et al., Random forest-based modelling to detect biomarkers for prostate cancer progression. Clin Epigenetics. (2019); Yan et al., Identification of candidate colon cancer biomarkers by applying a random forest approach on microarray data. Oncol Rep. (2012)). The results of this work generated a predictive feature constructed based on random forest (named "RF_9_20").

[0325] Create "RF_9_20" as a miRNA-based predictive biomarker for clinical response after VGX-3100 treatment of cervical HSIL. To create the random forest model named RF_9_20, microRNAs were downselected based on performance after multiple random forest generation loops. Then, the top 12 mirs ranked by variable importance were used to train the random forest model (R package randomForest v4.7-1) for classification.

[0326] The classifier was trained and tested on the same dataset, with the following precautions to mitigate overfitting: Use MWMOTE (Majority Weighted Minority Over-sampling Technique) to generate approximately 100 synthetic "responder" data points to balance the training dataset. The resulting oversampled training data was used to tune several hyperparameters (mtry, ntree, and maxnodes) of the random forest model. The final hyperparameters were selected based on the average model performance of 5-fold cross-validation.

[0327] After determining the optimal hyperparameters, the final random forest model was trained using the oversampled dataset. This model (RF_9_20) was performance-tested on the original intention-to-treat (ITT), modified intention-to-treat (mITT), and per-protocol (PP) datasets (removing the synthetic responder samples used during training for testing). Additionally, data from the placebo dataset was not used during model training, but these data were evaluated during testing.

[0328] Models were selected for further study based on overall accuracy, positive predictive value (PPV; defined here as the frequency of subjects who showed regression of HSIL and elimination of HPV16 / 18 infection among those predicted to respond by the classifier), and the number of miRNAs used to generate the model (with a soft upper limit of 15 miRNAs). Model RF_9_20 consists of 12 miRNAs on 8 trees. These 12 miRNAs are as follows:

[0329] hsa.miR.375.3p

[0330] hsa.miR.425.5p

[0331] hsa.miR.193a.5p

[0332] hsa.miR.199b.5p

[0333] hsa.miR.365a.3p

[0334] hsa.miR.223.5p

[0335] hsa.miR.148b.3p

[0336] hsa.miR.143.3p

[0337] hsa.miR.941

[0338] hsa.miR.340.5p

[0339] hsa.miR.151a.3p

[0340] hsa.miR.23a.3p

[0341] The miRNA sequences of the twelve miRNA species listed above are sequences that can be directly mapped to miRBase v22.1.

[0342] The following three tables respectively list the model performance in terms of positive predictive value (PPV), negative predictive value (NPV), accuracy, sensitivity, and specificity (as defined in the table legend) for subjects treated with VGX-3100 and placebo subjects in the intention-to-treat (ITT; all randomly assigned subjects), modified intention-to-treat (mITT; all randomly assigned and evaluable subjects), and per-protocol (PP; subjects who received all three doses of the study drug, did not violate the protocol, and were evaluable) populations.

[0343] As shown in Table 11, in the ITT population, the RF_9_20 model correctly predicted positive responses (HSIL regression concurrent with HPV16 / 18 infection elimination) to VGX-3100 treatment at two pre-treatment time points (labeled Day 0 and Screening in the table) in the range of 73.0% to 71.1%, while the prediction index for the placebo population (rated at Screening or Day 0 based on availability and labeled "Baseline" in the table) was 6.3%. The difference in predicted values and low variance indicate that this prediction is valuable in the context of VGX-3100 treatment and is robust when assessing day-to-day variations in the VGX-3100 treatment group.

[0344] Table 11: Performance of Model RF_9_20 in the ITT Population of REVEAL1 / HPV-301

[0345]

[0346] Responder = A study subject in REVEAL1 / HPV-301 who achieved the primary endpoint of HSIL lesion regression to LSIL or lower grade concurrent with HPV16 / HPV18 infection elimination Biomarker positive = A study subject in REVEAL1 / HPV-301 who was predicted to be a responder as defined above by the RF_9_20 model before administration of VGX-3100 or placebo is considered "biomarker positive"

[0347] PPV = Number of biomarker-positive subjects who are responders / Number of biomarker-positive subjects NPV = Number of biomarker-negative subjects who are responders / Number of biomarker-negative subjects Accuracy = Number of biomarker-positive subjects who are responders + Number of biomarker-negative subjects who are non-responders / Number of subjects

[0348] Sensitivity = Number of biomarker - positive subjects who are responders / Number of responders Specificity = Number of biomarker - negative subjects who are non - responders / Number of non - responders

[0349] Table 12 describes the performance of the RF_9_20 model in the mITT population. At two pre - treatment time points, the static value of the prediction accuracy for response to VGX - 3100 treatment was 75.0%, while the prediction index for the placebo population was 6.3%. As with the ITT population, the difference and low variance in the predicted values indicate that the prediction is valuable in the context of VGX - 3100 treatment and remains robust when assessing day - to - day changes in the VGX - 3100 treatment group.

[0350] Table 12: Performance of model RF_9_20 in the mITT population of REVEAL1 / HPV - 301

[0351]

[0352] Responders = Study subjects who achieved the primary endpoint of regression of HSIL lesions to LSIL or lower grade and elimination of HPV16 / HPV18 infection in REVEAL1 / HPV - 301 Biomarker - positive = Study subjects predicted by the RF_9_20 model to be responders as defined above before administration of VGX - 3100 or placebo in REVEAL1 / HPV - 301 are considered "biomarker - positive"

[0353] PPV = Number of biomarker - positive subjects who are responders / Number of biomarker - positive subjects NPV = Number of biomarker - negative subjects who are responders / Number of biomarker - negative subjects Accuracy = Number of biomarker - positive subjects who are responders+Number of biomarker - negative subjects who are non - responders / Number of subjects

[0354] Sensitivity = Number of biomarker - positive subjects who are responders / Number of responders Specificity = Number of biomarker - negative subjects who are non - responders / Number of non - responders

[0355] Table 13 describes the performance of the RF_9_20 model in the PP population, which is highly similar to the mITT population. In this case, at two pre - treatment time points, the range of prediction accuracy for response to VGX - 3100 treatment was 76.5% to 75.0%, while the prediction index for the placebo population was 6.3%. As with the ITT and mITT populations, the difference and low variance in the predicted values indicate that the prediction is valuable in the context of VGX - 3100 treatment and remains robust when assessing day - to - day changes in the VGX - 3100 treatment group.

[0356] Table 13: Performance of Model RF_9_20 in the PP Population of REVEAL1 / HPV-301

[0357]

[0358]

[0359] Responder = A study subject in REVEAL1 / HPV-301 who achieved the primary endpoint of regression of HSIL lesions to LSIL or lower grade and clearance of HPV16 / HPV18 infection. Biomarker positive = A study subject in REVEAL1 / HPV-301 who was predicted to be a responder as defined above by the RF_9_20 model before administration of VGX-3100 or placebo is considered "biomarker positive".

[0360] PPV = Number of biomarker-positive subjects who are responders / Number of biomarker-positive subjects. NPV = Number of biomarker-negative subjects who are responders / Number of biomarker-negative subjects. Accuracy = Number of biomarker-positive subjects who are responders + Number of biomarker-negative subjects who are non-responders / Number of subjects

[0361] Sensitivity = Number of biomarker-positive subjects who are responders / Number of responders. Specificity = Number of biomarker-negative subjects who are non-responders / Number of non-responders

[0362] Summary of "RF_9_20" as a miRNA-based predictive biomarker for clinical response after VGX-3100 treatment of cervical HSIL. The above RF_9_20 model was created using a common standard method for separating features from complex datasets. RF_9_20 clearly shows the ability to predict regression of cervical HSIL and clearance of HPV16 / 18 infection after VGX-3100 treatment. It has been proven that when this model is applied to the treatment group, the accuracy, PPV, NPV, sensitivity, and specificity are all superior compared to placebo. Specifically, compared to the placebo group, there are differences in PPV in all datasets for the VGX-3100 treatment group from REVEAL1 / HPV-301, indicating that this model can accurately identify women who are more likely to benefit from VGX-3100 treatment.

[0363] Examples

[0364] The following list of examples is intended to supplement rather than replace or supersede the previous description.

[0365] Example 1. A method for treating high-grade cervical intraepithelial neoplasia (HSIL) associated with human papillomavirus (HPV) type 16 or HPV type 18, the method comprising:

[0366] (a) evaluating the presence of a set of miRNAs and their isomiRs consisting of: hsa.miR.375.3p, hsa.miR.425.5p, hsa.miR.193a.5p, hsa.miR.199b.5p, hsa.miR.365a.3p, hsa.miR.223.5p, hsa.miR.148b.3p, hsa.miR.143.3p, hsa.miR.941, hsa.miR.340.5p, hsa.miR.151a.3p, hsa.miR.23a.3p in one or more biological samples from a subject with HSIL associated with HPV type 16 or HPV type 18;

[0367] (b) calculating the normalized level of each of the miRNAs and their isomiRs in the set;

[0368] (c) determining a miRNA signature based on the normalized levels of the miRNAs and their isomiRs in the set;

[0369] (d) classifying the subject as biomarker positive or biomarker negative based on the miRNA signature;

[0370] and

[0371] (e) if the subject is classified as biomarker positive, administering to the subject a therapeutically effective amount of VGX-3100.

[0372] Example 2. The method according to Example 1, wherein the presence of the miRNA is determined by RNA sequencing.

[0373] Example 3. The method according to Example 1 or Example 2, wherein the biological sample is a plasma sample.

[0374] Example 4. The method according to Example 3, wherein the plasma sample is isolated from the subject before administering VGX-3100.

[0375] Example 5. The method according to any one of the preceding examples, wherein the HSIL associated with HPV type 16 or HPV type 18 is determined by biopsy.

[0376] Example 6. The method according to any one of the preceding examples, wherein VGX-3100 is administered to the subject by intramuscular injection followed by electroporation.

[0377] Example 7. The method according to any one of the preceding examples, wherein VGX-3100 is administered to the subject at a dose of 6 mg.

[0378] Example 8. The method according to any one of the preceding examples, wherein VGX-3100 is administered to the subject three times over a period of 12 weeks.

[0379] Example 9. The method according to any one of the preceding examples, wherein VGX-3100 is formulated at a concentration of 6 mg / ml in 150 mM sodium chloride and 15 mM sodium citrate.

[0380] Example 10. The method according to any one of the preceding examples, wherein administration of VGX-3100 results in virological clearance of HPV-16 and / or HPV-18 and histopathological regression of cervical HSIL.

[0381] Example 11. The method according to any one of the preceding examples, wherein administration of VGX-3100 results in histopathological regression of cervical HSIL.

[0382] Example 12. The method according to any one of the preceding examples, wherein administration of VGX-3100 results in virological clearance of HPV-16 and / or HPV-18.

[0383] Example 13. The method according to any one of the preceding examples, wherein administration of VGX-3100 results in complete histopathological regression of cervical HSIL to normal.

[0384] Example 14. The method according to any one of the preceding examples, wherein administration of VGX-3100 results in complete histopathological regression of cervical HSIL to normal and virological clearance of HPV-16 and / or HPV-18.

[0385] Example 15. The method according to any one of the preceding examples, wherein administration of VGX-3100 results in no histopathological progression.

[0386] Example 16. The method according to any one of the preceding examples, wherein administration of VGX-3100 results in clearance of HPV-16 and / or HPV-18 infection from non-cervical anatomical sites.

[0387] Example 17. The method according to any one of the preceding examples, wherein administration of VGX-3100 results in improved humoral and cellular immune responses to VGX-3100 as assessed relative to baseline after the third administration of VGX-3100 and at 36 weeks after administration of VGX-3100.

[0388] Example 18. The method according to any one of Examples 10 to 17, wherein the results of VGX-3100 administration are evaluated at 36 weeks after administration of VGX-3100.

Claims

1. A method for treating high - grade cervical intraepithelial neoplasia (HSIL) associated with human papillomavirus (HPV) type 16 or HPV type 18, the method comprising: (a) assessing the presence of a set of miRNAs and their isomiRs consisting of: hsa.miR.375.3p, hsa.miR.425.5p, hsa.miR.193a.5p, hsa.miR.199b.5p, hsa.miR.365a.3p, hsa.miR.223.5p, hsa.miR.148b.3p, hsa.miR.143.3p, hsa.miR.941, hsa.miR.340.5p, hsa.miR.151a.3p, hsa.miR.23a.3p in one or more biological samples from a subject with HSIL associated with HPV type 16 or HPV type 18; (b) calculating the normalized level of each of the miRNAs and their isomiRs in the set; (c) determining a miRNA signature based on the normalized levels of the miRNAs and their isomiRs in the set; (d) classifying the subject as biomarker - positive or biomarker - negative based on the miRNA signature; and (e) if the subject is classified as biomarker - positive, administering to the subject a therapeutically effective amount of VGX - 3100.

2. The method according to claim 1, wherein the presence of the miRNA is determined by RNA sequencing.

3. The method according to claim 1 or claim 2, wherein the biological sample is a plasma sample.

4. The method according to claim 3, wherein the plasma sample is isolated from the subject before administering VGX - 3100.

5. The method according to any one of the preceding claims, wherein HPV type 16 or HPV type 18 - associated HSIL is determined by biopsy.

6. The method according to any one of the preceding claims, wherein VGX - 3100 is administered to the subject by intramuscular injection followed by electroporation.

7. The method according to any one of the preceding claims, wherein VGX - 3100 is administered to the subject at a dose of 6 mg.

8. The method according to any one of the preceding claims, wherein VGX - 3100 is administered to the subject three times over a 12 - week period.

9. The method according to any one of the preceding claims, wherein VGX - 3100 is formulated at a concentration of 6 mg / ml in 150 mM sodium chloride and 15 mM sodium citrate.

10. The method according to any one of the preceding claims, wherein administration of VGX - 3100 results in virological clearance of HPV - 16 and / or HPV - 18 and histopathological regression of cervical HSIL.

11. The method according to any one of the preceding claims, wherein administration of VGX - 3100 results in histopathological regression of cervical HSIL.

12. The method according to any one of the preceding claims, wherein administration of VGX-3100 results in virological clearance of HPV-16 and / or HPV-18.

13. The method according to any one of the preceding claims, wherein administration of VGX-3100 results in complete histopathological regression of cervical HSIL to normal.

14. The method according to any one of the preceding claims, wherein administration of VGX-3100 results in complete histopathological regression of cervical HSIL to normal and virological clearance of HPV-16 and / or HPV-18.

15. The method according to any one of the preceding claims, wherein administration of VGX-3100 results in no histopathological progression.

16. The method according to any one of the preceding claims, wherein administration of VGX-3100 results in clearance of HPV-16 and / or HPV-18 infection from non-cervical anatomical sites.

17. The method according to any one of the preceding claims, wherein administration of VGX-3100 results in improved humoral and cellular immune responses to VGX-3100 as assessed relative to baseline at 36 weeks after the third administration of VGX-3100 and at 36 weeks after administration of VGX-3100.

18. The method according to any one of claims 10 to 17, wherein the outcome of VGX-3100 administration is evaluated at 36 weeks after administration of VGX-3100.

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