Treatment methods for high-grade squamous intraepithelial lesions (HSIL)

Abstract

Methods for treating human papillomavirus (HPV) type 16- or HPV type 18-associated high-grade squamous intraepithelial lesions (HSIL) of the cervix are described

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 63 / 487,843, filed March 1, 2023, the disclosure of which is incorporated herein by reference in its entirety.

[0002] Methods for treating human papillomavirus (HPV) type 16 or HPV type 18-associated high-grade squamous intraepithelial lesions (HSIL) of the cervix are provided. [Background technology]

[0003] Infection with human papillomavirus (HPV) can result in malignant neoplasms localized in the anogenital and aerodigestive tracts in both men and women. HPV types affecting mucosal tissues are classified as high-risk (HR) or low-risk (LR) (which generally cause benign lesions) based on their potential to cause cancer. HPV-16 and HPV-18 are the most prominent high-risk types, as they are responsible for most HPV-induced cancers. In the United States alone, approximately 14 million new genital HPV infections occur annually, and approximately half of these infections involve high-risk HPV types. Up to 13,000 women are diagnosed with cervical cancer annually, resulting in an estimated 4,120 deaths annually in 2017. HPV-16 is the most common high-risk genotype; combined with HPV-18, these two genotypes are estimated to cause approximately 70% of all cervical cancers.

[0004] Incident HPV infection is characterized by ongoing viral replication and shedding and is associated with early histological changes (grade 1 cervical intraepithelial neoplasia) when a woman's cervix is ​​infected with HPV. While most cases of genital HPV infection resolve spontaneously, persistent infection with one or more oncogenic (high-risk) HPV genotypes can lead to the development of cervical precancerous, histologically high-grade squamous intraepithelial lesions (HSIL), including grades 2 and 3 cervical intraepithelial neoplasia (CIN2 / 3). Typically, over several years, cervical HSIL can progress to invasive cervical cancer. The basis for these changes is thought to be the viral proteins E6 and E7. Infected cells constitutively produce E6 and E7, which increase the degradation of the cell cycle regulatory proteins p53 and pRb, respectively, resulting in uncontrolled cell proliferation and neoplasia.

[0005] Treatment of HPV16- or HPV18-associated HSIL is indicated. Summary of the Invention

[0006] Provided herein are methods for treating human papillomavirus (HPV) type 16 or HPV type 18 associated high-grade cervical intraepithelial lesions (HSIL), the method comprising administering a therapeutically effective amount of VGX-3100 to a subject in need thereof.

[0007] In additional embodiments, cervical HPV16 or HPV18 associated HSIL is determined by biopsy.

[0008] In some embodiments, VGX-3100 is administered to a subject by intramuscular injection followed by electroporation. 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 yet further embodiments, VGX-3100 is formulated at a concentration of 6 mg / ml in 150 mM sodium chloride and 15 mM sodium citrate.

[0009] In certain embodiments, administration of VGX-3100 results in virologic 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 yet further embodiments, administration of VGX-3100 results in virologic 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 virologic clearance of HPV-16 and / or HPV-18. In yet further embodiments, administration of VGX-3100 results in histopathological non-progression. In certain embodiments, administration of VGX-3100 results in clearance of HPV-16 and / or HPV-18 infection from non-cervical anatomical locations. In further embodiments, administration of VGX-3100 results in improved humoral and cellular immune responses to VGX-3100 as assessed relative to baseline after the third dose of VGX-3100 and 36 weeks after administration of VGX-3100.

[0010] In certain embodiments, the results of administration of VGX-3100 are evaluated 36 weeks after administration of VGX-3100. DETAILED DESCRIPTION OF THE INVENTION

[0011] The disclosed methods may be more readily understood by reference to the following detailed description, which forms a part of this disclosure: It is to be understood that the disclosed methods are not limited to the specific methods described and / or illustrated herein, and that the terminology used herein is for the purpose of describing particular embodiments by way of example only, and is not intended to be a limitation of the claimed methods.

[0012] Unless specifically stated otherwise, any description of a possible mechanism or mode of action, or reason for improvement, is intended to be illustrative only, and the disclosed methods are not constrained by the accuracy or inaccuracy of any such proposed mechanism or mode of action, or reason for improvement.

[0013] When a range of values ​​is expressed, another embodiment includes from the one particular value and / or to the other particular value. Moreover, reference to values ​​stated in a range includes each and every value within that range. All ranges are inclusive and combinable. When values ​​are expressed as approximations, by use of the antecedent "about," it will be understood that the particular value forms another embodiment. Reference to a particular numerical value includes at least that particular value, unless the context clearly dictates otherwise.

[0014] It should be understood that certain features of the disclosed methods that are, for clarity, described herein in the context of separate embodiments, can also be provided in combination in a single embodiment. Conversely, various features of the disclosed methods that are, for brevity, described in the context of a single embodiment, can also be provided separately or in any subcombination.

[0015] Throughout this specification and claims, various terms relating to the embodiments of this specification are used. Such terms are to be given their ordinary meaning in the art unless otherwise indicated. Other specifically defined terms are to be interpreted in a manner consistent with the definitions provided herein.

[0016] As used herein, the singular forms "a", "an" and "the" include the plural forms.

[0017] Specific Terms As used herein, when used in reference to a numerical range, cutoff, or specific value, the term "about" is used to indicate that the recited value may vary by up to 10% from the recited value. Thus, the term "about" is used to encompass no more than ±10% variation, no more than ±5% variation, no more than ±1% variation, no more than ±0.5% variation, or no more than ±0.1% variation from the specified value.

[0018] As used herein, the term "at least one" means "one or more."

[0019] As used herein, the term "subject" as used herein refers to any animal, but particularly refers to humans. Thus, the present method is applicable to humans and non-human animals, but is most preferably used in humans. As used herein, "subject" and "patient" are used interchangeably.

[0020] As used herein, the term "comprising" is intended to include examples encompassed by the terms "consisting essentially of" and "consisting of," and similarly, the term "consisting essentially of" is intended to include examples encompassed by the term "consisting of."

[0021] As used herein, "treating" and similar terms refer to, for example, reducing the severity and / or frequency of cervical symptoms such as human papillomavirus (HPV) type 16 or HPV18-associated high-grade cervical intraepithelial lesions (HSIL), and cervical high-grade squamous intraepithelial lesion (HSIL) lesions, eliminating HPV16 or HPV18 infection symptoms, particularly HSIL lesions, and / or clearing the HPV16 or HPV18 virus from a subject, and / or resolution to cervical low-grade squamous intraepithelial lesions (LSIL) or normal tissue.

[0022] As used herein, the term "coding sequence" or "encoding nucleic acid" may be meant to refer to a nucleic acid (RNA or DNA molecule) comprising a nucleotide sequence that encodes a polypeptide. The coding sequence may further comprise initiation and termination signals operably linked to regulatory elements, including a promoter and polyadenylation signal, capable of directing expression in the cells of an individual or mammal to which the nucleic acid is administered. The coding sequence may further comprise a sequence encoding a signal peptide, e.g., an IgE leader sequence.

[0023] As used herein, the term "nucleic acid" or "oligonucleotide" or "polynucleotide" can refer to at least two nucleotides covalently linked together. A depiction of a single strand also defines the sequence of the complementary strand. Thus, a nucleic acid also encompasses the complementary strand of the depicted single strand. Many variants of a nucleic acid can be used for the same purpose as a given nucleic acid. Thus, a nucleic acid also encompasses substantially identical nucleic acids and their complements. A single strand provides a probe that can hybridize to a target sequence under stringent hybridization conditions. Thus, a nucleic acid also encompasses a probe that hybridizes under stringent hybridization conditions. A nucleic acid can be single-stranded or double-stranded, or can contain portions of both double-stranded and single-stranded sequences. Nucleic acids can be DNA, RNA, or hybrids, both genomic and cDNA, where the nucleic acid can contain combinations of deoxyribonucleotides and ribonucleotides, and combinations of bases including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine hypoxanthine, isocytosine, and isoguanine. Nucleic acids can be obtained by chemical synthesis or by recombinant methods.

[0024] As used herein, the term "operably linked" may mean that the expression of a gene is under the control of a promoter to which it is spatially connected. The promoter may be located 5' (upstream) or 3' (downstream) of the gene under its control. The distance between the promoter and the gene may be approximately the same as the distance between the promoter and the gene it controls in the gene from which the promoter is derived. As is known in the art, variations in this distance may be accommodated without loss of promoter function.

[0025] As used herein, the term "promoter" can refer to a synthetic or naturally occurring molecule that can confer, activate, or enhance expression of a nucleic acid in a cell. A promoter can contain one or more specific transcriptional regulatory sequences to further enhance expression and / or modify its spatial and / or temporal expression. A promoter can also contain distal enhancer or repressor elements, which can be located as much as several thousand base pairs from the start site of transcription. Promoters can be derived from sources including viruses, bacteria, fungi, plants, insects, and animals. A promoter can regulate expression of genetic 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.

[0026] As used herein, the term "vector" can refer to a nucleic acid sequence containing a replication origin.Vector can be a plasmid, bacteriophage, bacterial artificial chromosome, or yeast artificial chromosome.Vector can be a DNA or RNA vector.Vector can be either a self-replicating extrachromosomal vector or a vector that integrates into a host genome.

[0027] As used herein, the term "adverse event" (AE) is defined according to the Common Toxicity Criteria for Adverse Events (CTCAE) version 4.03 rating scale. The investigator assessed laboratory and clinical AEs for the relevant study population according to CTCAE version 4.03, with respect to the following levels of severity: Grade 1 (mild), Grade 2 (moderate), Grade 3 (severe), Grade 4 (potentially life-threatening), and Grade 5 (death).

[0028] As used herein, the term "responder" refers to study subjects who met the primary endpoint of the proportion of baseline biomarker-positive women with no evidence of cervical HSIL on histology samples and no evidence of HPV-16 and / or HPV-18 in cervical samples by type-specific HPV testing at the 36-week visit.

[0029] As used herein, the term "placebo" refers to the administration of a pharmaceutical composition that does not contain VGX-3100.

[0030] Treatment and Diagnostic Methods Provided herein are methods for treating human papillomavirus (HPV) type 16 or HPV type 18 associated high-grade cervical intraepithelial lesions (HSIL), the method comprising, consisting of, or consisting essentially of administering a therapeutically effective amount of VGX-3100 to a subject in need thereof.

[0031] Also provided herein is VGX-3100 for use in a method for treating human papillomavirus (HPV) type 16 or HPV type 18 associated high-grade cervical intraepithelial lesions (HSIL), the method comprising, consisting of, or consisting essentially of administering a therapeutically effective amount of VGX-3100 to a subject in need thereof.

[0032] Also provided herein is the use of VGX-3100 in the manufacture of a medicament for treating human papillomavirus (HPV) type 16 or HPV type 18 associated high-grade cervical intraepithelial lesions (HSIL), the method comprising, consisting of, or consisting essentially of administering a therapeutically effective amount of VGX-3100 to a subject in need thereof.

[0033] In additional embodiments, cervical HPV16 or HPV18 associated HSIL is determined by biopsy.

[0034] In certain embodiments, administration of VGX-3100 results in virologic 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 yet further embodiments, administration of VGX-3100 results in virologic 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 virologic clearance of HPV-16 and / or HPV-18. In yet further embodiments, administration of VGX-3100 results in histopathological non-progression. In certain embodiments, administration of VGX-3100 results in clearance of HPV-16 and / or HPV-18 infection from non-cervical anatomical locations. In further embodiments, administration of VGX-3100 results in improved humoral and cellular immune responses to VGX-3100 as assessed relative to baseline after the third dose of VGX-3100 and 36 weeks after administration of VGX-3100.

[0035] Also provided herein are methods for improving HPV-16 and / or HPV-18 virological clearance and cervical HSIL histopathological regression in a subject, comprising, consisting of, or consisting essentially of administering a therapeutically effective amount of VGX-3100 to a subject in need thereof. In certain embodiments, the improvement in HPV-16 and / or HPV-18 virological clearance and cervical HSIL histopathological regression is relative to administration of a placebo to a subject or a population of subjects. In certain embodiments, the improvement in HPV-16 and / or HPV-18 virological clearance and cervical HSIL histopathological regression is relative to no treatment of a subject or a population of subjects. In certain embodiments, the improvement in HPV-16 and / or HPV-18 virological clearance and cervical HSIL histopathological regression is relative to treatment of a subject in a population of subjects with standard therapy.

[0036] Also provided herein is a method for improving histopathological regression of cervical HSIL in a subject, comprising, consisting of, or consisting essentially of administering a therapeutically effective amount of VGX-3100 to a subject in need thereof. In certain embodiments, the improvement in histopathological regression of cervical HSIL is relative to administration of a placebo to a subject or a population of subjects. In certain embodiments, the improvement in histopathological regression of cervical HSIL is relative to no treatment of a subject or a population of subjects. In certain embodiments, the improvement in histopathological regression of cervical HSIL is relative to treatment of a subject in a population of subjects with standard therapy.

[0037] Also provided herein is a method for achieving complete histopathological regression of cervical HSIL to normal in a subject, comprising, consisting of, or consisting essentially of administering a therapeutically effective amount of VGX-3100 to a subject in need thereof. In certain embodiments, the achievement of complete histopathological regression of cervical HSIL to normal is relative to administration of a placebo to the subject or a population of subjects. In certain embodiments, the achievement of complete histopathological regression of cervical HSIL is relative to no treatment of the subject or population of subjects. In certain embodiments, the achievement of complete histopathological regression of cervical HSIL is relative to treatment of a subject in a population of subjects with standard therapy.

[0038] Also provided herein are methods for achieving complete histopathological regression of cervical HSIL to normal and virologic clearance of HPV-16 and / or HPV-18 in a subject, comprising, consisting of, or consisting essentially of administering a therapeutically effective amount of VGX-3100 to a subject in need thereof. In certain embodiments, the achievement of complete histopathological regression of cervical HSIL to normal and virologic clearance of HPV-16 and / or HPV-18 is relative to administration of a placebo to a subject or population of subjects. In certain embodiments, the achievement of complete histopathological regression of cervical HSIL to normal and virologic clearance of HPV-16 and / or HPV-18 is relative to no treatment of a subject or population of subjects. In certain embodiments, the achievement of complete histopathological regression of cervical HSIL to normal and virologic clearance of HPV-16 and / or HPV-18 is relative to treatment of a subject in a population of subjects with standard therapy.

[0039] Also provided herein are methods for improving histopathological non-progression in a subject, comprising, consisting of, or consisting essentially of administering a therapeutically effective amount of VGX-3100 to a subject in need thereof. In certain embodiments, the improvement in histopathological non-progression is relative to administration of a placebo to the subject or a population of subjects. In certain embodiments, the improvement in histopathological non-progression is relative to no treatment of the subject or population of subjects. In certain embodiments, the improvement in histopathological non-progression is relative to treatment of a subject in a population of subjects with standard therapy.

[0040] Also provided herein are methods for improving clearance of HPV-16 and / or HPV-18 infection from non-cervical anatomical locations in a subject, comprising, consisting of, or consisting essentially of administering a therapeutically effective amount of VGX-3100 to a subject in need thereof. In certain embodiments, the improvement in clearance of HPV-16 and / or HPV-18 infection from non-cervical anatomical locations is relative to administration of a placebo to a subject or population of subjects. In certain embodiments, the improvement in clearance of HPV-16 and / or HPV-18 infection from non-cervical anatomical locations is relative to no treatment of a subject or population of subjects. In certain embodiments, the improvement in clearance of HPV-16 and / or HPV-18 infection from non-cervical anatomical locations is relative to treatment of a subject in a population of subjects with standard therapy.

[0041] Provided herein are methods comprising, consisting of, or consisting essentially of administering a therapeutically effective amount of VGX-3100 to a subject in need thereof, for improving humoral and cellular immune responses to VGX-3100 after the third administration of VGX-3100 and 36 weeks after administration of VGX-3100, as assessed relative to baseline in the subject. In certain embodiments, the improvement in clearance of HPV-16 and / or HPV-18 infection from non-cervical anatomical locations is relative to administration of a placebo to the subject or population of subjects. In certain embodiments, the improvement in humoral and cellular immune responses to VGX-3100 after the third administration of VGX-3100 and 36 weeks after administration of VGX-3100, as assessed relative to baseline, is relative to no treatment in the subject or population of subjects. In certain embodiments, the improvement in humoral and cellular immune responses to VGX-3100 after the third administration of VGX-3100 and at 36 weeks after administration of VGX-3100, as assessed relative to baseline, is relative to treatment of subjects in a population of subjects with standard of care.

[0042] In certain embodiments, the results of or improvements achieved by VGX-3100 administration are evaluated 36 weeks after administration of VGX-3100.

[0043] Routes of Administration and Pharmaceutical Compositions VGX-3100 can be delivered using any of several well-known techniques, including DNA injection (also called DNA vaccination), recombinant vectors such as recombinant adenovirus, recombinant adenovirus-associated virus, and recombinant vaccinia.

[0044] Routes of administration include, but are not limited to, intramuscular, intranasal, intraperitoneal, intradermal, subcutaneous, intravenous, intraarterial, intraocular, and oral, as well as topical, transdermal, inhalation, or suppository administration, or into mucosal tissues, such as by lavage into vaginal, rectal, urethral, ​​buccal, and sublingual tissues. Preferred routes of administration include intramuscular, intraperitoneal, intradermal, and subcutaneous injection. The gene construct may be administered by means including, but not limited to, electroporation methods and devices, conventional syringes, needleless injection devices, or "microprojectile bombardment 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.

[0045] Examples of preferred electroporation devices and methods for enhancing delivery of DNA vaccines include those described in U.S. Patent No. 7,245,963 by Draghia-Akli et al., and U.S. Patent Publication No. 2005 / 0052630 filed by Smith et al., the contents of which are incorporated herein by reference in their entireties. Also preferred are electroporation devices and methods for enhancing delivery of DNA vaccines provided in co-pending and co-owned U.S. patent application Ser. No. 11 / 874,072, filed October 17, 2007, which claims benefit under 35 U.S.C. 119(e) to U.S. Provisional Application No. 60 / 852,149, filed October 17, 2006, and U.S. Provisional Application No. 60 / 978,982, filed October 10, 2007, all of which are incorporated herein by reference in their entireties. In certain embodiments, the electroporation apparatus is a CELLECTRA®-5P apparatus.

[0046] The following are examples of embodiments using electroporation techniques, discussed in more detail in the patent references mentioned above: The electroporation device can be configured to deliver pulses of energy to a desired tissue in a mammal, generating a constant current similar to a current input preset by a user. The electroporation device includes an electroporation component and an electrode assembly or handle assembly. The electroporation component can include or incorporate one or more of various elements of an electroporation device, including a controller, a current waveform generator, an impedance tester, a waveform logger, an input element, a status reporting element, a communication port, a memory component, a power supply, and a power switch. The electroporation component can function as one element of the electroporation device, with other elements being separate elements (or components) that communicate with the electroporation component. In some embodiments, the electroporation component can function as two or more elements of the electroporation device, which can communicate with yet other elements of the electroporation device that are separate from the electroporation component. The use of electroporation technology to deliver improved HPV vaccines is not limited by the elements of the electroporation device being part of a single electromechanical or mechanical device, as the elements can function as a single device or as separate elements that communicate with each other. The electroporation component can deliver pulses of energy that generate a constant current in the desired tissue and includes a feedback mechanism. The electrode assembly includes an electrode array having multiple electrodes in a spatial arrangement, and the electrode assembly receives the pulses of energy from the electroporation component and delivers them to the desired tissue through the electrodes. At least one of the multiple electrodes is neutral during delivery of the pulses of energy, measures the impedance at the desired tissue, and communicates the impedance to the electroporation component.A feedback mechanism can receive the measured impedance and can adjust the pulses of energy delivered by the electroporation component to maintain a constant current.

[0047] In some embodiments, the multiple electrodes can deliver pulses of energy in a decentralized pattern. In some embodiments, the multiple electrodes can deliver pulses of energy in a decentralized pattern through control of the electrodes under a programmed sequence, the programmed sequence being input into the electroporation component by a user. In some embodiments, the programmed sequence includes multiple pulses delivered sequentially, each pulse of the multiple pulses being delivered by at least two active electrodes with one indifferent electrode measuring impedance, and each subsequent pulse of the multiple pulses being delivered by a different one of the at least two active electrodes with one indifferent electrode measuring impedance.

[0048] In some embodiments, the feedback mechanism is implemented by either hardware or software. Preferably, the feedback mechanism is implemented by an analog closed-loop circuit. Preferably, this feedback occurs every 50 μg, 20 μg, 10 μg, or 1 μg, but is preferably real-time feedback or instantaneous (i.e., substantially instantaneous as determined by available techniques for determining response time). In some embodiments, an indifferent electrode measures the impedance in the desired tissue and communicates the impedance to the feedback mechanism, which responds to the impedance and adjusts the pulse of energy to maintain a constant current at a value similar to the preset current. In some embodiments, the feedback mechanism maintains a continuous, instantaneous constant current during delivery of the pulse of energy.

[0049] In some embodiments, the nucleic acid molecule is delivered to cells in conjunction with the administration of a polynucleotide function enhancer or a genetic vaccine facilitator. Polynucleotide function enhancers are described in U.S. Patent Nos. 5,593,972, 5,962,428, and International Patent Application No. PCT / US94 / 00899, filed January 26, 1994, each of which is incorporated herein by reference. The auxiliary agent administered in conjunction with the nucleic acid molecule may be administered as a mixture with the nucleic acid molecule, or may be administered separately and simultaneously before or after the administration of the nucleic acid molecule. In addition, other agents that may function as transfection and / or replication and / or inflammatory agents and may be co-administered with GVFs include growth factors, cytokines, and lymphokines, such as α-interferon, gamma-interferon, GM-CSF, platelet-derived growth factor (PDGF), TNF, epidermal growth factor (EGF), IL-1, IL-2, IL-4, IL-6, IL-10, IL-12, and IL-15, as well as fibroblast growth factors, surfactants, such as immune-stimulating complexes (ISCOMS), Freund's incomplete adjuvant, LPS analogs including monophosphoryl lipid A (WL), muramyl peptides, quinone analogs, and vesicles such as squalene and squalene; hyaluronic acid may also be used and administered in conjunction with gene constructs. In some embodiments, immunomodulatory proteins may be used as GVFs. In some embodiments, nucleic acid molecules are provided in association with PLG to enhance delivery / uptake.

[0050] Pharmaceutical compositions according to the present invention contain about 1 nanogram to about 2000 micrograms of DNA. In some preferred embodiments, pharmaceutical compositions according to the present invention contain about 5 nanograms to about 1000 micrograms of DNA. In some preferred embodiments, pharmaceutical compositions contain about 10 nanograms to about 800 micrograms of DNA. In some preferred embodiments, pharmaceutical compositions contain about 0.1 to about 500 micrograms of DNA. In some preferred embodiments, pharmaceutical compositions contain about 1 to about 350 micrograms of DNA. In some preferred embodiments, pharmaceutical compositions contain about 25 to about 250 micrograms of DNA. In some preferred embodiments, pharmaceutical compositions contain about 100 to about 200 micrograms of DNA. In certain embodiments, VGX-3100 is formulated at a concentration of 6 mg / ml.

[0051] Pharmaceutical compositions according to the present invention are formulated according to the mode of administration to be used. When the pharmaceutical compositions are injectable, they are sterile, pyrogen-free, and particulate-free. An isotonic formulation is preferably used. Common additives for isotonicity include sodium chloride, dextrose, mannitol, sorbitol, and lactose. In some cases, an isotonic solution such as phosphate-buffered saline is preferred. Stabilizers include gelatin and albumin. In some embodiments, a vasoconstrictor is added to the formulation. In some preferred embodiments, the pharmaceutical composition contains about 100 to about 200 micrograms of DNA. In one specific embodiment, VGX-3100 is formulated at a concentration of 6 mg / ml in 150 mM sodium chloride and 15 mM sodium citrate.

[0052] 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 yet further embodiments, a first dose of VGX-3100 is administered on day 0, a second dose of VGX-3100 is administered on week 4, and a third dose of VGX-3100 is administered on week 12. [Example]

[0053] It is understood that the examples and embodiments described herein are for illustrative purposes only, and that various modifications or changes will be suggested to those skilled in the art and are to be included within the spirit and scope of this application and the appended claims.

[0054] Example 1: REVEAL 2 - A Randomized, Double-Blind, Placebo-Controlled Phase 3 Study of VGX-3100 Delivered Intramuscularly Followed by Electroporation with CELLECTRA™ 5PSP for the Treatment of HPV-16 and / or HPV-18 Associated High-Grade Squamous Intraepithelial Lesions (HSIL) of the Cervix (HPV-303) Provided herein is a non-limiting example of a randomized, double-blind, placebo-controlled Phase 3 study of VGX-3100 delivered intramuscularly (IM) followed by electroporation with CELLECTRA™ 5PSP for the treatment of HPV-16 and / or HPV-18-associated high-grade squamous intraepithelial lesions (HSIL) of the cervix. The primary endpoint was the proportion of baseline biomarker-positive women with no evidence of cervical HSIL on histology samples and no evidence of HPV-16 and / or HPV-18 in cervical samples by type-specific HPV testing at the week 36 visit.

[0055] Clinical trial objectives and endpoints: Primary Objective: Determine the efficacy of VGX-3100 compared to placebo with respect to histopathologic regression of combined cervical HSIL and virologic clearance of HPV-16 and / or HPV-18 among baseline biomarker-positive women.

[0056] Primary endpoint: Proportion of baseline biomarker-positive women with no evidence of cervical HSIL on histology samples and no evidence of HPV-16 and / or HPV-18 in cervical samples by type-specific HPV testing at the 36-week visit.

[0057] Secondary objectives and relevant secondary endpoints are provided in Table 1 . [Table 1-1] [Table 1-2]

[0058] Exploratory objectives and relevant exploratory endpoints are provided in Table 2. [Table 2]

[0059] Study design Study treatment: 6 mg (1 ml) VGX-3100 intramuscular injection followed by EP with the CELLECTRA™ 5PSP device given on day 0, week 4, and week 12.

[0060] Study Design: To be eligible for the study, subjects must be at least 18 years of age and above the age of sexual consent, consent to participate at screening, and have undergone cervical biopsy(ies) for cervical lesion(s). Biopsy slides were sent blinded to the PAC to establish the presence of cervical HSIL at screening. To be eligible for randomization, the PAC must assign a histologic diagnosis of cervical HSIL. Subjects must also have a cervical specimen testing positive for HPV-16 / 18 by the cobas™ HPV test to be eligible to participate in the study.

[0061] A sample of approximately 198 subjects was randomized in a 2:1 ratio to receive either 6 mg (in 1 ml) of VGX-3100 or placebo, each administered intramuscularly followed by intraperitoneal administration. This sample size provides >90% statistical power to declare VGX-3100 superior to placebo among biomarker-positive women, assuming that the true proportion of subjects whose lesion(s) regress and whose HPV-16 / 18 clears is 66% and 15% for VGX-3100 and placebo, respectively, based on the HPV-301 study results, and the proportion of biomarker-positive women is 33%. Biomarker positivity in this context is defined as predicted lesion regression concomitant with HPV16 / 18 clearance following examination of a predefined microRNA (miRNA) signature in the subject's plasma, assessed prior to administration of VGX-3100 or placebo. Subjects who are not predicted to exhibit lesion regression and HVP16 / 18 clearance based on this signature prior to administration of VGX-3100 or placebo are considered biomarker negative.

[0062] Subjects were randomized stratified according to (a) CIN severity observed in biopsy specimens at screening (i.e., CIN2 vs. CIN3), (b) BMI category on day 0 (≦25 vs. >25 kg / m), and (c) age category on day 0 (<25 vs. ≧25 years). To ensure that CIN2 disease was not overrepresented in the study, the percentage of subjects enrolled with CIN2 did not exceed 50% of the total enrolled. Each country received consecutive allocation groups.

[0063] Efficacy Assessment: The primary endpoint of the Phase 3 trial (HPV-303) is based on the results of the Phase 2b trial (HPV-003) and the Phase 3 trial (HPV-301). Considering that HPV persistence is an important factor in clinical progression of cervical dysplasia, and based on the findings of the secondary objective of the HPV-003 trial, the responder definition for determining the HPV-303 primary endpoint will consider both histologic regression of cervical HSIL and clearance of high-risk HPV-16 and HPV-18.

[0064] Regression of cervical HSIL will be determined by histopathological evaluation of cervical tissue, which is considered the definitive method for diagnosing cervical dysplasia. Tissue analyzed for evidence of histopathological regression will be obtained at week 36. Cervical cytology samples will be obtained to characterize HPV infection at screening, day 0 (pre-dose), and weeks 8, 15, 28, and 36. In addition, if there is residual tissue in the paraffin block after the histologic diagnosis is given, unstained slides and / or relevant paraffin blocks may be collected for HPV-16 and / or HPV-18 testing. Vaginal, oropharyngeal, and optional anal samples will be obtained to characterize HPV infection at day 0 (pre-dose) and week 36 and to evaluate virologic response to treatment at non-cervical sites.

[0065] Immunogenicity Assessment: Humoral and cell-mediated immune responses in response to VGX-3100 treatment were assessed in blood samples taken at baseline (both pre-dose screening and day 0), and at weeks 15 and 36. Cervical tissue samples were analyzed for evidence of an elevated immune response at week 36 compared to baseline (screening).

[0066] Safety Assessment: The risk of HSIL progression to cancer or recurrence of HSIL is thought to be low and similar to rates observed after LEEP / CKC. Long-term follow-up planned for this HPV-303 trial will include safety, cytology, and HPV-16 and / or HPV-18 testing up to 7 months after the last dose of study treatment.

[0067] A Data Safety Monitoring Board (DSMB) will review safety data and histopathological regression results. The DSMB will be responsible for advising the sponsor if safety concerns appear to exist and if the proportion of subjects with histopathological regression in the VGX-3100 group appears unacceptably low compared to the placebo group. No formal interim analyses will be performed.

[0068] Study Population: This multi-center, global study recruited women aged 18 years and older. VGX-3100 is being developed as a non-surgical treatment option for the treatment of cervical cancer precursors, precancerous cervical HSIL, and underlying pathogenic HPV-16 and / or HPV-18 infection.

[0069] Inclusion Criteria—Each subject must meet all of the following criteria to be enrolled in the study: 1. Females aged 18 years or older who meet the minimum age of sexual consent according to local regulations. 2. Confirmed cervical infection with HPV types 16 and / or 18 as screened with the cobas™ HPV Test. 3. Cervical tissue specimens / slides provided to the study pathology review committee for diagnosis must be collected within 10 weeks of the anticipated date of the first dose of study medication. 4. Histologic evidence of cervical HSIL confirmed by a pathology review committee (PAC) at the time of screening. 5. Must be able to understand, agree to, and comply with the requirements of the protocol. Subjects must be willing and able to provide voluntary consent to participate and sign a consent form prior to any study-related activities. 6. Must be determined by the investigator to be an appropriate candidate for the required protocol-specific procedure(s) at Week 36. 7. Satisfactory colposcopy at screening, defined as complete visualization of the squamocolumnar epithelial junction (type I or type II transition zone) and complete visualization of the white epithelium or upper limit of suspicion for CIN disease. 8. Cervical lesion accessible for sampling with a biopsy instrument (e.g., Mini-Tischler device). 9. Cervical lesion of sufficient size to ensure that a visible lesion remains after screening biopsy. 10. With regard to fertility, one of the following criteria must be met: Postmenopausal as defined by spontaneous amenorrhea for more than 12 months b. Surgically sterile due to absent ovaries or bilateral tubal ligation / occlusion performed more than 12 months prior to screening. c. Women of childbearing potential (WOCBP) are willing to use a contraceptive method that has a failure rate of less than 1% per year when used consistently and correctly from screening through 36 weeks. The use of condoms alone or condoms with spermicide does not have a failure rate of <1% per year and therefore is not an acceptable form of contraception. The following methods are acceptable: Hormonal contraception: either combined or single progestins, including oral contraceptives, injectables, implants, vaginal rings, or transdermal patches. Hormonal contraceptives should not be used in subjects with a history of hypercoagulability (e.g., deep vein thrombosis, pulmonary embolism). · Abstinence from penile-vaginal intercourse when this is the mode of sexual activity in question. Intrauterine devices or systems, - Sterilization of the male partner at least 6 months prior to the female subject's participation in the study, and this male is the subject's only partner. 11. A normal screening electrocardiogram (ECG) or a screening ECG with no clinically significant findings as judged by the investigator.

[0070] Exclusion Criteria - Subjects meeting any of the following criteria will be excluded from enrollment or continuation in the study: 1. Microscopic or macroscopic evidence of adenocarcinoma in situ (AIS), high-grade vulvar, vaginal (including cervical HPV-associated lesions extending into the vaginal canal), or anal intraepithelial neoplasia or invasive carcinoma in any histopathologic specimen at screening. 2. Cervical lesion(s) that cannot be fully visualized by colposcopy due to extension into the high cervical canal at the time of screening. 3. ECC indicating potentially untreated cancer, untreated HSIL, indeterminate, or insufficient for diagnosis (ECC does not need to be performed as part of study screening). 4. Treatment for cervical HSIL within 4 weeks prior to screening. 5. Pregnant, nursing, or considering becoming pregnant through the 36-week visit. 6. History of previous therapeutic HPV vaccination (licensed prophylactic HPV vaccines, e.g., Gardasil™, Silgard™, Cervarix™, are acceptable). 7. Presence of any unresolved abnormal clinical screening laboratory value of Grade 1 or greater according to the Common Toxicity Criteria for Adverse Events (CTCAE) v4.03, deemed clinically significant by the investigator in the 60 days prior to Day 0. 8. Immunosuppression as a result of underlying disease or treatment, including: a) History of HIV or positive serology at screening (performed within 60 days prior to Day 0). b) Primary immunodeficiency. c) Long-term use (≥7 days) of oral or parenteral glucocorticoids at doses ≥20 mg / day prednisone equivalent. (The use of inhaled, otic, and ophthalmic corticosteroids is permitted.) d) Current or anticipated use of disease-modifying doses of antirheumatic drugs (e.g., azathioprine, cyclophosphamide, cyclosporine, methotrexate), and biologic disease-modifying drugs such as TNF-α inhibitors (e.g., infliximab, adalimumab, or etanercept). e) History of solid organ or bone marrow transplant. f) Any history of other clinically significant immunosuppressive or clinically diagnosed autoimmune disease that may necessitate therapy that would jeopardize the subject's safety or interfere with study or endpoint assessments, or otherwise affect the validity of the study results. g) Subjects who are malnourished (i.e., medically significant unintentional weight loss) based on screening laboratories, medical history, and physical examination according to the clinical judgment of the investigator. 9. Vaccination with any non-test, non-live vaccine within 2 weeks of administration. 10. Receipt of any non-trial, live vaccine (e.g., measles vaccine) within 4 weeks of administration. 11. Clinically significant, medically unstable current or history of illness that, in the judgment of the investigator, would jeopardize the subject's safety, interfere with study or endpoint assessment, or otherwise affect the validity of the study results (e.g., chronic renal failure, angina pectoris, myocardial ischemia or infarction, class 3 or greater congestive heart failure, cardiomyopathy, or clinically significant arrhythmia). 12. Malignancy or systemic treatment for malignancy within 2 years of screening (locally treated anogenital malignancies and superficial skin cancers are acceptable). 13. Presence of acute or chronic bleeding, or coagulopathy, that would contraindicate the use of IM injections or anticoagulants (e.g., anticoagulants or antiplatelet agents) within 2 weeks of Day 0. 14. History of seizures unless seizure-free for 5 years on one or less antiepileptic medication. 15. Sustained, manually confirmed, sitting systolic blood pressure >150mmHg or <90mmHg or diastolic blood pressure >95mmHg at screening or day 0. 16. Resting heart rate <50 bpm (unless due to exercise conditioning) or >100 bpm at screening or day 0. 17. Previous major surgery within 4 weeks of day 0. 18. Participation in an interventional study with a test compound or device (excluding HPV-301 REVEAL 1) within 30 days of signing the informed consent. Participation in observational studies is permitted. 19. Fewer than two acceptable sites available for IM injection, considering the deltoid and anterolateral quadriceps. 20. Tattoo, keloid, or hypertrophic scar located within 2 cm of the intended treatment area. 21. Cardiac defibrillator (to prevent life-threatening arrhythmias) or pacemaker located at the ipsilateral deltoid injection site (unless deemed acceptable by a cardiologist). 22. Metallic implants or implantable medical devices within the electroporation area. 23. Active drug or alcohol use or dependence that, in the opinion of the Investigator, would interfere with compliance with study requirements. 24. Prisoners or subjects who are involuntarily detained (involuntarily held) for treatment of either a mental or physical illness (i.e., a communicable disease). 25. Active duty military personnel. 26. Study-related staff or family members of study-related staff. 27. Any disease or condition that, in the opinion of the investigator, may affect the subject's safety or the evaluation of any study endpoint. Definitions of composite regression and clearance endpoint responders and non-responders are provided in Table 3: [Table 3]

[0071] Test product, dosage and mode of administration, batch number(s) Investigational Products Generic name: VGX-3100 Chemical name: Circular double-stranded deoxyribonucleic acid consisting of 3782 base pairs for the pGX3001 plasmid and 3824 base pairs for the pGX3002 plasmid.

[0072] Identification name: Eukaryotic expression plasmid containing transcription units encoding HPV 16 and 18-E6 and E7 controlled by a synthetic CMV promoter, and elements required for replication and selection in E. coli, namely, the pUC origin of replication (pUC Ori) and the kanamycin resistance gene (Kan R).

[0073] Detailed Description: VGX-3100, the HPV therapeutic vaccine, is a combination of two plasmids in equal amounts (i.e., a 6 mg dose delivers 3 mg of each pGX3001 and pGX3002 plasmid): a) pGX3001: p16ConE6E7, a plasmid encoding a synthetic HPV16 consensus E6 and E7 fusion gene ("consensus HPV16-6 and 7") into a pVAX1 backbone (Invitrogen, Carlsbad, CA) under the control of the cytomegalovirus immediate-early (CMV) promoter, and b) pGX3002: p18ConE6E7, a plasmid encoding an HPV18 consensus E6 and E7 fusion gene ("consensus HPV 18-6 and 7") into a pVAX1 backbone (Invitrogen, Carlsbad, CA) under the control of the cytomegalovirus immediate-early (CMV) promoter. VGX-3100 is described in WO2014 / 165291, which is incorporated by reference in its entirety. The nucleic acid and amino acid consensus sequences are provided in Tables 4 and 5, respectively. [Table 4] [Table 5]

[0074] In preparation for the Phase 3 program, a buffered, refrigerated (storage temperature: 2-8°C) formulation of VGX-3100 was developed using a saline sodium citrate (SSC) solution. The refrigerated formulation requires administration using the next-generation device, the CELLECTRA™ 5PSP. This refrigerated formulation of VGX-3100 was initially administered to 117 subjects in the Phase 1 clinical trial, HPV-101. Approximately half of the healthy volunteers in the HPV-101 trial received three 6 mg IM doses of VGX-3100 (refrigerated formulation), followed by EP. Based on analysis of the data, the refrigerated formulation was determined to be non-inferior to the frozen formulation based on a 2-fold increase in overall spot-forming units (SPUs) against the plasmid-encoded antigen per 10 peripheral blood mononuclear cells (PBMCs), measured from baseline to week 14 using an interferon-γ ELISpot assay.

[0075] Efficacy Results The study did not meet its primary endpoint of regression and clearance in the biomarker-positive population. The study met its secondary endpoints of regression and clearance in all patients. VGX-3100 was well tolerated, with no differences in the reported AE profile between the treatment and placebo groups. VGX-3100 was immunogenic, generating antigen-specific T cells in the periphery and T cells in tissues.

[0076] The study population is provided in Table 6. [Table 6]

[0077] HSIL regression and viral clearance are provided in Table 7. [Table 7]

[0078] HSIL regression or viral clearance is provided in Table 8 [Table 8]

[0079] Adverse events reported in >10% of patients indicated a favorable safety profile with no difference between the VGX-3100 and placebo groups. Adverse events are reported in Table 9. [Table 9]

[0080] Reported events of progression to cancer are provided in Table 10. [Table 10]

[0081] Conclusions: Although the primary endpoint of regression and clearance in the biomarker-positive population was not met, the trial met the secondary endpoints of regression and clearance in the overall population. The total number of progressive cases was 15 (7%): 8 (6%) in the treatment group and 7 (10%) in the placebo group. The adverse event profile was statistically comparable across groups. There was evidence of T-cell generation.

[0082] Embodiment The following list of embodiments is intended to complement, not replace or supersede, the previous description. Embodiment 1. A method of treating human papillomavirus (HPV) type 16 or HPV type 18 associated high-grade cervical intraepithelial lesion (HSIL), the method comprising administering a therapeutically effective amount of VGX-3100 to a subject in need thereof. Embodiment 2. The method of embodiment 1, wherein the HPV16 or HPV18 associated HSIL is determined by biopsy. Embodiment 3. The method of embodiment 1 or embodiment 2, wherein VGX-3100 is administered to the subject by intramuscular injection followed by electroporation. Embodiment 4. The method of any one of the preceding embodiments, wherein VGX-3100 is administered to the subject at a dose of 6 mg. Embodiment 5. The method of any one of the preceding embodiments, wherein VGX-3100 is administered to the subject three times over a 12-week period. Embodiment 6. The method of any one of the preceding embodiments, wherein VGX-3100 is formulated at a concentration of 6 mg / ml in 150 mM sodium chloride and 15 mM sodium citrate. Embodiment 7. The method of any one of the preceding embodiments, wherein administration of VGX-3100 results in virologic clearance of HPV-16 and / or HPV-18 and histopathologic regression of cervical HSIL. Embodiment 8. The method of any one of the preceding embodiments, wherein administration of VGX-3100 results in histopathological regression of cervical HSIL. Embodiment 9 The method of any one of the preceding embodiments, wherein administration of VGX-3100 results in virologic clearance of HPV-16 and / or HPV-18. Embodiment 10. The method of any one of the preceding embodiments, wherein administration of VGX-3100 results in complete histopathological regression of cervical HSIL to normal. Embodiment 11. The method of any one of the preceding embodiments, wherein administration of VGX-3100 results in complete histopathological regression of cervical HSIL to normal and virologic clearance of HPV-16 and / or HPV-18. Embodiment 12. The method of any one of the preceding embodiments, wherein administration of VGX-3100 results in histopathological non-progression. Embodiment 13. The method of any one of the preceding embodiments, wherein administration of VGX-3100 results in clearance of HPV-16 and / or HPV-18 infection from non-cervical anatomical locations. Embodiment 14. The method of any one of the preceding embodiments, wherein administration of VGX-3100 results in improved humoral and cellular immune responses to VGX-3100 as assessed relative to baseline after the third dose of VGX-3100, and at 36 weeks after administration of VGX-3100. Embodiment 15. The method of any one of the preceding embodiments, wherein the outcome of VGX-3100 administration is assessed 36 weeks after administration of VGX-3100.

Claims

1. 1. A method of treating human papillomavirus (HPV) type 16 or HPV type 18 associated high-grade cervical intraepithelial lesion (HSIL), said method comprising administering a therapeutically effective amount of VGX-3100 to a subject in need thereof.

2. 10. The method of claim 1, wherein HPV type 16 or HPV type 18 associated HSIL is determined by biopsy.

3. 3. The method of claim 1 or 2, wherein VGX-3100 is administered to the subject by intramuscular injection followed by electroporation.

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

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

6. 10. The method of 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.

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

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

9. 10. The method of any one of the preceding claims, wherein administration of VGX-3100 results in virologic clearance of HPV-16 and / or HPV-18.

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

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

12. 10. The method of any one of the preceding claims, wherein administration of VGX-3100 results in histopathological non-progression.

13. 10. The method of 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 locations.

14. 10. The method of 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 after the third administration of VGX-3100 and at 36 weeks after administration of VGX-3100.

15. 10. The method of any one of the preceding claims, wherein said outcome of VGX-3100 administration is assessed 36 weeks after administration of VGX-3100.

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