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

Abstract

Methods of treating human papilloma virus type 16 (HPV) or HPV type 18 associated high-level squamous intraepithelial lesions (HSIL) of the cervix are described.

Description

[0001] Cross-references to related applications

[0002] 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. Technical Field

[0003] Methods are provided for treating high-grade squamous intraepithelial lesions (HSIL) associated with human papillomavirus type 16 (HPV) or HPV type 18 in the cervix. Background Technology

[0004] Human papillomavirus (HPV) infection can lead to the formation of malignant tumors in both men and women, primarily occurring in the anogenital region and the gastrointestinal tract. Based on their carcinogenic potential, HPV types that target mucosal tissues are classified as high-risk (HR) and low-risk (LR) types (which typically cause benign lesions). HPV-16 and HPV-18 are the two most important high-risk types, as they cause the majority of HPV-related cancers. In the United States alone, there are approximately 14 million new cases of genital HPV infection each year, and about half of these infections involve high-risk HPV types. In the United States alone, up to 13,000 women are diagnosed with cervical cancer each year, with an estimated 4,120 deaths in 2017. HPV-16 is the most common high-risk genotype and, in combination with HPV-18, these two genotypes are estimated to cause approximately 70% of all cervical cancers.

[0005] Primary HPV infection is characterized by persistent viral replication and shedding, and when a woman's cervix is ​​infected with HPV, this infection is associated with early histological changes (grade 1 cervical intraepithelial neoplasia). Most cases of genital HPV infection clear spontaneously, but infection with one or more carcinogenic (high-risk) HPV genotypes can lead to the development of high-grade squamous intraepithelial lesions (HSIL), a precancerous histology of the cervix. HSIL includes grades 2 and 3 cervical intraepithelial neoplasia (CIN2 / 3). Over time, typically several years, cervical HSIL may progress to invasive cervical cancer. The basis for these changes is attributed to the viral proteins E6 and E7. Infected cells constitutively produce E6 and E7, which, respectively, increase the degradation of cell cycle regulatory proteins p53 and pRb, leading to uncontrolled cell growth and tumor formation.

[0006] There needs to be a treatment available for HPV16 or HPV18-related HSILs. Summary of the Invention

[0007] This article provides a method for treating high-grade cervical intraepithelial lesions (HSIL) associated with human papillomavirus (HPV) type 16 or HPV type 18, the method comprising administering a therapeutically effective amount of VGX-3100 to a subject in need of such treatment.

[0008] In another embodiment, cervical HPV type 16 or HPV type 18-associated HSIL is determined by biopsy.

[0009] In some embodiments, VGX-3100 is administered to the subject via intramuscular injection followed by electroporation. In some 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 12 weeks. 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.

[0010] In some 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 some embodiments, administration of VGX-3100 results in complete regression of cervical HSIL to normal histopathology. In further embodiments, administration of VGX-3100 results in complete regression of cervical HSIL to normal histopathology and virological clearance of HPV-16 and / or HPV-18. In still further embodiments, administration of VGX-3100 results in no progression of histopathology. In some embodiments, administration of VGX-3100 results in clearance of HPV-16 and / or HPV-18 infection from non-cervical anatomical locations. In a further embodiment, administration of VGX-3100 resulted in improved humoral and cellular immune responses to VGX-3100 relative to baseline at the third administration of VGX-3100 and at 36 weeks after administration of VGX-3100.

[0011] In some embodiments, the outcome of VGX-3100 administration is evaluated 36 weeks after administration. Detailed Implementation

[0012] The disclosed methods can be more readily understood by referring to the following detailed description, which forms part of this disclosure. It should be understood that the disclosed methods are not limited to the specific methods described and / or shown herein, and the terminology used herein is for the purpose of describing particular embodiments by way of example only and is not intended to limit the claimed methods.

[0013] Unless otherwise expressly stated, any description of possible mechanisms or modes of action or reasons for improvement is intended to be illustrative only, and the disclosed methods should not be limited by the correctness or incorrectness of any such suggested mechanisms or modes of action or reasons for improvement.

[0014] When expressing a series of numerical values, another embodiment includes from one specific value and / or to other specific values. Further, the values ​​described in the reference range include every value within that range. All ranges are inclusive and composable. When a value is expressed as an approximation using the antecedent “about,” it should be understood that the specific value forms another embodiment. Unless the context explicitly specifies otherwise, a reference to a specific numerical value includes at least that specific value.

[0015] It should be understood that, for clarity, certain features of the disclosed methods described herein in the context of individual embodiments may also be provided in combination in a single embodiment. Conversely, for brevity, the various features of the disclosed methods described above and below a single embodiment may also be provided individually or in any sub-combination.

[0016] Throughout the specification and claims, various terms are used in connection with different aspects of the specification. Unless otherwise stated, such terms shall have their ordinary meaning in the art. Other specifically defined terms will be interpreted in a manner consistent with the definitions provided herein.

[0017] As used in this article, the singular forms “a”, “an”, and “the” contain the plural.

[0018] certain terms

[0019] As used herein, the term “about” is used when referring to a range of reference figures, critical values, or specific values ​​to indicate that the stated value may differ from the listed value by as much as 10%. Therefore, the term “about” is used to cover variations of ±10%, ±5%, ±1%, ±0.5%, or ±0.1% or less from the specified value.

[0020] As used in this article, the term "at least one" means "one or more".

[0021] As used herein, the term "subject" refers to any animal, especially a human. Therefore, these methods are applicable to both human and non-human animals, but are best suited for humans. The terms "subject" and "patient" are used interchangeably in this document.

[0022] As used herein, the term “comprising” is intended to include instances covered by the terms “consisting essentially of” and “consisting of”; similarly, the term “essentially composed of” is intended to include instances covered by the term “composed of”.

[0023] As used herein, “treating” and similar terms refer to reducing symptoms of high-grade cervical intraepithelial lesions (HSIL) associated with human papillomavirus (HPV) type 16 or HPV type 18, such as the severity and / or frequency of HSIL lesions; eliminating symptoms of HPV type 16 or HPV type 18 infection, especially HSIL lesions; and / or clearing HPV type 16 or HPV type 18 virus from the subject; and / or regressing to low-grade cervical intraepithelial lesions (LSIL) or normal tissue.

[0024] As used herein, the term "coding sequence" or "coding nucleic acid" can mean a nucleic acid (RNA or DNA molecule) containing a nucleotide sequence encoding a polypeptide. The coding sequence may further include initiation and termination signals operatively linked to regulatory elements, including promoters and polyadenylation signals capable of directing the expression of the nucleic acid in an individual or mammalian cell. The coding sequence may further include a sequence encoding a signal peptide, such as an IgE leader sequence.

[0025] As used herein, the terms "nucleic acid," "oligonucleotide," or "polynucleotide" may refer to at least two nucleotides covalently linked together. The depiction of a single strand also defines the sequence of the complementary strand. Therefore, nucleic acids also include the complementary strand of the depicted single strand. Many variants of nucleic acids can be used as a given nucleic acid for the same purpose. Therefore, nucleic acids also include substantially the same nucleic acid and its complement. A single strand provides a probe that can hybridize with a target sequence under strict hybridization conditions. Therefore, nucleic acids also include probes that hybridize under strict hybridization conditions. Nucleic acids can be single-stranded or double-stranded, or may contain portions of both double-stranded and single-stranded sequences. Nucleic acids can be DNA (both genomic and cDNA), RNA, or a hybrid, wherein the nucleic acid may comprise 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 recombinant methods.

[0026] As used herein, the term "operably linked" can mean that gene expression is under the control of a promoter to which it is spatially linked. The promoter can be located at the 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 originates. As is known in the art, variations in this distance can be adapted without loss of promoter function.

[0027] As used herein, the term "promoter" can refer to a synthetic or naturally derived molecule that confers, activates, or enhances the expression of nucleic acids in a cell. A promoter may contain one or more specific transcriptional regulatory sequences to further enhance expression and / or alter its spatial and / or temporal expression. Promoters may also contain distal enhancer or repressor elements, which can be located up to thousands of base pairs from the transcription start site. Promoters can be derived from sources including viruses, bacteria, fungi, plants, insects, and animals. Promoters can constitutively or differentially regulate the expression of genomic molecules relative to the cell, tissue, or organ in which expression occurs, or relative to the developmental stage in which expression occurs, or in response to external stimuli such as physiological stress, pathogens, metal ions, or inducers.

[0028] As used herein, the term "vector" can refer to a nucleic acid sequence containing an origin of replication. Vectors can be plasmids, bacteriophages, bacterial artificial chromosomes, or yeast artificial chromosomes. Vectors can be DNA or RNA vectors. Vectors can be self-replicating extrachromosomal vectors or vectors integrated into the host genome.

[0029] As used herein, the term “adverse event” (AE) is defined according to the Common Toxicity Criteria for Adverse Events (CTCAE) version 4.03 grading criteria. Researchers grade laboratory and clinical AEs according to the following severity levels for the applicable subject population, based on CTCAE version 4.03: Grade 1 (mild), Grade 2 (moderate), Grade 3 (severe), Grade 4 (potentially life-threatening), and Grade 5 (fatal).

[0030] As used in this article, the term “responder” refers to the proportion of study subjects who were positive for the primary endpoint of baseline biomarkers at the 36-week visit, based on type-specific HPV testing, with no evidence of cervical HSIL in their histological samples and no evidence of HPV-16 and / or HPV-18 in their cervical samples.

[0031] As used herein, the term "placebo" refers to the administration of a pharmaceutical composition that does not contain VGX-3100.

[0032] Treatment and diagnostic methods

[0033] This article provides a method for treating high-grade cervical intraepithelial lesions (HSIL) associated with human papillomavirus (HPV) type 16 or HPV type 18, the method comprising, consisting of, or substantially consisting of administering a therapeutically effective amount of VGX-3100 to a subject in need of such treatment.

[0034] This article also provides VGX-3100 for use in methods of treating high-grade cervical intraepithelial lesions (HSIL) associated with human papillomavirus (HPV) type 16 or HPV type 18, said methods comprising, being, or substantially consisting of administering a therapeutically effective amount of VGX-3100 to a subject in need of it.

[0035] This article also provides the use of VGX-3100 in the manufacture of a medicament for the treatment of human papillomavirus (HPV) type 16 or HPV type 18 associated high-grade cervical intraepithelial lesion (HSIL), the method comprising, consisting of, or substantially consisting of administering a therapeutically effective amount of VGX-3100 to a subject in need of such treatment.

[0036] In another embodiment, cervical HPV type 16 or HPV type 18-associated HSIL is determined by biopsy.

[0037] In some 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 some embodiments, administration of VGX-3100 results in complete regression of cervical HSIL to normal histopathology. In further embodiments, administration of VGX-3100 results in complete regression of cervical HSIL to normal histopathology and virological clearance of HPV-16 and / or HPV-18. In still further embodiments, administration of VGX-3100 results in no progression of histopathology. In some embodiments, administration of VGX-3100 results in clearance of HPV-16 and / or HPV-18 infection from non-cervical anatomical locations. In a further embodiment, administration of VGX-3100 resulted in improved humoral and cellular immune responses to VGX-3100 relative to baseline at the third administration of VGX-3100 and at 36 weeks after administration of VGX-3100.

[0038] This article also provides a method for improving virological clearance of HPV-16 and / or HPV-18 and histopathological regression of cervical HSIL in subjects, the method comprising, consisting of, or substantially consisting of administering a therapeutically effective amount of VGX-3100 to subjects in need. In some embodiments, 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 subject population. In some embodiments, 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 subject population. In some embodiments, 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 subject population with standard care.

[0039] This article also provides a method for improving the histopathological regression of cervical HSIL in subjects, the method comprising, consisting of, or substantially consisting of administering a therapeutically effective amount of VGX-3100 to subjects in need. In some embodiments, improving the histopathological regression of cervical HSIL is relative to administering a placebo to the subject or subject population. In some embodiments, improving the histopathological regression of cervical HSIL is relative to not treating the subject or subject population. In some embodiments, improving the histopathological regression of cervical HSIL is relative to treating the subject or subject population with standard care.

[0040] This article also provides a method for achieving complete histopathological regression to normal for cervical HSIL in subjects, the method comprising, consisting of, or substantially consisting of administering a therapeutically effective amount of VGX-3100 to a subject in need. In some embodiments, achieving complete histopathological regression to normal for cervical HSIL is relative to administering a placebo to the subject or subject population. In some embodiments, achieving complete histopathological regression to normal for cervical HSIL is relative to not treating the subject or subject population. In some embodiments, achieving complete histopathological regression to normal for cervical HSIL is relative to treating the subject or subject population with standard care.

[0041] This article also provides a method for achieving complete histopathological regression to normal and virological clearance of HPV-16 and / or HPV-18 in subjects, the method comprising, consisting of, or substantially consisting of administering a therapeutically effective amount of VGX-3100 to a subject in need. In some embodiments, achieving complete histopathological regression to normal and virological clearance of HPV-16 and / or HPV-18 is relative to administering a placebo to the subject or subject population. In some embodiments, achieving complete histopathological regression to normal and virological clearance of HPV-16 and / or HPV-18 is relative to not treating the subject or subject population. In some embodiments, achieving complete histopathological regression to normal and virological clearance of HPV-16 and / or HPV-18 is relative to treating the subject or subject population with standard care.

[0042] This article also provides a method for improving histopathological progression-free status in subjects, the method comprising, consisting of, or substantially consisting of administering a therapeutically effective amount of VGX-3100 to a subject in need. In some embodiments, the improvement in histopathological progression-free status is relative to administration of a placebo to the subject or subject population. In some embodiments, the improvement in histopathological progression-free status is relative to no treatment received by the subject or subject population. In some embodiments, the improvement in histopathological progression-free status is relative to treatment received by the subject or subject population receiving standard care.

[0043] This article also provides a method for improving the clearance of HPV-16 and / or HPV-18 infection originating from non-cervical anatomical sites in subjects, the method comprising, consisting of, or substantially consisting of administering a therapeutically effective amount of VGX-3100 to a subject in need. In some embodiments, improved 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 subject population. In some embodiments, improved clearance of HPV-16 and / or HPV-18 infection from non-cervical anatomical sites is relative to no treatment of the subject or subject population. In some embodiments, improved clearance of HPV-16 and / or HPV-18 infection from non-cervical anatomical sites is relative to treatment of the subject or subject population with standard care.

[0044] This article also provides a method for improving humoral and cellular immune responses to VGX-3100 relative to baseline after a third administration of VGX-3100 and at 36 weeks after administration, the method comprising, consisting of, or substantially consisting of administering a therapeutically effective amount of VGX-3100 to a subject in need. In some embodiments, improved clearance of HPV-16 and / or HPV-18 infection from non-cervical anatomical sites is relative to administration of a placebo to a subject or subject population. In some embodiments, improved humoral and cellular immune responses to VGX-3100 relative to baseline after a third administration of VGX-3100 and at 36 weeks after administration of VGX-3100 is relative to no treatment of a subject or subject population. In some embodiments, the improved humoral and cellular immune response to VGX-3100 relative to baseline at the third administration of VGX-3100 and at 36 weeks after administration of VGX-3100 is relative to treatment of the subject or subject population with standard care.

[0045] In some embodiments, the results of VGX-3100 administration or the improvements achieved through VGX-3100 administration are evaluated 36 weeks after administration.

[0046] Route of administration and pharmaceutical composition

[0047] 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 adenovirus-associated virus, and recombinant vaccinia virus.

[0048] Routes of administration include, but are not limited to, intramuscular, intranasal, intraperitoneal, intradermal, subcutaneous, intravenous, intraarterial, intraocular, and oral administration, as well as local, transdermal, inhalation, suppository, or mucosal tissue administration (such as by irrigation into the vagina, rectum, urethra, oral cavity, and sublingual tissue). Preferred routes of administration include intramuscular, intraperitoneal, intradermal, and subcutaneous injection. The genetic construct can be administered via methods including, but not limited to, electroporation methods and instruments, conventional syringes, needle-free injection devices, or "microprojectile bombardment gone guns." In some embodiments, VGX-3100 is administered to the subject via intramuscular injection. In some embodiments, VGX-3100 is administered to the subject via intramuscular injection followed by electroporation.

[0049] Examples of preferred electroporation devices and methods for facilitating DNA vaccine delivery include those described in U.S. Patent No. 7,245,963 to Drghia-Akli et al., and U.S. Patent Publication No. 2005 / 0052630 to Smith et al., the contents of which are hereby incorporated herein by reference in their entirety. Also preferred are electroporation devices and methods for facilitating DNA vaccine delivery, provided in co-pending and co-owned U.S. Patent Application Serial No. 11 / 874072, 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, pursuant to 35 USC 1 19(e), all of which are hereby incorporated herein by reference in their entirety. In some embodiments, the electroporation device is -5P device.

[0050] The following are examples of embodiments using electroporation technology, discussed in more detail in the patent references discussed above: An electroporation device can be configured to deliver energy pulses to desired tissues in mammals, the pulses generating a constant current similar to a preset current input by the user. The electroporation device includes an electroporation component and an electrode assembly or handle assembly. The electroporation component may include and be incorporated into 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 supply, and a power switch. The electroporation component may function as one element of the electroporation device, and other elements are separate elements (or components) communicating with the electroporation component. In some embodiments, the electroporation component may function as more than one element of the electroporation device, communicating with other elements of the electroporation device separate from the electroporation component. The use of electroporation technology to deliver modified HPV vaccines is not limited to the elements of the electroporation device existing as part of an electromechanical or mechanical device, because these elements may function as a device or as separate elements communicating with each other. The electroporation component is capable of delivering energy pulses that generate a constant current in desired tissue and includes a feedback mechanism. The electrode assembly includes an electrode array having multiple electrodes arranged in a spatial configuration, wherein the electrode assembly receives energy pulses from the electroporation component and delivers them to the desired tissue via the electrodes. At least one of the multiple electrodes is neutral during energy pulse delivery and measures the impedance in the desired tissue, transmitting the impedance to the electroporation component. The feedback mechanism accepts the measured impedance and can adjust the energy pulses delivered by the electroporation component to maintain a constant current.

[0051] In some embodiments, multiple electrodes may deliver energy pulses in a distributed pattern. In some embodiments, multiple electrodes may deliver energy pulses in a distributed pattern 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, wherein each of the multiple pulses is delivered by at least two active electrodes with a neutral electrode having a measuring impedance, and wherein subsequent pulses of the multiple pulses are delivered by another of the at least two active electrodes with a neutral electrode having a measuring impedance.

[0052] In some embodiments, the feedback mechanism is implemented by hardware or software. Preferably, the feedback mechanism is implemented by an analog closed-loop circuit. Preferably, such feedback occurs once every 50 μg, 20 μg, 10 μg, or 1 μg, but real-time or instantaneous feedback (i.e., a basic instantaneous time determined by available techniques for determining response time) is preferred. In some embodiments, a 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 energy pulse delivery.

[0053] In some embodiments, nucleic acid molecules are delivered to cells together with the administration of polynucleotide enhancers or gene vaccine promoters. Polynucleotide enhancers are described in U.S. Serial Nos. 5,593,972, 5,962,428 and International Application Serial No. PCT / US94 / 00899, filed January 26, 1994, each of which is incorporated herein by reference. Adjuvants administered in conjunction with nucleic acid molecules may be administered as a mixture with the nucleic acid molecules, or separately, simultaneously with, before, or after the administration of the nucleic acid molecules. In addition, other agents 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 α-interferon, γ-interferon, GM-CSF, platelet-derived growth factor (PDGF), TNF, epidermal growth factor (EGF), IL-1, IL-2, IL-4, IL-6, IL-10, IL-12, and IL-15, and fibroblast growth factor; surfactants such as immunostimulatory complexes (ISCOM); Freund's incomplete adjuvant; LPS analogs including monophospholipid A (WL), cell wall peptides, quinone analogs, and vesicles (such as squalene and squalene); and hyaluronic acid can also be used for co-administration with gene constructs. In some embodiments, immunomodulatory proteins can be used as GVF. In some embodiments, nucleic acid molecules that bind to PLG are provided to enhance delivery / uptake.

[0054] The pharmaceutical composition according to the invention comprises about 1 nanogram to about 2000 micrograms of DNA. In some preferred embodiments, the pharmaceutical composition according to the invention comprises about 5 nanograms to about 1000 micrograms of DNA. In some preferred embodiments, the pharmaceutical composition comprises about 10 nanograms to about 800 micrograms of DNA. In some preferred embodiments, the pharmaceutical composition comprises about 0.1 to about 500 micrograms of DNA. In some preferred embodiments, the pharmaceutical composition comprises about 1 to about 350 micrograms of DNA. In some preferred embodiments, the pharmaceutical composition comprises about 25 to about 250 micrograms of DNA. In some preferred embodiments, the pharmaceutical composition comprises about 100 to about 200 micrograms of DNA. In some embodiments, VGX-3100 is formulated at a concentration of 6 mg / ml.

[0055] The pharmaceutical compositions according to the invention are formulated according to the intended administration method. In the case of injectable pharmaceutical compositions, they are sterile, pyrogen-free, and particulate-free. Isotonic formulations are preferred. Typically, isotonic additives may include sodium chloride, glucose, mannitol, sorbitol, and lactose. In some cases, isotonic solutions, such as phosphate-buffered saline, are preferred. Stabilizers comprise 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 some embodiments, VGX-3100 is formulated at a concentration of 6 mg / ml in 150 mM sodium chloride and 15 mM sodium citrate.

[0056] In some 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 12 weeks. In even further embodiments, a first dose of VGX-3100 is administered on day 0, a second dose of VGX-3100 is administered at week 4, and a third dose of VGX-3100 is administered at week 12.

[0057] Example

[0058] The examples and embodiments described herein are for illustrative purposes only, and various modifications or alterations suggested to those skilled in the art will be included within the spirit and scope of this application and the appended claims.

[0059] Example 1: Revealing 2-intramuscular delivery of VGX-3100 followed by CELLECTRA TM A randomized, double-blind, placebo-controlled phase 3 study (HPV-303) of 5PSP electroporation for the treatment of HPV-16 and / or HPV-18 associated high-grade squamous intraepithelial lesions (HSIL) of the cervix.

[0060] One intramuscular delivery (IM) of VGX-3100, followed by CELLECTRA TM This is a non-limiting example of a randomized, double-blind, placebo-controlled phase 3 study of 5PSP electroporation 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 women with baseline positive biomarkers who had no evidence of cervical HSIL in their histological samples and no evidence of HPV-16 and / or HPV-18 in their cervical samples by type-specific HPV testing at the 36-week visit.

[0061] Clinical trial goals and endpoints:

[0062] Primary objective: To determine the efficacy of VGX-3100 compared with placebo in the histopathological regression of cervical HSIL and the virological clearance of HPV-16 and / or HPV-18 in women with positive baseline biomarkers.

[0063] Primary endpoint: The proportion of women who were baseline biomarker-positive at the 36-week visit, with no evidence of cervical HSIL in histological samples and no evidence of HPV-16 and / or HPV-18 in cervical samples as determined by type-specific HPV testing.

[0064] Secondary objectives and related secondary endpoints are shown in Table 1.

[0065] Table 1.

[0066]

[0067]

[0068] The exploratory objectives and related exploratory endpoints are shown in Table 2.

[0069] Table 2.

[0070]

[0071] Research Design

[0072] Experimental treatment: 6 mg (1 ml) of VGX-3100 was administered intramuscularly on day 0, week 4, and week 12, followed by CELLECTRA. TM The 5PSP device performs EP.

[0073] Study Design: Participants must meet the following criteria: be eligible for this study, be at least 18 years of age and older than the age of consent, and have given prior consent to participate and undergo a cervical biopsy / biopsy of a cervical lesion site at the time of screening. Biopsy slides will be sent to the PAC in a blinded manner to determine the presence of cervical HSIL during the screening period. To be eligible for randomization, the PAC should specify a histological diagnosis of cervical HSIL. Participants must also undergo cobas TM Only those with a positive HPV test result for HPV-16 / 18 in their cervical specimen are eligible to participate in this study.

[0074] A sample of approximately 198 subjects was randomly assigned in a 2:1 ratio to receive either 6 mg (in 1 ml) of VGX-3100 or placebo, with EP performed after IM in each group. Based on the HPV-301 study results, assuming the true proportions of subjects with lesion regression and HPV-16 / 18 clearance in the VGX-3100 and placebo groups were 66% and 15%, respectively, and the proportion of biomarker-positive women was 33%, this sample size provided >90% power to demonstrate that VGX-3100 was superior to placebo in biomarker-positive women. In this context, a biomarker positivity was defined as the detection of a predefined microRNA (miRNA) signature in the plasma of a subject assessed prior to VGX-3100 or placebo, indicating anticipated lesion regression and HPV16 / 18 clearance. Subjects who, based on this signal, were not expected to experience lesion regression and HPV16 / 18 clearance prior to VGX-3100 or placebo were considered biomarker-negative.

[0075] Subjects were stratified and randomized according to the following criteria: (a) severity of CIN observed in biopsy specimens at screening (i.e., CIN2 vs. CIN3), (b) BMI classification on day 0 (≤25 vs. >25 kg / m2), and (c) age group on day 0 (<25 years vs. ≥25 years). To ensure that CIN2 did not constitute an excessive proportion of the study, the percentage of enrolled subjects with CIN2 did not exceed 50% of the total enrollment. Each country received a consecutive set of allocation numbers.

[0076] Efficacy assessment: The primary endpoint of the phase 3 study (HPV-303) was based on the results of the phase 2b study (HPV-003) and the phase 3 study (HPV-301). Given that HPV persistence is a significant factor in the clinical progression of cervical dysplasia, and also based on the results of the secondary objectives of the HPV-003 study, the responder definition for the primary endpoint of HPV-303 will consider both the histological regression of cervical HSILs and the clearance of high-risk HPV-16 and HPV-18.

[0077] Histopathological evaluation of cervical tissue is considered the definitive method for diagnosing cervical dysplasia to determine whether cervical HSIL has regressed. Tissue samples to be used for histopathological analysis of evidence of regression will be obtained at week 36. Cervical cytology samples will be obtained at screening, day 0 (before administration), and weeks 8, 15, 28, and 36 to identify HPV infection. Additionally, unstained sections and / or associated paraffin blocks may be obtained for HPV-16 and / or HPV-18 testing if residual tissue remains in the paraffin block after histological diagnosis has been completed. Vaginal, oropharyngeal, and optional intrarectal samples will be obtained at day 0 (before administration) and week 36 to identify HPV infection and assess virological response to treatment in sites other than the cervix.

[0078] Immunogenicity assessment: Humoral and cellular immune responses to VGX-3100 treatment were assessed in blood samples collected at baseline (at screening and on day 0 before dosing) and at weeks 15 and 36. Cervical tissue samples were analyzed at week 36 as evidence of an improved immune response compared to baseline (at screening).

[0079] Safety assessment: The risk of HSIL progressing to cancer or HSIL recurrence is considered low and comparable to the incidence observed after LEEP / CKC. Long-term follow-up for this HPV-303 study program will include safety, cytological examination, and HPV-16 and / or HPV-18 testing, continuing for up to 7 months after the last dose of study treatment.

[0080] The Data Safety Monitoring Board (DSMB) will review the safety data and histopathological regression results. If safety issues arise, and if the proportion of subjects with histopathological regression in the VGX-3100 group is unacceptably lower than in the placebo group, the DSMB will be responsible for making recommendations to the sponsor. No formal interim analysis will be conducted.

[0081] Trial population: This multicenter global trial enrolled women aged 18 years and older. VGX-3100 is being developed as a non-surgical treatment option for the treatment of precancerous cervical lesions, precancerous cervical HSIL, and potentially pathogenic HPV-16 and / or HPV-18 infections.

[0082] Inclusion criteria - Each participant must meet all of the following criteria to be enrolled in this study:

[0083] 1. Women aged 18 or older who meet the minimum age of consent according to local regulations;

[0084] 2. Screening is done via cobass TM HPV testing confirmed a cervical infection with HPV types 16 and / or 18;

[0085] 3. Cervical tissue specimens / sections provided to the study pathology review committee for diagnosis must be collected within 10 weeks prior to the expected date of the first dose of the study drug;

[0086] 4. During screening, histological evidence of cervical HSIL was confirmed by the Pathology Adjudication Committee (PAC);

[0087] 5. Participants must understand, agree to, and be able to comply with the requirements of the agreement; participants must be willing and able to provide voluntary consent and sign a consent form before participating in research-related activities;

[0088] 6. Must be determined by the researcher to be a suitable candidate as required by the protocol procedures at week 36;

[0089] 7. Satisfactory colposcopy during screening is defined as complete visibility of the squamocolumnar junction (type I or type II transformation zone) and complete visibility of the upper margin of acetowhite epithelium or suspected CIN lesions;

[0090] 8. Cervical lesions that can be sampled using biopsy instruments (e.g., the Mini-Tischler device);

[0091] 9. A sufficiently large cervical lesion to ensure that visible lesions remain after screening biopsy;

[0092] 10. Their reproductive capacity must meet one of the following criteria:

[0093] a. Postmenopause is defined as spontaneous amenorrhea lasting more than 12 months;

[0094] b. Surgical sterilization due to the absence of ovaries or due to bilateral tubal ligation / occlusion more than 12 months prior to screening;

[0095] c. Women of reproductive age (WOCBP) agree to consistently and correctly use a contraceptive method with an annual failure rate of less than 1% from screening to week 36. Condoms alone or spermicide-containing condoms cannot achieve an annual failure rate of <1%, and therefore they are not considered acceptable contraceptive methods. The following methods are acceptable:

[0096] ■ Hormonal contraceptives: including combination therapy or single progestin use, including oral contraceptives, injections, implants, vaginal rings, or transdermal patches. Hormonal contraceptives should not be used in subjects with a history of hypercoagulable states (e.g., deep vein thrombosis, pulmonary embolism);

[0097] ■ When penile-vaginal intercourse is the subject's sexual behavior, abstinence should be practiced;

[0098] ■Intrauterine device or intrauterine contraceptive system;

[0099] ■ Female participants must have had their male partners sterilized for at least 6 months prior to enrollment in this study, and the male partner must be the only partner of the participant.

[0100] 11. Normal screening electrocardiogram (ECG), or screening ECG with no clinically significant abnormalities as determined by the investigator.

[0101] Exclusion Criteria - Participants who meet any of the following criteria will not be allowed to participate in or continue to participate in this study:

[0102] 1. During screening, microscopic or gross evidence of any of the following conditions is found in any histopathological specimen: adenocarcinoma in situ (AIS), high-grade vulvar, vaginal (including cervical HPV-related lesions extending into the vaginal fornix), or anal intraepithelial neoplasia or invasive carcinoma.

[0103] 2. During screening, because the colposcopy extends too high into the cervical canal, the cervical lesions cannot be fully visualized.

[0104] 3. Indicate the possible presence of untreated cancer, untreated HSIL, or ECC with an unclear or inadequate diagnosis (ECC is not required as part of the study screening);

[0105] 4. Those who have received cervical HSIL treatment within 4 weeks prior to screening;

[0106] 5. During pregnancy, breastfeeding, or if pregnancy is anticipated before the 36th week visit;

[0107] 6. Previous history of therapeutic HPV vaccination (allowing licensed prophylactic HPV vaccines, such as Gardasil) TM Silgard TM Cervarix TM );

[0108] 7. According to Common Toxicity Criteria for Adverse Events (CTCAE) version 4.03, any unresolved Grade 1 or higher abnormal clinical screening laboratory value that was determined by the investigator to be clinically significant within 60 days prior to Day 0;

[0109] 8. Immunosuppression caused by underlying diseases or treatments, including:

[0110] a) Has a history of HIV or a positive HIV serological test at the time of screening (done within 60 days before day 0);

[0111] b) Primary immunodeficiency;

[0112] c) Long-term use (≥7 days) of oral or parenteral corticosteroids at a dose ≥20 mg / day, calculated as prednisone equivalent; (inhaled, ototropic and ophthalmic corticosteroids are permitted);

[0113] d) Antirheumatic drugs currently used or expected to be used at disease-reducing doses (e.g., azathioprine, cyclophosphamide, cyclosporine, methotrexate), and biological disease-reducing drugs, such as TNF-α inhibitors (e.g., infliximab, adalimumab, or etanercept).

[0114] e) History of solid organ or bone marrow transplantation;

[0115] f) Any other clinically significant history of immunosuppression or clinically diagnosed autoimmune disease that may jeopardize the safety of the subject, require treatment that interferes with the study evaluation or endpoint assessment, or otherwise affect the validity of the study results.

[0116] g) Subjects with malnutrition (i.e., unintentional weight loss of medical significance) were selected based on the investigator’s clinical judgment, laboratory tests, medical history and physical examination.

[0117] 9. Received any non-research, non-live vaccine within 2 weeks of administration;

[0118] 10. Administer any non-research live vaccine (e.g., measles vaccine) within 4 weeks of administration;

[0119] 11. Currently or previously suffering from a clinically significant, medically unstable disease that, in the investigator's opinion, would jeopardize the safety of the subject, interfere with the study evaluation or endpoint assessment, or otherwise affect the validity of the study results (e.g., chronic renal failure; angina, myocardial ischemia or myocardial infarction, grade 3 or higher congestive heart failure, cardiomyopathy, or clinically significant arrhythmia).

[0120] 12. Patients with malignant tumors or who have received systemic treatment for malignant tumors within the past 2 years (malignant tumors of the anus and genitals and superficial skin cancers that have received local treatment are allowed);

[0121] 13. Presence of acute or chronic bleeding or coagulation disorders that contraindicate IM injection, or use of blood thinners (e.g., anticoagulants or antiplatelet drugs) within 2 weeks prior to day 0;

[0122] 14. History of seizures, unless no seizures have occurred within 5 years of using one or fewer antiepileptic drugs;

[0123] 15. At the time of screening or on day 0, a consistently and manually confirmed sitting systolic blood pressure >150 mm Hg or <90 mm Hg, or a diastolic blood pressure >95 mm Hg;

[0124] 16. Resting heart rate <50 bpm (unless attributable to exercise-induced heart rate) or >100 bpm at screening or day 0;

[0125] 17. Had a major surgery within 4 weeks prior to day 0;

[0126] 18. Participation in interventional studies using investigational compounds or devices (excluding HPV-301REVEAL 1) within 30 days of signing the informed consent form; participation in observational studies is permitted.

[0127] 19. Considering the deltoid and anterolateral quadriceps muscles, there are fewer than two acceptable sites for IM injection;

[0128] 20. Tattoos, keloids, or hypertrophic scars located within 2 cm of the intended treatment site;

[0129] 21. A defibrillator or pacemaker located at the ipsilateral deltoid injection site (to prevent life-threatening arrhythmias) (unless deemed acceptable by a cardiologist);

[0130] 22. Metal implants or implantable medical devices within the electroporation area;

[0131] 23. Active drug use or dependence that researchers believe would interfere with adherence to research requirements;

[0132] 24. Detainees or subjects who are involuntarily imprisoned for treatment of mental or physical illness (i.e., infectious disease);

[0133] 25. Active military personnel;

[0134] 26. Research staff or their family members;

[0135] 27. Any disease or condition that, in the investigator's view, may affect the safety of the subjects or the assessment of any study endpoint.

[0136] The definitions of responders and non-responders for the combined extinction and clearance endpoints are provided in Table 3:

[0137] Table 3.

[0138]

[0139] Test product, dosage and administration method, batch number

[0140] Research products

[0141] Generic name: VGX-3100

[0142] Chemical name: Circular double-stranded deoxyribonucleic acid (DNA). For the pGX3001 plasmid, it consists of 3782 base pairs, and for the pGX3002 plasmid, it consists of 3824 base pairs.

[0143] Distinguishing names: Eukaryotic expression plasmid containing HPV 16 and 18-E6 & E7 encoding transcription units controlled by a synthetic CMV promoter, as well as elements required for replication and selection in E. coli, namely the pUC origin of replication (pUC Ori) and the kanamycin resistance gene (Kan R).

[0144] Detailed Description: VGX-3100 (HPV Therapeutic Vaccine) is a combination of two equal-volume plasmids (i.e., a 6 mg dose will deliver 3 mg of each pGX3001 and pGX3002 plasmid): a) pGX3001: p16ConE6E7, a plasmid that encodes the synthetic HPV16 consensus E6 and E7 fusion gene (“Consensus HPV 16-6&7”) into the pVAX1 backbone (Invitrogen, Carlsbad, CA) under the control of the cytomegalovirus Immediate Early (CMV) promoter; and b) pGX3002: p18ConE6E7, a plasmid that encodes the synthetic HPV18 consensus E6 and E7 fusion gene (“Consensus HPV 18-6&7”) into the 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 herein by reference in its entirety. Consensus sequences of nucleic acids and amino acids are shown in Tables 4 and 5, respectively.

[0145] Table 4. VGX-3100 Consensus Sequences (Nucleic Acids)

[0146]

[0147] Table 5. VGX-3100 consensus sequence (amino acids)

[0148]

[0149]

[0150] To prepare for the Phase 3 project, a buffered refrigerated formulation (2°C to 8°C storage) for VGX 3100 was developed using sodium citrate (SSC) solution. This refrigerated formulation requires the use of the next-generation CELLECTRA device. TMAdministered at 5 PSPs. In the Phase 1 clinical trial HPV-101, refrigerated formulation of VGX-3100 was first administered to 117 subjects. In the HPV-101 study, approximately half of the healthy volunteers received three 6 mg IM doses of VGX-3100 (refrigerated formulation) followed by EP. Based on the analysis of the data, the refrigerated formulation was determined to be non-inferior to the frozen formulation based on a 2-fold increase in total spotted units (SPUs) of plasmid-encoded antigens per 106 peripheral blood mononuclear cells (PBMCs) measured from baseline to week 14, as determined by interferon-γ ELISpot assay.

[0151] Efficacy Results

[0152] This trial did not meet the primary endpoint of regression and clearance in the biomarker-positive population. The trial met the secondary endpoint of regression and clearance in all patients. VGX-3100 was well tolerated and there was no difference in reported adverse event characteristics between the treatment and placebo groups. VGX-3100 is immunogenic and generated antigen-specific T cells in the periphery and T cells in the tissues.

[0153] The research group is provided in Table 6:

[0154] Table 6.

[0155]

[0156] The regression of HSIL and the clearance of the virus are shown in Table 7:

[0157] Table 7.

[0158]

[0159] The regression of HSIL or the clearance of the virus are shown in Table 8.

[0160] Table 8.

[0161] fade VGX-3100 placebo Difference (95% CI) biomarkers 8(38.1) 1(25) 13.1(-37.7,46) All patients 46(34.3) 15(21.7) 12.6(-0.8,24.5) Clear VGX-3100 placebo Difference (95% CI) biomarkers 7(33.3) 0 33.3(-20.1,55) All patients 50(37.3) 6(8.7) 28.6(17.1,38.7)

[0162] Adverse events reported in >10% of patients demonstrated a favorable safety profile, with no difference between the VGX-3100 group and the placebo group. Adverse events are shown in Table 9.

[0163] Table 9.

[0164] Adverse events VGX-3100 (%) Placebo (%) Injection site pain 91.8 86.6 Headache 49.3 55.2 fatigue 41.8 49.3 Myalgia 41.8 43.3 Swelling at the injection site 32.1 28.4 nausea 26.9 25.4 Itching at the injection site 24.6 32.8 Joint pain 21.6 20.9 Injection site contusion 15.7 16.4 discomfort 13.4 17.9 Hematoma at the injection site 7.5 10.4

[0165] The events that progressed to cancer are shown in Table 10.

[0166] Table 10.

[0167] From HSIL to cancer VGX-3100(N,%) Placebo (N, %) biomarkers 3(14％) 0 All patients 8(6％) 7(10％)

[0168] Conclusion: Although the primary endpoints of regression and viral clearance were not met in the biomarker-positive population, the trial met the secondary endpoints of regression and viral clearance in the overall population. The total number of cases of progression was 15 (7%), with 8 (6%) in the treatment group and 7 (10%) in the placebo group. The adverse event profile was statistically comparable between the groups. Evidence of T-cell production was present.

[0169] Example

[0170] The following list of examples is intended to supplement, rather than replace or supersede, the preceding description.

[0171] Example 1. A method for treating high-grade cervical intraepithelial lesion (HSIL) associated with human papillomavirus (HPV) type 16 or HPV type 18, the method comprising administering a therapeutically effective amount of VGX-3100 to a subject in need of the treatment.

[0172] Example 2. The method according to Example 1, wherein type 16 or type 18 HPV-related HSIL is determined by biopsy.

[0173] Example 3. The method according to Example 1 or Example 2, wherein VGX-3100 is administered to the subject via intramuscular injection followed by electroporation.

[0174] Example 4. The method according to any one of the preceding examples, wherein VGX-3100 is administered to the subject at a dose of 6 mg.

[0175] Example 5. The method according to any one of the preceding examples, wherein VGX-3100 is administered to the subject three times during a 12-week period.

[0176] Example 6. The method according to any one of the preceding examples, wherein VGX-3100 is prepared at a concentration of 6 mg / ml in 150 mM sodium chloride and 15 mM sodium citrate.

[0177] Example 7. The method according to any one of the preceding examples, wherein the administration of VGX-3100 results in virological clearance of HPV-16 and / or HPV-18 and histopathological regression of cervical HSIL.

[0178] Example 8. The method according to any one of the preceding examples, wherein the administration of VGX-3100 leads to histopathological regression of cervical HSIL.

[0179] Example 9: The method according to any one of the preceding examples, wherein the administration of VGX-3100 results in the virological clearance of HPV-16 and / or HPV-18.

[0180] Example 10. The method according to any one of the preceding examples, wherein the application of VGX-3100 resulted in complete histopathological regression of cervical HSIL to normal.

[0181] Example 11. The method according to any one of the preceding examples, wherein the administration of VGX-3100 results in complete histopathological regression of cervical HSIL to normal and virological clearance of HPV-16 and / or HPV-18.

[0182] Example 12. The method according to any one of the preceding examples, wherein the administration of VGX-3100 resulted in no histopathological progression.

[0183] Example 13. The method according to any one of the preceding examples, wherein the administration of VGX-3100 results in the clearance of HPV-16 and / or HPV-18 infection from non-cervical anatomical locations.

[0184] Example 14. The method according to any one of the preceding examples, wherein administration of VGX-3100 resulted in improved humoral and cellular immune responses to VGX-3100 relative to baseline at the third administration of VGX-3100 and at 36 weeks after administration of VGX-3100.

[0185] Example 15. The method according to any one of the preceding examples, wherein the results of VGX-3100 administration are evaluated 36 weeks after administration.

Claims

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

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

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

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

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

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

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

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

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

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

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

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

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

14. 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 relative to baseline at the third administration of VGX-3100 and at 36 weeks after administration of VGX-3100.

15. The method according to any one of the preceding claims, wherein the results of VGX-3100 administration are evaluated 36 weeks after administration.

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