WO2014085546A1

WO2014085546A1 - Use of ar-42 enhances e7-specific cd8+ t-cell mediated antitumor immunity in conjunction with dna vaccine

Info

Publication number: WO2014085546A1
Application number: PCT/US2013/072175
Authority: WO
Inventors: T.C. Wu; Chien-Fu Hung
Original assignee: The Johns Hopkins University
Priority date: 2012-11-29
Filing date: 2013-11-27
Publication date: 2014-06-05

Abstract

The present invention provides methods of treatment of HPV related cancers by administration and co-administration of the HDAC inhibitor AR-42 with CRT/E7 DNA vaccine which improves priming of tumor-specific CD8+ T cells by professional antigen presenting cells transfected by DNA vaccines. Tumor-bearing mice treated with the combination of AR-42 and CRT/E7 DNA vaccine experienced longer survival, decreased tumor growth, enhanced E7-specific immune response, and reduced immunosuppression compared to mice treated with AR-42 or CRT/E7 DNA vaccine alone. Treatment of TC-1 cells with the HDAC inhibitor AR-42 increased surface expression of MHC class I molecules and enhanced cytotoxicity of E7-specific T cells. The present invention shows that AR-42 is the ideal HDACi to be used in combination with a therapeutic HPV DNA vaccine in order to generate a potent therapeutic antitumor effect against E6/E7-expressing tumors

Description

USE OF AR-42 ENHANCES E7-SPECIFIC CD8+ T-CELL MEDIATED ANTITUMOR IMMUNITY IN CONJUNCTION WITH DNA VACCINE

REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Patent Application Nos. 61/731,225, filed on November 29, 2012, and 61/776,123, filed on March 11, 2013, both of which are hereby incorporated by reference for all purposes as if fully set forth herein.

STATEMENT OF GOVERNMENTAL INTEREST

[0002] This invention was made with government support under grant no. CA098252 awarded by the NIH. The government has certain rights in the invention.

INCORPORATION -BY-REFERENCE OF MATERIAL SUBMITTED

ELECTRONICALLY

[0003] The instant application contains a Sequence Listing which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on November 27, 2013, is named P 12118-04_ST25.txt and is 1,687 bytes in size.

BACKGROUND OF THE INVENTION

[0004] Cervical cancer is one of the leading causes of death in women worldwide. In 2008, there were an estimated 530,000 cases of cervical cancer with 275,000 mortalities. While therapeutic techniques such as chemotherapy, radiotherapy, and surgery are currently used to treat cervical cancer, the need for innovative methods is immense. Five-year survival rates for cervical cancer are estimated to drop from of 93% survival in stage 0 to 15% survival in stage IV. These threatening statistics validate the demand for novel therapeutic techniques to battle cervical cancer.

[0005] Attractive therapeutic techniques include targeting and inhibiting biological functions that promote tumor development. One such target, histone deacetylase (HDAC), is an enzyme responsible for the deacetylation of histones whose inhibition has been shown to produce antitumor effects. Currently, eighteen species of HDAC inhibitors (HDACi) are known which fall into three different classes based on their homology to yeast. Inhibition of these HDAC enzymes causes hyper-acetylation of core histones, which leads to the expression of suppressed genes and therefore regulation of abnormal cell growth. There is also evidence for histone-independent mechanisms altering the acetylation of non-histone proteins such as p53 and heat shock protein-90. These mechanisms produce antitumor effects including induced differentiation, cell growth arrest, and an increase in cellular apoptosis.

[0006] An innovative HDAC inhibitor, AR-42, was designed to enhance inhibitory activity by increasing contact with the HDAC active site. This novel phenylbutyrate-based hydroxamate-tethered HDAC inhibitor is a short-chain fatty acid that displays nanomolar potency. The inhibitor proves to have low efficacy if there is not contact with the zinc cation in the active site of the enzyme. The addition of a Zn²⁺ - chelating motif to the HDAC inhibitor permits the enzyme contact with the active site and shows a low IC5₀ and high inhibition efficacy. In the case of AR-42, hydroxamic acid serves as the Zn²⁺ chelating motif. This model is similar to the structure of Vorinostat, or suberoylanilide hydroxamic acid (SAHA), an FDA approved HDAC inhibitor used in the treatment of cutaneous T-cell lymphoma (CTCL). However, AR-42 was shown to have greater potency and antitumor effects against multiple myeloma, hepatocellular carcinoma and lung cancer than clinically available SAHA.

[0007] Vaccines for HPV-associated cervical cancer represent a unique opportunity to control of cancer through immunotherapy. It is now clear that two HPV-encoded oncogenic proteins, E6 and E7, are responsible for the malignant transformation of cervical cancer and are constantly expressed in HPV-associated cancer cells. Therefore, HPV E6 and E7 represent true tumor-specific antigens and are ideal targets for developing cancer

immunotherapy to control cervical cancer. In the past, we have created a potent DNA vaccine encoding calreticulin (CRT) linked to E7. Vaccination of the chimeric CRT/E7 DNA vaccine generates a significant E7-specific CD8+ T cell mediated immune response as well as a strong antitumor effect against an E7-expressing tumor model, TC-1, demonstrating the successful application of the HPV oncoprotein in generating a tumor-specific immune response.

[0008] Therefore, there still exists an unmet need to develop novel therapeutic modalities which can enhance the tumor killing effect of DNA vaccines in patients with cancer.

SUMMARY OF THE INVENTION [0009] In accordance with one or more embodiments, the present invention demonstrates that the HDAC inhibitor, AR-42, can further enhance CRT/E7 DNA vaccine potency. The potency of the synergistic effects of AR-42 administered orally and CRT/E7 DNA vaccine administered intradermally via gene gun was tested in tumor-bearing mice. It was found that treatment with AR-42 significantly increased the E7-specific CD8+ T cell immune response and the antitumor effects generated by CRT/E7 DNA vaccination.

[0010] In accordance with an embodiment, the present invention provides a

pharmaceutical composition comprising an effective amount of the compound AR-42, or a pharmaceutically acceptable salt, hydrate, solvate or stereoisomer thereof, and an effective amount of CRT/E7 vaccine, and a pharmaceutically acceptable carrier, suitable for use as a medicament, preferably for use as a medicament for treating a HPV-associated tumor in a subject.

[0011] In accordance with an embodiment, the present invention provides a method of treating an HPV-associated tumor in a subject comprising co-administering to the subject an effective amount of the compound AR-42, or a pharmaceutically acceptable salt, hydrate, solvate or stereoisomer thereof, in combination with administration to the subject of an effective amount of CRT/E7 vaccine.

[0012] In accordance with a further embodiment, the present invention provides for the use of the pharmaceutical compositions described herein for treating an HPV-associated tumor in a subject comprising a treatment regimen wherein AR-42, or a pharmaceutically acceptable salt, hydrate, solvate or stereoisomer thereof, is administered to the subject in combination with administration to the subject of an effective amount of CRT/E7 vaccine on day 1 of the regimen, followed by daily administration of AR-42, or a pharmaceutically acceptable salt, hydrate, solvate or stereoisomer thereof for up to 14 days; and wherein on day 5, day 8, and day 1 1 of the regimen, administration of CRT/E7 vaccine to the subject.

[0013] In accordance with another embodiment, the present invention provides a method of treating an HPV-associated tumor in a subject comprising co-administering to the subject an effective amount of the compound AR-42, or a pharmaceutically acceptable salt, hydrate, solvate or stereoisomer thereof, in combination with administration to the subject of an effective amount of CRT/E7 vaccine, and an effective amount of at least one or more additional biologically active agents.

[0014] In accordance with still another embodiment, the present invention provides a method of treating an HPV-associated tumor in a subject comprising a treatment regimen wherein AR-42, or a pharmaceutically acceptable salt, hydrate, solvate or stereoisomer thereof, is administered to the subject in combination with administration to the subject of an effective amount of CRT/E7 vaccine on day 1 of the regimen, followed by daily

administration of AR-42, or a pharmaceutically acceptable salt, hydrate, solvate or stereoisomer thereof for up to 14 days or longer; and wherein on day 5, day 8, day 11 of the regimen, administration of CRT/E7 vaccine to the subject.

[0015] In accordance with another embodiment, the present invention provides for the use of the pharmaceutical compositions described herein for increasing the amount of CD8+ T cells in a subject comprising co-administering to the subject an effective amount of the compound AR-42, or a pharmaceutically acceptable salt, hydrate, solvate or stereoisomer thereof, in combination with administration to the subject of an effective amount of CRT/E7 vaccine.

[0016] In accordance with a further embodiment, the present invention provides a method of increasing the amount of CD8+ T cells in a subject comprising co-administering to the subject an effective amount of the compound AR-42, or a pharmaceutically acceptable salt, hydrate, solvate or stereoisomer thereof, in combination with administration to the subject of an effective amount of CRT/E7 vaccine.

BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 shows the results of in vivo tumor treatment experiments. C57BL/6 mice (five per group) were subcutaneously (sc) challenged with 1 x 10⁵ TC-1 cells per mouse on Day 0. Tumor-bearing mice were treated with AR-42 and/or DNA vaccine (DNA encoding CRT/E7) beginning on day 5 as indicated in the time line. AR-42 was given daily at a dose of 30 mg/kg body weight via oral gavage. The DNA vaccine (2 μg/mouse) was given at 3- day intervals via gene gun. 1A depicts a schematic representation of the AR-42 and DNA vaccine treatment regimens. IB shows a graph depicting tumor volumes in the treated TC-1 tumor-bearing mice. Points, mean; bars, SD (P<0.01). 1C is a Kaplan-Meier survival analysis of the treated TC-1 tumor bearing mice (P<0.05).

[0018] Figure 2A shows peripheral blood samples (collected from the tail vein on day 21) were stained with PE-conjugated HPV16 H-2Db-RAHYNIVTF (SEQ ID NO: 7) tetramer reagent and FITC-conjugated CD8+ T cells. In Figure 2B, intracellular cytokine staining followed by flow cytometry analysis to determine the number of E7-specific CD8+ T cells in tumor-bearing mice treated with AR-42 and/or CRT/E7 DNA vaccine. On day 21 splenocytes from the treated tumor-bearing mice were harvested and incubated with E7 peptide overnight. E7-specific CD8+ T cells were counted using intracellular staining for IFN-γ followed by flow cytometry analysis (top right quadrant). Representative data shown. Bar graph depicting the numbers of E7-specific IFN-y-producing CD8+ T cells per 3 x 10⁵ pooled splenocytes. Columns, mean; bars, SD (*P<0.01).

[0019] Figure 3 depicts cell surface expression of MHC class I analyzed by flow cytometry. Expression levels of unstained control cells are shown as shaded peaks, A15 cells without treatment cell by the green line, and cells treated with AR-42 by the pink line. H- 2Kb expression was assessed by staining with FITC-conjugated anti-H-2Kb mouse monoclonal antibody and H-2Db expression was assessed by staining with PE-conjugated anti-H-2Db mouse monoclonal antibody (mAb; BD Pharmingen), followed by analysis on a FACScan cytometer. Note the enhanced expression of MHC class I on A15 cells treated with AR-42.

[0020] Figure 4 shows the HDAC inhibitor AR-42 enhanced the transcriptional activity of DC2.4 cells. DC2.4 cells were transfected with the expression vector for pcDNA3-GFP. The following day, the DC2.4 and pcDNA3-GFP-DC2.4 transfectants were seeded into a 24- well round-bottomed plates (1 x 10⁵ per well) and treated for 24 hours with 0, 0.1, 0.5, and 2.5 μΜ AR-42. FACSCalibur flow cytometry was used to determine the percentage of increased GFP-expression in each DC population. Bar graph representing the expression of GFP in DC2.4 populations treated with AR-42. Data shown are the means of two experiments performed. Columns, mean; bars, SD (*P<0.05).

[0021] Figure 5. 5A shows intracellular cytokine staining and flow cytometric analysis to determine the number of IFN-y-secreting E7-specific CD8+ T cells induced by non-treated TC-1 cells or 2.5 μΜ AR-42-treated TC-1 cells. TC-1 cells were incubated with E7-specific CD8+ T cells at 1 : 1 ratio for 15 hours. After incubation, cells were stained for CD8 and IFN- γ and analyzed by flow cytometry analysis. Bar graph representing the percentage of E7- specific CD8+ T-cells per 3xl0⁴ CD8 T cells. Data shown are the means of two experiments performed. Columns, mean; bars, SD (*P<0.05). 5B. In vitro cytotoxicity assay. Luciferase- expressing TC-1 tumor cells were seeded in 24-well plates (5xl0⁴ per well). The next day, the TC-1 tumor cells were not treated or treated with 0.5 μΜ AR-42, lxlO⁶ E7-specific CTLs, or with both 0.5 μΜ AR-42 and 1x106 E7-specific CTLs. The degree of CTL- mediated killing of the tumor cells is indicated by the decrease of luminescence activity and measured with the IVIS Luminescence Imaging System Series 200 (bioluminescence signals were acquired for 3 minutes). Representative luminescence images of the 24 well plates are shown. Bar graph depicting the quantification of luminescence intensity in tumor cells treated with AR-42 and/or E7-specific CTLs. Columns, mean; bars, SD (*P<0.05). RLU, relative luciferase unit.

[0022] Figure 6. Splenocytes were harvested on day 21 after tumor challenge. The isolated cells were characterized for CD1 lb⁺Grl⁺ myeloid suppressor cells using flow cytometry analysis. 6A depicts a treatment schema for flow cytometry analysis. 6B shows representative data from flow cytometry analysis showing the percentage of CD1 lb⁺Grl⁺ myeloid suppressor cells in the different treatment groups. Bar graph depicting the percentages of CD1 lb⁺Grl⁺ myeloid suppressor cells per 3xl0⁵ monocytes in the spleen. Columns, mean; bars, SD (*P<0.05).

[0023] Figure 7. Characterization of AR-42 compared to clinically available HDACi. Groups of C57BL/6 mice (five per group) were sc challenged with 1 x 10⁵ TC-1 tumor cells per mouse on day 0. Tumor-bearing mice were treated with romidepsin (1.5 mg/kg, intraperitoneal, twice a week), SAHA (30 mg/kg, per oral, ¾ο=26 μΜ) or AR-42 (30mg/kg, per oral, μΜ). CRTE6E7L2 DNA vaccine was administered as indicated in the time line. DNA vaccine was given via gene gun in the amount of 2 μg/mouse for a total of 4 vaccinations at 3 day intervals. 7A is a diagrammatic representation of the different treatment regimens of various HDACi and DNA vaccine. 7B is a line graph depicting the tumor volume in TC-1 tumor-bearing mice treated with the different treatment regimens. Points, mean; bars, SE (PO.01).

DETAILED DESCRIPTION OF THE INVENTION

[0024] In accordance with an embodiment, the present invention provides a method of treating an HPV-associated tumor in a subject comprising co-administering to the subject an effective amount of the compound AR-42, or a pharmaceutically acceptable salt, hydrate, solvate or stereoisomer thereof, in combination with administration to the subject of an effective amount of CRT/E7 vaccine.

[0025] The molecule AR-42 has the following formula:

[0026] The synthesis and characterization of the compound AR-42 is found in U.S. Patent No. 8,318,808, and incorporated by reference herein.

[0027] The terms "treat," and "prevent" as well as words stemming therefrom, as used herein, do not necessarily imply 100% or complete treatment or prevention. Rather, there are varying degrees of treatment or prevention of which one of ordinary skill in the art recognizes as having a potential benefit or therapeutic effect. In this respect, the inventive methods can provide any amount of any level of treatment or prevention of a disease in a mammal.

Furthermore, the treatment or prevention provided by the inventive method can include treatment or prevention of one or more conditions or symptoms of the disease being treated or prevented. Also, for purposes herein, "prevention" can encompass delaying the onset of the disease, or a symptom or condition thereof.

[0028] It is also contemplated that the present invention further includes AR-42 derivatives. In one embodiment, the term "derivative" includes, but is not limited to, ether derivatives, acid derivatives, amide derivatives, ester derivatives and the like. Methods of preparing these derivatives are known to a person skilled in the art. For example, ether derivatives are prepared by the coupling of the corresponding alcohols. Amide and ester derivatives are prepared from the corresponding carboxylic acid by a reaction with amines and alcohols, respectively. [0029] In accordance with an embodiment, the present invention provides a

[0030] In addition, this invention further includes hydrates of AR-42. The term "hydrate" includes but is not limited to hemihydrate, monohydrate, dihydrate, trihydrate and the like. Hydrates of AR-42 may be prepared by contacting AR-42 with water under suitable conditions to produce the hydrate of choice.

[0031] In another embodiment, the invention provides a metabolite of AR-42. In one embodiment, the term "metabolite" refers to any substance produced from another substance by metabolism or a through a metabolic process of a living cell or organ.

[0032] In an embodiment, the methods of the present invention can include AR-42 in conjunction with a carrier. The carrier is preferably a pharmaceutically acceptable carrier. With respect to pharmaceutical compositions, the carrier can be any of those conventionally used and is limited only by chemico-physical considerations, such as solubility and lack of reactivity with the active compound(s), and by the route of administration. The

pharmaceutically acceptable carriers described herein, for example, vehicles, adjuvants, excipients, and diluents, are well-known to those skilled in the art and are readily available to the public. It is preferred that the pharmaceutically acceptable carrier be one which is chemically inert to the active agent(s) and one which has no detrimental side effects or toxicity under the conditions of use.

[0033] The choice of carrier will be determined in part by the chemical properties of AR- 42 as well as by the particular method used to administer AR-42. Accordingly, there are a variety of suitable formulations of the pharmaceutical composition of the invention. The following formulations for parenteral, subcutaneous, intravenous, intramuscular, intraarterial, intrathecal, and interperitoneal administration are exemplary and are in no way limiting. More than one route can be used to administer AR-42, and in certain instances, a particular route can provide a more immediate and more effective response than another route.

[0034] Formulations suitable for parenteral administration of the compositions of the present invention include aqueous and non-aqueous, isotonic sterile injection solutions, which can contain anti-oxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives.

[0035] Oils, which can be used in parenteral formulations include petroleum, animal, vegetable, or synthetic oils. Specific examples of oils include peanut, soybean, sesame, cottonseed, corn, olive, petrolatum, and mineral. Suitable fatty acids for use in parenteral formulations include oleic acid, stearic acid, and isostearic acid. Ethyl oleate and isopropyl myristate are examples of suitable fatty acid esters.

[0036] Suitable soaps for use in parenteral formulations include fatty alkali metal, ammonium, and triethanolamine salts, and suitable detergents include (a) cationic detergents such as, for example, dimethyl dialkyl ammonium halides, and alkyl pyridinium halides, (b) anionic detergents such as, for example, alkyl, aryl, and olefin sulfonates, alkyl, olefin, ether, and monoglyceride sulfates, and sulfosuccinates, (c) nonionic detergents such as, for example, fatty amine oxides, fatty acid alkanolamides, and polyoxyethylenepolypropylene copolymers, (d) amphoteric detergents such as, for example, alkyl-P-aminopropionates, and 2-alkyl-imidazoline quaternary ammonium salts, and (e) mixtures thereof.

[0037] The parenteral formulations will typically contain from about 0.5% to about 25% by weight of the HDACi in solution. Preservatives and buffers may be used. In order to minimize or eliminate irritation at the site of injection, such compositions may contain one or more nonionic surfactants having a hydrophile-lipophile balance (HLB) of, for example, from about 12 to about 17. The quantity of surfactant in such formulations will typically range from, for example, about 5% to about 15% by weight. Suitable surfactants include polyethylene glycol sorbitan fatty acid esters, such as sorbitan monooleate and the high molecular weight adducts of ethylene oxide with a hydrophobic base, formed by the condensation of propylene oxide with propylene glycol. The parenteral formulations can be presented in unit-dose or multi-dose sealed containers, such as ampoules and vials, and can be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid excipient, for example, water, for injections, immediately prior to use.

Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules, and tablets.

[0038] Injectable formulations are in accordance with the present invention. The requirements for effective pharmaceutical carriers for injectable compositions are well- known to those of ordinary skill in the art (see, e.g., Pharmaceutics and Pharmacy Practice, J.B. Lippincott Company, Philadelphia, PA, Banker and Chalmers, eds., pages 238-250 (1982), and ASHP Handbook on Injectable Drugs, Trissel, 14th ed., (2007)).

[0039] For purposes of the invention, the amount or dose of AR-42 administered should be sufficient to effect, e.g., a therapeutic or prophylactic response, in the subject over a reasonable time frame. The dose will be determined by the efficacy of the particular HDACi and the condition of a human, as well as the body weight of a human to be treated.

[0040] Typically, the attending physician will decide the dosage of AR-42 with which to treat each individual patient, taking into consideration a variety of factors, such as age, body weight, general health, diet, sex, to be administered, route of administration, and the severity of the condition being treated. By way of example and not intending to limit the invention, the dose of AR-42 can be about 0.1 to about 100 mg/kg to the subject being treated, preferably from about 1 to about 50 mg/kg, more preferably between about 20 to about 40 mg/kg. In an embodiment, the dose of AR-42 administered is about 30 mg/kg.

[0041] For use in medicines, the salts of AR-42 will be pharmaceutically acceptable salts. Other salts may, however, be useful in the preparation of the compounds according to the invention or of their pharmaceutically acceptable salts. Suitable pharmaceutically acceptable salts of the compounds of the present invention include acid addition salts which may, for example, be formed by mixing a solution of the compound according to the invention with a solution of a pharmaceutically acceptable acid, such as hydrochloric acid, sulphuric acid, methanesulphonic acid, fumaric acid, maleic acid, succinic acid, acetic acid, benzoic acid, oxalic acid, citric acid, tartaric acid, carbonic acid or phosphoric acid.

[0042] As used herein, the term "CRT/E7" vaccine is a DNA vaccine first described in J. Clin. Invest, 108(5): 669-678 (2001) and in U.S. Patent No. 7,342,002, and incorporated by reference herein. The vaccine comprises a portion of the Calreticulin gene (CRT) with the E6 and E7 antigen proteins encoded in a pcDNA3 vector.

[0043] An active agent and a biologically active agent are used interchangeably herein to refer to a chemical or biological compound that induces a desired pharmacological and/or physiological effect, wherein the effect may be prophylactic or therapeutic. The terms also encompass pharmaceutically acceptable, pharmacologically active derivatives of those active agents specifically mentioned herein, including, but not limited to, salts, esters, amides, prodrugs, active metabolites, analogs and the like. When the terms "active agent,"

"pharmacologically active agent" and "drug" are used, then, it is to be understood that the invention includes the active agent per se as well as pharmaceutically acceptable, pharmacologically active salts, esters, amides, prodrugs, metabolites, analogs etc. The active agent can be a biological entity, such as a virus or cell, whether naturally occurring or manipulated, such as transformed.

[0044] The biologically active agent may vary widely with the intended purpose for the composition. The term active is art-recognized and refers to any moiety that is a biologically, physiologically, or pharmacologically active substance that acts locally or systemically in a subject. Examples of biologically active agents, that may be referred to as "drugs", are described in well-known literature references such as the Merck Index, the Physicians' Desk Reference, and The Pharmacological Basis of Therapeutics, and they include, without limitation, medicaments; vitamins; mineral supplements; substances used for the treatment, prevention, diagnosis, cure or mitigation of a disease or illness; substances which affect the structure or function of the body; or pro-drugs, which become biologically active or more active after they have been placed in a physiological environment.

[0045] Specific examples of useful biologically active agents the above categories include: anti-neoplastics such as androgen inhibitors, alkylating agents, nitrogen mustard alkylating agents, nitrosourea alkylating agents, antimetabolites, purine analog

antimetabolites, pyrimidine analog antimetabolites, hormonal antineoplastics, natural antineoplastics, antibiotic natural antineoplastics, carboplatin and cisplatin; nitrosourea alkylating antineoplastic agents, such as carmustine (BCNU); antimetabolite antineoplastic agents, such as methotrexate; pyrimidine analog antineoplastic agents, such as fluorouracil (5-FU) and gemcitabine; hormonal antineoplastics, such as goserelin, leuprolide, and tamoxifen; natural antineoplastics, such as aldesleukin, interleukin-2, docetaxel, etoposide, interferon; paclitaxel, other taxane derivatives, and tretinoin (ATRA); antibiotic natural antineoplastics, such as bleomycin, dactinomycin, daunorubicin, doxorubicin, and mitomycin; vinca alkaloid natural antineoplastics, such as vinblastine and vincristine.

[0046] Other biologically active agents can include peptides, proteins, and other large molecules, such as interleukins 1 through 18, including mutants and analogues; interferons a, γ, and which may be useful for cartilage regeneration, hormone releasing hormone (LHRH) and analogues, gonadotropin releasing hormone transforming growth factor (TGF); fibroblast growth factor (FGF); tumor necrosis factor-a); nerve growth factor (NGF); growth hormone releasing factor (GHRF), epidermal growth factor (EGF), connective tissue activated osteogenic factors, fibroblast growth factor homologous factor (FGFHF); hepatocyte growth factor (HGF); insulin growth factor (IGF); invasion inhibiting factor-2 (IFF -2); bone morphogenetic proteins 1-7 (BMP 1-7); somatostatin; thymosin-a-y-globulin; superoxide dismutase (SOD); and complement factors, and biologically active analogs, fragments, and derivatives of such factors, for example, growth factors.

[0047] In accordance with an embodiment, the present invention provides a method of treating a HPV-associated tumor in a subject comprising a treatment regimen wherein AR-42, or a pharmaceutically acceptable salt, hydrate, solvate or stereoisomer thereof, is

administered to the subject in combination with administration to the subject of an effective amount of CRT/E7 vaccine on day 1 of the regimen, followed by daily administration of AR- 42, or a pharmaceutically acceptable salt, hydrate, solvate or stereoisomer thereof for up to 14 days; and wherein on day 4, day 7, day 10 and day 13 of the regimen, administration of CRT/E7 vaccine to the subject.

[0048] In accordance with another embodiment, the present invention provides the use of the pharmaceutical compositions described herein for the treatment of a HPV-associated tumor in a subject comprising a treatment regimen wherein AR-42, or a pharmaceutically acceptable salt, hydrate, solvate or stereoisomer thereof, is administered to the subject in combination with administration to the subject of an effective amount of CRT/E7 vaccine on day 1 of the regimen, followed by daily administration of AR-42, or a pharmaceutically acceptable salt, hydrate, solvate or stereoisomer thereof for up to 14 days; and wherein on day 4, day 7, day 10 and day 13 of the regimen, administration of CRT/E7 vaccine to the subject.

[0049] It will be understood by those of ordinary skill in the art that the methods of the invention can be used in many variations of regimens, and should not be limited by any particular example. In an alternate embodiment, the HDAC inhibitor, AR-42, or a derivative thereof, is administered to the subject before administration of the CRT/E7 vaccine. It is contemplated that administration of AR-42 can be administered from 12 hours to 72 hours before the first administration of the CRT/E7 vaccine.

[0050] In other embodiments, the CRT/E7 vaccine is given with booster administration every 3 days, 4 days or 5 days. In certain embodiments, the effect of the vaccine

administration lasts at least 2 months or longer.

[0051] In accordance with one or more embodiments, the CRT/E7 vaccine is given via gene gun. In other embodiments, the vaccine can be given by injection, i.m., i.p., i.v., s.c, etc.

[0052] In accordance with one or more embodiments, the AR-42 compound or composition comprising same is administered orally. In other embodiments, the AR-42 compound or composition comprising same can be administered by any other acceptable route of administration.

[0053] In accordance with another embodiment, the present invention provides for the use of the pharmaceutical compositions described herein for increasing the amount of CD8+ T cells in a subject comprising co-administering to the subject an effective amount of the compound AR-42, or a pharmaceutically acceptable salt, hydrate, solvate or stereoisomer thereof, in combination with administration to the subject of an effective amount of CRT/E7 vaccine.

[0054] In accordance with an embodiment, the present invention provides a method of increasing the amount of CD8+ T cells in a subject comprising co-administering to the subject an effective amount of the compound AR-42, or a pharmaceutically acceptable salt, hydrate, solvate or stereoisomer thereof, in combination with administration to the subject of an effective amount of CRT/E7 vaccine.

[0055] As used herein, the term "subject" refers to any mammal, including, but not limited to, mammals of the order Rodentia, such as mice and hamsters, and mammals of the order Logomorpha, such as rabbits. It is preferred that the mammals are from the order Carnivora, including Felines (cats) and Canines (dogs). It is more preferred that the mammals are from the order Artiodactyla, including Bovines (cows) and Swines (pigs) or of the order Perssodactyla, including Equines (horses). It is most preferred that the mammals are of the order Primates, Ceboids, or Simoids (monkeys) or of the order Anthropoids (humans and apes). An especially preferred mammal is the human.

EXAMPLES

[0056] Mice. Five- to eight- week-old female C57BL/6 mice were purchased from the National Cancer Institute (Frederick, MD) and kept in the oncology animal facility of the Johns Hopkins Hospital (Baltimore, MD). All of the animal procedures were done according to approved protocols and in accordance with recommendations for the proper use and care of laboratory animals.

[0057] Cell lines. Primary C57BL/6 mouse lung epithelial cells were co-transformed with HPV-16 E6 and E7 and an activated ras oncogene as previously described to generate the TC-1 cells. The generation and characteristics of the MHC class I down-regulated tumor cell line TC-1 P3 (A15) have also been previously described. DC cell line DC2.4 was a gift from Dr. K.L. Rock. TC-1, TC-1 P3 (A 15) tumor cells and DC2.4 cells were grown in RPMI 1640 supplemented with 10% FBS, 50 U/ml penicillin/streptomycin, 2 mM L-glutamine, 1 mM sodium pyruvate, 2 mM nonessential amino acids and 2-mercaptothanol (2 -ME, 50 μΜ) at 37 °C with 5% C0₂.

[0058] DNA constructions. The generation of the DNA vaccine encoding CRT and E7 was described previously. For the generation of pcDNA3-CRT, CRT was first amplified by PCR using rabbit CRT cDNA as the template and the primers, 5'- CCGGTCTAGAATGCTGCTCCCTGTGCCGCT-3' (SEQ ID NO: 1) and 5'- CCGGGAATTCCAGCTCGTCCTTGGCCTGGC-3' (SEQ ID NO: 2). The amplified product was then cloned into the Xbal/EcoRI sites of pcDNA3 vector (Invitrogen Corp.). For the generation of pcDNA3-CRT/E7, E7 was first amplified with the set of primers, 5'- GGGGAATTCATGGAGATACACCTA-3 ' (SEQ ID NO: 3) and 5'- GGTGGATCCTTGAGAACAGATGG-3 ' (SEQ ID NO: 4), and cloned into the

EcoRI/BamHI site of pcDNA3-CRT. For the generation of pcDNA3-GFP, the DNA fragment encoding the GFP was amplified with the primers, 5'- ATCGGATCCATGGTGAGCAAGGGCGAGGAG-3' (SEQ ID NO: 5) and 5'- GGGAAGCTTTACTTGTACAGCTCGTCCATG-3 (SEQ ID NO: 6) and then cloned into the BamHI/Hindlll cloning sites of pcDNA3. The accuracy of the DNA constructs was confirmed by DNA sequencing.

[0059] DNA vaccination by gene gun. DNA-coated gold particles were prepared, and gene gun particle-mediated DNA vaccination was done according to a previously described protocol. The coated gold particles (1 μg DNA/bullet) were delivered to the shaved abdominal region of the mice with a helium-driven gene gun (Bio-Rad Laboratories, Inc.) at a discharge pressure of 400 lb/in². C57BL/6 mice (five per group) were immunized with 2 μg of the DNA vaccine and received four boosters with the same dose at 3 -day intervals.

Splenocytes were harvested on day 21 after the tumor challenge.

[0060] In vivo tumor treatment experiment. For in vivo tumor treatment, lxl0⁵ TC-l tumor cells per mouse were subcutaneously injected into the left flank area of 5- to 8-wk-old C57BL/6 mice. After 5 days, the mice were divided into five groups (five per group), each receiving a different treatment regimen: group 1 received no treatment after the TC-1 tumor challenge, group 2 was treated daily with AR-42 by gavage (30 mg/kg body weight) for 10 days, group 3 was immunized with the DNA vaccine schedule as described above, group 4 was both immunized and treated with AR-42. Mice were monitored twice a week by inspection and palpation. AR-42 (NSC 736012) was a gift from Dr. Ching-Shih Chen, and it is a novel hydroxamate-tethered phenylbutyrate derivative that has received IND approval from the FDA. AR-42 was prepared as suspensions in vehicle (0.5% methylcellulose, 0.1% Tween 80, in sterile water).

[0061] Intracellular cytokine staining and flow cytometry analysis. Pooled splenocytes from tumor-bearing mice that were treated with the various treatment regimens were harvested 7 days after the last treatment and incubated for 24 hours with 1 μg/mL of E7 peptide containing an MHC class I epitope (amino acids 49-57, RAHYNIVTF (SEQ ID NO: 7)) in the presence of GolgiPlug (BD Pharmingen). The stimulated splenocytes were then washed once with FACScan buffer and stained with phycoerythrin (PE)-conjugated monoclonal rat anti-mouse CD8a (clone 53.6.7). Cells were subjected to intracellular cytokine staining using the Cytofix/Cytoperm kit according to the manufacture's instruction (BD Pharmingen). Intracellular IFN-γ was stained with FITC-conjugated rat anti-mouse IFN-γ. All antibodies were purchased from BD Pharmigen. Flow cytometry analysis was done using FACSCalibur with CellQuest software (BD Bioscience).

[0062] Tetramer analysis of E7-specific CD8+ T cells. Peripheral blood samples (collected from the tail vein on day 21 after the tumor challenge) were depleted of RBC by ACK lysis and were stained with PE-conjugated HPV16 H-2Db-RAHYNIVTF tetramer reagent (NCI). Tetramer staining was combined with surface staining using FITC-conjugated anti-CD8 (BD PharMingen). Cells were analyzed on a BD FACSCalibur collecting 30,000 events.

[0063] In vitro CTL assays after AR-42 treatment. Luciferase-expressing TC-1 cells in medium were seeded into 24-well round-bottomed plates (5xl0⁴ cells per well). The following day, the medium was replaced with 2 mL of fresh medium containing 0.5 μΜ AR- 42 and incubated in 5% CO₂ for 24 hours at 37 °C. lxlO⁶ E7-specific CTLs from the spleens of tumor-bearing mice immunized with the DNA vaccine were added to each well to serve as effector cells. TC-1 cells expressing luciferase were used as target cells. After incubation, D-luciferin (potassium salt; Xenogen Corp.) was added to each well at a concentration of 15 μg/mL in medium. After 10 minutes the cells were imaged with the Xenogen IVIS 200 system.

[0064] Detection of green fluorescent protein (GFP)-positive dendritic cells. The DC2.4 cells were transfected with an expression vector for pcDNA3-GFP using lipofectamineTM 2000 (Invitrogen). DC2.4 and pcDNA3-GFP DC2.4 cells were seeded into a 24-well round- bottomed plates (lxlO⁵ per well), and then treated with 2 mL of fresh medium containing 0, 0.1, 0.5, or 2.5 μΜ AR-42. The cells were incubated in 5% C0₂ for 24 hours at 37 °C.

FACSCalibur flow cytometry was used to determine the percentage of GFP-positive DCs in each DC population.

[0065] Characterization of CD1 lb+ Grl+ myeloid derived suppressor cells. C57BL/6 mice (five per group) were inoculated with lxlO⁵ TC-1 tumor cells. Starting on day 5, mice were treated daily with AR-42 (30 mg/kg body weight) for 10 days. Splenocytes were harvested on day 21 and characterized for CD1 lb+ Grl+ myeloid derived suppressor cells using antibodies specific for CD1 lb and Grl (BD Pharmingen). The staining was characterized by flow cytometry analysis with FACSCalibur and CellQuest software.

[0066] Statistical analysis. All data expressed as mean ± SD are representative of at least two independent experiments. Comparisons between individual data points were made using Student's t-test. The Kaplan-Meier method was used to analyze the survival rate and the differences in survival among the groups was compared using the log rank test. Statistical analysis was performed using SPSS, version 17.0 with significance considered at p<0.05.

EXAMPLE 1

[0067] AR-42 given in combination with DNA vaccine results in reduced tumor growth and prolonged survival of TC-1 tumor-bearing mice. Previous studies have shown that tumor-bearing mice given intradermal administration of DNA encoding calreticulin linked to E7 (CRT/E7) via gene gun generate high numbers of E7-specific CD8+ T-cell immune responses and have reduced tumor growth rates. In the present invention, we tested if AR-42 administered via oral gavage could enhance these effects since histone deacetylase inhibitors have been shown to enhance MHC Class I and II expression on tumor cells and induce apoptotic cell death.

[0068] As shown in Figure 1A, mice were TC-1 tumor challenged and then began treatment five days later with AR-42 (daily for 10 days) and/or the CRT/E7 DNA vaccine (4 times with 3 -day intervals). The tumors of the mice receiving both the DNA vaccine and AR-42 consistently grew at a reduced rate compared to the mice in the other treatment groups (Figure IB). Those same mice, as shown in Figure 1C, lived much longer than the other groups of mice, suggesting that the combination therapy can enhance the antitumor effects generated by the CRT/E7 DNA vaccine. EXAMPLE 2

[0069] TC- 1 tumor-bearing mice receiving both AR-42 and CRT/E7 DNA generated greater numbers of E7-specific CD8+ T cells. On day 21, peripheral blood samples were collected from the tail vein of tumor-bearing mice in each treatment group, stained to identify E7-specific CD8+ T cells, and analyzed by flow cytometry. The mice receiving both treatments had more than twice the percentage of E7-specific CD8+ T cells than the group receiving only the DNA vaccine (17.78% vs. 7.89%) and more than 20 times the group receiving only AR-42 (17.78% vs. 0.83%) (Fig. 2A). Splenocytes from the treated tumor- bearing mice were also harvested on Day 21 and incubated with E7 peptide overnight. They were stained for intracellular IFN- γ and then analyzed by flow cytometry. Again, the group receiving both CRT/E7 DNA and AR-42 showed significantly more E7-specific IFN-γ- producing CD8+ T cells (Fig. 2B). The combination group had 691 per 3xl0⁵ pooled splenocytes, compared to 11 for the AR-42 group and 130 for CRT/E7 group. Collectively these data show that the AR-42 treatment enhances the generation of E7-specific CD8+ T cells.

EXAMPLE 3

[0070] TC-1 tumor cells treated with AR-42 show increased levels of cell surface expression of MHC class I. To test if administration of AR-42 can increase call surface expression of MHC class I molecules, TC-1 cells (referred to as A 15) were selected for the down-regulation of MHC class I. The A15 cells were incubated with AR-42 and cell surface expression of MHC class I was analyzed by flow cytometry. Figure 3 shows that TC-1 treated with an HDAC inhibitor increases cell surface expression of MHC class I in vitro.

EXAMPLE 4

[0071] AR-42 enhances transcriptional activity of DNA-encoded proteins in dendritic cells. To see what role AR-42 plays in increasing antigen presentation in dendritic cells, DC2.4 cells were transfected with the expression vector for pcDNA3-GFP. The DC2.4 and pcDNA3-GFP cells were incubated with different concentrations of AR-42 for 24 hours. Using flow cytometry, the amount of GFP expression was measured in each population. As shown in Figure 4, GFP expression levels increase with increasing AR-42 concentration, indicating that treatment with AR-42 can enhance immunity by increasing transcriptional activity in DCs.

EXAMPLE 5

[0072] AR-42 -treated TC-1 cells induce significant in vitro cytotoxicity. TC-1 cells and AR-42 -treated TC-1 cells were incubated with E7-specific CD8+ T cells for 15 hours, and then stained for CD8 and IFN- γ. In the in vitro cytotoxicity assay shown in Figure 5A, the luciferase-expressing TC-1 cells treated with both AR-42, and E7-specific CD8+ T cells, had a much greater decrease in luminescence than the luciferase-expressing TC-1 cells treated with either AR-42 or E7-specific CD8+ T cells alone. Flow cytometry analysis shows that the AR-42-treated TC-1 cells induced about twice as many IFN-y-secreting E7-specific CD 8+ T cells as the control TC-1 cells induced (Figure 5B).

EXAMPLE 6

[0073] AR-42 in combination with the DNA vaccine reduces immunosuppression of the tumor microenvironment. On day 21 , splenocytes from the mice of the different treatment groups (see Fig. 6A) were harvested and characterized for CD1 lb+Grl+ myeloid derived suppressor cells (MDSCs) by flow cytometry (Fig 6B). The percentage of CD1 lb+Grl+ MDSCs in the mice receiving both the HDAC inhibitor and DNA vaccine was significantly lower than in the mice receiving the DNA vaccine or HDAC inhibitor alone. This suggests that the AR-42 plays a role in moderating the immunosuppressive tumor microenvironment.

EXAMPLE 7

[0074] AR-42 treatment in combination with CRTE6E7L2 DNA vaccine generates a greater antitumor effect compared to various clinically available HDACi. Because there are two commercially available HDACi, Romidepsin and SAHA, it is important to compare AR- 42 with these HDACi for their ability to enhance the therapeutic antitumor effect generated by a therapeutic HPV DNA vaccine. Mice were subcutaneously challenged with TC-1 tumor cells followed by treatment with different HDACi five days later, including romidepsin, SAHA and AR-42 as indicated in Figure 7A. Concurrent with HDACi administration, mice were treated with a DNA vaccine encoding calreticulin linked to HPV-16 E6, E7 and L2 proteins (CRTE6E7L2) by gene gun. CRTE6E7L2 DNA vaccine has been shown to generate a potent therapeutic antitumor effect against HPV-16 E6/E7-expressing tumors. As shown in Figure 7B, treatment with CRTE6E7L2 DNA vaccine and AR-42 generated the best therapeutic antitumor effect in TC-1 tumor-bearing mice, among all treatment groups. This data indicates that AR-42 is the ideal HDACi to be used in combination with a therapeutic HPV DNA vaccine in order to generate a potent therapeutic antitumor effect against E6/E7- expressing tumors. Thus, this data has significant implications on the future clinical translation of AR-42 as a potent anti -cancer drug.

[0075] All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.

[0076] The use of the terms "a" and "an" and "the" and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms "comprising," "having," "including," and "containing" are to be construed as open-ended terms (i.e., meaning "including, but not limited to,") unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0077] Preferred embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

Claims

Claims:

1. A pharmaceutical composition comprising an effective amount of the compound AR-42, or a pharmaceutically acceptable salt, hydrate, solvate or stereoisomer thereof, and an effective amount of CRT/E7 vaccine, and a pharmaceutically acceptable carrier, suitable for use as a medicament, preferably for use as a medicament for treating a HPV-associated tumor in a subject.

2 The pharmaceutical composition of claim 1, wherein the amount of AR-42 administered to the subject is between about 1 mg/kg to about 100 mg/kg.

3. The pharmaceutical composition of either of claims 1 or 2, wherein the amount of CRT/E7 vaccine administered is between about 0.1 μg to about 10 μg of DNA per dose.

4. The pharmaceutical composition of any of claims 1 to 3, wherein the AR-42 is administered orally.

5. The pharmaceutical composition of any of claims 1 to 3, wherein the CRT/E7 vaccine is administered via gene gun.

6. The pharmaceutical composition of any of claims 1 to 5, wherein the HPV- associated tumor is cervical cancer.

7. The pharmaceutical composition of any of claims 1 to 6, wherein the composition further comprises administering to the subject at least one or more additional biologically active agents.

8. The pharmaceutical composition of claim 7, wherein the biologically active agent is a chemotherapeutic agent.

9. Use of the pharmaceutical composition of any of claims 1 to 8 for treating an HPV- associated tumor in a subject comprising a treatment regimen wherein AR-42, or a pharmaceutically acceptable salt, hydrate, solvate or stereoisomer thereof, is administered to the subject in combination with administration to the subject of an effective amount of CRT/E7 vaccine on day 1 of the regimen, followed by daily administration of AR-42, or a pharmaceutically acceptable salt, hydrate, solvate or stereoisomer thereof for up to 14 days; and wherein on day 5, day 8, and day 11 of the regimen, administration of CRT/E7 vaccine to the subject.

10. Use of the pharmaceutical composition of any of claims 1 to 8 for increasing the amount of CD8+ T cells in a subject comprising co-administering to the subject an effective amount of the compound AR-42, or a pharmaceutically acceptable salt, hydrate, solvate or stereoisomer thereof, in combination with administration to the subject of an effective amount of CRT/E7 vaccine.