Pharmaceutical composition containing F1 polypeptide and/or F3 polypeptide and use thereof

Abstract

A pharmaceutical composition containing F1 polypeptide and/or F3 polypeptide and a use thereof are provided. The pharmaceutical composition is made into a common dosage form, such as an emulsion and an ointment, and used for treating a disease, such as a wart associated with a HPV infection, or a solid tumor associated with or not associated with the HPV infection.

Claims

1. A pharmaceutical composition for treating a solid tumor or a disease associated with a HPV infection, comprising a F1 polypeptide, a F3 polypeptide, or a mixture thereof, and further comprising a Toll like receptor agonist, wherein the F1 polypeptide comprises SEQ ID NO: 1, and the F3 polypeptide comprises SEQ ID NO: 2.

2. The pharmaceutical composition of claim 1, wherein the pharmaceutical composition is prepared as an injection liquid suitable for parenteral administration or as an emulsion or an ointment suitable for percutaneous administration.

3. The pharmaceutical composition of claim 1, wherein a dose of the pharmaceutical composition comprises 3 μg to 30 μg of the F1 polypeptide, the F3 polypeptide, or a mixture thereof when used for tumor bearing mice.

4. The pharmaceutical composition of claim 1, wherein the Toll like receptor agonist is Imiquimod, CpG, or a mixture thereof.

5. The pharmaceutical composition of claim 1, further comprising a pharmaceutically acceptable excipient.

6. A method for treating a disease associated with a HPV infection, comprising administering to a subject in need thereof a pharmaceutical composition, wherein the pharmaceutical composition comprises a F1 polypeptide, a F3 polypeptide, or a mixture thereof and further comprises a Toll like receptor agonist, wherein the F1 polypeptide comprises SEQ ID NO: 1, and the F3 polypeptide comprises SEQ ID NO: 2.

7. The method of claim 6, wherein the disease is a wart.

8. The method of claim 7, wherein the disease is condyloma acuminate, verruca vulgaris, or verruca plana.

9. The method of claim 6, wherein the Toll like receptor agonist is Imiquimod, CpG, or a mixture thereof.

10. A method for treating a solid tumor, comprising administering to a subject in need thereof a pharmaceutical composition, wherein the pharmaceutical composition comprises a F1 polypeptide, a F3 polypeptide, or a mixture thereof and further comprises a Toll like receptor agonist, wherein the F1 polypeptide comprises SEQ ID NO: 1, and the F3 polypeptide comprises SEQ ID NO: 2.

11. The method of claim 10, wherein the solid tumor is cervical cancer, vulvar cancer, penile cancer, anal cancer, oral cancer, head and neck neoplasm, skin cancer, or melanocytoma.

12. The method of claim 10, wherein the Toll like receptor agonist is Imiquimod, CpG, or a mixture thereof.

13. The method of claim 10, wherein the pharmaceutical composition is administrated in combination with a therapeutic vaccine for the solid tumor.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 is a graph illustrating inhibiting effects of the F1 polypeptide and the F3 polypeptide on growth of TC-1 tumor cells in vitro according to an embodiment of the present disclosure.

(2) FIG. 2 is a graph illustrating a superimposed inhibiting effect of the F1 polypeptide in combination with the F3 polypeptide on growth of TC-1 tumor cells in vitro according to an embodiment of the present disclosure.

(3) FIG. 3 is a graph illustrating a superimposed inhibiting effect of the F1 polypeptide in combination with the F3 polypeptide on growth of human cervical carcinoma cells (HELA) in vitro according to an embodiment of the present disclosure.

(4) FIG. 4 is a graph illustrating a high throughput proteomic analysis for the F1 polypeptide and the F3 polypeptide according to an embodiment of the present disclosure.

(5) FIG. 5 is a graph illustrating an interaction between the F3 polypeptide and the TC-1 cell according to an embodiment of the present disclosure.

(6) FIG. 6 is a graph illustrating inhibiting effects of the F1 polypeptide, the F3 polypeptide, and Imiquimod administered separately and in combination on growth of TC-1 tumor cells in vitro according to an embodiment of the present disclosure.

(7) FIG. 7 is a graph illustrating inhibiting effects of different dosages of Imiquimod on growth of human cervical carcinoma cells (HELA) in vitro.

(8) FIG. 8 is a graph illustrating inhibiting effects of the F1 polypeptide, the F3 polypeptide, and Imiquimod administered separately and in combination on growth of human cervical carcinoma cells (HELA) in vitro.

(9) FIG. 9A to FIG. 9D are graphs illustrating expressions of CASP3, CASP9, p-AKT, and PI3K in Hela cells treated by a mixture of the F1 polypeptide and the F3 polypeptide (with a ratio of 1:1) in different concentrations.

(10) FIG. 10A is a graph illustrating inhibiting effects of the F1 polypeptide, the F3 polypeptide, and a mixture thereof on growth of TC-1 tumor cells in vitro at 24.sup.th hour, 48.sup.th hour, and 72.sup.th hour respectively; FIG. 10B is a graph showing inhibiting effects of the F1 polypeptide and the F3 polypeptide, which had been prepared and stored at room temperature for 1, 3, 5, and 7 days before using, on growth of TC-1 tumor cells in vitro; FIG. 10C is a graph showing inhibiting effects of the F1 polypeptide and the F3 polypeptide diluted by phosphate buffer saline which has different pH values or had been heated before dilatation.

(11) FIG. 11A is a graph illustrating proteins having significant changes in expression due to a F1 and/or F3 polypeptide treatment and gene functions of the proteins; FIG. 11B is a graph illustrating KEGG pathways on which proteins having significant changes in expression due to a F1 and/or F3 polypeptide treatment are concentrated; FIG. 11C is a graph illustrating an iTRAQ quantitative analysis for proteins secreted by TC-1 cells.

(12) FIG. 12A is a graph illustrating IL-6 levels (detected by ELISA) in TC-1 culture solutions treated by 5 μg/mL of P3 polypeptide, the F1 polypeptide, the F3 polypeptide, and the F1 and F3 polypeptides overnight, respectively; FIG. 12B is a graph illustrating IL-6 levels (detected by ELISA) in TC-1 culture solutions treated by 10 μm/mL of P3 polypeptide, the F1 polypeptide, the F3 polypeptide, and the F1 and F3 polypeptides overnight, respectively.

(13) FIG. 13A is a graph illustrating tumor sizes over time in C57BL/6 mice inoculated with TC-1 cells subcutaneously and injected with PBS, a mixture of F1 and F3 polypeptides, and P3 polypeptide; FIG. 13B is a graph illustrating tumor weights in C57BL/6 mice inoculated with TC-1 cells subcutaneously and injected with PBS, a mixture of F1 and F3 polypeptides, and P3 polypeptide.

(14) FIG. 13C is a graph illustrating tumor weights in C57BL/6 mice inoculated with TC-1 cells subcutaneously and injected with PBS, Imiquimod, a mixture of F1 and F3 polypeptides, and a mixture of F1 polypeptide, F3 polypeptide, and Imiquimod; FIG. 13D is a graph illustrating tumor weights in nude mice inoculated with TC-1 cells subcutaneously and injected with PBS, Imiquimod, a mixture of F1 and F3 polypeptides, and a mixture of F1 polypeptide, F3 polypeptide, and Imiquimod.

(15) FIG. 13E is a graph illustrating survival rates of C57BL/6 mice inoculated with TC-1 cells subcutaneously and injected with PBS, a mixture of the F1 polypeptide and the F3 polypeptide, and/or a vaccine containing HPV16E7 polypeptide, MPLA, and α-Interleukin(IR)-10R (EX/MPLA/α-IR-10R vaccine); FIG. 13F is a graph illustrating survival rates of C57BL/6 mice inoculated with TC-1 cells subcutaneously and injected with PBS, a mixture of the F1 polypeptide, the F3 polypeptide, and the EX/MPLA/α-IR-10R vaccine, and a mixture of the F1 polypeptide, the F3 polypeptide, and a vaccine containing HPV16E7 polypeptide and MPLA, (EX/MPLA vaccine).

(16) FIG. 14A to FIG. 14D are graphs illustrating infiltrations of immune cells into tissues of tumors after injecting PBS, a mixture of the F1 polypeptide and the F3 polypeptide, a immune vaccine (EX/MPLA/α-IR-10R vaccine), and a mixture of the F1 polypeptide, the F3 polypeptide, and the immune vaccine into tumor-bearing mice.

(17) FIG. 15A to FIG. 15B are graphs illustrating infiltrations of HPV16E7 specific CD8+T cells into spleen and tissues of tumors after injecting PBS, a mixture of the F1 polypeptide and the F3 polypeptide, the immune vaccine, and a mixture of the F1 polypeptide, the F3 polypeptide, and the immune vaccine into tumor-bearing mice.

(18) FIG. 16A to FIG. 16D are photos illustrating states of a thermosensitive gel prepared by the F1 polypeptide and/or the F3 polypeptide at different temperatures.

(19) FIG. 17A to FIG. 17D are graphs illustrating survival rates of TC-1 cells and Hela cells treated by P3 polypeptide, F1 polypeptide, F3 polypeptide, a mixture of F1 and F3, and gels thereof with different concentrations in a MTT assay.

(20) FIG. 18A to FIG. 18D are graphs illustrating IC50 values of P3 polypeptide, the F1 polypeptide, the F3 polypeptide, a mixture of F1 and F3, and gels thereof for TC-1 cells and Hela cells in the MTT assay.

(21) FIG. 19A is a graph illustrating tumor sizes in tumor-bearing mice inoculated with TC-1 cells subcutaneously and treated with the PBS, a mixture of F1 and F3, a gel having no active component, and a gel containing the F1 polypeptide and the F3 polypeptide; FIG. 19B is a graph illustrating tumor weights in tumor-bearing mice inoculated with TC-1 cells subcutaneously and treated with the PBS, the mixture of F1 and F3, a gel having no active component, and the gel containing the F1 polypeptide and the F3 polypeptide.

(22) FIG. 20A and FIG. 20B are graphs illustrating survival rates of C57BL/6 mice inoculated with TC-1 cells and treated with P3 polypeptide, F1 polypeptide, F3 polypeptide, a mixture of F1 and F3, and gels thereof with different concentrations, wherein the gels had been prepared and stored for 30 days before using.

(23) FIG. 21A to FIG. 21H are graphs illustrating weights and hematoxylin-eosin staining of tumors of nude mice treated with P3 polypeptide gel, Imiquimod, a gel having no active component, a gel containing the F1 polypeptide and the F3 polypeptide, the gel having no active component in combination with Imiquimod, and the gel containing the F1 polypeptide and the F3 polypeptide in combination with Imiquimod.

(24) FIG. 22A is a graph illustrating weights of tumors of C57BL/6 mice inoculated with TC-1 cells and treated by PBS, P3 polypeptide gel, and a gel containing the F1 polypeptide and the F3 polypeptide.

(25) FIG. 22B and FIG. 22C are graphs illustrating infiltrations of CD3+T cells and NK cells into tissues of tumors of C57BL/6 mice inoculated with TC-1 cells and treated with PBS, P3 polypeptide gel, and a gel containing the F1 polypeptide and the F3 polypeptide.

(26) FIG. 22D is a graph illustrating weights of tumors of nude mice inoculated with B16 cells and treated with PBS, P3 polypeptide gel, and a gel containing the F1 polypeptide and the F3 polypeptide.

(27) FIG. 22E is a graph illustrating weights of tumors of C57BL/6 mice inoculated with B16 cells and treated with PBS, P3 polypeptide gel, and a gel containing the F1 polypeptide and the F3 polypeptide.

DETAILED DESCRIPTION

(28) Process parameters herein can be modified appropriately by those skilled in the art with reference to this specification. It should be noted that all similar replacements and modifications will be apparent to those skilled in the art and thus should be included in the present invention.

(29) Types of instruments used in the present disclosure:

(30) High throughput high performance liquid chromatography-mass spectrometry: Shimadzu Prominance nano reversed phase high performance liquid chromatography-ABSCIEX triple time of flight mass spectrometer configured with nano electrospray ion source.

EXAMPLE 1

Preparation of F1 Polypeptide and F3 Polypeptide

(31) 1) firstly a tree frog was induced to secrete a thick liquid with a white appearance by stimulating with a direct current having a voltage of 9 volt, wherein the tree frog was from a coastal rainforest of eastern Australia;

(32) 2) the liquid was collected and transferred into a solution containing 20 wt % methanol in ultrapure water, and vibrated and then filtered by a PVDF membrane having a pore diameter of 0.45 μm;

(33) 3) a clear liquid was filtered out and dried in a low temperature vacuum centrifugal dryer to form a white solid powder;

(34) 4) the white solid powder was dissolved into a solution having 0.1 wt % trifluoroacetic acid, and a sequence of peptide therein and a post-translational modification possessed by the peptide were analyzed by a high throughput high performance liquid chromatography-mass spectrometry;

(35) 5) the F1 polypeptide or the F3 polypeptide was synthesized respectively by employing a solid phase peptide synthesis according the sequence of peptide and the post-translational modification analyzed, specifically comprising the steps of: a) on a CS336X peptide synthesizer, with a chloromethyl polystyrene resin as an insoluble solid state carrier, a C-terminal amino acid of the F1 polypeptide or the F3 polypeptide (an amino acid with its amino protected by a blocking group) was covalently attached to the solid state carrier; b) the protecting group of the amino was removed under an action of trifluoroacetic acid, so that a first amino acid was attached to the solid state carrier; c) a carboxyl of a second amino acid having its amino blocked is activated by N,N′-dicyclohexylcarbodiimide, (DCC, dicyclohexylcarbodiimide), and the second amino acid having its carboxyl activated by DCC is reacted with the amino of the first amino acid attached to the solid state carrier to form a peptide bond, so that a dipeptide with a protecting group was produced on the solid state carrier; d) the above peptide forming reaction was repeated to cause a peptide chain to grow from C-terminal to N-terminal until achieving a required chain length, finally the protecting group was removed, and a deprotection and depolymerization for the resin is performed spontaneously in a solution of TFA/DCM/H.sub.2O/TIPS with a volume ratio of 90:5:2.5:2.5; d) the F1 polypeptide or the F3 polypeptide was obtained by precipitating with 10 equivalents of cold either and purifying by a reversed phase high performance liquid chromatography.

(36) Particually, carboxyls on side chains of glutamic acid and serine were protected by t-Bu, and a side chain of histidine were protected by Boc group to prevent a racemization of the histidine.

EXAMPLE 2

Preparation of Ointment Composition (with an Amount of 10 g) Containing the F1 Polypeptide and the F3 Polypeptide

(37) Formula:

(38) F1 polypeptide: 10 mg;

(39) F3 polypeptide: 10 mg;

(40) PEG4000: 100 mg;

(41) arginine: 20 mg;

(42) PEG400: appropriate amount, added to reach 10 g.

(43) The ointment composition was prepared according to a conventional method and sub-packed into aluminum pipes for packing the ointment. Each pipe has 2 mg, 5 mg, or 10 mg of the ointment.

EXAMPLE 3

Preparation of Ointment Composition (with an Amount of 10 g) Containing F1 Polypeptide, F3 Polypeptide and Imiquimod

(44) Formula:

(45) F1 polypeptide: 2.5 mg;

(46) F3 polypeptide: 2.5 mg;

(47) Imiquimod: 30 mg;

(48) PEG3000: 100 mg;

(49) lysine: 5 mg;

(50) PEG200: appropriate amount, added to reach 10 g.

(51) The ointment composition was prepared according to a conventional method and sub-packed into aluminum pipes for packing the ointment. Each pipe has 2 mg, 5 mg, or 10 mg of the ointment.

EXAMPLE 4

Preparation of Emulsifiable Paste Composition (with an Amount of 10 g) Containing F1 Polypeptide and F3 Polypeptide

(52) Preparation of Matrix:

(53) 20 g of stearic acid, 1 g of ethylparaben, 2 g of white vaseline, 16 g of liquid paraffin, 5 g of glycerinum, 1 g of triethanolamine, 8 g of octadecanol, 6 g of wool fat, and an appropriate amount of distilled water were taken and formulated as 100 g of a matrix for later use.

(54) Formula:

(55) F1 polypeptide: 10 mg;

(56) F3 polypeptide: 10 mg;

(57) matrix: appropriate amount, added to reach 10 g.

(58) The emulsifiable paste composition was prepared according to a conventional method and sub-packed into aluminum pipes for packing the emulsifiable paste. Each pipe has 2 mg, 5 mg, or 10 mg of the emulsifiable paste.

EXAMPLE 5

Preparation of Emulsifiable Paste Composition (with an Amount of 10 g) Containing F1 Polypeptide, F3 Polypeptide and Imiquimod

(59) Formula:

(60) F1 polypeptide: 5 mg;

(61) F3 polypeptide: 5 mg;

(62) Imiquimod: 60 mg;

(63) matrix: appropriate amount, added to reach 10 g.

(64) The emulsifiable paste composition was prepared according to a conventional method and sub-packed into aluminium pipes for packing the emulsifiable paste. Each pipe has 2 mg, 5 mg, or 10 mg of the emulsifiable paste.

(65) Clinical Trial

(66) 1. Inhibiting effects of F1, F3, and F1+F3 on growth of HPV transformed cells (TC-1 cells) in vitro

(67) Referring to FIGS. 1A to 1D, the inhibiting effects of F1, F3, and F1+F3 on growth of HPV transformed cells (TC-1 cell) in vitro were studied.

(68) More specifically, in the method, 100 μL of 5×10.sup.3 TC-1 cells cultured in a RPMI medium containing 10% of calf serum were added into a 96-well cell culture plate, the F1 polypeptide (FIG. 1A), the F3 polypeptide (FIG. 1B), and control polypeptide P3 (FIG. 1C) with different concentrations were added respectively and incubated overnight in an incubator with 5% of CO.sub.2 at 37° C., and the cell proliferation was determined by a MTT assay.

(69) Immortalized normal cells NP69 were used in another experiment. More specifically, 100 μL of 5×10.sup.3 NP69 cells cultured in a RPMI medium containing 10% of calf serum were added into a 96-well cell culture plate, the F1 polypeptide, the F3 polypeptide, and control polypeptide P3 (FIG. 1D) with different concentrations were added respectively and incubated overnight in an incubator with 5% of CO.sub.2 at 37° C., and the cell proliferation was determined by a MTT assay.

(70) The results showed that the F1 polypeptide and the F3 polypeptide had obvious inhibiting effects on growth of TC-1 cells and the P3 polypeptide had no inhibiting effect. The F1 polypeptide, the F3 polypeptide, and the P3 polypeptide all had no inhibiting effect on NP69 cells.

(71) 2. mouse TC-1 tumor cells transformed by HPV16E7 can be killed better by Imiquimod in combination with the F1 polypeptide and the F3 polypeptide

(72) FIG. 2 is a graph showing a superimposed inhibiting effect of the F1 polypeptide in combination with the F3 polypeptide on growth of TC-1 tumor cells in vitro.

(73) 100 μL of 5×10.sup.3 TC-1 cells cultured in a RPMI medium containing 10% of calf serum were added into a 96-well cell culture plate, 5 μg/mL of F1 and/or F3 and control polypeptides P3 and F4 were added respectively and incubated overnight in an incubator with 5% of CO.sub.2 at 37° C., and the cell proliferation was determined by a MTT assay.

(74) The results showed that there was a synergistic inhibiting effect between the F1 polypeptide and the F3 polypeptide on growth of TC-1.

(75) 3. human cervical carcinoma cells (HELA) transformed by HPV18E7 can be killed better by Imiquimod in combination with the F1 polypeptide and the F3 polypeptide

(76) FIG. 3 is a graph showing a superimposed inhibiting effect of the F1 polypeptide in combination with the F3 polypeptide on growth of human cervical carcinoma cells (HELA).

(77) 100 μL of 5×10.sup.3 Hela cells cultured in a RPMI medium containing 10% of calf serum were added into a 96-well cell culture plate, F1 and/or F3 and control polypeptide P3 with different concentrations were added respectively and incubated overnight in an incubator with 5% of CO.sub.2 at 37° C., and the cell proliferation was determined by a MTT assay.

(78) The results showed that there was a synergistic inhibiting effect between the F1 polypeptide and the F3 polypeptide on growth of Hela cells.

(79) FIG. 4 shows a high throughput and quantitative proteomic analysis for the F1 polypeptide and the F3 polypeptide showing that the TEC kinase signaling pathway was activated by the superimposed effect.

(80) 1000 μL of 5×10.sup.5 TC-1 cells cultured in a RPMI medium containing 10% of calf serum were added into a 24-well cell culture plate, 1 μg/mL of a mixture of F1 and F3 polypeptides (with a ratio of 1:1) was added and incubated for 24 hours. The cells were collected, the protein was extracted and labeled by iTRAQ, and then the protein content in the cells was quantitatively analyzed by using reversed phase high performance liquid chromatography-mass spectrometer, the change in expression of protein was obtained by comparing with the cells cultured with no polypeptides F1 and F3, so as to analyze the changes of several signaling pathways in cells.

(81) FIG. 5A and FIG. 5B are graphs showing an interaction between the F3 polypeptide and TC-1 cell.

(82) 1000 μL of 5×10.sup.5 TC-1 cells cultured in a RPMI medium containing 10% of calf serum were added into a 24-well cell culture plate, 2 μg/mL of the F3 polypeptide labeled by FITC was added into the cells being incubated, cells at different time points of the incubation were collected and washed with PBS buffer solution, and then the relationship between the F3 polypeptide and TC-1 cell was observed by a flow cytometer (FIG. 5A) or a confocal microscopy (FIG. 5B).

(83) It was shown that the F3 polypeptide can be combined with the TC-1 cell, peak was achieved at 2.sup.nd hour, and the F3 polypeptide was distributed non-uniformly in the cytomembrane and the cytoplasm.

(84) FIG. 6 is a graph showing inhibiting effects of the F1 polypeptide, the F3 polypeptide, and Imiquimod administered separately and in combination on growth of TC-1 tumor cells in vitro.

(85) 100 μL of 5×10.sup.3 TC-1 cells cultured in a RPMI medium containing 10% of calf serum were added into a 96-well cell culture plate, 5 μg/mL of F1 and/or F3 and/or Imiquimod were added respectively and incubated overnight in an incubator with 5% of CO.sub.2 at 37° C., and the cell proliferation was determined by a MTT assay.

(86) The results showed that there were superimposed inhibiting effects between Imiquimod and F1 and/or F3 on growth of TC-1 cell.

(87) Inhibiting effects of the F1 polypeptide, the F3 polypeptide, and Imiquimod administered separately and in combination on growth of Hela tumor cells in vitro:

(88) 100 μL of 5×10.sup.3 Hela cells cultured in a RPMI medium containing 10% of calf serum were added into a 96-well cell culture plate, F1 and/or F3 and/or Imiquimod with different concentrations were added respectively and incubated overnight in an incubator with 5% of CO.sub.2 at 37° C., and the cell proliferation was determined by a MTT assay.

(89) The results showed that Imiquimod had an inhibiting effect on growth of Hela cells in vitro, as shown in FIG. 7.

(90) There were superimposed inhibiting effects between Imiquimod and F1 and/or F3 on growth of Hela cell in vitro, as shown in FIG. 8.

(91) Western Blot Analysis

(92) A mixture of the F1 polypeptide and the F3 polypeptide in a ratio of 1:1 was provided; 5×10.sup.5 Hela cells were treated with the mixture in different concentrations for 24 hours respectively; and expressions of CASP3, CASP9, p-AKT, and PI3K are analyzed by Western Blot. Referring to FIG. 9A, it can be seen that expression levels of CASP3 and CASP9 (which induce apoptosis) in Hela cells treated by the mixture were higher than in Hela cells untreated by the mixture, and the increases were related positively to the concentrations of the mixture. Referring to 9B and 9C, it can be seen that expressions of p-AKT and PI3K in Hela cells were significantly inhibited by the mixture in a concentration of 20 μg/mL, suggesting that the PI3K-AKT signal transduction pathway may be one of important pathways through which the F1 polypeptide and/or F3 polypeptide influence the cell functions.

(93) Stability in Vitro

(94) Test 1

(95) RPMI medium containing 10% of calf serum and 5×10.sup.3 TC-1 cells were added into a 96-well cell culture plate; 10 μg/ml of the F1 polypeptide, the F3 polypeptide, and a mixture of F1 and F3 in a ratio of 1:1 were added respectively into the medium and incubated for 72 hours in an incubator with 5% of CO.sub.2 at 37° C., and the cell proliferations at 24.sup.th hour, 48.sup.th hour, and 72.sup.th hour were determined respectively by the MTT assay. Referring to FIG. 10A, it can be seen that the F1 polypeptide and/or F3 polypeptide exhibited significant inhibiting effects on growth of TC-1 cells even at 72.sup.th hour.

(96) Test 2

(97) The steps in the Test 2 were substantially the same as those in the Test 1, except that the F1 polypeptide and the F3 polypeptide which had been prepared and stored for 0, 1, 3, 5, and 7 days at minus 20° C. respectively before using were added into the medium, and the incubation was performed for 24 hours. Referring to FIG. 10B, it can be seen that the activities of the F1 polypeptide and the F3 polypeptide were not reduced even after being prepared and stored for 7 days, suggesting that the F1 polypeptide and the F3 polypeptide have high stabilities.

(98) Test 3

(99) The steps in the Test 3 were substantially the same as those in the Test 1, except that the pH value of the phosphate buffer saline (PBS) in the medium was changed or the PBS is heated for 10 minutes at 100° C. before using, and the incubation was performed for 24 hours. Referring to FIG. 10C, it can be seen that the F1 polypeptide and the F3 polypeptide were still active even in the PBS with a pH value of 5.5 or heated at 100° C.

(100) Proteomic Analysis

(101) Changes in signal transduction induced by TC-1 cells treated by a mixture of the F1 polypeptide and the F3 polypeptide were analyzed by a bioinformatics method. The results showed that totally 16 pathways were activated or inhibited, including several important pathways associated with immunity, such as PI3K-AKT, signaling pathways of Tec family kinases, and LXR/RXR pathway, indicating that a variety of cytokines, such as IL-6, IL-1β, MCP-1, and MCP-3, may be secreted by TC-1 cells treated by the F1 and/or F3 (the secretion of IL-6 has been demonstrated by FIGS. 12A and 12B). Referring to FIG. 11C, it can be seen that NF-κB on the LXR/RXR pathway were significantly increased, thereby activating downstream iNOS, COX-2, IL-6/1β, MCP-1/3, and MMP9, which are important inflammatory mediators. LXR, RXR, and NCOR were significantly inhibited, suggesting that downstream control factors, including LPL, ABCA-1, ABCG-1/4, SREBP-1c, APO-C1/2/4, UGT1A3, and Arg-2, were activated. Consequently, cellular cholesterol transport and efflux and lipid synthesis were inhibited and inflammatory response was enhanced.

(102) Referring to FIG. 11A, it can be seen that the cell proliferation was significantly inhibited, the expression levels of some genes associated with the growth, attachment, and regeneration of cells were increased, suggesting that these functions may have been damaged by the F1 and/or F3. Referring to FIG. 11B, it can be seen that a majority of genes, associated with pathways related to cancer, are differentially expressed due to the F1 and/or F3 treatment. For example, microRNA, p53, or DNA in cancer cells were damaged or mis-controlled.

(103) FIGS. 12A and 12B are graphs illustrating IL-6 levels (detected by ELISA) in TC-1 culture solutions treated by 5 μg/mL and 10 μg/mL of the P3 polypeptide, the F1 polypeptide, the F3 polypeptide, and a mixture of F1 and F3 overnight, respectively. It can be seen that the IL-6 level in TC-1 culture solution treated by 5 μg/mL of the F1 polypeptide, the F3 polypeptide, or the mixture of F1 and F3 was significantly higher than that untreated solution or solution treated by the P3 polypeptide. IL-6 levels were decreased with increase of concentration of the F1 polypeptide, the F3 polypeptide, or the mixture of F1 and F3, which may be resulted from apoptosis of TC-1 cells induced by high concentration of the F1 polypeptide, the F3 polypeptide, or the mixture of F1 and F3.

(104) Inhibiting Effect on TC-1 Cell

(105) Test 4

(106) C57BL/6 mice were respectively inoculated with 5×10.sup.5 TC-1 cells subcutaneously. After 5 days of inoculation, tumors had grown and PBS, a mixture of the F1 polypeptide and the F3 polypeptide, and the P3 polypeptide were injected into the tumors respectively and consecutively for five days, after another five days, the tumors are isolated and weighted. The sizes of the tumors observed were shown in FIG. 13A and the weights of the tumors were shown in FIG. 13B. It can be seen that both the sizes and the weights of tumors of mice injected with the mixture of the F1 polypeptide and the F3 polypeptide were smaller those of mice injected with PBS or P3 polypeptide, suggesting that the mixture of the F1 polypeptide and the F3 polypeptide had higher inhibiting effect on the tumor as compared to PBS and P3 polypeptide.

(107) Test 5

(108) The steps in the Test 5 were substantially the same as those in the Test 4, except that both the C57BL/6 mice and the nude mice were subjected to the test, and PBS, Imiquimod, a mixture of F1 and F3 polypeptides, and a mixture of F1 polypeptide, F3 polypeptide, and Imiquimod were injected into the tumors. The results were shown in FIGS. 13C and 13D. It can be seen that the mixture of F1 and F3 polypeptides substantially had no inhibiting effect on tumor growths in nude mice (which has no specific immune cell), suggesting that the inhibiting effect on growth of TC-1 cells may caused by a specific immune response resulted from an apoptosis of TC-1 cell induced by the F1 polypeptide and/or the F3 polypeptide.

(109) It is noteworthy that the amounts of LPS in all of polypeptides had been measured and the values measured were between 0.03 EU/ml and 0.44 EU/ml, so the influence of LPS to the test 4 and test 5 can be neglected.

(110) Test 6

(111) The steps in the Test 6 were substantially the same as those in the Test 4, except that the C57BL/6 mice were replaced by the tumor-bearing mice, and the tumors were injected with PBS, a mixture of F1 and F3 polypeptides, and/or a vaccine containing HPV16E7 polypeptide, MPLA, and α-Interleukin(IR)-10R (EX/MPLA/α-IR-10R vaccine). The survival rates of C57BL/6 mice were shown in FIG. 13E.

(112) Test 7

(113) The steps in the Test 7 were substantially the same as those in the Test 4, except that the C57BL/6 mice were replaced by the tumor-bearing mice, and the tumors were injected with PBS, a mixture of the F1 polypeptide, the F3 polypeptide, and the EX/MPLA/α-IR-10R vaccine, and a mixture of the F1 polypeptide, the F3 polypeptide, and a vaccine containing HPV16E7 polypeptide and MPLA (EX/MPLA vaccine). The survival rates of C57BL/6 mice were shown in FIG. 13F.

(114) As shown in FIGS. 13E and 13F, the tumor-bearing mice injected only with the mixture of the F1 polypeptide and the F3 polypeptide or the vaccine have higher survival rate than the tumor-bearing mice injected with only the PBS. The tumor-bearing mice injected with both the mixture of the F1 polypeptide and the F3 polypeptide and the vaccine have significantly higher survival rate than the tumor-bearing mice injected with only the mixture of the F1 polypeptide and the F3 polypeptide or the vaccine. The tumor-bearing mice injected only with the EX/MPLA/α-IR-10R vaccine have higher survival rate than the tumor-bearing mice injected with only the PBS, and the tumor-bearing mice injected with the EX/MPLA/α-IR-10R vaccine have higher survival rate than the tumor-bearing mice injected with the EX/MPLA vaccine.

(115) Infiltrations of Immune Cells into Tumor Tissue

(116) FIGS. 14A to 14D are graphs illustrating infiltrations of immune cells into tissues of tumors after injecting PBS, a mixture of the F1 polypeptide and the F3 polypeptide, a immune vaccine (EX/MPLA/α-IR-10R vaccine), and a mixture of the F1 polypeptide, the F3 polypeptide, and the immune vaccine into tumor-bearing mice. It can be seen that more hematocytes including CD8+T cells, CD4+T cells, and NKT cells are attracted into the tissue of tumor treated by the mixture of the F1 polypeptide and the F3 polypeptide in combination with the vaccine as compared to that treated only by the mixture of the F1 polypeptide and the F3 polypeptide or the vaccine.

(117) FIGS. 15A to 15B are graphs illustrating infiltrations of HPV16E7 specific CD8+T cells into spleen (FIG. 15A) and tissues of tumors (FIG. 15B) after injecting PBS, a mixture of the F1 polypeptide and the F3 polypeptide, the immune vaccine, and a mixture of the F1 polypeptide, the F3 polypeptide, and the immune vaccine into tumor-bearing mice. It can be seen that more HPV16E7 specific CD8+T cells are attacked into spleen and tissue of tumor treated by the mixture of the F1 polypeptide and the F3 polypeptide in combination with the vaccine as compared to that treated only by the mixture of the F1 polypeptide and the F3 polypeptide or the vaccine.

(118) Thermosensitive Gel Prepared by the Caerin Polypeptide and Biological Activity Thereof

(119) Referring to FIGS. 16A to 16D, a thermosensitive gel is prepared by the F1 polypeptide and/or the F3 polypeptide. The thermosensitive gel is a colloidal solution at room temperature and 28-35° C. and is a gel at a physiological temperature.

(120) FIGS. 17A to 17D are graphs illustrating survival rates TC-1 cells and Hela cells treated by P3 polypeptide, F1 polypeptide, F3 polypeptide, a mixture of F1 and F3, and gels thereof with different concentrations in a MTT assay. FIGS. 18A to 18D are graphs illustrating IC50 values of P3 polypeptide, the F1 polypeptide, the F3 polypeptide, a mixture of F1 and F3, and gels thereof for TC-1 cells and Hela cells in the MTT assay. It can be seen that the gels prepared by the F1 polypeptide and/or F3 polypeptide have similar inhibiting effects on growths of Hela cells and TC-1 cells in vitro as the F1 polypeptide and/or F3 polypeptide.

(121) FIG. 19A is a graph illustrating tumor sizes in tumor-bearing mice inoculated with TC-1 cells subcutaneously and treated with the PBS, a mixture of F1 and F3, a gel having no active component, and a gel containing the F1 polypeptide and the F3 polypeptide. FIG. 19B is a graph illustrating tumor weights in tumor-bearing mice inoculated with TC-1 cells subcutaneously and treated with the PBS, the mixture of F1 and F3, a gel having no active component, and the gel containing the F1 polypeptide and the F3 polypeptide. It can be seen that the gel containing the F1 polypeptide and the F3 polypeptide has a similar inhibiting effects on growth of TC-1 cells in vivo as the F1 polypeptide and the F3 polypeptide.

(122) FIGS. 20A and 20B are graphs illustrating survival rates of C57BL/6 mice inoculated with TC-1 cells and treated with P3 polypeptide, F1 polypeptide, F3 polypeptide, a mixture of F1 and F3, and gels thereof with different concentrations in a MTT assay, wherein the gels had been prepared and stored for 30 days before using. It can be seen that the gel prepared by the F1 and/or F3 has high stability.

(123) C57BL/6 mice were inoculated with TC-1 cells and treated with P3 polypeptide gel, 5% Imiquimod, a gel having no active component, a gel containing the F1 polypeptide and the F3 polypeptide, the gel having no active component in combination of 5% Imiquimod, and the gel containing the F1 polypeptide and the F3 polypeptide in combination of 5% Imiquimod after 4 days of inoculation. The results are shown in FIGS. 21A to 21H. It can be seen that the smear of the gel containing the F1 polypeptide and the F3 polypeptide can significantly inhibit the growth of tumor. The gel containing the F1 polypeptide and the F3 polypeptide in combination with 5% Imiquimod has best inhibiting effect on growth of tumor.

(124) FIG. 22A is a graph illustrating weights of tumors of C57BL/6 mice inoculated with TC-1 cells smeared with PBS, P3 polypeptide gel, and a gel containing the F1 polypeptide and the F3 polypeptide; FIGS. 22B and 22C are graphs illustrating infiltrations of CD3+T cells and NK cells into tissues of tumors of C57BL/6 mice inoculated with TC-1 cells smeared with PBS, P3 polypeptide gel, and a gel containing the F1 polypeptide and the F3 polypeptide. It can be seen that more T cells and NK cells were infiltrated into tumors C57BL/6 mice smeared with gel containing the F1 polypeptide and the F3 polypeptide than that of C57BL/6 mice smeared with PBS or P3 polypeptide gel. FIG. 22D is a graph illustrating weights of tumors of nude mice inoculated with B16 cells intra-tumor injected with PBS, P3 polypeptide, and the F1 polypeptide and the F3 polypeptide, showing that the F1 and F3 polypeptide also inhibit B16 cell growth of nude mice. FIG. 22E is a graph illustrating weights of tumors of C57BL/6 mice inoculated with B16 cells intra-tumor injected with PBS, P3 polypeptide, and the F1 polypeptide and the F3 polypeptide, showing that the F1 and F3 polypeptides inhibit B16 cell growth in C57BL/6 mice.

(125) The present disclosure further provides a pharmaceutical composition including the F1 polypeptide and/or the F3 polypeptide. The pharmaceutical composition can be used in treating a disease associated with or not associated with a HPV infection. In one embodiment, the pharmaceutical composition can be used for treating a wart associated with the HPV infection, such as condyloma acuminate, verruca vulgaris, and verruca plana. In another embodiment, the pharmaceutical composition can be used for treating a solid tumor associated with the HPV infection, such as cervical cancer, vulvar cancer, penile cancer, and anal cancer, or not associated with the HPV infection, such as oral cancer, head and neck neoplasm, skin cancer, melanocytoma, etc. The pharmaceutical composition can be used for treating the disease in combination with a Toll like receptor agonist, such as Imiquimod, CpG, and a mixture thereof, or a therapeutic vaccine for tumor.

(126) Finally, it is to be understood that the above-described embodiments are intended to illustrate rather than limit the present disclosure. Variations may be made to the embodiments without departing from the spirit of the present disclosure as claimed. Elements associated with any of the above embodiments are envisioned to be associated with any other embodiments. The above-described embodiments illustrate the scope of the present disclosure but do not restrict the scope of the present disclosure.