Compositions and methods for enhancement of immune responses

Abstract

Provided is a pharmaceutical composition, including an antigen fusion protein which includes an antigen and an antagonist of an Fc gamma receptor. Also provided is a method of enhancing immunogenicity of an antigen, including conjugating the antigen with an antagonist of an Fc gamma receptor to form an antigen fusion protein. Also provided is a method of enhancing an immune response to an antigen in a subject, including administering to the subject an effective amount of an antigen fusion protein which includes an antigen and an antagonist of an Fc gamma receptor. The present invention may be applied in the development of potent vaccines based on targeting vaccine antigens to antigen-presenting cells via binding to Fc gamma receptors.

Claims

1. A pharmaceutical composition, comprising an antigen fusion protein comprising an antigen and an antagonist of an Fc gamma receptor, wherein the antagonist of the Fc gamma receptor is a formyl peptide receptor-like 1 inhibitory protein (FLIPr) or a FLIPr-like protein.

2. The pharmaceutical composition of claim 1, wherein the antigen is a polypeptide derived from a cancer cell or a virus.

3. The pharmaceutical composition of claim 2, wherein the polypeptide is selected from the group consisting of survivin, mesothelin, and Zika virus envelope protein domain III.

4. A method of enhancing immunogenicity of an antigen, comprising conjugating the antigen with an antagonist of an Fc gamma receptor to form an antigen fusion protein, wherein the antagonist of the Fc gamma receptor is a FLIPr or a FLIPr-like protein.

5. The method of claim 4, wherein the antigen is a polypeptide derived from a cancer cell or a virus.

6. The method of claim 5, wherein the polypeptide is selected from the group consisting of survivin, mesothelin, and Zika virus envelope protein domain III.

7. A method of enhancing an immune response to an antigen in a subject, comprising administering to the subject an effective amount of an antigen fusion protein, wherein the antigen fusion protein comprises the antigen and an antagonist of an Fc gamma receptor, wherein the antagonist of the Fc gamma receptor is a FLIPr or a FLIPr-like protein.

8. The method of claim 7, wherein the antigen is a polypeptide derived from a cancer cell or a virus.

9. The method of claim 8, wherein the polypeptide is selected from the group consisting of survivin, mesothelin, and Zika virus envelope protein domain III.

10. The method of claim 7, wherein the immune response comprises an increase in CD4.sup.+ T cells, CD8.sup.+ T cells, or combinations thereof.

11. The method of claim 7, wherein the immune response comprises secretion of a cytokine selected from the group consisting of interferon gamma, interleukin-2, interleukin-5, interleukin-17A, and combinations thereof.

12. The method of claim 7, wherein the immune response comprises production of an antigen-specific immunoglobulin G antibody.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) The present invention will be apparent to those skilled in the art from the following detailed description of the preferred embodiments, with reference to the attached drawings, in which:

(2) FIG. 1A shows an image of a polyacrylamide gel displaying purified recombinant proteins, including an ovalbumin (OVA) and two OVA fusion proteins fusing OVA with formyl peptide receptor-like 1 inhibitory protein (OVA-FLIPr) or with FLIPr-like protein (OVA-FLIPr-like), respectively;

(3) FIG. 1B shows detection of the purified recombinant proteins, including the OVA, the OVA-FLIPr fusion protein, and the OVA-FLIPr-like fusion protein, by western blotting using an anti-OVA antibody;

(4) FIG. 2A shows the binding of FcγRI to the recombinant proteins, including the OVA, the OVA-FLIPr fusion protein, and the OVA-FLIPr-like fusion protein;

(5) FIG. 2B shows the binding of FcγRIIa-H131 to the recombinant proteins, including the OVA, the OVA-FLIPr fusion protein, and the OVA-FLIPr-like fusion protein;

(6) FIG. 2C shows the binding of FcγRIIb to the recombinant proteins, including the OVA, the OVA-FLIPr fusion protein, and the OVA-FLIPr-like fusion protein;

(7) FIG. 2D shows the binding of FcγRIIIa-V158 to the recombinant proteins, including the OVA, the OVA-FLIPr fusion protein, and the OVA-FLIPr-like fusion protein;

(8) FIG. 2E shows the binding of FcγRIIIa-F158 to the recombinant proteins, including the OVA, the OVA-FLIPr fusion protein, and the OVA-FLIPr-like fusion protein;

(9) FIG. 3A shows the frequencies of IFN-γ-secreting CD4.sup.+ T cells in the peptide-pulsed splenocytes from mice immunized twice with the OVA, the OVA-FLIPr fusion protein, the OVA-FLIPr-like fusion protein, or phosphate buffered saline (PBS);

(10) FIG. 3B shows the frequencies of IFN-γ-secreting CD8.sup.+ T cells in the peptide-pulsed splenocytes from mice immunized twice with the OVA, the OVA-FLIPr fusion protein, the OVA-FLIPr-like fusion protein, or PBS;

(11) FIG. 4A shows carboxyfluorescein succinimidyl ester (CF SE) profiles of the OT-1 peptide-pulsed splenocytes labeled with high concentration of CFSE from mice immunized twice with the OVA, the OVA-FLIPr fusion protein, the OVA-FLIPr-like fusion protein, or PBS; the percentage shown in each panel represents the percent specific killing in one experiment;

(12) FIG. 4B shows the percent specific killing of the OT-1 peptide-pulsed splenocytes from mice immunized twice with the OVA, the OVA-FLIPr fusion protein, the OVA-FLIPr-like fusion protein, or PBS;

(13) FIG. 5 shows the estimated tumor volume in mice immunized twice with the OVA, the OVA-FLIPr fusion protein, the OVA-FLIPr-like fusion protein, or PBS, and then inoculated with EG7 tumor cells;

(14) FIGS. 6A-6B show an image of a polyacrylamide gel displaying purified recombinant proteins, including a survivin and a survivin-FLIPr (Sur-FLIPr) fusion protein;

(15) FIGS. 6C-6D show detection of the purified recombinant proteins, including the survivin and the Sur-FLIPr fusion protein, by western blotting using an anti-survivin antibody;

(16) FIG. 7A shows the frequencies of IFN-γ-secreting CD8.sup.+ T cells in the medium-pulsed splenocytes from mice immunized twice with the survivin, Sur-FLIPr fusion protein, or PBS;

(17) FIG. 7B shows the frequencies of IFN-γ-secreting CD8.sup.+ T cells in the RAH peptide-pulsed splenocytes from mice immunized twice with the survivin, Sur-FLIPr fusion protein, or PBS;

(18) FIG. 7C shows the frequencies of IFN-γ-secreting CD8.sup.+ T cells in the survivin21-29 peptide-pulsed splenocytes from mice immunized twice with the survivin, Sur-FLIPr fusion protein, or PBS;

(19) FIG. 7D shows the frequencies of IFN-γ-secreting CD8.sup.+ T cells in the survivin57-64 peptide-pulsed splenocytes from mice immunized twice with the survivin, Sur-FLIPr fusion protein, or PBS;

(20) FIG. 8 shows the estimated tumor volume in mice inoculated with EG7 tumor cells and then immunized twice with the survivin, the Sur-FLIPr fusion protein, or PBS;

(21) FIG. 9 shows the levels of proinflammatory cytokines secreted by helper T cells in the mesothelin-pulsed splenocytes from mice immunized twice with a recombinant mesothelin, a recombinant mesothelin-FLIPr fusion protein, or PBS; the cytokines include IFN-γ, IL-2, IL-5, and IL-17A;

(22) FIGS. 10A-10B show an image of a polyacrylamide gel displaying purified recombinant proteins, including a Zika virus envelope protein domain III (ZE3) and a ZE3-FLIPr fusion protein;

(23) FIGS. 10C-10D show detection of the purified recombinant proteins, including the ZE3 and the ZE3-FLIPr fusion protein, by western blotting using an anti-ZE3 antibody;

(24) FIG. 11 shows the binding of FcγRIIA to the recombinant proteins, including the ZE3 and the ZE3-FLIPr fusion protein; and

(25) FIG. 12 shows the titers of ZE3-specific IgG antibodies in the sera from mice immunized twice with the ZE3 or the ZE3-FLIPr fusion protein.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

(26) Definition

(27) Numerical quantities given herein are approximate, and experimental values may vary within 20 percent, preferably within 10 percent, and most preferably within 5 percent. Thus, the terms “about” and “approximately” refer to within 20 percent, preferably within 10 percent, and most preferably within 5 percent of a given value or range.

(28) As used herein, the term “immunogenicity” refers to the ability of an antigen to elicit or induce an immune response. An antigen which causes a greater immune response is of higher immunogenicity.

(29) The present invention relates to a pharmaceutical composition, which contains an antigen fusion protein including an antigen and an antagonist of an Fc gamma receptor, and a method of enhancing immune responses to an antigen in a subject, including administering to the subject an effective amount of the antigen fusion protein for immunization. In the following examples, ovalbumin (OVA), survivin (Sur), mesothelin, and Zika virus envelope protein domain III (ZE3) were used as exemplary antigens for investigation of the immune-augmenting effects of the antigen fusion protein of the present invention. The formyl peptide receptor-like 1 inhibitory protein (FLIPr; SEQ ID NO:1) and its homolog FLIPr-like (SEQ ID NO:2), which are potent FcγR antagonists secreted by Staphylococcus aureus to evade FcγR-mediated host immunity, are the exemplary antagonists of the FcγRs for preparation of the FLIPr- or FLIPr-like-containing fusion proteins.

(30) Methods and Materials

(31) Cloning and Expression of the Antigen Fusion Proteins

(32) The FLIPr or the FLIPr-like segment of the antigen fusion protein was preferably conjugated to the C-terminus of an antigen, such as OVA, survivin, and ZE3, via a peptide linker composed of three repeats of 4 glycine residues and 1 serine residue. According to the amino acid sequences of OVA (SEQ ID NO:3), OVA-FLIPr fusion protein (SEQ ID NO:4), OVA-FLIPr-like fusion protein (SEQ ID NO:5), survivin (SEQ ID NO:6), survivin-FLIPr (Sur-FLIPr) fusion protein (SEQ ID NO:7), mesothelin (SEQ ID NO:8), mesothelin-FLIPr fusion protein (SEQ ID NO:9), ZE3 (SEQ ID NO:10), and ZE3-FLIPr fusion protein (SEQ ID NO:11), the corresponding nucleotide sequences were determined based on Escherichia coli codon usage and the DNA with each of these nucleotide sequences were fully synthesized. The synthesized DNA was then amplified by polymerase chain reaction (PCR). The PCR products were cloned into the Ndel and Xhol sites of an expression vector pET-22b(+) to generate expression plasmids of the antigens or the antigen fusion proteins. These plasmids were used to produce the recombinant antigens and antigen fusion proteins.

(33) For preparation of the recombinant proteins, E. coli BL21 (DE3) was transformed with each of the abovementioned expression plasmids. The transformed cells were cultured at 37° C. overnight, and protein expression was induced by adding 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG), followed by incubation at 37° C. for 3 hours. Next, the transformed cells were lysed by a French press (Constant Systems, Daventry, UK) at 27 Kpsi in a homogenization buffer (20 mM Tris (pH 8.0), 50 mM sucrose, 500 mM NaCl, 10% glycerol). The cell lysate were then centrifuged at 80,000 xg for 40 minutes to obtain a cell pellet containing inclusion bodies. Most of the recombinant proteins were present in inclusion bodies and were solubilized with an extraction buffer (50 mM NaH.sub.2PO.sub.4, 5 mM ethylenediaminetetraacetic acid (EDTA), 200 mM NaCl, 0.5 M urea, 1% Triton X-100, pH 6.0). The recombinant proteins were purified by loading the extracted fraction onto an immobilized metal affinity chromatography column (QIagen, Hilden, Germany).

(34) Enzyme-Linked Immunosorbent Assay (ELISA) for FcγR Binding

(35) To detect the binding of the recombinant antigens or antigen fusion proteins to FcγR subclasses, 96-well plates were coated overnight at 4° C. with 0.1 mL recombinant proteins at 1 μg/mL in phosphate buffered saline (PBS; sodium chloride 137 mM, potassium chloride 2.7 mM, sodium hydrogen phosphate 10 mM, and potassium dihydrogen phosphate 1.8 mM, pH 7.4). The plates were washed three times with PBS supplemented with 0.05% v/v Tween 20 and incubated with a serial dilution of various biotin-conjugated recombinant FcγR proteins, including FcγRI, FcγRIIa-H131, FcγRIIb, FcγRIIIa-V158, FcγRIIIa-F158, and FcγRIIA. After incubation at room temperature for 2 hours, the plates were washed and incubated for 30 min with horseradish peroxidase (HRP)-conjugated streptavidin. For detection of the FcγR binding, 3, 3′, 5, 5′-tetramethylbenzidine (TMB) was added to the plates and the absorbance at 450 nm was measured with an ELISA reader.

(36) Animal Studies

(37) Female C57BL/6 mice were purchased from the National Laboratory Animal Center. All the mice were housed at the Laboratory Animal Center of the National Health Research Institutes (Taiwan). All the animal studies were approved and were performed in compliance with the guidelines of the Animal Committee of the National Health Research Institutes. For immunization, mice were arbitrarily assigned to groups and subcutaneously administered twice with the recombinant antigens or antigen fusion proteins at a two-week interval.

(38) Enzyme-Linked Immunospot (ELISPOT) Assay

(39) The number of IFN-γ-producing cells was determined using mouse IFN-γELISPOT kits. Briefly, 96-well plates with polyvinylidene difluoride (PVDF) membranes were first coated with capture antibody and incubated at 4° C. for 18 hours. The plates were washed twice and blocked with RPMI medium supplemented with 10% fetal bovine serum (FBS) for one hour to prevent nonspecific binding in later steps. Splenocytes from the immunized mice were seeded at a density of 5×10.sup.5 cells/well and stimulated with one of the indicated peptides.

(40) The splenocytes from the mice immunized with the OVA or the OVA fusion proteins were stimulated with an OVA peptide termed OT-1 (SIINFEKL; SEQ ID NO:12), another OVA peptide termed OT-2 (ISQAVHAAHAEINEAGR; SEQ ID NO:13), a control peptide for OT-1 peptide (RAHYNIVTF; SEQ ID NO:14), or a control peptide for OT-2 peptide (GRLITVNPIVTEKDS; SEQ ID NO:15) and incubated for two days.

(41) The splenocytes from the mice immunized with the survivin or the survivin fusion protein were stimulated with 10 μg/mL of a survivin peptide termed survivin21-29; SEQ ID NO:16), another survivin peptide termed survivin57-64; SEQ ID NO:17), or a control RAH peptide (SEQ ID NO:14) and incubated for three days.

(42) After incubation, the splenocytes were removed from the plates by washing three times with 0.05% (w/v) Tween 20 in PBS. A 100 μL aliquot of biotinylated detection antibody was added to each well. The plates were incubated at 37° C. for 2 hours. The washing steps were repeated as above, and after a 45-minute incubation at room temperature with the avidin-HRP complex reagent, the plates were washed three times with 0.05% (w/v) Tween 20 in PBS and then three times with PBS alone. A 100 μL aliquot of 3-amine-9-ethyl carbazole (Sigma-Aldrich) staining solution was added to each well to develop the spots. The reaction was stopped after one hour by placing the plates under tap water. The spots were counted using an ELISPOT reader (Cellular Technology Ltd.)

(43) In Vivo Cytotoxicity Assay

(44) Splenocytes from naive C57BL/6 mice were divided into two populations. One population was pulsed with 10 μM OT-1 peptide at 37° C. for 90 min. These cells were labeled with carboxyfluorescein succinimidyl ester (CFSE) at a final concentration of 10 μM (CFSE.sup.high) at 37° C. for 15 min. In parallel, the other population was pulsed with a control peptide for OT-1 (SEQ ID NO:14) and labeled with 1 μM CFSE (CFSE.sup.low). The two populations were mixed equally and 2×10.sup.7 cells were injected intravenously into the immunized mice. After 24 hours, single cells from spleen were isolated and CFSE intensities were analyzed using FACSCalibur flow cytometer and CellQuest Pro software. The percentage of specific killing was calculated as follows: % specific killing=[1-(% CFSE.sup.high/% CFSE.sup.high before injection)/(% CFSE.sup.low/% CFSE.sup.low before injection)].

(45) EG7 Tumor Model

(46) Tumor-bearing mice were established by inoculating EG7 cells into mice via subcutaneous injection before or after immunization with the antigen fusion protein. The EG7 tumor cells are derived from EL4 cells, a mouse lymphoma cell line. The presence or absence of tumor was assessed by visual inspection and palpation. Tumor size was measured three times per week with a caliper, and mice were sacrificed when tumor volume reached 3000 mm.sup.3. Tumor volume was estimated as follows: tumor volume=tumor width×tumor length×(tumor width+tumor length)/2.

Example 1

Preparation and FcγR Binding of the OVA Fusion Proteins

(47) The DNA encoding OVA, OVA-FLIPr fusion protein, or OVA-FLIPr-like fusion protein was synthesized, amplified by PCR, and cloned into a pET-22b-based vector to generate the expression plasmid pOVA, pOVA-FLIPr, or pOVA-FLIPr-like. E. coli were then transformed with each of the plasmids for protein expression. After purification with immobilized metal affinity chromatography, the successful production of the recombinant proteins was verified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting.

(48) As shown in FIG. 1A, the recombinant OVA, OVA-FLIPr fusion protein, and OVA-FLIPr-like fusion protein were visualized on the polyacrylamide gel stained with Coomassie brilliant blue. As shown in FIG. 1B, the recombinant OVA, OVA-FLIPr fusion protein, and OVA-FLIPr-like fusion protein were detected by western blotting using the anti-OVA antibody.

(49) To analyze the functional activity of the recombinant OVA-FLIPr fusion protein and OVA-FLIPr-like fusion protein, a capture ELISA was performed to confirm that both of the OVA fusion proteins directly interact with FcγR isoforms. As shown in FIGS. 2A-2E, the recombinant OVA-FLIPr fusion protein and OVA-FLIPr-like fusion protein bound all the tested FcγR isoforms, including FcγRI (FIG. 2A), FcγRIIa-H131 (FIG. 2B), FcγRIIb (FIG. 2C), FcγRIIIa-V158 (FIG. 2D), and FcγRIIIa-F158 (FIG. 2E). In contrast, there was no interaction between the recombinant OVA and the FcγR isoforms. The results were mean±standard error (SEM) of duplicate wells. These results indicate that the antigen fusion protein of the present invention can directly bind to different FcγR isoforms.

Example 2

Immunization with the OVA Fusion Protein Enhances OVA-Specific T-Cell Responses

(50) To ascertain whether the FcγR binding activity of the antigen fusion protein of the present invention associates with the immune responses in vivo, C57BL/6 mice were arbitrarily assigned to groups (6 mice per group) and subcutaneously administered twice with 10 μg of the OVA, the OVA-FLIPr fusion protein, or the OVA-FLIPr-like fusion protein at a two-week interval. The mice administered with PBS alone served as negative controls. One week after the second immunization, the splenocytes of the immunized mice were harvested and examined for the number of IFN-γ-secreting CD4.sup.+ and CD8.sup.+ T cells by ELISPOT assay.

(51) As shown in FIGS. 3A-3B, background levels of IFN-γ-secreting cells were detected from all of the splenocytes without stimulation (medium alone) or stimulated with control peptides. After stimulation with an OT-2 peptide (a CD4.sup.+ T cell epitope; FIG. 3A) or an OT-1 peptide (a CD8.sup.+ T cell epitope; FIG. 3B), low frequencies of the IFN-γ-secreting cells were observed in the splenocytes of OVA immunized mice. In contrast, the mice immunized with the OVA-FLIPr fusion protein or the OVA-FLIPr-like fusion protein induced high frequencies of IFN-γ-secreting cells. These results indicate that immunization with the antigen fusion protein of the present invention can effectively induce antigen-specific T-cell responses.

Example 3

Immunization with the OVA Fusion Protein Stimulates Cytotoxic Immunity

(52) To investigate whether the antigen fusion protein of the present invention stimulates cytotoxic immunity, an in vivo cytotoxicity assay was performed. C57BL/6 mice were arbitrarily assigned to groups (7-8 mice per group) and administered subcutaneously with 30 μg of the OVA, the OVA-FLIPr fusion protein, or the OVA-FLIPr-like fusion protein two times at a two-week interval. The mice administered with PBS alone served as negative controls. An equal mixture of OT-1 peptide-pulsed splenocytes labeled with high concentration of CFSE (CFSE.sup.hig) and control peptide-pulsed splenocytes labeled with lower concentration of CFSE (CFSE.sup.low) were then injected into the immunized mice via an intravenous route. The immunized mice were sacrificed after 24 hours and the killing of the peptide-pulsed splenocytes in spleen by cytotoxic T lymphocytes was analyzed using flow cytometry.

(53) FIG. 4A shows CFSE profiles of the peptide-pulsed splenocytes from each group of the immunized mice. FIG. 4B shows the percent specific killing of the peptide-pulsed splenocytes from each group of the immunized mice. According to FIGS. 4A-4B, less specific killing of the peptide-pulsed splenocytes was observed for the OVA immunized mice. In contrast, the mice immunized with the OVA-FLIPr fusion protein or the OVA-FLIPr-like fusion protein exhibited significantly more killing of the peptide-pulsed splenocytes. These results indicate that immunization with the antigen fusion protein of the present invention can effectively stimulate antigen-specific cytotoxic immunity.

Example 4

Immunization with the OVA Fusion Protein Induces Antitumor Responses

(54) To examine whether the antigen fusion protein of the present invention induces antitumor responses in vivo, C57BL/6 mice were arbitrarily assigned to groups (6 mice per group) and administered twice with 10 μg of the OVA, the OVA-FLIPr fusion protein, or the OVA-FLIPr-like fusion protein at a two-week interval. The mice administered with PBS alone served as negative controls. One week after the second immunization, mice were inoculated subcutaneously on the left flank with 5×10.sup.5 EG7 tumor cells transfected with an OVA gene and producing OVA constitutively, followed by measurement of the Tumor volume.

(55) As shown in FIG. 5, tumor growth was inhibited in the mice immunized with the OVA-FLIPr fusion protein or the OVA-FLIPr-like fusion protein. In contrast, the mice immunized with the OVA did not show significant reduction of tumor volume when compared with the mice immunized with PBS. These results indicate that immunization with the antigen fusion protein of the present invention can effectively induce antigen-specific antitumor responses in the tumor-bearing subjects.

Example 5

Preparation of the Survivin Fusion Protein

(56) According to the similar procedures described in Example 1, recombinant proteins of survivin, an inhibitor protein to apoptosis, and Sur-FLIPr fusion protein were prepared and analyzed by SDS-PAGE and western blotting.

(57) As shown in FIGS. 6A-6B, the recombinant survivin and Sur-FLIPr fusion protein were visualized on the polyacrylamide gel stained with Coomassie brilliant blue. As shown in FIGS. 6C-6D, the recombinant survivin and Sur-FLIPr fusion protein were detected by western blotting using the anti-survivin antibody.

Example 6

Immunization with the Survivin Fusion Protein Enhances Survivin-Specific T-Cell Responses

(58) C57BL/6 mice were arbitrarily assigned to groups (6 mice per group) and subcutaneously administered twice with 30 μg of the survivin or the Sur-FLIPr fusion protein at a two-week interval. The mice administered with PBS alone served as negative controls. One week after the second immunization, the splenocytes of the immunized mice were harvested and examined for the number of IFN-γ-secreting CD8.sup.+ T cells by ELISPOT assay.

(59) As shown in FIGS. 7A-7D, the background levels of IFN-γ-secreting cells were detected from all of the splenocytes without stimulation (medium alone) or stimulated with a control RAH peptide. After stimulation with a survivin21-29 peptide or a survivin57-64 peptide (CD8.sup.+ T cell epitopes), low frequencies of the IFN-γ-secreting cells were observed in the splenocytes of survivin immunized mice. In contrast, the mice immunized with the Sur-FLIPr fusion protein induced high frequencies of IFN-γ-secreting CD8.sup.+ T cells. These results indicate that immunization with the antigen fusion protein of the present invention can effectively induce antigen-specific T-cell responses.

Example 7

Immunization with the Survivin Fusion Protein Induces Antitumor Responses

(60) C57BL/6 mice subcutaneously inoculated with 5×10.sup.4 EG7 tumor cells were arbitrarily assigned to groups (9-11 mice per group) and administered twice with 30 mg of the survivin or the Sur-FLIPr fusion protein on day 3 and 10 after tumor inoculation. The mice administered with PBS alone served as negative controls. Tumor volume was measured to assess the antitumor activity of the Sur-FLIPr fusion protein.

(61) As shown in FIG. 8, tumor growth was inhibited in the mice immunized with the Sur-FLIPr fusion protein. In contrast, the mice immunized with the survivin did not show significant reduction of tumor volume when compared with the mice immunized with PBS. These results indicate that immunization with the antigen fusion protein of the present invention can effectively induce antitumor responses.

Example 8

Immunization with the Mesothelin Fusion Protein Enhances Mesothelin-Specific T-Cell Responses

(62) Recombinant proteins of mesothelin, a protein reported to be overexpressed in tumors such as mesothelioma and ovarian and pancreatic adenocarcinoma, and mesothelin-FLIPr fusion proteins were prepared according to the similar procedures described in Example 1 (data not shown). C57BL/6 mice were arbitrarily assigned to groups (3 mice per group) and subcutaneously administered twice with 10 μg of the mesothelin or the mesothelin-FLIPr fusion protein at a one-week interval. The mice administered with PBS alone served as negative controls. One week after the second immunization, the splenocytes of the immunized mice were isolated and stimulated with 10 μg/mL of the mesothelin for five days, and then the levels of IFN-γ, IL-2, IL-5, and IL-17A secreted into the culture medium were measured by ELISA.

(63) As shown in FIG. 9, high levels of proinflammatory cytokines, including IFN-γ, IL-2, IL-5, and IL-17, were secreted by helper T cells in the stimulated splenocytes from the mice immunized with the mesothelin-FLIPr fusion protein. In contrast, small amounts of these cytokines were detected in the splenocytes from the mesothelin or PBS-immunized mice. These results indicate that immunization with the antigen fusion protein of the present invention can effectively induce antigen-specific helper T-cell responses.

Example 9

Preparation and FcγR Binding of the ZE3 Fusion Protein

(64) According to the similar procedures described in Example 1, recombinant proteins of ZE3 and ZE3-FLIPr fusion protein were prepared and analyzed by SDS-PAGE and western blotting.

(65) As shown in FIGS. 10A-10B, the recombinant ZE3 and ZE3-FLIPr fusion protein were visualized on the polyacrylamide gel stained with Coomassie brilliant blue. As shown in FIGS. 10C-10D, the recombinant ZE3 and ZE3-FLIPr fusion protein were detected by western blotting using the anti-ZE3 antibody.

(66) To analyze the functional activity of the recombinant ZE3-FLIPr fusion protein, a capture ELISA was performed to confirm the interaction between FcγRIIA and the ZE3-FLIPr fusion protein. As shown in FIG. 11, the binding of FcγRIIA to the recombinant ZE3-FLIPr fusion protein was detected. In contrast, there was weak interaction between the recombinant ZE3 and FcγRIIA. This result demonstrates that the antigen fusion protein of the present invention can directly bind to FcγR.

Example 10

Immunization with the ZE3 Fusion Protein Enhances Antibody Production

(67) C57BL/6 mice were arbitrarily assigned to groups (4 mice per group) and subcutaneously administered twice with 10 μg of the ZE3 or ZE3-FLIPr fusion protein at a two-week interval. Four weeks after the first immunization, the sera of the immunized mice were collected for determination of the titers of anti-ZE3 IgG antibodies using ELISA.

(68) As shown in FIG. 12, superior production of the ZE3-specific IgG antibodies were observed in the mice immunized with the ZE3-FLIPr fusion protein. Comparatively, fewer ZE3-specific IgG were produced by the ZE3-immunized mice. These results indicate that immunization with the antigen fusion protein of the present invention can effectively induce antigen-specific antibody production.

(69) In conclusion, the antigen fusion protein of the present invention can induce higher antigen-specific immune responses in a subject than the antigen itself. Therefore, a pharmaceutical composition which contains such antigen fusion protein may be employed in the development of potent vaccines, such as antitumor and antivirus vaccines. The examples of the present invention also demonstrate a simple and direct method of enhancing immunogenicity of an antigen and a method of enhancing immune responses to an antigen in a subject. These methods may increase the efficacy of vaccine antigens and elicit efficient immune responses in subj ects with compromised immunity.