WO2007119895A1

WO2007119895A1 - Anti-cancer compositions

Info

Publication number: WO2007119895A1
Application number: PCT/KR2006/001447
Authority: WO
Inventors: Young Chul Sung; Hyun Tak Jin; Je-In Youn; Kumar Bajpai Arun
Original assignee: Postech Foundation; Genexine Co., Ltd
Priority date: 2006-04-18
Filing date: 2006-04-19
Publication date: 2007-10-25
Also published as: KR20070103254A; KR100788930B1

Abstract

The present invention relates to an anticancer composition comprising a recombinant vector containing a genetic construct encoding IL- 12M and a recombinant vector containing a gene encoding an immune regulatory factor or an anticancer composition comprising a recombinant vector containing a genetic construct encoding IL- 12M and an immune regulatory gene in combination, and a method for enhancing immune responses and suppressing proliferation of tumor cells, in which the anticancer composition is administered to a subject in need of anticancer treatment so as to express each gene in the subject. The present invention provides improved anticancer effects than the conventional anticancer composition.

Description

ANTI-CANCER COMPOSITIONS

[Technical Field]

The present invention relates to an anticancer composition comprising a recombinant vector containing a genetic construct encoding IL- 12M and an immune regulatory gene either individually or in combination, and to gene therapy using the same. [Background Art]

In cancer gene therapy, various methods have been employed such as a method for inducing apoptosis in cancer cells, immunotherapy using immune regulatory factors, and a method for regulating tumor specific gene expression. Further, a gene therapy using adenovirus, which can replicate only in cancer cells, has been currently developed, and it was proved that the therapy helps delaying the progress of cancer while treating cancer. However, there is a problem in that the transfer efficiency of the various clinical genes into cancer cells is low.

Cancer immunotherapy is a method for inducing immune responses to remove tumor cells, and is considered as a new method to overcome the limitation and side effect of conventional chemotherapy. The method is also a therapy that induces effective immune memory against tumor cells to prevent potential recurrence of cancer (Smyth MJ et al. Nat Immunol 2001 2; 293, Kaminski JM et al. Cancer Treat Rev 2003 29; 199-209).

Specifically, cancer immuno-gene therapy is a method for treating cancers, in which an immune-enhancing gene such as cytokine is directly injected so as to activate effective cellular immune responses(Colombo MP et al. Cytokine Growth Factor Rev 2002 13; 155-

168, Wang L et al. Cancer Gene Ther 2002 9; 819-824). IL-12 has been known as a cytokine showing strong antitumor effects among various immune-enhancing factors, since it activates cytotoxic T lymphocytes (CTLs), T helper Is (This), and NK cells and suppresses angiogenesis in tumors (Shurin MR et al. Chem Immunol 1997 68; 153-174,

Siders WM et al. J Immunol 1998 68; 5465-5474, Tannenbaum CS et al. J Immunol 1998 161 ; 927-932).

IL- 12 (Interleukin-12) is an IL-12p70 heterodimer, and composed of two subunits, that is, p35 and p40, which are covalently linked. Because of the structural characteristic, both of the two subunits, p35 and p40 are needed for gene therapy (Tahara H et al. Cancer Res 1994 54; 182-189, Rakhmilevich AL et al. PNAS 1996 93; 6291-6296). But excessive amount of p40 monomers or homodimers are secreted, as well as an IL-12p70 heterodimer which is a bioactive form of IL-12 (Trinchieri G Adv Immunol 1998 70; 83-243). It has been known that the p40 monomer or homodimer competes with IL-12p70 to bind a receptor of IL-12, thereby strongly inhibiting IL-12p70 activity (Gillessen S et al. Eur J Immunol 1995, 25; 200-206, Ling P et al. J Immunol 1995, 154; 116-127). Such characteristics have been thought to be an obstacle to be effective immunotherapy using IL-12 gene.

In order to overcome the above problem, a method for linking two subunits of p35 and p40 using a DNA sequence encoding a protein linker, which is generally used for producing an antibody, was proposed (Lieschke, GJ et al. Nat Biotechnol 1997 15; 35-40, Lode HN et al. PNAS 1998 95; 2475-2480, Lee YL et al. 1998 Human Gene Ther 1998 9; 457-465). The method made it possible to inhibit biological activity of IL-12p70 by an excessive amount of IL-12p40. However, the linkage of two subunits, p35 and p40 induced change of the structure. Subsequently, the activity of IL-12p70 was reduced by 5 to 100 times or more, such that the effects were insufficient. For effective treatment of cancer using IL-12 gene, a method for inducing a production of IL-12p70, of which activity is maintained without the secretion of IL-12p40, is now being developed. Accordingly, the present inventor disclosed a mutant of a human IL-12p40 subunit, and a mouse IL-12p40 subunit in Korean Patent Publication No. 2001-0103577, and constructed a recombinant vector comprising an IL-12p35 subunit and the mutant of IL-12p40 subunit (mouse p40-N220L or p40-N220Q, human p40-N222L or p40-N222Q) to confirm that the expression of p40 was significantly reduced in vivo and in vitro.

In the case of using the immune regulatory factors individually in immunotherapy, it has been known not to show strong antitumor effects of completely removing cancer cells. Therefore, expressing two or more immune regulatory genes selected from cytokines, chemokines, costimulatory factors in tumor cells and to improve anticancer immunoactivity has been tried. For example, a clinical trial to confirm anticancer effect of combination therapy with IL- 12 and IL-2 was performed. However, in the combination therapy, its toxicity was found to increase (Siders, J Immunol. 160:5465-5474). Further, in combination therapy with IL- 12 and IL-18, an anticancer effect was observed. However, it was not significant, as compared to monotherapy (Korean Patent Publication No. 2004-0060834).

Therefore, the present inventors have made extensive studies on an immune composition having excellent anticancer effects. They have found that combination therapy with a genetic construct encoding IL- 12M and an immune regulatory gene shows excellent anticancer effects, thereby completing the present invention.

[Disclosure] [Technical Solution]

It is an object of the present invention to provide an anticancer composition comprising a recombinant vector containing a genetic construct encoding IL- 12M and a recombinant vector containing an immune regulatory gene.

It is another object of the present invention to provide an anticancer composition comprising a recombinant vector containing a genetic construct encoding IL- 12M and an immune regulatory gene in combination. It is still another object of the present invention to provide a method for enhancing immune responses and a method for suppressing proliferation of tumor cells, in which the anticancer composition is administered to a subject in need of anticancer treatment, so as to induce expression of each gene in the subject. [Description of Drawings]

Fig. 1 is a diagram of an immune-enhancing gene, which is used for the construction of the recombinant virus of the present invention.

Fig. 2 is a drawing showing the mean volume of mice tumors from each group at day 24 after the combination-treatment, in which tumor nodes induced by mouse melanoma cell (B16F10) were combination-treated with various immune enhancing agents including rAd/IL-12M and rAd/hIL-2. Fig. 3 is a drawing showing a survival rate of mice according to suppression of tumor cell proliferation after combination-treating the tumor nodes induced by mouse melanoma cell (B16F10) with various immune enhancing agents including rAd/IL-12M and rAd/hIL-2.

[Best Mode]

In one embodiment, the present invention relates to an anticancer composition comprising a recombinant vector containing a genetic construct encoding IL- 12M and a recombinant vector containing an immune regulatory gene.

In another embodiment, the present invention relates to an anticancer composition comprising a recombinant vector containing a genetic construct encoding IL- 12M and an immune regulatory gene in combination.

In the present invention, the expression "a genetic construct encoding IL- 12M" is referred to a nucleic acid sequence expressing an IL-12p35 subunit and an IL-12p40 subunit, in which the asparagine of glycosylation site in a p40 subunit is substituted with other amino acids not to be glycosylated. The p35 subunit composing IL- 12M of the invention may be a wild-type p35 or a polypeptide having substantially the same biological activity as the wild-type p35.

A wild-type IL- 12 consists of the p35 and p40 subunits, and is secreted in the form of IL-12p40 monomer, IL-12p40 homodimer, and IL-12p70 heterodimer when expressed in vivo. However, only IL-12p70 heterodimer is involved in immunoactivity. Specifically, when IL- 12 is expressed, IL-12p40 is secreted 5 to 90 times more than IL- 12p70 and competes with IL-12p70 to bind a receptor of IL- 12, thereby strongly inhibiting immune response by IL-12p70 activity. The present inventor discloses a mutant IL- 12 (Hereinbelow, referred to as 'IL-12M'), in which the glycosylation site of p40 is modified to decrease the secretion of p40 and to relatively increase the expression of p70, in Korean Patent No. 0399728. The above mentioned "glycosylation" is referred to an N-glycoside bond of oligosaccharide to the side chain of asparagine. The glycosylation plays a crucial role in the secretion of IL- 12. Therefore, the control of IL- 12 secretion can be realized by mutating in the glycosylation site.

Accordingly, the p40 subunit of the invention is preferably a subunit in which the 127th, 141st, 222nd, and 303rd amino acids in the sequence of human p40 and the 220th amino acid in the sequence of mouse p40 are mutated not to be glycosylated, and more preferably a subunit in which the Asn-222 (asparagine at the 222nd position) in the sequence of human wild-type p40 and the Asn-220 (asparagine at the 220th position) in the sequence of mouse wild-type p40 are mutated not to be glycosylated. The mutation in the regions means that a codon ACA designating human Asn-222 and a codon AAC designating mouse Asn-220 are mutated to substitute the asparagine with other amino acids, preferably to substitute the asparagine with leucine, glutamine, and isoleucine, more preferably to substitute the asparagine with leucine, glutamine. Examples of the genetic construct comprising the mutated subunits, in which wild-type p35 and p40 subunits are substituted with other amino acids not to be glycosylated, include IL-12N220L (SEQ ID NO: 1), in which Asn-220 of mouse IL-12p40 subunit is substituted with leucine, IL- 12N220Q (SEQ ID NO: 2), in which Asn-220 of mouse IL-12p40 subunit is substituted with glutamine, IL-12N222L (SEQ ID NO: 3), in which Asn-222 of human IL-12p40 subunit is substituted with leucine, and IL-12N222Q (SEQ ID NO: 4) in which Asn-222 of human IL-12p40 subunit is substituted with glutamine. In the present invention, "the genetic construct encoding IL- 12M" may further include regulators capable of linking the genes or regulating the expression of the genes, in addition to genes encoding the p35 subunit, and genes encoding the p40 subunit mutated not to be glycosylated. The regulators are a nucleic acid sequence involved in the expression of the target genes, and play a role in regulating simultaneously the expression of the p35 subunit and the p40 subunit when the IL-12p35 gene and the mutated IL-12p40 gene are expressed. The regulators include an IRES (Internal ribosomal entry site), and a linker or the like, and more preferably IRES. The IRES is preferably derived from EMCV (encephalomyocarditis virus), but is not limited thereto. The regulator, in particular, the genetic construct comprising IRES can be arranged in the order of the gene encoding the p35 subunit, the IRES, and the gene encoding the mutated p40 subunit, or arranged in the order of the gene encoding the mutated p40 subunit, the IRES, and the gene encoding the p35 subunit, preferably in the order of the gene encoding the p35 subunit, the IRES, and the gene encoding the mutated p40 subunit.

In the present invention, the phrase "immune regulatory factor" is referred to as a material, which activates the cells involved in immune responses (for example, dendritic cell, etc.) to increase immune responses, such as a ligand, a cytokine, a cellular factor, and a peptide, excluding IL- 12 and IL- 12M. Examples of the immune regulatory factor include IL-2, IL-15, IL-23, CD40 ligand, Flt3 (FMS-like tyrosine kinase 3) ligand, HSP70 (heat shock protein 70), and GM-CSF (granulocyte-macrophage colony-stimulating factor). The IL-2 is secreted from the activated T cell to induce the proliferation of T cell and NK cell and the secretion of cytokine such as TNF-? and IL-I. The CD40 ligand (CD40L) is expressed in the activated CD4 T helper cell, and interacts with CD40 to play an important role in inducing humoral and cellular immune responses. GM-CSF is involved in the activation and maturation of dendritic cells and in the formation of NK cells and CTL (cytotoxic T lymphocyte). The HSP70 promotes the maturation and phagocytosis of dendritic cells and induces the activation of NK cell and CTL. The Flt3 ligand (Flt3L) is a growth factor of hematopoetic stem cell, and induces the proliferation of B cells, NK cells, and dendritic cells. The IL-23 induces the activation of memory T cell, and the IL- 15 induces the activation of NK cell and CTL to suppress cancers. In the present invention, at least one factor selected from the immune regulatory factors can be used, preferably used in combination of IL-2, CD40 ligand, and GM-CSF or in combination of IL-2 and CD40 ligand, or IL-2 and GM-CSF.

The genetic construct encoding IL- 12M and/or the immune regulatory gene of the invention can be modified to the optimized codon for a subject in order to increase the expression rate of the gene. The expression "modified to the optimized codon for a subject" as used herein is referred to increasing the expression rate of an amino acid or a protein by substituting the codon with host-preferred codons, which is a preferred codon according to a host among codons designating an amino acid on transcription and translation of DNA to protein in the host cell. Examples of the "subject" include mammals such as a human, a monkey, a mouse, a pig, a cow, and a rabbit, but are not limited thereto. The recombinant vector of the invention is a recombinant vector that is introduced into the host cell to express the IL- 12M and the immune regulatory factors. The vector can be usually prepared by the standard recombinant DNA technology, which includes a ligation of blunt end and cohesive end, a restriction enzyme treatment for providing a suitable end, a removal of phosphate group by alkaline phosphatase to prevent nonspecific binding, and an enzymatic linkage by T4 DNA ligase. Further, the recombinant vector of the invention may include a regulatory factor such as a promoter, an enhancer, a polyadenylation signal, and TPL, or a sequence designating the expression of target gene only in a specific host cell as a regulator for the expression.

The vector of the invention may be a phage particle or an RNA or DNA virus, which transforms a suitable host to integrate regardless of the host genome or to integrate into the host genome, replicates, and functions for its expression, or maybe a plasmid or a phage particle, which is originated from a microorganism for mass-culturing in the host microorganism, or a shuttle vector including both of the functions. The vector is preferably a retrovirus vector, an adenovirus vector, an adenovirus-associated vector, a herpes simplex vector or the like, and more preferably an adenovirus vector, but is not limited thereto.

IL- 12M has been known to be an excellent immune material showing the strong anticancer therapeutic effects. However, the combination therapy with IL- 12M and the immune regulatory factors of the invention promotes immune responses and suppresses the metastasis and angiogenesis of tumors and exhibits excellent tumor growth inhibition and therapeutic effects, as compared to the case of using IL- 12M alone. Further, the anticancer immune composition of the invention can induce its expression only in the tumor site, so as to reduce the toxicity that occurs on systemic administration of the recombinant protein, and thus its safety can be improved. The "combination therapy" is referred to a method for promoting immune responses of the body and thus suppressing the proliferation of tumor cells, in which each of IL- 12M and the immune regulatory factors is simultaneously expressed. In the specific embodiment, in the case of performing the combination therapy with the recombinant vector expressing IL- 12M and the recombinant vector expressing IL- 2, IL-23, CD40 ligand, Flt3 ligand, HSP70 or GM-CSF, the inhibition of tumor proliferation and the survival rate of mice were improved to exhibit stronger anticancer effects than the case of treating IL-12M alone.

In another embodiment, the present invention relates to a method for enhancing immune responses of a body and thus for suppressing proliferation of tumor cells, in which the anticancer composition is administered to a subject in need of anticancer treatment, so as to induce expression of each gene in the subject.

In the specific embodiment, the anticancer effect by combination therapy with the recombinant vector can be confirmed by the administration of the combination material to the tumor tissues of mouse, rat, dog, or the like. In a specific example, mice with tumors induced by melanoma cells were injected with the recombinant adenovirus, of which effects were examined with the size of the tumors and the survival rate of the mice.

In the invention, the "subject" is a mammal including a human, a monkey, a mouse, a pig, a cow, and a rabbit, but is not limited thereto.

The composition of the invention may additionally include at least one pharmaceutically acceptable composition. The pharmaceutical composition of the invention may include an excipient or a diluent. Examples thereof can be selected from the group consisting of saline, buffered saline, dextrose, water, glycerol or the like, but are not limited thereto. Further, a suitable drug delivery system can be employed in order to deliver the recombinant vector to tumor cells with safety and to reduce side effects in the body. For such purpose, hydrogel, collagen, PEG, PTD (protein transduction domain), or the like can be used, and the structural gene of the recombinant vector can be modified to improve the transfer efficiency into the tumor site. The composition of the invention may be systemically or topically administered.

In the case of administering topically, for example, an intratumoral injection of the composition can be performed into the tumor site. The dosage of the composition according to the invention is the conventional content being used in cancer gene therapy. For example, in the case of using the adenovirus vector, the recombinant virus particle can be injected to the maximum of IxIO¹² to IxIO₁₃, but is not limited thereto. The exact dosage of the composition may vary depending on the condition of patient, kind of disease, and drug combinations.

The composition of the invention can be employed in the treatment of common solid tumors such as skin cancer, liver cancer, breast cancer, cervical cancer, and colon cancer, as well as melanoma, but is not limited thereto.

*33Hereinafter, the present invention will be further described in detail with reference to Examples. However, these Examples are provided for the illustrative purpose only, and the invention is not intended to be limited thereto.

[Mode for Invention]

Example

Cell line culture

Each of a mouse melanoma cell line, Bl 6F10 cell, adenovirus production cell line, 293 cell, and monkey kidney fibroblast cells, COS-7 cell used to confirm the expression was purchased from ATCC (Manassas, VA20110-2209, USA) and cultured using DMEM

(Dulbecco's modified eagle's medium) containing 10% fetal bovine serum in a 5% CO₂ incubator.

Example 1: Production of recombinant adenovirus vector

The common methods used in molecular biology such as restriction enzyme treatment of DNA (New England Biolabs, Inc. USA), agarose gel electrophoresis, gel extraction kit, ligation of DNA fragment and E. coli transformation, purification of plasmid DNA, polymerase chain reaction were performed with minimal modifications of the methods introduced in Molecular Cloning (Sambrook et al., 2nd edition).

A gene, mIL-12M (SEQ ID NO: 1, Ha SJ et al. Nat Biotechnol 2002 20; 831) was digested with restriction enzymes, Xho I and Xba I, and the gene, mIL-23M (SEQ ID NO:

5, Ha SJ et al. J Immunol 2004 172; 525) was digested with restriction enzymes, Sal I and Not I. The gene mGM-CSF (SEQ ID NO: 7, in HT et al. Human Gene Ther 2005 16; 328) was digested with restriction enzymes, Not I and Xho I. Each of the digested genes was inserted into the site of Xho I/Xba I, Sal I/Not I, and Not I/Xho I in a pShuttle vector, which is an adenovirus shuttle vector. Genes, hIL-2 (SEQ ID NO: 6) and hIL-15 (SEQ ID NO: 11) were obtained by RT-PCR using mRNA from Jurkat cells, and a gene, mCD40L (SEQ ID NO: 8) was obtained by RT-PCR using mRNA from EL4 cells. Each of the obtained genes was inserted into the site of Kpn I/Not I in the pShuttle vector. Genes, HSP70 (SEQ ID NO: 9) and hFlt3L (SEQ ID NO: 10) were chemically synthesized to insert into the site of Not I/Hind III in the pShuttle vector. Subsequently, each pShuttle vector was transformed into competent cells. Each cloned pShuttle DNA was digested with restriction enzyme, Pme I to perform homologous recombination with a pAd/Easy vector, which is a replication-defective vector, in BJ5183 competent cells. Then, cells containing a recombinant adenovirus (rAd), in which a desirable gene expression cassette was integrated, were selected using Kanamycin, and the recombinant adenovirus vectors were isolated and purified from the selected cells. The purified recombinant adenovirus vectors were digested with Pac I and the DNA bands of 35 + 4.5 kb were confirmed. Subsequently, each of recombinant adenovirus DNA' s was transformed into the 293 cell line to obtain the virus vectors expressing the desirable gene at day 10 after the transformation (Fig. 1). The protein expression of the recombinant adenovirus was confirmed in the COS-

7 cell line. When the COS-7 cells covered 80% of the culture dish, the cells were infected with 200 MOI of each recombinant adenovirus for 2 hours and cultured for 48 hours in order to express its protein sufficiently after changing the culture media was changed with fresh media. The expressions of IL-12, IL-15, IL-2, GM-CSF, and IL-23 were confirmed by ELISA, and anti-IL-12 (R&D System, Minneapolis, MN), anti-IL-15 (R&D System, Minneapolis, MN), anti-IL-2, anti-GM-CSF (BD Pharmingen, Sandiego, CA), and anti-IL-23 (eBioscience, Sandiego, CA) were used as antibodies for ELISA. The expressions of CD40L, Flt3L, and HSP70 were confirmed by Western Blotting using anti-CD40L, anti-Flt3L (Santa Cruz Biotechnology, California, USA), and anti-HSP70 (StressGene Biotechnologies Corp. Victoria, BC, Canada).

Example 2: Effect of suppressing tumor by combination therapy with recombinant virus In order to confirm the effect of suppressing a tumor by combination therapy with recombinant virus, the melanoma-induced mice were administered with each recombinant virus of Example 1 to check the size of the tumors. The cell number of the cultured melanoma cell line, Bl 6F10 was counted. In the case where the live cells are 80% or more of the total cells, the cells were washed with IxPBS and diluted with IxPBS to be 5xlO⁶ cells/ml. Then, 100 μi (5x10⁵ of cell number) of cell containing media was subcutaneously injected to every C57BL/6 mouse to form tumor nodes for 7 to 9 days. The mice with tumor nodes were divided into 9 groups (12 mice per a group). When the diameter of the tumor nodes becomes 6 to 8 mm, 1 x10 pfu/100 μJL of the recombinant virus of Example 1, which is diluted with a I xPBS buffer solution, was injected into tumor nodes three times at two-day intervals, and the change of the tumor size was observed according to the time. The day of the first injection is marked as day 0, and a mean value of the volume (mm³), which is calculated as major axis ^χ minor axis x height/2, was taken as the tumor size. Fig. 2 is a graph showing the mean size of the tumors at day 24 after injecting 9 groups of mice with tumor nodes with each recombinant virus. As a control group, the recombinant virus, rAd/Mock with no expression was used, and all of the mice, which are treated with rAd/Mock, died at day 15 after performing the experiment, whereby the result is not shown in the graph. In the case of injecting rAd/IL-12M alone, the treatment effects on tumors were found to be excellent, as compared with the control group. In the case of combination therapy with other immune enhancing factors, the overall tumor size was significantly reduced, as compared to the group treated with rAd/IL-12M alone.

In particular, in the case of combination therapy with rAd/hIL-2, rAd/mGM-CSF, and rAd/mCD40L, the mean size of the tumors was 34 mm³, 52 mm³, and 44 mm³, respectively, which showed that the effects of suppressing tumor is 10 times, 7 times, 8 times better, as compared to the tumor size of about 350 mm³ of the group treated with rAd/IL-12M alone (Fig. 2).

Example 3: Survival rate of mice with melanoma by combination therapy with recombinant virus In order to confirm the effect of suppressing tumor proliferation by the recombinant virus, as well as the anticancer effect by the virus, the survival rate of the mice was examined.

The experiment was performed such that each of the nine groups of mice with melanoma was injected with each recombinant virus according to the method of Example 2, and then the survival period was observed over 50 days, which is the extended experiment of Example 2 (Table 1 and Fig. 3).

Overall, the survival rates, of which the mice with melanoma were treated with rAd/IL-12M individually or combination-treated with rAd/IL-12M and other immune regulatory factors, were much higher at 24 days, as compared to the control group, rAd/Mock with no expression. Moreover, it was found that the survival rates, of which the groups were combination-treated with rAd/mIL-12M and other immune regulatory factors, were excellent at day 45, as compared to the group treated with rAd/mIL-12M alone. In particular, the survival rates of the groups combination-treated with rAd/IL-2, rAd/CD40L, rAd/GM-CSF, rAd/HSP70, and rAd/hFlt3L were 66.4%, 49.8%, 33.3%, 16.6%, and 16.6%, respectively. That is, the anticancer effects by the combination therapy were found to be excellent, as the above mentioned (Table 1 and Fig. 3). Accordingly, in the case of combination therapy with IL- 12M and other immune regulatory factors such as IL-2, CD40L, GM-CSF, HSP70, hFlt3L, IL-23M, and IL-15, the effects of suppressing tumor proliferation were excellent and the survival rates of the tumor-induced mice were improved, as compared to the case of treating with IL- 12M individually.

[Table 1 ]

[Industrial Applicability] The anticancer composition comprising a recombinant vector containing a genetic construct encoding IL- 12M and a recombinant vector containing a gene encoding an immune regulatory factor and the anticancer composition comprising a recombinant vector containing a genetic construct encoding IL- 12M and an immune regulatory gene in combination in the invention show excellent effects of suppressing tumor proliferation, thereby being used as an effective anticancer therapeutic agent.

Claims

[CLAIMS] [Claim 1 ]

An anticancer composition comprising a recombinant vector containing a genetic construct encoding IL- 12M and a recombinant vector containing a gene encoding an immune regulatory factor. [Claim 2]

An anticancer composition comprising a recombinant vector containing a genetic construct encoding IL- 12M and a gene encoding an immune regulatory factor in combination. [Claim 3]

The anticancer composition according to claim 1 or 2, wherein the genetic construct comprises IRES (international ribosomal entry site). [Claim 4]

The anticancer composition according to claim 1 or 2, wherein the genetic construct is any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4. [Claim 5]

The anticancer composition according to claim 1 or 2, wherein the gene encoding the immune regulatory factor is one or more genes encoding any one IL-2, GM-CSF, CD40 ligand, Flt3 ligand, and HSP70. [Claim 6]

The anticancer composition according to claim 1 or 2, wherein the gene is modified to the optimized codon of a mammal in order to increase the expression rate. [Claim 7]

The anticancer composition according to claim 1 or 2, wherein the recombinant vector is a virus. [Claim 8] The anticancer composition according to claim 6, wherein the virus is an adenovirus. [Claim 9]

A method for enhancing immune responses of a body comprising: administrating the anticancer composition of claim 1 or 2 to a subject in need of anticancer treatment so as to express each gene in the subject. [Claim 10]

A method for suppressing proliferation of tumor cells comprising: administrating the anticancer composition of claim 1 or 2 to a subject in need of anticancer treatment so as to express each gene in the subject.