KR100788930B1 - Anti-Cancer Compositions - Google Patents

Abstract

The present invention comprises a recombinant vector comprising a gene construct encoding IL-12M and a recombinant vector comprising an immunoregulator gene or simultaneously combining the gene construct and the immunomodulatory factor gene in one vector. An anticancer composition comprising a recombinant vector to express and a method for administering the same to an individual in need of anticancer treatment and expressing each gene to enhance the body's immune response and inhibit the proliferation of tumor cells. The present invention provides an anticancer effect superior to conventional anticancer compositions.

Recombinant vector, anticancer immunotherapy, anticancer gene therapy, IL-12, immune modulator

Description

Anti-Cancer Compositions

1 is a schematic diagram of immune enhancing genes used in the production of recombinant virus of the present invention.

2 is a graph showing the average tumor volume of each group 24 days after the combination of other immune enhancers including rAd / IL-12M and rAd / hIL-2 in tumor nodules induced by melanoma cell line (B16F10). .

Figure 3 shows the survival curve of mice according to the inhibition of tumor cell proliferation after the combined administration of other immune enhancers, including rAd / IL-12M and rAd / hIL-2 in the tumor nodules of mice induced by melanoma cell line (B16F10) Figure shown.

The present invention relates to an anticancer composition comprising a recombinant vector comprising a gene construct encoding IL-12M and an immunoregulatory gene, respectively or simultaneously, and a gene therapy using the same.

Various methods such as inducing cancer cell death, immunotherapy using immune response modulators, and controlling expression of cancer cell specific genes are used for gene therapy of cancer. Recently, gene therapy using adenoviruses that can only proliferate in cancer cells has been developed, and effects of delaying and treating cancer by various gene treatments have been demonstrated. Delivery efficiency is a problem.

Anticancer immunotherapy with gene vaccines is a treatment that can remove tumor cells by inducing tumor specific immune responses, and has emerged as a new method to overcome the limitations and side effects of conventional anticancer treatments. It is a therapy that can induce immune memory and potentially prevent cancer recurrence (Smyth MJ et al. Nat Immunol 2001 2; 293, Kaminski JM et al. Cancer Treat Rev 2003 29; 199-209). In particular, anti-cancer immune gene therapy is a method of directly injecting an immune enhancing gene such as cytokine to activate an effective cellular immune response against tumor cells to treat cancer (Colombo MP et al. Cytokine Growth Factor Rev 2002 13; 155-168, Wang L et al. Cancer Gene Ther 2002 9; 819-824), among many immune enhancers, IL-12 is CD8 ⁺ cytotoxic T lymphocyte (CTL), T helper 1 (Th1), and It is known as a cytokine that has an excellent anticancer effect by activating NK cells and inhibiting 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 a heteropolymer of IL-12p70, which consists of two covalently linked subunits, p35 and p40 subunits. Because of these structural features, gene therapy requires both p35 and p40 subunits (Tahara H et al. Cancer Res 1994 54; 182-189, Rakhmilevich AL et al. PNAS 1996 93; 6291-6296), IL. IL-12p70 heteropolymer, a biologically active form of -12, as well as excess p40 monomer or homopolymer, is secreted (Trinchieri G Adv Immunol 1998 70; 83-243), resulting in IL-12 receptor It is known to competitively bind -12p70 to strongly inhibit the activity of IL-12p70 (Gillessen S et al. Eur J Immunol 1995, 25; 200-206, Ling P et al. J Immunol 1995, 154; 116-127 ). This property is considered to be an obstacle to effective immunotherapy using the IL-12 gene.

To overcome this, a method of linking p35 and p40 subunits using DNA sequences encoding protein linkers commonly used in antibody production has been 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). However, the above method was able to prevent the inhibition of the biological activity of IL-12p70 by excess IL-12p40, but the change of the structure caused by the structural change caused by linking two subunits of p35 and p40 Activity was reduced by more than 5 to 100 times, which did not have sufficient effect. Therefore, in order to efficiently treat cancer using IL-12 gene, a method of inducing the production of IL-12p70 in which activity is maintained without secreting IL-12p40 has been developed. In this regard, the present inventors have disclosed a mutant of human and mouse IL-12p40 subunits in Korean Patent Publication No. 2001-0103577, and the IL-12p35 subunit and IL-12p40 mutant subunits (rat p40-N220L or p40-N220Q). , A recombinant vector comprising human p40-N222L or p40-N222Q,) was produced to confirm that the expression of p40 is significantly reduced in vivo and in the laboratory.

In the case of chemotherapy with immunomodulators, the use of immunomodulators alone is not known to produce a potent anticancer effect that can completely remove tumor cells. Thus, attempts have been made to enhance anticancer immune activity by expressing two or more immunoregulatory genes selected from cytokines, chemokines, costimulatory factors, etc., in tumor cells. For example, a clinical trial was conducted to confirm the anti-cancer effect by combining IL-12 and IL-2. However, the body toxicity was increased during the combined treatment (Siders, J Immunol. 160: 5465-5474) . Anti-cancer effect was shown in the combination treatment of -12 and IL-18, but did not show a clear effect compared to the treatment alone (Korean Patent Publication No. 2004-0060834).

Accordingly, the present inventors have found that the anticancer effect is excellent when the gene construct encoding IL-12M and the immunoregulatory gene are combined while studying an immune composition having excellent anticancer effect, and completed the present invention. .

One object of the present invention is to provide an anticancer composition comprising a recombinant vector comprising a gene construct encoding IL-12M and a recombinant vector comprising an immunoregulatory gene.

It is another object of the present invention to provide an anticancer composition comprising a recombinant vector comprising a gene construct encoding IL-12M and an immunomodulator gene in one vector.

It is another object of the present invention to provide a method of administering the anticancer composition to a subject in need of anticancer treatment and expressing each gene in the subject to enhance the body's immune response and to inhibit the proliferation of tumor cells.

In one aspect, the present invention relates to an anticancer composition comprising a recombinant vector comprising a gene construct encoding IL-12M and a recombinant vector comprising an immunoregulatory gene.

In one aspect, the present invention relates to an anticancer composition comprising a recombinant vector comprising a gene construct encoding IL-12M and an immunomodulator gene in one vector.

In the present invention, the "gene construct encoding IL-12M" expresses IL-12p35 subunit and IL-12p40 subunit in which asparagine at the glycosylation site is substituted with another amino acid so that glycosylation does not occur at the p40 subunit. Refers to the nucleic acid sequence. The p35 constituting IL-12M of the present invention may be a polypeptide exhibiting substantially the same physiological activity as wild type p35 or wild type p35.

Wild type IL-12 is composed of p35 and p40 subunits and is secreted in the form of IL-12p40 monomer, IL-12p40 homopolymer and IL-12p70 isoform when expressed in vivo, Only is involved in immune activity. In particular, IL-12p40 is secreted 5 to 90 times more than IL-12p70 upon IL-12 expression, thereby strongly inhibiting the immune response activity by IL-12 by competitively binding IL-12p70 to the receptor of IL-12. The mutant IL-12 (hereinafter referred to as 'IL-12M') modified by the glycosylation site involved in reducing the secretion of p40 and relatively increasing the expression level of p70 at -12 0399728. The "glycosylation" refers to the N-glycosidic linkage of small sugars in the asparagine side chain. These glycosylations play an essential role in the secretion of IL-12, and the secretion of IL-12 can be regulated by mutating so that glycosylation does not occur at this glycosylation site. Thus, the p40 subunit of the present invention is preferably a subunit mutated so that glycosylation does not occur in SEQ ID NOs: 127, SEQ ID NO: 141, SEQ ID NO: 222, SEQ ID NO: 303, and SEQ ID NO: 220 of mouse p40, more preferably. Preferably, it is a subunit mutated so that glycosylation does not occur in Asn-222 (222nd asparagine) of human wild type p40 and Asn-220 (220th asparagine) of mouse wild type p40. The mutation at this site is a mutation of codon ACA designating human Asn-222 and codon AAC designating mouse Asn-220 to replace asparagine with another amino acid, preferably asparagine with leucine, glutamine, isoleucine. It is substituted with, More preferably, it replaces asparagine with leucine and glutamine. As a specific example of a gene construct comprising a subunit mutated to another amino acid so that glycosylation does not occur in the wild type p35 subunit and p40 subunit, asparagine, the 220th amino acid of the subunit of mouse IL-12p40, is mutated to leucine. IL-12N220L (SEQ ID NO: 1), IL-12N220Q (SEQ ID NO: 2), which transformed asparagine, the 220th amino acid of the mouse IL-12p40 subunit, to glutamine, and Asparagine, the 222nd amino acid, of the human IL-12p40 subunit. IL-12N222L (SEQ ID NO: 3) mutated to leucine and IL-12N222Q (SEQ ID NO: 4) mutated asparagine, the 222nd amino acid of the subunit of IL-12p40, to glutamine.

The "gene construct encoding the IL-12M" of the present invention is a regulator that connects or modulates expression of genes other than the gene encoding the p35 subunit and the gene encoding the p40 subunit mutated so that glycosylation does not occur. It may further comprise. The modulator is a nucleic acid sequence involved in the expression of a gene of interest, and means for simultaneously controlling the expression of the p35 subunit and p40 subunit upon expression of the IL-12p35 gene and the mutated IL-12p40 gene. The modulator includes an internal ribosomal entry site (IRES), a linker and the like, more preferably IRES. The IRES is not limited thereto, but it is preferable to use one derived from EMCV (encephalomyocarditis). The regulators, in particular the gene construct comprising the IRES, are arranged in the order of the gene encoding the p35 subunit, the gene encoding the IRES and the mutated p40 subunit, or the gene encoding the p40 subunit, IRES and p35 subunit. The genes encoding the monomers may be arranged in order, preferably the genes encoding the p35 subunits, the IRES and the genes encoding the mutated p40 subunits.

In the present invention, the 'immunoregulator' is a substance such as ligand, cytokine, cellular factor, peptide, etc. that activates cells (eg, dendritic cells, etc.) involved in the immune response to increase the immune response, and IL-12 and Refers to substances other than IL-12M. The immunomodulatory factors include, for example, 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). IL-2 is secreted from activated T cells to induce proliferation of T cells and NK cells and to induce cytokine secretion such as TNF-α and IL-1. CD40 ligand (CD40L) is expressed in activated CD4 T helper cells and plays an important role in inducing humoral and cellular immune responses through interaction with CD40. GM-CSF activates and matures dendritic cells and is involved in the formation of NK cells and cytotoxic T lymphocytes (CTLs). HSP70 stimulates the maturation and macrophages of dendritic cells to induce the activity of NK cells and CTLs, and Flt3 ligand (Flt3L) is a growth factor for hematopoietic stem cells and induces proliferation of B cells, NK cells, and dendritic cells. IL-23 induces the activity of memory T cells, and IL-15 induces the activity of NK cells and CTLs to inhibit cancer. In the present invention, one or more immunomodulators may be selected and used. Preferably, IL-2, CD40 ligand, and GM-CSF may be used in combination, or IL-2, CD40 ligand, or GM-CSF may be used in combination. Can be.

Gene constructs and / or immunoregulatory genes encoding IL-12M of the invention can be modified with codons optimized for the subject to increase the expression rate of the gene. As used herein, "to be transformed into an object-optimized codon" means that codons with high affinity depending on the host are exchanged between codons that command amino acids when DNA is transcribed and translated into proteins in host cells. It is said to increase the expression efficiency of the amino acid or protein encoded by the nucleic acid by substitution with these highly preferred codons. "Subject" includes, but is not limited to, mammals such as humans, monkeys, mice, pigs, cattle and rabbits.

The recombinant vector of the present invention was introduced into a host cell and recombined appropriately to express the IL-12M and immunoregulatory factor. Generally, vectors can be prepared by standard recombinant DNA techniques, which include blunt- and adhesive-terminal ligation, restriction enzyme treatment to provide appropriate ends, and alkaline phosphatase treatment to prevent inadequate binding. Phosphate group removal and enzymatic linkage by T4 DNA ligase. In addition, the recombinant vector of the present invention may include a promoter, an enhancer and a polyadenylation signal, a regulatory element such as TPL, or a sequence indicating expression of a gene of interest only in a specific host cell as a regulator for expression.

Vectors of the present invention are phage particles, RNA or DNA viruses that can be transformed into a suitable host for expression and integrated and replicated and function independently of the genome or into the genome itself, or in mass culture in a microbial host. For example, it may be a plasmid derived from a microorganism, phage particles, or the like, or a shuttle vector including both functions. Preferred vectors are retrovirus vectors, adenovirus vectors, adeno-associated virus vectors, herpes simplex vectors, and the like, and more preferably, adenoviruses may be selected, but are not limited thereto.

IL-12M is known to be an excellent immune substance with high anticancer effect. However, the combination therapy of IL-12M and immunomodulator of the present invention promotes an immune response and inhibits tumor metastasis and neovascularization than IL-12M alone, and has excellent tumor growth inhibition and therapeutic effects. In addition, the expression of the anticancer immune composition of the present invention can be induced only at the tumor site, thereby lowering the toxicity that may occur during systemic administration of the recombinant protein, thereby improving safety. The above "combination therapy" refers to a method of enhancing the body immune response by simultaneously expressing IL-12M or an immunomodulatory factor and thereby inhibiting the proliferation of tumor cells. In a specific embodiment, when the recombinant vector expressing IL-12M and the recombinant vector expressing IL-2, IL-23, CD40 ligand, Flt3 ligand, HSP70, or GM-CSF are treated, IL-12M alone is treated. Tumor growth inhibitory ability and survival rate of rats were improved than that of case, showing stronger anticancer effect.

In another aspect, the present invention relates to a method for administering the anticancer composition of the present invention to a subject in need of anticancer treatment and expressing each gene in the subject to enhance a body immune response and to inhibit the proliferation of tumor cells. will be.

In a specific embodiment, the anticancer effect by the combined treatment of the recombinant vector can be confirmed by injection into tumor tissues of mice, mice, dogs and the like which induced the tumor. In a specific embodiment of the present invention, the recombinant adenovirus was administered to mice inoculated with melanoma and tumor-induced, and the effects of each recombinant virus injection were investigated by the size of the cancer and the survival rate of the mice.

In the present invention, "individual" includes, but is not limited to, mammals such as humans, monkeys, mice, pigs, cattle and rabbits.

The composition of the present invention may additionally comprise one or more acceptable pharmacological compositions. The pharmaceutical composition may contain excipients or diluents. For example, one or more selected from the group consisting of saline, buffered saline, dextrose, water, glycerol, and the like, but is not limited thereto. Recombinant vectors can also be delivered to tumor cells more safely and appropriate drug delivery systems can be used to reduce side effects in the body. For this purpose, for example, hydrogel, collagen, PEG, PTD (protein transduction domain) and the like can be used, it is also possible to improve the delivery efficiency to the tumor site by modifying the structural gene of the recombinant vector.

The compositions of the present invention can be administered systemically and topically. In the case of topical administration, it may be administered, for example, by intratumoral injection into the tumor site. The amount of the composition according to the present invention is generally used for gene therapy of tumors using recombinant vectors. For example, when using adenovirus vectors, up to 1 × 10 ¹² to 1 × 10 ¹³ recombinant virus particles are injected. It may, but is not limited to. The exact dosage may be applied differently depending on the condition of the patient, the type of disease, and the drug being used.

The composition of the present invention can be applied to the treatment of general solid cancers, such as skin cancer, liver cancer, breast cancer, cervical cancer, colorectal cancer, etc., including but not limited to melanoma.

Hereinafter, the present invention will be described in more detail with reference to the following examples. These examples are only for illustrating the present invention more specifically, and the scope of the present invention is not limited to these examples according to the gist of the present invention.

Example

Culture of Cell Lines

The rat melanoma cell line B16F10, the adenovirus producing cell line 293, and the monkey kidney fibroblasts used for expression verification, COS-7 cells, were purchased from ATCC (Manassas, VA20110-2209, USA), respectively. Cultured in a 5% CO ₂ cell incubator using Dulbecco's modified eagle's medium (DMEM) medium containing fetal serum.

Example 1 Preparation of Recombinant Adenovirus Vectors

Common in molecular biology such as treatment of DNA restriction enzymes (New England Biolabs, Inc. USA), agarose gel electrophoresis, gel extraction kits, conjugation of DNA fragments and transformation of E. coli, pure plasmid DNA purification, and polymerase chain reaction. The methods used in the study were carried out with minimal modification to the methods introduced in Molecular Cloning (2nd edition) by Sambrook.

mIL-12M (SEQ ID NO: 1, Ha SJ et al. Nat Biotechnol 2002 20; 831) gene was cut with restriction enzymes Xho I and Xba I, and mIL-23M (SEQ ID NO: 5, Ha SJ et al. J Immunol 2004 172; 525) The gene was cut with restriction enzymes Sal I and Not I, and the mGM-CSF (SEQ ID NO: 7, in HT et al. Human Gene Ther 2005 16; 328) gene was cut and prepared with Not I and Xho I, which was then adenosed. The virus shuttle vector was inserted into the Xho I / Xba I, Sal I / Not I and Not I / Xho I sites of the pShuttle vector. hIL-2 (SEQ ID NO: 6) and hIL-15 (SEQ ID NO: 11) were obtained from Jurkat cells, and mCD40L (SEQ ID NO: 8) was obtained from RT4 PCR using mRNA obtained from EL4 cells. HSP70 (SEQ ID NO: 9) and hFlt3L (SEQ ID NO: 10) were chemically synthesized and inserted into the Not I / Hind III site of the pShuttle vector and then inserted into competent cells. Transformed. Each cloned pShuttle DNA was digested with restriction enzyme Pme I and homologous recombination was performed in a non-replicable pAd / Easy vector and BJ5183 competent cells. Then, using a kanamycin (Kanamycin) antibiotic, a strain containing a recombinant adenovirus (rAd) containing the desired gene expression cassette is selected, the recombinant adenovirus is isolated and purified, and digested with Pac I, 35 + 4.5 kb DNA band of was confirmed. Each recombinant adenovirus DNA was transformed into 293 cell lines to obtain a viral vector expressing a desired gene after 10 days (FIG. 1).

Expression confirmation of the protein of the recombinant adenovirus was performed in the COS-7 cell line. Each recombinant adenovirus was infected with 200 MOI for 2 hours in COS-7 cells cultured about 80% in a petri dish, and cultured for 48 hours after replacing the medium to sufficiently express the protein. IL-12, IL-15, IL-2, GM-CSF, IL-23 expression was confirmed by ELISA, anti-IL-12 (R & D System, Minneapolis, MN), anti-IL-15 as antibodies to (R & D System, Minneapolis, MN), anti-IL-2, anti-GM-CSF (BD Pharmingen, Sandiego, Calif.), Anti-IL-23 (eBioscience, Sandiego, Calif.) Were used. Expression of CD40L, Flt3L, HSP70 was confirmed by Western blot using anti-CD40L, anti-Flt3L (Santa Cruz Biotechnology, California, USA), anti-HSP70 (StressGene Biotechnologies Corp. Victoria, BC, Canada) It was.

Example 2 Tumor Suppression Effect by Combined Administration of Recombinant Virus

 In order to investigate the anticancer effect by the combined treatment of recombinant viruses, the size of cancer was investigated by administering the recombinant virus of Example 1 to the mice induced melanoma.

Cultured melanoma cell line B16F10 measures the number of cells, and if the living cells are more than 80% of the total, washed with 1 × PBS, suspended in 1 × PBS to 5 × 10 ⁶ cells / ml, C57BL / 6 mice 100 μl (5 × 10 ⁵ cells) per head were injected subcutaneously and tumor nodules formed for 7-9 days.

Tumor nodules were divided into 9 groups (12 animals per group), and when the tumor nodules were 6 to 8 mm in diameter, 1 × 10 ⁸ pfu of the recombinant virus of Example 1 diluted with 1 × PBS buffer solution. Injection into tumor nodules three times at two-day intervals in an amount of / 100 μl and tumor size change over time was observed. The day of the first injection was marked as day 0, and the tumor size was taken as the average value of the volume (mm ³ ) calculated as long axis × short axis × height / 2. Figure 2 graphically shows the mean tumor size of each group 24 days after injection of each recombinant virus into a group of mice with tumor nodules formed. As a control, a recombinant virus, rAd / Mock, which did not express anything, was used. All mice died about 15 days after the experiment and were not included in the graph. Even when rAd / IL-12M was injected alone, the tumor treatment effect was proven to be superior to that of the control group, and compared with the group treated with rAd / IL-12M alone (G2) in combination injection of other immune enhancing factors. The tumor size was greatly reduced. Especially, when combined with rAd / hIL-2, rAd / mGM-CSF, and rAd / mCD40L, the average tumor size of the group treated with rAd / IL-12M alone was about 350 mm ³ . Compared with the average tumor size of 34 mm ³ , 52 mm ³ , 44 mm ³ , 10 times, 7 times, and 8 times superior tumor growth inhibitory effects were shown (FIG. 2).

Example 3 Survival Investigation of Melanoma Mice by Combined Administration of Recombinant Virus

In order to confirm the anti-tumor effect of the virus as well as the tumor growth inhibitory effect of the recombinant virus, the survival rate of the mice was examined.

The experiment was performed by injecting each recombinant virus to each of the 9 groups of melanoma mice according to the method of Example 2 to observe the survival time of each mouse for 50 days or longer, and Example 2 was extended (Table 1 and FIG. 3). ).

Survival rate (%) day 24 day 45 rAd / Mock 0 0 rAd / mIL-12M 66.4 0 rAd / mIL-12M + rAd / hIL-2 82.5 66.4 rAd / mIL-12M + rAd / mIL-23M 82.5 0 rAd / mIL-12M + rAd / mGM-CSF 82.5 33.3 rAd / mIL-12M + rAd / mCD40L 82.5 49.8 rAd / mIL-12M + rAd / HSP70 66.4 16.6 rAd / mIL-12M + rAd / hFlt3L 82.5 16.6 rAd / mIL-12M + rAd / hIL-15 82.5 0

Overall, survival rates at day 24 in mice induced with melanoma were much higher in the treatment group treated with rAd / IL-12M alone or in combination with other immunomodulators compared to the control-free rAd / Mock with no expression. In addition, the survival rate at 45 days was superior to the group treated with rAd / mIL-12M alone and other immune enhancers compared to the group treated with rAd / mIL-12M alone (0%). In particular, the group treated with rAd / IL-2, rAd / CD40L, rAd / GM-CSF, rAd / HSP70, rAd / hFlt3L had a high survival rate of 66.4%, 49.8%, 33.3%, 16.6%, and 16.6%, respectively. It was confirmed that the anti-cancer increase effect by the combination treatment in order (Table 1 and Figure 3).

Therefore, compared with the treatment with IL-12M alone, the tumor cell growth inhibition effect when combined with other immunomodulators such as IL-2, CD40L, GM-CSF, HSP70, hFlt3L, IL-23M, IL-15 And improved survival of tumor-induced rats.

An anticancer composition comprising a recombinant vector comprising a gene construct encoding IL-12M of the present invention and a recombinant vector comprising a gene encoding an immunomodulatory factor, and a gene construct encoding IL-12M in one vector An anticancer composition comprising a recombinant vector comprising a gene encoding a truck and an immunomodulator may be used as an excellent anticancer agent because of its excellent tumor growth and inhibitory effect.

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Claims (10)

i) the codon encoding Asn-222 of the IL-12p35 subunit and the human IL-12p40 subunit or the codon encoding Asn-220 of the mouse IL-12p40 subunit consisting of leucine, glutamine and isoleucine A recombinant vector comprising a gene construct encoding an IL-12p40 subunit substituted with a codon encoding one amino acid selected from ii) IL-15, IL-2, GM-CSF, IL-23, CD40 An anticancer composition comprising a recombinant vector comprising a nucleotide encoding one or more immunomodulators selected from the group consisting of ligand, Flt3 ligand and HSP70. i) the codon encoding Asn-222 of the IL-12p35 subunit and the human IL-12p40 subunit or the codon encoding Asn-220 of the mouse IL-12p40 subunit consisting of leucine, glutamine and isoleucine A gene construct encoding an IL-12p40 subunit substituted with a codon encoding one amino acid selected from ii) IL-15, IL-2, GM-CSF, IL-23, CD40 ligand, Flt3 ligand and An anticancer composition comprising a recombinant vector comprising a nucleotide encoding one or more immunomodulators selected from the group consisting of HSP70. The anticancer composition according to claim 1 or 2, wherein the gene construct comprises an international ribosomal entry site (IRES). The anticancer composition according to claim 1 or 2, wherein the gene constructor is any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4. delete delete The anticancer composition according to claim 1 or 2, wherein the recombinant vector is a virus. The anticancer composition according to claim 7, wherein the virus is an adenovirus. delete delete

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