KR20010103577A - Genes of IL-12p40 subunit mutated for improving the activity of IL-12 and use thereof for DNA vaccine adjuvant - Google Patents

Abstract

The present invention is an IL-12p40 subunit mutant gene capable of producing highly active human and mouse Interleukin-12 (IL-12), an expression vector comprising the mutant gene, and use thereof as a DNA vaccine adjuvant. More specifically, the mutation of IL-12p40 by mutating the amino acid Asn-222 (human) or Asn-220 (mouse) of human or mouse IL-12p40, which acts as a competitive inhibitor of active IL-12 The IL-12p40 mutant gene allows IL-12p70, which has the immune activity of IL-12 to be secreted, to be secreted normally. The IL-12p40 subunit mutant gene of the present invention and IL-12p35 gene, another subunit of IL-12, can be used together for immunization with DNA vaccine against enveloped glycoprotein 2 (E2) of hepatitis C virus (HCV). In the case of HCV-E2 DNA alone or when IL-12p40 normal gene was used, it was found that it can induce and maintain an optimal cellular immune response for a longer time. Thus, these results confirm that IL-12p70, excluding IL-12p40 subunit, can elicit a long-term strong cellular immune response in vivo, and the IL-12p40 mutant gene of the present invention has a cellular immune response. It will be useful for gene therapy for the prevention and treatment of acquired immunodeficiency syndrome (AIDS), hepatitis C and B, cancer, flu, tuberculosis, malaria, etc., which are known to be indispensable.

Description

Gene of IL-12p40 subunit mutated for improving the activity of IL-12 and use about for DNA vaccine adjuvant}

The present invention relates to an IL-12p40 subunit mutant gene capable of producing human and mouse IL-12 having high activity, an expression vector comprising the mutant gene, and a use of the same as a DNA vaccine immunopotentiator. By mutating Asn-222 (human) or Asn-220 (mouse) amino acids of human or mouse IL-12p40 acting as a competitive inhibitor of active IL-12, the secretion of IL-12p40 is inhibited and IL-12p70 having immune activity is an IL-12p40 mutant gene which allows normal secretion; An expression vector comprising said mutant gene and used for enhancing DNA immunization; The mutant gene relates to the use of an immunoadjuvant for the immunization of DNA vaccines for diseases such as AIDS, hepatitis C and B, cancer, flu, tuberculosis, malaria, and an adjuvant for maintaining long-term immune activity.

IL-12 is secreted by antigen presenting cells (APCs), such as macrophages and monocytes, after proper stimulation and serves to regulate various immune responses in vivo. Specifically, IL-12 can differentiate T helper 1 (Th 1) cells and natural killer cell (NK) cells, regulate various cytokine production, elevate immune response by Th 1 cells, CD 8+ T cells Of differentiation, and hematopoietic cell proliferation (Hsieh, CS, et al., Science, 260: 547-549, 1993), especially CTL cells (cytotoxic T lymphocytes) ) And the hydrolytic capacity of NK cells (Robertson, MJ, and J. Ritz., Oncologist, 1: 88-97, 1999; Trinchieri, G., Annu. Rev. Immunol., 13: 251-276, 1995) By playing an important role in regulating the immune response. To date, reports of acquired immunodeficiency syndrome (AIDS) have reduced the synthesis of biologically active IL-12 by about five-fold (Chehimi, J. et al., J. Exp. Med., 179: 1361-). 1366, 1994), as well as a significant decrease in immunity to mycobacteria in people with an IL-12 receptor gene deletion (de Jong, R. et al., Science, 280: 1435-1438, 1998). Was observed. Since the action of IL-12 can induce a strong in vivo immune response against viruses, bacteria and various tumors early, the development of various therapeutic agents using the same is actively progressing.

Another reason why IL-12 may be used as an effective vaccine or therapeutic agent for a disease that requires the various cellular immune responses presented above is that IL-12 is a memory Th1 cell and a memory in vivo. It is based on the hypothesis that it is involved in the proliferation of CTL cells (memory CTL) (Stobie, L. et al., Proc. Natl. Acad. Sci. USA , 97: 8427-8432, 2000; Mortarini, R. et al. , C ancer Res. , 60: 3559-3568, 2000; Mbawuike, IN et al., J. Infect.Dis . , 180: 1477-1486, 1999). In particular, induction of the memory immune response is indispensable if we focus on the problem of pre- and relapse, which is the most problematic problem in the treatment of various tumors. However, the exact mechanism by which IL-12 exerts these effects is not known. However, some recent reports have shown that IL-12 is a CD4 + T cell, because increased IFN-γ may have an antiproliferative effect during the differentiation of T helper 1 by IL-12. Mechanisms have been suggested that memory immune responses can be induced by inhibiting apoptosis (Fuss, IJ et al., Gastroenteroloogy , 117: 1078-1088, 1999; Marth, T. et al., J.) . Immunol. 162: 7233-7240, 1999). In addition, IFN- [gamma] increased by IL-12 can stimulate the expression of IL-15 involved in potent and selective stimulation of memory CD8 + T cells (Zhang, X. et al., Immunity 8: 591-). 599, 1998) It is hypothesized that memory immune responses may be induced. Based on these reports, IL-12 may be involved not only in the initial immune response but also in the memory immune response, suggesting the possibility of being particularly useful for vaccine immunization.

The biologically active form of IL-12 is a heteropolymer, IL-12p70, 70 kDa, consisting of two covalently linked subunits, p35 and p40 subunits. The p40 subunit shares an amino acid sequence similar to the IL-6 receptor and thus belongs to the superfamily of cytokine receptors. The p35 subunit, on the other hand, is far from the cytokine receptor superfamily, but contains a cytokine family (Gearing, DP, and Cosman, D) containing IL-6 / granulocyte colony stimulating factor (GM-CSF). , Cell, 66: 9-10, 1991).

Upon expression of IL-12p70 in vitro and in vivo, the p40 subunit IL-12p40 is secreted together in large quantities in the form of a monomer and a homopolymer (Mattner, F). et al., Eur. J. Immunol., 23: 2202-2208, 1993; Heinzel, FP et al., Infect. Immun ., 62: 4244-4249, 1994), generally IL-12p40 is IL-12p70. It has been reported to secrete more than 5 to 90 times more and to inhibit IL-12p70 activity by competitively binding IL-12p70 to the receptor of IL-12 (Gillessen, S. et al., Eur. J. Immunol ., 25 : 200-206, 1995; Ling, P. et al., J. Immunol ., 154: 116-127, 1995). This fact has been shown to reduce the Th 1 response in mice transfected with IL-12p40 (Yoshimoto, T. et al., J. Immunol ., 160: 588-594, 1998) and to produce IL-12p40. Allogeneic rejection does not occur when transplanting myoblasts (Kato, K. et al., Proc. Natl. Acad. Sci , USA, 93: 9085-9089, 1996), expressing IL-12p40 Results of various in vivo experiments, including the inhibition of tumor reduction by IL-12p70 by adenovirus (Chen L. et al., J. Immunol., 159: 351-359, 1997) Backed by Recently, however, there have been reports of positive effects of IL-12p40 on the activity of IL-12p70, which induces the development of Th 1 that specifically responds to allogeneic antigens (Piccotti, JR et al., J. Immunol ., 157: 1951-1957, 1996), a novel function of IL-12p40 that acts as a chemotactic material of macrophages has been demonstrated by the inventors (Ha, SJ et al., J. Immunol ., 163: 2902-2908 , 1999).

Currently, various in vivo systems have been established to demonstrate the anticancer effects of IL-12p70. However, direct administration of human recombinant IL-12p70 protein to cancer patients presents severe toxicity that can lead to death (Robertson, MJ and Ritz. J., Oncologist, 1: 88-97, 1996; Cohen, J , Science, 270: 908, 1995). As a result of the prevention of these side effects and the search for an economically efficient method, many studies have been conducted on gene therapy using IL-12 genes. (Rakhmilevich, AL et al., Proc. Natl. Acad. Sci . USA, 93: 6291-6296, 1996; Tahara, H. et al., J. Immunol ., 154: 6466-6474, 1995; Lotze , MT et al., Ann. NY Acad. Sci., 795: 440-454, 1996).

For gene therapy with IL-12, production of IL-12p70, a biologically active form of IL-12 (Gubler, U. et al., Proc . Natl. Acad. Sci . USA, 88: 4143-4147, 1991) It is essential and therefore required to simultaneously express the cDNAs of p35 and p40 subunits in a cell. In order to simultaneously express two p35 and p40 subunits in one cell, they are arranged in succession in the same plasmid and using an expression cassette to express p35 and p40 genes respectively (Rakhmilevich, AL et al., Proc. Natl.Acad . Sci. USA, 93: 6291-6296, 1996) or methods of simultaneously expressing two genes using the internal ribosomal entry site (IRES) of encephalomyocarditis virus (EMVC) have been used. Although IL-12p70 can be successfully expressed in the cell, the continuous expression of an excessive amount of IL-12p40 as described above does not eliminate the possibility of interfering with the biological action of IL-12p70.

In order to overcome this, a method of linking p35 and p40 subunits by using a DNA sequence encoding a protein linker commonly used in antibody production has been proposed (Lieschke, GJ et al., Nat . Biotechnol , 15: 35-40, 1997; Lode, HN et al., Proc. Natl. Acad. Sci. USA, 95: 2475-2480, 1998; Lee, YL et al., Human Gene Ther ., 9: 457 -46534, 1998). Although this method prevented the inhibition of the biological activity of IL-12p70 by excess IL-12p40, the activity of IL-12p70 produced by the structural change caused by linking two subunits of p35 and p40 was Due to the problem of more than 5-100 times reduction, the effect was not sufficient. Therefore, in order to efficiently treat cancer or other diseases using the IL-12 gene, a method of inducing the production of IL-12p70 that maintains activity without secreting IL-12p40 is recognized as an essential task.

Glycosylation has been known to play an important role in the folding, secretion, composition, stability and biological role of proteins. The p35 and p40 subunits of human IL-12 express 219 and 328 amino acids, including amino acids with 56 and 22 lipophilic signal sequences, respectively, with three and four N-sugar chains present in the p35 and p40 subunits. A flammable moiety was identified through human IL-12 amino acid sequence analysis (Podlasky, FJ et al., Arch. Biochem. Biophys ., 294: 230-237, 1992). Stern et al. Reported that after treating human IL-12 with trifluoromethane-sulfonic acid or glycosidase F, the molecular weights of IL-12p35 and IL-12p40 showed reduced molecular weight, respectively. (Stern, AS et al., Arch. Biochem. Biophys ., 294: 230-237, 1992). This indicates that the p35 and p40 subunits of human IL-12 have carbohydrates as components. In the same report, the molecular weight of IL-12p35 was reduced when neuraminidase and internal α-N-acetyl-galactosaminidase were sequentially treated with IL-12p35. While reduced, it has been demonstrated that IL-12p40 is not affected by the same treatment, indicating that IL-12p35 has an O-linked sugar chain while IL-12p40 does not have an O-linked sugar chain. . In addition, N-glycosylation of the Asn-135 and Asn-222 amino acids of the four N-glycosylable moieties of the human IL-12p40 subunit was analyzed, revealing that N-glycosylation is actually occurring in the Asn-222 moiety. lost. However, N-glycosylation of the remainder of the human IL-12p40 subunit, human IL-12p35 subunit, and mouse IL-12 is not yet known, and glycosylation and glycosylation are also possible. The effect of moieties on the synthesis, secretion, biological activity, etc. of IL-12 has not been elucidated.

On the other hand, research on the use of the IL-12 gene is actively conducted because prevention and treatment of many viral or bacterial diseases and inhibition of cancer generation require an increase in cellular immune responses rather than humoral immune responses. It is becoming. Hepatitis C is a virus-borne disease, and more than 50% of HCV infections lead to chronic disease, which is the leading cause of diseases such as cirrhosis and liver cancer (Alter HJ et al., N. Engl). J. Med., 321: 1494-1500, 1989). Currently, alpha interferon is commonly used as the only treatment for them, but the therapeutic effect is only 10-30% (Weiland E. et al. J. Virol., 66: 3677-3682, 1992). There is an urgent need to develop an effective vaccine or treatment for the disease. Clinical trial reports in chimpanzees and humans have shown that HCV can produce both specific humoral and cellular immune responses (Prince AM et al., J. Infect. Dis., 165: 438-443, 1992). Structural proteins E1 and E2 of HCV have been identified as the most important antigens for inducing protective immunity (Choo QL et al., Proc. Natl. Acad. Sci. USA, 91: 1294-1298, 1994) . In addition, it has recently been reported that cell mediated immune responses including CTLs are essential for the removal of the virus (Cooper S. et al., Immunity, 10: 439-449, 1999; Rinaldo C.). et al., J. Virol., 69: 5838-5842, 1995).

Recently, a method of inducing such a cellular immune response has emerged as a DNA immune method. DNA immunization methods are distinguished from conventional immunization methods using some components of the pathogen or pathogen that have been attenuated or killed in that immunization is achieved by direct injection into the human body of DNA encoding a specific component of the pathogen, and the actual Since the production takes place in the host where the DNA is injected, there is an advantage that can eliminate the risk of infection that can occur when using live or dead bacteria. It has been reported that this DNA immunization method can induce strong immunity against various infectious viruses such as influenza virus, hepatitis B virus, human immunodeficiency virus (Ulmer JB et al., Science, 259). (1745-1749, 1993; Michel ML et al., Proc. Natl. Acad. Sci. USA, 92: 5307-5311, 1995; Irwin MJ et al., J. Virol., 68: 5306-5044, 1994) . In addition, it has been reported that the DNA immunization method was applied to HCV capsid protein (core) and E2 protein and thus induced specific immunity against them (Major ME et al., J. Virol., 69: 5798). -5805, 1995; Tedeschi V. et al., Hepatology, 25: 459-462, 1997).

However, this DNA immunization method has a limitation in that the immunized protein is not expressed in vivo and thus does not generate a strong immune response against a low immunity antigen. To enhance the effectiveness of DNA immunization methods, it may be necessary to use adjuvants such as several cytokine genes or costimulatory molecule genes required for immune cell activation (Geissler M. et al., J. Immunol). , 159: 5107-5113, 1997; Iwasaki A. et al., J. Immunol., 158: 4591-4601, 1997; Lee SW et al., J. Virol ., 72: 8430-8436, 1997) HCV Although there are examples of using IL-12 to efficiently induce an immune response against (Lasartte JJ et al., J. Immunol., 162: 270-5277, 1999), IL-12 can be used to target primates and humans. No positive results have been reported for DNA immunization (Boyer J. et al., Keystone Symposium on DNA vaccines April, 12-17, 1998).

With this in mind, the present inventors studied to manufacture genes capable of minimizing the secretion of IL-12p40, which expresses the active form of IL-12p70 and reduces immune activity by IL-12 through the regulation of glycosylation. As a result, the mutant genes obtained through the mutation of Asn-222 amino acid, the glycosylated portion of the human IL-12p40 subunit, and Asn-220 amino acid, the glycosylated portion of the mouse IL-12p40 subunit, were identified. We found that the secretion of IL-12p40 was increased while increasing expression, and that the mutant gene was immunized with the HCV E2 gene in a small animal model with DNA for optimal cell-mediated immune response, especially after a long period of time. Continuously induced facts confirm that the mutant gene of the present invention can be usefully used as an immunopotentiator in DNA vaccines. As the present invention has been completed.

It is an object of the present invention to reduce the secretion of IL-12p40, which inhibits the activity of IL-12p70 through competitive binding to the receptor of IL-12p70, to provide a glycosylation moiety essential for the secretion of IL-12p40 in humans or mice. Mutation provides an IL-12p40 mutant gene that activates IL-12 specific roles such as an increase in immune response through Th1 cells or activation of CTL cells.

It is still another object of the present invention to provide a use of the expression vector including the IL-12p40 mutant gene as an immunostimulator for enhancing and maintaining an antigen-specific cellular immune response by DNA immunization with a DNA vaccine.

1 compares the homology of the amino acid sequences of IL-12p40 in humans and mice,

Figure 2 shows the protein composition of the p40 and p35 subunits of the wild type human IL-12 and derivatives mutated N-glycosylated moieties of the two subunits in the present invention,

FIG. 3A shows the results of immunoblotting performed on cell lysate for wild-type human IL-12p35 and glycosylated partial mutations in the present invention, FIG .

3bIs wild type in the present invention Person Results of immunoblotting performed on cell lysates for IL-12p40 and glycosylated partial mutations,

Figure 3c is the result of immunoblotting performed in the cell culture supernatant for wild-type human IL-12p40 and glycosylated partial mutations in the present invention,

4 shows the expression vector of the present invention in which the envelope glycoprotein 2 (E2) gene of Hepatitis C Virus (HCV) and the normal or mutant gene of mouse IL-12 are inserted.

Figure 5a is a result of measuring the total IgG antigen titer against HCV E2 in the serum of mice immunized with the DNA vector of the present invention,

GI; Mice immunized with 200 μg mock plasmid pTV2,

GII; Mice immunized with 100 μg pTV2-HCV-gDsE2t and 100 μg pTV2,

GIII; Mice immunized with 100 μg pTV2-HCV-gDsE2t and 100 μg pTV2-mIL-12wt,

GIV; Mice immunized with 100 μg of pTV2-HCV-gDsE2t and pTV2-mIL-12mut

Figure 5b is the result of measuring the IgG1 antigen titer against HCV E2 in the serum of mice immunized with the DNA vector of the present invention,

GI; Mice immunized with 200 μg mock plasmid pTV2,

GII; Mice immunized with 100 μg pTV2-HCV-gDsE2t and 100 μg pTV2,

GIII; Mice immunized with 100 μg pTV2-HCV-gDsE2t and 100 μg pTV2-mIL-12wt,

GIV; Mice immunized with 100 μg of pTV2-HCV-gDsE2t and pTV2-mIL-12mut

Figure 5c is a result of measuring the IgG2a antigen titer against HCV E2 in the serum of mice immunized with the DNA vector of the present invention,

GI; Mice immunized with 200 μg mock plasmid pTV2,

GII; Mice immunized with 100 μg pTV2-HCV-gDsE2t and 100 μg pTV2,

GIII; Mice immunized with 100 μg pTV2-HCV-gDsE2t and 100 μg pTV2-mIL-12wt,

GIV; Mice immunized with 100 μg of pTV2-HCV-gDsE2t and pTV2-mIL-12mut

5D is a result of measuring the ratio (IgG2a / IgG1) of IgG2a antigen titer and IgG1 antigen titer to HCV E2 over time in the serum of mice immunized with the DNA vector of the present invention,

GI; Mice immunized with 200 μg mock plasmid pTV2,

GII; Mice immunized with 100 μg pTV2-HCV-gDsE2t and 100 μg pTV2,

GIII; Mice immunized with 100 μg pTV2-HCV-gDsE2t and 100 μg pTV2-mIL-12wt,

GIV; Mice immunized with 100 μg of pTV2-HCV-gDsE2t and pTV2-mIL-12mut

Figure 6a is a result of measuring the production level of IFN-γ produced from immune cells of the spleen stimulated by HCV E2 protein in the spleen of the mouse three weeks after immunization with the DNA vector of the present invention,

GI; Mice immunized with 200 μg mock plasmid pTV2,

GII; Mice immunized with 100 μg pTV2-HCV-gDsE2t and 100 μg pTV2,

GIII; Mice immunized with 100 μg pTV2-HCV-gDsE2t and 100 μg pTV2-mIL-12wt,

GIV; Mice immunized with 100 μg of pTV2-HCV-gDsE2t and pTV2-mIL-12mut

Figure 6b is a result of measuring the production level of IFN-γ produced from immune cells of the spleen stimulated by HCV E2 protein in the spleen of the mouse 6 weeks after immunization with the DNA vector of the present invention,

GI; Mice immunized with 200 μg mock plasmid pTV2,

GII; Mice immunized with 100 μg pTV2-HCV-gDsE2t and 100 μg pTV2,

GIII; Mice immunized with 100 μg pTV2-HCV-gDsE2t and 100 μg pTV2-mIL-12wt,

GIV; Mice immunized with 100 μg of pTV2-HCV-gDsE2t and pTV2-mIL-12mut

Figure 6c is a result of measuring the production level of IFN-γ produced from immune cells of the spleen stimulated by HCV E2 protein in the spleen of the mouse 10 weeks after immunization with the DNA vector of the present invention,

GI; Mice immunized with 200 μg mock plasmid pTV2,

GII; Mice immunized with 100 μg pTV2-HCV-gDsE2t and 100 μg pTV2,

GIII; Mice immunized with 100 μg pTV2-HCV-gDsE2t and 100 μg pTV2-mIL-12wt,

GIV; Mice immunized with 100 μg of pTV2-HCV-gDsE2t and pTV2-mIL-12mut

7 is a result of measuring the specific CTL response to HCV E2 using modified CT 26 tumor cells expressing hghE2t at various times after immunization in the spleen of mice immunized with the DNA vector of the present invention,

GI; Mice immunized with 200 μg mock plasmid pTV2,

GII; Mice immunized with 100 μg pTV2-HCV-gDsE2t and 100 μg pTV2,

GIII; Mice immunized with 100 μg pTV2-HCV-gDsE2t and 100 μg pTV2-mIL-12wt,

GIV; Mice immunized with 100 μg of pTV2-HCV-gDsE2t and pTV2-mIL-12mut,

a; Immediately after immunization (week 0), b; 3 weeks after immunization,

c; 6 weeks after immunization, d; 10 weeks after immunization,

e; 14 weeks after immunization, f; 14 weeks after immunization, control (CT26-neo cells)

FIG. 8A shows the levels of total IgG antigen against HCV E2 and its subsequences IgG1 and IgG2a in the serum of mice 2 weeks after injection of modified CT 26 tumor cells expressing hghE2t into mice immunized with the DNA vector of the present invention. Is the result of measuring

GI; Mice immunized with 200 μg mock plasmid pTV2,

GII; Mice immunized with 100 μg pTV2-HCV-gDsE2t and 100 μg pTV2,

GIII; Mice immunized with 100 μg pTV2-HCV-gDsE2t and 100 μg pTV2-mIL-12wt,

GIV; Mice immunized with 100 μg of pTV2-HCV-gDsE2t and pTV2-mIL-12mut

8B shows the ratio of IgG2a antigen titer and IgG1 antigen titer to HCV E2 in the serum of mice 2 weeks after injection of modified CT 26 tumor cells expressing hghE2t into mice immunized with the DNA vector of the present invention (IgG2a / IgG1 ) Is the result of measuring

GI; Mice immunized with 200 μg mock plasmid pTV2,

GII; Mice immunized with 100 μg pTV2-HCV-gDsE2t and 100 μg pTV2,

GIII; Mice immunized with 100 μg pTV2-HCV-gDsE2t and 100 μg pTV2-mIL-12wt,

GIV; Mice immunized with 100 μg of pTV2-HCV-gDsE2t and pTV2-mIL-12mut

Figure 8c is a result of measuring the volume change of the tumor after injection of modified CT 26 tumor cells expressing hghE2t in mice immunized with the DNA vector of the present invention,

GI; Mice immunized with 200 μg mock plasmid pTV2,

GII; Mice immunized with 100 μg pTV2-HCV-gDsE2t and 100 μg pTV2,

GIII; Mice immunized with 100 μg pTV2-HCV-gDsE2t and 100 μg pTV2-mIL-12wt,

GIV; Mice immunized with 100 μg of pTV2-HCV-gDsE2t and pTV2-mIL-12mut

Figure 8d is a result of measuring the survival rate of the mouse after injection of modified CT 26 tumor cells expressing hghE2t in the mice immunized with the DNA vector of the present invention,

GI; Mice immunized with 200 μg mock plasmid pTV2,

GII; Mice immunized with 100 μg pTV2-HCV-gDsE2t and 100 μg pTV2,

GIII; Mice immunized with 100 μg pTV2-HCV-gDsE2t and 100 μg pTV2-mIL-12wt,

GIV; Mice immunized with 100 μg of pTV2-HCV-gDsE2t and pTV2-mIL-12mut

9 is a schematic diagram showing the biological function of IL-12 in vivo.

▲; p35, ○; p40,

ㆀ; (p40) ₂ , iii; IL-12 (p70)

In order to achieve the above object, the present invention replaces the Asn-222 (human) or Asn-220 (mouse) moiety that plays an essential role in the secretion of genes encoding human or mouse IL-12p40 with other amino acids. Mutant genes of human or mouse IL-12p40 subunits are provided.

The present invention also provides a gene construct comprising an IRES sequence for the simultaneous expression of mutant genes and subunits of the human or mouse IL-12p40 subunit and an expression vector comprising the same.

In addition, the present invention provides a DNA vaccine vector containing the envelope glycoprotein 2 (E2) gene of HCV and a p40 subunit gene in which Asn-222 (human) and Asn-220 (mouse) portions are mutated. It provides a gene construct comprising and an expression vector comprising the same.

Finally, the present invention provides the use of the gene construct as an immunopotentiator for DNA vaccine immunization or gene therapy.

Hereinafter, the present invention will be described in detail.

The present invention relates to Asn-222 (human), which plays an essential role in the secretion of genes encoding human IL-12p40 having the nucleotide sequence of SEQ ID NO: 1 or mouse IL-12p40 having the nucleotide sequence of SEQ ID NO: 2 , or Mutant genes of human or mouse IL-12p40 subunits are substituted for the Asn-220 (mouse) portion with another amino acid.

Comparing the homology of the amino acid sequence to the mouse and human IL-12p40, the Asn of mouse IL-12p40 corresponding to Asn-222 of human IL-12p40 is located at amino acid sequence 220 and its surrounding amino acid sequences are very similar ( See FIG. 1 ).

Specifically, the codon AAC designating the 222nd Asn (Asn-222) of the human IL-12p40 subunit may be mutated to CUC, CAG, AUA, etc., wherein the corresponding amino acids are Leu, Gln, and Ile. In particular, in a preferred embodiment of the present invention, by changing AAC to CTC or CAG on the cDNA, AAC codons are replaced with CUC or CAG codons, resulting in the Asn-222 amino acid being substituted with Leu-222 or Gln-222 amino acids. (Hereinafter abbreviated as 'hp40-N222L' and 'hp40-N222Q').

In addition, even in the mouse IL-12p40 subunit homologous to the Asn-222 region of the human IL-12p40 subunit, the codon AAC designating the 220th Asn (Asn-220) may be mutated to CUC, CAG, AUA, or the like. In this case, the corresponding amino acids are Leu, Gln, and Ile. In particular, in a preferred embodiment of the present invention, by converting AAC to CTC on the cDNA, AAC codons are replaced with CUC codons, resulting in the Asn-220 amino acid being substituted with Leu-220 amino acid (hereinafter referred to as 'mp40-N220L'). Abbreviated).

The mutant genes hp40-N222L, hp40-N222Q and mp40-N220L of the present invention encode the amino acid sequences set forth in SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5 .

The present invention also provides a gene construct comprising an IRES sequence for the simultaneous expression of mutant genes and subunits of the human or mouse IL-12p40 subunit and an expression vector comprising the same.

First, the present invention provides hp40-N222L / IRES / hp35, hp40-hp35 / IRES / hp40-N222L and mp35 / IRES / mp40-N220L genes containing the genes. The hp40-N222L / IRES / hp35 gene and the hp35 / IRES / hp40-N222L gene include genes encoding the p35 subunit together with the p40 subunit gene of humans in which Asn-222 has been mutated to Leu-222, mp35 / The IRES / mp40-N220L gene contains a gene encoding the p35 subunit along with the p40 subunit gene in mice in which Asn-220 has been mutated to Leu-220, each of which is intended for simultaneous expression of subunits. It includes the IRES sequence. In the hp40-N222L / IRES / hp35, hp35 / IRES / hp40-N222L and mp35 / IRES / mp40-N220L genes, it is preferable to use IRES derived from encephalomyocarditis (EMV), but not limited thereto. IRES sequences are preferred for the simultaneous expression of genes encoding p40 subunits and p35 subunits upon expression of the two genes.

The present invention also provides pGX0-hp40-N222L / IRES / hp35, pGX0-, which are expression vectors comprising the hp40-N222L / IRES / hp35, hp35 / IRES / hp40-N222L and mp35 / IRES / mp40-N220L genes, respectively provided above. Available in hp35 / IRES / hp40-N222L and pTV2-mp35 / IRES / mp40-N220L.

Kanamycin resistant gene instead of the ampicillin resistant gene of the pTV2 DNA vaccine vector (Song, MK et al., J. Virol. , 74: 2920-2925, 2000) as one preferred embodiment in the present invention The genes hp40-N222L / IRES / hp35 and hp35 / IRES / hp40-N222L were inserted into the foreign gene insertion sites of the pGX0 plasmid, which is a DNA vaccine vector designed for clinical trials. Also, the mp35 / IRES / mp40-N220 gene was inserted into the foreign gene insertion site of the pTV2 DNA vaccine vector. However, since a variety of prokaryotic or eukaryotic cells vectors are known in addition to the plasmid used in the preparation of the expression vector, it will be apparent to those skilled in the art that other expression vectors other than the plasmid may be readily used depending on the purpose of expression. In addition, the size, nucleotide sequence, etc. of the gene inserted into the foreign gene insertion site of the expression vector can also be variously changed by known techniques.

The expression vectors pGX0-hp35 / IRES / hp40-N222L and pGX0-hp40-N222L / IRES / hp35 are dated February 26, 2001, and pTV2-mp35 / IRES / mp40-N220L are dated February 29, 2000. It was deposited with the Korea Biotechnology Research Institute Gene Bank (Accession No .: KCTC 0969BP, KCTC 0970BP and KCTC 0745BP).

Since co-expression of p35 and p40 subunits is essential for obtaining IL-12p70 which is biologically active, in the present invention In addition to pCIN-hp35 and pCIN-hp40 to express the hp35 and hp40 subunits, pGX0-hp40 / IRES / hp35 and pGX0-hp35 / IRES / hp40, which are bibictronic expression vectors using IRES from EMCV, were prepared. . To understand the effect of N-glycosylation on the synthesis, secretion, and specific activity of human IL-12, we first site-directed mutagenesis of the Asn residues of potential N-glycosylation moieties present in the p35 and p40 subunits. -directed mutagenesis) to substitute for other amino acid residues (2), Immunoblot of wild-type protein against mutant protein (3A, 3BAndFig 3c4) Asn-127, -141 of the hp35 subunit and Asn-222, -303 of the hp40 subunit are used as N-glycosylation moieties.

In addition, the inventors of the present invention, to investigate the N-glycosylation effect of hp35 or hp40 on the synthesis, heterodimerization and secretion of hIL-12p70, including each wild-type gene or its N-glycosylated mutant genes Cells were transformed with hIL-12 expression vectors and ELISA assays were performed using the culture supernatants and cell debris obtained therefrom.

As a result, it can be seen that Asn-127 of hp35 not only affects intracellular heterodimerization of IL-12p70 but also decreases its secretion. However, hp35 Asn-141 did not appear to have a significant effect on the heterodimerization and secretion of IL-12p40 and IL-12p70 compared to wild-type hp35 (see Table 1 ).

Mutations of Asn-135 and Asn-222 of hp40 did not affect the secretion of IL-12p70 but significantly reduced the secretion of IL-12p40. In particular, Asn-222 secretion of hp40 was reduced by about 12% compared to wild-type hp40. Although Asn-135 of hp40 was a part of N-glycosylation which resulted from immunoblotting, the effect was observed in the amino acid of Asn-135, which is asparagine (Asn) of Asn-135 itself to the secretion of hp40 It can be an important part. The amino acid of Asn-222 shows the same secretion pattern when it is replaced with leucine (Leu) in addition to glutamine (Gln), which is thought to be due to the loss of N-glycosylation of Asn-222 ( Table 1 Reference). Therefore, it can be seen that N-glycosylation in Asn-222 is required to secrete hIL-12p40 alone, not heterodimers hIL-12 and hIL-12p70.

Based on these results, we found that while N-glycosylation in Asn-222 is not required for heterodimerization and secretion of hIL-12p70, but plays an essential role in the secretion of hIL-12p40, hp35 N-sugar chain in Asn-127 It was confirmed that the painter plays an important role in heterodimerization and secretion of hIL-12p70.

In order to investigate the effect of glycosylation of hIL-12 on biological activity, the present inventors conducted the ELISA for the IFN-γ induction ability of wild type hIL-12 and its derivatives including mutations of potential N-glycosylation sites. Analyzed.

As a result, the culture supernatant obtained by simultaneously transforming the gene of the Asn-135 and / or Asn-222 mutated hp40 derivatives and the wild type hp35 gene between the wild type and all derivatives thereof in terms of IFN-γ inducing ability was wild type or It showed increased IFN- [gamma] induction compared to other hp40 mutants (see Table 1 ). In addition, when the same experiment was performed by supplementing the two supernatants with insufficient amount of hp40 compared to wild type hp40, it was confirmed that the increased IFN-γ inducing ability was reduced to a level similar to that of wild type (see Table 1 ). This result is because the level of hIL-12p40, known as antagonists of hIL-12p70, is relatively low compared to other mutants in the culture supernatant of mutants comprising hp40-N135Q and / or hp40-N222Q. It is shown that the hp70 produced by the mutation of Asn-135 and / or Asn-222 does not increase the activity by the mutation.

In addition, the present inventors have co-transformed cells with various amounts of wild-type hp35 DNA with expression vectors containing a constant amount of mutant genes to investigate how glycosylation of hp40 in Asn-222 reduces the secretion of hIL-12p40. After infection and culture, the amount of amplified human IFN-γ was measured by ELISA.

Generally, p35 subunits are not secreted alone and are bound to p40 and secreted in the form of IL-12p70, whereas p40 subunits are secreted in the form of monomers or homodimers, which are not p35 subunits. This suggests that the p40 subunit acts as a major factor in the secretion of IL-12p70. Consistent with this fact, the results of the experiment showed that the secretion of hIL-12p70 increased in proportion to the amount of transfected hp35 DNA in both wild type hp40 and hp40-N222Q mutations (see Table 1 ). These results indicate that the hp40 subunit with secretion defect is secreted in the form of IL-12p70 when combined with the hp35 subunit, suggesting that the hp35 subunit has another effect on the secretion of hIL-12p70. . Recently, studies indicating that conformational changes in hp40 are due to binding to hp35 have been reported, suggesting the potential contribution of hp35 subunits to IL-12p70 secretion (Yoon, C et al., EMBO J). , 19: 3530-3534, 2000). Taken together, the above study and the results of the present invention show that hp40, including glycosylation to Asn-222, is defective in itself but secreted in the form of hp70 due to morphological changes in the hp40 site through binding to hp35. It can be seen that.

In addition, the present inventors have found that the use of IRES rather than the cytomegalovirus promoter (CMV) in the vector used for the purpose of further reducing p40 secretion by inducing co-expression of p35 and p40 subunits in a cell. Due to the low expression level of the gene, placing the p40 gene behind the IRES induces lower p40 production, resulting in the lower hp40 / IRES / hp35 and hp35 / IRES / hp40 constructs. It was. In addition, hp40-N222L / IRES / hp35 and hp35 / IRES / hp40-N222L constructs were prepared in which each of the plasmids replaced the hp40-N222L gene instead of the hp40 gene (see Table 1 ). Comparing the N222L / IRES / hp35 constructs, the latter shows that the hp40 secretion is reduced by about 7%, which means that the hp40 secretion is 12 when the hp35 construct and the hp40-N222L construct are electroporated, respectively. Considering the percentages, the secretion is reduced. In other words, when electroporation of the hp35 construct and the hp40-N222L construct, both plasmids may not be transformed into a single cell. Therefore, if the hp40-N222L gene is expressed in a cell with the hp40-N222L gene alone, the hp40 is not secreted. Since a small amount of secretion, it can be assumed that the hp40 secretion is somewhat higher than the hp40-N222L / IRES / hp35 gene, which is the expression of both hp35 and hp40-N222L gene. In addition, when comparing the hp40 / IRES / hpp35 construct and the hp35 / IRES / hp40 construct, the expression and secretion of hp70 is not significant when the expression is lowered by arranging the hp40 gene at the back of IRES. It can be observed that the expression and secretion of hp40 was greatly reduced. In addition, when the hp35 / IRES / hp40-N222L construct was manufactured by introducing the hp40-N222L gene into the hp35 / IRES / hp40 construct instead of the hp40 gene, the secretion level of hp40 was further reduced to about 0.3%. . In this experiment, we found that the hp35 / IRES / hp40-N222L construct is a construct that minimizes the release of hp40 while maintaining the hp70 secretion level.

In addition, the present invention provides a gene vaccine comprising a DNA vaccine vector containing the E2 gene of HCV-1b and a p40 subunit gene in which Asn-222 (human) and Asn-220 (mouse) portions are mutated. Provide trucks.

First, we investigated the possibility of using the hIL-12 mutant gene of the present invention in gene theraphy, such as DNA vaccines, in a mouse IL-12p40 (mp40) similar to the Asn-222 of the hp40 gene. The base sequence of the gene was examined. As a result, the inventors confirmed that the Asn-220 amino acid of mouse p40 exists at a site very similar to Asn-222 of human p40 and its amino acid sequence is not known to be N-glycosylated until now (see FIG. 1 ). . Therefore, the present inventors have identified a pCIN containing a mouse IL-12p40 mutant gene which can be expressed in animal cells by mutating the mp40 gene, that is, the 220 th Asn of the amino acid sequence by Leu codon by site-directed mutagenesis. The mp40-N220L vector was completed.

In addition, the present inventors have expressed pTV2 vector (Lee et al.), Which is an eukaryotic expression vector that has already been used as a DNA vaccine vector in small animals to produce a vector that can be used for DNA immunization while simultaneously expressing p40 and p35 subunits. , J. Virol., 72: 8430-8436, 1998; Cho et al., Vaccine, 17: 1136-1144, 1999) to complete the pTV2-mp35 / IRES / mp40 vector by inserting the mp35 / IRES / mp40 fragment. It was. In addition, the present inventors observed that the hp35 / IRES / hp40-N222L gene can minimize the secretion of hp40 while maintaining the secretion of hp70, and based on this, Asn- of mouse IL-12p40 of the present invention The pTV2-mp35 / IRES / mp40-N220L vector was completed as a vector that simultaneously expresses p35 while including a 220 mutant gene.

The pTV2-mp35 / IRES / mp40-N220L vector was deposited with the Korea Biotechnology Research Institute Gene Bank on February 29, 2000 (Accession Number: KCTC 0745BP).

Finally, the inventors of the present invention describe simian virus 40 replication origin (SV40 ori), cytomegalovirus promoter (CMV), adenovirus to express HCV-E2 protein in eukaryotic cells. tripartite leader sequence (adenovirus), multiple cloning sequence (MCS), SV40 polyadenylation sequence (polyA) and ampicilin resistance gene (AmpR) A pTV2-HCV-E2 DNA vaccine vector was constructed, wherein the HCV-E2 gene was cloned into multiple cloning sequences (Song MK, et al., J. Virol., 74: 2920-2925, 2000) ( FIG. 4 ). The E2 gene used above facilitates the secretion of proteins by removing the carboxyl-terminal (C-terminal) region containing a hydrophobic amino acid residue, and the aminocyl-terminal. : The signal sequence (s) of the herpesvirus (HSV) envelope glycoprotein D (gD) was linked to the N-terminal to improve protein expression and cell secretion.

In order to confirm the secretion of IL-12p40 and IL-12p70 according to Asn-220 mutation of mouse IL-12p40 in the expression vector prepared as described above, cells were transformed into cells and cell lysates and cell culture medium were obtained. The amount of mouse IL-12 was measured by ELISA (Pharmigen). As a result, mp40-N220L mutants of the present invention showed almost similar characteristics to Asn-222 mutants of hp40 in terms of secretion of IL-12p40 and IL-12p70 and their biological activity (see Table 1 ). .

Finally, the present invention A gene construct comprising a p40 subunit gene in which Asn-222 (human) and Asn-220 (mouse) portions are mutated is provided for use as an immunopotentiator for DNA vaccine immunization or gene therapy.

The method can be usefully used for the prevention and treatment of diseases that can be cured by increased immunity in the human body, such as AIDS, hepatitis C and B, cancer, flu, tuberculosis, malaria.

According to previous reports, DNA immunization of plasmids encoding HCV-E2 antigen (pTV2-gDsE2t) resulted in antigen-specific humoral and cell-mediated immune responses (cell) three weeks after inoculation. Enough to induce a mediated immune response. The present inventors have therefore determined whether the mutant mIL-12 genes of the present invention can effectively affect antigen-specific immune responses in vivo compared to wild-type mIL-12 genes and how long such effects can be sustained. In order to investigate, after further inoculating the DNA vaccine vector of the present invention into mice, whether HCV-E2 specific antibody was produced or not was examined by ELISA using purified hGH-E2 protein.

As a result, the HCV E2 DNA vaccine induced systemic HCV E2-specific total IgG, IgG1 and IgG2a levels significantly higher than the negative control levels, and co-injection with the mutant mIL-12 gene or wild type mIL-12 gene It showed somewhat higher levels of total IgG levels compared to when the HCV E2 DNA vaccine was administered alone. The level of IgG1 was also somewhat higher compared to the group administered with HCV E2 DNA. In contrast, the level of IgG2a for HCV E2 was slightly increased in the wild type mIL-12 group and significantly increased in the mutant mIL-12 group compared to the HCV E2 DNA alone group. In addition, the ratio of IgG2a / IgG1 generally accepted as an indirect indicator of the Th1 immune response was highest in the mutant mIL-12 administration group with similar levels of IgG2 levels (see FIGS. 5A, 5B, 5C and 5D ). ). These results indicate that the mutant mIL-12 gene has a significant effect on the transfer of IgG subgroups from IgG1 to IgG2a in humoral immune responses compared to wild type mIL-12 or HCV E2 alone. Suggests an immune response. In addition, the effect was maintained even at 0, 3, 6, 10 weeks after DNA boosting.

In addition, the present inventors have carried out IFN-γ expression from spleen cells to investigate the effect of the mutant mIL-12 gene of the present invention on the Th1 immune response, one of the parameters that have been used to assess the titer of cell-mediated immune responses. Measured.

As a result, the group administered only HCV E2 DNA without the cytokine gene increased the level of IFN-γ in proportion to the hghE2t protein concentration, while the mock plasmid inoculation group was not increased. As expected, IFN-γ-induced levels in wild-type mIL-12-treated group were more enhanced than HCV E2 alone group, and mutant mIL-12-treated group showed 2-3 times higher IFN-γ production level than wild-type mIL-12-treated group. (See Figures 6A, 6B and 6C ). These results suggest that mIL-12p70 plays a role in increasing antigen-specific Th1 immune response and mIL-12p40 plays a role in inhibiting the induction of Th1 immune response by IL-12p70 in vivo. This effect begins at three weeks of boosting DNA and shows a similar pattern at 10 weeks, suggesting that mIL-12p70 not only induces an early Th1 immune response but also is involved in the continuation of this immune response.

As described above, the inventors of the present invention confirmed that the mutant mIL-12 gene of the present invention contributes to a long-term Th1 immune response in HCV E2 DNA immunization. Using spleen cells obtained from DNA immunized mice at various ages after booster vaccination to investigate whether the mutant mIL-12 gene is involved in maintenance of CTL activity in the DNA immunization model, linked to the immune response and major cell-mediated immune responses. CTL analysis was performed.

As a result, all the groups except the plasmid administration group showed very strong antigen-specific CTL activity after 2 weeks of inoculation, but showed little difference between the HCV E2 alone group, wild type mIL-12 group and mutant mIL-12 group. . The CTL response in the wild type mIL-12 group was higher than in the HCV E2 alone group throughout the experimental period, indicating that the mIL-12 gene plays an important role in increased CTL formation. Interestingly, the difference in CTL activity between the mutant mIL-12 group and the other two groups (HCVE2 group and wild type mIL-12 group) became more pronounced with time as the duration of the booster period increased. In particular, the CTL response after 10 weeks was very low in HCV E2 and wild-type mIL-12 groups because the frequency of antigen-specific CTLs was significantly reduced after a long period of time. On the other hand, the mutant mIL-12 group showed 5-10 times higher CTL activity than the other two groups while maintaining antigen-specific CTL response (see FIG. 7 ). As a control, no cell lysis was observed in all groups when CT26-neo cells were used as target cells, suggesting that the CTL activity observed above is HCV E2-specific.

In addition, the present inventors attempted to determine the effect of proliferation and maintenance of antigen-specific CD8 + T cells by mIL-12 administration by measuring the in vivo frequency of CD8 + T cells specific for HCV E2. To this end, two immunological assays were used. One method was to measure the number of cells that secrete IFN-γ in response to antigen-specific responses in CD8 + T cells. R-phycoerythrin (PE) conjugated anti-mouse IFN-γ antibody or control group after isolation of CD + 8 T cells to determine whether enhancement of CTL activity is manifested by the secretion of CD8 + T cells to a specific antigen. PE-conjugated isotype-matched antibody was added to double-stain the cells and stained cells were analyzed by flow cytometry (FACSCalibur flow cytometry) to observe the increase in IFN-γ.

As a result, the mice co-immunized with the mutant mIL-12 gene gradually secrete CD8 + IFN-γ-producing cells after 0, 3, 6, 10, 14 weeks, compared with HCV E2 alone and wild type mIL-12. It can be seen that the increase is similar to the result of CTL reaction. In the CTL response, however, CD8 + T cells were more frequent in wild-type mIL-12 group than HCV E2 alone group, and antigen-specific CD8 + T cells in mutant mIL-12 group were highest. This difference in frequency was markedly different at 14 weeks post booster, with almost no antigen-specific CD8 + T cells observed in HCV E2 alone and wild-type mIL-12 treated groups, whereas the frequency of these cells in mutant mIL-12 treated groups Was slightly reduced but retained (see Table 2 ). These results demonstrate that expression of the mutant mIL-12 gene maintains secretion of CD8 + IFN-γ producing T cells after DNA immunization for a long time.

The frequency of HCV E2-specific CD8 + T cells was measured by limiting dilution assay (LDA), another assay for determining antigen-specific CD8 + T cell frequency. In other words, the limiting dilution method is to limit the dilution of spleen cells of mice further inoculated with DNA to various concentrations, and then incubate with the splenocytes diluted as described above using CT26 cells expressing E2 as in CTL immune response observation. This method is for measuring CTL activity of specifically stimulated CD8 + T cells. As a result, the results similar to those obtained by intracellular IFN-γ staining were obtained in the limiting dilution method. In other words, the highest antigen-specific CD8 + T cell frequency was observed in the mutant mIL-12 group at the early immunization period, and the frequency was very low in the HCV E2 alone and wild type mIL-12 group even after 14 weeks of boosting, compared with the mutant mIL-12 group. It can be seen that the frequency is maintained continuously (see Table 2 ).

In addition, the present inventors also observed the frequency change of HCV E2 specific CD8 + T cells at each time after inoculation without additional inoculation of DNA. As a result, the frequency of antigen-specific CD8 + T cells was slightly reduced overall at the time of boosting, but even at one time, the highest frequency of CD8 + T cells was observed in the mutant mIL-12 administration group (see Table 2 ).

From this we have found that the role of IL-12p70 itself in the cell-mediated immune response is not only to induce the activity of Th1 and CTL from the beginning, but also to maintain it for a long time, whereas the role of IL-12p40 is in vivo As an antagonist, it was confirmed that it inhibits IL-12p70.

In order to confirm the Th1 and CTL immune responses induced in vivo by the mutant IL-12 gene of the present invention and to investigate the correlation between the Th1 and CTL immune responses and the protective immune response, the expression of hghE2t at 12 weeks of DNA boosting Tumor cells CT26-hghE2t were administered to mice. And two weeks after tumor injection, the levels of HCV E2 specific IgG and its subsidiary IgG1, IgG2a antigen and IgG2a / IgG1 ratio were examined from the serum of mice (see FIGS. 8A and 8B ). As a result, the highest IgG2a / IgG1 ratio was observed in the mutant mIL-12 administration group similarly before the tumor injection, indicating that the Th1 immune response was generated in the mutant mIL-12 administration group even during tumor injection.

In addition, as a result of measuring the tumor size for about 30 days after tumor injection, the group of mice immunized with wild type mIL-12 showed delayed tumor growth compared to the group immunized with pTV2-gDsE2t alone (see FIG. 8C ). In addition, when comparing the survival rate of the mice injected with the tumor between groups, it was observed that 90% of the mice survived for about 70 days in the group immunized with the mutant mIL-12. In comparison, the survival rate was only 20-30% in the group injected with E2 alone or the group injected with wild-type mIL-12 (see FIG. 8D ). Thus, these results suggest that the HCV E2-specific Th1 and CTL responses induced by the mutant mIL-12 genes of the present invention confer in vivo defense against challenge of antigen specific to the expression of modified tumor cells. .

Through the above experiments, the present inventors have found that the p40 subunit gene of IL-12 in human or mouse mutated codons encoding Asn-222 (human) or Asn-220 (mouse), which is essential for secretion of IL-12p40 of the present invention. Has been found to be useful for DNA vaccine immunization or gene therapy as an adjuvant. Accordingly, the present invention is an immunopotentiator for DNA immunization and gene therapy for preventing and treating diseases by enhancing antigen-specific cellular immune responses using the p40 subunit mutant gene of IL-12 of the human or mouse as an active ingredient. To provide.

The adjuvant enhances the hydrolytic capacity of cytotoxic T lymphocytes (CTL) cells when the p40 subunit gene of IL-12 is combined with a DNA vaccine, or interferon gamma (IFN-γ) of T helper cells. ) And enhance the immune response by increasing the secretion of interferon gamma (IFN-γ) of CD8 + cells.

EMBODIMENT OF THE INVENTION Hereinafter, this invention is demonstrated in detail based on an Example.

However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.

Example 1 Construction of Human IL-12 Expression Vector

<1-1> Construction of human IL-12 expression vector

1050 bp human p35 subunit and 1050 using reverse transcriptase-polymerase chain reaction (RT-PCR) (PCR System 2400, Perkin Elmer) method using complementary primers from NC37 cells, human B cells activated by PMA The cDNA of p40 subunit, bp, was cloned and amplified by PCR. The amplified cDNA was subcloned into the starting vector pSK plasmid (Stratagene). PSK-hp35 and pSK-hp40 were prepared by inserting the p35 and p40 subunit genes into the SmaI portion of pSK, respectively.

In order to construct a vector (bicistronic vector) that expresses genes encoding p40 and p35 subunits, the IRES gene of EMCV is first obtained by RT-PCR, and then inserted into the SmaI and PstI restriction enzyme sites of the pSK plasmid. -IRES vector was prepared. The vector was digested with EcoRV and pSK-hp40 / IRES was prepared by filling the ends of p40 DNA fragments obtained by treating pSK-hp40 with XbaI and BamHI with T4 DNA polymerase. Finally, p35 gene DNA fragments generated by treatment of restriction enzymes NcoI and SacI on pSK-hp35 were inserted into pSK-hp40 / IRES treated with NcoI and SacI, resulting in pSK-hp40 in which p40, IRES and p35 genes were arranged in sequence. / IRES / hp35 plasmid was prepared.

The pSK-IRES vector was digested with EcoRV, and the pSK-hp40 / IRES / hp35 plasmid was digested with NcoI and NotI, and the resulting hp35 DNA fragment was filled with T4 DNA polymerase and inserted into EcoSK. -hp35 / IRES plasmid was prepared. The pSK-hp35 / IRES / hp40 plasmid was completed by inserting an hp40 fragment obtained by cleaving pSK-hp40 with NcoI and BamHI to a site cut with BamHI and NcoI. By inserting the hp40 / IRES / hp35 gene and the hp35 / IRES / hp40 gene prepared by the above method into the restriction enzymes SpeI / NotI and XhoI / NotI portions of the pGX0 vector, respectively, the active type IL-12p70 can be expressed in mammalian cells. PGX0-hp40 / IRES / hp35 and pGX0-hp35 / IRES / hp40 expression vectors were obtained. The pGX0 vector cleaves the XmnI site in the empicillin resistance gene of the pTV2 vector (Song, MK et al., J. Virol. , 74: 2920-2925, 2000), a conventional DNA vaccine vector, to create both blunt ends. This was accomplished by inserting the kanamycin resistance gene in the pZErO-2 vector (Invitrogen) by PCR.

<1-2> Construction of p40 Subunit Expression Vector

To prepare the plasmid expressing the p40 subunit, the pCIN-hp40 / IRES / hp35 vector was digested with restriction enzymes SacII and NotI to remove the gene encoding the p35 subunit, followed by filling the ends with T4 DNA polymerase. It was self-ligation, which was named pCIN-hp40.

<1-3> Construction of p35 subunit expression vector

pC DNA fragments were obtained by digesting pCIN-hp40 / IRES / hp35 vector with NcoI, filling with T4 DNA polymerase, and then restriction enzyme NotI for the preparation of plasmid expressing p35 subunit, which was obtained by XhoI of pCI-neo vector. And NotI restriction enzymes were inserted, which was named pCIN-hp35.

<Example 2> Construction of the Mouse IL-12 Expression Vector

<2-1> Construction of Mouse IL-12 Expression Vector

To construct a vector that expresses the genes encoding the mouse p40 and p35 subunits together, first, the pSK-IRES vector containing the IRES of EMCV was digested with restriction enzymes NcoI and BamHI, and the mouse IL-12p40 PCR product ( PSK-IRES / mp40 was prepared by inserting a p40 DNA fragment resulting from cleavage of Schoenhaut DS et al., J. Immunol., 148: 3433-3440, 1999) with the same restriction enzyme. Subsequently, the mouse IL-12p35 product (Schoenhaut DS et al., J. Immunol., 148: 3433-3440, 1992) was treated with BamHI and terminated fragments using T4 DNA polymerase were subjected to ClaI and T4 DNA polymerase. By inserting into the pSK-IRES / mp40 treated with, as a result, a pSK-mp35 / IRES / mp40 plasmid with p35, IRES, p40 gene sequence was prepared. By inserting the mp35 / IRES / mp40 gene prepared by the above method into the restriction enzymes XhoI and NotI portions of the pCI-neo vector (Promega), pCIN-mp35 capable of expressing the active IL-12p70 in mammalian cells / IRES / mp40 expression vector was obtained.

<2-2> Construction of p40 Subunit Expression Vector

To prepare a plasmid expressing the wild-type mouse p40 subunit, pG fragments obtained by cleaving the pSK-mp35 / IRES / mp40 vector with restriction enzymes NcoI and SacI were treated with the same restriction enzyme pGEX-KG vector (Clontech, USA). ) Is inserted. The pGEX-KG-mp40 thus obtained was treated with EcoRI and NotI and inserted into the EcoRI and NotI portions of the pCI-neo vector to obtain a pCIN-mp40 expression vector.

<2-3> Construction of p35 subunit expression vector

To construct a plasmid expressing the wild type p35 subunit, the p35 fragment generated by cleaving the pSK-mp35 / IRES / mp40 vector with restriction enzymes XhoI and EcoRI was digested with XhoI and EcoRI restriction enzymes of the pCI-neo vector treated with the same restriction enzyme. The pCIN-mp35 expression vector was obtained by inserting in the part.

Example 3 Preparation of IL-12p40 and IL-12p70 with Partially Mutated Glycosylation

Seven Asn codons, which are expected to be used as N-glycosylation moieties of human p35 and p40 subunits, were replaced by unrelated codons by site-directed mutagenesis. To prepare a construct of the glutamine mutant gene for the site expected to be the N-glycosylation portion of the hp35 and hp40 subunits, Haraguchi et al. (Haraguchi, et al., J. Immunol ., 163: 2092-2098, Amino acid substitutions were performed using PCR according to the method described by 1999). At this time, the primers for mutagenesis T7 described in SEQ ID NO: 6 , T3 described in SEQ ID NO: 7 , hp40-N125Q (S) described in SEQ ID NO: 8 , hp40-N125Q (AS) described in SEQ ID NO: 9 , Hp40-N135Q (S) described in SEQ ID NO: 10 , hp40-N135Q (AS) described in SEQ ID NO: 11 , hp40-N222Q (S) described in SEQ ID NO: 12 , hp40-N222Q described in SEQ ID NO: 13 AS), hp40-N303Q (S) described in SEQ ID NO: 14 , hp40-N303Q (AS) described in SEQ ID NO: 15 , hp40-N127Q (S) described in SEQ ID NO: 16 , hp40- described in SEQ ID NO: 17 N127Q (AS), hp40-N141Q (S) set forth in SEQ ID NO: 18 , hp40-N141Q (AS) described in SEQ ID NO: 19 , hp40-N251Q (S) described in SEQ ID NO: 20 , set forth in SEQ ID NO: 21 Nucleotides of hp40-N251Q (AS) were synthesized and used (where S and AS refer to sense and antisense primers).

In order to prepare single glutamine mutant gene constructs of hp40 and hp35, pCIN-mp40 or pCIN-mp35 prepared in Examples <2-2> to <2-3> were used as the template, and the T7 primer and the respective sense PCR was performed using the primers. Similarly, PCR was performed using the T3 primer and each antisense primer. This resulted in the formation of two PCR fragments that shared a common site containing a mutational point. 2nd PCR was performed using the mixture of the fragments as a template and using flanking primers, as a result of which fusion products thereof were formed and the fragments were inserted into the pCI-neo vector. Similarly, double and triple mutant genes were constructed using single or double mutant genes as PCR templates. Mutant genes were identified through DNA sequencing.

To prepare a mouse IL-12p70 gene construct containing an N-glycosylation defect in Asn-220 of mp40, the pCIN-mp35 / IRES / prepared in Example 2-1 above was prepared. The mp40 gene region in mp40 was replaced with mp40-N222L. Mutation of mp40 to prepare pCIN-mp40-N222L, the nucleotides of mp40-N220L (S) as depicted in SEQ ID NO: 22 comprising SacI restriction enzyme sites and mp40-N220L (AS) as depicted in SEQ ID NO: 23 PCR was performed as described above. Mutant genes amplified therefrom were identified by restriction enzyme treatment and DNA sequencing of specific restriction enzyme recognition sites generated after mutation.

Figure 2 schematically shows the amino acid composition of the normal and mutant genes of the human IL-12p40 and IL-12p35 subunits used in the present invention. N-glycosylated moieties are indicated in Y form with the asparagine (Asn) amino acid number at that position, and the amino acids substituted in each variant gene are indicated in a rectangle.

Example 4 Effect of N-glycosylation of Asn-222 on Human IL-12p40 on Secretion of IL-12p40 Alone

Transformation into COS-7 cells (ATTC) incubated in DMEM medium (Dulbecco's modified Eagle's medium, GIBCO-BRL) containing 10% of heat-inactivated FBS (fetal bovine serum, GIBCO-BRL) was performed by electroporation. (electroporation, Biorad). 20 μg of a plasmid comprising 5 × 10 ⁶ COS-7 cells and pCIN-hp40 / IRES / hp35 of Example <1-1> or the mutant IL-12 gene of Example 3 in about 300 μl of the culture medium, In addition, 2 μg of pNEB-SEAP (pNEB-Secreted Alkaline phosphatase, New England Biolabs), which can act as a control by coding for alkaline phosphatase, was placed in a cuvette and electroporated at 250 V and 960 ㎌. Performed the law.

24 hours after the transformation by electroporation, the medium was changed to 1.5 ml of CHO-SFMII medium without GI serum (GIBCO-BRL), and tunicamycin (tunicamycin: Sigma) 1 Μg / ml was added. After 24 hours of medium exchange, cells were harvested, centrifuged, and the supernatants were used for measuring SEAP activity. The pellets were suspended in 200 μl of cell lysis solution (Promega). The levels of IL-12p70 and IL-12p40 in supernatants and pellets were measured by ELISA (R & D system). The culture supernatant and cell debris were electrophoresed with 10% and 12% sodium dodecyl-sulfate-polyacryl amide gel electrophoresis (SDS-PAGE) for immunoblotting, and the separated proteins were electrophoresed to nylon membrane (Amersham). Electrotransfer. Proteins adsorbed on the membranes include human IL-12 antibody (R & D system) with biotin, streptavidin (Pharmingen) with HRP (Horseradish peroxidase), and ECL kit (Amersham). The results were obtained and shown in Table 1 below. Table 1 shows the relative amounts of expression when the expression level of IL-12p70 and IL-12p40 in the wild type measured by ELISA as 100%.

a; A total of 20 µg of DNA constructs were used for electroporation transformation with COS-7 cells, and 10 µg of DNA each was used for simultaneous transformation of two DNA constructs.

b; The expression of IL-12p70 or p40 was measured by ELISA and expressed in relative proportion to the expression and secretion levels of p40 and p70 at 100% in the construct containing wild type IL-12.

c; The same amount of p70 present in each mutant supernatant was used for IFN-γ inducibility analysis. The level of induced IFN- [gamma] was measured by ELISA, and the level of IFN- [gamma] induced in experiments using the construct containing wild type IL-12 was expressed as 100% and expressed as a relative ratio.

d; IL-12p40 represents a monomeric or dimeric p40 form of IL-12p40 that is not the p40 portion of IL-12p70.

e; IL-12p70 represents a heterodimer consisting of IL-12p35 and IL-12p40.

f; PCI-neo was used as the plasmid prototype for the simultaneous transformation of two DNA constructs.

g; <1 means that a lower amount of protein is detected than the level of detection of ELISA.

h; When wild-type hp40 or hp40-N135Q and hp40-N222Q DNA were simultaneously transformed with hp35 DNA, and the amount of p70 detected in the supernatant was the same, transfection of hp40-N135Q and hp40-N222Q DNA was greater than that of wild-type DNA. The amount of p40 detected in the test supernatant is relatively low, which means that IFN-γ induction assay was performed by adding a shortage of hp40 (hp40 present in the supernatant obtained by transforming the hp40 construct) to the supernatant. .

i; Numbers in parentheses indicate the amount of plasmid hp40-N222Q and hp35 DNA transformed at the same time (μg). A total of 20 μg of DNA was used for transformation, the deficiency was supplemented with pCI-neo DNA.

j; The prototype of the plasmid used for the simultaneous expression of hIL-12p40 and its mutants and hIL-12p35 is the pGX0 vector.

k; The prototype of the plasmid used for the co-expression of mIL-12p40 and its mutants and mIL-12p35 is the pTV2 vector.

As shown in Table 1 , mutations in the Asn-141 and p40 Asn-303 glycosylable moieties of p35 had no effect on the secretion of IL-12p70 or IL-12p40, while mutations of p40 Asn-135 and p40 Mutations in the Asn-222 glycosylation moiety reduced only the secretion of IL-12p40. In particular, the amino acid of Asn-222 appears to exhibit the same secretion pattern when it is replaced by glutamine (Gln) in addition to leucine (Leu), which is thought to be due to the loss of N-glycosylation of Asn-222.

Figure 3a shows the results of immunoblotting performed on cell lysates after transformation in COS7 cells of normal human IL-12p35 gene and glycosylated partial mutant genes. Columns 1 and 2 are the results for cell lysates transformed with pCI-neo and pCIN-hp35, respectively, and columns 3, 4, 5 and 6 contain the mutant genes for the N-glycosylated portion of hp35. These results are for cell lysates transformed with the expression vectors pCIN-hp35-N127Q, pCIN-hp35-N141Q, pCIN-hp35-N251Q, and pCIN-hp35-N127,251Q. Bands 2 and 5 showed molecular weights on the order of 33.2 kDa, indicating a protein for the N-glycosylated p35 subunit, which was inhibited by tunicamycin to inhibit N-glycosylation of the p35 subunit. The resulting molecular weight 28 kDa band (column 7) appears in columns 3 and 4, indicating that the Asn-127 and Asn-141 amino acids are N-glycosylated.

Figure 3b shows the results of immunoblotting performed on cell lysates after COS7 intracellular transformation of normal human IL-12p40 gene and glycosylated partial mutant genes. Columns 1 and 2 are the results for cell lysates transformed with pCI-neo, pCIN-hp40, respectively, and columns 3, 4, 5 and 6 contain the mutant genes for the N-glycosylated portion of hp40. Results are for cell lysates transformed with the expression vectors pCIN-hp40-N125Q, pCIN-hp40-N135Q, pCIN-hp40-N222Q, pCIN-hp40-N303Q, and columns 7, 7, 8, 9 and 10 are each N- Results are for cell lysates transformed using double to triple mutations for glycosylable moieties following use. Column 11 is the result for cell lysates treated with tunicamycin, a reagent that inhibits N-glycosylation in cells transformed with pCIN-hp40.

Based on several reports, immunoblotting of IL-12p40 is known to result in 3-4 bands at molecular weights of 36-45 kDa. In cell lysates that express 12p35, bands are also found at molecular weights of about 36, 37.5, 40, and 43.1 kDa. In the cell lysate treated with the tunicamycin in column 11, the above three bands disappeared, leaving only a band of 36 kDa, indicating the p40 subunit without N-glycosylation. The Asn-125 and Asn-135 mutations in rows 3 and 4 did not differ significantly from the wild-type, and the band patterns were changing in the remaining Asn-222 and Asn-303 mutations in columns 5 and 6. This means that Asn amino acids at positions 135, 222, and 303 are used as N-glycosylation moieties.

Figure 3c shows the results of immunoblotting performed in cell culture supernatants after COS7 cell transformation of wild type human IL-12p40 gene and glycosylated partial mutant genes. Description of each column is the same as that of FIG. 3B . Similar to the results of cell lysates, 3-4 bands are shown at molecular weights 36-45 kDa and the Asn-125 and Asn-135 mutations in columns 3 and 4 show that the band pattern is not significantly different from the wild type, while the remaining 5 and Asn-222 and Asn-303 mutations in column 6 show a shift in band behavior. In particular, it can be seen that Asn-135 and Asn-222 amino acid mutations are rarely secreted into the cell culture unlike cell lysates, which is consistent with the result of quantification by ELISA.

The expression vectors pGX0-hp35 / IRES / hp40-N222L and pGX0-hp40, wherein the expression vector including the gene mutated to Leu-222, the glycosylated portion of the IL-12p40 gene, among the expression vectors including the mutant genes described above. -N222L / IRES / hp35 was deposited on February 29, 2001 with the Korea Biotechnology Research Institute Gene Bank (Accession No .: KCTC 0969BP and KCTC 0970BP).

<4-1> N-glycosylation effect of hp35 on synthesis, heterodimerization and secretion of hIL-12p70

In order to investigate the N-glycosylation effect of hp35 on the synthesis, heterodimerization and secretion of hIL-12p70, the inventors hIL comprising a wild type hp35 gene or its N-glycosylated mutant genes as described above. Cells were transformed with −12 expression vectors and then ELISA assays were performed using the culture supernatants and cell debris obtained therefrom.

As a result, removal of possible N-glycosylated residues of hp35 except Asn-127 had no effect on the synthesis, heterodimerization and secretion of hIL-12p70, as shown in Table 1 above. However, glycosylation of Asn-127 significantly reduced the heterodimerization and secretion of hIL-12p70, indicating that the expression levels of hp35-N127Q and hp35-N127,141Q, which showed normal transfection efficiency, differed on Western blots. This is because the expression level of the sieves is similar. Therefore, these results indicate that N-glycosylation of Asn-127 of hp35 rather than N-glycosylation of Asn-141 plays a very important role in heterodimerization and secretion of hIL-12p70.

<4-2> N-glycosylation effect of hp40 on synthesis, heterodimerization and secretion of hIL-12p70

The present inventors also determined the effect of N-glycosylation on the synthesis, heterodimerization and secretion of hIL-12p70, in the same manner as in Example <3-1>, in the same manner as in the wild type hp35 gene or its N-glycosylation mutation. The levels of hIL-12p70 and hIL-12p40 in culture supernatants and cell debris of cells transformed with hIL-12 expression vectors containing genes were analyzed by ELISA.

As a result, as shown in Table 1 , mutations of Asn-135 or Asn-222 had little effect on the secretion of hIL-12p70, which resulted in the extracellular level of hIL-12p70 in the mutants. It's almost like a wild type hp40. More interestingly, the secretion of hIL-12p40 was significantly reduced in Asn-135 and Asn-222 mutants, in particular the Asn-222 mutant showed about 15% reduced secretion levels compared to wild type hp40. These results indicate that N-glycosylation in Asn-222 is required for the secretion of hIL-12p40 alone, rather than the heterodimeric forms hIL-12 and hIL-12p70. In addition, double and triple mutants, including mutants in Asn-135 and / or Asn-222, also showed lower secretion levels than hIL-12p40 but not hIL-12p70.

In contrast, other mutants produced equivalent amounts of hIL-12p40 and hIL-12p70 in cell lysates compared to wild type hp40, with mutants of Asn-125 and Asn-303 expressing hIL-12p40 and hIL-12p70, There was a significant difference in heterodimerization and secretion.

Based on these results, we found that while N-glycosylation in Asn-222 is not required for heterodimerization and secretion of hIL-12p70, but plays an essential role in the secretion of hIL-12p40, hp35 N-sugar chain in Asn-127 It was confirmed that the painter plays an important role in heterodimerization and secretion of hIL-12p70. This is also in line with previous reports that N-glycosylation of hp35 is essential for the secretion of hIL-12p70 (Carra, G. et al., J. Immunol., 164: 4752-4761, 2000).

<4-3> Effect of glycosylation of hIL-12 on biological activity

To investigate the effect of glycosylation of hIL-12 on biological activity, we analyzed the IFN-γ induction ability of wild type hIL-12 and its derivatives including mutations of potential N-glycosylation sites. . To this end, culture supernatants containing the same amount of wild type hIL-12 and its mutants at 100 ng / ml were incubated with human PBLs, and then the level of IFN-γ induced in each culture supernatant was analyzed by ELISA.

As a result, as shown in Table 1 , there was no significant difference between the wild type and all derivatives thereof in terms of IFN-γ inducing ability. However, hp40 derivatives mutated in Asn-135 and / or Asn-222 showed slightly increased IFN-γ induction compared to wild type or other hp40 mutants. These results indicate that the level of hIL-12p40, known as antagonists of hIL-12p70, is relatively low compared to other mutants in the culture supernatant of mutants comprising hp40-N135Q and / or hp40-N222Q. Because.

Example 5 Role of IL-12p35 in the Secretion of IL-12p70 Mutated with Glycosylated Portions

To investigate how glycosylation of hp40 in Asn-222 reduces the secretion of hIL-12p40, we co-transfected cells with a constant amount of hp40-N222Q-expressing plasmid with various amounts of wild type hp35 DNA. .

To this end, human peripheral blood mononuclear cells (PBMCs) were isolated from blood by centrifugation using different concentrations of Ficoll-Hypaque (Sigma) and 10% FBS and penicillin / It was resuspended in RPMI-1640 medium (GIBCO-BRL) including streptomycin (GIBCO-BRL). For measurement of human IFN-γ amplification, 4 × 10 ⁵ human epidermal blood lymphocytes were incubated with culture supernatant containing 100 ng / ml IL-12p70 or mutant protein for 16 hours.

Meanwhile, in order to measure the mouse IFN-γ amplification, the spleen cells of 6 to 8 week old female BALB / c mice were obtained, and then 1 × 10 ⁵ splenocytes were obtained from 100 ng / ml mice for 24 hours. Culture supernatants containing IL-12p70 or mutant derivatives thereof were incubated. The amount of amplified human and mouse IFN-γ was measured using the human and mouse IFN-γELISA kit (R & D systems) and the results are shown in Table 1 . Figure 1 shows the relative value of 100% of the amount of IFN-γ amplification in the wild type measured by ELISA.

In general, p35 subunits are not secreted alone and are bound to p40 and secreted in the form of IL-12p70, whereas p40 subunits are secreted in the form of monomers or homodimers, which are not p35 subunits. This suggests that the p40 subunit acts as a major factor in the secretion of IL-12p70. As shown in Table 1 , the secretion of hIL-12p70 increased in proportion to the amount of hp35 DNA transfected in both wild type hp40 and hp40-N222Q mutations. These results indicate that hp40 subunits with secretion defects are secreted in the form of IL-12p70 in combination with hp35 subunits, suggesting that hp35 subunits also have another effect on the secretion of hIL-12p70. will be. Recently, studies indicating that conformational changes in hp40 subunits are due to binding to hp35 have been reported, suggesting the potential contribution of hp35 subunits to IL-12p70 secretion (Yoon, C et al., EMBO J. , 19: 3530-3534, 2000). Through the above studies and the results of the present invention, the inventors found that hp40, including glycosylation to Asn-222, is defective in its own secretion, but in the form of hp70 due to morphological changes of the hp40 site through binding to hp35. It was confirmed that it can be secreted.

Example 6 Preparation of Mutant Gene Expression Vector and HCV-E2 DNA Vaccine of Mouse IL-12p40 Asn-220 Glycosylation

<6-1> Preparation of pCIN-mp40-N220L

In order to investigate the possibility that the hIL-12 mutant gene of the present invention can be used for gene theraphy such as DNA vaccines, the inventors of the mouse IL-12p40 (mp40) gene similar to Asn-222 of the hp40 gene The sequence was examined. As a result, the present inventors confirmed that Asn-220 amino acid of mouse p40 exists at a site very similar to Asn-222 of human p40, and its amino acid sequence is not known to be N-glycosylated until now. Accordingly, the present inventors prepared mp40-N220L by substituting the Leu codon by mutation of the mp40 gene, that is, the 220 th Asn of the amino acid sequence by site-directed mutagenesis. At this time, the primer for mutagenesis was used to synthesize the nucleotides described in SEQ ID NO: 22 and SEQ ID NO: 23 in p40, the mutation includes a Sac I restriction enzyme recognition site for facilitating the identification of the gene. The mutations thus prepared were verified through restriction enzyme treatment and DNA sequence identification of specific restriction enzyme recognition sites generated after mutation. This completed the pCIN-mp40-N220L vector containing the mouse IL-12p40 mutant gene that can be expressed in animal cells.

<6-2> Preparation of pTV2-mp35 / IRES / mp40-N220L Vector

To produce a vector that can be used for DNA immunization while simultaneously coding and expressing p40 and p35 subunits, the pTV2 vector, a eukaryotic expression vector that has already been used as a DNA vaccine vector in small animals (Lee et al., J. Virol., 72: 8430-8436, 1998; Cho et al., Vaccine, 17: 1136-1144, 1999) are digested with restriction enzymes Asp718 and NotI, where pSK-mp35 / IRES / mp40 is digested with the same enzyme. PTV2-mp35 / IRES / mp40 vector was completed by inserting the resulting mp35 / IRES / mp40 fragment. In addition, for the preparation of a vector that simultaneously expresses p35 while including the Asn-220 mutant gene of mouse IL-12p40, the pSK-mp35 / IRES / mp40 vector is first cleaved with NcoI and NotI, and pCIN-mp40-N220L is identical thereto. The pSK-mp35 / IRES / mp40-N220L vector was produced by inserting the mp40-N220L fragment generated by the enzyme cleavage. The pTV2-mp35 / IRES / mp40 vector was cleaved with EcoRV and NotI to remove the mp40 fragment, and then the p402-mp35 / IRES was inserted by inserting the mp40-N220L fragment obtained by cleaving pSK-mp35 / IRES / mp40-N220L with the same enzyme. / mp40-N220L vector was completed.

The pTV2-mp35 / IRES / mp40-N220L vector was deposited on February 29, 2000 to the Genetic Research Institute of Biotechnology (Accession Number: KCTC 0745BP).

<6-3> Preparation of pTV2-HCV-E2 Vector

We have prepared a pTV2-HCV-E2 DNA vaccine vector capable of expressing HCV-E2 protein in eukaryotic cells (Song M. K., et al.,J. Virol.,74: 2920-2925, 2000).5As you can see The pTV2-HCV-E2 DNA vaccine vector contains the triplet sequence of the simian virus 40 replication origin (SV40 ori), the cytomegalovirus promoter (CMV), and the adenovirus (tripartite). leader sequence (TPL), multiple cloning sequence (MCS), SV40 polyadenylation sequence (polyA), and ampicillin resistance gene (ampicilin resistance gene (AmpR)) and the HCV-E2 gene It is cloned into multiple cloning sequences. E2 gene used in the present invention facilitates the secretion of proteins by removing the carboxyl-terminal (C-terminal) region containing a hydrophobic amino acid residue, aminocyl- The signal sequence (s) of the herpesvirus (HSV) envelope glycoprotein D (gD) was linked to the terminal: N-terminal to streamline protein expression and cell secretion.

<6-4> Identification of Secretion Patterns of IL-12p40 and IL-12p70 Following Mutation of Mouse IL-12p40 Asn-220

Embodiment was carried out for example to through the same method that was used at 4 after transfection vectors shown in Table 1 in COS-7 cells, cell debris and cell culture the obtained mouse IL-12 ELISA (Pharmingen Co.), ELISA in Table 1 The expression level of IL-12p70 and 40 in the wild type measured through the concentration is represented. A value of <1 in pCI-neo means a range that is not detected by ELISA.

As shown in Table 1 , the mp40-N220L mutants showed almost similar characteristics as the Asn-222 mutant of hp40 in terms of secretion of IL-12p40 and IL-12p70 and its biological activity.

In addition, the inventors of the present invention, in order to compare whether the mp40-N220L mutant gene can be applied as a DNA vaccine or the activity of the mp40-N220L mutant gene and wild-type mp40 gene in the induction of Th1 and CTL immune response, Mutant genes mp35 / IRES / mp40 (mIL-12wt) or mp35 / IRES / mp40-N220L (mIL-12mut) were inserted into the pTV2 DNA vaccine vector to observe their in vitro properties. As a result, as shown in Table 1 , the mIL-12mut gene mp40-N220L mutant in the pTV2 vector is almost similar to the Asn-222 mutant of hp40 in terms of secretion of IL-12p40 and IL-12p70 and its biological activity. Characteristics.

Example 7 Investigation of Effect of Mutant IL-12 on Antigen-Specific Humoral Immune Response

According to previous reports, DNA immunization of plasmids encoding HCV-E2 antigen (pTV2-gDsE2t) resulted in antigen-specific humoral and cell-mediated immune responses (cell) three weeks after inoculation. Enough to induce a mediated immune response. In order to investigate whether the mutant mIL-12 gene of the present invention can effectively affect the antigen-specific immune response in vivo compared to the wild type mIL-12 gene, the present inventors have performed pTV2-gDsE2t plasmid after initial immunization of mice. And further inoculated after 4 weeks with pTV2-mIL-12mut or pTV2-mIL-12wt.

Specifically, pTV2-gDsE2t DNA having pTV2, mutant mIL-12 or wild type mIL-12, in order to investigate the immune response enhanced by the DNA vaccine by the expression vector containing the mutant gene of the present invention prepared in Example 6 200 μg was dissolved in 100 μl phosphate-buffered saline solution and injected into the anterior tibialis muscles of female BALB / c mice about 6-8 weeks old. Four weeks after the first DNA injection, the same amount of DNA was further inoculated in the same manner. Approximately 0, 3, 6, and 10 weeks after the two additional DNA inoculations, blood was drawn from blood vessels near the eyeballs of the mice to separate serum, and ELISA was performed using purified hGH-E2 protein to generate HCV-E2-specific antibodies. Investigation was conducted. As the E2 protein used, hgh-E2 protein expressed from CHO cell line was used. 100 ng of hgh-E2 protein was coated in a 96-well plate, and serum obtained from the immunized mice was appropriately diluted to react with the protein. Thereafter, anti-mouse IgG antibody conjugated with horseradishperoxidase (HRP; Chemicon) conjugated to the antigen-specific IgG was reacted with colorant reaction by adding ABTS (Sigma) and 450 with an ELISA measuring instrument (BioTek). Analysis at nm. In addition, ELISA was performed using anti-mouse IgG antibodies that specifically bind to IgG1 and IgG2a instead of anti-mouse IgG antibodies in order to observe the relative ratios of IgG1 and IgG2a to the total IgG antibodies formed against E2 protein.

As a result, HCV E2 DNA vaccines induced systemic HCV E2-specific total IgG, IgG1 and IgG2a levels significantly higher than negative control levels, as shown in FIGS . 5A, 5B, 5C and 5D , and mutant mIL-12 Co-injection with the gene or wild type mIL-12 gene showed high levels of total IgG levels compared to when the HCV E2 DNA vaccine was administered alone. The level of IgG1 was somewhat higher compared to the group administered with HCV E2 DNA. In contrast, the level of IgG2a for HCV E2 was slightly increased in the wild type mIL-12 group and significantly increased in the mutant mIL-12 group compared to the HCV E2 DNA alone group. In addition, the ratio of IgG2a / IgG1 generally accepted as an indirect indicator of the Th1 immune response was highest in the mutant mIL-12 group with similar levels of IgG2 levels. These results indicate that the mutant mIL-12 gene is significantly involved in the transfer of IgG subtypes from IgG1 to IgG2a in the humoral immune response compared to wild type mIL-12 or HCV E2 alone. Suggests an immune response.

Example 8 Investigation of Cellular Immune Responses from Immunized Mice

Induction of Antigen-specific Th1 Immune Responses by Mutant IL-12

In order to investigate the effect of the mutant mIL-12 gene of the present invention on the Th1 immune response, one of the parameters that have been used to assess the titer of cell-mediated immune responses, we measured IFN-γ expression from spleen cells. It was. To this end, splenocytes (1 × 10 ⁵ ) obtained from the spleens of mice about 8 weeks after two DNA injections were added to U-bottomed 96-well plates. Thereafter, hgh-E2t protein purified from 1 to 5 μg of CHO cells was added to each well and the cells were activated by incubating for 3 days at 37 ° C., CO ₂ incubator. After obtaining the cell culture from it was used to measure the amount of IFN-γ present in the supernatant by ELISA. IFN-γ induced after activation with a specific antigen is produced from antigen-specific CD4 + T cells and can be used as an indicator of Th1 immune response.

As shown in FIG . 6 , the HCV E2 DNA-only group without the cytokine gene increased IFN-γ in proportion to the hghE2t protein concentration, whereas the mock plasmid inoculation group was not increased. As expected, IFN-γ-induced levels in wild-type mIL-12-treated group were more enhanced than HCV E2 alone group, and mutant mIL-12-treated group showed 2-3 times higher IFN-γ production level than wild-type mIL-12-treated group. . These results suggest that mIL-12p70 plays a role in increasing antigen-specific Th1 immune response and mIL-12p40 plays a role in inhibiting the induction of Th1 immune response by IL-12p70 in vivo. IFN-γ-induced levels between inoculated groups showed a difference from 3 weeks after booster dose and a similar difference after 10 weeks, suggesting that mIL-12p70 is involved in the induction and maintenance of Th1 immune response.

<8-2> Long-term Enhancing Effect of Antigen-specific CD8 + T Cell Function by Mutant IL-12

As described above, the inventors of the present invention confirmed that the mutant mIL-12 gene of the present invention contributes to a long-term Th1 immune response in HCV E2 DNA immunization. In order to investigate whether the mutant mIL-12 gene affects the maintenance of CTL activity in the DNA immunization model in association with the immune response and the major cell-mediated immune response, splenocytes obtained from DNA immunized mice at various weeks after boosting CTL analysis was performed.

To this end, mitomycin was expressed in CT26-hghE2t cells (Song MK et al., J. Virol., 74: 2920-2925, 2000), a HCV-E2 expressing cell line previously produced and reported by the present inventors, including Song. Cell division was inhibited through C (mitomycin C, 500 μg / ml) treatment and activated for about 5 days in virtro with splenocytes obtained from the spleen of mice about two weeks after two DNA injections. . Cytotoxicity of effector cells obtained therefrom was performed on different target cells such as CT26-hghE2t or CT26-neo labeled with ⁵¹ Cr. A variety of effector cells were coated in U-bottom well plates in triplicates at the desired E: T ratio. ⁵¹ Cr labeled target cells (5 × 10 ³ ) were added to each well of the well plate and incubated at 37 ° C. for 6 hours. From this, the supernatant was recovered and used, and the CTL immune response was investigated by measuring the release of ⁵¹ Cr obtained after the reaction with a γ-counter (γ-counter, Wallac, Turku, Finland). The percentage of specific lysis was calculated by Equation 1 below. At this time, minimal lysis was obtained by culturing the target cells only with the culture medium, and maximun lysis was obtained by exposing the target cells to 1% Nonidet-P40.

Specific hemolysis ratio = (experimental group hemolysis-minimum hemolysis) * 100 / (maximum hemolysis-minimum hemolysis)

As a result, as shown in FIG . 7 , very strong antigen-specific CTL activity was observed in all groups except the plasmid administration group after 2 weeks of inoculation, but the HCV E2 administration group, the wild type mIL-12 administration group, and the mutant mIL-12 administration group were observed. There was little difference between them. The CTL response in the wild type mIL-12 group was higher than the HCV E2 alone group throughout the experimental period, indicating that the mIL-12 gene plays an important role in the increased CTL formation. Interestingly, the difference in CTL activity between the mutant mIL-12 group and the other two groups (HCV E2 group and wild type mIL-12 group) became more pronounced with time as the duration of the booster period increased. In particular, the CTL response after 10 weeks was very low in HCV E2 and wild-type mIL-12 groups because the frequency of antigen-specific CTLs was significantly reduced after a long period of time. On the other hand, the mutant mIL-12 group showed 5-10 times higher CTL activity than the other two groups while maintaining the antigen-specific CTL response. As a control, no cell lysis was observed in all groups when CT26-neo cells were used as target cells, suggesting that the CTL activity observed above is HCV E2-specific.

<8-3> Flow Cytometry for Antigen-Specific IFN-γ Production in CD8 + Cells of Immunized Mice

Splenocytes obtained from the spleen of mice about two weeks after two DNA injections to determine if long-term enhancement of CTL activity is manifested by the secretion of CD8 + T cells to specific antigens and the frequency of CD8 + T cells in vivo And CT26-hghE2t cells treated with mitomycin C (500 µg / ml) were incubated for 40 hours. CD8 + T cells were isolated by magnetic cell sorting (MACS) using magnets, and then cultured with CT26-hghE2t cells (1 × 10 ⁶ ) and 10 U / ml recombinant IL-12. Addition was incubated for 40 hours. At this time, 4 μl GolgiStop (Pharmingen) was treated to inhibit the secretion of cytokines generated from CD8 + T cells, and the cells were further incubated at 37 ° C. for 8 hours.

To directly separate CD8 + T cells, the activated spleen cells were incubated with anti-CD8 microbeads (microbeads, Miltenyi Biotec, Inc.) and then passed through a column of the miniMACS system (microbeads, Miltenyi Biotec, Inc.). Remaining CD8 + T cells were isolated. To prevent nonspecific staining, cells were precultured with Fc Block ^™ (PharMingen) and stained with fluorescein isothiocyanate (FITC) conjugated anti CD8 antibody. After incubation, cells were first fixed with Cytofix / Cytoperm ^™ (PharMingen) and permeabilized, followed by PE (R-phycoerythrin) conjugated anti-mouse IFN-γ antibody or control PE-conjugated isotype-matched Antibodies were added to bistain the cells. Stained cells were analyzed using a flow cytometer (FACSCalibur flow cytomwtry, Becton Dickinson, Inc.) using CellQuest software, and the increase of IFN-γ was observed.

Frequency kinetics of HCV-E2-specific precursor CD + 8 T cells Inoculation frequency ^a Weeks Since Last Inoculation Distribution of HCV-E2 Specific Precursor CD + 8 T Cells Intracellular IFN-γ Staining Assay (%) Limited dilution method (No.) Group ^b Ⅰ Group II Group III Group IV Group II Group III Group IV 2 0 0.08 0.35 0.42 0.58 38.1 39.0 47.1 3 0.05 0.26 0.44 0.72 39.0 48.8 66.7 6 0.06 0.29 0.39 0.73 38.8 46.8 64.9 10 0.07 0.21 0.20 0.68 18.2 19.8 64.3 14 0.07 0.09 0.09 0.57 9.6 8.8 45.7 One 4 0.06 0.33 0.42 0.55 33.2 41.0 48.7 8 0.08 0.26 0.27 0.51 9.7 9.9 50.8 13 0.06 0.11 0.08 0.43 6.9 8.2 44.8

a; Female BALB / c mice 6 to 8 weeks of age were inoculated once or twice with various plasmids of the invention at 4 week intervals.

b; The expression of each group is as follows: group I; pTV2, group II; pTV2-HCV-E2t + pTV2, group III; pTV2-HCV-E2t + pTV2-mIL-12wt, group IV; pTV2-HCV-E2t + pTV2-mIL-12mut

c; Live CD8 + T cells were selected by gating using the FSC and CD8 plots, and live CD8 + T cells were selected by gating again using the CD8 and IFN-γ plots and then IFN-γ among the live CD8 + T cells in this plot. The percentage of CD8 + T cells producing was calculated in percent. This experiment was performed using 2-3 mice per group.

d; CTL frequency was calculated using the limiting dilution method by treating the wells of the plates exceeding the mean hemolysis value + 3 x standard deviation obtained from the GI mice as positive, and negative for each dilution of the response cell. negative) The number of wells was analyzed using a regression curve to show the frequency of CTL per 10 ⁷ spleen cells. This experiment was performed using 2-3 mice per group.

As shown in Table 2 , mice co-immunized with the mutant mIL-12 gene had CD8 + IFN-γ producing cells compared to HCV E2 alone and wild type mIL-12 administration groups at 0, 3, 6, 10 and 14 weeks after boosting. The secretion of was increased overall, which is consistent with the results of the CTL reaction. This difference is small in the early stages and gradually increases, and at 10 and 14 weeks, the frequency is hardly observed in the HCV E2 alone and wild-type mIL-12 administration groups, whereas the frequency is maintained in the mutant mIL-12 administration groups. In addition, the pattern between groups was similar when inoculated once without boosting DNA. These results demonstrate that expression of the mutant mIL-12 gene maintains the frequency of CD8 + IFNF-γ producing T cells after DNA immunization for an early and long period of time. From this, the inventors found that the in vivo role of IL-12p70 itself in cell-mediated immune response is to maintain the activity of Th1 and CTL for a long time, while the role of IL-12p40 is IL-12p70 as an antagonist in vivo It was confirmed that to suppress.

<8-4> Frequency analysis of antigen specific CD8 + T cells in spleen cells of immunized mice

Limiting dilution assay (LDA) to determine the frequency of different antigen-specific CD8 + T cells was performed to determine the frequency of antigen-specific CD8 + T cells using the hemolytic capacity of HCV E2-specific CD8 + T cells (Kuzushima, K). et al., Blood , 94: 3094-3100, 1999). In other words, spleen cells of mice further inoculated with DNA were diluted in various concentrations and allowed to have 20 wells per dilution in U-bottomed 96-well plates. CT26 cells expressing E2 were inhibited through mitomycin C (500 μg / ml) treatment as in CTL immune response observation and activated for about 5 days with diluted spleen cells. To measure the CTL frequency, one-third of the splenocytes of each well activated for 5 days were added to each well of the well plate with ⁵¹ Cr labeled target cells (5 × 10 ³ ) at 37 ° C. for 6 hours. Incubated. From this, the supernatant was recovered and used, and the CTL immune response was investigated by measuring the release of ⁵¹ Cr obtained after the reaction with a γ-counter (γ-counter, Wallac, Turku, Finland). If the level of specific hemolysis exceeds the mean of minimum hemolysis plus three times its standard deviation, the frequency of CTL was calculated as positive (Kuzushima, K. et al., Blood , 94: 3094-3100). , 1999).

Limited dilution yielded similar results to intracellular IFN-γ staining. In other words, the highest antigen-specific CD8 + T cell frequency was observed in the mutant mIL-12 administration group even at the initial immunization, and it was found that the frequency was maintained even after 14 weeks of boosting (see Table 2 ).

<8-4> Enhanced Analysis of Protective Immune Responses by Mutant IL-12

CT26-hghE2t, a tumor cell expressing hghE2t to identify Th1 and CTL immune responses induced in vivo by the mutant IL-12 gene of the present invention and to investigate the correlation between Th1 and CTL immune responses and protective immune responses Was administered to mice 12 weeks after DNA inoculation, and after 2 weeks thereafter, antigen-specific total IgG, IgG1, IgG2a and IgG2a / IgG1 ratios were determined from the serum as well as tumor size was measured for about 30 days. Specifically, local tumor growth every 3 days after subcutaneous injection of 1 × 10 ⁶ CT26-hghE2t cells in 0.1 μl serum-free medium 12 weeks after immunization into the hairy dorsal side of BALB / c mice. The determination was made by measuring the diameter and volume of the tumor using calipers. The mice were also observed for about 70 days to determine survival.

As shown in Figures 8A, 8B and 8C , a strong Th1 immune response was induced in the group immunized with mutant mIL-12 and the group of mice immunized with wild type mIL-12 was delayed compared to the group immunized with pTV2-gDsE2t alone. Tumor growth was shown. Groups immunized with mutant mIL-12 showed significantly delayed tumor growth. Most mice in the control group had tumors and died within 50 days, but in the mutant mIL-12 group about 90% of the mice were up to 70 days. Was alive. These results suggest that HCV E2-specific Th1 and CTL responses induced by the mutant mIL-12 genes of the present invention confer in vivo defense against challenge of modified tumor cell expression specific antigens. Although it is not easy to assess the relative effects of Th1 and CTL responses on tumor defense in vivo, this suggests that E2-specific CD8 + CTL and Th1 cells can kill CT26-hghE2t cells directly and in vitro in Th1 and CTL assays. As each observed, it is believed to assist the action of CTLs. In addition, IgG2a antibodies that bind strongly to FcγR on macrophages and natural killer cells mediate antibody-dependent cell mediated cytitoxicity.

As described above, the present invention is directed to a mutation in the amino acid Asn-222 (human) or Asn-220 (mouse) of human or mouse IL-12p40 that acts as a competitive inhibitor of active interleukin-12 (IL-12). Induces IL-12p40 mutant genes by inhibiting the secretion of IL-12p40 and allowing IL-12p70 with IL-12 immune activity to be secreted normally. When the IL-12p40 mutant gene of the present invention is used together for immunization with a DNA vaccine, it is possible to induce an optimal cellular immune response in the early and long periods. AIDS), hepatitis C and B, cancer, influenza, tuberculosis, malaria, etc. can be used as an immune enhancer for gene therapy for preventing and treating.

Claims (16)

A gene having a nucleotide sequence of SEQ ID NO: 1 encoding a p40 subunit of human IL-12, wherein the codon mutated codon encoding Asn-222 essential for secretion of IL-12p40. A gene having a nucleotide sequence of SEQ ID NO: 2 encoding a p40 subunit of mouse IL-12, wherein the codon mutated codon encoding Asn-220 essential for secretion of IL-12p40. The gene of claim 1, wherein the mutated human p40 subunit gene is substituted with a codon encoding Leu-222, Gln-222 or Ile-222 instead of the native Asn-222. 3. The gene of claim 2, wherein the mutated mouse p40 subunit gene is substituted with a codon encoding Leu-220 or Ile-220 instead of the native Asn-220. A gene construct comprising an internal ribosome entry site (IRES) between a gene of claim 1 and a gene encoding a human IL-12p35 subunit. A gene construct comprising an IRES between a gene of claim 2 and a gene encoding a mouse IL-12p35 subunit. An expression vector comprising the gene construct of claim 5. 8. The expression vector pGX0-hp35 / IRES / hp40-N222L of claim 7, wherein the gene construct comprises a gene in which the codon encoding Asn-222 of human IL-12p40 is mutated to Leu-222. (Accession number: KCTC 0969BP). 8. The expression vector pGX0-hp40-N222L / IRES / hp35 according to claim 7, wherein the gene construct comprises a gene in which the codon encoding Asn-222 of human IL-12p40 is mutated to Leu-222. (Accession number: KCTC 0970BP). An expression vector comprising the gene construct of claim 6. The expression vector pTV2-mp35 / IRES / mp40-N220L of claim 10, wherein the gene construct comprises a gene in which the codon encoding Asn-220 of IL-12p40 of the mouse is mutated to Leu-220. (Accession number: KCTC 0745BP). Prevention of disease, in which the codon encoding Asn-222 (human) or Asn-220 (mouse) essential for secretion of IL-12p40 is an active ingredient of the p40 subunit gene of IL-12 in mutated humans or mice Immunostimulators for DNA Immunization and Gene Therapy for Treatment. 13. The immunopotentiator according to claim 12, wherein the p40 subunit gene of IL-12 is administered with a DNA vaccine to enhance the immune response by enhancing the hydrolytic capacity of cytotoxic T lymphocytes (CTL) cells. The method of claim 12, wherein the p40 subunit gene of IL-12 is administered with a DNA vaccine to enhance the immune response by increasing the secretion of interferon gamma (IFN-γ) of T helper cells. Immunopotentiators. 13. The immunopotentiator according to claim 12, which enhances the immune response by increasing the secretion of interferon gamma (IFN-γ) of CD8 + cells when the p40 subunit gene of IL-12 is administered with a DNA vaccine. 13. The immunopotentiator according to claim 12, wherein the disease is cancer, AIDS, hepatitis C and B, flu, tuberculosis or malaria.