Novel cancer therapeutic agent comprising anticancer DNA vaccine and immune response-enhancing fusion protein

Abstract

The present invention relates to a cancer therapeutic agent, comprising: i) an anticancer DNA vaccine comprising a polynucleotide encoding at least one cancer antigen; and ii) a fusion protein comprising a CD80 protein and an IL -2 protein. The cancer therapeutic agent of the present invention very efficiently induces an antigen-specific T cell immune response compared to when a nucleic acid molecule encoding an antigen is administered alone, and thus can be efficiently used in the treatment of diseases requiring a T cell immune response, such as cancer.

Description

Novel cancer therapeutics including anticancer DNA vaccines and immune response-enhancing fusion proteins

[0001] The present invention relates to a novel cancer therapeutic agent, and more particularly, to a novel cancer therapeutic agent comprising an anticancer DNA vaccine and an immune response-enhancing fusion protein.

[0002] DNA vaccines are defined as purified plasmid DNA designed to be produced in an appropriate host system, containing genes encoding vaccine antigens and regulatory genes that trigger an immune response. However, the use of plasmid DNA as a vaccine has been less effective in humans than expected, hindering its practical application. It has been used in the development of some animal vaccines, and clinical trials of DNA vaccines have been conducted during the development of vaccines for diseases such as Zika and MERS. However, fully commercialized human vaccines remain rare.

[0003] To address these shortcomings, a technique called electroporation was introduced. This method involves placing a special injector on the skin and applying electrical stimulation to temporarily destabilize cell membranes, allowing the DNA contained in plasmids to be delivered through these gaps. Electroporation has significantly improved the efficiency of gene transfer into an individual.

[0004] Nevertheless, there are not many successful products using DNA vaccines because their gene transfer efficiency is still lower than that of viral vaccines or mRNA vaccines.

[0005] Meanwhile, cancer vaccines are an active immunotherapy method that enhances the body's immune function and eliminates cancer cells by administering tumor-specific antigens (TSAs) to cancer patients, thereby activating the immune system. Cytotoxic T lymphocyte (CTL) responses are crucial in the immune response to eliminate cancer cells. CTL activation allows them to destroy cancer cells through MHC class I molecules, which are antigen-recognizing molecules. CTL activation requires recognition of tumor-associated antigens (TAAs). However, none of the cancer antigens discovered to date are completely specific to cancer cells. While tumor antigens are primarily expressed on cancer cells, they are also expressed on normal cells. Therefore, stimulating the immune system to recognize these antigens could lead to attacks on normal cells as well, potentially triggering an autoimmune response.

[0006] Among the various cancers amenable to cancer vaccines, metastatic castrate-resistant prostate cancer (mCRPC) remains a significant unmet medical need, despite the approval of several new drugs for its treatment. Enzalutamide and abiraterone are the first- and second-line treatments for mCRPC, generating annual global sales of approximately 7 trillion won. Despite aggressive standard treatment, including these two drugs, the median survival time for mCRPC patients is 18-24 months, making extending life expectancy a critical need.

[0007] Abiraterone also inhibits the production of cortisol and androgens by inhibiting the enzyme CYP17A1. However, cortisol suppression leads to a relative increase in aldosterone levels, a mineralocorticoid hormone. Increased aldosterone levels can cause fluid retention, including hypertension, hypokalemia, and edema, so caution is advised in patients with a history of myocardial infarction or ventricular arrhythmia.

[0008] According to reports, mCRPC patients with cardiovascular disease experienced increased rates of hospitalization and mortality, Grade 3-5 systemic adverse reactions occurred in more than 50% of patients, and treatment discontinuation due to adverse reactions occurred in more than 10% of patients. Therefore, safety management is essential. Furthermore, resistance to both drugs typically develops around six months after administration, and cross-resistance between the two drugs has been observed in 30% of patients, significantly limiting treatment options.

[0009] Additionally, no significant improvement was observed in the quality of life assessment of prostate cancer patients, so the overall benefit-risk ratio of treatment is not significant.

[0010] Even immune checkpoint inhibitors, which have shown therapeutic efficacy in various cancer types, have failed to demonstrate significant therapeutic effects as monotherapy in clinical trials for patients with mCRPC. Combination therapy with pembrolizumab and enzalutamide has shown some clinical responses in patients with prostate cancer, but little objective benefit has been confirmed (Beer et al., J. Clin. Oncol. 35(1): 40-47, 2017; Graffet et al., Oncotarget7(33): 52810-52817, 2016; Slovinet et al., Ann. Oncol. 24(7): 1813-1821, 2013). The immunological characteristics of mCRPC patients can be summarized as follows: (1) a suppressive tumor microenvironment, (2) an impaired T cell immune system, and (3) a non-inflamed tumor environment. Approaches to overcoming these three factors are expected to be key factors in the treatment of mCRPC.

[0011] The present invention aims to address various issues, including the aforementioned ones, and aims to provide a more effective cancer treatment. However, the scope of the present invention is not limited by the aforementioned objectives.

[0012] According to one aspect of the present invention, there is provided an anticancer DNA vaccine comprising i) a polynucleotide encoding at least one cancer antigen and ii) a cancer therapeutic agent comprising a fusion protein comprising a CD80 protein and an IL-2 protein.

[0013] According to another aspect of the present invention, a method for treating cancer in a subject is provided, comprising administering to the subject the cancer treatment agent.

[0014] A cancer therapeutic agent according to one embodiment of the present invention can be effectively used in the treatment of diseases requiring a T cell immune response, such as cancer, by inducing an antigen-specific T cell immune response more efficiently than when administered alone as a nucleic acid molecule encoding an antigen.

[0015] Figure 1 is a schematic diagram illustrating the structure of SL-T10.

[0016] Figure 2 is a schematic diagram illustrating the structure of GI-101.

[0017] Figure 3 is a schematic diagram illustrating an experimental plan to confirm the anticancer effect of the combination of SL-T10 and GI-101 using a melanoma mouse model.

[0018] Figure 4 is a graph showing the extent of tumor growth during the administration period when SL-T10 and GI-101 were administered together in a melanoma mouse model.

[0019] Figure 5 is a graph showing the extent of tumor growth on the 21st day after combined administration of SL-T10 and GI-101 in a melanoma mouse model.

[0020] According to one aspect of the present invention, there is provided an anticancer DNA vaccine comprising i) a polynucleotide encoding at least one cancer antigen and ii) a cancer therapeutic agent comprising a fusion protein comprising a CD80 protein and an IL-2 protein.

[0021] In the above cancer therapeutic agent, the cancer antigen may be a tumor-specific antigen or a tumor-associated antigen. The cancer antigen may preferably be alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA), CA-125, MUC-1, epithelial tumor antigen (ETA), melanoma-associated antigen (MAGE), HPV E6 or E7, or a prostate cancer antigen. The prostate cancer antigen may be at least one selected from the group consisting of prostatic acid phosphatase (PAP), prostate-specific antigen (PSA), and prostate-specific membrane antigen (PSMA), and more preferably, may include all of prostatic acid phosphatase (PAP), prostate-specific antigen (PSA), and prostate-specific membrane antigen (PSMA). In this case, the prostatic acid phosphatase may include an amino acid sequence represented by SEQ ID NO: 7, the prostate-specific antigen may include an amino acid sequence represented by SEQ ID NO: 11, and the prostate-specific membrane antigen may include an amino acid sequence represented by SEQ ID NO: 14. The cancer antigen including all of PAP, PSA, and PSMA may include an amino acid sequence represented by SEQ ID NO: 16.

[0022] In the above cancer treatment agent, the anticancer DNA vaccine may further comprise one or more vaccine adjuvants and / or immunomodulators. The vaccine adjuvants may be polynucleotides encoding IL-12, IL-12p35, and IL-12p40, or one or more gene constructs comprising them, a combination administration agent of IL-12 and IL-21, or polynucleotides encoding IL-12p35, IL-12p40, and IL-21, or one or more gene constructs comprising the polynucleotides, and the immunomodulator may be a recombinant protein comprising IL-7, a fusion protein comprising the recombinant protein, or a polynucleotide encoding the recombinant protein or the fusion protein, or a gene construct comprising the same. The fusion protein may be one in which an Fc region of an antibody is added to the N- or C-terminus of IL-7, and the Fc region of the antibody may be a human IgG4 Fc region. Optionally, the immunomodulatory agent may be an immune checkpoint inhibitor, which may be an anti-PD-1 antibody, an anti-PD-1L antibody, an anti-CTLA antibody, or an anti-B7-H4 antibody.

[0023] In the above cancer treatment agent, the fusion protein includes CD80 protein and IL-2 protein.

[0024] As used herein, the term "CD80," also known as "B7-1," is a membrane protein present on dendritic cells, activated B cells, and monocytes. CD80 provides costimulatory signals essential for the activation and survival of T cells. CD80 is known to be a ligand for two different proteins present on the surface of T cells, CD28 and CTLA-4. CD80 consists of 288 amino acids, and may specifically have the amino acid sequence of SEQ ID NO: 36. Furthermore, as used herein, "CD80 protein" refers to full-length CD80 or a CD80 fragment.

[0025] As used herein, the term "CD80 fragment" refers to a truncated form of CD80. In addition, the CD80 fragment may be an extracellular domain of CD80. A specific example of the CD80 fragment may be one in which the 1st to 34th amino acids from the N-terminus, which is the signal sequence of CD80, are removed. Specifically, a specific example of the CD80 fragment may be a protein consisting of amino acids 35th to 288th of SEQ ID NO: 36. In addition, a specific example of the CD80 fragment may be a protein consisting of amino acids 35th to 242nd of SEQ ID NO: 36. In addition, a specific example of the CD80 fragment may be a protein consisting of amino acids 35th to 232nd of SEQ ID NO: 36. In addition, a specific example of the CD80 fragment may be a protein consisting of amino acids 35th to 139th of SEQ ID NO: 36. Additionally, one specific example of the CD80 fragment may be a protein consisting of amino acids 142 to 242 of SEQ ID NO: 36. In one embodiment, the CD80 fragment may have an amino acid sequence of SEQ ID NO: 20.

[0026] Additionally, the IL-2 protein and the CD80 protein may be linked by a linker or carrier. Specifically, the IL-2 or a variant thereof and the CD80 (B7-1) or a fragment thereof may be linked by a linker or carrier. In the present specification, the terms linker and carrier are used interchangeably.

[0027] The linker connects two proteins. A specific example of the linker may include 1 to 50 amino acids, albumin or a fragment thereof, or an Fc domain of an immunoglobulin. In this case, the Fc domain of the immunoglobulin refers to a protein that includes the heavy chain constant region 2 (CH2) and the heavy chain constant region 3 (CH3) of the immunoglobulin, and does not include the variable region of the heavy and light chains of the immunoglobulin and the light chain constant region 1 (CH1). The immunoglobulin may be IgG, IgA, IgE, IgD, or IgM, and preferably IgG4. In this case, the Fc domain of the wild-type immunoglobulin G4 may have the amino acid sequence of SEQ ID NO: 37.

[0028] In addition, the Fc domain of the immunoglobulin may be a wild-type Fc domain as well as an Fc domain variant. In addition, the term "Fc domain variant" as used herein may have a different glycosylation pattern from that of the wild-type Fc domain, or may have increased glycosylation compared to the wild-type Fc domain, decreased glycosylation compared to the wild-type Fc domain, or may have deglycosylated glycosylation. An aglycosylated Fc domain is also included. The Fc domain or variant may have a controlled number of sialic acid, fucosylation, or glycosylation through culture conditions or genetic manipulation of the host.

[0029] In addition, the sugar chain of the Fc domain of the immunoglobulin can be modified by conventional methods such as chemical methods, enzymatic methods, and genetic engineering methods using microorganisms. In addition, the Fc domain variant may be a form in which the Fc region of the immunoglobulin is mixed with the Fc region of IgG, IgA, IgE, IgD, or IgM. In addition, the Fc domain variant may be a form in which some amino acids of the Fc domain are substituted with other amino acids. A specific example of the Fc domain variant may have the amino acid sequence of SEQ ID NO: 24.

[0030] The fusion protein may have a structure in which CD80 and IL-2 proteins are linked to the N-terminus and C-terminus, respectively, of the Fc domain as a linker (or carrier), or IL-2 and CD80 are linked to each other. The linkage of the N-terminus or C-terminus of the Fc domain to CD80 or IL-2 may be arbitrarily achieved by a linker peptide.

[0031] Specifically, the fusion protein may be composed of the following structural formula (I) or (II):

[0032] N'-X-[linker (1)]n-Fc domain-[linker (2)]mYC' (I)

[0033] N'-Y-[linker (1)]n-Fc domain-[linker (2)]mXC' (II)

[0034] At this time, in the structural formulas (I) and (II),

[0035] The above N' is the N-terminus of the fusion protein,

[0036] The above C' is the C-terminus of the fusion protein,

[0037] The above X is CD80 protein,

[0038] The above Y is IL-2 protein,

[0039] The above linker (1) and linker (2) are peptide linkers,

[0040] The above n and m are each independently O or 1.

[0041] Preferably, the fusion protein may be composed of structural formula (I).

[0042] A peptide linker (1) may be included between the CD80 protein and the Fc domain. The peptide linker (1) may be comprised of 5 to 80 consecutive amino acids, 20 to 60 consecutive amino acids, 25 to 50 consecutive amino acids, or 30 to 40 amino acids. In one specific example, the peptide linker (1) may be comprised of 30 amino acids. In addition, the peptide linker (1) may include at least one cysteine. Specifically, it may include one, two, or three cysteines. In addition, the peptide linker (1) may be derived from the hinge of an immunoglobulin. In one specific example, the peptide linker (1) may be a peptide linker comprised of the amino acid sequence of SEQ ID NO: 22.

[0043] The above peptide linker (2) may be composed of 1 to 50 consecutive amino acids, or 3 to 30 consecutive amino acids, or 5 to 15 amino acids. In one specific example, the peptide linker (2) is (G4S) n (At this time, n can be an integer from 1 to 10). At this time, (G4S) n In , n may be 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In one embodiment, the peptide linker (2) may be a peptide linker consisting of an amino acid sequence of SEQ ID NO: 26.

[0044] As used herein, the term "IL-2" or "interleukin-2" means any wild-type IL-2 obtained from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise stated. The IL-2 may be obtained from animal cells, but also includes those obtained from recombinant cells capable of producing IL-2. Furthermore, the IL-2 may be wild-type IL-2 or a variant thereof.

[0045] In this specification, IL-2 or its variants are collectively referred to as "IL-2 protein" or "IL-2 polypeptide." IL-2, IL-2 protein, IL-2 polypeptide, and IL-2 variants specifically bind to, for example, the IL-2 receptor. This specific binding can be confirmed by methods known to those skilled in the art.

[0046] A specific example of the IL-2 may have the amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In addition, the IL-2 may be in a mature form. Specifically, the mature IL-2 may not include a signal sequence and may have the amino acid sequence of SEQ ID NO: 10. In this case, the IL-2 may be used as a concept that includes a fragment in which a portion of the N-terminus or C-terminus of the wild-type IL-2 is deleted (truncated).

[0047] In addition, the fragment of IL-2 may be in a form in which 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids are deleted consecutively from the N-terminus of the protein having the amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In addition, the fragment of the IL-2 may be in a form in which 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids are deleted consecutively from the C-terminus of the protein having the amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39.

[0048] As used herein, the term "IL-2 variant" refers to a form in which a portion of an amino acid sequence of full-length IL-2 or a fragment of the above-mentioned IL-2 is substituted. That is, the IL-2 variant may have an amino acid sequence different from that of wild-type IL-2 or a fragment thereof. However, the IL-2 variant may have an activity equivalent to or similar to that of wild-type IL-2. Here, "IL-2 activity" may mean, for example, specific binding to an IL-2 receptor, and this specific binding can be measured by a method known to those skilled in the art.

[0049] Specifically, the IL-2 variant may be one in which some of the amino acids of wild-type IL-2 are substituted. As a specific example of the IL-2 variant by amino acid substitution, at least one of the 38th, 42nd, 45th, 61st, and 72nd amino acids in the amino acid sequence of SEQ ID NO: 40 may be substituted.

[0050] Specifically, the IL-2 variant may be one in which at least one of the 38th, 42nd, 45th, 61st, or 72nd amino acids in the amino acid sequence of SEQ ID NO: 40 is substituted with another amino acid. In addition, when IL-2 is in a form in which a part of the N-terminus of the amino acid sequence of SEQ ID NO: 38 is deleted, the amino acid at the complementarily corresponding position in the amino acid sequence of SEQ ID NO: 40 may be substituted with another amino acid. For example, when IL-2 has the amino acid sequence of SEQ ID NO: 40, the IL-2 variant may be one in which at least one of the 58th, 62nd, 65th, 81st, or 92nd amino acids in the amino acid sequence of SEQ ID NO: 38 is substituted with another amino acid. These correspond to the 38th, 42nd, 45th, 61st, and 72nd amino acid residues of the amino acid sequence of SEQ ID NO: 40, respectively. In one embodiment, one, two, three, four, five, six, seven, eight, nine, or ten amino acids may be substituted, as long as IL-2 activity is maintained. In another embodiment, from one to five amino acids may be substituted.

[0051] In one specific example, the IL-2 variant may be a form in which two amino acids are substituted. Specifically, the IL-2 variant may be a form in which the 38th and 42nd amino acids in the amino acid sequence of SEQ ID NO: 40 are substituted. Furthermore, in one specific example, the IL-2 variant may be a form in which the 38th and 45th amino acids in the amino acid sequence of SEQ ID NO: 40 are substituted. Furthermore, in one specific example, the IL-2 variant may be a form in which the 38th and 61st amino acids in the amino acid sequence of SEQ ID NO: 40 are substituted. Furthermore, in one specific example, the IL-2 variant may be a form in which the 38th and 72nd amino acids in the amino acid sequence of SEQ ID NO: 40 are substituted. Furthermore, in one specific example, the IL-2 variant may be a form in which the 42nd and 45th amino acids in the amino acid sequence of SEQ ID NO: 40 are substituted. In addition, as a specific example, the IL-2 variant may be one in which the 42nd and 61st amino acids are substituted in the amino acid sequence of SEQ ID NO: 40. In addition, as a specific example, the IL-2 variant may be one in which the 42nd and 72nd amino acids are substituted in the amino acid sequence of SEQ ID NO: 40. In addition, as a specific example, the IL-2 variant may be one in which the 45th and 61st amino acids are substituted in the amino acid sequence of SEQ ID NO: 40. In addition, as a specific example, the IL-2 variant may be one in which the 45th and 72nd amino acids are substituted in the amino acid sequence of SEQ ID NO: 40. In addition, as a specific example, the IL-2 variant may be one in which the 61st and 72nd amino acids are substituted in the amino acid sequence of SEQ ID NO: 40.

[0052] Furthermore, the IL-2 variant may be a form in which three amino acids are substituted. Specifically, the IL-2 variant may be a form in which the 38th, 42nd, and 45th amino acids in the amino acid sequence of SEQ ID NO: 40 are substituted. In addition, as a specific example, the IL-2 variant may be a form in which the 38th, 42nd, and 61st amino acids in the amino acid sequence of SEQ ID NO: 40 are substituted. In addition, as a specific example, the IL-2 variant may be a form in which the 38th, 42nd, and 72nd amino acids in the amino acid sequence of SEQ ID NO: 40 are substituted. In addition, as a specific example, the IL-2 variant may be a form in which the 38th, 45th, and 61st amino acids in the amino acid sequence of SEQ ID NO: 40 are substituted. In addition, as a specific example, the IL-2 variant may be a form in which the 38th, 45th, and 72nd amino acids in the amino acid sequence of SEQ ID NO: 40 are substituted. In addition, as a specific example, the IL-2 variant may be one in which the 38th, 61st, and 72nd amino acids are substituted in the amino acid sequence of SEQ ID NO: 40. In addition, as a specific example, the IL-2 variant may be one in which the 42nd, 45th, and 61st amino acids are substituted in the amino acid sequence of SEQ ID NO: 40. In addition, as a specific example, the IL-2 variant may be one in which the 42nd, 45th, and 72nd amino acids are substituted in the amino acid sequence of SEQ ID NO: 40. In addition, as a specific example, the IL-2 variant may be one in which the 45th, 61st, and 72nd amino acids are substituted in the amino acid sequence of SEQ ID NO: 40.

[0053] In addition, the IL-2 variant may be a form in which four amino acids are substituted. Specifically, the IL-2 variant may be a form in which the 38th, 42nd, 45th, and 61st amino acids in the amino acid sequence of SEQ ID NO: 40 are substituted. In addition, as a specific example, the IL-2 variant may be a form in which the 38th, 42nd, 45th, and 72nd amino acids in the amino acid sequence of SEQ ID NO: 40 are substituted. In addition, as a specific example, the IL-2 variant may be a form in which the 38th, 45th, 61st, and 72nd amino acids in the amino acid sequence of SEQ ID NO: 40 are substituted. In addition, as a specific example, the IL-2 variant may be a form in which the 38th, 42nd, 61st, and 72nd amino acids in the amino acid sequence of SEQ ID NO: 40 are substituted. Additionally, as a specific example, the IL-2 variant may have amino acids at positions 42, 45, 61, and 72 substituted in the amino acid sequence of SEQ ID NO: 40.

[0054] Furthermore, the IL-2 variant may be a form in which five amino acids are substituted. Specifically, the IL-2 variant may be a form in which the 38th, 42nd, 45th, 61st, and 72nd amino acids in the amino acid sequence of SEQ ID NO: 40 are all substituted with different amino acids.

[0055] At this time, the “other amino acid” introduced by the substitution may be any one selected from the group consisting of alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. However, in the amino acid substitution of the IL-2 variant, the 38th position in the amino acid sequence of SEQ ID NO: 10 cannot be substituted with arginine, the 42nd position cannot be substituted with phenylalanine, the 45th position cannot be substituted with tyrosine, the 61st position cannot be substituted with glutamic acid, and the 72nd position cannot be substituted with leucine.

[0056] In the amino acid substitution of the IL-2 variant, arginine, which is the 38th amino acid in the amino acid sequence of SEQ ID NO: 40, may be substituted with an amino acid other than arginine. Preferably, in the amino acid substitution of the IL-2 variant, arginine, which is the 38th amino acid in the amino acid sequence of SEQ ID NO: 40, may be substituted with alanine (R38A).

[0057] In the amino acid substitution of the IL-2 variant, phenylalanine, which is the 42nd amino acid in the amino acid sequence of SEQ ID NO: 40, may be substituted with an amino acid other than phenylalanine. Preferably, in the amino acid substitution of the IL-2 variant, phenylalanine, which is the 42nd amino acid in the amino acid sequence of SEQ ID NO: 40, may be substituted with alanine (F42A).

[0058] In the amino acid substitution of the IL-2 variant, tyrosine, which is the 45th amino acid in the amino acid sequence of SEQ ID NO: 40, may be substituted with an amino acid other than tyrosine. Preferably, in the amino acid substitution of the IL-2 variant, tyrosine, which is the 45th amino acid in the amino acid sequence of SEQ ID NO: 40, may be substituted with alanine (Y45A).

[0059] In the amino acid substitution of the IL-2 variant, glutamic acid, which is the 61st amino acid in the amino acid sequence of SEQ ID NO: 40, may be substituted with an amino acid other than glutamic acid. Preferably, in the amino acid substitution of the IL-2 variant, glutamic acid, which is the 61st amino acid in the amino acid sequence of SEQ ID NO: 40, may be substituted with arginine (E61A).

[0060] In the amino acid substitution of the IL-2 variant, leucine, which is the 72nd amino acid in the amino acid sequence of SEQ ID NO: 40, may be substituted with an amino acid other than leucine. Preferably, in the amino acid substitution of the IL-2 variant, leucine, which is the 72nd amino acid in the amino acid sequence of SEQ ID NO: 10, may be substituted with glycine (L72G).

[0061] Specifically, the IL-2 variant may have at least one substitution selected from the group consisting of R38A, F42A, Y45A, E61R, and L72G in the amino acid sequence of SEQ ID NO: 40.

[0062] Specifically, the IL-2 variant may have amino acid substitutions at two, three, four or five positions selected from the group consisting of R38A, F42A, Y45A, E61R and L72G.

[0063] In addition, the IL-2 variant may be a form in which two amino acids are substituted. Specifically, the IL-2 variant may be a form in which R38A and F42A are substituted. In addition, as a specific example, the IL-2 variant may be a form in which R38A and Y45A are substituted. In addition, as a specific example, the IL-2 variant may be a form in which R38A and E61R are substituted. In addition, as a specific example, the IL-2 variant may be a form in which R38A and L72G are substituted. In addition, as a specific example, the IL-2 variant may be a form in which F42A and Y45A are substituted. In addition, as a specific example, the IL-2 variant may be a form in which F42A and E61R are substituted. In addition, as a specific example, the IL-2 variant may be a form in which F42A and L72G are substituted.

[0064] Additionally, as a specific example, the IL-2 variant may have substitutions of E61R and L72G.

[0065] Furthermore, the IL-2 variant may be a form in which three amino acids are substituted. Specifically, the IL-2 variant may be a form in which R38A, F42A, and Y45A are substituted. In addition, as a specific example, the IL-2 variant may be a form in which R38A, F42A, and E61R are substituted. In addition, as a specific example, the IL-2 variant may be a form in which R38A, F42A, and L72G are substituted. In addition, as a specific example, the IL-2 variant may be a form in which R38A, Y45A, and E61R are substituted. In addition, as a specific example, the IL-2 variant may be a form in which R38A, Y45A, and L72G are substituted. In addition, as a specific example, the IL-2 variant may be a form in which F42A, Y45A, and E61R are substituted. In addition, as a specific example, the IL-2 variant may be one in which substitutions occur at F42A, Y45A, and L72G. In addition, as a specific example, the IL-2 variant may be one in which substitutions occur at F42A, E61R, and L72G. In addition, as a specific example, the IL-2 variant may be one in which substitutions occur at Y45A, E61R, and L72G.

[0066] In addition, the IL-2 variant may be a form in which four amino acids are substituted. Specifically, the IL-2 variant may be a form in which substitutions occur at R38A, F42A, Y45A, and E61R. In addition, as a specific example, the IL-2 variant may be a form in which substitutions occur at R38A, F42A, Y45A, and L72G. In addition, as a specific example, the IL-2 variant may be a form in which substitutions occur at R38A, F42A, E61R, and L72G. In addition, as a specific example, the IL-2 variant may be a form in which substitutions occur at R38A, Y45A, E61R, and L72G. In addition, as a specific example, the IL-2 variant may be a form in which substitutions occur at F42A, Y45A, E61R, and L72G.

[0067] Furthermore, the IL-2 variant may have substitutions such as R38A, F42A, Y45A, E61R, and L72G.

[0068] Preferably, one specific example of the IL-2 variant may be one in which a substitution of any one of the following combinations (a) to (d) has occurred in the amino acid sequence of SEQ ID NO: 40 (human IL-2):

[0069] (a) R38A / F42A

[0070] (b) R38A / F42A / Y45A

[0071] (c) R38A / F42A / E61R

[0072] (d) R38A / F42A / L72G

[0073] At this time, if IL-2 has the amino acid sequence of SEQ ID NO: 38, it may have an amino acid substitution at a position complementarily corresponding to SEQ ID NO: 40. In addition, even if IL-2 is a fragment of the amino acid sequence of SEQ ID NO: 38, the amino acid at a position complementarily corresponding to SEQ ID NO: 40 may be substituted.

[0074] Specifically, the variant of IL-2 may have an amino acid sequence of SEQ ID NO: 28, 32, 41 or 42.

[0075] In addition, the IL-2 variant may be characterized by low toxicity in vivo. In this case, the low toxicity in vivo may be a side effect caused by IL-2 binding to the alpha chain of the IL-2 receptor (IL-2R). In particular, the IL-2 variant described in the present application has a low binding affinity to the alpha chain of the IL-2 receptor (IL-2R), and thus has lower in vivo toxicity than wild-type IL-2.

[0076] Specifically, the fusion protein may have an amino acid sequence of SEQ ID NO: 30, 34, 43, or 45. In another specific embodiment, the fusion protein comprises a polypeptide having 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 30, 34, 43, or 45. Here, the identity can be determined, for example, through percent homology, homology comparison software such as BlastN software of the National Center of Biotechnology Information (NCBI).

[0077] Information regarding IL-2 variants and fusion proteins containing the same that can be used in the present invention can be summarized in Table 1 below.

[0078] IL-2 mutant protein used in the present invention and fusion protein comprising the same Name Sequence number (nucleic acid) Amino acid sequence hIL-2v228(29)APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTAMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLThIL-2v332(33)APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTAMLTAKFYMPKKATELKHLQCLERELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLTWild type IL-238MYRMQLLSCI ALSLALVTNS APTSSSTKKT QLQLEHLLLDLQMILNGINN YKNPKLTRML TFKFYMPKKA TELKHLQCLEEELKPLEEVL NLAQSKNFHL RPRDLISNIN VIVLELKGSETTFMCEYADE TATIVEFLNR WITFCQSIIS TLTIL-2 Contains signal peptide39MDAMLRGLCC VLLLCGAVFV SPSHAAPTSS STKKTQLQLEHLLLDLQMIL NGINNYKNPK LTRMLTFKFY MPKKATELKHLQCLEEELKP LEEVLNLAQS KNFHLPRDL ISNINVIVLELKGSETTFMC EYADETATIV EFLNRWITFC QSIISTLThIL-240APTSSSTKKT QLQLEHLLLD LQMILNGINN YKNPKLTRMLTFKFYMPKKA TELKHLQCLE EELKPLEEVL NLAQSKNFHLRPRDLISNIN VIVLELKGSE TTFMCEYADE TATIVEFLNRWITFCQSIIS TLTIL-2 variant (3M, M45)41APTSSSTKKT QLQLEHLLLD LQMILNGINN YKNPKLTAMLTAKFAMPKKA TELKHLQCLE EELKPLEEVL NLAQSKNFHLRPRDLISNIN VIVLELKGSE TTFMCEYADE<h2 style=";text-align:left;direction:ltr">TATIVEFLNRWITFCQSIIS TLTIL-2 (3M, M72) 42APTSSSTKKT QLQLEHLLLD LQMILNGINN YKNPKLTAMLTAKFYMPKKA TELKHLQCLE EELKPLEEVL NGAQSKNFHLRPRDLISNIN VIVLELKGSE TTFMCEYADE TATIVEFLNRWITFCQSIIS TLTSU GI-10130(21)VIHVTKEVKE VATLSCGHNV SVEELAQTRI YWQKEKKMVLTMMSGDMNIW PEYKNRTIFD ITNNLSIVIL ALRPSDEGTYECVVLKYEKD AFKREHLAEV TLSVKADFPT PSISDFEIPTSNIRRIICST SGGFPEPHLS WLENGEELNA INTTVSQDPETELYAVSSKL DFNMTTNHSF MCLIKYGHLR VNQTFNWNTTKQEHFPDNGS GGGGSGGGGS GGGGSAESKY GPPCPPCPAPEEAAGGPSVFL FPPKPKDQLM ISRTPEVTCV VVDVSQEDPEVQFNWYVDGV EVHNAKTKPR EEQFNSTYRV VSVLTVLHQDWLNGKEYKCK VSNKGLPSSI EKTISKAKGQ PREPQVYTLLPPSQEEMTKNQ VSLTCLVKGF YPSDIAVEWE SNGQPENNYKTTPPVLDSDG SFFLYSRLTV DKSRWQEGNV FSCSVLHEALHNHYTQKSLS LSLGGGGGSA PTSSSTKKTQ LQLEHLLLDLQMILNGINNY KNPKLTAMLT AKFYMPKKAT ELKHLQCLEEELKPLEEVLN LAQSKNFHLR PRDLISNINV IVLELKGSETTFMCEYADET ATIVEFLNRW ITFCQSIIST LT융합 단백질 GI-102-M6134(35)VIHVTKEVKE VATLSCGHNV SVEELAQTRI YWQKEKKMVLTMMSGDMNIW PEYKNRTIFD ITNNLSIVIL ALRPSDEGTYECVVLKYEKD AFKREHLAEV TLSVKADFPTPSISDFEIPTSNIRRIICST SGGFPEPHLS WLENGEELNA INTTVSQDPETELYAVSSKL DFNMTTNHSF MCLIKYGHLR VNQTFNWNTTKQEHFPDNGS GGGGSGGGGS GGGGSAESKY GPPCPPCPAPEAAGGPSVFL FPPKPKDQLM ISRTPEVTCV VVDVSQEDPEVQFNWYVDGV EVHNAKTKPR EEQFNSTYRV VSVLTVLHQDWLNGKEYKCK VSNKGLPSSI EKTISKAKGQ PREPQVYTLPPSQEEMTKNQ VSLTCLVKGF YPSDIAVEWE SNGQPENNYKTTPPVLDSDG SFFLYSRLTV DKSRWQEGNV FSCSVLHEALHNHYTQKSLS LSLGGGGGSA PTSSSTKKTQ LQLEHLLLDLQMILNGINNY KNPKLTAMLT AKFYMPKKAT ELKHLQCLERELKPLEEVLN LAQSKNFHLR PRDLISNINV IVLELKGSETTFMCEYADET ATIVEFLNRW ITFCQSIIST LT융합 단백질GI-102-M4543(44)VIHVTKEVKE VATLSCGHNV SVEELAQTRI YWQKEKKMVLTMMSGDMNIW PEYKNRTIFD ITNNLSIVIL ALRPSDEGTYECVVLKYEKD AFKREHLAEV TLSVKADFPT PSISDFEIPTSNIRRIICST SGGFPEPHLS WLENGEELNA INTTVSQDPETELYAVSSKL DFNMTTNHSF MCLIKYGHLR VNQTFNWNTTKQEHFPDNGS GGGGSGGGGS GGGGSAESKY GPPCPPCPAPEAAGGPSVFL FPPKPKDQLM ISRTPEVTCV VVDVSQEDPEVQFNWYVDGV EVHNAKTKPR EEQFNSTYRV VSVLTVLHQDWLNGKEYKCK VSNKGLPSSI EKTISKAKGQ PREPQVYTLPPSQEEMTKNQ VSLTCLVKGF YPSDIAVEWE SNGQPENNYKTTPPVLDSDG SFFLYSRLTV<h2 style=";text-align:left;direction:ltr">LT융합 단백질GI-102-M7245(46)VIHVTKEVKE VATLSCGHNV SVEELAQTRI YWQKEKKMVL TMMSGDMNIW PEYKNRTIFD ITNNLSIVIL ALRPSDEGTY ECVVLKYEKD AFKREHLAEV TLSVKADFPT PSISDFEIPT SNIRRIICST SGGFPEPHLS WLENGEELNA INTTVSQDPE TELYAVSSKL DFNMTTNHSF MCLIKYGHLR VNQTFNWNTT KQEHFPDNGS GGGGSGGGGS GGGGSAESKY GPPCPPCPAP EAAGGPSVFL FPPKPKDQLM ISRTPEVTCV VVDVSQEDPE VQFNWYVDGV EVHNAKTKPR EEQFNSTYRV VSVLTVLHQD WLNGKEYKCK VSNKGLPSSI EKTISKAKGQ PREPQVYTLP PSQEEMTKNQ VSLTCLVKGF YPSDIAVEWE SNGQPENNYK TTPPVLDSDG SFFLYSRLTV DKSRWQEGNV FSCSVLHEAL HNHYTQKSLS LSLGGGGGSA PTSSSTKKTQ LQLEHLLLDL QMILNGINNY KNPKLTAMLT AKFYMPKKAT ELKHLQCLEE ELKPLEEVLN GAQSKNFHLR PRDLISNINV IVLELKGSET TFMCEYADET ATIVEFLNRW ITFCQSIIST LT<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0079] In the present invention, the fusion protein comprising a CD80 protein and an IL-2 protein may be a dimer in which two of each monomer are combined. The fusion protein comprising the IL-2 or a variant thereof and the CD80 or a fragment thereof is as described above. In this case, the bond between the fusion proteins constituting the dimer may be formed by a disulfide bond by a cysteine ​​present in the linker, but is not limited thereto. The fusion proteins constituting the dimer may be the same, but may be different fusion proteins. Preferably, the dimer may be a homodimer. One example of the fusion protein constituting the dimer may be a protein having the amino acid sequence of SEQ ID NO: 30.

[0080] The fusion protein of the present invention can activate immune cells such as natural killer cells due to the activity of IL-2. Therefore, it can be effectively utilized for cancer. In particular, it has been confirmed that an IL-2 variant having amino acid substitutions at two to five positions compared to the wild type, particularly an IL-2 variant having amino acid substitutions at two, three, four, or five positions selected from the group consisting of R38A, F42A, Y45A, E61R, and L72G in the amino acid sequence of SEQ ID NO: 10, exhibits characteristics that improve the pharmacological side effects of conventional IL-2 by lowering the binding affinity for the alpha chain of the IL-2 receptor. Therefore, such an IL-2 variant, when used alone or in the form of a fusion protein, can reduce the occurrence of vascular (or capillary) leak syndrome (VLS), which is a problem of conventional IL-2.

[0081] The term "adjuvant" as used in this document is used interchangeably with "vaccine adjuvant" and refers to a pharmaceutical or immunological preparation administered for the purpose of enhancing the immune response to a vaccine.

[0082] The term "immunomodulator" as used in this document refers to a substance that, when administered in vivo, plays a role in enhancing the humoral immune response and / or the innate immune response.

[0083] In the above cancer treatment agent, the polynucleotide may be operably linked to a promoter and may be inserted into a viral or non-viral vector.

[0084] In a more specific embodiment of the above cancer therapeutic agent, the anticancer DNA vaccine may comprise a gene construct comprising i) a polynucleotide encoding a fusion protein in which at least two proteins selected from the group consisting of PAP, PSA and PSMA are linked by a linker peptide; ii) a polynucleotide encoding at least two prostate cancer antigens selected from the group consisting of PAP, PSA and PSMA are operably linked to two or more promoters; or iii) a polynucleotide encoding at least two prostate cancer antigens selected from the group consisting of PAP, PSA and PSMA are linked by an internal ribosome entry site (IRES).

[0085] In the above cancer treatment agent, the vaccine adjuvant and / or immunomodulator may additionally include MIP-1α or a polynucleotide encoding the same.

[0086] In the above cancer treatment agent, the polynucleotide encoding IL-12 may be a composition comprising a polynucleotide encoding IL-12p35 and a polynucleotide encoding IL-12p40, a polynucleotide encoding a fusion protein in which the IL-12p35 and IL-12p40 are fused, or a polycistronic gene construct in which the polynucleotide encoding IL-12p35 and the polynucleotide encoding IL-12p40 are linked by an IRES.

[0087] In the above cancer therapeutic agent, the recombinant protein comprising IL-7 may be a peptide having 2 to 10 amino acids selected from the group consisting of glycine and methionine added to the N-terminus of the IL-7. In a preferred embodiment, the peptide may have the amino acid sequence of MGM, and the recombinant protein may include the amino acid sequence of SEQ ID NO: 19. In addition, the recombinant protein comprising IL-7 may be a wild-type hIL-7, a mutant IL-7 protein having at least 70 to 99% sequence homology with the wild-type hIL-7 and performing the same function. In the fusion protein, the Fc region of the antibody may be a hybrid Fc region in which portions of Fc of two or more isotypes are mixed, or a modified Fc region in which a specific amino acid is substituted, deleted, and / or inserted.

[0088] In the above cancer treatment agent, the anti-PD-1 antibody may be nivolumab, pembrolizumab, MEDI0608, AMP-224, PDR001, BGB-A317, or any combination thereof, and the anti-PD-L1 antibody may include BMS-936559, MPDL3280A, MEDI4736, MSB0010718C, or any combination thereof.

[0089] Instead of or in addition to the above-mentioned vaccine adjuvants and / or immunomodulators, conventionally used vaccine adjuvants may be included. Such vaccine adjuvants include aluminum hydroxide, aluminum phosphate, alum (potassium aluminum sulfate), MF59, virosome, AS04 [a mixture of aluminum hydroxide and monophosphoryl lipid A (MPL)], AS03 (a mixture of DL-α-tocopherol, squalene, and polysorbate 80, an emulsifier), CpG, Flagellin, Poly I:C, AS01, AS02, ISCOMs, and ISCOMMATRIX.

[0090] In the cancer treatment agent of the present invention, the immunomodulator may be administered simultaneously with the gene construct or 1 to 21 days after administration of the gene construct, more preferably 4 to 12 days after or 17 to 21 days after. Optionally, the gene construct may be administered once or in multiple doses. If administered in multiple doses, this may be viewed as a type of priming and boosting. In this case, the immunomodulator according to one embodiment of the present invention, particularly the recombinant protein comprising IL-7, may be administered before or after priming and / or before or after boosting. For example, after the first administration (priming) of the vaccine gene construct (first agent), an immunomodulator (second agent) may be administered after a certain period of time, followed by a second administration (boosting) of the first agent. It is also possible to additionally administer the second agent after boosting. The specific timing of administration of the first and second preparations can be adjusted depending on the type of antigen used or the patient's condition to maximize the therapeutic effect of the vaccine. If the gene construct and the immunomodulator are administered at different times, the gene construct and the immunomodulator are not formulated as a single composition but are formulated separately. Therefore, strictly speaking, it can be considered a kit containing each formulation rather than a composition. In this sense, the vaccine composition according to one embodiment of the present invention should be comprehensively interpreted as a concept that includes a vaccine kit comprising a formulation containing a gene construct and a formulation containing an immunomodulator.

[0091] As used herein, the term "operably linked to" means that a nucleic acid sequence of interest (e.g., in an in vitro transcription / translation system or in a host cell) is linked to a regulatory sequence in such a way that its expression can occur. The term "regulatory sequence" includes promoters, enhancers, and other regulatory elements (e.g., polyadenylation signals). Regulatory sequences include those that direct the constitutive expression of a nucleic acid of interest in many host cells, those that direct the expression of a nucleic acid of interest only in specific tissue cells (e.g., tissue-specific regulatory sequences), and those that direct expression to be induced by a specific signal (e.g., inducible regulatory sequences). It will be appreciated by those skilled in the art that the design of an expression vector may vary depending on factors such as the choice of host cell to be transformed and the level of protein expression desired. The plasmid DNA of the present invention can be introduced into a host cell to express the fusion protein. The host cell may be a eukaryotic cell, but may additionally contain a replication origin capable of propagating in a prokaryotic cell for amplification and production of the plasmid. Such replication origins are well known to those skilled in the art.

[0092] The first promoter and the second promoter may be the same or different, and each include a CMV promoter, a CMV-HSV thymidine kinase promoter, an SV40, an RSV-promoter (Rowe's sarcoma virus), a human elongation factor 1α-promoter, a glucocorticoid-inducible MMTV-promoter (Moloney mouse tumor virus), a metallothionein-inducible or a tetracycline-inducible promoter. Furthermore, the first promoter may be a neurofilament-promoter, a PGDF-promoter, an NSE-promoter, a PrP-promoter or a thy-1-promoter to enable expression in vivo. The promoters are known in the art and are described in the literature (Charron, J. Biol. Chem. 270: 25739-25745, 1995).

[0093] In the cancer therapeutic agent of the present invention, the plasmid DNA may additionally include a polynucleotide encoding a secretion signal in addition to the above components. These secretion signals are well known to those skilled in the art. Furthermore, depending on the expression system used, a leader sequence capable of directing the fusion protein into a cellular compartment is combined with the coding sequence of the polynucleotide according to one embodiment of the present invention. Preferably, this is a leader sequence capable of directly secreting the translated protein or its protein into the cytoplasm or extracellular medium.

[0094] Each agent or formulation within the above cancer therapeutic agent can be administered by various administration methods such as subcutaneous injection, intramuscular injection, intraperitoneal injection, intravenous injection, electroporation, gene gun, etc., but electroporation is more preferred. The electroporation can be performed by an electroporator, and the electroporator is a commercially available electroporator for injecting DNA drugs into the body, such as Glinporator from IGEA of Italy. TM, CUY21EDIT from JCBIO of Korea, SP-4a from Supertech of Switzerland, OrbiJector from SL Weizen (Korea) 　　 etc. can be used.

[0095] The voltage applied when using the electroporation method may be 25 to 40 V / cm, more preferably 26 to 39 V / cm, 27 to 38 V / cm, 28 to 37 V / cm, 29 to 36 V / cm, or 30 to 35 V / cm.

[0096] The cancer treatment agent of the present invention may additionally include a pharmaceutically acceptable auxiliary agent, excipient, or diluent in addition to the above-mentioned effective ingredient in each formulation or preparation.

[0097] The term "pharmaceutically acceptable" as used herein refers to a composition that is physiologically tolerable and does not typically cause allergic reactions such as gastrointestinal upset, dizziness, or similar reactions when administered to humans. Examples of such carriers, excipients, and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, and mineral oil. In addition, fillers, anticoagulants, lubricants, wetting agents, flavoring agents, emulsifiers, and preservatives may be additionally included.

[0098] Additionally, the cancer therapeutic agent according to one embodiment of the present invention can be formulated using methods known in the art to enable rapid, sustained, or delayed release of the active ingredient when administered to a mammal. The formulations include powders, granules, tablets, emulsions, syrups, aerosols, soft or hard gelatin capsules, sterile injectable solutions, and sterile powder forms.

[0099] Each agent in the cancer therapeutic agent according to one embodiment of the present invention may be formulated in a suitable form with a commonly used pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers include, for example, parenteral carriers such as water, suitable oils, saline solution, aqueous glucose, and glycol, and may further include stabilizers and preservatives. Suitable stabilizers include antioxidants such as sodium bisulfite, sodium sulfite, or ascorbic acid. Suitable preservatives include benzalkonium chloride, methyl- or propyl-paraben, and chlorobutanol. In addition, the composition according to the present invention may appropriately include suspending agents, solubilizing agents, stabilizers, isotonic agents, preservatives, antiadsorption agents, surfactants, diluents, excipients, pH adjusters, analgesics, buffers, antioxidants, and the like, if necessary, depending on the administration method or formulation. Pharmaceutically acceptable carriers and formulations suitable for the present invention, including those exemplified above, are described in detail in the literature [Remington's Pharmaceutical Sciences, latest edition].

[0100] According to another aspect of the present invention, a method for treating cancer in a subject is provided, comprising administering to the subject any one of the above cancer therapeutic agents.

[0101] In the above treatment method, the subject may be a human or a non-human animal.

[0102] The cancer may be selected from the group consisting of stomach cancer, liver cancer, lung cancer, colon cancer, breast cancer, prostate cancer, ovarian cancer, pancreatic cancer, cervical cancer, thyroid cancer, laryngeal cancer, acute myeloid leukemia, brain tumor, neuroblastoma, retinoblastoma, head and neck cancer, salivary gland cancer, melanoma, and lymphoma. Preferably, it may be prostate cancer.

[0103] The term "treatment" as used herein may be used to encompass both therapeutic treatment and preventative treatment. In this context, prevention may be used to mean alleviating or reducing a pathological condition or disease in a subject. In one embodiment, the term "treatment" encompasses any application or form of medication for treating a disease in mammals, including humans. Furthermore, the term encompasses inhibiting or slowing the progression of a disease or condition; restoring or repairing damaged or defective functions, thereby partially or completely alleviating a disease; or stimulating an inefficient process; or alleviating a serious disease. The active ingredients in the cancer therapeutic agent of the present invention are administered in a therapeutically effective amount.

[0104] The term "therapeutically effective amount" as used herein means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment, and the effective dosage level can be determined according to factors including the type and severity of the individual, age, sex, activity of the drug, sensitivity to the drug, time of administration, route of administration and excretion rate, duration of treatment, concurrently used drugs, and other factors well known in the medical field. The pharmaceutical composition of the present invention can be administered at a dosage of 0.1 mg / kg to 1 g / kg, and more preferably, it is administered at a dosage of 1 mg / kg to 500 mg / kg. Meanwhile, the dosage can be appropriately adjusted depending on the age, sex, and condition of the patient.

[0105] The route of administration, dosage, and frequency of administration of the composition of the present invention can be administered to a subject in various ways and amounts depending on the patient's condition and the presence or absence of side effects, and the optimal administration method, dosage, and frequency of administration can be selected within an appropriate range by a person skilled in the art.

[0106] Hereinafter, the present invention will be described in more detail through examples and experimental examples. However, the present invention is not limited to the examples and experimental examples disclosed below, but can be implemented in various different forms. The following examples and experimental examples are provided to ensure that the disclosure of the present invention is complete and to fully inform those skilled in the art of the present invention of the scope of the invention.

[0107] Example 1: Design of a DNA vaccine targeting prostate cancer.

[0108] The present inventors selected prostate-specific antigen (PSA), prostate-specific membrane antigen (PSMA), and prostatic acid phosphatase (PAP) as prostate cancer-specific antigens to produce a DNA vaccine for the treatment of prostate cancer. To produce an expression plasmid for expressing the same, a gene construct containing a polynucleotide encoding a fusion protein having the structure shown in Figure 1 was synthesized and inserted into the pGX27 backbone vector (Genexine, Korea), thereby producing a plasmid DNA for PSA / PSMA / PAP expression, which was named "SL-T10P," and a composition mixed with the vaccine adjuvant BD-121A was named "SL-T10." The antigen peptide of SL-T10 was linked to the tPA signal sequence to cause secretion from Flt3L. The components used in the manufacture of the above SL-T10P and their amino acid sequences and corresponding nucleic acid sequences are summarized in Table 2.

[0109] Structure of SL-T10 and components used in its manufacture Component Sequence number (nucleic acid) Amino acid sequence Signal peptide (tPA) 1(2) MDAMKRGLCCVLLLCGAVFVSPSHAFlt3L3(4) TQDCSFQHSPISSDFAVKIRELSDYLLQDYPVTVASNLQDEELCGGLWRLVLAQRWMERLKTVAGSKMQGLLERVNTEIHFVTKCAFQPPPSCLRFVQTNISRLLQETSEQLVALKPWITRQNFSRCLELQCQPDSSTLPPPWSPRPLEATAPTAP Linker #15(6)GSGSGSGSGSPAP7(8)GRAKELKFVTLVFRHGDRSPIDTFPTDPIKESSWPQGFGQLTQLGMEQHYELGEYIRKRYRKFLNESYKHEQVYIRSTDVDRTLMSAMTNLAALFPPEGVSIWNPILLWQPIPVHTVPLSEDQLLYLPFRNCPRFQELESETLKSEEFQKRLHPYKDFIATLGKLSGL HGQDLFGIWSKVYDPLYCESVHNFTLPSWATEDTMTKLRELSELSLSLSLYGIHKQKEKSRLQGGVLVNEILNHMKRATQIPSYKKLIMYSAHDTT VSGLQMALDVYNGLLPPYASCHLTELYFEKGEYFVEMYYRNETQHEPYPLMLPGCSPSCPLERFAELVGPVIPQDWSTECMTTNSHQGTEDSTDLinker #29(10)GGGGSGGGGSGGGGSGGGGSGGGGSPSA11(12)IVGGWECEKHSQPWQVLVASRGRAVCGGVLVHPQWVLTAAHCIRNKSVILLGRHSLFHPEDTGQVFQVSHSFPHPLYDMSLLKNRFLRPGDDSSHDLML LRLSEPAELTDAVKVMDLPTQEPALGTTCYASGWGSIEPEEFLTPKKLQCVDLHVISNDVCAQVHPQKVTKFMLCAGRWTGGKSTCSGDSGGPLVCNGVLQGITSWGSEPCALPERPSLYTKVVHYRKWIKDTIVANPLinker#39(13)GGGGSGGGGSGGGGSGGGGSGGGGSPSMA14(15)MWNLLHETDSAVATARRPRWLCAGALVLAGGFFLLGFLFGWFIKSSNEATNITPKHN MKAFLDELKAENIKKFLYNFTQIPHLAGTEQNFQLAKQIQSQWKEFGLDSVELAHYDVLLSYPNKTHPNYISIINEDGNEIFNTSLFEPPPPGYENVSD IVPPFSAFSPQGMPEGDLVYVNYARTEDFFKLERDMKINCSGKIVIARYGKVFRGNKVKNAQLAGAKGVILYSDPADYFAPGVKSYPDGWNLPGGGVQR GNILNLNGAGDPLTPGYPANEYAYRRGIAEAVGLPSIPVHPIGYYDAQKLLEKMGGSAPPDSSWRGSLKVPYNVGPGFTGNFSTQKVKMHIHSTNEVTR IYNVIGTLRGAVEPDRYVILGGHRDSWVFGGIDPQSGAAVVHEIVRSFGTLKKEGWRPRRTILFASWDAEEFGLLGSTEWAEENSRLLQERGVAYINADSSIEGNYTLRVDCTPLMYSLVHNLTKELKSPDEGFEGKSLYESWTKKSPSPEFSGMPRISKLGSGNDFEVFFQRLGIASGRARYTKNWETNKFSGYPLY HSVYETYELVEKFYDPMFKYHLTVAQVRGGMVFELANSIVLPFDCRDYAVVLRKYADKIYSISMKHPQEMKTYSVSFDSLFSAVKNFTEIASKFSERLQ DFDKSNPIVLRMMNDQLMFLERAFIDPLGLPDRPFYRHVIYAPSSHNKYAGESFPGIYDALFDIESKVDPSKAWGEVKRQIYVAAFTVQAAAETLSEVA

[0110] Example 2: Vaccine adjuvant

[0111] As a vaccine adjuvant, BD-121A (Korean Patent No. 10-2286397), a plasmid DNA vector containing polynucleotides encoding IL-12, IL-21, and MIP-1α, respectively, manufactured by the applicant, was used.

[0112] The basic structure of BD-121A is as shown in Fig. 1.

[0113]

[0114] Example 3: Immunomodulator

[0115] 3-1: Manufacturing of GI-101

[0116] The present inventors designed a fusion protein in which CD80 protein-IgG4 Fc region-IL-2 protein are sequentially linked as an immunomodulator (Fig. 3), and named it 'GI-101'.

[0117] Specifically, to produce a fusion protein comprising a human CD80 fragment, an Fc domain, and an IL-2 variant, a polynucleotide comprising a base sequence (SEQ ID NO: 31) encoding a fusion protein (SEQ ID NO: 30) comprising a tPA signal peptide (SEQ ID NO: 1), a CD80 fragment (SEQ ID NO: 20), an Ig hinge (SEQ ID NO: 22), an IgG4 Fc region (SEQ ID NO: 24), a linker peptide (SEQ ID NO: 26), and an IL-2 variant (2M) (R38A, F42A) (SEQ ID NO: 28) with two amino acids substituted in this order from the N-terminus was synthesized through the Invitrogen GeneArt Gene Synthesis service of Thermo Fisher Scientific and loaded into the pcDNA3.4 vector.

[0118] The components and amino acid sequence information of the above GI-101 are summarized in Table 3 below.

[0119] Structure of GI-101 and components used in its manufactureComponent Sequence number (nucleic acid) Amino acid sequence Signal peptide (tPA) 1(2) MDAMLRGLCCVLLLCGAVFVSPSHACD80(hB7-1:35-242) 20(21) VIHVTKEVKEVATLSCGHNVSVEELAQTRIYWQKEKKMVLTMMSGDMNIWPEYKNRTIFDITNNLSIVILALRPSDEGTYECVVLKYEKDAFKREHLAEVTLSVKADFPTPSISDFEIPTSNIRRIICSTSGGFPEPHLSWLENGEELNAINTTVSQDPETELYAVSSKLDFNMTTNHSFMCLIKYGHLRVNQTFNWNTTKQEHFPDN Hinge 22(23) GSGGGGSGGGGSGGGGSAESKYGPPCPPCPIgG4 Fc24(25)APEAAGGPSVFLFPPKPKDQLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGK EYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVD KSRWQEGNVFSCSVLHEALHNHYTQKSLSLSLGLinker26(27)GGGGShIL-2v228(29)APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLT AMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT

[0120] In addition, the above vector was expressed in CHO cells (Expi-CHO TM) was introduced to express a fusion protein having an amino acid sequence of sequence number 30. Feed medium was supplied to the stable cell line expressing the fusion protein at a ratio of 7.0% (v / v) every other day from the 3rd to the 7th day of culture in a fed-batch culture manner, and the culture medium was recovered on the 7th day. The fusion protein (GI-101) was purified from the recovered culture medium through chromatography.

[0121]

[0122] 3-2: Manufacturing of GI-102

[0123] To produce a fusion protein comprising a human CD80 fragment, an Fc domain, and an IL-2 variant (3M) with three amino acid substitutions (R38A, F42A, E61R) (GI101-M61), a polynucleotide comprising, in this order from the N-terminus, the base sequence (SEQ ID NO: 35) encoding a fusion protein (SEQ ID NO: 34) comprising a signal peptide (SEQ ID NO: 1), a CD80 fragment (SEQ ID NO: 20), an Ig hinge (SEQ ID NO: 22), an IgG4 Fc region (SEQ ID NO: 24), a linker peptide (SEQ ID NO: 26), and an IL-2 variant (SEQ ID NO: 32) was synthesized through the Invitrogen GeneArt Gene Synthesis service of Thermo Fisher Scientific and loaded into the pcDNA3.4 vector.

[0124] The components and amino acid sequence information of the above GI-101 are summarized in Table 4 below.

[0125] Components and sequence information of GI-102 Components Sequence number (nucleic acid) Amino acid sequence Signal peptide (tPA) 1 (2) MDAMLRGLCCVLLLCGAVFVSPSHACD80 (hB7-1:35-242) 20 (21) VIHVTKEVKEVATLSCGHNVSVEELAQTRIYWQKEKKMVLTMMSGDMNIWPEYKNRTIFDITNNLSIVILALRPSDEGTYECVVLKYEKDAFKREHLAEVTLSVKADFPTPSISDFEIPTSNIRRIICSTSGGFPEPHLSWLENGEELNAINTTVSQDPETELYAVSSKLDFNMTTNHSFMCLIKYGHLRVNQTFNWNTTKQEHFPDN Hinge 22 (23) GSGGGGSGGGGSGGGGSAESKYGPPCPPCPIgG4 Fc24(25)APEAAGGPSVFLFPPKPKDQLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGK EYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVD KSRWQEGNVFSCSVLHEALHNHYTQKSLLSLSLGLinker26(27)GGGGShIL-2v332(33)APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLT AMLTAKFYMPKKATELKHLQCLERELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT

[0126] In addition, the above vector was expressed in CHO cells (Expi-CHO TM ) was introduced to express the fusion protein of sequence number 34. Feed medium was supplied to the stable cell line expressing the fusion protein at a ratio of 7.0% (v / v) every other day from the 3rd to the 11th day of culture in a fed-batch manner, and the culture medium was recovered on the 12th day. The fusion protein (GI-102) was purified from the recovered culture medium through chromatography.

[0127]

[0128] Experimental Example 1: Establishment of a Melanoma Mouse Model

[0129] Cryopreservation vials of cryopreserved B16F10-PAP cells were gently shaken in a 37? water bath until the ice completely melted. The cryopreservation solution containing B16F10-PAP cells was transferred to a conical tube containing pre-warmed medium and centrifuged. The B16F10-PAP cell pellet was suspended in fresh culture medium and transferred to an appropriate flask. The resuspended B16F10-PAP cells were subcultured in a 37?, 5% CO2 cell incubator. The cultured B16F10-PAP cells were detached from the culture flask using Trypsin-EDTA, washed with DPBS, and prepared by resuspending them in DPBS. The prepared cells were 2 x 10 5 Cells / 100 L / head were administered subcutaneously once to 8-week-old female C57BL / 6 mice.

[0130] After tumor cell transplantation, the average tumor size is 50 to 100 mm. 3 When reached, the mice were ranked by tumor size and distributed according to body weight so that the average tumor size and body weight of each group were distributed within the margin of error.

[0131] After the first day of group separation and test substance administration (Day 0), the tumor length and short axis were measured twice a week using a caliper. Tumor volume was calculated using the following formula.

[0132] V (mean tumor volume) = (A x B 2 ) / 2 (A = major axis length, B = minor axis length)

[0133]

[0134] Experimental Example 2: Administration of Test Substance

[0135] Test substances were administered to mice administered B16F10-PAP cells according to the administration conditions in Table 5.

[0136] Test drug administration detailsExperimental groupGenderTest substanceAdministration conditionsDosageLiquid amount(μL)RouteElectroporationCycleG1MagneticPBS+hIgG4-50I.MIPw / EP-QWG2MagneticSL-T10(m)24 ㎍50I.Mw / EPBIWG3MagneticGI-1013 mg / kg100I.P.-QWG4MagneticSL-T10(m) +GI-10124 ㎍3 mg / kg50100I.MIPW / EP-BIWQW

[0137] 2-1: PBS

[0138] PBS was administered once a week (total of three times) after separation of the test groups. Mice were fixed using the femoral fixation method, and repeated injections were administered intramuscularly.

[0139] 2-2: hIgG4

[0140] hIgG4 was administered once a week (total of three times) after separation of the test groups. Mice were fixed using a dorsal skin fixation method, and the injection was administered intraperitoneally to the lower right or lower left side of the dorsal area centered on the linea alba.

[0141] 2-3: SL-T10(m)

[0142] SL-T10(m) was administered twice a week (total of 6 times) after separation of the test group. Mice were fixed using the femoral fixation method, and repeated injections were administered alternately to the left and right thigh muscles using an electroporator.

[0143] 2-4: GI-101

[0144] GI-101 was administered once a week (total of three times) after group separation. Mice were fixed using a transdorsal skin fixation technique, and the injection was administered intraperitoneally to the lower right or lower left area of ​​the back, centered on the linea alba.

[0145]

[0146] Experimental Example 3: Evaluation of Antitumor Efficacy

[0147] The present inventors created a tumor-bearing mice model by subcutaneously transplanting the melanoma cell line B16F10-PAP derived from C57BL / 6 into the right flank of C57BL / 6 mice and confirmed the solid tumor growth inhibitory effect through administration of the test substance SL-T10(m) alone or in combination with Gl-101 ​​(G1: Vehicle (PBS + hlgG4), G2: SL-T10(m), G3: Gl-101, G4: SL-T10(m) + Gl-101).

[0148] Tumor size was measured twice a week during the test period, and significant solid tumor growth inhibition was confirmed in G3 and G4 compared to the tumor size of G1 on the 21st day after administration of the test substance (Figs. 4 and 5).

[0149] The cancer treatment agent according to one embodiment of the present invention can be used to develop a treatment agent for treating malignant tumors very efficiently by improving the shortcomings of conventional anticancer vaccines.

Claims

1. i) An anticancer DNA vaccine comprising a polynucleotide encoding at least one cancer antigen and ii) a cancer therapeutic agent comprising a fusion protein comprising a CD80 protein and an IL-2 protein.

2. In paragraph 1, A cancer treatment agent, wherein the cancer antigen is a tumor-specific antigen or a tumor-associated antigen.

3. In paragraph 1, A cancer treatment agent wherein the cancer antigen is alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA), CA-125, MUC-1, epithelial tumor antigen (ETA), melanoma-associated antigen (MAGE), HPV E6 or E7, or prostate cancer antigen.

4. In paragraph 3, A cancer treatment agent, wherein the prostate cancer antigen is at least one selected from the group consisting of prostatic acid phosphatase (PAP), prostate-specific antigen (PSA), and prostate-specific membrane antigen (PSMA).

5. In paragraph 3, A cancer treatment agent, wherein the cancer antigen comprises all of the prostatic acid phosphatase (PAP), the prostate-specific antigen (PSA), and the prostate-specific membrane antigen (PSMA).

6. In paragraph 1, The above anticancer DNA vaccine is a cancer treatment agent that additionally comprises one or more vaccine adjuvants and / or immunomodulators.

7. In paragraph 6, A cancer treatment agent, wherein the vaccine adjuvant is a polynucleotide encoding IL-12, IL-12p35 and IL-12p40, or one or more gene constructs comprising the same, a combination agent of IL-12 and IL-21, or a polynucleotide encoding IL-12p35, IL-12p40 and IL-21, or one or more gene constructs comprising the polynucleotide.

8. In paragraph 1, A cancer treatment agent wherein the CD80 protein and IL-2 protein are bound by the Fc domain of immunoglobulin.

9. In paragraph 1, A cancer treatment agent, wherein the IL-2 protein is an IL-2 variant in which at least one of the amino acids at positions 38, 42, 45, 61, and 72 in the amino acid sequence of SEQ ID NO: 40 is substituted.

10. In paragraph 9, A cancer treatment agent, wherein the IL-2 variant has at least one substitution selected from the group consisting of R38A, F42A, Y45A, E61R, and L72G in the amino acid sequence of SEQ ID NO:

40.

11. In paragraph 9, The IL-2 variant is a cancer treatment agent in which any one of the following combinations (a) to (d) is substituted in the amino acid sequence of SEQ ID NO: 40: (a) R38A / F42A (b) R38A / F42A / Y45A (c) R38A / F42A / E61R (d) R38A / F42A / L72G.

12. In paragraph 1, A cancer treatment agent, wherein the CD80 protein is a fragment of the CD80 protein consisting of amino acids 35 to 242 of the amino acid sequence of SEQ ID NO:

36.

13. In paragraph 1, The above cancer DNA vaccine i) a polynucleotide encoding a fusion protein in which at least two proteins selected from the group consisting of PAP, PSA and PSMA are linked by a linker peptide; or ii) a polynucleotide encoding at least two prostate cancer antigens selected from the group consisting of PAP, PSA and PSMA, each operably linked to two or more promoters; or iii) A cancer therapeutic agent comprising a genetic construct comprising a polynucleotide encoding at least two prostate cancer antigens selected from the group consisting of PAP, PSA and PSMA, wherein the polynucleotide is linked by an internal ribosome entry site (IRES).

14. A method for treating cancer in a subject, comprising administering to the subject a cancer treatment agent of any one of claims 1 to 13.

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