TW201941786A - A novel polyvalent HPV vaccine composition - Google Patents

Abstract

Provided are a novel polyvalent human papillomavirus (HPV) DNA vaccine, a fusion protein used therein, and a polynucleotide encoding the fusion protein, and more specifically, a polyvalent HPV DNA vaccine composition including polynucleotides, which encode early protein antigen 6 (E6) of types 6, 11, 16, 18, 39, 45, and 56 HPV or immunogenic fragments thereof; and early protein antigen 7 (E7) of types 6, 11, 16, 18, 39, 45, and 56 human papillomavirus (HPV) or immunogenic fragments thereof, respectively.

Description

A new multivalent HPV vaccine ingredient

The invention relates to a novel multivalent human papilloma virus (hereinafter referred to as HPV) DNA vaccine, is a fusion protein used for multivalent HPV DNA vaccine, and a polynucleotide encoding a fusion protein.

Cervical cancer is one of the most deadly types of cancer in women worldwide (Einstein et al., Lancet Infect. Dis., 9: 347-356, 2009; Parkin and Bray, Vaccine 24 (3S): 11-25, 2007), 75% of the cases are caused by the most common high-risk HPV types of persistent infection (that is, HPV16 and HPV18) (Schiffman et al., Lancet, 370: 890-907, 2007; Forman et al. , Vaccine 30 (5S): F12-23, 2012). The persistence of HPV infection is usually related to the lack of clear HPV-specific T cell immunity, and virus-specific T cells found in premalignant and malignant patients are often reported as general dysfunction, sometimes even suppressive (Trimble, Cancer Immunol. Immunother. CII 59: 799-803, 2010). These results indicate that the functional damage of virus-specific T cells may be related to the occurrence of HPV-induced cervical cancer.

Cervical cancer occurs through high-risk HPV infection procedures, virus persistence, colony spread, and the continued alienation of infected cells, all the way to premalignant lesions, and the gradual transition to invasive cancer (Schiffman et al., Lancet 370: 890 -907, 2007). Stages 2 and 3 of premalignant cervical epithelioma, especially those that are HPV16 positive, are considered high-grade lesions and may have a 30% chance of developing invasive cancer (Moscicki et al., Vaccine 30 (5S ): F24-33, 2012). Therefore, there is an urgent need for an effective therapeutic vaccine that can prevent serious complications of persistent HPV infection and eradicate HPV-related tumors.

There are currently two commercial HPV vaccines in South Korea, a quadrivalent vaccine (Gardasil ^® ) and a bivalent vaccine (Cervarix ^® ). The four-valent vaccine includes L1 VLPs of HPV types 6, 11, 16, and 18, while the two-valent vaccine includes L1 VLPs of HPV types 16 and 18. Although HPV types 6 and 11 are the main cause of genital warts, they are low-risk HPVs that are not related to cervical cancer. Therefore, from the viewpoint of cervical cancer prevention, both vaccines correspond to the prevention of type 16 and 18 HPV bivalent vaccine.

At the same time, HPV E6 and E7 act like viral oncoproteins and function by linking and promoting tumor suppressor protein degradation (that is, p53 and retinoblastoma (pRb)) (Yugawa and Kiyono, Rev. Med. Virol., 19: 97-113, 2009). These viral oncoproteins are considered ideal targets for therapeutic vaccines against CIN2 / 3 and cervical cancer, not only because these proteins induce tumorigenesis, but also can be expressed with premalignant and malignant cells of HPV infection (Yugawa and Kiyono , Rev. Med. Virol., 19: 97-113, 2009). Because the recovery of cervical lesions is related to the presence of cellular immune responses, not humoral immune responses (Deligeoroglou et al., Infect. Dis. Obstet. Gynecol., 2013: 540850, 2013; Woo et al., Int. J. Cancer, 126: 133-141, 2010), a therapeutic vaccine capable of selectively inducing robust E6 / E7 specific T cell immunity is highly regarded by many.

Examples of vaccines currently using the HPV E6 / E7 antigen include DNA vaccine components using the HPV 16/18 E6 / E7 antigen disclosed in Korean Patent Application No. 2017-0045254. However, the two commercial vaccine components using the HPV 16/18 E6 / E7 and the DNA vaccine components target only the HPV types 16 and 18, which belong to the high-risk group, so these vaccine components can only cover all cervical cancers About 70%.

Therefore, there is an urgent need to develop a multivalent vaccine capable of triggering an immune response to various HPV types including cervical cancer.

The invention provides a multivalent HPV DNA vaccine, which can effectively prevent and treat cervical cancer and other diseases caused by HPV infection by inducing an immune response to fight against more types of HPV. However, the scope of application of the present invention is not limited to this.

In the embodiments of the present invention, a multivalent HPV DNA vaccine component including a polynucleotide is disclosed. The polynucleotide can be 6, 11, 16, 18, 39, 45, and 56 HPV types or their immunity, respectively. Encoding of early protein antigen 6 (E6) of the virogenic fragment; and encoding of early protein antigen 7 (E7) of HPV types 6, 11, 16, 18, 39, 45, and 56 or its immunogenic fragments.

In other embodiments of the present invention, an antigen-linked fusion protein is disclosed, in which at least four types of HPV antigen units are linked by a linking sequence, and the antigen unit is an N-terminal fragment including E6 and E7 and C The peptides of the terminal fragments were selected from the HPV group consisting of 6, 11, 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58 and 59 HPV types, respectively, with E6 (E6N) The N-terminal fragment-E7 (E7C) C-terminal fragment-E7 (E7N) N-terminal fragment-E6 (E6C) C-terminal fragment was derived in order.

In other embodiments of the invention, a polynucleotide encoding a fusion protein is disclosed.

In other embodiments of the invention, an expression vector is disclosed in which a polynucleotide is operably linked to an expression control sequence.

In other embodiments of the present invention, a 14-valent HPV DNA vaccine component is disclosed, including an expression vector in which a polynucleotide encoding a fusion protein is operably linked to a promoter, and a plurality of expression vectors to contain Polynucleotides encoding all 14 E6 / E7 antigen units.

In other embodiments of the present invention, a method for treating a disease caused by an HPV infection is disclosed, which includes administering to an individual a multivalent HPV DNA vaccine component or a 14-valent HPV DNA vaccine component.

The foregoing and other technical contents, features, and effects of the present invention will be clearly presented in the following detailed description of the preferred embodiments with reference to the drawings.

According to the viewpoint of the present invention, shown here is an HPV DNA vaccine component including an early protein antigen 6 (E6) that can encode HPV types 6, 11, 16, 18, 39, 45, and 56 respectively, or immunogenic fragments thereof. ), And polynucleotides encoding early protein antigen 7 (E7) of HPV types 6, 11, 16, 18, 39, 45, and 56 and immunogenic fragments, and among which E6 and E7 will not have wild-type functions.

According to the viewpoint of the present invention, the components of the multivalent HPV DNA vaccine may further include encoding at least one selected from the group consisting of 31, 33, 35, 51, 52, 58 and 59 HPV types or immunogenic fragments thereof. Early protein antigen 6 (E6); and at least one early protein antigen 7 (E6) selected from the group consisting of 31, 33, 35, 51, 52, 58 and 59 HPV types or immunogenic fragments thereof, and wherein The other E6 and E7 will not have wild-type functions.

Among the components of multivalent HPV DNA vaccines, E6 and E7 may be divided into N-terminal fragments and C-terminal fragments, and expressed in the form of random promiscuously ordered E6 / E7 antigen units, and E6 / E7 promiscuously ordered antigen units may be a The peptide will be connected to the N-terminal fragment of E6 and E7 in the order of the N-terminal fragment of E6 (E6N)-the C-terminal fragment of E7 (E7C)-the N-terminal fragment of E7 (E7N)-the C-terminal fragment of E6 (E6C) And C-terminal fragments.

Among the multivalent HPV DNA vaccine components, at least two, at least three, or at least four HPV E6 / E7 promiscuous sorted antigenic units may be expressed in the form of fusion proteins.

Among multivalent HPV DNA vaccine components, E6 / E7 promiscuously ordered antigenic units or fusion proteins may also include a signal sequence, while E6 / E7 promiscuously ordered antigenic units or fusion proteins may also include Flt3L.

Multivalent HPV DNA vaccine components may also include IL-7. The present inventors confirmed that when using a multivalent HPV DNA vaccine according to an embodiment of the present invention, using the HPV DNA vaccine in combination with IL-7 would significantly increase the anticancer effect (see FIG. 10B).

The multivalent HPV DNA of the present invention may also include at least one pharmaceutical adjuvant that is acceptable to the pharmaceutical industry.

Among the components of multivalent HPV DNA vaccines, the vaccine adjuvant may be a vaccine adjuvant used to stimulate the specific immune response of T lymphocytes, which contains IL-12 protein and IL-21 protein as active ingredients, or contains encoding IL Polynucleotide of -12 protein and polynucleotide encoding IL-21 protein are used as active ingredients.

Among multivalent HPV DNA vaccine components, a vaccine adjuvant used to stimulate a specific immune response on T lymphocytes may consist of at least one component selected from the following groups:

An IL-12 protein and an IL-21 protein, consisting of p35 (IL-12p35) and p40 (IL-12p40);

One to three vectors, each of which contains a polynucleotide encoding a p35 link (IL-12p35) and a p40 link (IL-12p40) constituting the IL-12 protein, respectively;

A polynucleotide encoding an IL-21 protein; and

An mRNA molecule, each encoding IL-12p35, IL-12p40, and IL-21 proteins.

Among the components of the multivalent HPV DNA vaccine, the IL-12p35 protein may have at least 90% sequence homology with the human IL-12p35 protein consisting of the amino acid sequence of SEQ ID NO: 1.

Among the components of the multivalent HPV DNA vaccine, the IL-12p40 protein may have at least 90% sequence homology with the human IL-12p40 protein consisting of the amino acid sequence of SEQ ID NO: 2.

Among the components of the multivalent HPV DNA vaccine, the IL-21 protein may have at least 90% sequence homology with the human IL-21 protein consisting of the amino acid sequence of SEQ ID NO: 3.

Among multivalent HPV DNA vaccine ingredients, vaccine adjuvants may consist of at least one ingredient selected from the following groups:

i) a MIP-1α protein;

ii) a MIP-1a gene structure in which a polynucleotide encoding a MIP-1a protein is operably linked to a promoter;

iii) A composite gene structure in which a polynucleotide encoding a MIP-1a protein is operably linked to at least one of IL-12p35, IL-12p40, and IL-21 proteins, and is entered by the polynucleotide encoding an internal ribosome Site (IRES) or linker peptide; and

iv) An mRNA molecule encoding a MIP-1a protein.

In the multivalent HPV DNA vaccine component, the structure of MIP-1a may be contained in a separate expression vector, or any one or more of one to three vectors, each of which includes: a separate coding for IL can be formed A p35 chain (IL-12p35) and a p40 chain (IL-12p40) of the -12 protein; and a polynucleotide encoding the IL-21 protein.

Among the components of the multivalent HPV DNA vaccine, the MIP-1a protein may have at least 90% sequence homology with the human MIP-1a protein consisting of the amino acid sequence of SEQ ID NO: 10.

In other embodiments of the present invention, an antigen-linked fusion protein is disclosed, in which at least four HPV antigenic units are linked by a linking sequence, wherein the antigenic unit comprises an N-terminal fragment of each of E6 and E7 and C E6 (E6N) N-terminal fragment-E7 (E7C) C-terminal fragment-E7 (E7N) N-terminal fragment-E7 (E7N) N-terminal fragment-E6 (E6C) The sequence of the C-terminal fragments is HPV selected from HPV types including 6, 11, 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58 and 59 types.

The term "HPV" used here to represent human papillomavirus is a DNA-based virus that is 52-55 nm in diameter and will be transferred to the skin or the subcutaneous layer of any animal, including humans. More than 130 types of HPV have been traced back (zur Hausen H., Vaccine 24 Suppl 3: S3, 2006). HPV can be transferred through the keratinocytes or mucosa of the skin. Most known HPVs do not show any signs in humans, but some HPVs can cause human papilloma. In addition, a small percentage of papilloma can develop into cancer (that is, cervical and testicular cancer). HPV 16 and HPV 18 are found in 70% of cervical cancer patients worldwide, and these HPVs are classified as high-risk groups. HPV DNA consists of 8,000 base pairs and is surrounded by a pentameric capsid protein instead of a non-lipid membrane. Capsid proteins containing two structural proteins (ie, L1 and L2) are expressed during viral replication Late in the cycle. In all HPV genomes, there are eight open reading frames (ORFs), and each ORF is divided into three functional regions. Early proteins E1-E7 (that is, the genes necessary to replicate the virus), L1-L2 (that is, a gene that expresses the structural proteins that make up the virus), and finally LCR controls the virus' replication and transcription.

The term "E6" as used herein represents an early-expressed protein necessary for HPV replication, which will connect to p53 and promote ubiquitination of p53, thereby inhibiting p53's function as a cancer tumor suppressor gene. In addition, E6 also induces the breakdown of BAK, a protein that promotes apoptosis. In addition, E6 also plays a role in activating the host cell cycle by activating telomerase.

The term "E7" used here represents an early-expressing protein necessary for HPV replication. It interacts with retinoblastoma (RB) and breaks down RB. Therefore, the transcriptional promoter that is inhibited by RB, E2F, will Be released. In addition, E7 will start cyclin E and cyclin A, which will act in the S phase of the cell cycle and thereby start the cell cycle of the host cell.

The term "immunogenic fragment" as used herein refers to a fragment of a full-length antigenic protein that can perform the function of an antigen, that is, a fragment can elicit an antigen-specific immune response.

In fusion proteins, the N-terminal and C-terminal fragments may overlap by 10 to 30 amino acids, and the antigenic unit functions as an antigen, because the antigenic unit is a heterogeneous sequence of proteins, but lacks the original wild-type E6 and E7 proteins. Intrinsic function (connecting p53 to pRb).

In the fusion protein, the antigen-linked fusion protein may additionally include fms-like tyrosine kinase-3 (Flt3) ligand (Flt3L) to its N-terminus, and the secretion signal sequence may be added here. The secretion signal sequence may induce the secretion of recombinant proteins expressed as extracellular cells, and may be a tissue plasminogen activator (tPA) signal sequence, a herpes simplex virus glycoprotein Ds (HSV gDs) signal sequence, or growth Hormone Signaling Series.

The fusion protein may also contain polynucleotides encoding one or two or more immune-enhancing peptides, and the immune-enhancing peptides may be CD28, inducible costimulatory molecules (ICOS), cytotoxic T lymphocytes Related protein 4 (CTLA4), programmed cell death protein 1 (PD1), B and T lymphocyte related protein (BTLA), death ligand 3 (DR3), 4-1BB, CD2, CD40, CD30, CD27, Signaling lymphocyte starter molecule (SLAM), 2B4 (CD244), natural killer cell group 2, member D (NKG2D) / DNAX starter protein 12 (DAP12), T cell immunoglobulin and mucin domain-containing protein 1 (TIM1 ), TIM2, TIM3, TIGIT, CD226, CD160, lymphocyte promoter gene 3 (LAG3), B7-1, B7-H1, induced glucocorticoid TNFR family-related protein (GITR), fms-like tyrosine kinase 3 Body (Flt3 ligand), flagellin, herpesvirus entry regulatory factor (HVEM), or cytoplasmic protein domain of OX40L [ligand of CD134 (OX40) and CD252], or a linker sequence of at least two of them.

In fusion proteins, the linker sequence is preferably a linker peptide, and the linker peptide may contain (G ₄ S) _n (unit: SEQ ID NO: 32, n is an integer from 1 to 10), (GS) _n (n is (1 to 10 integers), (GSSGGS) _n (unit: SEQ ID NO: 33, n is an integer from 1 to 10), KESGSVSSEQLAQFRSLD (SEQ ID NO: 34), EGKSSGSGSESKST (SEQ ID NO: 35), GSAGSAAGSGEF (SEQ ID NO: 36), (EAAAK) _n (unit: SEQ ID NO: 37, n is an integer from 1 to 10), CRRRRRREAEAC (SEQ ID NO: 38), A (EAAAK) ₄ ALEA (EAAAK) ₄ A (SEQ ID NO: 39), GGGGGGGG (SEQ ID NO: 40), GGGGGG (SEQ ID NO: 41), AEAAAKEAAAAKA (SEQ ID NO: 42), PAPAP (SEQ ID NO: 43), (Ala-Pro) _n (n Is an integer from 1 to 10), VSQTSKLTRAETVFPDV (SEQ ID NO: 44), PLGLWA (SEQ ID NO: 45), TRHRQPRGWE (SEQ ID NO: 46), AGNRVRRSVG (SEQ ID NO: 47), RRRRRRRR (SEQ ID NO: 48), GFLG (SEQ ID NO: 49), GSSGGSGSSGGSGGGDEADGSRGSQKAGVDE (SEQ ID NO: 50), and the like.

The term "fusion protein" as used herein refers to a recombinant protein in which two or more proteins or protein domains are responsible for a specific function when the proteins are linked to each other. Traditionally, a flexible linker peptide can be inserted between two or more proteins or protein domains, but any flexible linker peptide that does not limit the intrinsic function of the peptide will be linked together and will not inhibit the fusion protein that may be used. expression. Specific embodiments are described above.

In other aspects of the invention, a polynucleotide encoding a fusion protein is disclosed.

Polynucleotides may be deoxyribonucleic acid (DNA) or ribonucleic acid (RNA).

According to other aspects of the invention, an expression vector is disclosed in which a polynucleotide is operably linked to a control sequence.

The term "operably linked to" as used herein means that the nucleic acid of interest (eg, in an in vitro transcription / transfer system or in a subject cell) is linked to a control sequence so that a heterologous nucleic acid sequence can be expressed.

The term "control sequence" is a term that includes promoters, enhancers, and other control sequences, such as polyadenylation signals. Control sequences include: one will direct the heterologous nucleic acid to be expressed in many host cells, one will direct the heterologous nucleic acid to be expressed only in cells of a specific tissue (such as tissue-specific control sequences), and one will direct specific signals to induce expression (Such as induction control sequences). A common technique in the art is to be able to understand that the expression vector may depend on certain factors, such as the selection of the subject cell to be transferred, the desired protein expression level, and the like. The expression vector of the present invention may be introduced into a host cell to express a fusion protein. Control sequences that permit expression in eukaryotic and prokaryotic cells are techniques well known to experts in the art. As described above, these control sequences include those that are normally responsible for initiating transcription and a polyadenylation signal that is responsible for selectively terminating transcription and stabilization. Other control sequences may include translation enhancement factors in addition to transcriptional control factors and / or native combinations or heterologous promoter regions, such as those that may include those that may include the CMV-HSV thymidine kinase promoter, SV40, RSV promoter (Laboratory Sarcoma virus), human elongation factor 1α promoter, glucocorticoid MMTV promoter (Moroni mouse tumor virus), metallothionein or tetracycline promoter, or an amplification agent, such as a CMV amplification agent or The mammalian host cells expressing the control sequence of the SV40 amplifier. For expression in neurons, nerve fiber filament promoters, PGDF promoters, NSE promoters, PrP promoters, or thy-1 promoters are all considered for use. These promoters are well known to experts in the art and described in the literature (Charron, J. Biol. Chem. 270: 25739-25745, 1995). For the expression of prokaryotic cells, including the lac promoter, tac Promoters, as well as many promoters including the trp promoter, have been revealed. In addition to these factors that can initiate transcription, these control sequences may also include a transcription termination signal (such as the SV40 polyadenylation region or TK polyadenylation region) that exists downstream of the polynucleotide according to the embodiment of the present invention. ). In the present invention, suitable expression vectors are well known in the art, for example, Okayama-Berg cDNA expression vectors pcDV1 (Pharmacia), pRc / CMV, pcDNA1, pcDNA3 (in vitro genes), pSPORT1 (GIBCO BRL), pGX27 ( Korean Patent No. 1442254), pX (Pagano (1992) Science 255, 1144-1147), yeast double hybrid vectors, such as pEG202 and dpJG4-5 (Gyuris (1995) Cell 75, 791-803), or Eukaryotic expression vectors, such as lambda gt11 or pGEX (Amersham-Pharmacia). In addition to the nucleic acid molecule of the present invention, the vector may also include a polynucleotide encoding a secretory signal peptide, which is well known to experts in the art. In addition, depending on the expression system used, there is a leader sequence that can direct the fusion protein to the cell compartment according to the embodiment of the present invention, combined with the polynucleotide sequence, and the leader sequence is preferably secreted directly. A translated protein or a protein that enters the peripheral cytoplasm or extracellular medium.

In addition, the vectors of the present invention can also be formulated using, for example, standard recombinant DNA techniques. Examples of standard recombinant DNA techniques include blunt-end and stick-end junctions, restriction enzyme therapies to provide appropriate ends, alkaline phosphatase therapy to remove phosphate groups to prevent inappropriate bonding, and the use of T4 DNA ligase to produce Enzymatic ligation sequence. According to the embodiment of the present invention, the vector of the present invention can be prepared by chemical synthesis or genetic recombination technology or DNA encoding a fusion protein to obtain a DNA encoding a signal peptide to obtain a vector containing an appropriate control sequence. The vector containing the control sequence can be purchased or produced through a commercial transaction. In the embodiment of the present invention, pGX27 (Korean Patent No. 1442254), a vector for formulating a DNA vaccine, is used.

The expression vector according to the embodiment of the present invention may be an expression vector capable of expressing a fusion protein, and the expression vector may represent any form of plastid vector, virus vector, plastid vector, phage vector, artificial human chromosome, and the like.

In accordance with other aspects of the present invention, a 14-valent HPV DNA vaccine component is disclosed, including an expression vector comprising a polynucleotide encoding a fusion protein operably linked to a promoter, and including all of E14 encoding all 14 types Multiple expression vector of a / E7 antigen unit polynucleotide.

The 14-valent HPV DNA vaccine component may include three expression vectors whose structure allows the polynucleotides encoding each of the three fusion proteins to link and replicate four to five E6 / E7 antigen units of 14 HPVs, respectively.

The three expression vectors may be constructed as follows, but the structure of the three expression vectors is not limited to these three, but may also be different types of combinations:

i) the first expression vector includes the first gene, and its structure allows the first nucleic acid molecule encoding the first fusion protein to be operably linked to the promoter, and the first fusion protein is the HPV16 E6 / E7 antigen unit, The N-terminal and C-terminal fragments of each early antigen 6 (E6) and early antigen 7 (E7) of type 16 HPV (HPV16) will be the N-terminal fragments of E6 (16E6N)-the C-terminal fragment of E7 (16E7C) -N-terminal fragment of E7 (16E7N)-C-terminal fragment of E6 (16E6C) This sequence is connected; an HPV18 E6 / E7 antigen unit, in which each of HPV 18 (HPV18) early antigen 6 (E6) and early antigen 7 The N-terminal fragment and C-terminal fragment of (E7) will be the N-terminal fragment of E6 (18E6N)-the C-terminal fragment of E7 (18E7C)-the N-terminal fragment of E7 (18E7N)-the C-terminal fragment of E6 (18E6C) Sequential linkage; an HPV35 E6 / E7 antigenic unit in which the N-terminal and C-terminal fragments of each early antigen 6 (E6) and early antigen 7 (E7) of type 35 HPV (HPV35) will be N-terminal to E6 (35E6N) Fragment-C-terminal fragment of E7 (35E7C)-N-terminal fragment of E7 (35E7N)-C-terminal fragment of E6 (35E6C) This sequence is linked; an HPV45 E6 / E7 antigen unit, each of which is type 45 HPV (HPV45) Early antigen 6 ( N6 and C-terminal fragments of E6) and early antigen 7 (E7) will be N-terminal fragments of E6 (45E6N)-C-terminal fragments of E7 (45E7C)-N-terminal fragments of E7 (45E7N)-E6 (45E6C) C-terminal fragments of this sequence are linked; and an HPV58 E6 / E7 antigen unit, in which HPV58 (HPV58) each N-terminal fragment and C-terminal fragment of early antigen 6 (E6) and early antigen 7 (E7) The N-terminal fragment of E6 (58E6N)-the C-terminal fragment of E7 (58E7C)-the N-terminal fragment of E7 (58E7N)-the C-terminal fragment of E6 (58E6C) are linked in this order by a linker peptide;

ii) the second expression vector includes a second gene whose architecture allows a second nucleic acid molecule encoding a second fusion protein to be operably linked to a promoter, and the second fusion protein is an HPV31 E6 / E7 antigen unit, The N-terminal and C-terminal fragments of each early antigen 6 (E6) and early antigen 7 (E7) of type 31 HPV (HPV31) will be the N-terminal fragments of E6 (31E6N)-the C-terminal fragment of E7 (31E7C) -The N-terminal fragment of E7 (31E7N)-The C-terminal fragment of E6 (31E6C) is connected in this order; an HPV33 E6 / E7 antigenic unit in which each type 33 HPV (HPV33) early antigen 6 (E6) and early antigen 7 The N-terminal fragment and C-terminal fragment of (E7) will be the N-terminal fragment of E6 (33E6N)-the C-terminal fragment of E7 (33E7C)-the N-terminal fragment of E7 (33E7N)-the C-terminal fragment of E6 (33E6C) Sequential linkage; an HPV6 E6 / E7 antigenic unit, in which the N-terminal and C-terminal fragments of each early antigen 6 (E6) and early antigen 7 (E7) of type 6 HPV (HPV6) will be N-terminal to E6 (6E6N) Fragment-C-terminal fragment of E7 (6E7C)-N-terminal fragment of E7 (6E7N)-C-terminal fragment of E6 (6E6C) This sequence is linked; an HPV11 E6 / E7 antigen unit, of which type 11 HPV (HPV11) each Early antigen 6 (E6) and The N-terminal fragment and C-terminal fragment of early antigen 7 (E7) will be the N-terminal fragment of E6 (11E6N)-the C-terminal fragment of E7 (11E7C)-the N-terminal fragment of E7 (11E7N)-the C-terminal of E6 (11E6C) The fragments are linked in this order; and an HPV52 E6 / E7 antigen unit, in which the N-terminal and C-terminal fragments of each of the early antigen 6 (E6) and early antigen 7 (E7) of type 52 HPV (HPV52) will be E6 (52E6N ) N-terminal fragment-E7 (52E7C) C-terminal fragment-E7 (52E7N) N-terminal fragment-E6 (52E6C) C-terminal fragment is linked in this order by a linker peptide; and

The third expression vector includes a third gene whose architecture allows a third nucleic acid molecule encoding a third fusion protein to be operably linked to a promoter. The third fusion protein is the HPV39 E6 / E7 antigen unit, of which 39 The N-terminal and C-terminal fragments of each early antigen 6 (E6) and early antigen 7 (E7) of HPV (HPV39) will be the N-terminal fragment of E6 (39E6N)-the C-terminal fragment of E7 (39E7C)-E7 N-terminal fragment of (39E7N)-the C-terminal fragment of E6 (39E6C) is connected in this order; an HPV51 E6 / E7 antigen unit, in which type 51 HPV (HPV51) each of early antigen 6 (E6) and early antigen 7 (E7 The N-terminal fragment and the C-terminal fragment of E) (E6 (51E6N))-the C-terminal fragment of E7 (51E7C)-the N-terminal fragment of E7 (51E7N)-the C-terminal fragment of E6 (51E6C) ; An HPV56 E6 / E7 antigen unit, in which HPV56 (HPV56) each N-terminal fragment and C-terminal fragment of early antigen 6 (E6) and early antigen 7 (E7) will be the N-terminal fragment of E6 (56E6N)- The C-terminal fragment of E7 (56E7C)-the N-terminal fragment of E7 (56E7N)-the C-terminal fragment of E6 (56E6C) are connected in this order; and an HPV59 E6 / E7 antigenic unit, of which HPV 59 (HPV59) each early anti- N-terminal and C-terminal fragments of 6 (E6) and early antigen 7 (E7) will be N-terminal fragments of E6 (59E6N)-C-terminal fragments of E7 (59E7C)-N-terminal fragments of E7 (59E7N)-E6 ( The C-terminal fragments of 59E6C) are linked in this order by a linker peptide.

In the vaccine component, the first fusion protein to the third fusion protein may have a secretion signal sequence and Flt3L added to the N-terminus, and the secretion signal sequence is as described above.

The vaccine component may contain at least one vaccine adjuvant that is acceptable to the pharmaceutical industry. Aluminum hydroxide, aluminum phosphate, alum (potassium aluminum sulfate), MF59, virus particles, AS04 [a mixture of aluminum hydroxide and monophosphoryl lipid A (MPL)], AS03 (a DL-a-tocopherol, horn shark A mixture of ene and polysorbate (an emulsifier), CpG, flagellin, Poly I: C, AS01, AS02, ISCOMs, ISCOMMATRIX, etc. may all be used as vaccine adjuvants.

The terms "adjuvant" or "vaccine adjuvant" as used herein represent a pharmaceutical or immunological formula for the purpose of improving the immune response of a vaccine.

In addition, the vaccine adjuvant may also be a vaccine adjuvant used to stimulate the specific immune response of T lymphocytes. It contains an IL-12 protein and an IL-21 protein as active ingredients, or it contains a polynucleus encoding the IL-12 protein. Glycosides and a polynucleotide encoding the IL-21 protein serve as active ingredients.

In particular, vaccine adjuvants may contain at least one ingredient selected from the group consisting of:

An IL-12 protein comprising p35 (IL-12p35) and p40 (IL-12p40) and IL-21 protein;

One to three vectors, each of which contains a polynucleotide encoding a p35 link (IL-12p35) and a p40 link (IL-12p40), respectively, that construct the IL-12 protein;

A polynucleotide encoding an IL-21 protein; and

An mRNA molecule, each encoding IL-12p35, IL-12p40, and IL-21 proteins.

In addition, it is possible that in the vaccine agents described above, parts of IL-21p35, IL-12p40 and IL-21 will also be included as proteins, and other parts will be used as heterogeneous molecules. Like a mixture of expression vectors and / or mRNA molecules.

In particular, one to three vectors may contain a genetic structure in which a polynucleotide is operably linked to a control sequence (such as a promoter) so that IL-21p35, IL-12p40, and IL-21 can all be expressed. Vaccine adjuvants may be constructed from one to three vectors, each allowing insertion of polynucleotides encoding IL-21p35, IL-12p40, and IL-21 into individual expression vectors (a triple vector system), or to insert One or two expression vectors (a single vector system or a dual vector system). A specific example of such a single carrier system to a triple carrier system is as follows:

i) There is a fourth expression vector including a 4th gene structure to a 6th gene structure, in which the polynucleotides encoding IL-21p35, IL-12p40 and IL-21 proteins, respectively, each of which can be operably linked to A promoter

ii) there is a 5th to 7th expression vector containing a 4th gene structure to a 6th gene structure;

iii) There is an 8th expression vector and a 9th expression vector, each of which includes two 4th to 6th gene structures, and the other includes one gene structure;

iv) there is a fusion protein in which IL-21 is linked to any of IL-12p35 and IL-12p40; and the peptide between IL-12p35 and IL-12p40 is not included in the fusion protein;

v) There is a 10th expression vector containing a 7th gene structure, in which iv) a polynucleotide encoding a fusion protein is operably linked to a promoter, and an 8th gene structure, in which iv) encodes a peptide A polynucleotide operably linked to a promoter;

vi) an 11th expression vector and a 12th expression vector, which respectively include a 7th expression vector and an 8th expression vector;

vii) A 13th expression vector includes: a 9th gene structure in which at least two polynucleotides encoding IL-21p35, IL-12p40 and IL-21 proteins are operably linked to an IRES, and there is a An option for the 10th gene structure in which the remaining polynucleotides that are not contained in the 9th gene structure among the three polynucleotides are operably linked to a promoter; and

viii) a 14th expression vector contains a 9th gene structure; and a 15th expression vector can optionally include a 10th gene structure;

In the vaccine component, the IL-12p35 protein may be composed of amino acid sequences, at least 90%, preferably 95% of the sequences are homologous to human IL-12p35 composed of the amino acid sequence SEQ ID NO: 1, and It is also possible to use IL-12p35 derived from non-humans (such as primates or apes), which have a high degree of homology but do not induce any immune response in the human body. The IL-12p40 protein may consist of at least 90%, preferably 95% of the sequence, and the human IL-12p40 homologous amino acid sequence consisting of the amino acid sequence SEQ ID NO: 2, and it is also possible to use IL-12p40 derived from non-humans (such as primates or simians) is highly homologous but does not induce any immune response in the human body. It is also possible to use IL-12p35 and IL-12p40 as the sequence described in Korean Patent No. 0399728. The IL-21 protein may consist of at least 90%, preferably 95% of the sequence, and the amino acid sequence of human IL-21, which is composed of the amino acid sequence SEQ ID NO: 3, and it is also possible to use Derived from non-humans (such as primates or simians), IL-21 is highly homologous but does not induce any immune response in the human body.

Among vaccine components, vaccine adjuvants may also include at least one component selected from the group consisting of:

i) a MIP-1α protein;

ii) a MIP-1α gene structure in which a polynucleotide encoding a MIP-1α protein is operably linked to a promoter;

iii) A composite gene structure in which a polynucleotide encoding a MIP-1α protein can be operably linked to a gene encoding IL-21p35, IL-12p40, and IL-21 through a polynucleotide encoding an IRES or a linker peptide. At least one polynucleotide in a polynucleotide; and

iv) An mRNA molecule encoding a MIP-1α protein.

The polynucleotide encoding the MIP-1α protein can be operably linked to the polynucleotide encoding IL-21p35, IL-12p40, and IL-21 by encoding a polynucleotide or an IRES of a linker peptide, or can be used independently Gene structure is provided. In addition, the structure of the MIP-1α gene may also be contained in at least one of the three or more vectors, which include p35 (IL-12p35) and p40 (IL-12p40) encoding the IL-12 protein. ); And a polynucleotide encoding the IL-21 protein, that is, the structure of the MIP-1α gene may be included from the 4th expression vector described in the example about vaccine adjuvants to At least one expression vector selected from the group consisting of the 15th expression vector.

In the vaccine component, the MIP-1α protein may consist of at least 90%, preferably 95% of the sequences. These sequences are homologous to the MIP-1α protein consisting of the amino acid sequence SEQ ID NO: 10, It is also possible to use MIP-1α proteins derived from non-humans (such as primates or simians) that have a high degree of homology but do not induce any immune response in the human body.

Vaccine components may also include IL-7.

Vaccine ingredients may also include pharmaceutically acceptable adjuvants, excipients or diluents in addition to carriers.

The term "pharmaceutically acceptable" as used herein refers to a physiologically acceptable, but generally does not cause allergic reactions (such as gastrointestinal upset, dizziness, etc.) or similar reactions in humans. Examples of carriers, excipients and diluents may include lactose, glucose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, locust rubber, sodium alginate, gelatin, calcium phosphate , Calcium silicate, cellulose, methylcellulose, polyvinylpyrrolidone, water, methyl paraben, hypromellose, talc, magnesium stearate and mineral oil. It may also include fillers, anticoagulants, lubricants, humectants, fragrances, emulsifiers, preservatives, etc.

In addition to the adjuvants mentioned above, vaccine components may also include a vaccine adjuvant traditionally used. May use aluminum hydroxide, aluminum phosphate, alum (potassium aluminum sulfate), MF59, virus particles, AS04 [a mixture of aluminum hydroxide and monophosphoryl lipid A (MPL)], AS03 (a DL-a tocopherol, Squalene and polyxyester 80 (an emulsifier)), CpG, flagellin, Poly I: C, AS01, AS02, ISCOMs, ISCOMMATRIX, etc.) are traditionally used as vaccine adjuvants.

In addition, the vaccine components according to the embodiments of the present invention may be produced according to a formula well known in the art, so as to be able to be quickly released, or maintained, or to delay the release of the main components when administered to mammals. These formulations may include powders, granules, tablets, emulsions, syrups, nebulizers, soft or hard gelatin capsules, injectable germicidal solutions, and germicidal powders.

The vaccine components according to the present invention may be administered in a variety of different ways including, for example, oral, parenteral (e.g., suppositories, transdermal diffusion, intravenous, intraperitoneal, intramuscular, intralesional, intranasal, spinal canal), or It can be administered as an implanted device for continuous, continuous, or repeated release. The number of medications can be a single medication, or several times a day within the expected range, and the medication cycle time is not particularly limited.

The vaccine component according to the embodiment of the present invention may be administered in a traditional systematic or typical manner (for example, intramuscular or intravenous). If a DNA vaccine component is provided, the most preferred injection method for the vaccine component is electroporation. When using electroporation, DNA may be used a commercially available drug injected into the body (e.g. the Italian IGEA linporator ^TM, the Korean JCBIO CUY21EDIT, Switzerland Supertech of SP-4a, Korea SLVAXiGEN the OrbiJector ^®, etc.) method.

The medication route according to the embodiment of the present invention may be administered through any conventional route, as long as the vaccine components can reach the target tissue. The route of administration may include, but is not limited to, administration via the digestive tract (eg, peritoneal, intravenous, intramuscular, subcutaneous, and intrathecal).

In addition, the multivalent HPV DNA vaccine component according to the embodiment of the present invention may be administered in combination with an IL-7 protein or a polynucleotide encoding the IL-7 protein. Traditionally, DNA drugs and protein drugs may be administered in separate formulations, but they may also be packaged in separate formulations and then administered through different routes because they may be administered in different ways. For example, DNA vaccine components may be administered in different ways. Electroporation is administered by intramuscular injection, and IL-7 protein may be administered by ordinary protein medication, such as traditional intramuscular or intravenous or peritoneal injection.

The vaccine components according to the embodiments of the present invention may be formulated in a suitable form that is acceptable to the general pharmaceutical industry. The pharmaceutical industry's acceptable methods may include, for example, water, suitable oils, physiological saline, parenteral vehicles (eg, aqueous glucose, ethylene glycol, etc.), and may also include stabilizers and preservatives. Suitable stabilizers may include antioxidants (such as sodium bisulfite, sodium sulfite, or ascorbic acid). Suitable preservatives may include hydroxychloroaniline, methyl or parabens and chlorobutanol. In addition, the ingredients according to the present invention may suitably contain suspending agents, dissolving adjuvants, stabilizers, isotonic agents, preservatives, adsorption inhibitors, surfactants, diluents, excipients, pH adjusters, analgesics, relief Agents, antioxidants, etc., if necessary, depending on the method of preparation and the method of preparation. Examples of suitable pharmaceutical industry acceptable carriers and formulations suitable for the present invention, including the examples cited above, are described in detail in the literature [Remington's Pharmacy, New Edition].

The dosage of vaccine components for a patient may depend on many factors, including the patient's height, body surface area, age, the particular combination of medications used, gender, time and route of administration, general health status, and simultaneous administration. Other drugs. The pharmaceutically effective DNA may range from 100 ng / body weight (kg) to 10 mg / body weight (kg), preferably from 1 mg / kg to 500 mg / kg (body weight), preferably from 5 mg / kg kg to 50 mg / kg (body weight), and the dosage may be adjusted in consideration of various factors.

In addition, the vaccine component of the present invention may be administered in a therapeutically effective amount.

The term "therapeutically effective amount" as used herein represents a sufficient amount to treat a disease at a reasonable benefit / risk ratio when applied in medical treatment, and the level of the effective dose may be determined based on the following factors, including the type of article and the severity of the disease , Age, gender, drug activity, drug sensitivity, time of administration, route of administration and dissolution rate, length of treatment, factors including concurrent medication, and other factors that are well known in the medical field. The vaccine component of the present invention may be administered at a dose of 0.1 mg / kg to 1 g / kg, and it is better to use a dose from 1 mg / kg to 500 mg / kg, and the dose may be 1 mg, 2 mg, 3 Unit doses of mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, etc. are used. At the same time, the dosage may be adjusted appropriately according to age, gender and the health status of the patient.

According to another aspect of the present invention, a method for treating a disease caused by HPV infection is disclosed, which includes medication, for individuals, a multivalent HPV DNA vaccine component or a 14-valent HPV DNA vaccine component.

In the above treatment methods, the diseases caused by HPV infection may be squamous cell carcinoma (SCC), adenocarcinoma, adenosquamous epithelial carcinoma, small cell carcinoma, neuroendocrine tumor (NET), clear cell carcinoma, villous adenocarcinoma ( VGA), non-cancerous malignancy, melanoma, lymphoma, or cervical intraepithelial neoplasia (CIN).

In the above treatment methods, the vaccine components may be administered by in vivo electroporation.

In order to solve the disadvantage that the traditional HPV vaccine cannot be widely used in HPV infection diseases such as cervical cancer, the present invention formulates a multivalent HPV DNA vaccine composed of 3 plastids, of which one multivalent DNA vaccine covers all 6 , 11, 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58 and 59 HPV types, which are mainly prevalent in cervical intraepithelial neoplasia (CIN), Cervical cancer, vulvar intraepithelial neoplasia (VIN), vulvar cancer, anal genital warts, and condyloma acuminata, each HPV hybrid sequence E6 / E7 can be expressed in the form of a single giant fusion protein every 4 or 5 types, And experiments using multivalent HPV DNA vaccines were performed. As a result, it can be confirmed experimentally that the multivalent HPV DNA vaccine can induce T cell-specific immune responses for the two representative HPV types, HPV16 and HPV18, and other HPV types. These results are very encouraging. The use of expressable multiple hybrid sorted antigen proteins The expression vector can achieve these results. Among them, there are several promiscuously sequenced proteins instead of a single promiscuously sequenced protein, which will only be linked by the linker sequence, and the promiscuously sequenced antigen protein does not even need to use the original antigenic protein. In addition, it is surprising that the multivalent HPV DNA vaccine according to the embodiment of the present invention not only elicits a specific T cell immune response to HPV16 and HPV18 similar to the traditional bivalent vaccine, but also only uses a quarter of the traditional bivalent vaccine. A small dose, but in cases of types 6, 11, 39, 45, and 56, multivalent HPV DNA vaccines can also elicit a higher T-cell-specific immune response than HPV16 or HPV18. Therefore, the multivalent HPV DNA vaccine according to the embodiment of the present invention can be used very effectively as a vaccination that can prevent 7 types of HPV at the same time. In addition, the multivalent HPV DNA vaccine can also be effectively used as a preventable vaccine containing 31, Groups 33, 35, 51, 52, 58 and 59 selected at least one infection in the high-risk group and a vaccine to treat the infection.

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 described below, but can be implemented in various other forms. The following examples and experimental examples can disclose the present invention, so as to fully and completely convey the scope of the present invention to those skilled in the art to which the present invention belongs.

Example 1: Formulation of multivalent HPV DNA vaccine

In order to formulate a multivalent HPV DNA vaccine, the present invention provides polynucleotides that can encode N-terminal and C-terminal fragments of various types of E6 and E7 obtained by PCR. This is for expressing E6 and E7 antigens in the form of a hybrid sorted protein. , 6, 11, 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58 and 59 HPV E6 and E7 early-expressing proteins that belong to the high-risk group cannot display wild-type E6 and E7 Function, and the ligated polynucleotides will be obtained by the linking methods disclosed in Figure 1 and Table 1, thereby preparing three gene structures, each of which is inserted into the pGX27 vector, thereby preparing an HPV DNA structure Each of the three gene structures is named BD-14A, BD-14B, and BD-14C, and the component containing these three vectors is called BD-14. The reason why the multivalent HPV DNA vaccine structure is formulated into a triple vector system is that pGX-27 will have insufficient capacity when the size of the inserted gene structure is too large.

As shown in Figure 1 and Table 1, construct the structure of various HPV type E6 and E7 antigens in which the antigen is divided into N-terminal fragments and C-terminal fragments, and the local sequences (20 aa) in these fragment ends overlap, respectively, and then there is a fusion polypeptide, Wherein the C-terminal fragment of E7 (E7C) is connected to the N-terminal end of E6 (E6N), and there is a fusion polypeptide, in which the C-terminal fragment of E6 (E6C) is connected to the N-terminal fragment of E7 (E7N), it will be determined by (GS ) 5 Ligation peptides are ligated again, allowing 4 to 5 antigenic units (GS) of each type to be ligated to the ligation sequence for inclusion in a single vector. The species of each seed type are inserted into an expression vector exemplified in Table 1, but these are for illustration purposes only and may be formulated in virtually any other order.

[Table 1] Construction method of the multivalent HPV DNA vaccine structure of the present invention

Example 2: Preparation of human IL-2 and IL-21 expression vectors

2-1: Single carrier system

The present invention designs a single vector system, which enables IL-12 and IL-21 to be expressed in a single vector.

To this end, the present invention specifically formulates a gene structure by linking a hIL-12p35 polypeptide composed of a human IL-12 protein (that is, an amino acid sequence of SEQ ID NO: 1) and an amino acid sequence of SEQ ID The iL-12p40 polypeptide consisting of NO: 2) is derived from one of the two subunits of the polynucleotide (SEQ ID NOs: 4 and 5) to EMCV and has the internal ribosome entry site of the nucleic acid sequence SEQ ID NO: 6 ( IRES); then the RSV promoter (pRSV) of SEQ ID NO: 7 was sequentially linked to a polynucleoside encoding human IL-21 protein (hIL-21) consisting of the amino acid sequence SEQ ID NO: 3 Acid (SEQ ID NO: 8), and then to the 3 'end of the polynucleotide encoding the hIL-12p40 polypeptide; then insert the gene structure into the multiple replication site of the pGX-27 vector (Korean Patent No. 1442254), thereby A carrier according to an embodiment of the present invention is formulated. The vector formulated in this way is called "hBD-121" (Figure 2A).

2-2: Dual carrier system

The present invention designed a dual vector system to express the ability to insert IL-12 and IL-21 into separate vectors.

The dual vector system is prepared as follows. A polynucleotide encoding the hIL-12p35 polypeptide (SEQ ID NO: 1) and a polynucleotide encoding the hIL-12p40 polypeptide (SEQ ID NO: 2) are linked to a nucleic acid sequence. SQE ID NO: 6 of EMCV-IRES; and its composition will be inserted into the multiple replication site of pGX-27 vector; then, in a similar manner, it encodes human IL-21 consisting of the amino acid sequence SEQ ID NO: 3 The polynucleotide (SEQ ID NO: 8) of the protein (hIL-21) will also be inserted into the multiple replication site of pGX-27; thereby obtaining a vector expressing each IL-12 and IL-21. Vectors formulated in this way are called "'hBD-12" and "hBD-21".

2-3: Triple carrier system

Because IL-12 is a dimeric protein consisting of hIL-12p35 polypeptide and hIL-12p40 polypeptide, hIL-12p35 polypeptide and hIL12p40 polypeptide can be expressed from separate vectors. In this way, according to the embodiment of the present invention, hIL-12p35 polypeptide, hIL-12p40 polypeptide, and IL-21 may be expressed by any of three independent construction vectors, and for convenience, this will be named " 'Triple carrier system'.

The triple carrier system may be formulated as follows.

Polynucleotides encoding the hiL-12p35 polypeptide, hIL-12p40 polypeptide, and hIL-21 (SEQ ID NOS: 4, 5, and 8) will be inserted into various replication sites of the pGX-27 vector, thereby preparing a triple vector system .

Example 3: Preparation of human IL-12, IL21 and MIP-α expression vectors

The gene structure is linked by a polynucleotide encoding any one of the hIL-12p35 polypeptide consisting of the amino acid sequence SEQ ID NO: 1 and the hIL-12p40 polypeptide consisting of the amino acid sequence of SEQ ID NO: 2 ( (SEQ ID NOS: 4 and 5) to an EMCV-derived internal ribosome entry site (IRES) having the nucleic acid sequence SEQ ID NO: 6; and a human EF-1α promoter consisting of the nucleic acid sequence SEQ ID NO: 9 in sequence ( pEF-1α) and a polynucleotide (SEQ ID NO: 11) encoding the human protein MIP-1α (hMIP-1α) consisting of the amino acid sequence SEQ ID NO: 10, which are sequentially linked to SEQ ID NO: 7 RSV promoter (pRSV) and a polynucleotide (SEQ ID NO: 8) encoding a human IL-21 protein consisting of the amino acid sequence SEQ ID NO: 3; and inserting the gene structure into pGX-27 Multiple replication sites of the vector; the vector thus formulated was named "'hBD-121A" (Figure 2B).

Example 4: Preparation of IL-12 and IL-21 expression vectors for mice

A genetic structure is prepared by encoding a mouse IL-12 protein (that is, a mIL-12p35 polypeptide consisting of an amino acid sequence of SEQ ID NO: 12 and a mIL-12p40 consisting of an amino acid sequence of SEQ ID NO: 13). Polypeptide) (SEQ ID NOS: 14 and 15) of either of the two subunits is linked to an EMCV-derived internal ribosome entry site (IRES) with a nucleic acid sequence of SEQ ID NO: 6; The RSV promoter (pRSV) of SEQ ID NO: 7 and the 3 'encoding the IL-21 protein (mIL-21) consisting of the amino acid sequence SEQ ID NO: 16 are linked to 3' of the polynucleotide encoding the mIL-12p40 polypeptide The gene structure was then inserted into the multiple replication site of the pGX-27 vector to prepare a vector according to an embodiment of the present invention; the vector thus prepared was named "mBD-121" (Fig. 2A).

Example 5: Formulation of expression vectors for mouse IL-12, IL-21 and MIP-1α

A genetic structure is prepared by encoding a polynucleoside of any one of the mIL-12p35 polypeptide consisting of the amino acid sequence SEQ ID NO: 12 and the mIL-12p40 polypeptide consisting of the amino acid sequence SEQ ID NO: 13 Acid (SEQ ID NOS: 14 and 15), linked to the EMCV-derived internal ribosome entry site (IRES) with the nucleic acid sequence SEQ ID NO: 6; and human EF- consisting of the nucleic acid sequence SEQ ID NO: 9 in sequence The 1α promoter (pEF-1α) and a polynucleotide (SEQ ID NO: 19) encoding the human protein MIP-1α (hMIP-1α) consisting of the amino acid sequence SEQ ID NO: 18 are sequentially linked to The 3 'end of the polynucleotide encoding mIL-12p40 polypeptide is connected in sequence to the RSV promoter (pRSV) of SEQ ID NO: 7 and mouse IL-21 consisting of the amino acid sequence SEQ ID NO: 16 Polynucleotide (SEQ ID NO: 17) of protein (mIL-21); then insert the gene structure into various replication sites of pGX-27; the vector prepared from this was named "'mBD-121A" (Figure 2B ).

Experimental Example 1: Analysis of BD-14 Expression

1-1: ELISA assay

In order to confirm whether BD-14 (that is, the multivalent HPV DNA vaccine prepared according to the embodiment of the present invention in Example 1) can be normally expressed when introduced into mammalian cells, the present invention respectively transforms the cells with BD-14 (that is, BD14A). , BD-14B and BD-14C) mammalian cells expressing three kinds of expression vectors, and the Flt3L specific antigen contained in each structure is used to analyze the presence of protein expression.

In particular, COS-7 cell lines will be seeded in each 100 mm petri dish and cultured for 16 hours, and transformed with empty vectors (mimetic plastid DNA), while BD14A, BD-14B, and BD-14C plastid DNA are Liposomes will be formulated in Example 1. Each transformant was cultured in an incubator (37 ° C, CO ₂ ) for 3 days, and the culture supernatant of COS-7 cells was recovered under various conditions and used as samples. Since the protein presented in each sample was in the form of Flt3L and protein binding, the protein was quantified using the Flt3L ELISA reagent (Figure 3A).

As shown in the results shown in Fig. 3A, it was confirmed that all BD14A, BD-14B and BD-14C had good expression.

1-2: Western blot analysis

In the present invention, the cell lysate contained in Experimental Example 1-1 was used to perform SDS-PAGE electrophoresis, converted to a nylon membrane, and an anti-Flt3L antibody was used to perform immunoblot analysis (Abcam, Cat # ab52648) (Figure 3B).

As shown in the results shown in FIG. 3B, it was confirmed that all the BD14A, BD-14B, and BD-14C can express the fusion protein at a desired size.

Experimental example 2: Analysis of the immune response in BD-14

2-1: Analysis of T cell specific immune response

The present inventors used BD14 as a medicine in experimental animals according to the embodiment of the present invention, and used the method of counting the number of spleen cells that showed an immune response for each antigen to check whether BD-14 can induce a T-cell specific immune response. In particular, C57BL / 6 mice (that is, experimental animals) are divided into a group of drugs administered with an empty vector (control group) (n = 5), and a group of drugs administered with a traditional bivalent HPV DNA vaccine (Korea Patent Publication No. 10-2017 -0045254) (n = 5), and a group of BD-14 medications according to an embodiment of the present invention (n = 5). Then every 2 mg of plastid DNA (0.67 mg for BD-14A to BD-14C) was administered to the femoral muscle by electroporation in vivo twice in two week intervals. Two weeks after the last administration, the animals were sacrificed, their spleens were removed, and the number of spleen immune cells responding to each E6 / E7 antigen was calculated by ELISPOT analysis (Figures 4A and 4B).

As shown in the results shown in FIG. 4B, in the BD-14 embodiment according to the experimental example of the present invention, all types of HPV E6 / E7 antigens successfully elicited T cell-specific immune responses, although the T cell-specific immune response against HPV 35 Types 52, 59, and 59 are relatively weak, but at the same time, no significant difference was observed in the response capabilities of HPV 16 and 18 E6 / E7 compared with traditional bivalent vaccines. These results confirm that although the BD-14 HPV type 16 and type 18 E6 / E7 antigen ratios are only one-third of the traditional bivalent HPV DNA vaccine according to the examples of the present invention, HPV type 16 and type 18 E6 / E7 Antigens still have equivalent effects. In particular, it was confirmed that HPV 6, 11, 39, 45, and 56 type reactions were stronger than those caused by HPV type 16 and type 18.

2-2: Analysis of antitumor effects

The present invention has performed in vivo anticancer activity analysis using tumor animal mimics to confirm whether BD-14 according to an embodiment of the present invention is effective for the prevention and treatment of cancer caused by HPV infection.

In particular, C57BL / 6 mice (that is, experimental animals) are divided into a group of drugs administered with an empty vector (control group) (n = 8), and a group of drugs administered with a traditional bivalent HPV DNA vaccine (Korea Patent Publication No. 10-2017 -0045254) (n = 8), and a group of BD-14 medications according to an embodiment of the present invention (n = 8). Is then derived from C57BL / 6 mouse lung epithelial cells and TC-1 cells to convert the expression of HPV16 E6 / E7 antigens, in volume 5 ^'105 cells were injected subcutaneously into the back to induce tumors. Three days after cancer cells were administered, femoral bone was electroporated at two-week intervals using OrbiJector ^® (SLVAXiGEN, South Korea) with 2 mg of plastid DNA each (0.67 mg for BD-14A to BD-14C). Take the muscle twice. Tumor size and survival of experimental animals will be examined at intervals of 3 to 4 days from the 7th day of tumor injection (Figures 5A and 5C).

As shown in the results shown in FIG. 5B, the BD-14 according to the embodiment of the present invention not only significantly reduced the tumor size compared with the control group, but also exhibited an anti-tumor effect similar to that of the conventional divalent HPV DNA vaccine. In addition, as shown in FIG. 5C, compared with the results of tumor size analysis, the grouping with the BD14 medication according to the embodiment of the present invention also showed a higher survival rate than the grouping with the conventional bivalent HPV DNA vaccine medication. Based on a comprehensive review of these results, it can be confirmed that the components of the BD-14 vaccine according to the embodiment of the present invention, although the dose is only one third of the high-risk group (such as HPV16 or HPV18), the components of the BD-14 vaccine and traditional Compared with the 2-valent DNA vaccine, it not only shows equivalent or better anti-cancer performance, but also the BD-14 vaccine is a highly innovative vaccine component. It induces specific immune responses of T cells and even targets other types of HPV. Can increase the ability to prevent cervical cancer prevention by up to 90% or more.

Experimental Example 3: Effect Analysis of BD-121 as a Vaccine Adjuvant

3-1: ELISA assay

The present invention converts the hBD-121 structure and the mBD-121 structure according to the embodiment of the present invention into cells in Examples 2 and 4, respectively, and checks whether IL-12 and IL-21 are normally expressed in the transformed cells. In particular, the COS-7 cell line will be seeded in each 100 mm petri dish and cultured for 16 hours. The empty vector (simulated plastid DNA), the hBD-121 plastid DNA prepared in Example 2-1 and Example 4 will be used. The mBD-121 plastid DNA prepared here was transformed with liposomes. Each transformant was cultured in an incubator (37 ° C, CO ₂ ) for 3 days, and the culture supernatant of COS-7 cells was recovered under various conditions and used as samples. The IL-12 and IL-21 proteins presented in each sample use antibodies that specifically recognize IL-12 and IL-21 (IL-12: R & D Systems, Cat # D1200, IL-21: BioLegend, Cat # 433808) was quantified by ELISA assay (Figures 6A and 6B).

As shown in the results in FIG. 6A, when hIL-12 was introduced at a DNA content of 4 mg, the protein expression of the hBD-121 plastid DNA sample was greater than 4,000 pg / mL, so it was confirmed that these proteins were normally expressed. When hIL-21 was introduced in 4 mg DNA, the protein expression in the sample was as high as 200 ng / mL, so it was confirmed that these proteins can be expressed to a high degree. In addition, as shown in Figure 6B, the mouse structure showed results similar to the human structure. At the same time, neither of the two proteins was expressed in the control group introduced with the empty vector, so it was confirmed that the vaccine adjuvant expression system of the present invention functions normally.

3-2: Western blot analysis

The present invention uses cell lysates obtained in Experimental Example 3-1 to perform SDS-PAGE electrophoresis, converts to nylon membrane, and then performs immunoblot analysis with anti-IL-12A antibody, anti-IL-12B antibody and anti-IL-21 antibody (Figure 6C).

As shown in the results of FIG. 6C, it was confirmed that both IL-12 and IL-21 can be transfected with BD-121 plastid DNA according to the embodiment of the present invention.

Experimental example 4: Analysis of the effect of BD-121A as an adjuvant

4-1: Analysis of T cell specific immune response

The present invention performs an analysis of whether BD-121A can improve the function of a BD-14 vaccine as a vaccine adjuvant.

For this purpose, the inventors used BD-14 according to the embodiment of the present invention and mBD-121A prepared in Example 5 to test animals, and used the method of calculating the number of spleen cells showing the immune response of each antibody to check whether the drug would induce T cell-specific immune response. In particular, C57BL / 6 mice (that is, experimental animals) will be divided into a group administered with an empty carrier (control group) (n = 3), a group administered only with BD-14 (n = 5), and a group administered according to this The BD-14 and mBD-121A in the embodiment of the invention are commonly used (n = 5), and then the group treated with BD-14 only uses each plastid DNA (BD-14A, BD-14B, and BD-14C). OrbiJector ^® (SLVAXiGEN, South Korea) was administered in a dose of 1.3 mg, while the group co-administered with BD-14 and mBD-121A was administered with 1 mg of each plastid DNA and in vivo electroporation. On the femoral muscle head. After two weeks of treatment, the animals were slaughtered and the spleen was removed. The ELISPOT analysis was then used to calculate the number of spleen immune cells on various E6 / E7 antibodies (Figures 7A and 7B).

As shown in the results of FIG. 7B, it was confirmed that mBD-121A according to the embodiment of the present invention strengthened E6 / E7 specific T cell responses of various HPV types compared with BD-14 alone, especially HPV type 16 (that is, high (Risk group) showed at least a two-fold increase in T-cell immune responses, with HPV31, 33, 51, and 58 types showing significantly increased immune responses. These results confirm that BD-121A according to the embodiment of the present invention is a very effective vaccine adjuvant for a multivalent HPV DNA vaccine.

4-2: Analysis of antitumor manifestations

In particular, C57BL / 6 mice (that is, experimental animals) are divided into a group administered with an empty vector (control group) (n = 10), and a group administered with a traditional bivalent HPV DNA vaccine (Korean Patent Bulletin Issue No. 10-2017 -0045254) (n = 13), and a group of BD-14 and BD-121 medications according to an embodiment of the present invention (n = 13). Examples 2-2 are then used for the experiments TC-1 cells to the number of 5 ^'105 cells were injected subcutaneously into the back to induce tumors. Seven days after injection of TC-1 cells, femoral bone was electroporated at a two-week interval using OrbiJector ^® (SLVAXiGEN, Korea) with 4 mg of plastid DNA (1 mg each for BD-14A to BD-14C). Take the muscle twice. Tumor size and survival of experimental animals will be examined at intervals of 3 to 4 days from the 7th day of tumor injection (Figures 8A and 8C).

As shown in the confirmation results of FIGS. 8B and 8C, it was confirmed that the multivalent vaccine according to the embodiment of the present invention showed significant anti-cancer performance compared with the negative control group (grouped with PBS), and was more positive than the positive control group (that is, Traditional bivalent vaccines) have equivalent or similar or better anti-cancer performance. Taking into account the portion of the antigen (HP616 E6 / E7) is only about a quarter of the traditional bivalent vaccine, the above results mean that the antigen shows similar anti-cancer performance. In addition, although there are many (quantity) antigens administered at the same time, they can also induce various immune responses, and the anti-cancer performance of the special type (HPV16) will not be impaired.

Experimental example 5: Analysis of vaccine action mechanism

In order to examine the mechanism through which the multivalent HPV vaccine according to the embodiment of the present invention may function, the inventors prepared the experiment by removing CD-4 T cells and CD-8 T cells with anti-CD4 or anti-CD8 antibodies. Animals, and a comparative analysis on the anti-cancer effect of the HPV DNA vaccine according to the examples of the present invention was performed.

In particular, the experiment will divide C57BL / 6 mice (that is, experimental animals) into four groups for execution: one group uses the empty vector medicine as the control group (n = 9), and one group uses the vaccine according to the embodiment of the present invention plus a vaccine Adjuvant (BD14A + BD121A) was used as the control group (that is, subtype antibody (isotype)) (n = 9), one group was administered with anti-CD4 antibody (n = 9); and one group was administered with anti-CD8 antibody Medication (n = 9). Cheng experiment plan as follows: Experimental Example 2-2 TC-1 in cancer cell weight per animal 5 '10 ⁵ cells were inoculated subcutaneously 7 times, and antibodies (isotype, anti-CD4 and anti-CD8 antibodies) the On the first day of the day when the cancer cells were inoculated, the drug was administered intraperitoneally at a dose of 200 mg / injection / mouse at intervals of 7 days, and the vaccine components (BD-14A and BD- 121) From the 3rd day after the cancer cell inoculation, at a 7-day interval, electroporation was used at the dose of 8 mg / injection / mouse to the femoral muscle hindlimb site 3 times (Fig. 9A). The tumor size and survival rate of the experimental animals were examined at intervals of 3 to 4 days from the 9th day of tumor inoculation (Figs. 9B to 9C).

As shown in the results confirmed in Figures 9B and 9C, it was observed that the anticancer effect was maintained in mice with CD4 T cells removed, while the anticancer effect in mice with CD8 T cells removed was better than that in the control group (mimics and subtypes). Antibody) is much lower. These results show that the multivalent HPV DNA vaccine according to the embodiment of the present invention exhibits an anti-cancer immune effect by CD8 T cells, and these results are very important for the known fact that CD8 T cells are very important for the effect of anti-cancer therapeutic vaccines stable.

Experimental example 6: Analysis of the effect of co-administration with IL-7

Interleukin 7 (IL-7) is a cytokine that is well known to promote the differentiation of pluripotent stem cells into lymphoptic cells, and plays an important role in the formation of B cells and T cells. The well-known role is to promote the malignant transformation of blood cancer (that is, acute lymphoblastic leukemia or T-cell lymphoma). In general solid tumors, IL-7 is known to interfere with the homeostasis of CD8 and CD4 cells, thereby reducing CD4. ⁺ CD25 ⁺ Foxp3 ⁺ regulates the proportion of T cells, and clinical phase 1 and phase 2 trials are already underway for certain cancers. From the results of Experimental Example 5, IL-7 tendency affects the balance between CD4 T cells and CD8 T cells, and causes CD8 T cells to increase. Therefore, the inventor assumes that IL-7 may have a synergistic effect when used in combination with the HPV DNA vaccine of the embodiment of the present invention. In order to confirm this hypothesis, the inventors used the vaccine alone or combined with IL-7 according to the embodiment of the present invention, and analyzed its anticancer effect.

In particular, the experiment will divide C57BL / 6 mice (that is, experimental animals) into three groups to perform: one group uses the empty vector as the control group (n = 8), and one group uses the vaccine according to the embodiment of the present invention (BD14A) Medication; a group of medications with a vaccine and IL-7 (BD14A + IL-7) according to an embodiment of the invention. Experimental Examples 2-2 to TC-1 cancer cell per animal weight 1 '10 ⁵ cells were injected into the vagina, carcinoma in situ formulated to simulate tumor-induced body, and the vaccine (4 mg) or vaccine (4 mg ) And IL-7 (50 mg) are administered 3 times intramuscularly from the day the cancer cells are inoculated (that is, on the 7th, 14th, and 28th days), and will be administered for 7 days from the day the cancer cells are inoculated Tumor volume was measured at intervals (Figure 10A).

As shown in the results confirmed in FIG. 10B, it was confirmed that the administration of BD14 when administered in situ tumor mimics significantly improved the anticancer effect compared with the group administered with PBS. This confirms that the results are consistent with those obtained from experiments using subcutaneous injections of ectopic tumors. What's more interesting is that when BD14 and IL-7 are co-administered, the anti-cancer effect is significantly improved compared with BD14 alone. This point shows that it is possible to co-administer the HPV DNA vaccine according to the embodiment of the present invention and a cytokine having a mechanism of action different from that of T cells.

As described above, BD-14 (that is, the multivalent HPV DNA vaccine according to the embodiment of the present invention), although has a complex structure, can still express the E6 / E7 hybrid sequenced protein (that is, the antigen contained therein). Protein) and can successfully elicit actual T-cell specific immune responses of various types of HPV E6 / E7 antigens. As a result of the analysis of the anti-cancer effect of a cancer model expressing the HPV16 E6 / E7 antigen, BD-14 showed the same or better anti-cancer effect than the conventional divalent DNA vaccine. In addition, the co-administration of the multivalent HPV DNA vaccine according to the embodiment of the present invention and BD-121A (that is, the vaccine adjuvant according to the embodiment of the present invention) can significantly increase the T-cell specific immune response of the HPV E6 / E7 antigen, and also Will show a more significant anti-cancer effect.

Therefore, a vaccine component containing a multivalent HPV DNA vaccine according to an embodiment of the present invention, a DNA vaccine, and BD-121 or BD-121A as a vaccine adjuvant may be effectively used to prevent various HPV infections, and to treat diseases that have a cause such as cervical cancer. Symptoms of HPV infection at risk of a fatal disease such as.

Although the components of the new multivalent HPV vaccine have been described with reference to specific examples, those are only suitable for illustrative purposes. Therefore, those skilled in the art understand that various modifications and changes can be made without departing from the spirit and scope of the invention as defined by the scope of the added patent application. Therefore, the actual protection scope of the invention should be determined by the scope of the accompanying patent application. The scope of technology is determined.

FIG. 1 is a series of schematic diagrams showing the structures of three fusion proteins (BD-14A, BD-14B, and BD-14C) included in a multivalent HPV DNA vaccine according to an embodiment;

2A is a schematic diagram showing a structure of a vaccine adjuvant BD-121 according to an embodiment;

2B is a schematic diagram showing a structure of a vaccine adjuvant BD-121A according to the embodiment;

FIG. 3A is a series of graphs showing the expression of fms-like tyrosine kinase-3 (hereinafter referred to as Flt3L) after converting COS-7 with each of BD-14A, BD-14B and BD-14C plastids according to an embodiment The amount of ELISA assay;

FIG. 3B is an image showing the results of an immunoblotting analysis performed on the cell lysate that disrupted the cells of FIG. 3A, using an anti-Flt3L antibody;

FIG. 4A shows a vaccination schedule of an experiment for performing an immunological response analysis of a multivalent HPV DNA vaccine (BD-14) according to an embodiment; FIG.

FIG. 4B is a graph showing the results of an enzyme-linked immunospot analysis, in which spleen cells extracted from mice vaccinated with a multivalent HPV DNA vaccine (BD-14) according to an example and a conventional bivalent HPV DNA vaccine are analyzed These vaccines will specifically respond to E6 / E7 of various types of HPV;

5A shows an experimental vaccination schedule based on an analysis of anti-cancer effects of an example of a multivalent HPV DNA vaccine (BD-14) against HPV-induced cancer;

FIG. 5B is a chart showing the volume change of tumor tissues over time in tumor xenograft mice. These mice received empty-body (pGX27), multivalent HPV DNA vaccine (BD-14) according to the embodiment , And traditional 2-valent HPV DNA vaccines;

FIG. 5C is a graph showing survival rates recorded by time of tumor xenograft mice, which were vaccinated (pGX27), multivalent HPV DNA vaccine (BD-14) according to the example, and conventional 2 HPV DNA vaccine;

FIG. 6A is a series of graphs showing the concentration of IL-12 and IL-21 in the culture supernatant of COS-7 cells transferred with a vaccine adjuvant (that is, an hBD-121 construct) according to an example ELISA test results;

FIG. 6B is a series of diagrams illustrating IL-12 and IL- in culture supernatants of COS-7 cells transferred with a vaccine adjuvant (that is, an mBD-121 construct) according to an example 21 concentration ELISA test results

FIG. 6C shows the expression levels of IL-12 and IL-21 in a cell lysate transferred to COS-7 cells with a vaccine adjuvant (that is, an hBD-121 construct and an mBD-121 construct) according to an example. Immunoblotting results;

7A shows a vaccination schedule for confirming the anti-cancer effect of the DNA vaccine component according to the embodiment;

FIG. 7B is a chart showing that when the multivalent HPV DNA vaccine (BD-14) is administered alone or in combination with a vaccine adjuvant (BD-121A) according to the examples, some E6 / E7 exhibiting an immune response particularly for various HPVs ELISA spot analysis results of spleen cells;

8A shows a vaccination schedule for comparison of anti-cancer effects between a DNA vaccine component and a conventional bivalent vaccine component according to an embodiment;

FIG. 8B is a graph showing comparison results of changes in tumor tissue volume over time when the multivalent HPV DNA vaccine components are administered according to the embodiment; FIG.

8C is a graph showing comparison results of survival rates in experimental animals over time when the multivalent HPV DNA vaccine components are administered according to the embodiment;

9A shows a vaccination schedule of an animal experiment confirming the mechanism of action of a multivalent HPV DNA vaccine component according to an embodiment;

FIG. 9B is a graph showing that when the multivalent HPV DNA vaccine component is administered according to the embodiment, the experimental animals use anti-CD4 antibody and anti-CD8 antibody to remove CD4 T cells and CD8 T cells, respectively. Changes in tumor volume over time;

FIG. 10A shows a vaccination schedule of an animal experiment for examining anti-cancer effects when the HPV DNA vaccine component according to the embodiment is combined with IL-7; and

FIG. 10B is a series of graphs showing the results of examination of changes in the volume of cancer cells over time when they are administered alone or in combination with IL-7 according to the HPV DNA vaccine component according to the embodiment.

Claims (20)

A multivalent human papilloma virus (HPV) DNA vaccine component, comprising polynucleotides that can encode HPV type 6, 11, 16, 18, 39, 45, and 56 early protein antigens 6 (E6) or their immunogens, respectively Sexual fragments; and early protein antigens 7 (E7) or immunogenic fragments thereof of HPV types 6, 11, 16, 18, 39, 45, and 56, wherein E6 and E7 do not have wild-type functions. The multivalent human papillomavirus (HPV) DNA vaccine component according to claim 1, further comprising a polynucleotide, which can encode HPV types 31, 33, 35, 51, 52, 58 and 59 or their immunity, respectively. At least one early protein antigen 6 (E6) selected from the group consisting of original fragments; and at least one selected from the group consisting of HPV types 31, 33, 35, 51, 52, 58 and 59 or immunogenic fragments thereof Early protein antigen 7 (E7), of which E6 and E7 do not have wild-type functions. The multivalent human papilloma virus (HPV) DNA vaccine component as described in claim 1 or claim 2, wherein E6 and E7 are each divided into N-terminal fragment and C-terminal fragment, and the E6 / E7 promiscuously ordered antigens are sorted randomly Unit form expression. The multivalent human papillomavirus (HPV) DNA vaccine component as described in claim 3, wherein the E6 / E7 promiscuous sequencing antigen unit is a polypeptide, and the N-terminal fragment and the C-terminal fragment of E6 and E7 are the N of E6 (E6N). The sequence of the E-terminal fragment-the C-terminal fragment of E7 (E7C)-the N-terminal fragment of E7 (E7N)-the C-terminal fragment of E6 (E6C) are mutually connected. The multivalent human papilloma virus (HPV) DNA vaccine component as described in claim 3, wherein at least two E6 / E7 promiscuous sequencing antigen units of human papilloma virus are linked to each other in the form of an expressed fusion protein. The multivalent human papilloma virus (HPV) DNA vaccine component according to claim 5, wherein the E6 / E7 promiscuously ordered antigenic unit or fusion protein further comprises a signal sequence. The multivalent human papilloma virus (HPV) DNA vaccine component as described in claim 5, wherein the E6 / E7 promiscuous sequencing antigen unit or fusion protein further comprises Flt3L. The multivalent human papilloma virus (HPV) DNA vaccine component as described in claim 1, which also contains IL-7. The multivalent human papilloma virus (HPV) DNA vaccine component according to claim 1, further comprising at least one vaccine adjuvant acceptable to the pharmaceutical industry. The multivalent human papilloma virus (HPV) DNA vaccine component as described in claim 9, wherein the vaccine adjuvant is a vaccine adjuvant for simulating a specific immune response of T lymphocytes, comprising IL-12 protein and IL-21 The protein is used as an active ingredient, or a polynucleotide encoding an IL-12 protein or a polynucleotide encoding an IL-21 protein is used as an active ingredient. The multivalent human papilloma virus (HPV) DNA vaccine component as described in claim 10, wherein the vaccine adjuvant for simulating a specific immune response of T lymphocytes comprises at least one component selected from the group consisting of: -An IL-12 protein and an IL-21 protein consisting of p35 (IL-12p35) and p40 (IL-12p40); One to three vectors, each containing a polynucleotide encoding a p35 link (IL-12p35) and a p40 link (IL-12p40), respectively, that can construct the IL-12 protein; A polynucleotide encoding an IL-21 protein; and mRNA molecules, each encoding IL-12p35, IL-12p40 and IL-21 proteins. The multivalent human papillomavirus (HPV) DNA vaccine component according to claim 10, wherein the IL-12p35 protein is human IL-12p35 consisting of an amino acid sequence of SEQ ID NO: 1. The multivalent human papilloma virus (HPV) DNA vaccine component according to claim 10, wherein the IL-12p40 protein is human IL-12p40 consisting of an amino acid sequence of SEQ ID NO: 2. The multivalent human papilloma virus (HPV) DNA vaccine component according to claim 10, wherein the IL-21 protein is human IL-21 composed of an amino acid sequence of SEQ ID NO: 3. The multivalent human papilloma virus (HPV) DNA vaccine component according to claim 10, wherein the vaccine adjuvant further comprises at least one component selected from the group consisting of: i) a MIP-1α protein; ii) a MIP-1α gene structure in which a polynucleotide encoding a MIP-1α protein is operably linked to a promoter; iii) A composite gene structure in which a polynucleotide encoding a MIP-1α protein is operably linked to IL-12p35, IL-12p40 through a polynucleotide or a linker that encodes a ribosome entry site (IRES) And at least one of the IL-21 proteins; and iv) An mRNA molecule encoding a MIP-1α protein. The multivalent human papilloma virus (HPV) DNA vaccine component as described in claim 15, wherein the MIP-1α gene structure is contained in a separate expression vector, or one to three each contains a gene encoding the IL-12 protein. Any one or more of the polynucleotides in the p35 (IL-12p35) and p40 (IL-12p40) vectors; and a polynucleotide encoding the IL-21 protein. The multivalent human papilloma virus (HPV) DNA vaccine component according to claim 16, wherein the MIP-1α protein is a human MIP-1α protein consisting of an amino acid sequence of SEQ ID NO: 10. A method for treating a disease caused by a human papillomavirus (HPV) infection, comprising administering to a person a multivalent HPV DNA vaccine according to Request 1 to Request 17 items. A method for treating a disease caused by human papillomavirus (HPV) infection as described in claim 18, wherein the disease caused by HPV infection is squamous cell carcinoma (SCC), adenocarcinoma, adenosquamous carcinoma, small cell carcinoma , Neuroendocrine tumor (NET), clear cell carcinoma, villous adenocarcinoma (VGA), non-cancerous malignancy, melanoma, lymphoma or cervical intraepithelial neoplasia (CIN). A method for treating a disease caused by human papillomavirus (HPV) infection as described in claim 18, wherein the vaccine component is administered by an in vivo electroporation method.