WO2019151760A1

WO2019151760A1 - Novel multivalent hpv vaccine composition

Info

Publication number: WO2019151760A1
Application number: PCT/KR2019/001263
Authority: WO
Inventors: 서용복; 심상희
Original assignee: 주식회사 에스엘백시젠
Priority date: 2018-02-02
Filing date: 2019-01-30
Publication date: 2019-08-08
Also published as: TWI731300B; KR102222582B1; TW201941786A; KR20190094106A

Abstract

The present invention relates to a novel multivalent HPV DNA vaccine, a fusion protein used therein, and a polynucleotide for encoding the fusion protein and, more particularly, to a multivalent HPV DNA vaccine composition comprising polynucleotides for encoding early protein antigen 6 (E6) of 6, 11, 16, 18, 39, 45 and 56-type human papillomaviruses (HPVs) or an immunogenic fragment thereof, and early protein antigen 7 (E7) or an immunogenic fragment thereof, respectively.

Description

New Multivalent HPV Vaccine Compositions

This application claims priority to Korean Patent Application No. 2018-0013328, filed February 2, 2018. This patent application is incorporated herein by reference in its entirety. The present invention relates to novel multivalent HPV DNA vaccines and fusion proteins used therein and to polynucleotides encoding them.

Cervical cancer is one of the leading causes of cancer death in women worldwide (Einstein et al ., Lancet Infect. Dis ., 9: 347-356, 2009; Parkin and Bray, Vaccine 24 (3S): 11-25 , 2007), about 75% of cases are caused by persistent infection with the most common high risk human papillomavirus (HPV) type, HPV16 and HPV18 (Schiffman et al ., Lancet , 370: 890-907, 2007; Forman et al ., Vaccine 30 (5S): F 12-23, 2012). HPV infection persistence is commonly associated with a lack of apparent HPV-specific T-cell immunity, and virus-specific T cells found in pre-malignant and malignant tumor patients are generally reported to be dysfunctional and sometimes even inhibitory (Trimble, Cancer Immunol. Immunother . CII 59: 799-803, 2010). These results suggest that impaired function of virus-specific T cells may be associated with the development of HPV-induced cervical cancer.

Cervical cancer occurs through the process of high risk HPV infection, viral persistence, clonal expansion and differentiation of persistently infected cells into pre-malignant lesions, and their progressive transformation into invasive cancer (Schiffman et al ., Lancet 370: 890-907, 2007). Pre-malignant cervical epithelial tumors 2 and 3 (CIN2 and 3), especially those tumors positive for HPV16, are considered high-grade lesions with a 30% chance of developing invasive cancer (Moscicki et al ., Vaccine 30 (5S): F 24-33, 2012). Therefore, there is an urgent need for an effective therapeutic vaccine to prevent serious complications of persistent HPV infection and to root out HPV-associated tumors.

Two HPV vaccines currently on the market in Korea are tetravalent vaccine (gadsil) and divalent vaccine (subvarix), which contain L1 VLPs of HPV6, 11, 16, and 18 types of vaccine. Contains L1 VLPs of

HPV

16 and 18. However, HPV6 and 11 are the main causes of genital warts, but HPV6 and 11 are low-risk HPVs that are not related to cervical cancer. Therefore, both vaccines are bivalent vaccines that prevent

HPV types

16 and 18.

HPV E6 and E7, on the other hand, act as viral oncoproteins by binding to and inhibiting tumor suppressor proteins p53 and retinoblastoma (pRb), respectively (Yugawa and Kiyono, Rev. Med. Virol ., 19: 97- 113, 2009). The viral oncoproteins are considered ideal targets for therapeutic vaccines against CIN2 / 3 and cervical cancer because these proteins not only induce tumorigenesis but also they are constitutively expressed in HPV-infected pre-malignant and malignant cells. Yugawa and Kiyono, Rev. Med. Virol ., 19: 97-113, 2009). Degeneration of cervical lesions is associated with cellular immune responses but not humoral immune responses (Deligeoroglou et al ., Infect. Dis. Obstet.Gynecol ., 2013: 540850, 2013; Woo et al ., Int. J. Cancer , 126: 133-141, 2010), therapeutic vaccines capable of selectively inducing potent E6 / E7-specific T-cell immunity are highly preferred.

As a vaccine currently being developed using the HPV E6 / E7 antigen, there is a DNA vaccine composition using the HPV 16/18 E6 / E7 antigen disclosed in Korean Patent Publication No. 2017-0045254. However, since both the commercial vaccine composition and the DNA vaccine composition using the HPV 16/18 E6 / E7 antigen target only the

highest risk type

16 and 18 HPV, they cover only about 70% of all cervical cancers. .

Therefore, there is an urgent need to develop a multivalent vaccine capable of eliciting an immune response against various HPV types, which poses a risk of almost all cervical cancers.

Accordingly, an object of the present invention is to provide a multivalent HPV DNA vaccine that is effective for the prevention and treatment of diseases caused by cervical cancer and other HPV infection by inducing an immune response against more various types of HPV. However, the scope of the present invention is not limited by the above object.

According to one aspect of the invention, early protein antigen 6 (E6) or immunogenic fragments of 6, 11, 16, 18, 39, 45 and 56 human papillomavirus (HPV), and early protein antigen 7 (E7) or Multivalent HPV DNA vaccine compositions are provided, comprising polynucleotides each encoding an immunogenic fragment thereof.

According to one aspect of the invention, human papilloma virus selected from the group consisting of 6, 11, 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58 and 59 type N-terminal fragments and C-terminal fragments of early protein 6 (E6) and early protein antigen 7 (E7), respectively, of the N-terminal fragment (E6N) -E7's C-terminal fragment (E7C) -E7's N -Terminal fragment (E7N)-polypeptides linked in sequence of C-terminal fragments (E6C) of E6 are E6 / E7 antigenic units, wherein at least four types of HPV antigenic units of the HPVs are linked by a linker Fusion proteins are provided.

According to one aspect of the invention there is provided a polynucleotide encoding the fusion protein.

According to another aspect of the invention there is provided an expression vector operably linked to the expression regulatory region of the polynucleotide.

According to another aspect of the invention, the polynucleotide encoding the fusion protein comprises an expression vector operably linked to a promoter, a plurality of polynucleotides to include all the polynucleotides encoding the E6 / E7 antigen unit of the 14 type A 14-valent HPV DNA vaccine composition comprising an expression vector is provided.

According to another aspect of the invention, there is provided a method of treating a disease caused by HPV infection comprising administering the multivalent HPV DNA vaccine composition or the 14 valent HPV DNA vaccine composition to a subject.

The multivalent HPV DNA vaccine according to an embodiment of the present invention normally expressed the E6 / E7 shuffled protein, which is an antigen protein contained therein, despite the complex structure, and actually T cell specificity for various types of HPV E6 / E7 antigens. The immune response was successfully induced and the anticancer activity analysis of the cancer model expressing the high risk group HPV16 E6 / E7 antigen showed the same or better anticancer effect than the conventional bivalent DNA vaccine.

1 is a schematic diagram showing the structure of three fusion proteins (BD-14A, BD-14B and BD-14C) included in a multivalent HPV DNA vaccine according to an embodiment of the present invention.

Figure 2a is a schematic diagram showing the structure of the vaccine immunoadjuvant BD-121 according to an embodiment of the present invention, Figure 2b is a schematic diagram showing the structure of the vaccine immunoadjuvant BD-121A according to an embodiment of the present invention.

Figure 3a is a graph showing the results of analyzing the expression level of Flt3L by ELISA after transfecting COS-7 cells with BD-14A, BD-14B and BD-14C plasmid according to an embodiment of the present invention, 3b is a photograph showing the results of Western blot analysis using the anti-Flt3L antibody to the cell lysate in which the cells of FIG. 3a were crushed.

4A shows the vaccination schedule of an experiment for analyzing the immune response of a multivalent HPV DNA vaccine (BD-14) according to one embodiment of the present invention.

4B shows the E6 / E of each type of HPV in splenocytes extracted from mice vaccinated with a mock vector, a multivalent HPV DNA vaccine (BD-14) according to one embodiment of the invention and a conventional bivalent HPV DNA vaccine. It is a graph showing the result of ELISPOT analysis of the number of splenocytes specifically responding to E7.

Figure 5a shows the vaccination schedule of the experiment to analyze the anticancer effect of HPV-induced cancer of the multivalent HPV DNA vaccine (BD-14) according to an embodiment of the present invention.

Figure 5b shows the volume of tumor tissue over time of xenograft mice inoculated with the empty vector (pGX27), a multivalent HPV DNA vaccine (BD-14) according to one embodiment of the present invention and a conventional bivalent HPV DNA vaccine. It is a graph recording the change of.

Figure 5c is a graph recording the survival rate over time of xenograft mice vaccinated with the empty vector (pGX27), a multivalent HPV DNA vaccine (BD-14) and a conventional bivalent HPV DNA vaccine according to an embodiment of the present invention. to be.

Figure 6a is a graph showing the results of ELISA analysis of the concentration of IL-12 and IL-21 in the culture supernatant of COS-7 cells transfected with a vaccine adjuvant hBD-121 construct according to an embodiment of the present invention.

Figure 6b is a graph showing the results of analysis by ELISA the concentration of IL-12 and IL-21 in the culture supernatant of COS-7 cells transfected with a vaccine immunoadjuvant mBD-121 construct according to an embodiment of the present invention.

Figure 6c Western blot analysis of the expression level of IL-12 and IL-21 in the cell lysate of the vaccine immunoadjuvant hBD-121 construct and COS-7 cells transfected with mBD-121 according to an embodiment of the present invention It shows the result confirmed through.

Figure 7a shows the vaccination schedule for confirming the anticancer effect of the DNA vaccine composition according to an embodiment of the present invention.

Figure 7b shows the E6 / E7 specific immune response of each type of HPV upon administration of the multivalent HPV DNA vaccine (BD-14) alone or in combination with the BD-14 and the vaccine adjuvant BD-121A according to one embodiment of the present invention. It is a graph which shows the result of analyzing the number of splenocytes by ELISPOT analysis.

Figure 8a shows the vaccination schedule for comparing the anticancer effect of the DNA vaccine composition and the conventional bivalent vaccine composition according to an embodiment of the present invention.

Figure 8b is a graph showing the change in volume of the tumor tissue over time when administering the multivalent HPV DNA vaccine composition according to an embodiment of the present invention compared with the conventional bivalent vaccine composition.

Figure 8c is a graph showing the survival rate over time of the test animal compared to the conventional bivalent vaccine composition when administering the multivalent HPV DNA vaccine composition according to an embodiment of the present invention.

Figure 9a shows the vaccination schedule of the animal experiment to confirm the mechanism of action of the multivalent HPV DNA vaccine composition according to an embodiment of the present invention.

Figure 9b shows the time course when the multivalent HPV DNA vaccine composition according to one embodiment of the present invention is administered to an experimental animal deficient in CD4 T cells and CD8 T cells by administering an anti-CD4 antibody or an anti-CD8 antibody, respectively. The graph showing the volume of the tumor according to.

Figure 10a shows the vaccination schedule of the animal experiment to investigate the anticancer activity when used in combination with the multivalent HPV DNA vaccine composition according to an embodiment of the present invention IL-7.

Figure 10b is a graph showing the results of examining the volume of cancer cells over time when the multivalent HPV DNA vaccine composition according to an embodiment of the present invention alone or in combination with IL-7.

According to one aspect of the invention, early protein antigen 6 (E6) or immunogenic fragments of 6, 11, 16, 18, 39, 45 and 56 human papillomavirus (HPV), and early protein antigen 7 (E7) or Polyvalent HPV DNA vaccine compositions are provided, each comprising a polynucleotide encoding an immunogenic fragment thereof, wherein E6 and E7 do not have a wild-type function.

The multivalent HPV DNA vaccine composition comprises one or more HPV early protein antigen 6 (E6) selected from the group consisting of 31, 33, 35, 51, 52, 58 and 59 human papillomavirus (HPV) or Immunogenic fragments, and polynucleotides encoding early protein antigen 7 (E7) or immunogenic fragments thereof, respectively, which further add E6 and E7 also do not have wild-type function.

In the multivalent HPV DNA vaccine composition, the E6 and E7 can be expressed in the form of randomly shuffled E6 / E7 shuffled antigenic units divided into N-terminal fragments and C-terminal fragments, respectively, and the E6 / E7 shuffle. De antigenic monomers are N-terminal fragments of E6 and E7 and C-terminal fragments of N-terminal fragment of E6 (E6N) -C7-terminal fragment of E7 (E7C) -N7-terminal fragment of E7 (E7N) -E6 It may be a polypeptide linked in the order of the C-terminal fragment (E6C) of.

In the multivalent HPV DNA vaccine composition, at least two, three or more, or four or more E6 / E7 shuffled antigenic units of human papillomaviruses may be linked and expressed in the form of a fusion protein.

In the multivalent HPV DNA vaccine composition, the E6 / E7 shuffled antigenic unit or the fusion protein may further comprise a signal sequence, and the E6 / E7 shuffled antigenic unit or the fusion protein further comprises Flt3L. can do.

The multivalent HPV DNA vaccine composition may further comprise IL-7. The present inventors confirmed that the anti-cancer effect is significantly increased when IL-7 is administered in combination with the multivalent HPV DNA vaccine according to an embodiment of the present invention (see FIG. 10B).

The multivalent HPV DNA vaccine composition of the present invention may further comprise one or more pharmaceutically acceptable vaccine adjuvants.

In the multivalent HPV DNA vaccine composition, the vaccine immunoadjuvant includes IL-12 protein and IL-21 protein as an active ingredient or polynucleotide encoding the IL-12 protein and polynucleotide encoding the IL-21 protein. It may be a vaccine immunoadjuvant for promoting T lymphocyte specific immune response comprising as an active ingredient.

In the multivalent HPV DNA vaccine composition, the T lymphocyte specific immune response promoting vaccine immunoadjuvant may include one or more selected from the group consisting of:

IL-12 protein and IL-21 protein consisting of p35 chain (IL-12p35) and p40 chain (IL-12p40);

One to three vectors comprising a polynucleotide encoding the p35 chain (IL-12p35) and the p40 chain (IL-12p40) constituting the IL-12, and a polynucleotide encoding the IL-21 protein, respectively; And

MRNA molecules encoding the IL-12p35, IL-12p40 and IL-21 proteins, respectively.

In the multivalent HPV DNA vaccine composition, the IL-12p35 protein may be 90% or more homologous to human IL-12p35 consisting of the amino acid sequence represented by SEQ ID NO: 1.

In the multivalent HPV DNA vaccine composition, the IL-12p40 protein may be 90% or more in sequence homology with human IL-12p40 composed of the amino acid sequence represented by SEQ ID NO: 2.

In the multivalent HPV DNA vaccine composition, the IL-21 protein may be 90% or more of sequence homology with human IL-21 consisting of the amino acid sequence of SEQ ID NO.

In the multivalent HPV DNA vaccine composition, the vaccine immunoadjuvant may further comprise one or more selected from the group consisting of:

i) MIP-1α protein;

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

iii) a complex gene construct in which the polynucleotide encoding the MIP-1α protein is operably linked to any one or more of the IL-12p35, IL-12p40 and IL-21 proteins by a polynucleotide encoding an IRES or linker peptide T; And

iv) mRNA molecule encoding MIP-1α protein.

In the multivalent HPV DNA vaccine composition, the MIP-1α gene construct is included in a separate expression vector, or encodes the p35 chain (IL-12p35) and p40 chain (IL-12p40) constituting the IL-12, respectively. It may be included in any one or more of one to three vectors containing a polynucleotide and a polynucleotide encoding the IL-21 protein, respectively.

In the multivalent HPV DNA vaccine composition, the MIP-1α protein may be 90% or more homologous to a human MIP-1α protein consisting of an amino acid sequence of SEQ ID NO: 10.

As used herein, the term "HPV" is a human papilloma virus, a DNA-based virus with a diameter of 52-55 nm, which infects the skin or subcutaneous of humans and other animals. To date, more than 130 HPV have been identified (zur Hausen H., Vaccine 24 Suppl 3: S3, 2006). HPV is transmitted through keratinocytes and mucous membranes of the skin. Most of the known HPVs do not show any signs in humans, but some human papillomaviruses (HPVs) can cause papilloma in humans. In addition, a few human papillomaviruses cause cancers such as cervical cancer and testicular cancer. Human papillomavirus 16 (HPV 16) and human papillomavirus 18 (HPV 18) are found in 70% of cervical cancer patients worldwide. Such HPV viruses are classified as high risk. The DNA of HPV contains 8,000 base pairs and is surrounded by a capsid protein of a pentameric rather than lipid membrane, which consists of two structural proteins, L1 and L2, which are expressed later in the viral replication cycle. . There are eight open reading frames (ORFs) in every HPV genome, and each ORF is divided into three functional sites. It consists of early proteins E1-E7, genes required for virus replication, genes L1-L2, which express structural proteins constituting virions, and finally LCR, which regulates virus replication and transcription.

As used herein, the term “E6” is one of the early expression proteins required for the replication of HPV, which binds to p53 and promotes ubiquitination of p53, thereby inhibiting the function of p53 as a cancer tumor suppressor gene. It also induces the degradation of BAK, a pro-apoptotic protein. In addition, it plays a role of activating the cell cycle of the host cell through the activation of telomerase.

As used herein, the term "E7" is one of the early expression proteins required for the replication of HPV, and interacts with retinoblastoma (RB) to degrade RB. This releases E2F, a transcriptional promoter that has been inhibited by RB. Moreover, cyclin E (cycilin E) and cyclin A (cycilin A) acting on the cell cycle S phase activates the cell cycle of the host cell.

As used herein, the term "immunogenic fragment" refers to a fragment of a fragment of the full-length antigenic protein that is capable of functioning as an antigen, ie, capable of eliciting an antigen-specific immune response.

In the fusion protein, the N-terminal fragment and the C-terminal fragment may overlap 10 to 30 aa, and since the antigen unit is a shuffled protein, it retains its function as an antigen. It lacks the intrinsic functions (p53 and pRb binding functions) of wild type E6 and E7 proteins.

In the fusion protein, the antigen-linked protein may be added with Flt3 (fms-like tyrosine kinase-3) ligand (Flt3L) at the N-terminus and secretory signal sequence may be added. The secretory signal sequence induces the secretion of the recombinant protein expressed in the cell out of the cell, and may be a tissue plasminogen activator (tPA) signal sequence, HSV gDs (herpes simplex virus glycoprotein Ds) signal sequence, or growth hormone signal sequence. .

The fusion protein may further comprise polynucleotides encoding one or more immune enhancing peptides, wherein the immune enhancing peptides are CD28, inducible costimulator (ICOS), cytotoxic T lymphocyte associated protein 4 (CTLA4), or programmed (PD1). cell death protein 1), B and T lymphocyte associated protein (BTLA), death receptor 3 (DR3), 4-1BB, CD2, CD40, CD30, CD27, signaling lymphocyte activation molecule (SLAM), 2B4 (CD244), NKG2D ( natural-killer group 2, member D) / DAP12 (DNAX-activating protein 12), TIM1 (T-Cell immunoglobulin and mucin domain containing protein 1), TIM2, TIM3, TIGIT, CD226, CD160, LAG3 (lymphocyte activation gene 3) , B7-1, B7-H1, glucocorticoid-induced TNFR family related protein (GITR), Flt3 ligand (fms-like tyrosine kinase 3 ligand), flagellin, herpesvirus entry mediator (HVEM) or OX40L [ligand for CD134] (OX40), CD252] or a linkage of two or more thereof.

In the fusion protein, the linker is preferably a linker peptide, which includes (G ₄ S) _n (unit: SEQ ID NO: 32, n is an integer of 1 to 10), (GS) _n (n is 1 To integers from 10), (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), and GSSGGSGSSGGSGGGDEADGSRGSQKAGVDE (SEQ ID NO: 50).

As used herein, the term "fusion protein" refers to a recombinant protein in which two or more proteins or domains responsible for specific functions within the protein are linked. A linker peptide having a generally flexible structure may be inserted between the two or more proteins or domains, provided that the flexible peptide linker does not limit the function of the polypeptide to which it is linked and does not inhibit the expression of the fusion protein. Any may be used, and specific examples are as described above.

The polynucleotide may be deoxyribonucleic acid (DNA) or ribonucleic acid (RNA).

According to another aspect of the present invention, an expression vector is provided in which the polynucleotide is operably linked to a regulatory sequence.

As used herein, the term “operably linked to” refers to such nucleic acid sequence (eg, in an in vitro transcription / translation system or in a host cell) in such a way that its expression can be achieved. It is linked to a sequence.

The term "regulatory sequence" is meant to include promoters, enhancers and other regulatory elements (eg, polyadenylation signals). The regulatory sequence indicates that the target nucleic acid is constantly expressed in many host cells, that the target nucleic acid is expressed only in specific tissue cells (eg, tissue specific regulatory sequences), and Directing expression (eg, inducible regulatory sequences) by specific signals is included. It will be appreciated by those skilled in the art that the design of the 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 expression vector of the present invention can be introduced into a host cell to express the fusion protein. Regulatory sequences that allow expression in the eukaryotic and prokaryotic cells are well known to those skilled in the art. As mentioned above, these usually include regulatory sequences responsible for transcription initiation and, optionally, poly-A signals responsible for transcription termination and stabilization of the transcript. Additional regulatory sequences may include translation enhancers and / or naturally-combined or heterologous promoter regions in addition to transcriptional regulators. For example, possible regulatory sequences that allow expression in mammalian host cells include the CMV-HSV thymidine kinase promoter, SV40, RSV-promoter (Louse sarcoma virus), human kidney urea 1α-promoter, glucocorticoid-induced MMTV- Promoters (molony mouse tumor virus), metallothionein-induced or tetracycline-induced promoters or amplification agents such as CMV amplifiers or SV40-amplifiers. For expression in neurons, it is contemplated that nerve microfiber-promoter, PGDF-promoter, NSE-promoter, PrP-promoter or thy-1-promoter may be used. Such promoters are known in the art and described in Charron, J. Biol. Chem. 270: 25739-25745, 1995. For prokaryotic expression, a number of promoters have been disclosed, including lac-promoters, tac-promoters or trp promoters. In addition to factors that can initiate transcription, the regulatory sequences include transcription termination signals, such as the SV40-poly-A site or the TK-poly-A site, downstream of the polynucleotide according to one embodiment of the invention. You may. In this document, suitable expression vectors are known in the art, for example, the Okayama-Berg cDNA expression vector pcDV1 (Parmacia), pRc / CMV, pcDNA1, pcDNA3 (In-vitrogene), pSPORT1 (GIBCO BRL). ), pGX27 (Patent No. 1442254), pX (Pagano (1992) Science 255, 1144-1147), yeast two-hybrid vectors, such as pEG202 and dpJG4-5 (Gyuris (1995) Cell 75, 791 -803) or prokaryotic expression vectors such as lambda gt11 or pGEX (Amersham-Pharmacia). In addition to the nucleic acid molecules of the invention, the vector may further comprise a polynucleotide encoding a secretory signal peptide. The secretory signal peptides are well known to those skilled in the art. And, depending on the expression system used, a leader sequence that can lead the fusion protein to the cell compartment is combined with the coding sequence of the polynucleotide according to one embodiment of the invention, preferably the translated protein or its It is a leader sequence capable of secreting proteins directly around the cytoplasm or into extracellular media.

In addition, the vectors of the present invention can be prepared, for example, by standard recombinant DNA techniques, which include, for example, blunt- and adhesive-terminal ligation, restriction enzyme treatment to provide appropriate ends, and inappropriate. In order to prevent binding, phosphate group removal by alkaline four-stage treatment and enzymatic linkage by T4 DNA ligase are included. The vector of the present invention may be prepared by recombining a DNA encoding a signal peptide obtained by chemical synthesis or genetic recombination technology, or a DNA encoding a fusion protein according to an embodiment of the present invention into a vector containing an appropriate regulatory sequence. have. The vector containing the control sequence can be purchased or produced commercially, in one embodiment of the present invention was used pGX27 (Korean Patent No. 1442254), a vector for preparing a DNA vaccine.

The expression vector according to an embodiment of the present invention may be an expression vector capable of expressing the fusion protein in a host cell, wherein the expression vector is a plasmid vector, a viral vector, a cosmid vector, a phagemid vector, an artificial human It may be in any form such as a chromosome.

According to another aspect of the present invention, a polynucleotide encoding the fusion protein comprises an expression vector operably linked to a promoter, and a plurality of polynucleotides each encoding all the E6 / E7 antigenic units of the 14 types. A 14-valent HPV DNA vaccine composition comprising an expression vector of is provided.

The 14-valent HPV DNA vaccine composition includes three expression vectors each constructed by cloning a polynucleotide encoding three fusion proteins linked to 4 to 5 E6 / E7 antigen units of the 14-type HPV, respectively. can do.

The three expression vectors may be configured as follows, but are not limited thereto, and various HPV types may be combined:

i) N-terminal fragment and C-terminal fragment of early antigen 6 (E6) and early antigen 7 (E7) of type 16 human papillomavirus (HPV16), respectively, the C-terminal of N-terminal fragment (16E6N) -E7 of E6 HPV16 E6 / E7 antigenic unit, linked to N-terminal fragment (16E7N) -E6 C-terminal fragment (16E6C) of fragment (16E7C) -E7, early antigen 6 (E6) of type 18 human papillomavirus (HPV18), and N-terminal fragment and C-terminal fragment of each of early antigen 7 (E7) are N-terminal fragment (18E6N) -E7 C-terminal fragment (18E7C) -E7 N-terminal fragment (18E7N) -E6 N-terminal fragments and C-terminal fragments of HPV18 E6 / E7 antigenic units linked in the order of C-terminal fragments (18E6C), early antigen 6 (E6) and early antigen 7 (E7), respectively, of type 35 human papillomavirus (HPV35) HPV35 E6 / E7 antigenic monomers concatenated in this order of N-terminal fragment (35E6N) -E7 C-terminal fragment (35E7C) -E7 N-terminal fragment (35E7N) -E6 C-terminal fragment (35E6C) , N- of early antigen 6 (E6) and early antigen 7 (E7) of type 45 human papillomavirus (HPV45) The short and C-terminal fragments are in the order of the N-terminal fragment of E6 (45E6N) -E7 C-terminal fragment (45E7C) -E7 of N-terminal fragment (45E7N) -E6 C-terminal fragment (45E6C) N-terminal fragments and C-terminal fragments of linked HPV45 E6 / E7 antigenic units and early antigen 6 (E6) and early antigen 7 (E7) of type 58 human papillomavirus (HPV58), respectively, are N-terminal fragments of E6 (58E6N C-terminal fragment (58E7C) -E7 N-terminal fragment (58E7N) -E6 C-terminal fragment (58E6C) in the sequence of the first fusion linked HPV58 E6 / E7 antigen unit linked by linker peptide A first expression vector comprising a first gene construct in which a first nucleic acid molecule encoding a protein is operably linked to a promoter;

ii) the N-terminal fragment and the C-terminal fragment of early antigen 6 (E6) and early antigen 7 (E7) of type 31 human papillomavirus (HPV31), respectively, the C-terminal of N-terminal fragment (31E6N) -E7 of E6 HPV31 E6 / E7 antigenic unit, linked to N-terminal fragment (31E7N) -E6 C-terminal fragment (31E6C) of fragment (31E7C) -E7, early antigen 6 (E6) of type 33 human papillomavirus (HPV33), and The N-terminal fragment and C-terminal fragment of each of early antigen 7 (E7) are N-terminal fragment (33E6N) of E6-C-terminal fragment (33E7C) of E7-N-terminal fragment (33E7N) of E7 -E6 N-terminal fragments and C-terminal fragments of HPV33 E6 / E7 antigenic units linked in the order of C-terminal fragments (33E6C), early antigen 6 (E6) and early antigen 7 (E7), respectively, of type 6 human papillomavirus (HPV6) HPV6 E6 / E7 antigenic monomers concatenated in this order of N-terminal fragment (6E6N) -E7 C-terminal fragment (6E7C) -E7 N-terminal fragment (6E7N) -E6 C-terminal fragment (6E6C) Terminal of early antigen 6 (E6) and early antigen 7 (E7) of type 11 human papillomavirus (HPV11) The fragments and C-terminal fragments are connected in the order of N-terminal fragment (11E6N) of E6-C-terminal fragment (11E7C) of E7-N-terminal fragment (11E7N) of E7-C-terminal fragment (11E6C) of E6 N-terminal fragments and C-terminal fragments of HPV11 E6 / E7 antigenic monomer, and early antigen 6 (E6) and early antigen 7 (E7) of type 52 human papillomavirus (HPV52), respectively, are N-terminal fragments of E6 (52E6N) C-terminal fragment (52E7C) of E7 -N-terminal fragment (52E7N) of E7-Second fusion protein in which the HPV52 E6 / E7 antigenic unit connected in sequence of C-terminal fragment (52E6C) of E6 is linked by a linker peptide A second expression vector comprising a second gene construct, the second nucleic acid molecule encoding a second nucleic acid molecule operably connected to the promoter; And

iii) the N-terminal fragment and C-terminal fragment of early antigen 6 (E6) and early antigen 7 (E7) of type 39 human papillomavirus (HPV39), respectively, and the C-terminal of N-terminal fragment (39E6N) -E7 of E6 HPV39 E6 / E7 antigenic unit, linked to N-terminal fragment (39E7N) -E6 C-terminal fragment (39E6C) of fragment (39E7C) -E7, early antigen 6 (E6) of type 51 human papillomavirus (HPV51), and N-terminal fragment and C-terminal fragment of each of early antigen 7 (E7) are N-terminal fragment (51E6N) of E6-C-terminal fragment (51E7C) of E7-N-terminal fragment (51E7N) of E7 -E6 N-terminal fragments and C-terminal fragments of HPV51 E6 / E7 antigenic units linked in the order of C-terminal fragments (51E6C), early antigen 6 (E6) and early antigen 7 (E7), respectively, of type 56 human papillomavirus (HPV56) HPV56 E6 / E7 antigenic monomers concatenated in this order of N-terminal fragment (56E6N) -E7 C-terminal fragment (56E7C) -E7 N-terminal fragment (56E7N) -E6 C-terminal fragment (56E6C) Early Antigen 6 (E6) and Early Antigen 7 (E7) of,, and Type 59 human papillomavirus (HPV59), respectively. N-terminal fragment and C-terminal fragment of N-terminal fragment (59E6N) -E7 C-terminal fragment (59E7C) -E7 N-terminal fragment (59E7N) -E7 C-terminal fragment (59E6C) A third expression vector comprising a third gene construct in which a third nucleic acid molecule encoding a third fusion protein linked in sequence by a linked HPV59 E6 / E7 antigenic unit is linked to a promoter.

In the vaccine composition, the first to third fusion proteins may be the secretion signal sequence and Flt3L added to the N-terminal. The secretion signal sequence is as described above.

The vaccine composition may comprise one or more pharmaceutically acceptable vaccine adjuvant. The vaccine adjuvant includes aluminum hydroxide, aluminum phosphate, alum (potassium aluminum sulfate), MF59, virosome, AS04 [a mixture of aluminum hydroxide and monophosphoryl lipid A (MPL)], AS03 (DL-α- mixtures of tocopherol, squalene and emulsifier polysorbate 80), CpG, Flagellin, Poly I: C, AS01, AS02, ISCOMs and ISCOMMATRIX.

As used herein, the term "adjuvant" or "vaccine adjuvant" refers to a pharmaceutical or immunological agent administered for the purpose of enhancing the immune response of the vaccine.

In addition, the vaccine immunoadjuvant includes IL-12 protein and IL-21 protein as an active ingredient or polynucleotides encoding the IL-12 protein and T lymphocytes comprising the polynucleotide encoding the IL-21 protein as an active ingredient. It may be a vaccine adjuvant for promoting a specific immune response.

In this case, the vaccine immunoadjuvant may include one or more selected from the group consisting of:

In addition, the above-described vaccine immunoadjuvant may include some of the IL-21p35, IL-12p40, and IL-21 as proteins, and the other may be used in combination with heterologous molecules such as expression vectors and / or mRNA molecules.

In this case, the one to three vectors may include a gene construct in which the polynucleotide is operably linked to a regulatory sequence such as a promoter to express the IL-12p35, IL-12p40 and IL-21. The vaccine adjuvant is a polynucleotide encoding the IL-12p35, IL-12p40 and IL-21, respectively, is inserted into a separate expression vector (3 vector system) or in one or two expression vectors (single vector or double vector) System) may consist of one to three vectors. Specific embodiments of such single to triple vector systems are as follows:

i) a fourth expression vector comprising fourth to sixth gene constructs to which the polynucleotides encoding the IL-12p35, IL-12p40 and IL-21 proteins are operably linked, respectively, to a promoter;

ii) fifth to seventh expression vectors each comprising the fourth to sixth gene constructs;

iii) an eighth expression vector and a ninth expression vector including two and the other of the fourth to sixth gene constructs, respectively;

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

v) a seventh gene construct in which the polynucleotide encoding the fusion protein of iv is operably linked to a promoter and an eighth gene construct in which the polynucleotide encoding the peptide of iv is operably linked to a promoter A tenth expression vector;

vi) an eleventh and twelfth expression vector comprising the seventh and eighth gene constructs, respectively;

vii) a ninth gene construct in which at least two of the polynucleotides encoding the IL-12p35, IL-12p40 and IL-21 proteins, respectively, are linked to an internal ribosomal entry site (IRES) and operably linked to a promoter ; And optionally a thirteenth expression vector comprising a tenth gene construct, wherein a polynucleotide not included in the ninth gene construct of the three polynucleotides is operably linked to a promoter; And

viii) a fourteenth expression vector comprising the ninth gene construct and optionally a fifteenth expression vector comprising the tenth gene construct.

In the vaccine composition, the IL-12p35 protein may be composed of an amino acid sequence of at least 90%, preferably at least 95%, of sequence homology with human IL-12p35 consisting of the amino acid sequence represented by SEQ ID NO: 1 It is also possible to use IL-12p35 from non-humans such as primates or apes that have a high degree of homology that will not induce an immune response. The IL-12p40 protein may be composed of an amino acid sequence of at least 90%, preferably at least 95%, of sequence homology with human IL-12p40 consisting of the amino acid sequence of SEQ ID NO: 2, inducing an immune response in the human body. It is also possible to use IL-12p40 from non-humans such as primates or apes with a high degree of homology that would not. The IL-12p35 and IL-12p40 may also be used in the sequence described in Korean Patent No. 0399728. The IL-21 protein may be composed of at least 90%, preferably at least 95%, amino acid sequence sequence homology with human IL-21 consisting of the amino acid sequence of SEQ ID NO: 3, induces an immune response in the human body It is also possible to use IL-21 from nonhumans, such as primates or apes with a high degree of homology that would not.

In the vaccine composition, the vaccine adjuvant may further comprise one or more selected from the group consisting of:

i) MIP-1α protein;

iii) the polynucleotide encoding the MIP-1α protein is operable by a polynucleotide encoding an IRES or linker peptide to any one or more of the polynucleotides encoding the IL-12p35, IL-12p40 and IL-21 proteins, respectively Complex genetic constructs; And

iv) mRNA molecule encoding MIP-1α protein.

The polynucleotide encoding the MIP-1α protein is operably linked to a polynucleotide encoding the IL-12p35, IL-12p40 and IL-21 proteins, respectively, or a separate gene operably linked through a polynucleotide encoding a peptide linker or an IRES. It may be provided in the form of a construct. In addition, the MIP-1α gene construct comprises a polynucleotide encoding the p35 chain (IL-12p35) and the p40 chain (IL-12p40) constituting the IL-12 and a polynucleotide encoding the IL-21 protein, respectively. It may be included in any one or more of one to three vectors containing. That is, the MIP-1α gene construct may be included in any one or more expression vectors selected from the group consisting of the fourth to fifteenth expression vectors described in the embodiment of the vaccine immunoadjuvant.

In the vaccine composition, the MIP-1α protein may be composed of an amino acid sequence of 90% or more, preferably 95% or more of sequence homology with the MIP-1α protein consisting of the amino acid sequence of SEQ ID NO: 10, the human body It is also possible to use non-humans such as primates or apes-derived MIP-1α proteins with a high degree of homology that will not induce an immune response.

The vaccine composition may further comprise IL-7.

The vaccine composition may further include a pharmaceutically acceptable adjuvant, excipient or diluent in addition to the carrier.

As used herein, the term "pharmaceutically acceptable" refers to a composition that is physiologically acceptable and does not normally cause an allergic reaction, such as gastrointestinal disorders, dizziness, or the like when administered to a human. Examples of such carriers, excipients and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, Polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. In addition, fillers, anti-coagulants, lubricants, wetting agents, fragrances, emulsifiers and preservatives may be further included.

The vaccine composition may further comprise a vaccine adjuvant commonly used in addition to the vaccine adjuvant, such vaccine adjuvant, such as aluminum hydroxide, aluminum phosphate, alum (potassium aluminum sulfate), MF59, virosome, AS04 [ Mixture of aluminum hydroxide and monophosphoryl lipid A (MPL)], AS03 (mixture of DL-α-tocopherol, squalene and polysorbate 80 as an emulsifier), CpG, Flagellin, Poly I: C, AS01, AS02, ISCOMs and ISCOMMATRIX and the like can be used.

In addition, the vaccine composition according to an embodiment of the present invention may be formulated using a method known in the art to enable rapid release, or sustained or delayed release of the active ingredient when administered to a mammal. Formulations include powders, granules, tablets, emulsions, syrups, aerosols, soft or hard gelatin capsules, sterile injectable solutions, sterile powder forms.

Vaccine compositions according to one embodiment of the invention can be administered by a variety of routes, for example, oral, parenteral, such as suppositories, transdermal, intravenous, intraperitoneal, intramuscular, intralesional, nasal, spinal administration It can also be administered in a sustained release or using an implantable device for continuous or repeated release. The frequency of administration can be administered once a day or divided into several times within the desired range, the administration period is not particularly limited.

The vaccine composition according to an embodiment of the present invention may be administered by general systemic administration or topical administration, for example, by intramuscular injection or intravenous injection, but, when provided as a DNA vaccine composition, most preferably an electroporator It can be injected using. The electroporator is a commercially available electroporator for injecting DNA drugs, such as Glinporator ^™ of IGEA, Italy, CUY21EDIT of JCBIO of Korea, SP-4a of Supertech of Switzerland, OrbiJector of SLVAXiGEN of Korea And the like can be used.

The route of administration of the vaccine composition according to an embodiment of the present invention may be administered via any general route as long as it can reach the target tissue. Such administration route may be, but is not limited to, parenteral administration, eg, intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intravitreal administration.

In addition, the multivalent HPV DNA vaccine composition according to one embodiment of the present invention may be administered in combination with an IL-7 protein or a polynucleotide encoding the same. Typically, since a DNA drug and a protein drug may have different administration routes, It may be administered in the formulation of, but may be packaged in a separate formulation and administered via other routes. For example, the DNA vaccine composition may be administered by intramuscular injection by electroporation, and the IL-7 protein may be administered according to a general protein drug administration method such as general intramuscular injection or intravenous administration, intraperitoneal administration. .

Vaccine compositions according to one embodiment of the invention may be formulated in a suitable form with a pharmaceutically acceptable carrier generally used. Pharmaceutically acceptable carriers include, for example, water, suitable oils, saline, carriers for parenteral administration such as aqueous glucose and glycols, and the like, and may further include stabilizers and preservatives. Suitable stabilizers include antioxidants such as sodium hydrogen sulfite, 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, if necessary according to the administration method or dosage form, suspensions, dissolution aids, stabilizers, isotonic agents, preservatives, adsorption agents, surfactants, diluents, excipients, pH adjusters, analgesics, buffers, Antioxidant etc. can be contained suitably. Pharmaceutically acceptable carriers and formulations suitable for the present invention, including those exemplified above, are described in detail in Remington's Pharmaceutical Sciences, latest edition.

Dosage to the patient of the vaccine composition depends on many factors including the patient's height, body surface area, age, the specific compound administered, sex, time and route of administration, general health, and other drugs administered simultaneously. Pharmaceutically active DNA can be administered in an amount of 100 ng / kg body weight (kg)-10 mg / kg body weight, more preferably from 1 to 500 μg / kg body weight, most Preferably from 5 to 50 μg / kg (body weight), the dosage can be adjusted in consideration of the above factors.

In addition, the vaccine composition of the present invention is administered in a therapeutically effective amount.

As used herein, the term “therapeutically effective amount” means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment, with an effective dose level of individual type and severity, age, sex, drug Can be determined according to the activity of the drug, the sensitivity to the drug, the time of administration, the route of administration and the rate of release, the duration of treatment, factors including the drug used concurrently and other factors well known in the medical field. The vaccine composition of the present invention may be administered at a dose of 0.1 mg / kg to 1 g / kg, more preferably at a dose of 1 mg / kg to 500 mg / kg and in a unit dose of 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg and the like. Meanwhile, the dosage may be appropriately adjusted according to the age, sex and condition of the patient.

In the treatment method, the disease caused by HPV infection is squamous cell carcinoma (SCC), adenocarcinoma, adenosquamous cell carcinoma, small cell carcinoma, neuroendocrine tumor (NET), free cell carcinoma, chorionic adenocarcinoma (VGA), Non-carcinoma malignancy, melanoma, lymphoma, or cervical epithelial tumor (CIN).

In the method of treatment, the vaccine composition may be administered by in vivo electroporation.

In order to solve the problem that conventional HPV vaccines cannot be universally applied to HPV-infected diseases such as cervical cancer, the present inventors have performed cervical intraepithelial neoplasia (CIN), cervical cancer, and vulvar intraepithelial cancer. Neoplasia (VIN), Vulvar cancer, Viginal intraepithelial neoplasia, Viginal cancer, and warts (Anogenital warts, genital warts) have major prevalences. , DNA, to cover all 18, 31, 33, 35, 39, 45, 51, 52, 56, 58 and 59 human papilloma virus (HPV) The vaccine was made of a multivalent HPV DNA vaccine consisting of three plasmids in which the E6 / E7 shuffled protein of each HPV could be expressed in the form of one large fusion protein per four or five types. All antigen, HPV16 and HPV18 as well as with respect to other types of HPV was experimentally proven to induce a T cell specific immune response. This result is very encouraging in that it is achieved using a shuffled antigenic protein that is not using the original antigenic protein, or an expression vector where multiple shuffled proteins express multiple shuffled antigenic proteins linked by only a linker. will be. Moreover, surprisingly, the multivalent HPV DNA vaccine according to one embodiment of the present invention is administered at a very low dose of 1/4 of the conventional bivalent vaccine, even though T cell specific immune responses against HPV16 and HPV18 similar to the conventional bivalent vaccine. In addition to,, 6, 11, 39, 45 and 56 type induced higher T cell specific immune response than that against HPV16 or HPV18. Therefore, the multivalent HPV DNA vaccine according to one embodiment of the present invention can be used very effectively as a prophylactic vaccine that can simultaneously prevent the infection of the seven types of HPV, as well as 31, 33, 35, 51, It can be efficiently used as a vaccine for the prevention of infection and the treatment of infection of one or more high risk HPVs selected from the group consisting of 52, 58 and 59 types.

Hereinafter, the present invention will be described in more detail with reference to 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 forms, and the following Examples and Experimental Examples to complete the disclosure of the present invention, the present invention It is provided to fully inform the person skilled in the art the scope of the invention.

실시예 1: 다가 HPV DNA 백신의 제조Example 1 Preparation of Multivalent HPV DNA Vaccines

In order to manufacture multivalent HPV DNA vaccines, the inventors of the

high risk group

6, 11, 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 56 E6 antigens of each type for expressing the early expression proteins E6 and E7 antigens of human,

type

58 and 59 human papilloma virus (HPV) in the form of shuffled proteins that do not exhibit the functions of wild type E6 and E7 And polynucleotides encoding the N-terminal fragment and the C-terminal fragment of the E7 antigen through PCR reaction, and then linked in the order shown in FIG. 1 and Table 1 to prepare three gene constructs. HPV DNA vaccine constructs were prepared by inserting them into the pGX-27 vector, respectively, named BD-14A, BD-14B, and BD-14C, and the composition comprising the three vectors was named BD-14. . The reason why the multivalent HPV DNA vaccine construct was prepared using the 3 vector system as described above is that when the size of the gene construct to be inserted is too large, the capacity of the pGX-27 vector is inefficient.

As shown in FIG. 1 and Table 1 below, each type of HPV E6 and E7 antigens are divided into N-terminal fragments and C-terminal fragments in which some sequences (20 aa) are overlapped, respectively, and then N-terminal fragments of E6 ( E6N) followed by a fusion polypeptide linked to the C-terminal fragment of E7 (E7C) by a (GS) ₅ linker peptide followed by an N-terminal fragment of E7 (E7N) followed by a C-terminal fragment of E6 (E6C). The structure was connected again, and four to five antigenic units of each type were constructed to be linked to the (GS) ₅ linker and included in one vector. The type of each subtype inserted into one expression vector is exemplarily described in Table 1, but this is merely exemplary and may be prepared in any other order.

Construction of the Multivalent HPV DNA Vaccine Construct of the Present Invention

구성요소Component		구체적인 구조Concrete structure	서열번호SEQ ID NO:		기원origin
구성요소Component		구체적인 구조Concrete structure	단백질protein	핵산Nucleic acid	기원origin
공통요소Common factor	링커Linker	(GS)₅ (GS) ₅	2020	2121	N/AN / A
	tPAtPA	tPA_1-22 tPA _1-22	2222	2323	Uniprot: P00750Uniprot: P00750
	Flt3LFlt3L	Flt3L_{27-182, △1-26} Flt3L _{27-182, △ 1-26}	2424	2525	Uniprot: P49771Uniprot: P49771
BD-14ABD-14A	16 E6E716 E6E7	16E6N_1-85-16E7C_41-105-(GS)₅-16E7N_1-60-16E6C_66-158 16E6N _1-85 -16E7C _41-105- (GS) ₅ -16E7N _1-60 -16E6C _66-158	2626	2727	GenBank: K02718.1GenBank: K02718.1
	18 E6E718 E6E7	18E6N_1-85-18E7C_41-98-(GS)₅-18E7N_1-60-18E6C_66-158 18E6N _1-85- 18E7C _41-98- (GS) ₅ -18E7N _1-60 -18E6C _66-158			GenBank: X05015.1GenBank: X05015.1
	35 E6E735 E6E7	35E6N_1-78-35E7C_42-99-(GS)₅-35E7N_1-61-35E6C_59-149 35E6N _1-78- 35E7C _42-99- (GS) ₅ -35E7N _1-61 -35E6C _59-149			GenBank: X74477.1GenBank: X74477.1
	45 E6E745 E6E7	45E6N_1-85-45E7C_41-106-(GS)₅-45E7N_1-60-45E6C_66-158 _{_{45E6N 1-85 -45E7C 41-10 6- (GS)}} 5 -45E7N 1-60 -45E6C 66-158			GenBank: X74479.1GenBank: X74479.1
	58 E6E758 E6E7	58E6N_1-85-58E7C_41-98-(GS)₅-58E7N_1-60-58E6C_66-149 58E6N _1-85 -58E7C _41-98- (GS) ₅ -58E7N _1-60 -58E6C _66-149			GenBank: D90400.1GenBank: D90400.1
BD-14BBD-14B	31 E6E731 E6E7	31E6N_1-85-31E7C_42-98-(GS)₅-31E7N_1-61-31E6C_66-149 31E6N _1-85- 31E7C _42-98- (GS) ₅ -31E7N _1-61 -31E6C _66-149	2828	2929	GenBank: J04353.1GenBank: J04353.1
	33 E6E733 E6E7	33E6N_1-85-33E7C_42-96-(GS)₅-33E7N_1-61-33E6C_66-149 33E6N _1-85 -33E7C _42-96- (GS) ₅ -33E7N _1-61 -33E6C _66-149			GenBank: M12732.1GenBank: M12732.1
	06 E6E706 E6E7	6E6N_1-85-6E7C_42-98-(GS)₅-6E7N_1-61-6E6C_66-150 6E6N _1-85 -6E7C _42-98- (GS) ₅ -6E7N _1-61 -6E6C _66-150			GenBank: X00203.1GenBank: X00203.1
	11 E6E711 E6E7	11E6N_1-85-11E7C_42-98-(GS)₅-11E7N_1-61-11E6C_66-150 11E6N _1-85 -11E7C _42-98- (GS) ₅ -11E7N _1-61 -11E6C _66-150			GenBank: M14119.1GenBank: M14119.1
	52 E6E752 E6E7	52E6N_1-85-52E7C_41-99-(GS)₅-52E7N_1-60-52E6C_66-148 52E6N _1-85 -52E7C _41-99- (GS) ₅ -52E7N _1-60 -52E6C _66-148			GenBank: X74481.1GenBank: X74481.1
BD-14CBD-14C	39 E6E739 E6E7	39E6N_1-85-39E7C_44-109-(GS)₅-39E7N_1-63-51E6C_66-158 39E6N _1-85 -39E7C _44-109- (GS) ₅ -39E7N _1-63 -51E6C _66-158	3030	3131	GenBank: M62849.1GenBank: M62849.1
	51 E6E751 E6E7	51E6N_1-83-51E7C_45-101-(GS)₅-51E7N_1-64-51E6C_64-151 51E6N _1-83 -51E7C _45-101- (GS) ₅ -51E7N _1-64 -51E6C _64-151			Uniprot : P26554(E6), P26558(E7)Uniprot: P26554 (E6), P26558 (E7)
	56 E6E756 E6E7	56E6N_1-86-56E7C_48-105-(GS)₅-56E7N_1-67-56E6C_67-155 56E6N _1-86 -56E7C _48-105- (GS) ₅ -56E7N _1-67 -56E6C _67-155			Uniprot : P24836(E6), P36833(E7)Uniprot: P24836 (E6), P36833 (E7)
	59 E6E759 E6E7	59E6N_1-85-59E7C_50-107-(GS)₅-59E7N_1-69-59E6C_66-160 59E6N _1-85 -59E7C _50-107- (GS) ₅ -59E7N _1-69 -59E6C _66-160			GenBank: CAA54849.1, CAA54850.1GenBank: CAA54849.1, CAA54850.1

실시예 2: 인간 IL-12 및 IL-21 발현 벡터의 제조Example 2: Preparation of Human IL-12 and IL-21 Expression Vectors

2-1: 단일벡터 시스템2-1: Single Vector System

We have designed a single vector system such that IL-12 and IL-21 are expressed through one vector.

For this purpose, the present inventors specifically describe hIL-12p35 consisting of the amino acid sequence of SEQ ID NO: 1, two subunits of human IL-12 protein, and hIL-12p40 polypeptide consisting of the amino acid sequence of SEQ ID NO: 2, respectively. The encoding polynucleotides (SEQ ID NOS: 4 and 5) were linked to an EMCV-derived internal ribosome entry site (IRES) having the nucleic acid sequence set forth in SEQ ID NO: 6, encoding the hIL-12p40 polypeptide. A polynucleotide encoding the human IL-21 protein (hIL-21) consisting of an RSV promoter (pRSV) as set out in SEQ ID NO: 7 and an amino acid sequence as set out in SEQ ID NO: 3 at the 3'-end of the polynucleotide (SEQ ID NO: 8) After the gene constructs were sequentially connected, the gene construct was converted into pGX-27 vector (Korean Patent No. 14422254). A vector according to one embodiment of the present invention inserted into the cloning site were prepared and named it as 'hBD-121' (Fig. 2a).

1-2: 이중벡터 시스템1-2: Dual Vector System

The inventors have devised a double vector system so that the IL-12 and IL-21 are inserted and expressed in separate vectors.

The double vector system is prepared as follows. The polynucleotide encoding the hIL-12p35 polypeptide (SEQ ID NO: 1) and the polynucleotide encoding the hIL-12p40 polypeptide (SEQ ID NO: 2) are linked to an EMCV-IRES having a nucleic acid sequence set forth in SEQ ID NO: 6. The polynucleotide (SEQ ID NO: 8), which is inserted into the multicloning site of the pGX-27 vector and similarly consists of the amino acid sequence set forth in SEQ ID NO: 3 (hIL-21), also comprises Inserted into the multicloning site to prepare vectors expressing IL-12 and IL-21, respectively. It was named 'hBD-12 and hBD-21'.

1-3: 삼중벡터 시스템1-3: Triple Vector System

Since IL-12 is a dimeric protein consisting of a hIL-12p35 polypeptide and a hIL-12p40 polypeptide, the hIL-12p35 polypeptide and hIL-12p40 polypeptide can be expressed from an independent vector. As such, according to one embodiment of the present invention, the hIL-12p35 polypeptide, hIL-12p40 polypeptide, and IL-21 may be expressed through three vectors each independently configured. The present inventors named it as a "triple vector system" for convenience.

The triple vector system can be prepared as follows:

The triple vector system was prepared by inserting the hIL-12p35 polypeptide, the hIL-12p40 polypeptide, and polynucleotides encoding SEQ ID NOs: 4, 5, and 8, respectively, into the multicloning site of the pGX-27 vector.

실시예 3: 인간 IL-12, IL-21 및 MIP-α 발현 벡터의 제조Example 3: Preparation of Human IL-12, IL-21 and MIP-α Expression Vectors

A polynucleotide (SEQ ID NOs: 4 and 5) encoding the hIL-12p35 polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 1 and the hIL-12p40 polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 2 is represented by SEQ ID NO: 6 RSV as described in SEQ ID NO: 7 at the 3'-terminus of the polynucleotide encoding the hIL-12p40 polypeptide, linked to an EMCV-derived internal ribosome entry site (IRES) having a nucleic acid sequence as described The polynucleotide (SEQ ID NO: 8) encoding the promoter (pRSV) and the human IL-21 protein (hIL-21) consisting of the amino acid sequence set forth in SEQ ID NO: 3 is represented by SEQ ID NO: 9 Human MIP- consisting of human EF-1α promoter (pEF-1α) consisting of nucleic acid sequences and amino acid sequence set forth in SEQ ID NO: 10 Gene constructs in which the polynucleotides encoding the 1α protein (hMIP-1α) (SEQ ID NO: 11) were sequentially connected to each other were prepared and inserted into the multicloning site of the pGX-27 vector, which was named hBD-121A (Fig. 2b).

실시예 4: 마우스 IL-12 및 IL-21 발현 벡터의 제조Example 4: Preparation of Mouse IL-12 and IL-21 Expression Vectors

A polynucleotide encoding the mIL-12p35 polypeptide consisting of the amino acid sequence of SEQ ID NO: 12, the two subunits of the mouse IL-12 protein, and the mIL-12p40 polypeptide consisting of the amino acid sequence of SEQ ID NO: 13, respectively Nos. 14 and 15) were linked to an EMCV-derived internal ribosome entry site (IRES) having the nucleic acid sequence set forth in SEQ ID NO: 6, and 3'- of the polynucleotide encoding the mIL-12p40 polypeptide. Sequentially connecting a polynucleotide (SEQ ID NO: 17) encoding a mouse IL-21 protein (mIL-21) consisting of an RSV promoter (pRSV) as shown in SEQ ID NO: 7 and an amino acid sequence as shown in SEQ ID NO: 16 at the end; After preparing a gene construct, the gene construct was inserted into the multicloning site of the pGX-27 vector to provide a gene construct. A vector was prepared according to the example and named 'mBD-121' (FIG. 2A).

실시예 5: 마우스 IL-12, IL-21 및 MIP-1α 발현 벡터의 제조Example 5: Preparation of Mouse IL-12, IL-21 and MIP-1α Expression Vectors

Polynucleotides (SEQ ID NOs: 14 and 15) encoding the mIL-12p35 polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 12 and the mIL-12p40 polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 13 are represented by SEQ ID NO: 6, respectively. RSV as described in SEQ ID NO: 7 at the 3'-terminus of the polynucleotide encoding the mIL-12p40 polypeptide, linked to an EMCV-derived internal ribosome entry site (IRS) having a nucleic acid sequence as described A polynucleotide (SEQ ID NO: 17) encoding a promoter (pRSV) and a mouse IL-21 protein (mIL-21) consisting of the amino acid sequence set forth in SEQ ID NO: 16 is set forth in SEQ ID NO: 9 Human EF-1α promoter (pEF-1α) consisting of a nucleic acid sequence and the amino acid sequence of SEQ ID NO: 18 Gene constructs in which polynucleotides (SEQ ID NO: 19) encoding U.S. MIP-1α protein (mMIP-1α) were sequentially connected were prepared and inserted into the multicloning site of the pGX-27 vector, which was named mBD-121A. (FIG. 2B).

실험예 1: BD-14의 발현 분석Experimental Example 1: Expression Analysis of BD-14

1-1: ELISA 분석1-1: ELISA analysis

The inventors of the present invention, in order to check whether the BD-14, a multivalent HPV DNA vaccine according to an embodiment of the present invention prepared in Example 1, is normally expressed upon introduction into mammalian cells, Three expression vectors BD-14A, BD-14B, and BD-14C included in the transgenic cells were transduced, respectively, and analyzed for protein expression using an antigen specific for Flt3L included in each construct.

Specifically, the COS-7 cell line was inoculated in a 100 mm culture dish, followed by 16 hours of incubation, and the mock plasmid DNA and BD-14A, BD14-B and BD-14C plasmid DNA prepared in Example 1 using Lipofectamine 2000. and were each transfected and infected, used as the sample to recover the culture supernatant of COS-7 cells in each condition, after cultured at 37 ℃ CO ₂ incubator for three days. Proteins present in the sample were quantified using the Flt3L ELISA kit because Flt3L is bound (FIG. 3a).

As a result, as shown in Figure 3a, it was confirmed that the BD-14A, BD-14B and BD-14C are all well expressed.

1-2: 웨스턴 블랏 분석1-2: Western blot analysis

The inventors performed SDS-PAGE electrophoresis on the cell lysate of the cells obtained in Experimental Example 1-1, and after transcribing into nylon membrane, anti-Flt3L antibody (Abcam, Cat # ab52648). Western blot analysis was performed using () (FIG. 3B).

As a result, as confirmed in Figure 3b, it was confirmed that all of BD-14A, BD-14B and BD-14C can normally express the fusion protein of the expected size.

실험예 2: BD-14의 생체내 면역반응 분석Experimental Example 2: In vivo Immune Response Analysis of BD-14

2-1: T 세포 특이적 면역반응 분석2-1: T Cell Specific Immune Response Analysis

The present inventors have provided a method of counting the number of spleen immune cells that respond to each antigen whether or not to induce a T cell specific immune response after administration of BD-14 according to one embodiment of the present invention to an experimental animal. It was investigated using. Specifically, C57BL / 6 mice, which are experimental animals, were treated with empty vector administration group (n = 5), conventional bivalent HPV DNA vaccine (Korean Patent Publication No. 10-2017-0045254) administration group (n = 5), and one of the present invention. Divided into BD-14 administration group (n = 5) according to the embodiment, each 2 μg of plasmid DNA (0.67 μg each for BD-14A to BD-14C) to OrbiJector (SLVAXiGEN, Korea) Spleen immune cells responding to each type of E6 / E7 antigen by administering an in vivo electroporator twice at two-week intervals, sacrificing experimental animals two weeks after the last administration, and extracting the spleen. Were counted using ELISPOT analysis (FIGS. 4A and 4B).

As a result, as shown in Figure 4b, the BD-14 according to an embodiment of the present invention, although relatively weak for 35, 52 and 59 HPV type for all types of HPV E6 / E7 antigen It has been successful in inducing T cell specific immune responses. On the other hand, there was no significant difference from the conventional bivalent DNA vaccine in the reactivity with the

type

16 and 18 type HPV E6 / E7 antigen. This proves that even though the ratio of the 16-type and 18-type HPV E6 / E7 antigens in BD-14 according to one embodiment of the present invention is only about one third of the conventional bivalent HPV DNA vaccines, they are equally effective. . In particular, it was confirmed that for 6, 11, 39, 45, and 56 HPV, a much stronger response was induced than the immune response induced by 16, 18 HPV.

2-2: 항-종양 효과 분석2-2: Anti-Tumor Effect Analysis

The present inventors performed in vivo anticancer activity analysis using tumor model animals to determine whether BD-14 according to an embodiment of the present invention is effective in the prevention and treatment of cancer caused by the infection of human papillomavirus.

Specifically, C57BL / 6 mice, which are experimental animals, are the empty vector administration group (n = 8), the conventional bivalent HPV DNA vaccine (Korean Patent No. 2017-0045254) administration group (n = 8), and one embodiment of the present invention. TC-1 cells 5x10 ⁵ derived from lung epithelial cells of C57BL / 6 mice and transformed to express the E6 / E7 antigen of HPV16, divided into BD-14 administration groups (n = 8) according to an example After injecting the dog by subcutaneous injection into the dorsal tumor, 3 days after administration of the cancer cells, 2 μg of plasmid DNA (0.67 μg each for BD-14A to BD-14C) was added to OrbiJector (SLVAXiGEN, Korea). In vivo electroporation was administered twice at intervals of 2 weeks, and tumor size and survival rate of the experimental animals were examined at intervals of 3 to 4 days from day 7 of tumor injection (FIGS. 5A to 5C).

As a result, as shown in Figure 5b, BD-14 according to an embodiment of the present invention not only significantly reduced the tumor size compared to the control group, but showed similar anti-tumor efficacy as the conventional bivalent HPV DNA vaccine. In addition, as shown in Figure 5c, in terms of survival, the survival rate of the BD-14 administration group according to an embodiment of the present invention, in contrast to the tumor size analysis results, was shown to be higher than the conventional bivalent HPV DNA vaccine administration group. Comprehensive review of these results, BD-14 vaccine composition according to an embodiment of the present invention is equivalent to or better than the conventional bivalent DNA vaccine despite the 1/3 dose for high risk groups such as HPV16 or HPV18 Not only does it show anti-cancer activity, but it also proves to be a very innovative vaccine composition that can raise the protection range for cervical cancer by more than 90% by inducing T cell specific immune responses against other types of HPV.

실험예 3: 백신 보조제로서의 BD-121의 효과 분석Experimental Example 3: Analysis of the effect of BD-121 as a vaccine adjuvant

3-1: ELISA 분석3-1: ELISA analysis

The present inventors transduced the hBD-121 construct and mBD-121 construct according to an embodiment of the present invention prepared in Examples 2 and 4 to the cells, and then IL-12 and Whether IL-21 is normally expressed was examined. Specifically, the COS-7 cell line was inoculated in a 100 mm culture dish and cultured for 16 hours, followed by mock plasmid DNA and hBD-121 plasmid DNA prepared in Example 2-1 and mBD-121 plasmid prepared in Example 4. Each DNA was transfected with Lipofectamine 2000, and cultured supernatant of COS-7 cells in each condition was recovered after 3 days of incubation in a 37 ° C. CO ₂ incubator and used as a sample. The IL-12 and IL-21 proteins present in the sample are antibodies that specifically recognize IL-12 and IL-21, respectively (IL-12: R & D Systems, Cat # D1200, IL-21: BioLegend, Cat # 433808). Quantitation was performed using the ELISA assay method (FIGS. 6A and 6B).

As a result, as shown in Figure 6a, it was confirmed that the expression level of the protein in the sample introduced with hBD-121 plasmid DNA is more than 4,000 pg / ml when introduced 4 ㎍ DNA in hIL-12, hIL-21 Also, when the 4 ㎍ DNA was introduced, it was confirmed that the expression is very high, showing a value close to 200 ng / ml. In addition, as shown in Figure 6b, the mouse constructs showed similar results as the human constructs. On the other hand, in the case of the control group introduced with the empty vector, both proteins were not expressed at all, and thus the vaccine immunoadjuvant expression system of the present invention was confirmed to operate normally.

3-2: 웨스턴블랏 분석3-2: Western blot analysis

The inventors performed SDS-PAGE electrophoresis on the cell lysate of the cells obtained in Experimental Example 3-1, and then transferred to nylon membrane, followed by anti-IL-12A antibody and anti-IL-12B antibody. And Western blot analysis using anti-IL-21 antibody (FIG. 6C).

As a result, as shown in Figure 6c, it was confirmed that both IL-12 and IL-21 is normally expressed by the transduction of BD-121 plasmid DNA according to an embodiment of the present invention.

실험예 4: 백신 보조제로서의 BD-121A의 효과 분석Experimental Example 4: Analysis of the effect of BD-121A as a vaccine adjuvant

4-1: T 세포 특이적 면역반응 분석4-1: T Cell Specific Immune Response Analysis

We analyzed whether BD-121A as a vaccine adjuvant improves the vaccine performance of BD-14.

To this end, the present inventors have immunized with each antigen whether BD-14 and the mBD-121A prepared in Example 5 induce an T cell specific immune response after administration to experimental animals according to an embodiment of the present invention. The method was counted by counting the number of spleen immune cells responding. Specifically, C57BL / 6 mice, which are experimental animals, were treated with an empty vector administration group (n = 3), BD-14 alone administration group (n = 5), and BD-14 and mBD-121A administration groups according to an embodiment of the present invention (n = 5), 1.3 μg of each plasmid DNA (BD-14A, BD-14B and BD-14C) for the BD-14 alone group and plasmid DNA 1 for the BD-14 and mBD-121A combination dose groups. Splenocytes were administered to femoral muscles once using OrbiJector (SLVAXiGEN, Korea) in vivo electroporator, and after 2 weeks of administration, the animals were sacrificed and spleens were extracted to respond to each type of E6 / E7 antigen. Cells were counted using the ELISPOT assay (FIGS. 7A and 7B).

As a result, as shown in Figure 7b, mBD-121A according to an embodiment of the present invention was shown to enhance the E6 / E7-specific T cell response of various HPV types than when administered BD-14 alone. In particular, the high-risk type 16 HPV showed more than twofold increase in T cell immune responses, and

type

31, 33, 51 and 58 showed significant elevations in immune responses. This demonstrates that BD-121A according to one embodiment of the present invention is a highly efficient vaccine immunoadjuvant against multivalent HPV DNA vaccines.

4-2: 항-종양 활성분석4-2: Anti-Tumor Activity Assay

Specifically, C57BL / 6 mice, which are experimental animals, were treated with the empty vector administration group (n = 10), the conventional bivalent HPV DNA vaccine (Korean Patent No. 2017-0045254) administration group (n = 13), and one embodiment of the present invention. After dividing into BD-14 and BD-121 administration group according to an example (n = 13), 5x10 ⁵ TC-1 cells used in Experimental Example 2-2 were injected by subcutaneous injection into the dorsal side to induce a tumor, and After 7 days of TC-1 cell inoculation, 4 μg of plasmid DNA (1 μg each for BD-14A to BD-14C and BD-121) was injected into the femoral muscle using an OrbiJector (SLVAXiGEN, Korea) in vivo electroporator. Two doses were administered at intervals of two weeks, and tumor size and survival rate of experimental animals were examined at intervals of 3 to 4 days from day 7 of tumor inoculation (FIGS. 8A to 8C).

As a result, as shown in Figure 8b and 8c, the multivalent vaccine according to an embodiment of the present invention showed a significant anti-cancer efficacy compared to the negative control group (PBS administration group), compared with the conventional bivalent vaccine as a positive control Equivalent or more than equivalent anticancer efficacy was confirmed. This indicates similar anti-cancer efficacy when the amount of antigen administered (E6 / E7 of HPV16) is 1/4 of that of the conventional 2 vaccine, and at the same time a large number of antigens are administered. Nevertheless, it is possible to induce a variety of immune responses, as well as means that the anti-cancer efficacy for a particular type (HPV16) is not compromised.

실험예 5: 백신의 작용 기전 분석Experimental Example 5: Analysis of the mechanism of action of the vaccine

In order to investigate the mechanism through which the multivalent HPV DNA vaccine according to one embodiment of the present invention functions, we administered anti-CD4 or anti-CD8 antibodies to CD-4 T cells and CD-8 T cells, respectively. The experimental animal was removed to compare the anticancer activity of the HPV DNA vaccine according to an embodiment of the present invention.

Specifically, experiments were performed by dividing the experimental animals C57BL / 6 mice into four groups, using the empty vector administration group (n = 9) as a control group, the vaccine and vaccine adjuvant (BD14A + BD121A) according to an embodiment of the present invention. The control group was divided into isotype administration group (n = 9), anti-CD4 antibody administration group (n = 9) and anti-CD8 antibody administration group (n = 9) as a control group. The experimental schedule is as follows: After the TC-1 cancer cells of Experimental Example 2-2 were subcutaneously inoculated at ⁵ × 10 ⁵ cells per head, the antibodies (isotype, anti-CD4 and anti-CD8 antibodies) were from day 1 from cancer cell inoculation. Intraperitoneal administration was performed 7 times at a dose of 200 μg / injection / mouse at 7-day intervals, and the vaccine composition (BD-14A and BD-121) according to one embodiment of the present invention was separated from the 3 days to 7 days from the date of inoculation of cancer cells As an intramuscular administration to the hind limb muscle at a dose of 8 μg / injection / mouse using three electroporation methods (FIG. 9A). 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 a result, as shown in FIGS. 9B and 9C, anti-cancer efficacy was maintained in the mice from which the CD4 T cells were removed, but anti-cancer efficacy was worse in the mice from which the CD8 T cells were removed than the control (mock and homologous antibodies). It could be observed. This shows that the multivalent HPV DNA vaccine according to one embodiment of the present invention exhibits anti-cancer immunity through CD8 T cells, which is consistent with the known that CD8 T cells are very important for the efficacy of anti-cancer therapeutic vaccines. to be.

실험예 6: IL-7과의 병용 투여 효과 분석Experimental Example 6: Analysis of the combined administration effect with IL-7

IL-7 (Interleukin 7) is a cytokine that promotes the differentiation of pluripotent hematopoietic stem cells into lymphoid progenitor cells and is known to play an important role in the development of B cells and T cells. IL-7 is known to promote the malignancy of hematologic malignancies such as acute lymphocytic leukemia and T cell lymphoma. However, in general solid cancer, the homeostasis of CD8 and CD4 cells is disrupted to decrease the ratio of CD4 ⁺ CD25 ⁺ Foxp3 ⁺ regulatory T cells. It is known to reduce, and currently, Phase 1 and Phase 2 trials are in progress for some cancers. The inventors of the present invention, since IL-7 tends to affect the balance between CD4 T cells and CD8 T cells from the results of Experimental Example 5 and cause an increase of CD8 T cells, according to one embodiment of the present invention We hypothesized that synergy with HPV DNA vaccines could be expected. In order to confirm whether this hypothesis is correct, the present inventors analyzed the anticancer activity after administering the vaccine composition according to one embodiment of the present invention alone or in combination with IL-7.

Specifically, the present inventors performed experiments by dividing the experimental animals C57BL / 6 mice into three groups, the empty vector administration group (n = 8) as a control group, the vaccine (BD14) alone administration group (n) according to an embodiment of the present invention. = 8), and a vaccine and an IL-7 administration group (BD14 + IL-7) according to an embodiment of the present invention were used, and TC-1 cancer cells of Experimental Example 2-2 were injected intravaginally at 1 × 10 ⁵ cells per head. (i.va) After preparing a stereotactic tumor model that caused the tumor, 4 μg of the vaccine or 4 μg of the vaccine and 50 μg of the vaccine were administered intramuscularly three times on the 7th, 14th and 28th days from the date of cancer cell inoculation. The tumor volume was measured at intervals of 7 days from the date of inoculation of cancer cells (FIG. 10A).

As a result, as shown in Figure 10b, as a result of using an orthotopic tumor (orthotopic tumor) model, it was confirmed that the anticancer efficacy significantly improved by the BD14 administration compared to the PBS administration group. This was found to be consistent with the results confirmed in the experiment using the ectopic tumor model following subcutaneous injection. More interestingly, when combined with IL-7, anticancer efficacy was significantly improved compared to BD14 alone. This can be said to show the possibility of using a multivalent HPV DNA vaccine according to an embodiment of the present invention and other cytokines having a mechanism of action on T cells.

As described above, BD-14, a multivalent HPV DNA vaccine according to an embodiment of the present invention, normally expressed the E6 / E7 shuffled protein, which is an antigen protein contained therein, despite the complicated structure, and actually various types of HPV. Successfully induced T cell specific immune responses against the E6 / E7 antigen and anticancer activity assays for cancer models expressing the high-risk HPV16 E6 / E7 antigen resulted in equal or better anticancer effects than conventional bivalent DNA vaccines. Indicated. Furthermore, the multivalent HPV DNA vaccine according to one embodiment of the present invention is a vaccine immune vaccine adjuvant and is a T cell-specific immune response against HPV E6 / E7 antigen when co-administered with BD-121A according to one embodiment of the present invention. Not only increased significantly, but also showed a significant effect on anticancer effects.

Therefore, the multivalent HPV DNA vaccine and the vaccine composition including the DNA vaccine and the BD-121 or BD-121A vaccine adjuvant according to an embodiment of the present invention prevent various HPV infections with the risk of causing fatal diseases such as cervical cancer. And very effectively in the treatment of HPV infection.

Although the present invention has been described with reference to the above-described examples and experimental examples, these are merely exemplary, and those skilled in the art will understand that various modifications and equivalent other embodiments are possible. Therefore, the true technical protection scope of the present invention will be defined by the technical spirit of the appended claims.

Multivalent HPV DNA vaccine composition according to an embodiment of the present invention can be used very effectively as a medicament for the prevention of various HPV infections and the treatment of HPV infections with the risk of causing fatal diseases such as cervical cancer.

SEQ ID NO: 1 is the amino acid sequence of a human IL-12p35 polypeptide.

SEQ ID NO: 2 is the amino acid sequence of a human IL-12p40 polypeptide.

SEQ ID NO: 3 is the amino acid sequence of a human IL-21 polypeptide.

SEQ ID NO: 4 is the nucleic acid sequence of a polynucleotide encoding the human IL-12p35 polypeptide.

SEQ ID NO: 5 is the nucleic acid sequence of a polynucleotide encoding the human IL-12p40 polypeptide.

SEQ ID NO: 6 is the nucleic acid sequence at the EMCV-derived internal ribosome entry site.

SEQ ID NO: 7 is the nucleic acid sequence of the RSV promoter.

SEQ ID NO: 8 is the nucleic acid sequence of a polynucleotide encoding the human IL-12 polypeptide.

SEQ ID NO: 9 is the nucleic acid sequence of the human EF-1α promoter.

SEQ ID NO: 10 is the amino acid sequence of a human MIP-1α polypeptide.

SEQ ID NO: 11 is a polynucleotide encoding the human MIP-1α polypeptide is a nucleic acid sequence.

SEQ ID NO: 12 is the amino acid sequence of the mouse IL-12p35 polypeptide.

SEQ ID NO: 13 is the amino acid sequence of a mouse IL-12p40 polypeptide.

SEQ ID NO: 14 is the nucleic acid sequence of a polynucleotide encoding the mouse IL-12p35 polypeptide.

SEQ ID NO: 15 is the nucleic acid sequence of a polynucleotide encoding the mouse IL-12p40 polypeptide.

SEQ ID NO: 16 is the amino acid sequence of the mouse IL-21 polypeptide.

SEQ ID NO: 17 is the nucleic acid sequence of a polynucleotide encoding the mouse IL-21 polypeptide.

SEQ ID NO: 18 is the amino acid sequence of a mouse MIP-1α polypeptide.

SEQ ID NO: 19 is the nucleic acid sequence of a polynucleotide encoding the mouse MIP-1α polypeptide.

SEQ ID NO: 20 is the amino acid sequence of the (GS) ₅ linker peptide.

SEQ ID NO: 21 is the nucleic acid sequence of a polynucleotide encoding the (GS) ₅ linker peptide.

SEQ ID NO: 22 is the amino acid sequence of a tPA leader peptide

SEQ ID NO: 23 is the nucleic acid sequence of a polynucleotide encoding the tPA leader peptide.

SEQ ID NO: 24 is the amino acid sequence of a Flt3L polypeptide with a cleaved signal sequence.

SEQ ID NO: 25 is the nucleic acid sequence of a polynucleotide encoding the Flt3L polypeptide.

SEQ ID NOs: 26, 28 and 30 are amino acid sequences of shuffled antigen fusion proteins included in the DNA vaccine construct according to one embodiment of the present invention, respectively.

SEQ ID NOs: 27, 29, and 31 are the nucleic acid sequences of the polynucleotides encoding the shuffled antigen fusion proteins, respectively.

SEQ ID NOs: 32 to 50 are amino acid sequences of various linker peptides that may be used in the present invention.

Claims

Encoding early protein antigen 6 (E6) or immunogenic fragments of 6, 11, 16, 18, 39, 45, and 56 human papillomavirus (HPV), and early protein antigen 7 (E7) or immunogenic fragments thereof, respectively A polyvalent HPV DNA vaccine composition comprising polynucleotides, wherein E6 and E7 do not have wild type functions.
The method of claim 1,

One or more HPV early protein antigen 6 (E6) or immunogenic fragments thereof and early protein antigen 7 selected from the group consisting of 31, 33, 35, 51, 52, 58 and 59 human papillomavirus (HPV) (E7) or a polynucleotide each encoding an immunogenic fragment thereof, wherein said E6 and E7 do not have a wild-type function.
The method according to claim 1 or 2,

Wherein E6 and E7 are divided into N-terminal fragment and C-terminal fragment, respectively, is expressed in the form of randomly shuffled E6 / E7 shuffled antigen unit, multivalent HPV DNA vaccine composition.
The method of claim 3,

The E6 / E7 shuffled antigenic unit is composed of N-terminal fragments of E6 and E7 and C-terminal fragments of N-terminal fragment of E6 (E6N) -C7-terminal fragment of E7 (E7C) -E7 N-terminal fragment The multivalent HPV DNA vaccine composition, which is a polypeptide linked in the order of the C-terminal fragment (E6C) of (E7N) -E6.
The method of claim 3,

A multivalent HPV DNA vaccine composition, wherein at least two E6 / E7 shuffled antigenic units of human papillomavirus are linked and expressed in the form of a fusion protein.
The method of claim 5,

The E6 / E7 shuffled antigen unit or the fusion protein further comprises a signal sequence, multivalent HPV DNA vaccine composition.
The method of claim 5,

The E6 / E7 shuffled antigen unit or the fusion protein further comprises Flt3L, multivalent HPV DNA vaccine composition.
The method of claim 1,

The multivalent HPV DNA vaccine composition further comprising IL-7.
The method of claim 1,

The multivalent HPV DNA vaccine composition further comprising one or more pharmaceutically acceptable vaccine adjuvant.
The method of claim 9,

The vaccine immunoadjuvant includes IL-12 protein and IL-21 protein as an active ingredient or polynucleotides encoding the IL-12 protein and T lymphocyte specificity comprising a polynucleotide encoding the IL-21 protein as an active ingredient. Multivalent HPV DNA vaccine composition, which is a vaccine immunoadjuvant for promoting an immune response.
The method of claim 10,

The T lymphocyte specific immune response promoting vaccine immunoadjuvant comprises one or more selected from the group consisting of, multivalent HPV DNA vaccine composition:

IL-12 protein and IL-21 protein consisting of p35 chain (IL-12p35) and p40 chain (IL-12p40);

One to three vectors comprising a polynucleotide encoding the p35 chain (IL-12p35) and the p40 chain (IL-12p40) constituting the IL-12, and a polynucleotide encoding the IL-21 protein, respectively; And

MRNA molecules encoding the IL-12p35, IL-12p40 and IL-21 proteins, respectively.
The method of claim 10,

The IL-12p35 protein is human IL-12p35 consisting of the amino acid sequence set forth in SEQ ID NO: 1, multivalent HPV DNA vaccine composition.
The method of claim 10,

The IL-12p40 protein is human IL-12p40 consisting of the amino acid sequence set forth in SEQ ID NO: 2, multivalent HPV DNA vaccine composition.
The method of claim 10,

The IL-21 protein is human IL-21 consisting of the amino acid sequence set forth in SEQ ID NO: 3, multivalent HPV DNA vaccine composition.
The method of claim 10,

The vaccine immunoadjuvant further comprises one or more selected from the group consisting of: Multivalent HPV DNA vaccine composition:

i) MIP-1α protein;

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

iii) a complex gene construct in which the polynucleotide encoding the MIP-1α protein is operably linked to any one or more of the IL-12p35, IL-12p40 and IL-21 proteins by a polynucleotide encoding an IRES or linker peptide T; And

iv) mRNA molecule encoding MIP-1α protein.
The method of claim 15,

The MIP-1α gene construct is included in a separate expression vector or polynucleotides encoding the p35 chain (IL-12p35) and p40 chain (IL-12p40) constituting the IL-12, respectively, and the IL-21 protein. A multivalent HPV DNA vaccine composition comprising one or more of one to three vectors comprising polynucleotides each encoding a.
The method of claim 16,

The MIP-1α protein is a human MIP-1α protein consisting of the amino acid sequence set forth in SEQ ID NO: 10, a multivalent HPV DNA vaccine composition.
A method of treating a disease caused by an HPV infection comprising administering to the subject a multivalent HPV DNA vaccine of any one of claims 1 to 17.
The method of claim 18,

Diseases caused by HPV infection include squamous cell carcinoma (SCC), adenocarcinoma, adenosquamous cell carcinoma, small cell carcinoma, neuroendocrine tumor (NET), free cell carcinoma, chorionic adenocarcinoma (VGA), non-carcinoma malignancy, The method of treatment is melanoma, lymphoma, or cervical epithelial tumor (CIN).
The method of claim 18,

The vaccine composition is administered by in vivo electroporation.