KR102222582B1 - A novel polyvalent HPV vaccine composition - Google Patents

Abstract

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

Description

A novel polyvalent HPV vaccine composition

The present invention relates to a novel polyvalent HPV DNA vaccine, a fusion protein used therein, and a polynucleotide encoding the same.

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 those cases are caused by persistent infection with the most common high-risk human papillomavirus (HPV) types, namely HPV16 and HPV18 (Schiffman et al ., Lancet , 370:890-907, 2007; Forman et al ., Vaccine 30(5S): F12-23, 2012). HPV infection persistence is usually associated with a lack of apparent HPV-specific T-cell immunity, and virus-specific T cells found in patients with pre-malignant and malignant tumors 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 occurrence of HPV-induced cervical cancer.

Cervical cancer develops through the process of high-risk HPV infection, viral persistence, clonal expansion and differentiation of persistently infected cells into pre-malignant lesions, and its gradual transformation into invasive cancer (Schiffman et al ., Lancet 370: 890-907, 2007). Pre-malignant cervical intraepithelial tumors 2 and 3 (CIN2 and 3), particularly those tumors that are positive for HPV16, are considered high-grade lesions with an approximately 30% likelihood of developing invasive cancer (Moscicki et al ., Vaccine 30). (5S): F24-33, 2012). Therefore, there is an urgent need for an effective therapeutic vaccine capable of preventing serious complications of persistent HPV infection and eradicating HPV-related tumors.

Currently, the two HPV vaccines on the market in Korea are tetravalent vaccine (Gadasil) and bivalent vaccine (Servarix). The tetravalent vaccine contains HPV6, 11, 16, 18 types of L1 VLP, and the bivalent vaccine In case, it contains HPV 16 and 18 L1 VLPs. However, HPV 6 and 11 types are the main causes of genital warts, but since they are low-risk HPVs that are not related to cervical cancer, both vaccines are bivalent vaccines that prevent HPV types 16 and 18 in terms of preventing cervical cancer.

On the other hand, HPV E6 and E7 act as viral oncoproteins by binding each of the tumor suppressor proteins p53 and retinoblastoma (pRb) and promoting their degradation (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 because these proteins not only induce tumorigenesis, but they are also constitutively expressed in HPV-infected pre-malignant and malignant cells. Yugawa and Kiyono, Rev. Med. Virol ., 19: 97-113, 2009). Regression of cervical lesions is associated with a cellular immune response but not a humoral immune response (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 strong E6/E7-specific T-cell immunity are highly desirable.

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 commercially available vaccine composition and the DNA vaccine composition using the HPV 16/18 E6/E7 antigen target only the highest risk groups, type 16 and type 18 HPV, only about 70% of all cervical cancers are covered. .

Therefore, there is an urgent need to develop a multivalent vaccine capable of inducing an immune response against various HPV types, which are at risk of inducing almost all cervical cancer.

Accordingly, an object of the present invention is to provide a multivalent HPV DNA vaccine that is effective in preventing and treating diseases caused by cervical cancer and other HPV infections by inducing an immune response to various types of HPV. However, the scope of the present invention is not limited by the above object.

According to one aspect of the present invention, early protein antigen 6 (E6) or immunogenic fragment thereof of human papillomavirus type 6, 11, 16, 18, 39, 45 and 56 (HPV), and early protein antigen 7 (E7) or A multivalent HPV DNA vaccine composition comprising a polynucleotide encoding each of its immunogenic fragments is provided.

According to one aspect of the present invention, human papilloma virus selected from the group consisting of types 6, 11, 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58 and 59 N-terminal fragment and C-terminal fragment of each of early protein 6 (E6) and early protein antigen 7 (E7) of E6 are N-terminal fragment of E6 (E6N)-C-terminal fragment of E7 (E7C)-N of E7 -Terminal fragment (E7N)-E6 C-terminal fragment (E6C) is an E6/E7 antigen unit, and at least 4 types of HPV antigen units are connected by a linker. A fusion protein is provided.

A polynucleotide encoding the fusion protein according to an aspect of the present invention is provided.

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

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

According to another aspect of the present invention, there is provided a method for treating diseases caused by HPV infection, comprising administering the multivalent HPV DNA vaccine composition or the 14-valent HPV DNA vaccine composition to an individual.

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 adjuvant BD-121 according to an embodiment of the present invention, Figure 2b is a schematic diagram showing the structure of the vaccine adjuvant BD-121A according to an embodiment of the present invention.
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 plasmids according to an embodiment of the present invention, respectively. 3b is a photograph showing the results of Western blot analysis using an anti-Flt3L antibody on the cell lysate obtained by disrupting the cells of FIG. 3A.
Figure 4a shows the vaccination schedule of the experiment for analyzing the immune response of the multivalent HPV DNA vaccine (BD-14) according to an embodiment of the present invention.
Figure 4b is a blank vector (Mock), the multivalent HPV DNA vaccine (BD-14) according to an embodiment of the present invention and the E6 of each type of HPV among splenocytes extracted from mice inoculated with a conventional bivalent HPV DNA vaccine. This is a graph showing the results of ELISPOT analysis, which analyzed the number of splenocytes specifically responding to E7.
5A shows an experimental vaccination schedule for analyzing the anticancer effect of the multivalent HPV DNA vaccine (BD-14) on HPV-induced cancer according to an embodiment of the present invention.
Figure 5b is a blank vector (pGX27), a multivalent HPV DNA vaccine (BD-14) according to an embodiment of the present invention, and the volume of tumor tissue over time of xenograft mice inoculated with a conventional bivalent HPV DNA vaccine It is a graph that records the change of.
Figure 5c is a graph recording the survival rate over time of xenograft mice inoculated with an 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.
6A is a graph showing the results of analyzing the concentrations of IL-12 and IL-21 in the culture supernatant of COS-7 cells transfected with the vaccine adjuvant hBD-121 construct according to an embodiment of the present invention by ELISA.
6B is a graph showing the results of analyzing the concentrations of IL-12 and IL-21 in the culture supernatant of COS-7 cells transfected with the mBD-121 construct, a vaccine adjuvant according to an embodiment of the present invention, by ELISA.
6C is a Western blot analysis of the expression levels of IL-12 and IL-21 in the cell lysate of COS-7 cells transfected with the vaccine adjuvant hBD-121 construct and mBD-121 according to an embodiment of the present invention. Shows the results confirmed through.
7A shows a vaccination schedule for confirming the anticancer effect of the DNA vaccine composition according to an embodiment of the present invention.
7B is an E6/E7 specific immune response of each type of HPV upon administration of a multivalent HPV DNA vaccine (BD-14) alone or in combination with BD-14 and BD-121A, a vaccine adjuvant according to an embodiment of the present invention. It is a graph showing the result of analyzing the number of splenocytes by ELISPOT analysis.
Figure 8a shows a 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.
8B is a graph showing a change in volume of tumor tissue over time when administering a multivalent HPV DNA vaccine composition according to an embodiment of the present invention compared with a conventional bivalent vaccine composition.
8C is a graph showing the survival rate over time of an experimental animal when administered a multivalent HPV DNA vaccine composition according to an embodiment of the present invention compared with a conventional divalent vaccine composition.
Figure 9a shows the vaccination schedule of an animal experiment to confirm the mechanism of action of the multivalent HPV DNA vaccine composition according to an embodiment of the present invention.
9B shows the passage of time when the multivalent HPV DNA vaccine composition according to an embodiment of the present invention was administered to an experimental animal in which an anti-CD4 antibody or an anti-CD8 antibody was administered, respectively, and CD4 T cells and CD8 T cells were deficient. It is a graph showing the volume of the tumor according to.
Figure 10a shows the vaccination schedule of an animal experiment to investigate anticancer activity when a multivalent HPV DNA vaccine composition according to an embodiment of the present invention is used in combination with IL-7.
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 is administered alone or in combination with IL-7.

According to one aspect of the present invention, early protein antigen 6 (E6) or immunogenic fragment thereof of human papillomavirus type 6, 11, 16, 18, 39, 45 and 56 (HPV), and early protein antigen 7 (E7) or A multivalent HPV DNA vaccine composition is provided that includes a polynucleotide encoding each of its immunogenic fragments, wherein E6 and E7 do not have a wild-type function.

The multivalent HPV DNA vaccine composition is one or more HPV early protein antigen 6 (E6) selected from the group consisting of 31, 33, 35, 51, 52, 58, and 59 type human papillomavirus (HPV) or its It may further comprise an immunogenic fragment, and a polynucleotide encoding each of the early protein antigen 7 (E7) or an immunogenic fragment thereof, wherein the added E6 and E7 also do not have a wild-type function.

In the multivalent HPV DNA vaccine composition, the E6 and E7 can be expressed in the form of randomly mixed E6/E7 shuffled antigen units divided into N-terminal fragments and C-terminal fragments, respectively, and the E6/E7 shuffle De antigenic units are the N-terminal fragments and C-terminal fragments of E6 and E7 are the N-terminal fragment of E6 (E6N)-C-terminal fragment of E7 (E7C)-E7 N-terminal fragment (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 or more, three or more, or four or more human papillomavirus E6/E7 shuffled antigen units may be linked and expressed in the form of a fusion protein.

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

The multivalent HPV DNA vaccine composition may further contain IL-7. The present inventors confirmed that when the multivalent HPV DNA vaccine according to an embodiment of the present invention is administered, the anticancer effect is remarkably increased when IL-7 is co-administered (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 adjuvant contains an IL-12 protein and an IL-21 protein as active ingredients, or a polynucleotide encoding the IL-12 protein and a polynucleotide encoding the IL-21 protein It may be a vaccine adjuvant for promoting a T lymphocyte-specific immune response comprising as an active ingredient.

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

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

One to three vectors comprising a polynucleotide encoding each of the p35 chain (IL-12p35) and the p40 chain (IL-12p40) constituting the IL-12, and a polynucleotide encoding each of the IL-21 protein; 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 have 90% or more sequence homology with human IL-12p35 composed of the amino acid sequence described in SEQ ID NO: 1.

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

In the multivalent HPV DNA vaccine composition, the IL-21 protein may have 90% or more sequence homology with human IL-21 consisting of an amino acid sequence represented by SEQ ID NO: 3.

In the multivalent HPV DNA vaccine composition, the vaccine adjuvant 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 a linker peptide T; And

Iv) an mRNA molecule encoding the 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 the p40 chain (IL-12p40) constituting the IL-12, respectively It may be included in any one or more of one to three vectors comprising a polynucleotide that encodes the IL-21 protein and a polynucleotide that encodes each of the IL-21 protein.

In the multivalent HPV DNA vaccine composition, the MIP-1α protein may have 90% or more sequence homology with the human MIP-1α protein composed of the amino acid sequence described in SEQ ID NO: 10.

According to one aspect of the present invention, human papilloma virus selected from the group consisting of types 6, 11, 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58 and 59 N-terminal fragment and C-terminal fragment of each of early protein 6 (E6) and early protein antigen 7 (E7) of E6 are N-terminal fragment of E6 (E6N)-C-terminal fragment of E7 (E7C)-N of E7 -Terminal fragment (E7N)-E6 C-terminal fragment (E6C) is an E6/E7 antigen unit, and at least 4 types of HPV antigen units are connected by a linker. A fusion protein is provided.

The term "HPV" used in this document is a human papilloma virus, which is a DNA-based virus with a diameter of 52 to 55 nm, and infects the skin or subcutaneous of several animals including humans. To date, more than 130 types of HPV have been discovered (zur Hausen H., Vaccine 24 Suppl 3: S3, 2006), and HPV is infected through keratinocytes or mucous membranes of the skin. Most of the known HPVs do not show any signs in people, but some human papillomaviruses (HPV) can cause papillomas in humans. In addition, a small number of 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. These human papillomaviruses are classified as high-risk groups. HPV DNA contains 8,000 base pairs and is surrounded by a pentameric capsid protein, not a lipid membrane. The capsid protein consists of two structural proteins, L1 and L2, which are expressed at the end of the viral replication cycle. . In all HPV genomes, there are 8 open reading frames (ORFs), and each ORF is divided into 3 functional sites. It consists of early protein E1-E7, a gene required for viral replication, L1-L2, a gene that expresses structural proteins that make up virion, and LCR, which regulates viral replication and transcription.

The term "E6" as used herein is one of the early expression proteins required for HPV replication, and inhibits the function of p53 as a cancer tumor suppressor gene by binding to p53 and promoting ubiquitination of p53. It also induces the degradation of BAK, a pro-apoptotic protein. In addition, it plays a role of activating the cell cycle of host cells through activation of telomerase.

The term "E7" used in this document is one of the early expression proteins required for HPV replication, and it degrades RB by interacting with RB (retinoblastoma). Through this, it releases E2F, a transcription-promoting factor, which has been inhibited by RB. Moreover, it activates the cell cycle of the host cell by activating cyclin E and cyclin A, which act in the S phase of the cell cycle.

As used herein, the term "immunogenic fragment" refers to a fragment capable of functioning as an antigen among fragments of a full-length antigen protein, that is, a fragment capable of inducing 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 the antigenic unit is a shuffled protein, thus retaining the function as an antigen, but originally It lacks the intrinsic function (p53 and pRb binding function) of wild-type E6 and E7 proteins.

In the fusion protein, Flt3 (fms-like tyrosine kinase-3) ligand (Flt3L) may be added to the N-terminus of the antigen conjugate protein, and a secretion signal sequence may be added. The secretion 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, an HSV gDs (herpes simplex virus glycoprotein Ds) signal sequence, or a growth hormone signal sequence. .

The fusion protein may further include a polynucleotide encoding one or more immune enhancing peptides, and the immune enhancing peptides include CD28, ICOS (inducible costimulator), CTLA4 (cytotoxic T lymphocyte associated protein 4), PD1 (programmed cell death protein 1), BTLA(B and T lymphocyte associated protein), DR3(death receptor 3), 4-1BB, CD2, CD40, CD30, CD27, SLAM(signaling lymphocyte activation molecule), 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, GITR (glucocorticoid-induced TNFR family related protein), Flt3 ligand (fms-like tyrosine kinase 3 ligand), flagellin, HVEM (herpesvirus entry mediator) or OX40L (ligand for CD134) (OX40), CD252] or a linker of two or more of them.

In the fusion protein, the linker is preferably a linker peptide, and the linker peptide includes (G ₄ S) _n (unit: SEQ ID NO: 32, n is an integer of 1 to 10), (GS) _n (n is 1 To 10), (GSSGGS) _n (unit: SEQ ID NO: 33, n is an integer of 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 of 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 of 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).

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

A polynucleotide encoding the fusion protein according to an aspect of the present invention is provided.

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

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

The term "operably linked to" as used in this document is regulated in such a way that the target nucleic acid sequence (eg, in an in vitro transcription/translation system or in a host cell) can be expressed. It means that it is linked to a sequence.

The term "regulatory sequence" is meant to include promoters, enhancers and other regulatory elements (eg, polyadenylation signals). Regulation sequences are those that direct the expression of the nucleic acid of interest in many host cells, and that the nucleic acid of interest can be expressed only in specific tissue cells (e.g., tissue-specific regulatory sequences), and It includes directing expression to be induced by a specific signal (eg, an inducible regulatory sequence). It can be understood by those skilled in the art that the design of the expression vector may vary depending on factors such as the selection of host cells to be transformed and the level of desired protein expression. The expression vector of the present invention can be introduced into a host cell to express the fusion protein. Regulatory sequences that enable expression in eukaryotic and prokaryotic cells are well known to those skilled in the art. As described above, they usually contain regulatory sequences responsible for initiation of transcription and, optionally, a poly-A signal responsible for transcription termination and stabilization of the transcript. In addition to the transcriptional regulator, additional regulatory sequences may include a translation enhancer and/or a naturally-combined or heterologous promoter region. For example, possible regulatory sequences that allow expression in mammalian host cells include the CMV-HSV thymidine kinase promoter, SV40, RSV-promoter (Rous sarcoma virus), human kidney element 1α-promoter, glucocorticoid-inducible MMTV- A promoter (Moloni mouse tumor virus), a metallothionein-inducible or tetracycline-inducible promoter, or an amplifying agent such as a CMV amplifying agent or an SV40-amplifying agent. For expression in neurons, it is contemplated that neurofibrillation-promoter, PGDF-promoter, NSE-promoter, PrP-promoter or thy-1-promoter can be used. Such promoters are known in the art and are described in Charron, J. Biol. Chem. 270: 25739-25745, 1995. For expression in prokaryotic cells, a number of promoters have been disclosed, including the lac-promoter, tac-promoter or trp promoter. In addition to factors capable of initiating transcription, the regulatory sequences include transcription termination signals such as SV40-poly-A site or TK-poly-A site downstream of the polynucleotide according to an embodiment of the present invention. You may. In this document, suitable expression vectors are known in the art, examples of which are 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 present invention, the vector may further contain a polynucleotide encoding a secretion signal peptide. The secretion signal peptides are well known to those skilled in the art. And, depending on the expression system used, a leader sequence capable of leading the fusion protein to the cell compartment is combined with the coding sequence of the polynucleotide according to an embodiment of the present invention, preferably the translated protein or its It is a leader sequence that can secrete proteins directly into the periplasm or extracellular medium.

In addition, the vector of the present invention can be prepared by, for example, standard recombinant DNA techniques, and standard recombinant DNA techniques include, for example, ligation of blunt ends and adhesive ends, treatment with restriction enzymes to provide appropriate ends, and inappropriate In order to prevent binding, phosphate group removal by alkaline postage treatment and enzymatic linkage by T4 DNA ligase are included. The vector of the present invention can be prepared by recombining the DNA encoding the signal peptide obtained by chemical synthesis or gene recombination technology and the DNA encoding the 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 manufactured commercially, and in an embodiment of the present invention, pGX27 (Korean Patent No. 1442254), a vector for preparing a DNA vaccine, was used.

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, and the expression vector is a plasmid vector, a viral vector, a cosmid vector, a phagemid vector, an artificial human It can be any form such as chromosome.

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

The 14-valent HPV DNA vaccine composition includes three expression vectors constructed by cloning polynucleotides each encoding three fusion proteins to which 4 to 5 E6/E7 antigen units of the 14 types of HPV are linked, respectively, into an expression vector. can do.

The three expression vectors may be constructed as follows, but are not limited thereto, and a combination of various HPV types is possible:

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

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

Iii) N-terminal fragment and C-terminal fragment of each of early antigen 6 (E6) and early antigen 7 (E7) of type 39 human papillomavirus (HPV39) are the N-terminal fragment of E6 (39E6N)-C-terminal of E7 Fragment (39E7C)-E7 N-terminal fragment (39E7N)-E6 C-terminal fragment (39E6C) linked in the sequence of HPV39 E6/E7 antigen units, type 51 human papillomavirus (HPV51) early antigen 6 (E6), and Early antigen 7 (E7) N-terminal fragment and C-terminal fragment of each of E6 N-terminal fragment (51E6N)-E7 C-terminal fragment (51E7C)-E7 N-terminal fragment (51E7N)-E6 HPV51 E6/E7 antigen units linked in the order of C-terminal fragment (51E6C), N-terminal fragment and C-terminal fragment of each of early antigen 6 (E6) and early antigen 7 (E7) of type 56 human papillomavirus (HPV56) HPV56 E6/E7 antigen units linked in the order of the N-terminal fragment of E6 (56E6N)-E7 C-terminal fragment (56E7C)-E7 N-terminal fragment (56E7N)-E6 C-terminal fragment (56E6C) , And the N-terminal fragment and C-terminal fragment of each of the early antigen 6 (E6) and early antigen 7 (E7) of the human papillomavirus (HPV59) type 59 is the N-terminal fragment of E6 (59E6N)-the C-terminal of E7 The third nucleic acid encoding the third fusion protein in which the HPV59 E6/E7 antigen units linked in the order of the N-terminal fragment (59E7N)-E6 C-terminal fragment (59E6C) of the fragment (59E7C)-E7 are linked by a linker peptide. A third expression vector comprising a third gene construct in which the molecule is operably linked to a promoter.

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

The vaccine composition may contain one or more pharmaceutically acceptable vaccine adjuvants. The vaccine adjuvants include aluminum hydroxide, aluminum phosphate, alum (potassium aluminum sulfate), MF59, virosome, AS04 [a mixture of aluminum hydroxide and monophosphoryl lipid A (MPL)], AS03 (DL-α- a mixture of tocopherol, squalene and polysorbate 80 as an emulsifier), CpG, Flagellin, Poly I:C, AS01, AS02, ISCOMs and ISCOMMATRIX, and the like can be used.

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 a vaccine.

In addition, the vaccine adjuvant contains IL-12 protein and IL-21 protein as active ingredients, or T lymphocytes containing polynucleotides encoding the IL-12 protein and polynucleotides encoding the IL-21 protein as active ingredients. It may be a vaccine adjuvant for promoting a specific immune response.

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

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

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

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

In addition, in the above-described vaccine adjuvant, some of the IL-21p35, IL-12p40 and IL-21 are included as proteins, and the rest may be used by mixing heterologous molecules such as expression vectors and/or mRNA molecules.

At this time, 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 so as to express the IL-12p35, IL-12p40 and IL-21. The vaccine adjuvant is a polynucleotide encoding each of the IL-12p35, IL-12p40 and IL-21 is inserted into individual expression vectors (3 vector system) or inserted into one or two expression vectors (single vector or double vector System) It can be composed of one to three vectors. Specific embodiments of such a single vector to a triple vector system are as follows:

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

Ii) fifth to seventh expression vectors each including the fourth to sixth gene constructs

Iii) an eighth expression vector and a ninth expression vector each including two and the other one of the fourth to sixth gene constructs;

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 among the 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 the eighth gene construct in which the polynucleotide encoding the peptide of iv is operably linked to a promoter The tenth expression vector to be;

Vi) an eleventh expression vector and a twelfth expression vector each including the seventh gene construct and the eighth gene construct;

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

Viii) a 14th expression vector including the ninth gene construct and optionally a 15th expression vector including the 10th gene construct.

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

In the vaccine composition, the vaccine adjuvant may further include 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) The polynucleotide encoding the MIP-1α protein is operable by a polynucleotide encoding an IRES or a linker peptide in any one or more of the polynucleotides encoding the IL-12p35, IL-12p40 and IL-21 proteins, respectively. Complex gene constructs linked together; And

Iv) an mRNA molecule encoding the MIP-1α protein.

The polynucleotide encoding the MIP-1α protein is operably linked to the polynucleotide encoding the IL-12p35, IL-12p40 and IL-21 proteins respectively through a polynucleotide encoding a peptide linker or an IRES or a separate gene It can be provided in the form of a construct. In addition, the MIP-1α gene construct comprises a polynucleotide encoding each of the p35 chain (IL-12p35) and p40 chain (IL-12p40) constituting the IL-12, and a polynucleotide encoding each of the IL-21 protein. It may be included in any one or more of the included one to three vectors. 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 embodiments of the vaccine adjuvant.

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 composed of the amino acid sequence described in SEQ ID NO: 10, and the human body It is also possible to use MIP-1α protein derived from non-humans such as primates or apes with a high level of homology that does not induce an immune response within the body.

The vaccine composition may further include IL-7.

The vaccine composition may additionally 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, when administered to a human, does not usually cause allergic reactions such as gastrointestinal disorders, dizziness, or similar reactions. Examples of the carrier, excipient and diluent include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, Polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oils. In addition, fillers, anti-aggregating agents, lubricants, wetting agents, flavoring agents, emulsifying agents, and preservatives may additionally be included.

The vaccine composition may further include a commonly used vaccine adjuvant in addition to the vaccine adjuvant. Such vaccine adjuvants include 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 or 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, and sterile powder forms.

The vaccine composition according to an embodiment of the present invention may be administered by various routes, for example, oral, parenteral, for example, suppository, transdermal, intravenous, intraperitoneal, intramuscular, intralesional, nasal, intrathecal administration It may be administered as a sustained release or using an implantable device for continuous or repeated release. The number of administration may be administered once a day or divided into several times a day within a desired range, and 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 local administration, such as intramuscular injection or intravenous injection, but when provided as a DNA vaccine composition, most preferably, an electroporator Can be injected using The electroporator is a commercially available electroporator for injecting DNA drugs, such as Glinporator ^TM of IGEA of Italy, CUY21EDIT of JCBIO of Korea, SP-4a of Supertech of Switzerland, OrbiJector of SLVAXiGEN of Korea.Etc. can be used.

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

In addition, the multivalent HPV DNA vaccine composition according to an embodiment of the present invention may be administered in combination with an IL-7 protein or a polynucleotide encoding it. In general, since the DNA drug and the protein drug may have different routes of administration, one Although it may be administered as a preparation of, it may be packaged in a separate formulation and administered through a different route. 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 method of administration of protein drugs such as general intramuscular injection, intravenous administration, or intraperitoneal administration. .

The vaccine composition according to an embodiment of the present invention may be formulated in a suitable form together with a generally used pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers include, for example, carriers for parenteral administration such as water, suitable oils, saline, aqueous glucose and glycol, and the like, and may further include stabilizers and preservatives. Suitable stabilizers are antioxidants such as sodium hydrogen sulfite, sodium sulfite or ascorbic acid. Suitable preservatives are benzalkonium chloride, methyl- or propyl-paraben and chlorobutanol. In addition, the composition according to the present invention is a suspension agent, a solubilizer, a stabilizer, an isotonic agent, a preservative, an adsorption inhibitor, a surfactant, a diluent, an excipient, a pH adjuster, a painless agent, a buffer agent, if necessary depending on the administration method or formulation. Antioxidants and the like may be appropriately included. Pharmaceutically acceptable carriers and formulations suitable for the present invention, including those exemplified above, are described in detail in Remington's Pharmaceutical Sciences, the latest edition.

The dosage of the vaccine composition to a patient 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. The pharmaceutically active DNA may be administered in an amount of 100 ng/body weight (kg)-10 mg/body weight (kg), more preferably 1 to 500 μg/kg (body weight). Preferably, it may be administered at 5 to 50 μg/kg (body weight), and the dosage may 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 with a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level refers to the type and severity of the subject, age, sex, drug. Activity, sensitivity to drugs, time of administration, route of administration and rate of excretion, duration of treatment, factors including drugs 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 1 mg/kg to 500 mg/kg, and 1 mg as a unit dose, It may be administered in 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, etc. Meanwhile, the dosage may be appropriately adjusted according to the age, sex and condition of the patient.

According to another aspect of the present invention, there is provided a method for treating diseases caused by HPV infection, comprising administering the multivalent HPV DNA vaccine composition or the 14-valent HPV DNA vaccine composition to an individual.

In the treatment method, the diseases caused by the HPV infection are squamous cell carcinoma (SCC), adenocarcinoma, adenosquamous cell carcinoma, small cell carcinoma, neuroendocrine tumor (NET), free cell carcinoma, chorionic adenocarcinoma (VGA), It may be a non-carcinoma malignancy, melanoma, lymphoma, or cervical intraepithelial tumor (CIN).

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

In order to solve the problem that conventional HPV vaccines cannot be applied universally to HPV-infected diseases such as cervical cancer, the present inventors have developed cervical intraepithelial neoplasia (CIN), cervical cancer, and vulvar intraepithelial cancer. Neoplasia (VIN), vulvar cancer, vaginal intraepithelial neoplasia, vaginal cancer, and warts (Anogenital warts, genital warts). , Type 18, Type 31, Type 33, Type 35, Type 39, Type 45, Type 51, Type 52, Type 56, Type 58, and Type 59 Multivalent DNA to cover all human papilloma virus (HPV) As a vaccine, a multivalent HPV DNA vaccine was prepared consisting of three plasmids in which the E6/E7 shuffled protein of each HPV can be expressed in the form of one giant fusion protein per four or five types. It was experimentally demonstrated that antigens HPV16 and HPV18 as well as other types of HPV induce T cell-specific immune responses. This result is very encouraging in that it was achieved by using an expression vector expressing multiple shuffled antigen proteins in which several shuffled proteins are connected only by a linker. will be. Moreover, surprisingly, even if the multivalent HPV DNA vaccine according to an embodiment of the present invention is administered at a very low dose of 1/4 compared to the conventional divalent vaccine, T cell specific immune responses to HPV16 and HPV18 similar to the conventional divalent vaccine In addition, in the case of types 6, 11, 39, 45, and 56, higher T cell-specific immune responses were induced than those for HPV16 or HPV18. Therefore, the multivalent HPV DNA vaccine according to an embodiment of the present invention can be used very efficiently as a preventive vaccine capable of simultaneously preventing infection against the 7 types of HPV, as well as 31, 33, 35, 51, It can be effectively used as a vaccine for preventing infection and treating infectious diseases of at least one high-risk HPV selected from the group consisting of 52, 58 and 59 types.

Hereinafter, the present invention will be described in more detail through examples and experimental examples. However, the present invention is not limited to the embodiments and experimental examples disclosed below, but can be implemented in various different forms, and the following examples and experimental examples are intended to complete the disclosure of the present invention, and to which the present invention belongs. It is provided to fully inform the scope of the invention to those of ordinary skill in the art.

Example 1: Preparation of multivalent HPV DNA vaccine

In order to prepare a multivalent HPV DNA vaccine, the inventors of the present invention are 6 type, 11 type, 16 type, 18 type, 31 type, 33 type, 35 type, 39 type, 45 type, 51 type, 52 type, 56 type belonging to high risk group , E6 antigens of each type to express the early expression proteins E6 and E7 antigens of type 58 and type 59 human papilloma virus (HPV) in the form of shuffled proteins mixed so as not to exhibit the function of wild type E6 and E7. And polynucleotides encoding the N-terminal fragment and the C-terminal fragment of the E7 antigen were obtained through a PCR reaction, and then ligated as described in Fig. 1 and Table 1 to prepare three gene constructs, HPV DNA vaccine constructs were prepared by inserting these into the pGX-27 vector, respectively, and these were designated as BD-14A, BD-14B and BD-14C, respectively, and the composition containing the three vectors was designated as BD-14. . The reason for producing a multivalent HPV DNA vaccine construct with a three vector system as described above is that the capacity of the pGX-27 vector is inefficient when the size of the inserted gene construct is too large.

1 and Table 1 below, each type of HPV E6 and E7 antigen is divided into an N-terminal fragment and a C-terminal fragment in which some sequences (20 aa) overlap, respectively, and then the N-terminal fragment of E6 ( E6N) is followed by a fusion polypeptide to which the C-terminal fragment (E7C) of E7 is connected (GS) is a fusion polypeptide to which the C-terminal fragment (E6C) of E6 is connected to the N-terminal fragment (E7N) of E7 as a _{5 linker peptide.} It has a re-linked structure, and 4 to 5 antigen units of each of these types _{were constructed to be linked to the (GS) 5} linker to be included in one vector. The types of each subtype inserted into one expression vector are exemplarily described in Table 1, but these are exemplary and may be prepared in any other order.

Construction of the multivalent HPV DNA vaccine construct of the present invention Component Concrete structure Sequence number origin protein Nucleic acid common
Element Linker (GS) ₅ 20 21 N/A tPA tPA _1-22 22 23 Uniprot: P00750 Flt3L Flt3L _{27-182, △1-26} 24 25 Uniprot: P49771 BD-14A 16 E6E7 16E6N _1-85 -16E7C _41-105 -(GS) ₅ -16E7N _1-60 -16E6C _66-158 26 27 GenBank: K02718.1 18 E6E7 18E6N _1-85- 18E7C _41-98 -(GS) ₅ -18E7N _1-60 -18E6C _66-158 GenBank: X05015.1 35 E6E7 35E6N _1-78- 35E7C _42-99 -(GS) ₅ -35E7N _1-61 -35E6C _59-149 GenBank: X74477.1 45 E6E7 _{45E6N 1-85 -45E7C 41-10 6- (GS)} 5 -45E7N 1-60 -45E6C 66-158 GenBank: X74479.1 58 E6E7 58E6N _1-85 -58E7C _41-98 -(GS) ₅ -58E7N _1-60 -58E6C _66-149 GenBank: D90400.1 BD-14B 31 E6E7 31E6N _1-85- 31E7C _42-98 -(GS) ₅ -31E7N _1-61 -31E6C _66-149 28 29 GenBank: J04353.1 33 E6E7 33E6N _1-85 -33E7C _42-96 -(GS) ₅ -33E7N _1-61 -33E6C _66-149 GenBank: M12732.1 06 E6E7 6E6N _1-85 -6E7C _42-98 -(GS) ₅ -6E7N _1-61 -6E6C _66-150 GenBank: X00203.1 11 E6E7 11E6N _1-85 -11E7C _42-98 -(GS) ₅ -11E7N _1-61 -11E6C _66-150 GenBank: M14119.1 52 E6E7 52E6N _1-85 -52E7C _41-99 -(GS) ₅ -52E7N _1-60 -52E6C _66-148 GenBank: X74481.1 BD-14C 39 E6E7 39E6N _1-85 -39E7C _44-109 -(GS) ₅ -39E7N _1-63 -51E6C _66-158 30 31 GenBank: M62849.1 51 E6E7 51E6N _1-83 -51E7C _45-101 -(GS) ₅ -51E7N _1-64 -51E6C _64-151 Uniprot: P26554(E6),
P26558(E7) 56 E6E7 56E6N _1-86 -56E7C _48-105 -(GS) ₅ -56E7N _1-67 -56E6C _67-155 Uniprot: P24836(E6), P36833(E7) 59 E6E7 59E6N _1-85 -59E7C _50-107 -(GS) ₅ -59E7N _1-69 -59E6C _66-160 GenBank: CAA54849.1(E6), CAA54850.1(E7)

Example 2: Preparation of human IL-12 and IL-21 expression vectors

2-1: single vector system

The present inventors designed a single vector system so that IL-12 and IL-21 are expressed through one vector.

Specifically, for this purpose, the present inventors prepared hIL-12p35 consisting of the amino acid sequence described in SEQ ID NO: 1, which is two subunits of human IL-12 protein, and hIL-12p40 polypeptide consisting of the amino acid sequence described in SEQ ID NO: 2, respectively. Encoding polynucleotides (SEQ ID NOs: 4 and 5) were ligated to an EMCV-derived internal ribosome entry site (IRS) having a nucleic acid sequence represented by SEQ ID NO: 6, and the hIL-12p40 polypeptide encoding Polynucleotide encoding human IL-21 protein (hIL-21) consisting of the RSV promoter (pRSV) shown in SEQ ID NO: 7 at the 3'-end of the polynucleotide and the amino acid sequence shown in SEQ ID NO: 3 (SEQ ID NO: 8) After preparing a gene construct by connecting sequentially, the gene construct is inserted into the multicloning site of the pGX-27 vector (Korean Patent No. 1442254), and the vector according to an embodiment of the present invention. Was prepared and named it'hBD-121' (FIG. 2A).

1-2: double vector system

The present inventors designed 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 EMCV-IRES having the nucleic acid sequence described in SEQ ID NO: 6, and this The polynucleotide (SEQ ID NO: 8) which is inserted into the multicloning site of the pGX-27 vector and encodes the human IL-21 protein (hIL-21) composed of the amino acid sequence shown in SEQ ID NO: 3 is also used in the pGX-27 vector. By inserting into the multicloning site, a vector expressing IL-12 and IL-21, respectively, is prepared. This was named'hBD-12 and hBD-21'.

1-3: triple vector system

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

The triple vector system can be prepared as follows:

The hIL-12p35 polypeptide, the hIL-12p40 polypeptide, and the polynucleotides (SEQ ID NOs: 4, 5 and 8) respectively encoding hIL-21 are inserted into the multicloning site of the pGX-27 vector to prepare a triple vector system.

Example 3: Preparation of human IL-12, IL-21 and MIP-α expression vectors

Polynucleotides (SEQ ID NOs: 4 and 5) each encoding the hIL-12p35 polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1 and the hIL-12p40 polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 2 are identified as SEQ ID NO: 6 RSV described in SEQ ID NO: 7 at the 3'-end of the polynucleotide encoding the hIL-12p40 polypeptide and ligated to an EMCV-derived internal ribosome entry site (IRS) having a nucleic acid sequence described as A promoter (pRSV), and a polynucleotide encoding a human IL-21 protein (hIL-21) consisting of the amino acid sequence shown in SEQ ID NO: 3 (SEQ ID NO: 8) is sequentially linked to a polynucleotide described in SEQ ID NO: 9 Polynucleotide (SEQ ID NO: 11) encoding human EF-1α protein (hMIP-1α) consisting of the amino acid sequence shown in SEQ ID NO: 10 and the human EF-1α promoter (pEF-1α) consisting of a nucleic acid sequence is sequentially The linked gene construct was prepared and inserted into the multicloning site of the pGX-27 vector, which was named hBD-121A (Fig. 2b).

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

Polynucleotides encoding the mIL-12p35 polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 12, which are two subunits of the mouse IL-12 protein, and the mIL-12p40 polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 13, respectively (SEQ ID NO: Numbers 14 and 15) were ligated to an EMCV-derived internal ribosome entry site (IRS) having a nucleic acid sequence represented by SEQ ID NO: 6, and 3'- of the polynucleotide encoding the mIL-12p40 polypeptide. At the end, a polynucleotide encoding a mouse IL-21 protein (mIL-21) consisting of the RSV promoter (pRSV) shown in SEQ ID NO: 7 and the amino acid sequence shown in SEQ ID NO: 16 (SEQ ID NO: 17) is sequentially linked After preparing one gene construct, the gene construct was inserted into the multiple cloning site of the pGX-27 vector to prepare a vector according to an embodiment of the present invention, and it was named'mBD-121' (Fig. 2a).

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

Polynucleotides (SEQ ID NOs: 14 and 15) each encoding the mIL-12p35 polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 12 and the mIL-12p40 polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 13 are identified as SEQ ID NO: 6 RSV described in SEQ ID NO: 7 at the 3'-end of the polynucleotide encoding the mIL-12p40 polypeptide and ligated to an EMCV-derived internal ribosome entry site (IRS) having a nucleic acid sequence described as A promoter (pRSV) and a polynucleotide encoding a mouse IL-21 protein (mIL-21) consisting of the amino acid sequence shown in SEQ ID NO: 16 (SEQ ID NO: 17) are sequentially linked to a polynucleotide described in SEQ ID NO: 9 A polynucleotide encoding a human EF-1α promoter (pEF-1α) consisting of a nucleic acid sequence and a mouse MIP-1α protein (mMIP-1α) consisting of the amino acid sequence shown in SEQ ID NO: 18 (SEQ ID NO: 19) sequentially The linked gene construct was prepared and inserted into the multicloning site of the pGX-27 vector, which was named mBD-121A (FIG. 2B).

Experimental Example 1: Analysis of expression of BD-14

1-1: ELISA analysis

In order to confirm whether the multivalent HPV DNA vaccine BD-14 according to an embodiment of the present invention prepared in Example 1 is normally expressed when introduced into mammalian cells, the present inventors After transduction of the three expression vectors BD-14A, BD-14B and BD-14C included in each of the three expression vectors, the protein expression was analyzed using an antigen specific to Flt3L included in each construct.

Specifically, the COS-7 cell line was inoculated into a 100 mm culture dish and cultured for 16 hours, and then the blank vector (mock plasmid DNA) and the BD-14A, BD14-B and BD-14C plasmid DNA prepared in Example 1 were used with Lipofectamine 2000. Each was transfected, and _{after incubation for 3 days in a 37° C. CO 2} incubator, the culture supernatant of each condition of COS-7 cells was recovered and used as a sample. Since the proteins present in the sample are in a form to which Flt3L is bound, they were quantified using the Flt3L ELISA kit (Fig. 3a).

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

1-2: Western blot analysis

The present inventors performed SDS-PAGE electrophoresis on the cell lysate of the cells obtained in Experimental Example 1-1, transferred to a nylon membrane, and then anti-Flt3L antibody (Abcam, Cat# ab52648 ) Was used to perform western blot analysis (Fig. 3b).

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

Experimental Example 2: In vivo immune response analysis of BD-14

2-1: T cell specific immune response analysis

The present inventors investigated whether or not a T cell-specific immune response was induced after administering BD-14 according to an embodiment of the present invention to an experimental animal, and a method of counting the number of splenic immune cells exhibiting an immune response to each antigen. It was investigated using. Specifically, C57BL/6 mice, which are experimental animals, were used as a control group with an empty vector administration group (n=5), a conventional bivalent HPV DNA vaccine (Korean Patent Laid-Open Patent No. 10-2017-0045254) administration group (n=5) and an aspect of the present invention. Divided into the BD-14 administration group (n=5) according to the example, 2 µg of plasmid DNA (0.67 µg each in the case of BD-14A to BD-14C) was applied to the femoral muscle.(SLVAXiGEN, Korea) Spleen immune cells responding to each type of E6/E7 antigen by sacrificing the experimental animal after two weeks of administration using an in vivo electroporator and two weeks after the last administration. Were counted using ELISPOT analysis (FIGS. 4A and 4B ).

As a result, as shown in Figure 4b, in the case of the BD-14 according to an embodiment of the present invention, although relatively weak against 35 type, 52 type and 59 type HPV, all types of HPV E6 / E7 antigen It succeeded in inducing a T cell-specific immune response. On the other hand, there was no significant difference from the conventional bivalent DNA vaccine in reactivity to type 16 and type 18 HPV E6/E7 antigens. This is to prove that even though the ratio of type 16 and type 18 HPV E6/E7 antigens in BD-14 according to an embodiment of the present invention is only about 1/3 of the conventional 2 HPV DNA vaccine, equivalent efficacy is possible. . In particular, it was confirmed that a much stronger response was induced for 6, 11, 39, 45, and 56 type HPV than the immune response induced by 16, 18 type HPV.

2-2: Anti-tumor effect analysis

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

Specifically, an experimental animal C57BL/6 mouse as a control group was administered with an empty vector (n=8), a conventional bivalent HPV DNA vaccine (Korean Patent Publication No. 2017-0045254) administered group (n=8) and an implementation of the present invention. Divided into the BD-14 administration group according to the example (n=8), TC-1 cells derived from lung epithelial cells of C57BL/6 mice and transformed to express the E6/E7 antigen of HPV16 ^{5×10 5} After inducing a tumor by injecting a dog by subcutaneous injection into the back, 2 µg of plasmid DNA (0.67 µg each in the case of BD-14A to BD-14C) was added to the femoral muscle 3 days after administration of the cancer cells. OrbiJector (SLVAXiGEN, Korea ) The in vivo electroporation was administered twice at intervals of 2 weeks, and the size of the tumor and the survival rate of the experimental animals were investigated at intervals of 3 to 4 days from the 7th day of tumor injection (FIGS. 5A to 5C ).

As a result, as shown in Fig. 5b, BD-14 according to an embodiment of the present invention not only significantly reduced the tumor size compared to the control group, but also showed anti-tumor efficacy similar to that of the conventional bivalent HPV DNA vaccine. In addition, as shown in Fig. 5c, in terms of survival rate, contrary to the results of the tumor size analysis, the survival rate of the BD-14-administered group according to an embodiment of the present invention was higher than that of the conventional 2 HPV DNA vaccine-administered group. Comprehensive review of these results, the BD-14 vaccine composition according to an embodiment of the present invention is equivalent to or better than the conventional divalent DNA vaccine, although the dose is 1/3 for the high-risk group such as HPV16 or HPV18. It has been proven to be a very innovative vaccine composition that not only exhibits anticancer activity, but can also increase the prevention range for cervical cancer to 90% or more by inducing a T cell-specific immune response to other types of HPV.

Experimental Example 3: Analysis of the effect of BD-121 as a vaccine adjuvant

3-1: ELISA analysis

The present inventors transduced cells with the hBD-121 construct and the mBD-121 construct according to an embodiment of the present invention prepared in Examples 2 and 4, respectively, and then IL-12 and Whether or not IL-21 is normally expressed was investigated. Specifically, the COS-7 cell line was inoculated into a 100 mm culture dish and cultured for 16 hours, followed by mock plasmid DNA, hBD-121 plasmid DNA prepared in Example 2-1, and mBD-121 plasmid prepared in Example 4 DNA was transfected using Lipofectamine 2000, respectively, and _{after incubation for 3 days in a 37°C CO 2} incubator, the culture supernatant of COS-7 cells under each condition was recovered and used as a sample. 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) It was quantified using the ELISA analysis method using (Fig. 6a and 6b).

As a result, as shown in Fig. 6a, it was confirmed that the protein expression level in the sample into which the hBD-121 plasmid DNA was introduced exceeded 4,000 pg/ml when 4 μg DNA was introduced in the case of hIL-12, and was normally expressed. Also, when 4 ㎍ DNA was introduced, it was confirmed that it was very high expression by showing a value close to 200 ng/ml. In addition, as shown in Figure 6b, the mouse construct also showed similar results to the human construct. On the other hand, in the case of the control group to which the empty vector was introduced, neither protein was expressed at all, and it was confirmed that the vaccine adjuvant expression system of the present invention operates normally.

3-2: Western blot analysis

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

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

Experimental Example 4: Analysis of the effect of BD-121A as a vaccine adjuvant

4-1: T cell specific immune response analysis

The present inventors conducted an analysis on whether BD-121A as a vaccine adjuvant improves the vaccine performance of BD-14.

To this end, the present inventors immunize against each antigen to determine whether to induce a T cell-specific immune response after administering BD-14 according to an embodiment of the present invention and mBD-121A prepared in Example 5 to an experimental animal. It was investigated using a method of counting the number of splenic immune cells exhibiting a response. Specifically, C57BL/6 mice, which are experimental animals, were used as a control group with an empty vector (n=3), BD-14 alone (n=5), and BD-14 and mBD-121A administered groups according to an embodiment of the present invention (n =5), respectively, 1.3 µg of each plasmid DNA (BD-14A, BD-14B, and BD-14C) for the group administered with BD-14 alone, and each plasmid DNA 1 for the combined group administered with BD-14 and mBD-121A. Μg was administered once to the femoral muscle using an in vivo electroporator of OrbiJector (SLVAXiGEN, Korea), and two weeks after the administration, the experimental animals were sacrificed and the spleen was excised to respond to each type of E6/E7 antigen Cells were counted using ELISPOT assay (FIGS. 7A and 7B ).

As a result, as shown in FIG. 7B, mBD-121A according to an embodiment of the present invention was found to enhance the E6/E7-specific T cell responses of various HPV types than when administered with BD-14 alone. In particular, in the case of high-risk type 16 HPV, the T cell immune response was more than doubled, and the 31, 33, 51 and 58 types showed a marked increase in the immune response. This is a result of demonstrating that BD-121A according to an embodiment of the present invention is a highly effective vaccine adjuvant for a multivalent HPV DNA vaccine.

4-2: Anti-tumor activity assay

Specifically, an experimental animal C57BL/6 mouse as a control group was administered with an empty vector (n=10), a conventional bivalent HPV DNA vaccine (Korean Patent Publication No. 2017-0045254) administration group (n=13) and an implementation of the present invention. After dividing into the BD-14 and BD-121 administration groups according to the example (n=13), ⁵ ×10 5 TC-1 cells used in Experimental Example 2-2 were injected into the back by subcutaneous injection to induce tumors, and then the above 7 days after inoculation of TC-1 cells, 4 μg of plasmid DNA (1 μg each in the case of BD-14A to BD-14C and BD-121) was applied to the femoral muscle by using an OrbiJector (SLVAXiGEN, Korea) in vivo electroporator. The tumor was administered twice at intervals of 2 weeks, and the size of the tumor and the survival rate of the experimental animals were investigated at intervals of 3 to 4 days from the 7th day of tumor inoculation (FIGS. 8A to 8C ).

As a result, as can be seen in Figures 8b and 8c, the multivalent vaccine according to an embodiment of the present invention showed significant anticancer efficacy compared to the negative control group (PBS administration group), when compared to the conventional bivalent vaccine, which is a positive control group. It was confirmed that it had an anticancer efficacy equal to or equal to or higher than that. This means that when the amount of antigen to be administered (E6/E7 of HPV16) is 1/4 level than that of the conventional 2 vaccine, it shows similar anticancer efficacy, and at the same time, even when many types (number) of antigens are administered. Nevertheless, it means that it can induce various immune responses and that the anticancer efficacy against a specific type (HPV16) is not impaired.

Experimental Example 5: Analysis of the mechanism of action of the vaccine

In order to investigate the mechanism by which the multivalent HPV DNA vaccine according to an embodiment of the present invention works, the present inventors administered anti-CD4 or anti-CD8 antibodies to CD-4 T cells and CD-8 T cells, respectively. An experimental animal from which was removed was prepared, and the anticancer activity of the HPV DNA vaccine according to an embodiment of the present invention was compared and analyzed.

Specifically, the experiment was conducted by dividing the experimental animal C57BL/6 mice into four groups, and an empty vector administration group (n=9) was used as a control, and a vaccine and vaccine adjuvant (BD14A+BD121A) according to an embodiment of the present invention. ) The administration group was divided into an isotype administration group (n=9), an anti-CD4 antibody administration group (n=9), and an anti-CD8 antibody administration group (n=9) as a control group. The experimental schedule is as follows: After subcutaneous inoculation of the TC-1 cancer cells of Experimental Example 2-2 at ⁵ ×10 5 cells per head, antibodies (isotype, anti-CD4 and anti-CD8 antibodies) were used from day 1 from the date of inoculation of cancer cells. Intraperitoneally administered 7 times at a dose of 200 μg/injection/mouse every 7 days, the vaccine composition (BD-14A and BD-121) according to an embodiment of the present invention is 7 days apart from the 3rd day from the date of inoculation of cancer cells. As a result, it was intramuscularly administered to the hind femoral muscle at a dose of 8 μg/injection/mouse using electroporation three times (FIG. 9A). From the 9th day of tumor inoculation, the size of the tumor and the survival rate of the experimental animals were examined at intervals of 3 to 4 days (FIGS. 9B to 9C ).

As a result, as shown in Figs. 9b and 9c, the anticancer efficacy was maintained in the mouse from which the CD4 T cells were removed, but the anticancer efficacy in the mouse from which the CD8 T cells were removed was worse than that of the control group (mock and homologous antibody). Could be observed. This shows that the multivalent HPV DNA vaccine according to an embodiment of the present invention exhibits anti-cancer immunity through CD8 T cells, which is consistent with the fact that CD8 T cells are very important to the efficacy of anti-cancer therapeutic vaccines as previously known. to be.

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

IL-7 (Interleukin 7) is a cytokine that promotes the differentiation of pluripotent hematopoietic stem cells into lymphocytic 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 malignantization of hematologic cancers such as acute lymphocytic leukemia and T cell lymphoma, but in general solid cancer, it disrupts the homeostasis of CD8 and CD4 cells, thereby reducing ^{the ratio of CD4 +} CD25 ⁺ Foxp3 ⁺ regulatory T cells. It is known to reduce, and is currently undergoing phase 1 and phase 2 clinical trials for several cancers. From the results of Experimental Example 5, the present inventors found that IL-7 affects the balance between CD4 T cells and CD8 T cells and tends to cause an increase in CD8 T cells, according to an embodiment of the present invention. A hypothesis was established that synergistic effects could be expected when administered concurrently with HPV DNA vaccine. In order to confirm whether this hypothesis is correct, the present inventors analyzed anticancer activity after administering the vaccine composition according to an embodiment of the present invention alone or in combination with IL-7.

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

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

As described above, the multivalent HPV DNA vaccine BD-14 according to an embodiment of the present invention normally expresses the E6/E7 shuffled protein, which is an antigen protein contained therein, despite its complex structure, and actually various types of HPV T cell-specific immune response to the E6/E7 antigen was successfully induced, and as a result of an anticancer activity assay for a cancer model expressing the high-risk HPV16 E6/E7 antigen, the anticancer effect was equal to or better than the conventional divalent DNA vaccine Showed. Furthermore, the multivalent HPV DNA vaccine according to an embodiment of the present invention is a vaccine adjuvant, and a T cell-specific immune response to HPV E6/E7 antigen when administered in combination with BD-121A according to an embodiment of the present invention Not only was it remarkably increased, but it also showed a more remarkable effect in anticancer effect.

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

The present invention has been described with reference to the above-described embodiments and experimental examples, but these are merely exemplary, and those of ordinary skill in the art will appreciate that various modifications and equivalent other embodiments are possible therefrom. Therefore, the true technical protection scope of the present invention should be determined by the technical spirit of the appended claims.

<110> SL VAXIGEN, Inc. <120> A novel polyvalent HPV vaccine compostion <130> PD18-5742 <150> KR 10-2018-0013328 <151> 2018-02-02 <160> 50 <170> KoPatentIn 3.0 <210> 1 <211> 253 <212> PRT <213> Homo sapiens <400> 1 Met Trp Pro Pro Gly Ser Ala Ser Gln Pro Pro Pro Ser Pro Ala Ala 1 5 10 15 Ala Thr Gly Leu His Pro Ala Ala Arg Pro Val Ser Leu Gln Cys Arg 20 25 30 Leu Ser Met Cys Pro Ala Arg Ser Leu Leu Leu Val Ala Thr Leu Val 35 40 45 Leu Leu Asp His Leu Ser Leu Ala Arg Asn Leu Pro Val Ala Thr Pro 50 55 60 Asp Pro Gly Met Phe Pro Cys Leu His His Ser Gln Asn Leu Leu Arg 65 70 75 80 Ala Val Ser Asn Met Leu Gln Lys Ala Arg Gln Thr Leu Glu Phe Tyr 85 90 95 Pro Cys Thr Ser Glu Glu Ile Asp His Glu Asp Ile Thr Lys Asp Lys 100 105 110 Thr Ser Thr Val Glu Ala Cys Leu Pro Leu Glu Leu Thr Lys Asn Glu 115 120 125 Ser Cys Leu Asn Ser Arg Glu Thr Ser Phe Ile Thr Asn Gly Ser Cys 130 135 140 Leu Ala Ser Arg Lys Thr Ser Phe Met Met Ala Leu Cys Leu Ser Ser 145 150 155 160 Ile Tyr Glu Asp Leu Lys Met Tyr Gln Val Glu Phe Lys Thr Met Asn 165 170 175 Ala Lys Leu Leu Met Asp Pro Lys Arg Gln Ile Phe Leu Asp Gln Asn 180 185 190 Met Leu Ala Val Ile Asp Glu Leu Met Gln Ala Leu Asn Phe Asn Ser 195 200 205 Glu Thr Val Pro Gln Lys Ser Ser Leu Glu Glu Pro Asp Phe Tyr Lys 210 215 220 Thr Lys Ile Lys Leu Cys Ile Leu Leu His Ala Phe Arg Ile Arg Ala 225 230 235 240 Val Thr Ile Asp Arg Val Met Ser Tyr Leu Asn Ala Ser 245 250 <210> 2 <211> 328 <212> PRT <213> Homo sapiens <400> 2 Met Cys His Gln Gln Leu Val Ile Ser Trp Phe Ser Leu Val Phe Leu 1 5 10 15 Ala Ser Pro Leu Val Ala Ile Trp Glu Leu Lys Lys Asp Val Tyr Val 20 25 30 Val Glu Leu Asp Trp Tyr Pro Asp Ala Pro Gly Glu Met Val Val Leu 35 40 45 Thr Cys Asp Thr Pro Glu Glu Asp Gly Ile Thr Trp Thr Leu Asp Gln 50 55 60 Ser Ser Glu Val Leu Gly Ser Gly Lys Thr Leu Thr Ile Gln Val Lys 65 70 75 80 Glu Phe Gly Asp Ala Gly Gln Tyr Thr Cys His Lys Gly Gly Glu Val 85 90 95 Leu Ser His Ser Leu Leu Leu Leu His Lys Lys Glu Asp Gly Ile Trp 100 105 110 Ser Thr Asp Ile Leu Lys Asp Gln Lys Glu Pro Lys Asn Lys Thr Phe 115 120 125 Leu Arg Cys Glu Ala Lys Asn Tyr Ser Gly Arg Phe Thr Cys Trp Trp 130 135 140 Leu Thr Thr Ile Ser Thr Asp Leu Thr Phe Ser Val Lys Ser Ser Arg 145 150 155 160 Gly Ser Ser Asp Pro Gln Gly Val Thr Cys Gly Ala Ala Thr Leu Ser 165 170 175 Ala Glu Arg Val Arg Gly Asp Asn Lys Glu Tyr Glu Tyr Ser Val Glu 180 185 190 Cys Gln Glu Asp Ser Ala Cys Pro Ala Ala Glu Glu Ser Leu Pro Ile 195 200 205 Glu Val Met Val Asp Ala Val His Lys Leu Lys Tyr Glu Asn Tyr Thr 210 215 220 Ser Ser Phe Phe Ile Arg Asp Ile Ile Lys Pro Asp Pro Pro Lys Asn 225 230 235 240 Leu Gln Leu Lys Pro Leu Lys Asn Ser Arg Gln Val Glu Val Ser Trp 245 250 255 Glu Tyr Pro Asp Thr Trp Ser Thr Pro His Ser Tyr Phe Ser Leu Thr 260 265 270 Phe Cys Val Gln Val Gln Gly Lys Ser Lys Arg Glu Lys Lys Asp Arg 275 280 285 Val Phe Thr Asp Lys Thr Ser Ala Thr Val Ile Cys Arg Lys Asn Ala 290 295 300 Ser Ile Ser Val Arg Ala Gln Asp Arg Tyr Tyr Ser Ser Ser Trp Ser 305 310 315 320 Glu Trp Ala Ser Val Pro Cys Ser 325 <210> 3 <211> 155 <212> PRT <213> Homo sapiens <400> 3 Met Glu Arg Ile Val Ile Cys Leu Met Val Ile Phe Leu Gly Thr Leu 1 5 10 15 Val His Lys Ser Ser Ser Gln Gly Gln Asp Arg His Met Ile Arg Met 20 25 30 Arg Gln Leu Ile Asp Ile Val Asp Gln Leu Lys Asn Tyr Val Asn Asp 35 40 45 Leu Val Pro Glu Phe Leu Pro Ala Pro Glu Asp Val Glu Thr Asn Cys 50 55 60 Glu Trp Ser Ala Phe Ser Cys Phe Gln Lys Ala Gln Leu Lys Ser Ala 65 70 75 80 Asn Thr Gly Asn Asn Glu Arg Ile Ile Asn Val Ser Ile Lys Lys Leu 85 90 95 Lys Arg Lys Pro Pro Ser Thr Asn Ala Gly Arg Arg Gln Lys His Arg 100 105 110 Leu Thr Cys Pro Ser Cys Asp Ser Tyr Glu Lys Lys Pro Pro Lys Glu 115 120 125 Phe Leu Glu Arg Phe Lys Ser Leu Leu Gln Lys Met Ile His Gln His 130 135 140 Leu Ser Ser Arg Thr His Gly Ser Glu Asp Ser 145 150 155 <210> 4 <211> 759 <212> DNA <213> Homo sapiens <400> 4 atgtggcccc ctgggtcagc ctcccagcca ccgccctcac ctgccgcggc cacaggtctg 60 catccagcgg ctcgccctgt gtccctgcag tgccggctca gcatgtgtcc agcgcgcagc 120 ctcctccttg tggctaccct ggtcctcctg gaccacctca gtttggccag aaacctcccc 180 gtggccactc cagacccagg aatgttccca tgccttcacc actcccaaaa cctgctgagg 240 gccgtcagca acatgctcca gaaggccaga caaactctgg aattttaccc ttgcacttct 300 gaagagattg atcatgaaga tatcacaaaa gataaaacga gcacagtgga ggcctgttta 360 ccattggaat taaccaagaa tgagagttgc ctaaattcca gagagacctc tttcataact 420 aatgggagtt gcctggcctc cagaaagacc tcttttatga tggccctgtg ccttagtagt 480 atttatgaag acttgaagat gtaccaggtg gagttcaaga ccatgaatgc aaagcttctg 540 atggatccta agaggcagat ctttctggat caaaacatgc tggcagttat tgatgagctg 600 atgcaggccc tgaatttcaa cagtgagact gtgccacaaa aatcctccct tgaagaaccg 660 gatttttata aaactaaaat caagctctgc atacttcttc atgctttcag gattcgggca 720 gtgactattg atagagtgat gagctatctg aatgcttcc 759 <210> 5 <211> 987 <212> DNA <213> Homo sapiens <400> 5 atgtgccacc agcagctggt catcagctgg ttctccctgg tctttctggc ttctcctctg 60 gtggcaattt gggagctgaa gaaagacgtg tacgtggtcg aactggactg gtatccagat 120 gcccccggag agatggtggt cctgacctgc gacacaccag aggaagatgg catcacttgg 180 accctggacc agagctccga ggtcctggga agcggcaaga cactgactat tcaggtgaaa 240 gaattcgggg atgctggaca gtacacatgt cataagggcg gggaggtgct gtcccactct 300 ctgctgctgc tgcataagaa agaagatggc atctggtcta ctgacattct gaaggatcag 360 aaagagccca agaacaaaac cttcctgaga tgcgaagcca agaattatag cgggaggttt 420 acctgttggt ggctgaccac aatctctact gacctgacct ttagtgtgaa gtctagtagg 480 gggtcaagcg atcctcaggg agtgacctgc ggagcagcta cactgagcgc agagcgggtc 540 agaggagaca acaaggagta cgaatattcc gtggagtgcc aggaagattc tgcatgtccc 600 gcagccgagg aatccctgcc tatcgaagtg atggtggacg ccgtgcacaa gctgaaatac 660 gaaaactaca catcctcttt ctttatccgg gacatcatta agccagatcc ccctaaaaac 720 ctgcagctga agcccctgaa aaattcacga caggtggagg tcagctggga ataccctgat 780 acatggagca ctccacattc ttatttcagt ctgacttttt gcgtgcaggt ccagggcaag 840 agtaaacgag agaagaaaga ccgggtcttc accgataaga catccgctac tgtgatctgt 900 cggaaaaacg ccagtatttc agtgagggct caggaccgct actatagttc aagctggtca 960 gagtgggcaa gcgtgccctg ttcctag 987 <210> 6 <211> 579 <212> DNA <213> Artificial Sequence <220> <223> ECMV IRES <400> 6 cccctctccc tccccccccc ctaacgttac tggccgaagc cgcttggaat aaggccggtg 60 tgcgtttgtc tatatgttat tttccaccat attgccgtct tttggcaatg tgagggcccg 120 gaaacctggc cctgtcttct tgacgagcat tcctaggggt ctttcccctc tcgccaaagg 180 aatgcaaggt ctgttgaatg tcgtgaagga agcagttcct ctggaagctt cttgaagaca 240 aacaacgtct gtagcgaccc tttgcaggca gcggaacccc ccacctggcg acaggtgcct 300 ctgcggccaa aagccacgtg tataagatac acctgcaaag gcggcacaac cccagtgcca 360 cgttgtgagt tggatagttg tggaaagagt caaatggctc tcctcaagcg tattcaacaa 420 ggggctgaag gatgcccaga aggtacccca ttgtatggga tctgatctgg ggcctcggtg 480 cacatgcttt acatgtgttt agtcgaggtt aaaaaaacgt ctaggccccc cgaaccacgg 540 ggacgtggtt ttcctttgaa aaacacgatg ataatatgg 579 <210> 7 <211> 527 <212> DNA <213> Artificial Sequence <220> <223> RSV promoter <400> 7 ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg cgagcaaaat ttaagctaca 60 acaaggcaag gcttgaccga caattgcatg aagaatctgc ttagggttag gcgttttgcg 120 ctgcttcgcg atgtacgggc cagatatacg cgtatctgag gggactaggg tgtgtttagg 180 cgaaaagcgg ggcttcggtt gtacgcggtt aggagtcccc tcaggatata gtagtttcgc 240 ttttgcatag ggagggggaa atgtagtctt atgcaatact cttgtagtct tgcaacatgg 300 taacgatgag ttagcaacat gccttacaag gagagaaaaa gcaccgtgca tgccgattgg 360 tggaagtaag gtggtacgat cgtgccttat taggaaggca acagacgggt ctgacatgga 420 ttggacgaac cactgaattc cgcattgcag agatattgta tttaagtgcc tagctcgata 480 caataaacgc catttgacca ttcaccacat tggtgtgcac ctccaag 527 <210> 8 <211> 468 <212> DNA <213> Homo sapiens <400> 8 atggaacgga ttgtcatttg cctgatggtc atttttctgg gaaccctggt ccacaagtca 60 agcagtcagg gccaggatag gcacatgatt aggatgcgcc agctgatcga cattgtggat 120 cagctgaaga actacgtgaa tgacctggtc cctgagtttc tgcctgcacc agaggatgtc 180 gaaacaaact gcgaatggag cgccttctcc tgttttcaga aggcccagct gaaatccgct 240 aacaccggca acaatgagcg aatcatcaac gtgagcatca agaagctgaa gcggaaaccc 300 cctagcacta atgctgggcg gagacagaaa catagactga cctgcccctc ttgtgacagt 360 tatgaaaaga aaccacccaa ggagttcctg gaacgcttta aaagtctgct gcagaaaatg 420 attcaccagc acctgtcctc cagaactcac gggtccgaag attcctaa 468 <210> 9 <211> 1189 <212> DNA <213> Artificial Sequence <220> <223> hEF-1alpha promoter <400> 9 cgtgaggctc cggtgcccgt cagtgggcag agcgcacatc gcccacagtc cccgagaagt 60 tggggggagg ggtcggcaat tgaaccggtg cctagagaag gtggcgcggg gtaaactggg 120 aaagtgatgt cgtgtactgg ctccgccttt ttcccgaggg tgggggagaa ccgtatataa 180 gtgcagtagt cgccgtgaac gttctttttc gcaacgggtt tgccgccaga acacaggtaa 240 gtgccgtgtg tggttcccgc gggcctggcc tctttacggg ttatggccct tgcgtgcctt 300 gaattacttc cacgcccctg gctgcagtac gtgattcttg atcccgagct tcgggttgga 360 agtgggtggg agagttcgag gccttgcgct taaggagccc cttcgcctcg tgcttgagtt 420 gaggcctggc ctgggcgctg gggccgccgc gtgcgaatct ggtggcacct tcgcgcctgt 480 ctcgctgctt tcgataagtc tctagccatt taaaattttt gatgacctgc tgcgacgctt 540 tttttctggc aagatagtct tgtaaatgcg ggccaagatc tgcacactgg tatttcggtt 600 tttggggccg cgggcggcga cggggcccgt gcgtcccagc gcacatgttc ggcgaggcgg 660 ggcctgcgag cgcggccacc gagaatcgga cgggggtagt ctcaagctgg ccggcctgct 720 ctggtgcctg gcctcgcgcc gccgtgtatc gccccgccct gggcggcaag gctggcccgg 780 tcggcaccag ttgcgtgagc ggaaagatgg ccgcttcccg gccctgctgc agggagctca 840 aaatggagga cgcggcgctc gggagagcgg gcgggtgagt cacccacaca aaggaaaagg 900 gcctttccgt cctcagccgt cgcttcatgt gactccacgg agtaccgggc gccgtccagg 960 cacctcgatt agttctcgag cttttggagt acgtcgtctt taggttgggg ggaggggttt 1020 tatgcgatgg agtttcccca cactgagtgg gtggagactg aagttaggcc agcttggcac 1080 ttgatgtaat tctccttgga atttgccctt tttgagtttg gatcttggtt cattctcaag 1140 cctcagacag tggttcaaag tttttttctt ccatttcagg tgtcgtgaa 1189 <210> 10 <211> 92 <212> PRT <213> Homo sapiens <400> 10 Met Gln Val Ser Thr Ala Ala Leu Ala Val Leu Leu Cys Thr Met Ala 1 5 10 15 Leu Cys Asn Gln Phe Ser Ala Ser Leu Ala Ala Asp Thr Pro Thr Ala 20 25 30 Cys Cys Phe Ser Tyr Thr Ser Arg Gln Ile Pro Gln Asn Phe Ile Ala 35 40 45 Asp Tyr Phe Glu Thr Ser Ser Gln Cys Ser Lys Pro Gly Val Ile Phe 50 55 60 Leu Thr Lys Arg Ser Arg Gln Val Cys Ala Asp Pro Ser Glu Glu Trp 65 70 75 80 Val Gln Lys Tyr Val Ser Asp Leu Glu Leu Ser Ala 85 90 <210> 11 <211> 279 <212> DNA <213> Homo sapiens <400> 11 atgcaggtgt caaccgccgc cctggctgtc ctgctgtgca ctatggctct gtgcaatcag 60 ttttccgcaa gtctggccgc tgatactccc accgcctgct gtttctctta cacaagtagg 120 cagatccctc agaacttcat tgctgactat tttgagacta gctcccagtg cagcaagccc 180 ggcgtgatct ttctgaccaa gcggagccgg caggtctgtg ccgatccctc cgaagaatgg 240 gtgcagaagt atgtctccga cctggaactg tcagcataa 279 <210> 12 <211> 215 <212> PRT <213> Mus musculus <400> 12 Met Cys Gln Ser Arg Tyr Leu Leu Phe Leu Ala Thr Leu Ala Leu Leu 1 5 10 15 Asn His Leu Ser Leu Ala Arg Val Ile Pro Val Ser Gly Pro Ala Arg 20 25 30 Cys Leu Ser Gln Ser Arg Asn Leu Leu Lys Thr Thr Asp Asp Met Val 35 40 45 Lys Thr Ala Arg Glu Lys Leu Lys His Tyr Ser Cys Thr Ala Glu Asp 50 55 60 Ile Asp His Glu Asp Ile Thr Arg Asp Gln Thr Ser Thr Leu Lys Thr 65 70 75 80 Cys Leu Pro Leu Glu Leu His Lys Asn Glu Ser Cys Leu Ala Thr Arg 85 90 95 Glu Thr Ser Ser Thr Thr Arg Gly Ser Cys Leu Pro Pro Gln Lys Thr 100 105 110 Ser Leu Met Met Thr Leu Cys Leu Gly Ser Ile Tyr Glu Asp Leu Lys 115 120 125 Met Tyr Gln Thr Glu Phe Gln Ala Ile Asn Ala Ala Leu Gln Asn His 130 135 140 Asn His Gln Gln Ile Ile Leu Asp Lys Gly Met Leu Val Ala Ile Asp 145 150 155 160 Glu Leu Met Gln Ser Leu Asn His Asn Gly Glu Thr Leu Arg Gln Lys 165 170 175 Pro Pro Val Gly Glu Ala Asp Pro Tyr Arg Val Lys Met Lys Leu Cys 180 185 190 Ile Leu Leu His Ala Phe Ser Thr Arg Val Val Thr Ile Asn Arg Val 195 200 205 Met Gly Tyr Leu Ser Ser Ala 210 215 <210> 13 <211> 335 <212> PRT <213> Mus musculus <400> 13 Met Cys Pro Gln Lys Leu Thr Ile Ser Trp Phe Ala Ile Val Leu Leu 1 5 10 15 Val Ser Pro Leu Met Ala Met Trp Glu Leu Glu Lys Asp Val Tyr Val 20 25 30 Val Glu Val Asp Trp Thr Pro Asp Ala Pro Gly Glu Thr Val Asn Leu 35 40 45 Thr Cys Asp Thr Pro Glu Glu Asp Asp Ile Thr Trp Thr Ser Asp Gln 50 55 60 Arg His Gly Val Ile Gly Ser Gly Lys Thr Leu Thr Ile Thr Val Lys 65 70 75 80 Glu Phe Leu Asp Ala Gly Gln Tyr Thr Cys His Lys Gly Gly Glu Thr 85 90 95 Leu Ser His Ser His Leu Leu Leu His Lys Lys Glu Asn Gly Ile Trp 100 105 110 Ser Thr Glu Ile Leu Lys Asn Phe Lys Asn Lys Thr Phe Leu Lys Cys 115 120 125 Glu Ala Pro Asn Tyr Ser Gly Arg Phe Thr Cys Ser Trp Leu Val Gln 130 135 140 Arg Asn Met Asp Leu Lys Phe Asn Ile Lys Ser Ser Ser Ser Ser Pro 145 150 155 160 Asp Ser Arg Ala Val Thr Cys Gly Met Ala Ser Leu Ser Ala Glu Lys 165 170 175 Val Thr Leu Asp Gln Arg Asp Tyr Glu Lys Tyr Ser Val Ser Cys Gln 180 185 190 Glu Asp Val Thr Cys Pro Thr Ala Glu Glu Thr Leu Pro Ile Glu Leu 195 200 205 Ala Leu Glu Ala Arg Gln Gln Asn Lys Tyr Glu Asn Tyr Ser Thr Ser 210 215 220 Phe Phe Ile Arg Asp Ile Ile Lys Pro Asp Pro Pro Lys Asn Leu Gln 225 230 235 240 Met Lys Pro Leu Lys Asn Ser Gln Val Glu Val Ser Trp Glu Tyr Pro 245 250 255 Asp Ser Trp Ser Thr Pro His Ser Tyr Phe Ser Leu Lys Phe Phe Val 260 265 270 Arg Ile Gln Arg Lys Lys Glu Lys Met Lys Glu Thr Glu Glu Gly Cys 275 280 285 Asn Gln Lys Gly Ala Phe Leu Val Glu Lys Thr Ser Thr Glu Val Gln 290 295 300 Cys Lys Gly Gly Asn Val Cys Val Gln Ala Gln Asp Arg Tyr Tyr Asn 305 310 315 320 Ser Ser Cys Ser Lys Trp Ala Cys Val Pro Cys Arg Val Arg Ser 325 330 335 <210> 14 <211> 648 <212> DNA <213> Mus musculus <400> 14 atgtgccaga gcagatacct gctgttcctg gccaccctgg ccctgctgaa ccacctgagc 60 ctggccagag tgatccccgt gagcggcccc gccagatgcc tgagccagag cagaaacctg 120 ctgaagacaa ccgacgacat ggtgaagacc gccagagaga agctgaagca ctacagctgc 180 accgccgagg acatcgacca cgaggacatc accagagacc agaccagcac cctgaagacc 240 tgcctgcccc tggagctgca caagaacgag agctgcctgg ccacaagaga gaccagcagc 300 accacaagag gcagctgcct gcctccccag aagaccagcc tgatgatgac cctgtgcctg 360 ggcagcatct acgaggacct gaagatgtac cagaccgagt tccaggccat caacgctgcc 420 ctgcagaacc acaatcacca gcagatcatc ctggacaagg gcatgctggt ggccatcgac 480 gagctgatgc agagcctgaa ccacaacggc gagaccctga gacagaagcc ccctgtgggc 540 gaggccgatc cctacagagt gaagatgaag ctgtgcatcc tgctgcacgc cttcagcacc 600 agagtggtga ccatcaacag agtgatgggc tacctgagca gcgcctga 648 <210> 15 <211> 1008 <212> DNA <213> Mus musculus <400> 15 atgtgccccc agaagctgac catcagctgg ttcgccatcg tgctgctggt gagccccctg 60 atggccatgt gggagctgga gaaggacgtg tacgtggtgg aggtggactg gacccccgac 120 gcccccggcg agaccgtgaa cctgacctgc gacacccccg aggaggacga catcacctgg 180 accagcgacc agaggcacgg cgtgatcggc agcggcaaga ccctgaccat caccgtgaag 240 gagttcctgg acgccggcca gtacacctgc cacaagggcg gcgagaccct gagccacagc 300 cacctgctgc tgcacaagaa ggagaacggc atctggagca ccgagatcct gaagaacttc 360 aagaacaaga ccttcctgaa gtgcgaggcc cccaactaca gcggcaggtt cacctgcagc 420 tggctggtgc agaggaacat ggacctgaag ttcaacatca agagcagcag cagcagcccc 480 gacagcaggg ccgtgacctg cggcatggcc agcctgagcg ccgagaaggt gaccctggac 540 cagagggact acgagaagta cagcgtgagc tgccaggagg acgtgacctg ccccaccgcc 600 gaggagaccc tgcccatcga gctggccctg gaggccaggc agcagaacaa gtacgagaac 660 tacagcacca gcttcttcat cagggacatc atcaagcccg acccccccaa gaacctgcag 720 atgaagcccc tgaagaacag ccaggtggag gtgagctggg agtaccccga cagctggagc 780 accccccaca gctacttcag cctgaagttc ttcgtgagaa tccagaggaa gaaggagaag 840 atgaaggaga ccgaggaggg ctgcaaccag aagggcgcct tcctggtgga gaagaccagc 900 accgaggtgc agtgcaaggg cggcaacgtg tgcgtgcagg cccaggacag gtactacaac 960 agcagctgca gcaagtgggc ctgcgtgccc tgcagggtga ggagctaa 1008 <210> 16 <211> 146 <212> PRT <213> Mus musculus <400> 16 Met Glu Arg Thr Leu Val Cys Leu Val Val Ile Phe Leu Gly Thr Val 1 5 10 15 Ala His Lys Ser Ser Pro Gln Gly Pro Asp Arg Leu Leu Ile Arg Leu 20 25 30 Arg His Leu Ile Asp Ile Val Glu Gln Leu Lys Ile Tyr Glu Asn Asp 35 40 45 Leu Asp Pro Glu Leu Leu Ser Ala Pro Gln Asp Val Lys Gly His Cys 50 55 60 Glu His Ala Ala Phe Ala Cys Phe Gln Lys Ala Lys Leu Lys Pro Ser 65 70 75 80 Asn Pro Gly Asn Asn Lys Thr Phe Ile Ile Asp Leu Val Ala Gln Leu 85 90 95 Arg Arg Arg Leu Pro Ala Arg Arg Gly Gly Lys Lys Gln Lys His Ile 100 105 110 Ala Lys Cys Pro Ser Cys Asp Ser Tyr Glu Lys Arg Thr Pro Lys Glu 115 120 125 Phe Leu Glu Arg Leu Lys Trp Leu Leu Gln Lys Met Ile His Gln His 130 135 140 Leu Ser 145 <210> 17 <211> 441 <212> DNA <213> Mus musculus <400> 17 atggagagaa cactggtctg cctcgtggtc atcttcctgg gtactgtggc tcataaatcc 60 tcacctcagg gtcccgatag actgctgatc aggctgcggc acctcatcga cattgtggag 120 cagctcaaaa tctacgaaaa cgacctggac cccgagctgc tctctgcccc ccaggatgtc 180 aaggggcact gcgaacatgc cgctttcgca tgttttcaga aggccaaact gaagcccagc 240 aatcctggca acaataagac cttcatcatt gacctggtgg ctcagctcag gagacggctg 300 ccagcacgac gaggaggaaa gaaacagaaa catatcgcta agtgccctag ctgtgattcc 360 tatgagaaaa gaacaccaaa ggagttcctc gaaaggctca aatggctcct ccagaagatg 420 attcaccagc acctctccta a 441 <210> 18 <211> 92 <212> PRT <213> Mus musculus <400> 18 Met Lys Val Ser Thr Thr Ala Leu Ala Val Leu Leu Cys Thr Met Thr 1 5 10 15 Leu Cys Asn Gln Val Phe Ser Ala Pro Tyr Gly Ala Asp Thr Pro Thr 20 25 30 Ala Cys Cys Phe Ser Tyr Ser Arg Lys Ile Pro Arg Gln Phe Ile Val 35 40 45 Asp Tyr Phe Glu Thr Ser Ser Leu Cys Ser Gln Pro Gly Val Ile Phe 50 55 60 Leu Thr Lys Arg Asn Arg Gln Ile Cys Ala Asp Ser Lys Glu Thr Trp 65 70 75 80 Val Gln Glu Tyr Ile Thr Asp Leu Glu Leu Asn Ala 85 90 <210> 19 <211> 279 <212> DNA <213> Mus musculus <400> 19 atgaaggtga gcaccaccgc tctggctgtg ctgctctgca ccatgaccct ctgcaaccag 60 gtgttctcag ctccctacgg cgctgatacc cccaccgcct gctgcttcag ctacagccgg 120 aagatccccc ggcagttcat cgtggactac ttcgaaacca gcagcctgtg cagccagccc 180 ggcgtgatct tcctgaccaa acggaaccgg cagatctgcg ctgacagcaa agagacctgg 240 gtgcaggaat acatcaccga cctggaactg aacgcctaa 279 <210> 20 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> (GS)5 linker <400> 20 Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser 1 5 10 <210> 21 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> polynucleotide encoding (GS)5 linker <400> 21 ggatcaggca gtggttcagg atcaggtagt 30 <210> 22 <211> 25 <212> PRT <213> Artificial Sequence <220> <223> tPA signal sequence <400> 22 Met Asp Ala Met Lys Arg Gly Leu Cys Cys Val Leu Leu Leu Cys Gly 1 5 10 15 Ala Val Phe Val Ser Pro Ser His Ala 20 25 <210> 23 <211> 75 <212> DNA <213> Artificial Sequence <220> <223> polynucleotide encoding tPA signal sequence <400> 23 atggacgcca tgaagagagg cctgtgctgc gtgctgctgc tgtgcggcgc cgtgttcgtg 60 agccccagcc acgcc 75 <210> 24 <211> 156 <212> PRT <213> Artificial Sequence <220> <223> Flt3L_27-182 <400> 24 Thr Gln Asp Cys Ser Phe Gln His Ser Pro Ile Ser Ser Asp Phe Ala 1 5 10 15 Val Lys Ile Arg Glu Leu Ser Asp Tyr Leu Leu Gln Asp Tyr Pro Val 20 25 30 Thr Val Ala Ser Asn Leu Gln Asp Glu Glu Leu Cys Gly Gly Leu Trp 35 40 45 Arg Leu Val Leu Ala Gln Arg Trp Met Glu Arg Leu Lys Thr Val Ala 50 55 60 Gly Ser Lys Met Gln Gly Leu Leu Glu Arg Val Asn Thr Glu Ile His 65 70 75 80 Phe Val Thr Lys Cys Ala Phe Gln Pro Pro Pro Ser Cys Leu Arg Phe 85 90 95 Val Gln Thr Asn Ile Ser Arg Leu Leu Gln Glu Thr Ser Glu Gln Leu 100 105 110 Val Ala Leu Lys Pro Trp Ile Thr Arg Gln Asn Phe Ser Arg Cys Leu 115 120 125 Glu Leu Gln Cys Gln Pro Asp Ser Ser Thr Leu Pro Pro Pro Trp Ser 130 135 140 Pro Arg Pro Leu Glu Ala Thr Ala Pro Thr Ala Pro 145 150 155 <210> 25 <211> 468 <212> DNA <213> Artificial Sequence <220> <223> polynucleotide encoding Flt3L_27-182 <400> 25 acccaggact gcagcttcca gcacagcccc atcagcagcg acttcgccgt gaagatcaga 60 gagctgagcg actacctgct gcaggactac cccgtgaccg tggccagcaa cctgcaggac 120 gaggagctgt gcggcggcct gtggagactg gtgctggccc agagatggat ggagagactg 180 aagaccgtgg ccggcagcaa gatgcagggc ctgctggaga gagtgaacac cgagatccac 240 ttcgtgacca agtgcgcctt ccagcctccc cccagctgcc tgaggttcgt gcagaccaac 300 atcagcagac tgctgcagga gaccagcgag cagctggtgg ccctgaagcc ctggatcacc 360 agacagaact tcagcagatg cctggagctg cagtgccagc ccgacagcag caccctgccc 420 cctccctgga gccccagacc cctggaggcc accgctccca cagcccct 468 <210> 26 <211> 1957 <212> PRT <213> Artificial Sequence <220> <223> BD-14A polypeptide <400> 26 Asp Ala Met Lys Arg Gly Leu Cys Cys Val Leu Leu Leu Cys Gly Ala 1 5 10 15 Val Phe Val Ser Pro Ser His Ala Thr Gln Asp Cys Ser Phe Gln His 20 25 30 Ser Pro Ile Ser Ser Asp Phe Ala Val Lys Ile Arg Glu Leu Ser Asp 35 40 45 Tyr Leu Leu Gln Asp Tyr Pro Val Thr Val Ala Ser Asn Leu Gln Asp 50 55 60 Glu Glu Leu Cys Gly Gly Leu Trp Arg Leu Val Leu Ala Gln Arg Trp 65 70 75 80 Met Glu Arg Leu Lys Thr Val Ala Gly Ser Lys Met Gln Gly Leu Leu 85 90 95 Glu Arg Val Asn Thr Glu Ile His Phe Val Thr Lys Cys Ala Phe Gln 100 105 110 Pro Pro Pro Ser Cys Leu Arg Phe Val Gln Thr Asn Ile Ser Arg Leu 115 120 125 Leu Gln Glu Thr Ser Glu Gln Leu Val Ala Leu Lys Pro Trp Ile Thr 130 135 140 Arg Gln Asn Phe Ser Arg Cys Leu Glu Leu Gln Cys Gln Pro Asp Ser 145 150 155 160 Ser Thr Leu Pro Pro Pro Trp Ser Pro Arg Pro Leu Glu Ala Thr Ala 165 170 175 Pro Thr Ala Pro Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser Met Asp 180 185 190 Ala Met Lys Arg Gly Leu Cys Cys Val Leu Leu Leu Cys Gly Ala Val 195 200 205 Phe Val Ser Pro Ser His Ala Thr Gln Asp Cys Ser Phe Gln His Ser 210 215 220 Pro Ile Ser Ser Asp Phe Ala Val Lys Ile Arg Glu Leu Ser Asp Tyr 225 230 235 240 Leu Leu Gln Asp Tyr Pro Val Thr Val Ala Ser Asn Leu Gln Asp Glu 245 250 255 Glu Leu Cys Gly Gly Leu Trp Arg Leu Val Leu Ala Gln Arg Trp Met 260 265 270 Glu Arg Leu Lys Thr Val Ala Gly Ser Lys Met Gln Gly Leu Leu Glu 275 280 285 Arg Val Asn Thr Glu Ile His Phe Val Thr Lys Cys Ala Phe Gln Pro 290 295 300 Pro Pro Ser Cys Leu Arg Phe Val Gln Thr Asn Ile Ser Arg Leu Leu 305 310 315 320 Gln Glu Thr Ser Glu Gln Leu Val Ala Leu Lys Pro Trp Ile Thr Arg 325 330 335 Gln Asn Phe Ser Arg Cys Leu Glu Leu Gln Cys Gln Pro Asp Ser Ser 340 345 350 Thr Leu Pro Pro Pro Trp Ser Pro Arg Pro Leu Glu Ala Thr Ala Pro 355 360 365 Thr Ala Pro Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser Met His Gln 370 375 380 Lys Arg Thr Ala Met Phe Gln Asp Pro Gln Glu Arg Pro Arg Lys Leu 385 390 395 400 Pro Gln Leu Cys Thr Glu Leu Gln Thr Thr Ile His Asp Ile Ile Leu 405 410 415 Glu Cys Val Tyr Cys Lys Gln Gln Leu Leu Arg Arg Glu Val Tyr Asp 420 425 430 Phe Ala Phe Arg Asp Leu Cys Ile Val Tyr Arg Asp Gly Asn Pro Tyr 435 440 445 Ala Val Cys Asp Lys Cys Leu Lys Phe Tyr Ser Lys Ile Ser Glu Tyr 450 455 460 Arg His Pro Ala Gly Gln Ala Glu Pro Asp Arg Ala His Tyr Asn Ile 465 470 475 480 Val Thr Phe Cys Cys Lys Cys Asp Ser Thr Leu Arg Leu Cys Val Gln 485 490 495 Ser Thr His Val Asp Ile Arg Thr Leu Glu Asp Leu Leu Met Gly Thr 500 505 510 Leu Gly Ile Val Cys Pro Ile Cys Ser Gln Lys Pro Gly Ser Gly Ser 515 520 525 Gly Ser Gly Ser Gly Ser Met His Gly Asp Thr Pro Thr Leu His Glu 530 535 540 Tyr Met Leu Asp Leu Gln Pro Glu Thr Thr Asp Leu Tyr Cys Tyr Glu 545 550 555 560 Gln Leu Asn Asp Ser Ser Glu Glu Glu Asp Glu Ile Asp Gly Pro Ala 565 570 575 Gly Gln Ala Glu Pro Asp Arg Ala His Tyr Asn Ile Val Thr Phe Cys 580 585 590 Cys Lys Pro Tyr Ala Val Cys Asp Lys Cys Leu Lys Phe Tyr Ser Lys 595 600 605 Ile Ser Glu Tyr Arg His Tyr Cys Tyr Ser Val Tyr Gly Thr Thr Leu 610 615 620 Glu Gln Gln Tyr Asn Lys Pro Leu Cys Asp Leu Leu Ile Arg Cys Ile 625 630 635 640 Asn Cys Gln Lys Pro Leu Cys Pro Glu Glu Lys Gln Arg His Leu Asp 645 650 655 Lys Lys Gln Arg Phe His Asn Ile Arg Gly Arg Trp Thr Gly Arg Cys 660 665 670 Met Ser Cys Cys Arg Ser Ser Arg Thr Arg Arg Glu Thr Gln Leu Gly 675 680 685 Ser Gly Ser Gly Ser Gly Ser Gly Ser Met Ala Arg Phe Glu Asp Pro 690 695 700 Thr Arg Arg Pro Tyr Lys Leu Pro Asp Leu Cys Thr Glu Leu Asn Thr 705 710 715 720 Ser Leu Gln Asp Ile Glu Ile Thr Cys Val Tyr Cys Lys Thr Val Leu 725 730 735 Glu Leu Thr Glu Val Phe Glu Phe Ala Phe Lys Asp Leu Phe Val Val 740 745 750 Tyr Arg Asp Ser Ile Pro His Ala Ala Cys His Lys Cys Ile Asp Phe 755 760 765 Tyr Ser Arg Ile Arg Glu Leu Arg His Tyr Ser Asp Ser Val Ile Asp 770 775 780 Gly Val Asn His Gln His Leu Pro Ala Arg Arg Ala Glu Pro Gln Arg 785 790 795 800 His Thr Met Leu Cys Met Cys Cys Lys Cys Glu Ala Arg Ile Glu Leu 805 810 815 Val Val Glu Ser Ser Ala Asp Asp Leu Arg Ala Phe Gln Gln Leu Phe 820 825 830 Leu Ser Thr Leu Ser Phe Val Cys Pro Trp Cys Ala Ser Gln Gln Gly 835 840 845 Ser Gly Ser Gly Ser Gly Ser Gly Ser Met Tyr Gly Pro Lys Ala Thr 850 855 860 Leu Gln Asp Ile Val Leu His Leu Glu Pro Gln Asn Glu Ile Pro Val 865 870 875 880 Asp Leu Leu Cys His Glu Gln Leu Ser Asp Ser Glu Glu Glu Asn Asp 885 890 895 Glu Ile Asp Gly Val Asn His Gln His Leu Pro Ala Arg Arg Ala Glu 900 905 910 Pro Gln Arg His Thr His Lys Cys Ile Asp Phe Tyr Ser Arg Ile Arg 915 920 925 Glu Leu Arg His Tyr Ser Asp Ser Val Tyr Gly Asp Thr Leu Glu Lys 930 935 940 Leu Thr Asn Thr Gly Leu Tyr Asn Leu Leu Ile Arg Cys Leu Arg Cys 945 950 955 960 Gln Lys Pro Leu Asn Pro Ala Glu Lys Leu Arg His Leu Asn Glu Lys 965 970 975 Arg Arg Phe His Asn Ile Ala Gly His Tyr Arg Gly Gln Cys His Ser 980 985 990 Cys Cys Asn Arg Ala Arg Gln Glu Arg Leu Gln Arg Arg Arg Glu Thr 995 1000 1005 Gln Val Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser Leu Glu Met Phe 1010 1015 1020 Gln Asp Pro Ala Glu Arg Pro Tyr Lys Leu His Asp Leu Cys Asn Glu 1025 1030 1035 1040 Val Glu Glu Ser Ile His Glu Ile Cys Leu Asn Cys Val Tyr Cys Lys 1045 1050 1055 Gln Glu Leu Gln Arg Ser Glu Val Tyr Asp Phe Ala Cys Tyr Asp Leu 1060 1065 1070 Cys Ile Val Tyr Arg Glu Gly Gln Pro Tyr Gly Val Cys Met Lys Cys 1075 1080 1085 Leu Lys Phe Tyr Ser Lys Ile Ser Glu Tyr Arg Trp Pro Ala Gly Gln 1090 1095 1100 Ala Lys Pro Asp Thr Ser Asn Tyr Asn Ile Val Thr Ser Cys Cys Lys 1105 1110 1115 1120 Cys Glu Ala Thr Leu Arg Leu Cys Val Gln Ser Thr His Ile Asp Ile 1125 1130 1135 Arg Lys Leu Glu Asp Leu Leu Met Gly Thr Phe Gly Ile Val Cys Pro 1140 1145 1150 Gly Cys Ser Gln Arg Ala Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser 1155 1160 1165 Met His Gly Glu Ile Thr Thr Leu Gln Asp Tyr Val Leu Asp Leu Glu 1170 1175 1180 Pro Glu Ala Thr Asp Leu Tyr Cys Tyr Glu Gln Leu Cys Asp Ser Ser 1185 1190 1195 1200 Glu Glu Glu Glu Asp Thr Ile Asp Gly Pro Ala Gly Gln Ala Lys Pro 1205 1210 1215 Asp Thr Ser Asn Tyr Asn Ile Val Thr Ser Cys Cys Lys Pro Tyr Gly 1220 1225 1230 Val Cys Met Lys Cys Leu Lys Phe Tyr Ser Lys Ile Ser Glu Tyr Arg 1235 1240 1245 Trp Tyr Arg Tyr Ser Val Tyr Gly Glu Thr Leu Glu Lys Gln Cys Asn 1250 1255 1260 Lys Gln Leu Cys His Leu Leu Ile Arg Cys Ile Thr Cys Gln Lys Pro 1265 1270 1275 1280 Leu Cys Pro Val Glu Lys Gln Arg His Leu Glu Glu Lys Lys Arg Phe 1285 1290 1295 His Asn Ile Gly Gly Arg Trp Thr Gly Arg Cys Met Ser Cys Trp Lys 1300 1305 1310 Pro Thr Arg Arg Glu Thr Glu Val Gly Ser Gly Ser Gly Ser Gly Ser 1315 1320 1325 Gly Ser Val Glu Gly Ser Met Ala Arg Phe Asp Asp Pro Lys Gln Arg 1330 1335 1340 Pro Tyr Lys Leu Pro Asp Leu Cys Thr Glu Leu Asn Thr Ser Leu Gln 1345 1350 1355 1360 Asp Val Ser Ile Ala Cys Val Tyr Cys Lys Ala Thr Leu Glu Arg Thr 1365 1370 1375 Glu Val Tyr Gln Phe Ala Phe Lys Asp Leu Cys Ile Val Tyr Arg Asp 1380 1385 1390 Cys Ile Ala Tyr Ala Ala Cys His Lys Cys Ile Asp Phe Tyr Ser Arg 1395 1400 1405 Ile Arg Glu Leu Arg Tyr Tyr Ser Asn Ser Val Glu Ala Asp Gly Val 1410 1415 1420 Ser His Ala Gln Leu Pro Ala Arg Arg Ala Glu Pro Gln Arg His Lys 1425 1430 1435 1440 Ile Leu Cys Val Cys Cys Lys Cys Asp Gly Arg Ile Asp Leu Thr Val 1445 1450 1455 Glu Ser Ser Ala Asp Asp Leu Arg Thr Leu Gln Gln Leu Phe Leu Ser 1460 1465 1470 Thr Leu Ser Phe Val Cys Pro Trp Cys Ala Thr Asn Gln Gly Ser Gly 1475 1480 1485 Ser Gly Ser Gly Ser Gly Ser Met His Gly Pro Arg Ala Thr Leu Gln 1490 1495 1500 Glu Ile Val Leu His Leu Glu Pro Gln Asn Glu Leu Asp Pro Val Asp 1505 1510 1515 1520 Leu Leu Cys Tyr Glu Gln Leu Ser Glu Ser Glu Glu Glu Asn Asp Glu 1525 1530 1535 Ala Asp Gly Val Ser His Ala Gln Leu Pro Ala Arg Arg Ala Glu Pro 1540 1545 1550 Gln Arg His His Lys Cys Ile Asp Phe Tyr Ser Arg Ile Arg Glu Leu 1555 1560 1565 Arg Tyr Tyr Ser Asn Ser Val Tyr Gly Glu Thr Leu Glu Lys Ile Thr 1570 1575 1580 Asn Thr Glu Leu Tyr Asn Leu Leu Ile Arg Cys Leu Arg Cys Gln Lys 1585 1590 1595 1600 Pro Leu Asn Pro Ala Glu Lys Arg Arg His Leu Lys Asp Lys Arg Arg 1605 1610 1615 Phe His Ser Ile Ala Gly Gln Tyr Arg Gly Gln Cys Asn Thr Cys Cys 1620 1625 1630 Asp Gln Ala Arg Gln Glu Arg Leu Arg Arg Arg Arg Glu Thr Gln Val 1635 1640 1645 Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser Met Phe Gln Asp Ala Glu 1650 1655 1660 Glu Lys Pro Arg Thr Leu His Asp Leu Cys Gln Ala Leu Glu Thr Ser 1665 1670 1675 1680 Val His Glu Ile Glu Leu Lys Cys Val Glu Cys Lys Lys Thr Leu Gln 1685 1690 1695 Arg Ser Glu Val Tyr Asp Phe Val Phe Ala Asp Leu Arg Ile Val Tyr 1700 1705 1710 Arg Asp Gly Asn Pro Phe Ala Val Cys Lys Val Cys Leu Arg Leu Leu 1715 1720 1725 Ser Lys Ile Ser Glu Tyr Arg His Tyr Asn Tyr Ser Leu Tyr Gly Arg 1730 1735 1740 Pro Asp Gly Gln Ala Gln Pro Ala Thr Ala Asn Tyr Tyr Ile Val Thr 1745 1750 1755 1760 Cys Cys Tyr Thr Cys Asp Thr Thr Val Arg Leu Cys Ile Asn Ser Thr 1765 1770 1775 Thr Thr Asp Val Arg Thr Leu Gln Gln Leu Leu Met Gly Thr Cys Thr 1780 1785 1790 Ile Val Cys Pro Ser Cys Ala Gln Gln Gly Ser Gly Ser Gly Ser Gly 1795 1800 1805 Ser Gly Ser Met Arg Gly Asn Asn Pro Thr Leu Arg Glu Tyr Ile Leu 1810 1815 1820 Asp Leu His Pro Glu Pro Thr Asp Leu Phe Cys Tyr Glu Gln Leu Cys 1825 1830 1835 1840 Asp Ser Ser Asp Glu Asp Glu Ile Gly Leu Asp Arg Pro Asp Gly Gln 1845 1850 1855 Ala Gln Pro Ala Thr Ala Asn Tyr Tyr Ile Val Thr Cys Cys Tyr Cys 1860 1865 1870 Leu Arg Leu Leu Ser Lys Ile Ser Glu Tyr Arg His Tyr Asn Tyr Ser 1875 1880 1885 Leu Tyr Gly Asp Thr Leu Glu Gln Thr Leu Lys Lys Cys Leu Asn Glu 1890 1895 1900 Ile Leu Ile Arg Cys Ile Ile Cys Gln Arg Pro Leu Cys Pro Gln Glu 1905 1910 1915 1920 Lys Lys Arg His Val Asp Leu Asn Lys Arg Phe His Asn Ile Ser Gly 1925 1930 1935 Arg Trp Thr Gly Arg Cys Ala Val Cys Trp Arg Pro Arg Arg Arg Gln 1940 1945 1950 Thr Gln Val Gly Ser 1955 <210> 27 <211> 5304 <212> DNA <213> Artificial Sequence <220> <223> polynucleotide encoding BD-14A polypeptide <400> 27 atggacgcca tgaagagagg cctgtgctgc gtgctgctgc tgtgcggcgc cgtgttcgtg 60 agccccagcc acgccaccca ggactgcagc ttccagcaca gccccatcag cagcgacttc 120 gccgtgaaga tcagagagct gagcgactac ctgctgcagg actaccccgt gaccgtggcc 180 agcaacctgc aggacgagga gctgtgcggc ggcctgtgga gactggtgct ggcccagaga 240 tggatggaga gactgaagac cgtggccggc agcaagatgc agggcctgct ggagagagtg 300 aacaccgaga tccacttcgt gaccaagtgc gccttccagc ctccccccag ctgcctgagg 360 ttcgtgcaga ccaacatcag cagactgctg caggagacca gcgagcagct ggtggccctg 420 aagccctgga tcaccagaca gaacttcagc agatgcctgg agctgcagtg ccagcccgac 480 agcagcaccc tgccccctcc ctggagcccc agacccctgg aggccaccgc tcccacagcc 540 cctggcagcg ggtccggaag tgggtctgga tctatgcacc agaagagaac cgccatgttc 600 caggaccccc aggagagacc cagaaagctg ccccagctgt gcaccgagct gcagaccaca 660 atccacgaca tcatcctgga gtgcgtgtac tgcaagcagc agctgctgag aagagaggtg 720 tacgacttcg ccttcagaga cctgtgcatc gtgtacagag atggcaaccc ttatgctgtc 780 tgtgataaat gtctcaaatt ttattccaaa attagtgaat ataggcatcc agcaggacag 840 gctgaaccag atagggctca ttataatatt gtcacatttt gttgtaaatg cgacagcacc 900 ctgagactgt gcgtgcagag cacccacgtg gacatcagaa ccctggagga cctgctgatg 960 ggcaccctgg gcatcgtgtg ccccatctgc agccagaagc ctggcagcgg ctctggctcc 1020 ggcagtggct caatgcacgg cgacacaccc accctgcacg agtacatgct ggacctgcag 1080 cccgagacta ccgacctgta ctgctacgag cagctgaacg acagcagcga ggaagaggac 1140 gagatcgacg gccctgctgg ccaggccgag cccgacagag cccactacaa catcgtgacc 1200 ttctgctgca agccctacgc cgtgtgcgac aagtgcctga agttctacag caagatcagc 1260 gagtacagac actactgcta cagcgtgtac ggcaccaccc tggagcagca gtacaacaag 1320 cccctgtgcg acctgctgat cagatgcatc aactgccaga agcccctgtg ccccgaggag 1380 aagcagagac acctggacaa gaagcagaga ttccacaaca tcagaggcag atggaccggc 1440 agatgcatga gctgctgcag aagcagcaga accagaagag agacccagct gggatctggc 1500 agtggatctg gaagcggctc tatggccaga ttcgaagatc ccaccagaag accctacaag 1560 ctgcccgacc tgtgcaccga gctgaacacc agcctgcagg acatcgagat cacctgcgtg 1620 tactgcaaga ccgtgctgga gctgaccgag gtgttcgagt tcgccttcaa ggacctgttc 1680 gtggtgtaca gagacagcat cccccacgct gcctgccata aatgtattga tttttattcc 1740 aggattaggg aactcaggca ttatagtgat tctgtcattg atggtgtcaa tcatcagcat 1800 ctcccagcta ggagggctga acctcagagg cataccatgc tgtgcatgtg ctgcaagtgc 1860 gaggccagaa tcgagctggt ggtggagagc agcgccgacg acctgagagc cttccagcag 1920 ctgttcctga gcaccctgag cttcgtgtgc ccctggtgcg ccagccagca gggctcagga 1980 tctggcagcg gaagtggatc tatgtacggc cccaaggcta ccctgcagga catcgtgctg 2040 cacctggagc cccagaacga gatccccgtg gacctgctgt gccacgagca gctgagcgac 2100 agcgaggaag aaaacgacga gatcgacggc gtgaaccacc agcacctgcc tgccagaaga 2160 gccgagcccc agagacacac ccacaagtgc atcgacttct acagcagaat cagagagctg 2220 agacactaca gcgacagcgt gtacggcgac accctggaga agctgaccaa caccggcctg 2280 tacaacctgc tgatcagatg cctgagatgc cagaagcccc tgaaccctgc cgagaagctg 2340 agacacctga acgagaagag aagattccac aacatcgccg gccactacag aggccagtgc 2400 cacagctgct gcaacagagc cagacaggag agactgcaga gaagaagaga gacccaggtg 2460 ggatctggca gcggctctgg ctccggctca ctcgagatgt tccaggaccc tgccgaaaga 2520 ccctacaagc tgcatgatct gtgcaatgaa gtcgaagaga gtatccatga aatctgtctg 2580 aattgcgtgt actgtaagca ggagctgcag cgcagtgaag tctacgactt cgcctgctat 2640 gacctgtgca tcgtgtaccg agagggacag ccatatggcg tctgcatgaa gtgtctgaag 2700 ttctactcta agatcagtga atataggtgg ccagccggcc aggctaaacc cgacacttcc 2760 aactataata ttgtgacctc ttgctgtaaa tgcgaggcta ccctgagact gtgcgtgcag 2820 agcacacaca tcgacattag gaagctggag gacctgctga tggggacctt cggaatcgtg 2880 tgcccaggat gttcccagcg agctggatct ggcagtgggt caggaagcgg ctccatgcat 2940 ggagagatta ccacactgca ggactacgtc ctggatctgg agcctgaagc aactgacctg 3000 tactgctatg aacagctgtg cgatagctcc gaggaagagg aagacaccat cgatggccct 3060 gcagggcagg ccaagccaga tacaagtaac tacaacatcg tgacttcatg ctgtaaaccc 3120 tacggcgtct gcatgaaatg tctgaaattc tactcaaaga tcagcgagta tcggtggtac 3180 agatatagcg tgtacgggga gacactggaa aagcagtgca acaaacagct gtgccacctg 3240 ctgatccggt gcattacttg tcagaagccc ctgtgccctg tggagaaaca gcgacacctg 3300 gaggaaaaga aacggtttca taatattggc gggaggtgga caggccgctg catgagctgt 3360 tggaagccta ccagacggga gaccgaagtg ggcagcggca gtgggagcgg aagcgggagt 3420 gtcgagggat ctatggccag attcgacgac cccaagcaga gaccctacaa gctgcccgac 3480 ctgtgcaccg agctgaacac cagcctgcag gacgtgagca tcgcctgcgt gtactgcaag 3540 gccaccctgg agagaaccga ggtgtaccag ttcgccttca aggacctgtg catcgtgtac 3600 agagactgca tcgcctacgc cgcctgccat aaatgtattg atttttattc caggattcgg 3660 gagctccgct attattctaa tagtgtcgaa gctgatggag tcagtcatgc tcagctccct 3720 gctcggaggg cagaacctca gaggcataag atcctgtgcg tgtgctgcaa gtgcgacggc 3780 agaatcgacc tgaccgtgga gagcagcgcc gacgacctga gaaccctgca gcagctgttc 3840 ctgagcaccc tgagcttcgt gtgcccctgg tgcgccacca accagggcag cggaagcgga 3900 agcggcagcg gcagcatgca cggccccaga gccaccctgc aggagatcgt gctgcacctg 3960 gagccccaga acgagctgga ccccgtggac ctgttgtgct acgagcagct gagcgaaagc 4020 gaggaagaga acgacgaggc cgacggcgtg agccacgccc agctgcccgc cagaagagcc 4080 gagccccaga gacaccacaa gtgcatcgac ttctacagca gaatcagaga gctgagatac 4140 tacagcaaca gcgtgtacgg cgagaccctg gagaagatca ccaacaccga gctgtacaac 4200 ctgttgatca gatgcctgag atgccagaag cccctgaacc ccgccgagaa gagaagacac 4260 ctgaaggaca agagaagatt ccacagcatc gccggccagt acagaggcca gtgcaacacc 4320 tgctgcgacc aggccagaca ggagagactg agaaggagga gagagaccca ggtgggatca 4380 ggaagtggat ctgggtccgg cagcatgttc caggacgccg aggagaagcc cagaaccctg 4440 cacgacctgt gccaggccct ggagaccagc gtgcacgaga tcgagctgaa gtgcgtggag 4500 tgcaagaaga ccctgcagag aagcgaggtg tatgacttcg tgttcgccga cctgagaatc 4560 gtgtatagag acggcaaccc cttcgccgtg tgcaaggtgt gtttgaggct cctctccaaa 4620 atttctgaat atcggcatta taactattcc ctctatggaa ggcctgatgg acaggctcag 4680 ccagctacag caaattatta tattgtcaca tgttgctata cctgcgacac caccgtgaga 4740 ctgtgcatca acagcaccac aaccgacgtg agaaccctgc agcagctgct gatgggcacc 4800 tgcaccatcg tgtgccccag ctgcgcccag cagggctcag gcagcggctc cggcagcgga 4860 tctatgagag gcaacaaccc caccctgaga gagtacatcc tggacctgca ccccgagccc 4920 accgacctgt tctgctacga gcagctgtgc gacagcagcg acgaggacga gatcggcctg 4980 gacagacccg acggccaggc ccagcccgcc accgccaact actacatcgt gacctgctgc 5040 tactgcctga gactgctgag caagatcagc gagtacagac actacaacta cagcctgtac 5100 ggcgacaccc tggagcagac cctgaagaag tgcctgaacg agatcctgat cagatgcatc 5160 atctgccaga gacccctgtg cccccaggag aagaagagac acgtggacct gaacaagaga 5220 ttccacaaca tcagcggcag atggaccggc agatgcgccg tgtgctggag acccagaagg 5280 agacagaccc aggtgggatc ctaa 5304 <210> 28 <211> 1909 <212> PRT <213> Artificial Sequence <220> <223> BD-14B polypeptide <400> 28 Asp Ala Met Lys Arg Gly Leu Cys Cys Val Leu Leu Leu Cys Gly Ala 1 5 10 15 Val Phe Val Ser Pro Ser His Ala Thr Gln Asp Cys Ser Phe Gln His 20 25 30 Ser Pro Ile Ser Ser Asp Phe Ala Val Lys Ile Arg Glu Leu Ser Asp 35 40 45 Tyr Leu Leu Gln Asp Tyr Pro Val Thr Val Ala Ser Asn Leu Gln Asp 50 55 60 Glu Glu Leu Cys Gly Gly Leu Trp Arg Leu Val Leu Ala Gln Arg Trp 65 70 75 80 Met Glu Arg Leu Lys Thr Val Ala Gly Ser Lys Met Gln Gly Leu Leu 85 90 95 Glu Arg Val Asn Thr Glu Ile His Phe Val Thr Lys Cys Ala Phe Gln 100 105 110 Pro Pro Pro Ser Cys Leu Arg Phe Val Gln Thr Asn Ile Ser Arg Leu 115 120 125 Leu Gln Glu Thr Ser Glu Gln Leu Val Ala Leu Lys Pro Trp Ile Thr 130 135 140 Arg Gln Asn Phe Ser Arg Cys Leu Glu Leu Gln Cys Gln Pro Asp Ser 145 150 155 160 Ser Thr Leu Pro Pro Pro Trp Ser Pro Arg Pro Leu Glu Ala Thr Ala 165 170 175 Pro Thr Ala Pro Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser Met Asp 180 185 190 Ala Met Lys Arg Gly Leu Cys Cys Val Leu Leu Leu Cys Gly Ala Val 195 200 205 Phe Val Ser Pro Ser His Ala Thr Gln Asp Cys Ser Phe Gln His Ser 210 215 220 Pro Ile Ser Ser Asp Phe Ala Val Lys Ile Arg Glu Leu Ser Asp Tyr 225 230 235 240 Leu Leu Gln Asp Tyr Pro Val Thr Val Ala Ser Asn Leu Gln Asp Glu 245 250 255 Glu Leu Cys Gly Gly Leu Trp Arg Leu Val Leu Ala Gln Arg Trp Met 260 265 270 Glu Arg Leu Lys Thr Val Ala Gly Ser Lys Met Gln Gly Leu Leu Glu 275 280 285 Arg Val Asn Thr Glu Ile His Phe Val Thr Lys Cys Ala Phe Gln Pro 290 295 300 Pro Pro Ser Cys Leu Arg Phe Val Gln Thr Asn Ile Ser Arg Leu Leu 305 310 315 320 Gln Glu Thr Ser Glu Gln Leu Val Ala Leu Lys Pro Trp Ile Thr Arg 325 330 335 Gln Asn Phe Ser Arg Cys Leu Glu Leu Gln Cys Gln Pro Asp Ser Ser 340 345 350 Thr Leu Pro Pro Pro Trp Ser Pro Arg Pro Leu Glu Ala Thr Ala Pro 355 360 365 Thr Ala Pro Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser Met Phe Lys 370 375 380 Asn Pro Ala Glu Arg Pro Arg Lys Leu His Glu Leu Ser Ser Ala Leu 385 390 395 400 Glu Ile Pro Tyr Asp Glu Leu Arg Leu Asn Cys Val Tyr Cys Lys Gly 405 410 415 Gln Leu Thr Glu Thr Glu Val Leu Asp Phe Ala Phe Thr Asp Leu Thr 420 425 430 Ile Val Tyr Arg Asp Asp Thr Pro Tyr Gly Val Cys Thr Lys Cys Leu 435 440 445 Arg Phe Tyr Ser Lys Val Ser Glu Phe Arg Trp Tyr Arg Tyr Ser Val 450 455 460 Tyr Gly Pro Ala Gly Gln Ala Lys Pro Asp Thr Ser Asn Tyr Asn Ile 465 470 475 480 Val Thr Phe Cys Cys Gln Cys Glu Ser Thr Leu Arg Leu Cys Val Gln 485 490 495 Ser Thr Gln Val Asp Ile Arg Ile Leu Gln Glu Leu Leu Met Gly Ser 500 505 510 Phe Gly Ile Val Cys Pro Asn Cys Ser Thr Arg Leu Gly Ser Gly Ser 515 520 525 Gly Ser Gly Ser Gly Ser Met Arg Gly Glu Thr Pro Thr Leu Gln Asp 530 535 540 Tyr Val Leu Asp Leu Gln Pro Glu Ala Thr Asp Leu His Cys Tyr Glu 545 550 555 560 Gln Leu Pro Asp Ser Ser Asp Glu Glu Asp Val Ile Asp Ser Pro Ala 565 570 575 Gly Gln Ala Lys Pro Asp Thr Ser Asn Tyr Asn Ile Val Thr Phe Cys 580 585 590 Cys Gln Cys Leu Arg Phe Tyr Ser Lys Val Ser Glu Phe Arg Trp Tyr 595 600 605 Arg Tyr Ser Val Tyr Gly Thr Thr Leu Glu Lys Leu Thr Asn Lys Gly 610 615 620 Ile Cys Asp Leu Leu Ile Arg Cys Ile Thr Cys Gln Arg Pro Leu Cys 625 630 635 640 Pro Glu Glu Lys Gln Arg His Leu Asp Lys Lys Lys Arg Phe His Asn 645 650 655 Ile Gly Gly Arg Trp Thr Gly Arg Cys Ile Val Cys Trp Arg Arg Pro 660 665 670 Arg Thr Glu Thr Gln Val Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser 675 680 685 Met Phe Gln Asp Thr Glu Glu Lys Pro Arg Thr Leu His Asp Leu Cys 690 695 700 Gln Ala Leu Glu Thr Thr Ile His Asn Ile Glu Leu Gln Cys Val Glu 705 710 715 720 Cys Lys Asn Pro Leu Gln Arg Ser Glu Val Tyr Asp Phe Ala Phe Ala 725 730 735 Asp Leu Thr Val Val Tyr Arg Glu Gly Asn Pro Phe Gly Ile Cys Lys 740 745 750 Leu Cys Leu Arg Phe Leu Ser Lys Ile Ser Glu Tyr Arg His Tyr Asn 755 760 765 Tyr Ser Val Tyr Gly Pro Asp Gly Gln Ala Gln Pro Ala Thr Ala Asp 770 775 780 Tyr Tyr Ile Val Thr Cys Cys His Thr Cys Asn Thr Thr Val Arg Leu 785 790 795 800 Cys Val Asn Ser Thr Ala Ser Asp Leu Arg Thr Ile Gln Gln Leu Leu 805 810 815 Met Gly Thr Val Asn Ile Val Cys Pro Thr Cys Ala Gln Leu Gly Ser 820 825 830 Gly Ser Gly Ser Gly Ser Gly Ser Met Arg Gly His Lys Pro Thr Leu 835 840 845 Lys Glu Tyr Val Leu Asp Leu Tyr Pro Glu Pro Thr Asp Leu Tyr Cys 850 855 860 Tyr Glu Gln Leu Ser Asp Ser Ser Asp Glu Asp Glu Gly Leu Asp Arg 865 870 875 880 Pro Asp Gly Gln Ala Gln Pro Ala Thr Ala Asp Tyr Tyr Ile Val Thr 885 890 895 Cys Cys His Thr Cys Leu Arg Phe Leu Ser Lys Ile Ser Glu Tyr Arg 900 905 910 His Tyr Asn Tyr Ser Val Tyr Gly His Thr Leu Glu Gln Thr Val Lys 915 920 925 Lys Pro Leu Asn Glu Ile Leu Ile Arg Cys Ile Ile Cys Gln Arg Pro 930 935 940 Leu Cys Pro Gln Glu Lys Lys Arg His Val Asp Leu Asn Lys Arg Phe 945 950 955 960 His Asn Ile Ser Gly Arg Trp Ala Gly Arg Cys Ala Ala Cys Trp Arg 965 970 975 Ser Arg Arg Arg Glu Thr Ala Leu Gly Ser Gly Ser Gly Ser Gly Ser 980 985 990 Gly Ser Met Glu Ser Ala Asn Ala Ser Thr Ser Ala Thr Thr Ile Asp 995 1000 1005 Gln Leu Cys Lys Thr Phe Asn Leu Ser Met His Thr Leu Gln Ile Asn 1010 1015 1020 Cys Val Phe Cys Lys Asn Ala Leu Thr Thr Ala Glu Ile Tyr Ser Tyr 1025 1030 1035 1040 Ala Tyr Lys His Leu Lys Val Leu Phe Arg Gly Gly Tyr Pro Tyr Ala 1045 1050 1055 Ala Cys Ala Cys Cys Leu Glu Phe His Gly Lys Ile Asn Gln Tyr Arg 1060 1065 1070 His Phe Asp Tyr Ala Gly Tyr Asp Gly Gln Asp Ser Gln Pro Leu Lys 1075 1080 1085 Gln His Tyr Gln Ile Val Thr Cys Cys Cys Gly Cys Asp Ser Asn Val 1090 1095 1100 Arg Leu Val Val Gln Cys Thr Glu Thr Asp Ile Arg Glu Val Gln Gln 1105 1110 1115 1120 Leu Leu Leu Gly Thr Leu Asn Ile Val Cys Pro Ile Cys Ala Pro Lys 1125 1130 1135 Thr Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser Met His Gly Arg His 1140 1145 1150 Val Thr Leu Lys Asp Ile Val Leu Asp Leu Gln Pro Pro Asp Pro Val 1155 1160 1165 Gly Leu His Cys Tyr Glu Gln Leu Val Asp Ser Ser Glu Asp Glu Val 1170 1175 1180 Asp Glu Val Asp Gly Gln Asp Ser Gln Pro Leu Lys Gln His Tyr Gln 1185 1190 1195 1200 Ile Val Thr Cys Cys Cys Gly Cys Cys Leu Glu Phe His Gly Lys Ile 1205 1210 1215 Asn Gln Tyr Arg His Phe Asp Tyr Ala Gly Tyr Ala Thr Thr Val Glu 1220 1225 1230 Glu Glu Thr Lys Gln Asp Ile Leu Asp Val Leu Ile Arg Cys Tyr Leu 1235 1240 1245 Cys His Lys Pro Leu Cys Glu Val Glu Lys Val Lys His Ile Leu Thr 1250 1255 1260 Lys Ala Arg Phe Ile Lys Leu Asn Cys Thr Trp Lys Gly Arg Cys Leu 1265 1270 1275 1280 His Cys Trp Thr Thr Cys Met Glu Asp Met Leu Pro Gly Ser Gly Ser 1285 1290 1295 Gly Ser Gly Ser Gly Ser Met Glu Ser Lys Asp Ala Ser Thr Ser Ala 1300 1305 1310 Thr Ser Ile Asp Gln Leu Cys Lys Thr Phe Asn Leu Ser Leu His Thr 1315 1320 1325 Leu Gln Ile Gln Cys Val Phe Cys Arg Asn Ala Leu Thr Thr Ala Glu 1330 1335 1340 Ile Tyr Ala Tyr Ala Tyr Lys Asn Leu Lys Val Val Trp Arg Asp Asn 1345 1350 1355 1360 Phe Pro Phe Ala Ala Cys Ala Cys Cys Leu Glu Leu Gln Gly Lys Ile 1365 1370 1375 Asn Gln Tyr Arg His Phe Asn Tyr Ala Ala Tyr Asp Lys Gln Asp Ser 1380 1385 1390 Gln Pro Leu Thr Gln His Tyr Gln Ile Leu Thr Cys Cys Cys Gly Cys 1395 1400 1405 Asp Ser Asn Val Arg Leu Val Val Glu Cys Thr Asp Gly Asp Ile Arg 1410 1415 1420 Gln Leu Gln Asp Leu Leu Leu Gly Thr Leu Asn Ile Val Cys Pro Ile 1425 1430 1435 1440 Cys Ala Pro Lys Pro Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser Met 1445 1450 1455 His Gly Arg Leu Val Thr Leu Lys Asp Ile Val Leu Asp Leu Gln Pro 1460 1465 1470 Pro Asp Pro Val Gly Leu His Cys Tyr Glu Gln Leu Glu Asp Ser Ser 1475 1480 1485 Glu Asp Glu Val Asp Lys Val Asp Lys Gln Asp Ser Gln Pro Leu Thr 1490 1495 1500 Gln His Tyr Gln Ile Leu Thr Cys Cys Cys Gly Cys Cys Leu Glu Leu 1505 1510 1515 1520 Gln Gly Lys Ile Asn Gln Tyr Arg His Phe Asn Tyr Ala Ala Tyr Ala 1525 1530 1535 Pro Thr Val Glu Glu Glu Thr Asn Glu Asp Ile Leu Lys Val Leu Ile 1540 1545 1550 Arg Cys Tyr Leu Cys His Lys Pro Leu Cys Glu Ile Glu Lys Leu Lys 1555 1560 1565 His Ile Leu Gly Lys Ala Arg Phe Ile Lys Leu Asn Asn Gln Trp Lys 1570 1575 1580 Gly Arg Cys Leu His Cys Trp Thr Thr Cys Met Glu Asp Leu Leu Pro 1585 1590 1595 1600 Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser Met Phe Glu Asp Pro Ala 1605 1610 1615 Thr Arg Pro Arg Thr Leu His Glu Leu Cys Glu Val Leu Glu Glu Ser 1620 1625 1630 Val His Glu Ile Arg Leu Gln Cys Val Gln Cys Lys Lys Glu Leu Gln 1635 1640 1645 Arg Arg Glu Val Tyr Lys Phe Leu Phe Thr Asp Leu Arg Ile Val Tyr 1650 1655 1660 Arg Asp Asn Asn Pro Tyr Gly Val Cys Ile Met Cys Leu Arg Phe Leu 1665 1670 1675 1680 Ser Lys Ile Ser Glu Tyr Arg His Tyr Gln Tyr Ser Leu Tyr Gly Asp 1685 1690 1695 Arg Pro Asp Gly Gln Ala Glu Gln Ala Thr Ser Asn Tyr Tyr Ile Val 1700 1705 1710 Thr Tyr Cys His Ser Cys Asp Ser Thr Leu Arg Leu Cys Ile His Ser 1715 1720 1725 Thr Ala Thr Asp Leu Arg Thr Leu Gln Gln Met Leu Leu Gly Thr Leu 1730 1735 1740 Gln Val Val Cys Pro Gly Cys Ala Arg Leu Gly Ser Gly Ser Gly Ser 1745 1750 1755 1760 Gly Ser Gly Ser Met Arg Gly Asp Lys Ala Thr Ile Lys Asp Tyr Ile 1765 1770 1775 Leu Asp Leu Gln Pro Glu Thr Thr Asp Leu His Cys Tyr Glu Gln Leu 1780 1785 1790 Gly Asp Ser Ser Asp Glu Glu Asp Thr Asp Gly Val Asp Arg Pro Asp 1795 1800 1805 Gly Gln Ala Glu Gln Ala Thr Ser Asn Tyr Tyr Ile Val Thr Tyr Cys 1810 1815 1820 Cys Leu Arg Phe Leu Ser Lys Ile Ser Glu Tyr Arg His Tyr Gln Tyr 1825 1830 1835 1840 Ser Leu Tyr Gly Lys Thr Leu Glu Glu Arg Val Lys Lys Pro Leu Ser 1845 1850 1855 Glu Ile Thr Ile Arg Cys Ile Ile Cys Gln Thr Pro Leu Cys Pro Glu 1860 1865 1870 Glu Lys Glu Arg His Val Asn Ala Asn Lys Arg Phe His Asn Ile Met 1875 1880 1885 Gly Arg Trp Thr Gly Arg Cys Ser Glu Cys Trp Arg Pro Arg Pro Val 1890 1895 1900 Thr Gln Val Gly Ser 1905 <210> 29 <211> 5160 <212> DNA <213> Artificial Sequence <220> <223> polynucleotide encoding BD-14B polypeptide <400> 29 atggacgcca tgaagagagg cctgtgctgc gtgctgctgc tgtgcggcgc cgtgttcgtg 60 agccccagcc acgccaccca ggactgcagc ttccagcaca gccccatcag cagcgacttc 120 gccgtgaaga tcagagagct gagcgactac ctgctgcagg actaccccgt gaccgtggcc 180 agcaacctgc aggacgagga gctgtgcggc ggcctgtgga gactggtgct ggcccagaga 240 tggatggaga gactgaagac cgtggccggc agcaagatgc agggcctgct ggagagagtg 300 aacaccgaga tccacttcgt gaccaagtgc gccttccagc ctccccccag ctgcctgagg 360 ttcgtgcaga ccaacatcag cagactgctg caggagacca gcgagcagct ggtggccctg 420 aagccctgga tcaccagaca gaacttcagc agatgcctgg agctgcagtg ccagcccgac 480 agcagcaccc tgccccctcc ctggagcccc agacccctgg aggccaccgc tcccacagcc 540 cctggcagcg ggtccggaag tgggtctgga tctatgttca agaaccccgc cgagagaccc 600 agaaagctgc acgagctgag cagcgccctg gagatcccct acgacgagct gagactgaac 660 tgcgtgtact gcaagggcca gctgaccgag accgaggtgc tggacttcgc cttcaccgac 720 ctgaccatcg tgtacagaga cgacaccccc tacggcgtgt gcaccaagtg tctcaggttt 780 tatagtaaag tctctgaatt taggtggtat aggtattccg tctatggtcc tgcaggacag 840 gctaaacctg atacaagtaa ttataatatt gtcacatttt gttgtcagtg tgagagcacc 900 ctgagactgt gcgtgcagag cacccaggtg gacatcagaa tcctgcagga gctgctgatg 960 ggcagcttcg gcatcgtgtg ccccaactgc agcaccagac tgggcagtgg aagcggctca 1020 ggaagcggca gcatgagagg cgagaccccc accctgcagg actacgtgct ggacctgcag 1080 cccgaggcca ccgacctgca ctgctacgag cagctgcccg acagcagcga cgaggaggat 1140 gtgatcgaca gccccgccgg ccaggccaag cccgacacca gcaactacaa catcgtgacc 1200 ttctgctgcc agtgcctgag attctacagc aaggtgagcg agttcagatg gtacagatac 1260 agcgtgtacg gcaccaccct ggagaagctg accaacaagg gcatctgcga cctgctgatc 1320 agatgcatca cctgccagag acccctgtgc cccgaggaga agcagagaca cctggacaag 1380 aagaaaagat tccacaacat cggcggcaga tggaccggca gatgcatcgt gtgctggaga 1440 agacccagaa ccgagaccca ggtgggcagc ggctccggat caggcagcgg aagtatgttc 1500 caggacaccg aggagaagcc cagaaccctg cacgacctgt gccaggccct ggagaccacc 1560 atccacaaca tcgagctgca gtgcgtggag tgcaagaacc ccctgcagag aagcgaggtg 1620 tacgacttcg ccttcgccga cctgaccgtg gtgtacagag agggcaaccc cttcggcatc 1680 tgcaagctgt gtctcaggtt tctcagtaaa atttctgaat ataggcatta taattattcc 1740 gtctatggac ctgatggaca ggctcagcct gctacagcag attattatat tgtcacatgt 1800 tgtcatacat gtaacaccac cgtgagactg tgcgtgaaca gcaccgccag cgatctgaga 1860 accatccagc agctgctgat gggcaccgtg aacatcgtgt gccccacctg cgcccagctg 1920 ggctcaggaa gtggaagcgg ctctggatct atgagaggcc acaagcccac cctgaaggag 1980 tacgtgctgg acctgtaccc cgagcccacc gacctgtact gctacgagca gctgagcgat 2040 agcagcgacg aggacgaggg cctggacaga cccgatggcc aggcccagcc cgccaccgcc 2100 gactactaca tcgtgacctg ctgccacacc tgcctgagat tcctgagcaa gatcagcgag 2160 tacagacact acaactacag cgtgtacggc cacaccctgg agcagaccgt gaagaagccc 2220 ctgaacgaga tcctgatcag atgcatcatc tgccagagac ccctgtgccc ccaggagaag 2280 aagagacacg tggacctgaa caagagattc cacaacatca gcggcagatg ggccggcaga 2340 tgcgccgcct gctggagaag cagaagaaga gagaccgccc tgggcagcgg ctctggctcc 2400 ggctcaggat ctatggagtc tgctaacgct tccacatccg ctacaactat cgaccagctg 2460 tgcaagactt tcaacctcag catgcacacc ttgcagatca actgtgtgtt ttgcaaaaac 2520 gccctgacca cagcagaaat ttacagttac gcctacaaac atctgaaggt gctctttcgg 2580 gggggctatc cctacgccgc atgcgcttgt tgcttggaat ttcatggaaa aatcaaccag 2640 tatcggcatt tcgattatgc cggatacgat gggcaggata gtcagcctct gaaacagcac 2700 tatcagattg tgacctgttg ctgtggatgt gacagcaacg tgaggctggt cgtgcagtgt 2760 acagaaacag acatcagaga ggtgcagcag cttcttctgg gcactctcaa catcgtgtgt 2820 cccatctgcg ctccaaaaac cgggtccggc agcggatctg gaagcggctc catgcacggg 2880 cggcacgtga cacttaaaga catcgtcctg gaccttcagc cccctgatcc tgtcggcttg 2940 cactgttacg agcagctggt ggactcatct gaggatgagg tggacgaagt ggacggacag 3000 gattcacagc ctctgaaaca gcattaccag attgtgacct gctgctgcgg ctgttgtctt 3060 gagttccatg gaaaaatcaa ccagtacaga catttcgatt atgccggata cgcaacaacc 3120 gtcgaagagg agactaaaca ggacatcctc gacgtcctga ttcgctgcta cctgtgtcac 3180 aaaccactgt gtgaggtcga aaaggtgaaa cacattctta ccaaggcaag attcatcaaa 3240 ctcaattgta cctggaaggg acggtgcctg cactgttgga ctacatgcat ggaagatatg 3300 cttccaggaa gtgggagcgg ctcaggaagc gggagcatgg aaagtaaaga cgcttccaca 3360 agtgccactt caatcgacca gctctgtaag acattcaact tgagtctgca caccctgcag 3420 atccagtgcg tgttttgcag aaacgcactc acaaccgctg agatttacgc ctatgcttac 3480 aagaacctca aggtcgtgtg gagggataat ttccccttcg ctgcctgcgc ctgttgcctg 3540 gaactgcagg ggaaaatcaa tcagtatcgg catttcaact atgctgctta cgacaaacag 3600 gattctcagc ctctgaccca gcactaccag attctcacct gctgctgcgg ctgcgatagt 3660 aatgtgaggc tcgtggtcga gtgtaccgac ggcgacatta ggcagctcca ggatcttctc 3720 cttggcacac tgaatatcgt gtgtcctatt tgtgccccaa aacccgggtc tggaagtggc 3780 tccggatctg ggagtatgca tggacgcctc gtgacactga aggatattgt gctcgatctg 3840 cagccacctg atcccgtggg cctccactgt tatgagcagc tggaggattc ctcagaagat 3900 gaggtggata aagtggacaa acaggactcc cagcctctta cccagcatta tcagatcctg 3960 acctgctgct gcggatgttg tctggaattg cagggcaaaa tcaaccagta tagacatttc 4020 aattatgctg catacgcccc tacagtcgag gaggaaacca atgaagacat cctcaaggtg 4080 ctgatcagat gttacctctg tcacaagcct ctttgcgaaa tcgagaaact gaagcatatc 4140 ctgggaaagg ctcgctttat caagcttaac aatcagtgga aaggcaggtg cctgcactgc 4200 tggaccacct gtatggaaga cctgctgccc gggtccggct caggaagcgg ctccggctct 4260 atgtttgaag acccagccac caggccaaga acattgcacg agctttgcga agtcctcgaa 4320 gagagtgtgc atgagattag gctccagtgt gtgcagtgca agaaggaact tcagcgcaga 4380 gaggtctaca agttcttgtt tacagacctg cggatcgtgt acagggataa taatccctat 4440 ggcgtctgca ttatgtgtct taggttcctg tccaagattt cagagtacag acattaccag 4500 tattcactgt atggggacag gccagatggc caggccgagc aggctactag taactactac 4560 attgtgacct actgtcactc ctgcgactca accctccggc tgtgcattca cagcaccgcc 4620 accgaccttc gcactctgca gcagatgctg ctcggcacct tgcaggtggt gtgtcccgga 4680 tgcgccaggt tgggcagcgg gagtgggtcc ggaagcggca gtatgagagg cgataaggca 4740 accatcaagg actacatcct ggacctgcag cctgagacca ctgatttgca ttgctacgaa 4800 cagctgggag actcaagcga tgaagaagac actgatggcg tggacaggcc cgacggacag 4860 gccgaacagg ccaccagtaa ctattatatc gtcacctatt gctgcctgag gtttctcagt 4920 aaaatttctg agtacagaca ctatcagtac tcactttacg gcaagacatt ggaggagagg 4980 gtgaagaagc ctctgtccga gatcactatt aggtgcatca tctgtcagac tcccctgtgt 5040 cctgaggaaa aggagcggca tgtcaatgct aacaagagat tccacaacat catgggacgg 5100 tggacaggcc gctgctctga atgctggcgc cccaggccag tgactcaggt gggatcctaa 5160 5160 <210> 30 <211> 1467 <212> PRT <213> Artificial Sequence <220> <223> BD-14C polypeptide <400> 30 Asp Ala Met Lys Arg Gly Leu Cys Cys Val Leu Leu Leu Cys Gly Ala 1 5 10 15 Val Phe Val Ser Pro Ser His Ala Thr Gln Asp Cys Ser Phe Gln His 20 25 30 Ser Pro Ile Ser Ser Asp Phe Ala Val Lys Ile Arg Glu Leu Ser Asp 35 40 45 Tyr Leu Leu Gln Asp Tyr Pro Val Thr Val Ala Ser Asn Leu Gln Asp 50 55 60 Glu Glu Leu Cys Gly Gly Leu Trp Arg Leu Val Leu Ala Gln Arg Trp 65 70 75 80 Met Glu Arg Leu Lys Thr Val Ala Gly Ser Lys Met Gln Gly Leu Leu 85 90 95 Glu Arg Val Asn Thr Glu Ile His Phe Val Thr Lys Cys Ala Phe Gln 100 105 110 Pro Pro Pro Ser Cys Leu Arg Phe Val Gln Thr Asn Ile Ser Arg Leu 115 120 125 Leu Gln Glu Thr Ser Glu Gln Leu Val Ala Leu Lys Pro Trp Ile Thr 130 135 140 Arg Gln Asn Phe Ser Arg Cys Leu Glu Leu Gln Cys Gln Pro Asp Ser 145 150 155 160 Ser Thr Leu Pro Pro Pro Trp Ser Pro Arg Pro Leu Glu Ala Thr Ala 165 170 175 Pro Thr Ala Pro Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser Met Ala 180 185 190 Arg Phe His Asn Pro Ala Glu Arg Pro Tyr Lys Leu Pro Asp Leu Cys 195 200 205 Thr Thr Leu Asp Thr Thr Leu Gln Asp Ile Thr Ile Ala Cys Val Tyr 210 215 220 Cys Arg Arg Pro Leu Gln Gln Thr Glu Val Tyr Glu Phe Ala Phe Ser 225 230 235 240 Asp Leu Tyr Val Val Tyr Arg Asp Gly Glu Pro Leu Ala Ala Cys Gln 245 250 255 Ser Cys Ile Lys Phe Tyr Ala Lys Ile Arg Glu Leu Arg Tyr Tyr Ser 260 265 270 Asp Ser Val Gln Leu Leu Ala Arg Arg Asp Glu Pro Gln Arg His Thr 275 280 285 Ile Gln Cys Ser Cys Cys Lys Cys Asn Asn Thr Leu Gln Leu Val Val 290 295 300 Glu Ala Ser Arg Asp Thr Leu Arg Gln Leu Gln Gln Leu Phe Met Asp 305 310 315 320 Ser Leu Gly Phe Val Cys Pro Trp Cys Ala Thr Ala Asn Gln Gly Ser 325 330 335 Gly Ser Gly Ser Gly Ser Gly Ser Met Arg Gly Pro Lys Pro Thr Leu 340 345 350 Gln Glu Ile Val Leu Asp Leu Cys Pro Tyr Asn Glu Ile Gln Pro Val 355 360 365 Asp Leu Val Cys His Glu Gln Leu Gly Glu Ser Glu Asp Glu Ile Asp 370 375 380 Glu Pro Asp His Ala Val Asn His Gln His Gln Leu Leu Ala Arg Arg 385 390 395 400 Asp Glu Pro Gln Arg His Thr Ile Gln Cys Ser Cys Cys Lys Gln Ser 405 410 415 Cys Ile Lys Phe Tyr Ala Lys Ile Arg Glu Leu Arg Tyr Tyr Ser Asp 420 425 430 Ser Val Tyr Ala Thr Thr Leu Glu Asn Ile Thr Asn Thr Lys Leu Tyr 435 440 445 Asn Leu Leu Ile Arg Cys Met Cys Cys Leu Lys Pro Leu Cys Pro Ala 450 455 460 Glu Lys Leu Arg His Leu Asn Ser Lys Arg Arg Phe His Lys Ile Ala 465 470 475 480 Gly Ser Tyr Thr Gly Gln Cys Arg Arg Cys Trp Thr Thr Lys Arg Glu 485 490 495 Asp Arg Arg Leu Thr Arg Arg Glu Thr Gln Val Gly Ser Gly Ser Gly 500 505 510 Ser Gly Ser Gly Ser Met Phe Glu Asp Lys Arg Glu Arg Pro Arg Thr 515 520 525 Leu His Glu Leu Cys Glu Ala Leu Asn Val Ser Met His Asn Ile Gln 530 535 540 Val Val Cys Val Tyr Cys Lys Lys Glu Leu Cys Arg Ala Asp Val Tyr 545 550 555 560 Asn Val Ala Phe Thr Glu Ile Lys Ile Val Tyr Arg Asp Asn Asn Pro 565 570 575 Tyr Ala Val Cys Lys Gln Cys Leu Leu Phe Tyr Ser Lys Ile Arg Glu 580 585 590 Tyr Arg Arg Tyr Ser Arg Ser Val Leu Pro Glu Arg Arg Ala Gly Gln 595 600 605 Ala Thr Cys Tyr Arg Ile Glu Ala Pro Cys Cys Arg Cys Ser Ser Val 610 615 620 Val Gln Leu Ala Val Glu Ser Ser Gly Asp Thr Leu Arg Val Val Gln 625 630 635 640 Gln Met Leu Met Gly Glu Leu Ser Leu Val Cys Pro Cys Cys Ala Asn 645 650 655 Asn Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser Met Arg Gly Asn Val 660 665 670 Pro Gln Leu Lys Asp Val Val Leu His Leu Thr Pro Gln Thr Glu Ile 675 680 685 Asp Leu Gln Cys Tyr Glu Gln Phe Asp Ser Ser Glu Glu Glu Asp Glu 690 695 700 Val Asp Asn Met Arg Asp Gln Leu Pro Glu Arg Arg Ala Gly Gln Ala 705 710 715 720 Thr Cys Tyr Arg Ile Glu Ala Pro Cys Cys Arg Lys Gln Cys Leu Leu 725 730 735 Phe Tyr Ser Lys Ile Arg Glu Tyr Arg Arg Tyr Ser Arg Ser Val Tyr 740 745 750 Gly Thr Thr Leu Glu Ala Ile Thr Lys Lys Ser Leu Tyr Asp Leu Ser 755 760 765 Ile Arg Cys His Arg Cys Gln Arg Pro Leu Gly Pro Glu Glu Glu Lys Gln 770 775 780 Lys Leu Val Asp Glu Lys Lys Arg Phe His Glu Ile Ala Gly Arg Trp 785 790 795 800 Thr Gly Gln Cys Ala Asn Cys Trp Gln Arg Thr Arg Gln Arg Asn Glu 805 810 815 Thr Gln Val Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser Met Glu Pro 820 825 830 Gln Phe Asn Asn Pro Gln Glu Arg Pro Arg Ser Leu His His Leu Ser 835 840 845 Glu Val Leu Glu Ile Pro Leu Ile Asp Leu Arg Leu Ser Cys Val Tyr 850 855 860 Cys Lys Lys Glu Leu Thr Arg Ala Glu Val Tyr Asn Phe Ala Cys Thr 865 870 875 880 Glu Leu Lys Leu Val Tyr Arg Asp Asp Phe Pro Tyr Ala Val Cys Arg 885 890 895 Val Cys Leu Leu Phe Tyr Ser Lys Val Arg Lys Tyr Arg Tyr Tyr Asp 900 905 910 Tyr Ser Val Gln Ala Arg Gln Ala Lys Gln His Thr Cys Tyr Leu Ile 915 920 925 His Val Pro Cys Cys Glu Cys Lys Phe Val Val Gln Leu Asp Ile Gln 930 935 940 Ser Thr Lys Glu Asp Leu Arg Val Val Gln Gln Leu Leu Met Gly Ala 945 950 955 960 Leu Thr Val Thr Cys Pro Leu Cys Ala Ser Ser Asn Gly Ser Gly Ser 965 970 975 Gly Ser Gly Ser Gly Ser Met His Gly Lys Val Pro Thr Leu Gln Asp 980 985 990 Val Val Leu Glu Leu Thr Pro Gln Thr Glu Ile Asp Leu Gln Cys Asn 995 1000 1005 Glu Gln Leu Asp Ser Ser Glu Asp Glu Asp Glu Asp Glu Val Asp His 1010 1015 1020 Leu Gln Glu Arg Pro Gln Gln Ala Arg Gln Ala Lys Gln His Thr Cys 1025 1030 1035 1040 Tyr Leu Ile His Val Pro Cys Cys Glu Arg Val Cys Leu Leu Phe Tyr 1045 1050 1055 Ser Lys Val Arg Lys Tyr Arg Tyr Tyr Asp Tyr Ser Val Tyr Gly Ala 1060 1065 1070 Thr Leu Glu Ser Ile Thr Lys Lys Gln Leu Cys Asp Leu Leu Ile Arg 1075 1080 1085 Cys Tyr Arg Cys Gln Ser Pro Leu Thr Pro Glu Glu Lys Gln Leu His 1090 1095 1100 Cys Asp Arg Lys Arg Arg Phe His Leu Ile Ala His Gly Trp Thr Gly 1105 1110 1115 1120 Ser Cys Leu Gly Cys Trp Arg Gln Thr Ser Arg Glu Pro Arg Glu Ser 1125 1130 1135 Thr Val Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser Met Ala Arg Phe 1140 1145 1150 Glu Asp Pro Thr Gln Arg Pro Tyr Lys Leu Pro Asp Leu Ser Thr Thr 1155 1160 1165 Leu Asn Ile Pro Leu His Asp Ile Arg Ile Asn Cys Val Phe Cys Lys 1170 1175 1180 Gly Glu Leu Gln Glu Arg Glu Val Phe Glu Phe Ala Phe Asn Asp Leu 1185 1190 1195 1200 Phe Ile Val Tyr Arg Asp Cys Thr Pro Tyr Ala Ala Cys Leu Lys Cys 1205 1210 1215 Ile Ser Phe Tyr Ala Arg Val Arg Glu Leu Arg Tyr Tyr Arg Asp Ser 1220 1225 1230 Val Leu Leu Leu Ala Arg Arg Ala Glu Pro Gln Arg His Asn Ile Val 1235 1240 1245 Cys Val Cys Cys Lys Cys Asn Asn Gln Leu Gln Leu Val Val Glu Thr 1250 1255 1260 Ser Gln Asp Gly Leu Arg Ala Leu Gln Gln Leu Phe Met Asp Thr Leu 1265 1270 1275 1280 Ser Phe Val Cys Pro Leu Cys Ala Ala Asn Gln Gly Ser Gly Ser Gly 1285 1290 1295 Ser Gly Ser Gly Ser Met His Gly Pro Lys Ala Thr Leu Cys Asp Ile 1300 1305 1310 Val Leu Asp Leu Glu Pro Gln Asn Tyr Glu Glu Val Asp Leu Val Cys 1315 1320 1325 Tyr Glu Gln Leu Pro Asp Ser Asp Ser Glu Asn Glu Lys Asp Glu Pro 1330 1335 1340 Asp Gly Val Asn His Pro Leu Leu Leu Ala Arg Arg Ala Glu Pro Gln 1345 1350 1355 1360 Arg His Asn Ile Val Cys Val Cys Cys Lys Leu Lys Cys Ile Ser Phe 1365 1370 1375 Tyr Ala Arg Val Arg Glu Leu Arg Tyr Tyr Arg Asp Ser Val Tyr Gly 1380 1385 1390 Glu Thr Leu Glu Ala Glu Thr Lys Thr Pro Leu His Glu Leu Leu Ile 1395 1400 1405 Arg Cys Tyr Arg Cys Leu Lys Pro Leu Cys Pro Thr Asp Lys Leu Lys 1410 1415 1420 His Ile Thr Glu Lys Arg Arg Phe His Asn Ile Ala Gly Ile Tyr Thr 1425 1430 1435 1440 Gly Gln Cys Arg Gly Cys Arg Thr Arg Ala Arg His Leu Arg Gln Gln 1445 1450 1455 Arg Gln Ala Arg Ser Glu Thr Leu Val Gly Ser 1460 1465 <210> 31 <211> 4407 <212> DNA <213> Artificial Sequence <220> <223> polynucleotide encoding BD-14C polypeptide <400> 31 atggacgcca tgaagagagg cctgtgctgc gtgctgctgc tgtgcggcgc cgtgttcgtg 60 agccccagcc acgccaccca ggactgcagc ttccagcaca gccccatcag cagcgacttc 120 gccgtgaaga tcagagagct gagcgactac ctgctgcagg actaccccgt gaccgtggcc 180 agcaacctgc aggacgagga gctgtgcggc ggcctgtgga gactggtgct ggcccagaga 240 tggatggaga gactgaagac cgtggccggc agcaagatgc agggcctgct ggagagagtg 300 aacaccgaga tccacttcgt gaccaagtgc gccttccagc ctccccccag ctgcctgagg 360 ttcgtgcaga ccaacatcag cagactgctg caggagacca gcgagcagct ggtggccctg 420 aagccctgga tcaccagaca gaacttcagc agatgcctgg agctgcagtg ccagcccgac 480 agcagcaccc tgccccctcc ctggagcccc agacccctgg aggccaccgc tcccacagcc 540 cctggcagcg ggtccggaag tgggtctgga tctatggcta gatttcataa ccccgccgag 600 cgcccttaca aactgcccga cctgtgcacc actctggata ccactctgca ggacatcact 660 atcgcatgcg tgtactgtcg gagaccactg cagcagaccg aggtctatga gttcgccttt 720 tccgacctgt acgtggtcta tagagatggc gagcccctgg ccgcttgcca gtcttgtatc 780 aagttttacg ctaagatcag ggagctgcgc tactatagcg actccgtgca gctgctggca 840 aggcgcgatg aaccccagag gcacaccatc cagtgctcct gctgtaagtg taacaataca 900 ctgcagctgg tggtcgaggc ctcacgcgac actctgcgac agctgcagca gctgtttatg 960 gatagcctgg ggttcgtgtg cccttggtgt gccactgcta accagggctc tgggagtgga 1020 tcaggcagcg ggtccatgcg aggaccaaag cctaccctgc aggagatcgt gctggacctg 1080 tgcccctaca acgaaattca gcctgtggat ctggtctgtc acgagcagct gggcgaaagc 1140 gaggatgaaa tcgacgagcc agatcatgca gtgaatcacc agcatcagct gctggcccga 1200 cgggacgaac cacagcggca cacaattcag tgcagctgct gtaagcagtc ctgtatcaag 1260 ttctacgcaa aaattcgaga gctgcggtac tattctgata gcgtgtacgc caccacactg 1320 gaaaacatca ctaataccaa actgtataac ctgctgatta gatgcatgtg ctgtctgaag 1380 ccactgtgcc ccgccgagaa actgaggcac ctgaatagca agagaaggtt tcataaaatc 1440 gctgggtcct acaccggaca gtgccgccga tgttggacta ccaagagaga ggaccggaga 1500 ctgaccaggc gcgaaacaca agtgggatca ggcagcgggt ccggatctgg cagtatgttc 1560 gaggataaac gggaaagacc aaggacactg cacgagctgt gcgaagccct gaacgtgtcc 1620 atgcataata ttcaggtggt ctgcgtctac tgtaagaaag aactgtgccg cgcagacgtg 1680 tataatgtcg cctttactga gatcaagatc gtgtaccggg ataacaatcc ctatgccgtc 1740 tgcaagcagt gtctgctgtt ctactctaaa atccgcgaat accgacggta ttcacggagc 1800 gtgctgcctg agagaagggc aggccaggcc acttgctata gaattgaggc cccatgctgt 1860 aggtgtagct ccgtggtcca gctggctgtg gaatctagtg gagacaccct gagagtggtc 1920 cagcagatgc tgatgggaga gctgagcctg gtgtgcccat gctgtgccaa caatgggtcc 1980 ggatctggca gtgggtcagg aagcatgagg ggcaacgtgc cacagctgaa ggacgtggtc 2040 ctgcacctga ctccacagac cgagatcgac ctgcagtgct acgaacagtt tgattcaagc 2100 gaggaagagg acgaagtgga taatatgcga gatcagctgc cagagcgccg agctggacag 2160 gcaacctgct accgcatcga ggcaccttgc tgtcggaaac agtgcctgct gttctattcc 2220 aaaattagag agtaccggcg gtacagccgg agcgtgtacg gcacaactct ggaagctatc 2280 acaaagaaat ctctgtatga cctgagtatt agatgccaca ggtgtcagcg ccctctggga 2340 ccagaagaga agcagaaact ggtggatgag aagaaacgct ttcatgaaat cgcaggccgg 2400 tggaccggac agtgcgctaa ctgttggcag cgcacacgac agcggaatga gactcaagtg 2460 ggcagtgggt caggaagcgg ctccgggtct atggagcccc agttcaacaa tcctcaggaa 2520 agaccaaggt cactgcacca tctgagcgag gtgctggaaa tccctctgat tgacctgaga 2580 ctgagctgcg tgtactgtaa gaaagagctg acaagggctg aagtctataa ctttgcatgc 2640 actgagctga agctggtgta ccgcgacgat tttccctatg ccgtgtgccg ggtctgtctg 2700 ctgttctact ccaaggtgcg aaaataccgg tactatgatt atagtgtcca ggcccgccag 2760 gctaaacagc acacatgcta tctgatccat gtgccatgct gtgagtgtaa gttcgtggtc 2820 cagctggaca ttcagagcac taaagaggac ctgcgggtgg tccagcagct gctgatggga 2880 gctctgacag tgacttgccc cctgtgcgca tcctctaacg gaagtggctc agggagcggc 2940 agcggctcta tgcacggcaa ggtgcccaca ctgcaggacg tggtcctgga gctgacacct 3000 cagactgaaa tcgacctgca gtgcaatgag cagctggata gttcagagga cgaagatgag 3060 gacgaagtgg atcatctgca ggaaagacct cagcaggcaa ggcaggccaa gcagcacacc 3120 tgctacctga ttcacgtccc atgctgtgag cgcgtctgtc tgctgtttta cagcaaggtg 3180 agaaaatata ggtactatga ctacagtgtc tatggcgcca ctctggagtc aatcaccaag 3240 aaacagctgt gcgatctgct gattcgatgc taccggtgcc agagcccact gacccctgaa 3300 gagaagcagc tgcactgcga cagaaaaagg cgcttccacc tgatcgccca tggatggaca 3360 ggcagctgcc tgggctgttg gaggcagact tcccgggagc ctagagaatc taccgtgggg 3420 agtggatcag gcagcgggtc cggatctatg gctagatttg aggaccccac acagaggcct 3480 tacaagctgc ccgacctgag caccaccctg aacattccac tgcatgacat ccgcattaat 3540 tgcgtcttct gtaaaggcga gctgcaggag cgggaagtgt tcgaatttgc cttcaacgac 3600 ctgtttatcg tgtacaggga ttgcaccccc tatgcagcct gcctgaagtg tatttccttc 3660 tacgctcgcg tgcgagagct gaggtactat cgcgattctg tcctgctgct ggctcgacgg 3720 gcagaacctc agcgccacaa tatcgtgtgc gtctgctgta aatgtaacaa tcagctgcag 3780 ctggtcgtgg agaccagcca ggacggactg cgggccctgc aacaactgtt tatggataca 3840 ctgagcttcg tgtgccctct gtgcgctgca aaccaaggca gtgggtcagg aagcggctcc 3900 gggtctatgc atggaccaaa ggccaccctg tgcgacatcg tgctggatct ggaaccccag 3960 aattacgaag aggtggacct ggtctgttat gagcagctgc ctgatagtga ctcagagaac 4020 gaaaaagacg aaccagatgg cgtgaatcac ccactgctgc tggccagaag ggctgagcca 4080 cagagacata acatcgtgtg cgtctgctgc aagctgaaat gtattagttt ttacgctcgg 4140 gtgagagaac tgcgatacta tcgggactct gtctatgggg agactctgga ggcagaaacc 4200 aagacacccc tgcacgagct gctgatcaga tgctacaggt gtctgaaacc tctgtgcccc 4260 accgataagc tgaaacacat tacagagaaa cgccgattcc ataatatcgc cggaatctac 4320 accggccagt gcagggggtg tagaacacga gcaaggcatc tgaggcagca gcggcaggca 4380 aggtccgaga ctctggtggg atcctaa 4407 <210> 32 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> (GGGGS)n linker peptide unit <400> 32 Gly Gly Gly Gly Ser 1 5 <210> 33 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> (GSSGGS)n linker peptide unit <400> 33 Gly Ser Ser Gly Gly Ser 1 5 <210> 34 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> linker peptide <400> 34 Lys Glu Ser Gly Ser Val Ser Ser Glu Gln Leu Ala Gln Phe Arg Ser 1 5 10 15 Leu Asp <210> 35 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> linker peptide <400> 35 Glu Gly Lys Ser Ser Gly Ser Gly Ser Glu Ser Lys Ser Thr 1 5 10 <210> 36 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> linker peptide <400> 36 Gly Ser Ala Gly Ser Ala Ala Gly Ser Gly Glu Phe 1 5 10 <210> 37 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> (EAAAK)n linker peptide unit <400> 37 Glu Ala Ala Ala Lys 1 5 <210> 38 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> linker peptide <400> 38 Cys Arg Arg Arg Arg Arg Arg Glu Ala Glu Ala Cys 1 5 10 <210> 39 <211> 46 <212> PRT <213> Artificial Sequence <220> <223> linker peptide <400> 39 Ala Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys 1 5 10 15 Glu Ala Ala Ala Lys Ala Leu Glu Ala Glu Ala Ala Ala Lys Glu Ala 20 25 30 Ala Ala Lys Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Ala 35 40 45 <210> 40 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> linker peptide <400> 40 Gly Gly Gly Gly Gly Gly Gly Gly 1 5 <210> 41 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> linker peptide <400> 41 Gly Gly Gly Gly Gly Gly 1 5 <210> 42 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> linker peptide <400> 42 Ala Glu Ala Ala Ala Lys Glu Ala Ala Ala Ala Lys Ala 1 5 10 <210> 43 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> linker peptide <400> 43 Pro Ala Pro Ala Pro 1 5 <210> 44 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> linker peptide <400> 44 Val Ser Gln Thr Ser Lys Leu Thr Arg Ala Glu Thr Val Phe Pro Asp 1 5 10 15 Val <210> 45 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> linker peptide <400> 45 Pro Leu Gly Leu Trp Ala 1 5 <210> 46 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> linker peptide <400> 46 Thr Arg His Arg Gln Pro Arg Gly Trp Glu 1 5 10 <210> 47 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> linker peptide <400> 47 Ala Gly Asn Arg Val Arg Arg Ser Val Gly 1 5 10 <210> 48 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> linker peptide <400> 48 Arg Arg Arg Arg Arg Arg Arg Arg 1 5 <210> 49 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> linker peptide <400> 49 Gly Phe Leu Gly One <210> 50 <211> 31 <212> PRT <213> Artificial Sequence <220> <223> linker peptide <400> 50 Gly Ser Ser Gly Gly Ser Gly Ser Ser Gly Gly Ser Gly Gly Gly Asp 1 5 10 15 Glu Ala Asp Gly Ser Arg Gly Ser Gln Lys Ala Gly Val Asp Glu 20 25 30

Claims (20)

Early protein antigen 6 (E6) of human papillomavirus types 6, 11, 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58 and 59 (HPV), and early protein antigen 7 (E7) Each contains a polynucleotide encoding, wherein E6 and E7 are divided into N-terminal fragments and C-terminal fragments, respectively, and expressed in the form of randomly shuffled E6/E7 shuffled antigen units, and p53 and pRb binding functions It is lacking, 14-valent HPV DNA vaccine composition. delete delete The method of claim 1,
The E6/E7 shuffled antigenic unit is the N-terminal fragment and C-terminal fragment of E6 and E7 are the N-terminal fragment of E6 (E6N)-C-terminal fragment of E7 (E7C)-N-terminal fragment of E7 (E7N)-E6 C-terminal fragment (E6C) is a polypeptide linked in order, a 14-valent HPV DNA vaccine composition. The method of claim 1,
A 14-valent HPV DNA vaccine composition wherein at least two or more human papillomavirus E6/E7 shuffled antigen units are ligated and expressed in the form of a fusion protein. The method of claim 5,
The E6/E7 shuffled antigenic unit or the fusion protein further comprises a signal sequence, a 14-valent HPV DNA vaccine composition. The method of claim 5,
The E6/E7 shuffled antigen unit or the fusion protein further comprises Flt3L, a 14-valent HPV DNA vaccine composition. The method of claim 1,
A 14-valent HPV DNA vaccine composition further comprising IL-7. The method of claim 1,
14. A 14-valent HPV DNA vaccine composition further comprising one or more pharmaceutically acceptable vaccine adjuvants. The method of claim 9,
The vaccine adjuvant contains IL-12 protein and IL-21 protein as active ingredients, or T lymphocyte-specific comprising a polynucleotide encoding the IL-12 protein and a polynucleotide encoding the IL-21 protein as an active ingredient. 14-valent HPV DNA vaccine composition, which is a vaccine adjuvant for promoting the immune response. The method of claim 10,
The vaccine adjuvant for promoting the T lymphocyte-specific immune response comprises at least one selected from the group consisting of the following 14-valent HPV DNA vaccine composition:
IL-12 protein and IL-21 protein composed of p35 chain (IL-12p35) and p40 chain (IL-12p40);
One to three vectors comprising a polynucleotide encoding each of the p35 chain (IL-12p35) and the p40 chain (IL-12p40) constituting the IL-12, and a polynucleotide encoding each of the IL-21 protein; And
MRNA molecules encoding the IL-12p35, IL-12p40 and IL-21 proteins, respectively. The method of claim 11,
The IL-12p35 protein is human IL-12p35 consisting of the amino acid sequence shown in SEQ ID NO: 1, 14-valent HPV DNA vaccine composition. The method of claim 11,
The IL-12p40 protein is human IL-12p40 consisting of the amino acid sequence shown in SEQ ID NO: 2, 14-valent HPV DNA vaccine composition. The method of claim 10,
The IL-21 protein is human IL-21 consisting of the amino acid sequence shown in SEQ ID NO: 3, 14-valent HPV DNA vaccine composition. The method of claim 11,
The vaccine adjuvant is a 14-valent HPV DNA vaccine composition further comprising at least one 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 a linker peptide T; And
Iv) an mRNA molecule encoding the MIP-1α protein. The method of claim 15,
The MIP-1α gene construct is included in a separate expression vector, or a polynucleotide encoding each of the p35 chain (IL-12p35) and p40 chain (IL-12p40) constituting the IL-12 and the IL-21 protein A 14-valent HPV DNA vaccine composition contained in any one or more vectors of one to three vectors comprising a polynucleotide encoding each. The method of claim 16,
The MIP-1α protein is a human MIP-1α protein consisting of an amino acid sequence represented by SEQ ID NO: 10, a 14-valent HPV DNA vaccine composition. delete delete delete

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