KR20120053627A - E6 or e7 fusion immunoglobulin from human papillomavirus type 16 - Google Patents

Abstract

The present invention relates to a recombinant HPV 16 E6 or E7 fusion immunoglobulin, more specifically, HPV 16 E6 protein having an amino acid sequence of SEQ ID NO: 2 or HPV 16 E7 protein having an amino acid sequence of SEQ ID NO: 4 and human A recombinant HPV type 16 E6 or E7 fusion immunoglobulin consisting of an Fc region of an immunoglobulin (human IgG).
According to the present invention, the recombinant HPV type E7 or E7 fusion immunoglobulin of the present invention effectively induces the maturation of dendritic cells, and treatment of the fusion immunoglobulin in mice results in antigen-specific T helper 1 cells. Cause active activity. This, in turn, offers the potential for the use of new antigen-specific dendritic cell therapy during HPV type 16 infection in humans, and cervical cancer containing the fusion immunoglobulin of the present invention as an active ingredient. The therapeutic vaccine composition may be selected as one of the effective therapeutic agents for cervical cancer patients in place of prophylactic HPV vaccines that are not available to patients already diagnosed with cervical cancer.

Description

Recombinant HPP type E6 or E7 fusion immunoglobulin {E6 or E7 fusion immunoglobulin from human papillomavirus type 16}

The present invention relates to a recombinant HPV 16 E6 or E7 fusion immunoglobulin, more specifically, HPV 16 E6 protein having an amino acid sequence of SEQ ID NO: 2 or HPV 16 E7 protein having an amino acid sequence of SEQ ID NO: 4 and human A recombinant HPV type 16 E6 or E7 fusion immunoglobulin consisting of an Fc region of an immunoglobulin (human IgG).

Human papillomavirus (HPV) belongs to the Papillomavirus family, and 200 types of HPVs are classified as dermal HPV or mucosal HPV depending on where the target tissue is [zur Hausen H, (1999) Papillomaviruses causing cancer : evasion from host-cell control in early events in carcinogenesis. J Natl Cancer Inst 92: 690-698. The target tissue of dermal HPV is the squamous epithelium of the skin, and HPV forms warts there. Mucosal HPV targets the mucous membranes of the genital, oral and conjunctival papillomas, where HPV induces epithelial cell proliferation and malignant tumor formation. [Orth G and Favre M, (1985) Human papillomaviruses. Biochemical and biologic properties. Clin Dermatol 3: 27-42.

The four types of mucosal HPV (HPV 16, HPV 18, HPV 31 and HPV 45) are closely related to the malignant cells found in cervical cancer, so mucosal HPV is one of the most dangerous One of the sources of infection. In particular, if HPV 16 alone or co-infection with three other types of HPV (HPV 18, HPV 31 and HPV 45), it is considered a definitive primary factor for uterine cancer [Harro CD, Pang YY, Roden] RB, Hildesheim A, Wang Z, Reynolds MJ, Mast TC, Robinson R, Murphy BR, Karron RA, Dillner J, Schiller JT, and Lowy DR, (2001) Safety and immunogenicity trial in adult volunteers of a human papillomarvirus 16 L1 virus -like particle vaccine. J Natl Cancer Inst 93: 284-292. Cervical cancer is one of the most common cancers among women worldwide. In fact, cervical cancer is the second most prevalent cancer species in women worldwide and is the cause of death [Harro CD et al., (2001) Safety and immunogenicity trial in adult volunteers of a human papillomarvirus 16 L1 virus- like particle vaccine. J Natl Cancer Inst 93: 284-292.

Tumorogenesis after HPV 16 infection is largely dependent on the activity of the E6 and E7 proteins due to HPV 16. While HPV 16 invades the host, the functional role of these proteins is to disrupt cell proliferation and promote virus proliferation. [SyrjSM and SyrjKJ (1999) New concepts on the role of human papillomavirus in cell cycle regulation. Ann Med 31: 175-187. Symptoms in host cells are that E6 protein binds to p53, mediates the rapid degradation of p53, stops the cell cycle in the G1 phase, promotes apoptosis, and prevents DNA repair in host cells [SyrjSM et al. , (1999) New concepts on the role of human papillomavirus in cell cycle regulation. Ann Med 31: 175-187. In addition, the binding of E7 protein to phosphorylated pRB causes the function of phosphorylated pRB to not function properly, leading to the activity of E2F-1. And E2F-1 initiates the transcription of several genes essential for the host cell to enter S phase. Thus, E6 and E7 proteins generally promote cell proliferation and introduce tumorigenesis [SyrjSM et al., (1999) New concepts on the role of human papillomavirus in cell cycle regulation. Ann Med 31: 175-187.

Recently, two vaccines (Gardasil and Cervarix) for cervical cancer introduced by HPV are commercially available [Astbury K and Turner MJ (2009) Human papillomavirus vaccination in the prevention of cervical neoplasia. Int J Gynecol Cancer 19: 1610-1613. Gardasil is the commercial name for a quadrivalent HPV vaccine that protects against HPV VI, HPV 11, HPV 16 and HPV 18, and Cevarix is 2 for HPV 16 and HPV 18 A commercial name for a bivalent vaccine [Astbury K et al., (2009) Human papillomavirus vaccination in the prevention of cervical neoplasia. Int J Gynecol Cancer 19: 1610-1613. However, commercially available prophylactic HPV vaccines are not available for patients already diagnosed with cervical cancer.

Accordingly, the present inventors have made a research effort to overcome the problems of the prior art, as a result of linking the immunoglobulin and HPV 16 E6 or E7 protein to prepare a recombinant HPV 16 E6 or E7 fusion immunoglobulin, When the fused immunoglobulin was treated to immature dendritic cells, it was confirmed that not only effectively induces the maturation of dendritic cells but also shows the antigen specific activity of Th1 cells, thereby completing the present invention.

Accordingly, a primary object of the present invention is to provide a recombinant HPV 16 E6 or E7 fusion immunoglobulin consisting of the HPV 16 E6 or E7 protein and the Fc region of human immunoglobulin.

Another object of the present invention to provide a vaccine composition for treating cervical cancer containing the fusion immunoglobulin as an active ingredient.

In another aspect, the present invention provides an antigen-specific dendritic cell therapeutic composition comprising a dendritic cell activated by the treatment of the fusion immunoglobulin as an active ingredient.

According to one aspect of the invention, the invention provides an HPC 16 E6 protein having the amino acid sequence of SEQ ID NO: 2 or an HPC 16 E7 protein having the amino acid sequence of SEQ ID NO: 4 and an Fc region of human immunoglobulin (human IgG) And a recombinant HPV 16 E6 or E7 fusion immunoglobulin consisting of (Fc regions).

The recombinant HPV 16 E6 or E7 fusion immunoglobulin is prepared by linking an immunoglobulin (Immunoglobulin, Ig) having a constant region of the heavy chain of the HPV 16 E6 or E7 protein and antibody. It was.

In the present invention, the recombinant HPV 16 E6 or E7 fusion immunoglobulin is characterized by having the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 6.

The Fc region of the human immunoglobulin may be any region other than a fragment antigen binding region in which human immunoglobulins bind to leukocytes or macrophages. The sequence can be obtained from CAA75032 of NCBI.

In addition, a hinge sequence which is a flexible region may be included between the HPV type 16 E6 protein or the E7 protein and the human immunoglobulin Fc region. In the present invention, the hinge sequence is connected to the Fc region of the human immunoglobulin as it is. Used.

In addition, the recombinant HPV 16 E6 or E7 fusion immunoglobulin is characterized in that it further comprises an Ig leader peptide (Ig leader peptide) at the N 'end of the HPV 16 E6 protein or E7 protein.

The term "Ig leader peptide" of the present invention refers to a sequence present at the N-terminus of the secretory protein precursor and also not present in the native mature protein, and the leader peptide refers to a peptide cleaved from the protein precursor. . In general, leader sequences are cleaved by proteases (or signal peptides) upon secretion into the cell. Although such leader peptides have certain common sequence characteristics across species, leader peptides that secrete to one species do not necessarily exert secretory functions in other species. In the prepro protein or free protein, the leader peptide may be from a different protein or may be a leader peptide naturally present in the protein of interest, preferably derived from a secreted protein of the host used. Alternatively, the codon may be modified to have an optimal codon according to the codon usage frequency of the host. In addition, leader peptides that can be used for the purposes of the present invention may contain a portion of the N-terminal amino acid sequence of the native mature protein derived therefrom. In the present invention, a leader peptide having an amino acid sequence of "METDTLLLWVLLLWVPGSTGD" was specifically used.

According to another aspect of the invention, the invention provides a recombinant expression vector comprising an HPV type 16 E6 or E7 gene encoding the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4 and the Fc gene of human immunoglobulin (human IgG) to provide.

In the present invention, the HPV 16 E6 or E7 gene is characterized in that it has a nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 3.

The recombinant expression vector of the present invention may be any expression vector capable of expressing the HPV E6 type 16 or E7 fusion immunoglobulin, but preferably HPV16E6-Ig / pLNCX2 or HPV16E7 having a cleavage map of FIG. 2B. -Ig / pLNCX2.

According to another aspect of the present invention, the ball invention provides a host cell transformed with the recombinant expression vector.

The host cell of the present invention may be any animal cell capable of expressing and secreting the HPV 16 E6 or E7 fusion immunoglobulin, but preferably CHO (chinese hamster ovary) cells, COS-7 (Monkey, African green) ) Cells, NIH / 3T3 (Mouse fibroblast) cells, BHK-21 (Baby Hamster kidney, Syrian golden) cells, Hela (Human Carcinoma) cells, Vero (Monkey kidney, African green) cells, and C127 (Mouse) cells It is preferable to use any one cell selected from the group, more preferably using CHO cells due to the high transformation rate. In the embodiment of the present invention was transformed using 293GPG cells of the Tetracycline off system and CHO cells which are mammalian-derived cell lines. When transformed through the 293GPG cells, the expression was more stably maintained and transformed into CHO cells via the 293GPG cells because they had the advantage of little toxicity to the target cells.

According to an aspect of the present invention, the present invention provides a vaccine composition for treating cervical cancer containing the recombinant HPV type 16 E6 or E7 fusion immunoglobulin as an active ingredient.

The composition of the present invention is characterized in that by the recombinant HPV type 16 E6 or E7 fusion immunoglobulin results in the activation of antigen-specific dendritic cells and the activation of antigen-specific Th1 (T helper 1) lymphocytes. do.

The Th1 pattern is superior in HPV-positive women, and this Th1 cytokine response is associated with the elimination of subsequent HPV infection in the cervix [Scott M, Stites DP, Moscicki AB. Th1 cytokine patterns in cervical human papillomavirus infection. Clin Diagn Lab Immunol . 1999 Sep; 6 (5): 751-5.

In a specific embodiment of the present invention using the recombinant water-soluble HPV E6 or E7 fusion immunoglobulin protein confirmed the expression in mammalian cells was confirmed whether the activity of dendritic cells and antigen specific Th1 lymphocyte activity.

When immature dendritic cells were treated with the recombinant water-soluble HPV E6 or E7 fusion immunoglobulin protein of the present invention, mRNA levels of pro-inflammatory cytokines observed only in mature dendritic cells were measured, as well as increased expression of co-stimulatory molecules. It was confirmed. As a result, it can be seen that the fusion immunoglobulin of the present invention activates dendritic cells. In addition, after immature dendritic cells of mice were treated with recombinant water-soluble HPV E6 or E7 fusion immunoglobulins of the present invention, the expression of IL-2 and IFN-γ was confirmed from splenocytes of mice. Compared with the group, the secretion of Th1 cytokine IL-2 and IFN-γ was significantly increased in the group treated with the fusion immunoglobulin of the present invention. Therefore, it can be seen that the recombinant water-soluble HPV E6 or E7 fusion immunoglobulin of the present invention induces activation of Th1 lymphocytes.

In order to use the vaccine composition for treating cervical cancer of the present invention as a medicament, it may be prepared by a method known in the pharmaceutical field, in which case it includes a pharmaceutically acceptable carrier in addition to the active ingredient of the present invention as an active ingredient. Pharmaceutically acceptable carriers included in the pharmaceutical compositions of the present invention are those commonly used in the preparation, such as lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, phosphorus calcium, alginate, gelatin, silicic acid Calcium, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oils, and the like. It is not. In addition to the above components, the pharmaceutical composition of the present invention may further include a lubricant, a humectant, a sweetener, a flavoring agent, an emulsifier, a suspending agent, a preservative, and the like. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).

The pharmaceutical compositions of the present invention may be prepared in unit dose form by being formulated with a pharmaceutically acceptable carrier or excipient according to methods which can be easily carried out by those skilled in the art. It can be prepared by incorporation into a multi-dose container. The formulation may be in the form of a solution, suspension or emulsion in oil or medium, or may be in the form of extracts, powders, granules, tablets, capsules or injections, and may further comprise dispersants or stabilizers. They may be parenterally administered (eg, applied intravenously, subcutaneously, intraperitoneally or topically) or orally.

Appropriate dosages of the pharmaceutical compositions of the present invention may be appropriately determined by such factors as formulation method, mode of administration, age, weight, sex, health condition, degree of disease symptom, food, time of administration, method of administration and response to reaction. It may be selected, preferably 0.01 to 100 mg per day of adult can be administered.

According to another aspect of the present invention, the present invention provides a method for activating antigen specific dendritic cells comprising treating the recombinant HPV type E6 or E7 fusion immunoglobulin with dendritic cells in vitro. .

In the present invention, the "activation of dendritic cells" means that dendritic cells can transmit antigen signals and costimulatory molecule signals to T cells, thereby inducing antigen-specific immune responses of T cells.

The present invention also provides an antigen-specific dendritic cell therapy composition comprising the antigen-specific dendritic cells activated according to the above as an active ingredient.

In a specific embodiment of the present invention, treatment of recombinant HPV 16 E6 or E7 fusion immunoglobulin to immature dendritic cells induces differentiation into mature dendritic cells to induce activation of antigen specific dendritic cells (see FIG. 4). Therefore, the antigen-specific dendritic cells activated by the recombinant HPV 16 E6 or E7 fusion immunoglobulin of the present invention can be usefully used as a cell therapeutic agent for diseases caused by HPV infection.

The cell therapeutic composition of the present invention may be administered parenterally and may be administered by intraperitoneal injection, subcutaneous injection, intravenous injection or intramuscular injection. In addition, the cell therapy agent of the present invention can be used alone or in combination with methods for using surgery, hormone therapy, drug treatment and biological response modifiers for the individual for the prevention and treatment of cervical cancer.

The preferred dosage of the cell therapy of the present invention depends on the condition and weight of the patient, the extent of the disease, the form of the drug, the route of administration and the duration, and may be appropriately selected by those skilled in the art. However, for the desired effect, the cell therapy composition of the present invention may be administered to humans once to several times, and the dosage may be 1 × 10 ³ cells / kg to 1 × 10 ⁹ cells / kg, preferably 1 ×. 10 ⁴ pcs / kg-1 × 10 ⁸ pcs / kg.

As described above, according to the present invention, recombinant HPV 16 E6 or E7 fusion immunoglobulins can be prepared using HPV 16 E6 or E7 protein.

The recombinant HPV 16 E6 or E7 fusion immunoglobulin of the present invention effectively induces the maturation of dendritic cells, and treatment of the fusion immunoglobulin in mice results in antigen-specific activity of T helper 1 cells. This, in turn, offers the potential for the use of new antigen-specific dendritic cell therapy during HPV type 16 infection in humans, and cervical cancer containing the fusion immunoglobulin of the present invention as an active ingredient. The therapeutic vaccine composition may be selected as one of the effective therapeutic agents for cervical cancer patients in place of prophylactic HPV vaccines that are not available to patients already diagnosed with cervical cancer.

Figure 1 shows a gene construction schematic of recombinant HPV 16 E6 or E7 fusion immunoglobulin.
Figure 2a shows a cleavage map of the expression vector pLNCX2, Figure 2b is a recombinant vector HPV16E6-Ig / pLNCX2 comprising a gene encoding a recombinant HPV type 16 E6 or E7 fusion immunoglobulin protein ((A) of Figure 2b) Or a cleavage map of HPV16E7-Ig / pLNCX2 (FIG. 2B (B)).
3 shows the results of purification of recombinant HPV 16 E6 or E7 fusion immunoglobulin proteins.
4A shows the results of RT-PCR maturation of immature dendritic cells following treatment with recombinant HPV type 16 E6 or E7 fusion immunoglobulin, and FIG. 4B shows dendritic cells by analyzing the material on the cell surface using a flow cytometer. Results of the maturation of (iDC; immature dendritic cells, LPS; immature dendritic cells treated with LPS, E6 Ig; HPV type E6 fused Ig treated immature dendritic cells, E7 Ig; HPV type E7 fused Ig treated immature dendritic cells) .
FIG. 5 shows the results of antigen specific Th1 lymphocyte activation following treatment with recombinant HPV 16 E6 or E7 fusion immunoglobulins (control Ig; human IgG1 treatment used as negative control, E6 Ig; HPV 16 E6 fusion Ig) Treatment, E7 Ig; HPV 16 E7 fusion Ig treatment).

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention, and the scope of the present invention is not to be construed as being limited by these examples.

Example  1. Recombinant Water Soluble HPV  16 inch E6  or E7  fusion Immunoglobulins  Gene Cloning

Cloning of the recombinant water soluble HPV E6 and E7 immunoglobulin genes of the present invention was performed using RT-PCR.

First, Caski cells (cat # CRL-1550) were purchased from ATCC (American Type Culture Collection, Manassas, VA, USA) as HPV type 16 positive cell lines and cultured. To obtain cDNAs of HPV type 16 E6 and E7, 200 μl of chloroform (Sigma) was added per 1 ml of Trizol (Invitrogen), and the total RNA was isolated from the Caski cells, followed by extraction of 2 μg of mRNA. The cDNA was cloned by reverse transcription chain polymerization (RT-PCR) as a template. Using the cDNA as a template, each primer of Table 1 was prepared according to the nucleotide sequence obtained from a gene database, and cDNAs of HPV 16 E6 and E7 were obtained through chain polymerization. At this time, to facilitate gene cloning, the Xho I site was included in the forward primer, and the Sma I site was included in the reverse primer. The site is the underlined portion of the primer below. In addition, reverse transcriptase was used as Superscript III (Invitrogen) and polymerase was used as tag polymerase (Gene craft). As a result, a PCR product was obtained, and the sizes of the PCR products were 492 and 312 bp, respectively.

Table 1. HPV Type 16 Primers

Example  2. Recombinant Water Soluble HPV  16 inch E6  or E7  fusion Immunoglobulins  Expression Plasmid Preparation

To express the protein from the HPV 16 E6 or E7 fusion immunoglobulin gene, the HPV 16 E6 or E7 gene was cloned into pLNCX2 (FIG. 2A, CLONTECH), a mammalian expression vector.

First, a primer is prepared to synthesize a cleavage site sequence of restriction enzyme Xho I at the 5 'end, and a restriction enzyme cleavage site is formed by polymerase chain reaction, and a primer is made to synthesize a cleavage site sequence of restriction enzyme Sma I at the 3' end. Restriction cleavage sites were made by chain reaction. The cDNA of the Fc region of human immunoglobulin (human Ig) was then fused to the 3 'end of the HPV E6 or E7 gene. A gene schematic of each fused immunoglobulin is shown in FIG. 1. Restriction enzyme sites used in the fusion was used as restriction enzyme SmaI at the 5 'end, restriction enzyme NotI at the 3' end (see Figure 1). The base sequence of the restriction enzyme cleavage site is as follows.

XhoⅠ: CTC GAG

SmaⅠ: CCC GGG

NotⅠ: GCG GCC GC

As a result, the recombinant vector HPV16E6-Ig / pLNCX2 (FIG. 2B (A)) or HPV16E7-Ig / pLNCX2 (FIG. 2B) expressing the recombinant HPV type 16 E6 Ig or E7 Ig fusion immunoglobulin having the structure of FIG. (B)) was completed.

Example  3. Transformed cell line  making

Recombinant soluble HPV E6 and E7 Ig genes were cloned and transformed using 293GPG cells of the Tetracycline off system and CHO (Chinese Hamster Ovary) cells, a mammalian cell line, respectively. First, 293GPG cells were seeded in 6-well tissue culture plates at 1 × 10 ⁵ cells per well, followed by incubation for 24 hours in DMEM medium containing 10% calf serum. After incubation, when cells grow to cover 70-80% of the well surface, 1-2 μg of recombinant water-soluble HPV E6 or E7 fusion immunoglobulin (Ig) expression vector in serum-free DMEM medium. DNA was introduced into 293GPG cells using 6-8 μg of Lipofectamine plus (Invitrogen). Thereafter, 293GPG cells into which the expression vector was introduced were incubated for 24 hours, and then cultured host cells (CHO cells) for 4 to 5 hours using a culture supernatant containing a virus (retrovirus, retrovirus), and then serum. This 10% medium was replaced with the culture for 24 hours, and this process was repeated three times. The CHO cells were treated with G418 antibiotic 1.5 mg / ml to obtain the transduced cells to obtain cells expressing the gene.

Example  4. Recombinant Water Soluble HPV  16 inch E6  or E7  fusion Immunoglobulins  Protein Expression and Purification

To confirm that the recombinant water soluble HPV E6 or E7 fusion immunoglobulin protein is overexpressed in mammalian cells, a cell lysis buffer (20 mM Tris pH 7.4, 10 mM EDTA, 0.5% Triton X-100) was used. Cell lysates were obtained from the transformed CHO cells. After electrophoresis on a 10% polyacrylamide gel, the proteins were transferred to a membrane (Amersham Hybond-p, GE Healthcare), followed by a goat ammonium as a primary antibody. The globulin antibody was bound, the HRP conjugated anti-chlorimmunoglobulin antibody was bound with a secondary antibody, and then developed with Chemiluminescent Substrate (Thermo, 34080) to the Kodak film (KODAK Biomax Light Film, 868 9358). It was exposed. CHO cells in which only the vector was introduced into the negative comparison group were used. In addition, the transformed cells were identified through ELISA kit (R & D systems) to confirm the secreted proteins expressed in the culture supernatant.

Expression of recombinant water-soluble HPV E6 or E7 fusion immunoglobulin protein was confirmed by the above method, and Protein A agarose kit (553-50-00) was used to purify the expressed protein from the culture supernatant obtained by culturing the identified transformed cells. , KPL). First, pass the wash / binding buffer (0.1 M sodium phosphate, 0.15 M NaCl, pH 7.4) to the Protein A agarose column, and then pass the culture supernatant containing the recombinant water-soluble HPV E6 or E7 fused immunoglobulin protein. The binding force of A and IgG was used to adsorb the column. Adsorbed proteins were purified by elution again using Elution buffer (0.2 M Glycine, pH 3 ± 1.85). The eluted samples were subjected to SDS-polyacrylamide gel electrophoresis and stained with coomassie blue to identify the recombinant water-soluble HPV E6 or E7 fusion immunoglobulin protein expressed. The protein purification results are shown in FIG. 3.

The protein size of E6 fusion immunoglobulin is 48.5 kDa because of its monomeric state under reducing conditions and 97 kDa because it forms a dimer under non-reducing conditions. In the case of E7 fusion immunoglobulin, the k7a is 40 kDa under reducing conditions and 80 kDa under non-reducing conditions. The identified proteins were dialyzed in PBS, stored at −70 ° C. and used for subsequent experiments.

Example 5 Activation of Antigen-specific Dendritic Cells Using Recombinant Water Soluble HPV Type 16 E6 or E7 Fusion Immunoglobulin Proteins

Mouse dendritic cells were derived from bone marrow cells of C57BL / 6 mice, cultured for one week with 20 ng / ml IL-4 and 20 ng / ml GM-CSF, and induced into immature DCs. . Thereafter, 100 ng / ml LPS was added as a positive control and cultured for one week to induce differentiation of immature dendritic cells into mature DCs. In addition, 100 ng / ml of HPV E6 or E7 fusion immunoglobulin protein was added to the cell culture medium in the same manner as LPS to induce differentiation of immature dendritic cells into mature dendritic cells. Then, RT-PCR and flow cytometry were used to verify the activation of antigen specific dendritic cells using recombinant water-soluble HPV E6 or E7 fusion immunoglobulins.

RT-PCR was performed in the same manner as described in Example 1, wherein the primers used were as follows. And processing LPS, a recombinant HPV E6 or E7 fusion immunoglobulin protein made of LPS, and measuring FITC in order to measure activation of antigen specific dendritic cells using recombinant water soluble HPV E6 or E7 fusion immunoglobulin using a flow cytometer. Stained with anti-mouse CD86 antibody (BDpharmingen) labeled with the fluorescence intensity was measured by flow cytometry. The measurement results are shown in FIGS. 4A and 4B.

Table 2. Primers

As a result of RT-PCR, when the HPV E6 or E7 fusion immunoglobulin protein was treated to immature dendritic cells, mRNA transcription levels of major pro-inflammatory cytokine (IL-1β, IL-6, IL-12) were observed only in mature dendritic cells. Confirmed (see FIG. 4A). In addition, flow cytometry analysis showed that the treatment of HPV E6 or E7 fusion immunoglobulin recombinant protein in immature dendritic cells increased the expression of CD80 and CD86, the major co-stimulatory molecules that increase cell surface expression in mature dendritic cells. (See FIG. 4B). As a result of this, treatment of immature dendritic cells with recombinant water-soluble HPV E6 or E7 fusion immunoglobulin protein may indicate that activation of antigen-specific dendritic cells occurs.

Example 6 Antigen Specific Th1 Lymphocyte Activation Using Recombinant Water Soluble HPV Type E6 or E7 Fusion Immunoglobulin Proteins

To measure antigen specific Th1 lymphocyte activation using recombinant water soluble HPV E6 or E7 fusion immunoglobulins, HPV E6 or E7 fusion immunoglobulin recombinant proteins were applied to immature dendritic cells (1 × 10 ⁶ cells) of C57BL / 6 mice. After treatment, they were administered to the tail vein of C57BL / 6 mice with the same genetic background. At this time, the same number of dendritic cells treated with human IgG1 as a negative control were administered to the tail vein of C57BL / 6 mice. Seven days after administration of the dendritic cells, splenocytes were isolated from the spleen of donor mice, inoculated into 1 × 10 ⁶ cells in a 96-well plate, and then 100 ng / ml. HPV E6 or E7 fusion immunoglobulin protein or 100 ng / ml of human IgG1 (negative control, negative control) were treated. After 3 days of treatment, IL-2 EH was measured by the sandwich ELISA kit (Invitrogen) secretion of IFN-γ.

As a result, it was found that the amount of secretion of IL-2 or IFN-γ, which is a Th1 cytokine, was significantly increased in the HPV E6 or E7 fusion immunoglobulin protein treated group compared to the human IgG1 treated group, which was negative control (see FIG. 5). As a result, it can be seen that the recombinant water-soluble HPV E6 or E7 fusion immunoglobulin of the present invention induces antigen-specific Th1 lymphocyte activation.

<110> Korea University Industrial & Academic Collaboration Foundation <120> E6 or E7 fusion immunoglobulin from human papillomavirus type 16 <160> 6 <170> Kopatentin 1.71 <210> 1 <211> 474 <212> DNA <213> Human papillomavirus type 16 E6 <400> 1 atgcaccaaa agagaactgc aatgtttcag gacccacagg agcgacccag aaagttacca 60 cagttatgca cagagctgca aacaactata catgatataa tattagaatg tgtgtactgc 120 aagcaacagt tactgcgacg tgaggtatat gactttgctt ttcgggattt atgcatagta 180 tatagagatg ggaatccata tgctgtatgt gataaatgtt taaagtttta ttctaaaatt 240 agtgagtata gacattattg ttatagtttg tatggaacaa cattagaaca gcaatacaac 300 aaaccgttgt gtgatttgtt aattaggtgt attaactgtc aaaagccact gtgtcctgaa 360 gaaaagcaaa gacatctgga caaaaagcaa agattccata atataagggg tcggtggacc 420 ggtcgatgta tgtcttgttg cagatcatca agaacacgta gagaaaccca gctg 474 <210> 2 <211> 158 <212> PRT <213> Human papillomavirus type 16 E6 <400> 2 Met His Gln Lys Arg Thr Ala Met Phe Gln Asp Pro Gln Glu Arg Pro   1 5 10 15 Arg Lys Leu Pro Gln Leu Cys Thr Glu Leu Gln Thr Thr Ile His Asp              20 25 30 Ile Ile Leu Glu Cys Val Tyr Cys Lys Gln Gln Leu Leu Arg Arg Glu          35 40 45 Val Tyr Asp Phe Ala Phe Arg Asp Leu Cys Ile Val Tyr Arg Asp Gly      50 55 60 Asn Pro Tyr Ala Val Cys Asp Lys Cys Leu Lys Phe Tyr Ser Lys Ile  65 70 75 80 Ser Glu Tyr Arg His Tyr Cys Tyr Ser Leu Tyr Gly Thr Thr Leu Glu                  85 90 95 Gln Gln Tyr Asn Lys Pro Leu Cys Asp Leu Leu Ile Arg Cys Ile Asn             100 105 110 Cys Gln Lys Pro Leu Cys Pro Glu Glu Lys Gln Arg His Leu Asp Lys         115 120 125 Lys Gln Arg Phe His Asn Ile Arg Gly Arg Trp Thr Gly Arg Cys Met     130 135 140 Ser Cys Cys Arg Ser Ser Arg Thr Arg Arg Glu Thr Gln Leu 145 150 155 <210> 3 <211> 294 <212> DNA <213> Human papillomavirus type 16 E7 <400> 3 atgcatggag atacacctac attgcatgaa tatatgttag atttgcaacc agagacaact 60 gatctctact gttatgagca attaaatgac agctcagagg aggaggatga aatagatggt 120 ccagctggac aagcagaacc ggacagagcc cattacaata ttgtaacctt ttgttgcaag 180 tgtgactcta cgcttcggtt gtgcgtacaa agcacacacg tagacattcg tactttggaa 240 gacctgttaa tgggcacact aggaattgtg tgccccatct gttctcagaa acca 294 <210> 4 <211> 98 <212> PRT <213> Human papillomavirus type 16 E7 <400> 4 Met His Gly Asp Thr Pro Thr Leu His Glu Tyr Met Leu Asp Leu Gln   1 5 10 15 Pro Glu Thr Thr Asp Leu Tyr Cys Tyr Glu Gln Leu Asn Asp Ser Ser              20 25 30 Glu Glu Glu Asp Glu Ile Asp Gly Pro Ala Gly Gln Ala Glu Pro Asp          35 40 45 Arg Ala His Tyr Asn Ile Val Thr Phe Cys Cys Lys Cys Asp Ser Thr      50 55 60 Leu Arg Leu Cys Val Gln Ser Thr His Val Asp Ile Arg Thr Leu Glu  65 70 75 80 Asp Leu Leu Met Gly Thr Leu Gly Ile Val Cys Pro Ile Cys Ser Gln                  85 90 95 Lys pro         <210> 5 <211> 413 <212> PRT <213> Artificial Sequence <220> <223> E6 fusion immunoglobulin from human papillomavirus type 16 <400> 5 Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro   1 5 10 15 Gly Ser Thr Gly Asp Met His Gln Lys Arg Thr Ala Met Phe Gln Asp              20 25 30 Pro Gln Glu Arg Pro Arg Lys Leu Pro Gln Leu Cys Thr Glu Leu Gln          35 40 45 Thr Thr Ile His Asp Ile Ile Leu Glu Cys Val Tyr Cys Lys Gln Gln      50 55 60 Leu Leu Arg Arg Glu Val Tyr Asp Phe Ala Phe Arg Asp Leu Cys Ile  65 70 75 80 Val Tyr Arg Asp Gly Asn Pro Tyr Ala Val Cys Asp Lys Cys Leu Lys                  85 90 95 Phe Tyr Ser Lys Ile Ser Glu Tyr Arg His Tyr Cys Tyr Ser Leu Tyr             100 105 110 Gly Thr Thr Leu Glu Gln Gln Tyr Asn Lys Pro Leu Cys Asp Leu Leu         115 120 125 Ile Arg Cys Ile Asn Cys Gln Lys Pro Leu Cys Pro Glu Glu Lys Gln     130 135 140 Arg His Leu Asp Lys Lys Gln Arg Phe His Asn Ile Arg Gly Arg Trp 145 150 155 160 Thr Gly Arg Cys Met Ser Cys Cys Arg Ser Ser Arg Thr Arg Arg Glu                 165 170 175 Thr Gln Leu Pro Gly Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys             180 185 190 Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu         195 200 205 Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu     210 215 220 Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys 225 230 235 240 Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys                 245 250 255 Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu             260 265 270 Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys         275 280 285 Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys     290 295 300 Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser 305 310 315 320 Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys                 325 330 335 Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln             340 345 350 Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly         355 360 365 Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln     370 375 380 Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn 385 390 395 400 His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys                 405 410 <210> 6 <211> 353 <212> PRT <213> Artificial Sequence <220> <223> E7 fusion immunoglobulin from human papillomavirus type 16 <400> 6 Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro   1 5 10 15 Gly Ser Thr Gly Asp Met His Gly Asp Thr Pro Thr Leu His Glu Tyr              20 25 30 Met Leu Asp Leu Gln Pro Glu Thr Thr Asp Leu Tyr Cys Tyr Glu Gln          35 40 45 Leu Asn Asp Ser Ser Glu Glu Glu Asp Glu Ile Asp Gly Pro Ala Gly      50 55 60 Gln Ala Glu Pro Asp Arg Ala His Tyr Asn Ile Val Thr Phe Cys Cys  65 70 75 80 Lys Cys Asp Ser Thr Leu Arg Leu Cys Val Gln Ser Thr His Val Asp                  85 90 95 Ile Arg Thr Leu Glu Asp Leu Leu Met Gly Thr Leu Gly Ile Val Cys             100 105 110 Pro Ile Cys Ser Gln Lys Pro Pro Gly Glu Pro Lys Ser Cys Asp Lys         115 120 125 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro     130 135 140 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 145 150 155 160 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Ser Glu Asp                 165 170 175 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn             180 185 190 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val         195 200 205 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu     210 215 220 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 225 230 235 240 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr                 245 250 255 Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr             260 265 270 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu         275 280 285 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu     290 295 300 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 305 310 315 320 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu                 325 330 335 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly             340 345 350 Lys    

Claims (11)

Recombinant HPV 16 E6 consisting of the HPV 16 E6 protein having the amino acid sequence of SEQ ID NO: 2 or HPV 16 E7 protein having the amino acid sequence of SEQ ID NO: 4 and the Fc region of human immunoglobulin (human IgG) Or E7 fusion immunoglobulin.
The recombinant HPV 16 E6 or E7 fusion immunoglobulin according to claim 1, wherein the recombinant HPV 16 E6 or E7 fusion immunoglobulin has an amino acid sequence represented by SEQ ID NO: 5 or SEQ ID NO: 6.
A recombinant expression vector comprising an HPV type 16 E6 or E7 gene encoding the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4 and an Fc gene of human immunoglobulin (human IgG).
The recombinant expression vector of claim 3, wherein the HPV 16 E6 or E7 gene has a nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 3.
The recombinant expression vector of claim 3, wherein the recombinant expression vector has HPV16E6-Ig / pLNCX2 or HPV16E7-Ig / pLNCX2 having a cleavage map of FIG. 2B.
A host cell transformed with the recombinant expression vector of claim 3.
7. The transformed host cell of claim 6, wherein said host cell is a CHO cell.
The vaccine composition for treating cervical cancer containing the recombinant HPV type 16 E6 or E7 fusion immunoglobulin of claim 1 as an active ingredient.
9. The vaccine composition of claim 8, wherein the recombinant HPV 16 E6 or E7 fusion immunoglobulin results in the activation of antigen-specific dendritic cells and the activation of antigen-specific Th1 lymphocytes.
A method of activating an antigen specific dendritic cell comprising treating the dendritic cell in vitro with a recombinant HPV type 16 E6 or E7 fusion immunoglobulin according to claim 1.
An antigen-specific dendritic cell therapy composition comprising an antigen-specific dendritic cell activated according to claim 10 as an active ingredient.

Images