WO2010032696A1 - がんの診断方法と治療方法 - Google Patents
がんの診断方法と治療方法 Download PDFInfo
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- WO2010032696A1 WO2010032696A1 PCT/JP2009/065955 JP2009065955W WO2010032696A1 WO 2010032696 A1 WO2010032696 A1 WO 2010032696A1 JP 2009065955 W JP2009065955 W JP 2009065955W WO 2010032696 A1 WO2010032696 A1 WO 2010032696A1
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Definitions
- the present invention relates to a method for diagnosing cancer, a diagnostic agent used therefor, a method for treating cancer, an anticancer agent used therefor, and a cancer vaccine.
- Human endogenous retrovirus Human endogenous retrovirus (Human endogenous retrovirus; HERV) and other viral LTR-like sequences constitute about 8% of the DNA of the genome. More than 20 types of HERV have been identified in the human genome, but most of them have mutations in gag, pol, and env genes, but they are derived from placenta, teratocarcinoma cell line, germ cell Some genes have been detected in other tumors and breast cancer cell lines.
- the HERV-H gene group forms the largest group, and there are about 100 copies of full-length sequences, 800-900 copies of deletion mutations, and about 1000 copies of LTR sequences.
- the HERV-H env gene is reported to be highly expressed in placenta, skeletal muscle, spleen, and thymus, but not in other normal tissues. In addition, expression is widely detected in tumor cell lines derived from various tissues (Yi, J-M, Kim, H-M, and Kim, H-S (2006) Cancer Letters vol.231, pp.228-239).
- the present invention relates to a method for diagnosing cancer and a method for treating cancer using the HERV-H env gene and gene product, in particular, a method for diagnosing cancer by detecting the expression of the HERV-H env gene and the method used therefor Diagnostic agents, methods of treating cancer by suppressing the function of the HERV-H env gene, anticancer agents used therefor, peptides with a specific sequence of the HERV-H env protein, etc.
- the purpose is to provide a method of treatment / prevention and a cancer vaccine used therefor.
- Zinc-finger transcription factor Snail is a cancer malignant factor, and it has been known that the higher the expression of Snail, the more malignant the cancer becomes (Nature-Rev Cancer 7, 415-428, 2007).
- Snail suppressed the expression of intercellular adhesion molecules such as E-cadherin, resulting in loss of gastrulation and tissue and organ development during ontogeny, normal tissues and cells.
- EMT epithelial-mesenchymal transition
- metastasis and metastasis of cancer cells Nature Rev Cancer 7, 415-428, 2007
- the present inventors have intensively studied the mechanism of EMT in cancer caused by overexpression of Snail, and found that the action of Snail is mediated by HERV-H env protein. From this, it was demonstrated that HERV-H env gene and its product can be used to diagnose and treat cancer, and the present invention has been completed.
- the cancer diagnostic agent according to the present invention contains a PCR primer pair or an anti-HERV-Henv antibody that can detect the expression of the HERV-Henv gene.
- the PCR primer pair may have SEQ ID NO: 1 and SEQ ID NO: 2.
- the cancer may be pancreatic cancer, colon cancer, melanoma, lung cancer, leukemia, esophageal cancer.
- the cancer diagnostic kit according to the present invention includes any one of the above diagnostic agents.
- the pharmaceutical composition according to the present invention contains a function inhibitor that suppresses the function of the HERV-H env gene.
- the function-suppressing substance may suppress the expression of the HERV-H env gene. Further, the function suppressing substance may be siRNA.
- the anticancer agent according to the present invention includes any one of the above pharmaceutical compositions.
- the peptide according to the present invention has any one of SEQ ID NOs: 3 to 5.
- the antigen-presenting cell according to the present invention is a cell in which a peptide having any one of SEQ ID NOs: 3 to 5 is presented on the cell surface.
- the T cell according to the present invention may be a cytotoxic T cell that recognizes a cancer cell that is induced by the antigen-presenting cell and expresses the HERV-H env antigen.
- the cancer vaccine according to the present invention contains one or more peptides selected from SEQ ID NOs: 3 to 5, an expression vector that expresses the peptide, any of the antigen-presenting cells, or any of the T cells. . It may be a cancer vaccine against cancer cells expressing Snail protein or HERV-H env antigen. Further, it may be a cancer vaccine containing one or more peptides selected from SEQ ID NOs: 3 to 5 and cancer antigen peptides other than the peptides.
- the cancer treatment / prevention method according to the present invention is a method using any one of the above cancer vaccines against humans or non-human vertebrates.
- the human endogenous retrovirus® HVHERV-H / env60 proviral copy clone 734E12 (amino acid sequence: SEQ ID NO: 1, base sequence: SEQ ID NO: 2) described in Accession No. AJ289710 is referred to as “HERV-H env gene”.
- cancer in the present specification means a tumor generally called a malignant tumor, such as a cancer derived from epithelial cells, a sarcoma derived from non-epithelial cells, or a blood cancer. Origin does not matter.
- FIG. In one Example of the present invention, the results of RT-PCR examining the expression of the HERV-H env gene in human normal tissue (A), tumor cell line (A), and human advanced colorectal cancer tissue (B).
- FIG. In one Example of this invention, it is the table
- gamma interferon production when HERV-H env specific CTL induced by HERV-H env peptide is restimulated with HERV-H env peptide in HLA-A24 expressing gene-modified mice It is a graph which shows the result of having measured quantity.
- HERV-H env-specific CTLs induced by HERV-H env peptide using HLA-A24 positive healthy human peripheral blood mononuclear cells HERV-H env-expressing tumor cells It is a graph which shows the result of having measured the killing rate (A and C) of the tumor cell when making it contact.
- HERV-H env specific CTL induced by HERV-H env peptide using HLA-A24 positive healthy peripheral blood mononuclear cells was restimulated with HERV-H env peptide It is a graph which shows the result of having measured the gamma interferon production amount (B and D).
- HERV-H env-specific CTLs induced by HERV-H env peptide using HLA-A02 positive healthy peripheral blood mononuclear cells, HERV-H env-expressing tumor cells It is a graph which shows the result of having measured the killing rate (E) of the tumor cell when making it contact.
- HERV-H ⁇ env peptide when a HERV-H ⁇ env peptide is mixed with another cancer antigen peptide and used as a cancer vaccine, it is a graph showing the results of synergistic enhancement of immune activity.
- HERV-H env-specific CD8 + CTL induced by HERV-H env peptide using HLA-A24-positive healthy human peripheral blood mononuclear cells HLA-A24-positive healthy human peripheral blood mononuclear cells It is the result of the flow cytometry which shows the proliferation of the CD8 + cell in a pancreas and a peripheral blood when a nuclear cell and a HERV-H env peptide are co-administered to an immunodeficient mouse.
- the expression level of epithelial-mesenchymal transition control gene group when HERV-H env gene expression is suppressed in human colon cancer cell line SW837 cells expressing endogenous HERV-H env It is a figure which shows the change of (A) and the cell membrane infiltrating cell number (B).
- epithelial-mesenchymal transition control when HERV-H env gene expression and Snail gene expression are suppressed in human pancreatic cancer cell line MIAPaca cells expressing endogenous HERV-H env and Snail It is a figure which shows the change (A) of the expression level of a gene group, and the number of cell membrane infiltrating cells (B).
- the types of cancer that can be diagnosed are not particularly limited, such as neuroma, kidney cancer, liver cancer, pancreatic cancer, sarcoma, colon cancer, melanoma, lung cancer, esophageal cancer, uterine cancer, testicular cancer, ovarian cancer, leukemia, lymphoma, myeloma, etc. It can be solid or hematological cancer, but it is derived from a tissue whose expression is not detected in normal tissue, brain tumor, neuroma, kidney cancer, thymoma, spleen tumor, liver cancer, osteosarcoma, lymphoma, myeloma And the like. Even cancer types whose expression is detected in normal tissues can be diagnosed as cancer by comparing the intensity of expression with normal tissues and enhancing expression.
- the diagnostic method is not particularly limited, and as long as it is a method that can detect the expression of the HERV-H ⁇ ⁇ ⁇ ⁇ ⁇ env gene, either a protein or RNA may be detected.
- a method using an antibody for example, ELISA
- a method using PCR for example, RT-PCR
- diagnostic kits that enable easy detection include diagnostic agents such as anti-HERV-H env antibodies that can detect HERV-H env proteins and PCR primer pairs that can detect HERV-H env gene expression. You may make a kit. In addition to antibodies and primers, ELISA reagents and plates, PCR reagents and the like may be attached to this kit.
- HERV-Henv In cancers that overexpressed Snail, when the function of HERV-Henv is suppressed, the function of EMT-related genes is suppressed, and as a result, cancer metastasis and proliferation ability can be suppressed. In fact, in cancers that overexpressed Snail, suppressing the function of HERV-H env reduces the number of infiltrating cells and suppresses proliferation. Therefore, a pharmaceutical composition containing a function inhibitor that suppresses the function of the HERV-H env protein can be used as an anticancer agent.
- the function inhibitor that inhibits the function of HERV-H-env protein is not particularly limited, and may be anti-HERV-H env antibody, siRNA, antisense RNA, etc. It may be inhibited at the transcription level, at the translation level, or the protein function may be suppressed.
- the cancer to be treated is not particularly limited as long as it expresses Snail protein or HERV-H env antigen.
- Neuroma, renal cancer, liver cancer, pancreatic cancer, sarcoma, colon cancer, melanoma, lung cancer, esophageal cancer , Uterine cancer, testicular cancer, ovarian cancer, leukemia, lymphoma, myeloma, solid cancer or blood cancer may be used, but pancreatic cancer, colon cancer, melanoma, lung cancer, leukemia, esophageal cancer are preferred, Most preferred is colon cancer or pancreatic cancer.
- the method of using the anticancer agent may be determined as appropriate, but is preferably administered systemically or directly to the tumor site or its vicinity in the patient.
- cytotoxic T cells established by stimulation of each peptide efficiently recognize cancer cells expressing the HERV-H env protein
- one or more peptides selected from SEQ ID NOs: 3 to 5 Antigen-presenting cells presenting peptides on the cell surface, cytotoxic T cells induced by antigen-presenting cells and recognizing cancer cells expressing HERV-H env antigen are used as cancer vaccines to treat cancer ⁇ Can be used for prevention.
- the tumor to be treated or prevented is not particularly limited as long as it is a cancer expressing HERV-H ⁇ env.
- the main treatment target is a human patient having such a tumor, but it may be a vertebrate other than a human having a tumor.
- the cancer vaccine of the present invention may contain a peptide having SEQ ID NOs: 3 to 5.
- a cancer vaccine when administered to a patient having a tumor to be treated, it is preferable to examine the patient's HLA class I type in advance.
- the peptides of SEQ ID NOs: 3 to 5 are administered.
- the peptide to be administered may be one type or a plurality of types.
- intradermal administration, subcutaneous administration, intravenous administration, intraperitoneal administration, and the like can be considered, and there is no particular limitation.
- peptides when administering, you may administer a peptide with the adjuvant etc. which improve immunity induction ability.
- the peptide to be administered may be modified so as not to be degraded in vivo.
- they instead of the peptides themselves, they may be administered as gene vaccines using expression vectors incorporating genes encoding these peptides.
- the cancer vaccine may contain antigen-presenting cells presenting peptides having SEQ ID NOs: 3 to 5.
- the peptide displayed on the cell surface may be the peptide itself having SEQ ID NOs: 3 to 5, or may be modified with sugar or phosphate.
- chemical synthesis may be used, and expression may be performed using a vector incorporating a gene encoding these peptides.
- antigen-presenting cells include macrophages, B cells, and tumor cells forcibly expressing T cell stimulating factors such as B7 and 4-1BBL by gene transfer (pseudoantigen presenting cells)
- dendritic cells are preferred because of their high antigen presenting ability.
- an example of a method for isolating dendritic cells will be described.
- mononuclear cells are isolated from the peripheral blood of vertebrate individuals.
- the mononuclear cells are preferably separated from the individual to be treated, but may be isolated from other individuals.
- the mononuclear cells are preferably CD14 positive or CD11c positive.
- the isolated mononuclear cells are cultured with GM-CSF and IL-4 for 7 days later, they can be induced to differentiate into immature dendritic cells.
- the dendritic cells thus induced to differentiate highly express MHC molecules that are antigen-presenting molecules.
- the immature dendritic cell is examined for HLA class I type, and if it is A24 or A02, the following peptides of SEQ ID NOs: 3 to 5 are added.
- HERV-H # 1 SYLHHTINL (SEQ ID NO: 3)
- HERV-H # 2 FYSLLLYSL (SEQ ID NO: 4)
- HERV-H # 3 NYAEPPWPL (SEQ ID NO: 5)
- the addition is not limited to artificially synthesized peptides, but may be extracts (extracts or lysates) or purified products of cells in which peptides are expressed.
- the antigen-presenting dendritic cells thus obtained are administered to an individual having a tumor.
- intradermal administration, subcutaneous administration, intravenous administration, intralymphatic administration, etc. are conceivable, and although there is no particular limitation, a physiological antitumor immune reaction including antigen presentation of dendritic cells
- the treatment is performed in a tumor tissue or in the vicinity of a tumor such as a regional lymph node, direct administration into a tumor tissue or a lymph node is preferable.
- the cancer vaccine may contain T cells established by stimulation of antigen-presenting cells presenting peptides derived from SEQ ID NOs: 3 to 5. T cells are co-cultured with antigen-presenting cells presenting peptides derived from SEQ ID NOs: 3 to 5, and stimulated with antigen-presenting cells.
- the T cells thus established may be administered to an individual having a tumor.
- the T cell here is preferably a cytotoxic T cell, but may be a helper T cell or the like.
- intradermal administration, subcutaneous administration, intravenous administration, intratumoral administration, and the like are conceivable. Although there is no particular limitation, in the case of cytotoxic T cells, cells expressing the antigen can be directly attacked. Therefore, intratumoral administration is preferred.
- these cancer vaccines may be administered together with other cancer vaccines.
- these peptides derived from SEQ ID NOs: 3 to 5 are used as cancer vaccines, since these peptides are virus-derived antigens, when co-administered with other cancer antigen peptides that can act as cancer vaccines, Since it also has the effect of acting as an adjuvant that enhances antigenicity, it can exhibit a synergistic effect.
- Example 1 Expression of HERV-H env gene in normal human tissues and tumor cells
- endogenous retrovirus HERV-H env gene is hardly expressed in human normal tissues, but tumor cells It is highly expressed in strains and tumor tissues.
- FIG. 1 shows the expression of the HERV-H env gene, and the lower part shows the expression of the GAPDH gene as a control of the expression level.
- HERV-H env primer Forward 5'- GGATCCTCTACCTACATGTGTC -3 '(SEQ ID NO: 6) Reverse 5'- TCAAGGGAATTAGTGGAATAAC -3 '(SEQ ID NO: 7)
- Primer for GAPDH Forward 5'- GTCAACGGATTTGGTCGTATT -3 '(SEQ ID NO: 8) Reverse 5'- ATCACTGCCACCCAGAAGACT -3 '(SEQ ID NO: 9)
- FIG. 1A in normal human tissues, weak HERV-H env gene expression was detected in the heart, spleen, pancreas, testis, and placenta. However, no expression was detected in the brain, kidney, liver, liver, thymus, muscle, and bone marrow. Strong HERV-H ⁇ ⁇ ⁇ env expression was detected in a wide range of cancer types (pancreatic cancer, colon cancer, melanoma, lung cancer, leukemia, esophageal cancer) and human tumor cell lines. In addition, as shown in FIG. 1B, a higher level of expression was observed in the cancer tissue (Tu) of colorectal cancer than in the normal tissue (N).
- Tu cancer tissue
- N normal tissue
- HERV-H env gene expression can be used for cancer diagnosis, and therapies targeting HERV-H env as a cancer antigen have few side effects on various organs and a wide range of cancer types. Applicable to any patient.
- Example 2 Function of HERV-H env expressed in tumor cells and its suppression
- HERV-H env is shown to be involved in the invasion of cancer cells, and by suppressing the expression of HERV-H env gene It shows that infiltration of cancer cells can be suppressed.
- snail cDNA (CDS 71-865, 795 bp) was amplified by PCR from Panc-1 cells stimulated with TGF-beta, known as one of EMT inducers. And inserted into the restriction enzyme EcoR I-Xho I site of a pcDNA3.1 (+) plasmid vector (Invitrogen) having a G418 resistance gene. This was introduced into a tumor cell line by electroporation, cultured for 2 weeks, drug-resistant cells were selected with G418 (2 mg / mL), and the cells were cloned.
- FIG. 2 summarizes the phenotypes of the snail transgenic cell lines in a table.
- F3 cells As controls, F3 cells (Control) introduced with control oligonucleotides manufactured by Invitrogen, F3 cells (siRNA-snail) introduced with the following snail gene-specific siRNA (SiRNA-snail # 1), and Panc-1 cell line (Parent) was used.
- siRNA-HERVH # 1 Sense: CCAAUCUUAUGCCACCCUUdTdT (SEQ ID NO: 10) Antisense: AAGGGUGGCAUAAGAUUGGdTdT (SEQ ID NO: 11)
- siRNA-HERVH # 2 Sense: CCAAUUCUUAGUCCUUUAAdTdT (SEQ ID NO: 12) Antisense: UUAAAGGACUAAGAAUUGGdTdT (SEQ ID NO: 13)
- siRNA-HERVH # 3 Sense: CCAGGCCAUCACCGAUCAUdTdT (SEQ ID NO: 14) Antisense: AUGAUCGGUGAUGGCCUGGdTdT (SEQ ID NO: 15)
- siRNA-HERVH # 4 Sense: GGAGGACUCUGUAUAUUCUdTdT (SEQ ID NO: 16) Antisense: AGAAUAUACAGAGUCCUCCCCdTdT (SEQ ID NO: 17) siRNA-sna
- HERV-H env protein functions downstream of the snail protein, by suppressing the function of the HERV-H env protein, forced expression of the snail gene
- the effect of can be countered.
- the ability of HERV-H env to be suppressed in cancer cells that overexpress the snail gene by suppressing the function of HERV-H env, thereby reducing the ability to proliferate cells and infiltrate cells.
- a function-suppressing substance that can be used is useful as an anticancer agent (a growth inhibitor or an anti-metastatic agent).
- Example 3 Induction of HERV-H env-specific CTL using HLA-A24-expressing gene-modified mice
- genetically-modified mice that express HLA-A24 with HLA-A24-restricted HERV-H env peptide ( A24-Tg) (purchased from SLC, Reference: International J. Cancer 100: 5565-5570, 2002) and HERV-H env is immunogenic, ie HERV-H env specific CTL Indicates that it is navigable.
- dendritic cell progenitor cells were isolated from bone marrow cells of A24-Tg mice using LineagePanel Streptavidin Plus Magnetic Particles-DM (BD Biosciences) for 6 days in the presence of GM-CSF (10 ng / mL, Peprotech). Cultured to differentiate. The dendritic cells were cultured for 6 hours in the presence of a peptide having the following sequence (10 ⁇ g / mL, synthesized by Invitrogen), and then 5 ⁇ 10 6 cells per mouse were subcutaneously subcutaneously inserted into HLA-A24-expressing gene-modified mice. Immunized by inoculation.
- SAGE which has already been identified as a cancer testis antigen and has an established HLA-A24-restricted peptide, was used as a positive control (Cancer Research 60: 3848-3855, 2000) and dendritic cells not stimulated with the peptide The case of using was used as a negative control.
- HERV-H # 1 SYLHHTINL (SEQ ID NO: 3)
- HERV-H # 2 FYSLLLYSL (SEQ ID NO: 4)
- HERV-H # 3 NYAEPPWPL (SEQ ID NO: 5)
- CD8 + cells (1x10 6 ) in the presence of APC (fresh spleen cells inactivated with mitomycin C, 1x10 7 cells collected from HLA-A24 expressing gene-modified mice) to obtain stronger cytokine production was stimulated with 0.08-10 ⁇ g / mL of the same peptide for an additional 24 hours.
- the gamma interferon value contained in the culture supernatant was measured using a Cytometric Bead Array kit for mice (BD Biosciences).
- FIG. 4A shows the amount of gamma interferon produced when CD8 + cells were stimulated with 10 ⁇ g / mL peptide.
- any of the three HERV-H ⁇ ⁇ ⁇ ⁇ env peptides used can induce CD8 + cells that produce high levels of gamma interferon in response to HERV-H env antigen, and its activity is SAGE It was superior to the peptide.
- FIG. 4B shows the amount of gamma interferon produced when CD8 + cells were stimulated at a concentration of 0.08 to 10 ⁇ g / mL for each peptide. Also, in the upper left of the figure, the amount of gamma and interferon produced by stimulation at each peptide concentration is converted (standardized) with the amount of gamma and interferon produced by 10 ⁇ g / mL peptide stimulation as 100%. Indicates.
- HERV-H # 2 Comparing each HERV-H env peptide, HERV-H # 2 has the highest activity, but HERV-H # 2 and HERV-H # 3 have extremely low activity when the concentration is less than 2 ⁇ g / mL On the other hand, the activity of HERV-H # 1 was kept constant even at low concentrations. From this, it was shown that the TCR binding affinity of HERV-H # 1 was significantly higher (P ⁇ 0.001, t-test test) than other peptides.
- Example 4 Induction of HERV-H env specific CTL using healthy human peripheral blood mononuclear cells HERV-H env peptide having any one of SEQ ID NOs: 3 to 5 is found only in HLA-A24 expressing gene-modified mice In addition, HERV-H env-specific CTL can be induced in humans.
- PBMC mononuclear cell fraction
- COLO320 tumor cells 50: 1
- MBL Immunocyto Cytotoxity Detection Kit
- CD8 + cells capable of sufficiently killing COLO320 tumor cells were able to be induced by HERV-H ⁇ env peptide in PBMC derived from any healthy person, equivalent to or higher than SAGE peptide.
- the tumor cytotoxic activity was highest in HERV-H # 1-induced CTL.
- gamma interferon-producing ability was also evaluated, and 1x10 6 CD8 + cells were inactivated by IL-2 (100 U / mL, Peprotech) and APC (fresh PBMC collected from healthy individuals with mitomycin C) , 5 ⁇ 10 6 ) in the presence of 1 or 10 ⁇ g / mL of each peptide for 24 hours, and the gamma interferon value contained in the culture supernatant is measured using a human Cytometric Bead Array kit (BD Biosciences) The results are graphed in FIG. 5B.
- HERV-H env peptides of SEQ ID NOs: 3 to 5 are used. Stimulation can induce CD8 + cells that can sufficiently kill the human pancreatic cancer cell line Panc-1 (HLA-A24 - HLA-A02 + SAGE + HERV-H env + ). -Highest in H # 1-induced CTL.
- the HERV-H env peptide having any one of SEQ ID NOs: 3 to 5 can induce HERV-H env-specific CTLs in both HLA-A24 positive and HLA-A02 positive humans,
- the peptide HERV-H # 1 of SEQ ID NO: 3 was most effective in both the tumor cytotoxic activity and the ability to produce gamma interferon.
- peptide HERV-H # 1 of SEQ ID NO: 3 was compared with the following HLA-A24-restricted cancer antigen peptide for tumor cytotoxic activity and gamma interferon production ability. These are peptides that are used clinically in modern peptide vaccine therapies.
- NY-ESO-1 LLMWITQCF (SEQ ID NO: 23)
- CEA TYACFWSNL (SEQ ID NO: 24)
- WT1 CMTWNQMNL (SEQ ID NO: 25)
- the ratio of CD8 + cells: COLO320 tumor cells was 6.25: 1, 12.5: 1, 25: 1, 50: 1, and the results were graphed in FIG. 5C.
- CD8 + cells were stimulated with each peptide of 0.1, 1 or 10 ⁇ g / mL in the presence of IL-2 and APC, and the results are plotted in FIG. 5D.
- NY-ESO-1-induced CTL showed the highest production at high concentrations of 1 ⁇ g / mL or higher, but NY-ESO-1 induction was observed at low concentrations (0.1 ⁇ g / mL). The activity of CTL was extremely reduced.
- the peptide HERV-H # 1 of SEQ ID NO: 3 showed an excellent effect even on peptides currently used clinically.
- Example 5 Usefulness of HERV-H env peptide as an adjuvant
- HERV-H env peptide when HERV-H env peptide is mixed with other cancer antigen peptides and used as a cancer vaccine, it is synergistically enhanced. It shows that the immunized activity exhibited.
- PBMCs were collected from healthy individuals in the same manner as in Example 4 and stimulated with HERV-H # 1, NY-ESO-1, CEA, WT1 peptides (final concentration 10 ⁇ g / mL) for 6 days, and then isolated CD8 + cells ( 1x10 6 ) in the presence of IL-2 and APC in addition to HERV-H # 1, NY-ESO-1, CEA, WT1 peptides (final concentration 1.0 ⁇ g / mL) and HERV-H # 1 (final)
- the concentration of 0.5 g / mL was stimulated for 24 hours, and the gamma interferon value contained in the culture supernatant was measured with the Cytometric Bead Array kit for humans (that is, HERV-H # in the HERV-H env group) 1 is a single stimulus, and the final peptide concentration is 1.5 ⁇ g / mL, including the other groups).
- the results are graphed in FIG. As
- the ability to produce gamma interferon was synergistically enhanced by adding HERV-H env peptide to each peptide compared to when stimulated with each peptide alone.
- the HERV-H env peptide is additionally added with existing peptide vaccine therapy, it is shown to be useful as an adjuvant.
- Example 6 Inhibition of HERV-H env-specific CTL induction by anti-HLA antibody
- HERV-H env peptide is inhibited by anti-HLA antibody.
- the cells were mixed and cultured for 6 hours, and the killed tumor cells were detected using Immunocyto Cytotoxity Detection Kit (MBL), and the tumor-specific injury rate was calculated according to the attached protocol.
- MBL Immunocyto Cytotoxity Detection Kit
- an anti-HLA antibody non-added group treated in the same manner was used as a control except that stimulation culture was performed using a medium not added with anti-HLA antibody.
- the killed tumor cell rate was significantly reduced as compared to the non-added group (control).
- Example 7 Proliferation of CD8 + cells in vivo by HERV-H env peptide
- stimulation of HERV-H env peptide shows that CD8 + cells self-proliferate in immunodeficient mice.
- CD8 + cells were obtained by stimulating PBMC with HERV-H # 1 (final concentration 10 ⁇ g / mL).
- CD8 + cells in the control group were obtained by stimulation culture with NY-ESO-1 peptide (SEQ ID NO: 23) instead of HERV-H # 1 at the same concentration.
- the CD8 + cells were mixed with a fluorescent dye PKH67 (Sigma) and fluorescently labeled by incubating for 10 minutes.
- PKH67-labeled CD8 + cells 2x10 6 PKH67-labeled CD8 + cells, PBMC collected from the same healthy person (2x10 7 ), and peptide (HERV-H env or NY-ESO-1 (control group), 100 ⁇ g per animal) Administration was performed from the tail vein of a mouse (SCID mouse, CLEA Japan). Five days after administration, mouse spleen cells and peripheral blood cells were collected, and the ratio of PKH67-labeled CD8 + cells was measured by flow cytometry (BD). A mouse treated with the same treatment except that no peptide was administered was used as a control.
- CD8 + cells induced with HERV-H env peptide were more proliferative than CD8 + cells induced with NY-ESO-1 peptide. That is, it was considered that CD8 + cells induced with HERV-H env peptide proliferate more antigen-specifically than CD8 + cells induced with NY-ESO-1 peptide. This tendency was observed in both spleen and peripheral blood.
- Example 8 Function and suppression of HERV-H env and Snail in tumor cells expressing endogenous HERV-H env and Snail.
- This example is for tumor cells expressing endogenous HERV-H env and Snail. It shows that HERV-H env and Snail are involved in cell invasion, and shows that invasion of colon cancer cells can be suppressed by suppressing expression of HERV-H env gene or Snail gene.
- siRNA SEQ ID NO: 10 to 19
- siRNA that specifically inhibits HERV-H env or snail
- a control oligonucleotide Invitrogen
- control oligonucleotide incorporated by Invitrogen
- PEI complex method Polyplus
- RT-PCR was carried out using the following primers according to “RT-PCR gene expression analysis method” in Example 1, and HERV-H env, Snail, Slug, Twist, E-cadherin, Fibronectin, GAPDH (internal standard) Each gene expression was detected.
- HERV-H env primer Forward 5'- GGATCCTCTACCTACATGTGTC -3 '(SEQ ID NO: 6) Reverse 5'- TCAAGGGAATTAGTGGAATAAC -3 '(SEQ ID NO: 7)
- Snail primer Forward 5'-CAGATGAGGACAGTGGGAAAGG -3 '(SEQ ID NO: 26) Reverse 5'-ACTCTTGGTGCTTGTGGAGCAG-3 '(SEQ ID NO: 27)
- Slug primer Forward 5'-AGCGAACTGGACACACATAC -3 '(SEQ ID NO: 28) Reverse 5'-TCTAGACTGGGCATCGCAG-3 '(SEQ ID NO: 29) Twist primer: Forward 5'-GCAAGCTTAGAGATGATGCAGGACG -3 '(SEQ ID NO: 30) Reverse 5'-GACTCGAGGTGGGACGCGGACATGGA -3 '(SEQ ID NO: 31) Primer for E-cadherin
- the human colon cancer cell line COLO320 cells cultured for 2 to 3 days after the introduction of each siRNA were collected, and the number of cells infiltrated in the lower layer (membranes) according to “Method for measuring cell invasion ability” of Example 1 The number of infiltrating cells) was counted.
- HERV-H env As shown in FIG. 9A, the expression of HERV-H env was detected in the siRNA non-introduced group (None), and it was confirmed that COLO320 cells expressed endogenous HERV-H env.
- HERV-H env expression was inhibited by siRNA (HERV # 1-4), HERV-H env gene expression decreased, and at the same time, Snail, Slug, Twist, and Fibronectin expression decreased. The expression level of -cadherin increased.
- HERV-H env When siRNA that inhibits the expression of HERV-HSenv or Snail was introduced, the number of membrane infiltrating cells decreased.
- a function inhibitor that can suppress the function of HERV-H env is useful as an anticancer agent.
- Example 9 Function and suppression of HERV-H env in tumor cells that express endogenous HERV-H env
- This example is for tumor cells that highly express endogenous HERV-H env but do not express Snail. It shows that HERV-H env is involved in cell invasion and shows that invasion of colon cancer cells can be suppressed by suppressing the expression of HERV-H env gene.
- siRNA that specifically inhibits HERV-H ⁇ env SEQ ID NOs: 10 to 19
- control oligonucleotides SEQ ID NO: SEQ ID NO :
- SW837 cells are cell lines that do not express Snail but express HERV-H env.
- RT-PCR gene expression analysis method in Example 1, was performed using the primers of SEQ ID NOs: 6-9 and 26-35 described in Example 8, and HERV-H-env, Snail, Slug , Twist, E-cadherin, Fibronectin, GAPDH (internal standard) gene expression was detected.
- human colon cancer cell line SW837 cells cultured for 2 to 3 days after the introduction of each siRNA as described above were collected, and the cell membrane infiltrated into the lower layer according to “Method for measuring cell invasion ability” of Example 1. The number of cells (number of membrane infiltrating cells) was counted.
- the expression of HERV-H env was detected in the siRNA non-introduced group (None), and it was confirmed that SW837 cells expressed HERV-H.
- HERV-H expression was inhibited by siRNA (HERV # 1-4)
- the gene expression level of HERV-H env decreased, and simultaneously the expression levels of Slug, Twist, and Fibronectin decreased, and E-cadherin The expression level increased.
- HERV-H env inhibiting the expression of HERV-H env significantly reduces the cell invasion ability of the cancer cells. Therefore, a function inhibitor that can suppress the function of HERV-H env is useful as an anticancer agent.
- Example 10 Function and suppression of HERV-H env and Snail in tumor cells expressing endogenous HERV-H env and Snail.
- This example is for tumor cells expressing endogenous HERV-H env and Snail. It shows that HERV-H env and Snail are involved in cell invasion, and shows that invasion of pancreatic cancer cells can be suppressed by suppressing the expression of HERV-H env gene or Snail gene.
- siRNA that specifically inhibits HERV-H env (SEQ ID NOs: 10 to 19) or siRNA that specifically inhibits snail (SiRNA-snail) is applied to human pancreatic cancer cell line MIAPaca cells by the PEI complex method (Polyplus). # 2) (SEQ ID NOs: 36 and 37) or control oligonucleotides (SEQ ID NOs: 20 and 21) manufactured by Invitrogen were introduced and cultured for 2 to 3 days. MIAPaca cells are a cell line that expresses endogenous HERV-H env and Snail.
- siRNA-snail # 2 Sense: CCCACUCAGAUGUCAAGAAdTdT (SEQ ID NO: 36)
- Antisense UUCUUGACAUCUGAGUGGGdTdT (SEQ ID NO: 37)
- RT-PCR gene expression analysis method in Example 1, was performed using the primers of SEQ ID NOs: 6-9 and 26-35 described in Example 8, and HERV-H-env, Snail, Slug , Twist, E-cadherin, Fibronectin, GAPDH (internal standard) gene expression was detected.
- the human pancreatic cancer cell line MIAPaca cells cultured for 2 to 3 days after the introduction of each siRNA as described above are collected, and the cell membrane is infiltrated in the lower layer according to “Method for measuring cell invasion ability” of Example 1 The number (number of membrane infiltrating cells) was counted.
- HERV-H env and Snail were detected in the siRNA non-introduced group (None), confirming that MIAPaca cells expressed endogenous HERV-H env and Snail. .
- siRNA siRNA non-introduced group
- HERV # 1-4 siRNA non-introduced group
- HERV-H env gene expression level is decreased and at the same time it controls epithelial-mesenchymal transition.
- the expression level of the gene group (Snail, Slug, Twist, Fibronectin) decreased, and the expression level of E-cadherin increased.
- a function inhibitor that can suppress the function of HERV-H env is useful as an anticancer agent.
- a method for diagnosing and treating cancer using HERV-H env gene and protein in particular, a method for diagnosing cancer by detecting the expression of HERV-H env gene and a diagnostic agent used therefor, HERV A method of treating cancer by inhibiting the function of -H env gene, a method of treating cancer by administering an anticancer agent used therefor, a peptide having a specific sequence of HERV-H env protein, and the like It has become possible to provide cancer vaccines for this purpose.
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Abstract
Description
本出願は、平成20年9月18日付で出願した日本国特許出願第2008-239943に基づく優先権を主張するものであり、当該基礎出願を引用することにより、本明細書に含めるものとする。
HERV-H env遺伝子は、正常組織には、ほとんど発現が検出されず、一部の組織で弱い発現があるだけである。しかしながら、ヒト腫瘍細胞株では広範ながん種と腫瘍株において強い発現が検出され、実際、臨床患者のがん組織においてもHERV-H envが高率に発現するため、生検などによって採取された組織や細胞で、HERV-H env遺伝子の発現を調べ、強いHERV-H env遺伝子の発現が検出された場合に、がんという診断をすることができる。
上述したように、がんにおけるSnailの過剰発現は、EMT関連遺伝子(例えば、E-カドヘリン)の発現を増強させ、がん細胞の転移や浸潤を促進し、がんを悪性化すると考えられている。Snailを過剰発現したがんにおいては、HERV-H env遺伝子の発現も亢進しているが、ここでHERV-H envタンパク質の機能を抑制すると、Snailの過剰発現による下流遺伝子発現の増強が抑制される。従って、遺伝子発現制御の流れの中で、Snail遺伝子が最も上流にあり、その下流にHERV-H env遺伝子があり、さらに下流にEMT関連遺伝子が存在する。
HERV-H envタンパク質の有するアミノ酸配列の一部である配列番号3~5を有するペプチドを抗原提示細胞である樹状細胞に添加したとき、HLAクラスI分子に結合することにより細胞表面に提示され、細胞障害性T細胞に認識されることで、HERV-H env特異的な細胞障害性T細胞を誘導できる。また各ペプチドの刺激により樹立された細胞障害性T細胞がHERV-H envタンパク質を発現するがん細胞を効率よく認識することから、配列番号3~5から選択される一つ以上のペプチド、そのペプチドを細胞表面に提示した抗原提示細胞、抗原提示細胞によって誘導され、HERV-H env抗原を発現しているがん細胞を認識する細胞障害性T細胞は、がんワクチンとして、がんの治療・予防に利用することができる。
まず、本発明のがんワクチンは、配列番号3~5を有するペプチドを含有してもよい。この場合、治療対象となる腫瘍を有する患者に対してがんワクチンを投与する際、あらかじめ患者のHLAクラスIのタイプを調べるのが好ましい。ここでは、患者のHLAクラスIタイプがA24またはA02である場合に、配列番号3~5のペプチドを投与する。投与するペプチドは、一種類であっても複数種類であってもよい。投与部位に関しては、皮内投与、皮下投与、静脈内投与、腹腔内投与などが考えられ、特に限定されることはない。また、投与する際には、免疫誘導能を高めるアジュバントなどとともにペプチドを投与してもよい。また、投与されるペプチドは、生体内で分解されにくくするような修飾が施されていてもよい。さらには、ペプチドそのものではなく、これらのペプチドをコードする遺伝子を組み込んだ発現ベクターなどを用いた遺伝子ワクチンとして投与してもよい。
HERV-H#1: SYLHHTINL(配列番号3)
HERV-H#2: FYSLLLYSL(配列番号4)
HERV-H#3: NYAEPPWPL(配列番号5)
本実施例では、内因性レトロウイルスHERV-H env遺伝子が、ヒト正常組織にはほとんど発現していないが、腫瘍細胞株及び腫瘍組織では高発現していることを示す。
RNeasykit(Qiagen社)を用いて、ヒト正常組織、種々のヒト腫瘍細胞株、及びヒト大腸癌(図1参照)からRNAを抽出し、AMVで逆転写してcDNAを得た。次に、下記のプライマーを用いてiCycler(Biorad社)で遺伝子を増幅し、電気泳動にて遺伝子発現を検出した。
HERV-H env用プライマー:
Forward 5'- GGATCCTCTACCTACATGTGTC -3'(配列番号6)
Reverse 5'- TCAAGGGAATTAGTGGAATAAC -3'(配列番号7)
GAPDH用プライマー:
Forward 5'- GTCAACGGATTTGGTCGTATT -3'(配列番号8)
Reverse 5'- ATCACTGCCACCCAGAAGACT -3'(配列番号9)
本実施例では、HERV-H envががん細胞の浸潤に関与することを示し、HERV-H env遺伝子の発現抑制によってがん細胞の浸潤を抑制できることを示す。
まず、EMT誘導剤の一つとして知られるTGF-betaで刺激したPanc-1細胞からsnail cDNA (CDS 71-865, 795 bp)をPCRで増幅し、G418耐性遺伝子を有するpcDNA3.1(+)プラスミドベクター(Invitrogen社)の制限酵素EcoR I-Xho Iサイトに挿入した。これを腫瘍細胞株にエレクトロポレーションによって導入し、2週間培養した後、G418(2mg/mL)で薬剤耐性細胞を選択し、細胞をクローニングした。
ここでは、クローンF3において、HERV-H env遺伝子発現を抑制し、細胞浸潤能及び増殖能を測定した。
次に、回収した細胞をメンブレンがmatrigelでコートされたInvasion Chamber(ポアサイズ8μm, BD Bioscience社)の上層に入れて4時間培養した (37℃, 5%CO2)。メンブレン上の細胞を完全に除去後、下層へ浸潤した細胞をクリスタルバイオレット溶液で固定・染色して顕微鏡下で計数することにより、細胞浸潤能を測定した。
siRNA-HERVH#1:
Sense:CCAAUCUUAUGCCACCCUUdTdT(配列番号10)
Antisense:AAGGGUGGCAUAAGAUUGGdTdT(配列番号11)
siRNA-HERVH#2:
Sense:CCAAUUCUUAGUCCUUUAAdTdT(配列番号12)
Antisense:UUAAAGGACUAAGAAUUGGdTdT(配列番号13)
siRNA-HERVH#3:
Sense:CCAGGCCAUCACCGAUCAUdTdT(配列番号14)
Antisense:AUGAUCGGUGAUGGCCUGGdTdT(配列番号15)
siRNA-HERVH#4:
Sense:GGAGGACUCUGUAUAUUCUdTdT(配列番号16)
Antisense:AGAAUAUACAGAGUCCUCCdTdT(配列番号17)
siRNA-snail#1:
Sense: GCGAGCUGCAGGACUCUAAdTdT(配列番号18)
Antisense: UUAGAGUCCUGCAGCUCGCdTdT(配列番号19)
コントロールオリゴヌクレオチド:
Sense: GGAUCAGUCUAUUAGGUCUdTdT (配列番号20)
Antisense: AGACCUAAUAGACUGAUCCdTdT(配列番号21)
本実施例では、HLA-A24拘束性HERV-H envペプチドでHLA-A24を発現する遺伝子改変マウス(A24-Tg)(SLC社より購入、参考文献:International J. Cancer 100: 5565-5570, 2002)を免疫し、HERV-H envが免疫原性を有すること、すなわち、HERV-H env特異的CTLが誘導可能であることを示す。
HERV-H#1: SYLHHTINL(配列番号3)
HERV-H#2: FYSLLLYSL(配列番号4)
HERV-H#3: NYAEPPWPL(配列番号5)
SAGE: LYKPDSNEF(配列番号22)
配列番号3~5のいずれかを有するHERV-H envペプチドが、HLA-A24発現遺伝子改変マウスにおいてのみならず、ヒトにおいてもHERV-H env特異的CTLを誘導できることを示す。
NY-ESO-1: LLMWITQCF(配列番号23)
CEA: TYACFWSNL(配列番号24)
WT1: CMTWNQMNL(配列番号25)
本実施例では、他のがん抗原ペプチドにHERV-H envペプチドを混合してがんワクチンとして用いた場合、相乗的に増強された免疫活性が発揮されることを示す。
本実施例では、HERV-H envペプチドによる特異的CTL誘導が抗HLA抗体によって阻害されることを示す。
本実施例では、HERV-H envペプチドの刺激によって、免疫不全マウス体内において、CD8+細胞が自己増殖することを示す。
本実施例は、内因性HERV-H envとSnailを発現する腫瘍細胞において、HERV-H envとSnailが細胞浸潤に関与することを示し、HERV-H env遺伝子またはSnail遺伝子の発現抑制によって大腸癌細胞の浸潤を抑制できることを示す。
HERV-H env用プライマー:
Forward 5'- GGATCCTCTACCTACATGTGTC -3'(配列番号6)
Reverse 5'- TCAAGGGAATTAGTGGAATAAC -3'(配列番号7)
Snail用プライマー:
Forward 5'-CAGATGAGGACAGTGGGAAAGG -3'(配列番号26)
Reverse 5'-ACTCTTGGTGCTTGTGGAGCAG -3'(配列番号27)
Slug用プライマー:
Forward 5'-AGCGAACTGGACACACATAC -3'(配列番号28)
Reverse 5'-TCTAGACTGGGCATCGCAG -3'(配列番号29)
Twist用プライマー:
Forward 5'-GCAAGCTTAGAGATGATGCAGGACG -3'(配列番号30)
Reverse 5'-GACTCGAGGTGGGACGCGGACATGGA -3'(配列番号31)
E-cadherin用プライマー:
Forward 5'-TTCCTCCCAATACATCTCCCTTCACAGCAG -3'(配列番号32)
Reverse 5'-CGAAGAAACAGCAAGAGCAGCAGAATCAGA -3'(配列番号33)
Fibronectin用プライマー:
Forward 5'-CCGTGGGCAACTCTGTC -3'(配列番号34)
Reverse 5'-TGCGGCAGTTGTCACAG -3'(配列番号35)
GAPDH用プライマー:
Forward 5'- GTCAACGGATTTGGTCGTATT -3'(配列番号8)
Reverse 5'- ATCACTGCCACCCAGAAGACT -3'(配列番号9)
本実施例は、内因性HERV-H envを高発現するがSnailを発現しない腫瘍細胞において、HERV-H envが細胞浸潤に関与することを示し、HERV-H env遺伝子の発現抑制によって大腸癌細胞の浸潤を抑制できることを示す。
本実施例は、内因性HERV-H envとSnailを発現する腫瘍細胞において、HERV-H envとSnailが細胞浸潤に関与することを示し、HERV-H env遺伝子またはSnail遺伝子の発現抑制によって膵癌細胞の浸潤を抑制できることを示す。
siRNA-snail#2:
Sense: CCCACUCAGAUGUCAAGAAdTdT(配列番号36)
Antisense: UUCUUGACAUCUGAGUGGGdTdT(配列番号37)
Claims (16)
- HERV-H env遺伝子の機能を抑制する機能抑制物質を含有することを特徴とする医薬組成物。
- 前記機能抑制物質がHERV-H env遺伝子の発現を抑制することを特徴とする請求項1に記載の医薬組成物。
- 前記機能抑制物質がsiRNAであることを特徴とする請求項2に記載の医薬組成物。
- 請求項1~3のいずれかの医薬組成物を含む抗がん剤。
- 配列番号3~5のいずれかの配列を有するペプチド。
- 配列番号3~5のいずれかの配列を有するペプチドを細胞表面に提示した抗原提示細胞。
- 請求項6に記載の抗原提示細胞によって誘導され、HERV-H env抗原を発現しているがん細胞を認識するT細胞。
- 細胞障害性T細胞であることを特徴とする請求項7に記載のT細胞。
- 配列番号3~5から選択される一つ以上のペプチド、前記ペプチドを発現する発現ベクター、請求項6に記載の抗原提示細胞、または請求項7または8に記載のT細胞を含有するがんワクチン。
- Snailタンパク質またはHERV-H env抗原を発現するがん細胞に対するがんワクチンであることを特徴とする請求項9に記載のがんワクチン。
- 配列番号3~5から選択される一つ以上のペプチド、及び前記ペプチド以外のがん抗原ペプチドを含有するがんワクチン。
- ヒト以外の脊椎動物に対し、請求項9~11のいずれか1項に記載のがんワクチンを用いたがんの治療・予防方法。
- HERV-H env遺伝子の発現を検出し得るPCR用プライマー・ペアまたは抗HERV-H env抗体を含有するがんの診断剤。
- 前記PCR用プライマー・ペアが、配列番号6及び配列番号7を有することを特徴とする請求項13に記載の診断剤。
- 前記がんが、膵癌、大腸癌、メラノーマ、肺癌、白血病、食道癌であることを特徴とする請求項13または14に記載の診断剤。
- 請求項13~15のいずれか1項に記載の診断剤を含むがんの診断キット。
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CN2009801459017A CN102215870B (zh) | 2008-09-18 | 2009-09-11 | 癌症的诊断方法和治疗方法 |
US13/119,645 US8323659B2 (en) | 2008-09-18 | 2009-09-11 | Method for diagnosing and/or treating tumor |
JP2010529742A JP5504163B2 (ja) | 2008-09-18 | 2009-09-11 | がんの診断方法と治療方法 |
EP09814541A EP2340851A4 (en) | 2008-09-18 | 2009-09-11 | DIAGNOSTIC METHOD AND THERAPEUTIC METHOD FOR CANCER |
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EP (1) | EP2340851A4 (ja) |
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Cited By (7)
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WO2011122611A1 (ja) * | 2010-03-30 | 2011-10-06 | 学校法人慶應義塾 | がんワクチン |
WO2014041784A1 (en) * | 2012-09-11 | 2014-03-20 | Oncotherapy Science, Inc. | Ube2t peptides and vaccines containing the same |
KR20140109958A (ko) * | 2011-12-20 | 2014-09-16 | 비오메리으 | 대장암의 시험관내 진단 또는 예후 예측 방법 |
CN104169434A (zh) * | 2011-12-20 | 2014-11-26 | 拜奥默里克斯公司 | 一种用于卵巢癌的体外诊断或预后的方法 |
WO2016159377A1 (ja) * | 2015-04-03 | 2016-10-06 | 国立大学法人京都大学 | がんの治療薬のスクリーニング方法 |
WO2021044009A1 (en) * | 2019-09-04 | 2021-03-11 | Deutsches Zentrum Für Neurodegenerative Erkrankungen E.V. (Dzne) | Herv inhibitors for use in treating tauopathies |
JP2021059550A (ja) * | 2015-05-01 | 2021-04-15 | 国立研究開発法人科学技術振興機構 | 腫瘍細胞の悪性化抑制剤及び抗腫瘍剤 |
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WO2011122611A1 (ja) * | 2010-03-30 | 2011-10-06 | 学校法人慶應義塾 | がんワクチン |
JP2011207832A (ja) * | 2010-03-30 | 2011-10-20 | Keio Gijuku | がんワクチン |
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RU2663350C2 (ru) * | 2012-09-11 | 2018-08-03 | Онкотерапи Сайенс, Инк. | Пептиды ube2t и содержащие их вакцины |
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Also Published As
Publication number | Publication date |
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EP2340851A1 (en) | 2011-07-06 |
JPWO2010032696A1 (ja) | 2012-02-09 |
JP5504163B2 (ja) | 2014-05-28 |
US20110217326A1 (en) | 2011-09-08 |
EP2340851A9 (en) | 2013-05-08 |
EP2340851A4 (en) | 2012-09-26 |
CN102215870B (zh) | 2013-11-20 |
CN102215870A (zh) | 2011-10-12 |
US8323659B2 (en) | 2012-12-04 |
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