WO2017209053A1 - 新規な遺伝子組換えワクシニアウイルス - Google Patents
新規な遺伝子組換えワクシニアウイルス Download PDFInfo
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- WO2017209053A1 WO2017209053A1 PCT/JP2017/019921 JP2017019921W WO2017209053A1 WO 2017209053 A1 WO2017209053 A1 WO 2017209053A1 JP 2017019921 W JP2017019921 W JP 2017019921W WO 2017209053 A1 WO2017209053 A1 WO 2017209053A1
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- cancer
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Definitions
- the present invention relates to a novel genetically engineered vaccinia virus.
- Recombinant vaccinia virus which lacks the functions of viral proteins vaccinia virus growth factor (VGF) and O1L and specifically grows in cancer cells and destroys cancer cells, is going to be used for cancer treatment Technology has been reported. It has also been described that foreign genes such as marker genes and therapeutic genes encoding products having cytotoxicity or immunostimulatory effects may be introduced into the virus into genes that are not essential for the life cycle of vaccinia virus, What is specifically verified in the Examples is the introduction of a marker gene luciferase-green fluorescent protein (GFP) fusion gene or DsRed expression cassette. The introduction of therapeutic genes has not been verified. There is no suggestion of combining a plurality of therapeutic genes (Patent Document 2).
- GFP luciferase-green fluorescent protein
- human recombinant interleukin-7 (IL-7) protein alone has a level of interferon-gamma (detectable by CD8 + T cells). Although it does not induce IFN- ⁇ ) production, it has been reported that the combination of human recombinant IL-7 protein and human recombinant IL-12 protein synergistically promotes IFN- ⁇ production ( The Journal of Immunology, 1995, Vol.154, p.5093-5102). With regard to oncolytic vaccinia viruses that express immunostimulatory molecules, it has been reported that the virus may be cleared early due to a strong immune response. It has also been described that a strong immune response can be an enemy and ally for vaccinia virus-mediated cancer therapy (Molecular Therapy, 2005, Vol. 11, No. 2, p. 180-195).
- An object of the present invention is to provide a genetically modified vaccinia virus (particularly an oncolytic vaccinia virus), a pharmaceutical composition and a combination kit for treating or preventing cancer.
- vaccinia virus comprising a polynucleotide encoding IL-7 or vaccinia virus comprising a polynucleotide encoding IL-12
- IL- A vaccinia virus comprising two polynucleotides, a polynucleotide encoding 7 and a polynucleotide encoding IL-12
- Example 2 1) encoding a polynucleotide encoding IL-7 and IL-12
- a mixture of two vaccinia viruses comprising a vaccinia virus comprising two polynucleotides, and 2) a vaccinia virus comprising a polynucleotide encoding IL-7 and a vaccinia virus comprising a polynucleotide encoding IL-12
- a vaccinia virus comprising two polynucleotides, a polynucleotide encoding IL-7 and a polynucleotide encoding IL-12, exhibits tumor regression in a tumor-bearing humanized mouse model (Example 6).
- a complete remission was exhibited in a cancer mouse model (Example 7), and further, the acquired immunity was induced and the antitumor effect could be sustained (Example 8).
- the present invention may include the following inventions as medically or industrially useful substances or methods.
- Vaccinia virus comprising the following (1) and (2): (1) a polynucleotide encoding interleukin-7 (IL-7); and (2) a polynucleotide encoding interleukin-12 (IL-12).
- composition selected from the following (1) or (2): (1) a pharmaceutical composition comprising a vaccinia virus comprising a polynucleotide encoding IL-12 for use in combination with a pharmaceutical composition comprising a vaccinia virus comprising a polynucleotide encoding IL-7; or (2) IL- A pharmaceutical composition comprising a vaccinia virus comprising a polynucleotide encoding IL-7 for use in combination with a pharmaceutical composition comprising a vaccinia virus comprising a polynucleotide encoding 12.
- a combination kit comprising the following vaccinia viruses (1) and (2): (1) a vaccinia virus comprising a polynucleotide encoding IL-7; and (2) a vaccinia virus comprising a polynucleotide encoding IL-12.
- the vaccinia virus according to [1] which lacks the function of vaccinia virus growth factor (VGF).
- VVF vaccinia virus growth factor
- the vaccinia virus according to [1] lacking the function of O1L.
- the vaccinia virus according to [1] which lacks VGF and O1L functions.
- the vaccinia virus according to [1] wherein an SCR (short consensus repeat) domain of the B5R extracellular region is deleted.
- a pharmaceutical composition comprising the vaccinia virus according to any one of [1] and [4] to [9] and a pharmaceutically acceptable excipient.
- Cancer is malignant melanoma, lung adenocarcinoma, lung cancer, small cell lung cancer, lung squamous cell carcinoma, renal cancer, bladder cancer, head and neck cancer, breast cancer, esophageal cancer, glioblastoma, neuroblastoma
- the pharmaceutical composition or kit according to [18] which is myeloma, ovarian cancer, colon cancer, pancreatic cancer, prostate cancer, hepatocellular carcinoma, mesothelioma, cervical cancer, or gastric cancer.
- a method for preventing or treating cancer comprising the step of administering the vaccinia virus according to any one of [1] and [4] to [9] to a subject in need of cancer prevention or treatment .
- Cancer is malignant melanoma, lung adenocarcinoma, lung cancer, small cell lung cancer, lung squamous cell carcinoma, renal cancer, bladder cancer, head and neck cancer, breast cancer, esophageal cancer, glioblastoma, neuroblastoma
- the method according to [20] which is myeloma, ovarian cancer, colon cancer, pancreatic cancer, prostate cancer, hepatocellular carcinoma, mesothelioma, cervical cancer, or stomach cancer.
- the vaccinia virus according to any one of [1] and [4] to [9] for use in the prevention or treatment of cancer.
- Cancer is malignant melanoma, lung adenocarcinoma, lung cancer, small cell lung cancer, lung squamous cell carcinoma, renal cancer, bladder cancer, head and neck cancer, breast cancer, esophageal cancer, glioblastoma, neuroblastoma
- the vaccinia virus according to [22] which is myeloma, ovarian cancer, colon cancer, pancreatic cancer, prostate cancer, hepatocellular carcinoma, mesothelioma, cervical cancer, or stomach cancer.
- Cancer is malignant melanoma, lung adenocarcinoma, lung cancer, small cell lung cancer, lung squamous cell carcinoma, renal cancer, bladder cancer, head and neck cancer, breast cancer, esophageal cancer, glioblastoma, neuroblastoma
- vaccinia virus comprising a polynucleotide encoding IL-7; and (2) vaccinia virus comprising a polynucleotide encoding IL-12.
- a method for preventing or treating cancer comprising a step.
- Cancer is malignant melanoma, lung adenocarcinoma, lung cancer, small cell lung cancer, lung squamous cell carcinoma, renal cancer, bladder cancer, head and neck cancer, breast cancer, esophageal cancer, glioblastoma, neuroblastoma
- the method according to [26] wherein myeloma, ovarian cancer, colon cancer, pancreatic cancer, prostate cancer, hepatocellular carcinoma, mesothelioma, cervical cancer, or stomach cancer.
- Vaccinia virus selected from the following (1) or (2): (1) a vaccinia virus comprising a polynucleotide encoding IL-7 for use in the prevention or treatment of cancer in combination with a pharmaceutical composition comprising a vaccinia virus comprising a polynucleotide encoding IL-12; or (2) A vaccinia virus comprising a polynucleotide encoding IL-12 for use in the prevention or treatment of cancer in combination with a pharmaceutical composition comprising a vaccinia virus comprising a polynucleotide encoding IL-7.
- Cancer is malignant melanoma, lung adenocarcinoma, lung cancer, small cell lung cancer, lung squamous cell carcinoma, renal cancer, bladder cancer, head and neck cancer, breast cancer, esophageal cancer, glioblastoma, neuroblastoma
- the vaccinia virus according to [28] which is myeloma, ovarian cancer, colon cancer, pancreatic cancer, prostate cancer, hepatocellular carcinoma, mesothelioma, cervical cancer, or stomach cancer.
- vaccinia virus selected from the following (1) or (2): (1) A vaccinia containing a polynucleotide encoding IL-7 for producing a pharmaceutical composition for preventing or treating cancer to be used in combination with a pharmaceutical composition containing a vaccinia virus containing a polynucleotide encoding IL-12 Use of a virus; or (2) encodes IL-12 for producing a pharmaceutical composition for preventing or treating cancer in combination with a pharmaceutical composition comprising a vaccinia virus comprising a polynucleotide encoding IL-7 Use of a vaccinia virus comprising a polynucleotide.
- Cancer is malignant melanoma, lung adenocarcinoma, lung cancer, small cell lung cancer, lung squamous cell carcinoma, renal cancer, bladder cancer, head and neck cancer, breast cancer, esophageal cancer, glioblastoma, neuroblastoma
- the vaccinia virus of the present invention and the vaccinia virus contained in the pharmaceutical composition and the combination kit of the present invention exhibit an oncolytic action, and are expressed in IL-12 polypeptide encoded by a polynucleotide loaded on the virus in cancer cells and It expresses IL-7 polypeptide, resulting in complete remission and acquired immunity, and the vaccinia virus, pharmaceutical composition and combination kit of the present invention can be used for prevention or treatment of cancer.
- FIG. 5 shows the oncolytic properties of recombinant vaccinia virus (LC16mO ⁇ SCR VGF-SP-IL12 / O1L-SP-IL7).
- the vertical axis shows cancer cell survival rate (%). Error bars indicate standard deviation.
- FIG. 5 shows the oncolytic properties of recombinant vaccinia virus (LC16mO ⁇ SCR VGF-SP-IL12 / O1L-SP-IL7).
- the vertical axis shows cancer cell survival rate (%). Error bars indicate standard deviation.
- FIGS. 5-1 and 5-2 are obtained under the same experimental conditions except that the measured cell types are different.
- FIG. 2 shows the oncolytic properties of two mixtures of recombinant vaccinia viruses (LC16mO ⁇ SCR VGF-SP-IL12 / O1L-SP-LacZ and LC16mO ⁇ SCR VGF-SP-IL7 / O1L-SP-LacZ).
- the vertical axis shows cancer cell survival rate (%). Error bars indicate standard deviation.
- FIGS. 5-3 and 5-4 were obtained under the same experimental conditions except that the measured cell types were different.
- the present invention provides a vaccinia virus comprising the following (1) and (2): (1) a polynucleotide encoding IL-7; and (2) a polynucleotide encoding IL-12.
- a vaccinia virus comprising the following (1) and (2): (1) a polynucleotide encoding IL-7; and (2) a polynucleotide encoding IL-12.
- the vaccinia virus is also referred to as “vaccinia virus of the present invention”.
- the pharmaceutical composition is also referred to as “the pharmaceutical composition for combined use of the present invention”, and IL-7 contained in the combined pharmaceutical composition of the present invention described in (1) or (2) above)
- IL-7 contained in the combined pharmaceutical composition of the present invention described in (1) or (2) above
- a vaccinia virus containing a polynucleotide encoding or a vaccinia virus containing a polynucleotide encoding IL-12 is also referred to as a “combined vaccinia virus”.
- the present invention also provides a combination kit comprising the following vaccinia viruses (1) and (2): (1) a vaccinia virus comprising a polynucleotide encoding IL-7; and (2) a vaccinia virus comprising a polynucleotide encoding IL-12.
- the combination kit is also referred to as “the combination kit of the present invention”, and each vaccinia virus contained in the combination kit of the present invention is also referred to as “the vaccinia virus for the combination kit”.
- a polypeptide comprising an amino acid sequence that is 90% or more identical to the amino acid sequence shown in NP_000873.2, and A polypeptide having the function of human IL-12 and (3) (1-a) Accession No.
- the method for introducing the transfer vector plasmid DNA into the cell is not particularly limited, and for example, a calcium phosphate method, an electroporation method, or the like can be used.
- the vaccinia virus of the present invention, the combined vaccinia virus, or the combined kit vaccinia virus does not have a drug selection marker gene.
- the expression and / or propagation of the vaccinia virus of the present invention, the vaccinia virus for combined use, or the vaccinia virus for combination kit can be carried out by infecting host cells with the vaccinia virus and culturing the infected host cells.
- Vaccinia virus can be expressed and / or propagated by methods known in the art.
- a host cell used for expressing or propagating the vaccinia virus of the present invention the vaccinia virus for combined use, or the vaccinia virus for combined kit
- the vaccinia virus of the present invention, the vaccinia virus for combined use, or the vaccinia virus for combined kit is expressed. As long as it can proliferate, it is not particularly limited.
- the pharmaceutical composition of the present invention includes a pharmaceutical composition comprising the vaccinia virus of the present invention and a pharmaceutically acceptable excipient.
- the pharmaceutical composition of the present invention includes the pharmaceutical composition for combined use of the present invention.
- the combination pharmaceutical composition of the present invention comprises a pharmaceutically acceptable excipient.
- the pharmaceutical composition of the present invention can be prepared by a commonly used method using an excipient usually used in the art, that is, a pharmaceutical excipient or a pharmaceutical carrier.
- dosage forms of these pharmaceutical compositions include parenteral agents such as injections and infusions, and can be administered by intravenous administration, subcutaneous administration, intratumoral administration, or the like.
- parenteral agents such as injections and infusions
- excipients, carriers, additives or the like corresponding to these dosage forms can be used within a pharmaceutically acceptable range.
- the present invention includes cancer, for example, malignant melanoma, lung adenocarcinoma, which includes the step of administering the vaccinia virus of the present invention to a subject (for example, patient) in need of cancer prevention or treatment.
- cancer for example, malignant melanoma, lung adenocarcinoma
- Lung cancer small cell lung cancer, squamous cell lung cancer, renal cancer, bladder cancer, head and neck cancer, breast cancer, esophageal cancer, glioblastoma, neuroblastoma, myeloma, ovarian cancer, colon cancer, pancreatic cancer, prostate cancer
- a method for preventing or treating cancer selected from the group consisting of hepatocellular carcinoma, mesothelioma, cervical cancer, gastric cancer and the like is included.
- a vaccinia virus comprising a polynucleotide encoding IL-7; and (2) a vaccinia virus comprising a polynucleotide encoding IL-12.
- the two vaccinia viruses may be administered to the subject simultaneously, separately, sequentially or at intervals.
- the present invention also includes cancers such as malignant melanoma, lung adenocarcinoma, lung cancer, small cell lung cancer, lung squamous cell carcinoma, kidney cancer, bladder cancer, head and neck cancer, breast cancer, esophageal cancer, glioblastoma.
- cancers such as malignant melanoma, lung adenocarcinoma, lung cancer, small cell lung cancer, lung squamous cell carcinoma, kidney cancer, bladder cancer, head and neck cancer, breast cancer, esophageal cancer, glioblastoma.
- cancer selected from the group consisting of neuroblastoma, myeloma, ovarian cancer, colon cancer, pancreatic cancer, prostate cancer, hepatocellular carcinoma, mesothelioma, cervical cancer, and stomach cancer
- a vaccinia virus of the invention is included.
- the present invention also includes cancers such as malignant melanoma, lung adenocarcinoma, lung cancer, small cell lung cancer, lung squamous cell carcinoma, kidney cancer, bladder cancer, head and neck cancer, breast cancer, esophageal cancer, glioblastoma.
- cancers such as malignant melanoma, lung adenocarcinoma, lung cancer, small cell lung cancer, lung squamous cell carcinoma, kidney cancer, bladder cancer, head and neck cancer, breast cancer, esophageal cancer, glioblastoma.
- cancers such as malignant melanoma, lung adenocarcinoma, lung cancer, small cell lung cancer, lung squamous cell carcinoma, kidney cancer, bladder cancer, head and neck cancer, breast cancer, esophageal cancer, glioblastoma.
- cancer selected from the group consisting of neuroblastoma, myeloma, ovarian cancer, colon cancer, pancreatic cancer, prostate cancer
- the present invention includes cancers such as malignant melanoma, lung adenocarcinoma, lung cancer, small cell lung cancer, lung squamous cell carcinoma, renal cancer, bladder cancer, head and neck cancer, breast cancer, esophageal cancer, glioblastoma.
- cancers such as malignant melanoma, lung adenocarcinoma, lung cancer, small cell lung cancer, lung squamous cell carcinoma, renal cancer, bladder cancer, head and neck cancer, breast cancer, esophageal cancer, glioblastoma.
- a vaccinia virus comprising a polynucleotide encoding IL-7 and a vaccinia virus comprising a polynucleotide encoding IL-12 to produce a combination kit.
- the pharmaceutical composition or combination kit of the present invention is a cancer such as malignant melanoma, lung adenocarcinoma, lung cancer, small cell lung cancer, lung squamous cell carcinoma, kidney cancer, bladder cancer, head and neck cancer, breast cancer, esophageal cancer,
- cancer selected from the group consisting of glioblastoma, neuroblastoma, myeloma, ovarian cancer, colon cancer, pancreatic cancer, prostate cancer, hepatocellular carcinoma, mesothelioma, cervical cancer, gastric cancer, etc. It can be used in combination with various therapeutic agents showing efficacy.
- the combination may be administered simultaneously or separately in succession or at desired time intervals.
- the pharmaceutical composition of the present invention may be formulated or separately formulated for simultaneous administration.
- the vaccinia virus of the present invention the pharmaceutical composition of the present invention, the combination kit of the present invention, the method of preventing or treating cancer of the present invention, or the cancer to which the use of the present invention is applied, an organ other than the primary lesion.
- organ other than the primary lesion examples include metastatic cancers such as lymph nodes and liver.
- pTN-VGF-P-DsRed Transfer Vector Plasmid DNA A pUC19-VGF vector was prepared based on International Publication No. 2015/076422. More specifically, for the production of the pUC19-VGF vector, the genomic DNA of the LC16mO strain (Accession No. AY678277.1) was used as a template, and the pUC19 vector (product code: 54357) from Invitrogen was used. The prepared pUC19-VGF vector was cleaved with the restriction enzyme AccI, and the ends were made blunt.
- a transfer vector plasmid DNA was constructed by inserting a DNA fragment (SEQ ID NO: 22) containing a p7.5k promoter and a DsRed fragment into this cleavage site.
- the constructed plasmid DNA is referred to as pTN-VGF-P-DsRed.
- (2) Construction of pTN-VGF-SP-IL12 and pTN-VGF-SP-IL7 transfer vector plasmid DNA Two primers (SEQ ID NO: 1 and SEQ ID NO: 1) using DNA of pTagBFP-N vector (FP172, Evrogen) as a template The BFP gene region was amplified by 2).
- the PCR product was cleaved with restriction enzymes SfiI and EcoRI, and the pTK-SP-LG vector (International Publication No. WO 2015/076422.
- LC16mO strain genomic DNA accesion No. AY67877.1
- Invitrogen was used.
- the same restriction of the pUC19 vector (product code: 54357) was used, and the pVNC110-Luc / IRES / EGFP plasmid used was the pVNC110-Luc / IRES / EGFP described in WO 2011/125469.
- a polynucleotide encoding human IL-12 (human IL-12 subunit p40, a polynucleotide comprising an internal ribosome introduction site and human IL-12 subunit ⁇ .
- SEQ ID NO: 7 human IL-12 subunit p40, a polynucleotide comprising an internal ribosome introduction site and human IL-12 subunit ⁇ .
- SEQ ID NO: 7 human IL-12
- human IL-7 human IL-7
- each polynucleotide includes a restriction enzyme site accgggtcccccacc (SEQ ID NO: 16) on the 5 ′ side and a restriction enzyme site gctagcgaattc (SEQ ID NO: 17) on the 3 ′ side
- AgeI Cleaved with NheI is a restriction enzyme site accgggtcccccacc (SEQ ID NO: 16) on the 5 ′ side and a restriction enzyme site gctagcgaat
- Transfer vector plasmid DNA was constructed by cloning each polynucleotide fragment into the same restriction enzyme sites of pTN-VGF-SP-BFP.
- the constructed plasmid DNAs are referred to as pTN-VGF-SP-IL12 and pTN-VGF-SP-IL7, respectively.
- the O1L gene region was inserted into the XbaI site of the pUC19 vector.)
- the restriction enzyme Xb By cloning the ends were digested with I to smoothed site, to prepare a transfer vector plasmid DNA ( Figure 1).
- the prepared plasmid DNA is referred to as pTN-O1L-SP-BFP.
- a polynucleotide comprising the E.
- the B4R gene region was amplified with two primers (SEQ ID NO: 3 and SEQ ID NO: 4) using the DNA of (used) as a template.
- the DsRed gene region was amplified with two primers (SEQ ID NO: 5 and SEQ ID NO: 6).
- the former PCR product was cleaved with restriction enzymes NotI and FspI, and the latter PCR product was cleaved with restriction enzymes FspI and MfeI.
- a recombinant vaccinia virus having the viral genome shown in FIG. 2 was recovered.
- the collection procedure will be specifically described.
- LC16mO VGF-SP-LucGFP / O1L-p7.5-DsRed MOI (Multiplicity of Infection) on CV1 cells (ATCC CCL-70) or RK13 cells (ATCC CCL-37) cultured in 6-well dishes at 80% confluence 0.02 to 0.1 and allowed to adsorb for 1 hour at room temperature.
- PTN-O1L-SP-BFP constructed in Example 1 (3) was mixed with FuGENE® HD Transfection Reagent (Roche), added to cells according to the manual, and taken up at 37 ° C. in the presence of 5% CO 2.
- LC16mO VGF-SP-LucGFP / O1L-SP-BFP a virus clone that was confirmed by direct sequencing that the base sequence of the PCR product was correct
- LC16mO VGF-SP-LucGFP / O1L-SP-BFP a virus clone that was confirmed by direct sequencing that the base sequence of the PCR product was correct
- the recombinant virus was recovered.
- the recovered virus is referred to as LC16mO ⁇ -DsRed VGF-SP-LucGFP / O1L-SP-BFP (FIG. 2).
- Example 2 A recombinant virus lacking the four SCR domains of the B5R protein was recovered. Specifically, BFP expression using LC16mO ⁇ -DsRed VGF-SP-LucGFP / O1L-SP-BFP prepared in Example 2 (1) and pTN-B5R ⁇ 1-4 constructed in Example 1 (4) Recombinant virus was recovered in the same manner as in Example 2 (1) using DsRed expression loss as an index instead of. The recovered virus is referred to as LC16mO ⁇ SCR VGF-SP-LucGFP / O1L-SP-BFP (FIG. 2).
- Example 1 using the prepared LC16mO ⁇ SCR VGF-SP-LucGFP / O1L-SP-BFP and pTN-VGF-P-DsRed constructed in Example 1 (1), using DsRed expression instead of BFP expression as an indicator Recombinant virus was recovered in the same manner as in Example 2 (1).
- the recovered virus is referred to as LC16mO ⁇ SCR VGF-p7.5-DsRed / O1L-SP-BFP (FIG. 2).
- BFP expression disappears instead of BFP expression.
- the recombinant virus was recovered in the same manner as in Example 2 (1).
- the collected virus is referred to as LC16mO ⁇ SCR VGF-p7.5-DsRed / O1L-SP-LacZ (FIG. 2).
- Example 3 The SCR region-deleted recombinant vaccinia virus expressing the therapeutic gene and marker gene having the viral genome shown in FIG. 3 was recovered. Specifically, the LC16mO ⁇ SCR VGF-p7.5-DsRed / O1L-SP-LacZ prepared in Example 2 (2) and the transfer vector plasmid DNA constructed in Example 1 (2) (pTN-VGF-SP- Using IL12 and pTN-VGF-SP-IL7), each recombinant virus was recovered in the same manner as in Example 2 (1), using DsRed expression loss as an index instead of BFP expression.
- Each of the recovered viruses was LC16mO ⁇ SCR VGF-SP-IL12 / O1L-SP-LacZ (hereinafter referred to as “hIL12-loaded vaccinia virus”), LC16mO ⁇ SCR VGF-SP-IL7 / O1L-SP-LacZ (hereinafter “ (referred to as “hIL7-loaded vaccinia virus”) (FIG. 3).
- hIL12-loaded vaccinia virus LC16mO ⁇ SCR VGF-SP-IL7 / O1L-SP-LacZ
- FIG. 3 For purification, A549 cells or RK13 cells were infected with each recombinant virus. After culturing at 37 ° C. in the presence of 5% CO 2 for 2 to 5 days, the infected cells were collected. Cells were freeze-thawed and sonicated. It refine
- a SCR domain-deleted recombinant vaccinia virus expressing a polynucleotide encoding human IL-7 and a polynucleotide encoding human IL-12 having the viral genome shown in FIG. 4 was recovered.
- (4-1) Specifically, the LC16mO ⁇ SCR VGF-p7.5-DsRed / O1L-SP-BFP prepared in Example 2 (2) and the transfer vector plasmid DNA pTN- constructed in Example 1 (2) Using VGF-SP-IL12, each recombinant virus was recovered in the same manner as in Example 2 (1), using DsRed expression loss as an index instead of BFP expression.
- the recovered virus is referred to as LC16mO ⁇ SCR VGF-SP-IL12 / O1L-SP-BFP.
- Example 2 4-2)
- LC16mO ⁇ SCR VGF-SP-IL12 / O1L-SP-BFP prepared in Example 2 (4-1) and transfer vector plasmid DNA pTN-O1L- constructed in Example 1 (3)
- SP-IL7 transfer vector plasmid DNA pTN-O1L- constructed in Example 1 (3)
- the collected virus is referred to as LC16mO ⁇ SCR VGF-SP-IL12 / O1L-SP-IL7 (hereinafter, also referred to as “hIL12 and hIL7-loaded vaccinia virus” in the examples below) (FIG. 4).
- the virus titer of each virus was measured in RK13 cells.
- Example 3 Oncolytic property of recombinant vaccinia virus
- the hIL12 and hIL7-loaded vaccinia viruses prepared in Example 2 were evaluated for lysis ability (cell killing ability) in various human cancer cells.
- the lytic ability in various human cancer cells was similarly evaluated for the two kinds of combination mixtures of the vaccinia virus loaded with hIL12 and the vaccinia virus loaded with hIL7 prepared in Example 2.
- vaccinia virus loaded with hIL12 and vaccinia loaded with hIL7 100 ⁇ L of each cell suspended to 1 ⁇ 10 4 cells / mL was added. After overnight culture, 1) a vaccinia virus loaded with hIL12 and hIL7, and 2) a mixture of vaccinia virus loaded with hIL12 and vaccinia virus loaded with hIL7 in a one-to-one concentration (hereinafter referred to as “vaccinia virus loaded with hIL12 and vaccinia loaded with hIL7”).
- Example 4 Protein production from cancer cells infected with recombinant vaccinia virus
- concentrations of human IL-7 protein and human IL-12 protein produced from the cancer cells were measured. Further, for the mixture of vaccinia virus loaded with hIL12 and vaccinia virus loaded with hIL7, the concentrations of human IL-7 protein and human IL-12 protein produced from cancer cells when infected with cancer cells are also measured. did.
- human IL-12 protein and human IL-7 protein can be produced from cells to which hIL12 and vaccinia virus loaded with hIL12 are added, and from cells to which a mixture of vaccinia virus loaded with hIL12 and vaccinia virus loaded with hIL7 is added. It became clear (Tables 2-1 and 2-2).
- coli LacZ gene (SEQ ID NO: 9), a polynucleotide encoding the luciferase Luc2 gene (Accession No. DQ188840, 100th to 1752th) was used. This polynucleotide fragment was cloned into pTN-O1L-SP-BFP.
- Example 2 (2) Recombinant virus was recovered according to the method of Example 2 (2).
- the method of Example 2 (2) instead of LC16mO ⁇ SCR VGF-p7.5-DsRed / O1L-SP-BFP and pTN-O1L-SP-LacZ, pTN-O1L- produced in Example 5 (2) SP-Luc2 was used.
- the collected virus is referred to as LC16mO ⁇ SCR VGF-p7.5-DsRed / O1L-SP-Luc2 (hereinafter, this virus is also referred to as “control vaccinia virus”).
- Example 2 The recombinant virus was recovered according to the method of Example 2 (3).
- LC16mO ⁇ SCR VGF-p7.5-DsRed / O1L-SP produced in Example 5 (3) instead of LC16mO ⁇ SCR VGF-p7.5-DsRed / O1L-SP-LacZ in the method of Example 2 (3)
- -Luc2 and pTN-VGF-SP-mIL12 prepared in Example 5 (1) were used instead of pTN-VGF-SP-IL12.
- the collected virus is referred to as LC16mO ⁇ SCR VGF-SP-mIL12 / O1L-SP-Luc2 (hereinafter also referred to as “mIL12-equipped vaccinia virus”).
- Recombinant virus was recovered according to the method of Example 2 (4-1).
- pTN- prepared in Example 5 (1) was used.
- VGF-SP-mIL12 was used.
- the recovered virus is referred to as LC16mO ⁇ SCR VGF-SP-mIL12 / O1L-SP-BFP.
- the recombinant virus was recovered according to the method of Example 2 (4-2).
- Example 2 In the method of Example 2 (4-2), instead of LC16mO ⁇ SCR VGF-SP-IL12 / O1L-SP-BFP, LC16mO ⁇ SCR VGF-SP-mIL12 / O1L-SP-BFP prepared above and pTN-O1L -SP-IL7 was used.
- the recovered virus is referred to as LC16mO ⁇ SCR VGF-SP-mIL12 / O1L-SP-IL7 (hereinafter also referred to as “mIL12 and hIL7-loaded vaccinia virus”).
- Example 6 Anti-tumor effect of recombinant vaccinia virus in tumor-bearing humanized mice.
- humanized mice transplanted with human cancer cells mice in which the immune system was replaced with human immune cells by transferring human hematopoietic stem cells to severely immunodeficient mice. Tumor effect was evaluated.
- a NOG mouse NOD / Shi-scidIL-2R ⁇ KO Jic, female, 6 weeks old
- X-ray irradiation apparatus 3 ⁇ 10 4 human umbilical cord blood-derived hematopoietic stem cells (Lonza) were injected from the tail vein and transferred.
- human lung cancer cells NCI-H1373 ATCC CRL-5866 suspended at 3 ⁇ 10 7 cells / mL in PBS were injected by subcutaneous injection at 100 ⁇ L into the right dorsal side of mice.
- the tumor diameter is measured with a caliper, and the groups are divided so that the average value of the tumor volume (minor axis mm ⁇ minor axis mm ⁇ major axis mm ⁇ 0.52) of each group is 37 mm 3 to 47 mm 3. It was.
- 20 ⁇ L of vaccinia virus loaded with hIL12 and hIL7 diluted to a concentration of 1.0 ⁇ 10 8 PFU / mL with PBS was injected into the tumor (referred to as “VIL administration group loaded with hIL12 and hIL7” in the table).
- a group in which 20 ⁇ L of PBS was administered into the tumor was designated as a solvent (PBS) administration group.
- a significant difference (p value ⁇ 0.05 was considered significant) was found, it was determined that there was a tumor regression effect.
- the same injection volume (20 ⁇ L) and diluted solution (PBS) were used as a control vaccinia virus (2 ⁇ 10 6 PFU / Individual), vaccinia virus loaded with hIL12 (2 ⁇ 10 6 PFU / individual), or vaccinia virus loaded with hIL7 (2 ⁇ 10 6 PFU / individual) (in the table, “control VV administration group”, “hIL12 loaded”, respectively) VV administration group ”and“ hIL7 loaded VV administration group ”).
- the average value of the tumor volume change rate on the 14th day after virus administration was less than 100%. Furthermore, when the tumor volume on the 14th day after virus administration and the tumor volume on the virus administration day were tested by Paired t-test, a significant difference was observed, so that it was determined that there was a tumor regression effect (Table 3). Therefore, it became clear that the administration of hIL12 and hIL7-loaded vaccinia virus has a tumor regression effect. On the other hand, the tumor regression effect was not confirmed in the group administered with either the hIL12-loaded vaccinia virus or the hIL7-loaded vaccinia virus (Table 3).
- Example 7 Complete remission effect of genetically engineered vaccinia virus in a syngeneic tumor-bearing mouse model
- LLC1 mouse lung cancer cells LL / 2 (LLC1) prepared to 4 ⁇ 10 6 cells / mL with PBS were placed on the right ventral side of C57BL / 6J mice (male, 5-7 weeks old, Charles River Japan). ) (ATCC CRL-1642) (hereinafter referred to as LLC1) 50 ⁇ L was implanted subcutaneously. Tumor volumes were calculated by the same method as in Example 6, and the groups were divided so that the average value of the tumor volumes in each group was 50 mm 3 to 60 mm 3 .
- Virus, mIL12 loaded vaccinia virus, or hIL7 loaded vaccinia virus each 2 ⁇ 10 7 PFU / dose, 3 doses (in the table, “control VV administration group”, “mIL12 loaded VV administration group”, respectively) , “HIL7 loaded VV administration group”).
- mice that achieved complete remission by administration of vaccinia virus loaded with mIL12 and hIL7 are mice that achieved complete remission by administration of vaccinia virus loaded with mIL12 and hIL7
- Mixture of vaccinia virus loaded with mIL12 and vaccinia virus loaded with hIL7 One-to-one mixture of vaccinia virus loaded with mIL12 and vaccinia virus loaded with hIL7 (hereinafter referred to as “mixture of vaccinia virus loaded with mIL12 and vaccinia virus loaded with hIL7”) The effect of introducing complete remission in vivo was evaluated using syngeneic cancer-bearing mice.
- mouse lung cancer cells LLC1 suspended at 8 ⁇ 10 6 cells / mL were transplanted. Furthermore, instead of 30 ⁇ L of mIL12 and hIL7 loaded vaccinia virus (2 ⁇ 10 7 PFU) diluted to a concentration of 6.7 ⁇ 10 8 PFU / mL, a mixture of vaccinia virus loaded with mIL12 and vaccinia virus loaded with hIL7 (in PBS, 30 ⁇ L of each virus diluted to 6.7 ⁇ 10 8 PFU / mL) (2 ⁇ 10 7 PFU each / dose, 3 times administration) was used (in the table, “mixture of mV12 loaded VH and hIL7 loaded VV” "Administration group").
- Example 8 Acquired immune effect of recombinant vaccinia virus in a syngeneic tumor-bearing mouse model (tumor rejection effect by acquired immunity)
- About vaccinia virus carrying mIL12 and hIL7 Mice that had undergone complete remission as a result of treatment with vaccinia virus loaded with mIL12 and hIL7 were subjected to re-transplantation of the same cancer cells to evaluate the acquired immune effect of the virus.
- LLC1 tumor-bearing mice were prepared according to Example 7, and mIL12 and hIL7-loaded vaccinia virus were intratumorally administered thereto (however, intratumoral injection of the virus was performed 2 days and 4 days after the first administration). In addition, the test was carried out 1 day and 3 days later (total 5 times), which is referred to as “mV12 and hIL7 loaded VV administration group” in the table).
- mice completely remissioned by administration of mIL12 and hIL7 loaded vaccinia virus a mixture of mIL12 loaded vaccinia virus and hIL7 loaded vaccinia virus was administered according to Example 7 (2) (1 day, 3 days after grouping, After 5 days, mice were used in complete remission for 3 times (in the table, referred to as “administration group of mV12 loaded VV and hIL7 loaded VV”). Retransplantation of cancer cells was performed 74 days after the last dose of virus (complete remission determined 24 days after the last dose).
- the vaccinia virus, pharmaceutical composition and combination kit of the present invention are useful for the prevention or treatment of various cancers.
- the description of “Artificial Sequence” is described in the numerical heading ⁇ 223> of the Sequence Listing.
- the nucleotide sequences shown in SEQ ID NOs: 1-6 and 10-15 are primers.
- the nucleotide sequences represented by SEQ ID NOs: 7, 8, and 9 are a polynucleotide containing a human IL-12 gene, a polynucleotide containing a human IL-7 gene, and a polynucleotide containing an E. coli LacZ gene, respectively.
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Abstract
Description
1)IL-7をコードするポリヌクレオチド及びIL-12をコードするポリヌクレオチドの2つのポリヌクレオチドを含むワクシニアウイルスは、担癌ヒト化マウスモデルにおいて腫瘍退縮作用を示し(実施例6)、同系担癌マウスモデルにおいて完全寛解を示し(実施例7)、さらに、獲得免疫を誘導して抗腫瘍効果を持続させ得た(実施例8)。また、2)IL-7をコードするポリヌクレオチドを含むワクシニアウイルスとIL-12をコードするポリヌクレオチドを含むワクシニアウイルスとの2つのワクシニアウイルスの混合物は、同系担癌マウスモデルにおいて完全寛解を示し(実施例7)、さらに、獲得免疫を誘導して抗腫瘍効果を持続させ得た(実施例8)。
[1]以下の(1)及び(2)を含む、ワクシニアウイルス:
(1)インターロイキン-7(IL-7)をコードするポリヌクレオチド;及び
(2)インターロイキン-12(IL-12)をコードするポリヌクレオチド。
[2]以下の(1)又は(2)から選択される医薬組成物:
(1)IL-7をコードするポリヌクレオチドを含むワクシニアウイルスを含む医薬組成物と併用するための、IL-12をコードするポリヌクレオチドを含むワクシニアウイルスを含む医薬組成物;又は
(2)IL-12をコードするポリヌクレオチドを含むワクシニアウイルスを含む医薬組成物と併用するための、IL-7をコードするポリヌクレオチドを含むワクシニアウイルスを含む医薬組成物。
[3]以下の(1)及び(2)のワクシニアウイルスを含む、組合せキット:
(1)IL-7をコードするポリヌクレオチドを含む、ワクシニアウイルス;及び
(2)IL-12をコードするポリヌクレオチドを含む、ワクシニアウイルス。
[4]ワクシニアウイルス増殖因子(VGF)の機能を欠損している、[1]に記載のワクシニアウイルス。
[5]O1Lの機能を欠損している、[1]に記載のワクシニアウイルス。
[6]VGF及びO1Lの機能を欠損している、[1]に記載のワクシニアウイルス。
[7]B5R細胞外領域のSCR(short consensus repeat)ドメインを欠失している、[1]に記載のワクシニアウイルス。
[8]VGF及びO1Lの機能を欠損し、かつ、B5R細胞外領域のSCRドメインを欠失している、[1]に記載のワクシニアウイルス。
[9]LC16mO株である、[1]及び[4]~[8]のいずれかに記載のワクシニアウイルス。
[10][1]及び[4]~[9]のいずれかに記載のワクシニアウイルス及び薬学的に許容される賦形剤を含む、医薬組成物。
[11]ワクシニアウイルスがVGFの機能を欠損している、[2]に記載の医薬組成物又は[3]に記載のキット。
[12]ワクシニアウイルスがO1Lの機能を欠損している、[2]に記載の医薬組成物又は[3]に記載のキット。
[13]ワクシニアウイルスがVGF及びO1Lの機能を欠損している、[2]に記載の医薬組成物又は[3]に記載のキット。
[14]ワクシニアウイルスがB5R細胞外領域のSCRドメインを欠失している、[2]に記載の医薬組成物又は[3]に記載のキット。
[15]ワクシニアウイルスがVGF及びO1Lの機能を欠損し、かつ、B5R細胞外領域のSCRドメインを欠失している、[2]に記載の医薬組成物又は[3]に記載のキット。
[16]ワクシニアウイルスがLC16mO株である、[2]及び[11]~[15]のいずれかに記載の医薬組成物又は[3]及び[11]~[15]のいずれかに記載のキット。
[17]薬学的に許容される賦形剤を含む、[2]及び[11]~[16]のいずれかに記載の医薬組成物又は[3]及び[11]~[16]のいずれかに記載のキット。
[18]がんの予防又は治療用である、[10]~[17]のいずれかに記載の医薬組成物又はキット。
[19]がんが、悪性黒色腫、肺腺癌、肺癌、小細胞肺癌、肺扁平上皮癌、腎癌、膀胱癌、頭頸部癌、乳癌、食道癌、グリア芽細胞腫、神経芽細胞腫、骨髄腫、卵巣癌、大腸癌、膵癌、前立腺癌、肝細胞癌、中皮腫、子宮頸癌、又は、胃癌である、[18]に記載の医薬組成物又はキット。
[20]がんの予防又は治療を必要とする対象に[1]及び[4]~[9]のいずれかに記載のワクシニアウイルスを投与する工程を包含する、がんを予防又は治療する方法。
[21]がんが、悪性黒色腫、肺腺癌、肺癌、小細胞肺癌、肺扁平上皮癌、腎癌、膀胱癌、頭頸部癌、乳癌、食道癌、グリア芽細胞腫、神経芽細胞腫、骨髄腫、卵巣癌、大腸癌、膵癌、前立腺癌、肝細胞癌、中皮腫、子宮頸癌、又は、胃癌である、[20]記載の方法。
[22]がんの予防又は治療に使用するための、[1]及び[4]~[9]のいずれかに記載のワクシニアウイルス。
[23]がんが、悪性黒色腫、肺腺癌、肺癌、小細胞肺癌、肺扁平上皮癌、腎癌、膀胱癌、頭頸部癌、乳癌、食道癌、グリア芽細胞腫、神経芽細胞腫、骨髄腫、卵巣癌、大腸癌、膵癌、前立腺癌、肝細胞癌、中皮腫、子宮頸癌、又は、胃癌である、[22]記載のワクシニアウイルス。
[24]がんの予防又は治療用医薬組成物を製造するための、[1]及び[4]~[9]のいずれかに記載のワクシニアウイルスの使用。
[25]がんが、悪性黒色腫、肺腺癌、肺癌、小細胞肺癌、肺扁平上皮癌、腎癌、膀胱癌、頭頸部癌、乳癌、食道癌、グリア芽細胞腫、神経芽細胞腫、骨髄腫、卵巣癌、大腸癌、膵癌、前立腺癌、肝細胞癌、中皮腫、子宮頸癌、又は、胃癌である、[24]記載の使用。
[26]がんの予防又は治療を必要とする対象に
(1)IL-7をコードするポリヌクレオチドを含むワクシニアウイルス;及び
(2)IL-12をコードするポリヌクレオチドを含むワクシニアウイルス
を投与する工程を包含する、がんを予防又は治療する方法。
[27]がんが、悪性黒色腫、肺腺癌、肺癌、小細胞肺癌、肺扁平上皮癌、腎癌、膀胱癌、頭頸部癌、乳癌、食道癌、グリア芽細胞腫、神経芽細胞腫、骨髄腫、卵巣癌、大腸癌、膵癌、前立腺癌、肝細胞癌、中皮腫、子宮頸癌、又は、胃癌である、[26]記載の方法。
[28]以下の(1)又は(2)から選択される、ワクシニアウイルス:
(1)IL-12をコードするポリヌクレオチドを含むワクシニアウイルスを含む医薬組成物と併用してがんの予防又は治療に使用するための、IL-7をコードするポリヌクレオチドを含むワクシニアウイルス;又は
(2)IL-7をコードするポリヌクレオチドを含むワクシニアウイルスを含む医薬組成物と併用してがんの予防又は治療に使用するための、IL-12をコードするポリヌクレオチドを含むワクシニアウイルス。
[29]がんが、悪性黒色腫、肺腺癌、肺癌、小細胞肺癌、肺扁平上皮癌、腎癌、膀胱癌、頭頸部癌、乳癌、食道癌、グリア芽細胞腫、神経芽細胞腫、骨髄腫、卵巣癌、大腸癌、膵癌、前立腺癌、肝細胞癌、中皮腫、子宮頸癌、又は、胃癌である、[28]記載のワクシニアウイルス。
[30]以下の(1)又は(2)から選択される、ワクシニアウイルスの使用:
(1)IL-12をコードするポリヌクレオチドを含むワクシニアウイルスを含む医薬組成物と併用するがんの予防又は治療用医薬組成物を製造するための、IL-7をコードするポリヌクレオチドを含むワクシニアウイルスの使用;又は
(2)IL-7をコードするポリヌクレオチドを含むワクシニアウイルスを含む医薬組成物と併用するがんの予防又は治療用医薬組成物を製造するための、IL-12をコードするポリヌクレオチドを含むワクシニアウイルスの使用。
[31]がんが、悪性黒色腫、肺腺癌、肺癌、小細胞肺癌、肺扁平上皮癌、腎癌、膀胱癌、頭頸部癌、乳癌、食道癌、グリア芽細胞腫、神経芽細胞腫、骨髄腫、卵巣癌、大腸癌、膵癌、前立腺癌、肝細胞癌、中皮腫、子宮頸癌、又は、胃癌である、[30]記載の使用。
[32]がんの予防又は治療用の組合せキットを製造するための、(1)IL-7をコードするポリヌクレオチドを含むワクシニアウイルス及び(2)IL-12をコードするポリヌクレオチドを含むワクシニアウイルスの使用。
[33]がんが、悪性黒色腫、肺腺癌、肺癌、小細胞肺癌、肺扁平上皮癌、腎癌、膀胱癌、頭頸部癌、乳癌、食道癌、グリア芽細胞腫、神経芽細胞腫、骨髄腫、卵巣癌、大腸癌、膵癌、前立腺癌、肝細胞癌、中皮腫、子宮頸癌、又は、胃癌である、[32]記載の使用。
本発明は、以下の(1)及び(2)を含む、ワクシニアウイルスを提供する:
(1)IL-7をコードするポリヌクレオチド;及び
(2)IL-12をコードするポリヌクレオチド。
(本明細書中、当該ワクシニアウイルスを「本発明のワクシニアウイルス」ともいう。)
(1)IL-7をコードするポリヌクレオチドを含むワクシニアウイルスを含む医薬組成物と併用するための、IL-12をコードするポリヌクレオチドを含むワクシニアウイルスを含む医薬組成物;又は
(2)IL-12をコードするポリヌクレオチドを含むワクシニアウイルスを含む医薬組成物と併用するための、IL-7をコードするポリヌクレオチドを含むワクシニアウイルスを含む医薬組成物。
(本明細書中、当該医薬組成物を「本発明の併用用医薬組成物」ともいい、上記(1)又は(2)に記載の本発明の併用用医薬組成物にそれぞれ含まれるIL-7をコードするポリヌクレオチドを含むワクシニアウイルス又はIL-12をコードするポリヌクレオチドを含むワクシニアウイルスを、「併用用ワクシニアウイルス」ともいう。)
(1)IL-7をコードするポリヌクレオチドを含む、ワクシニアウイルス;及び
(2)IL-12をコードするポリヌクレオチドを含む、ワクシニアウイルス。
(本明細書中、当該組合せキットを「本発明の組合せキット」ともいい、本発明の組合せキットに含まれる各ワクシニアウイルスを、「組合せキット用ワクシニアウイルス」ともいう。)
(1)Accession No.NP_000871.1に示されるアミノ酸配列を含み、かつ、ヒトIL-7の機能を有するポリペプチド、
(2)Accession No.NP_000871.1に示されるアミノ酸配列において、1~10個のアミノ酸が欠失、置換、挿入、及び/又は付加されたアミノ酸配列からなり、かつ、ヒトIL-7の機能を有するポリペプチド、並びに
(3)Accession No.NP_000871.1に示されるアミノ酸配列との同一性が90%以上であるアミノ酸配列を含み、かつ、ヒトIL-7の機能を有するポリペプチド。
ここで、ヒトIL-7の機能とは、各種ヒト免疫細胞に対しての生存、増殖及び分化作用をいう。
(1)(1-a)Accession No.NP_002178.2に示されるアミノ酸配列を含むポリペプチド、(1-b)Accession No.NP_002178.2に示されるアミノ酸配列において1~10個のアミノ酸が欠失、置換、挿入、及び/若しくは付加されたアミノ酸配列からなるポリペプチド、又は、(1-c)Accession No.NP_002178.2に示されるアミノ酸配列との同一性が90%以上であるアミノ酸配列を含むポリペプチド、並びに、
(2-a)Accession No.NP_000873.2に示されるアミノ酸配列を含むポリペプチド、(2-b)Accession No.NP_000873.2に示されるアミノ酸配列において1~10個のアミノ酸が欠失、置換、挿入、及び/若しくは付加されたアミノ酸配列からなるポリペプチド、又は、(2-c)Accession No.NP_000873.2に示されるアミノ酸配列との同一性が90%以上であるアミノ酸配列を含むポリペプチドを含み、かつ、
ヒトIL-12の機能を有するポリペプチド、
(2)(1-a)Accession No.NP_002178.2に示されるアミノ酸配列からなるポリペプチド、並びに、
(2-a)Accession No.NP_000873.2に示されるアミノ酸配列を含むポリペプチド、(2-b)Accession No.NP_000873.2に示されるアミノ酸配列において1~10個のアミノ酸が欠失、置換、挿入、及び/若しくは付加されたアミノ酸配列からなるポリペプチド、又は(2-c)Accession No.NP_000873.2に示されるアミノ酸配列との同一性が90%以上であるアミノ酸配列を含むポリペプチドを含み、かつ、
ヒトIL-12の機能を有するポリペプチド、並びに、
(3)(1-a)Accession No.NP_002178.2に示されるアミノ酸配列を含むポリペプチド、(1-b)Accession No.NP_002178.2に示されるアミノ酸配列において1~10個のアミノ酸が欠失、置換、挿入、及び/若しくは付加されたアミノ酸配列からなるポリペプチド、又は、(1-c)Accession No.NP_002178.2に示されるアミノ酸配列との同一性が90%以上であるアミノ酸配列を含むポリペプチド、並びに、
(2-a)Accession No.NP_000873.2に示されるアミノ酸配列からなるポリペプチドを含み、かつ、
ヒトIL-12の機能を有するポリペプチド。
ここで、ヒトIL-12の機能とは、T細胞やNK細胞の活性化作用、及び/又は分化誘導作用をいう。IL-12サブユニットp40及びIL-12サブユニットαは直接結合することでIL-12を形成することができる。また、IL-12サブユニットp40及びIL-12サブユニットαをリンカーを介して結合させることができる。
Gap penalty = 10
Extend penalty = 0.5
Matrix = EBLOSUM62
本発明の医薬組成物には、本発明のワクシニアウイルス及び薬学的に許容される賦形剤を含む医薬組成物が含まれる。本発明の医薬組成物には、本発明の併用用医薬組成物も含まれる。1つの実施形態において、本発明の併用用医薬組成物は、薬学的に許容される賦形剤を含む。
本発明の医薬組成物は、がん、例えば、悪性黒色腫、肺腺癌、肺癌、小細胞肺癌、肺扁平上皮癌、腎癌、膀胱癌、頭頸部癌、乳癌、食道癌、グリア芽細胞腫、神経芽細胞腫、骨髄腫、卵巣癌、大腸癌、膵癌、前立腺癌、肝細胞癌、中皮腫、子宮頸癌、及び、胃癌等からなる群から選択されるがんの予防又は治療剤として用いることができる。
(1)IL-7をコードするポリヌクレオチドを含むワクシニアウイルス;及び
(2)IL-12をコードするポリヌクレオチドを含むワクシニアウイルス。
当該2つのワクシニアウイルスは、同時に、別々に、連続して、あるいは間隔をあけて、対象に投与してよい。
(1)IL-12をコードするポリヌクレオチドを含むワクシニアウイルスを含む医薬組成物と併用してがんの予防又は治療に使用するための、IL-7をコードするポリヌクレオチドを含むワクシニアウイルス;又は
(2)IL-7をコードするポリヌクレオチドを含むワクシニアウイルスを含む医薬組成物と併用してがんの予防又は治療に使用するための、IL-12をコードするポリヌクレオチドを含むワクシニアウイルス。
(1)IL-12をコードするポリヌクレオチドを含むワクシニアウイルスを含む医薬組成物と併用するがんの予防又は治療用医薬組成物を製造するための、IL-7をコードするポリヌクレオチドを含むワクシニアウイルスの使用;又は
(2)IL-7をコードするポリヌクレオチドを含むワクシニアウイルスを含む医薬組成物と併用するがんの予防又は治療用医薬組成物を製造するための、IL-12をコードするポリヌクレオチドを含むワクシニアウイルスの使用。
相同組換え法により組換えワクシニアウイルスを作製するために使用するトランスファーベクタープラスミドDNAを以下の通りに作製した。
国際公開第2015/076422号に基づいてpUC19-VGFベクターを作製した。より具体的には、pUC19-VGFベクター作製のために、LC16mO株のゲノムDNA(Accession No.AY678277.1)を鋳型に使用し、Invitrogen社のpUC19ベクター(製品コード:54357)を使用した。作製したpUC19-VGFベクターを制限酵素AccIで切断した後、末端を平滑化した。この切断部位に、p7.5kプロモーターとDsRed断片とを含むDNA断片(配列番号22)を挿入することで、トランスファーベクタープラスミドDNAを構築した。構築したプラスミドDNAをpTN-VGF-P-DsRedと称する。
(2)pTN-VGF-SP-IL12及びpTN-VGF-SP-IL7トランスファーベクタープラスミドDNAの構築
pTagBFP-N vector(FP172、Evrogen社)のDNAを鋳型として、2つのプライマー(配列番号1及び配列番号2)によってBFP遺伝子領域を増幅した。そのPCR産物を制限酵素SfiI及びEcoRIで切断し、pTK-SP-LGベクター(国際公開第2015/076422号。但し、LC16mO株のゲノムDNA(Accession No.AY678277.1)を鋳型に使用し、Invitrogen社のpUC19ベクター(製品コード:54357)を使用した。また、pVNC110-Luc/IRES/EGFPプラスミドは国際公開第2011/125469号に記載のpVNC110-Luc/IRES/EGFPを使用した。)の同じ制限酵素部位にクローニングし、合成ワクシニアウイルスプロモーター(Journal of Virological Methods、1997、Vol.66、p.135-138)下にBFPを連結したpTK-SP-BFPを構築した。次に、pTK-SP-BFPを制限酵素SphI及びEcoRIで切断し、末端を平滑化した。こうして得られたDNA断片を、pUC19-VGFベクターを制限酵素AccIで切断し末端を平滑化した部位へクローニングすることで、pTN-VGF-SP-BFPを構築した(図1)。次に、ヒトIL-12をコードするポリヌクレオチド(ヒトIL-12サブユニットp40、内部リボソーム導入部位及びヒトIL-12サブユニットαを含むポリヌクレオチド。配列番号7)、及びヒトIL-7をコードするポリヌクレオチド(配列番号8)(各ポリヌクレオチドは、5’側に制限酵素サイトaccggtcgccacc(配列番号16)及び3’側に制限酵素サイトgctagcgaattc(配列番号17)をそれぞれ含む)を制限酵素AgeIとNheIで切断した。各ポリヌクレオチド断片をpTN-VGF-SP-BFPの同じ制限酵素部位にクローニングすることで、トランスファーベクタープラスミドDNAを構築した。構築したプラスミドDNAをそれぞれpTN-VGF-SP-IL12及びpTN-VGF-SP-IL7と称する。
(3)pTN-O1L-SP-BFP、pTN-O1L-SP-LacZ、pTN-O1L-SP-IL12、及びpTN-O1L-SP-IL7トランスファーベクタープラスミドDNAの構築
上記(2)と同様の方法で、pTK-SP-BFPを制限酵素SphI及びEcoRIで切断し、末端を平滑化して得られたDNA断片を、pUC19-O1Lベクター(国際公開第2015/076422号。但し、pUC19-VGFベクター作製と同様に、LC16mO株のゲノムDNA(Accession No.AY678277.1)を鋳型に使用し、Invitrogen社のpUC19ベクター(製品コード:54357)を使用した。O1L遺伝子領域はpUC19ベクターのXbaIサイトに挿入した。)を制限酵素XbaIで切断し末端を平滑化した部位へクローニングすることで、トランスファーベクタープラスミドDNAを作製した(図1)。作製したプラスミドDNAをpTN-O1L-SP-BFPと称する。次に、ヒトに対してコドンを最適化した大腸菌LacZ遺伝子を含むポリヌクレオチド(配列番号9)、ヒトIL-12をコードするポリヌクレオチド(配列番号7)、及びヒトIL-7をコードするポリヌクレオチド(配列番号8)を制限酵素AgeIとNheIで切断した。LacZ、IL-12、又はIL-7をコードする各ポリヌクレオチド断片をpTN-O1L-SP-BFPベクターの同じ制限酵素部位(AgeI部位及びNheI部位)にクローニングすることで、トランスファーベクタープラスミドDNAを構築した。構築したプラスミドDNAをそれぞれpTN-O1L-SP-LacZ、pTN-O1L-SP-IL12、及びpTN-O1L-SP-IL7と称する。
pB5R(国際公開第2011/125469号。但し、LC16mO株のゲノムDNA(Accession No.AY678277.1)を鋳型に使用した)のDNAを鋳型として、2つのプライマー(配列番号3及び配列番号4)によってB4R遺伝子領域を増幅した。また、pDsRed-Express-N1(Clontech社)のDNAを鋳型として、2つのプライマー(配列番号5及び配列番号6)によってDsRed遺伝子領域を増幅した。前者のPCR産物を制限酵素NotI及びFspIで、後者のPCR産物を制限酵素FspI及びMfeIで切断した。この2種類のDNA断片を、制限酵素NotI及びMfeIで切断したpB5Rにクローニングすることで、トランスファーベクタープラスミドDNAを作製した。作製したプラスミドDNAをpTN-DsRed(B5R-)と称する。一方、pB5Rを制限酵素NotI及びNspIで、又は制限酵素NspI及びSacIで切断した。この2種類のDNA断片を、制限酵素NotIとSacIで切断したpB5Rにクローニングすることで、トランスファーベクタープラスミドDNAを構築した。作製したプラスミドDNAをpTN-B5RΔ1-4と称する。pTN-B5RΔ1-4には4つのSCRドメインが欠失したB5R蛋白質がコードされる。当該アミノ酸配列は配列番号18に示した配列となる。
(4-1)具体的には、実施例2(2)で作製したLC16mO ΔSCR VGF-p7.5-DsRed/O1L-SP-BFP及び実施例1(2)で構築したトランスファーベクタープラスミドDNA pTN-VGF-SP-IL12を用いて、BFP発現の代わりにDsRed発現消失を指標に、実施例2(1)と同様の方法にて各組換えウイルスを回収した。回収したウイルスをLC16mO ΔSCR VGF-SP-IL12/O1L-SP-BFPと称する。
実施例2で作製されたhIL12及びhIL7搭載ワクシニアウイルスについて、各種ヒトがん細胞における溶解能力(細胞死滅能力)を評価した。また、実施例2で作製されたhIL12搭載ワクシニアウイルスとhIL7搭載ワクシニアウイルスとの2種類の組合せ混合物についても、同様に各種ヒトがん細胞における溶解能力を評価した。
hIL12及びhIL7搭載ワクシニアウイルスをがん細胞に感染させた際に、がん細胞から産生されるヒトIL-7蛋白質及びヒトIL-12蛋白質の濃度を測定した。さらに、hIL12搭載ワクシニアウイルスとhIL7搭載ワクシニアウイルスとの混合物についても同様に、がん細胞に感染させた際にがん細胞から産生されるヒトIL-7蛋白質及びヒトIL-12蛋白質の濃度を測定した。
(1)実施例1(2)に記載の方法に従い、トランスファーベクタープラスミドDNA pTN-VGF-SP-mIL12を構築した。実施例1(2)に記載の方法において、ヒトIL-12をコードするポリヌクレオチド(配列番号7)に代えて、マウスIL-12をコードするポリヌクレオチド(マウスIL-12サブユニットp40、内部リボソーム導入部位及びマウスIL-12サブユニットαを含むポリヌクレオチド。配列番号23)を用い、このポリヌクレオチド断片をpTN-VGF-SP-BFPにクローニングした。
(2)実施例1(3)に記載の方法に従い、トランスファーベクタープラスミドDNA pTN-O1L-SP-Luc2を構築した。実施例1(3)に記載の方法において、大腸菌LacZ遺伝子を含むポリヌクレオチド(配列番号9)に代えて、ルシフェラーゼLuc2遺伝子をコードするポリヌクレオチド(Accession No.DQ188840の100番目から1752番目)を用い、このポリヌクレオチド断片をpTN-O1L-SP-BFPにクローニングした。
さらに、実施例2(4-2)の方法に従い、組換えウイルスを回収した。実施例2(4-2)の方法において、LC16mO ΔSCR VGF-SP-IL12/O1L-SP-BFPに代えて上記で作製したLC16mO ΔSCR VGF-SP-mIL12/O1L-SP-BFPと、pTN-O1L-SP-IL7とを用いた。回収したウイルスをLC16mO ΔSCR VGF-SP-mIL12/O1L-SP-IL7と称する(以下、「mIL12及びhIL7搭載ワクシニアウイルス」とも称する。)。
hIL12及びhIL7搭載ワクシニアウイルスについて、ヒト癌細胞を移植したヒト化マウス(重度免疫不全マウスにヒト造血幹細胞を移入することで免疫系をヒト免疫細胞に置き換えたマウス)を用いて、生体内における抗腫瘍効果を評価した。
ウイルス投与後14日の腫瘍体積変化率(%)=100(%)×ウイルス投与後14日の腫瘍体積(mm3)/ウイルス投与日の腫瘍体積(mm3)。
ウイルス投与後14日の腫瘍体積変化率(%)の各群の平均値が100を下回り、かつ、各群のウイルス投与後14日の腫瘍体積とウイルス投与日の腫瘍体積をPaired t-testにて検定し有意差(p値<0.05を有意差ありとする)が認められる場合に、腫瘍退縮効果ありと判定した。
なお本実施例では、hIL12及びhIL7搭載ワクシニアウイルス(2×106PFU/個体)に対する比較ウイルスとして、同じ注射量(20μL)及び希釈溶液(PBS)で、対照ワクシニアウイルス(2×106PFU/個体)、hIL12搭載ワクシニアウイルス(2×106PFU/個体)、又はhIL7搭載ワクシニアウイルス(2×106PFU/個体)を用いた(表中、それぞれ、「対照VV投与群」、「hIL12搭載VV投与群」、「hIL7搭載VV投与群」とする。)。
mIL12及びhIL7搭載ワクシニアウイルスについて、同系のマウス癌細胞株を皮下移植したマウス(同系担癌マウス)を用いて生体における完全寛解導入効果を評価した。なお、ヒトIL-12はマウスの免疫細胞に対して反応しないことが知られているため、ヒトIL-12をコードするポリヌクレオチドに代えてマウスIL-12をコードするポリヌクレオチドを搭載した遺伝子組換えワクシニアウイルス(実施例5で作製)を用いた。
ノギスで腫瘍径を週に2回測定して腫瘍体積を算出した。最終的にウイルス初回投与の27日後の観察で、触診によって腫瘍が認められない場合を完全寛解と定義し、完全寛解を示した個体の数を計測した。試験期間中に群の平均腫瘍体積が1700mm3を超えた群に関しては、動物倫理上の観点から安楽死させた。なお本実施例では、mIL12及びhIL7搭載ワクシニアウイルス(2×107PFU/回、3回投与)に対する比較ウイルスとして、同じ注射量(30μL/1回投与)及び希釈溶液(PBS)で、対照ワクシニアウイルス、mIL12搭載ワクシニアウイルス、又はhIL7搭載ワクシニアウイルス(それぞれ、2×107PFU/回、3回投与)を用いた(表中、それぞれ、「対照VV投与群」、「mIL12搭載VV投与群」、「hIL7搭載VV投与群」とする。)。
mIL12搭載ワクシニアウイルスとhIL7搭載ワクシニアウイルスとの1対1混合物(以下、「mIL12搭載ワクシニアウイルスとhIL7搭載ワクシニアウイルスとの混合物」)について、同系担癌マウスを用いて生体における完全寛解導入効果を評価した。
(1)mIL12及びhIL7搭載ワクシニアウイルスについて:
mIL12及びhIL7搭載ワクシニアウイルスを処置した結果完全寛解したマウスに対して同癌細胞の再移植実験を行い、ウイルスによる獲得免疫効果を評価した。
mIL12搭載ワクシニアウイルスとhIL7搭載ワクシニアウイルスとの混合物を処置した結果完全寛解したマウスに対して同癌細胞の再移植実験を行い、獲得免疫効果を評価した。
したがって本実施例により、mIL12搭載ワクシニアウイルスとhIL7搭載ワクシニアウイルスとの混合物の投与による獲得免疫効果が確認された。
配列番号1~6及び10~15で示されるヌクレオチド配列はプライマーである。
配列番号7、8及び9で示される塩基配列は、それぞれ、ヒトIL-12遺伝子を含むポリヌクレオチド、ヒトIL-7遺伝子を含むポリヌクレオチド及び大腸菌LacZ遺伝子を含むポリヌクレオチドである。配列番号7において、14番目~1000番目の塩基配列がIL-12のp40サブユニットをコードする領域に対応し、1606番目~2367番目の塩基配列がIL-12のサブユニットαをコードする領域に対応する。
配列番号16及び17で示されるヌクレオチド配列は配列番号7~9の各遺伝子コード領域に連結された制限酵素サイトである。
配列番号18で示されるアミノ酸配列は、4つのSCRドメインが欠失したB5R蛋白質である。
配列番号19、20で示される塩基配列は、LC16mO VGF-SP-LucGFP/O1L-p7.5-DsRedの両末端ループ配列の配列である。
配列番号21で示される塩基配列はLC16mO VGF-SP-LucGFP/O1L-p7.5-DsRedの両末端ループ配列を除く配列である。
配列番号22で示される塩基配列はp7.5kプロモーターとDsRed断片とを含むDNA断片である。
配列番号23で示される塩基配列はマウスIL-12遺伝子を含むポリヌクレオチドである。
Claims (33)
- 以下の(1)及び(2)を含む、ワクシニアウイルス:
(1)インターロイキン-7(IL-7)をコードするポリヌクレオチド;及び
(2)インターロイキン-12(IL-12)をコードするポリヌクレオチド。 - 以下の(1)又は(2)から選択される医薬組成物:
(1)IL-7をコードするポリヌクレオチドを含むワクシニアウイルスを含む医薬組成物と併用するための、IL-12をコードするポリヌクレオチドを含むワクシニアウイルスを含む医薬組成物;又は
(2)IL-12をコードするポリヌクレオチドを含むワクシニアウイルスを含む医薬組成物と併用するための、IL-7をコードするポリヌクレオチドを含むワクシニアウイルスを含む医薬組成物。 - 以下の(1)及び(2)のワクシニアウイルスを含む、組合せキット:
(1)IL-7をコードするポリヌクレオチドを含む、ワクシニアウイルス;及び
(2)IL-12をコードするポリヌクレオチドを含む、ワクシニアウイルス。 - ワクシニアウイルス増殖因子(VGF)の機能を欠損している、請求項1に記載のワクシニアウイルス。
- O1Lの機能を欠損している、請求項1に記載のワクシニアウイルス。
- VGF及びO1Lの機能を欠損している、請求項1に記載のワクシニアウイルス。
- B5R細胞外領域のSCR(short consensus repeat)ドメインを欠失している、請求項1に記載のワクシニアウイルス。
- VGF及びO1Lの機能を欠損し、かつ、B5R細胞外領域のSCRドメインを欠失している、請求項1に記載のワクシニアウイルス。
- LC16mO株である、請求項1及び4~8のいずれかに記載のワクシニアウイルス。
- 請求項1及び4~9のいずれかに記載のワクシニアウイルス及び薬学的に許容される賦形剤を含む、医薬組成物。
- ワクシニアウイルスがVGFの機能を欠損している、請求項2に記載の医薬組成物又は請求項3に記載のキット。
- ワクシニアウイルスがO1Lの機能を欠損している、請求項2に記載の医薬組成物又は請求項3に記載のキット。
- ワクシニアウイルスがVGF及びO1Lの機能を欠損している、請求項2に記載の医薬組成物又は請求項3に記載のキット。
- ワクシニアウイルスがB5R細胞外領域のSCRドメインを欠失している、請求項2に記載の医薬組成物又は請求項3に記載のキット。
- ワクシニアウイルスがVGF及びO1Lの機能を欠損し、かつ、B5R細胞外領域のSCRドメインを欠失している、請求項2に記載の医薬組成物又は請求項3に記載のキット。
- ワクシニアウイルスがLC16mO株である、請求項2及び11~15のいずれかに記載の医薬組成物又は請求項3及び11~15のいずれかに記載のキット。
- 薬学的に許容される賦形剤を含む、請求項2及び11~16のいずれかに記載の医薬組成物又は請求項3及び11~16のいずれかに記載のキット。
- がんの予防又は治療用である、請求項10~17のいずれかに記載の医薬組成物又はキット。
- がんが、悪性黒色腫、肺腺癌、肺癌、小細胞肺癌、肺扁平上皮癌、腎癌、膀胱癌、頭頸部癌、乳癌、食道癌、グリア芽細胞腫、神経芽細胞腫、骨髄腫、卵巣癌、大腸癌、膵癌、前立腺癌、肝細胞癌、中皮腫、子宮頸癌、又は、胃癌である、請求項18に記載の医薬組成物又はキット。
- がんの予防又は治療を必要とする対象に請求項1及び4~9のいずれかに記載のワクシニアウイルスを投与する工程を包含する、がんを予防又は治療する方法。
- がんが、悪性黒色腫、肺腺癌、肺癌、小細胞肺癌、肺扁平上皮癌、腎癌、膀胱癌、頭頸部癌、乳癌、食道癌、グリア芽細胞腫、神経芽細胞腫、骨髄腫、卵巣癌、大腸癌、膵癌、前立腺癌、肝細胞癌、中皮腫、子宮頸癌、又は、胃癌である、請求項20記載の方法。
- がんの予防又は治療に使用するための、請求項1及び4~9のいずれかに記載のワクシニアウイルス。
- がんが、悪性黒色腫、肺腺癌、肺癌、小細胞肺癌、肺扁平上皮癌、腎癌、膀胱癌、頭頸部癌、乳癌、食道癌、グリア芽細胞腫、神経芽細胞腫、骨髄腫、卵巣癌、大腸癌、膵癌、前立腺癌、肝細胞癌、中皮腫、子宮頸癌、又は、胃癌である、請求項22記載のワクシニアウイルス。
- がんの予防又は治療用医薬組成物を製造するための、請求項1及び4~9のいずれかに記載のワクシニアウイルスの使用。
- がんが、悪性黒色腫、肺腺癌、肺癌、小細胞肺癌、肺扁平上皮癌、腎癌、膀胱癌、頭頸部癌、乳癌、食道癌、グリア芽細胞腫、神経芽細胞腫、骨髄腫、卵巣癌、大腸癌、膵癌、前立腺癌、肝細胞癌、中皮腫、子宮頸癌、又は、胃癌である、請求項24記載の使用。
- がんの予防又は治療を必要とする対象に
(1)IL-7をコードするポリヌクレオチドを含むワクシニアウイルス;及び
(2)IL-12をコードするポリヌクレオチドを含むワクシニアウイルスを投与する工程を包含する、がんを予防又は治療する方法。 - がんが、悪性黒色腫、肺腺癌、肺癌、小細胞肺癌、肺扁平上皮癌、腎癌、膀胱癌、頭頸部癌、乳癌、食道癌、グリア芽細胞腫、神経芽細胞腫、骨髄腫、卵巣癌、大腸癌、膵癌、前立腺癌、肝細胞癌、中皮腫、子宮頸癌、又は、胃癌である、請求項26記載の方法。
- 以下の(1)又は(2)から選択される、ワクシニアウイルス:
(1)IL-12をコードするポリヌクレオチドを含むワクシニアウイルスを含む医薬組成物と併用してがんの予防又は治療に使用するための、IL-7をコードするポリヌクレオチドを含むワクシニアウイルス;又は
(2)IL-7をコードするポリヌクレオチドを含むワクシニアウイルスを含む医薬組成物と併用してがんの予防又は治療に使用するための、IL-12をコードするポリヌクレオチドを含むワクシニアウイルス。 - がんが、悪性黒色腫、肺腺癌、肺癌、小細胞肺癌、肺扁平上皮癌、腎癌、膀胱癌、頭頸部癌、乳癌、食道癌、グリア芽細胞腫、神経芽細胞腫、骨髄腫、卵巣癌、大腸癌、膵癌、前立腺癌、肝細胞癌、中皮腫、子宮頸癌、又は、胃癌である、請求項28記載のワクシニアウイルス。
- 以下の(1)又は(2)から選択される、ワクシニアウイルスの使用:
(1)IL-12をコードするポリヌクレオチドを含むワクシニアウイルスを含む医薬組成物と併用するがんの予防又は治療用医薬組成物を製造するための、IL-7をコードするポリヌクレオチドを含むワクシニアウイルスの使用;又は
(2)IL-7をコードするポリヌクレオチドを含むワクシニアウイルスを含む医薬組成物と併用するがんの予防又は治療用医薬組成物を製造するための、IL-12をコードするポリヌクレオチドを含むワクシニアウイルスの使用。 - がんが、悪性黒色腫、肺腺癌、肺癌、小細胞肺癌、肺扁平上皮癌、腎癌、膀胱癌、頭頸部癌、乳癌、食道癌、グリア芽細胞腫、神経芽細胞腫、骨髄腫、卵巣癌、大腸癌、膵癌、前立腺癌、肝細胞癌、中皮腫、子宮頸癌、又は、胃癌である、請求項30記載の使用。
- がんの予防又は治療用の組合せキットを製造するための、(1)IL-7をコードするポリヌクレオチドを含むワクシニアウイルス及び(2)IL-12をコードするポリヌクレオチドを含むワクシニアウイルスの使用。
- がんが、悪性黒色腫、肺腺癌、肺癌、小細胞肺癌、肺扁平上皮癌、腎癌、膀胱癌、頭頸部癌、乳癌、食道癌、グリア芽細胞腫、神経芽細胞腫、骨髄腫、卵巣癌、大腸癌、膵癌、前立腺癌、肝細胞癌、中皮腫、子宮頸癌、又は、胃癌である、請求項32記載の使用。
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