WO2012157408A1 - 細胞傷害性t細胞誘導剤 - Google Patents
細胞傷害性t細胞誘導剤 Download PDFInfo
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- WO2012157408A1 WO2012157408A1 PCT/JP2012/060910 JP2012060910W WO2012157408A1 WO 2012157408 A1 WO2012157408 A1 WO 2012157408A1 JP 2012060910 W JP2012060910 W JP 2012060910W WO 2012157408 A1 WO2012157408 A1 WO 2012157408A1
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Definitions
- the present invention relates to a cytotoxic T cell (CTL) inducer and a method for preventing or treating a viral disease or cancer using the same.
- CTL cytotoxic T cell
- Simian virus 40 is a kind of monkey polyomavirus belonging to the family of papovaviridae, and is a small tumor virus without an envelope.
- the outer shell of the SV40 virus is composed of a major capsid protein called VP1.
- VP1 a major capsid protein
- VLPs virus-like particles
- Patent Document 2 describes a CTL vaccine containing a VLP to which a CTL epitope peptide is bound.
- virus-derived peptides are used as vaccines.
- peptides have poor retention and are easily degraded, they are usually administered with an adjuvant.
- adjuvants have side effects and are problematic for administration to humans.
- the present invention has been made under the above technical background, and an object of the present invention is to provide means for allowing a peptide to exert a sufficient effect as a vaccine without an adjuvant.
- VLP composed of a modified VP1 in which a T cell epitope peptide is inserted into a DE loop or HI loop exhibits strong CTL inducibility.
- Patent Document 1 A VLP composed of a modified VP1 in which a foreign peptide is inserted into the DE loop or HI loop is disclosed in Patent Document 1.
- Patent Document 1 an exogenous peptide is inserted exclusively to change the cell tropism of VLP, and the idea of inducing CTL using an exogenous peptide is not disclosed in Patent Document 1.
- claim 10 of Patent Document 1 describes the use of an antigenic epitope as a foreign peptide, and this antigenic epitope is used to allow VLP to bind to an antibody. Is. That is, the “epitope” described in Patent Document 1 is an epitope recognized by an antibody, not an epitope recognized by a T cell (T cell epitope).
- Patent Document 2 A CTL vaccine containing a VLP to which a CTL epitope peptide is bound is disclosed in Patent Document 2 (claim 182 and the like).
- Patent Document 2 does not disclose that a CTL epitope peptide is inserted into the DE loop and / or HI loop of VP1 of simian virus 40.
- the CTL inducer of the present invention has a sufficient CTL inducing effect even without an immunostimulatory substance, but the vaccine disclosed in Citation 2 is an immunostimulatory substance such as an unmethylated CpG-containing oligonucleotide. (Claim 1), and this point is also different from the CTL inducer of the present invention.
- the present invention has been completed based on the above findings.
- the present invention provides the following (1) to (10).
- a CTL inducer comprising a virus-like particle composed of VP1 of simian virus 40, wherein a T cell epitope peptide is inserted into the DE loop and / or HI loop of the VP1.
- CTL inducer (2)
- the T cell epitope peptide is a peptide derived from influenza virus HA, NA, M1, M2, NP, NS1, NS2, PA, PB1, PB2, or PB1-F2
- the T cell epitope peptide is a peptide derived from Melan-A / MART-1, gp100, MAGE-A3, MAGE-A10, CEA, HER2 / new, NY-E50-1, WT-1, or hTERT
- a virus-like particle composed of VP1 of simian virus 40 wherein a virus protein-derived T-cell epitope peptide is inserted into the VP1 DE loop and / or HI loop
- a method for preventing or treating a viral disease in an animal other than a human characterized by administration to an animal other than the above.
- a method for preventing or treating a human viral disease characterized by comprising (10) A virus-like particle composed of VP1 of simian virus 40, wherein a T cell epitope peptide derived from a protein specific to cancer cells is inserted into the DE loop and / or HI loop of VP1.
- a method for preventing or treating human cancer comprising administering the like particle to a human.
- the VLP contained in the CTL inducer of the present invention has a strong CTL inducing effect even without an adjuvant. Therefore, the CTL inducer of the present invention can prevent or treat viral diseases and cancer without causing side effects.
- the lysate of M1-DE-VLP was lanes 1-4 in the left figure, and lanes 1 and 2 did not contain CpG adjuvant, and lanes 3 and 4 developed samples containing CpG adjuvant.
- M1-DE-VLP pellets are lanes 5-6 in the left figure, lanes 5 and 6 were not containing CpG adjuvant, and lanes 7 and 8 were samples containing CpG adjuvant.
- the lysate of M1-HI-VLP was lanes 1-4 in the right figure, lanes 1 and 2 were not containing CpG adjuvant, and lanes 3 and 4 were samples containing CpG adjuvant.
- the M1-HI-VLP pellets are lanes 5-6 shown in the right figure, lanes 5 and 6 are those containing no CpG adjuvant, and lanes 7 and 8 are samples containing CpG adjuvant.
- the transgenic mice were immunized and analyzed by ICS two weeks later.
- CpG ( ⁇ ) indicates that no CpG adjuvant is added
- CpG + indicates that a CpG adjuvant is added.
- peptide pulse (M1 flu M58-66) (-) is not added with CTL epitope sequence (GILGFVFTL)
- peptide pulse (M1 flu M58-66) + is reinduced by adding the above sequence. It shows that you are doing.
- the dots in the upper right frame of each figure represent CD8 positive / intracellular IFN- ⁇ positive cells, and the numbers in the frame indicate the percentage of these cells.
- the figure which shows the ICS analysis result in the different immune pathway of M1-DE-VLP and M1-HI-VLP.
- M1-DE-VLP left
- M1-HI-VLP right
- pellet solution resuspended by sonication
- ICS analysis was performed.
- peptide pulse (M1 flu M58-66) (-) is not added with CTL epitope sequence (GILGFVFTL), peptide pulse (M1 flu M58-66) + reinduces CTL by adding the above sequence It shows that.
- Spleen indicates lymphocytes extracted from the spleen
- lung indicates lymphocytes extracted from the lung.
- the dots in the upper right frame of each figure represent CD8 positive / intracellular IFN- ⁇ positive cells, and the numbers in the frame indicate the percentage of these cells.
- the figure which shows the CD107-ICS analysis result in the immunity of M1-DE-VLP and M1-HI-VLP.
- M1-DE-VLP (left) and M1-HI-VLP (right) pellet solution (resuspended by sonication) were immunized by intramuscular and intraperitoneal routes, and one week later CD107-ICS Analysis was performed.
- peptide pulse (M1 flu M58-66) (-) is not added with CTL epitope sequence (GILGFVFTL), peptide pulse (M1 flu M58-66) + reinduces CTL by adding the above sequence It shows that.
- Spleen indicates lymphocytes extracted from the spleen.
- the dots in the upper right frame of each figure represent CD8 positive / intracellular IFN- ⁇ positive cell lines (upper figure) and CD107 positive / intracellular IFN- ⁇ positive cell lines (lower figure) in CD8 positive cells, respectively. The number indicates the percentage of cells.
- the figure which shows the 51 Chrome release analysis result in the immunity of M1-DE-VLP and M1-HI-VLP.
- M1-DE-VLP left and M1-HI-VLP (right) pellet solutions (resuspended by sonication) were immunized by intramuscular and intraperitoneal routes, and one week later 51 Chrome release analysis For this purpose, lymphocytes were taken out and cultured, and after one week of culture, 51 Chrome release analysis was performed.
- M1-DE-VLP (left) and M1-HI-VLP (right) pellet solution (resuspended by sonication) were immunized by intramuscular and intraperitoneal routes, and one week later, stained with CFSE
- the same number of lymphocytes (CFSE low) mixed with CTL epitope sequence (GILGFVFTL) and non-mixed lymphocytes (CFSE low) were introduced by intravenous injection, and one day later, In vivo CTL analysis was performed.
- Each figure shows the CFSE intensity (horizontal axis) and cell number (vertical axis) of CFSE-stained cells in mouse lymphocytes. (-) In the figure shows the case of using non-immunized mouse lymphocytes.
- M1-DE-VLP and M1-HI-VLP are M1-DE-VLP and M1-HI-VLP, respectively.
- mouth is used is shown.
- the numbers in the boxes indicate the specific injury rate for lymphocytes calculated by the following formula.
- Specific injury rate (%) (1-r (control) / r (immunized)) x100
- r (control) is the number of lymphocytes in CFSE low / the number of lymphocytes in CFSE high in non-immunized mice
- R (immunized) represents (CFSE (low lymphocyte count / CFSE high lymphocyte count) in the immunized mouse.
- M1-DE-VLP and M1-HI-VLP pellet solutions were immunized by intramuscular and intraperitoneal routes, and one week later, influenza A virus ( a / H1N1 / PR8 and a / H3N2 / Aichi, concentration: 100 TCID 50/20 ⁇ l, inoculum size: extract 20 [mu] l), after anesthetized mice immunized, were intranasally infected, from the lungs after 4 days A liquid was prepared, and the amount of influenza virus contained therein was calculated from the distribution of erythrocyte aggregation at a tissue culture influenza virus infectious dose (TCID 50 ).
- TCID 50 tissue culture influenza virus infectious dose
- the left table shows the results of influenza virus load in the mouse lung infected with A / H1N1 / PR8, and the right table shows the result of influenza virus load in the mouse lung infected with A / H3N2 / Aichi. immunized with (-) is an unimmunized mouse
- M1-DE-VLP is a mouse immunized with M1-DE-VLP
- M1-HI-VLP is a lung extract of a mouse immunized with M1-HI-VLP 1
- TCID 50 tissue culture influenza virus infectious dose
- M1-DE-VLP and M1-HI-VLP pellet solutions were immunized by intramuscular and intraperitoneal routes.
- influenza A virus a / H1N1 / PR8, density: 100 TCID 50/20 ⁇ l, inoculum size: a 20 [mu] l
- influenza A virus after anesthetized mice immunized, were intranasally infected, the body weight of mice at that time as 100%, 2 weeks The weight change of the mice was analyzed daily.
- FIG. 9-a The figure which shows the result of the CBB dyeing
- the WT1WT-DE-VLP lysate is lane 3
- the WT1WT-DE-VLP pellet is lane 4
- the WT1WT-HI-VLP lysate is lane 5
- the WT1WT-HI-VLP pellet is lane 6.
- FIG. 9-b The figure which shows the ICS analysis result in the different immune pathway of WT1WT-DE-VLP and WT1WT-HI-VLP.
- WT1WT-DE-VLP Fig. 9-b, top
- WT1WT-HI-VLP Fig.
- M1 An emulsion obtained by suspending a peptide of CTL58-66 CTL epitope sequence (GILGFVFTL) ⁇ with IFA once through the route of the subcutaneous vagina (sc) (upper left figure), and one week after the immunization one week
- GILGFVFTL CTL58-66 CTL epitope sequence
- peptide pulse (M1: 58-66) (-) is the one without CTL epitope sequence (GILGFVFTL) added, peptide pulse (M1: 58-66) + reinduces CTL by adding the above sequence It shows that.
- Spleen indicates lymphocytes extracted from the spleen.
- the dots in the upper right frame of each figure represent CD8 positive / intracellular IFN- ⁇ positive cells, and the numbers in the frame indicate the percentage of these cells.
- the optiprep on the left of the table shows only the solvent dissolving VLP, wild-type-VLP is a wild-type VLP without CTL epitope inserted, and LPS is incubated with lymphocytes extracted from the spleen with lipopolysaccharide Yes.
- the middle shows the incubation time, and the right shows the concentration of IL-12 in the culture supernatant of the cultured lymphocytes calculated by ELISA.
- the CTL inducer of the present invention contains virus-like particles composed of VP1 of simian virus 40, and a T cell epitope peptide is inserted into the DE loop and / or HI loop of VP1. It is characterized by.
- the T cell epitope peptide used in the present invention is not particularly limited as long as it can induce CTL.
- T cell epitope peptides peptides derived from viral proteins, peptides derived from proteins specific to cancer cells, and the like are known, and any of these peptides can be used in the present invention.
- the “protein specific to cancer cells” means a protein that is not expressed in normal cells and expressed only in cancer cells, or a protein whose expression is increased in cancer cells compared to normal cells.
- peptides derived from viral proteins include peptides derived from proteins such as influenza virus HA, NA, M1, M2, NP, NS1, NS2, PA, PB1, PB2, and PB1-F2.
- proteins such as influenza virus M1, NP, NS1, PA, PB1, and PB2 are preferred.
- Specific peptides include peptides derived from influenza virus M1 derived peptides GILGFVFTL (SEQ ID NO: 1), ASCMGLIY (SEQ ID NO: 2), SIIPSGPLK (SEQ ID NO: 3), ILGFVFTLTV (SEQ ID NO: 4), and NP.
- CTELKLSDY (SEQ ID NO: 5), LPFEKSTVM (SEQ ID NO: 6), ILRGSVAHK (SEQ ID NO: 7), ELRSRYWAI (SEQ ID NO: 8), SRYWAIRTR (SEQ ID NO: 9), PB1-derived peptides NMLSTVLGV (SEQ ID NO: 10), RIFLAMITYI ( SEQ ID NO: 11), VSDGGPNLY (SEQ ID NO: 12), PB2-derived peptide VLTGNLQTL (SEQ ID NO: 13), NS1-derived peptide IILKANFSV (SEQ ID NO: 14), and the like.
- Peptides derived from proteins of viruses other than influenza viruses include HIV Gag, Pol, Env, Tat, Nef, Rev derived peptides, Hepatitis C virus (HCV) E1, E2, Core, NS2, NS3, NS4, Examples include NS5-derived peptides, human papillomavirus (HPV) E6, E7-derived peptides, and the like.
- Protein-specific peptides derived from cancer cells include Melan-A / MART-1, gp100, MAGE-A3, MAGE-A10, CEA, HER2 / new, NY-E50-1, WT-1, and hTERT.
- a protein-derived peptide can be exemplified. Among these, peptides derived from proteins such as HER2 / new, WT-1, and MAGE-A3 are preferable.
- Specific peptides include ARCRWGLLL (SEQ ID NO: 15), QLFEDNYAL (SEQ ID NO: 16), KIFGSLAFL (SEQ ID NO: 17), ILHNGAYSL (SEQ ID NO: 18), IISAVVGIL (SEQ ID NO: 19), which are peptides derived from HER2 / new.
- VVLGVVFGI SEQ ID NO: 20
- RLLQETELV SEQ ID NO: 21
- VMAGVGSPYV SEQ ID NO: 22
- CLTSTVQLV SEQ ID NO: 23
- QLMPYGCLL SEQ ID NO: 24
- YLEDVRLV SEQ ID NO: 25
- VLVKSPNHV SEQ ID NO: 26
- YMIMVKCWMI SEQ ID NO: 27
- ELVSEFSRM SEQ ID NO: 28
- VLRENTSPK SEQ ID NO: 29
- RWGLLLALL SEQ ID NO: 30
- TYLPTNASL SEQ ID NO: 31
- PYVSRLLGI SEQ ID NO: 32
- RMFPNAPYL a peptide derived from WT-1 (SEQ ID NO: 33), SLGEQQYSV (SEQ ID NO: 34), CMTWNQMNL (SEQ ID NO: 35), YMFPNAPYL (SEQ ID NO: 36), CMTWNQMNL (S
- the length of the T cell epitope peptide to be inserted into the loop is not limited as long as it can induce CTL, but is usually 3 to 27 amino acids, preferably 8 to 11 amino acids, more preferably 9 amino acids.
- the DE loop is at positions 127-146 counting from the N-terminus of VP1, and the HI loop is at positions 268-277 (International Publication 2006/088229).
- the T cell epitope peptide may be inserted into any of these loops, but for the DE loop it is preferred to insert the amino acid sequence at positions 137-138 with a T cell epitope peptide, and for the HI loop it is 273.
- the amino acid sequence at position -274 is preferably inserted so as to be replaced with a T cell epitope peptide.
- one to several spacer amino acids such as glycine, alanine and serine are usually inserted at both ends of the T cell epitope peptide inserted into the loop (International Publication 2006/088229).
- the number of spacer amino acids is usually 1 to 9, preferably 1 to 6, and more preferably 3 to 6.
- the T cell epitope peptide may be inserted into both the DE loop and the HI loop, or may be inserted only into one. When both are inserted, the same T cell epitope peptide may be inserted, or different T cell epitope peptides may be inserted. Furthermore, two or more T cell epitope peptides may be inserted in series in the loop.
- a method for producing a virus-like particle composed of VP1 having a foreign peptide inserted in a loop is described in International Publication 2006/088229, according to this method, from VP1 having a T cell epitope peptide inserted in a loop Constructed virus-like particles can be made.
- a modified VP1 gene in which a base sequence encoding a T cell epitope peptide is inserted is prepared, and this modified VP1 gene is highly expressed in insect cells using a baculovirus vector or the like.
- Virus-like particles can be made.
- the CTL inducer of the present invention is in the form of a general pharmaceutical composition depending on the properties of the substance to be the active ingredient, and direct delivery of the composition is generally performed by parenteral injection (eg, subcutaneous injection, intraperitoneal injection). , Intravenous injection, intramuscular injection, injection into the space of tissues, etc.). Other methods of administration include mucosal administration (eg, oral, nasal, or lung), administration through the eye, transdermal administration, and suppositories.
- parenteral injection eg, subcutaneous injection, intraperitoneal injection.
- Intravenous injection intramuscular injection, injection into the space of tissues, etc.
- Other methods of administration include mucosal administration (eg, oral, nasal, or lung), administration through the eye, transdermal administration, and suppositories.
- a dosage form such as an injection, a nasal agent, a topical agent (such as a transdermal agent), or a rectal agent.
- injections include sterile solutions or suspensions, emulsions, and the like, and specifically water, water-propylene glycol solutions, buffered solutions, 0.4% physiological saline, and the like.
- injections include sterile solutions or suspensions, emulsions, and the like, and specifically water, water-propylene glycol solutions, buffered solutions, 0.4% physiological saline, and the like.
- when it is set as a liquid formulation it can preserve
- topical administration agent include creams, ointments, lotions, transdermal agents, and the like.
- the oral preparation or rectal administration agent include capsules, tablets, pills, powders
- compositions and additives are formulated together with pharmaceutically acceptable excipients and additives by techniques generally used in the art.
- Pharmaceutically acceptable excipients and additives include carriers, binders, fragrances, buffers, thickeners, colorants, stabilizers, emulsifiers, dispersants, suspending agents, preservatives, pH adjustment Agents, tonicity modifiers, infiltrant and the like.
- Examples of the pharmaceutically acceptable carrier include magnesium carbonate, lactose, pectin, starch, methylcellulose and the like.
- the CTL inducer of the present invention has a sufficient CTL inducing effect without an adjuvant, and therefore may not contain an adjuvant, but may contain it.
- the adjuvant to be used include aluminum hydroxide gel, complete Freund's adjuvant, incomplete Freund's adjuvant, pertussis adjuvant, poly (I, C), CpG-DNA and the like.
- the dose of T cell epitope peptide in the preparation and the number of administrations of the preparation vary depending on the type of T cell epitope peptide, the symptom of the administration subject, age, body weight, dosage form, etc., but usually 0.01 ⁇ g to 1 mg, preferably 0.1 ⁇ g. It is preferably ⁇ 500 ⁇ g, more preferably 1.0 ⁇ g to 100 ⁇ g, and this is preferably administered once every several days to several months.
- initial immunization involves administering 1.0 ⁇ g to 500 ⁇ g of peptide for an adult patient, depending on the patient's response and condition by measuring specific CTL activity in the patient's blood, depending on the patient's response and condition This is followed by a boosting dose of 1.0 ⁇ g to 100 ⁇ g of peptide according to boosting therapy from weeks to months.
- the CTL inducer of the present invention can be used as a vaccine for preventing or treating viral diseases and a vaccine for preventing or treating various cancers.
- the CTL inducer of the present invention can be administered to humans and non-human animals.
- animals other than humans include livestock (eg, cows, horses, pigs, chickens, etc.), pets (eg, dogs, cats, etc.), laboratory animals (mouse, rats, etc.), and the like.
- the CTL inducer of the present invention can also be used in the following preventive or therapeutic methods.
- a virus-like particle composed of VP1 of simian virus 40 in which a virus protein-derived T cell epitope peptide is inserted into the DE loop and / or HI loop of VP1 to humans A method of administering or preventing or treating a human viral disease.
- B Virus-like particles composed of VP1 of simian virus 40, wherein virus-like particles in which a T protein epitope peptide derived from a viral protein is inserted into the DE loop and / or HI loop of VP1 are non-human To prevent or treat viral diseases in animals other than humans.
- D A virus-like particle composed of VP1 of simian virus 40, wherein a T cell epitope peptide derived from a protein specific to cancer cells is inserted into the DE loop and / or HI loop of VP1.
- Example 1 Production of M40 CTL epitope inserted SV40 VP1 gene CTL epitope sequence (GILGFVFTL) of influenza virus particle internal protein M1 (this epitope sequence is an epitope sequence for HLA-A * 0201 of human MHC class I ), Inserted into SV40 VP1.
- GILGFVFTL gene CTL epitope sequence of influenza virus particle internal protein M1
- the DE loop region of VP1 (137-138 amino acid region, classically the fourth Ala of the VP1 gene is amino acid number 1) or the HI loop region (273-274 amino acid region) is replaced with GILGFVFTL, and M1 CTL epitope insertion SV40 VP1 insertion mutants were prepared (M1-DE-VP1, M1-HI-VP1).
- the preparation was performed by Overhang PCR using pFastBac1-SV40 wild type VP1 encoding SV40 VP1 as a template. The following primers were used.
- PCR was incubated at 98 ° C for 60 seconds for both 1st and 2nd rounds, then the next cycle was repeated 25 times (98 ° C for 15 seconds, 59 ° C for 15 seconds, 74 ° C for 30 seconds), and finally at 74 ° C for 1 minute. Incubated for 30 seconds and transferred to 4 ° C.
- the PCR fragment prepared by Overhang PCR was cleaved with restriction enzymes KpnI and SalI and inserted between the KpnI and SalI sites of the pFastBac-1 plasmid vector to obtain M1-DE-VP1 and M1-HI-VP1 plasmids.
- Example 2 Preparation of baculovirus expressing M1-DE-VP1 and M1-HI-VP1 M1-DE-VP1 and M1-HI-VP1 plasmids were introduced into E. coli DH10bac (invitrogen) carrying the baculovirus genome.
- the recombinant baculovirus genome incorporating M1-DE-VP1 and M1-HI-VP1 was prepared.
- the recombinant baculovirus genome was transfected into Sf-9 cells, and three days later, the supernatant was collected to obtain a solution containing the recombinant baculovirus. A part of this solution was again infected with Sf-9 cells to raise the recombinant baculovirus titer to obtain a recombinant baculovirus stock solution.
- MOI multiplicity of infection
- Example 4 Immunization of M1-DE-VLP and M1-HI-VLP The lysate solution (100 ⁇ l) and pellet solution (100 ⁇ l) containing the above M1-DE-VLP and M1-HI-VLP are used as an adjuvant.
- CpG-olygodeoxyncleotide5002 (TCCATGACGTTCTTGATGTT: SEQ ID NO: 52) Adjuvant was added in an amount of 5 ⁇ g, and 8-week-old transgenic mice were immunized by the footpad, hip joint, intravenous, intraperitoneal, intramuscular, and intranasal routes.
- mice were used that expressed C57BL / 6 as a background and a HLA-A * 0201 and H-2Db chimera fused with human ⁇ 2m. Since this mouse knocks out mouse ⁇ 2m and H-2Db, it is considered that the mouse-derived MHC class I is not exposed on the cell surface.
- Example 5 Preparation of lymphocytes from spleen and lungs of immunized mice
- the spleens and lungs were removed from the mice and each was placed in a ⁇ 6 cm dish containing 5 ml of RPMI-1640 medium. placed.
- tweezers thoroughly spleen the spleen in the medium, transfer the solution containing lymphocytes eluted in the medium to a 15 ml tube, wash the ⁇ 6 cm dish with 5 ml of RPMI-1640 medium, and remove the supernatant.
- the total volume was 10 ml.
- the supernatant was transferred again to a new 15 ml tube and centrifuged at 1,200 rpm for 5 minutes at room temperature to transfer the lymphocytes to the pellet. After removing the supernatant and loosening the pellet, add 250 ⁇ l NH 4 Cl-Tris solution and stir to remove red blood cells, then quickly add 10 ml of RPMI-1640 medium, room temperature, 1,2000 rpm For 5 minutes to transfer lymphocytes to pellets.
- lymphocytes To count lymphocytes, add 10 ⁇ l of the above suspension to 490 ⁇ l of 2% acetic acid solution, calculate the number of cells using the Bürkerturk hemocytometer, and obtain 10% to obtain 2 ⁇ 10 7 cells / ml. It was diluted with FCS-contaminated RPMI-1640 medium or concentrated.
- the removed lungs are transferred to a ⁇ 6 cm dish without medium, chopped well with scissors, suspended in 10 ml of 50 unit / ml Collagenase Type 1, 10% FCS-contained RPMI-1640 medium for 1 hour. Incubation was performed at 37 ° C. in a 5% CO 2 incubator. After incubation, the supernatant was collected into a 50 ml tube through a 100 ⁇ m Nylon cell strainer (BD Falcon), and the tissue pieces remaining in the strainer were ground with a cell scraper, and then further 10 ml of 10% FCS-contaminated RPMI-1640 The medium was passed through and a 20 ml total solution was collected in a 50 ml tube.
- BD Falcon 100 ⁇ m Nylon cell strainer
- the medium was passed through and a 20 ml total solution was collected in a 50 ml tube.
- the collected solution was centrifuged at 1,200 rpm for 5 minutes at room temperature, the supernatant was removed, the pellet was loosened, 10 ml RPMI-1640 medium was added, and the suspension was transferred to a 15 ml tube. Centrifuge the transferred 15 ml tube at room temperature at 1,200 rpm for 5 minutes, remove the supernatant, loosen the pellet, add 10 ml RPMI-1640 medium, and centrifuge at room temperature at 1,200 rpm for 5 minutes. Was washed.
- ICS analysis To examine the presence of CTL induced in response to the CTL epitope sequence of M1 (GILGFVFTL) in lymphocytes collected from the spleen and lung by the above method, ICS analysis was performed. went. 96-well round-bottom plates in 10% FCS mixed RPMI-1640 medium 25-fold diluted BD GolgiPlug TM and (BD) 1 per well 5 [mu] l added, 20 [mu] M of diluted with further 10% FCS mixed RPMI-1640 medium therein 100 ⁇ l of M1 CTL epitope peptide (GILGFVFTL, Operon) was added. As a negative control, 100 ⁇ l of 10% RCS-contaminated RPMI-1640 medium containing no peptide was added.
- the wells were incubated at 37 ° C. in a 5% CO 2 incubator for 5 hours.
- the ratio of the number of CD8 positive / intracellular IFN- ⁇ positive cells was increased by incubating the CTL epitope peptide in any measurement result. Moreover, the result that the ratio of the number of CD8 positive / intracellular IFN- ⁇ positive cells increases was obtained when CpG was not added. From these, it was found that M1-DE-VLP and M1-HI-VLP could induce CTL without an adjuvant.
- CD107-ICS analysis The presence of CTL having cytotoxic activity induced in response to the CTL epitope sequence of M1 (GILGFVFTL) in lymphocytes collected from the spleen by the method of Example 5 To investigate, CD107-ICS analysis was performed.
- BD GolgiPlug TM (BD) diluted 25-fold with 10% FCS-contaminated RPMI-1640 medium in a 96-well round-bottom plate, 5 ⁇ l per well, and BD GolgiStop TM diluted 25-fold with 10% FCS-contained RPMI-1640 medium
- 100 ⁇ l of peptide (GILGFVFTL, Operon) was added.
- As a negative control 100 ⁇ l of 10% RCS-contaminated RPMI-1640 medium containing no peptide was added.
- the wells were incubated in the dark for 6 hours at 37 ° C. in a 5% CO 2 incubator.
- Fig. 4 shows the results of two-dimensional analysis of CD107-ICS.
- CD8-positive, CD107-positive, and intracellular IFN- ⁇ -positive cells when the ratio of CD8-positive / intracellular IFN- ⁇ -positive cells is increased by incubation of the CTL epitope peptide in any measurement result It was found that the number ratio also increased.
- CD8-positive, CD107-positive, and intracellular IFN- ⁇ -positive cells have cytotoxic activity, so immunization with M1-DE-VLP and M1-HI-VLP may induce cytotoxic CTLs I found out.
- Example 8 51 Chrome release analysis In order to show that the CD8 positive / CD107 positive / intracellular IFN- ⁇ positive cells shown in Example 7 are cytotoxic, 51 Chrome release analysis was performed.
- Spleen lymphocytes were prepared from a non-immunized mouse in the same manner as in Example 5. Add 4 ⁇ l of 10 mM M1 CTL epitope peptide (GILGFVFTL, Operon) to spleen lymphocytes 2.4 ⁇ 10 8 cells of non-immunized mice in 2 ml of RPMI-1640 medium containing 10% FCS, and add 15 ⁇ l tube Incubated for 2 hours at 37 ° C. with 5% CO 2 incubator.
- GILGFVFTL 10 mM M1 CTL epitope peptide
- Immunized lymphocytes do not remove blood, so leave the tissue fragment immediately at the bottom of the 15 ml tube, transfer the supernatant again to a new 15 ml tube, centrifuge at 1,200 rpm for 5 minutes at room temperature, The spheres were transferred to pellets. After removing the supernatant and loosening the pellet, add 2 ml of 10% FCS-contaminated RPMI-1640 medium and add 10 ⁇ l of lymphocyte solution to 490 ⁇ l of 2% acetic acid solution to count lymphocytes. The number of cells was calculated using a Bürkerturk hemocytometer, and diluted with 10% FCS-contained RPMI-1640 medium to 5 ⁇ 10 6 cells / ml.
- X-irradiated lymphocytes 5x10 6 cells / ml, 500 ⁇ l, and immunized lymphocytes 5x10 6 cells / ml, 500 ⁇ l was mixed. 24 wells were prepared per sample. After mixing, the cells were incubated for 7 days at 37 ° C. in a 5% CO 2 incubator.
- lymphocytes in 24 wells were collected into a single 50 ml tube and centrifuged at 1,200 rpm for 5 minutes at room temperature to transfer the lymphocytes to a pellet. After removing the supernatant and loosening the pellet, add 2 ml of 10% FCS-contaminated RPMI-1640 medium and count 20 ⁇ l lymphocytes in 20 ⁇ l 0.4% trypan blue solution (Gibco) to count lymphocytes. The solution was added, the number of cells was calculated with a Bürkerturk hemocytometer, and diluted or concentrated in RPMI-1640 medium containing 10% FCS to 7.5 ⁇ 10 6 cells / ml. The prepared lymphocytes were used as effector cells.
- 1 ml of 1x10 6 cells / ml RMA-HHD cultured cells are dispensed into two 15 ml tubes, and 10 mM M1 CTL epitope peptide (GILGFVFTL, Operon) is placed in one tube.
- 10 mM M1 CTL epitope peptide GILGFVFTL, Operon
- 5 ⁇ l was added and incubated for 2 hours at 37 ° C. in a 5% CO 2 incubator. No peptide was added to the other tube.
- the cells were centrifuged at 1,000 rpm for 5 minutes at room temperature to transfer the cells to a pellet. After removing the supernatant and loosening the pellet, 100 ⁇ Ci (microsievert) of Na 2 51 CrO 4 solution was added and incubated for 30 minutes at 37 ° C.
- 10 ⁇ l (7.5 ⁇ 10 4 cells) of effector cells and 90 ⁇ l of RPMI-1640 medium mixed with 10% FCS were added so that the ratio was 1.
- Triton-X 100 solution 100 ⁇ l of 5% Triton-X 100 solution was added as a positive control, and 100 ⁇ l of 10% FCS-contaminated RPMI-1640 medium was added as a negative control. After mixing, the cells were incubated for 4 hours at 37 ° C. in a 5% CO 2 incubator.
- lymphocytes prepared by immunization with M1-DE-VLP and M1-HI-VLP show cells that present a peptide of the CTL epitope of M1 (GILGFVFTL) (M1: 58 in FIG. 5).
- GILGFVFTL CTL epitope of M1
- -66 induces the release of 51 Cr by selectively damaging the M1-DE-VLP and M1-HI-VLP immunizations by presenting a peptide of the M1 CTL epitope (GILGFVFTL) It was found that CTL can be induced to selectively injure cells.
- Example 9 In vivo CTL analysis CTLs that selectively damage cells presenting the peptide of the CTL epitope of M1 (GILGFVFTL) shown in Example 8 are actually In vivo CTL analysis was performed to analyze selective damage to cells presenting the peptide (GILGFVFTL). About 50 ⁇ g of M1-DE-VLP was immunized by intramuscular injection and about 50 ⁇ g of M1-HI-VLP was injected by intraperitoneal injection.
- spleens were removed from 3 non-immunized mice and placed in a ⁇ 6 cm dish containing 5 ml of RPMI-1640 medium. Using tweezers, thoroughly spleen the spleen in the medium, transfer the solution containing lymphocytes eluted in the medium to a 15 ml tube, wash the ⁇ 6 cm dish with 5 ml of RPMI-1640 medium, and remove the supernatant. In addition to the 15 ml tube, the total volume was 10 ml.
- Unimmunized lymphocytes do not remove blood, so leave the tissue fragment immediately at the bottom of the 15 ml tube, transfer the supernatant again to a new 15 ml tube, and centrifuge at 1,200 rpm for 5 minutes at room temperature. The lymphocytes were transferred to a pellet. After removing the supernatant and loosening the pellet, 2 ml of 10% FCS-contaminated RPMI-1640 medium was added. Dispense 1 ml of non-immunized lymphocytes prepared in two 15 ml tubes, and add 1 ⁇ l of 10 mM M1 CTL epitope peptide (GILGFVFTL, Operon) to one side for 1 hour. Incubation was performed at 37 ° C.
- CFSE succinimidyl ester
- mice about 50 ⁇ g M1-DE-VLP by intramuscular injection, and about 50 ⁇ g M1-HI-VLP by intraperitoneal injection, each one week passed each mouse were prepared as above 200 ⁇ l of CFSE-labeled lymphocyte solution was intravenously injected for one day.
- the spleen was extracted from the mouse and placed in a ⁇ 6 cm dish containing 5 ml of RPMI-1640 medium. Disperse the spleen well in the medium using tweezers, transfer the solution containing the lymphocytes eluted in the medium to a 15 ml tube, wash the 6 cm dish again with 5 RP ml RPMI-1640 ⁇ medium, and remove the supernatant. In addition to the 15 ml tube, the total volume was 10 ml.
- FIG. 6 shows the results of In vivo CTL analysis.
- lymphocytes mixed with CTL epitope sequences shown in the low position in the figure
- CTL epitope sequences were mixed.
- the number of cells in the sphere was almost the same.
- CTLs were compared to lymphocytes not mixed with CTL epitope sequences (shown at the low position in the figure).
- the number of lymphocytes mixed with the epitope sequence (shown at the high position in the figure) was small. Therefore, CTLs induced by immunization with M1-DE-VLP or M1-HI-VLP can selectively damage cells that present peptides of M1 CTL epitopes in animals and others. I understood.
- Example 10 Analysis of maturation of lymphocytes
- lymphocytes from which erythrocytes were removed from the spleen of C57BL / 6 mice were purified and mixed with 10% FCS so as to be 1x10 7 cells / ml. Dilute with RPMI-1640 medium.
- FACS buffer 20% FCS, 0.1% sodium azide, 1xPBS (-)
- FACS fixation buffer 1% formaldehyde, 1x FACS buffer
- FIG. 7 shows the result of the histogram.
- Example 11 Virus challenge experiment
- HLA-A2 transgenic mice HHD mice were immunized with about 50 ⁇ g M1-DE-VLP by intramuscular injection and about 50 ⁇ g M1-HI-VLP by intraperitoneal injection.
- influenza virus (A / H1N1 / PR8 and,, A / H3N2 / Aichi) the tissue culture influenza virus infectious dose so that 100 TCID 50/20 ⁇ l with (TCID 50) PBS - diluted () Then, 20 ⁇ l of this diluted solution was anaesthetized after immunization and then nasally infected.
- tissue homogenizer with glass homogenizer, with warts, 10 ml, 812-770-02, Rika Ikemoto
- the solution was transferred to a 15 ml tube. Centrifugation was performed at 4 ° C. and 2000 rpm for 5 minutes, and the supernatant was transferred to a 2 ml tube with a screw cap.
- TCID 50 tissue culture influenza virus infectious dose
- influenza virus titer in the lungs of mice immunized with M1-DE-VLP and M1-HI-VLP is approximately 10 times lower compared to mice that did not receive immunization, thus -VLP and M1-HI-VLP immunity was suggested to have the effect of suppressing the growth of influenza virus in the lung.
- Example 12 VLP immunization with cancer CTL epitope inserted
- WT1 HLA-A2 known as a cancer antigen VLPs into which a CTL epitope sequence (RMFPNAPYL) was inserted were prepared (WT1WT-DE-VLP, WT1WT-HI-VLP) (FIG. 9-a).
- WT1WT-DE-VLP, WT1WT-HI-VLP (FIG. 9-a).
- HLA-A2 transgenic mice were immunized, lymphocytes were prepared, and ICS analysis was performed on the CTL epitope sequence of WT1 (FIG. 9-b).
- 51 Chrome release analysis was performed on the CTL epitope sequence of WT1 by the same method as in Example 8.
- a VLP into which a WT1 CTL epitope sequence was inserted was prepared in the same manner as a VLP into which a CTL epitope sequence of M1 was inserted (FIG. 9-a).
- the CTL epitope sequence of WT1 It was revealed that CTL against RMFPNAPYL was induced (FIG. 9-b), and that the CTL against the induced CTL epitope of WT1 selectively impaired cells presenting the CTL epitope sequence of WT1 (FIG. 9).
- VLP is a useful carrier capable of inducing CTL against various CTL epitope sequences.
- Example 13 ICS analysis of CTL induction by M1 CTL peptide suspension IFA emulsion
- M1-DE-VLP Fig. 10, upper right figure
- M1-HI-VLP The ICS analysis of Fig. 10, lower right figure
- Immunization was performed by injecting 100 ⁇ l of a pellet solution of M1-DE-VLP and M1-HI-VLP prepared in the same manner as in Example 4 subcutaneously (subcutaneous, sc).
- the IFA emulsion in which the peptide of CTL epitope (GILGFVFTL) of M1 is suspended is obtained by adding 50 ⁇ l of GILGFVFTL peptide (Operon) 50 ⁇ l dissolved in DMSO solvent at a concentration of 20 ⁇ g / ⁇ l to 450 ⁇ l PBS (-). It was prepared by mixing with 500 ⁇ l of incomplete freund's adjuvant IF (IFA) (Sigma) T and two-syringe method (GP syringe connector, NIPRO) (Inject 2 ml, B. Immunization was performed by injecting 100 ⁇ l of the prepared emulsion into the subcutaneous, s.c.
- Example 14 ELISA analysis of IL-12 secretion amount of spleen lymphocytes
- lymphocytes from which erythrocytes were removed from the spleen of HLA-A2 transgenic mice were purified, and 1x10 7 cells / It was diluted with 10% FCS-contaminated RPMI-1640 medium to a volume of ml.
- LPS LPS
- RPMI-1640 medium containing 10% FCS 50 ⁇ l of the lymphocytes purified above.
- the plate was incubated for 6 or 24 hours at 37 ° C., 5% CO 2 incubator. After incubation, the supernatant was spun down at 1400 rpm at 4 ° C. and collected in an Eppendorf tube, and the supernatant was spun down at 15,000 rpm at 4 ° C. and collected in a new Eppendorf tube.
- IL-12 enzyme-linked immunosorbent assay (Mouse Total IL-12 ELISA Kit, Thermo SCIENTIFIC) according to the protocol of the kit, and IL-12 in the culture supernatant was analyzed. The concentration was calculated ( Figure 11).
- the present invention is useful as a prophylactic or therapeutic agent for viral diseases and cancer, it can be used in industrial fields such as pharmaceuticals.
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Abstract
Description
(1)シミアンウイルス40のVP1から構成されるウイルス様粒子を含有するCTL誘導剤であって、前記VP1のDEループ及び/又はHIループ中にT細胞エピトープペプチドが挿入されていることを特徴とするCTL誘導剤。
(2)T細胞エピトープペプチドが、ウイルスタンパク質由来のペプチドであることを特徴とする(1)に記載のCTL誘導剤。
(3)T細胞エピトープペプチドが、インフルエンザウイルスのHA、NA、M1、M2、NP、NS1、NS2、PA、PB1、PB2、又はPB1-F2由来のペプチドであることを特徴とする(1)に記載のCTL誘導剤。
(4)T細胞エピトープペプチドが、癌細胞に特異的なタンパク質由来のペプチドであることを特徴とする(1)に記載のCTL誘導剤。
(5)T細胞エピトープペプチドが、Melan-A/MART-1、gp100、MAGE-A3、MAGE-A10、CEA、HER2/new、NY-E50-1、WT-1、又はhTERT由来のペプチドあることを特徴とする(1)に記載のCTL誘導剤。
(6)T細胞エピトープペプチドが、配列番号1乃至39のいずれかの配列番号に記載されたアミノ酸配列からなるペプチドであることを特徴とする(1)に記載のCTL誘導剤。
(7)シミアンウイルス40のVP1から構成されるウイルス様粒子であって、前記VP1のDEループ及び/又はHIループ中にウイルスタンパク質由来のT細胞エピトープペプチドが挿入されているウイルス様粒子を、ヒト以外の動物に投与することを特徴とするヒト以外の動物のウイルス性疾患の予防又は治療方法。
(8)シミアンウイルス40のVP1から構成されるウイルス様粒子であって、前記VP1のDEループ及び/又はHIループ中に癌細胞に特異的なタンパク質由来のT細胞エピトープペプチドが挿入されているウイルス様粒子を、ヒト以外の動物に投与することを特徴とするヒト以外の動物の癌の予防又は治療方法。
(9)シミアンウイルス40のVP1から構成されるウイルス様粒子であって、前記VP1のDEループ及び/又はHIループ中にウイルスタンパク質由来のT細胞エピトープペプチドが挿入されているウイルス様粒子を、ヒトに投与することを特徴とするヒトのウイルス性疾患の予防又は治療方法。
(10)シミアンウイルス40のVP1から構成されるウイルス様粒子であって、前記VP1のDEループ及び/又はHIループ中に癌細胞に特異的なタンパク質由来のT細胞エピトープペプチドが挿入されているウイルス様粒子を、ヒトに投与することを特徴とするヒトの癌の予防又は治療方法。
(A)シミアンウイルス40のVP1から構成されるウイルス様粒子であって、前記VP1のDEループ及び/又はHIループ中にウイルスタンパク質由来のT細胞エピトープペプチドが挿入されているウイルス様粒子をヒトに投与し、ヒトのウイルス性疾患を予防又は治療する方法。
(B)シミアンウイルス40のVP1から構成されるウイルス様粒子であって、前記VP1のDEループ及び/又はHIループ中にウイルスタンパク質由来のT細胞エピトープペプチドが挿入されているウイルス様粒子をヒト以外の動物に投与し、ヒト以外の動物のウイルス性疾患を予防又は治療する方法。
(C)シミアンウイルス40のVP1から構成されるウイルス様粒子であって、前記VP1のDEループ及び/又はHIループ中に癌細胞に特異的なタンパク質由来のT細胞エピトープペプチドが挿入されているウイルス様粒子をヒトに投与し、ヒトの癌を予防又は治療する方法。
(D)シミアンウイルス40のVP1から構成されるウイルス様粒子であって、前記VP1のDEループ及び/又はHIループ中に癌細胞に特異的なタンパク質由来のT細胞エピトープペプチドが挿入されているウイルス様粒子をヒト以外の動物に投与し、ヒト以外の動物の癌を予防又は治療する方法。
〔実施例1〕 M1 CTLエピトープ挿入SV40 VP1遺伝子の作製
インフルエンザウイルス粒子内部タンパク質M1のCTLエピトープ配列 (GILGFVFTL)を(このエピトープ配列は、ヒトのMHC class IのHLA-A*0201に対するエピトープ配列である。)、SV40 VP1に挿入した。具体的には、VP1のDEループ領域 (137-138アミノ酸領域、古典的にVP1遺伝子の4番目のAlaをアミノ酸番号1として) またはHIループ領域 (273-274アミノ酸領域) をGILGFVFTLで置き換え、M1 CTL エピトープ挿入SV40 VP1挿入変異体を作製した (M1-DE-VP1、M1-HI-VP1)。作製方法は、SV40 VP1をコードするpFastBac 1-SV40 wild type VP1をテンプレートして、Overhang PCR法にて作製した。プライマーは以下のものを用いた。
1st round
5'-SalI-Kozac-SV40 VP1
AAAAGTCGACACCATGAAGATGGCCCCAACAAAAAG(配列番号40)
3'-DE2 loop (M1)
CGTGAACACAAAGCCCAAAATGCCGCCACCGCCATGAGTTTTTTGTGTCCCTGAATG(配列番号41)
5'-DE2 loop (M1)
CATTTTGGGCTTTGTGTTCACGTTGGGCGGCGGTGGTGCTGGAAAACCCATTCAAG(配列番号42)
3'-KpnI-SV40 VP1
AAAAGGTACCTCACTGCATTCTAGTTGTGGTTTG(配列番号43)
5'-SalI-Kozac-SV40 VP1
AAAAGTCGACACCATGAAGATGGCCCCAACAAAAAG(配列番号44)
3'-KpnI-SV40 VP1
AAAAGGTACCTCACTGCATTCTAGTTGTGGTTTG(配列番号45)
1st round
5'-SalI-Kozac-SV40 VP1
AAAAGTCGACACCATGAAGATGGCCCCAACAAAAAG(配列番号46)
3'-HI1 loop (M1)
CGTGAACACAAAGCCCAAAATGCCGCCACCGCCGTTGGTAAACAGCCCACAAATG(配列番号47)
5'-HI1 loop (M1)
CATTTTGGGCTTTGTGTTCACGTTGGGCGGCGGTGGAACACAGCAGTGGAAGGG(配列番号48)
3'-KpnI-SV40 VP1
AAAAGGTACCTCACTGCATTCTAGTTGTGGTTTG(配列番号49)
5'-SalI-Kozac-SV40 VP1
AAAAGTCGACACCATGAAGATGGCCCCAACAAAAAG(配列番号50)
3'-KpnI-SV40 VP1
AAAAGGTACCTCACTGCATTCTAGTTGTGGTTTG(配列番号51)
バキュロウイルスゲノムを保持する大腸菌DH10bac (invitrogen) にM1-DE-VP1及びM1-HI-VP1プラスミドを導入して形質転換し、M1-DE-VP1及びM1-HI-VP1を組込んだ組換えバキュロウイルスゲノムを調製した。組換えバキュロウイルスゲノムをSf-9細胞にトランスフェクションし、三日後にその上清を回収して、組換えバキュロウイルスを含む溶液とした。この溶液の一部を再び、Sf-9細胞に感染させることで、組換えバキュロウイルスタイターを上昇させて、組換えバキュロウイルスのストック溶液とした。
M1-DE-VP1及びM1-HI-VP1を組込んだ組換えバキュロウイルスを、M.O.I. (multiplicity of infection) = 0.05~0.2で3x107個のSf-9細胞に感染させた。感染3日後にSf-9細胞を回収、PBS(-)で洗浄した後、細胞を200 μlの50% Opti-prepTM 溶液 (20 mM Tris-HCl(pH7.9), 50% Opti-prepTM) で再懸濁し、超音波破砕して、ライセート溶液とした (図1左図、レーン1-4、 M1-DE-VP1及び右図、レーン1-4、M1-HI-VP1)。一方、細胞を1 mlのVP1 超音波処理 溶液 (20 mM Tris-HCl(pH7.9), 1% sodium deoxycholate) で再懸濁し、超音波破砕した後、15,000 rpm、4℃で5分間遠心して上清とペレットに分画し、上清を除いた。残ったペレットに50% Opti-prepTM 溶液 (20 mM Tris-HCl(pH7.9), 50%Opti-prepTM) を加えて、ピペッティング、または、超音波処理することで再懸濁し、ペレット溶液とした (図1左図、レーン5-8、 M1-DE-VP1及び右図、レーン5-8、M1-HI-VP1)。
上記のM1-DE-VLP及びM1-HI-VLPを含むライセート溶液 (100 μl) 及びペレット溶液 (100 μl)にアジュバントとしてはCpG-olygodeoxyncleotide5002 (TCCATGACGTTCTTGATGTT:配列番号52) アジュバントを5 μg加えて、8週齢のトランスジェニックマウスに足蹠、踵関節、静脈内、腹腔内、筋肉内、鼻腔内の経路で免疫した。トランスジェニックマウスは、C57BL/6をバックグラウンドとし、HLA-A*0201とH-2Dbのキメラでさらにヒトのβ2mを融合したものを発現しているものを使用した。このマウスは、マウスのβ2mおよびH-2Dbをノックアウトしているため、マウス由来のMHC class Iは細胞表面に露出していないと考えられる。
免疫1週間または2週間後、マウスから脾臓及び肺を摘出し、それぞれ5 mlのRPMI-1640 培地の入ったφ6 cm ディッシュに置いた。ピンセットを用いて脾臓を培地内で、良く揉みほぐし、培地に溶出されたリンパ球を含む溶液を15 ml チューブに移し、もう一度、φ6 cm ディッシュを5 mlのRPMI-1640 培地で洗浄し、上清をその15 ml チューブに加え、総量 10 mlとした。すぐに15 ml チューブの底にたまる組織断片を残して、上清を再び新しい15 ml チューブに移し、室温、1,200 rpmで5分間遠心し、リンパ球をペレットに移行させた。上清を除いて、ペレットをほぐした後、赤血球を除くために、250 μlのNH4Cl-トリス溶液を加えかき混ぜた後、すばやく10 mlのRPMI-1640 培地を加え、室温、1,2000 rpmで5分間遠心し、リンパ球をペレットに移行させた。上清を除いて、ペレットをほぐした後、再び10 mlのRPMI-1640 培地を加え、変性した赤血球を吸わないように、ピペットで新しい15 ml チューブに移して、再び室温、1,200 rpmで5分間遠心した。上清を除いた後、ペレットをほぐしてもう一度10 mlのRPMI-1640 培地で懸濁し、室温、1,200 rpmで5分間遠心した。上清を除いて、最後に2 mlの10% FCS混入RPMI-1640 培地で懸濁した。リンパ球を数えるために、490 μlの2%酢酸溶液に10 μlの上記懸濁溶液を加え、ビュルケルチュルク血球計算版で、細胞数を算出し、2x107 cells/mlとなるように10%FCS混入RPMI-1640 培地で希釈、または、濃縮した。
上記の方法で脾臓及び肺から回収したリンパ球中に、M1のCTLエピトープ配列 (GILGFVFTL)に反応して誘導されるCTLが存在することを調べるために、ICS解析を行った。96 ウェル丸底プレートに10% FCS混入RPMI-1640 培地で25倍希釈したBD GolgiPlugTM (BD) を1 ウェル当り5 μl加え、そこにさらに10% FCS混入RPMI-1640 培地で希釈した20 μMのM1のCTLエピトープのペプチド(GILGFVFTL、Operon)を100 μl加えた。ネガティブコントロールとして、ペプチドを含まない10% RCS混入RPMI-1640 培地を100 μl加えた。
実施例5の方法で脾臓から回収したリンパ球中に、M1のCTLエピトープ配列 (GILGFVFTL)に反応して誘導される細胞傷害活性を有するCTLが存在することを調べるために、CD107-ICS解析を行った。96 ウェル丸底プレートに10% FCS混入RPMI-1640 培地で25倍希釈したBD GolgiPlugTM (BD) を1 ウェル当り5 μl、及び、10% FCS混入RPMI-1640 培地で25倍希釈したBD GolgiStopTM (BD) を1 ウェル当り5 μl、FITC Rat Anti-Mouse CD107a Clone: 1D4B (BD Pharmingen)を0.8 μg加え、 そこにさらに10% FCS混入RPMI-1640 培地で希釈した20 μMのM1のCTLエピトープのペプチド(GILGFVFTL、Operon)を100 μl加えた。ネガティブコントロールとして、ペプチドを含まない10% RCS混入RPMI-1640 培地を100 μl加えた。
実施例7で示した、CD8陽性・CD107陽性・細胞内IFN-γ陽性細胞が、細胞傷害することを示すために、51Chrome release解析を行った。免疫をしていないマウスから脾臓のリンパ球を実施例5と同様の方法で調製した。2 mlの10% FCS混入RPMI-1640 培地中の免疫していないマウスの脾臓リンパ球2.4x108 cellsに、10 mMのM1のCTLエピトープのペプチド(GILGFVFTL、Operon)を4 μl加え、15 mlチューブ中で、2時間、37℃、5% CO2 incubatorでインキュベートした。インキュベート後、20 Gy (グレイ) のX線を照射した。照射後、室温、1,2000 rpmで5分間遠心し、リンパ球をペレットに移行させた。上清を除いて、ペレットをほぐした後、2 mlの10% FCS混入RPMI-1640 培地を加え、リンパ球を数えるために、490 μlの2%酢酸溶液に10 μlのリンパ球溶液を加え、ビュルケルチュルク血球計算版で、細胞数を算出し、5x106 cells/mlとなるように10%FCS混入RPMI-1640 培地で希釈した。
実施例8で示した、M1のCTLエピトープのペプチド(GILGFVFTL)を提示している細胞を選択的に傷害するCTLが、実際に動物体内でM1のCTLエピトープのペプチド(GILGFVFTL)を提示している細胞を選択的に傷害することを解析するために、In vivo CTL解析を行った。筋肉注射によって約50 μgのM1-DE-VLP、及び、腹腔注射によって約50 μgのM1-HI-VLPを免疫した。
実施例5と同様の方法で、C57BL/6マウスの脾臓から赤血球を除いたリンパ球を精製し、1x107 cells/mlとなるように10% FCS混入RPMI-1640 培地で希釈した。96 ウェルプレートに、a)何も加えない50% Opti-prepTM 溶液 (20 mM Tris-HCl(pH7.9), 50% Opti-prepTM)を5 μl、b)実施例3で調製したM1-DE-VLP溶液を5 μl、もしくは、c)M1-HI-VLP溶液を5 μl加え、そこに、上記で調製したリンパ球を50 μl加え、1 ウェル当りの総量が200 μlとなるように、10% FCS混入RPMI-1640 培地を加えた。ピペッティングで混合した後、このプレートを6時間、または24時間、37℃、5% CO2 incubatorでインキュベートした。
M1のCTLエピトープのペプチド(GILGFVFTL)を提示している細胞を選択的に傷害するCTLが、インフルエンザウイルス感染に与える影響を解析するために、ウイルスチャレンジ実験を行った。筋肉注射によって約50 μgのM1-DE-VLP、及び、腹腔注射によって約50 μgのM1-HI-VLPをHLA-A2トランスジェニックマウス (HHDマウス) に免疫した。
実施例1から3と同じ方法で、M1のCTLエピトープ配列(GILGFVFTL)の代わりに、がん抗原として知られているWT1のHLA-A2に対するCTLエピトープ配列(RMFPNAPYL)を挿入したVLPを調製した (WT1WT-DE-VLP、WT1WT-HI-VLP) (図9-a) 。実施例4から6と同じ方法で、HLA-A2トランスジェニックマウスに免疫し、リンパ球を調製、WT1のCTLエピトープ配列に対するICS解析を行った (図9-b) 。また、実施例8と同様の方法により、WT1のCTLエピトープ配列に対する51Chrome release解析を行った。
実施例3から6と同様の方法によって、M1-DE-VLP (図10, 右上図) 及びM1-HI-VLP (図10, 右下図) のICS解析を行った。免疫は、実施例4と同様に調製した100 μlのM1-DE-VLP及びM1-HI-VLPのペレット溶液を鼠蹊部皮下 (subcutaneous, s.c.) に注射することによって行った。
実施例5と同様の方法で、HLA-A2トランスジェニックマウスの脾臓から赤血球を除いたリンパ球を精製し、1x107 cells/mlとなるように10% FCS混入RPMI-1640 培地で希釈した。96 ウェルプレートに、a)何も加えない50% Opti-prepTM 溶液 (20 mM Tris-HCl(pH7.9), 50% Opti-prepTM) を5 μl、b)高度に精製された野生型SV40 VP1-VLP (500 ng/μl) を5μl、c)実施例3で調製したM1-DE-VLP溶液を5 μl、もしくは、d)M1-HI-VLP溶液を5 μl加え、そこに、上記で調製したリンパ球を50 μl加え、1 ウェル当りの総量が200 μlとなるように、10%FCS混入RPMI-1640 培地を加えた。または、上記で精製したリンパ球50 μlに10% FCS混入RPMI-1640 培地で100 ng/μlとなるように希釈したLPS (Sigma) を150 μl加えた。ピペッティングで混合した後、このプレートを6時間、または24時間、37℃、5% CO2 incubatorでインキュベートした。インキュベート後4℃、1,400 rpmでスピンダウンして上清をエッペンドルフチューブに回収し、4℃、15,000 rpmでスピンダウンして上清を新しいエッペンドルフチューブに回収した。回収した培養上清の50 μlをIL-12のenzyme-linked immunosorbent assay (ELISA) (Mouse Total IL-12 ELISA Kit, Thermo SCIENTIFIC) でキットのプロトコールに従って解析し、培養上清中のIL-12の濃度を算出した (図11)。
Claims (10)
- シミアンウイルス40のVP1から構成されるウイルス様粒子を含有する細胞傷害性T細胞誘導剤であって、前記VP1のDEループ及び/又はHIループ中にT細胞エピトープペプチドが挿入されていることを特徴とする細胞傷害性T細胞誘導剤。
- T細胞エピトープペプチドが、ウイルスタンパク質由来のペプチドであることを特徴とする請求項1に記載の細胞傷害性T細胞誘導剤。
- T細胞エピトープペプチドが、インフルエンザウイルスのHA、NA、M1、M2、NP、NS1、NS2、PA、PB1、PB2、又はPB1-F2由来のペプチドであることを特徴とする請求項1に記載の細胞傷害性T細胞誘導剤。
- T細胞エピトープペプチドが、癌細胞に特異的なタンパク質由来のペプチドであることを特徴とする請求項1に記載の細胞傷害性T細胞誘導剤。
- T細胞エピトープペプチドが、Melan-A/MART-1、gp100、MAGE-A3、MAGE-A10、CEA、HER2/new、NY-E50-1、WT-1、又はhTERT由来のペプチドあることを特徴とする請求項1に記載の細胞傷害性T細胞誘導剤。
- T細胞エピトープペプチドが、配列番号1乃至39のいずれかの配列番号に記載されたアミノ酸配列からなるペプチドであることを特徴とする請求項1に記載の細胞傷害性T細胞誘導剤。
- シミアンウイルス40のVP1から構成されるウイルス様粒子であって、前記VP1のDEループ及び/又はHIループ中にウイルスタンパク質由来のT細胞エピトープペプチドが挿入されているウイルス様粒子を、ヒト以外の動物に投与することを特徴とするヒト以外の動物のウイルス性疾患の予防又は治療方法。
- シミアンウイルス40のVP1から構成されるウイルス様粒子であって、前記VP1のDEループ及び/又はHIループ中に癌細胞に特異的なタンパク質由来のT細胞エピトープペプチドが挿入されているウイルス様粒子を、ヒト以外の動物に投与することを特徴とするヒト以外の動物の癌の予防又は治療方法。
- シミアンウイルス40のVP1から構成されるウイルス様粒子であって、前記VP1のDEループ及び/又はHIループ中にウイルスタンパク質由来のT細胞エピトープペプチドが挿入されているウイルス様粒子を、ヒトに投与することを特徴とするヒトのウイルス性疾患の予防又は治療方法。
- シミアンウイルス40のVP1から構成されるウイルス様粒子であって、前記VP1のDEループ及び/又はHIループ中に癌細胞に特異的なタンパク質由来のT細胞エピトープペプチドが挿入されているウイルス様粒子を、ヒトに投与することを特徴とするヒトの癌の予防又は治療方法。
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CN (1) | CN103747799A (ja) |
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US10994007B2 (en) | 2015-11-30 | 2021-05-04 | Saitama Medical University | Immunity inducer |
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CN104277094A (zh) * | 2014-07-04 | 2015-01-14 | 文康医疗技术(深圳)有限公司 | Dc靶向肽及其应用 |
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WO2006004173A1 (ja) | 2004-07-01 | 2006-01-12 | The Circle For The Promotion Of Science And Engineering | 生理的条件下でのウイルス粒子様構造体及びその形成方法 |
WO2006088229A1 (ja) | 2005-02-16 | 2006-08-24 | The Circle For The Promotion Of Science And Engineering | 改変されたウイルスカプシドタンパク質及びその使用 |
JP2009197001A (ja) | 2001-09-14 | 2009-09-03 | Cytos Biotechnology Ag | ウィルス様粒子中への免疫賦活物質のパッケージ化:調製法および使用法 |
JP2011107874A (ja) | 2009-11-16 | 2011-06-02 | Aisin Aw Co Ltd | 施設情報提供装置 |
JP2011191208A (ja) | 2010-03-15 | 2011-09-29 | Toru Mino | 土壌水分測定方法及び土壌水分測定装置 |
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WO2008040060A1 (en) * | 2006-10-04 | 2008-04-10 | The University Of Queensland | Vlp based vaccine delivery system |
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- 2012-04-24 WO PCT/JP2012/060910 patent/WO2012157408A1/ja active Application Filing
- 2012-04-24 EP EP12786584.8A patent/EP2708238A4/en not_active Withdrawn
- 2012-04-24 JP JP2013515059A patent/JPWO2012157408A1/ja active Pending
- 2012-04-24 CN CN201280031425.8A patent/CN103747799A/zh active Pending
- 2012-04-24 KR KR1020137033082A patent/KR20140056184A/ko not_active Application Discontinuation
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JP2009197001A (ja) | 2001-09-14 | 2009-09-03 | Cytos Biotechnology Ag | ウィルス様粒子中への免疫賦活物質のパッケージ化:調製法および使用法 |
WO2006004173A1 (ja) | 2004-07-01 | 2006-01-12 | The Circle For The Promotion Of Science And Engineering | 生理的条件下でのウイルス粒子様構造体及びその形成方法 |
WO2006088229A1 (ja) | 2005-02-16 | 2006-08-24 | The Circle For The Promotion Of Science And Engineering | 改変されたウイルスカプシドタンパク質及びその使用 |
JP2011107874A (ja) | 2009-11-16 | 2011-06-02 | Aisin Aw Co Ltd | 施設情報提供装置 |
JP2011191208A (ja) | 2010-03-15 | 2011-09-29 | Toru Mino | 土壌水分測定方法及び土壌水分測定装置 |
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Cited By (1)
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US10994007B2 (en) | 2015-11-30 | 2021-05-04 | Saitama Medical University | Immunity inducer |
Also Published As
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US20140286978A1 (en) | 2014-09-25 |
KR20140056184A (ko) | 2014-05-09 |
CN103747799A (zh) | 2014-04-23 |
JPWO2012157408A1 (ja) | 2014-07-31 |
EP2708238A4 (en) | 2014-12-24 |
EP2708238A1 (en) | 2014-03-19 |
CA2836028A1 (en) | 2012-11-22 |
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