US20180057531A1 - Muc1-derived peptide, and pharmaceutical composition for treatment or prevention of cancer, immunity-inducing agent and method for manufacturing antigen presenting cell using same - Google Patents
Muc1-derived peptide, and pharmaceutical composition for treatment or prevention of cancer, immunity-inducing agent and method for manufacturing antigen presenting cell using same Download PDFInfo
- Publication number
- US20180057531A1 US20180057531A1 US15/556,766 US201615556766A US2018057531A1 US 20180057531 A1 US20180057531 A1 US 20180057531A1 US 201615556766 A US201615556766 A US 201615556766A US 2018057531 A1 US2018057531 A1 US 2018057531A1
- Authority
- US
- United States
- Prior art keywords
- peptide
- amino acid
- hla
- cancer
- antigen presenting
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
Links
- 108090000765 processed proteins & peptides Proteins 0.000 title claims abstract description 140
- 210000000612 antigen-presenting cell Anatomy 0.000 title claims description 53
- 238000000034 method Methods 0.000 title claims description 49
- 206010028980 Neoplasm Diseases 0.000 title claims description 48
- 201000011510 cancer Diseases 0.000 title claims description 42
- 239000008194 pharmaceutical composition Substances 0.000 title claims description 37
- 230000001939 inductive effect Effects 0.000 title claims description 27
- 230000036039 immunity Effects 0.000 title claims description 26
- 239000003795 chemical substances by application Substances 0.000 title claims description 23
- 238000004519 manufacturing process Methods 0.000 title claims description 11
- 230000002265 prevention Effects 0.000 title claims description 11
- 101100346932 Mus musculus Muc1 gene Proteins 0.000 title 1
- 125000000539 amino acid group Chemical group 0.000 claims abstract description 16
- 150000001413 amino acids Chemical group 0.000 claims description 58
- 229940024606 amino acid Drugs 0.000 claims description 36
- 210000001151 cytotoxic T lymphocyte Anatomy 0.000 claims description 30
- 229960005486 vaccine Drugs 0.000 claims description 11
- 238000000338 in vitro Methods 0.000 claims description 9
- ROHFNLRQFUQHCH-UHFFFAOYSA-N Leucine Chemical group CC(C)CC(N)C(O)=O ROHFNLRQFUQHCH-UHFFFAOYSA-N 0.000 claims description 8
- ROHFNLRQFUQHCH-YFKPBYRVSA-N L-leucine Chemical group CC(C)C[C@H](N)C(O)=O ROHFNLRQFUQHCH-YFKPBYRVSA-N 0.000 claims description 7
- FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical group CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 0.000 claims description 7
- KZSNJWFQEVHDMF-UHFFFAOYSA-N Valine Chemical group CC(C)C(N)C(O)=O KZSNJWFQEVHDMF-UHFFFAOYSA-N 0.000 claims description 7
- 229930182817 methionine Chemical group 0.000 claims description 7
- 239000004474 valine Chemical group 0.000 claims description 7
- COLNVLDHVKWLRT-QMMMGPOBSA-N L-phenylalanine Chemical group OC(=O)[C@@H](N)CC1=CC=CC=C1 COLNVLDHVKWLRT-QMMMGPOBSA-N 0.000 claims description 6
- QIVBCDIJIAJPQS-VIFPVBQESA-N L-tryptophane Chemical group C1=CC=C2C(C[C@H](N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-VIFPVBQESA-N 0.000 claims description 6
- QIVBCDIJIAJPQS-UHFFFAOYSA-N Tryptophan Chemical group C1=CC=C2C(CC(N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-UHFFFAOYSA-N 0.000 claims description 6
- 230000005847 immunogenicity Effects 0.000 claims description 6
- COLNVLDHVKWLRT-UHFFFAOYSA-N phenylalanine Chemical group OC(=O)C(N)CC1=CC=CC=C1 COLNVLDHVKWLRT-UHFFFAOYSA-N 0.000 claims description 6
- 125000001433 C-terminal amino-acid group Chemical group 0.000 claims description 5
- KZSNJWFQEVHDMF-BYPYZUCNSA-N L-valine Chemical group CC(C)[C@H](N)C(O)=O KZSNJWFQEVHDMF-BYPYZUCNSA-N 0.000 claims description 4
- ZDXPYRJPNDTMRX-VKHMYHEASA-N L-glutamine Chemical group OC(=O)[C@@H](N)CCC(N)=O ZDXPYRJPNDTMRX-VKHMYHEASA-N 0.000 claims description 3
- AGPKZVBTJJNPAG-WHFBIAKZSA-N L-isoleucine Chemical group CC[C@H](C)[C@H](N)C(O)=O AGPKZVBTJJNPAG-WHFBIAKZSA-N 0.000 claims description 3
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Chemical group OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 claims description 3
- 229960000310 isoleucine Drugs 0.000 claims description 3
- AGPKZVBTJJNPAG-UHFFFAOYSA-N isoleucine Chemical group CCC(C)C(N)C(O)=O AGPKZVBTJJNPAG-UHFFFAOYSA-N 0.000 claims description 3
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 claims description 3
- 125000001493 tyrosinyl group Chemical group [H]OC1=C([H])C([H])=C(C([H])=C1[H])C([H])([H])C([H])(N([H])[H])C(*)=O 0.000 claims description 3
- 125000003275 alpha amino acid group Chemical group 0.000 abstract 1
- 210000004027 cell Anatomy 0.000 description 58
- 210000004443 dendritic cell Anatomy 0.000 description 28
- 102000004196 processed proteins & peptides Human genes 0.000 description 24
- 102000007298 Mucin-1 Human genes 0.000 description 21
- 108010008707 Mucin-1 Proteins 0.000 description 21
- 230000027455 binding Effects 0.000 description 20
- 102000040430 polynucleotide Human genes 0.000 description 20
- 108091033319 polynucleotide Proteins 0.000 description 20
- 239000002157 polynucleotide Substances 0.000 description 20
- 102100028972 HLA class I histocompatibility antigen, A alpha chain Human genes 0.000 description 14
- 108010075704 HLA-A Antigens Proteins 0.000 description 14
- 108010021727 HLA-A*24:02 antigen Proteins 0.000 description 13
- 238000002474 experimental method Methods 0.000 description 12
- 102210042925 HLA-A*02:01 Human genes 0.000 description 11
- 239000000427 antigen Substances 0.000 description 11
- 108091007433 antigens Proteins 0.000 description 11
- 102000036639 antigens Human genes 0.000 description 11
- 239000001963 growth medium Substances 0.000 description 11
- 108090000623 proteins and genes Proteins 0.000 description 11
- 241000282414 Homo sapiens Species 0.000 description 9
- 230000002163 immunogen Effects 0.000 description 7
- 238000002347 injection Methods 0.000 description 7
- 239000007924 injection Substances 0.000 description 7
- 239000000047 product Substances 0.000 description 7
- 238000012286 ELISA Assay Methods 0.000 description 6
- 239000002671 adjuvant Substances 0.000 description 6
- 230000006698 induction Effects 0.000 description 6
- 238000001638 lipofection Methods 0.000 description 6
- 102000015728 Mucins Human genes 0.000 description 5
- 108010063954 Mucins Proteins 0.000 description 5
- 238000002659 cell therapy Methods 0.000 description 5
- 230000001472 cytotoxic effect Effects 0.000 description 5
- 230000000694 effects Effects 0.000 description 5
- 102000004169 proteins and genes Human genes 0.000 description 5
- 238000002560 therapeutic procedure Methods 0.000 description 5
- 206010006187 Breast cancer Diseases 0.000 description 4
- 102100037850 Interferon gamma Human genes 0.000 description 4
- 108010074328 Interferon-gamma Proteins 0.000 description 4
- 206010061902 Pancreatic neoplasm Diseases 0.000 description 4
- 238000010790 dilution Methods 0.000 description 4
- 239000012895 dilution Substances 0.000 description 4
- 230000002998 immunogenetic effect Effects 0.000 description 4
- 238000009169 immunotherapy Methods 0.000 description 4
- 208000015486 malignant pancreatic neoplasm Diseases 0.000 description 4
- 201000002528 pancreatic cancer Diseases 0.000 description 4
- 208000008443 pancreatic carcinoma Diseases 0.000 description 4
- 239000013641 positive control Substances 0.000 description 4
- 238000002360 preparation method Methods 0.000 description 4
- BFSVOASYOCHEOV-UHFFFAOYSA-N 2-diethylaminoethanol Chemical compound CCN(CC)CCO BFSVOASYOCHEOV-UHFFFAOYSA-N 0.000 description 3
- 208000026310 Breast neoplasm Diseases 0.000 description 3
- 102000004127 Cytokines Human genes 0.000 description 3
- 108090000695 Cytokines Proteins 0.000 description 3
- 229920002307 Dextran Polymers 0.000 description 3
- 238000002965 ELISA Methods 0.000 description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 3
- DNIAPMSPPWPWGF-UHFFFAOYSA-N Propylene glycol Chemical compound CC(O)CO DNIAPMSPPWPWGF-UHFFFAOYSA-N 0.000 description 3
- 210000001744 T-lymphocyte Anatomy 0.000 description 3
- 230000015572 biosynthetic process Effects 0.000 description 3
- 239000001506 calcium phosphate Substances 0.000 description 3
- 229910000389 calcium phosphate Inorganic materials 0.000 description 3
- 235000011010 calcium phosphates Nutrition 0.000 description 3
- 238000003163 cell fusion method Methods 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 3
- 238000010494 dissociation reaction Methods 0.000 description 3
- 238000004520 electroporation Methods 0.000 description 3
- 210000001808 exosome Anatomy 0.000 description 3
- 238000009472 formulation Methods 0.000 description 3
- 238000011534 incubation Methods 0.000 description 3
- 210000004698 lymphocyte Anatomy 0.000 description 3
- 210000002540 macrophage Anatomy 0.000 description 3
- 238000000520 microinjection Methods 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 210000001616 monocyte Anatomy 0.000 description 3
- 239000013642 negative control Substances 0.000 description 3
- 238000000746 purification Methods 0.000 description 3
- 150000003839 salts Chemical class 0.000 description 3
- QORWJWZARLRLPR-UHFFFAOYSA-H tricalcium bis(phosphate) Chemical compound [Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O QORWJWZARLRLPR-UHFFFAOYSA-H 0.000 description 3
- 125000002987 valine group Chemical group [H]N([H])C([H])(C(*)=O)C([H])(C([H])([H])[H])C([H])([H])[H] 0.000 description 3
- WRMNZCZEMHIOCP-UHFFFAOYSA-N 2-phenylethanol Chemical compound OCCC1=CC=CC=C1 WRMNZCZEMHIOCP-UHFFFAOYSA-N 0.000 description 2
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 2
- 150000008574 D-amino acids Chemical class 0.000 description 2
- 102000003886 Glycoproteins Human genes 0.000 description 2
- 108090000288 Glycoproteins Proteins 0.000 description 2
- 102100039620 Granulocyte-macrophage colony-stimulating factor Human genes 0.000 description 2
- 101001136981 Homo sapiens Proteasome subunit beta type-9 Proteins 0.000 description 2
- 108010050904 Interferons Proteins 0.000 description 2
- 102000014150 Interferons Human genes 0.000 description 2
- 108010002350 Interleukin-2 Proteins 0.000 description 2
- 108090000978 Interleukin-4 Proteins 0.000 description 2
- 102100035764 Proteasome subunit beta type-9 Human genes 0.000 description 2
- 102100030086 Receptor tyrosine-protein kinase erbB-2 Human genes 0.000 description 2
- 101710100968 Receptor tyrosine-protein kinase erbB-2 Proteins 0.000 description 2
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 description 2
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 2
- 102000016266 T-Cell Antigen Receptors Human genes 0.000 description 2
- 108010092262 T-Cell Antigen Receptors Proteins 0.000 description 2
- 108060008682 Tumor Necrosis Factor Proteins 0.000 description 2
- 230000009471 action Effects 0.000 description 2
- 150000003862 amino acid derivatives Chemical class 0.000 description 2
- UCMIRNVEIXFBKS-UHFFFAOYSA-N beta-alanine Chemical compound NCCC(O)=O UCMIRNVEIXFBKS-UHFFFAOYSA-N 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 229940022399 cancer vaccine Drugs 0.000 description 2
- 238000009566 cancer vaccine Methods 0.000 description 2
- OSASVXMJTNOKOY-UHFFFAOYSA-N chlorobutanol Chemical compound CC(C)(O)C(Cl)(Cl)Cl OSASVXMJTNOKOY-UHFFFAOYSA-N 0.000 description 2
- XVOYSCVBGLVSOL-UHFFFAOYSA-N cysteic acid Chemical compound OC(=O)C(N)CS(O)(=O)=O XVOYSCVBGLVSOL-UHFFFAOYSA-N 0.000 description 2
- 230000005593 dissociations Effects 0.000 description 2
- 238000011156 evaluation Methods 0.000 description 2
- 230000013595 glycosylation Effects 0.000 description 2
- 238000006206 glycosylation reaction Methods 0.000 description 2
- 230000008105 immune reaction Effects 0.000 description 2
- 229940079322 interferon Drugs 0.000 description 2
- 210000000265 leukocyte Anatomy 0.000 description 2
- 239000002502 liposome Substances 0.000 description 2
- 230000003211 malignant effect Effects 0.000 description 2
- 238000001840 matrix-assisted laser desorption--ionisation time-of-flight mass spectrometry Methods 0.000 description 2
- LXCFILQKKLGQFO-UHFFFAOYSA-N methylparaben Chemical compound COC(=O)C1=CC=C(O)C=C1 LXCFILQKKLGQFO-UHFFFAOYSA-N 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 229940051875 mucins Drugs 0.000 description 2
- 210000005259 peripheral blood Anatomy 0.000 description 2
- 239000011886 peripheral blood Substances 0.000 description 2
- 239000002243 precursor Substances 0.000 description 2
- 239000003755 preservative agent Substances 0.000 description 2
- 230000002335 preservative effect Effects 0.000 description 2
- 238000010532 solid phase synthesis reaction Methods 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 238000001890 transfection Methods 0.000 description 2
- LWIHDJKSTIGBAC-UHFFFAOYSA-K tripotassium phosphate Chemical compound [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 description 2
- 102000003390 tumor necrosis factor Human genes 0.000 description 2
- 239000003799 water insoluble solvent Substances 0.000 description 2
- 239000003021 water soluble solvent Substances 0.000 description 2
- KQMBIBBJWXGSEI-ROLXFIACSA-N (2s)-2-amino-3-hydroxy-3-(1h-imidazol-5-yl)propanoic acid Chemical compound OC(=O)[C@@H](N)C(O)C1=CNC=N1 KQMBIBBJWXGSEI-ROLXFIACSA-N 0.000 description 1
- AJFGLTPLWPTALJ-SSDOTTSWSA-N (2s)-2-azaniumyl-2-(fluoromethyl)-3-(1h-imidazol-5-yl)propanoate Chemical compound FC[C@@](N)(C(O)=O)CC1=CN=CN1 AJFGLTPLWPTALJ-SSDOTTSWSA-N 0.000 description 1
- MSECZMWQBBVGEN-LURJTMIESA-N (2s)-2-azaniumyl-4-(1h-imidazol-5-yl)butanoate Chemical compound OC(=O)[C@@H](N)CCC1=CN=CN1 MSECZMWQBBVGEN-LURJTMIESA-N 0.000 description 1
- 125000003088 (fluoren-9-ylmethoxy)carbonyl group Chemical group 0.000 description 1
- JCIIKRHCWVHVFF-UHFFFAOYSA-N 1,2,4-thiadiazol-5-amine;hydrochloride Chemical compound Cl.NC1=NC=NS1 JCIIKRHCWVHVFF-UHFFFAOYSA-N 0.000 description 1
- FUOOLUPWFVMBKG-UHFFFAOYSA-N 2-Aminoisobutyric acid Chemical compound CC(C)(N)C(O)=O FUOOLUPWFVMBKG-UHFFFAOYSA-N 0.000 description 1
- GOJUJUVQIVIZAV-UHFFFAOYSA-N 2-amino-4,6-dichloropyrimidine-5-carbaldehyde Chemical group NC1=NC(Cl)=C(C=O)C(Cl)=N1 GOJUJUVQIVIZAV-UHFFFAOYSA-N 0.000 description 1
- 108700028369 Alleles Proteins 0.000 description 1
- LSNNMFCWUKXFEE-UHFFFAOYSA-M Bisulfite Chemical compound OS([O-])=O LSNNMFCWUKXFEE-UHFFFAOYSA-M 0.000 description 1
- 208000003174 Brain Neoplasms Diseases 0.000 description 1
- 210000001239 CD8-positive, alpha-beta cytotoxic T lymphocyte Anatomy 0.000 description 1
- 201000009030 Carcinoma Diseases 0.000 description 1
- 102000019034 Chemokines Human genes 0.000 description 1
- 108010012236 Chemokines Proteins 0.000 description 1
- 102000007644 Colony-Stimulating Factors Human genes 0.000 description 1
- 108010071942 Colony-Stimulating Factors Proteins 0.000 description 1
- -1 Fmoc amino acids Chemical class 0.000 description 1
- 201000003741 Gastrointestinal carcinoma Diseases 0.000 description 1
- 101150118346 HLA-A gene Proteins 0.000 description 1
- 102000015789 HLA-DP Antigens Human genes 0.000 description 1
- 108010010378 HLA-DP Antigens Proteins 0.000 description 1
- 101000599940 Homo sapiens Interferon gamma Proteins 0.000 description 1
- 208000008839 Kidney Neoplasms Diseases 0.000 description 1
- AHLPHDHHMVZTML-BYPYZUCNSA-N L-Ornithine Chemical compound NCCC[C@H](N)C(O)=O AHLPHDHHMVZTML-BYPYZUCNSA-N 0.000 description 1
- 150000008575 L-amino acids Chemical class 0.000 description 1
- LRQKBLKVPFOOQJ-YFKPBYRVSA-N L-norleucine Chemical compound CCCC[C@H]([NH3+])C([O-])=O LRQKBLKVPFOOQJ-YFKPBYRVSA-N 0.000 description 1
- 206010058467 Lung neoplasm malignant Diseases 0.000 description 1
- 206010025538 Malignant ascites Diseases 0.000 description 1
- 101000767152 Mus musculus General vesicular transport factor p115 Proteins 0.000 description 1
- 101001093920 Mus musculus SEC14-like protein 2 Proteins 0.000 description 1
- 230000004989 O-glycosylation Effects 0.000 description 1
- 102000015636 Oligopeptides Human genes 0.000 description 1
- 108010038807 Oligopeptides Proteins 0.000 description 1
- AHLPHDHHMVZTML-UHFFFAOYSA-N Orn-delta-NH2 Natural products NCCCC(N)C(O)=O AHLPHDHHMVZTML-UHFFFAOYSA-N 0.000 description 1
- UTJLXEIPEHZYQJ-UHFFFAOYSA-N Ornithine Natural products OC(=O)C(C)CCCN UTJLXEIPEHZYQJ-UHFFFAOYSA-N 0.000 description 1
- 206010033128 Ovarian cancer Diseases 0.000 description 1
- 206010061535 Ovarian neoplasm Diseases 0.000 description 1
- 239000004372 Polyvinyl alcohol Substances 0.000 description 1
- 206010060862 Prostate cancer Diseases 0.000 description 1
- 208000000236 Prostatic Neoplasms Diseases 0.000 description 1
- 206010038389 Renal cancer Diseases 0.000 description 1
- 238000012952 Resampling Methods 0.000 description 1
- 208000000453 Skin Neoplasms Diseases 0.000 description 1
- 102220497176 Small vasohibin-binding protein_T47D_mutation Human genes 0.000 description 1
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 description 1
- DWAQJAXMDSEUJJ-UHFFFAOYSA-M Sodium bisulfite Chemical compound [Na+].OS([O-])=O DWAQJAXMDSEUJJ-UHFFFAOYSA-M 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 150000001298 alcohols Chemical class 0.000 description 1
- HRRYYCWYCMJNGA-ZETCQYMHSA-N alpha-methyl-L-histidine Chemical compound OC(=O)[C@](N)(C)CC1=CN=CN1 HRRYYCWYCMJNGA-ZETCQYMHSA-N 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- WNROFYMDJYEPJX-UHFFFAOYSA-K aluminium hydroxide Chemical compound [OH-].[OH-].[OH-].[Al+3] WNROFYMDJYEPJX-UHFFFAOYSA-K 0.000 description 1
- ILRRQNADMUWWFW-UHFFFAOYSA-K aluminium phosphate Chemical compound O1[Al]2OP1(=O)O2 ILRRQNADMUWWFW-UHFFFAOYSA-K 0.000 description 1
- 229910021529 ammonia Inorganic materials 0.000 description 1
- 229960000510 ammonia Drugs 0.000 description 1
- 230000030741 antigen processing and presentation Effects 0.000 description 1
- 239000002246 antineoplastic agent Substances 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 229960000686 benzalkonium chloride Drugs 0.000 description 1
- CADWTSSKOVRVJC-UHFFFAOYSA-N benzyl(dimethyl)azanium;chloride Chemical compound [Cl-].C[NH+](C)CC1=CC=CC=C1 CADWTSSKOVRVJC-UHFFFAOYSA-N 0.000 description 1
- 229940000635 beta-alanine Drugs 0.000 description 1
- 229910021538 borax Inorganic materials 0.000 description 1
- 201000008274 breast adenocarcinoma Diseases 0.000 description 1
- 159000000007 calcium salts Chemical class 0.000 description 1
- 150000001732 carboxylic acid derivatives Chemical group 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 229960004926 chlorobutanol Drugs 0.000 description 1
- 239000002299 complementary DNA Substances 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 239000012228 culture supernatant Substances 0.000 description 1
- 229940127089 cytotoxic agent Drugs 0.000 description 1
- 230000002950 deficient Effects 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 238000010511 deprotection reaction Methods 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- VHJLVAABSRFDPM-QWWZWVQMSA-N dithiothreitol Chemical compound SC[C@@H](O)[C@H](O)CS VHJLVAABSRFDPM-QWWZWVQMSA-N 0.000 description 1
- 239000012636 effector Substances 0.000 description 1
- 238000003114 enzyme-linked immunosorbent spot assay Methods 0.000 description 1
- 210000002919 epithelial cell Anatomy 0.000 description 1
- 230000001747 exhibiting effect Effects 0.000 description 1
- 230000006870 function Effects 0.000 description 1
- 210000003714 granulocyte Anatomy 0.000 description 1
- 201000005787 hematologic cancer Diseases 0.000 description 1
- 208000024200 hematopoietic and lymphoid system neoplasm Diseases 0.000 description 1
- 206010073071 hepatocellular carcinoma Diseases 0.000 description 1
- 108010007811 human immunodeficiency virus p17 gag peptide Proteins 0.000 description 1
- 230000028993 immune response Effects 0.000 description 1
- 208000026278 immune system disease Diseases 0.000 description 1
- 238000003018 immunoassay Methods 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 108010061181 influenza matrix peptide (58-66) Proteins 0.000 description 1
- 238000001802 infusion Methods 0.000 description 1
- 201000002313 intestinal cancer Diseases 0.000 description 1
- 238000007918 intramuscular administration Methods 0.000 description 1
- 238000010255 intramuscular injection Methods 0.000 description 1
- 239000007927 intramuscular injection Substances 0.000 description 1
- 238000001990 intravenous administration Methods 0.000 description 1
- 201000010982 kidney cancer Diseases 0.000 description 1
- 125000001909 leucine group Chemical group [H]N(*)C(C(*)=O)C([H])([H])C(C([H])([H])[H])C([H])([H])[H] 0.000 description 1
- 239000007791 liquid phase Substances 0.000 description 1
- 208000014018 liver neoplasm Diseases 0.000 description 1
- 201000005202 lung cancer Diseases 0.000 description 1
- 208000020816 lung neoplasm Diseases 0.000 description 1
- 159000000003 magnesium salts Chemical class 0.000 description 1
- 230000036210 malignancy Effects 0.000 description 1
- 238000004949 mass spectrometry Methods 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 235000010270 methyl p-hydroxybenzoate Nutrition 0.000 description 1
- 239000004292 methyl p-hydroxybenzoate Substances 0.000 description 1
- 125000000325 methylidene group Chemical group [H]C([H])=* 0.000 description 1
- 229960002216 methylparaben Drugs 0.000 description 1
- 230000009149 molecular binding Effects 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 210000004400 mucous membrane Anatomy 0.000 description 1
- 230000004719 natural immunity Effects 0.000 description 1
- 238000006386 neutralization reaction Methods 0.000 description 1
- 210000004798 organs belonging to the digestive system Anatomy 0.000 description 1
- 229960003104 ornithine Drugs 0.000 description 1
- 230000002611 ovarian Effects 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 210000003819 peripheral blood mononuclear cell Anatomy 0.000 description 1
- 210000003800 pharynx Anatomy 0.000 description 1
- 229940067107 phenylethyl alcohol Drugs 0.000 description 1
- 229940096826 phenylmercuric acetate Drugs 0.000 description 1
- PDTFCHSETJBPTR-UHFFFAOYSA-N phenylmercuric nitrate Chemical compound [O-][N+](=O)O[Hg]C1=CC=CC=C1 PDTFCHSETJBPTR-UHFFFAOYSA-N 0.000 description 1
- 230000026731 phosphorylation Effects 0.000 description 1
- 238000006366 phosphorylation reaction Methods 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- 229920002451 polyvinyl alcohol Polymers 0.000 description 1
- 229940068984 polyvinyl alcohol Drugs 0.000 description 1
- XAEFZNCEHLXOMS-UHFFFAOYSA-M potassium benzoate Chemical compound [K+].[O-]C(=O)C1=CC=CC=C1 XAEFZNCEHLXOMS-UHFFFAOYSA-M 0.000 description 1
- 229910000160 potassium phosphate Inorganic materials 0.000 description 1
- 235000011009 potassium phosphates Nutrition 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 238000004007 reversed phase HPLC Methods 0.000 description 1
- 238000013207 serial dilution Methods 0.000 description 1
- 201000000849 skin cancer Diseases 0.000 description 1
- 239000001632 sodium acetate Substances 0.000 description 1
- 235000017281 sodium acetate Nutrition 0.000 description 1
- 235000017557 sodium bicarbonate Nutrition 0.000 description 1
- 229910000030 sodium bicarbonate Inorganic materials 0.000 description 1
- WBHQBSYUUJJSRZ-UHFFFAOYSA-M sodium bisulfate Chemical compound [Na+].OS([O-])(=O)=O WBHQBSYUUJJSRZ-UHFFFAOYSA-M 0.000 description 1
- 229910000342 sodium bisulfate Inorganic materials 0.000 description 1
- 229910000029 sodium carbonate Inorganic materials 0.000 description 1
- 235000017550 sodium carbonate Nutrition 0.000 description 1
- 235000010267 sodium hydrogen sulphite Nutrition 0.000 description 1
- 239000001488 sodium phosphate Substances 0.000 description 1
- 229910000162 sodium phosphate Inorganic materials 0.000 description 1
- 235000011008 sodium phosphates Nutrition 0.000 description 1
- 159000000000 sodium salts Chemical class 0.000 description 1
- 235000010339 sodium tetraborate Nutrition 0.000 description 1
- AKHNMLFCWUSKQB-UHFFFAOYSA-L sodium thiosulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=S AKHNMLFCWUSKQB-UHFFFAOYSA-L 0.000 description 1
- 235000019345 sodium thiosulphate Nutrition 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 230000000638 stimulation Effects 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 230000001839 systemic circulation Effects 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- RTKIYNMVFMVABJ-UHFFFAOYSA-L thimerosal Chemical compound [Na+].CC[Hg]SC1=CC=CC=C1C([O-])=O RTKIYNMVFMVABJ-UHFFFAOYSA-L 0.000 description 1
- 229940033663 thimerosal Drugs 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- BSVBQGMMJUBVOD-UHFFFAOYSA-N trisodium borate Chemical compound [Na+].[Na+].[Na+].[O-]B([O-])[O-] BSVBQGMMJUBVOD-UHFFFAOYSA-N 0.000 description 1
- RYFMWSXOAZQYPI-UHFFFAOYSA-K trisodium phosphate Chemical compound [Na+].[Na+].[Na+].[O-]P([O-])([O-])=O RYFMWSXOAZQYPI-UHFFFAOYSA-K 0.000 description 1
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
- C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
- C07K14/4727—Mucins, e.g. human intestinal mucin
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/0005—Vertebrate antigens
- A61K39/0011—Cancer antigens
- A61K39/001169—Tumor associated carbohydrates
- A61K39/00117—Mucins, e.g. MUC-1
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/46—Cellular immunotherapy
- A61K39/461—Cellular immunotherapy characterised by the cell type used
- A61K39/4615—Dendritic cells
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/46—Cellular immunotherapy
- A61K39/462—Cellular immunotherapy characterized by the effect or the function of the cells
- A61K39/4622—Antigen presenting cells
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/46—Cellular immunotherapy
- A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
- A61K39/4643—Vertebrate antigens
- A61K39/4644—Cancer antigens
- A61K39/464469—Tumor associated carbohydrates
- A61K39/46447—Mucins, e.g. MUC-1
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/10—Cells modified by introduction of foreign genetic material
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
- A61K2039/572—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2 cytotoxic response
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
Definitions
- the present invention relates to an MUC1-derived peptide, more specifically an immunogenic peptide that binds to a human leukocyte antigen and thus presents an antigen to a T cell, and a pharmaceutical composition for treatment or prevention of cancer, an immunity-inducing agent, a method for manufacturing an antigen presenting cell and the like, using the same.
- HLA human leukocyte antigen
- lymphocyte a human leukocyte antigen derived from cancer cells.
- HLA molecules which are major histocompatible antigens, are broadly divided into class-I molecules (HLA-A, -B, and -C) and class-II molecules (HLA-DP, -DQ, and -DR).
- T cell antigen receptors (TCRs) on cytotoxic T cells (CTLs) specifically recognize cancer antigens (CTL epitopes) consisting of 8 to 11 amino acids presented to HLA class-I molecules on the surfaces of cancer cells, and thus a reaction in which cancer cells are eliminated by the CTLs is induced.
- JP H8-151396A discloses that oligopeptides having specific amino acid sequences have an HLA-binding property.
- Patent Document 1 JP H8-151396A
- peptides having an HLA-binding property are known, further peptides that can be used for treatment or prevention of various cancers are required.
- the gene for HLA is rich in polymorphism, and therefore, multi-type immunogenic peptides that can be applied to a plurality of types of HLAs are also required.
- the present invention has been achieved in light of the aforementioned circumstances, and an object thereof is to provide an immunogenic peptide that binds to an HLA class-I molecule, particularly a peptide that can induce CTLs, and a pharmaceutical composition for treatment or prevention of cancer, an immunity-inducing agent, a method for manufacturing an antigen presenting cell and the like, using the same.
- the present invention encompasses the following aspects of the invention.
- a pharmaceutical composition for treatment or prevention of cancer comprising the peptide according to any one of aspects (1) to (3).
- composition according to aspect (4) or (5), wherein the peptide can bind to one or more types of HLA molecules.
- An immunity-inducing agent comprising the peptide according to any one of aspects (1) to (3).
- a method for manufacturing an antigen presenting cell having a CTL-inducing activity comprising a step of bringing the peptide according to any one of aspects (1) to (3) into contact with an antigen presenting cell in vitro.
- the peptide of the present invention has a high HLA-binding property and a high ability to induce CTLs, and is thus strongly expected to be useful as a cancer vaccine. Moreover, it is likely that the peptide of the present invention can be applied to various immunotherapies, particularly to a dendritic cell therapy.
- Mucin is a large glycoprotein that is expressed in various epithelial cells including normal cells and malignant cells (Devine P L and McKenzie I F: Mucins: structure, function, and associations with malignancy. Bioessays 14: 619-625, 1992).
- MUC1 which is one of mucin polypeptides, is a unique glycoprotein that is expressed passing through the cell membrane on a cell surface (Gendler S J, Lancaster C A, Taylor-Papadimitriou J, Duhig T, Peat N, Burchell J, Pemberton L, Lalani E N and Wilson D: Molecular cloning and expression of human tumor-associated polymorphic epithelial mucin. J Biol Chem 265: 15286-15293, 1990).
- MUC1 of cancer cells undergoes incomplete glycosylation (Hanisch F G, Stadie T R, Deutzmann F and Peter-Katalinic J: MUC1 glycoforms in breast cancer—cell line T47D as a model for carcinoma-associated alterations of O-glycosylation. Eur J Biochem 236: 318-327, 1996), and it has been reported that killer T cells against MUC1 are induced in pancreatic cancer, breast cancer, ovarian cancer, and the like (20 Barnd D L, Lan M S, Metzgar R S and Finn O J: Specific, major histocompatibility complex-unrestricted recognition of tumorassociated mucins by human cytotoxic T cells. Proc Natl Acad Sci USA 86: 7159-7163, 1989.
- Specific aspects of the peptide of the present invention can bind to a plurality of types of HLAs. Therefore, with the peptide of the present invention, a cancer vaccine, a dendritic cell therapy, and the like that cover an extremely broad group of cancer patients can be provided.
- FIG. 1 shows results (the number of IFN- ⁇ producing cells) of ELISA assay when samples from a patient (HLA type: 24:02/33:03) that has undergone a dendritic cell/CTL therapy against MUC1 are stimulated using peptides of Sequence ID Nos. 1, 3, 5, and 10.
- FIG. 2 shows results (the number of IFN- ⁇ producing cells) of ELISA assay when samples from a patient (HLA type: 24:02/33:03) that has undergone a dendritic cell/CTL therapy against MUC1 are stimulated using peptides of Sequence ID Nos. 1, 3, 5, and 10.
- FIG. 3 shows results (the number of IFN- ⁇ producing cells) of ELISA assay when samples from a patient (HLA type: 02:06/24:02) that has undergone a dendritic cell/CTL therapy against MUC1 are stimulated using peptides of Sequence ID Nos. 1, 3, 5, and 10.
- FIG. 4 shows results (the number of IFN- ⁇ producing cells) of ELISA assay when samples from a patient (HLA type: 02:01/02:06) that has undergone a dendritic cell/CTL therapy against MUC1 are stimulated using peptides of Sequence ID Nos. 1, 2, 3, 5, 10, and 11.
- a peptide according to the present invention is a peptide that includes eight or more consecutive amino acid residues of the amino acid sequence of one of Sequence ID Nos. 1 to 12, and that consists of eleven or less amino acids, preferably ten or less amino acids, and more preferably nine or less amino acids, in total.
- the peptide of the present invention may also have the amino acid sequence of one of Sequence ID Nos. 1 to 12.
- the peptide of the present invention is derived from mucin 1 (MUC1), which is one of tumor-associated antigens.
- Selected were the amino acid sequences that were based on the amino acid sequence of MUC1 and whose HLA molecule-binding properties predicted based on the hypothesis obtained by using an active learning experiment method (JP H11-316754A) were 3 or more in terms of a ⁇ log Kd value.
- Table 1 shows the amino acid sequences for the peptide of the present invention, and the predicted HLA-binding scores.
- the peptide of the present invention has an HLA-binding property and immunogenicity (also referred to merely as “HLA peptide” or “immunogenic peptide” hereinafter).
- Immunogenicity as used herein means an ability to induce an immune reaction, and, for example, means “having a CTL-inducing activity and thus a cytotoxic activity against cancer cells”.
- the peptide of the present invention is a multi-HLA peptide that can bind to a plurality of types of alleles of the HLA-A gene A.
- the peptide of Sequence ID No. 10 strongly binds to an HLA-A*24:02 gene product (HLA-A*24:02 molecule), an HLA-A*02:01 gene product (HLA-A*02:01 molecule), and an HLA-A*02:06 gene product (HLA-A*02:06 molecule), and has a high immunogenicity.
- HLA subtypes to which the peptide of the present invention can bind are not limited to the HLA-A*24:02, the HLA-A*02:01, and the HLA-A*02:06.
- about 85% of the Oriental including Japanese and about 55% of the Occidental have these HLA subtypes, and therefore, it is thought that the multi-HLA peptide has a high patient cover ratio in an immunotherapy or the like.
- amino acid residues of the amino acid sequences of Sequence ID Nos. 1 to 12 or portions thereof may be modified as long as the peptide of the present invention retains immunogenicity.
- the amino acid sequences of Sequence ID Nos. 1 to 12 are intended to be presented on antigen presenting cells, but, when being administered directly to the body, the peptide of the present invention may undergo a modification such as digestion of its terminus in the digestive organs depending on the administration route.
- the peptide of the present invention may exist in a precursor form in which one or more amino acid residues and the like are added to the N-terminus and/or C-terminus such that the peptide of the present invention retains the amino acid residues of Sequence ID Nos. 1 to 12 when binding to a predetermined HLA class-I molecule on the antigen presenting cell.
- amino acids are used herein in their broadest sense, and include artificial amino acid variants and artificial amino acid derivatives in addition to natural amino acids.
- amino acids examples include natural protein L-amino acids; D-amino acids; chemically modified amino acids such as amino acid variants and amino acid derivatives; non-protein natural amino acids such as norleucine, ⁇ -alanine, and ornithine; and chemically synthesized compounds having characteristics known in the art as features of amino acids.
- non-natural amino acids include ⁇ -methylamino acids (e.g., ⁇ -methylalanine), D-amino acids, histidine-like amino acids (e.g., ⁇ -hydroxy-histidine, homohistidine, ⁇ -fluoromethyl-histidine, and ⁇ -methyl-histidine), amino acids with side chains including additional methylenes (i.e., “homo-” amino acids), and substituted amino acids (e.g., cysteic acid) in which a carboxylic acid functional group in a side chain is substituted with a sulfonic acid.
- ⁇ -methylamino acids e.g., ⁇ -methylalanine
- D-amino acids e.g., D-amino acids
- histidine-like amino acids e.g., ⁇ -hydroxy-histidine, homohistidine, ⁇ -fluoromethyl-histidine, and ⁇ -methyl-histidine
- the amino acid at the 2-position of the peptide may be substituted with tyrosine, phenylalanine, methionine, or tryptophan, and/or the C-terminal amino acid may be substituted with phenylalanine, leucine, isoleucine, tryptophan, or methionine.
- the amino acid at the 2-position may be substituted with leucine or methionine, and/or the C-terminal amino acid may be substituted with valine or leucine.
- the amino acid at the 2-position may be substituted with valine or glutamine, and/or the C-terminal amino acid may be substituted with valine or leucine.
- the peptide of the present invention can be manufactured using methods known to a person skilled in the art.
- the peptide of the present invention may be synthesized artificially using a solid phase method such as the Fmoc method or the tBoc method, or a liquid phase method.
- a desired peptide may also be manufactured by expressing a polynucleotide coding for the peptide of the present invention or a recombinant vector including the polynucleotide. All of the thus obtained peptides can be identified using a method known to a person skilled in the art.
- the peptides can be identified using the Edman degradation method, mass spectrometry, or the like.
- a pharmaceutical composition for treatment or prevention of cancer according to the present invention contains, as an active component, a peptide that includes eight or more consecutive amino acid residues of one or more amino acid sequences selected from the group consisting of Sequence ID Nos. 1 to 12, and that consists of eleven or less amino acids, preferably ten or less amino acids, and more preferably nine or less amino acids, in total, for example.
- the peptide contained in the pharmaceutical composition may also have the amino acid sequence of one of Sequence ID Nos. 1 to 12.
- the peptide is as defined above.
- the peptide of the present invention is presented on an antigen presenting cell and thus induces CTLs, and the induced CTLs injure cancer cells. Therefore, the active component of the pharmaceutical composition of the present invention is not limited to the peptide of the present invention, and may be a component that can induce CTLs directly or indirectly.
- a polynucleotide coding for the peptide or a vector including the polynucleotide, an antigen presenting cell that presents a complex between the peptide and an HLA molecule on its surface, or an exosome secreted by the antigen presenting cell, or combinations thereof may be used.
- the antigen presenting cell to be used examples include a macrophage and a dendritic cell, and it is preferable to use a dendritic cell, which has a high ability to induce CTLs.
- the pharmaceutical composition of the present invention may also contain other components known to be used for treatment of cancer, such as a chemokine, a cytokine, a tumor necrosis factor, and a chemotherapeutic agent.
- a peptide dosage may be about 1 to 10 mg per day, for example, when a patient is an adult. However, the dosage varies depending on the age and weight of a patient, an administration method, and the like, and therefore, a person skilled in the art determines the dosage as appropriate.
- the pharmaceutical composition of the present invention is not construed as being limited, but is thought to be useful for killing or the like of cancer cells due to the following action mechanism.
- the pharmaceutical composition of the present invention is administered to a specific cancer patient, the peptide in the pharmaceutical composition is presented on the surface of the antigen presenting cell in a state in which the peptide binds to an HLA molecule.
- the CTLs recognize the peptide on such an antigen presenting cell and is thus activated, followed by proliferation and systemic circulation.
- CTLs that are specific to the peptide enter the cancer tissue, the CTLs recognize the same peptide derived from the specific cancer antigen that naturally binds to the HLA molecule present on the surface of the cancer cell, and then kill the cancer cell.
- the pharmaceutical composition can contribute to treatment of cancer due to this action.
- the pharmaceutical composition can be used for not only treatment of cancer but also prevention of cancer.
- the pharmaceutical composition of the present invention when administered to a healthy human body, CTLs are induced, and the induced CTLs remain in the body. Therefore, when a specific cancer occurs, it is possible to injure the cancer cells. Similarly, a recurrence of cancer may be prevented by the administration to the human body that has undergone treatment of cancer.
- All cancers in which MUC1 is expressed are assumed as cancer to be treated or prevented. More specifically, examples of target cancers include pancreatic cancer, hepatocellular cancer, prostate cancer, lung cancer, breast cancer, bowel cancer, blood cancer, brain tumor, kidney cancer, and skin cancer, but are not construed as being limited.
- MUC1 from which the peptide of the present invention is derived is overexpressed in pancreatic cancer, and therefore, it is thought that the peptide of the present invention is effective in treatment or prevention of pancreatic cancer.
- the pharmaceutical composition of the present invention can contain a plurality of types of active components such as an immunogenic peptide.
- the pharmaceutical composition of the present invention can be administered to a patient in a form of a preparation of a pharmaceutically acceptable salt obtained by dissolving the pharmaceutical composition in a water-soluble solvent.
- a pharmaceutically acceptable salt examples include forms of a physiologically acceptable water-soluble salt such as a sodium salt, a potassium salt, a magnesium salt, or a calcium salt that is buffered at a physiological pH.
- a water-insoluble solvent can also be used in addition to a water-soluble solvent, and examples of the water-insoluble solvent include alcohols such as ethanol and propylene glycol.
- a preparation containing the pharmaceutical composition of this embodiment can contain agents for various purposes, and examples of such agents include a preservative and a buffer.
- the preservative include sodium bisulfite, sodium bisulfate, sodium thiosulfate benzalkonium chloride, chlorobutanol, thimerosal, phenylmercuric acetate, phenylmercuric nitrate, methylparaben, polyvinyl alcohol, phenylethyl alcohol, ammonia, dithiothreitol, and ⁇ -mercaptoethanol.
- the buffer include sodium carbonate, sodium borate, sodium phosphate, sodium acetate, and sodium bicarbonate. These agents can be present in such an amount that the pH of a system can be maintained between 2 and 9, and preferably between 4 and 8.
- the formulation of the pharmaceutical composition of the present invention includes injections (an intramuscular injection, a hypodermic injection, an intradermal injection), oral formulations, and nasal formulations when a form of vaccine is used.
- injections an intramuscular injection, a hypodermic injection, an intradermal injection
- oral formulations an intradermal injection
- nasal formulations when a form of vaccine is used.
- a mixed cocktail vaccine containing a plurality of types of active components may be used.
- such vaccine can contain a plurality of types of active components that is a combination of any two or more peptides of Sequence ID Nos. 1 to 12 or other active components.
- the vaccine of the present invention may be inert component-containing vaccine containing a component other than the pharmaceutical composition, the component being inert itself and having an effect of further improving the effect of the pharmaceutical composition as vaccine.
- the inert component include an adjuvant and a toxoid.
- the adjuvant include sedimentary adjuvants such as aluminum hydroxide, aluminum phosphate, and potassium phosphate, and oily adjuvants such as Freund's complete adjuvant and Freund's incomplete adjuvant.
- the pharmaceutical composition of the present invention is present in a form of vaccine
- a dose thereof can be set to be between such an amount that a cytotoxic T cell can be significantly induced and such an amount that a significant number of non-cancer cells are not injured.
- the pharmaceutical composition of the present invention is intended to be not only administered to a human body but also used outside the body. More specifically, the pharmaceutical composition of the present invention may be used to stimulate an antigen presenting cell in vitro or ex vivo and increase a CTL-inducing activity.
- the following is description of an example of a case where the pharmaceutical composition of the present invention is used in a dendritic cell therapy for cancer.
- the pharmaceutical composition of the present invention can be administered to a patient requiring treatment or prevention of cancer by bringing the pharmaceutical composition into contact with an antigen presenting cell such as a dendritic cell derived from the patient in advance, and then putting the antigen presenting cell back to the body of the patient.
- the peptide contained in the pharmaceutical composition can be introduced into the antigen presenting cell using a lipofection method, an injection method, or the like.
- a polynucleotide coding for the peptide of the present invention is used for such an application, the polynucleotide can be introduced into the antigen presenting cell using a method known in the art.
- the antigen presenting cell derived from a patient may be transformed in vitro, for example, with the target polynucleotide or a vector coding for the polynucleotide using a lipofection method, an electroporation method, a microinjection method, a cell fusion method, a DEAE dextran method, a calcium phosphate method, or the like.
- An immunity-inducing agent contains, as an active component, a peptide that includes eight or more consecutive amino acid residues of one or more amino acid sequences selected from the group consisting of Sequence ID Nos. 1 to 12, and that consists of eleven or less amino acids, preferably ten or less amino acids, and more preferably nine or less amino acids, in total, for example.
- the peptide contained in the immunity-inducing agent may also have the amino acid sequence of one of Sequence ID Nos. 1 to 12.
- the peptide is as defined above.
- the active component of the immunity-inducing agent of the present invention is not limited to the peptide of the present invention, and may be a component that can induce immunity directly or indirectly.
- a polynucleotide coding for the peptide of the present invention or a vector including the polynucleotide, an antigen presenting cell that presents a complex between the peptide and an HLA molecule on its surface, or an exosome secreted by the antigen presenting cell, or combinations thereof may be used.
- the antigen presenting cell to be used include a macrophage and a dendritic cell, and it is preferable to use a dendritic cell, which has a high ability to induce CTLs.
- the immunity-inducing agent of the present invention is intended to be not only administered to a human body but also used outside the body. More specifically, the immunity-inducing agent of the present invention may be used to stimulate an antigen presenting cell in vitro or ex vivo and increase a CTL-inducing activity. For example, the following is description of an example of a case where the immunity-inducing agent of the present invention is used in a dendritic cell therapy.
- the immunity-inducing agent of the present invention can be administered to a patient requiring immunity induction by bringing the immunity-inducing agent into contact with an antigen presenting cell such as a dendritic cell derived from the patient in advance, and then putting the antigen presenting cell back to the body of the patient.
- the peptide contained in the immunity-inducing agent can be introduced into the antigen presenting cell using transfection via a liposome (lipofection method), an injection method, or the like.
- liposome lipofection method
- the polynucleotide can be introduced into the antigen presenting cell using a method known in the art.
- the antigen presenting cell derived from a patient may be transformed in vitro, for example, with the target polynucleotide or a vector expressing the polynucleotide using a lipofection method, an electroporation method, a microinjection method, a cell fusion method, a DEAE dextran method, a calcium phosphate method, or the like.
- the “immunity induction” as used herein means that an immune reaction is induced, and a CTL-inducing activity of the antigen presenting cell as well as a cytotoxic activity of CTLs against cancer cells, for example, are thus increased.
- the “CTL induction” as used herein means that, in vitro or in vivo, CTLs that specifically recognize a certain antigen are induced or proliferated, or naive T cells are differentiated into effector cells that has an ability (cytotoxic activity) to kill target cells such as cancer cells, and/or the cytotoxic activity of CTLs is increased, due to the peptide of the present invention being presented on the surface of an antigen presenting cell.
- the CTL-inducing activity can be measured by evaluating the production of a cytokine (e.g., interferon (IFN)- ⁇ ) by CTLs.
- a cytokine e.g., interferon (IFN)- ⁇
- the CTL-inducing activity may be measured by evaluating, using a known highly sensitive immunoassay such as ELISPOT (Enzyme-Linked ImmunoSpot) or ELISA (Enzyme-Linked ImmunoSorbent Assay), an increase in the number of cytokine-producing cells that have been induced from precursor cells by an antigen presenting cell such as a peripheral blood mononuclear cell stimulated by the peptide of the present invention.
- the cytotoxic activity can also be measured using a known method such as a 51 Cr releasing method.
- a method for manufacturing an antigen presenting cell according to the present invention includes a step of bringing a peptide that includes eight or more consecutive amino acid residues of one or more amino acid sequences selected from the group consisting of Sequence ID Nos. 1 to 12, and that consists of eleven or less amino acids, preferably ten or less amino acids, and more preferably nine or less amino acids, in total into contact with an antigen presenting cell in vitro, for example.
- the peptide used in the manufacturing method of the present invention may also have the amino acid sequence of one of Sequence ID Nos. 1 to 12.
- the peptide is as defined above.
- a component to be brought into contact with an antigen presenting cell is not limited to the peptide of the present invention, and may be a component that can induce CTLs directly or indirectly.
- a polynucleotide coding for the peptide or a vector including the polynucleotide, an antigen presenting cell that presents a complex between the peptide and an HLA molecule on its surface, or an exosome secreted by the antigen presenting cell, or combinations thereof may be used.
- the antigen presenting cell to be used include a macrophage and a dendritic cell, and it is preferable to use a dendritic cell, which has a high ability to induce CTLs.
- the antigen presenting cell manufactured using the manufacturing method of the present invention is intended to be used not only as an active component of the above-mentioned pharmaceutical composition or immunity-inducing agent but also in an immunotherapy or the like.
- the following is description of an example of a case where the manufactured antigen presenting cell is used in a dendritic cell therapy for cancer.
- the manufactured antigen presenting cell can be administered to a patient requiring immunity induction by bringing the manufactured antigen presenting cell into contact with an antigen presenting cell such as a dendritic cell that are derived from the patient and has a low ability to induce CTLs in advance, and then putting the manufactured antigen presenting cell back to the body of the patient.
- the peptide of the present invention can be introduced into the antigen presenting cell using transfection via a liposome (lipofection method), an injection method, or the like.
- liposome lipofection method
- the polynucleotide can be introduced into the antigen presenting cell using a method known in the art.
- the antigen presenting cell derived from a patient may be transformed in vitro, for example, with the target polynucleotide or a vector coding for the polynucleotide using a lipofection method, an electroporation method, a microinjection method, a cell fusion method, a DEAE dextran method, a calcium phosphate method, or the like.
- a trial of a lower-order learning algorithm which will be described later, was carried out once. That is, a plurality of hypotheses were generated by random resampling from accumulated data, and with regard to randomly expressed candidate query points (peptides), a point that showed the largest distribution of predicted values was selected as a query point to be subjected to an experiment.
- the peptide at the selected query point was prepared by a synthesis and purification method, which will be described later, and the actual binding ability was measured by an experiment, which will be described later, and added to the accumulated data.
- Peptides having amino acid sequences of Sequence ID Nos. 1 to 12 were manually synthesized with the Merrifield solid-phase method using Fmoc amino acids. After deprotection, reverse phase HPLC purification was carried out using a C18 column to give a purity of 95% or higher.
- MALDI-TOF mass spectrometry (AB SCIEX MALDI-TOF/TOF5800) was carried out to identify the peptides and confirm their purities. The peptides were quantified with Micro BCA assay (Thermo Scientific) using BSA as a standard protein.
- C1R-A24 cells were exposed to acidic conditions at a pH of 3.3 for 30 seconds, and thus an endogenous peptide that originally binds to the HLA-A*24:02 molecule, and a light chain ⁇ 2m, which is associated with HLA class-I molecules in common, were dissociated and removed. After neutralization, a purified ⁇ 2m was added to C1R-A24 cells, and the obtained product was added to a series of dilutions of the peptide and incubated on ice for 4 hours.
- Staining was carried out using a fluorescently labeled monoclonal antibody 17A12, which recognizes association (MHC-pep) of the three members, that is, a HLA-A*24:02 molecule, a peptide, and ⁇ 2m, which had reassociated during the incubation.
- MHC-pep monoclonal antibody 17A12, which recognizes association (MHC-pep) of the three members, that is, a HLA-A*24:02 molecule, a peptide, and ⁇ 2m, which had reassociated during the incubation.
- the MHC-pep count per C1R-A24 cell (proportional to the intensity of fluorescence of the above-mentioned fluorescent antibody) was quantitatively measured using a fluorescence-activated cell sorter FACScan (Becton-Dickinson).
- FACScan Fluorescence-activated cell sorter
- a binding-dissociation constant Kd value between the HLA-A*24:02 molecule and the peptide was calculated from the average intensity of fluorescence per cell using a method that was reported in a paper (Udaka et al., Immunogenetics, 51, 816-828, 2000) by the inventors of the present invention.
- the T2 cells and purified ⁇ 2m were added to a series of serial dilutions of the peptide whose binding ability would be measured, and then incubation was carried out at 37° C. for 4 hours.
- the HLA-A*02:01 molecule that had increased in an expression amount in a peptide-concentration-dependent manner up to this point was stained using a fluorescently labeled monoclonal antibody BB7.2, which is specific to an association type.
- HLA-A*02:06 molecule which is an HLA-A*02:06 gene product
- RA2.6 cells cell line that had been newly produced in Kochi University
- TAP transporter associated with antigen processing
- RA2.6 cells were cultured at 26° C. overnight. After HLA-A*02:06 molecules to which the peptide did not bind accumulated on the cell surface, a series of dilutions of the peptide were added thereto, and binding was carried out at 26° C. for 60 minutes.
- sequence ID Nos. 1 to 12 are derived from the entire sequence (Sequence ID No. 13) (>sp
- peripheral blood monocytes obtained through pheresis from a patient (0) that had undergone a dendritic cell/CTL therapy against MUC1 a cell fraction that adhered to a culture flask was cultured in an AIM-CM culture medium (manufactured by Gibco) at 37° C. for 10 days.
- AIM-CM culture medium manufactured by Gibco
- 15 ⁇ l of IL-4, 30 ⁇ l of GM-CSF, and 75 ⁇ l of a tumor necrosis factor (TFN)- ⁇ were added thereto on Day 5.
- GM-CSF granulocyte monocyte colony-stimulating factor
- Induced dendritic cells were collected in a new AIM-CM culture medium, and the peptide of the present invention (Sequence ID Nos. 1 to 12) was added thereto to a concentration of 20 ⁇ g/ml. Thereafter, the culture medium containing the dendritic cells was cultured at 37° C. for 2 hours. The following peptides were used as positive controls and negative controls.
- HLA-A*24:02 EBV LMP2, 419-427: TYGPVFMCL (Sequence ID No. 14)
- Negative control for HLA-A*24:02 HLA env gp160, 584-592: RYLRDQQLL (Sequence ID No. 15)
- Positive control for HLA-A*02:01 Flu A MP, 58-66: GILGFVFTL (Sequence ID No. 16)
- Negative control for HLA-A*02:01 HLA gap p17, 77-85: SLYNTVATL (Sequence ID No.
- the dendritic cells were collected and washed three or more times using a sufficient amount of an AIM-CM culture medium, and then the number of cells was counted.
- peripheral blood monocytes obtained through pheresis from a patient that had undergone treatment using the above-mentioned vaccine twice or more a floating cell fraction (containing lymphocytes) that did not adhere to a culture flask was cultured in an AIM-CM culture medium (manufactured by Gibco) at 37° C. for 10 days. During the culture, 40 ⁇ l of IL-2 was added to the culture medium on Day 4 and Day 6.
- CD8T cells were isolated from the culture medium using a CD8 negative selection kit (manufactured by Miltenyi), and the number of cells was counted.
- the dendritic cells and CD8T cells obtained in the above (1) and (2) were cocultured in an AIM-CM culture medium at 37° C. in the following conditions.
- the culture supernatant of the coculture of the T cells and the dendritic cells pulsed with the above-mentioned peptides on Day 7 was diluted to four steps, that is, ⁇ 1, ⁇ 5, ⁇ 25, and ⁇ 125, and the dilution step within a measurement limit was identified using Human IFN- ⁇ ELISA MAX Deluxe Set (manufactured by BioLegend). Thereafter, at the identified dilution step, each sample was measured three times.
- 1 to 4 respectively show typical results of ELISA assay from a patient with an HLA type of 24:02/33:03, a patient with an HLA type of 24:02/33:03, a patient with an HLA type of 11:01/24:02, a patient with an HLA type of 02:06/24:02, and a patient having an HLA type of 02:01/02:06.
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Organic Chemistry (AREA)
- Immunology (AREA)
- Genetics & Genomics (AREA)
- Cell Biology (AREA)
- Animal Behavior & Ethology (AREA)
- Pharmacology & Pharmacy (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Epidemiology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Mycology (AREA)
- Biochemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Zoology (AREA)
- Biophysics (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Gastroenterology & Hepatology (AREA)
- Toxicology (AREA)
- Biomedical Technology (AREA)
- Biotechnology (AREA)
- Wood Science & Technology (AREA)
- Oncology (AREA)
- General Engineering & Computer Science (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Peptides Or Proteins (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The present invention provides a peptide that includes eight or more consecutive amino acid residues of amino acid sequence of one of Sequence ID Nos. 1 to 12 and that consists of eleven or less amino acid residues.
Description
- The present invention relates to an MUC1-derived peptide, more specifically an immunogenic peptide that binds to a human leukocyte antigen and thus presents an antigen to a T cell, and a pharmaceutical composition for treatment or prevention of cancer, an immunity-inducing agent, a method for manufacturing an antigen presenting cell and the like, using the same.
- It is thought that cancer cells always occur accidentally in living bodies. It is likely that a reaction in which specific cancer antigens derived from cancer cells are eliminated by natural immunity usually occurs, and then a specific immune response is induced, so that a reaction in which the cancer cells are eliminated by lymphocytes and the like occurs.
- The formation of a complex between a human leukocyte antigen (HLA) that is present on the cell surface and a lymphocyte is necessary for recognition of antigens derived from cancer cells. HLA molecules, which are major histocompatible antigens, are broadly divided into class-I molecules (HLA-A, -B, and -C) and class-II molecules (HLA-DP, -DQ, and -DR). T cell antigen receptors (TCRs) on cytotoxic T cells (CTLs) specifically recognize cancer antigens (CTL epitopes) consisting of 8 to 11 amino acids presented to HLA class-I molecules on the surfaces of cancer cells, and thus a reaction in which cancer cells are eliminated by the CTLs is induced.
- Nowadays, a search for immunogenic peptides is made with the expectation of the application to treatment or prevention of various immunological diseases. For example, JP H8-151396A discloses that oligopeptides having specific amino acid sequences have an HLA-binding property.
- Patent Document 1: JP H8-151396A
- Although a large number of peptides having an HLA-binding property are known, further peptides that can be used for treatment or prevention of various cancers are required. The gene for HLA is rich in polymorphism, and therefore, multi-type immunogenic peptides that can be applied to a plurality of types of HLAs are also required.
- The present invention has been achieved in light of the aforementioned circumstances, and an object thereof is to provide an immunogenic peptide that binds to an HLA class-I molecule, particularly a peptide that can induce CTLs, and a pharmaceutical composition for treatment or prevention of cancer, an immunity-inducing agent, a method for manufacturing an antigen presenting cell and the like, using the same.
- That is, the present invention encompasses the following aspects of the invention.
- (1) A peptide including eight or more consecutive amino acid residues of amino acid sequence of one of Sequence ID Nos. 1 to 12, and consisting of eleven or less amino acid residues.
- (2) The peptide according to aspect (1), wherein one or several amino acids in the amino acid sequence are substituted, inserted, deleted, or added, and the peptide has immunogenicity.
- (3) The peptide according to aspect (2), wherein an amino acid at a 2-position of the amino acid sequence is substituted with tyrosine, phenylalanine, methionine, tryptophan, valine, leucine or glutamine, and/or a C-terminal amino acid is substituted with phenylalanine, leucine, isoleucine, tryptophan, methionine, or valine.
- (4) A pharmaceutical composition for treatment or prevention of cancer, comprising the peptide according to any one of aspects (1) to (3).
- (5) The pharmaceutical composition according to aspect (4), which is in a form of vaccine.
- (6) The pharmaceutical composition according to aspect (4) or (5), wherein the peptide can bind to one or more types of HLA molecules.
- (7) An immunity-inducing agent comprising the peptide according to any one of aspects (1) to (3).
- (8) The immunity-inducing agent according to aspect (7), which is used to induce a cytotoxic T cell.
- (9) The immunity-inducing agent according to aspect (7) or (8), wherein the peptide can bind to one or more types of HLA molecules.
- (10) A method for manufacturing an antigen presenting cell having a CTL-inducing activity, the method comprising a step of bringing the peptide according to any one of aspects (1) to (3) into contact with an antigen presenting cell in vitro.
- In recent years, an immunotherapy has been attracting attention as treatment of cancer. The peptide of the present invention has a high HLA-binding property and a high ability to induce CTLs, and is thus strongly expected to be useful as a cancer vaccine. Moreover, it is likely that the peptide of the present invention can be applied to various immunotherapies, particularly to a dendritic cell therapy.
- Mucin is a large glycoprotein that is expressed in various epithelial cells including normal cells and malignant cells (Devine P L and McKenzie I F: Mucins: structure, function, and associations with malignancy. Bioessays 14: 619-625, 1992). MUC1, which is one of mucin polypeptides, is a unique glycoprotein that is expressed passing through the cell membrane on a cell surface (Gendler S J, Lancaster C A, Taylor-Papadimitriou J, Duhig T, Peat N, Burchell J, Pemberton L, Lalani E N and Wilson D: Molecular cloning and expression of human tumor-associated polymorphic epithelial mucin. J Biol Chem 265: 15286-15293, 1990).
- MUC1 of cancer cells undergoes incomplete glycosylation (Hanisch F G, Stadie T R, Deutzmann F and Peter-Katalinic J: MUC1 glycoforms in breast cancer—cell line T47D as a model for carcinoma-associated alterations of O-glycosylation. Eur J Biochem 236: 318-327, 1996), and it has been reported that killer T cells against MUC1 are induced in pancreatic cancer, breast cancer, ovarian cancer, and the like (20 Barnd D L, Lan M S, Metzgar R S and Finn O J: Specific, major histocompatibility complex-unrestricted recognition of tumorassociated mucins by human cytotoxic T cells. Proc Natl Acad Sci USA 86: 7159-7163, 1989.
- 21 Jerome K R, Barnd D L, Bendt K M, Boyer C M, Taylor-Papadimitriou J, McKenzie I F, Bast R C Jr and Finn O J: Cytotoxic T-lymphocytes derived from patients with breast adenocarcinoma recognize an epitope present on the protein core of a mucin molecule preferentially expressed by malignant cells. Cancer Res 51: 2908-2916, 1991.
- 22 Ioannides C G, Fisk B, Fan D, Biddison W E, Wharton J T and O'Brian C A: Cytotoxic T cells isolated from ovarian malignant ascites recognize a peptide derived from the HER-2/neu protooncogene. Cell Immunol 151: 225-234, 1993.).
- Specific aspects of the peptide of the present invention can bind to a plurality of types of HLAs. Therefore, with the peptide of the present invention, a cancer vaccine, a dendritic cell therapy, and the like that cover an extremely broad group of cancer patients can be provided.
-
FIG. 1 shows results (the number of IFN-γ producing cells) of ELISA assay when samples from a patient (HLA type: 24:02/33:03) that has undergone a dendritic cell/CTL therapy against MUC1 are stimulated using peptides of Sequence ID Nos. 1, 3, 5, and 10. -
FIG. 2 shows results (the number of IFN-γ producing cells) of ELISA assay when samples from a patient (HLA type: 24:02/33:03) that has undergone a dendritic cell/CTL therapy against MUC1 are stimulated using peptides of Sequence ID Nos. 1, 3, 5, and 10. -
FIG. 3 shows results (the number of IFN-γ producing cells) of ELISA assay when samples from a patient (HLA type: 02:06/24:02) that has undergone a dendritic cell/CTL therapy against MUC1 are stimulated using peptides of Sequence ID Nos. 1, 3, 5, and 10. -
FIG. 4 shows results (the number of IFN-γ producing cells) of ELISA assay when samples from a patient (HLA type: 02:01/02:06) that has undergone a dendritic cell/CTL therapy against MUC1 are stimulated using peptides of Sequence ID Nos. 1, 2, 3, 5, 10, and 11. - 1. Immunogenic Peptide
- A peptide according to the present invention is a peptide that includes eight or more consecutive amino acid residues of the amino acid sequence of one of Sequence ID Nos. 1 to 12, and that consists of eleven or less amino acids, preferably ten or less amino acids, and more preferably nine or less amino acids, in total. The peptide of the present invention may also have the amino acid sequence of one of Sequence ID Nos. 1 to 12. The peptide of the present invention is derived from mucin 1 (MUC1), which is one of tumor-associated antigens.
- Selected were the amino acid sequences that were based on the amino acid sequence of MUC1 and whose HLA molecule-binding properties predicted based on the hypothesis obtained by using an active learning experiment method (JP H11-316754A) were 3 or more in terms of a −log Kd value.
- Table 1 below shows the amino acid sequences for the peptide of the present invention, and the predicted HLA-binding scores.
-
TABLE 1 Amino acid sequence Predicted binding score (Sequence ID No.) Location in MUC1 to A*24:02 to A*02:01 to A*02:06 FLGLSNIKF (Seq. ID No. 1) 1086 5.0955 4.4235 4.1939 SVPVTRPAL (Seq. ID No. 2) 100 5.0767 4.4656 5.8224 GVPGWGIAL (Seq. ID No. 3) 1155 4.9066 4.5398 5.3254 AFREGTINV (Seq. ID No. 4) 1106 4.6737 4.6098 4.4007 AASRYNLTI (Seq. ID No. 5) 1128 4.5613 4.2522 4.1161 LQRDISEMF (Seq. ID No. 6) 1069 5.4895 3.9363 3.812 HHSDTPTTL (Seq. ID No. 7) 997 5.2785 3.8534 4.0609 SFFFLSFHI (Seq. ID No. 8) 1041 5.0343 4.5581 3.958 TLAFREGTI (Seq. ID No. 9) 1104 4.8467 4.8911 3.9425 STGVSFFFL (Seq. ID No. 10) 1037 4.7519 4.0289 5.2728 GQDVTSVPV (Seq. ID No. 11) 95 3.7866 5.1026 6.1919 FSAQSGAGV (Seq. ID No. 12) 1148 3.0319 5.2427 4.0462 - The peptide of the present invention has an HLA-binding property and immunogenicity (also referred to merely as “HLA peptide” or “immunogenic peptide” hereinafter). “Immunogenicity” as used herein means an ability to induce an immune reaction, and, for example, means “having a CTL-inducing activity and thus a cytotoxic activity against cancer cells”.
- In a preferred aspect, the peptide of the present invention is a multi-HLA peptide that can bind to a plurality of types of alleles of the HLA-A gene A. For example, the peptide of Sequence ID No. 10 strongly binds to an HLA-A*24:02 gene product (HLA-A*24:02 molecule), an HLA-A*02:01 gene product (HLA-A*02:01 molecule), and an HLA-A*02:06 gene product (HLA-A*02:06 molecule), and has a high immunogenicity.
- HLA subtypes to which the peptide of the present invention can bind are not limited to the HLA-A*24:02, the HLA-A*02:01, and the HLA-A*02:06. However, about 85% of the Oriental including Japanese and about 55% of the Occidental have these HLA subtypes, and therefore, it is thought that the multi-HLA peptide has a high patient cover ratio in an immunotherapy or the like.
- Amino acid residues of the amino acid sequences of Sequence ID Nos. 1 to 12 or portions thereof may be modified as long as the peptide of the present invention retains immunogenicity. The amino acid sequences of Sequence ID Nos. 1 to 12 are intended to be presented on antigen presenting cells, but, when being administered directly to the body, the peptide of the present invention may undergo a modification such as digestion of its terminus in the digestive organs depending on the administration route. Therefore, prior to being taken up by an antigen presenting cell, the peptide of the present invention may exist in a precursor form in which one or more amino acid residues and the like are added to the N-terminus and/or C-terminus such that the peptide of the present invention retains the amino acid residues of Sequence ID Nos. 1 to 12 when binding to a predetermined HLA class-I molecule on the antigen presenting cell.
- Furthermore, in the peptide of the present invention, one or several amino acid residues of the peptide of the present invention may be substituted, inserted, deleted, or added, and/or the peptide of the present invention may undergo modifications such as glycosylation, side-chain oxidation and/or phosphorylation. The “amino acids” are used herein in their broadest sense, and include artificial amino acid variants and artificial amino acid derivatives in addition to natural amino acids. In this specification, examples of the amino acids include natural protein L-amino acids; D-amino acids; chemically modified amino acids such as amino acid variants and amino acid derivatives; non-protein natural amino acids such as norleucine, β-alanine, and ornithine; and chemically synthesized compounds having characteristics known in the art as features of amino acids. Examples of non-natural amino acids include α-methylamino acids (e.g., α-methylalanine), D-amino acids, histidine-like amino acids (e.g., β-hydroxy-histidine, homohistidine, α-fluoromethyl-histidine, and α-methyl-histidine), amino acids with side chains including additional methylenes (i.e., “homo-” amino acids), and substituted amino acids (e.g., cysteic acid) in which a carboxylic acid functional group in a side chain is substituted with a sulfonic acid.
- Regarding the substitution of amino acid residues and the like, a person skilled in the art could substitute the amino acids of the peptide of the present invention as appropriate in consideration of the regularity of peptide sequences exhibiting an HLA-binding property (J. Immunol., 152: p 3913, 1994; Immunogenetics, 41: p 178, 1995; J. Immunol., 155: p 4307, 1994).
- More specifically, in the case of a peptide that binds to the HLA-A*24:02 molecule, the amino acid at the 2-position of the peptide may be substituted with tyrosine, phenylalanine, methionine, or tryptophan, and/or the C-terminal amino acid may be substituted with phenylalanine, leucine, isoleucine, tryptophan, or methionine. In the case of a peptide that binds to the HLA-A*02:01 molecule, the amino acid at the 2-position may be substituted with leucine or methionine, and/or the C-terminal amino acid may be substituted with valine or leucine. Furthermore, in the case of a peptide that binds to the HLA-A*02:06 molecule, the amino acid at the 2-position may be substituted with valine or glutamine, and/or the C-terminal amino acid may be substituted with valine or leucine.
- All aspects of the peptide of the present invention can be manufactured using methods known to a person skilled in the art. For example, the peptide of the present invention may be synthesized artificially using a solid phase method such as the Fmoc method or the tBoc method, or a liquid phase method. Moreover, a desired peptide may also be manufactured by expressing a polynucleotide coding for the peptide of the present invention or a recombinant vector including the polynucleotide. All of the thus obtained peptides can be identified using a method known to a person skilled in the art. For example, the peptides can be identified using the Edman degradation method, mass spectrometry, or the like.
- 2. Pharmaceutical Composition
- A pharmaceutical composition for treatment or prevention of cancer according to the present invention contains, as an active component, a peptide that includes eight or more consecutive amino acid residues of one or more amino acid sequences selected from the group consisting of Sequence ID Nos. 1 to 12, and that consists of eleven or less amino acids, preferably ten or less amino acids, and more preferably nine or less amino acids, in total, for example. The peptide contained in the pharmaceutical composition may also have the amino acid sequence of one of Sequence ID Nos. 1 to 12. The peptide is as defined above.
- The peptide of the present invention is presented on an antigen presenting cell and thus induces CTLs, and the induced CTLs injure cancer cells. Therefore, the active component of the pharmaceutical composition of the present invention is not limited to the peptide of the present invention, and may be a component that can induce CTLs directly or indirectly. For example, a polynucleotide coding for the peptide or a vector including the polynucleotide, an antigen presenting cell that presents a complex between the peptide and an HLA molecule on its surface, or an exosome secreted by the antigen presenting cell, or combinations thereof may be used. Examples of the antigen presenting cell to be used include a macrophage and a dendritic cell, and it is preferable to use a dendritic cell, which has a high ability to induce CTLs. The pharmaceutical composition of the present invention may also contain other components known to be used for treatment of cancer, such as a chemokine, a cytokine, a tumor necrosis factor, and a chemotherapeutic agent. A peptide dosage may be about 1 to 10 mg per day, for example, when a patient is an adult. However, the dosage varies depending on the age and weight of a patient, an administration method, and the like, and therefore, a person skilled in the art determines the dosage as appropriate.
- The pharmaceutical composition of the present invention is not construed as being limited, but is thought to be useful for killing or the like of cancer cells due to the following action mechanism. When the pharmaceutical composition of the present invention is administered to a specific cancer patient, the peptide in the pharmaceutical composition is presented on the surface of the antigen presenting cell in a state in which the peptide binds to an HLA molecule. The CTLs recognize the peptide on such an antigen presenting cell and is thus activated, followed by proliferation and systemic circulation. When CTLs that are specific to the peptide enter the cancer tissue, the CTLs recognize the same peptide derived from the specific cancer antigen that naturally binds to the HLA molecule present on the surface of the cancer cell, and then kill the cancer cell. The pharmaceutical composition can contribute to treatment of cancer due to this action.
- The pharmaceutical composition can be used for not only treatment of cancer but also prevention of cancer. For example, when the pharmaceutical composition of the present invention is administered to a healthy human body, CTLs are induced, and the induced CTLs remain in the body. Therefore, when a specific cancer occurs, it is possible to injure the cancer cells. Similarly, a recurrence of cancer may be prevented by the administration to the human body that has undergone treatment of cancer.
- All cancers in which MUC1 is expressed are assumed as cancer to be treated or prevented. More specifically, examples of target cancers include pancreatic cancer, hepatocellular cancer, prostate cancer, lung cancer, breast cancer, bowel cancer, blood cancer, brain tumor, kidney cancer, and skin cancer, but are not construed as being limited. For example, MUC1 from which the peptide of the present invention is derived is overexpressed in pancreatic cancer, and therefore, it is thought that the peptide of the present invention is effective in treatment or prevention of pancreatic cancer. When a plurality of types of cancers to be treated or prevented are present, the pharmaceutical composition of the present invention can contain a plurality of types of active components such as an immunogenic peptide.
- The pharmaceutical composition of the present invention can be administered to a patient in a form of a preparation of a pharmaceutically acceptable salt obtained by dissolving the pharmaceutical composition in a water-soluble solvent. Examples of the form of such a pharmaceutically acceptable salt include forms of a physiologically acceptable water-soluble salt such as a sodium salt, a potassium salt, a magnesium salt, or a calcium salt that is buffered at a physiological pH. A water-insoluble solvent can also be used in addition to a water-soluble solvent, and examples of the water-insoluble solvent include alcohols such as ethanol and propylene glycol.
- A preparation containing the pharmaceutical composition of this embodiment can contain agents for various purposes, and examples of such agents include a preservative and a buffer. Examples of the preservative include sodium bisulfite, sodium bisulfate, sodium thiosulfate benzalkonium chloride, chlorobutanol, thimerosal, phenylmercuric acetate, phenylmercuric nitrate, methylparaben, polyvinyl alcohol, phenylethyl alcohol, ammonia, dithiothreitol, and β-mercaptoethanol. Examples of the buffer include sodium carbonate, sodium borate, sodium phosphate, sodium acetate, and sodium bicarbonate. These agents can be present in such an amount that the pH of a system can be maintained between 2 and 9, and preferably between 4 and 8.
- There is no particular limitation on the formulation of the pharmaceutical composition of the present invention, but examples thereof include injections (an intramuscular injection, a hypodermic injection, an intradermal injection), oral formulations, and nasal formulations when a form of vaccine is used. When the pharmaceutical composition of the present invention is in a form of vaccine, a mixed cocktail vaccine containing a plurality of types of active components may be used. For example, such vaccine can contain a plurality of types of active components that is a combination of any two or more peptides of Sequence ID Nos. 1 to 12 or other active components.
- The vaccine of the present invention may be inert component-containing vaccine containing a component other than the pharmaceutical composition, the component being inert itself and having an effect of further improving the effect of the pharmaceutical composition as vaccine. Examples of the inert component include an adjuvant and a toxoid. Examples of the adjuvant include sedimentary adjuvants such as aluminum hydroxide, aluminum phosphate, and potassium phosphate, and oily adjuvants such as Freund's complete adjuvant and Freund's incomplete adjuvant.
- When the pharmaceutical composition of the present invention is present in a form of vaccine, it is preferable to administer the pharmaceutical composition of the present invention to the body by an injection or infusion through intradermal administration, hypodermic administration, intravenous administration, intramuscular administration, or the like, or by transdermal inhalation or inhalation through a mucous membrane of the nose, the pharynx, or the like. A dose thereof can be set to be between such an amount that a cytotoxic T cell can be significantly induced and such an amount that a significant number of non-cancer cells are not injured.
- The pharmaceutical composition of the present invention is intended to be not only administered to a human body but also used outside the body. More specifically, the pharmaceutical composition of the present invention may be used to stimulate an antigen presenting cell in vitro or ex vivo and increase a CTL-inducing activity. For example, the following is description of an example of a case where the pharmaceutical composition of the present invention is used in a dendritic cell therapy for cancer. The pharmaceutical composition of the present invention can be administered to a patient requiring treatment or prevention of cancer by bringing the pharmaceutical composition into contact with an antigen presenting cell such as a dendritic cell derived from the patient in advance, and then putting the antigen presenting cell back to the body of the patient. The peptide contained in the pharmaceutical composition can be introduced into the antigen presenting cell using a lipofection method, an injection method, or the like. When a polynucleotide coding for the peptide of the present invention is used for such an application, the polynucleotide can be introduced into the antigen presenting cell using a method known in the art. The antigen presenting cell derived from a patient may be transformed in vitro, for example, with the target polynucleotide or a vector coding for the polynucleotide using a lipofection method, an electroporation method, a microinjection method, a cell fusion method, a DEAE dextran method, a calcium phosphate method, or the like.
- 3. Immunity-Inducing Agent
- An immunity-inducing agent according to the present invention contains, as an active component, a peptide that includes eight or more consecutive amino acid residues of one or more amino acid sequences selected from the group consisting of Sequence ID Nos. 1 to 12, and that consists of eleven or less amino acids, preferably ten or less amino acids, and more preferably nine or less amino acids, in total, for example. The peptide contained in the immunity-inducing agent may also have the amino acid sequence of one of Sequence ID Nos. 1 to 12. The peptide is as defined above.
- It is thought that the peptide of the present invention is presented on an antigen presenting cell and thus induces immunity. Therefore, the active component of the immunity-inducing agent of the present invention is not limited to the peptide of the present invention, and may be a component that can induce immunity directly or indirectly. For example, a polynucleotide coding for the peptide of the present invention or a vector including the polynucleotide, an antigen presenting cell that presents a complex between the peptide and an HLA molecule on its surface, or an exosome secreted by the antigen presenting cell, or combinations thereof may be used. Examples of the antigen presenting cell to be used include a macrophage and a dendritic cell, and it is preferable to use a dendritic cell, which has a high ability to induce CTLs.
- The immunity-inducing agent of the present invention is intended to be not only administered to a human body but also used outside the body. More specifically, the immunity-inducing agent of the present invention may be used to stimulate an antigen presenting cell in vitro or ex vivo and increase a CTL-inducing activity. For example, the following is description of an example of a case where the immunity-inducing agent of the present invention is used in a dendritic cell therapy. The immunity-inducing agent of the present invention can be administered to a patient requiring immunity induction by bringing the immunity-inducing agent into contact with an antigen presenting cell such as a dendritic cell derived from the patient in advance, and then putting the antigen presenting cell back to the body of the patient. The peptide contained in the immunity-inducing agent can be introduced into the antigen presenting cell using transfection via a liposome (lipofection method), an injection method, or the like. When a polynucleotide coding for the peptide of the present invention is used for such an application, the polynucleotide can be introduced into the antigen presenting cell using a method known in the art. The antigen presenting cell derived from a patient may be transformed in vitro, for example, with the target polynucleotide or a vector expressing the polynucleotide using a lipofection method, an electroporation method, a microinjection method, a cell fusion method, a DEAE dextran method, a calcium phosphate method, or the like.
- The “immunity induction” as used herein means that an immune reaction is induced, and a CTL-inducing activity of the antigen presenting cell as well as a cytotoxic activity of CTLs against cancer cells, for example, are thus increased. The “CTL induction” as used herein means that, in vitro or in vivo, CTLs that specifically recognize a certain antigen are induced or proliferated, or naive T cells are differentiated into effector cells that has an ability (cytotoxic activity) to kill target cells such as cancer cells, and/or the cytotoxic activity of CTLs is increased, due to the peptide of the present invention being presented on the surface of an antigen presenting cell. The CTL-inducing activity can be measured by evaluating the production of a cytokine (e.g., interferon (IFN)-γ) by CTLs. For example, the CTL-inducing activity may be measured by evaluating, using a known highly sensitive immunoassay such as ELISPOT (Enzyme-Linked ImmunoSpot) or ELISA (Enzyme-Linked ImmunoSorbent Assay), an increase in the number of cytokine-producing cells that have been induced from precursor cells by an antigen presenting cell such as a peripheral blood mononuclear cell stimulated by the peptide of the present invention. The cytotoxic activity can also be measured using a known method such as a 51Cr releasing method. When the above-mentioned activities increase significantly, for example, by 5% or more, 10% or more, or 20% or more, or preferably 50% or more, compared with control samples, immunity and CTLs can be evaluated as being induced.
- 4. Method for Manufacturing Antigen Presenting Cell
- A method for manufacturing an antigen presenting cell according to the present invention includes a step of bringing a peptide that includes eight or more consecutive amino acid residues of one or more amino acid sequences selected from the group consisting of Sequence ID Nos. 1 to 12, and that consists of eleven or less amino acids, preferably ten or less amino acids, and more preferably nine or less amino acids, in total into contact with an antigen presenting cell in vitro, for example. The peptide used in the manufacturing method of the present invention may also have the amino acid sequence of one of Sequence ID Nos. 1 to 12. The peptide is as defined above.
- It is thought that the peptide used in the manufacturing method of the present invention binds to an HLA class-I molecule on the surface of an antigen presenting cell and is presented to CTLs as an antigen peptide, and the CTL activity of the antigen presenting cell is thus induced. Therefore, a component to be brought into contact with an antigen presenting cell is not limited to the peptide of the present invention, and may be a component that can induce CTLs directly or indirectly. For example, a polynucleotide coding for the peptide or a vector including the polynucleotide, an antigen presenting cell that presents a complex between the peptide and an HLA molecule on its surface, or an exosome secreted by the antigen presenting cell, or combinations thereof may be used. Examples of the antigen presenting cell to be used include a macrophage and a dendritic cell, and it is preferable to use a dendritic cell, which has a high ability to induce CTLs.
- The antigen presenting cell manufactured using the manufacturing method of the present invention is intended to be used not only as an active component of the above-mentioned pharmaceutical composition or immunity-inducing agent but also in an immunotherapy or the like. For example, the following is description of an example of a case where the manufactured antigen presenting cell is used in a dendritic cell therapy for cancer. The manufactured antigen presenting cell can be administered to a patient requiring immunity induction by bringing the manufactured antigen presenting cell into contact with an antigen presenting cell such as a dendritic cell that are derived from the patient and has a low ability to induce CTLs in advance, and then putting the manufactured antigen presenting cell back to the body of the patient. The peptide of the present invention can be introduced into the antigen presenting cell using transfection via a liposome (lipofection method), an injection method, or the like. When a polynucleotide coding for the peptide of the present invention is used for such an application, the polynucleotide can be introduced into the antigen presenting cell using a method known in the art. The antigen presenting cell derived from a patient may be transformed in vitro, for example, with the target polynucleotide or a vector coding for the polynucleotide using a lipofection method, an electroporation method, a microinjection method, a cell fusion method, a DEAE dextran method, a calcium phosphate method, or the like.
- Hereinafter, the present invention will be described more specifically by use of examples, but the present invention is not limited to the examples.
- Specifically, procedures of prediction, experiment, and evaluation in these examples were carried out based on the active learning experiment design described in WO 2006/004182, and rules were constructed by repeating the following steps as a whole.
- (1) A trial of a lower-order learning algorithm, which will be described later, was carried out once. That is, a plurality of hypotheses were generated by random resampling from accumulated data, and with regard to randomly expressed candidate query points (peptides), a point that showed the largest distribution of predicted values was selected as a query point to be subjected to an experiment.
- (2) The peptide at the selected query point was prepared by a synthesis and purification method, which will be described later, and the actual binding ability was measured by an experiment, which will be described later, and added to the accumulated data.
- In accordance with such an active learning method, the number of repetitions of the binding experiment for peptides consisting of 9 amino acid residues, which would otherwise have to be carried out for the 500 billion (=209) or more combinations of all the candidates for HLA-binding peptides, could be reduced.
- Based on the rules as described above, the amino acid sequences of Sequence ID Nos. 1 to 12 were extracted.
- Synthesis and Purification of Peptide
- Peptides having amino acid sequences of Sequence ID Nos. 1 to 12 were manually synthesized with the Merrifield solid-phase method using Fmoc amino acids. After deprotection, reverse phase HPLC purification was carried out using a C18 column to give a purity of 95% or higher. MALDI-TOF mass spectrometry (AB SCIEX MALDI-TOF/TOF5800) was carried out to identify the peptides and confirm their purities. The peptides were quantified with Micro BCA assay (Thermo Scientific) using BSA as a standard protein.
- Experiment of Binding Peptide to HLA-A*24:02 Molecule
- The ability of each of the peptides to bind to an HLA-A*24:02 molecule, which is an HLA-A*24:02 gene product, was measured using C1R-A24 cells expressing the HLA-A*24:02 molecule (cells prepared by Professor Masafumi TAKIGUCHI, Kumamoto University being supplied with permission by Assistant Professor Masaki YASUKAWA, Ehime University).
- First, C1R-A24 cells were exposed to acidic conditions at a pH of 3.3 for 30 seconds, and thus an endogenous peptide that originally binds to the HLA-A*24:02 molecule, and a light chain β2m, which is associated with HLA class-I molecules in common, were dissociated and removed. After neutralization, a purified β2m was added to C1R-A24 cells, and the obtained product was added to a series of dilutions of the peptide and incubated on ice for 4 hours. Staining was carried out using a fluorescently labeled monoclonal antibody 17A12, which recognizes association (MHC-pep) of the three members, that is, a HLA-A*24:02 molecule, a peptide, and β2m, which had reassociated during the incubation.
- Thereafter, the MHC-pep count per C1R-A24 cell (proportional to the intensity of fluorescence of the above-mentioned fluorescent antibody) was quantitatively measured using a fluorescence-activated cell sorter FACScan (Becton-Dickinson). A binding-dissociation constant Kd value between the HLA-A*24:02 molecule and the peptide was calculated from the average intensity of fluorescence per cell using a method that was reported in a paper (Udaka et al., Immunogenetics, 51, 816-828, 2000) by the inventors of the present invention.
- Experiment of Binding Peptide to HLA-A*02:01 Molecule
- The ability of each of the peptides to bind to an HLA-A*02:01 molecule, which is an HLA-A*02:01 gene product, was measured using a cell line T2 (purchased from ATCC) expressing the HLA-A*02:01 molecule.
- The T2 cells and purified β2m were added to a series of serial dilutions of the peptide whose binding ability would be measured, and then incubation was carried out at 37° C. for 4 hours. The HLA-A*02:01 molecule that had increased in an expression amount in a peptide-concentration-dependent manner up to this point was stained using a fluorescently labeled monoclonal antibody BB7.2, which is specific to an association type.
- Thereafter, the amount of fluorescence per cell was measured using a flow cytometer, and a dissociation constant Kd value was calculated using a method that was reported in a paper (Udaka et al., Immunogenetics, 51, 816-828, 2000) by the inventors of the present invention.
- Experiment of Binding Peptide to HLA-A*02:06 Molecule
- The ability of each of the peptides to bind to an HLA-A*02:06 molecule, which is an HLA-A*02:06 gene product, was measured using RA2.6 cells (cell line that had been newly produced in Kochi University) produced by introducing the cDNA of the HLA-A*02:06 gene into a mouse TAP (transporter associated with antigen processing)-deficient cell line RMAS.
- First, RA2.6 cells were cultured at 26° C. overnight. After HLA-A*02:06 molecules to which the peptide did not bind accumulated on the cell surface, a series of dilutions of the peptide were added thereto, and binding was carried out at 26° C. for 60 minutes.
- Thereafter, the cells were cultured at 35° C. for 4 hours. As a result, blank HLA-A*02:06 molecules to which the peptide did not bind were denatured, and thus their conformation was lost. A fluorescently labeled monoclonal antibody BB7.2, which specifically recognizes a peptide-binding HLA-A*02:06 molecule, was added thereto, incubation was carried out on ice for 20 minutes, and thus the cells were stained.
- Thereafter, the amount of fluorescence per cell was measured using a flow cytometer, and a dissociation constant Kd value was calculated using a method that was reported in a paper (Udaka et al., Immunogenetics, 51, 816-828, 2000) by the inventors of the present invention.
- Evaluation Results of Binding Experiments
- As a result, as shown in a table below, the data from the experiment of binding the peptides of the present invention to the HLA molecules were obtained.
-
TABLE 2 Amino acid sequence Location in Data of binding experiment (Sequence ID No.) MUC1 to A*24:02 to A*02:01 to A*02:06 FLGLSNIKF (Seq. ID No. 1) 1086 −7.073261692 −5.390853526 −4.386007431 SVPVTRPAL (Seq. ID No. 2) 100 −7.241740934 −3.903379214 −5.627441171 GVPGWGIAL (Seq. ID No. 3) 1155 −5.927200496 −4.54796761 −5.763029942 AFREGTINV (Seq. ID No. 4) 1106 −5.834149762 >−3 >−3 AASRYNLTI (Seq. ID No. 5) 1128 −6.148024331 −4.811130083 −3.824201259 LQRDISEMF (Seq. ID No. 6) 1069 −6.646212952 >−3 −4.260522119 HHSDTPTTL (Seq. ID No. 7) 997 −5.867440069 >−3 >−3 SFFFLSFHI (Seq. ID No. 8) 1041 −7.065109329 >−4 −3.980495237 TLAFREGTI (Seq. ID No. 9) 1104 −5.973982835 >−3 −3.08622966 STGVSFFFL (Seq. ID No. 10) 1037 −5.171414811 −5.121912366 −5.208079724 GQDVTSVPV (Seq. ID No. 11) 95 >−3 −6.437064471 −6.349002291 FSAQSGAGV (Seq. ID No. 12) 1148 >−3 −4.74018 −5.661074048 - It should be noted that the amino acid sequences of Sequence ID Nos. 1 to 12 are derived from the entire sequence (Sequence ID No. 13) (>sp|P15941|MUC1_HUMAN Mucin-1 OS=Homo sapiens GN=MUC1 PE=1 SV=3) of a predetermined MUC1 genome protein registered in GENBANK.
- Peptide Immunity Induction Testing
- (1) Preparation of Peptide-Stimulated Dendritic Cell
- Day 0 to 9 (Induction of Dendritic Cell)
- Of the peripheral blood monocytes obtained through pheresis from a patient (0) that had undergone a dendritic cell/CTL therapy against MUC1, a cell fraction that adhered to a culture flask was cultured in an AIM-CM culture medium (manufactured by Gibco) at 37° C. for 10 days. During the culture, 15 μl of IL-4 and 30 μl of a granulocyte monocyte colony-stimulating factor (GM-CSF) added to the culture medium on Day 0 and Day 3, and 15 μl of IL-4, 30 μl of GM-CSF, and 75 μl of a tumor necrosis factor (TFN)-α were added thereto on Day 5.
- Day 10 (Peptide Stimulation and Dendritic Cell Collection)
- Induced dendritic cells were collected in a new AIM-CM culture medium, and the peptide of the present invention (Sequence ID Nos. 1 to 12) was added thereto to a concentration of 20 μg/ml. Thereafter, the culture medium containing the dendritic cells was cultured at 37° C. for 2 hours. The following peptides were used as positive controls and negative controls.
-
Positive control for HLA-A*24:02 (EBV LMP2, 419-427: TYGPVFMCL (Sequence ID No. 14)) Negative control for HLA-A*24:02 (HIV env gp160, 584-592: RYLRDQQLL (Sequence ID No. 15)) Positive control for HLA-A*02:01 (Flu A MP, 58-66: GILGFVFTL (Sequence ID No. 16)) Negative control for HLA-A*02:01 (HIV gap p17, 77-85: SLYNTVATL (Sequence ID No. 17)) Positive control for HLA-A*02:06 (EBV LMP2 453-461: LTAGFLIFL (Sequence ID No. 18)) Negative conrol for HLA-A*02:06 (HIV gap p24 341-349: ATLEEMMTA (Sequence ID No. 19)) - The dendritic cells were collected and washed three or more times using a sufficient amount of an AIM-CM culture medium, and then the number of cells was counted.
- (2) Preparation of CD8T Cells
- Day 0 to 9
- Of the peripheral blood monocytes obtained through pheresis from a patient that had undergone treatment using the above-mentioned vaccine twice or more, a floating cell fraction (containing lymphocytes) that did not adhere to a culture flask was cultured in an AIM-CM culture medium (manufactured by Gibco) at 37° C. for 10 days. During the culture, 40 μl of IL-2 was added to the culture medium on Day 4 and Day 6.
- Day 10
- CD8T cells were isolated from the culture medium using a CD8 negative selection kit (manufactured by Miltenyi), and the number of cells was counted.
- (3) Coculture
- The dendritic cells and CD8T cells obtained in the above (1) and (2) were cocultured in an AIM-CM culture medium at 37° C. in the following conditions.
-
- CD8T cell: 5×105 cells/well
- Dendritic cell: 2×105 cells/well
- Day 12 or 13
- To the above-mentioned culture medium, 0.4 ml/well of an AIM-CM culture medium containing 20 U/ml of IL-2 was added.
- (5) ELISA Assay
- Day 17
- The culture supernatant of the coculture of the T cells and the dendritic cells pulsed with the above-mentioned peptides on Day 7 was diluted to four steps, that is, ×1, ×5, ×25, and ×125, and the dilution step within a measurement limit was identified using Human IFN-γ ELISA MAX Deluxe Set (manufactured by BioLegend). Thereafter, at the identified dilution step, each sample was measured three times.
FIGS. 1 to 4 respectively show typical results of ELISA assay from a patient with an HLA type of 24:02/33:03, a patient with an HLA type of 24:02/33:03, a patient with an HLA type of 11:01/24:02, a patient with an HLA type of 02:06/24:02, and a patient having an HLA type of 02:01/02:06. - The present invention has been described based on the examples. The examples are merely exemplary, and a person skilled in the art would understand that various modified examples are possible, and such modified examples are also within a scope of the present invention.
Claims (10)
1. A peptide comprising eight or more consecutive amino acid residues of amino acid sequence of one of Sequence ID Nos. 1 to 12, and consisting of eleven or less amino acid residues.
2. The peptide according to claim 1 , wherein one or several amino acids in the amino acid sequence are substituted, inserted, deleted, or added, and the peptide has immunogenicity.
3. The peptide according to claim 2 , wherein an amino acid at a 2-position of the amino acid sequence is substituted with tyrosine, phenylalanine, methionine, tryptophan, valine, leucine or glutamine, and/or a C-terminal amino acid is substituted with phenylalanine, leucine, isoleucine, tryptophan, methionine, or valine.
4. A pharmaceutical composition for treatment or prevention of cancer, comprising the peptide according to claim 1 .
5. The pharmaceutical composition according to claim 4 , which is in a form of vaccine.
6. The pharmaceutical composition according to claim 4 , wherein the peptide can bind to one or more types of HLA molecules.
7. An immunity-inducing agent comprising the peptide according to claim 1 .
8. The immunity-inducing agent according to claim 7 , which is used to induce a cytotoxic T cell.
9. The immunity-inducing agent according to claim 7 , wherein the peptide can bind to one or more types of HLA molecules.
10. A method for manufacturing an antigen presenting cell having a CTL-inducing activity, the method comprising a step of bringing the peptide according to claim 1 into contact with an antigen presenting cell in vitro.
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP2015-046462 | 2015-03-09 | ||
JP2015046462 | 2015-03-09 | ||
PCT/JP2016/057349 WO2016143814A1 (en) | 2015-03-09 | 2016-03-09 | Peptide derived from muc1, pharmaceutical composition for treating or preventing cancer using same, immunity inducer, and method for producing antigen-presenting cells |
Related Parent Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/JP2016/057349 A-371-Of-International WO2016143814A1 (en) | 2015-03-09 | 2016-03-09 | Peptide derived from muc1, pharmaceutical composition for treating or preventing cancer using same, immunity inducer, and method for producing antigen-presenting cells |
Related Child Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US16/795,391 Continuation US11618770B2 (en) | 2015-03-09 | 2020-02-19 | MUC1-derived peptide, and pharmaceutical composition for treatment or prevention of cancer, immunity-inducing agent and method for manufacturing antigen presenting cell using same |
Publications (1)
Publication Number | Publication Date |
---|---|
US20180057531A1 true US20180057531A1 (en) | 2018-03-01 |
Family
ID=56880033
Family Applications (2)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US15/556,766 Abandoned US20180057531A1 (en) | 2015-03-09 | 2016-03-09 | Muc1-derived peptide, and pharmaceutical composition for treatment or prevention of cancer, immunity-inducing agent and method for manufacturing antigen presenting cell using same |
US16/795,391 Active 2036-09-19 US11618770B2 (en) | 2015-03-09 | 2020-02-19 | MUC1-derived peptide, and pharmaceutical composition for treatment or prevention of cancer, immunity-inducing agent and method for manufacturing antigen presenting cell using same |
Family Applications After (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US16/795,391 Active 2036-09-19 US11618770B2 (en) | 2015-03-09 | 2020-02-19 | MUC1-derived peptide, and pharmaceutical composition for treatment or prevention of cancer, immunity-inducing agent and method for manufacturing antigen presenting cell using same |
Country Status (5)
Country | Link |
---|---|
US (2) | US20180057531A1 (en) |
EP (1) | EP3269726A4 (en) |
JP (1) | JP6481873B2 (en) |
TW (1) | TW201639868A (en) |
WO (1) | WO2016143814A1 (en) |
Families Citing this family (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2023163094A1 (en) * | 2022-02-25 | 2023-08-31 | 日本電気株式会社 | Pharmaceutical composition for treatment or prevention of adult t-cell leukemia |
Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2001018035A2 (en) * | 1999-09-08 | 2001-03-15 | Transgene S.A. | Muc-1 derived peptides |
US20110318380A1 (en) * | 2008-10-01 | 2011-12-29 | Dako Denmark A/S | MHC Multimers in Cancer Vaccines and Immune Monitoring |
CN104211796A (en) * | 2013-11-09 | 2014-12-17 | 深圳市康尔诺生物技术有限公司 | Compound formed by pancreas cancer related peptide and heat shock protein and applications thereof |
Family Cites Families (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPH08151396A (en) | 1994-11-28 | 1996-06-11 | Teijin Ltd | Hla-binding oligopeptide and immunomodulating agent containing the compound |
JPH11316754A (en) | 1998-05-06 | 1999-11-16 | Nec Corp | Experimental design and recording medium recording experimental design program |
WO2006004182A1 (en) | 2004-07-07 | 2006-01-12 | Nec Corporation | Arrangement prediction system |
EP1982992B1 (en) | 2006-02-07 | 2010-10-27 | NEC Corporation | Hla-binding peptide, precursor thereof, dna fragment encoding the same and recombinant vector |
KR20150091311A (en) * | 2012-12-04 | 2015-08-10 | 온코세라피 사이언스 가부시키가이샤 | Sema5b peptides and vaccines containing the same |
-
2016
- 2016-03-09 EP EP16761781.0A patent/EP3269726A4/en active Pending
- 2016-03-09 TW TW105107134A patent/TW201639868A/en unknown
- 2016-03-09 JP JP2017505372A patent/JP6481873B2/en active Active
- 2016-03-09 US US15/556,766 patent/US20180057531A1/en not_active Abandoned
- 2016-03-09 WO PCT/JP2016/057349 patent/WO2016143814A1/en active Application Filing
-
2020
- 2020-02-19 US US16/795,391 patent/US11618770B2/en active Active
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2001018035A2 (en) * | 1999-09-08 | 2001-03-15 | Transgene S.A. | Muc-1 derived peptides |
US20110318380A1 (en) * | 2008-10-01 | 2011-12-29 | Dako Denmark A/S | MHC Multimers in Cancer Vaccines and Immune Monitoring |
CN104211796A (en) * | 2013-11-09 | 2014-12-17 | 深圳市康尔诺生物技术有限公司 | Compound formed by pancreas cancer related peptide and heat shock protein and applications thereof |
Also Published As
Publication number | Publication date |
---|---|
TW201639868A (en) | 2016-11-16 |
WO2016143814A1 (en) | 2016-09-15 |
JPWO2016143814A1 (en) | 2018-01-18 |
JP6481873B2 (en) | 2019-03-13 |
US20200181195A1 (en) | 2020-06-11 |
US11618770B2 (en) | 2023-04-04 |
EP3269726A4 (en) | 2018-12-05 |
EP3269726A1 (en) | 2018-01-17 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US10875900B2 (en) | Peptide derived from GPC3, pharmaceutical composition for treatment or prevention of cancer using same, immunity inducer, and method for producing antigen-presenting cells | |
US20220193213A1 (en) | Hsp70-derived peptide, pharmaceutical composition for treating or preventing cancer using same, immunity inducer, and method for producing antigen presenting cell | |
US11618770B2 (en) | MUC1-derived peptide, and pharmaceutical composition for treatment or prevention of cancer, immunity-inducing agent and method for manufacturing antigen presenting cell using same | |
WO2023163094A1 (en) | Pharmaceutical composition for treatment or prevention of adult t-cell leukemia |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
AS | Assignment |
Owner name: NEC CORPORATION, JAPAN Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:MIYAKAWA, TOMOYA;OKA, MASAAKI;HAZAMA, SHOICHI;AND OTHERS;REEL/FRAME:043535/0103 Effective date: 20170822 |
|
STPP | Information on status: patent application and granting procedure in general |
Free format text: NON FINAL ACTION MAILED |
|
STPP | Information on status: patent application and granting procedure in general |
Free format text: RESPONSE TO NON-FINAL OFFICE ACTION ENTERED AND FORWARDED TO EXAMINER |
|
STPP | Information on status: patent application and granting procedure in general |
Free format text: NON FINAL ACTION MAILED |
|
STCB | Information on status: application discontinuation |
Free format text: ABANDONED -- FAILURE TO RESPOND TO AN OFFICE ACTION |