WO2014157704A1 - Erap1によるトリミング機能をきっかけとしたコンジュゲートワクチン - Google Patents
Erap1によるトリミング機能をきっかけとしたコンジュゲートワクチン Download PDFInfo
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- WO2014157704A1 WO2014157704A1 PCT/JP2014/059352 JP2014059352W WO2014157704A1 WO 2014157704 A1 WO2014157704 A1 WO 2014157704A1 JP 2014059352 W JP2014059352 W JP 2014059352W WO 2014157704 A1 WO2014157704 A1 WO 2014157704A1
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- cancer antigen
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- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
- A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
- A61K47/646—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent the entire peptide or protein drug conjugate elicits an immune response, e.g. conjugate vaccines
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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- 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
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
- C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
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- 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
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/62—Medicinal preparations containing antigens or antibodies characterised by the link between antigen and carrier
- A61K2039/627—Medicinal preparations containing antigens or antibodies characterised by the link between antigen and carrier characterised by the linker
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
- C12Y207/10—Protein-tyrosine kinases (2.7.10)
- C12Y207/10001—Receptor protein-tyrosine kinase (2.7.10.1)
Definitions
- the present invention belongs to the field of cancer immunotherapy, and efficiently treats cytotoxic T cells comprising a cancer antigen peptide precursor complexed via a sulfur-sulfur covalent bond, which can be trimmed by a specific peptide-degrading enzyme. It relates to a conjugate vaccine to be guided.
- CTL cytotoxic T lymphocytes, cytotoxic T-lymphocyte, cytotoxic T-cell, hereinafter referred to as CTL
- CTLs are produced by the differentiation and proliferation of precursor T cells that recognize complexes formed by cancer antigen protein-derived antigenic peptides (cancer antigen peptides) and MHC class I molecules, and attack cancer cells.
- MHC is called human leukocyte antigen (HLA) in humans, and HLA-A, B, Cw, F and G are known.
- the cancer antigen peptide is most suitable for degradation of cancer antigen protein synthesized in cancer cells (processing), ie, protein denaturation by reduction of sulfur-sulfur covalent bond, degradation by proteosome or protease, trimming enzyme in endoplasmic reticulum
- processing ie, protein denaturation by reduction of sulfur-sulfur covalent bond, degradation by proteosome or protease, trimming enzyme in endoplasmic reticulum
- a peptide produced by cleavage to length generally consisting of 8 to 12 amino acid residues.
- helper T cells In cancer immunotherapy, activation of helper T cells is also important for enhancing the function of other T cells including CTLs.
- an antigen protein is degraded by intracellular lysosomes, and a part of a fragment peptide composed of a peptide consisting of amino acids of about 13 to 17 residues binds to an MHC class II molecule as an antigen peptide, and the helper T cell.
- the helper T cell is activated by being presented to the TCR / CD3 complex.
- HLA-DR, DQ, DP and the like are known as HLA.
- Cancer antigen proteins themselves or antigen peptides derived from cancer antigen proteins are mainly used as cancer vaccine antigens (see Non-Patent Document 1). Since cancer vaccines using proteins usually contain various cancer antigen peptides, multiple CTLs and helper T cells can be induced simultaneously. However, it has problems in stable supply and quality control. On the other hand, although a cancer vaccine using a peptide can be easily manufactured and quality controlled, it is mainly composed of a single MHC class I-presenting peptide antigen. In recent years, it has been pointed out that improvement is necessary (see Non-Patent Document 2).
- Such peptide cancer vaccines include cocktail vaccines in which a plurality of peptide antigens presented in MHC class I and class II are mixed, and long-chain peptide vaccines in which peptide antigens presented in MHC class I and class II are linked by amide bonds
- cocktail vaccines since each peptide antigen composed of various amino acids exhibits various physical properties, it is often difficult to develop an optimal formulation that efficiently induces CTLs corresponding to them.
- long-chain peptide vaccines there are cases where there are problems in the production thereof as in the case of proteins.
- long-chain peptide vaccines bind peptide antigens presented in class I and class II via an arbitrary peptide spacer. Therefore, it is difficult to control and predict the cleavage site by intracellular enzymes.
- a peptide dimer in which two peptide monomers are bonded to each other by a disulfide bond has been reported (see Patent Document 1). Unlike a cocktail vaccine, these are homodimers to which two single peptides are bound, and thus have a single physical property and can be easily produced.
- cancer antigen peptides need to contain cysteine in their amino acid sequences, and their versatility is low.
- Endoplasmic reticulum aminopeptidase 1 (hereinafter referred to as ERAP1) is one of the trimming enzymes within the endoplasmic reticulum (hereinafter referred to as ER) and has a specific antigen peptide sequence and peptide length. It is reported that the cancer antigen peptide precursor is cleaved from the N-terminus and adjusted to an optimal length for binding to MHC class I (see Non-Patent Document 3).
- An object of the present invention is to provide a conjugate vaccine that efficiently induces CTL.
- cysteine is added to a cancer antigen peptide while studying the use of a conjugate vaccine. It is possible to cleave cysteines extended from the N-terminus of various cancer antigen peptides generated by reductive cleavage of disulfide bonds of ribonucleic acid and efficiently convert them into cancer antigen peptides, such as pharmacological tests in in vivo animal models. By confirming that the results were strongly suggested, the present inventors completed the present invention by reaching the discovery of creating a multivalent antigen peptide-presenting conjugate vaccine capable of inducing CTLs in the body.
- the necessary cysteine can be converted to N-terminal without affecting antigen presentation to MHC class I when complexing two different cancer antigen peptides.
- a method of introducing an arbitrary position into the C-terminal was conceived.
- a peptide in which 0 to 5 amino acids including cysteine were introduced at the N-terminus of the cancer antigen peptide, and a conjugate containing a disulfide bond via cysteine of the peptide were created.
- the present inventors have confirmed for the first time that the peptide or conjugate is easily subjected to trimming by ERAP1 in vitro and / or in vivo, and as a result, produces a cancer antigen peptide, and the present invention has been completed. It was.
- the development of a novel multivalent antigen peptide-presenting peptide cancer vaccine that is easy to manufacture, excellent in versatility, and efficiently induces CTLs has been desired.
- the conjugates invented by the present inventors enable CTLs to be produced. It has become possible to develop a multivalent antigen peptide-presenting peptide cancer vaccine that is efficiently derived, excellent in physicochemical properties, easy to manufacture, easy to manage, and versatile.
- the present invention relates to the following.
- X a and Y a are independently a divalent group of a peptide consisting of amino acids of a single bond or 1 to 4 residues, amino acid residues of the amino acid residues and Y a of X a
- Cancer antigen peptide A represents MHC class I-restricted cancer antigen peptide consisting of amino acids 7 to 30 residues, the amino group of the N-terminal amino acid of the cancer antigen peptide A bound to Y a in the formula (1), The carbonyl group of the C-terminal amino acid of cancer antigen peptide A is bonded to the hydroxyl group in formula (1), R 1 is a hydrogen atom, Formula (2):
- the cancer antigen peptide B has a sequence different from that of the cancer antigen peptide A and is an MHC class I-restricted cancer antigen peptide consisting of 7 to 30 amino acids or an MHC class II-restricted cancer antigen consisting of 7 to 30 amino acids represents a peptide, the amino group of the N-terminal amino acids of the cancer antigen peptide B bound to Y b in the formula (2), the carbonyl group of the C-terminal amino acids of the cancer antigen peptide B bound to the hydroxyl group in the formula (2) ,
- the thioether group in formula (2) is bonded to the thioether group in formula (1).
- X c and Y c independently represent a single bond or a divalent group of a peptide consisting of 1 to 4 amino acids, and the number of amino acid residues of X c and the number of amino acid residues of Y c
- Cancer antigen peptide C represents an MHC class II-restricted cancer antigen peptide consisting of 7 to 30 amino acids, and the carbonyl group of the C-terminal amino acid of cancer antigen peptide C is bonded to X c in formula (3)
- the amino group of the N-terminal amino acid of cancer antigen peptide C is bonded to the hydrogen atom in formula (3)
- the thioether group in formula (3) is bonded to the thioether group in formula (1).
- Cancer antigen peptide D is an MHC class I-restricted cancer antigen peptide consisting of 7 to 30 amino acids containing one cysteine residue or MHC class II consisting of 7 to 30 amino acids containing one cysteine residue This represents a restricted cancer antigen peptide, and the thioether group of the cysteine residue of cancer antigen peptide D is bonded to the thioether group in formula (1).
- R 1 is a hydrogen atom
- the sequence of the compound represented by formula (1) is not the same as the partial sequence of the cancer antigen protein.
- X a is a divalent group of a peptide consisting of two amino acids and Y a is a single bond, or X a and Y a are independently a divalent group of a peptide consisting of one amino acid Or X a is a single bond and Y a is a divalent group of a peptide consisting of two amino acids, or X a is a divalent group of a peptide consisting of one amino acid and Y a is or a single bond, or X a is a divalent radical of a peptide and Y a is a single bond comprises the amino acid of one residue, or X a and Y a is a single bond, according to claim 1 A compound, or a pharmaceutically acceptable salt thereof;
- Item 3 The compound according to Item 1 or 2, or a pharmaceutically acceptable salt thereof, wherein X a is a single bond and Y a is a single bond, an alanine residue, a leucine residue or a methionine residue;
- Item 4 The compound according to Item 1 or 2, or a pharmaceutically acceptable salt thereof, wherein X a is a single bond, alanine residue, glycine residue, serine residue or tyrosine residue, and Y a is a single bond;
- Item 5 The compound according to any one of Items 1 to 4, wherein X a and Y a are a single bond, or a pharmaceutically acceptable salt thereof;
- Item 6 The compound according to any one of Items 1 to 5, wherein the cancer antigen peptide A is an amino acid having 7 to 15 residues and is an HLA-A, HLA-B or HLA-Cw-restricted cancer antigen peptide; A pharmaceutically acceptable salt;
- Cancer antigen peptide A is composed of 7 to 15 amino acids and is HLA-A1, HLA-A2, HLA-A3, HLA-A11, HLA-A24, HLA-A28, HLA-A29, HLA-A31, HLA-A33 , HLA-A34, HLA-A68, HLA-B7, HLA-B13, HLA-B35, HLA-B37, HLA-B44, HLA-B45, HLA-B51, HLA-B52, HLA-B53, HLA-Cw2, HLA The compound according to any one of Items 1 to 6, or a pharmaceutically acceptable salt thereof, which is a Cw3, HLA-Cw6, HLA-Cw7, HLA-Cw8 or HLA-Cw16 restricted cancer antigen peptide;
- Cancer antigen peptide A is MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A6, MAGE-A10, MAGE-A12, BAGE, DAM-6, DAM-10, GAGE-1, GAGE- 2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7B, GAGE-8, NA88-A, NY-ESO-1, NY-ESO-1a, MART-1 / Melan-A, MC1R, Gp100, PSA, PSM, Tyrosinase, Proteinase 3, TRP-1, TRP-2, ART-4, CAMEL, CEA, Ep-CAM, Cyp-B, Her2 / neu, VEGFR, hTERT, hTRT, iCE, MUC1 , MUC2, PRAME, P15, RU1, RU2, SART-1, ART-2, SART-3, AFP, ⁇ -Catenin, Caspase-8, CDK-4, ELF2, G
- Cancer antigen peptide A has the following amino acid sequence: NYKHCCFEI (SEQ ID NO: 3), EYLQLVFGI (SEQ ID NO: 11), FLWGPRALV (SEQ ID NO: 13), GLYDGMEHL (SEQ ID NO: 19), SLLMWITCQC (SEQ ID NO: 26), QLSLLMWIT (SEQ ID NO: 27), AAGIGILTV (SEQ ID NO: 29), LIYRRRLLMK (SEQ ID NO: 33), YMDGTMSQV (SEQ ID NO: 40), AFLPWHHRLF (SEQ ID NO: 41), VLQELNVTV (SEQ ID NO: 43), YLSGANLNL (SEQ ID NO: 50), KIFGSLAFL (SEQ ID NO: 53), AYIDFEMKI (SEQ ID NO: 66), AYACNTSTL (SEQ ID NO: 83), KWFPSCQFLL (SEQ ID NO: 84) and GYDQIMPKK (SEQ ID NO: 85
- Cancer antigen peptide A has the following amino acid sequence: GLYDGMEHL (SEQ ID NO: 19), VLQELNVTV (SEQ ID NO: 43) and KIFGSLAFL (SEQ ID NO: 53) Item 10.
- GLYDGMEHL SEQ ID NO: 19
- VLQELNVTV SEQ ID NO: 43
- KIFGSLAFL SEQ ID NO: 53
- Item 11 The compound according to any one of Items 1 to 10, wherein R 1 is a hydrogen atom, or a pharmaceutically acceptable salt thereof;
- the compound represented by the formula (1) has the following amino acid sequence: CAGLYDGMEHL (SEQ ID NO: 89), CLGLYDGMEHL (SEQ ID NO: 90), CMGLYDGMEHL (SEQ ID NO: 91), CAVLQELNVTV (SEQ ID NO: 92), CLVLQELNVTV (SEQ ID NO: 93), CMVLQELNVTV (SEQ ID NO: 94), CAKIFGSLAFL (SEQ ID NO: 95), CLKIFGSLAFL (SEQ ID NO: 96) and CMKIFGSLAFL (SEQ ID NO: 97) Item 12.
- the compound represented by the formula (1) has the following amino acid sequence: CGLYDGMEHL (SEQ ID NO: 98), CVLQELNVTV (SEQ ID NO: 99) and CKIFGSLAFL (SEQ ID NO: 100) Item 12.
- Item 14 The compound according to any one of Items 1 to 10, wherein R 1 is a group represented by Formula (2), or a pharmaceutically acceptable salt thereof;
- X b is a divalent group of a peptide consisting of two amino acids and Y b is a single bond, or X b and Y b are independently a divalent group of a peptide consisting of one amino acid Or X b is a single bond and Y b is a divalent group of a peptide consisting of two amino acids, or X b is a divalent group of a peptide consisting of one amino acid and Y b is Item 1-10, wherein X is a single bond, X b is a single bond and Y b is a divalent group of a peptide consisting of one amino acid, or X b and Y b are a single bond 14.
- Item 16 The compound according to any one of Items 1 to 10 and 14 to 15, wherein X b is a single bond and Y b is a single bond, an alanine residue, a leucine residue or a methionine residue, or a pharmaceutically acceptable salt thereof Acceptable salts;
- Item 17 The compound according to any one of Items 1 to 10 and 14 to 15, wherein X b is a single bond or a divalent group of a peptide consisting of a single-residue amino acid, and Y b is a single bond, or a pharmaceutical thereof Top acceptable salts;
- Item 18 The compound according to any one of Items 1 to 10 and 14 to 17, or a pharmaceutically acceptable salt thereof, wherein X b and Y b are a single bond;
- Item 19 The compound according to any one of Items 1 to 10 and 14 to 18, or a pharmaceutically acceptable salt thereof, wherein the cancer antigen peptide B is an MHC class I-restricted cancer antigen peptide consisting of 7 to 15 amino acids salt;
- Item 20 The cancer antigen peptide B according to any one of Items 1 to 10 and 14 to 19, wherein the cancer antigen peptide B consists of amino acids of 7 to 15 residues and is an HLA-A, HLA-B or HLA-Cw restricted cancer antigen peptide.
- Cancer antigen peptide B is composed of 7 to 15 amino acids and is HLA-A1, HLA-A2, HLA-A3, HLA-A11, HLA-A24, HLA-A28, HLA-A29, HLA-A31, HLA-A33 , HLA-A34, HLA-A68, HLA-B7, HLA-B13, HLA-B35, HLA-B37, HLA-B44, HLA-B45, HLA-B51, HLA-B52, HLA-B53, HLA-Cw2, HLA The compound according to any one of Items 1 to 10 and 14 to 20, which is a Cw3, HLA-Cw6, HLA-Cw7, HLA-Cw8 or HLA-Cw16 restricted cancer antigen peptide, or a pharmaceutically acceptable salt thereof Salt;
- Cancer antigen peptide B is MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A6, MAGE-A10, MAGE-A12, BAGE, DAM-6, DAM-10, GAGE-1, GAGE- 2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7B, GAGE-8, NA88-A, NY-ESO-1, NY-ESO-1a, MART-1 / Melan-A, MC1R, Gp100, PSA, PSM, Tyrosinase, Proteinase 3, TRP-1, TRP-2, ART-4, CAMEL, CEA, Ep-CAM, Cyp-B, Her2 / neu, VEGFR, hTERT, hTRT, iCE, MUC1 , MUC2, PRAME, P15, RU1, RU2, SART-1, ART-2, SART-3, AFP, ⁇ -Catenin, Caspase-8, CDK-4, ELF2,
- Cancer antigen peptide B has the following amino acid sequence: NYKHCCFEI (SEQ ID NO: 3), EYLQLVFGI (SEQ ID NO: 11), FLWGPRALV (SEQ ID NO: 13), GLYDGMEHL (SEQ ID NO: 19), SLLMWITCQC (SEQ ID NO: 26), QLSLLMWIT (SEQ ID NO: 27), AAGIGILTV (SEQ ID NO: 29), LIYRRRLLMK (SEQ ID NO: 33), YMDGTMSQV (SEQ ID NO: 40), AFLPWHHRLF (SEQ ID NO: 41), VLQELNVTV (SEQ ID NO: 43), YLSGANLNL (SEQ ID NO: 50), KIFGSLAFL (SEQ ID NO: 53), AYIDFEMKI (SEQ ID NO: 66), AYACNTSTL (SEQ ID NO: 83), KWFPSCQFLL (SEQ ID NO: 84) and GYDQIMPKK (SEQ ID NO:
- Cancer antigen peptide B has the following amino acid sequence: GLYDGMEHL (SEQ ID NO: 19), VLQELNVTV (SEQ ID NO: 43) and KIFGSLAFL (SEQ ID NO: 53) 24.
- X b is a divalent group of a peptide consisting of two amino acids containing a cysteine residue and Y b is a single bond, or X b is a single bond and Y b contains a cysteine residue 2
- the thioether group in the cysteine residue of X b or the thioether group in the cysteine residue of Y b is represented by the formula (20):
- X e and Y e independently represent a single bond or a divalent group of a peptide consisting of 1 to 4 amino acids, wherein X e is the number of amino acid residues and Y e is the number of amino acid residues.
- the sum of is an integer from 0 to 4
- Cancer antigen peptide E represents an MHC class II-restricted cancer antigen peptide consisting of 7 to 30 amino acids, and the carbonyl group of the C-terminal amino acid of cancer antigen peptide E binds to X e in formula (20),
- the amino group of the N-terminal amino acid of cancer antigen peptide E is bonded to the hydrogen atom in formula (20).
- X b is one and Y b is a divalent radical of a dipeptide consisting of CA is a single bond, or X b is a divalent radical of a dipeptide and Y b is a single bond consists CA, according to claim 26 Or a pharmaceutically acceptable salt thereof;
- Item 28 The compound according to any one of Items 26 to 27, or a pharmaceutically acceptable salt thereof, wherein the cancer antigen peptide B is an MHC class I-restricted cancer antigen peptide consisting of 7 to 15 amino acids.
- Item 29 The compound according to any one of Items 26 to 28, wherein the cancer antigen peptide B consists of 7 to 15 amino acids and is an HLA-A, HLA-B or HLA-Cw restricted cancer antigen peptide, or a compound thereof A pharmaceutically acceptable salt;
- Cancer antigen peptide B is composed of 7 to 15 amino acids and is HLA-A1, HLA-A2, HLA-A3, HLA-A11, HLA-A24, HLA-A28, HLA-A29, HLA-A31, HLA-A33 , HLA-A34, HLA-A68, HLA-B7, HLA-B13, HLA-B35, HLA-B37, HLA-B44, HLA-B45, HLA-B51, HLA-B52, HLA-B53, HLA-Cw2, HLA The compound according to any one of Items 26 to 29, or a pharmaceutically acceptable salt thereof, which is a Cw3, HLA-Cw6, HLA-Cw7, HLA-Cw8 or HLA-Cw16 restricted cancer antigen peptide;
- Cancer antigen peptide B is MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A6, MAGE-A10, MAGE-A12, BAGE, DAM-6, DAM-10, GAGE-1, GAGE- 2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7B, GAGE-8, NA88-A, NY-ESO-1, NY-ESO-1a, MART-1 / Melan-A, MC1R, Gp100, PSA, PSM, Tyrosinase, Proteinase 3, TRP-1, TRP-2, ART-4, CAMEL, CEA, Ep-CAM, Cyp-B, Her2 / neu, VEGFR, hTERT, hTRT, iCE, MUC1 , MUC2, PRAME, P15, RU1, RU2, SART-1, ART-2, SART-3, AFP, ⁇ -Catenin, Caspase-8, CDK-4, ELF2,
- Cancer antigen peptide B has the following amino acid sequence: NYKHCCFEI (SEQ ID NO: 3), EYLQLVFGI (SEQ ID NO: 11), FLWGPRALV (SEQ ID NO: 13), GLYDGMEHL (SEQ ID NO: 19), SLLMWITCQC (SEQ ID NO: 26), QLSLLMWIT (SEQ ID NO: 27), AAGIGILTV (SEQ ID NO: 29), LIYRRRLLMK (SEQ ID NO: 33), YMDGTMSQV (SEQ ID NO: 40), AFLPWHHRLF (SEQ ID NO: 41), VLQELNVTV (SEQ ID NO: 43), YLSGANLNL (SEQ ID NO: 50), KIFGSLAFL (SEQ ID NO: 53), AYIDFEMKI (SEQ ID NO: 66), AYACNTSTL (SEQ ID NO: 83), KWFPSCQFLL (SEQ ID NO: 84) and GYDQIMPKK (SEQ ID NO:
- Cancer antigen peptide B has the following amino acid sequence: GLYDGMEHL (SEQ ID NO: 19), VLQELNVTV (SEQ ID NO: 43) and KIFGSLAFL (SEQ ID NO: 53) Item 33.
- X e is a divalent group of a peptide consisting of two amino acids and Y e is a single bond, or X e and Y e are independently a divalent group of a peptide consisting of one amino acid Or X e is a single bond and Y e is a divalent group of a peptide consisting of two amino acids, or X e is a divalent group of a peptide consisting of one amino acid and Y e is Item 26 to 33 are a single bond, X e is a single bond and Y e is a divalent group of a peptide consisting of one amino acid, or X e and Y e are a single bond A compound according to any one of the above, or a pharmaceutically acceptable salt thereof;
- Item 35 The compound according to any one of Items 26 to 34, or a pharmaceutically acceptable salt thereof, wherein X e is a single bond and Y e is a single bond, an alanine residue, a leucine residue or a methionine residue. ;
- Item 36 The compound according to any one of Items 26 to 34, or a pharmaceutically acceptable salt thereof, wherein X e is a single bond or a divalent group of a peptide consisting of an amino acid having one residue, and Y e is a single bond. salt;
- Item 37 The compound according to any one of Items 26 to 36, or a pharmaceutically acceptable salt thereof, wherein X e and Y e are a single bond;
- Item 38 The compound according to any one of Items 26 to 37, or a pharmaceutically acceptable salt thereof, wherein the cancer antigen peptide E is an MHC class II-restricted cancer antigen peptide consisting of 7 to 15 amino acids.
- Item 39 39.
- the cancer antigen peptide E consists of amino acids of 7 to 15 residues and is an HLA-DR, HLA-DQ, or HLA-DP restricted cancer antigen peptide; A pharmaceutically acceptable salt;
- Item 40 40.
- Cancer antigen peptide E has the following amino acid sequence: AKFVAAWTLKAAA (SEQ ID NO: 101) and aKFVAAAWTLKAAa (SEQ ID NO: 102) Or a modified amino acid sequence containing a modified amino acid residue in any of the amino acid sequences selected from SEQ ID NOs: 101 and 102, and Item 41.
- Cancer antigen peptide E has the following amino acid sequence: AKFVAAWTLKAAA (SEQ ID NO: 101) and aKFVAAAWTLKAAa (SEQ ID NO: 102) 42.
- Item 44 The compound according to any one of Items 1 to 10 and 14 to 18, or a pharmaceutically acceptable salt thereof, wherein the cancer antigen peptide B is an MHC class II-restricted cancer antigen peptide consisting of 7 to 15 amino acids salt;
- Item 45 The cancer antigen peptide B according to any one of Items 1 to 10, 14 to 18, and 26, wherein the cancer antigen peptide B consists of 7 to 15 amino acids and is an HLA-DR, HLA-DQ, or HLA-DP restricted cancer antigen peptide.
- Item 46 The compound according to any one of Items 1 to 10, 14 to 18, and 44 to 45, wherein the cancer antigen peptide B is an HLA-DR-restricted universal cancer antigen peptide consisting of 13 to 15 amino acids, or A pharmaceutically acceptable salt thereof;
- Cancer antigen peptide B has the following amino acid sequence: AKFVAAWTLKAAA (SEQ ID NO: 101) and aKFVAAAWTLKAAa (SEQ ID NO: 102) Or a modified amino acid sequence containing a modified amino acid residue in any of the amino acid sequences selected from SEQ ID NOs: 101 and 102, and 50.
- Cancer antigen peptide B has the following amino acid sequence: AKFVAAWTLKAAA (SEQ ID NO: 101) and aKFVAAAWTLKAAa (SEQ ID NO: 102) 48.
- Item 50 The compound according to any one of Items 1 to 10, wherein R 1 is a group represented by Formula (3), or a pharmaceutically acceptable salt thereof;
- X c is a divalent group of a peptide consisting of two amino acids and Y c is a single bond, or X c and Y c are independently a divalent group of a peptide consisting of one amino acid Or X c is a single bond and Y c is a divalent group of a peptide consisting of two amino acids, or X c is a divalent group of a peptide consisting of one amino acid and Y c is Item 1-10, wherein X is a single bond, X c is a single bond and Y c is a divalent group of a peptide consisting of one amino acid, or X c and Y c are a single bond 50.
- Item 52 The compound according to any one of Items 1 to 10 and 50 to 51, or a pharmaceutically acceptable salt thereof, wherein X c is a single bond and Y c is a single bond, an alanine residue, a leucine residue or a methionine residue. Acceptable salts;
- Item 53 The compound according to any one of Items 1 to 10 and 50 to 51, or a pharmaceutical thereof, wherein X c is a single bond or a divalent group of a peptide consisting of an amino acid having one residue, and Y c is a single bond.
- Top acceptable salts
- Item 54 The compound according to any one of Items 1 to 10 and 50 to 53, or a pharmaceutically acceptable salt thereof, wherein X c and Y c are a single bond;
- Item 55 The compound according to any one of Items 1 to 10 and 50 to 54, or a pharmaceutically acceptable salt thereof, wherein the cancer antigen peptide C is an MHC class II-restricted cancer antigen peptide consisting of 7 to 15 amino acids salt;
- Item 56 The cancer antigen peptide C according to any one of Items 1 to 10 and 50 to 55, which comprises an amino acid having 7 to 15 residues and is an HLA-DR, HLA-DQ, or HLA-DP-restricted cancer antigen peptide.
- Item 57 The compound according to any one of Items 1 to 10 and 50 to 56, or a pharmaceutically acceptable salt thereof, wherein the cancer antigen peptide C is an HLA-DR-restricted universal cancer antigen peptide consisting of 13 to 15 amino acids Salt to be made;
- Cancer antigen peptide C has the following amino acid sequence: AKFVAAWTLKAAA (SEQ ID NO: 101) and aKFVAAAWTLKAAa (SEQ ID NO: 102) Or a modified amino acid sequence containing a modified amino acid residue in any of the amino acid sequences selected from SEQ ID NOs: 101 and 102, and 58.
- Cancer antigen peptide C has the following amino acid sequence: AKFVAAWTLKAAA (SEQ ID NO: 101) and aKFVAAAWTLKAAa (SEQ ID NO: 102)
- AKFVAAWTLKAAA SEQ ID NO: 101
- aKFVAAAWTLKAAa SEQ ID NO: 102
- Item 61 The compound according to any one of Items 1 to 10, wherein R 1 is cancer antigen peptide D, or a pharmaceutically acceptable salt thereof;
- Item 62 The compound according to any one of Items 1 to 10 and 61, or a pharmaceutically acceptable salt thereof, wherein the cancer antigen peptide D is an MHC class I-restricted cancer antigen peptide consisting of 7 to 15 amino acids ;
- the cancer antigen peptide D is an amino acid having 7 to 15 residues and is an HLA-A, HLA-B or HLA-Cw restricted cancer antigen peptide, Or a pharmaceutically acceptable salt thereof;
- the cancer antigen peptide D is composed of 7 to 15 amino acids and HLA-A1, HLA-A2, HLA-A3, HLA-A11, HLA-A24, HLA-A28, HLA-A29, HLA-A31, HLA-A33 , HLA-A34, HLA-A68, HLA-B7, HLA-B13, HLA-B35, HLA-B37, HLA-B44, HLA-B45, HLA-B51, HLA-B52, HLA-B53, HLA-Cw2, HLA The compound according to any one of Items 1 to 10 and 61 to 63, which is a Cw3, HLA-Cw6, HLA-Cw7, HLA-Cw8 or HLA-Cw16 restricted cancer antigen peptide, or a pharmaceutically acceptable salt thereof Salt;
- the cancer antigen peptide D is MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A6, MAGE-A10, MAGE-A12, BAGE, DAM-6, DAM-10, GAGE-1, GAGE- 2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7B, GAGE-8, NA88-A, NY-ESO-1, NY-ESO-1a, MART-1 / Melan-A, MC1R, Gp100, PSA, PSM, Tyrosinase, Proteinase 3, TRP-1, TRP-2, ART-4, CAMEL, CEA, Ep-CAM, Cyp-B, Her2 / neu, VEGFR, hTERT, hTRT, iCE, MUC1 , MUC2, PRAME, P15, RU1, RU2, SART-1, ART-2, SART-3, AFP, ⁇ -Catenin, Caspase-8, CDK-4, ELF
- Cancer antigen peptide D has the following amino acid sequence: VYGFVRRACL (SEQ ID NO: 87) and SLLMWITC (SEQ ID NO: 88)
- a peptide comprising any amino acid sequence selected from among, or including a modified amino acid sequence containing a modification of an amino acid residue in any amino acid sequence selected from SEQ ID NOs: 87 and 88, and The compound according to any one of Items 1 to 10 and 61 to 65, or a pharmaceutically acceptable salt thereof, which is a peptide having CTL-inducing activity;
- Cancer antigen peptide D has the following amino acid sequence: VYGFVRRACL (SEQ ID NO: 87) and SLLMWITC (SEQ ID NO: 88)
- VYGFVRRACL SEQ ID NO: 87
- SLLMWITC SEQ ID NO: 88
- Item 69 The compound according to any one of Items 1 to 10 and 61, or a pharmaceutically acceptable salt thereof, wherein the cancer antigen peptide D is an MHC class II-restricted cancer antigen peptide consisting of 7 to 30 amino acids. ;
- Item 70 Item 68.
- the cancer antigen peptide D according to any one of Items 1 to 10, 61, and 69, wherein the cancer antigen peptide D is composed of 13 to 15 amino acids and is an HLA-DR, HLA-DQ, or HLA-DP-restricted cancer antigen peptide.
- Item 71 The compound according to any one of Items 1 to 10, 61 and 69 to 70, or a pharmacology thereof, wherein the cancer antigen peptide D is an HLA-DR-restricted universal cancer antigen peptide consisting of amino acids of 13 to 15 residues. Top acceptable salts;
- Cancer antigen peptide D has the following amino acid sequence: aK-Cha-VAAWTLKAAa-Ahx-C (SEQ ID NO: 103)
- the peptide comprising the amino acid sequence of SEQ ID NO: 103 or a peptide comprising a modified amino acid sequence containing an amino acid residue modification in the amino acid sequence of SEQ ID NO: 103 and having a helper T cell inducing activity,
- Cancer antigen peptide D has the following amino acid sequence: aK-Cha-VAAWTLKAAa-Ahx-C (SEQ ID NO: 103)
- Cancer antigen peptide D is MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A6, NY-ESO-1, MART-1 / Melan-A, Gp100, PSA, Tyrosinase, CEA, HER-2 / neu, Item 70.
- Cancer antigen peptide D has the following amino acid sequence: AADHRQLQLSISSCLQQL (SEQ ID NO: 104), RNGYRALMDKSLHVGTQCALTR (SEQ ID NO: 105), KKLQCVQLHVISM (SEQ ID NO: 106) and GSYVSRLLGICL (SEQ ID NO: 107) Or a modified amino acid containing a modified amino acid residue in any amino acid sequence selected from SEQ ID NOs: 104, 105, 106 and 107 Item 76.
- Cancer antigen peptide D has the following amino acid sequence: AADHRQLQLSISSCLQQL (SEQ ID NO: 104), RNGYRALMDKSLHVGTQCALTR (SEQ ID NO: 105), KKLQCVQLHVISM (SEQ ID NO: 106) and GSYVSRLLGICL (SEQ ID NO: 107)
- Item 78 The compound according to any one of Items 1 to 77, wherein the cancer antigen peptide A, cancer antigen peptide B, cancer antigen peptide C, and / or cancer antigen peptide D is not a WT1 protein-derived cancer antigen peptide; A pharmaceutically acceptable salt;
- Item 79 A pharmaceutical composition comprising the compound according to any one of Items 1 to 78 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier;
- Item 80 The pharmaceutical composition according to Item 79, which is used as a cancer vaccine;
- Item 81 Use of the compound according to any one of Items 1 to 78 or a pharmaceutically acceptable salt thereof for producing a cancer vaccine;
- Item 82 A method for treating or preventing cancer, wherein the cancer is in need of an effective amount for treating or preventing the compound or pharmaceutically acceptable salt thereof according to any one of Items 1 to 78.
- a method comprising administering to an antigen protein positive cancer patient,
- MHC class I-restricted epitopes Two different MHC class I-restricted epitopes, or MHC class I-restricted epitope and MHC class, by reacting the compound according to any one of Items 1 to 78 or a pharmaceutically acceptable salt thereof with ERAP1 A method of obtaining a II-restricted epitope; and
- a method for synthesizing a compound comprising the following steps: (1) A carbonyl group of a C-terminal amino acid of C (Mmt) A and a cancer antigen peptide using Fmoc-C (Mmt) A-SBn and a cancer antigen peptide B in which one cysteine residue is bonded to the N-terminus A step of synthesizing a peptide in which the amino group of the N-terminal amino acid bonded to the N-terminus of B is bound, wherein the cancer antigen peptide B is an MHC class I-restricted cancer antigen peptide consisting of 7 to 15 amino acids.
- This is a step of synthesizing a peptide in which a thioether group of a cysteine residue bonded to the N-terminus of cancer antigen peptide B in the peptide and a thioether group of a cysteine residue bonded to the N-terminus of cancer antigen peptide A are combined.
- Cancer antigen peptide A represents a MHC class I-restricted cancer antigen peptide consisting of 7 to 30 amino acids, and (3) protected with the peptide obtained in step (2) and the SPy group.
- Cysteine bound to the N-terminus of cancer antigen peptide A in the peptide obtained in the step (2) using cancer antigen peptide E in which one cysteine residue is bound to the C-terminus Synthesizes a peptide in which a thioether group of a cysteine residue bonded to the C-terminus of a cancer antigen peptide E is bound, wherein the cancer antigen peptide E consists of 7 to 30 amino acid residues Representing an MHC class II restricted cancer antigen peptide; About.
- the present invention it has become possible to provide a compound represented by the above formula (1) useful as a cancer immunotherapeutic agent (hereinafter sometimes referred to as the compound of the present invention).
- the compounds of the present invention have made it possible to provide cancer vaccines and cancer immunotherapeutic agents that efficiently induce CTLs in vivo and in vitro.
- the compounds of the present invention may be used to provide two MHC class I-restricted cancer antigen peptides having different sequences or two MHC class I-restricted cancer antigen epitopes having different sequences, MHC class I cancer antigen-restricted peptides, and MHC class II-restricted peptides.
- the present invention can be combined with a peptide of A02 type (A-0201, A0206, etc.) and a peptide of A24 type (A-2402, etc.)
- the compound (conjugate) is particularly preferred.
- HLA-A0201 type or HLA-A0206 type In the Caucasian, Population that is HLA-A0201 type or HLA-A0206 type is the largest with about 47%, then HLA-A2402 type is about 13%, and the sum of these types is duplicated (ie, Excluding humans with both types, accounting for about 56% (Human Immunol. 62: 1009; 2001). On the other hand, in Japanese and other countries, the population with HLA-A2402 is the largest with about 60%, followed by HLA-A0201 or HLA-A0206 with about 39%. Excluding humans having a double count), it accounts for about 81% (www.bmdc.irc.or.jp/GF-A.htm).
- the advantages of the compounds of the present invention include, in particular, the advantage of covering a larger population with one compound of the present invention, and it is essential to select the patient's HLA subtype prior to administration.
- the compound of the present invention makes it possible to provide an active ingredient of a cancer vaccine that is excellent in physicochemical properties and stability and can be easily produced. This also facilitated the formulation of cancer vaccines.
- the physicochemical properties include solubility, viscosity of the solution, concomitant ease of purification, ease of handling after lyophilization, concomitant ease of purification, and the like.
- Stability includes stability after salt substitution, hygroscopicity, thermal stability, stability after emulsion formation, and the like.
- examples of the pharmacological activity include drug efficacy as a cancer vaccine, differences caused by API (Active Pharmaceutical Ingredient), interaction with additives in the preparation, and the like.
- the difference caused by API is a difference as a cancer vaccine by API.
- APIs having greatly different solubilities APIs having low solubilities tend to precipitate, and are essential for pharmaceuticals. The possibility that sterilization by filtration of a certain membrane filter cannot be expected is sufficiently expected.
- FIG. 1 is a diagram showing the results of testing the time-dependent changes in N-terminal amino acid trimming by ERAP1 for each peptide of SEQ ID NOs: 100, 99 and 98 synthesized in Examples 1, 21, and 25 in Test Example 2. is there.
- FIG. 2 is a diagram showing the results of testing the time-dependent change in trimming of the N-terminal amino acid by ERAP1 for each peptide of SEQ ID NOs: 95, 92 and 89 synthesized in Examples 2, 22, and 26 in Test Example 2. is there.
- FIG. 1 is a diagram showing the results of testing the time-dependent changes in N-terminal amino acid trimming by ERAP1 for each peptide of SEQ ID NOs: 100, 99 and 98 synthesized in Examples 1, 21, and 25 in Test Example 2. is there.
- FIG. 2 is a diagram showing the results of testing the time-dependent change in trimming of the N-terminal amino acid by ERAP1 for each peptide of SEQ ID NOs: 95, 92 and 89 synthe
- FIG. 3 is a diagram showing the results of testing the time-dependent change in trimming of the N-terminal amino acid by ERAP1 for each peptide of SEQ ID NOs: 96, 93 and 90 synthesized in Examples 11, 23 and 27 in Test Example 2. is there.
- FIG. 4 is a diagram showing the results of testing the time-dependent change in trimming of the N-terminal amino acid by ERAP1 for each of the peptides of SEQ ID NOs: 97, 94 and 91 synthesized in Examples 12, 24 and 28 in Test Example 2. is there.
- FIG. 4 is a diagram showing the results of testing the time-dependent change in trimming of the N-terminal amino acid by ERAP1 for each of the peptides of SEQ ID NOs: 97, 94 and 91 synthesized in Examples 12, 24 and 28 in Test Example 2. is there.
- FIG. 5 shows the results of testing the in vivo CTL inducibility by IFN ⁇ ELISPOT assay using the HLA-A0201 transgenic mouse in the peptide of SEQ ID NO: 100 synthesized in Example 1 in Test Example 3.
- FIG. FIG. 6 shows the results of testing the in vivo CTL inducing ability of the peptide of SEQ ID NO: 99 synthesized in Example 21 in Test Example 3 using IFN ⁇ ELISPOT assay using HLA-A0201 transgenic mice.
- FIG. FIG. 7 shows the results of testing the in vivo CTL inducing ability of the peptide of SEQ ID NO: 98 synthesized in Example 25 in Test Example 3 using IFN ⁇ ELISPOT assay using HLA-A0201 transgenic mice.
- FIG. 8 shows the in vivo CTL inducing ability of the compound represented by the formula (4) synthesized in Example 29 in Test Example 4 using IFN ⁇ ELISPOT assay using HLA-A0201 transgenic mice. It is a figure which shows a result.
- FIG. 9 shows the in vivo CTL inducing ability of the compound represented by the formula (10) synthesized in Example 30 in Test Example 5 by IFN ⁇ ELISPOT assay using HLA-A0201 transgenic mice. It is a figure which shows a result.
- FIG. 10 shows the in vivo CTL inducing ability of the compound represented by the formula (10) synthesized in Example 30 in Test Example 5 by IFN ⁇ ELISPOT assay using HLA-A2402 transgenic mice.
- FIG. 11 shows that in the test example 13, the compound represented by the formula (11) synthesized in the example 109 was tested for the in vivo CTL inducing ability by the IFN ⁇ ELISPOT assay using HLA-A0201 transgenic mice. It is a figure which shows a result.
- FIG. 12 shows the in vivo CTL inducing ability of the compound represented by the formula (5) synthesized in Example 110 in Test Example 14 by IFN ⁇ ELISPOT assay using HLA-A0201 transgenic mice. It is a figure which shows a result.
- FIG. 13 shows the in vivo CTL inducibility of IFN ⁇ ELISPOT assay using HLA-A0201 transgenic mice in Test Example 19 for the compound represented by Formula (16) synthesized in Example 112. It is a figure which shows a result.
- FIG. 14 shows the in vivo CTL inducing ability of the compound represented by the formula (17) synthesized in Example 113 in Test Example 20 using IFN ⁇ ⁇ ⁇ ⁇ ELISPOT assay using HLA-A0201 transgenic mice. It is a figure which shows a result.
- FIG. 15 shows the in vivo CTL inducing ability of the compound represented by the formula (18) synthesized in Example 147 in Test Example 21 using IFN ⁇ ELISPOT assay using HLA-A0201 transgenic mice.
- FIG. 16 shows the in vivo CTL inducing ability of the compound represented by the formula (15) synthesized in Example 111 in Test Example 22 using IFN ⁇ 22ELISPOT assay using HLA-A0201 transgenic mice. It is a figure which shows a result.
- FIG. 17 shows that, in Test Example 23, HLA-A0201 transgenic mice in which the compound of Formula (19) synthesized in Example 149 was pulsed or non-pulsed with the peptide of SEQ ID NO: 19 were used. It is a figure which shows the result of having tested the CTL inducibility in vivo by IFN ⁇ ELISPOT assay.
- FIG. 16 shows the in vivo CTL inducing ability of the compound represented by the formula (15) synthesized in Example 111 in Test Example 22 using IFN ⁇ 22ELISPOT assay using HLA-A0201 transgenic mice. It is a figure which shows a result.
- FIG. 17 shows that, in Test Example 23, HLA-A0201 transgenic mice in which the compound of
- FIG. 19 shows that, in Test Example 23, the compound represented by the formula (19) synthesized in Example 149 was used as an HLA-A0201 transgenic mouse in which the peptide of SEQ ID NO: 53 was pulsed or non-pulsed. It is a figure which shows the result of having tested the CTL inducibility in vivo by IFN ⁇ ELISPOT assay.
- FIG. 19 shows the introduction of HLA-A0201 gene in the pulse of non-pulse of the peptide of SEQ ID NO: 19 with respect to the peptide represented by SEQ ID NO: 231 and 232 synthesized in Reference Examples 2 and 3 in Comparative Example 1.
- FIG. 20 shows the introduction of HLA-A0201 gene in the pulse of the peptide of SEQ ID NO: 53 with or without pulse for the peptide represented by SEQ ID NO: 231 and 232 synthesized in Reference Examples 2 and 3 in Comparative Example 1. It is a figure which shows the result of having tested the CTL inducibility in vivo by IFN (gamma) * ELISPOT assay using a mouse
- FIG. 20 shows the introduction of HLA-A0201 gene in the pulse of the peptide of SEQ ID NO: 53 with or without pulse for the peptide represented by SEQ ID NO: 231 and 232 synthesized in Reference Examples 2 and 3 in Comparative Example 1. It is a figure which shows the result of having tested the CTL inducibility in vivo by IFN (gamma) * ELISPOT assay using a mouse
- FIG. 21 shows the HLA-A0201 gene introduction in the pulse of the peptide of SEQ ID NO: 19 or the non-pulse for the peptide represented by SEQ ID NO: 233 and 234 synthesized in Reference Examples 4 and 5 in Comparative Example 2. It is a figure which shows the result of having tested the CTL inducibility in vivo by IFN (gamma) * ELISPOT assay using a mouse
- FIG. 22 shows HLA-A0201 gene introduction in the pulse of the peptide of SEQ ID NO: 53 or non-pulse with respect to the peptide represented by SEQ ID NO: 233 and 234 synthesized in Reference Examples 4 and 5 in Comparative Example 2. It is a figure which shows the result of having tested the CTL inducibility in vivo by IFN (gamma) * ELISPOT assay using a mouse
- the “amino acid residue” means a part corresponding to one unit of amino acid constituting the peptide or protein on the peptide or protein molecule.
- “Amino acid residue” includes a natural or non-natural ⁇ -amino acid residue, ⁇ -amino acid residue, ⁇ -amino acid residue or ⁇ -amino acid residue. Specific examples include natural ⁇ -amino acid residues, ornithine residues, homoserine residues, homocysteine residues, ⁇ -alanine, ⁇ -aminobutanoic acid or ⁇ -aminopentanoic acid.
- the “amino acid residue” may have an optically active form, it may be either L-form or D-form, but L-form is preferred.
- amino acid residue in the present invention is indicated by an abbreviation, it is described by the following abbreviation.
- Ala or A alanine residue a: D-alanine residue Arg or R: Arginine residue Asn or N: Asparagine residue Asp or D: Aspartic acid residue Cys or C: Cysteine residue Gln or Q: Glutamine residue Glu or E: Glutamic acid residue Gly or G: Glycine residue His or H: histidine residue Ile or I: isoleucine residue Leu or L: Leucine residue Lys or K: Lysine residue Met or M: methionine residue Phe or F: phenylalanine residue Pro or P: proline residue Ser or S: Serine residue Thr or T: Threonine residue Trp or W: Tryptophan residue Tyr or Y: tyrosine residue Val or V: valine residue Abu: 2-aminobutyric acid residue (also referred to as ⁇ -aminobutyric acid residue
- the amino acid sequence of the “peptide” in the present invention is described according to a conventional method such that the amino acid residue of the N-terminal amino acid is located on the left side and the amino acid residue of the C-terminal amino acid is located on the right side.
- the amino group of the amino acid residue of the N-terminal amino acid is bonded to a hydrogen atom
- the carbonyl group of the amino acid residue of the C-terminal amino acid is bonded to a hydroxyl group.
- the divalent group of the peptide means a group bonded through the amino group of the amino acid residue of the N-terminal amino acid and the carbonyl group of the amino acid residue of the C-terminal amino acid.
- the amino group of the amino acid residue of the N-terminal amino acid is a hydrogen atom unless otherwise specified for the peptide corresponding to the partial structure.
- the carbonyl group of the amino acid residue of the C-terminal amino acid is bonded to the hydroxyl group.
- X a and “Y a ” independently represent a single bond or a divalent group of a peptide composed of 1 to 4 amino acids.
- the sum of the number of amino acid residues amino acid residues and Y a of X a is an integer of 0-4.
- the sum being an integer of 0 means that X a and Y a are a single bond.
- the sum is an integer of 4
- X a and Y a are independently a divalent group of a peptide consisting of 2 amino acids
- X a consists of 3 amino acids.
- Y a is a divalent group of the peptide is a divalent radical of a peptide consisting of amino acids 1 residue
- a divalent radical of a peptide X a comprises the amino acid 4 residues and Y a is a single bond
- the integer of the sum is preferably 0 to 2, more preferably 0 to 1, and most preferably 0. That is, X a and Y a are most preferably a single bond. In the case where the integer of the sum is 2, when X a is a divalent group of a peptide consisting of two residues of amino acid and Y a is a single bond, X a and Y a are independently one residue.
- X a is a divalent group of a peptide consisting of one amino acid and Y a is a single bond
- Y a is a single bond
- X a is a single bond
- Y a Is a divalent group of a peptide consisting of an amino acid having one residue.
- X a is a single bond and Y a is an alanine residue, leucine residue or methionine residue, or X a is an alanine residue, glycine residue, serine residue or tyrosine residue. And a case where Y a is a single bond.
- the “cancer antigen peptide A” in the present invention is an MHC class I-restricted cancer antigen peptide consisting of 7 to 30 amino acids.
- Cancer antigen peptide A in the formula (1) the amino group of the N-terminal amino acid is bound to Y a in the formula (1), the carbonyl group of the C-terminal amino acid is bound to a hydroxyl group in the formula (1).
- MHC class I restriction in the present invention means a property of inducing CTL by binding to an MHC class I molecule which is a class I of a major histocompatibility complex (MHC).
- MHC is called human leukocyte type antigen (HLA) in humans.
- HLA corresponding to MHC class I molecules is classified into subtypes such as HLA-A, B, Cw, F and G.
- the “MHC class I restriction” preferably includes HLA-A restriction, HLA-B restriction, or HLA-Cw restriction.
- HLA-A polymorphism include 27 or more types such as HLA-A1, HLA-A0201, and HLA-A24.
- HLA-B polymorphism include HLA-B7, HLA-B40, HLA-B4403, etc.
- the HLA-Cw polymorphism includes 10 or more types such as HLA-Cw0301, HLA-Cw0401, and HLA-Cw0602. Among these polymorphisms, HLA-A0201 and HLA-A24 are preferable.
- the cancer antigen peptide A is preferably an amino acid having 7 to 15 residues and HLA-A, HLA-B or HLA-Cw restricted cancer antigen peptide, preferably 7 to 15 amino acids and HLA-A1, HLA-A2, HLA-A3, HLA-A11, HLA-A24, HLA-A28, HLA-A29, HLA-A31, HLA-A33, HLA-A34, HLA-A68, HLA-B7, HLA-B13, HLA- B35, HLA-B37, HLA-B44, HLA-B45, HLA-B51, HLA-B52, HLA-B53, HLA-Cw2, HLA-Cw3, HLA-Cw6, HLA-Cw7, HLA-Cw8 or HLA-Cw16 More preferred is a sex cancer antigen peptide.
- the “cancer antigen peptide” in the present invention means a known partial peptide of human cancer antigen protein. Specifically, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A6, MAGE-A10, MAGE-A12, BAGE, DAM-6, DAM-10, GAGE-1, GAGE-2 , GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7B, GAGE-8, NA88-A, NY-ESO-1, NY-ESO-1a, MART-1 / Melan-A, MC1R , Gp100, PSA, PSM, Tyrosinase, Proteinase 3, TRP-1, TRP-2, ART-4, CAMEL, CEA, Ep-CAM, Cyp-B, Her2 / neu, VEGFR, hTERT, hTRT, iCE, MUC1, MUC2, PRAME, P15, RU1, RU2, SART-1, SART- 2, SART-3,
- cancer antigen peptide in the present invention does not include human WT1 protein (Cell, 60: 509, 1990, GenBank Acc. No. A38080). That is, in the compound of the present invention, cancer antigen peptide A, cancer antigen peptide B, cancer antigen peptide C, and / or cancer antigen peptide D are not cancer antigen peptides derived from WT1 protein.
- the cancer antigen peptide A, cancer antigen peptide B, cancer antigen peptide C or cancer antigen peptide D is not a cancer antigen peptide derived from the WT1 protein, and the cancer antigen peptide A, cancer antigen peptide B, cancer antigen peptide C and cancer More preferably, the antigen peptide D is not a cancer antigen peptide derived from the WT1 protein.
- the MHC class I restriction wherein the cancer antigen peptide A is composed of 7 to 30 amino acids continuous in the amino acid sequence of the cancer antigen protein.
- R 1 is a group represented by the formula (2), unlike the cancer antigen peptide derived from the WT1 protein, 7 to 30 amino acids that are continuous in the amino acid sequence of the cancer antigen protein Unlike MHC class I or MHC class II-restricted cancer antigen peptides and are and WT1 protein derived from a cancer antigen peptide consisting of, R 1 is a group represented by the formula (3), a cancer antigen peptide C cancer A WT1 tamper which is an MHC class II-restricted cancer antigen peptide consisting of 7 to 30 amino acids continuous in the amino acid sequence of the antigen protein
- the cancer antigen peptide D is an MHC class I comprising 7 to 30 amino acids that are continuous in the amino acid sequence of the cancer antigen protein. It is preferably an MHC class II-restricted cancer antigen peptide and different from a cancer antigen peptide derived from WT1 protein.
- the “MHC class I-restricted cancer antigen peptide” in the present invention is a peptide that binds to an MHC class I antigen and is presented as a complex in vitro and / or in vivo, and the complex is a precursor. It means a peptide that induces CTL as a result of being recognized by T cells.
- the number of amino acid residues of the “MHC class I-restricted cancer antigen peptide” is 7-30, preferably 7-15, more preferably 8-12, still more preferably 8-11, Preferably 8 or 9.
- MHC class I-restricted cancer antigen peptide consisting of 7 to 12 or preferably 9 amino acids is also referred to as “MHC class I-restricted cancer antigen epitope”.
- the “MHC class I-restricted cancer antigen epitope” in the present invention means a peptide itself that binds to an MHC class I antigen and is presented as a complex. That is, the “MHC class I-restricted cancer antigen peptide” can be produced in vitro and / or in vivo by a proteosome such as Gamma-Interferon-inductive Lysosomal Thiol Reductase (GILT, GLT) and / or protease in cells.
- GILT Gamma-Interferon-inductive Lysosomal Thiol Reductase
- the C-terminal amino acid of “MHC class I-restricted cancer antigen epitope” is generated as a result of degradation by proteosome and / or protease, and then the result of trimming (cleavage) by ERAP1 is “MHC class I-restricted cancer antigen”
- the generation process of the N-terminal amino acid of the “epitope” is mainly considered. However, in the generation, a process other than the generation process can be passed.
- ERAP1 is also called ERAAP (ER aminopeptidase associated with antigen presentation), and was once also called A-LAP, PILS-AP, or ARTS-1.
- the “MHC class I-restricted cancer antigen peptide” includes an amino acid having 1 to 23 residues in the carbonyl group of the C-terminal amino acid of the “MHC class I-restricted cancer antigen epitope” consisting of 7 to 12 amino acids. Peptides consisting of 7 to 30 amino acids produced by addition are preferred.
- Examples of the “MHC class I-restricted cancer antigen peptide” include the peptides listed in Tables 1 to 9.
- the peptide of SEQ ID NO: 8 and the peptide of SEQ ID NO: 9 in Table 1 are the same peptide having the same amino acid sequence.
- the peptide is an HLA-Cw3 restricted cancer antigen peptide and also an HLA-Cw16 restricted cancer antigen peptide.
- the “MHC class I-restricted cancer antigen peptide” is preferably the following amino acid sequence: NYKHCCFEI (SEQ ID NO: 3), EYLQLVFGI (SEQ ID NO: 11), FLWGPRALV (SEQ ID NO: 13), GLYDGMEHL (SEQ ID NO: 19), SLLMWITCQC (SEQ ID NO: 26), QLSLLMWIT (SEQ ID NO: 27), AAGIGILTV (SEQ ID NO: 29), LIYRRRLLMK (SEQ ID NO: 33), YMDGTMSQV (SEQ ID NO: 40), AFLPWHHRLF (SEQ ID NO: 41), VLQELNVTV (SEQ ID NO: 43), YLSGANLNL (SEQ ID NO: 50), KIFGSLAFL (SEQ ID NO: 53), AYIDFEMKI (SEQ ID NO: 66), AYACNTSTL (SEQ ID NO: 83), KWFPSCQFLL (SEQ ID NO: 84), GYD
- MHC class I-restricted cancer antigen peptide SEQ ID NOs: 3, 11, 13, 19, 26, 27, 29, 33, 40, 41, 43, 50, 53, 66, 83, 84, 85, 87 And a peptide consisting of any amino acid sequence selected from 88, more preferably a peptide consisting of any amino acid sequence selected from SEQ ID NOs: 19, 43 and 53.
- the “peptide containing an amino acid sequence” means a peptide in which an additional amino acid is added to the N-terminal amino acid and / or C-terminal amino acid of the amino acid sequence as usual.
- MHC class I-restricted cancer antigen peptide in “cancer antigen peptide A” and “cancer antigen peptide B” is added, a peptide added to the C-terminal side is preferable.
- a peptide containing a modified amino acid sequence containing a modified amino acid residue in the amino acid sequence and having CTL-inducing activity is also referred to as a “modified killer peptide”.
- the modified killer peptide means a peptide that consists of an amino acid sequence in which 1 to 3 amino acids are deleted, substituted and / or added in the amino acid sequence, binds to MHC class I, and induces CTL.
- Examples of the substitution position of the amino acid to be substituted include 1-position (N-terminal), 2-position, 3-position and 9-position in the case of a peptide consisting of 9-residue amino acids.
- the number of amino acids to be added (including insertion) is preferably 1 or 2, more preferably 1.
- a preferred addition position includes the C-terminus.
- the number of amino acids to be deleted is preferably 1.
- the added amino acid or the substituted amino acid may be an unnatural amino acid other than the 20 kinds of amino acids encoded by the gene.
- R 1 in the present invention represents a hydrogen atom, a group represented by the above formula (2), a group represented by the above formula (3), or a cancer antigen peptide D, preferably the above formula ( The group represented by 2), the group represented by the formula (3), or the cancer antigen peptide D.
- the sequence is not identical to the partial sequence of the cancer antigen protein.
- the requirement “the sequence is not identical to the partial sequence of the cancer antigen protein” in the formula (1) means that MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A6, MAGE-A10, MAGE-A12 , BAGE, DAM-6, DAM-10, GAGE-1, GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7B, GAGE-8, NA88-A, NY-ESO -1, NY-ESO-1a, MART-1 / Melan-A, MC1R, Gp100, PSA, PSM, Tyrosinase, Proteinase 3, TRP-1, TRP-2, ART-4, CAMEL, CEA, Ep-CAM, Cyp-B, Her2 / neu, VEGFR, hTERT, hT
- the compound of the formula (1) in which R 1 is a hydrogen atom is not a partial peptide consisting of 8- to 35-amino acid residues in the amino acid sequence of the cancer antigen protein.
- R 1 is a hydrogen atom
- the HER2 / neu 369-377 peptide has a KIFGSLAFL (SEQ ID NO: 53) amino acid sequence, and is a partial peptide consisting of 9 consecutive amino acids at positions 369 to 377 in the amino acid sequence of human HER2 / neu protein. It is.
- the HER2 / neu 367-377 peptide corresponds to a partial peptide consisting of 11 consecutive amino acids in the amino acid sequence of the HER2 / neu protein.
- R 1 is a hydrogen atom, portions not a peptide consisting of amino acids 8-35 contiguous residues in the amino acid sequence of tumor antigen protein
- R 1 When the cancer antigen peptide A is HER2 / neu 369-377 KIFGSLAFL (SEQ ID NO: 53) in the compound of formula (1) in which is a hydrogen atom, the HER2 / neu 367-377 peptide (CKKIFGSLAFL) is the present invention. Therefore, X a is a single bond and Ya does not become a lysine residue.
- the HER2 / neu 367-377 peptide (CKKIFGSLAFL) (SEQ ID NO: 86) is a reference example of the present invention and is not an example, as described later.
- MHC class I-restricted cancer antigen epitope Preferred as “MHC class I-restricted cancer antigen epitope” of the present invention, SEQ ID NOs: 3, 11, 13, 19, 26, 27, 29, 33, 40, 41, 43, 50, 53, 66, 83, 84 Tables 10 to 11 show the 5-residue consecutive amino acids on the N-terminal side in the amino acid sequence of the corresponding cancer antigen protein for each of peptides 85 and 85.
- X a -Cys-Y a in the above formula (1-1) is expressed as, for example, Table 10
- the compound of the formula (1) in which the requirement “R 1 is a hydrogen atom” according to the present invention by comparing the amino acid sequence of the corresponding cancer antigen protein shown in Table 11 with the 5-residue consecutive amino acids on the N-terminal side Is not a partial peptide consisting of 8- to 35-amino acid residues in the amino acid sequence of a cancer antigen protein.
- R 1 is a hydrogen atom
- CAGLYDGMEHL SEQ ID NO: 89
- CLGLYDGMEHL SEQ ID NO: 90
- CMGLYDGMEHL SEQ ID NO: 91
- CAVLQELNVTV SEQ ID NO: 92
- CLVLQELNVTV SEQ ID NO: 93
- CMVLQELNVTV SEQ ID NO: 94
- CAKIFGSLAFL SEQ ID NO: 95
- CLKIFGSLAFL SEQ ID NO: 96
- CMKIFGSLAFL SEQ ID NO: 97
- a peptide consisting of any amino acid sequence selected from among these is preferable, and a peptide consisting of any amino acid sequence selected from SEQ ID NOs: 89 to 91 is even more preferable.
- R 1 is a hydrogen atom
- the following amino acid sequence CGLYDGMEHL (SEQ ID NO: 98), CVLQELNVTV (SEQ ID NO: 99) and CKIFGSLAFL (SEQ ID NO: 100)
- a peptide consisting of any amino acid sequence selected from among these is also preferred, and a peptide consisting of the amino acid sequence of SEQ ID NO: 98 is even more preferred.
- X b and “Y b ” independently represent a single bond or a divalent group of a peptide consisting of amino acids of 1 to 4 residues.
- the sum of the number of amino acid residues amino acid residues and Y b of X b is an integer of 0-4.
- the sum being an integer of 0 means that X b and Y b are single bonds.
- the sum is an integer of 4
- X b and Y b are independently a divalent group of a peptide consisting of 2 amino acids
- X b consists of 3 amino acids.
- a divalent radical of a peptide X b comprises the amino acid 4 residues and Y b is a single bond
- the integer of the sum is preferably 0 to 2, more preferably 0 to 1, and most preferably 0. That is, X b and Y b are most preferably a single bond. In the case where the integer of the sum is 2, when X b is a divalent group of a peptide consisting of two amino acids and Y b is a single bond, X b and Y b independently represent one residue.
- the integer of the sum is 1, X b is a divalent group of a peptide consisting of one amino acid and Y b is a single bond, or X b is a single bond and Y b Is a divalent group of a peptide consisting of an amino acid having one residue.
- the case where X b is a single bond and Y b is an alanine residue, a leucine residue or a methionine residue is preferable.
- cancer antigen peptide B in the present invention is an MHC class I-restricted cancer antigen peptide consisting of 7 to 30 amino acids or an MHC class II-restricted cancer antigen peptide consisting of 7 to 30 amino acids.
- cancer antigen peptide A and cancer antigen peptide B are not simultaneously the same peptide. That is, cancer antigen peptide B is limited by the requirement that “unlike cancer antigen peptide A”.
- the compound of the formula (1) in which R 1 is a group represented by the above formula (2) is assumed to be X a and X Even if b is the same and Y a and Y b are the same, it is not a homodimer but a heterodimer.
- a homodimer means a dimer in which the same peptide monomer is dimerized
- a heterodimer means a dimer in which a different peptide monomer is dimerized.
- N-terminal amino group of the amino acid bound to Y b is in formula (2) (i.e. bound to the formula (1-2) Of these Y b), C-terminal carbonyl group of the amino acid has the formula (2 ) In the group (that is, it binds to the hydroxyl group in the formula (1-2)).
- cancer antigen peptide B in the present invention is an MHC class I-restricted cancer antigen peptide consisting of 7 to 30 amino acids
- the “MHC class I-restricted cancer antigen peptide” has the same meaning as described above. is there.
- the compound of formula (1) when R 1 is a group represented by formula (2) and cancer antigen peptide B is an MHC class I-restricted cancer antigen peptide consisting of 7 to 30 amino acids is preferably Formula (4):
- X b is a divalent group of a peptide composed of two amino acids including a cysteine residue and Y b is a single bond, or X b is a single bond and Y b is a cysteine residue. More preferably, when X b is a divalent group of a dipeptide consisting of CA and Y b is a single bond, or X b is a single bond And Y b is a divalent group of a dipeptide consisting of CA, the compound of formula (1) has a thioether group in the cysteine residue of X b or a thioether group in the cysteine residue of Y b (20):
- bonded with the thioether group in it may be sufficient.
- X e and “Y e ” independently represent a single bond or a divalent group of a peptide consisting of amino acids of 1 to 4 residues.
- the sum of the number of amino acid residues amino acid residues and element Y e X e is an integer of 0-4.
- that the sum is an integer of 0 means that X e and Y e are single bonds.
- the sum is an integer of 4
- X e and Y e are independently a divalent group of a peptide consisting of 2 amino acids
- X e is composed of 3 amino acids.
- Y e is a divalent group of the peptide is a divalent radical of a peptide consisting of amino acids 1 residue
- a divalent radical of a peptide X e is composed of amino acids 4 residues and a single bond is Y e
- the integer of the sum is preferably 0 to 2, more preferably 0 to 1, and most preferably 0. That is, X e and Y e are most preferably a single bond. In the case where the integer of the sum is 2, when X e is a divalent group of a peptide consisting of two amino acids and Y e is a single bond, X e and Y e independently represent one residue.
- the “cancer antigen peptide E” in the present invention is an MHC class II restricted cancer antigen peptide consisting of 7 to 30 amino acids.
- the cancer antigen peptide E has a carbonyl group of the C-terminal amino acid bonded to Xe in the formula (20), and an amino group of the N-terminal amino acid of the cancer antigen peptide E is a hydrogen atom in the formula (20).
- MHC class II-restricted cancer antigen peptide in the present invention has the same definition as “MHC class II-restricted cancer antigen peptide” in “cancer antigen peptide B” described later.
- HLA corresponding to MHC class II molecules is classified into subtypes such as HLA-DR, DQ and DP.
- the “MHC class II restriction” preferably includes HLA-DR restriction, HLA-DQ restriction or HLA-DP restriction.
- the “MHC class II-restricted cancer antigen peptide” in the present invention means a peptide that binds to MHC class II antigen and induces helper T cells in vitro and / or in vivo.
- the number of amino acid residues of the “MHC class II restricted cancer antigen peptide” is 7-30, preferably 14-30.
- cancer antigen peptide E is an MHC class II-restricted cancer antigen peptide consisting of 7 to 30 amino acids
- the number of amino acid residues is preferably 9 to 15, and more preferably 13 to 15.
- the cancer antigen peptide E is more preferably an HLA-DR-restricted universal cancer antigen peptide consisting of 13 to 15 amino acids.
- universal cancer antigen peptide in the present invention means a cancer antigen peptide or epitope that binds to a plurality of types of MHC class II molecules and induces helper T cells regardless of the types of MHC class II subtypes or polymorphisms. To do.
- the “HLA-DR restricted universal cancer antigen peptide” is also referred to as a universal cancer antigen peptide pan DR-binding peptide.
- cancer antigen peptide E in the same manner as the amino acid sequence mentioned in “cancer antigen peptide B” described later, the following amino acid sequence: AKFVAAWTLKAAA (SEQ ID NO: 101) and aKFVAAAWTLKAAa (SEQ ID NO: 102) Peptides having any amino acid sequence selected from
- X b is a divalent group of a peptide composed of two amino acids including a cysteine residue and Y b is a single bond, or X b is a single bond
- Y b is a divalent group of a peptide composed of two amino acids including a cysteine residue
- X b is preferably a divalent group of a dipeptide composed of CA and Y b is a single bond.
- the thioether group in the cysteine residue of X b or the thioether group in the cysteine residue of Y b has the formula
- the compound bonded to the thioether group in (20) is preferably the formula (19):
- cancer antigen peptide B in “cancer antigen peptide B” in the present invention will be described below.
- MHC class II-restricted means the property of inducing helper T cells by binding to MHC class II molecules.
- HLA corresponding to MHC class II molecules is classified into subtypes such as HLA-DR, DQ and DP.
- the “MHC class II restriction” preferably includes HLA-DR restriction, HLA-DQ restriction or HLA-DP restriction.
- the “MHC class II-restricted cancer antigen peptide” in the present invention means a peptide that binds to MHC class II antigen and induces helper T cells in vitro and / or in vivo.
- the number of amino acid residues of the “MHC class II restricted cancer antigen peptide” is 7-30, preferably 14-30.
- cancer antigen peptide B is an MHC class II-restricted cancer antigen peptide consisting of 7 to 30 amino acids
- the number of amino acid residues is preferably 9 to 15, and more preferably 13 to 15.
- the cancer antigen peptide B is more preferably an HLA-DR-restricted universal cancer antigen peptide consisting of 13 to 15 amino acids.
- universal cancer antigen peptide in the present invention means a cancer antigen peptide or epitope that binds to a plurality of types of MHC class II molecules and induces helper T cells regardless of the types of MHC class II subtypes or polymorphisms. To do.
- the “HLA-DR restricted universal cancer antigen peptide” is also referred to as a universal cancer antigen peptide pan DR-binding peptide.
- HLA-DR restricted universal cancer antigen peptide examples include the peptides listed in Table 12.
- a peptide comprising any amino acid sequence selected from SEQ ID NOs: 101 and 102, or an amino acid residue in any amino acid sequence selected from SEQ ID NOs: 101 and 102 A peptide comprising a modified amino acid sequence containing the above-mentioned modification and having helper T cell inducing activity is preferred, and a peptide comprising any amino acid sequence selected from SEQ ID NOs: 101 and 102 is more preferred.
- the “including amino acid sequence” has the same meaning as described above, and “a peptide having a modified amino acid sequence containing a modified amino acid residue in the amino acid sequence and having helper T cell inducing activity” is also referred to as “modified helper peptide”. be called.
- the modified helper peptide means a peptide that consists of an amino acid sequence in which 1 to 3 amino acids are deleted, substituted and / or added in the amino acid sequence, binds to MHC class II, and induces helper T cells. .
- the number of amino acids to be added (including insertion) is preferably 1 to 3.
- the number of amino acids to be deleted is preferably 1-5.
- the added amino acid or the substituted amino acid may be an unnatural amino acid other than the 20 kinds of amino acids encoded by the gene.
- the compound of formula (1) when R 1 is a group represented by formula (2) and cancer antigen peptide B is an MHC class II-restricted cancer antigen peptide consisting of 7 to 30 amino acids is preferably Equation (6):
- X c and “Y c ” independently represent a single bond or a divalent group of a peptide consisting of amino acids of 1 to 4 residues.
- the sum of the number of amino acid residues of X c and the number of amino acid residues of Y c is an integer of 0-4.
- the sum being an integer of 0 means that X c and Y c are a single bond.
- the sum is an integer of 4
- X c and Y c are independently a divalent group of a peptide consisting of 2 amino acids
- X c consists of 3 amino acids.
- X c is a peptide divalent group of four residues of amino acid and Y c is a single bond
- the integer of the sum is preferably 0 to 2, more preferably 0 to 1, and most preferably 0. That is, X c and Y c are most preferably a single bond. In the case where the integer of the sum is 2, when X c is a divalent group of a peptide consisting of two amino acids and Y c is a single bond, X c and Y c independently represent one residue.
- the integer of the sum is 1, X c is a divalent group of a peptide consisting of one amino acid and Y c is a single bond, or X c is a single bond and Y c Is a divalent group of a peptide consisting of an amino acid having one residue.
- the case where X c is a single bond and Y c is an alanine residue, a leucine residue or a methionine residue is preferable.
- the “cancer antigen peptide C” in the present invention is an MHC class II-restricted cancer antigen peptide consisting of 7 to 30 amino acids.
- the “MHC class II restricted cancer antigen peptide” has the same meaning as described above.
- the “cancer antigen peptide C” in the present invention is an MHC class II-restricted cancer antigen peptide consisting of 7 to 30 amino acids, the number of amino acid residues is preferably 9 to 15, and more preferably 13 to 15.
- the cancer antigen peptide C is more preferably an HLA-DR-restricted universal cancer antigen peptide consisting of 13 to 15 amino acids.
- the “HLA-DR-restricted universal cancer antigen peptide” has the same meaning as described above.
- C carbonyl group of the terminal amino acid bound to X c is in the formula (3) (i.e. bound to the formula (1-3) Among these X c), the amino group of the N-terminal amino acid has the formula (3 ) In the formula (1-3).
- the compound of formula (1) when R 1 is a group represented by formula (3) and cancer antigen peptide C is an MHC class II-restricted cancer antigen peptide consisting of 7 to 30 amino acids is preferably Formula (8):
- R 1 is cancer antigen peptide D
- the thioether group of the cysteine residue of cancer antigen peptide D is bonded to the thioether group in formula (1).
- the amino group of the N-terminal amino acid is bonded to a hydrogen atom
- the carbonyl group of the C-terminal amino acid is bonded to a hydroxyl group.
- Cancer antigen peptide D is an MHC class I-restricted cancer antigen peptide consisting of 7 to 30 amino acids containing one cysteine residue or MHC class II consisting of 7 to 30 amino acids containing one cysteine residue Represents a restricted cancer antigen peptide.
- the amino acid sequence of the peptide includes at least one cysteine residue
- the number of cysteine residues contained is preferably 1 to 3, more preferably 1 to 2, and most preferably 1.
- the “MHC class I-restricted cancer antigen peptide” has the same meaning as described above.
- the compound of the formula (1) in which R 1 is “MHC class I-restricted cancer antigen peptide consisting of 7 to 30 amino acids including one cysteine residue” is also not a homodimer but a heterodimer.
- MHC class I-restricted cancer antigen peptide consisting of 7 to 30 amino acids including one cysteine residue examples include the peptides listed in Table 13.
- MHC class I-restricted cancer antigen peptide comprising 7 to 30 amino acids including one cysteine residue
- VYGFVRRACL SEQ ID NO: 87
- SLLMWITC SEQ ID NO: 88
- a peptide comprising any amino acid sequence selected from among the above, or a modified amino acid sequence containing a modification of an amino acid residue in any amino acid sequence selected from among SEQ ID NOs: 87 and 88, and CTL-inducing activity
- a peptide consisting of any amino acid sequence selected from SEQ ID NOs: 87 and 88 is used.
- the “including the amino acid sequence” and the “peptide containing a modified amino acid sequence containing an amino acid residue modification in the amino acid sequence and having CTL-inducing activity” are as defined above.
- R 1 is “an MHC class I-restricted cancer antigen peptide consisting of 7 to 30 amino acids including one cysteine residue”, preferably the formula (10):
- MHC class II-restricted cancer antigen peptide consisting of 7 to 30 amino acids including one cysteine residue
- the amino acid sequence of the peptide includes at least one cysteine residue
- the number of cysteine residues contained is preferably 1 to 3, more preferably 1 to 2, and most preferably 1.
- the “MHC class II restricted cancer antigen peptide” has the same meaning as described above.
- the number of amino acid residues is preferably 9 to 15, more preferably 13 to 15, and MHC class II.
- the restraint is preferably HLA-DR restraint, HLA-DQ restraint or HLA-DP restraint.
- cancer antigen peptide D is “MHC class II-restricted cancer antigen peptide consisting of 13 to 15 amino acids including one cysteine residue”
- HLA is designated as “MHC class II-restricted cancer antigen peptide”.
- a cancer antigen peptide derived from a cancer antigen protein selected from the group consisting of -2 / neu, hTERT, MUC1 and SART-3 is preferred.
- HLA-DR-restricted universal cancer antigen peptide consisting of 13 to 15 amino acids including one cysteine residue examples include the peptides listed in Table 14.
- the peptide comprising the amino acid sequence of SEQ ID NO: 103 or the amino acid sequence of SEQ ID NO: 103
- a peptide having a modified amino acid sequence containing a modified amino acid residue and having T helper cell induction activity is preferred, and a peptide consisting of the amino acid sequence of SEQ ID NO: 103 is more preferred.
- the “including the amino acid sequence” and the “peptide containing a modified amino acid sequence containing an amino acid residue modification in the amino acid sequence and having a helper T cell-inducing activity” are as defined above.
- R 1 is “an HLA-DR-restricted universal cancer antigen peptide consisting of 13 to 15 amino acids including one cysteine residue”, preferably the compound of the formula (12) :
- the cancer antigen peptide derived from a cancer antigen protein selected from the group consisting of -3 and consisting of 13 to 15 amino acids including one cysteine residue is described in, for example, Table 15 Peptides are mentioned.
- a cancer antigen peptide derived from a cancer antigen protein selected from the group consisting of -3 and consisting of 13 to 15 amino acids including one cysteine residue A peptide comprising any selected amino acid sequence, or a modified amino acid sequence containing a modified amino acid residue in any amino acid sequence selected from SEQ ID NOs: 104 to 107, and having T helper cell induction activity Having a peptide selected from SEQ ID NOs: 104 to 107 Peptide comprising the amino acid sequence of Zureka is more preferable.
- the invention also disulfides two different MHC class I-restricted cancer antigen peptides and MHC class II-restricted cancer antigen peptides, or two different MHC class I-restricted cancer antigen epitopes and MHC class II-restricted cancer antigen epitopes, respectively.
- Methods of synthesizing compounds linked through a bond are provided.
- the method of the present invention includes the following steps (1) to (3).
- the C-terminal amino acid of C (Mmt) A is prepared using cancer antigen peptide B in which Fmoc-C (Mmt) A-SBn and one cysteine residue are bound to the N-terminus.
- a peptide in which the amino group of the N-terminal amino acid bonded to the N-terminus of cancer antigen peptide B is bound is synthesized.
- “Cancer antigen peptide B” has the same definition as “cancer antigen peptide B”. “Fmoc” represents a 9-fluorenylmethoxycarbonyl group. “Mmt” represents a monomethoxytrityl group. “SBn” represents a thiobenzyl group.
- the peptide obtained in the step (1) and the cancer antigen peptide A in which one cysteine residue protected with an SPy group is bonded to the N-terminus are used.
- the thioether group of the cysteine residue bonded to the N terminus of the cancer antigen peptide B and the thioether group of the cysteine residue bonded to the N terminus of the cancer antigen peptide A were bonded. Synthesize peptides.
- Carcer antigen peptide A has the same definition as “cancer antigen peptide A”.
- SPy represents a 2-pyridyl sulfide group.
- the peptide obtained in the step (2) and the cancer antigen peptide E in which one cysteine residue protected with an SPy group is bonded to the C-terminus are used.
- the thioether group of the cysteine residue bonded to the N-terminus of the cancer antigen peptide A in the peptide obtained in (2) and the thioether group of the cysteine residue bonded to the C-terminus of the cancer antigen peptide E were combined. Synthesize peptides.
- Cancer antigen peptide E has the same definition as “cancer antigen peptide E”.
- the compounds and peptides of the present invention and peptides corresponding to intermediates thereof can be produced according to the methods described in the examples of the present specification or the methods used in usual peptide synthesis.
- literature Peptide Synthesis, Interscience, New York, 1966; The Proteins, Vol 2, Academic Press Inc., New York, 1976; Peptide Synthesis, Maruzen (1976) ), 1975; peptide synthesis basics and experiments, Maruzen Co., Ltd., 1985; Development of pharmaceuticals, Vol. 14, Peptide synthesis, Hirokawa Shoten, 1991).
- a method of producing by a solid phase synthesizer using the Fmoc method or Boc method and a method of producing by sequentially condensing Boc-amino acid or Z-amino acid by a liquid phase synthesis method
- Fmoc is 9-fluorene
- Nylmethoxycarbonyl group, Boc represents t-butoxycarbonyl group
- Z represents benzyloxycarbonyl group
- functional groups such as amino group, carboxy group, mercapto group and the like are protected with an appropriate protecting group using a protection or deprotection technique as necessary. Can be protected.
- protecting groups include an acetamidomethyl group or a trityl group.
- the disulfide bond between two different peptides containing a cysteine residue or between a peptide containing a cysteine residue and a cysteine can be formed.
- Methods for forming disulfide bonds are described in the literature (Peptide Synthesis, Interscience, New York, 1966; The Proteins, Vol 2, Academic Press, Inc., New York, Maruichi Peptide; (Inc.), 1975; Basics and experiments of peptide synthesis, Maruzen Co., Ltd., 1985; Pharmaceutical Development, Vol. 14, Peptide Synthesis, Hirokawa Shoten, 1991).
- disulfide when a peptide has one cysteine residue, disulfide is obtained by removing all protecting groups including a mercapto group protecting group on the cysteine side chain and then oxidizing in an inert solvent.
- a compound having a bond (disulfide compound) can be produced.
- it can manufacture by mixing two intermediates which have a mercapto group in a suitable solvent, and oxidizing.
- oxidation method a known method for forming a disulfide bond by ordinary peptide synthesis may be appropriately selected.
- iodine oxidation a method of subjecting to an air oxidation reaction under alkaline conditions, or a method of forming a disulfide bond by adding an oxidizing agent under alkaline or acidic conditions.
- the oxidizing agent include iodine, dimethyl sulfoxide (DMSO), potassium ferricyanide and the like.
- DMSO dimethyl sulfoxide
- the solvent water, acetic acid, methanol, chloroform, DMF or DMSO, or a mixture thereof can be used. Oxidation reactions often give a mixture of symmetrical, asymmetrical disulfide compounds.
- the target asymmetric disulfide compound can be obtained by purification by various chromatography or recrystallization.
- a selective disulfide bond can be formed by mixing an intermediate having an activated mercapto group and an intermediate having a mercapto group.
- the intermediate having an activated mercapto group include a mercapto group to which an Npys group (3-nitro-2-pyridinesulfenyl group) is bonded.
- a selective disulfide bond is formed by adding the other intermediate after activating the mercapto group in advance by mixing one intermediate with, for example, 2,2′-dithiobis (5-nitropyridine). (Tetrahedron Letters. Vol. 37. No. 9, pp. 1347-1350).
- a dimer in which a disulfide bond is formed between the target cysteine residues can be obtained by combining the protecting groups of the cysteine side chains in a specific combination.
- Combinations of the protecting groups include MeBzl (methylbenzyl) group and Acm (acetamidomethyl) group, Trt (trityl) group and Acm group, Npys (3-nitro-2-pyridylthio) group and Acm group, S-Bu- and t (S-tert-butyl) group and Acm group.
- the obtained compounds, peptides and intermediates of the present invention can be purified according to methods known to those skilled in the art and methods used in ordinary peptide chemistry. For example, it can be purified by various chromatography (for example, silica gel column chromatography, ion exchange column chromatography, gel filtration, or reverse phase chromatography) or recrystallization.
- chromatography for example, silica gel column chromatography, ion exchange column chromatography, gel filtration, or reverse phase chromatography
- the recrystallization solvent includes alcohol solvents such as methanol, ethanol or 2-propanol, ether solvents such as diethyl ether, ester solvents such as ethyl acetate, aromatic hydrocarbon solvents such as benzene or toluene, acetone A ketone solvent such as hexane, a hydrocarbon solvent such as hexane, an aprotic solvent such as dimethylformamide or acetonitrile, water, or a mixed solvent thereof can be used.
- alcohol solvents such as methanol, ethanol or 2-propanol
- ether solvents such as diethyl ether
- ester solvents such as ethyl acetate
- aromatic hydrocarbon solvents such as benzene or toluene
- acetone A ketone solvent such as hexane
- a hydrocarbon solvent such as hexane
- an aprotic solvent such as dimethylformamide or acetonitrile
- the compound of the present invention has one or more asymmetric points, it can be produced by using a raw material (amino acid) having the asymmetric point according to a usual method.
- a raw material amino acid
- optical resolution or the like may be performed at an appropriate stage of the production process.
- the compound of the present invention or an intermediate thereof in an inert solvent for example, alcohol solvents such as methanol, ethanol or 2-propanol, ether solvents such as diethyl ether, ester solvents such as ethyl acetate) , Hydrocarbon solvents such as toluene, or aprotic solvents such as acetonitrile, and mixed solvents thereof), optically active acids (for example, monocarboxylic acids such as mandelic acid, N-benzyloxyalanine, or lactic acid, tartaric acid) , Dicarboxylic acid such as o-diisopropylidene tartaric acid or malic acid, or sulfonic acid such as camphor sulfonic acid or bromocamphor sulfonic acid), and the like.
- an inert solvent for example, alcohol solvents such as methanol, ethanol or 2-propanol, ether solvents such as diethyl ether, ester solvents such as
- a salt is formed with an optically active amine (for example, an organic amine such as ⁇ -phenethylamine, quinine, quinidine, cinchonidine, cinchonine, strychnine).
- an optically active amine for example, an organic amine such as ⁇ -phenethylamine, quinine, quinidine, cinchonidine, cinchonine, strychnine.
- the optical division can also be performed.
- the temperature for forming the salt is selected from the range from room temperature to the boiling point of the solvent. In order to improve the optical purity, it is desirable to raise the temperature once to near the boiling point of the solvent. When the precipitated salt is collected by filtration, the yield can be improved by cooling as necessary.
- the amount of the optically active acid or amine used is in the range of about 0.5 to about 2.0 equivalents, preferably in the range of about 1 equivalent, relative to the substrate. Crystals in an inert solvent as necessary (for example, alcohol solvents such as methanol, ethanol, 2-propanol, ether solvents such as diethyl ether, ester solvents such as ethyl acetate, hydrocarbon solvents such as toluene, acetonitrile, etc. And a high-purity optically active salt can be obtained. Further, if necessary, the optically resolved salt can be treated with an acid or base by a conventional method to obtain a free form.
- “pharmaceutically acceptable salts” include acid addition salts and base addition salts.
- the acid addition salts include inorganic acid salts such as hydrochloride, hydrobromide, sulfate, hydroiodide, nitrate and phosphate, citrate, oxalate, acetate, formate , Propionate, benzoate, trifluoroacetate, maleate, tartrate, methanesulfonate, benzenesulfonate, paratoluenesulfonate, and other organic acid salts.
- inorganic acid salts such as hydrochloride, hydrobromide, sulfate, hydroiodide, nitrate and phosphate, citrate, oxalate, acetate, formate , Propionate, benzoate, trifluoroacetate, maleate, tartrate, methanesulfonate, benzenesulfonate, paratoluenesulfonate, and
- Inorganic base salts such as sodium salt, potassium salt, calcium salt, magnesium salt, ammonium salt, organic base salts such as triethylammonium salt, triethanolammonium salt, pyridinium salt, diisopropylammonium salt, and the like, and arginine, Examples thereof include amino acid salts such as basic or acidic amino acids such as aspartic acid and glutamic acid.
- hydrates of the compounds of the present invention or pharmaceutically acceptable salts thereof are also included in the present invention.
- solvates such as ethanol solvates.
- the present invention also includes all diastereomers, all enantiomers and the like of the compound represented by the formula (1), and all forms of crystal forms.
- amino acid-deficient peptides were decomposed by hydrolysis, oxidation, etc. during the steps of condensing optically active ⁇ -amino acids, removing various protecting groups, or cleaving peptides from resins.
- Various by-products such as peptides and peptides racemized from amino acids are produced.
- these impurities can be removed to combine high purity peptides and compounds by combining various chromatographies (eg silica gel column chromatography, ion exchange column chromatography, gel filtration, or reverse phase chromatography). Obtainable. However, it is not easy to obtain highly pure peptides and compounds on an industrial scale to provide them as pharmaceuticals.
- the compound of the present invention also has the property that it can be mass-produced as an active pharmaceutical ingredient in its physicochemical properties. Specifically, it has properties such as high solubility, excellent stability in solution, or difficulty in gelation when concentrated, and is a large scale in the purification process by column chromatography such as reverse phase HPLC. Can be easily produced as a drug substance with high purity.
- the compound of the present invention thus produced has excellent stability against oxidizing agents in solution due to the cysteine residue forming a disulfide bond, and has a certain quality as a pharmaceutical raw material. It retains efficient CTL inducing activity.
- the compound of the present invention is useful as an active ingredient of a CTL inducer in cancer immunotherapy, as an active ingredient of a cancer vaccine, and as an active ingredient of a pharmaceutical composition. That is, the compound of the present invention has excellent immunogenicity and can efficiently exhibit excellent CTL-inducing activity as shown in the Examples of the present specification.
- the CTL induced by the compound of the present invention can surprisingly recognize a natural partial peptide of a cancer antigen protein originally possessed by a cancer cell.
- CTL inducing activity is determined by measuring the number of CTLs by the HLA tetramer method (Int. J. Cancer: 100, 565-570 (2002)) or the limiting dilution method (Nat. Med .: 4, 321-327 (1998)). This can be confirmed.
- HLA-A24-restricted CTL inducing activity WO 02/47474 and Int. J. et al. Cancer: 100, 565-570 (2002) can be examined by using the HLA-A24 model mouse described.
- the compound of the present invention can be used as a therapeutic or preventive drug (recurrence preventive drug) for cancer in which cancer antigen protein is expressed or cancer accompanied by an increased expression level of cancer antigen protein gene.
- cancer include blood cancer such as leukemia, myelodysplastic syndrome, multiple myeloma or malignant lymphoma, or gastric cancer, colon cancer, lung cancer, breast cancer, germ cell cancer, liver cancer, skin cancer, bladder cancer, prostate cancer.
- Solid cancer such as uterine cancer, cervical cancer, ovarian cancer or brain tumor.
- the compound of the present invention or a pharmaceutically acceptable salt thereof is made into an appropriate form according to each compound or each salt, so that the active ingredient of the CTL inducer, the active ingredient of the cancer vaccine or the cancer vaccine / And can be an active ingredient of a pharmaceutical composition.
- the cellular immunity of the compound of the present invention can be administered together with a pharmaceutically acceptable carrier, for example, an appropriate adjuvant.
- a pharmaceutically acceptable carrier for example, an appropriate adjuvant.
- an appropriate adjuvant those described in the literature (Clin. Microbiol. Rev., 7: 277-289, 1994) and the like can be applied, and specifically, bacterial cell-derived components, GM-CSF, interleukin-2 , Cytokines such as interleukin-7 or interleukin-12, plant-derived components, marine organism-derived components, mineral gels such as aluminum hydroxide, surfactants such as lysolecithin and pluronic polyols, polyanions, peptides, or oil emulsions (Emulsion formulation).
- lipid A As the cell-derived component, lipid A (lipid A), its derivative monophosphoryl lipid A (monophosphoryl lipid A), bacteria (including Mycobacterium bacteria such as BCG bacteria), bacteria-derived proteins, Examples include polynucleotides, Freund's Incomplete Adjuvant, Freund's Complete Adjuvant, cell wall skeleton components (for example, BCG-CWS), trehalose dimycolate (TDM), etc. .
- the compound of the present invention can also be administered in the form of a liposome preparation, a particulate preparation bound to beads having a diameter of several ⁇ m, a preparation bound to lipid, and the like.
- the compounds (conjugates) of the invention can be administered with MHC class II restricted peptides (ie helper peptides).
- MHC class II restricted peptides ie helper peptides
- the cocktail formulation comprises a conjugate capable of producing an MHC class I restricted peptide (ie, a killer peptide) and an MHC class II restricted peptide (ie, a helper peptide).
- helper T cell activation of a helper T cell (helper T cell) that is important for enhancing the function of other T cells including CTLs can be achieved by administering this cocktail preparation containing a helper peptide as a cancer vaccine in cancer immunotherapy. It becomes possible, and the function and medicinal effect (cellular immunity etc.) of the conjugate can be improved.
- the MHC class II-restricted peptide ie, helper peptide
- this cocktail preparation had improved efficacy as a cancer vaccine such as cellular immunity.
- the dosage of the compound of the present invention in the preparation can be appropriately adjusted depending on the disease to be treated, the age, weight, etc. of the patient, but is usually 0.0001 mg to 1000 mg, preferably 0.001 mg to 1000 mg, more preferably. 0.1 mg to 10 mg.
- Examples of the administration method include intradermal administration, subcutaneous administration, intramuscular administration, intravenous administration, and transdermal administration. Intradermal administration and subcutaneous administration that efficiently induce CTL are preferred.
- the number of administrations and the administration interval can be appropriately adjusted depending on the disease for treatment or prevention and individual differences among patients, but it is usually a plurality of times and preferably administered once every several days to several months.
- Example 1 The following amino acid sequence: CKIFGSLAFL (SEQ ID NO: 100) Synthesis of peptides consisting of
- Step 1 and Step 2 were sequentially performed using the amino acids shown below.
- Step 2 was repeated three times, and the resulting resin was washed with DMF and then unreacted amino group using 25% Ac 2 O (acetic anhydride).
- Step 1 the deprotection operation in Step 1 was performed to obtain H-Lys (Boc) -Ile-Phe-Gly-Ser (tBu) -Leu-Ala-Phe-Leu-Alko-Resin.
- a solution of 340.4 mg of Fmoc-Cys (tBu) -OH, 248.2 mg of HBTU and 92.9 mg of HOBT in 10 ml of DMF was added, and 0.2 ml of DIEA was further added and shaken at room temperature for 3 hours. A coupling reaction was performed.
- the Fmoc group was cleaved by treatment with 10 ml of 30% Pip / DMF (10 minutes ⁇ 1 time and 5 minutes ⁇ 2 times).
- 100 ml of TFA cocktail (2.5% tetraisopropylsilane / 2.5% dodecanthiol / 2.5% H 2 O / 92.5% TFA solution) was added and stirred at room temperature for 2.0 hours.
- diethyl ether was added, it filtered with the glass filter, and the TFA cocktail and diethyl ether were removed as a filtrate.
- the filtered product was washed with diethyl ether to obtain 269.5 mg of a crude peptide (CKIFGSLAFL (SEQ ID NO: 100)).
- Examples 2 to 20 Each peptide consisting of the amino acid sequences of SEQ ID NOs: 95 to 97 and 108 to 123 was synthesized in the same manner as in Example 1. Tables 16 to 17 show the mass analysis results of the synthesized peptides.
- R 1 is a hydrogen atom
- X a is a single bond
- Y a is a divalent group of a peptide consisting of one amino acid
- the cancer antigen peptide A is HER2 / neu 369-377 peptide (KIFGSLAFL) (SEQ ID NO: 53) which is a partial peptide of the cancer antigen protein HER2 / neu.
- Reference example 1 A peptide consisting of the amino acid sequence of SEQ ID NO: 86 was synthesized in the same manner as in Example 1. Table 18 shows the results of mass spectrometry of the synthesized peptides. Since the peptide of SEQ ID NO: 86 is not a compound of the present invention as described above, it was described as Reference Example 1.
- Examples 21-24 In the same manner as in Example 1, peptides having the amino acid sequences of SEQ ID NOs: 92 to 94, 99 were synthesized. Table 19 shows the mass analysis results of each synthesized peptide.
- R 1 is a hydrogen atom
- X a is a single bond
- Y a is a single bond or a divalent group of a peptide consisting of one amino acid.
- the cancer antigen peptide A is a compound of the present invention which is Proteinase-3 169-177 peptide (VLQELNVTV) (SEQ ID NO: 43), which is a partial peptide of cancer antigen protein Proteinase-3.
- Examples 25-28 In the same manner as in Example 1, each peptide consisting of the amino acid sequences of SEQ ID NOs: 98 and 89 to 91 was synthesized. Table 20 shows the results of mass spectrometry of each synthesized peptide.
- R 1 is a hydrogen atom
- X a is a single bond
- Y a is a single bond or a divalent group of a peptide consisting of one amino acid.
- the cancer antigen peptide A is MAGE-A10 254-262 peptide (GLYDGMEHL) (SEQ ID NO: 19) which is a partial peptide of the cancer antigen protein MAGE-A10.
- Test Example 1 Trimming test of N-terminal amino acid by ERAP1 Evaluation of trimming efficiency of N-terminal amino acid of peptides synthesized in Examples 1 to 20 using ERAP1 (PLoS One November 2008, vol. 3, Issue 11, e3658) did. 50 ⁇ l of ERAP1 (50 ng / ml) pH 8.0 20 mM Tris ⁇ HCl—100 mM NaCl buffer (Tris ⁇ HCl buffer) solution was added to 142 ⁇ l of Tris ⁇ HCl buffer. 8.0 ⁇ l of 2.5 mM peptide aqueous solution was added to the above-mentioned ERAP1 solution, mixed well, and allowed to stand at room temperature.
- ERAP1 50 ⁇ l of ERAP1 (50 ng / ml) pH 8.0 20 mM Tris ⁇ HCl—100 mM NaCl buffer (Tris ⁇ HCl buffer) solution was added to 142 ⁇ l of Tris
- Test Example 2 Change with time of trimming of N-terminal amino acid by ERAP1 For each peptide synthesized in Examples 1-2, 11-12, and 21-28, changes with time of trimming of N-terminal amino acid by ERAP1 were evaluated. . 20 ⁇ l of ERAP1 (0.5 mg / ml) Tris ⁇ HCl buffer solution was added to 172 ⁇ l of Tris ⁇ HCl buffer. 8.0 ⁇ l of a 10 mM DMSO solution of each peptide was added to the above ERAP1 solution, mixed well, and allowed to stand at room temperature.
- Test Example 3 Evaluation of CTL inducing ability in vivo using HLA-A0201 transgenic mice The CTL inducing ability of each peptide (Cys-extended peptide) obtained by extending cysteine (Cys) shown in Table 23 is shown in HLA-A0201. Evaluation was performed by an in vivo CTL induction test using transgenic mice.
- HLA-A0201 transgenic mice C57BL / 6CrHLA-A2.1DR1 lack mouse MHC, and human HHC HLA-A0201 and mouse MHC H-2D b chimeric HLA and HLA-DRB1 * 0101 By using this mouse, it is possible to select a peptide capable of inducing CTL in an HLA-A02 positive human (Eur J Immunol. 2004; 34: 3060-9).
- each peptide with extended Cys (SEQ ID NO: 100, 99 or 98) induces CTL against the peptide (SEQ ID NO: 53, 43 or 19) endogenously presented to cancer cells Produces IFN ⁇ when spleen cells derived from the above-mentioned mice administered with a peptide with extended Cys (SEQ ID NO: 100, 99 or 98) are restimulated with the peptide (SEQ ID NO: 53, 43 or 19). It was judged by measuring.
- the peptide (SEQ ID NO: 53, 43 or 19) endogenously presented to the cancer cells shown in Table 24 is also referred to as a Cys non-extending peptide in this test example.
- each peptide (SEQ ID NO: 19, 43, 53, 98, 99 or 100) is dissolved in dimethyl sulfoxide (DMSO) at 200 mg / mL, and further with phosphate buffered saline (PBS, pH 5). After diluting to 2 mg / mL, it was mixed with an equal volume of incomplete Freund's adjuvant (IFA) and emulsified. The emulsified peptide was administered into the ridge skin of the mouse at 2 sites at 50 ⁇ g / site. One week later, the mouse was euthanized with CO 2 gas, and then the spleen was removed and spleen cells were prepared.
- DMSO dimethyl sulfoxide
- PBS phosphate buffered saline
- IFN ⁇ ELISPOT assay kit was used to measure IFN ⁇ production.
- the ELISPOT plate was treated with anti-mouse IFN ⁇ antibody and blocked on RPMI 1640 medium containing 10% FBS on the day.
- the prepared spleen cells derived from HLA-A0201 transgenic mice were seeded on a blocked ELISPOT plate at 2.5 ⁇ 10 5 cells / well.
- Each peptide (SEQ ID NO: 19, 43, 53, 98, 99 or 100) was dissolved in DMSO to 40 mg / mL, and further diluted to 40 ⁇ g / mL in RPMI 1640 medium containing 10% FBS.
- Spleen cells derived from mice administered with the peptide represented by SEQ ID NO: 53 or 100 were pulsed with a diluted peptide of both of them (final concentration 10 ⁇ g / mL), and mice administered with the peptide represented by SEQ ID NO: 43 or 99
- the derived spleen cells were pulsed with both of these diluted peptides (final concentration 10 ⁇ g / mL), and the spleen cells derived from mice administered with the peptide represented by SEQ ID NO: 19 or 98 were both diluted peptides (final concentration).
- 10 ⁇ g / mL was pulsed, and the peptide restimulation in vitro was performed by culturing at 37 ° C. under 5% CO 2 for 20 hours.
- the vertical axis indicates the number of cells that reacted in the number of seeded cells.
- the value of the white bar indicating the result in the case of non-pulse is hardly recognized. This indicates that the spleen cells of each transgenic mouse did not react at all with no peptide restimulation.
- the difference between the value of the color bar and the white bar indicates the number of CTLs specific to the peptide used at the time of in vitro re-stimulation, indicating that it was induced in the mouse body by administration of each peptide.
- the color bar means a black bar indicating a result when pulsed with a Cys non-extended peptide, or a gray bar indicating a result when pulsed with a Cys extended peptide.
- FIG. 5 shows the result of administering the peptide represented by SEQ ID NO: 53 or 100
- FIG. 6 shows the result of administering the peptide represented by SEQ ID NO: 43 or 99
- FIG. 7 shows the result represented by SEQ ID NO: 19 or 98.
- Cys-extended peptide can induce CTLs that recognize peptides endogenously presented to cancer cells (Cys non-extended peptide), and the Cys-extended peptide is appropriately trimmed by ERAP1 in the mouse body. In response, it was strongly suggested that it was actually produced into a Cys non-extended peptide.
- Test Example 4 Evaluation of CTL inducibility in vivo using HLA-A0201 transgenic mice
- the bond between C and C represents a disulfide bond.
- the compound represented by formula (I) is particularly a compound in which the cancer antigen peptide A is GLYDGMEHL (SEQ ID NO: 19) and the cancer antigen peptide B is KIFGSLAFL (SEQ ID NO: 53).
- GLYDGMEHL (SEQ ID NO: 19) and KIFGSLAFL (SEQ ID NO: 53) are HLA-A0201-restricted cancer antigen peptides.
- HLA-A0201 transgenic mice C57BL / 6CrHLA-A2.1DR1 lack mouse MHC, and human HHC HLA-A0201 and mouse MHC H-2D b chimeric HLA and HLA-DRB1 * 0101 By using this mouse, it is possible to select a peptide capable of inducing CTL in an HLA-A02 positive human (Eur J Immunol. 2004; 34: 3060-9).
- the compound represented by formula (4) was introduced into the HLA-A0201 gene. Administered to mice. That is, whether or not the mouse-derived spleen cells administered with the compound represented by formula (4) were re-stimulated with the peptide (SEQ ID NO: 19, 53) was confirmed by whether or not IFN ⁇ was produced. .
- the compound represented by formula (4) was diluted to 10 mg / mL with water for injection, and then mixed with an equal amount of incomplete Freund's adjuvant (IFA) to form an emulsion.
- IFA incomplete Freund's adjuvant
- the emulsified compound was administered into the ridge skin of the mouse at two sites at 250 ⁇ g / site.
- the mouse was euthanized with CO 2 gas, and then the spleen was removed and spleen cells were prepared.
- An IFN ⁇ ELISPOT assay kit was used to measure IFN ⁇ production.
- the ELISPOT plate was treated with anti-mouse IFN ⁇ antibody and blocked on RPMI 1640 medium containing 10% FBS on the day.
- the prepared spleen cells derived from HLA-A0201 transgenic mice were seeded on a blocked ELISPOT plate at 0.25 ⁇ 10 6 cells / well.
- the peptide (SEQ ID NO: 19, 53) was dissolved in DMSO to 40 mg / mL, and further diluted to 40 ⁇ g / mL in RPMI 1640 medium containing 10% FBS.
- Spleen cells derived from HLA-A0201 transgenic mice are pulsed with a diluted peptide represented by SEQ ID NO: 19 or SEQ ID NO: 53 (final concentration 10 ⁇ g / mL) and cultured for 20 hours at 37 ° C. under 5% CO 2.
- a diluted peptide represented by SEQ ID NO: 19 or SEQ ID NO: 53 final concentration 10 ⁇ g / mL
- FIG. 8 The results of IFN ⁇ ELISPOT assay using HLA-A0201 transgenic mice are shown in FIG. In FIG. 8, the vertical axis indicates the number of cells that reacted in the number of seeded cells.
- the black bars and hatched bars in FIG. 8 show the results of culturing spleen cells derived from HLA-A0201 transgenic mice while pulsing each peptide represented by SEQ ID NOs: 19 and 53, and the white bars are cultured without pulse. Results are shown. That is, the difference between the values of black bars or diagonal bars and white bars indicates the number of peptide-specific CTLs, which are represented by SEQ ID NOs: 19 and 53 in the mouse in vivo by administration of the compound represented by formula (4).
- the compound represented by the formula (4) can induce each peptide-specific CTL represented by SEQ ID NOs: 19, 53.
- the compound represented by the formula (4) is actually produced into the peptides represented by SEQ ID NOs: 19 and 53 after undergoing disulfide bond cleavage and appropriate trimming by ERAP-1 in the mouse body. It was strongly suggested. That is, the compound represented by formula (4), which is an example of the compound of the present invention, is a conjugate in which two different peptides are complexed via a disulfide bond as shown in formula (1). It became clear that this is a cancer antigen peptide conjugate vaccine that can actually induce two different CTLs in vivo.
- Test Example 5 Evaluation of CTL inducibility in vivo using HLA-A0201 transgenic mice and HLA-A2402 transgenic mice
- the compound represented by formula (I) is particularly a compound in which the cancer antigen peptide A is GLYDGMEHL (SEQ ID NO: 19) and the cancer antigen peptide D is VYGFVRACL (SEQ ID NO: 87).
- GLYDGMEHL SEQ ID NO: 19
- VYGFVRACL SEQ ID NO: 87
- HLA-A24-restricted cancer antigen peptide is an HLA-A24-restricted cancer antigen peptide.
- the HLA-A0201 transgenic mouse is as described in Test Example 4.
- the HLA-A2402 transgenic mouse (C57BL / 6CrHLA-A2402 / K b ) is a mouse that expresses a chimeric HLA of human MHC HLA-A2402 and mouse MHC H-2K b. By using it, it is possible to select peptides capable of inducing CTL in HLA-A24 positive humans (Int J Cancer. 2002; 100: 565-70).
- the compound represented by formula (10) was introduced into the HLA-A0201 gene. Mice and HLA-A2402 transgenic mice were administered. That is, whether or not the mouse-derived spleen cells administered with the compound represented by the formula (10) were restimulated with the peptide (SEQ ID NO: 19, 87) was confirmed by whether or not IFN ⁇ was produced. .
- the compound represented by the formula (10) is dissolved in dimethyl sulfoxide (DMSO) at 80 mg / mL, further diluted to 10 mg / mL with water for injection, and then an equivalent amount of incomplete Freund's adjuvant (IFA). And emulsified.
- DMSO dimethyl sulfoxide
- IFA incomplete Freund's adjuvant
- the emulsified compound was administered into the ridge skin of the mouse at two sites at 250 ⁇ g / site. One week later, the mouse was euthanized with CO 2 gas, and then the spleen was removed and spleen cells were prepared.
- An IFN ⁇ ELISPOT assay kit was used to measure IFN ⁇ production.
- the ELISPOT plate was treated with anti-mouse IFN ⁇ antibody and blocked on RPMI 1640 medium containing 10% FBS on the day.
- the prepared spleen cells derived from HLA-A0201 transgenic mice were blocked at 0.25 ⁇ 10 6 cells / well and spleen cells derived from HLA-A2402 transgenic mice were blocked at 0.5 ⁇ 10 6 cells / well. Sowing.
- the peptide (SEQ ID NO: 19, 87) was dissolved in DMSO to 40 mg / mL, and further diluted to 40 ⁇ g / mL in RPMI 1640 medium containing 10% FBS.
- Spleen cells derived from HLA-A0201 transgenic mice were pulsed with a diluted peptide represented by SEQ ID NO: 19 (final concentration 10 ⁇ g / mL), and splenocytes derived from HLA-A2402 transgenic mice were represented by SEQ ID NO: 87.
- In vitro peptide restimulation was applied by pulsing with diluted peptide (final concentration 10 ⁇ g / mL) and incubating for 20 hours at 37 ° C., 5% CO 2 . After incubation, the supernatant was removed and the ELISPOT plate was developed according to the attached protocol. The number of spots developed was measured by an ImmunoSpot Analyzer (manufactured by C.T.L.).
- the results of IFN ⁇ ELISPOT assay using HLA-A0201 transgenic mice are shown in FIG. 9, and the results of IFN ⁇ ELISPOT assay using HLA-A2402 transgenic mice are shown in FIG.
- the vertical axis indicates the number of cells that reacted in the number of seeded cells.
- the black bars and white bars in FIG. 9 show the results of culturing splenocytes derived from HLA-A0201 transgenic mice while pulsing and non-pulsing the peptide represented by SEQ ID NO: 19, and the black bars and white bars in FIG.
- FIG. 7 shows the results of culturing splenocytes derived from HLA-A2402 transgenic mice while pulsing and non-pulsing the peptide represented by SEQ ID NO: 87. That is, the difference between the value of the black bar and the white bar indicates the number of peptide-specific CTLs, and each peptide represented by SEQ ID NOs: 19 and 87 is administered in vivo in the mouse by administration of the compound represented by formula (10). It shows that specific CTL was induced. In particular, in FIG. 9, the value of the white bar is not recognized. This indicates that the splenocytes of HLA-A0201 transgenic mice did not react at all in the absence of the target peptide.
- IFN ⁇ production specific to the peptide represented by SEQ ID NO: 19 was produced in splenocytes derived from HLA-A0201 transgenic mice, and SEQ ID NO: in splenocytes derived from HLA-A2402 transgenic mice.
- the production of peptide-specific IFN ⁇ represented by 87 was confirmed.
- the compound represented by the formula (10) can induce CTL specific to each peptide represented by SEQ ID NOs: 19, 87.
- the compound represented by the formula (10) is actually produced into the peptides represented by SEQ ID NOs: 19 and 87 after undergoing disulfide bond cleavage and appropriate trimming by ERAP-1 in the mouse body. It was strongly suggested. That is, the compound represented by the formula (10), which is an example of the compound of the present invention, is a conjugate in which two different peptides are complexed via a disulfide bond as shown in the formula (1). It became clear that this is a cancer antigen peptide conjugate vaccine that can actually induce two different CTLs in vivo.
- Examples 31-49 In the same manner as in Example 1, each peptide consisting of the amino acid sequences of SEQ ID NOs: 124 to 142 was synthesized. Tables 25 to 26 show the mass analysis results of each synthesized peptide.
- R 1 is a hydrogen atom
- X a is a divalent group of a peptide consisting of one amino acid
- Y a is a single bond
- the cancer antigen peptide A is HER2 / neu 369-377 peptide (KIFGSLAFL) (SEQ ID NO: 53) which is a partial peptide of the cancer antigen protein HER2 / neu.
- Examples 50-68 In the same manner as in Example 1, each peptide consisting of the amino acid sequence of SEQ ID NOs: 143 to 161 was synthesized. Tables 27 to 28 show the mass analysis results of each synthesized peptide.
- R 1 is a hydrogen atom
- X a is a divalent group of a peptide consisting of one amino acid
- Y a is a single bond
- the cancer antigen peptide A is Proteinase-3 169-177 peptide (VLQELNVTV) (SEQ ID NO: 43), which is a partial peptide of cancer antigen protein Proteinase-3.
- Examples 69-84 In the same manner as in Example 1, peptides having the amino acid sequences of SEQ ID NOs: 162 to 177 were synthesized. Tables 29 to 30 show the mass spectrometry results of the synthesized peptides.
- R 1 is a hydrogen atom
- X a is a single bond
- Y a is a divalent group of a peptide consisting of one amino acid
- the cancer antigen peptide A is MAGE-A10 254-262 peptide (GLYDGMEHL) (SEQ ID NO: 19) which is a partial peptide of the cancer antigen protein MAGE-A10.
- Examples 85-103 Each peptide consisting of the amino acid sequences of SEQ ID NOs: 178 to 196 was synthesized in the same manner as in Example 1.
- Tables 31 to 32 show the results of mass spectrometry of each synthesized peptide.
- R 1 is a hydrogen atom
- X a is a divalent group of a peptide consisting of one amino acid
- Y a is a single bond
- the cancer antigen peptide A is MAGE-A10 254-262 peptide (GLYDGMEHL) (SEQ ID NO: 19) which is a partial peptide of the cancer antigen protein MAGE-A10.
- Test Example 6 N-terminal amino acid trimming test using ERAP1
- the N-terminal amino acid trimming efficiency of the peptides synthesized in Examples 31 to 49 was evaluated using ERAP1.
- 50 ⁇ l of ERAP1 (50 ⁇ g / ml) pH 8.0 20 mM Tris • HCl—100 mM NaCl buffer (Tris • HCl buffer) solution was added to 142 ⁇ l of Tris • HCl buffer.
- 2.0 ⁇ l of 10 mM peptide DMSO solution and 6.0 ⁇ L of DMSO were added to the above ERAP1 solution, mixed well, and then allowed to stand at 30 ° C.
- HPLC measurement condition column Chemcopack Quicksorb (4.6 mm ⁇ ⁇ 150 mm, 5 ⁇ m) Mobile phase manufactured by Chemco Corporation: A solution; 0.1% TFA water, B solution; 0.1% TFA acetonitrile solution Column temperature: room temperature flow rate : 1mL / min Detection wavelength: UV254nm, 230nm (2 wavelength detection) Sample injection volume: 10 ⁇ L
- Examples 104-105 Each peptide consisting of the amino acid sequences of SEQ ID NOs: 197 and 198 was synthesized in the same manner as in Example 1.
- Table 36 shows the mass analysis results of each synthesized peptide.
- Xb is a single bond
- Yb is a single bond
- cancer antigen peptide B is an HLA-DR-restricted universal cancer antigen peptide (SEQ ID NO: 101 and 102) of the compounds of the present invention.
- Examples 106-107 In the same manner as in Example 1, each peptide consisting of the amino acid sequences of SEQ ID NOs: 199 and 200 was synthesized. Table 37 shows the mass analysis results of each synthesized peptide. In any of the peptides in Table 37, in Formula (3), X c is a single bond, Y c is a single bond, and cancer antigen peptide C is HLA-DR-restricted universal cancer antigen peptide (SEQ ID NO: 101 and 102) of the compounds of the present invention.
- Examples 114-143 A peptide having the amino acid sequence of SEQ ID NOs: 201 to 230 was synthesized in the same manner as in Example 1.
- Tables 39 to 41 show the results of mass spectrometry of each synthesized peptide.
- R 1 is a hydrogen atom
- X a is a single bond
- Y a is a single bond
- cancer antigen peptide A is represented in Tables 1 to 9 It is the compound of this invention which is the MHC class I restriction cancer antigen peptide shown in above.
- Test Example 8 With respect to the compounds (conjugates) represented by the formulas (5), (11) and (15) to (17) synthesized in Examples 109 to 113, the solubility measurement shown in Test Example 7 was performed, and each solubility was measured. Are shown in Table 42.
- Test Example 9 Trimming test of N-terminal amino acid by ERAP1 The trimming efficiency of the N-terminal amino acid of the peptides synthesized in Examples 69 to 84 was evaluated using ERAP1. 50 ⁇ l of ERAP1 (10 ⁇ g / ml) pH 8.0 20 mM Tris ⁇ HCl—100 mM NaCl buffer (Tris ⁇ HCl buffer) solution was added to 142 ⁇ l of Tris ⁇ HCl buffer. 2.0 ⁇ l of 10 mM peptide DMSO solution and 6.0 ⁇ L of DMSO were added to the above ERAP1 solution, mixed well, and then allowed to stand at 30 ° C.
- Test Example 10 Trimming Test for N-terminal Amino Acid Using ERAP1 The N-terminal amino acid trimming efficiency of the peptides synthesized in Examples 85 to 103 was evaluated using ERAP1. 50 ⁇ l of ERAP1 (10 ⁇ g / ml) pH 8.0 20 mM Tris ⁇ HCl—100 mM NaCl buffer (Tris ⁇ HCl buffer) solution was added to 142 ⁇ l of Tris ⁇ HCl buffer. 2.0 ⁇ l of 10 mM peptide DMSO solution and 6.0 ⁇ L of DMSO were added to the above ERAP1 solution, mixed well, and then allowed to stand at 30 ° C.
- Test Example 11 N-terminal amino acid trimming test using ERAP1
- the N-terminal amino acid trimming efficiency of the peptides synthesized in Examples 50, 56, 64 and 67 was evaluated using ERAP1.
- 50 ⁇ l of ERAP1 (10 ⁇ g / ml) pH 8.0 20 mM Tris ⁇ HCl—100 mM NaCl buffer (Tris ⁇ HCl buffer) solution was added to 142 ⁇ l of Tris ⁇ HCl buffer.
- 2.0 ⁇ l of 10 mM peptide DMSO solution and 6.0 ⁇ L of DMSO were added to the above ERAP1 solution, mixed well, and then allowed to stand at 30 ° C.
- Test Example 12 Trimming test of N-terminal amino acid by ERAP1 The trimming efficiency of the N-terminal amino acid of peptides synthesized in Examples 114 to 115, 118 to 122, 126, 129, 132, 137, 139 and 141 to 143 Evaluated. 50 ⁇ l of ERAP1 (10 ⁇ g / ml) pH 8.0 20 mM Tris ⁇ HCl—100 mM NaCl buffer (Tris ⁇ HCl buffer) solution was added to 142 ⁇ l of Tris ⁇ HCl buffer.
- Test Example 13 Evaluation of CTL inducibility in vivo using HLA-A0201 transgenic mice
- the bond between C and C represents a disulfide bond.
- the compound represented by the formula (1) is a compound in which the cancer antigen peptide A is GLYDGMEHL (SEQ ID NO: 19) and the cancer antigen peptide D is SLLMWITC (SEQ ID NO: 88).
- GLYDGMEHL (SEQ ID NO: 19) and SLLMWITQC (SEQ ID NO: 88) are HLA-A0201-restricted cancer antigen peptides.
- the HLA-A0201 transgenic mouse is as described in Test Example 4.
- the compound represented by formula (11) was introduced into HLA-A0201 gene. Administered to mice. That is, whether or not the mouse-derived spleen cells administered with the compound represented by the formula (11) were restimulated with a peptide (SEQ ID NO: 19, 88) was confirmed by whether or not IFN ⁇ was produced. .
- the compound represented by the formula (11) is dissolved in dimethyl sulfoxide (DMSO) at 80 mg / mL, further diluted to 10 mg / mL with water for injection, and then an equal amount of incomplete Freund's adjuvant (IFA). And emulsified.
- DMSO dimethyl sulfoxide
- IFA incomplete Freund's adjuvant
- the emulsified compound was administered into the ridge skin of the mouse at two sites at 250 ⁇ g / site. One week later, the mouse was euthanized with CO 2 gas, and then the spleen was removed and spleen cells were prepared.
- An IFN ⁇ ELISPOT assay kit was used to measure IFN ⁇ production.
- the ELISPOT plate was treated with anti-mouse IFN ⁇ antibody and blocked on RPMI 1640 medium containing 10% FBS on the day.
- the prepared spleen cells derived from HLA-A0201 transgenic mice were seeded on a blocked ELISPOT plate at 0.25 ⁇ 10 6 cells / well.
- the peptide (SEQ ID NO: 19, 88) was dissolved in DMSO to 40 mg / mL, and further diluted to 40 ⁇ g / mL in RPMI 1640 medium containing 10% FBS.
- Spleen cells derived from HLA-A0201 transgenic mice are pulsed with a diluted peptide represented by SEQ ID NO: 19 or SEQ ID NO: 88 (final concentration 10 ⁇ g / mL) and cultured for 17 hours at 37 ° C. under 5% CO 2. Thus, peptide re-stimulation in vitro was added. After incubation, the supernatant was removed and the ELISPOT plate was developed according to the attached protocol. The number of spots developed was measured by an ImmunoSpot Analyzer (manufactured by C.T.L.).
- FIG. 11 The results of IFN ⁇ ELISPOT assay using HLA-A0201 transgenic mice are shown in FIG. In FIG. 11, the vertical axis represents the number of cells that reacted in the number of seeded cells.
- the black bars and hatched bars in FIG. 11 show the results of culturing spleen cells derived from HLA-A0201 transgenic mice while pulsing each peptide represented by SEQ ID NOs: 19, 88, and the white bars are cultured without pulse. Results are shown. That is, the difference between the values of black bars or diagonal bars and white bars indicates the number of peptide-specific CTLs, which are represented by SEQ ID NOs: 19, 88 in the mouse body by administration of the compound represented by formula (11).
- the compound represented by the formula (11) can induce CTL specific to each peptide represented by SEQ ID NOs: 19, 88.
- the compound represented by the formula (11) is actually produced into the peptides represented by SEQ ID NOs: 19 and 88 after undergoing disulfide bond cleavage and appropriate trimming by ERAP-1 in vivo in mice. It was strongly suggested. That is, the compound represented by the formula (11), which is an example of the compound of the present invention, is a conjugate in which two different peptides are complexed via a disulfide bond as shown in the formula (1). It became clear that this is a cancer antigen peptide conjugate vaccine that can actually induce two different CTLs in vivo.
- Test Example 14 Evaluation of CTL inducibility in vivo using HLA-A0201 transgenic mice
- the compound represented by the formula (1) is a compound in which the cancer antigen peptide A is GLYDGMEHL (SEQ ID NO: 19) and the cancer antigen peptide A is VLQELNVTV (SEQ ID NO: 43).
- GLYDGMEHL (SEQ ID NO: 19) and VLQELNVTV (SEQ ID NO: 43) are HLA-A0201-restricted cancer antigen peptides.
- the HLA-A0201 transgenic mouse is as described in Test Example 4.
- the compound represented by formula (5) was introduced into HLA-A0201 gene. Administered to mice. That is, whether or not the mouse-derived spleen cells administered with the compound represented by formula (5) were restimulated with a peptide (SEQ ID NO: 19, 43) was confirmed by whether or not IFN ⁇ was produced. .
- the compound represented by formula (5) is dissolved in dimethyl sulfoxide (DMSO) at 80 mg / mL, further diluted to 10 mg / mL with water for injection, and then an equal amount of incomplete Freund's adjuvant (IFA). And emulsified.
- DMSO dimethyl sulfoxide
- IFA incomplete Freund's adjuvant
- the emulsified compound was administered into the ridge skin of the mouse at two sites at 250 ⁇ g / site. One week later, the mouse was euthanized with CO 2 gas, and then the spleen was removed and spleen cells were prepared.
- An IFN ⁇ ELISPOT assay kit was used to measure IFN ⁇ production.
- the ELISPOT plate was treated with anti-mouse IFN ⁇ antibody and blocked on RPMI 1640 medium containing 10% FBS on the day.
- the prepared spleen cells derived from HLA-A0201 transgenic mice were seeded on a blocked ELISPOT plate at 0.25 ⁇ 10 6 cells / well.
- the peptide (SEQ ID NO: 19, 43) was dissolved in DMSO to 40 mg / mL, and further diluted to 40 ⁇ g / mL in RPMI 1640 medium containing 10% FBS.
- Spleen cells derived from HLA-A0201 transgenic mice are pulsed with a diluted peptide represented by SEQ ID NO: 19 or SEQ ID NO: 43 (final concentration 10 ⁇ g / mL) and cultured for 17 hours at 37 ° C. under 5% CO 2. Thus, peptide re-stimulation in vitro was added. After incubation, the supernatant was removed and the ELISPOT plate was developed according to the attached protocol. The number of spots developed was measured by an ImmunoSpot Analyzer (manufactured by C.T.L.).
- FIG. 12 The results of IFN ⁇ ELISPOT assay using HLA-A0201 transgenic mice are shown in FIG. In FIG. 12, the vertical axis represents the number of cells that reacted in the number of seeded cells.
- the black bars and hatched bars in FIG. 12 show the results of culturing spleen cells derived from HLA-A0201 transgenic mice while pulsing each peptide represented by SEQ ID NOs: 19 and 43, and the white bars are cultured without pulse. Results are shown. That is, the difference between the value of the black bar or the hatched bar and the white bar indicates the number of peptide-specific CTLs, which are represented by SEQ ID NOs: 19 and 43 in the mouse body by administration of the compound represented by formula (5).
- the compound represented by the formula (5) can induce CTL specific to each peptide represented by SEQ ID NOs: 19 and 43.
- the compound represented by the formula (5) is actually produced into the peptides represented by SEQ ID NOs: 19 and 43 after undergoing disulfide bond cleavage and appropriate trimming by ERAP-1 in the mouse body. It was strongly suggested. That is, the compound represented by formula (5), which is an example of the compound of the present invention, is a conjugate in which two different peptides are complexed via a disulfide bond as shown in formula (1). It became clear that this is a cancer antigen peptide conjugate vaccine that can actually induce two different CTLs in vivo.
- Test Example 15 Trimming test of N-terminal amino acid by ERAP1 The trimming efficiency of N-terminal amino acid of peptides synthesized in Examples 123 to 125, 130, 134 to 135 and 138 was evaluated using ERAP1.
- 50 ⁇ l of ERAP1 (50 ⁇ g / ml) pH 8.0 20 mM Tris • HCl—100 mM NaCl buffer (Tris • HCl buffer) solution was added to 142 ⁇ l of Tris • HCl buffer.
- 2.0 ⁇ l of 10 mM peptide DMSO solution and 6.0 ⁇ L of DMSO were added to the above ERAP1 solution, mixed well, and then allowed to stand at 30 ° C.
- Test Example 16 Trimming test of N-terminal amino acid by ERAP1 The trimming efficiency of the N-terminal amino acid of the peptide synthesized in Example 140 was evaluated using ERAP1.
- 50 ⁇ l of ERAP1 (50 ⁇ g / ml) pH 8.0 20 mM Tris • HCl—100 mM NaCl buffer (Tris • HCl buffer) solution was added to 142 ⁇ l of Tris • HCl buffer.
- 2.0 ⁇ l of 10 mM peptide DMSO solution and 6.0 ⁇ L of DMSO were added to the above ERAP1 solution, mixed well, and then allowed to stand at 30 ° C.
- Test Example 17 Trimming test of N-terminal amino acid by ERAP1 The trimming efficiency of the N-terminal amino acid of the peptide synthesized in Example 131 was evaluated using ERAP1. 50 ⁇ l of ERAP1 (100 ⁇ g / ml) pH 8.0 20 mM Tris ⁇ HCl—100 mM NaCl buffer (Tris ⁇ HCl buffer) solution was added to 142 ⁇ l of Tris ⁇ HCl buffer. 8.0 ⁇ l of 10 mM peptide DMSO solution was added to the above ERAP1 solution, mixed well, and then allowed to stand at 30 ° C.
- Test Example 18 Trimming test of N-terminal amino acid by ERAP1 The trimming efficiency of the N-terminal amino acid of the peptide synthesized in Example 136 was evaluated using ERAP1. 50 ⁇ l of ERAP1 (100 ⁇ g / ml) pH 8.0 20 mM Tris ⁇ HCl—100 mM NaCl buffer (Tris ⁇ HCl buffer) solution was added to 142 ⁇ l of Tris ⁇ HCl buffer. 8.0 ⁇ l of 10 mM peptide DMSO solution was added to the above ERAP1 solution, mixed well, and then allowed to stand at 30 ° C.
- the peptides represented by SEQ ID NOs: 233, 234, 237 and 238 shown in Table 56 were synthesized with reference to non-patent documents Cancer Science January 2012, Vol. 103, no. 1, 150-153..
- Step 1 Synthesis of Fmoc-Cys (Mmt) -Ala-SBn (Mmt is 4-Methoxytrityl) (Synthesis of Fmoc-C (Mmt) A-SBn) Fmoc-Cys (Mmt) -OH (4.80 g), N, N-diisopropylethylamine (2.56 mL), hexafluorophosphoric acid (benzotriazol-1-yloxy) tripyrrolidinophosphonium (4.50 g) and the known A solution of H-Ala-SBn in chloroform (20 mL) synthesized by a method (for example, Journal of Organic Chemistry, Vol. 64, No.
- Step 2 Synthesis of H-Cys (Mmt) -Ala-Cys-Gly-Leu-Tyr-Asp-Gly-Met-Glu-His-Leu-OH (Synthesis of C (Mmt) ACGLYDGMEHL) Fmoc-Cys (Mmt) -Ala-SBn (11 mg) obtained in Step 1 and H-Cys-Gly-Leu-Tyr-Asp-Gly-Met-Glu-His-Leu-OH (16 mg) synthesized in Example 25 ), N, N-diisopropylethylamine (200 ⁇ L), 3,3 ′, 3 ′′ -phosphanetryl tripropanoic acid hydrochloride (1 mg), 4-mercaptophenylacetic acid (1 mg) and 0.1 M sodium phosphate buffer (PH 7.5, 200 ⁇ L) in DMF (400 ⁇ L) was stirred at room temperature for 4 hours.
- Test Example 19 Evaluation of CTL inducibility in vivo using HLA-A0201 transgenic mice
- the compound represented by formula (I) is particularly a compound in which cancer antigen peptide A is KIFGSLAFL (SEQ ID NO: 53) and cancer antigen peptide B is aKFVAAWTLKAAa (SEQ ID NO: 102).
- KIFGSLAFL SEQ ID NO: 53
- aKFVAAWTLKAAa SEQ ID NO: 102
- HLA-DR-restricted universal cancer antigen peptide ie, helper peptide.
- the HLA-A0201 transgenic mouse is as described in Test Example 4. By using this mouse, it is possible to select a peptide capable of inducing CTL in HLA-A02 positive humans, and in addition, a helper peptide capable of inducing helper T cells by binding to human HLA-DRB1 * 0101 It is also possible to evaluate the effect of enhancing CTL induction.
- CTL against the target peptide (SEQ ID NO: 53) is induced by administration of the compound represented by the formula (16) is determined based on the spleen derived from the mouse administered with the compound represented by the formula (16). It was judged by measuring whether IFN ⁇ was produced when the cells were restimulated with the peptide (SEQ ID NO: 53). Whether or not the helper peptide (SEQ ID NO: 102) is acting in vivo is represented by the splenocytes derived from the mouse administered with the compound represented by formula (16) and SEQ ID NO: 53. Spleen cells derived from the above mice administered with the compound were judged by comparing the number of IFN ⁇ producing cells when restimulated with the peptide (SEQ ID NO: 53).
- the compound represented by the formula (16) is dissolved in dimethyl sulfoxide (DMSO) at 80 mg / mL, further diluted to 5.1 mg / mL with water for injection, and then an equivalent amount of incomplete Freund's adjuvant ( IFA) and emulsified.
- DMSO dimethyl sulfoxide
- IFA incomplete Freund's adjuvant
- the emulsified compound was administered into the ridge skin of the mouse at two sites at 130 ⁇ g / site. One week later, the mouse was euthanized with CO 2 gas, and then the spleen was removed and spleen cells were prepared.
- An IFN ⁇ ELISPOT assay kit was used to measure IFN ⁇ production.
- the ELISPOT plate was treated with anti-mouse IFN ⁇ antibody and blocked on RPMI 1640 medium containing 10% FBS on the day.
- Spleen cells derived from the prepared HLA-A0201 transgenic mouse were seeded on a blocked ELISPOT plate at 0.125 ⁇ 10 6 cells / well.
- the peptide (SEQ ID NO: 53) was dissolved in DMSO to 40 mg / mL, and further diluted to 40 ⁇ g / mL in RPMI 1640 medium containing 10% FBS.
- Spleen cells derived from HLA-A0201 transgenic mice were pulsed with a diluted peptide represented by SEQ ID NO: 53 (final concentration 10 ⁇ g / mL) and cultured for 19 hours at 37 ° C. under 5% CO 2. Peptide restimulation in vitro was added. After incubation, the supernatant was removed and the ELISPOT plate was developed according to the attached protocol. The number of spots developed was measured by an ImmunoSpot Analyzer (manufactured by C.T.L.).
- FIG. 13 The results of IFN ⁇ ELISPOT assay using HLA-A0201 transgenic mice are shown in FIG.
- the vertical axis represents the number of cells reacted in the number of seeded cells
- the horizontal axis represents the compound or peptide administered to the mouse.
- the black bars in FIG. 13 show the results of culturing splenocytes derived from HLA-A0201 transgenic mice while pulsing the peptide represented by SEQ ID NO: 53, and the white bars show the results of non-pulse culture.
- the difference between the value of the black bar and the white bar indicates the number of peptide-specific CTLs, and the administration of the peptide represented by SEQ ID NO: 53 or the compound represented by formula (16) in the mouse in vivo CTL specific to the peptide represented by 53 was induced.
- the value of the white bar is not recognized. This indicates that the spleen cells of HLA-A0201 transgenic mice did not react at all when the peptide of interest was not pulsed.
- production of IFN ⁇ specific for the peptide represented by SEQ ID NO: 53 was confirmed in splenocytes derived from HLA-A0201 transgenic mice.
- the compound represented by the formula (16) can induce CTL specific for the peptide represented by SEQ ID NO: 53.
- IFN ⁇ -producing cells specific for the peptide represented by SEQ ID NO: 53 are compared with the case where the peptide represented by SEQ ID NO: 53 is administered. Most often observed is that a cell reactive to the helper peptide represented by SEQ ID NO: 102 generated from the compound represented by formula (16) was induced, and thus represented by SEQ ID NO: 53. It was assumed that the induction of CTL specific for the peptide was enhanced.
- the compound represented by the formula (16) is actually produced into the peptides represented by SEQ ID NOs: 53 and 102 after undergoing disulfide bond cleavage and appropriate trimming by ERAP-1 in the mouse body. It was strongly suggested. That is, the compound represented by the formula (16), which is an example of the compound of the present invention, is a conjugate in which two different peptides are complexed via a disulfide bond as shown in the formula (1). It has become apparent that this is a cancer antigen peptide conjugate vaccine that can actually induce CTL and helper peptide reactive cells in vivo.
- Test Example 20 Evaluation of CTL inducibility in vivo using HLA-A0201 transgenic mice
- the compound represented by formula (I) is particularly a compound in which the cancer antigen peptide A is KIFGSLAFL (SEQ ID NO: 53) and the cancer antigen peptide C is aKFVAAWTLKAAa (SEQ ID NO: 102).
- KIFGSLAFL SEQ ID NO: 53
- aKFVAAWTLKAAa SEQ ID NO: 102
- HLA-DR-restricted universal cancer antigen peptide ie, helper peptide.
- HLA-A0201 transgenic mice are as described in Test Examples 4 and 19.
- CTL against the target peptide (SEQ ID NO: 53) is induced by administration of the compound represented by the formula (17) is determined based on the spleen derived from the mouse administered with the compound represented by the formula (17). It was judged by measuring whether IFN ⁇ was produced when the cells were restimulated with the peptide (SEQ ID NO: 53). Whether or not the helper peptide (SEQ ID NO: 102) is acting in vivo is represented by the splenocyte derived from the mouse administered with the compound represented by formula (17) and SEQ ID NO: 53. Spleen cells derived from the above mice administered with the compound were judged by comparing the number of IFN ⁇ producing cells when restimulated with the peptide (SEQ ID NO: 53).
- FIG. 14 The results of IFN ⁇ ELISPOT assay using HLA-A0201 transgenic mice are shown in FIG.
- the vertical axis represents the number of cells reacted in the number of seeded cells
- the horizontal axis represents the compound or peptide administered to the mouse.
- the black bars in FIG. 14 show the results of culturing spleen cells derived from HLA-A0201 transgenic mice while pulsing the peptide represented by SEQ ID NO: 53, and the white bars show the results of non-pulse culture.
- the difference between the value of the black bar and the white bar indicates the number of peptide-specific CTLs, and the administration of the peptide represented by SEQ ID NO: 53 or the compound represented by formula (17) in the mouse in vivo CTL specific to the peptide represented by 53 was induced.
- the value of the white bar is not recognized. This indicates that the spleen cells of HLA-A0201 transgenic mice did not react at all when the peptide of interest was not pulsed.
- production of IFN ⁇ specific for the peptide represented by SEQ ID NO: 53 was confirmed in splenocytes derived from HLA-A0201 transgenic mice.
- the compound represented by the formula (17) is actually produced into the peptides represented by SEQ ID NOs: 53 and 102 after undergoing disulfide bond cleavage and appropriate trimming by ERAP-1 in the mouse body. It was strongly suggested. That is, the compound represented by the formula (17), which is an example of the compound of the present invention, is a conjugate in which two different peptides are complexed via a disulfide bond as shown in the formula (1). It has become apparent that this is a cancer antigen peptide conjugate vaccine that can actually induce CTL and helper peptide reactive cells in vivo.
- Test Example 21 Evaluation of CTL inducibility in vivo using HLA-A0201 transgenic mice
- the compound represented by formula (I) is particularly a compound in which the cancer antigen peptide A is KIFGSLAFL (SEQ ID NO: 53) and the cancer antigen peptide C is AKFVAAWTLKAAA (SEQ ID NO: 101).
- KIFGSLAFL SEQ ID NO: 53
- AKFVAAWTLKAAA SEQ ID NO: 101
- HLA-DR restricted universal cancer antigen peptide ie, helper peptide
- HLA-A0201 transgenic mice are as described in Test Examples 4 and 19.
- CTL against the target peptide (SEQ ID NO: 53) is induced by administration of the compound represented by the formula (18) is determined based on the spleen derived from the mouse administered with the compound represented by the formula (18). It was judged by measuring whether IFN ⁇ was produced when the cells were restimulated with the peptide (SEQ ID NO: 53). Whether or not the helper peptide (SEQ ID NO: 101) acts in vivo is represented by the above mouse-derived splenocytes administered with the compound represented by formula (18), and SEQ ID NO: 53. Spleen cells derived from the above mice administered with the compound were judged by comparing the number of IFN ⁇ producing cells when restimulated with the peptide (SEQ ID NO: 53).
- FIG. 15 The results of IFN ⁇ ELISPOT assay using HLA-A0201 transgenic mice are shown in FIG.
- the vertical axis represents the number of cells reacted in the number of seeded cells
- the horizontal axis represents the compound or peptide administered to the mouse.
- the black bars in FIG. 15 show the results of culturing spleen cells derived from HLA-A0201 transgenic mice while pulsing the peptide represented by SEQ ID NO: 53, and the white bars show the results of non-pulse culture.
- the difference between the values of the black bars and the white bars indicates the number of peptide-specific CTLs, and the administration of the peptide represented by SEQ ID NO: 53 or the compound represented by formula (18) in the mouse in vivo CTL specific to the peptide represented by 53 was induced.
- the value of the white bar is not recognized. This indicates that the spleen cells of HLA-A0201 transgenic mice did not react at all when the peptide of interest was not pulsed.
- production of IFN ⁇ specific for the peptide represented by SEQ ID NO: 53 was confirmed in splenocytes derived from HLA-A0201 transgenic mice.
- the compound represented by the formula (18) can induce CTL specific for the peptide represented by SEQ ID NO: 53.
- IFN ⁇ -producing cells specific for the peptide represented by SEQ ID NO: 53 are compared with the case where the peptide represented by SEQ ID NO: 53 is administered. Most often observed is that a cell reactive to the helper peptide represented by SEQ ID NO: 101 generated from the compound represented by formula (18) was induced, and thus represented by SEQ ID NO: 53. It was assumed that the induction of CTL specific for the peptide was enhanced.
- the compound represented by formula (18) is actually produced into the peptides represented by SEQ ID NOs: 53 and 101 after undergoing disulfide bond cleavage and appropriate trimming by ERAP-1 in the mouse body. It was strongly suggested. That is, the compound represented by formula (18), which is an example of the compound of the present invention, is a conjugate in which two different peptides are complexed via a disulfide bond as shown in formula (1). It has become apparent that this is a cancer antigen peptide conjugate vaccine that can actually induce CTL and helper peptide reactive cells in vivo.
- Test Example 22 Evaluation of CTL inducibility in vivo using HLA-A0201 transgenic mice
- the compound represented by formula (I) is particularly a compound in which the cancer antigen peptide A is KIFGSLAFL (SEQ ID NO: 53) and the cancer antigen peptide B is AKFVAAATLKAAA (SEQ ID NO: 101).
- KIFGSLAFL SEQ ID NO: 53
- AKFVAAWTLKAAA SEQ ID NO: 101
- HLA-DR restricted universal cancer antigen peptide ie, helper peptide
- HLA-A0201 transgenic mice are as described in Test Examples 4 and 19.
- CTL against the target peptide (SEQ ID NO: 53) is induced by administration of the compound represented by the formula (15) is determined based on the spleen derived from the mouse administered with the compound represented by the formula (15). It was judged by measuring whether IFN ⁇ was produced when the cells were restimulated with the peptide (SEQ ID NO: 53). Whether or not the helper peptide (SEQ ID NO: 101) is acting in vivo is represented by the splenocyte derived from the mouse administered with the compound represented by formula (15) and SEQ ID NO: 53. Spleen cells derived from the above mice administered with the compound were judged by comparing the number of IFN ⁇ producing cells when restimulated with the peptide (SEQ ID NO: 53).
- FIG. 16 The results of IFN ⁇ ELISPOT assay using HLA-A0201 transgenic mice are shown in FIG. In FIG. 16, the vertical axis represents the number of cells reacted in the number of seeded cells, and the horizontal axis represents the compound or peptide administered to the mouse.
- the black bars in FIG. 16 show the results of culturing spleen cells derived from HLA-A0201 transgenic mice while pulsing the peptide represented by SEQ ID NO: 53, and the white bars show the results of non-pulse culture.
- the difference between the value of the black bar and the white bar indicates the number of peptide-specific CTLs, and the administration of the peptide represented by SEQ ID NO: 53 or the compound represented by formula (15) in the mouse in vivo CTL specific to the peptide represented by 53 was induced.
- the value of the white bar is not recognized. This indicates that the spleen cells of HLA-A0201 transgenic mice did not react at all when the peptide of interest was not pulsed.
- production of IFN ⁇ specific for the peptide represented by SEQ ID NO: 53 was confirmed in splenocytes derived from HLA-A0201 transgenic mice.
- the number of IFN ⁇ -producing cells specific for the peptide represented by SEQ ID NO: 53 induced by administration of the compound represented by formula (15) in FIG. The number was similar to the number of peptide-specific IFN ⁇ -producing cells induced by administration.
- cancer antigen peptide C is AKFVAAWTLKAAA (SEQ ID NO: 101) in the formula (1). It was suggested that cancer antigen peptide B is a more preferred embodiment of the invention compared to AKFVAAWTLKAAA (SEQ ID NO: 101).
- KIFGSLAFL SEQ ID NO: 53
- GLYDGMEHL SEQ ID NO: 19
- HLA-DR restricted universal It is a cancer antigen peptide (ie, helper peptide).
- HLA-A0201 transgenic mice are as described in Test Examples 4 and 19.
- CTL against the target peptide (SEQ ID NOs: 19 and 53) is induced by administration of the compound represented by the formula (19) is derived from the above mouse administered with the compound represented by the formula (19) It was determined by measuring whether spleen cells produced IFN ⁇ when restimulated with a peptide (SEQ ID NO: 19, 53). Whether or not the helper peptide (SEQ ID NO: 102) is acting in vivo is represented by the spleen cell derived from the mouse administered with the compound represented by formula (19) and formula (4). Spleen cells derived from the above mice administered with the compound were judged by comparing the number of IFN ⁇ producing cells when restimulated with the peptide (SEQ ID NO: 19, 53).
- the compound represented by the formula (4) is dissolved in dimethyl sulfoxide (DMSO) at 80 mg / mL, further diluted to 2 mg / mL with water for injection, and then an equal amount of incomplete Freund's adjuvant (IFA). And emulsified.
- the emulsified compound was administered into the ridge skin of mice at 100 ⁇ g / site at two sites.
- an equivalent amount of incomplete Freund's adjuvant (IFA) and Emulsified by mixing is added to dissolving the compound represented by the formula (19) at 80 mg / mL with dimethyl sulfoxide (DMSO) and further diluting to 3.45 mg / mL with water for injection.
- the emulsified compound was administered into the ridge skin of the mouse at 173 ⁇ g / site at two sites.
- the amount of the compound of the formula (4) contained in the dose per mouse of the compound represented by the formula (19) is included in the dose of the compound represented by the formula (4) per mouse.
- the amount was adjusted to be equal to the amount of substance.
- the DMSO concentration contained in each emulsion was also made constant.
- An IFN ⁇ ELISPOT assay kit was used to measure IFN ⁇ production.
- the ELISPOT plate was treated with anti-mouse IFN ⁇ antibody and blocked on RPMI 1640 medium containing 10% FBS on the day.
- the prepared spleen cells derived from HLA-A0201 transgenic mice were seeded on a blocked ELISPOT plate at 0.25 ⁇ 10 6 cells / well.
- the peptide (SEQ ID NO: 19, 53) was dissolved in DMSO to 40 mg / mL, and further diluted to 40 ⁇ g / mL in RPMI 1640 medium containing 10% FBS.
- the diluted peptide (SEQ ID NO: 19, 53) was added to splenocytes derived from HLA-A0201 transgenic mice at a final concentration of 10 ⁇ g / mL.
- In vitro peptide re-stimulation was applied by culturing the splenocytes to which the peptide had been added for 18 hours at 37 ° C. under 5% CO 2 . After incubation, the supernatant was removed and the ELISPOT plate was developed according to the attached protocol. The number of spots developed was measured by an ImmunoSpot Analyzer (manufactured by C.T.L.).
- FIG. 17 The results of IFN ⁇ ELISPOT assay using HLA-A0201 transgenic mice are shown in FIG. 17 and FIG. In each figure, the vertical axis represents the number of cells that reacted in the number of seeded cells.
- the black and white bars in FIG. 17 show the results of culturing splenocytes derived from HLA-A0201 transgenic mice in the presence and absence of the peptide of interest represented by SEQ ID NO: 19, The white bars show the results of culturing spleen cells derived from HLA-A0201 transgenic mice in the presence and absence of the peptide of interest represented by SEQ ID NO: 53.
- the difference between the values of the black bars and the white bars indicates the number of each peptide-specific CTL of interest induced in the mouse body by administration of the compounds represented by the formulas (4) and (19).
- the value of the white bar is not recognized in each figure. This indicates that the splenocytes of the respective transgenic mice did not react in the absence of the target peptide.
- the number of IFN ⁇ -producing cells specific for the peptides represented by SEQ ID NOs: 19, 53 induced by administration of the compound represented by formula (19) is represented by formula (4). More than the number of peptide-specific IFN ⁇ producing cells induced by compound administration.
- the compound represented by the formula (19) is actually generated into the peptide represented by SEQ ID NOs: 19, 53 and 102 after undergoing disulfide bond cleavage and appropriate trimming by ERAP-1 in the mouse body. It was strongly suggested that That is, the compound represented by the formula (19), which is an example of the compound of the present invention, is a conjugate in which three different peptides are complexed via a disulfide bond. Actually, in vivo, CTL and helper It was revealed that this is a cancer antigen peptide conjugate vaccine capable of inducing peptide reactive cells.
- the compounds represented by are as described in Test Example 4.
- the peptides represented by SEQ ID NOs: 231 and 232 were obtained by amide bond between GLYDGMEHL (SEQ ID NO: 19) which is a cancer antigen peptide A restricted by HLA-A0201 and KIFGSLAFL (SEQ ID NO: 53) which is a cancer antigen peptide B. It is a linked long chain peptide.
- the HLA-A0201 transgenic mouse is as described in Test Example 4.
- the compound represented by formula (4) is dissolved in dimethyl sulfoxide (DMSO) at 80 mg / mL, further diluted to 10 mg / mL with water for injection, and then an equal amount of incomplete Freund's adjuvant (IFA). And emulsified.
- the emulsified compound was administered into the ridge skin of the mouse at two sites at 250 ⁇ g / site.
- the peptide represented by SEQ ID NO: 231, 232 was dissolved in dimethyl sulfoxide (DMSO) at 80 mg / mL, further diluted to 9 mg / mL with water for injection, and then with an equal amount of incomplete Freund's adjuvant (IFA). Mix and emulsify.
- the emulsified compound was administered at 225 ⁇ g / site at two sites in the ridge skin of mice.
- the mouse was euthanized with CO 2 gas, and then the spleen was removed and spleen cells were prepared.
- An IFN ⁇ ELISPOT assay kit was used to measure IFN ⁇ production.
- the ELISPOT plate was treated with anti-mouse IFN ⁇ antibody and blocked on RPMI 1640 medium containing 10% FBS on the day.
- the prepared spleen cells derived from HLA-A0201 transgenic mice were seeded on a blocked ELISPOT plate at 0.25 ⁇ 10 6 cells / well.
- the peptide (SEQ ID NO: 19, 53) was dissolved in DMSO to 40 mg / mL, and further diluted to 40 ⁇ g / mL in RPMI 1640 medium containing 10% FBS.
- the diluted peptide (SEQ ID NO: 19, 53) was added to splenocytes derived from HLA-A0201 transgenic mice at a final concentration of 10 ⁇ g / mL.
- In vitro peptide re-stimulation was applied by culturing the splenocytes to which the peptide had been added for 18 hours at 37 ° C. under 5% CO 2 . After incubation, the supernatant was removed and the ELISPOT plate was developed according to the attached protocol. The number of spots developed was measured by an ImmunoSpot Analyzer (manufactured by C.T.L.).
- the vertical axis represents the number of cells that reacted in the number of seeded cells.
- the black bars and white bars in FIG. 19 show the results of culturing splenocytes derived from HLA-A0201 transgenic mice in the presence and absence of the peptide of interest represented by SEQ ID NO: 19.
- the white bars show the results of culturing spleen cells derived from HLA-A0201 transgenic mice in the presence and absence of the peptide of interest represented by SEQ ID NO: 53.
- the difference between the values of the black bars and the white bars is specific to each peptide of interest induced in the mouse by administration of the compound represented by formula (4) and the peptide represented by SEQ ID NO: 231,232. Indicates the number of CTLs. The value of the white bar is not recognized in each figure. This indicates that the splenocytes of each transgenic mouse did not react at all in the absence of the target peptide. As a result of this test, the target peptide-specific IFN ⁇ production represented by SEQ ID NOs: 19 and 53 was confirmed in splenocytes derived from HLA-A0201 transgenic mice administered with the compound represented by formula (4). It was.
- the target peptide-specific IFN ⁇ production represented by SEQ ID NO: 19 was observed in the spleen cells derived from mice administered with the peptide represented by SEQ ID NO: 231, it was expressed by the formula (4). The number was small compared with splenocytes derived from mice administered with the compound. Spleen cells derived from mice administered with the peptide represented by SEQ ID NO: 232 confirmed the production of the target peptide-specific IFN ⁇ represented by SEQ ID NO: 19.
- the compound represented by the formula (4) of the present invention efficiently induces both the peptide-specific CTL represented by SEQ ID NO: 19 and the peptide-specific CTL represented by SEQ ID NO: 53. It became clear to get. On the other hand, the long-chain peptides represented by SEQ ID NOs: 231 and 232 could not efficiently induce both the peptide-specific CTLs represented by SEQ ID NO: 19 and SEQ ID NO: 53.
- the compounds represented by are as described in Test Example 4.
- the peptides represented by SEQ ID NOs: 233 and 234 were obtained by combining GLYDGMEHL (SEQ ID NO: 19) which is a cancer antigen peptide A restricted with HLA-A0201 and KIFGSLAFL (SEQ ID NO: 53) which is a cancer antigen peptide B with a peptide spacer.
- GLYDGMEHL SEQ ID NO: 19
- KIFGSLAFL SEQ ID NO: 53
- the HLA-A0201 transgenic mouse is as described in Test Example 4.
- the compound represented by the formula (4) is dissolved in dimethyl sulfoxide (DMSO) at 80 mg / mL, further diluted to 10 mg / mL with water for injection, and then an equivalent amount of incomplete Freund's adjuvant (IFA). And emulsified.
- the emulsified compound was administered into the ridge skin of the mouse at two sites at 250 ⁇ g / site.
- the peptide represented by SEQ ID NOs: 233 and 234 was dissolved in dimethyl sulfoxide (DMSO) at 80 mg / mL, further diluted to 10.5 mg / mL with water for injection, and then an equivalent amount of incomplete Freund's adjuvant (IFA And emulsified.
- the emulsified compound was administered at two sites at 265 ⁇ g / site in the ridge skin of mice. Thereafter, the same operation as in Comparative Example 1 was performed.
- the vertical axis represents the number of cells that reacted in the number of seeded cells.
- the black bars and white bars in FIG. 21 show the results of culturing splenocytes derived from HLA-A0201 transgenic mice in the presence and absence of the peptide of interest represented by SEQ ID NO: 19,
- the white bars show the results of culturing spleen cells derived from HLA-A0201 transgenic mice in the presence and absence of the peptide of interest represented by SEQ ID NO: 53.
- the difference between the values of the black bar and the white bar is specific to each peptide of interest induced by administration of the compound represented by the formula (4) and the peptide represented by SEQ ID NOs: 233, 234. Indicates the number of CTLs.
- the value of the white bar is not recognized in each figure. This indicates that the splenocytes of each transgenic mouse did not react at all in the absence of the target peptide.
- the target peptide-specific IFN ⁇ production represented by SEQ ID NOs: 19 and 53 was confirmed in splenocytes derived from HLA-A0201 transgenic mice administered with the compound represented by formula (4). It was.
- the target peptide-specific IFN ⁇ production represented by SEQ ID NO: 19 was also confirmed in splenocytes derived from mice administered with the peptides represented by SEQ ID NO: 233 and 234.
- the expression (4) The number was very small compared to splenocytes from mice administered with the compounds represented.
- the compound represented by the formula (4) of the present invention efficiently induces both the peptide-specific CTL represented by SEQ ID NO: 19 and the peptide-specific CTL represented by SEQ ID NO: 53. It became clear to get. On the other hand, the long-chain peptides represented by SEQ ID NOs: 233 and 234 could not efficiently induce both the peptide-specific CTLs represented by SEQ ID NO: 19 and SEQ ID NO: 53.
- An example of creating a vaccine containing two antigenic peptides is a cocktail vaccine that combines two different peptides into one formulation.
- the physical properties of the cancer antigen peptides to be mixed are a problem.
- Table 35 and Table 42 when two antigenic peptides are cocktailed, two peptides having different solubility, that is, physical properties, are combined into one preparation.
- the conjugate of the present invention is a compound in which two antigenic peptides are linked by a disulfide bond, and exhibits a single solubility, that is, a physical property.
- the conjugate of the present invention has a single physical property and has a property of responding to two antigenic peptides as shown in Test Example 4.
- the conjugate of the present invention is a compound capable of causing a response to two antigenic peptides without considering the interaction between the two antigenic peptides like a cocktail vaccine. .
- Reference Example 10 A peptide having the amino acid sequence of SEQ ID NOs: 239 to 242 was synthesized in the same manner as in Example 1. Table 57 shows the mass analysis results of each synthesized peptide. Since SEQ ID NOs: 239 to 242 are not compounds of the present invention, they are described as reference examples.
- the peptides represented by SEQ ID NOs: 241 and 242 shown in Table 57 were synthesized with reference to non-patent documents Cancer Science January 2012, Vol. 103, No. 1, and 150-153.
- Test Example 24 After filtering, evaluation of CTL inducibility in vivo using HLA-A2401 transgenic mice.
- a homodimer of SEQ ID NO: 4 formed via a disulfide bond and a compound represented by formula (5) Dissolve in water for injection to 10 mg / mL.
- the pharmacological activity of each compound is evaluated using HLA-A2402 transgenic mice (C57BL / 6CrHLA-A2402 / K b ) using CTL inducing activity as an index.
- the compound dissolved in water for injection is sterilized by filtration with a low protein binding filter (grade membrane filter for the purpose of sterilization of injections), then incomplete Freund's adjuvant And emulsified.
- the emulsified compound is administered into the ridge skin of HLA-A2402 transgenic mice.
- the mouse is euthanized with CO 2 gas, and then the spleen or groin lymph node is removed to prepare spleen cells or lymph node cells.
- IFN ⁇ ELISPOT assay kit is used for the measurement of IFN ⁇ production.
- ELISPOT plates are treated with anti-mouse IFN ⁇ antibody and blocked on RPMI 1640 medium containing 10% FBS on the day.
- the prepared mouse-derived cells are seeded on a blocked ELISPOT plate.
- the peptide (SEQ ID NO: 4) is dissolved in DMSO to 40 mg / mL, and further diluted to 40 ⁇ g / mL in RPMI 1640 medium containing 10% FBS.
- the diluted peptide (SEQ ID NO: 4) is added to spleen cells or lymph node cells derived from HLA-A2402 transgenic mice at a final concentration of 10 ⁇ g / mL.
- In vitro peptide re-stimulation is applied by culturing cells supplemented with peptides for 16-20 hours at 37 ° C., 5% CO 2 . After removing the supernatant after incubation, the ELISPOT plate is developed according to the attached protocol. The number of spots developed is measured with an ImmunoSpot Analyzer (manufactured by C.T.L.).
- the compound of the present invention is useful as an active ingredient of a cancer vaccine that induces CTL efficiently and is easy to produce.
- This application is based on Japanese Patent Application Nos. 2013-074411 (filing date: March 29, 2013) and 2013-158386 (filing date: July 31, 2013) filed in Japan. All contents are intended to be included herein.
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Abstract
Description
具体的には、上記課題の解決策を検討する過程で、異なる2つの癌抗原ペプチドを複合化する際に、必要なシステインを、MHCクラスIへの抗原提示に影響することなく、N-末端またはC-末端へ任意の位置へ導入する方法を着想した。更なる検討の結果、癌抗原ペプチドのN末端にシステインを含む0~5個のアミノ酸を導入したペプチドや、当該ペプチドのシステインを介したジスルフィド結合を含むコンジュゲート体を創製した。そして、当該ペプチドやコンジュゲート体が、in vitroおよび/またはin vivoでERAP1によるトリミングを受け易く、その結果癌抗原ペプチドを生成することを、本発明者らが初めて確認し、本発明が完成された。
製造容易で、汎用性に優れ、かつCTLを効率良く誘導する新規な多価抗原ペプチド提示型ペプチド癌ワクチンの開発が望まれていたところ、本発明者らが発明したコンジュゲート体により、CTLが効率よく誘導され、物理化学的性質に優れ、製造が容易で、製造の管理も容易で、汎用性に優れた多価抗原ペプチド提示型ペプチド癌ワクチンを開発することが可能となった。
癌抗原ペプチドAは、7~30残基のアミノ酸からなるMHCクラスI拘束性癌抗原ペプチドを表し、癌抗原ペプチドAのN末端アミノ酸のアミノ基が式(1)中のYaと結合し、癌抗原ペプチドAのC末端アミノ酸のカルボニル基が式(1)中の水酸基と結合し、
R1は、水素原子、式(2):
癌抗原ペプチドBは、癌抗原ペプチドAとは配列が異なり且つ7~30残基のアミノ酸からなるMHCクラスI拘束性癌抗原ペプチドまたは7~30残基のアミノ酸からなるMHCクラスII拘束性癌抗原ペプチドを表し、癌抗原ペプチドBのN末端アミノ酸のアミノ基が式(2)中のYbと結合し、癌抗原ペプチドBのC末端アミノ酸のカルボニル基が式(2)中の水酸基と結合し、
式(2)中のチオエーテル基が、式(1)中のチオエーテル基と結合する。)
で表される基、式(3):
癌抗原ペプチドCは、7~30残基のアミノ酸からなるMHCクラスII拘束性癌抗原ペプチドを表し、癌抗原ペプチドCのC末端アミノ酸のカルボニル基が式(3)中のXcと結合し、癌抗原ペプチドCのN末端アミノ酸のアミノ基が式(3)中の水素原子と結合し、
式(3)中のチオエーテル基が、式(1)中のチオエーテル基と結合する。)
で表される基、または癌抗原ペプチドDを表し、
癌抗原ペプチドDは、1つのシステイン残基を含む7~30残基のアミノ酸からなるMHCクラスI拘束性癌抗原ペプチドまたは1つのシステイン残基を含む7~30残基のアミノ酸からなるMHCクラスII拘束性癌抗原ペプチドを表し、癌抗原ペプチドDのシステイン残基のチオエーテル基が式(1)中のチオエーテル基と結合する。
但し、R1が水素原子である場合、式(1)で表される化合物の配列は癌抗原タンパク質の部分配列と同一ではない。]
で表される化合物、またはその薬学上許容される塩;
NYKHCFPEI (配列番号:3)、
EYLQLVFGI (配列番号:11)、
FLWGPRALV (配列番号:13)、
GLYDGMEHL (配列番号:19)、
SLLMWITQCFL (配列番号:26)、
QLSLLMWIT (配列番号:27)、
AAGIGILTV (配列番号:29)、
LIYRRRLMK (配列番号:33)、
YMDGTMSQV (配列番号:40)、
AFLPWHRLF (配列番号:41)、
VLQELNVTV (配列番号:43)、
YLSGANLNL (配列番号:50)、
KIFGSLAFL (配列番号:53)、
AYIDFEMKI (配列番号:66)、
AYACNTSTL (配列番号:83)、
KWFPSCQFLL (配列番号:84)および
GYDQIMPKK (配列番号:85)
の中から選ばれるいずれかのアミノ酸配列を含むペプチドであるか、または配列番号:3、11、13、19、26、27、29、33、40、41、43、50、53、66、83、84および85の中から選ばれるいずれかのアミノ酸配列中にアミノ酸残基の改変を含有する改変アミノ酸配列を含み且つCTL誘導活性を有するペプチドである、項1~8のいずれか一項に記載の化合物、またはその薬学上許容される塩;
GLYDGMEHL (配列番号:19)、
VLQELNVTV (配列番号:43)および
KIFGSLAFL (配列番号:53)
の中から選ばれるいずれかのアミノ酸配列からなるペプチドである、項1~9のいずれか一項に記載の化合物、またはその薬学上許容される塩;
CAGLYDGMEHL (配列番号:89)、
CLGLYDGMEHL (配列番号:90)、
CMGLYDGMEHL (配列番号:91)、
CAVLQELNVTV (配列番号:92)、
CLVLQELNVTV (配列番号:93)、
CMVLQELNVTV (配列番号:94)、
CAKIFGSLAFL (配列番号:95)、
CLKIFGSLAFL (配列番号:96)および
CMKIFGSLAFL (配列番号:97)
の中から選ばれるいずれかのアミノ酸配列からなるペプチドである、項1~3および6~11のいずれか一項に記載の化合物、またはその薬学上許容される塩;
CGLYDGMEHL (配列番号:98)、
CVLQELNVTV (配列番号:99)および
CKIFGSLAFL (配列番号:100)
の中から選ばれるいずれかのアミノ酸配列からなるペプチドである、項1~11のいずれか一項に記載の化合物、またはその薬学上許容される塩;
NYKHCFPEI (配列番号:3)、
EYLQLVFGI (配列番号:11)、
FLWGPRALV (配列番号:13)、
GLYDGMEHL (配列番号:19)、
SLLMWITQCFL (配列番号:26)、
QLSLLMWIT (配列番号:27)、
AAGIGILTV (配列番号:29)、
LIYRRRLMK (配列番号:33)、
YMDGTMSQV (配列番号:40)、
AFLPWHRLF (配列番号:41)、
VLQELNVTV (配列番号:43)、
YLSGANLNL (配列番号:50)、
KIFGSLAFL (配列番号:53)、
AYIDFEMKI (配列番号:66)、
AYACNTSTL (配列番号:83)、
KWFPSCQFLL (配列番号:84)および
GYDQIMPKK (配列番号:85)
の中から選ばれるいずれかのアミノ酸配列を含むペプチドであるか、または配列番号:3、11、13、19、26、27、29、33、40、41、43、50、53、66、83、84および85の中から選ばれるいずれかのアミノ酸配列中にアミノ酸残基の改変を含有する改変アミノ酸配列を含み且つCTL誘導活性を有するペプチドである、項1~10および14~22のいずれか一項に記載の化合物、またはその薬学上許容される塩;
GLYDGMEHL (配列番号:19)、
VLQELNVTV (配列番号:43)および
KIFGSLAFL (配列番号:53)
の中から選ばれるいずれかのアミノ酸配列からなるペプチドである、項1~10および14~23のいずれか一項に記載の化合物、またはその薬学上許容される塩;
で表される化合物、または式(5):
で表される化合物である、項1~10および14~24のいずれか一項に記載の化合物、またはその薬学上許容される塩;
癌抗原ペプチドEは、7~30残基のアミノ酸からなるMHCクラスII拘束性癌抗原ペプチドを表し、癌抗原ペプチドEのC末端アミノ酸のカルボニル基が式(20)中のXeと結合し、癌抗原ペプチドEのN末端アミノ酸のアミノ基が式(20)中の水素原子と結合する。)
中のチオエーテル基と結合している、項1~10、14~15のいずれか一項に記載の化合物、またはその薬学上許容される塩;
NYKHCFPEI (配列番号:3)、
EYLQLVFGI (配列番号:11)、
FLWGPRALV (配列番号:13)、
GLYDGMEHL (配列番号:19)、
SLLMWITQCFL (配列番号:26)、
QLSLLMWIT (配列番号:27)、
AAGIGILTV (配列番号:29)、
LIYRRRLMK (配列番号:33)、
YMDGTMSQV (配列番号:40)、
AFLPWHRLF (配列番号:41)、
VLQELNVTV (配列番号:43)、
YLSGANLNL (配列番号:50)、
KIFGSLAFL (配列番号:53)、
AYIDFEMKI (配列番号:66)、
AYACNTSTL (配列番号:83)、
KWFPSCQFLL (配列番号:84)および
GYDQIMPKK (配列番号:85)
の中から選ばれるいずれかのアミノ酸配列を含むペプチドであるか、または配列番号:3、11、13、19、26、27、29、33、40、41、43、50、53、66、83、84および85の中から選ばれるいずれかのアミノ酸配列中にアミノ酸残基の改変を含有する改変アミノ酸配列を含み且つCTL誘導活性を有するペプチドである、項26~31のいずれか一項に記載の化合物、またはその薬学上許容される塩;
GLYDGMEHL (配列番号:19)、
VLQELNVTV (配列番号:43)および
KIFGSLAFL (配列番号:53)
の中から選ばれるいずれかのアミノ酸配列からなるペプチドである、項26~32のいずれか一項に記載の化合物、またはその薬学上許容される塩;
AKFVAAWTLKAAA (配列番号:101)および
aKFVAAWTLKAAa (配列番号:102)
の中から選ばれるいずれかのアミノ酸配列を含むペプチドであるか、または配列番号:101および102の中から選ばれるいずれかのアミノ酸配列中にアミノ酸残基の改変を含有する改変アミノ酸配列を含み且つヘルパーT細胞誘導活性を有するペプチドである、項26~40のいずれか一項に記載の化合物、またはその薬学上許容される塩;
AKFVAAWTLKAAA (配列番号:101)および
aKFVAAWTLKAAa (配列番号:102)
の中から選ばれるいずれかのアミノ酸配列からなるペプチドである、項26~41のいずれか一項に記載の化合物、またはその薬学上許容される塩;
で表される化合物、または式(21):
で表される化合物である、項1~10、14~15および26~42のいずれか一項に記載の化合物、またはその薬学上許容される塩;
AKFVAAWTLKAAA (配列番号:101)および
aKFVAAWTLKAAa (配列番号:102)
の中から選ばれるいずれかのアミノ酸配列を含むペプチドであるか、または配列番号:101および102の中から選ばれるいずれかのアミノ酸配列中にアミノ酸残基の改変を含有する改変アミノ酸配列を含み且つヘルパーT細胞誘導活性を有するペプチドである、項1~10、14~18および44~46のいずれか一項に記載の化合物、またはその薬学上許容される塩;
AKFVAAWTLKAAA (配列番号:101)および
aKFVAAWTLKAAa (配列番号:102)
の中から選ばれるいずれかのアミノ酸配列からなるペプチドである、項1~10、14~18および44~47のいずれか一項に記載の化合物、またはその薬学上許容される塩;
で表される化合物、式(7):
で表される化合物、式(15):
で表される化合物、または式(16):
で表される化合物である、項1~10、14~18および44~48のいずれか一項に記載の化合物、またはその薬学上許容される塩;
AKFVAAWTLKAAA (配列番号:101)および
aKFVAAWTLKAAa (配列番号:102)
の中から選ばれるいずれかのアミノ酸配列を含むペプチドであるか、または配列番号:101および102の中から選ばれるいずれかのアミノ酸配列中にアミノ酸残基の改変を含有する改変アミノ酸配列を含み且つヘルパーT細胞誘導活性を有するペプチドである、項1~10および50~57のいずれか一項に記載の化合物、またはその薬学上許容される塩;
AKFVAAWTLKAAA (配列番号:101)および
aKFVAAWTLKAAa (配列番号:102)
の中から選ばれるいずれかのアミノ酸配列からなるペプチドである、項1~10および50~58のいずれか一項に記載の化合物、またはその薬学上許容される塩;
で表される化合物、式(9):
で表される化合物、式(18):
で表される化合物、または式(17):
で表される化合物である、項1~10および50~59のいずれか一項に記載の化合物、またはその薬学上許容される塩;
VYGFVRACL (配列番号:87)および
SLLMWITQC (配列番号:88)
の中から選ばれるいずれかのアミノ酸配列を含むペプチドであるか、または配列番号:87および88の中から選ばれるいずれかのアミノ酸配列中にアミノ酸残基の改変を含有する改変アミノ酸配列を含み且つCTL誘導活性を有するペプチドである、項1~10および61~65のいずれか一項に記載の化合物、またはその薬学上許容される塩;
VYGFVRACL (配列番号:87)および
SLLMWITQC (配列番号:88)
の中から選ばれるいずれかのアミノ酸配列からなるペプチドである、項1~10および61~66のいずれか一項に記載の化合物、またはその薬学上許容される塩;
で表される化合物または式(11):
で表される化合物である、項1~10および61~67のいずれか一項に記載の化合物、またはその薬学上許容される塩;
aK-Cha-VAAWTLKAAa-Ahx-C (配列番号:103)
のアミノ酸配列を含むペプチドであるか、または配列番号:103のアミノ酸配列中にアミノ酸残基の改変を含有する改変アミノ酸配列を含み且つヘルパーT細胞誘導活性を有するペプチドである、項1~10、61および69~71のいずれか一項に記載の化合物、またはその薬学上許容される塩;
aK-Cha-VAAWTLKAAa-Ahx-C (配列番号:103)
のアミノ酸配列からなるペプチドである、項1~10、61および69~72のいずれか一項に記載の化合物、またはその薬学上許容される塩;
で表される化合物である、項1~10、61および69~73のいずれか一項に記載の化合物、またはその薬学上許容される塩;
AADHRQLQLSISSCLQQL (配列番号:104)、
RNGYRALMDKSLHVGTQCALTRR (配列番号:105)、
KKLQCVQLHVISM (配列番号:106)および
GSYVSRLLGICL (配列番号:107)
の中から選ばれるいずれかのアミノ酸配列を含むペプチドであるか、または配列番号:104、105、106および107の中から選ばれるいずれかのアミノ酸配列中にアミノ酸残基の改変を含有する改変アミノ酸配列を含み且つヘルパーT細胞誘導活性を有するペプチドである、項1~10、61、69~70および75のいずれか一項に記載の化合物、またはその薬学上許容される塩;
AADHRQLQLSISSCLQQL (配列番号:104)、
RNGYRALMDKSLHVGTQCALTRR (配列番号:105)、
KKLQCVQLHVISM (配列番号:106)および
GSYVSRLLGICL (配列番号:107)
の中から選ばれるいずれかのアミノ酸配列からなるペプチドである、項1~10、61、69~70および75~76のいずれか一項に記載の化合物、またはその薬学上許容される塩;
(1)Fmoc-C(Mmt)A-SBnおよび1つのシステイン残基がN末端に結合している癌抗原ペプチドBを用いて、C(Mmt)AのC末端アミノ酸のカルボニル基と癌抗原ペプチドBのN末端に結合しているN末端アミノ酸のアミノ基が結合したペプチドを合成する工程であって、癌抗原ペプチドBが7~15残基のアミノ酸からなるMHCクラスI拘束性癌抗原ペプチドを表す工程、
(2)前記工程(1)で得られたペプチドおよびSPy基で保護された1つのシステイン残基がN末端に結合している癌抗原ペプチドAを用いて、前記工程(1)で得られたペプチド中の癌抗原ペプチドBのN末端に結合しているシステイン残基のチオエーテル基と癌抗原ペプチドAのN末端に結合しているシステイン残基のチオエーテル基が結合したペプチドを合成する工程であって、癌抗原ペプチドAは、7~30残基のアミノ酸からなるMHCクラスI拘束性癌抗原ペプチドを表す工程、および
(3)前記工程(2)で得られたペプチドおよびSPy基で保護された1つのシステイン残基がC末端に結合している癌抗原ペプチドEを用いて、前記工程(2)で得られたペプチド中の癌抗原ペプチドAのN末端に結合しているシステイン残基のチオエーテル基と癌抗原ペプチドEのC末端に結合しているシステイン残基のチオエーテル基が結合したペプチドを合成する工程であって、癌抗原ペプチドEは、7~30残基のアミノ酸からなるMHCクラスII拘束性癌抗原ペプチドを表す工程、
に関する。
中でも、配列の異なる2つのMHCクラスI拘束性ペプチドのHLAのサブタイプに関し、A02タイプ(A-0201、A0206など)のペプチドとA24タイプ(A-2402など)のペプチドとと組み合わせ得られる本発明の化合物(コンジュゲート体)が、特に好ましい。欧米人(Caucasian)においては、HLA-A0201タイプまたはHLA-A0206タイプであるPopulationが約47%と最も多く、次いでHLA-A2402タイプが約13%であり、これらのタイプの総和は、重複(すなわち両方のタイプを有するヒトを二重に計算すること)を除くと約56%を占める(Human Immunol. 62:1009;2001)。一方日本人などにおいては、HLA-A2402であるPopulationが約60%と最も多く、次いでHLA-A0201またはHLA-A0206が約39%であり、これらのタイプの総和は、重複(すなわち両方のタイプを有するヒトを二重に計算すること)を除くと約81%を占める(www.bmdc.irc.or.jp/GF-A.htm)。従って、本発明の化合物の利点としては、具体的には、より大きなPopulationを一つの本発明の化合物でカバーするという利点、および必ずしも投与の事前に患者のHLAのサブタイプを選別することが必須ではなくなり得るという利点などが挙げられる。
また、本発明の化合物により、物理化学的性質や安定性に優れ、製造が容易な癌ワクチンの有効成分を提供することが可能となった。これにより、癌ワクチンの製剤化も容易となった。
具体的には、物理化学的性質としては、溶解度、溶液の粘度、これに伴う精製の容易さ、凍結乾燥後の取り扱い易さ、これに伴う精製の容易さなどが挙げられる。安定性として、塩置換した後の安定性、吸湿性、熱安定性、エマルション形成後の安定性などが挙げられる。さらに薬理活性としては、癌ワクチンとしての薬効、API(Active Pharmaceutical Ingredient、医薬品有効成分)によって生じる違い、製剤中の添加剤との相互作用などが挙げられる。このうちAPIによって生じる違いとは、APIによる癌ワクチンとしての違いであり、具体的には、溶解度が大きく異なる2つのAPIにおいては、溶解度が小さいAPIの場合析出し易く、医薬品には必須要件であるメンブランフィルターの濾過による滅菌処理ができなくなる可能性が十分予想される。または、辛うじて溶解度が小さいAPIの濾過による滅菌処理を行っても、濾液に含まれるAPIの量が大きく減少し、癌ワクチンとして必須のCTL誘導能も著しく低下すると考えられる。よって、溶解度が小さいAPIでは、生産上の効率が著しく低下するというデメリットが容易に予想される。
AlaまたはA:アラニン残基
a:D-アラニン残基
ArgまたはR:アルギニン残基
AsnまたはN:アスパラギン残基
AspまたはD:アスパラギン酸残基
CysまたはC:システイン残基
GlnまたはQ:グルタミン残基
GluまたはE:グルタミン酸残基
GlyまたはG:グリシン残基
HisまたはH:ヒスチジン残基
IleまたはI:イソロイシン残基
LeuまたはL:ロイシン残基
LysまたはK:リジン残基
MetまたはM:メチオニン残基
PheまたはF:フェニルアラニン残基
ProまたはP:プロリン残基
SerまたはS:セリン残基
ThrまたはT:スレオニン残基
TrpまたはW:トリプトファン残基
TyrまたはY:チロシン残基
ValまたはV:バリン残基
Abu:2-アミノ酪酸残基(α-アミノ酪酸残基とも言う)
Orn:オルニチン残基
Cit:シトルリン残基
Cha:シクロヘキシルアラニン残基(Cyclohexylalanine残基)
Ahx:2-アミノヘキサン酸残基(2-Aminohexanoic acid残基)
本発明の化合物において、たとえば式(4)~(12)で表される化合物において、その部分構造にあたるペプチドについても、特に断りの無い限り、N末端アミノ酸のアミノ酸残基のアミノ基は水素原子と結合し、C末端アミノ酸のアミノ酸残基のカルボニル基は水酸基と結合している。
当該和の整数として、好ましくは0~2が挙げられ、より好ましくは0~1が挙げられ、最も好ましくは0が挙げられる。すなわち、XaおよびYaとしては、共に単結合である場合が最も好ましい。
当該和の整数が2である場合としては、Xaが2残基のアミノ酸からなるペプチドの二価基であり且つYaが単結合である場合、XaおよびYaが独立して1残基のアミノ酸からなるペプチドの二価基である場合、またはXaが単結合であり且つYaが2残基のアミノ酸からなるペプチドの二価基である場合が挙げられる。
当該和の整数が1である場合としては、Xaが1残基のアミノ酸からなるペプチドの二価基であり且つYaが単結合である場合、またはXaが単結合であり且つYaが1残基のアミノ酸からなるペプチドの二価基である場合が挙げられる。このうち好ましくは、Xaが単結合であり且つYaがアラニン残基、ロイシン残基またはメチオニン残基である場合、あるいははXaがアラニン残基、グリシン残基、セリン残基またはチロシン残基であり且つYaが単結合である場合が挙げられる。
HLAの各サブタイプについて、多型(対立遺伝子)が知られている。HLA-Aの多型としては、HLA-A1、HLA-A0201、HLA-A24などの27種以上が挙げられ、HLA-Bの多型としては、HLA-B7、HLA-B40、HLA-B4403などの59種以上が挙げられ、HLA-Cwの多型としては、HLA-Cw0301、HLA-Cw0401、HLA-Cw0602などの10種以上が挙げられる。これら多型の中、好ましくは、HLA-A0201やHLA-A24が挙げられる。
但し、本発明における「癌抗原ペプチド」には、ヒトWT1タンパク質(Cell,60:509,1990、GenBank Acc.No.A38080)は含まれない。すなわち、本発明の化合物においては、癌抗原ペプチドA、癌抗原ペプチドB、癌抗原ペプチドC、または/および癌抗原ペプチドDが、WT1タンパク質由来の癌抗原ペプチドではない。癌抗原ペプチドA、癌抗原ペプチドB、癌抗原ペプチドCまたは癌抗原ペプチドDがWT1タンパク質由来の癌抗原ペプチドではないことがより好ましく、癌抗原ペプチドA、癌抗原ペプチドB、癌抗原ペプチドCおよび癌抗原ペプチドDが、WT1タンパク質由来の癌抗原ペプチドではないことがさらに好ましい。
具体的には、前記の項1に示した式(1)で表される化合物において、癌抗原ペプチドAが癌抗原タンパク質のアミノ酸配列において連続する7~30残基のアミノ酸からなるMHCクラスI拘束性癌抗原ペプチドであり且つWT1タンパク質由来の癌抗原ペプチドとは異なり、R1が式(2)で表される基である場合、癌抗原タンパク質のアミノ酸配列において連続する7~30残基のアミノ酸からなるMHCクラスIまたはMHCクラスII拘束性癌抗原ペプチドであり且つWT1タンパク質由来の癌抗原ペプチドとは異なり、R1が式(3)で表される基である場合、癌抗原ペプチドCが癌抗原タンパク質のアミノ酸配列において連続する7~30残基のアミノ酸からなるMHCクラスII拘束性癌抗原ペプチドであり且つWT1タンパク質由来の癌抗原ペプチドとは異なり、またR1が癌抗原ペプチドDである場合、癌抗原ペプチドDが癌抗原タンパク質のアミノ酸配列において連続する7~30残基のアミノ酸からなるMHCクラスIまたはMHCクラスII拘束性癌抗原ペプチドであり且つWT1タンパク質由来の癌抗原ペプチドとは異なることが好ましい。
表1中の配列番号:8のペプチドと配列番号:9のペプチドは、同じアミノ酸配列からなる同一のペプチドである。当該ペプチドは、HLA-Cw3拘束性癌抗原ペプチドであり、またHLA-Cw16拘束性癌抗原ペプチドでもある。
NYKHCFPEI (配列番号:3)、
EYLQLVFGI (配列番号:11)、
FLWGPRALV (配列番号:13)、
GLYDGMEHL (配列番号:19)、
SLLMWITQCFL (配列番号:26)、
QLSLLMWIT (配列番号:27)、
AAGIGILTV (配列番号:29)、
LIYRRRLMK (配列番号:33)、
YMDGTMSQV (配列番号:40)、
AFLPWHRLF (配列番号:41)、
VLQELNVTV (配列番号:43)、
YLSGANLNL (配列番号:50)、
KIFGSLAFL (配列番号:53)、
AYIDFEMKI (配列番号:66)、
AYACNTSTL (配列番号:83)、
KWFPSCQFLL (配列番号:84)、
GYDQIMPKK (配列番号:85)、
VYGFVRACL (配列番号:87)および
SLLMWITQC (配列番号:88)
の中から選ばれるいずれかのアミノ酸配列を含むペプチド、または配列番号:3、11、13、19、26、27、29、33、40、41、43、50、53、66、83、84、85、87および88の中から選ばれるいずれかのアミノ酸配列中にアミノ酸残基の改変を含有する改変アミノ酸配列を含み且つCTL誘導活性を有するペプチドが挙げられる。「MHCクラスI拘束性癌抗原ペプチド」として、配列番号:3、11、13、19、26、27、29、33、40、41、43、50、53、66、83、84、85、87および88の中から選ばれるいずれかのアミノ酸配列からなるペプチドがより好ましく、配列番号:19、43および53の中から選ばれるいずれかのアミノ酸配列からなるペプチドが更により好ましい。
で表される化合物すなわちペプチドである。
CAGLYDGMEHL (配列番号:89)、
CLGLYDGMEHL (配列番号:90)、
CMGLYDGMEHL (配列番号:91)、
CAVLQELNVTV (配列番号:92)、
CLVLQELNVTV (配列番号:93)、
CMVLQELNVTV (配列番号:94)、
CAKIFGSLAFL (配列番号:95)、
CLKIFGSLAFL (配列番号:96)および
CMKIFGSLAFL (配列番号:97)
の中から選ばれるいずれかのアミノ酸配列からなるペプチドが好ましく、さらに配列番号:89~91の中から選ばれるいずれかのアミノ酸配列からなるペプチドが更により好ましい。
CGLYDGMEHL (配列番号:98)、
CVLQELNVTV (配列番号:99)および
CKIFGSLAFL (配列番号:100)
の中から選ばれるいずれかのアミノ酸配列からなるペプチドも好ましく、さらに配列番号:98のアミノ酸配列からなるペプチドが更により好ましい。
当該和の整数として、好ましくは0~2が挙げられ、より好ましくは0~1が挙げられ、最も好ましくは0が挙げられる。すなわち、XbおよびYbとしては、共に単結合である場合が最も好ましい。
当該和の整数が2である場合としては、Xbが2残基のアミノ酸からなるペプチドの二価基であり且つYbが単結合である場合、XbおよびYbが独立して1残基のアミノ酸からなるペプチドの二価基である場合、またはXbが単結合であり且つYbが2残基のアミノ酸からなるペプチドの二価基である場合が挙げられる。
当該和の整数が1である場合としては、Xbが1残基のアミノ酸からなるペプチドの二価基であり且つYbが単結合である場合、またはXbが単結合であり且つYbが1残基のアミノ酸からなるペプチドの二価基である場合が挙げられる。このうち好ましくは、Xbが単結合であり且つYbがアラニン残基、ロイシン残基またはメチオニン残基である場合である場合が挙げられる。
癌抗原ペプチドAと癌抗原ペプチドBが同時に同じペプチドであることはないことから、R1が前記の式(2)で表される基である式(1)の化合物は、仮にXaおよびXbが同じで且つYaおよびYbが同じであっても、ホモダイマーではなく、ヘテロダイマーである。ホモダイマーは、同じペプチド単量体が二量体化された二量体を意味し、ヘテロダイマーは、異なるペプチド単量体が二量体化された二量体を意味する。
で表される化合物または式(5):
で表される化合物が挙げられる。
当該和の整数として、好ましくは0~2が挙げられ、より好ましくは0~1が挙げられ、最も好ましくは0が挙げられる。すなわち、XeおよびYeとしては、共に単結合である場合が最も好ましい。
当該和の整数が2である場合としては、Xeが2残基のアミノ酸からなるペプチドの二価基であり且つYeが単結合である場合、XeおよびYeが独立して1残基のアミノ酸からなるペプチドの二価基である場合、またはXeが単結合であり且つYeが2残基のアミノ酸からなるペプチドの二価基である場合が挙げられる。
当該和の整数が1である場合としては、Xeが1残基のアミノ酸からなるペプチドの二価基であり且つYeが単結合である場合、またはXeが単結合であり且つYeが1残基のアミノ酸からなるペプチドの二価基である場合が挙げられる。このうち好ましくは、Xeが単結合であり且つYeがアラニン残基、ロイシン残基またはメチオニン残基である場合が挙げられる。
AKFVAAWTLKAAA (配列番号:101)および
aKFVAAWTLKAAa (配列番号:102)
の中から選ばれるいずれかのアミノ酸配列からなるペプチドが挙げられる。
で表される化合物、または式(21):
で表される化合物が挙げられる。
当該「アミノ酸配列を含む」は、前記と同義である「アミノ酸配列中にアミノ酸残基の改変を含有する改変アミノ酸配列を含み且つヘルパーT細胞誘導活性を有するペプチド」は、「改変ヘルパーペプチド」とも呼ばれる。当該改変ヘルパーペプチドは、アミノ酸配列において、1~3個のアミノ酸が、欠失、置換および/または付加されたアミノ酸配列からなり、MHCクラスIIに結合し、ヘルパーT細胞を誘導するペプチドを意味する。付加(挿入も包含される)されるアミノ酸の数は好ましくは1~3である。欠失されるアミノ酸の数は好ましくは1~5である。改変において、付加されるアミノ酸または置換されるアミノ酸は、遺伝子によりコードされる20種類のアミノ酸以外の非天然アミノ酸であってもよい。
で表される化合物、式(7):
で表される化合物、式(15):
で表される化合物、または式(16):
で表される化合物が挙げられる。
当該和の整数として、好ましくは0~2が挙げられ、より好ましくは0~1が挙げられ、最も好ましくは0が挙げられる。すなわち、XcおよびYcとしては、共に単結合である場合が最も好ましい。
当該和の整数が2である場合としては、Xcが2残基のアミノ酸からなるペプチドの二価基であり且つYcが単結合である場合、XcおよびYcが独立して1残基のアミノ酸からなるペプチドの二価基である場合、またはXcが単結合であり且つYcが2残基のアミノ酸からなるペプチドの二価基である場合が挙げられる。
当該和の整数が1である場合としては、Xcが1残基のアミノ酸からなるペプチドの二価基であり且つYcが単結合である場合、またはXcが単結合であり且つYcが1残基のアミノ酸からなるペプチドの二価基である場合が挙げられる。このうち好ましくは、Xcが単結合であり且つYcがアラニン残基、ロイシン残基またはメチオニン残基である場合である場合が挙げられる。
本発明における「癌抗原ペプチドC」が7~30残基のアミノ酸からなるMHCクラスII拘束性癌抗原ペプチドである場合、アミノ酸残基数は、9~15が好ましく、13~15がより好ましい。癌抗原ペプチドCとしては、13~15残基のアミノ酸からなるHLA-DR拘束性のユニバーサル癌抗原ペプチドがさらに好ましい。当該「HLA-DR拘束性のユニバーサル癌抗原ペプチド」については、前記と同義である。
で表される化合物、式(9):
で表される化合物、式(18):
で表される化合物、または式(17):
で表される化合物が挙げられる。
癌抗原ペプチドDは、1つのシステイン残基を含む7~30残基のアミノ酸からなるMHCクラスI拘束性癌抗原ペプチドまたは1つのシステイン残基を含む7~30残基のアミノ酸からなるMHCクラスII拘束性癌抗原ペプチドを表す。
VYGFVRACL (配列番号:87)および
SLLMWITQC (配列番号:88)
の中から選ばれるいずれかのアミノ酸配列を含むペプチド、または配列番号:87および88の中から選ばれるいずれかのアミノ酸配列中にアミノ酸残基の改変を含有する改変アミノ酸配列を含み且つCTL誘導活性を有するペプチドが挙げられ、最も好ましくは、配列番号:87および88の中から選ばれるいずれかのアミノ酸配列からなるペプチドが挙げられる。当該「アミノ酸配列を含む」および「アミノ酸配列中にアミノ酸残基の改変を含有する改変アミノ酸配列を含み且つCTL誘導活性を有するペプチド」は、前記と同義である。
で表される化合物または式(11):
で表される化合物が挙げられる。
で表される化合物が挙げられる。
例えば、Fmoc法もしくはBoc法を用いて固相合成機で製造する方法や、Boc-アミノ酸もしくはZ-アミノ酸を液相合成法で逐次縮合させて製造する方法が挙げられる(Fmocは9-フルオレニルメトキシカルボニル基、Bocはt-ブトキシカルボニル基、Zはベンジルオキシカルボニル基をそれぞれ表わす)。
本発明の化合物を製造するための中間体において、アミノ基、カルボキシ基、メルカプト基などの官能基は、必要に応じて保護、脱保護の技術を用い、適当な保護基で保護し、また脱保護することができる。好適な保護基、保護する方法、および脱保護する方法としては、「Protective Groups in Organic Synthesis 2nd Edition (John Wiley & Sons, Inc.;1990)」などに詳細に記載されている。たとえば、メルカプト基の保護基としてはアセトアミドメチル基またはトリチル基などが挙げられる。
ジスルフィド化合物の精製方法は、文献(ペプタイド・シンセシス(Peptide Synthesis),Interscience,New York,1966;ザ・プロテインズ(The Proteins),Vol 2,Academic Press Inc.,New York,1976;ペプチド合成,丸善(株),1975;ペプチド合成の基礎と実験、丸善(株),1985;医薬品の開発 続 第14巻・ペプチド合成,広川書店,1991)などに記載されている。中でも、HPLCが好ましい。
本発明の化合物は、その物理化学的性質においても、医薬品原薬として大量製造可能な性質を有する。具体的には、溶解性が高い、溶液中安定性に優れている、または濃縮された際にゲル化しにくいなどの性質を有し、逆相HPLCなどのカラムクロマトグラフィーによる精製工程で、大量スケールにおいても容易に高純度の化合物を原薬として製造することができる。
本発明の化合物は、癌免疫療法におけるCTL誘導剤の有効成分として、癌ワクチンの有効成分として、また医薬組成物の有効成分として有用である。すなわち本発明の化合物は、本明細書の実施例に示すとおり、優れた免疫原性を有し、優れたCTL誘導活性を効率よく示すことができる。また、本発明の化合物によって誘導されるCTLは、驚くべきことに、癌細胞が本来保有する癌抗原タンパクの天然型部分ペプチドを認識することができる。
CTL誘導活性は、HLAテトラマー法(Int.J.Cancer:100,565-570(2002))または限界希釈法(Nat.Med.:4,321-327(1998))によりCTLの数を測定することにより確認することができる。あるいは、例えばHLA-A24拘束性のCTL誘導活性の場合、国際公開第02/47474号およびInt.J.Cancer:100,565-570(2002)に記述されたHLA-A24モデルマウスを用いることなどにより調べることができる。
また本発明の化合物は、リポソーム製剤、直径数μm のビーズに結合させた粒子状の製剤、リピッドを結合させた製剤などにして投与することもできる。
さらに本発明の化合物(コンジュゲート体)は、MHCクラスII拘束性ペプチド(すなわちヘルパーペプチド)と共に投与することができる。共に投与する方法としては、コンジュゲート体とヘルパーペプチドを個別に投与することも可能であるが、一つの医薬組成物の中にコンジュゲート体とヘルパーペプチドを含むカクテル製剤(カクテル剤、カクテル)がより好ましい。本カクテル製剤は、MHCクラスI拘束性ペプチド(すなわちキラーペプチド)を生じ得るコンジュゲート体およびMHCクラスII拘束性ペプチド(すなわちヘルパーペプチド)を含むものである。よって、癌免疫療法における癌ワクチンとして、ヘルパーペプチドを含有した本カクテル製剤を投与することによって、CTLを含む他のT cellの機能亢進に重要であるヘルパーT細胞(helper T cell)の活性化も可能となり、コンジュゲート体の機能・薬効(細胞性免疫能など)を向上させることができる。
MHCクラスII拘束性ペプチド(すなわちヘルパーペプチド)については、本願明細書中に記載したとおりである。本カクテル製剤は、細胞性免疫能などの癌ワクチンとしての薬効が向上されたものであることを、一例として本願明細書の実施例や試験例として示したように、確認できた。
投与方法としては、皮内投与、皮下投与、筋肉内投与、静脈内投与、経皮投与などが挙げられる。CTLを効率良く誘導する皮内投与や皮下投与が好ましい。投与回数および投与間隔は、治療または予防目的の疾患、患者の個体差により適宜調整することができるが、通常複数回であり、数日ないし数月に1回投与するのが好ましい。
このような本発明の化合物を有効成分とする医薬組成物を癌抗原タンパク陽性の患者に投与することにより、癌を治療または予防するための方法を提供することができる。
以下のアミノ酸配列:
CKIFGSLAFL (配列番号:100)
からなるペプチドの合成
Fmoc-Leu-Alko-樹脂(Alkoはp-アルコキシベンジルアルコール)8.25g(渡辺化学製;0.77mmol/g)を1.0Lガラス製反応容器内に入れ、30%Pip(ピペリジン)/DMF(ジメチルホルムアミド)150mL溶液で処理(10分×1回、5分×2回:合計450mL)し、Fmoc基を除去した(工程1)。DMFおよびジエチルエーテルで樹脂を洗浄した後、Fmoc-Phe-OH 6.2g(5当量)、HBTU(O-ベンゾトリアゾール-1-イル-N,N,N’,N’-テトラメチルウロニウムヘキサフルオロホスファート)6.04g(5当量)およびHOBT(1-ヒドロキシベンゾトリアゾール)2.46g(5当量)を加え、次いでDMF 150mlとDIEA(N,N-ジイソプロピルエチルアミン)5.52ml(5当量)を加えて3時間室温撹拌を行った(工程2)。DMFで樹脂を2回洗浄し、Fmoc-Phe-Leu-Alko樹脂を合成した。
Fmoc-Ala-OH 5.14g、Fmoc-Leu-OH 5.69g、Fmoc-Ser(tBu)-OH 6.18g、Fmoc-Gly-OH 4.97g、Fmoc-Phe-OH 6.63g、Fmoc-Ile-OH 5.98g、Fmoc-Lys(Boc)-OH 7.83g。
但しFmoc-Ser(tBu)-OHについては工程2を3回繰り返して行った後、得られた樹脂をDMFで洗浄後、25%Ac2O(無水酢酸)を用いて、未反応のアミノ基をキャッピングした(15分×2回)。最後に、DMF洗浄操作を実施し、Fmoc-Lys(Boc)-Ile-Phe-Gly-Ser(tBu)-Leu-Ala-Phe-Leu-Alko-Resin 14.14gを得た。
上記操作によって得られた保護ペプチド樹脂Fmoc-Lys(Boc)-Ile-Phe-Gly-Ser(tBu)-Leu-Ala-Phe-Leu-Alko-Resin 503mgを25mlガラス製反応容器に入れ国産化学社製ロータリーシェーカーN-500で振とうしながら、工程1の脱保護操作を行い、H-Lys(Boc)-Ile-Phe-Gly-Ser(tBu)-Leu-Ala-Phe-Leu-Alko-Resinとした。Fmoc-Cys(tBu)-OH 340.4mgとHBTU 248.2mg、HOBT 92.9mgをDMF 10mlに溶解した溶液を加え、更にDIEA 0.2mlを加えて 3時間室温で振とうし、工程2のカップリング反応を行った。DMF 10mlで4回洗浄した後に、30%Pip/DMF 10mlで処理(10分×1回及び5分×2回)してFmoc基を切断した。TFAカクテル(2.5%テトライソプロピルシラン/2.5%ドデカンチオール/2.5%H2O/92.5%TFA溶液)100mlを加え、室温で2.0時間攪拌した。その後、ジエチルエーテルを加え、グラスフィルターで濾過を行い、TFAカクテルとジエチルエーテルを濾液として除いた。濾上物をジエチルエーテルで洗浄し、粗ペプチド(CKIFGSLAFL(配列番号:100))269.5mgを得た。
得られた粗ペプチド269.5mgを、1液=H2O/0.1%TFAと2液=CH3CN/0.1%TFAの溶出液で、2液=CH3CN/0.1%TFAの濃度を24.3%で平衡化しているDaiso社製 Daiso-Pak ODS 30 I.D.x250mmのカラムをセットしたHPLC(Shimadzu製;LC8AD型)にチャージした。220nmのUVでモニターしながら2液の濃度44.3%まで80分間で上昇させ、目的ペプチドを含む分画を集めた。得られた溶液を凍結乾燥することによって目的とする精製品49mgを得た。
ポンプ:Shimadzu製;LC-8A型
カラム:ODS Daiso-Pak ODS 30 I.D. cmφ×cmL
溶出液1:H2O/0.1%TFA
溶出液2:CH3CN/0.1%TFA
流速:20ml/min
カラム温度:40℃
検出:UV220nm
質量分析:LC-ESI/MS m/z =1098.9 [M+1]+(理論値=1098.4)
実施例1と同様の方法で、配列番号:95~97、108~123のアミノ酸配列からなる各ペプチドを合成した。表16~17に、合成した各ペプチドの質量分析結果を示した。
表16~17の何れのペプチドも、式(1)において、R1が水素原子であり、Xaが単結合であり、Yaが1残基のアミノ酸からなるペプチドの二価基であり、且つ癌抗原ペプチドAが癌抗原タンパクHER2/neuの部分ペプチドであるHER2/neu369-377ペプチド(KIFGSLAFL)(配列番号:53)である、本発明の化合物である。
実施例1と同様の方法で、配列番号:86のアミノ酸配列からなるペプチドを合成した。表18に、合成したペプチドの質量分析結果を示した。
配列番号:86のペプチドは、前記のとおり、本発明の化合物ではないことから、参考例1として記した。
実施例1と同様の方法で、配列番号:92~94、99のアミノ酸配列からなる各ペプチドを合成した。表19に、合成した各ペプチドの質量分析結果を示した。
表19の何れのペプチドも、式(1)において、R1が水素原子であり、Xaが単結合であり、Yaが単結合または1残基のアミノ酸からなるペプチドの二価基であり、且つ癌抗原ペプチドAが癌抗原タンパクProteinase-3の部分ペプチドであるProteinase-3169-177ペプチド(VLQELNVTV)(配列番号:43)である本発明の化合物である。
実施例1と同様の方法で、配列番号:98、89~91のアミノ酸配列からなる各ペプチドを合成した。表20に、合成した各ペプチドの質量分析結果を示した。
表20の何れのペプチドも、式(1)において、R1が水素原子であり、Xaが単結合であり、Yaが単結合または1残基のアミノ酸からなるペプチドの二価基であり、且つ癌抗原ペプチドAが癌抗原タンパクMAGE-A10の部分ペプチドであるMAGE-A10254-262ペプチド(GLYDGMEHL)(配列番号:19)である本発明の化合物である。
実施例1~20にて合成したペプチドのN末端アミノ酸のトリミング効率をERAP1(PLoS One November 2008, vol.3, Issue 11, e3658)を用いて評価した。50μlのERAP1(50ng/ml)pH8.0 20mM Tris・HCl- 100mM NaCl バッファー(Tris・HClバッファー)溶液を142μlのTris・HClバッファーに加えた。2.5mMのペプチド水溶液8.0μlを上述のERAP1溶液に加えて、良く混和した後に室温で静置した。1.0時間後に50μlUFLC(分析条件は以下に示す。)に打ち込み、目的とするペプチドのAUCを求めた。トリミングによって得られるペプチドを別に化学的に合成し、酵素の無い同様の条件で分析して得られたAUCを基にして、トリミングによって得られたペプチド〔HER2/neu369-377ペプチド(KIFGSLAFL)(配列番号:53)〕の生成率を求め、表21に示した。
分析条件
ポンプ:Shimadzu社製 UFLC
カラム:Shim-pack XR-ODS 3.0mmi.d.x75mm
溶液:0.05% TFA H2O(A)― 0.05% TFA CH3CN(B)
オーブン温度:40℃
流速:1.0ml/min
検出波長:λ=220nm
グラジエント:0.0minから5.0minでB液濃度を10%から60%まで上昇
実施例1~2、11~12、21~28にて合成した各ペプチドについて、ERAP1によるN末端アミノ酸のトリミングの時間経過による変化を評価した。20μlのERAP1(0.5mg/ml)Tris・HClバッファー溶液を172μlのTris・HClバッファーに加えた。10mMの各ペプチドのDMSO溶液8.0μlを上述のERAP1溶液に加えて、良く混和した後に室温で静置した。1.0、2.0、4.0および8.0時間後に50μlのサンプルを150μlのMeOHに加えて反応を停止し、25μlをUFLC(分析条件は以下に示す。)に打ち込み、目的とするペプチドのAUCを求めた。トリミングによって得られるペプチドを別に化学的に合成し、酵素の無い同様の条件で分析して得られたAUCを基にして、トリミングによって得られたペプチドの生成率を求め、表22に示した。
分析条件
ポンプ:Shimadzu社製 UFLC
カラム:Shim-pack XR-ODS 3.0mmi.d.x75mm
溶液:0.05% TFA H2O(A)― 0.05% TFA CH3CN(B)
オーブン温度:40℃
流速:1.0ml/min
検出波長:λ=220nm
グラジエント: 0.0minから5.0minでB液濃度を1.0%から70%まで上昇
目的とするペプチド:
ERAP1によるN末端アミノ酸のトリミングにより得られるペプチドは、
配列番号:100および95~97の各ペプチドの場合、HER2/neu369-377ペプチド(KIFGSLAFL)(配列番号:53)であり、
配列番号:99および92~94の各ペプチドの場合、Proteinase-3169-177ペプチド(VLQELNVTV)(配列番号:43)であり、また
配列番号:98および89~91のペプチドの場合、MAGE-A10254-262ペプチド(GLYDGMEHL)(配列番号:19)である。
表23に示すシステイン(Cys)を延長した各ペプチド(Cys延長ペプチド)のCTL誘導能を、HLA-A0201遺伝子導入マウスを用いたin vivo CTL誘導試験によって評価した。
Cysを延長した各ペプチド(配列番号:100、99または98)の投与により、がん細胞に内因性に提示されるペプチド(配列番号:53、43または19)に対するCTLが誘導されるか否かは、Cysを延長したペプチド(配列番号:100、99または98)を投与した上記マウス由来の脾細胞をペプチド(配列番号:53、43または19)で再刺激を行った場合にIFNγを産生するか測定することで判断した。表24に示したがん細胞に内因性に提示されるペプチド(配列番号:53、43または19)は、本試験例においては、Cys非延長ペプチドともいう。
IFNγ ELISPOT assayの結果は図5~7に示す。各図において、縦軸は播種細胞数中に反応した細胞数を示している。何れの図でも非パルスの場合の結果を示す白棒の値は、殆ど認められていない。このことはペプチド非再刺激ではそれぞれの遺伝子導入マウスの脾細胞は全く反応しなかったことを示している。即ち、色棒と白棒の値の差がin vitro再刺激時に利用したペプチドに特異的なCTLの数を示しており、各ペプチドの投与によってマウス生体内で誘導されたことを示す。ここで、色棒とは、Cys非延長ペプチドでパルスした場合の結果を示す黒棒、またはCys延長ペプチドでパルスした場合の結果を示す灰色棒を意味する。図5は配列番号:53あるいは100で示されるペプチドを投与した結果を、図6は配列番号:43あるいは99で示されるペプチドを投与した結果を、図7は配列番号:19あるいは98で示されるペプチドを投与した結果を示す。これより、HLA-A0201遺伝子導入マウス由来の脾細胞において、in vivo投与したペプチドのCysの延長の有無に関らずCys非延長ペプチドあるいはCys延長ペプチドによるin vitroの刺激に対して同程度のIFNγ産生が確認され、Cys延長ペプチド(配列番号:100、99および98)がin vivoにおいてCTL誘導能を持つことが判明した。即ち、Cys延長ペプチドの投与(配列番号:100、99または98)により、Cys非延長ペプチド(配列番号:53、43または19)を認識できるCTLを誘導し得ることが判明した。このことは、Cys延長ペプチドはがん細胞に内因性に提示されるペプチド(Cys非延長ペプチド)を認識するCTLを誘導できることを示しており、マウス生体内でCys延長ペプチドがERAP1による適切なトリミングを受けて、Cys非延長ペプチドへ実際に生成されることが強く示唆された。
式(4)
で表される化合物の合成
〔すなわち式(13):
実施例25で得たH-Cys-Gly-Leu-Tyr-Asp-Gly-Met-Glu-His-Leu-OH(配列番号:98)274mgの2.74mLの20%w/w酢酸水溶液に、2,2’-ビスピリジルジスルフィドのイソプロパノール溶液(0.2M)を1.20mL加えた後、室温にて30分攪拌した。反応液を逆相HPLCにて精製した。
ポンプ:Shimadzu製;LC-8A型
カラム:YMC ODS-A 3cmφ×25cmL, 10μm
溶出液1:H2O/0.1%TFA
溶出液2:CH3CN/0.1%TFA
流速:20ml/min
検出:UV220nm
2液濃度10%で平衡化させたカラムに当該反応液を注入した。その後10分間かけて、2液濃度を32%まで上昇させた後、更に2液濃度を1分間あたり0.25%の割合で上昇させた。目的物を含む画分を集め凍結乾燥を行うことで、H-Cys(Pys)-Gly-Leu-Tyr-Asp-Gly-Met-Glu-His-Leu-OH(すなわち式(13)で表される化合物)230mgを得た。
質量分析:LC-ESI/MS m/z =1246.7[M+H]1+ (理論値=1246.5)
〔すなわち式(4)
で表される化合物の合成〕
工程1で得たH-Cys(Pys)-Gly-Leu-Tyr-Asp-Gly-Met-Glu-His-Leu-OH(すなわち式(13)で表される化合物)27mgと実施例1で得たH-Cys-Lys-Ile-Phe-Gly-Ser-Leu-Ala-Phe-Leu-OH(配列番号:100)23mgとを混合し、20%v/v酢酸水を2mL加え、室温にて30分間攪拌した。反応液を逆相HPLCにて精製した。
ポンプ:Shimadzu製;LC-8A型
カラム:YMC ODS-A 3cmφ×25cmL, 10μm
溶出液1:H2O/0.1%TFA
溶出液2:CH3CN/0.1%TFA
流速:20ml/min
検出:UV220nm
2液濃度10%で平衡化させたカラムに当該反応液を注入した。その後2液濃度を10分間かけて34%まで上昇させた後、更に1分間あたり0.25%の割合で上昇させた。目的物を含む画分を集め凍結乾燥し、目的とする式(4)で表される化合物21mgを得た。
質量分析:LC-ESI/MS m/z =1118.0[M+2H]2+ (理論値=1117.8)
式(10)
で表される化合物の合成
質量分析:LC-ESI/MS m/z =1082.2[M+2H]2+ (理論値=1082.3)
で表される化合物は、前記の式(1)に照らすと、特に癌抗原ペプチドAがGLYDGMEHL(配列番号:19)であり且つ癌抗原ペプチドBがKIFGSLAFL(配列番号:53)である化合物である。GLYDGMEHL(配列番号:19)およびKIFGSLAFL(配列番号:53)はHLA-A0201拘束性癌抗原ペプチドである。
図8において、縦軸は播種細胞数中に反応した細胞数を示す。図8の黒棒、斜線棒はHLA-A0201遺伝子導入マウス由来の脾細胞を配列番号:19、53で表される各ペプチドをパルスしながら培養した結果を示し、白棒は非パルスで培養した結果を示す。即ち、黒棒または斜線棒と白棒の値の差がペプチド特異的CTLの数を示し、式(4)で表される化合物の投与によってマウス生体内において配列番号:19、53で表される各ペプチドに特異的なCTLが誘導されたことを示す。
図8において白棒の値は認められていない。このことは目的のペプチドをパルスしない場合にはHLA-A0201遺伝子導入マウスの脾細胞は全く反応しなかったことを示している。本試験の結果、HLA-A0201遺伝子導入マウス由来の脾細胞において配列番号:19、53で表されるペプチドに特異的なIFNγの産生が確認された。
すなわち、本発明の化合物の一例である式(4)で表される化合物は、異なる2種のペプチドを式(1)に示されたようなジスルフィド結合を介して複合化されたコンジュゲート体であり、実際にin vivoにおいて異なる2種のCTLを誘導できる癌抗原ペプチドコンジュゲートワクチンであることが、明らかとなった。
で表される化合物は、前記の式(1)に照らすと、特に癌抗原ペプチドAがGLYDGMEHL(配列番号:19)であり且つ癌抗原ペプチドDがVYGFVRACL(配列番号:87)である化合物である。GLYDGMEHL(配列番号:19)はHLA-A0201拘束性癌抗原ペプチドであり、VYGFVRACL(配列番号:87)はHLA-A24拘束性癌抗原ペプチドである。
各図において、縦軸は播種細胞数中に反応した細胞数を示す。図9の黒棒、白棒はHLA-A0201遺伝子導入マウス由来の脾細胞を配列番号:19で表されるペプチドのパルスおよび非パルスしながら培養した結果を、図10の黒棒、白棒はHLA-A2402遺伝子導入マウス由来の脾細胞を配列番号:87で表されるペプチドのパルスおよび非パルスしながら培養した結果を示す。即ち、黒棒と白棒の値の差がペプチド特異的CTLの数を示し、式(10)で表される化合物の投与によってマウス生体内において配列番号:19、87で表される各ペプチドに特異的なCTLが誘導されたことを示す。
特に図9において白棒の値は認められていない。このことは目的のペプチド非存在下ではHLA-A0201遺伝子導入マウスの脾細胞は全く反応しなかったことを示している。本試験の結果、HLA-A0201遺伝子導入マウス由来の脾細胞においては配列番号:19で表されるペプチドに特異的なIFNγの産生が、HLA-A2402遺伝子導入マウス由来の脾細胞においては配列番号:87で表されるペプチド特異的なIFNγの産生が確認された。
すなわち、本発明の化合物の一例である式(10)で表される化合物は、異なる2種のペプチドを式(1)に示されたようなジスルフィド結合を介して複合化されたコンジュゲート体であり、実際にin vivoにおいて異なる2種のCTLを誘導できる癌抗原ペプチドコンジュゲートワクチンであることが、明らかとなった。
実施例1と同様の方法で、配列番号:124~142のアミノ酸配列からなる各ペプチドを合成した。表25~26に、合成した各ペプチドの質量分析結果を示した。
表25~26の何れのペプチドも、式(1)において、R1が水素原子であり、Xaが1残基のアミノ酸からなるペプチドの二価基であり、Yaが単結合であり、且つ癌抗原ペプチドAが癌抗原タンパクHER2/neuの部分ペプチドであるHER2/neu369-377ペプチド(KIFGSLAFL)(配列番号:53)である、本発明の化合物である。
実施例1と同様の方法で、配列番号:143~161のアミノ酸配列からなる各ペプチドを合成した。表27~28に、合成した各ペプチドの質量分析結果を示した。
表27~28の何れのペプチドも、式(1)において、R1が水素原子であり、Xaが1残基のアミノ酸からなるペプチドの二価基であり、Yaが単結合であり、且つ癌抗原ペプチドAが癌抗原タンパクProteinase-3の部分ペプチドであるProteinase-3169-177ペプチド(VLQELNVTV)(配列番号:43)である、本発明の化合物である。
実施例1と同様の方法で、配列番号:162~177のアミノ酸配列からなる各ペプチドを合成した。表29~30に、合成した各ペプチドの質量分析結果を示した。
表29~30の何れのペプチドも、式(1)において、R1が水素原子であり、Xaが単結合であり、Yaが1残基のアミノ酸からなるペプチドの二価基であり、且つ癌抗原ペプチドAが癌抗原タンパクMAGE-A10の部分ペプチドであるMAGE-A10254-262ペプチド(GLYDGMEHL)(配列番号:19)である、本発明の化合物である。
実施例1と同様の方法で、配列番号:178~196のアミノ酸配列からなる各ペプチドを合成した。表31~32に、合成した各ペプチドの質量分析結果を示した。
表31~32の何れのペプチドも、式(1)において、R1が水素原子であり、Xaが1残基のアミノ酸からなるペプチドの二価基であり、Yaが単結合であり、且つ癌抗原ペプチドAが癌抗原タンパクMAGE-A10の部分ペプチドであるMAGE-A10254-262ペプチド(GLYDGMEHL)(配列番号:19)である、本発明の化合物である。
実施例31~49にて合成したペプチドのN末端アミノ酸のトリミング効率をERAP1を用いて評価した。50μlのERAP1(50μg/ml)pH8.0 20mM Tris・HCl- 100mM NaCl バッファー(Tris・HClバッファー)溶液を142μlのTris・HClバッファーに加えた。10mMのペプチドDMSO溶液2.0μlおよびDMSO6.0μLを上述のERAP1溶液に加えて、良く混和した後に30℃で静置した。1.0時間後に50μlUFLC(分析条件は以下に示す。)に打ち込み、目的とするペプチドのAUCを求めた。トリミングによって得られるペプチドを別に化学的に合成し、酵素の無い同様の条件で分析して得られたAUCを基にして、トリミングによって得られたペプチド〔HER2/neu369-377ペプチド(KIFGSLAFL)(配列番号:53)〕の生成率を求め、表33に示した。
分析条件
ポンプ:Shimadzu社製 UFLC
カラム:Shim-pack XR-ODS 3.0mmi.d.x75mm
溶液:0.05% TFA H2O(A)― 0.05% TFA CH3CN(B)
オーブン温度:40℃
流速:1.0ml/min
検出波長:λ=220nm
グラジエント:0.0minから5.0minでB液濃度を10%から70%まで上昇
工程1.等張緩衝液の調製
1.75%リン酸水素二ナトリウム水溶液と5.53%クエン酸水溶液を混合し、pH6.0および7.4の緩衝液を調製した。
工程2.試験溶液の調製
被験物を1mg程度秤量し、等張緩衝液を0.5mL加えこれを試験溶液とした。調製した試験溶液は室温にて90分間振とう(振とう条件: TAITEC社製RECIPRO SHAKER SR-1N, Speed=8)後、遠心分離(15000rpm、5分間、室温)を行い、遠心分離後の上清を試験溶液とした。
工程3.標準液の調製
被験物約1mgを精秤し、0.1%TFA水/アセトニトリル=1/1にて溶解し、全量を10mLとし、これを被験物の標準液として用いた。
工程4.被験物の濃度測定
被験物の標準液及び試験溶液をHPLC(分析条件は表34に記載)にて分析を行い、標準液のピーク面積比より被験物の溶解度を算出する。
HPLC測定条件
カラム:Chemcopack Quicksorb(4.6 mmφ×150 mm, 5μm) ケムコ株式会社製
移動相:A液;0.1%TFA水、B液;0.1%TFAアセトニトリル溶液
カラム温度:室温
流速:1mL/min
検出波長:UV254nm,230nm(2波長検出)
サンプル注入量:10μL
実施例1と同様の方法で、配列番号197および198のアミノ酸配列からなる各ペプチドを合成した。表36に、合成した各ペプチドの質量分析結果を示した。
表36の何れのペプチドも、式(2)において、Xbが単結合であり、Ybが単結合であり、且つ癌抗原ペプチドBがHLA-DR拘束性のユニバーサル癌抗原ペプチド(配列番号:101および102)である、本発明の化合物である。
実施例1と同様の方法で、配列番号199および200のアミノ酸配列からなる各ペプチドを合成した。表37に、合成した各ペプチドの質量分析結果を示した。
表37の何れのペプチドも、式(3)において、Xcが単結合であり、Ycが単結合であり、且つ癌抗原ペプチドCがHLA-DR拘束性のユニバーサル癌抗原ペプチド(配列番号:101および102)である、本発明の化合物である。
式(14)
質量分析:LC-ESI/MS m/z =1208.1[M+H]+ (理論値=1208.5)
実施例29工程2記載の合成法及び実施例108記載の合成法を用いて式(5)、(11)および(15)~(17)で表される各化合物(コンジュゲート体)を合成した。表38に質量分析結果を示した。(式中、CとCの間の結合はジスルフィド結合を表す。)
実施例1と同様の方法で、配列番号:201~230のアミノ酸配列からなるペプチドを合成した。表39~41に、合成した各ペプチドの質量分析結果を示した。
表39~41の何れのペプチドも、式(1)において、R1が水素原子であり、Xaが単結合であり、Yaが単結合であり、且つ癌抗原ペプチドAが表1~9に示したMHCクラスI拘束性癌抗原ペプチドである、本発明の化合物である。
実施例109~113にて合成した式(5)、(11)および(15)~(17)で表される化合物(コンジュゲート体)について、試験例7に示した溶解度測定を行い、各溶解度を表42に示した。
実施例69~84にて合成したペプチドのN末端アミノ酸のトリミング効率をERAP1を用いて評価した。50μlのERAP1(10μg/ml)pH8.0 20mM Tris・HCl- 100mM NaCl バッファー(Tris・HClバッファー)溶液を142μlのTris・HClバッファーに加えた。10mMのペプチドDMSO溶液2.0μlおよびDMSO6.0μLを上述のERAP1溶液に加えて、良く混和した後に30℃で静置した。1.0時間後に50μlUFLC(分析条件は以下に示す。)に打ち込み、目的とするペプチドのAUCを求めた。トリミングによって得られるペプチドを別に化学的に合成し、酵素の無い同様の条件で分析して得られたAUCを基にして、トリミングによって得られたペプチド〔MAGE‐A10254‐262ペプチド(GLYDGMEHL)(配列番号:19)〕の生成率を求め、表43および44に示した。
分析条件
ポンプ:Shimadzu社製 UFLC
カラム:Kinetex 2.6u C18 100A 3.0mmi.d.x75mm
溶液:0.1% TFA H2O(A)― 0.1% TFA CH3CN(B)
オーブン温度:40℃
流速:1.0ml/min
検出波長:λ=220nm
グラジエント:0.0minから8.5minでB液濃度を10%から50%まで上昇
実施例85~103にて合成したペプチドのN末端アミノ酸のトリミング効率をERAP1を用いて評価した。50μlのERAP1(10μg/ml)pH8.0 20mM Tris・HCl- 100mM NaCl バッファー(Tris・HClバッファー)溶液を142μlのTris・HClバッファーに加えた。10mMのペプチドDMSO溶液2.0μlおよびDMSO6.0μLを上述のERAP1溶液に加えて、良く混和した後に30℃で静置した。1.0時間後に50μlUFLC(分析条件は以下に示す。)に打ち込み、目的とするペプチドのAUCを求めた。トリミングによって得られるペプチドを別に化学的に合成し、酵素の無い同様の条件で分析して得られたAUCを基にして、トリミングによって得られたペプチド〔MAGE‐A10254‐262ペプチド(GLYDGMEHL)(配列番号:19)〕の生成率を求め、表45および46に示した。
分析条件
ポンプ:Shimadzu社製 UFLC
カラム:Kinetex 2.6u C18 100A 3.0mmi.d.x75mm
溶液:0.1% TFA H2O(A)― 0.1% TFA CH3CN(B)
オーブン温度:40℃
流速:1.0ml/min
検出波長:λ=220nm
グラジエント:0.0minから8.5minでB液濃度を10%から50%まで上昇
実施例50、56、64及び67にて合成したペプチドのN末端アミノ酸のトリミング効率をERAP1を用いて評価した。50μlのERAP1(10μg/ml)pH8.0 20mM Tris・HCl- 100mM NaCl バッファー(Tris・HClバッファー)溶液を142μlのTris・HClバッファーに加えた。10mMのペプチドDMSO溶液2.0μlおよびDMSO6.0μLを上述のERAP1溶液に加えて、良く混和した後に30℃で静置した。1.0時間後に50μlUFLC(分析条件は以下に示す。)に打ち込み、目的とするペプチドのAUCを求めた。トリミングによって得られるペプチドを別に化学的に合成し、酵素の無い同様の条件で分析して得られたAUCを基にして、トリミングによって得られたペプチド〔Proteinase-3169-177ペプチド(VLQELNVTV)(配列番号:43)〕の生成率を求め、表47に示した。
分析条件
ポンプ:Shimadzu社製 UFLC
カラム:Kinetex 2.6u C18 100A 3.0mmi.d.x75mm
溶液:0.1% TFA H2O(A)― 0.1% TFA CH3CN(B)
オーブン温度:40℃
流速:1.0ml/min
検出波長:λ=220nm
グラジエント:0.0minから8.5minでB液濃度を10%から50%まで上昇
実施例114~115、118~122、126、129、132、137、139および141~143にて合成したペプチドのN末端アミノ酸のトリミング効率をERAP1を用いて評価した。50μlのERAP1(10μg/ml)pH8.0 20mM Tris・HCl- 100mM NaCl バッファー(Tris・HClバッファー)溶液を142μlのTris・HClバッファーに加えた。10mMのペプチドDMSO溶液2.0μlおよびDMSO6.0μLを上述のERAP1溶液に加えて、良く混和した後に30℃で静置した。1.0時間後に50μlUFLC(分析条件は以下に示す。)に打ち込み、目的とするペプチドのAUCを求めた。トリミングによって得られるペプチドを別に化学的に合成し、酵素の無い同様の条件で分析して得られたAUCを基にして、トリミングによって得られたペプチドの生成率を求め、表48~49に示した。
分析条件
ポンプ:Shimadzu社製 UFLC
カラム:Kinetex 2.6u C18 100A 3.0mmi.d.x75mm
溶液:0.1% TFA H2O(A)― 0.1% TFA CH3CN(B)
オーブン温度:40℃
流速:1.0ml/min
検出波長:λ=220nm
グラジエント:0.0minから8.5minでB液濃度を10%から50%まで上昇
実施例29と同様の方法で、式(7)~(9)で表される各化合物(コンジュゲート体)を合成した。表50に質量分析結果を示した。(式中、CとCの間の結合はジスルフィド結合を表す。)
で表される化合物は、前記の式(1)に照らすと、特に癌抗原ペプチドAがGLYDGMEHL(配列番号:19)であり且つ癌抗原ペプチドDがSLLMWITQC(配列番号:88)である化合物である。GLYDGMEHL(配列番号:19)およびSLLMWITQC(配列番号:88)はHLA-A0201拘束性癌抗原ペプチドである。
図11において、縦軸は播種細胞数中に反応した細胞数を示す。図11の黒棒、斜線棒はHLA-A0201遺伝子導入マウス由来の脾細胞を配列番号:19、88で表される各ペプチドをパルスしながら培養した結果を示し、白棒は非パルスで培養した結果を示す。即ち、黒棒または斜線棒と白棒の値の差がペプチド特異的CTLの数を示し、式(11)で表される化合物の投与によってマウス生体内において配列番号:19、88で表される各ペプチドに特異的なCTLが誘導されたことを示す。
図11において白棒の値は認められていない。このことは目的のペプチドをパルスしない場合にはHLA-A0201遺伝子導入マウスの脾細胞は全く反応しなかったことを示している。本試験の結果、HLA-A0201遺伝子導入マウス由来の脾細胞において配列番号:19、88で表されるペプチドに特異的なIFNγの産生が確認された。
すなわち、本発明の化合物の一例である式(11)で表される化合物は、異なる2種のペプチドを式(1)に示されたようなジスルフィド結合を介して複合化されたコンジュゲート体であり、実際にin vivoにおいて異なる2種のCTLを誘導できる癌抗原ペプチドコンジュゲートワクチンであることが、明らかとなった。
で表される化合物は、前記の式(1)に照らすと、特に癌抗原ペプチドAがGLYDGMEHL(配列番号:19)であり且つ癌抗原ペプチドAがVLQELNVTV(配列番号:43)である化合物である。GLYDGMEHL(配列番号:19)およびVLQELNVTV(配列番号:43)はHLA-A0201拘束性癌抗原ペプチドである。
図12において、縦軸は播種細胞数中に反応した細胞数を示す。図12の黒棒、斜線棒はHLA-A0201遺伝子導入マウス由来の脾細胞を配列番号:19、43で表される各ペプチドをパルスしながら培養した結果を示し、白棒は非パルスで培養した結果を示す。即ち、黒棒または斜線棒と白棒の値の差がペプチド特異的CTLの数を示し、式(5)で表される化合物の投与によってマウス生体内において配列番号:19、43で表される各ペプチドに特異的なCTLが誘導されたことを示す。
図12において白棒の値は認められていない。このことは目的のペプチドをパルスしない場合にはHLA-A0201遺伝子導入マウスの脾細胞は全く反応しなかったことを示している。本試験の結果、HLA-A0201遺伝子導入マウス由来の脾細胞において配列番号:19、43で表されるペプチドに特異的なIFNγの産生が確認された。
すなわち、本発明の化合物の一例である式(5)で表される化合物は、異なる2種のペプチドを式(1)に示されたようなジスルフィド結合を介して複合化されたコンジュゲート体であり、実際にin vivoにおいて異なる2種のCTLを誘導できる癌抗原ペプチドコンジュゲートワクチンであることが、明らかとなった。
実施例123~125、130、134~135及び138にて合成したペプチドのN末端アミノ酸のトリミング効率をERAP1を用いて評価した。50μlのERAP1(50μg/ml)pH8.0 20mM Tris・HCl- 100mM NaCl バッファー(Tris・HClバッファー)溶液を142μlのTris・HClバッファーに加えた。10mMのペプチドDMSO溶液2.0μlおよびDMSO6.0μLを上述のERAP1溶液に加えて、良く混和した後に30℃で静置した。1.0時間後に50μlUFLC(分析条件は以下に示す。)に打ち込み、目的とするペプチドのAUCを求めた。トリミングによって得られるペプチドを別に化学的に合成し、酵素の無い同様の条件で分析して得られたAUCを基にして、トリミングによって得られたペプチドの生成率を求め、表51に示した。
分析条件
ポンプ:Shimadzu社製 UFLC
カラム:Kinetex 2.6u C18 100A 3.0mmi.d.x75mm
溶液:0.1% TFA H2O(A)― 0.1% TFA CH3CN(B)
オーブン温度:40℃
流速:1.0ml/min
検出波長:λ=220nm
グラジエント:0.0minから8.5minでB液濃度を10%から50%まで上昇
実施例140にて合成したペプチドのN末端アミノ酸のトリミング効率をERAP1を用いて評価した。50μlのERAP1(50μg/ml)pH8.0 20mM Tris・HCl- 100mM NaCl バッファー(Tris・HClバッファー)溶液を142μlのTris・HClバッファーに加えた。10mMのペプチドDMSO溶液2.0μlおよびDMSO6.0μLを上述のERAP1溶液に加えて、良く混和した後に30℃で静置した。1.0時間後に50μlUFLC(分析条件は以下に示す。)に打ち込み、目的とするペプチドのAUCを求めた。トリミングによって得られるペプチドを別に化学的に合成し、酵素の無い同様の条件で分析して得られたAUCを基にして、トリミングによって得られたペプチドの生成率を求め、表52に示した。
分析条件
ポンプ:Shimadzu社製 UFLC
カラム:Kinetex 2.6u C18 100A 3.0mmi.d.x75mm
溶液:0.1% TFA H2O(A)― 0.1% TFA CH3CN(B)
オーブン温度:40℃
流速:1.0ml/min
検出波長:λ=220nm
グラジエント:0.0minから8.5minでB液濃度を1%から30%まで上昇
実施例131にて合成したペプチドのN末端アミノ酸のトリミング効率をERAP1を用いて評価した。50μlのERAP1(100μg/ml)pH8.0 20mM Tris・HCl- 100mM NaCl バッファー(Tris・HClバッファー)溶液を142μlのTris・HClバッファーに加えた。10mMのペプチドDMSO溶液8.0μlを上述のERAP1溶液に加えて、良く混和した後に30℃で静置した。1.0時間後に10μlUFLC(分析条件は以下に示す。)に打ち込み、目的とするペプチドのAUCを求めた。トリミングによって得られるペプチドを別に化学的に合成し、酵素の無い同様の条件で分析して得られたAUCを基にして、トリミングによって得られたペプチドの生成率を求め、表53に示した。
分析条件
ポンプ:Shimadzu社製 UFLC
カラム:Kinetex 2.6u C18 100A 3.0mmi.d.x75mm
溶液:0.1% TFA H2O(A)― 0.1% TFA CH3CN(B)
オーブン温度:40℃
流速:1.0ml/min
検出波長:λ=220nm
グラジエント:0.0minから8.5minでB液濃度を10%から50%まで上昇
実施例136にて合成したペプチドのN末端アミノ酸のトリミング効率をERAP1を用いて評価した。50μlのERAP1(100μg/ml)pH8.0 20mM Tris・HCl- 100mM NaCl バッファー(Tris・HClバッファー)溶液を142μlのTris・HClバッファーに加えた。10mMのペプチドDMSO溶液8.0μlを上述のERAP1溶液に加えて、良く混和した後に30℃で静置した。1.0時間後に10μlUFLC(分析条件は以下に示す。)に打ち込み、目的とするペプチドのAUCを求めた。トリミングによって得られるペプチドを別に化学的に合成し、酵素の無い同様の条件で分析して得られたAUCを基にして、トリミングによって得られたペプチドの生成率を求め、表54に示した。
分析条件
ポンプ:Shimadzu社製 UFLC
カラム:Kinetex 2.6u C18 100A 3.0mmi.d.x75mm
溶液:0.1% TFA H2O(A)― 0.1% TFA CH3CN(B)
オーブン温度:40℃
流速:1.0ml/min
検出波長:λ=220nm
グラジエント:0.0minから8.5minでB液濃度を1%から30%まで上昇
実施例29と同様の方法で、式(12)及び(18)で表される各化合物(コンジュゲート体)を合成した。表55に質量分析結果を示した。(式中、CとCの間の結合はジスルフィド結合を表す。)
実施例1と同様の方法で、配列番号:231~238のアミノ酸配列からなるペプチドを合成した。表56に、合成した各ペプチドの質量分析結果を示した。
配列番号:231~238は本発明の化合物ではないことから参考例として記載した。
式(19)
で表される化合物の合成
(Fmoc-C(Mmt)A-SBnの合成)
Fmoc-Cys(Mmt)-OH(4.80g)、N,N-ジイソプロピルエチルアミン(2.56mL)、ヘキサフルオロリン酸(ベンゾトリアゾール-1-イルオキシ)トリピロリジノホスホニウム(4.50g)及び公知の方法(例えばJournal of Organic Chemistry, Vol. 64, No. 24 8761-8769)にて合成されたH-Ala-SBnのクロロホルム(20mL)溶液を室温にて1時間撹拌した。反応液をカラムクロマトグラフィー(溶出溶媒はヘキサン/酢酸エチル)にて精製することで目的化合物であるFmoc-C(Mmt)A-SBn(2.80g)を得た。
NMR:1H NMR (CDCl3)δ 7.72 (t, J = 7.6 Hz, 2H), 7.54 (d, J = 7.2 Hz, 1H), 7.38-7.34 (m, 7H), 7.29-7.25 (m, 6H), 7.23-7.15 (m, 7H), 6.76 (d, J = 8.8 Hz, 2H), 6.15 (d, J = 8.0 Hz, 1H), 4.95 (d, J = 7.2 Hz, 1H), 4.57 (quin, J = 7.6 Hz, 1H), 4.35 (d, J = 6.8 Hz, 2H) 4.19-4.17 (m, 1H), 4.04 (s, 2H), 3.73 (s, 3H), 2.72 (dd, J = 13.2, 8.4 Hz, 1H), 2.61 (d, J = 9.6 Hz, 1H), 1.31 (d, J = 7.2 Hz, 3H).
(C(Mmt)ACGLYDGMEHLの合成)
工程1で得たFmoc-Cys(Mmt)-Ala-SBn(11mg)と実施例25で合成したH-Cys-Gly-Leu-Tyr-Asp-Gly-Met-Glu-His-Leu-OH(16mg)、N,N-ジイソプロピルエチルアミン(200μL)、3,3',3’’-フォスファネトリルトリプロパン酸塩酸塩(1mg)、4-メルカプトフェニル酢酸(1mg)及び0.1Mリン酸ナトリウム緩衝液(pH7.5、200μL)のDMF(400μL)溶液を室温にて4時間撹拌した。反応液にジエチルアミン(200μL)を加え更に15分撹拌した。反応液を逆相HPLCにて精製することで、目的化合物であるC(Mmt)ACGLYDGMEHL(6mg)を得た。
質量分析:LC-ESI/MS m/z=792.7 [M+2H]2+ (理論値=792.9)
〔すなわち式(20):
で表される化合物の合成〕
工程2で得たH-Cys(Mmt)-Ala-Cys-Gly-Leu-Tyr-Asp-Gly-Met-Glu-His-Leu-OH(19mg)と実施例108で得た(H-Cys(Pys)-Lys-Ile-Phe-Gly-Ser-Leu-Ala-Phe-Leu-OH(15mg)のDMF(1mL)溶液を室温にて2時間撹拌した。反応液を逆相HPLCにて精製することで目的化合物である(H-Cys(Mmt)-Ala-Cys-Gly-Leu-Tyr-Asp-Gly-Met-Glu-His-Leu-OH)(H-Cys-Lys-Ile-Phe-Gly-Ser-Leu-Ala-Phe-Leu-OH) disulfide bond〔すなわち式(20)で示される化合物〕を19mg得た。
質量分析:LC-ESI/MS m/z=803.3 [M-Mmt+3H]2+ (理論値=803.3)
aKFVAAWTLKAAaC(Pys)の合成
実施例107で得たaKFVAAWTLKAAaC(138mg)と2,2’-ジピリジルビスルフィド(0.2Mイソプロパノール溶液、718μL)の(20%w/w酢酸水)/(アセトニトリル)=1/1(5mL)溶液を室温にて2時間撹拌した。2,2’-ジピリジルビスルフィド(0.2Mイソプロパノール溶液、350μL)を更に加え、2時間撹拌した。反応液を逆相HPLCにて精製することで目的化合物であるaKFVAAWTLKAAaC(Pys)を34mg得た。
質量分析:LC-ESI/MS m/z= 520.5[M+3H]3+ (理論値=521.0)
工程3で得た(H-Cys(Mmt)-Ala-Cys-Gly-Leu-Tyr-Asp-Gly-Met-Glu-His-Leu-OH)(H-Cys-Lys-Ile-Phe-Gly-Ser-Leu-Ala-Phe-Leu-OH) disulfide bond〔すなわち式(20)で示される化合物〕(40mg)、工程4で得たaKFVAAWTLKAAaC(Pys)(35mg)及びトリイソプロピルシラン(30μL)のトリフルオロ酢酸(570μL)溶液を室温にて30分撹拌した。反応液を逆相HPLCにて精製することで目的化合物である式(19)で表される化合物を5mg得た。
質量分析:LC-ESI/MS m/z= 1285.8[M+3H]3+ (理論値=1286.5)
で表される化合物は、前記の式(1)に照らすと、特に癌抗原ペプチドAがKIFGSLAFL(配列番号:53)であり且つ癌抗原ペプチドBがaKFVAAWTLKAAa(配列番号:102)である化合物である。KIFGSLAFL(配列番号:53)はHLA-A0201拘束性癌抗原ペプチドであり、aKFVAAWTLKAAa(配列番号:102)はHLA-DR拘束性のユニバーサル癌抗原ペプチド(すなわちヘルパーペプチド)である。
すなわち、本発明の化合物の一例である式(16)で表される化合物は、異なる2種のペプチドを式(1)に示されたようなジスルフィド結合を介して複合化されたコンジュゲート体であり、実際にin vivoにおいてCTLおよびヘルパーペプチド反応性細胞を誘導できる癌抗原ペプチドコンジュゲートワクチンであることが、明らかとなった。
で表される化合物は、前記の式(1)に照らすと、特に癌抗原ペプチドAがKIFGSLAFL(配列番号:53)であり且つ癌抗原ペプチドCがaKFVAAWTLKAAa(配列番号:102)である化合物である。KIFGSLAFL(配列番号:53)はHLA-A0201拘束性癌抗原ペプチドであり、aKFVAAWTLKAAa(配列番号:102)はHLA-DR拘束性のユニバーサル癌抗原ペプチド(すなわちヘルパーペプチド)である。
すなわち、本発明の化合物の一例である式(17)で表される化合物は、異なる2種のペプチドを式(1)に示されたようなジスルフィド結合を介して複合化されたコンジュゲート体であり、実際にin vivoにおいてCTLおよびヘルパーペプチド反応性細胞を誘導できる癌抗原ペプチドコンジュゲートワクチンであることが、明らかとなった。
で表される化合物は、前記の式(1)に照らすと、特に癌抗原ペプチドAがKIFGSLAFL(配列番号:53)であり且つ癌抗原ペプチドCがAKFVAAWTLKAAA(配列番号:101)である化合物である。KIFGSLAFL(配列番号:53)はHLA-A0201拘束性癌抗原ペプチドであり、AKFVAAWTLKAAA(配列番号:101)はHLA-DR拘束性のユニバーサル癌抗原ペプチド(すなわちヘルパーペプチド)である。
すなわち、本発明の化合物の一例である式(18)で表される化合物は、異なる2種のペプチドを式(1)に示されたようなジスルフィド結合を介して複合化されたコンジュゲート体であり、実際にin vivoにおいてCTLおよびヘルパーペプチド反応性細胞を誘導できる癌抗原ペプチドコンジュゲートワクチンであることが、明らかとなった。
で表される化合物は、前記の式(1)に照らすと、特に癌抗原ペプチドAがKIFGSLAFL(配列番号:53)であり且つ癌抗原ペプチドBがAKFVAAWTLKAAA(配列番号:101)である化合物である。KIFGSLAFL(配列番号:53)はHLA-A0201拘束性癌抗原ペプチドであり、AKFVAAWTLKAAA(配列番号:101)はHLA-DR拘束性のユニバーサル癌抗原ペプチド(すなわちヘルパーペプチド)である。
HLA-A0201遺伝子導入マウスを用いた、in vivoでのCTL誘導能の評価
で表される化合物に含まれるKIFGSLAFL(配列番号:53)およびGLYDGMEHL(配列番号:19)はHLA-A0201拘束性癌抗原ペプチドであり、aKFVAAWTLKAAa(配列番号:102)はHLA-DR拘束性のユニバーサル癌抗原ペプチド(すなわちヘルパーペプチド)である。
各図中において白棒の値は認められていない。このことは目的のペプチド非存在下ではそれぞれの遺伝子導入マウスの脾細胞は反応しなかったことを示している。本試験の結果、式(4)および式(19)で表される化合物を投与したHLA-A0201遺伝子導入マウス由来の脾細胞において配列番号:19、53で表される目的のペプチド特異的なIFNγ産生がそれぞれ確認された。また、各図において式(19)で表される化合物の投与によって誘導された配列番号:19、53で表されるペプチドに特異的なIFNγ産生細胞の数は、式(4)で表される化合物の投与によって誘導されたペプチド特異的なIFNγ産生細胞の数より多かった。
すなわち、本発明の化合物の一例である式(19)で表される化合物は、異なる3種のペプチドをジスルフィド結合を介して複合化されたコンジュゲート体であり、実際にin vivoにおいてCTLおよびヘルパーペプチド反応性細胞を誘導できる癌抗原ペプチドコンジュゲートワクチンであることが明らかとなった。
HLA-A0201遺伝子導入マウスを用いた、in vivoでのCTL誘導能の評価
で表される化合物については、試験例4に記したとおりである。配列番号:231および232で表されるペプチドは、HLA-A0201拘束性の癌抗原ペプチドAであるGLYDGMEHL(配列番号:19)および癌抗原ペプチドBであるKIFGSLAFL(配列番号:53)をアミド結合で連結した長鎖ペプチドである。
各図中において白棒の値は認められていない。このことは目的のペプチド非存在下ではそれぞれの遺伝子導入マウスの脾細胞は全く反応しなかったことを示している。本試験の結果、式(4)で表される化合物を投与したHLA-A0201遺伝子導入マウス由来の脾細胞において配列番号:19、53で表される目的のペプチド特異的なIFNγ産生がそれぞれ確認された。一方で、配列番号:231で表されるペプチドを投与したマウス由来の脾細胞においては配列番号:19で表される目的のペプチド特異的なIFNγ産生は認められたものの、式(4)で表される化合物を投与したマウス由来の脾細胞と比較するとその数は少なかった。配列番号:232で表されるペプチドを投与したマウス由来の脾細胞においては配列番号:19で表される目的のペプチド特異的なIFNγ産生が確認された。また、配列番号:231、232で表されるペプチドを投与したマウス由来の脾細胞においては配列番号:53で表される目的のペプチド特異的なIFNγ産生は認められたものの、式(4)で表される化合物を投与したマウス由来の脾細胞と比較するとその数は非常に少なかった。
HLA-A0201遺伝子導入マウスを用いた、in vivoでのCTL誘導能の評価
で表される化合物については、試験例4に記したとおりである。配列番号:233および234で表されるペプチドは、HLA-A0201拘束性の癌抗原ペプチドAであるGLYDGMEHL(配列番号:19)および癌抗原ペプチドBであるKIFGSLAFL(配列番号:53)を、ペプチドスペーサーとして6個のグリシンを介してアミド結合で連結した長鎖ペプチドである。
各図中において白棒の値は認められていない。このことは目的のペプチド非存在下ではそれぞれの遺伝子導入マウスの脾細胞は全く反応しなかったことを示している。本試験の結果、式(4)で表される化合物を投与したHLA-A0201遺伝子導入マウス由来の脾細胞において配列番号:19、53で表される目的のペプチド特異的なIFNγ産生がそれぞれ確認された。一方で、配列番号:233、234で表されるペプチドを投与したマウス由来の脾細胞においても配列番号:19で表される目的のペプチド特異的なIFNγ産生が確認された。しかし、配列番号:233、234で表されるペプチドを投与したマウス由来の脾細胞において、配列番号:53で表される目的のペプチド特異的なIFNγ産生は認められたものの、式(4)で表される化合物を投与したマウス由来の脾細胞と比較するとその数は非常に少なかった。
実施例1と同様の方法で、配列番号:239~242のアミノ酸配列からなるペプチドを合成した。表57に、合成した各ペプチドの質量分析結果を示した。
配列番号:239~242は本発明の化合物ではないことから参考例として記載した。
フィルター濾過を経た後に、HLA-A2401遺伝子導入マウスを用いた、in vivoでのCTL誘導能の評価
ジスルフィド結合を介して形成した配列番号4のホモダイマーおよび式(5)で表される化合物を3-10mg/mLとなるように注射用水にて溶解する。各化合物の薬理活性を、CTL誘導活性を指標としてHLA-A2402遺伝子導入マウス (C57BL/6CrHLA-A2402/Kb)を利用して評価する。HLA-A2402遺伝子導入マウスに投与するにあたり、注射用水で溶解した化合物をタンパク質低結合性のフィルター(注射剤の滅菌処理を目的としたグレードのメンブランフィルター)にて濾過滅菌したのち、不完全フロイントアジュバントと混合しエマルション化させる。
エマルション化させた化合物はHLA-A2402遺伝子導入マウスの尾根部皮内に投与する。投与1週間後、マウスをCO2ガスにより安楽死させたのち脾臓あるいは鼠蹊部リンパ節を摘出し、脾細胞あるいはリンパ節細胞を調製する。IFNγ産生の測定には、IFNγ ELISPOT assay kitを用いる。細胞調製の前日に、ELISPOTプレートを抗マウスIFNγ抗体で処理し、当日に10%FBSを含むRPMI1640培地でブロッキングする。調製したマウス由来の細胞をブロッキングしたELISPOTプレートに播種する。ペプチド(配列番号:4)をDMSOで40mg/mLに溶解し、さらに10%FBSを含むRPMI1640培地で40μg/mLに希釈する。希釈したペプチド(配列番号:4)を、最終濃度10μg/mLでHLA-A2402遺伝子導入マウス由来の脾細胞あるいはリンパ節細胞に添加する。ペプチドを添加した細胞を、16-20時間、37℃、5% CO2下で培養することで、in vitroにおけるペプチド再刺激を加える。培養後に上清を除いたのち、ELISPOTプレートを、添付のプロトコールに従って発色させる。発色したスポット数は、ImmunoSpot Analyzer(C.T.L.社製)によって測定する。
Claims (19)
- 式(1):
癌抗原ペプチドAは、7~30残基のアミノ酸からなるMHCクラスI拘束性癌抗原ペプチドを表し、癌抗原ペプチドAのN末端アミノ酸のアミノ基が式(1)中のYaと結合し、癌抗原ペプチドAのC末端アミノ酸のカルボニル基が式(1)中の水酸基と結合し、
R1は、水素原子、式(2):
癌抗原ペプチドBは、癌抗原ペプチドAとは異なり且つ7~30残基のアミノ酸からなるMHCクラスI拘束性癌抗原ペプチドまたは7~30残基のアミノ酸からなるMHCクラスII拘束性癌抗原ペプチドを表し、癌抗原ペプチドBのN末端アミノ酸のアミノ基が式(2)中のYbと結合し、癌抗原ペプチドBのC末端アミノ酸のカルボニル基が式(2)中の水酸基と結合し、
式(2)中のチオエーテル基が、式(1)中のチオエーテル基と結合する。)
で表される基、式(3):
癌抗原ペプチドCは、7~30残基のアミノ酸からなるMHCクラスII拘束性癌抗原ペプチドを表し、癌抗原ペプチドCのC末端アミノ酸のカルボニル基が式(3)中のXcと結合し、癌抗原ペプチドCのN末端アミノ酸のアミノ基が式(3)中の水素原子と結合し、
式(3)中のチオエーテル基が、式(1)中のチオエーテル基と結合する。)
で表される基、または癌抗原ペプチドDを表し、
癌抗原ペプチドDは、1つのシステイン残基を含む7~30残基のアミノ酸からなるMHCクラスI拘束性癌抗原ペプチドまたは1つのシステイン残基を含む7~30残基のアミノ酸からなるMHCクラスII拘束性癌抗原ペプチドを表し、癌抗原ペプチドDのシステイン残基のチオエーテル基が式(1)中のチオエーテル基と結合する。
但し、R1が水素原子である場合、式(1)の配列は癌抗原タンパクの部分配列と同一ではない。]
で表される化合物、またはその薬学上許容される塩。 - Xaが単結合であり、Yaが単結合、アラニン残基、ロイシン残基またはメチオニン残基である、請求項1に記載の化合物、またはその薬学上許容される塩。
- Xaが単結合、アラニン残基、グリシン残基、セリン残基またはチロシン残基であり、Yaが単結合である、請求項1に記載の化合物、またはその薬学上許容される塩。
- XaおよびYaが単結合である、請求項1~3のいずれか一項に記載の化合物、またはその薬学上許容される塩。
- 癌抗原ペプチドAが、以下のアミノ酸配列:
GLYDGMEHL (配列番号:19)、
VLQELNVTV (配列番号:43)および
KIFGSLAFL (配列番号:53)
の中から選ばれるいずれかのアミノ酸配列からなるペプチドである、請求項1~4のいずれか一項に記載の化合物、またはその薬学上許容される塩。 - R1が水素原子である、請求項1~5のいずれか一項に記載の化合物、またはその薬学上許容される塩。
- R1が式(2)で表される基である、請求項1~5のいずれか一項に記載の化合物、またはその薬学上許容される塩。
- XbおよびYbが単結合である、請求項1~5および7のいずれか一項に記載の化合物、またはその薬学上許容される塩。
- 癌抗原ペプチドBが、以下のアミノ酸配列:
GLYDGMEHL (配列番号:19)、
VLQELNVTV (配列番号:43)および
KIFGSLAFL (配列番号:53)
の中から選ばれるいずれかのアミノ酸配列からなるペプチドである、請求項1~5および7~8のいずれか一項に記載の化合物、またはその薬学上許容される塩。 - 癌抗原ペプチドBが、以下のアミノ酸配列:
AKFVAAWTLKAAA (配列番号:101)および
aKFVAAWTLKAAa (配列番号:102)
の中から選ばれるいずれかのアミノ酸配列からなるペプチドである、請求項1~5および7~8のいずれか一項に記載の化合物、またはその薬学上許容される塩。 - R1が式(3)で表される基である、請求項1~5のいずれか一項に記載の化合物、またはその薬学上許容される塩。
- XcおよびYcが単結合である、請求項1~5および11のいずれか一項に記載の化合物、またはその薬学上許容される塩。
- 癌抗原ペプチドCが、以下のアミノ酸配列:
AKFVAAWTLKAAA (配列番号:101)および
aKFVAAWTLKAAa (配列番号:102)
の中から選ばれるいずれかのアミノ酸配列からなるペプチドである、請求項1~5および11~12のいずれか一項に記載の化合物、またはその薬学上許容される塩。 - R1が癌抗原ペプチドDである、請求項1~5のいずれか一項に記載の化合物、またはその薬学上許容される塩。
- 癌抗原ペプチドDが、以下のアミノ酸配列:
VYGFVRACL (配列番号:87)、および
SLLMWITQC (配列番号:88)
の中から選ばれるいずれかのアミノ酸配列からなるペプチドである、請求項1~5および14のいずれか一項に記載の化合物、またはその薬学上許容される塩。 - 癌抗原ペプチドDが、以下のアミノ酸配列:
aK-Cha-VAAWTLKAAa-Ahx-C (配列番号:103)
のアミノ酸配列からなるペプチドである、請求項1~5および14のいずれか一項に記載の化合物、またはその薬学上許容される塩。 - 癌抗原ペプチドDが、以下のアミノ酸配列:
AADHRQLQLSISSCLQQL (配列番号:104)、
RNGYRALMDKSLHVGTQCALTRR (配列番号:105)、
KKLQCVQLHVISM (配列番号:106)および
GSYVSRLLGICL (配列番号:107)
の中から選ばれるいずれかのアミノ酸配列からなるペプチドである、請求項1~5および14のいずれか一項に記載の化合物、またはその薬学上許容される塩。 - 請求項1~17のいずれか一項に記載の化合物またはその薬学上許容される塩、および薬学的に許容される担体を含有する医薬組成物。
- 癌を治療または予防するための方法であって、請求項1~17のいずれか一項に記載の化合物またはその薬学上許容される塩の治療または予防に有効な量を、それを必要としている癌抗原タンパク質陽性の癌患者に投与することからなる方法。
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US10588952B2 (en) | 2020-03-17 |
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US20200171136A1 (en) | 2020-06-04 |
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