WO2016021508A1 - Cdca1由来ペプチドおよびそれを含むワクチン - Google Patents
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- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
- C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
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- G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
Definitions
- the present invention relates to the field of biological science, more specifically to the field of cancer therapy.
- the present invention relates to a novel peptide effective as a cancer vaccine, a method for treating or preventing tumors using the peptide, or both, and a pharmaceutical composition containing the peptide.
- TTL Cytotoxic T lymphocytes
- TAA tumor-associated antigens
- MHC major histocompatibility complex
- Non-patent Document 3 HarrisHCC, J Natl). Cancer Inst 1996 Oct 16, 88 (20): 1442-55; Non-patent document 4: Butterfield LH et al., Cancer Res 1999 Jul 1, 59 (13): 3134-42; Non-patent document 5: Vissers JL et al ., ⁇ Cancer Res 1999 Nov 1, 59 (21): 5554-9; Non-patent document 6: van der Burg SH et al., J Immunol 1996 May 1, 156 (9) 3308-14; F et al., Cancer Res 1997 Oct 15, 57 (20): 4465-8; Non-patent document 8: Fujie T et al., Int J Cancer 1999 Jan 18, 80 (2): 169-72; 9: Kikuchi M et al., Int J Cancer 1999 May 5, 81 (3): 439-66; Non-patent
- Non-Patent Document 11 Belli F et al., J Clin Oncol 2002 Oct 15, 20 (20): 4169-80; 12: Coulie PG et al., Immunol Rev 2002 Oct, 188: 33-42; Non-Patent Document 13: Rosenberg SA et al., Nat Med 2004 Sep, 10 (9): 909-15). Therefore, there remains a need for the identification of novel CTL epitopes that can be used for cancer immunotherapy.
- CDCA1 cell division cycle 1 associated; NUF2, NDC80 centromere complex component (NUF2, NDC80, kinetochore complex) component: also described as Nuf2 .; reference sequence: GeneBank accession number NM_145697 (SEQ ID NO: 81) or GeneBank accession number NM_031423 (SEQ ID NO: 83)) was identified as a member of a gene co-expressed with CDC2, cyclin, topoisomerase II and other cell cycle genes (Non-Patent Document 14: Walker et al., Curr Cancer Drug Targets 2001 May; 1 (1): 73-83).
- CDCA1 has been found to be related to the centromere of Hela cells during mitosis and is considered to be a functional homologue of yeast Nuf2 (Non-patent document 15: Wigge PA et al., J Cell Biol 2001) Jan 22; (152 (2): 349-60).
- CDCA1 was identified as a gene that is up-regulated in non-small cell lung cancer by gene expression profile analysis using a genome-wide cDNA microarray containing 23,040 genes (Non-patent Document 16: Hayama et al., Cancer Res) 2006 Nov 1; 66 (21): 10339-48; Patent Document 1: WO2007 / 013480; Patent Document 2: WO2005 / 089735).
- CDCA1 expression was up-regulated in both tumors and tumor cell lines, but expression was not detected in normal organs other than testis (Non-patent Document 16; Patent Document 1). Furthermore, down-regulation of CDCA1 expression by siRNA caused cell growth suppression in lung cancer cell lines expressing CDCA1 (Non-patent Document 16; Patent Document 1-2).
- Non-patent Document 17 Harao et al., Int J Cancer. 2008: 123 (11): 2616-25; Patent Literature 3: WO2009 / 025117) and HLA -A24-restricted CTL epitope peptide (Patent Document 4: WO2009 / 153992) has been identified. These peptides are effective in cancer patients having HLA-A2 type or HLA-A24 type, but cannot be expected to be effective in cancer patients not having these HLA types.
- the present invention relates to a peptide capable of inducing cytotoxic T cells (CTL) specific to cells expressing CDCA1.
- CTL cytotoxic T cells
- APC antigen-presenting cells
- HLA human leukocyte antigens
- CTLs exhibiting specific cytotoxic activity against CDCA1-expressing cells Be guided.
- the CDCA1-derived CTL-inducing peptides that have been identified so far are HLA-A2-restricted or HLA-A24-restricted peptides.
- CTL is I can't guide you. Therefore, conventional peptides are not suitable when immunotherapy is performed in subjects who do not have these HLA.
- HLA-A11 and HLA-A33 are alleles commonly found in Asians (Sette A, Sidney J., Immunogenetics 1999, 50: 201-12), and HLA-A03 is an allele commonly found in whites. (Cao et al., Hum Immunol. 2001; 62 (9): 1009-30). HLA-A11 restricted peptides for HLA-A11 positive subjects, HLA-A33 restricted peptides for HLA-A33 positive subjects, and HLA-A03 for HLA-A03 positive subjects It is desirable to administer a restrictive peptide.
- the present invention relates to a CDCA1-derived peptide restricted to HLA-A11, HLA-A33 or HLA-A03 and having a CTL inducing ability.
- the peptide of the present invention is an epitope peptide that can induce a strong and specific immune response against cells expressing CDCA1 and HLA-A11, HLA-A33 or HLA-A03. It is proved that there is.
- CDCA1-derived peptide capable of inducing CTL in a HLA-A11, HLA-A33 or HLA-A03-restricted manner.
- These peptides can be used to induce CTL in vitro, ex vivo or in vivo, or can be used to administer to a subject for the purpose of inducing an immune response against cancer cells expressing CDCA1.
- Preferred peptides are SEQ ID NOs: 3, 5-7, 9, 10, 12-14, 17, 19, 21, 30, 35, 38-40, 45, 53, 56, 58, 27, 60, 28, 67, The amino acid sequence selected from 69 and 47, more preferably a nonapeptide or a decapeptide, and still more preferably SEQ ID NOs: 3, 5-7, 9, 10, 12-14, 17, 19, A peptide comprising an amino acid sequence selected from 21, 30, 35, 38 to 40, 45, 53, 56, 58, 27, 60, 28, 67, 69 and 47.
- the peptide of the present invention is a peptide in which one, two, or more amino acids are substituted, deleted, inserted and / or added as long as the resulting modified peptide retains the CTL-inducing ability of the original peptide Is also included.
- the present invention also provides an isolated polynucleotide encoding any one of the peptides of the present invention. These polynucleotides, like the peptides of the present invention, can be used to induce APCs capable of inducing CTLs, or are administered to a subject to induce an immune response against cancer cells that express CDCA1. be able to.
- the present invention also presents one or more peptides of the present invention, one or more polynucleotides encoding one or more peptides of the present invention, an APC of the present invention, and a peptide of the present invention.
- Compositions comprising exosomes and / or CTLs of the invention are provided.
- the composition of the present invention is preferably a pharmaceutical composition.
- the pharmaceutical composition of the present invention can be used for treatment and / or prevention of cancer and prevention of its recurrence after surgery. It can also be used to induce an immune response against cancer.
- the peptides of the invention are presented on the surface of the APC, thereby inducing CTL targeting the peptide.
- compositions for inducing CTL comprising one or more peptides of the present invention, one or more polynucleotides encoding one or more peptides of the present invention. It is a further object of the invention to provide a composition comprising APC and / or an exosome presenting a peptide of the invention.
- a method for inducing APC having the ability to induce CTL comprising contacting APC with one or more peptides of the present invention, or introducing a polynucleotide encoding any one of the peptides of the present invention into APC It is a further object of the present invention to provide a method comprising the steps of:
- the present invention also comprises a step of co-culturing a CD8-positive T cell with APC that presents a complex of the HLA antigen and the peptide of the present invention on its surface, and the CD8 positive T cell is converted to an HLA antigen and the peptide of the present invention.
- a T cell receptor (TCR) capable of binding to the peptide of the present invention presented by the HLA antigen on the cell surface,
- a method for inducing CTL comprising introducing a vector comprising a polynucleotide encoding each subunit into a CD8-positive T cell.
- a preferred HLA antigen in the present invention is HLA-A11, HLA-A33 or HLA-A03.
- the present invention provides an isolated APC that presents a complex of the HLA antigen and the peptide of the present invention on its surface.
- the invention further provides an isolated CTL that targets the peptide of the invention.
- These APCs and CTLs can be used for immunotherapy against cancers that express CDCA1.
- the cancer to be subjected to immunotherapy is a cancer of a patient having HLA-A11, HLA-A33 or HLA-A03, for example, homo or hetero. That is, the present invention provides immunotherapy for cancer expressing CDCA1 and at least one HLA antigen selected from HLA-A11, HLA-A33 and HLA-A03.
- a method for inducing an immune response against cancer in a subject comprising the peptide of the present invention or a polynucleotide encoding the peptide, the APC of the present invention, the exosome presenting the peptide of the present invention, and / or the CTL of the present invention. It is another object of the present invention to provide a method comprising the step of administering a composition comprising it to the subject. Furthermore, a method for treating and / or preventing cancer in a subject and preventing its recurrence after surgery, comprising the peptide of the present invention, a polynucleotide encoding the peptide, the APC of the present invention, and the peptide of the present invention. It is another object of the present invention to provide a method comprising the step of administering to a subject an exosome presenting and / or a CTL of the present invention.
- FIG. 1 is composed of photographs (a) to (v) showing the results of an IFN- ⁇ enzyme-linked immunospot (ELISPOT) assay in CTL induced with a peptide derived from CDCA1.
- Well number # 7 (a) using CDCA1-A11-9-123 (SEQ ID NO: 3), Well number # 8 (b) using CDCA1-A11-9-105 (SEQ ID NO: 5), CDCA1- Well number # 1 (c) using A11-9-419 (SEQ ID NO: 6), well number # 4 (d) using CDCA1-A11-9-219 (SEQ ID NO: 7), CDCA1-A11- Well number # 2 (e) using 9-439 (SEQ ID NO: 9), well number # 2 (f) using CDCA1-A11-9-343 (SEQ ID NO: 10), CDCA1-A11-9- Well number # 8 (g) using 21 (SEQ ID NO: 12), well number # 3 (h) using CDCA1-A11-9-157 (SEQ ID NO:
- CDCA1-A11-10-391 (SEQ ID NO: 33) (v) is shown as an example of typical negative data that did not show specific IFN- ⁇ production.
- “+” indicates IFN- ⁇ production for target cells pulsed with an appropriate peptide
- “ ⁇ ” indicates IFN- ⁇ production for target cells not pulsed with any peptide.
- FIG. 2 shows CDCA1-A11-9-123 (SEQ ID NO: 3) (a), CDCA1-A11-9-105 (SEQ ID NO: 5) (b), CDCA1-A11-9-419 (SEQ ID NO: 6 ) (C), CDCA1-A11-9-219 (SEQ ID NO: 7) (d), CDCA1-A11-9-439 (SEQ ID NO: 9) (e), CDCA1-A11-9-157 (SEQ ID NO: 13) (f), CDCA1-A11-9-25 (SEQ ID NO: 21) (g), CDCA1-A11-10-339 (SEQ ID NO: 30) (h) and CDCA1-A11-10-358 (SEQ ID NO: : 58) Consists of a series of line graphs (a) to (i) showing the results of IFN- ⁇ enzyme-linked immunosorbent assay (ELISA), which in turn demonstrates IFN- ⁇ production of the CTL strain stimulated in (i) The These results demonstrate that CTL lines established by
- the R / S ratio indicates the ratio of the number of cells of the CTL line that is the responder cell (Responder cells) and the number of target cells that are the stimulator cells (Stimulator cell).
- FIG. 3 shows CDCA1-A11-9-105 (SEQ ID NO: 5) (a), CDCA1-A11-9-419 (SEQ ID NO: 6) (b) and CDCA1-A11-9-219 (SEQ ID NO: 7 ) Consists of a series of line graphs (a) to (c) showing IFN- ⁇ production of CTL clones established by limiting dilution from the CTL strain stimulated in (c). These results demonstrate that CTL clones established by stimulation with each peptide show strong IFN- ⁇ production compared to controls.
- “+” indicates IFN- ⁇ production for target cells pulsed with an appropriate peptide
- ⁇ indicates IFN- ⁇ production for target cells not pulsed with any peptide.
- the R / S ratio indicates the ratio of the number of CTL clone cells that are responder cells and the number of target cells that are stimulator cells.
- FIG. 4 is a line graph showing specific CTL activity against target cells expressing both CDCA1 and HLA-A * 1101.
- COS7 cells transfected with either HLA-A * 1101 or full-length CDCA1 gene were prepared as controls.
- CTL clones established using CDCA1-A11-9-219 (SEQ ID NO: 7) showed specific CTL activity against COS7 cells transfected with both CDCA1 and HLA-A * 1101 (black) Diamond).
- FIG. 5 is composed of photographs (a) to (h) showing the results of the IFN- ⁇ ELISPOT assay in CTL induced using a peptide derived from CDCA1.
- Well number # 2 (a) using CDCA1-A33-9-43 (SEQ ID NO: 27), Well number # 1 (b) using CDCA1-A33-9-123 (SEQ ID NO: 3), CDCA1- Well number # 3 (c) using A33-9-108 (SEQ ID NO: 60), well number # 2 (d) using CDCA1-A33-9-261 (SEQ ID NO: 28), CDCA1-A33- Well number # 6 (e) using 9-105 (SEQ ID NO: 5), well number # 8 (f) using CDCA1-A33-10-10 (SEQ ID NO: 67) and CDCA1-A33-10- CTL in well # 5 (g) using 260 (SEQ ID NO: 69) showed strong IFN- ⁇ production compared to the control.
- CDCA1-A33-10-122 (SEQ ID NO: 68) (h) is shown as an example of typical negative data that did not show specific IFN- ⁇ production.
- “+” indicates IFN- ⁇ production for target cells pulsed with an appropriate peptide
- “ ⁇ ” indicates IFN- ⁇ production for target cells not pulsed with any peptide.
- FIG. 6 shows CDCA1-A33-9-43 (SEQ ID NO: 27) (a), CDCA1-A33-9-123 (SEQ ID NO: 3) (b), CDCA1-A33-10-10 (SEQ ID NO: 67). ) (C) and CDCA1-A33-10-260 (SEQ ID NO: 69) (d) A series of line graphs showing the results of an IFN- ⁇ ELISA that sequentially demonstrates IFN- ⁇ production of CTL strains stimulated with (d) Is composed of (d). These results demonstrate that CTL lines established by stimulation with each peptide show strong IFN- ⁇ production compared to controls.
- the R / S ratio indicates the ratio of the number of cells of the CTL line that is the responder cell (Responder cells) and the number of target cells that are the stimulator cells (Stimulator cell).
- FIG. 7 was established by limiting dilution from CTL lines stimulated with CDCA1-A33-9-43 (SEQ ID NO: 27) (a), and CDCA1-A33-9-123 (SEQ ID NO: 3) (b) Consists of a series of line graphs (a)-(b) showing IFN- ⁇ production of CTL clones.
- These results demonstrate that CTL clones established by stimulation with each peptide show strong IFN- ⁇ production compared to controls.
- “+” indicates IFN- ⁇ production for target cells pulsed with an appropriate peptide
- ⁇ indicates IFN- ⁇ production for target cells not pulsed with any peptide.
- the R / S ratio indicates the ratio of the number of CTL clone cells that are responder cells and the number of target cells that are stimulator cells.
- FIG. 8 is a line graph showing specific CTL activity against target cells expressing both CDCA1 and HLA-A * 3303.
- COS7 cells transfected with either HLA-A * 3303 or full length CDCA1 gene were prepared as controls.
- the CTL clone established using CDCA1-A33-9-43 (SEQ ID NO: 27) showed specific CTL activity against COS7 cells transfected with both CDCA1 and HLA-A * 3303 (black) Diamond).
- no significant specific CTL activity was shown against target cells transfected with either HLA-A * 3303 (white triangle) or CDCA1 (white circle).
- FIG. 9 is composed of photographs (a) to (d) showing the results of an IFN- ⁇ ELISPOT assay in CTL induced using a peptide derived from CDCA1.
- Well number # 3 (a) using CDCA1-A03-9-219 (SEQ ID NO: 7), well number # 1 (b) using CDCA1-A03-10-400 (SEQ ID NO: 38) and CDCA1- CTL in well number # 2 (c) using A03-10-257 (SEQ ID NO: 47) showed strong IFN- ⁇ production compared to the control.
- the squares on the wells in these photographs indicate that cells from the corresponding wells were grown to establish a CTL line.
- CDCA1-A03-9-343 (SEQ ID NO: 10) (d) is shown as an example of typical negative data that did not show specific IFN- ⁇ production.
- “+” indicates IFN- ⁇ production for target cells pulsed with an appropriate peptide
- “ ⁇ ” indicates IFN- ⁇ production for target cells not pulsed with any peptide.
- FIG. 10 shows CDCA1-A03-9-219 (SEQ ID NO: 7) (a), CDCA1-A03-10-400 (SEQ ID NO: 38) (b) and CDCA1-A03-10-257 (SEQ ID NO: 47).
- These results demonstrate that CTL lines established by stimulation with each peptide show strong IFN- ⁇ production compared to controls.
- “+” indicates IFN- ⁇ production for target cells pulsed with an appropriate peptide
- ⁇ indicates IFN- ⁇ production for target cells not pulsed with any peptide.
- the R / S ratio indicates the ratio of the number of cells of the CTL line that is the responder cell (Responder cells) and the number of target cells that are the stimulator cells (Stimulator cell).
- FIG. 11 shows CDCA1-A03-9-219 (SEQ ID NO: 7) (a), CDCA1-A03-10-400 (SEQ ID NO: 38) (b) and CDCA1-A03-10-257 (SEQ ID NO: 47).
- FIG. 12 consists of a series of line graphs (a)-(b) showing specific CTL activity against target cells expressing both CDCA1 and HLA-A * 0301.
- COS7 cells transfected with either HLA-A * 0301 or full-length CDCA1 gene were prepared as controls.
- (a) and CDCA1-A03-10-400 SEQ ID NO: 38)
- b) are CDCA1 and HLA-A * 0301 Both showed specific CTL activity against COS7 cells transfected with both (black diamonds).
- no significant specific CTL activity was shown against target cells transfected with either HLA-A * 0301 (white triangles) or CDCA1 (white circles).
- an isolated or purified peptide refers to a peptide that is substantially free of other cellular material from the cell or tissue source from which the peptide is derived, such as carbohydrates, lipids, and other contaminating proteins.
- an isolated or purified peptide refers to a peptide that is substantially free of precursor material or other chemicals.
- substantially free of cellular material includes preparations of a peptide in which the peptide is separated from the cellular components of the cell from which it was isolated or recombinantly produced.
- a peptide that is substantially free of cellular material contains less than about 30%, 20%, 10%, or 5%, 3%, 2%, or 1% (on a dry weight basis) of other cellular material, Peptide preparations are included.
- an isolated or purified peptide is substantially free of culture medium, and a peptide substantially free of culture medium is about 20% of the volume of peptide preparation, Peptide preparations containing less than 10%, or 5%, 3%, 2% or 1% (dry weight basis) are included.
- the isolated or purified peptide is substantially free of precursor material and other chemicals, and the peptide substantially free of precursor material and other chemicals is Peptide preparations containing precursor substances and other chemicals in less than about 30%, 20%, 10%, 5%, 3%, 2% or 1% (dry weight basis) of the volume of the peptide preparation Is included.
- a particular peptide preparation is an isolated or purified peptide, for example by the appearance of a single band after sodium dodecyl sulfate (SDS) -polyacrylamide gel electrophoresis and coomassie brilliant blue staining of the gel. Can be confirmed.
- SDS sodium dodecyl sulfate
- the peptides and polynucleotides of the invention are isolated or purified.
- polypeptide refers to a polymer of amino acid residues. This term applies to non-natural amino acid polymers containing one or more non-natural amino acid residues as well as natural amino acid polymers. Non-natural amino acids include amino acid analogs and amino acid mimetics.
- amino acid refers to natural amino acids, as well as amino acid analogs and amino acid mimetics that function similarly to natural amino acids.
- Natural amino acids are amino acids encoded by the genetic code and amino acids that are post-translationally modified in cells (eg, hydroxyproline, ⁇ -carboxyglutamic acid, O-phosphoserine, etc.).
- amino acid analog has the same basic chemical structure as a natural amino acid (hydrogen, carboxy group, amino group, and alpha carbon attached to the R group), but has a modified R group or modified backbone Refers to compounds such as homoserine, norleucine, methionine sulfoxide and methionine methylsulfonium.
- amino acid mimetic refers to a compound having a structure different from that of a general amino acid, but having a function similar to that of an amino acid.
- the amino acid may be either an L-amino acid or a D-amino acid, but the peptide of the present invention is preferably an L-amino acid polymer.
- polynucleotide oligonucleotide
- nucleic acid refers to a polymer of nucleotides.
- composition is intended to encompass products containing a specific amount of a specific component, and any product that results directly or indirectly from a combination of a specific amount of a specific component. Is done.
- composition refers to a product comprising an active ingredient and an inert ingredient, as well as a combination, complexation or aggregation of any two or more ingredients. From any dissociation of one or more components or from other types of reactions or interactions of one or more components is intended to encompass any product.
- the pharmaceutical compositions of the present invention encompass any composition made by admixing a compound or cell of the present invention and a pharmaceutically or physiologically acceptable carrier.
- pharmaceutically acceptable carrier or “physiologically acceptable carrier” includes liquid or solid fillers, diluents, excipients, solvents and encapsulating materials. It means a pharmaceutically or physiologically acceptable material, composition, substance, or vehicle that is not limited thereto.
- the term “cancer” refers to a cancer that overexpresses the CDCA1 gene, including bladder cancer, breast cancer, cervical cancer, cholangiocellular cancer, chronic myelogenous leukemia (CML). ), Esophageal cancer, stomach cancer, non-small cell lung cancer, lymphoma, osteosarcoma, prostate cancer, kidney cancer, small cell lung cancer, head and neck cancer, soft tissue tumor, and colon cancer, etc. It is not limited to.
- the “cancer” is a cancer that expresses CDCA1 and HLA-A11, HLA-A33 and / or HLA-A03.
- cytotoxic T lymphocyte refers to a sub-group of T lymphocytes that can recognize tumor / cancer cells, virus-infected cells) and induce the death of such cells.
- HLA-A11 refers to the HLA-A11 type, including subtypes such as HLA-A * 1101, HLA-A * 1102, HLA-A * 1103, HLA-A * 1104 and the like.
- HLA-A33 refers to the HLA-A33 type including subtypes such as HLA-A * 3303, HLA-A * 3301, and HLA-A * 3304.
- HLA-A03 refers to the HLA-A03 type, including subtypes such as HLA-A * 0301, HLA-A * 0302, and HLA-A * 0305.
- HLA-A11 HLA-A11
- MHC Major Histocompatibility Complex
- subject (or patient) HLA antigen is HLA-A33
- subject (or patient) HLA antigen is HLA-A03
- the subject or patient carries the HLA-A33 antigen gene homozygously or heterozygously as an MHC (major histocompatibility complex) class I molecule
- the HLA-A33 antigen is an HLA antigen in the subject or patient cell Expressed
- the subject or patient carries the HLA-A03 antigen gene as a MHC (major histocompatibility complex) class I molecule either homozygously or heterozygously
- the HLA-A03 antigen is subject to the subject or patient It is expressed as an HLA antigen in cells.
- treatment is of clinical benefit, eg, reduced cancer size, spread, or metastatic potential in the subject, progression of cancer.
- Treatment is considered “effective” if it results in delay, alleviation of clinical symptoms of cancer, prolongation of survival, suppression of postoperative recurrence, etc.
- “effective” means that the treatment delays or prevents the formation of cancer or prevents or alleviates the clinical symptoms of the cancer. Efficacy is determined in connection with any known method for diagnosing or treating a particular tumor type.
- prevention is used herein to describe any function that reduces the burden of mortality or morbidity from cancer. including.
- Prevention can take place at “primary, secondary, and tertiary prevention levels”. Primary prevention avoids the development of disease, whereas secondary and tertiary level prevention, in addition to preventing disease progression and the appearance of symptoms, restores function and is disease-related Including the aim of reducing the adverse effects of existing diseases by reducing the complications of Alternatively, prophylaxis can include a wide range of prophylactic treatment aimed at reducing the severity of a particular disorder, eg, reducing tumor growth and metastasis.
- the treatment and / or prevention of cancer and / or prevention of its recurrence after surgery is the inhibition of cancer cell growth, tumor regression or regression, induction of remission and development of cancer. It includes any of the following events: suppression, tumor regression, and reduction or inhibition of metastasis, suppression of postoperative recurrence of cancer, and prolonged survival.
- Effective treatment and / or prevention of cancer reduces mortality, improves the prognosis of individuals with cancer, decreases the level of tumor markers in the blood, and detectable symptoms associated with cancer To ease.
- symptom relief or amelioration constitutes effective treatment and / or prevention, including 10%, 20%, 30%, or more relief or symptom stability.
- antibody refers to immunoglobulins and fragments thereof that react specifically with the designated protein or peptide thereof.
- Antibodies can include human antibodies, primatized antibodies, chimeric antibodies, bispecific antibodies, humanized antibodies, antibodies fused with other proteins or radiolabels, and antibody fragments.
- antibody is used in a broad sense, specifically, an intact monoclonal antibody, a polyclonal antibody, a multispecific antibody formed from two or more intact antibodies (for example, a bispecific antibody). And antibody fragments as long as they exhibit the desired biological activity.
- An “antibody” may be an antibody of any class (eg, IgA, IgD, IgE, IgG, and IgM).
- HLA-A11 and HLA-A33 are alleles that are common in Asians (Sette A, Sidney J., Immunogenetics 1999, 50: 201-12), and HLA-A03 is common in whites Allyl (Cao et al., Hum Immunol. 2001; 62 (9): 1009-30). Therefore, by providing a CDCA1-derived CTL-inducing peptide that is restricted by HLA-A11, HLA-A33, or HLA-A03, many Asians or Caucasians can effectively treat cancer that expresses CDCA1. Can be provided.
- the present invention provides CDCA1-derived peptides that can induce CTLs in a HLA-A11, HLA-A33 or HLA-A03 restricted manner.
- the peptide of the present invention is a CDCA1-derived peptide capable of inducing CTL in a HLA-A11, HLA-A33 or HLA-A03-restricted manner.
- Peptides that can induce CTL in a HLA-A11-restricted manner include SEQ ID NOs: 3, 5-7, 9, 10, 12-14, 17, 19, 21, 30, 35, 38-40, 45, Peptides having an amino acid sequence selected from 53, 56 and 58 are mentioned.
- Peptides that can induce CTL in a HLA-A33-restricted manner include peptides having an amino acid sequence selected from SEQ ID NOs: 27, 3, 60, 28, 5, 67 and 69.
- Peptides capable of inducing CTL in an HLA-A03 restricted manner include peptides having an amino acid sequence selected from SEQ ID NOs: 7, 38 and 47.
- CTLs having cytotoxic activity specific to these peptides can be established by in vitro stimulation of T cells by dendritic cells (DC) pulsed with these peptides. Established CTLs show specific cytotoxic activity against target cells pulsed with each peptide.
- DC dendritic cells
- CDCA1 gene is a cancer cell such as bladder cancer, breast cancer, cervical cancer, cholangiocellular carcinoma, chronic myelogenous leukemia (CML), esophageal cancer, stomach cancer, non-small cell lung cancer, lymphoma, osteosarcoma, It is overexpressed in cancer cells such as prostate cancer, kidney cancer, small cell lung cancer, head and neck cancer, soft tissue tumor, and colon cancer, but it is not expressed in most normal organs. Is an excellent target for. Therefore, the peptide of the present invention can be suitably used for cancer immunotherapy.
- CML chronic myelogenous leukemia
- Preferred peptides are nonapeptides (peptides consisting of 9 amino acid residues) or decapeptides (peptides consisting of 10 amino acid residues), SEQ ID NOs: 3, 5-7, 9, 10, 12-14, 17, Preference is given to peptides consisting of amino acid sequences selected from among 19, 21, 30, 35, 38 to 40, 45, 53, 56, 58, 27, 60, 28, 67, 69 and 47.
- the peptide having the amino acid sequence set forth in SEQ ID NO: 7 is suitable for induction of CTLs that show specific cytotoxic activity against cells expressing HLA-A11 and CDCA1, and is HLA-A11 positive patient Can be suitably used for cancer immunotherapy.
- the peptide having the amino acid sequence set forth in SEQ ID NO: 27 is suitable for inducing CTLs showing specific cytotoxic activity against cells expressing HLA-A33 and CDCA1, and is HLA-A33 positive It can be suitably used for immunotherapy of cancer in patients.
- a peptide having an amino acid sequence selected from SEQ ID NOs: 7 and 38 is suitable for inducing CTLs having specific cytotoxic activity against cells expressing HLA-A03 and CDCA1. It can be suitably used for cancer immunotherapy in HLA-A03 positive patients.
- the peptide of the present invention is a peptide consisting of an amino acid sequence selected from among SEQ ID NOs: 7, 27 and 38.
- the peptide of the present invention can be flanked by additional amino acid residues to the amino acid sequence of the peptide of the present invention. Additional amino acid residues can be composed of any type of amino acid as long as they do not impair the ability of the original peptide to induce CTL. Therefore, the peptide of the present invention has SEQ ID NOs: 3, 5 to 7, 9, 10, 12 to 14, 17, 19, 21, 30, 35, 38 to 40, 45, 53, 56, 58, 27, 60, A peptide having an ability to induce CTL, comprising an amino acid sequence selected from among 28, 67, 69 and 47 is included.
- Such peptides are, for example, less than about 40 amino acids, often less than about 20 amino acids, and usually less than about 15 amino acids. Therefore, if the original peptide is a nonapeptide, the peptide of the present invention includes a peptide having a length of 10 amino acids or a length of 11 to 40 amino acids generated by flanking an additional amino acid to the peptide. In addition, if the original peptide is a decapeptide, it includes a peptide having a length of 11 to 40 amino acids. Such peptides can be, for example, peptides that are 11-20 amino acids in length, and can be peptides that are 11-15 amino acids in length.
- an additional amino acid residue is an amino acid residue adjacent to the amino acid sequence of the peptide of the present invention in the full-length amino acid sequence of CDCA1 (eg, SEQ ID NO: 82 or 84). Therefore, the peptide of the present invention has SEQ ID NOs: 3, 5-7, 9, 10, 12-14, 17, 19, 21, 30, 35, 38-40, 45, 53, 56, 58, 27, 60. , 28, 67, 69 and 47, which includes a peptide fragment of CDCA1, which comprises a peptide capable of inducing CTL.
- modification of one, two, or more amino acids in a peptide does not affect the function of the peptide and in some cases even enhances the desired function of the original peptide.
- a modified peptide ie, one, two, or a few amino acid residues were modified (ie, substituted, deleted, inserted and / or added) compared to the original reference sequence
- a peptide composed of an amino acid sequence is known to retain the biological activity of the original peptide (Mark et al., Proc Natl Acad Sci USA 1984, 81: 5662-6; Zoller and Smith, Nucleic Acids) Res 1982, 10: 6487-500; Dalbadie-McFarland et al., Proc Natl Acad Sci USA 1982, 79: 6409-13).
- the peptide of the invention has SEQ ID NOs: 3, 5-7, 9, 10, 12-14, 17, 19, 21, 30, 35, 38-40, 45, 53, 56, 58 , 27, 60, 28, 67, 69 and 47, an amino acid sequence in which one, two, or several amino acids are substituted, deleted, inserted and / or added to the amino acid sequence. And a peptide having an ability to induce CTL.
- Examples of properties of functionally similar amino acid side chains include, for example, hydrophobic amino acids (A, I, L, M, F, P, W, Y, V), hydrophilic amino acids (R, D, N, C, E, Q, G, H, K, S, T), as well as side chains having the following functional groups or characteristics in common: aliphatic side chains (G, A, V, L, Hydroxyl group-containing side chains (S, T, Y); sulfur atom-containing side chains (C, M); carboxylic acid and amide-containing side chains (D, N, E, Q); base-containing side chains (R, K, H); and aromatic-containing side chains (H, F, Y, W).
- hydrophobic amino acids A, I, L, M, F, P, W, Y, V
- hydrophilic amino acids R, D, N, C, E, Q, G, H, K, S, T
- side chains having the following functional groups or characteristics in common aliphatic side chains (G, A, V,
- the following eight groups each contain amino acids recognized in the art as conservative substitutions for each other: 1) Alanine (A), Glycine (G); 2) Aspartic acid (D), glutamic acid (E); 3) Asparagine (N), glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), leucine (L), methionine (M), valine (V); 6) phenylalanine (F), tyrosine (Y), tryptophan (W); 7) serine (S), threonine (T); and 8) Cysteine (C), methionine (M) (see, for example, Creighton, Proteins 1984).
- Such conservatively modified peptides are also included in the peptides of the present invention.
- the peptide of the present invention is not limited to these, and may contain non-conservative modifications as long as the modified peptide retains the CTL-inducing ability of the original peptide.
- the modified peptides do not exclude CDCA1 polymorphic variants, interspecies homologs, and allele-derived CTL inducible peptides.
- the term “several” means 5 or fewer amino acids, such as 4 or 3 or fewer.
- the percentage of amino acids to be modified is preferably 20% or less, more preferably 15% or less, even more preferably 10% or less, or 1-5%.
- the peptides of the present invention are presented on the surface of cells or exosomes, preferably as a complex with HLA antigens. Therefore, the peptide of the present invention preferably has a high binding affinity for the HLA antigen. Therefore, the peptide may be modified by amino acid residue substitution, deletion, insertion and / or addition to obtain a modified peptide with improved binding affinity.
- the second amino acid from the N-terminal and the C-terminal amino acid are often anchor residues involved in binding to HLA class I (Rammensee HG, et al., Immunogenetics. 1995; 41 (4): 178-228.).
- HLA-A11 threonine, valine, isoleucine, leucine, phenylalanine and tyrosine are the second amino acids from the N-terminus, lysine and arginine are the C-terminal amino acids, and anchor residues with high binding affinity for HLA-A11.
- HLA-A11 has auxiliary anchor residues at the 3rd and 7th positions from the N-terminus, and the 3rd amino acid from the N-terminus is preferably leucine, phenylalanine, tyrosine, isoleucine, and alanine, It is known that leucine, isoleucine, tyrosine, valine and phenylalanine are preferred as the seventh amino acid from the N-terminus (Falk, et al., Immunogenetics 1994 40 232-41; Chujoh, et al., Tissue Antigens 1998: 52: 501-9).
- the second amino acid from the N-terminus is replaced with threonine, valine, isoleucine, leucine, phenylalanine or tyrosine, and / or the C-terminal amino acid is lysine.
- the third amino acid from the N-terminus may be replaced with leucine, phenylalanine, tyrosine, isoleucine, or alanine
- / or the seventh amino acid from the N-terminus may be replaced with leucine, isoleucine, tyrosine, valine, or phenylalanine. May be desirable.
- N-terminal second amino acid is replaced with threonine, valine, isoleucine, leucine, phenylalanine or tyrosine
- N-terminal third amino acid is replaced with leucine, phenylalanine, tyrosine, isoleucine, alanine
- N-terminal A peptide having an CTL-inducing ability comprising an amino acid sequence, wherein the seventh amino acid from is substituted with leucine, isoleucine, tyrosine, valine or phenylalanine and / or the C-terminal amino acid is substituted with lysine or arginine, Included in the peptides of the present invention.
- the peptide of the invention is in SEQ ID NOs: 3, 5-7, 9, 10, 12-14, 17, 19, 21, 30, 35, 38-40, 45, 53, 56 and 58.
- the second amino acid from the N-terminus is substituted with threonine, valine, isoleucine, leucine, phenylalanine or tyrosine
- the third amino acid from the N-terminus is leucine, phenylalanine, tyrosine, isoleucine or alanine
- the peptide of the present invention is selected from SEQ ID NOs: 3, 5-7, 9, 10, 12-14, 17, 19, 21, 30, 35, 38-40, 45, 53, 56 and 58
- a peptide having CTL-inducing ability including an amino acid sequence having one or more substitutions selected from the following (a) to (d): (A) the second amino acid from the N-terminus is substituted with threonine, valine, isoleucine, leucine, phenylalanine or tyrosine; (B) the third amino acid from the N-terminus is substituted with leucine, phenylalanine, tyrosine, isoleucine or alanine; (C) the seventh amino acid from the N-terminus is substituted with leucine, isoleucine, tyrosine, valine or phenylalanine; and (d) the C-terminal amino acid is substituted with lysine or arginine.
- the peptide of the invention is in SEQ ID NOs: 3, 5-7, 9, 10, 12-14, 17, 19, 21, 30, 35, 38-40, 45, 53, 56 and 58.
- a peptide having an ability to induce CTLs comprising an amino acid sequence in which one or more substitutions selected from the above (a) to (d) are substituted for an amino acid sequence selected from (a) to (d).
- the preferred number of substitutions is 1, 2, 3 or 4 substitutions selected from the above (a) to (d).
- the peptide of the present invention is selected from SEQ ID NOs: 3, 5, 7, 9, 10, 12, 14, 17, 19, 21, 30, 35, 38-40, 45, 53, 56, and 58.
- Amino acid sequence wherein the second amino acid from the N-terminus is substituted with threonine, valine, isoleucine, leucine, phenylalanine or tyrosine, and / or the C-terminal amino acid is substituted with lysine or arginine
- the peptide of the present invention is selected from SEQ ID NOs: 3, 5-7, 9, 10, 12-14, 17, 19, 21, 30, 35, 38-40, 45, 53, 56 and 58.
- the peptide of the present invention is selected from SEQ ID NOs: 3, 5-7, 9, 10, 12-14, 17, 19, 21, 30, 35, 38-40, 45, 53, 56 and 58
- a peptide having an ability to induce CTL comprising an amino acid sequence having one or more substitutions selected from the following (a) and (b) with respect to the amino acid sequence to be selected: (A) the second amino acid from the N-terminus is substituted with threonine, valine, isoleucine, leucine, phenylalanine or tyrosine; and (b) the C-terminal amino acid is substituted with lysine or arginine.
- the peptide of the invention is in SEQ ID NOs: 3, 5-7, 9, 10, 12-14, 17, 19, 21, 30, 35, 38-40, 45, 53, 56 and 58.
- a peptide having the ability to induce CTLs comprising an amino acid sequence selected from the above (a) to (b) and having one or more substitutions.
- the second amino acid from the N-terminus is substituted with threonine, valine, isoleucine or leucine.
- HLA-A33 phenylalanine, tyrosine, alanine, isoleucine, leucine and valine are known as the second amino acids from the N-terminus, and arginine and lysine are known as anchor residues with high binding affinity for HLA-A33 as C-terminal amino acids.
- arginine and lysine are known as anchor residues with high binding affinity for HLA-A33 as C-terminal amino acids.
- the first amino acid residue from the N-terminus also functions as an anchor residue
- aspartic acid and glutamic acid are known to be preferred as the first amino acid from the N-terminus.
- the first amino acid from the N terminus is replaced with aspartic acid or glutamic acid
- the second amino acid from the N terminus is phenylalanine, tyrosine, alanine, isoleucine
- the first amino acid from the N-terminus is substituted with aspartic acid or glutamic acid, and 2 from the N-terminus.
- a peptide having an CTL-inducing ability including an amino acid sequence, in which the second amino acid is substituted with phenylalanine, tyrosine, alanine, isoleucine, leucine or valine and / or the C-terminal amino acid is substituted with arginine or lysine, Included in the peptides of the present invention.
- the peptide of the present invention has an amino acid sequence selected from SEQ ID NOs: 27, 3, 60, 28, 5, 67 and 69, wherein the first amino acid from the N-terminus is substituted with aspartic acid or glutamic acid. Consisting of an amino acid sequence in which the second amino acid from the N-terminus is substituted with phenylalanine, tyrosine, alanine, isoleucine, leucine or valine, and / or the C-terminal amino acid is substituted with arginine or lysine It may be a peptide having the ability to induce CTL.
- the peptide of the present invention is selected from the following (a) to (c) with respect to an amino acid sequence selected from SEQ ID NOs: 27, 3, 60, 28, 5, 67 and 69.
- the peptide of the present invention is selected from the above (a) to (c) with respect to an amino acid sequence selected from SEQ ID NOs: 27, 3, 60, 28, 5, 67 and 69. It can be a peptide having the ability to induce CTL, consisting of an amino acid sequence having one or more substitutions. In the present invention, the preferred number of substitutions is 1, 2 or 3 substitutions selected from the above (a) to (c).
- the peptide of the present invention is the amino acid sequence selected from SEQ ID NOs: 27, 3, 60, 28, 5, 67 and 69, wherein the second amino acid from the N-terminus is phenylalanine, tyrosine, alanine, isoleucine, It may be a peptide having an ability to induce CTL, including an amino acid sequence, substituted with leucine or valine and / or substituted with arginine or lysine at the C-terminal amino acid.
- the peptide of the present invention has an amino acid sequence selected from SEQ ID NOs: 27, 3, 60, 28, 5, 67 and 69, wherein the second amino acid from the N-terminus is phenylalanine, tyrosine, alanine, isoleucine.
- a peptide capable of inducing CTLs consisting of an amino acid sequence, substituted with leucine or valine, and / or substituted with arginine or lysine at the C-terminal amino acid. That is, the peptide of the present invention is selected from the following (a) and (b) with respect to an amino acid sequence selected from SEQ ID NOs: 27, 3, 60, 28, 5, 67 and 69.
- a peptide capable of inducing CTLs comprising an amino acid sequence with one or more substitutions: (A) the second amino acid from the N-terminus is substituted with phenylalanine, tyrosine, alanine, isoleucine, leucine or valine; and (b) the C-terminal amino acid is substituted with arginine or lysine.
- the peptide of the present invention is selected from among (a) and (b) above with respect to an amino acid sequence selected from SEQ ID NOs: 27, 3, 60, 28, 5, 67 and 69. It can be a peptide having the ability to induce CTL, consisting of an amino acid sequence having one or more substitutions.
- the second amino acid from the N-terminus is substituted with phenylalanine or tyrosine.
- binding affinity for HLA-A03 is leucine, methionine, valine, alanine, isoleucine, serine, and threonine as the second amino acids from the N-terminus, and arginine, lysine, tyrosine, and phenylalanine as the C-terminal amino acids. It is known as a highly anchor residue (Kubo RT. Et al., J Immunol. 1994 Apr 15; 152 (8): 3913-24; Sidney J et al., Hum Immunol. 1996 Feb; 45 (2 ): 79-93; Gambacorti-Passerini C et al., Clin Cancer Res. 1997 May; 3 (5): 675-83).
- the second amino acid from the N-terminus is replaced with leucine, methionine, valine, alanine, isoleucine, serine, or threonine, and / or at the C-terminus. It may be desirable to replace amino acids with arginine, lysine, tyrosine, or phenylalanine.
- the second amino acid from the N-terminus is substituted with leucine, methionine, valine, alanine, isoleucine, serine, or threonine, and / or
- peptides having the ability to induce CTLs including amino acid sequences in which the C-terminal amino acid is substituted with arginine, lysine, tyrosine, or phenylalanine are included in the peptides of the present invention.
- the peptide of the present invention has a leucine, methionine, valine, alanine, isoleucine, serine, or threonine in which the second amino acid from the N-terminus in the amino acid sequence selected from SEQ ID NOs: 7, 38 and 47 And / or a C-terminal amino acid substituted with arginine, lysine, tyrosine, or phenylalanine. That is, the peptide of the present invention has one or more substitutions selected from the following (a) and (b) with respect to the amino acid sequence selected from SEQ ID NOs: 7, 38 and 47.
- peptides having the ability to induce CTLs including amino acid sequences: (A) the second amino acid from the N-terminus is replaced with leucine, methionine, valine, alanine, isoleucine, serine, or threonine; and (b) the C-terminal amino acid is replaced with arginine, lysine, tyrosine, or phenylalanine. Has been.
- the peptide of the present invention has one or more substitutions selected from the above (a) and (b) to an amino acid sequence selected from SEQ ID NOs: 7, 38 and 47. It can be a peptide having the ability to induce CTLs, which consists of a sequenced amino acid sequence.
- the preferred number of substitutions is one or two substitutions selected from the above (a) and (b).
- the second amino acid from the N-terminus is substituted with leucine, methionine, or valine.
- Substitutions can be introduced not only at the amino acid at the anchor site, but also at the potential T cell receptor (TCR) recognition site of the peptide.
- TCR T cell receptor
- Some studies have shown that peptides with amino acid substitutions, such as CAP1, p53 (264-272) , Her-2 / neu (369-377) , or gp100 (209-217) , are as active as the original peptide. Have demonstrated or may have superior activity (Zaremba et al. Cancer Res. 57, 4570-7, 1997, TK Hoffmann et al. J Immunol. (2002) Feb 1; 168 (3 ): 1338-47., SO Dionne et al. Cancer Immunol immunother. (2003) 52: 199-206, and SO Dionne et al. Cancer Immunology, Immunotherapy (2004) 53, 307-14).
- the present invention also includes peptides of the present invention (eg, SEQ ID NOs: 3, 5-7, 9, 10, 12-14, 17, 19, 21, 30, 35, 38-40, 45, 53, 56, 58 , 27, 60, 28, 67, 69 and 47), one, two, or several amino acids may be added to the N-terminus and / or C-terminus of the peptide. Contemplate what you can do. Such modified peptides that retain CTL inducibility are also included in the present invention. For example, a peptide having one, two, or several amino acids added to the N-terminal and / or C-terminal of a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 7 is brought into contact with APC.
- SEQ ID NOs: 3 5-7, 9, 10, 12-14, 17, 19, 21, 30, 35, 38-40, 45, 53, 56, 58 , 27, 60, 28, 67, 69 and 47 one, two, or several amino acids may be added to the N-terminus and / or C
- the peptide is incorporated and processed, and becomes a peptide consisting of the amino acid sequence shown in SEQ ID NO: 7. Thereafter, CTL can be induced by being presented on the cell surface of APC via an antigen presentation pathway. That is, the peptide of the present invention can be a peptide in which one, two, or several amino acids are added to either or both of the N-terminal and C-terminal.
- the amino acid sequence of the peptide is identical to part of the amino acid sequence of an endogenous or exogenous protein with different functions, side effects such as autoimmune disorders and / or allergic symptoms to certain substances can be induced There is sex. Therefore, in order to avoid a situation in which the amino acid sequence of the peptide matches the amino acid sequence of another protein, it is preferable to perform a homology search using an available database. If the homology search reveals that there is not even a peptide that differs by one or two amino acids compared to the peptide of interest, the HLA antigen and The peptide of interest can be modified to increase its binding affinity and / or to increase its ability to induce CTLs.
- the “peptide having the ability to induce CTL” refers to a peptide from which CTL is induced by APC stimulated with the peptide.
- “Induction of CTL” includes differentiation induction into CTL, induction of CTL activation, induction of CTL proliferation, induction of CTL cytotoxic activity, induction of lysis of target cells by CTL, and increase of CTL IFN- ⁇ production Includes induction.
- APC eg, B lymphocytes, macrophages, and dendritic cells
- HLA antigens eg, B lymphocytes, macrophages, and dendritic cells
- APC can preferably use dendritic cells derived from human peripheral blood mononuclear leukocytes.
- a transgenic animal prepared to express an HLA antigen can also be used.
- the target cells are radiolabeled with 51 Cr or the like, and the cytotoxic activity of CTL induced by the peptide can be calculated from the radioactivity released from the target cells.
- the ability of CTL to be induced can be measured by measuring IFN- ⁇ produced and released by CTL and visualizing the inhibition zone on the medium using anti-IFN- ⁇ monoclonal antibody. Can be evaluated.
- the peptides of the present invention can be linked to other peptides as long as the resulting linked peptide retains the ability to induce CTL.
- suitable peptides to be linked to the peptides of the present invention include CTL inducible peptides derived from TAA.
- the peptides of the present invention can be linked together. Suitable linkers that can be used to link peptides are known in the art, such as AAY (P. M. Daftarian et al., J Trans Med 2007, 5:26), AAA, NKRK (SEQ ID NO: 85) (R. P. M. Sutmuller et al., J Immunol.
- Peptides can be linked in various configurations (eg, linked, overlapping, etc.), and more than two peptides can be linked.
- the peptide of the present invention can also be linked to other substances as long as the resulting linked peptide retains the ability to induce CTL.
- suitable substances linked to the peptides of the present invention include, for example, peptides, lipids, sugars or sugar chains, acetyl groups, and natural or synthetic polymers.
- the peptide of the present invention can be modified such as glycosylation, side chain oxidation, or phosphorylation as long as the CTL inducing ability is not impaired. Such types of modifications can be made to provide additional functions (eg, targeting and delivery functions) or to stabilize the peptide.
- Peptide stability can be assayed in several ways. For example, peptidases and various biological media such as human plasma and serum can be used to test the stability (see, eg, Verhoef et al., Eur J Drug Metab Pharmacokin 1986, 11: 291-302). I want to be)
- the modified peptide in which one, two, or several amino acid residues are substituted, deleted, inserted and / or added is the same as or compared with the original peptide. Those having higher activity can be screened or selected. Accordingly, the present invention also provides a method for screening or selecting for a modified peptide having the same or higher activity compared to the original.
- the present invention provides a method for screening a peptide having the ability to induce CTL, comprising the following steps: (A) SEQ ID NOs: 3, 5-7, 9, 10, 12-14, 17, 19, 21, 30, 35, 38-40, 45, 53, 56, 58, 27, 60, 28, 67, An amino acid sequence in which one, two, or several amino acid residues are substituted, deleted, inserted, and / or added to the original amino acid sequence consisting of an amino acid sequence selected from 69 and 47 Creating a candidate sequence comprising: (B) selecting a candidate sequence having no significant homology (sequence identity) with any known human gene product other than CDCA1 from the candidate sequences prepared in (a); (C) contacting the peptide comprising the candidate sequence selected in (b) with APC; (D) a step of contacting the APC of (c) with a CD8-positive T cell; and (e) a step of selecting a peptide having a CTL inducing ability equivalent to or higher than that of a
- the peptide of the present invention is also described as “CDCA1 peptide” or “CDCA1 polypeptide”.
- the peptides of the invention can be prepared using well-known techniques.
- the peptides of the invention can be prepared using recombinant DNA technology or chemical synthesis.
- the peptides of the present invention can be synthesized individually or as longer polypeptides comprising two or more peptides.
- the peptides of the invention can be isolated from the host cell or synthesis reaction. That is, the peptides of the invention can be purified or isolated so that they are substantially free of other host cell proteins and fragments thereof, or any other chemicals.
- the peptide of the present invention may contain modifications such as glycosylation, side chain oxidation, or phosphorylation, as long as the modification does not impair the biological activity of the original peptide.
- modifications include the incorporation of D-amino acids or other amino acid mimetics that can be used, for example, to increase the serum half-life of the peptide.
- the peptide of the present invention can be obtained by chemical synthesis based on the selected amino acid sequence.
- Examples of conventional peptide synthesis methods that can be adapted for the synthesis include those described in the literature as follows: (I) Peptide Synthesis, Interscience, New York, 1966; (Ii) The Proteins, Vol. 2, Academic Press, New York, 1976; (Iii) “Peptide Synthesis” (Japanese), Maruzen, 1975; (Iv) “Basics and Experiments of Peptide Synthesis” (Japanese), Maruzen, 1985; (V) "Development of drugs” (Japanese), Vol.
- any known genetic engineering method for producing a peptide can be adapted to obtain a peptide of the invention (eg, Morrison J, J Bacteriology 1977, 132: 349-51; Clark-Curtiss & Curtiss, Methods in Enzymology (Wu et al.) 1983, 101: 347-62).
- an appropriate vector containing a polynucleotide encoding the peptide of the present invention in an expressible form eg, downstream of a regulatory sequence corresponding to a promoter sequence
- the host cell is then cultured to produce the peptide of the invention.
- the peptides of the present invention can be generated in vitro using an in vitro translation system.
- polynucleotides that encode any of the peptides of the present invention. These include polynucleotides derived from the native CDCA1 gene (eg, GenBank accession number NM_145697 (SEQ ID NO: 81) or GenBank accession number NM_031423 (SEQ ID NO: 83)), and conservatively modified nucleotide sequences thereof. The polynucleotide having is included. As used herein, the phrase “conservatively modified nucleotide sequence” refers to sequences that encode the same or essentially the same amino acid sequence. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any particular protein.
- the codons GCA, GCC, GCG, and GCU all encode the amino acid alanine.
- the codon can be changed to any of the corresponding codons without changing the encoded polypeptide.
- Such nucleic acid mutations are “silent mutations” and are a type of mutation conservatively modified. Every nucleic acid sequence herein that encodes a peptide also represents every possible silent variation of the nucleic acid. Those skilled in the art will be able to modify each codon in the nucleic acid (except AUG, which is usually the only codon for methionine, and TGG, which is usually the only codon for tryptophan) to obtain a functionally identical molecule. You will recognize. Accordingly, each silent variation of a nucleic acid that encodes a peptide is implicitly described in each disclosed sequence.
- the polynucleotide of the present invention can be composed of DNA, RNA, and derivatives thereof.
- DNA is appropriately composed of bases such as A, T, C, and G, and in RNA, T is replaced with U.
- the polynucleotide of the present invention can encode a plurality of peptides of the present invention with or without an intervening amino acid sequence in between.
- the intervening amino acid sequence can provide a cleavage site (eg, an enzyme recognition sequence) for the polynucleotide or translated peptide.
- the polynucleotide may comprise any additional sequence to the coding sequence that encodes a peptide of the invention.
- the polynucleotide may be a recombinant polynucleotide containing regulatory sequences necessary for expression of the peptide, or an expression vector (eg, a plasmid) having a marker gene or the like.
- such recombinant polynucleotides can be prepared, for example, by manipulating the polynucleotide by conventional recombinant techniques using polymerases and endonucleases.
- the polynucleotide of the present invention can be produced using any of recombinant techniques and chemical synthesis techniques.
- a polynucleotide can be made by inserting into an appropriate vector, which can be expressed when transfected into competent cells.
- polynucleotides can be amplified using PCR techniques or expression in a suitable host (see, eg, Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York, 1989) I want to be)
- polynucleotides can be synthesized using the solid phase technique described in Beaucage SL & Iyer RP, Tetrahedron 1992, 48: 2223-311; Matthes et al., EMBO J 1984, 3: 801-5. it can.
- Exosomes The present invention further provides intracellular vesicles, called exosomes, that present complexes formed between the peptides of the present invention and HLA antigens on their surface. Exosomes can be prepared using, for example, the methods detailed in JP-A-11-510507 and WO99 / 03499, and prepared using APC obtained from a patient to be treated and / or prevented can do. The exosomes of the present invention can be vaccinated as a vaccine in the same manner as the peptides of the present invention.
- HLA-A11 eg, HLA-A * 1101
- HLA-A33 eg, HLA-A * 3303
- HLA-A03 eg, HLA-A * 0301
- an antigen that has a high level of binding affinity for or is mediated by a specific HLA antigen by pre-examining the type of HLA antigen in the patient in need of treatment in the clinic Appropriate selection of peptides having the ability to induce CTLs by presentation becomes possible.
- the exosome of the present invention presents a complex of the peptide of the present invention and HLA-A11, HLA-A33 or HLA-A03 on its surface.
- HLA-A11 the peptide of the present invention is represented by SEQ ID NOs: 3, 5, 7, 9, 10, 12, 14, 17, 19, 21, 30, It is preferably a peptide having an amino acid sequence selected from 35, 38-40, 45, 53, 56 and 58 or a modified peptide thereof.
- the peptide of the present invention is an amino acid selected from SEQ ID NOs: 27, 3, 60, 28, 5, 67 and 69. It is preferably a peptide having a sequence or a modified peptide thereof, more preferably a peptide consisting of an amino sequence selected from SEQ ID NOs: 27, 3, 60, 28, 5, 67 and 69 or a modified peptide thereof. preferable.
- the peptide of the present invention is a peptide having an amino acid sequence selected from SEQ ID NOs: 7, 38 and 47 or a modified peptide thereof It is preferable that it is a peptide consisting of an amino acid sequence selected from SEQ ID NOs: 7, 38 and 47 or a modified peptide thereof.
- the present invention also provides an APC that presents a complex formed between the HLA antigen and the peptide of the present invention on its surface.
- the present invention provides an APC having on its cell surface a complex formed between an HLA antigen and a peptide of the present invention.
- the APC of the present invention can be an isolated APC.
- isolated when used in reference to a cell (APC, CTL, etc.) refers to the cell being separated from other types of cells.
- the APC of the present invention may be derived from an APC derived from a patient to be treated and / or prevented, and alone or another drug containing the peptide, exosome, or CTL of the present invention Can be used as a vaccine.
- the APC of the present invention is not limited to a specific type of cell, but is known to present proteinaceous antigens on its cell surface as recognized by lymphocytes, such as dendritic cells (dendric cells). : DC), Langerhans cells, macrophages, B cells, and activated T cells. Since DC is a representative APC having the strongest CTL inducing action among APCs, DC can be preferably used as the APC of the present invention. In the present invention, preferred DC is human-derived isolated DC.
- the APC of the present invention may be a mixture of a plurality of types of cells having an antigen presenting function, or may be a mixture of APCs each presenting a different peptide of the present invention.
- the APCs of the present invention can be obtained by isolating DCs from peripheral blood mononuclear cells and then stimulating them in vitro with the peptides of the present invention.
- the peptide of the present invention is administered to a subject, APC presenting the peptide of the present invention is induced in the subject's body. Therefore, the APC of the present invention can be obtained by administering the peptide of the present invention to a subject and then recovering the APC from the subject.
- the APC of the present invention can be obtained by contacting APC recovered from a subject with the peptide of the present invention.
- the APC of the invention is administered to the subject alone or in combination with other drugs, including peptides, exosomes, or CTLs of the invention, to induce an immune response against cancer cells that express CDCA1 can do.
- ex vivo administration may include the following steps: (A) recovering APC from the first subject; (B) contacting the APC of step (a) with a peptide; and (c) administering the APC of step (b) to a second subject.
- the first object and the second object may be the same individual or different individuals.
- the HLA of the first object and the second object is preferably the same type.
- the APC obtained by the above step b can be a vaccine for treating and / or preventing cancer.
- the APC of the present invention obtained by the method as described above has the ability to induce CTL.
- CTL inducing ability refers to the ability of APC to induce CTL when contacted with CD8 positive T cells.
- CTL inducing ability includes APC ability to induce CTL activation, APC ability to induce CTL proliferation, APC ability to promote lysis of target cells by CTL, and IFN- ⁇ production by CTL Includes APC ability to increase.
- the CTL induced by the APC of the present invention is a CTL specific for CDCA1, and exhibits a specific cytotoxic activity against CDCA1-expressing cells.
- the APC of the present invention can also be prepared by introducing a polynucleotide encoding the peptide of the present invention into APC in vitro.
- the polynucleotide to be introduced may be in the form of DNA or RNA.
- introduction methods include, but are not limited to, various methods conventionally practiced in the art, such as lipofection, electroporation, and the calcium phosphate method. More specifically, it is described in Cancer Res 1996, 56: 5672-7; J Immunol 1998, 161: 5607-13; J Exp Med 1996, 184: 465-72; Published Patent Publication No. 2000-509281 Such a method can be used.
- the polynucleotide By introducing a polynucleotide encoding the peptide of the present invention into APC, the polynucleotide undergoes transcription, translation, etc. in the cell, and then the resulting peptide is processed by MHC class I and passed through the presentation pathway to the main pathway.
- the peptides of the invention are presented on the cell surface of APC.
- the APC of the present invention is HLA-A11 (more preferably HLA-A * 1101), HLA-A33 (more preferably HLA-A * 3303) or HLA-A03 (more preferably HLA-A * 0301).
- HLA-A11 the HLA that forms a complex with the peptide of the present invention is HLA-A11
- the peptide of the present invention is represented by SEQ ID NOs: 3, 5, 7, 9, 10, 12, 14, 17, 19, 21, 30, It is preferably a peptide having an amino acid sequence selected from 35, 38-40, 45, 53, 56 and 58 or a modified peptide thereof.
- the peptide of the present invention has an amino sequence selected from SEQ ID NOs: 27, 3, 60, 28, 5, 67 and 69.
- a modified peptide thereof more preferably a peptide consisting of an amino sequence selected from SEQ ID NOs: 27, 3, 60, 28, 5, 67 and 69.
- the peptide of the present invention is a peptide having an amino acid sequence selected from SEQ ID NOs: 7, 38 and 47 or a modified peptide thereof It is preferably a peptide consisting of an amino acid sequence selected from SEQ ID NOs: 7, 38 and 47.
- the APC of the present invention is preferably an APC derived by a method comprising the steps described in the following (a) or (b): (A) selected from HLA-A11 (more preferably HLA-A * 1101), HLA-A33 (more preferably HLA-A * 3303) and HLA-A03 (more preferably HLA-A * 0301) Contacting an APC expressing at least one HLA with a peptide of the invention; (B) selected from HLA-A11 (more preferably HLA-A * 1101), HLA-A33 (more preferably HLA-A * 3303) and HLA-A03 (more preferably HLA-A * 0301) Introducing a polynucleotide encoding the peptide of the present invention into an APC expressing at least one HLA.
- the peptide of the present invention to be contacted with APC expressing HLA-A11 is SEQ ID NO: 3, 5-7, 9, 10, 12-14, 17, 19, 21, 30, 35, 38-40, 45 , 53, 56 and 58, or a modified peptide thereof, preferably SEQ ID NO: 3, 5-7, 9, 10, 12-14, 17, 19, 21, More preferred is a peptide consisting of an amino acid sequence selected from 30, 35, 38 to 40, 45, 53, 56 and 58.
- the peptide of the present invention to be contacted with APC expressing HLA-A33 is a peptide having an amino sequence selected from SEQ ID NOs: 27, 3, 60, 28, 5, 67 and 69 or a modified peptide thereof.
- it is a peptide consisting of an amino sequence selected from SEQ ID NOs: 27, 3, 60, 28, 5, 67 and 69.
- the peptide of the present invention to be contacted with APC expressing HLA-A03 is preferably a peptide having an amino acid sequence selected from SEQ ID NOs: 7, 38 and 47 or a modified peptide thereof, and SEQ ID NO: More preferred is a peptide consisting of an amino acid sequence selected from 7, 38 and 47.
- the present invention uses of the peptide of the present invention for producing a pharmaceutical composition for inducing APC having CTL inducing ability is provided.
- the present invention provides a method or process for producing a pharmaceutical composition that induces APC having the ability to induce CTL.
- the present invention also provides a peptide of the present invention for inducing APC having CTL inducibility.
- the CTL induced by the peptide of the present invention can be used as a vaccine in the same manner as the peptide of the present invention because it enhances the immune response targeting cancer cells expressing CDCA1 in vivo. Accordingly, the present invention provides CTL induced or activated by the peptides of the present invention.
- the CTL of the present invention is a CTL that targets the peptide of the present invention, and is a CTL that can bind to a complex of the peptide of the present invention and an HLA antigen. Binding of CTL to the complex is performed via a T cell receptor (TCR) present on the cell surface of CTL.
- TCR T cell receptor
- the CTL of the present invention can be an isolated CTL.
- a preferred CTL is an isolated CTL from human origin.
- the CTL of the present invention may be a mixture of CTLs targeting different peptides of the present invention.
- the CTL of the present invention comprises (1) administering the peptide of the present invention to a subject, or (2) subject-derived APC and CD8 positive T cells, or peripheral blood mononuclear cells (PBMC). Stimulating in vitro with a peptide of the invention, or (3) contacting a CD8 positive T cell or PBMC in vitro with an APC or exosome that presents a complex of the HLA antigen and the peptide of the invention on its surface Or (4) introducing into a CD8-positive T cell a vector comprising a polynucleotide encoding each subunit of the T cell receptor (TCR) capable of binding to the peptide of the present invention presented by the HLA antigen on the cell surface Can be obtained by The exosome and APC used in the above method (2) or (3) can be prepared by the methods described in the chapters “V. Exosomes” and “VI. Antigen-presenting cells (APC)”, respectively. Details of the method of (4) are described in the chapter “VIII. T cell receptor
- the CTL of the present invention can be administered alone to a patient to be treated and / or prevented, or used in combination with other drugs including the peptide, APC or exosome of the present invention for the purpose of regulating the effect. Can be administered.
- the CTL of the present invention can be a CTL derived from a CD8-positive T cell derived from a patient to whom the CTL is administered.
- the CTL of the present invention specifically acts on a target cell presenting the peptide of the present invention, for example, the same peptide used for induction of the CTL of the present invention.
- the target cell may be a cell that endogenously expresses CDCA1, such as a cancer cell, or a cell that has been transfected with the CDCA1 gene.
- the target cells of the CTL of the present invention are preferably HLA-A11 (more preferably HLA-A * 1101), HLA-A33 (more preferably HLA-A * 3303) and HLA-A03 (more preferably HLA -A * 0301) is a positive cell.
- the CTL of the present invention comprises HLA-A11 (more preferably HLA-A * 1101), HLA-A33 (more preferably HLA-A * 3303) and HLA-A03 (more preferably HLA-A * 0301). ) Specifically targeting cells expressing both HLA and CDCA1 selected from.
- the cell targeted by CTL can be a cell having any one of HLA-A11, HLA-A33 and HLA-A03 alleles in homo or hetero.
- CTL “targets” a cell means that CTL recognizes a cell presenting a complex of HLA and the peptide of the present invention on the cell surface, and cytotoxicity against the cell. Indicates activity. Further, “specifically target” means that CTL exhibits cytotoxic activity against the cell but does not exhibit cytotoxic activity against other cells. In the context of CTL, the term “recognize a cell” is specific to the cell that binds to the complex of HLA presented on the cell surface and the peptide of the present invention via its TCR. It shows that it shows a cytotoxic activity.
- the CTL of the present invention is preferably HLA-A11 (more preferably HLA-A * 1101), HLA-A33 (more preferably HLA-A * 3303) or HLA-A03 (displayed on the cell surface). More preferably, it is a CTL that can bind to a complex formed between HLA-A * 0301) and the peptide of the present invention via TCR.
- the CTL of the present invention is preferably a CTL induced by a method comprising the steps described in the following (a) or (b):
- (A) CD8-positive T cells are treated with HLA-A11 (more preferably HLA-A * 1101), HLA-A33 (more preferably HLA-A * 3303) or HLA-A03 (more preferably HLA-A * 0301) Contacting in vitro with an APC or exosome presenting a complex of the peptide of the invention with a peptide of the invention on its surface;
- B To CD8 positive T cells, HLA-A11 (more preferably HLA-A * 1101), HLA-A33 (more preferably HLA-A * 3303) or HLA-A03 (more preferably HLA- Introducing a polynucleotide encoding each subunit of TCR that can bind to the peptides of the invention presented by A * 0301).
- T cell receptor The present invention also provides a composition comprising a polynucleotide encoding each subunit of TCR capable of binding to the peptide of the present invention presented by the HLA antigen on the cell surface, and a method of using the same.
- the polynucleotide expresses specificity for cancer cells expressing CDCA1 on CD8-positive T cells by expressing TCR capable of binding to the peptide of the present invention presented on the cell surface by the HLA antigen on the cell surface.
- PCR primers for analysis can be used as a primer set for amplification by combining the following 5′-side primer with the following 3′-side primer, but are not limited thereto.
- TCR formed by introducing the identified polynucleotide into a CD8-positive T cell can bind with high binding force to a target cell presenting the peptide of the present invention, and presents the peptid
- the polynucleotide encoding each subunit of TCR can be incorporated into an appropriate vector, for example, a retroviral vector. These vectors are well known in the art.
- the polynucleotide or a vector containing them in an expressible form can be introduced into a CD8-positive T cell, eg, a patient-derived CD8-positive T cell.
- the present invention provides a ready-made modified T cell having excellent cancer cell killing properties by rapid modification of a patient's own T cell (or T cell derived from another subject).
- a composition is provided.
- the specific TCR specifically indicates a complex of the peptide of the present invention and the HLA antigen presented on the surface of a target cell when the TCR is present on the surface of a CD8-positive T cell. It is a TCR that can recognize and confer specific cytotoxic activity on target cells. Specific recognition of the complex can be confirmed by any known method, and preferred examples include HLA multimer staining analysis using HLA molecules and peptides of the present invention, and ELISPOT assay. By performing the ELISPOT assay, it can be confirmed that the T cell introduced with the polynucleotide specifically recognizes the target cell by TCR and that the signal is transmitted intracellularly.
- TCR When the TCR is present on the surface of a CD8-positive T cell, confirmation that the TCR can impart target cell-specific cytotoxic activity to the CD8-positive T cell should also be performed by a known method. Can do. Preferred methods include measuring cytotoxic activity against target cells, such as by a chromium release assay.
- the present invention relates to HLA-A11, for example, SEQ ID NOs: 3, 5-7, 9, 10, 12-14, 17, 19, 21, 30, 35, 38-40, 45, 53, 56.
- CTL prepared by transducing CD8 positive T cells with a polynucleotide encoding each subunit of TCR that binds to a peptide having an amino acid sequence selected from among 58 and 58.
- the present invention also relates to each subunit of TCR that binds to a peptide having an amino acid sequence selected from, for example, SEQ ID NOs: 27, 3, 60, 28, 5, 67, and 69 in the context of HLA-A33.
- CTLs prepared by transducing CD8 positive T cells with the encoding polynucleotide are provided.
- the present invention also relates to a polynucleotide encoding each subunit of TCR that binds to a peptide having an amino acid sequence selected from, for example, SEQ ID NOs: 7, 38, and 47 in the context of HLA-A03. CTLs prepared by transducing T cells are provided.
- Transduced CTL can be homed in vivo and can be propagated by well-known in vitro culture methods (eg, Kawakami et al., J Immunol., 142, 2-3452-61 (1989)).
- the CTLs of the invention can be used to form immunogenic compositions useful for the treatment or prevention of disease in patients in need of treatment or prevention, the contents of which are hereby incorporated by reference. (See WO2006 / 031221).
- compositions or pharmaceutical composition comprising at least one active ingredient selected from: (A) the peptide of the present invention; (B) a polynucleotide encoding the peptide of the present invention in an expressible form; (C) APC of the present invention; (D) the exosome of the present invention; (E) CTL of the present invention.
- the pharmaceutical composition of the present invention may contain carriers, excipients and the like that are usually used for pharmaceuticals as necessary, in addition to the above-mentioned active ingredients.
- carriers that can be used in the pharmaceutical composition of the present invention include sterilized water, physiological saline, phosphate buffer, culture solution, and the like.
- the present invention also provides a pharmaceutical composition comprising at least one active ingredient selected from the following (a) to (e) and a pharmaceutically acceptable carrier: (A) the peptide of the present invention; (B) a polynucleotide encoding the peptide of the present invention in an expressible form; (C) APC of the present invention; (D) the exosome of the present invention; (E) CTL of the present invention.
- the pharmaceutical composition of the present invention may contain a stabilizer, a suspension, a preservative, a surfactant, a solubilizing agent, a pH adjuster, an aggregation inhibitor, and the like, if necessary.
- CDCA1 expression is significantly elevated in cancer cells compared to normal tissues. Therefore, the peptide of the present invention or a polynucleotide encoding the peptide can be used for treatment and / or prevention of cancer and / or prevention of recurrence after surgery. Accordingly, the present invention provides a pharmaceutical composition for treating and / or preventing cancer and / or preventing its recurrence after surgery, wherein one or more of the peptides or polynucleotides of the present invention are effective.
- compositions comprising as ingredients are provided.
- the peptides of the invention can be presented on the surface of exosomes or APCs for use as pharmaceutical compositions.
- a CTL of the present invention that targets any one of the peptides of the present invention can also be used as an active ingredient of the pharmaceutical composition of the present invention.
- the pharmaceutical composition of the present invention may contain a therapeutically effective amount or a pharmaceutically effective amount of the above-mentioned active ingredient.
- the pharmaceutical composition of the present invention may also be used as a vaccine.
- the phrase “vaccine” also referred to as “immunogenic composition” refers to a composition having the function of inducing an immune response that results in an anti-tumor effect when inoculated into an animal. Point to. Accordingly, the pharmaceutical compositions of the present invention can be used to induce an immune response that provides an anti-tumor effect.
- the immune response induced by the peptides, polypeptides, APCs, CTLs and pharmaceutical compositions of the present invention is not particularly limited as long as it is an immune response that brings about an antitumor effect, but illustratively specific to cancer cells. Induction of CTLs and cytotoxic activity specific to cancer cells.
- the pharmaceutical composition of the invention can be used to treat and / or prevent cancer and / or prevent its recurrence after surgery in a human subject or patient.
- the pharmaceutical composition of the present invention can be preferably used for a subject positive for at least one HLA selected from HLA-A11, HLA-A33 and HLA-A03.
- the pharmaceutical composition of the present invention is used to treat and / or prevent a cancer expressing at least one HLA selected from HLA-A11, HLA-A33 and HLA-A03 and CDCA1, and / or Or in order to prevent the recurrence after an operation, it can use preferably.
- the present invention also provides the use of an active ingredient selected from among the following in the manufacture of a pharmaceutical composition for treating or preventing cancer: (A) the peptide of the present invention; (B) a polynucleotide encoding the peptide of the present invention in an expressible form; (C) APC presenting the peptide of the invention on its surface; (D) an exosome presenting a peptide of the invention on its surface; and (e) a CTL of the invention.
- an active ingredient selected from among the following in the manufacture of a pharmaceutical composition for treating or preventing cancer: (A) the peptide of the present invention; (B) a polynucleotide encoding the peptide of the present invention in an expressible form; (C) APC presenting the peptide of the invention on its surface; (D) an exosome presenting a peptide of the invention on its surface; and (e) a CTL of the invention.
- the present invention further provides an active ingredient selected from among the following for use in the treatment or prevention of cancer: (A) the peptide of the present invention; (B) a polynucleotide encoding the peptide of the present invention in an expressible form; (C) APC presenting the peptide of the invention on its surface; (D) an exosome presenting a peptide of the invention on its surface; and (e) a CTL of the invention.
- the present invention further relates to a method or process for producing a pharmaceutical composition for treating or preventing cancer, comprising at least one active ingredient selected from the following, pharmaceutically or Provided is a method or process comprising formulating a physiologically acceptable carrier: (A) the peptide of the present invention; (B) a polynucleotide encoding the peptide of the present invention in an expressible form; (C) APC presenting the peptide of the invention on its surface; (D) an exosome presenting a peptide of the invention on its surface; and (e) a CTL of the invention.
- a physiologically acceptable carrier (A) the peptide of the present invention; (B) a polynucleotide encoding the peptide of the present invention in an expressible form; (C) APC presenting the peptide of the invention on its surface; (D) an exosome presenting a peptide of the invention on its surface; and (e) a CTL of the invention.
- the present invention also provides a method or process for producing a pharmaceutical composition for treating or preventing cancer, wherein the active ingredient selected from the following is pharmaceutically or physiologically
- a method or process comprising the step of admixing with a chemically acceptable carrier is provided: (A) the peptide of the present invention; (B) a polynucleotide encoding the peptide of the present invention in an expressible form; (C) APC presenting the peptide of the invention on its surface; (D) an exosome presenting a peptide of the invention on its surface; and (e) a CTL of the invention.
- the present invention also provides a method for treating or preventing cancer comprising administering to a subject at least one active ingredient selected from the following: (A) the peptide of the present invention; (B) a polynucleotide encoding the peptide of the present invention in an expressible form; (C) APC presenting the peptide of the invention on its surface; (D) an exosome presenting a peptide of the invention on its surface; and (e) a CTL of the invention.
- active ingredient selected from the following: (A) the peptide of the present invention; (B) a polynucleotide encoding the peptide of the present invention in an expressible form; (C) APC presenting the peptide of the invention on its surface; (D) an exosome presenting a peptide of the invention on its surface; and (e) a CTL of the invention.
- the amino acid sequence selected from SEQ ID NOs: 3, 5 to 7, 9, 10, 12 to 14, 17, 19, 21, 30, 35, 38 to 40, 45, 53, 56 and 58 Have been found as HLA-A11 restricted epitope peptides capable of inducing strong and specific immune responses. Therefore, it has an amino acid sequence selected from SEQ ID NOs: 3, 5-7, 9, 10, 12-14, 17, 19, 21, 30, 35, 38-40, 45, 53, 56 and 58
- a pharmaceutical composition of the invention comprising at least one of the peptides is particularly suitable for administration to a subject having HLA-A11 (eg, HLA-A * 1101) as the HLA antigen.
- compositions containing the targeted CTL ie, the CTLs of the present invention. That is, it has an amino acid sequence selected from SEQ ID NOs: 3, 5-7, 9, 10, 12-14, 17, 19, 21, 30, 35, 38-40, 45, 53, 56 and 58
- a pharmaceutical composition comprising an active ingredient associated with a peptide is suitable for administration to a subject having HLA-A11 (ie, an HLA-A11 positive subject).
- the pharmaceutical composition of the present invention is a pharmaceutical composition comprising a peptide having the amino acid sequence of SEQ ID NO: 7.
- a peptide having an amino acid sequence selected from SEQ ID NOs: 27, 3, 60, 28, 5, 67 and 69 can induce a strong and specific immune response.
- the pharmaceutical composition of the present invention comprising at least one peptide having an amino acid sequence selected from SEQ ID NOs: 27, 3, 60, 28, 5, 67 and 69, has HLA-A33 as the HLA antigen.
- Particularly suitable for administration to subjects with eg, HLA-A * 3303).
- compositions containing the targeted CTL ie, the CTLs of the present invention. That is, a pharmaceutical composition comprising an active ingredient related to a peptide having an amino acid sequence selected from SEQ ID NOs: 27, 3, 60, 28, 5, 67 and 69 is a subject having HLA-A33 (ie Suitable for administration to HLA-A33-positive subjects).
- the pharmaceutical composition of the present invention is a pharmaceutical composition comprising a peptide having the amino acid sequence of SEQ ID NO: 27.
- a peptide having an amino acid sequence selected from among SEQ ID NOs: 7, 38 and 47 is found as an HLA-A03 restricted epitope peptide capable of inducing a strong and specific immune response. It was. Accordingly, the pharmaceutical composition of the present invention comprising at least one peptide having an amino acid sequence selected from SEQ ID NOs: 7, 38 and 47, contains HLA-A03 (eg, HLA-A * 0301) as an HLA antigen. Are particularly suitable for administration to subjects having.
- compositions containing the targeted CTL ie, the CTLs of the present invention. That is, a pharmaceutical composition comprising an active ingredient related to a peptide having an amino acid sequence selected from SEQ ID NOs: 7, 38 and 47 is a subject having HLA-A03 (ie, an HLA-A03 positive subject) Suitable for administration to.
- the pharmaceutical composition of the invention is a pharmaceutical composition comprising a peptide having the amino acid sequence of SEQ ID NO: 7 or 38.
- the cancer to be treated and / or prevented by the pharmaceutical composition of the present invention is not particularly limited as long as it expresses CDCA1, and includes various cancers, bladder cancer, breast cancer, cervical cancer, and bile duct cells. Cancer, chronic myelogenous leukemia (CML), esophageal cancer, stomach cancer, non-small cell lung cancer, lymphoma, osteosarcoma, prostate cancer, kidney cancer, small cell lung cancer, head and neck cancer, soft tissue tumor, and large intestine Including.
- the pharmaceutical composition of the present invention is preferably used for a subject having HLA allele selected from HLA-A11, HLA-A33 and HLA-A03 in homo or hetero.
- the pharmaceutical composition of the present invention comprises, in addition to the above-mentioned active ingredients, other peptides having the ability to induce CTLs against cancer cells (for example, other CTL-inducing peptides derived from TAA), the other peptides Other polynucleotides encoding the other, other cells presenting the other peptides, and the like.
- the pharmaceutical composition of the present invention may optionally contain other therapeutic substances as an active ingredient as long as the antitumor effect of the active ingredient such as the peptide of the present invention is not inhibited.
- the pharmaceutical composition of the present invention may optionally include an anti-inflammatory composition, an analgesic, a chemotherapeutic agent and the like.
- the pharmaceutical composition of the present invention may also be administered sequentially or simultaneously with one or more other pharmaceutical compositions. it can.
- the dosage of the pharmaceutical composition of the present invention and other pharmaceutical compositions depends, for example, on the type of pharmaceutical composition used, the disease to be treated, and the schedule and route of administration.
- compositions of the invention may also contain other ingredients customary in the art in view of the type of formulation. Should.
- the present invention also provides a product or kit comprising the pharmaceutical composition of the present invention.
- the product or kit of the present invention may comprise a container containing the pharmaceutical composition of the present invention.
- suitable containers include, but are not limited to, bottles, vials, and test tubes.
- the container can be formed from a variety of materials such as glass or plastic.
- a label may be attached to the container, and the label can describe a disease or a disease state in which the pharmaceutical composition of the present invention is to be used.
- the label may also indicate directions for administration and the like.
- the product or kit of the present invention may optionally further comprise a second container containing a pharmaceutically acceptable diluent in addition to the container containing the pharmaceutical composition of the present invention.
- the product or kit of the present invention further includes other materials desirable from a commercial and user standpoint, such as other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use. May be included.
- the pharmaceutical composition of the present invention can be provided in a pack or dispenser device that may contain one or more unit dosage forms containing the active ingredient.
- the pack may include metal foil or plastic foil, such as a blister pack.
- the pack or dispenser device can be accompanied by instructions for administration.
- composition containing a peptide as an active ingredient can be formulated by a conventional formulation method, if necessary.
- the pharmaceutical composition of the present invention can contain carriers, excipients and the like that are commonly used in pharmaceuticals as needed without particular limitation.
- carriers that can be used in the pharmaceutical composition of the present invention include sterile water (for example, water for injection), physiological saline, phosphate buffer, phosphate buffered saline, Tris buffered saline, 0.3% A glycine, a culture solution, etc. are mentioned.
- the pharmaceutical composition of the present invention may contain a stabilizer, a suspension, a preservative, a surfactant, a solubilizing agent, a pH adjuster, an aggregation inhibitor, and the like, if necessary. Since the pharmaceutical composition of the present invention can induce specific immunity against cancer cells expressing CDCA1, it can be used for the treatment or prevention of cancer.
- the pharmaceutical composition of the present invention is pharmaceutically or physiologically acceptable such as sterile water (for example, water for injection), physiological saline, phosphate buffer, phosphate buffered saline, Tris buffered saline, and the like. Dissolved in a water-soluble carrier, and if necessary, after adding stabilizers, suspensions, preservatives, surfactants, solubilizers, pH adjusters, aggregation inhibitors, etc. It can be prepared by sterilization.
- the method for sterilizing the peptide solution is not particularly limited, but it is preferably performed by filter sterilization. Filtration sterilization can be performed, for example, using a filter sterilization filter having a pore size of 0.22 ⁇ m or less.
- the pharmaceutical composition of the present invention may be prepared as a lyophilized preparation by lyophilizing the above peptide solution.
- the peptide solution prepared as described above is filled in an appropriate container such as an ampoule, vial, or plastic container, freeze-dried, and sealed with a sterilized and washed rubber stopper after returning to pressure.
- the lyophilized preparation is pharmaceutically or physiologically acceptable prior to administration, such as sterile water (eg, water for injection), physiological saline, phosphate buffer, phosphate buffered saline, Tris buffered saline, etc.
- the pharmaceutical composition of the present invention includes such a filter sterilized peptide solution injection and a lyophilized preparation obtained by lyophilizing the peptide solution.
- a kit containing such a lyophilized preparation and a redissolved solution is also encompassed in the present invention.
- the kit containing the container in which the freeze-dried formulation which is the pharmaceutical composition of this invention is accommodated, and the container in which the redissolved solution is accommodated is also included by this invention.
- the pharmaceutical composition of the present invention can also contain a combination of two or more kinds of the peptides of the present invention.
- the peptide combination may take the form of a cocktail in which the peptides are mixed, or the peptides may be linked together using standard techniques.
- the peptides may be chemically conjugated or expressed as a single fusion polypeptide sequence.
- APC eg, DC
- APCs that present any of the peptides of the invention on their cell surface.
- APCs can be administered again to the subject to induce CTL in the subject, resulting in increased aggressiveness against cancer cells that express CDCA1.
- the pharmaceutical composition of the present invention may also include an adjuvant known to efficiently establish cellular immunity.
- An adjuvant refers to a compound that, when administered with (or sequentially) an antigen having immunological activity, enhances the immune response to the antigen.
- the adjuvant for example, known ones described in documents such as Clin Microbiol Rev 1994, 7: 277-89 can be used.
- Suitable adjuvants include aluminum salts (aluminum phosphate, aluminum hydroxide, aluminum oxyhydroxide, etc.), alum, cholera toxin, salmonella toxin, incomplete Freund's adjuvant (IFA), complete Freund's adjuvant (CFA), ISCOMatrix , GM-CSF and other immunostimulatory cytokines, oligodeoxynucleotides containing CpG motifs (CpG7909, etc.), oil-in-water emulsions, saponins or their derivatives (QS21, etc.), lipopolysaccharides such as lipid A or its derivatives (MPL, RC529) , GLA, E6020, etc.), lipopeptides, lactoferrin, flagellin, double-stranded RNA or derivatives thereof (poly IC, etc.), bacterial DNA, imidazoquinoline (Imiquimod, R848, etc.), C-type lectin lig
- the adjuvant may be contained in a container separate from the pharmaceutical composition containing the peptide of the present invention.
- the adjuvant and the pharmaceutical composition may be administered to the subject sequentially or may be mixed immediately before administration to the subject.
- a kit containing a pharmaceutical composition containing such a peptide of the present invention and an adjuvant is also provided by the present invention.
- the kit may further contain a redissolved solution.
- this invention provides the kit containing the container in which the pharmaceutical composition of this invention is accommodated, and the container in which the adjuvant is accommodated.
- the kit may further include a container in which a redissolved solution is stored, if necessary.
- the pharmaceutical composition of the present invention may be prepared as an emulsion.
- the emulsion can be prepared, for example, by mixing and stirring the peptide solution prepared as described above and an oily adjuvant.
- the peptide solution may be redissolved after lyophilization.
- the emulsion may be either a W / O emulsion or an O / W emulsion, but is preferably a W / O emulsion in order to obtain a high immune response enhancing effect.
- IFA can be preferably used as the oil-based adjuvant, but is not limited thereto.
- the preparation of the emulsion may be performed immediately before administration to a subject.
- the pharmaceutical composition of the present invention may be provided as a kit containing the peptide solution of the present invention and an oily adjuvant.
- the kit may further contain a redissolved solution.
- the pharmaceutical composition of the present invention is a liposome preparation encapsulating the peptide of the present invention, a granule preparation in which the peptide is bound to beads having a diameter of several micrometers, or a preparation in which a lipid is bound to the peptide. Also good.
- the peptide of the invention may also be administered in the form of a pharmaceutically acceptable salt.
- salts include salts with alkali metals (lithium, potassium, sodium, etc.), salts with alkaline earth metals (calcium, magnesium, etc.), and other metals (copper, iron, zinc, manganese, etc.).
- compositions comprising pharmaceutically acceptable salts of the peptides of the present invention are also encompassed by the present invention.
- the “peptide of the present invention” includes not only a free peptide but also a pharmaceutically acceptable salt thereof.
- the pharmaceutical composition of the present invention may further comprise an ingredient that stimulates CTL.
- Lipids have been identified as substances that can stimulate CTLs in vivo against viral antigens.
- palmitic acid residues can be attached to the ⁇ and ⁇ amino groups of lysine residues and then linked to the peptides of the invention.
- the lipidated peptide can then be administered directly in the form of micelles or particles, administered in liposomes, or emulsified in an adjuvant.
- lipid stimulation of the CTL response E.
- coli lipoproteins such as tripalmitoyl-S-glycerylcysteinyl-seryl-serine (P3CSS) when covalently bound to the appropriate peptide Can be used to stimulate CTL (see, eg, Deres et al., Nature 1989, 342: 561-4).
- P3CSS tripalmitoyl-S-glycerylcysteinyl-seryl-serine
- suitable methods of administration of the peptides or pharmaceutical compositions of the invention include oral, intradermal, subcutaneous, intramuscular, intraosseous, peritoneal and intravenous injection, and the like, as well as systemic administration or local to the target site. Administration is included, but not limited to.
- a preferable administration method includes subcutaneous injection in the vicinity of a lymph node such as an axilla or axilla. Administration can be by a single dose or can be boosted by multiple doses.
- the peptide of the present invention is a therapeutically or pharmaceutically effective amount for treating cancer or a therapeutically or pharmaceutically effective amount for inducing immunity (more specifically, CTL) against cancer cells expressing CDCA1. Can be administered to the subject.
- the dose of the peptide of the present invention can be appropriately adjusted according to the disease to be treated, the age, weight, administration method, etc. of the patient, and this is usually 0.001 mg to 1000 mg, for example 0.01 mg, for each peptide of the present invention. It can be ⁇ 100 mg, such as 0.1 mg to 30 mg, such as 0.1 mg to 10 mg, for example 0.5 mg to 5 mg. Further, the administration interval can be once every several days to several months. For example, administration can be performed once a week. Those skilled in the art can appropriately select an appropriate dose (dose).
- the pharmaceutical composition of the invention comprises a therapeutically effective amount of a peptide of the invention and a pharmaceutically or physiologically acceptable carrier.
- the pharmaceutical composition of the invention comprises a therapeutically effective amount of a peptide of the invention, a pharmaceutically or physiologically acceptable carrier, and an adjuvant.
- the pharmaceutical composition of the present invention contains 0.001 mg to 1000 mg, preferably 0.01 mg to 100 mg, more preferably 0.1 mg to 30 mg, more preferably 0.1 mg to 10 mg, such as 0.5 mg to 5 mg, of the peptide of the present invention. Can do.
- the peptide of the present invention is added in an amount of 0.001 mg / ml to 1000 mg / ml, preferably 0.01 mg / ml to 100 mg / ml, more preferably 0.1 mg / ml. It can be included at a concentration of ml-30 mg / ml, more preferably 0.1 mg / ml-10 mg / ml, for example 0.5 mg / ml-5 mg / ml. In this case, for example, 0.1 to 5 ml, preferably 0.5 to 2 ml of the pharmaceutical composition of the present invention can be administered to a subject by injection.
- the present invention provides for treating and / or preventing cancer and / or after surgery comprising administering to a subject a therapeutically effective amount of a peptide of the present invention or a pharmaceutical composition of the present invention.
- a method for preventing recurrence As described above, the peptide of the present invention is usually 0.001 mg to 1000 mg, such as 0.01 mg to 100 mg, such as 0.1 mg to 30 mg, such as 0.1 mg to 10 mg, for example, 0.5 mg to 5 mg administered to a subject in a single administration. can do.
- the peptide of the invention is administered to a subject with an adjuvant.
- the administration interval can be once every several days to several months, preferably once every several days to one month, for example, once a week or once every two weeks. be able to.
- composition comprising polynucleotide as active ingredient
- the pharmaceutical composition of the present invention may also comprise a polynucleotide encoding the peptide of the present invention in a form capable of expression.
- the phrase “in an expressible form” means that the peptide of the invention is expressed when the polynucleotide is introduced into a cell.
- the sequence of the polynucleotide of the invention includes regulatory elements necessary for expression of the peptide of the invention.
- the polynucleotides of the invention can be provided with the sequences necessary to achieve stable insertion into the genome of the target cell (for a description of homologous recombination cassette vectors, see eg, Thomas KR & Capecchi MR, Cell 1987, 51: 503-12). See, for example, Wolff et al., Science 1990, 247: 1465-8; US Pat. Nos. 5,580,859; 5,589,466; 5,804,566; 5,739,118; 5,736,524; 5,679,647; and WO98 / 04720 I want to be.
- DNA-based delivery technologies include “naked DNA”, facilitated (bupivacaine, polymer, peptide-mediated) delivery, cationic lipid complexes, and particle-mediated (“gene gun”) or pressure-mediated Delivery is included (see, eg, US Pat. No. 5,922,687).
- the peptide of the present invention can also be expressed by a viral vector or a bacterial vector.
- expression vectors include attenuated viral hosts such as vaccinia virus or fowlpox virus.
- vaccinia virus can be used as a vector for expressing the peptide of the present invention.
- vaccinia virus When introduced into a host, recombinant vaccinia virus expresses an immunogenic peptide, thereby eliciting an immune response.
- Vaccinia vectors and methods useful for immunization protocols are described, for example, in US Pat. No. 4,722,848.
- Another vector is BCG (Bacilli Calmette Guerin). BCG vectors are described in Stover et al., Nature 1991, 351: 456-60.
- adenovirus vectors and adeno-associated virus vectors such as adenovirus vectors and adeno-associated virus vectors, retrovirus vectors, Salmonella typhi vectors, detoxified anthrax toxin vectors, etc.
- retrovirus vectors such as Salmonella typhi vectors, detoxified anthrax toxin vectors, etc.
- Salmonella typhi vectors such as Salmonella typhi vectors, detoxified anthrax toxin vectors, etc.
- Delivery of the polynucleotide of the present invention into a patient may be direct, and in this case, the patient can be directly exposed to a vector carrying the polynucleotide of the present invention.
- it may be indirect, in which case the cells are first transformed in vitro with a vector carrying the polynucleotide of the invention and then the cells are transplanted into the patient.
- Each of these two approaches is known as in vivo and ex vivo gene therapy.
- polynucleotide administration may be performed by oral, intradermal, subcutaneous, intravenous, intramuscular, intraosseous, and / or peritoneal injection.
- the administration of the polynucleotide may be systemic administration or local administration in the vicinity of the target site. Administration can be by a single dose or can be boosted by multiple doses.
- the polynucleotide of the present invention has a therapeutically or pharmaceutically effective amount for treating cancer or a therapeutically or pharmaceutically effective for inducing immunity (more specifically, CTL) against cancer cells expressing CDCA1. The amount can be administered to a subject.
- the dosage of the polynucleotide in a suitable carrier, or the dosage of the polynucleotide in a cell transformed with a polynucleotide encoding a peptide of the invention will depend on the disease being treated, the age, weight of the patient, method of administration, etc. This can usually be 0.001 mg to 1000 mg, such as 0.01 mg to 100 mg, such as 0.1 mg to 30 mg, such as 0.1 mg to 10 mg, such as 0.5 mg to 5 mg.
- the dosing interval can be once every few days to once every several months, for example, once a week. Those skilled in the art can appropriately select an appropriate dose (dose).
- peptides and polynucleotides of the present invention can be used to induce APCs and CTLs.
- CTLs can also be induced using the exosomes and APCs of the present invention.
- the peptides, polynucleotides, exosomes, and APCs of the present invention can be used in combination with any other compound as long as their ability to induce CTLs is not inhibited. Therefore, a CTL of the present invention can be induced using a pharmaceutical composition comprising any of the peptide, polynucleotide, APC and exosome of the present invention.
- APC of this invention can be induced
- the present invention provides a method for inducing APC having CTL inducing ability using the peptide or polynucleotide of the present invention.
- the method of the invention comprises contacting APC with a peptide of the invention in vitro, ex vivo or in vivo.
- a method of contacting APC with the peptide ex vivo may comprise the following steps: (A) recovering APC from the subject; and (b) contacting the APC of step (a) with the peptide of the present invention.
- the APC is not limited to a particular type of cell, but is known to present proteinaceous antigens on its cell surface as recognized by lymphocytes, DCs, Langerhans cells, macrophages, B cells, and Activated T cells can be used. Since DC has the strongest CTL inducing ability among APCs, DC can be preferably used.
- Any peptide of the invention can be used alone or with other peptides of the invention.
- the peptide of the present invention can be used in combination with other CTL-inducing peptides (for example, other TAA-derived CTL-inducing peptides).
- the methods of the invention can include the step of administering to the subject a peptide of the invention.
- the polynucleotide of the present invention is administered to a subject in an expressible form, the peptide of the present invention is expressed in vivo, which comes into contact with APC in vivo, and as a result, APC having a high CTL inducing ability is produced. Induced in the subject's body.
- the present invention can also include the step of administering a polynucleotide of the present invention to a subject.
- the present invention can also include the step of introducing the polynucleotide of the present invention into APC in order to induce APC having CTL inducing ability.
- the method may include the following steps: (A) recovering APC from the subject; and (b) introducing a polynucleotide encoding the peptide of the present invention into the APC in step (a).
- Step (b) can be performed as described above in section "VI. Antigen-presenting cells (APC)".
- the present invention provides a method for inducing APC having CTL inducing ability, comprising the following steps (a) or (b): (A) contacting APC with a peptide of the invention; (B) introducing a polynucleotide encoding the peptide of the present invention into APC.
- the present invention also provides a method for preparing APC having CTL inducing ability, comprising the following steps (a) or (b): (A) contacting APC with a peptide of the invention; (B) introducing a polynucleotide encoding the peptide of the present invention into APC.
- the above method can be performed in vitro, ex vivo, or in vivo, but is preferably performed in vitro or ex vivo.
- the APC used in the above method may be derived from a subject to whom administration of induced APC is scheduled, but may be derived from a different subject.
- the HLA type of the subject and the donor needs to be the same.
- the peptides of the present invention are represented by SEQ ID NOs: 3, 5-7, 9, 10, 12-14, 17, 19, 21, 30, 35, 38-40, 45, 53, 56 and 58.
- the HLA types of the administration subject and the donor are both HLA-A11 (more preferably HLA-A * 1101).
- the APC used in the above method is preferably an APC expressing HLA-A11 (more preferably HLA-A * 1101).
- the administration subject and donor The HLA is preferably HLA-A33 (more preferably HLA-A * 3303).
- the APC used in the above method is preferably an APC expressing HLA-A33 (more preferably HLA-A * 3303).
- the HLA types of the administration subject and the donor are both HLA- A03 (more preferably HLA-A * 0301) is preferred.
- the APC used in the above method is preferably an APC expressing HLA-A03 (more preferably HLA-A * 0301).
- APC can be prepared from blood collected from a donor by specific gravity centrifugation or the like, and then prepared from the PBMC using a known method.
- the present invention also provides a pharmaceutical composition for inducing ACTL having the ability to induce CTL, comprising the peptide of the present invention or a polynucleotide encoding the peptide.
- the present invention further provides use of the peptide of the present invention or a polynucleotide encoding the peptide in the manufacture of a pharmaceutical composition for inducing APC having CTL inducing ability.
- the present invention further provides a peptide of the present invention or a polynucleotide encoding the peptide for use in the induction of APC having the ability to induce CTL.
- the present invention further relates to a method or process for producing a pharmaceutical composition for inducing APC comprising a peptide of the present invention or a polynucleotide encoding the peptide, pharmaceutically or physiologically.
- a method or process is provided that includes the step of formulating an acceptable carrier.
- the present invention also provides a method or process for producing a pharmaceutical composition for inducing APC having CTL inducing ability, comprising the peptide of the present invention or a polynucleotide encoding the peptide.
- a method or process is provided that comprises admixing with a pharmaceutically or physiologically acceptable carrier.
- APC induced by the method of the present invention can induce CTL specific for CDCA1 (ie, CTL of the present invention).
- the present invention also provides a method for inducing CTL using the peptide, polynucleotide, exosome, or APC of the present invention.
- the invention also provides one or more polynucleotides that encode a polypeptide (ie, TCR subunit) that can form a T cell receptor (TCR) that can recognize a complex of a peptide of the invention and an HLA antigen.
- the method of inducing CTL comprises at least one step selected from the following: (A) contacting a CD8 positive T cell with an antigen presenting cell that presents a complex of the HLA antigen and the peptide of the present invention on its surface; (B) contacting a CD8 positive T cell with an exosome presenting a complex of the HLA antigen and the peptide of the present invention on its surface; and (c) a complex of the peptide of the present invention and the HLA antigen.
- the methods of the invention can include administering to the subject a peptide, polynucleotide, APC, or exosome of the invention.
- CTLs can be induced by using them in vitro or ex vivo.
- the method of the present invention may include the following steps: (A) recovering APC from the subject; (B) contacting the APC of step (a) with the peptide of the present invention; and (c) co-culturing the APC of step (b) with CD8 positive T cells. The induced CTL may then be returned to the subject.
- the APC co-cultured with the CD8-positive T cell in the above step (c) introduces the polynucleotide encoding the peptide of the present invention into the APC as described above in the section “VI. Antigen-presenting cells (APC)”. Can also be prepared.
- APC Antigen-presenting cells
- the APC used in the method of the present invention is not limited to this, and any APC that presents a complex of the HLA antigen and the peptide of the present invention on its surface can be used.
- an exosome that presents a complex of the HLA antigen and the peptide of the present invention on its surface can be used instead of such APC. That is, the method of the present invention can include the step of co-culturing exosomes that present a complex of the HLA antigen and the peptide of the present invention on its surface.
- exosomes can be prepared by the methods described above in the section “V. Exosomes”.
- CTL can be induced by introducing a vector containing a polynucleotide encoding each subunit of TCR capable of binding to the peptide of the present invention presented by the HLA antigen on the cell surface into CD8-positive T cells. it can.
- Such transduction can be performed as described above in the section “VIII. T Cell Receptor (TCR)”.
- the present invention provides a method for inducing CTL comprising the step selected from: (A) co-culturing CD8 positive T cells with APC presenting a complex of HLA antigen and the peptide of the present invention on its surface; (B) co-culturing a CD8 positive T cell with an exosome that presents a complex of HLA antigen and the peptide of the present invention on its surface; and (c) a book presented by HLA antigen on the cell surface.
- the above method can be performed in vitro, ex vivo, or in vivo, but is preferably performed in vitro or ex vivo.
- the APC or exosome and CD8 positive T cell used in the above method may be derived from a subject to whom the induced CTL is to be administered, or may be derived from a different subject. Good.
- APC or exosome derived from a subject (donor) different from the subject to be administered, and CD8 positive T cells the HLA type of the subject and the donor needs to be the same.
- the peptide of the present invention is selected from SEQ ID NOs: 3, 5 to 7, 9, 10, 12 to 14, 17, 19, 21, 30, 35, 38 to 40, 45, 53, 56 and 58
- the HLA types of the administration subject and the donor are both HLA-A11 (more preferably HLA-A * 1101).
- the APC or exosome used in the above method is HLA-A11 (more preferably HLA-A * 1101) and the peptide of the present invention (SEQ ID NOs: 3, 5-7, 9, 10, 12-14, 17 , 19, 21, 30, 35, 38 to 40, 45, 53, 56 and 58, a peptide having an amino acid sequence selected from the above, or a modified peptide thereof) Exosome is preferred.
- the induced CTL exhibits specific cytotoxic activity against cells that present a complex of HLA-A11 and the peptide of the present invention (for example, HLA-A11 positive cells that express CDCA1). .
- the HLA of the administration subject and the donor are preferably HLA-A33 (more preferably HLA-A * 3303).
- the APC or exosome used in the above method is HLA-A33 (more preferably HLA-A * 3303) and the peptide of the present invention (SEQ ID NOs: 27, 3, 60, 28, 5, 67 and 69). It is preferably an APC or exosome that presents a complex with a peptide having an amino acid sequence selected from or a modified peptide thereof on its surface.
- the induced CTL exhibits specific cytotoxic activity against cells that present a complex of HLA-A33 and the peptide of the present invention (for example, HLA-A33 positive cells that express CDCA1). .
- the HLA types of the administration subject and the donor are both HLA-A03. (More preferably, HLA-A * 0301) is preferable.
- the APC or exosome used in the above method has an amino acid sequence selected from HLA-A03 (more preferably HLA-A * 0301) and the peptide of the present invention (SEQ ID NOs: 7, 38 and 47) Peptides or their modified peptides) are preferably APCs or exosomes that present complexes on their surface.
- the induced CTL exhibits specific cytotoxic activity against cells that present a complex of HLA-A03 and the peptide of the present invention (for example, HLA-A03 positive cells expressing CDCA1). .
- the present invention also provides a composition or pharmaceutical composition for inducing CTL comprising at least one active ingredient selected from the following: (A) the peptide of the present invention; (B) a polynucleotide encoding the peptide of the present invention in an expressible form; (C) APC presenting the peptides of the present invention on its surface; and (d) exosomes presenting the peptides of the present invention on its surface.
- the present invention also provides the use of an active ingredient selected from among the following in the manufacture of a composition or pharmaceutical composition for inducing CTL: (A) the peptide of the present invention; (B) a polynucleotide encoding the peptide of the present invention in an expressible form; (C) APC presenting the peptides of the present invention on its surface; and (d) exosomes presenting the peptides of the present invention on its surface.
- an active ingredient selected from among the following in the manufacture of a composition or pharmaceutical composition for inducing CTL: (A) the peptide of the present invention; (B) a polynucleotide encoding the peptide of the present invention in an expressible form; (C) APC presenting the peptides of the present invention on its surface; and (d) exosomes presenting the peptides of the present invention on its surface.
- the present invention further provides an active ingredient selected from among the following for use in the induction of CTL: (A) the peptide of the present invention; (B) a polynucleotide encoding the peptide of the present invention in an expressible form; (C) APC presenting the peptides of the present invention on its surface; and (d) exosomes presenting the peptides of the present invention on its surface.
- the present invention further relates to a method or process for producing a composition or pharmaceutical composition for inducing CTLs, comprising an active ingredient selected from the following, pharmaceutically or physiologically
- a method or process comprises formulating an acceptable carrier with: (A) the peptide of the present invention; (B) a polynucleotide encoding the peptide of the present invention in an expressible form; (C) APC presenting the peptides of the present invention on its surface; and (d) exosomes presenting the peptides of the present invention on its surface.
- the present invention also provides a method or process for producing a composition or pharmaceutical composition for inducing CTL, wherein the active ingredient selected from the following is pharmaceutically or physiologically
- a method or process comprising the step of admixing with a chemically acceptable carrier is provided: (A) the peptide of the present invention; (B) a polynucleotide encoding the peptide of the present invention in an expressible form; (C) APC presenting the peptides of the present invention on its surface; and (d) exosomes presenting the peptides of the present invention on its surface.
- the present invention further provides a method for inducing an immune response against a cancer that expresses CDCA1.
- Applicable cancers include bladder cancer, breast cancer, cervical cancer, cholangiocarcinoma, chronic myelogenous leukemia (CML), esophageal cancer, stomach cancer, non-small cell lung cancer, lymphoma, osteosarcoma, prostate , Renal cancer, small cell lung cancer, head and neck cancer, soft tissue tumor, and colorectal cancer.
- the cancer preferably expresses at least one HLA selected from HLA-A11, HLA-A33 and HLA-A03.
- the present invention also provides a method for inducing an immune response against a cancer cell expressing CDCA1.
- CDCA1 is found to be overexpressed in various cancers as described above. Therefore, when an immune response against a cancer cell that expresses CDCA1 is induced, growth of the cancer cell is inhibited as a result. Accordingly, the present invention also provides a method of inhibiting the growth of cancer cells that express CDCA1.
- the method of the present invention is particularly suitable for inhibiting the growth of cancer cells expressing at least one HLA selected from CDCA1 and HLA-A11, HLA-A33 and HLA-A03.
- the method of the present invention may comprise the step of administering a composition comprising any of the peptides of the present invention or a polynucleotide encoding them.
- the methods of the present invention also contemplate administration of exosomes or APCs that present any of the peptides of the present invention.
- exosomes and APCs that can be used in the methods of the invention to induce an immune response include the aforementioned “V. exosomes”, “VI. Antigen presenting cells (APCs)”, and “X. It is described in detail in the sections (1) and (2) of “Method Using Exosome, APC, and CTL”.
- the present invention also provides a pharmaceutical composition or vaccine for inducing an immune response against a cancer expressing CDCA1, comprising an active ingredient selected from the following: Or provide a vaccine: (A) the peptide of the present invention; (B) a polynucleotide encoding the peptide of the present invention in an expressible form; (C) APC presenting the peptide of the invention on its surface; (D) an exosome presenting a peptide of the invention on its surface; and (e) a CTL of the invention.
- a vaccine comprising an active ingredient selected from the following: Or provide a vaccine: (A) the peptide of the present invention; (B) a polynucleotide encoding the peptide of the present invention in an expressible form; (C) APC presenting the peptide of the invention on its surface; (D) an exosome presenting a peptide of the invention on its surface; and (e) a CTL of the invention.
- the present invention further relates to a pharmaceutical composition or vaccine for inducing an immune response against a cancer cell expressing CDCA1, which comprises an active ingredient selected from the following: I will provide a: (A) the peptide of the present invention; (B) a polynucleotide encoding the peptide of the present invention in an expressible form; (C) APC presenting the peptide of the invention on its surface; (D) an exosome presenting a peptide of the invention on its surface; and (e) a CTL of the invention.
- a pharmaceutical composition or vaccine for inducing an immune response against a cancer cell expressing CDCA1 which comprises an active ingredient selected from the following: I will provide a: (A) the peptide of the present invention; (B) a polynucleotide encoding the peptide of the present invention in an expressible form; (C) APC presenting the peptide of the invention on its surface; (D) an exosome presenting a peptide of the
- the present invention further relates to a pharmaceutical composition or vaccine for inhibiting the growth of cancer cells expressing CDCA1, wherein the pharmaceutical composition or vaccine comprises an active ingredient selected from the following: provide: (A) the peptide of the present invention; (B) a polynucleotide encoding the peptide of the present invention in an expressible form; (C) APC presenting the peptide of the invention on its surface; (D) an exosome presenting a peptide of the invention on its surface; and (e) a CTL of the invention.
- an active ingredient selected from the following: provide: (A) the peptide of the present invention; (B) a polynucleotide encoding the peptide of the present invention in an expressible form; (C) APC presenting the peptide of the invention on its surface; (D) an exosome presenting a peptide of the invention on its surface; and (e) a CTL of the invention.
- the present invention also provides the use of an active ingredient selected from among the following in the manufacture of a pharmaceutical composition or vaccine for inducing an immune response against a cancer expressing CDCA1: (A) the peptide of the present invention; (B) a polynucleotide encoding the peptide of the present invention in an expressible form; (C) APC presenting the peptide of the invention on its surface; (D) an exosome presenting a peptide of the invention on its surface; and (e) a CTL of the invention.
- an active ingredient selected from among the following in the manufacture of a pharmaceutical composition or vaccine for inducing an immune response against a cancer expressing CDCA1: (A) the peptide of the present invention; (B) a polynucleotide encoding the peptide of the present invention in an expressible form; (C) APC presenting the peptide of the invention on its surface; (D) an exosome presenting a peptide of the invention on its surface; and (
- the present invention further provides the use of an active ingredient selected from among the following in the manufacture of a pharmaceutical composition or vaccine for inducing an immune response against cancer cells expressing CDCA1: (A) the peptide of the present invention; (B) a polynucleotide encoding the peptide of the present invention in an expressible form; (C) APC presenting the peptide of the invention on its surface; (D) an exosome presenting a peptide of the invention on its surface; and (e) a CTL of the invention.
- an active ingredient selected from among the following in the manufacture of a pharmaceutical composition or vaccine for inducing an immune response against cancer cells expressing CDCA1: (A) the peptide of the present invention; (B) a polynucleotide encoding the peptide of the present invention in an expressible form; (C) APC presenting the peptide of the invention on its surface; (D) an exosome presenting a peptide of the invention on its surface; and (e)
- the present invention further provides the use of an active ingredient selected from among the following in the manufacture of a pharmaceutical composition or vaccine for inhibiting the growth of cancer cells expressing CDCA1: (A) the peptide of the present invention; (B) a polynucleotide encoding the peptide of the present invention in an expressible form; (C) APC presenting the peptide of the invention on its surface; (D) an exosome presenting a peptide of the invention on its surface; and (e) a CTL of the invention.
- an active ingredient selected from among the following in the manufacture of a pharmaceutical composition or vaccine for inhibiting the growth of cancer cells expressing CDCA1: (A) the peptide of the present invention; (B) a polynucleotide encoding the peptide of the present invention in an expressible form; (C) APC presenting the peptide of the invention on its surface; (D) an exosome presenting a peptide of the invention on its surface; and (e) a CTL
- the present invention also provides a method or process for producing a pharmaceutical composition that induces an immune response against a cancer expressing CDCA1, wherein the peptide or polynucleotide of the present invention is mixed with a pharmaceutically acceptable carrier.
- a method is provided that may include the step of formulating.
- the present invention provides a method for inhibiting the growth of cancer cells expressing CDCA1, comprising the step of administering to a subject a vaccine or pharmaceutical composition comprising an active ingredient selected from: Providing a method of inducing an immune response against a cancer that expresses: (A) the peptide of the present invention; (B) a polynucleotide encoding the peptide of the present invention in an expressible form; (C) APC presenting the peptide of the invention on its surface; (D) an exosome presenting a peptide of the invention on its surface; and (e) a CTL of the invention.
- a cancer expressing CDCA1 can be treated by administering the peptide, polypeptide, APC, exosome and / or CTL of the present invention.
- an immune response against a cancer expressing CDCA1 can be induced by administering the peptide, polypeptide, APC, exosome and / or CTL of the present invention.
- cancers include bladder cancer, breast cancer, cervical cancer, cholangiocarcinoma, chronic myelogenous leukemia (CML), esophageal cancer, stomach cancer, non-small cell lung cancer, lymphoma, osteosarcoma, Examples include, but are not limited to, prostate cancer, kidney cancer, small cell lung cancer, head and neck cancer, soft tissue tumor, and colon cancer.
- an immune response against a cancer cell expressing CDCA1 can be induced by administering the peptide, polypeptide, APC, exosome and / or CTL of the present invention. Therefore, it is preferable to confirm whether the expression level of CDCA1 at the disease site to be treated is enhanced before administering a vaccine or pharmaceutical composition containing the above active ingredient.
- the invention provides a method of treating a cancer in a patient in need of treatment for a cancer that expresses CDCA1, and such method comprises the following steps: i) measuring the expression level of CDCA1 in a biological sample collected from a disease site of a subject having cancer; ii) identifying a subject having a cancer that expresses CDCA1 based on the expression level of CDCA1 measured in i); and iii) at least selected from the group consisting of (a) to (e) above Administering one component to a subject with a cancer that overexpresses CDCA1 compared to a normal control.
- the present invention also provides a vaccine or pharmaceutical composition comprising at least one active ingredient selected from the group consisting of (a) to (e) above for administration to a subject having a cancer that expresses CDCA1.
- the present invention further provides a method for identifying or selecting a subject to be treated with at least one active ingredient selected from the group consisting of (a) to (e) above, which method comprises the following steps: : i) measuring the expression level of CDCA1 in a biological sample collected from a disease site of a subject having cancer; ii) identifying a subject having a cancer that expresses CDCA1 based on the expression level of CDCA1 measured in i); and iii) identifying the subject identified in ii) above (a) to (e Identifying or selecting as a subject that can be treated with at least one active ingredient selected from the group consisting of:
- the biological sample collected from the subject in order to measure the expression level of CDCA1 in the above method is not particularly limited.
- a tissue sample containing cancer cells collected by biopsy or the like can be preferably used.
- the expression level of CDCA1 in a biological sample can be measured by a known method, for example, a method of detecting a transcription product of CDCA1 gene by a probe or PCR method (for example, cDNA microarray method, Northern blot method, RT-PCR method) Etc.), a method for detecting the translation product of the CDCA1 gene with an antibody or the like (for example, Western blotting, immunostaining, etc.) and the like can be used.
- the biological sample may be a blood sample.
- the blood level of an antibody against CDCA1 or a fragment thereof is measured, and the expression level of CDCA1 at the disease site is evaluated based on the blood level. Also good.
- the blood level of the antibody against CDCA1 can be measured using a known method. For example, enzyme immunoassay (EIA) using the CDCA1 protein or the peptide of the present invention as an antigen, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and the like can be used.
- EIA enzyme immunoassay
- ELISA enzyme-linked immunosorbent assay
- RIA radioimmunoassay
- Whether or not the cancer of the subject expresses CDCA1 is determined based on the same kind of biological material collected from the non-cancerous part of the subject or the same kind of living body collected from the subject not having cancer. You may carry out by the comparison with the measurement result in a material (normal control sample). That is, when the level in the biological sample to be tested is increased compared to the level of the measurement target in the normal control sample (normal control level), the subject cancer expresses CDCA1 It can be judged. For example, when the detected amount of the measurement object is increased by at least 10% or more compared to the normal control level, it may be determined that the cancer of the object expresses CDCA1.
- the detected amount of the measurement object is increased more than 25% or more, more preferably 50% or more than the normal control level.
- the detected amount of transcription product or translation product of CDCA1 can be evaluated by normalizing to the detected amount of known housekeeping genes such as ⁇ -actin, glyceraldehyde 3-phosphate dehydrogenase and ribosomal protein P1. Good.
- an HLA-A11 positive subject as an administration subject of the active ingredient related to the peptide.
- an HLA-A33 positive subject should be selected as an administration subject of an active ingredient related to a peptide having an amino acid sequence selected from SEQ ID NOs: 27, 3, 60, 28, 5, 67 and 69.
- an HLA-A33 positive subject should be selected as an administration subject of an active ingredient related to a peptide having an amino acid sequence selected from SEQ ID NOs: 7, 38 and 47.
- the present invention also provides a complex of the peptide of the present invention and HLA.
- the complex of the present invention may be a monomer or a multimer.
- the number of polymerizations is not particularly limited, and can be a multimer of any number of polymerizations. Examples include, but are not limited to, tetramer, pentamer, hexamer and the like.
- Dextramers WO2002 / 072631
- streptamers KnabelnaM et al.,. Nat Med. 2002 Jun; 8 (6): 631-7.
- the complex of the peptide of the present invention and HLA can be prepared according to a known method (for example, Altman JD et al., Science.1996,274 (5284): 94-6, WO2002 / 072631, WO2009 / 003492 Knabel M et al., Nat Med. 2002 Jun; 8 (6): 631-7.
- the complex of the present invention can be used, for example, for quantification of CTL specific for the peptide of the present invention.
- a blood sample is collected from a subject administered with the pharmaceutical composition of the present invention, PBMCs are separated, CD4 negative cells are prepared, and the complex of the present invention conjugated with a fluorescent dye and the CD4 negative cells Make contact.
- the ratio of CTL specific for the peptide of the present invention can be measured by analyzing by flow cytometry. For example, by measuring CTL specific for the peptide of the present invention before, during and / or after administration of the pharmaceutical composition of the present invention, the immune response inducing effect of the pharmaceutical composition of the present invention can be increased. Can be monitored.
- the present invention further provides antibodies that bind to the peptides of the present invention.
- Preferred antibodies specifically bind to peptides of the invention and do not bind (or bind weakly) to non-peptides of the invention.
- such antibodies may include antibodies that recognize peptides in the context of HLA molecules, ie, antibodies that bind to peptide-MHC complexes.
- the binding specificity of the antibody can be confirmed by an inhibition test. That is, when the binding between the antibody to be analyzed and the full-length CDCA1 polypeptide is inhibited in the presence of the peptide of the present invention, this indicates that the antibody specifically binds to the peptide of the present invention.
- the antibodies against the peptides of the present invention can be used in disease diagnosis and prognostic assays, as well as in the selection of subjects for administration of the pharmaceutical compositions of the present invention and monitoring of the pharmaceutical compositions of the present invention.
- the present invention also provides various immunological assay methods for detecting and / or quantifying the peptides of the present invention or fragments thereof.
- immunological assays include, but are not limited to, radioimmunoassay, immunochromatography, enzyme-linked immunosorbent assay (ELISA), enzyme-linked immunofluorescence assay (ELIFA), etc. It is performed within a variety of immunological assay formats well known in the art.
- the antibody of the present invention can be used in an immunological imaging method capable of detecting a disease expressing CDCA1, and examples thereof include a radioscintigraphic imaging method using the labeled antibody of the present invention.
- the present invention is not limited to this.
- Such assays are used clinically in the detection, monitoring, and prognosis of cancers that express CDCA1, and examples of such cancers include bladder cancer, breast cancer, cervical cancer, bile ducts Cell cancer, chronic myelogenous leukemia (CML), esophageal cancer, stomach cancer, non-small cell lung cancer, lymphoma, osteosarcoma, prostate cancer, kidney cancer, small cell lung cancer, head and neck cancer, soft tissue tumor, and Examples include, but are not limited to, colorectal cancer.
- CML chronic myelogenous leukemia
- the antibody of the present invention can be used in any form such as a monoclonal antibody or a polyclonal antibody, for example, antiserum obtained by immunizing animals such as rabbits with the peptide of the present invention, all classes of polyclonal antibodies and monoclonal antibodies. It may further include antibodies, human antibodies, and chimeric and humanized antibodies produced by genetic recombination.
- the peptide of the present invention or a fragment thereof used as an antigen for obtaining an antibody can be obtained by chemical synthesis or genetic engineering techniques based on the amino acid sequence disclosed in the present specification.
- the peptide used as an immunizing antigen may be a peptide of the present invention or a fragment of the peptide of the present invention.
- KLH keyhole limpet hemocyanin
- Methods for conjugating KLH and peptides are also well known in the art.
- Rodentia any mammal can be immunized with the antigen, but when producing a monoclonal antibody, it is preferable to take into consideration compatibility with the parent cell used for cell fusion.
- Rodent animals include, for example, mice, rats, and hamsters.
- Rabbits include, for example, rabbits.
- Primatological animals include, for example, monkeys of the Catarrhini (Old World monkey) such as cynomolgus monkeys (Macaca fascicularis), rhesus monkeys, baboons, and chimpanzees.
- antigen Intraperitoneal or subcutaneous injection of antigen is a standard method for immunizing mammals. More specifically, the antigen is diluted with an appropriate amount of phosphate buffered saline (PBS), physiological saline or the like and suspended. If desired, the antigen suspension can be mixed with an appropriate amount of a standard adjuvant, such as Freund's complete adjuvant, emulsified and administered to a mammal. Thereafter, the antigen mixed with an appropriate amount of Freund's incomplete adjuvant is preferably administered several times every 4 to 21 days. A suitable carrier may be used for immunization. After immunization as described above, serum can be examined by standard methods for increasing amounts of the desired antibody.
- PBS phosphate buffered saline
- a suitable carrier may be used for immunization. After immunization as described above, serum can be examined by standard methods for increasing amounts of the desired antibody.
- Polyclonal antibodies against the peptides of the present invention can be prepared by collecting blood from mammals that have been confirmed to have increased desired antibody levels in serum after immunization and separating the serum from blood by any conventional method. it can.
- the polyclonal antibody may be a serum containing a polyclonal antibody, and a fraction containing the polyclonal antibody may be isolated from the serum.
- Immunoglobulin G or M is obtained from a fraction that recognizes only the peptide of the present invention by using, for example, an affinity column to which the peptide of the present invention is bound, and further using this protein A or protein G column. It can be purified and prepared.
- immune cells are collected from the mammal and subjected to cell fusion.
- the immune cells used for cell fusion can be preferably obtained from the spleen.
- a myeloma cell of a mammal preferably a myeloma cell that has acquired the characteristics for selection of a fusion cell by a drug can be used.
- the above immune cells and myeloma cells can be fused.
- Hybridomas obtained by cell fusion can be selected by culturing them in a standard selective medium such as HAT medium (medium containing hypoxanthine, aminopterin, and thymidine). Cell culture typically continues in HAT medium for a period of time sufficient to kill all other cells (non-fused cells) except the desired hybridoma (eg, days to weeks). Thereafter, standard limiting dilution can be performed to screen and clone hybridoma cells producing the desired antibody.
- HAT medium medium containing hypoxanthine, aminopterin, and thymidine
- human lymphocytes such as lymphocytes infected with EB virus can be transformed with peptides, cells expressing the peptides, or lysates thereof. It is also possible to immunize in vitro. Subsequently, the lymphocyte after immunization is fused with an infinitely-dividable human-derived myeloma cell such as U266 to obtain a hybridoma that produces a desired human antibody that can bind to the peptide (Japanese Patent Application Laid-Open No. Sho A). 63-17688).
- the obtained hybridoma is transplanted into the abdominal cavity of the mouse, and ascites is extracted.
- the obtained monoclonal antibody can be purified by, for example, ammonium sulfate precipitation, protein A or protein G column, DEAE ion exchange chromatography, or an affinity column to which the peptide of the present invention is bound.
- immune cells that produce antibodies can be immortalized by oncogenes and used to prepare monoclonal antibodies.
- the monoclonal antibodies thus obtained can also be prepared recombinantly using genetic engineering techniques (see, for example, Borrebaeck and Larrick, Therapeutic Monoclonal Antibodies, published in the UK by MacMillan Publishers LTD (1990)).
- Wanna For example, DNA encoding an antibody is cloned from an immune cell such as an antibody-producing hybridoma or immunized lymphocyte, inserted into an appropriate vector, and then introduced into a host cell to prepare a recombinant antibody. Can do.
- the present invention also provides a recombinant antibody prepared as described above.
- the antibody of the present invention may be an antibody fragment or a modified antibody as long as it binds to the peptide of the present invention.
- the antibody fragment desirably contains an antigen-binding site of an antibody.
- the antibody fragment may be Fab, F (ab ′) 2 , Fv, or a single chain Fv (scFv) in which Fv fragments derived from H and L chains are linked by an appropriate linker.
- an antibody fragment can be prepared by treating an antibody with an enzyme such as papain or pepsin.
- a gene encoding the antibody fragment can be constructed, inserted into an expression vector and expressed in a suitable host cell (eg, Co et al., J Immunol 152: 2968-76 (1994); Better and Horwitz, Methods Enzymol 178: 476-96 (1989); Pluckthun and Skerra, Methods Enzymol 178: 497-515 (1989); Lamoyi, Methods Enzymol 121: 652-63 (1986); Rousseaux et al., Methods Enzymol 121: 663-9 (1986); see Bird and Walker, Trends Biotechnol 9: 132-7 (1991)).
- Antibodies can be modified by binding with various molecules such as polyethylene glycol (PEG).
- PEG polyethylene glycol
- the present invention provides such modified antibodies.
- Modified antibodies can be obtained by chemically modifying antibodies. These modification methods are routine in the art.
- the antibody of the present invention is a chimeric antibody between a variable region derived from a non-human antibody and a constant region derived from a human antibody, or a complementarity determining region (CDR) derived from a non-human antibody and a human antibody. It can also be obtained as a humanized antibody comprising a framework region (FR) derived from and a constant region.
- Such antibodies can be prepared according to known techniques. Humanization can be performed by replacing the corresponding sequence of a human antibody with the CDR sequence of a non-human antibody (see, for example, Verhoeyen et al., Science 239: 1534-6 (1988)). Accordingly, such humanized antibodies are chimeric antibodies in which substantially less than a human variable domain has been replaced by the corresponding sequence from a non-human species.
- human antibodies containing human variable regions can also be used.
- Such antibodies can be generated using various techniques known in the art. For example, in vitro methods include the use of recombinant libraries of human antibody fragments displayed on bacteriophages (eg, Hoogenboom & Winter, J. Mol. Biol. 227: 381 1991 (1991)).
- human antibodies can be made by introducing human immunoglobulin loci into transgenic animals, such as mice, in which the endogenous immunoglobulin genes are partially or completely inactivated. This approach is described, for example, in US Pat. Nos. 6,150,584, 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425;
- the antibody obtained as described above may be purified to homogeneity.
- separation and purification of antibodies can be performed according to separation and purification methods used for general proteins. For example, but not limited to, appropriately selecting and combining the use of column chromatography such as affinity chromatography, filters, ultrafiltration, salting out, dialysis, SDS polyacrylamide gel electrophoresis, and isoelectric focusing
- column chromatography such as affinity chromatography, filters, ultrafiltration, salting out, dialysis, SDS polyacrylamide gel electrophoresis, and isoelectric focusing
- Protein A and protein G columns can be used as affinity columns.
- Exemplary protein A columns to be used include, for example, HyperD, POROS, and Sepharose F.F. (Pharmacia).
- Exemplary chromatography includes, in addition to affinity chromatography, for example, ion exchange chromatography, hydrophobic chromatography, gel filtration, reverse phase chromatography, adsorption chromatography, etc. (Strategies for Protein Purification and Characterization: A Laboratory Course Manual. Ed Daniel R. Marshak et al., Cold Spring Harbor Laboratory Press (1996)). Chromatographic procedures can be performed by liquid phase chromatography such as HPLC and FPLC.
- ELISA enzyme-linked immunosorbent assay
- EIA enzyme immunoassay
- RIA radioimmunoassay
- IF immunofluorescence
- the antibody of the present invention is immobilized on a plate, the peptide of the present invention is added to the plate, and then a sample containing a desired antibody such as a culture supernatant of antibody-producing cells or a purified antibody is added.
- a secondary antibody that recognizes the primary antibody and is labeled with an enzyme such as alkaline phosphatase is then added and the plate is incubated.
- an enzyme substrate such as p-nitrophenyl phosphate is added to the plate, the absorbance is measured, and the antigen binding activity of the sample is evaluated.
- a peptide fragment such as a C-terminal or N-terminal fragment may be used as an antigen.
- BIAcore Pharmacia
- the present invention By exposing the antibody of the present invention to a sample considered to contain the peptide of the present invention, and detecting or measuring an immune complex formed by the antibody and the peptide, the present invention can be obtained by the method as described above. Detection or measurement of the peptide.
- the antibodies of the present invention can also be used to detect the peptides of the present invention present in a blood sample (eg, a serum sample) of interest.
- the antibody of the present invention present in the blood sample (eg, serum sample) of the subject can be detected using the peptide of the present invention.
- the result of measuring the peptide of the present invention or the antibody of the present invention in the blood sample of the subject can be useful for selecting the administration target of the pharmaceutical composition of the present invention or monitoring the effect of the pharmaceutical composition of the present invention. it can.
- patients with antibodies to peptides administered as vaccines for example, may be highly responsive to vaccines. Therefore, when the peptide of the present invention is administered as a vaccine, the peptide of the present invention can be used as an immunoassay antigen for selecting by using an antibody of a highly responsive patient as an index.
- the present invention also provides a vector comprising a polynucleotide encoding the peptide of the present invention and a host cell into which the vector has been introduced.
- the vectors of the present invention can be used to retain a polynucleotide of the present invention in a host cell, to express a peptide of the present invention in a host cell, or to administer a polynucleotide of the present invention for gene therapy. .
- E. coli is the host cell and the vector is amplified in E. coli (eg, JM109, DH5 ⁇ , HB101, or XL1-Blue) and produced in large quantities, the vector is the “origin of replication” for amplification in E. coli.
- a marker gene for example, a drug resistance gene selected by a drug such as ampicillin, tetracycline, kanamycin, chloramphenicol, etc.
- M13 vectors, pUC vectors, pBR322, pBluescript, pCR-Script and the like can be used.
- an expression vector can be used.
- an expression vector to be expressed in E. coli must have the above characteristics in order to amplify in E. coli.
- the vector may be a promoter that can efficiently express a desired gene in E. coli, such as a lacZ promoter (Ward et al., Nature 341).
- the vector may contain a signal sequence for peptide secretion.
- An exemplary signal sequence that causes the peptide to be secreted into the periplasm of E. coli is the pelB signal sequence (Lei et al., J Bacteriol 169: 4379 (1987)).
- Means for introducing the vector into the target host cell include, for example, the calcium chloride method and the electroporation method.
- expression vectors derived from mammals for example, pcDNA3 (Invitrogen), and pEGF-BOS (Nucleic Acids Res 18 (17): 5322 (1990)
- pEF pCDM8
- insect cell-derived expression Vectors eg, “Bac-to-BAC baculovirus expression system” (GIBCO BRL), pBacPAK8)
- plant-derived expression vectors eg, pMH1, pMH2
- animal virus-derived expression vectors eg, pHSV, pMV, pAdexLcw
- Retrovirus-derived expression vectors eg, pZIpneo
- yeast-derived expression vectors eg, “Pichia expression kit” (Invitrogen), pNV11, SP-Q01
- Bacillus subtilis Expression vectors eg, pPL608, pKTH50
- pPL608, pKTH50 Bacillus subtilis Expression vectors
- the vector In order for a vector to be expressed in animal cells such as CHO, COS, or NIH3T3 cells, the vector must be a promoter required for expression in such cells, such as the SV40 promoter (Mulligan et al., Nature 277: 108 (1979 )), MMLV-LTR promoter, EF1 ⁇ promoter (Mizushima et al., Nucleic Acids Res 18: 5322 (1990)), CMV promoter and the like, and preferably a marker gene for selecting a transformant (eg, drug (eg, , Neomycin, G418).
- a promoter required for expression in such cells such as the SV40 promoter (Mulligan et al., Nature 277: 108 (1979 )), MMLV-LTR promoter, EF1 ⁇ promoter (Mizushima et al., Nucleic Acids Res 18: 5322 (1990)), CMV promoter and the like, and preferably a
- a peptide of less than 15 amino acids having an ability to induce cytotoxic T cells comprising an amino acid sequence selected from the following group: (A) SEQ ID NOs: 3, 5-7, 9, 10, 12-14, 17, 19, 21, 30, 35, 38-40, 45, 53, 56, 58, 27, 60, 28, 67, An amino acid sequence selected from the group consisting of 69 and 47; and (b) SEQ ID NOs: 3, 5-7, 9, 10, 12-14, 17, 19, 21, 30, 35, 38-40, 45, For amino acid sequences selected from the group consisting of 53, 56, 58, 27, 60, 28, 67, 69 and 47, 1, 2 or several amino acids are substituted, deleted, inserted and / or Or an added amino acid sequence.
- CTL cytotoxic T cells
- a composition comprising a pharmaceutically acceptable carrier and at least one component selected from the group consisting of the following (a) to (e): (A) one or more peptides according to any one of [1] to [3]; (B) one or more polynucleotides encoding the peptide according to any one of [1] to [3] in a form capable of being expressed; (C) an antigen-presenting cell (APC) that presents a complex of the peptide according to any one of [1] to [3] and an HLA antigen on its cell surface; (D) an exosome that presents a complex of the peptide according to any one of [1] to [3] and an HLA antigen on its cell surface; and (e) any of [1] to [3] A CTL targeting the peptide according to any one of the above.
- APC antigen-presenting cell
- D an exosome that presents a complex of the peptide according to any one of [1] to [3] and an HLA anti
- APC antigen-presenting cell
- composition according to [5] which is a pharmaceutical composition.
- a pharmaceutical composition for one or more uses selected from the group consisting of (i) cancer treatment, (ii) cancer prevention, and (iii) prevention of recurrence after cancer surgery The composition according to [7], wherein [9] The composition according to [7] for inducing an immune response against cancer.
- Cancer is bladder cancer, breast cancer, cervical cancer, cholangiocarcinoma, chronic myelogenous leukemia (CML), esophageal cancer, stomach cancer, non-small cell lung cancer, lymphoma, osteosarcoma, prostate cancer [8] or [9], wherein the composition is selected from the group consisting of: renal cancer, small cell lung cancer, head and neck cancer, soft tissue tumor, and colon cancer.
- a method for inducing APC having CTL inducing ability comprising a step selected from the group consisting of: (A) contacting APC with the peptide according to any one of [1] to [3] in vitro, ex vivo, or in vivo, and (b) any one of [1] to [3] A step of introducing a polynucleotide encoding the peptide described in 1 into APC.
- a method for inducing CTL comprising a step selected from the group consisting of the following (a) to (c): (A) co-culturing a CD8-positive T cell with APC presenting on its surface a complex of the HLA antigen and the peptide according to any one of [1] to [3], (B) co-culturing a CD8-positive T cell with an exosome that presents a complex of the HLA antigen and the peptide according to any one of [1] to [3] on its surface; and (c ) CD8 positive polynucleotide encoding each subunit of T cell receptor (TCR) capable of binding to the peptide according to any one of [1] to [3] presented by HLA antigen on the cell surface Introducing into T cells.
- TCR T cell receptor
- a method for inducing an immune response against cancer comprising administering to a subject at least one component selected from the group consisting of the following (a) to (e): (A) one or more peptides according to any one of [1] to [3]; (B) one or more polynucleotides encoding the peptide according to any one of [1] to [3] in a form capable of being expressed; (C) an antigen-presenting cell (APC) that presents a complex of the peptide according to any one of [1] to [3] and an HLA antigen on its cell surface; (D) an exosome that presents a complex of the peptide according to any one of [1] to [3] and an HLA antigen on its cell surface; and (e) any of [1] to [3] A CTL targeting the peptide according to any one of the above.
- APC antigen-presenting cell
- D an exosome that presents a complex of the peptide according to any one of [1] to
- Treating and / or preventing cancer and / or its recurrence after surgery comprising administering to a subject at least one component selected from the group consisting of (a) to (e) below: How to prevent: (A) one or more peptides according to any one of [1] to [3]; (B) one or more polynucleotides encoding the peptide according to any one of [1] to [3] in a form capable of being expressed; (C) an antigen-presenting cell (APC) that presents a complex of the peptide according to any one of [1] to [3] and an HLA antigen on its cell surface; (D) an exosome that presents a complex of the peptide according to any one of [1] to [3] and an HLA antigen on its cell surface; and (e) any of [1] to [3] A CTL targeting the peptide according to any one of the above.
- APC antigen-presenting cell
- D an exosome that presents a complex of
- a method for screening a peptide having CTL inducing ability comprising the following steps: (A) SEQ ID NOs: 3, 5-7, 9, 10, 12-14, 17, 19, 21, 30, 35, 38-40, 45, 53, 56, 58, 27, 60, 28, 67, An amino acid in which one, two, or several amino acid residues are substituted, deleted, inserted, and / or added to the original amino acid sequence consisting of an amino acid sequence selected from the group consisting of 69 and 47 Creating a candidate sequence of sequences; (B) selecting a candidate sequence having no significant homology (sequence identity) with any known human gene product other than CDCA1 from the candidate sequences prepared in (a); (C) contacting the peptide comprising the candidate sequence selected in (b) with APC; (D) a step of contacting the APC of (c) with a CD8-positive T cell; and (e) a step
- [22] Use of at least one active ingredient selected from the group consisting of the following (a) to (e) in the manufacture of a composition for inducing an immune response against cancer: (A) one or more peptides according to any one of [1] to [3]; (B) one or more polynucleotides encoding the peptide according to any one of [1] to [3] in a form capable of being expressed; (C) an antigen-presenting cell (APC) that presents a complex of the peptide according to any one of [1] to [3] and an HLA antigen on its cell surface; (D) an exosome that presents a complex of the peptide according to any one of [1] to [3] and an HLA antigen on its cell surface; and (e) any of [1] to [3] A CTL targeting the peptide according to any one of the above.
- a CTL antigen-presenting cell
- [23] At least selected from the group consisting of the following (a) to (e) in the manufacture of a pharmaceutical composition for the treatment and / or prevention of cancer and / or prevention of its recurrence after surgery: Use of one ingredient: (A) one or more peptides according to any one of [1] to [3]; (B) one or more polynucleotides encoding the peptide according to any one of [1] to [3] in a form capable of being expressed; (C) an antigen-presenting cell (APC) that presents a complex of the peptide according to any one of [1] to [3] and an HLA antigen on its cell surface; (D) an exosome that presents a complex of the peptide according to any one of [1] to [3] and an HLA antigen on its cell surface; and (e) any of [1] to [3] A CTL targeting the peptide according to any one of the above.
- APC antigen-presenting cell
- [24] Use of at least one component selected from the group consisting of the following (a) to (e) for inducing an immune response against cancer: (A) one or more peptides according to any one of [1] to [3]; (B) one or more polynucleotides encoding the peptide according to any one of [1] to [3] in a form capable of being expressed; (C) an antigen-presenting cell (APC) that presents a complex of the peptide according to any one of [1] to [3] and an HLA antigen on its cell surface; (D) an exosome that presents a complex of the peptide according to any one of [1] to [3] and an HLA antigen on its cell surface; and (e) any of [1] to [3] A CTL targeting the peptide according to any one of the above.
- APC antigen-presenting cell
- D an exosome that presents a complex of the peptide according to any one of [1] to [3] and an
- [25] Use of at least one component selected from the group consisting of (a) to (e) below for treating and / or preventing cancer and / or preventing its recurrence after surgery: (A) one or more peptides according to any one of [1] to [3]; (B) one or more polynucleotides encoding the peptide according to any one of [1] to [3] in a form capable of being expressed; (C) an antigen-presenting cell (APC) that presents a complex of the peptide according to any one of [1] to [3] and an HLA antigen on its cell surface; (D) an exosome that presents a complex of the peptide according to any one of [1] to [3] and an HLA antigen on its own cell surface; and (e) any of [1] to [4] A CTL targeting the peptide according to any one of the above.
- APC antigen-presenting cell
- D an exosome that presents a complex of the peptide
- a method for inducing cytotoxic activity against cells expressing CDCA1, comprising the step of administering to a subject at least one component selected from the group consisting of the following (a) to (e): (A) one or more peptides according to any one of [1] to [3]; (B) one or more polynucleotides encoding the peptide according to any one of [1] to [3] in a form capable of being expressed; (C) an antigen-presenting cell (APC) that presents a complex of the peptide according to any one of [1] to [3] and an HLA antigen on its cell surface; (D) an exosome that presents a complex of the peptide according to any one of [1] to [3] and an HLA antigen on its cell surface; and (e) any of [1] to [3] A CTL targeting the peptide according to any one of the above.
- APC antigen-presenting cell
- a freeze-dried preparation comprising one or more peptides according to any one of [1] to [3].
- a pharmaceutical composition prepared by a method comprising dissolving one or more peptides according to any one of [1] to [3] in a water-soluble carrier and sterilizing by filtration. .
- An aqueous solution comprising one or more peptides according to any one of [1] to [3] and a water-soluble carrier, which is sterilized by filtration.
- An emulsion comprising one or more peptides according to any one of [1] to [3], a water-soluble carrier, and an oily adjuvant.
- a kit comprising a container containing the composition according to any one of [5] to [11] and a container containing an adjuvant.
- a container containing a freeze-dried preparation containing the peptide according to any one of [1] to [3], a container containing an adjuvant, and a redissolved solution for the freeze-dried preparation A kit containing a container that is stored.
- Example 1 Materials and methods Cell line HLA-A and HLA-B negative human B lymphoblastoid cell line C1R, and African green monkey kidney cell line COS7 were purchased from ATCC.
- HLA-A * 1101 Creating stably expressing stimulator cells with HLA-A * 1101 the C1R stably expressing (C1R-A11) were used as stimulator cells.
- a cDNA encoding the open reading frame of HLA-A * 1101 was amplified by PCR and cloned into an expression vector.
- C1R cells were transformed with the expression vector and then selected for 2 weeks using G418 (Invitrogen). G418-selected cells were seeded into the wells of a 96-well plate containing a culture medium supplemented with G418 and further cultured for 30 days. Expression of exogenous HLA-A * 1101 in C1R cells was confirmed by flow cytometry analysis.
- Candidate selection of peptides derived from CDCA1 9mer and 10mer peptides derived from CDCA1 that bind to HLA-A * 1101 molecules are linked to the binding prediction server ⁇ NetMHC 3.2 '' (www.cbs.dtu.dk/services/NetMHC/) (Buus et al., Tissue Antigens. 2003 Nov, 62 (5): 378-84; Nielsen et al., Protein Sci. 2003 May, 12 (5): 1007-17, Bioinformatics. 2004 Jun 12:20 (9): 1388 -97).
- CTL-derived monocyte-derived dendritic cells were used as antigen presenting cells to induce cytotoxic T lymphocyte (CTL) responses to peptides presented on human leukocyte antigen (HLA).
- CTL cytotoxic T lymphocyte
- HLA human leukocyte antigen
- DCs were generated in vitro as described elsewhere (Nakahara S et al., Cancer Res 2003, 63 (14): 4112-8). Specifically, peripheral blood mononuclear cells isolated from healthy volunteers (HLA-A * 1101 positive) with Ficoll-Paque plus (Pharmacia) solution are attached to plastic tissue culture dishes (Becton Dickinson) And concentrated as monocyte fractions.
- AIM-V medium Invitrogen
- AS heat-inactivated autoserum
- a population enriched for monocytes was cultured in the presence of 4 (R & D System). After 7 days of culture, cytokine-induced DCs were pulsed with 20 ⁇ g / ml of each synthetic peptide in AIM-V medium at 37 ° C. for 3 hours in the presence of 3 ⁇ g / ml ⁇ 2-microglobulin.
- the generated cells appeared to express DC related molecules such as CD80, CD83, CD86, and HLA class II on their cell surface (data not shown).
- DC related molecules such as CD80, CD83, CD86, and HLA class II on their cell surface (data not shown).
- These peptide-pulsed DCs were then inactivated by X-ray irradiation (20 Gy) and mixed with autologous CD8 + T cells obtained by positive selection using CD8 Positive Isolation Kit (Dynal) at a ratio of 1:20 . These cultures were seeded in 48-well plates (Corning). Each well was filled with 1.5 x 10 4 peptide-pulsed DC, 3 x 10 5 CD8 + T cells and 10 ng / ml IL-7 (R & D System) in 0.5 ml AIM-V / 2% AS medium. ) Included.
- IL-2 (CHIRON) was added to these cultures to a final concentration of 20 IU / ml.
- T cells were further stimulated with peptide-pulsed autologous DCs.
- DC was prepared each time by the same method as above.
- C1R-A11 cells pulsed with peptides were tested for CTL in human interferon (IFN) - ⁇ enzyme-linked immunospot (ELISPOT) assay (Tanaka H et al., Br J Cancer 2001, 84 (1): 94-9; Umano Y et al., Br J Cancer 2001, 84 (8): 1052-7; Uchida N et al., Clin Cancer Res 2004, 10 (24): 8577-86; Suda T et al., Cancer Sci 2006, 97 (5): 411-9; Watanabe T et al., Cancer Sci 2005, 96 (8): 498-506).
- IFN interferon
- ELISPOT enzyme-linked immunospot
- CTL proliferation procedure described by Riddell et al. (Walter EA et al., N Engl J Med 1995, 333 (16): 1038-44; Riddell SR et al., Nat Med 1996, 2 (2): 216-23) CTL were grown in culture using methods similar to those used. CTL was combined with 2 human B lymphoblastoid cell lines treated with 5 ⁇ 10 6 cells / flask mitomycin C, and 40 ng / ml anti-CD3 antibody for a total of 25 ml of 5% AS-containing AIM-V Co-cultured in (AIM-V / 5% AS) medium. One day after the start of culture, 120 IU / ml IL-2 was added to the culture.
- CTL clones Dilution was carried out so that there were 0.3, 1, and 3 cells / well of CTL in a 96 round bottom microtiter plate (Nalge Nunc International). CTL in a total volume of 150 ⁇ l with 2 human B lymphoblastoid cell lines treated with 1 ⁇ 10 4 cells / well mitomycin C, 30 ng / ml anti-CD3 antibody, and 125 IU / ml IL-2 / Well in AIM-V / 5% AS medium. Ten days later, 50 ⁇ l / well of IL-2 was added to the medium so that the final concentration of IL-2 reached 125 IU / ml.
- IFN- ⁇ ELISPOT assay IFN- ⁇ enzyme-linked immunosorbent assay (ELISA) were performed. Specifically, peptide-pulsed C1R-A11 (1 ⁇ 10 4 cells / well) was prepared as a stimulator cell. Induced CTL, ie CTL lines and CTL clones, were used as responder cells. IFN- ⁇ ELISPOT assay and IFN- ⁇ ELISA were performed according to the manufacturer's procedure.
- the target gene and a target cell in which HLA-A * 1101 was forcibly expressed or a cDNA encoding the open reading frame of HLA-A * 1101 was amplified by PCR.
- the PCR amplification product was cloned into an expression vector.
- Lipofectamine 2000 (Invitrogen) according to the manufacturer's recommended procedure, transfect one or both of the target gene and HLA-A * 1101 expression vectors into the target gene and HLA negative cell line COS7. I did it.
- Tables 1a and 1b show CDCA1-derived 9mer peptides and 10mer peptides predicted to bind to HLA-A * 1101 in descending order of binding affinity. A total of 58 peptides potentially having HLA-A * 1101 binding ability were selected and examined to determine the epitope peptides.
- CDCA1-derived predicted peptide CTL against CDCA1-derived peptide was prepared according to the protocol described in “Materials and Methods”. Peptide-specific CTL activity was measured by IFN- ⁇ ELISPOT assay (FIG. 1).
- CDCA1-A11-9-123 showed peptide specific CTL activity in established IFN-gamma ELISPOT assay CTL lines and clones to-restricted CDCA1 derived peptide (SEQ ID NO: 3) wells using Number # 7 (a), well number # 8 using CDCA1-A11-9-105 (SEQ ID NO: 5), well number # using CDCA1-A11-9-419 (SEQ ID NO: 6) 1 (c), well number # 4 (d) using CDCA1-A11-9-219 (SEQ ID NO: 7), well number # 4 using CDCA1-A11-9-439 (SEQ ID NO: 9) e), well number # 3 (f) using CDCA1-A11-9-157 (SEQ ID NO: 13), well number # 4 (g) using CDCA1-A11-9-25 (SEQ ID NO: 21) CTL in well number # 3 (h) using CDCA1-A11-10-339 (SEQ ID NO: 30), well number # 2 (
- CTL activity of these CTL lines was measured by IFN- ⁇ ELISA (FIG. 2). These CTL lines showed strong IFN- ⁇ production against target cells pulsed with the corresponding peptide compared to target cells that were not pulsed with the peptide.
- CTL clones were established by limiting dilution from CTL lines as described in the “Materials and Methods” section above, and IFN- ⁇ production from CTL clones against peptide-pulsed C1R-A11 was determined by IFN- ⁇ ELISA. It was measured.
- CDCA1-A11-9-105 (SEQ ID NO: 5) (a), CDCA1-A11-9-419 (SEQ ID NO: 6) (b) and CDCA1-A11-9-219 (SEQ ID NO: 7) (c) Strong IFN- ⁇ production was observed from CTL clones stimulated with (Fig. 3).
- CTL clone established against CDCA1-A11-9-219 expresses CDCA1 and HLA-A * 1101 molecules The ability to recognize target cells was examined.
- COS7 cells (specific model of target cells expressing CDCA1 and HLA-A * 1101 genes) transfected with both full-length CDCA1 and HLA-A * 1101 genes were prepared as target cells.
- COS7 cells transfected with either full length CDCA1 or HLA-A * 1101 were prepared as controls.
- CDCA1-A11-9-219 (SEQ ID NO: 7) is a peptide generated by the endogenous processing of CDCA1 and is presented on target cells with HLA-A * 1101 molecules and recognized by CTL. It was clearly demonstrated that These results indicate that CDCA1-A11-9-219 (SEQ ID NO: 7) may be suitable as a cancer vaccine for patients with cancer that express CDCA1.
- CDCA1-A11-9-123 SEQ ID NO: 3
- CDCA1-A11-9-105 SEQ ID NO: 5
- CDCA1-A11-9-419 SEQ ID NO: 6
- CDCA1- A11-9-219 SEQ ID NO: 7
- CDCA1-A11-9-439 SEQ ID NO: 9
- CDCA1-A11-9-343 SEQ ID NO: 10
- CDCA1-A11-9-21 SEQ ID NO: 9) : 12
- CDCA1-A11-9-157 SEQ ID NO: 13
- CDCA1-A11-9-244 SEQ ID NO: 14
- CDCA1-A11-9-213 SEQ ID NO: 17
- CDCA1-A11- 9-335 SEQ ID NO: 19
- CDCA1-A11-9-25 SEQ ID NO: 21
- CDCA1-A11-10-339 SEQ ID NO: 30
- CDCA1-A11-10-452 SEQ ID NO: 35
- CDCA1-A11-9-123 (SEQ ID NO: 3), CDCA1-A11-9-105 (SEQ ID NO: 5), CDCA1-A11-9-419 (SEQ ID NO: 6), CDCA1-A11 -9-219 (SEQ ID NO: 7), CDCA1-A11-9-439 (SEQ ID NO: 9), CDCA1-A11-9-343 (SEQ ID NO: 10), CDCA1-A11-9-21 (SEQ ID NO: 12), CDCA1-A11-9-157 (SEQ ID NO: 13), CDCA1-A11-9-244 (SEQ ID NO: 14), CDCA1-A11-9-213 (SEQ ID NO: 17), CDCA1-A11-9 -335 (SEQ ID NO: 19), CDCA1-A11-9-25 (SEQ ID NO: 21) CDCA1-A11-10-339 (SEQ ID NO: 30), CDCA1-A11-10-452 (SEQ ID NO: 35), CDCA1-A11-10-400 (SEQ ID NO: 5
- CDCA1-A11-9-123 (SEQ ID NO: 3), CDCA1-A11-9-105 (SEQ ID NO: 5), CDCA1-A11-9-419 (SEQ ID NO: 6), CDCA1-A11-9 -219 (SEQ ID NO: 7), CDCA1-A11-9-439 (SEQ ID NO: 9), CDCA1-A11-9-343 (SEQ ID NO: 10), CDCA1-A11-9-21 (SEQ ID NO: 12) , CDCA1-A11-9-157 (SEQ ID NO: 13), CDCA1-A11-9-244 (SEQ ID NO: 14), CDCA1-A11-9-213 (SEQ ID NO: 17), CDCA1-A11-9-335 (SEQ ID NO: 19), CDCA1-
- Example 2 Materials and methods Cell line HLA-A and HLA-B negative human B lymphoblastoid cell line C1R, and African green monkey kidney cell line COS7 were purchased from ATCC.
- HLA-A * 3303 Creating stably expressing stimulator cells with HLA-A * 3303 the C1R stably expressing (C1R-A33) were used as stimulator cells.
- a cDNA encoding the open reading frame of HLA-A * 3303 was amplified by PCR and cloned into an expression vector.
- C1R cells were transformed with the expression vector and then selected for 2 weeks using G418 (Invitrogen). G418-selected cells were seeded into the wells of a 96-well plate containing a culture medium supplemented with G418 and further cultured for 30 days. Expression of exogenous HLA-A * 3303 in C1R cells was confirmed by flow cytometric analysis.
- Peptide synthesis Peptides were synthesized by Biosynthesis (Lewisville, Texas) according to standard solid phase synthesis and purified by reverse phase high performance liquid chromatography (HPLC). The purity (> 90%) and identity of the peptides were analyzed by analytical HPLC and mass spectrometry, respectively. The peptide was dissolved in dimethyl sulfoxide at 20 mg / ml and stored at -80 ° C.
- CTL-derived monocyte-derived dendritic cells were used as antigen presenting cells to induce cytotoxic T lymphocyte (CTL) responses to peptides presented on human leukocyte antigen (HLA).
- CTL cytotoxic T lymphocyte
- HLA human leukocyte antigen
- DCs were generated in vitro as described elsewhere (Nakahara S et al., Cancer Res 2003, 63 (14): 4112-8).
- peripheral blood mononuclear cells isolated from healthy volunteers HLA-A * 3303 positive
- Ficoll-Paque plus (Pharmacia) solution are attached to plastic tissue culture dishes (Becton Dickinson) And concentrated as monocyte fractions.
- AIM-V medium Invitrogen
- AS heat-inactivated autoserum
- a population enriched for monocytes was cultured in the presence of 4 (R & D System). After 7 days of culture, cytokine-induced DCs were pulsed with 20 ⁇ g / ml of each synthetic peptide in AIM-V medium at 37 ° C. for 3 hours in the presence of 3 ⁇ g / ml ⁇ 2-microglobulin.
- the generated cells appeared to express DC related molecules such as CD80, CD83, CD86, and HLA class II on their cell surface (data not shown).
- DC related molecules such as CD80, CD83, CD86, and HLA class II on their cell surface (data not shown).
- These peptide-pulsed DCs were then inactivated by X-ray irradiation (20 Gy) and mixed with autologous CD8 + T cells obtained by positive selection using CD8 Positive Isolation Kit (Dynal) at a ratio of 1:20 . These cultures were seeded in 48-well plates (Corning). Each well was filled with 1.5 x 10 4 peptide-pulsed DC, 3 x 10 5 CD8 + T cells and 10 ng / ml IL-7 (R & D System) in 0.5 ml AIM-V / 2% AS medium. ) Included.
- IL-2 (CHIRON) was added to these cultures to a final concentration of 20 IU / ml.
- T cells were further stimulated with peptide-pulsed autologous DCs.
- DC was prepared each time by the same method as above.
- CTL proliferation procedure described by Riddell et al. (Walter EA et al., N Engl J Med 1995, 333 (16): 1038-44; Riddell SR et al., Nat Med 1996, 2 (2): 216-23) CTL were grown in culture using methods similar to those used. CTL was combined with 2 human B lymphoblastoid cell lines treated with 5 ⁇ 10 6 cells / flask mitomycin C, and 40 ng / ml anti-CD3 antibody in a total volume of 25 ml of 5% AS-containing AIM-V ( AIM-V / 5% AS) was co-cultured in the medium. One day after the start of culture, 120 IU / ml IL-2 was added to the culture.
- CTL clones Dilution was carried out so that there were 0.3, 1, and 3 cells / well of CTL in a 96 round bottom microtiter plate (Nalge Nunc International). CTL in a total volume of 150 ⁇ l with 2 human B lymphoblastoid cell lines treated with 1 ⁇ 10 4 cells / well mitomycin C, 30 ng / ml anti-CD3 antibody, and 125 IU / ml IL-2 / Well in AIM-V / 5% AS medium. Ten days later, 50 ⁇ l / well of IL-2 was added to the medium so that the final concentration of IL-2 reached 125 IU / ml.
- IFN- ⁇ ELISPOT assay IFN- ⁇ enzyme-linked immunosorbent assay (ELISA) were performed. Specifically, peptide-pulsed C1R-A33 (1 ⁇ 10 4 cells / well) was prepared as a stimulator cell. Induced CTL, ie CTL lines and CTL clones, were used as responder cells. IFN- ⁇ ELISPOT assay and IFN- ⁇ ELISA were performed according to the manufacturer's procedure.
- the target gene and the target cell in which HLA-A * 3303 was forcibly expressed or a cDNA encoding the open reading frame of HLA-A * 3303 was amplified by PCR.
- the PCR amplification product was cloned into an expression vector.
- Lipofectamine 2000 (Invitrogen) according to the manufacturer's recommended procedure, transfect one or both of the target gene and HLA-A * 3303 expression vectors into the target gene and HLA negative cell line COS7 I did it.
- Tables 2a and 2b show CDCA1-derived 9mer and 10mer peptides predicted to bind to HLA-A * 3303 in descending order of binding affinity. A total of 32 peptides with the potential to bind HLA-A * 3303 were selected and examined to determine the epitope peptides.
- CDCA1-derived predictive peptide CTL against CDCA1-derived peptide was prepared according to the protocol described in “Materials and Methods”. Peptide-specific CTL activity was measured by IFN- ⁇ ELISPOT assay (FIG. 5).
- Well number # 2 (a) using CDCA1-A33-9-43 (SEQ ID NO: 27), Well number # 1 (b) using CDCA1-A33-9-123 (SEQ ID NO: 3), CDCA1- Well number # 3 (c) using A33-9-108 (SEQ ID NO: 60), well number # 2 (d) using CDCA1-A33-9-261 (SEQ ID NO: 28), CDCA1-A33- Well number # 6 (e) using 9-105 (SEQ ID NO: 5), well number # 8 (f) using CDCA1-A33-10-10 (SEQ ID NO: 67) and CDCA1-A33-10- CTL in well number # 5 (g) using 260 (SEQ ID NO: 69) showed strong IFN- ⁇ production compared to the subject.
- CTL lines showed strong IFN- ⁇ production against target cells pulsed with the corresponding peptide compared to target cells that were not pulsed with the peptide.
- CTL clones were established by limiting dilution from CTL lines as described in the “Materials and Methods” section above, and IFN- ⁇ production from CTL clones against peptide-pulsed C1R-A33 was determined by IFN- ⁇ ELISA. It was measured. Strong IFN- ⁇ production was observed from CTL clones stimulated with CDCA1-A33-9-43 (SEQ ID NO: 27) (a) and CDCA1-A33-9-123 (SEQ ID NO: 3) (b) ( Figure 7).
- CTL clone established against CDCA1-A33-9-43 expresses CDCA1 and HLA-A * 3303 molecules The ability to recognize target cells was examined.
- COS7 cells (specific model of target cells expressing CDCA1 and HLA-A * 3303 genes) transfected with both full-length CDCA1 and HLA-A * 3303 genes were prepared as target cells.
- COS7 cells transfected with either full-length CDCA1 or HLA-A * 3303 were prepared as controls.
- CDCA1-A33-9-43 (SEQ ID NO: 27) is a peptide generated by the endogenous processing of CDCA1 and is presented on target cells with HLA-A * 3303 molecules and recognized by CTL. Is clearly demonstrated. These results indicate that CDCA1-A33-9-43 (SEQ ID NO: 27) may be suitable as a cancer vaccine for patients with cancer that express CDCA1.
- CDCA1-A33-9-43 SEQ ID NO: 27
- CDCA1-A33-9-123 SEQ ID NO: 3
- CDCA1-A33-9-108 SEQ ID NO: 60
- CDCA1- A33-9-261 SEQ ID NO: 28
- CDCA1-A33-9-105 SEQ ID NO: 5
- CDCA1-A33-10-10 SEQ ID NO: 67
- CDCA1-A33-10-260 SEQ ID NO: : 69
- CDCA1-A33-9-43 (SEQ ID NO: 27), CDCA1-A33-9-123 (SEQ ID NO: 3), CDCA1-A33-9-108 (SEQ ID NO: 60), CDCA1-A33-9 -261 (SEQ ID NO: 28), CDCA1-A33-9-105 (SEQ ID NO: 5), CDCA1-A33-10-10 (SEQ ID NO: 67) and CDCA1-A33-10-260 (SEQ ID NO: 69) No sequence having significant homology to the above sequence was observed.
- Example 3 Materials and methods Cell line HLA-A and HLA-B negative human B lymphoblastoid cell line C1R, and African green monkey kidney cell line COS7 were purchased from ATCC.
- HLA-A * 0301 Creating stably expressing stimulator cells with HLA-A * 0301 the C1R stably expressing (C1R-A03) were used as stimulator cells.
- a cDNA encoding the open reading frame of HLA-A * 0301 was amplified by PCR and cloned into an expression vector.
- C1R cells were transformed with the expression vector and then selected for 2 weeks using G418 (Invitrogen). G418-selected cells were seeded into the wells of a 96-well plate containing a culture medium supplemented with G418 and further cultured for 30 days. Expression of exogenous HLA-A * 0301 in C1R cells was confirmed by flow cytometry analysis.
- Candidate selection of peptide derived from CDCA1 9 -mer and 10-mer peptides derived from CDCA1 that bind to HLA-A * 0301 molecules are linked to the binding prediction server ⁇ NetMHC 3.2 '' (www.cbs.dtu.dk/services/NetMHC/) (Buus et al., Tissue Antigens. 2003 Nov, 62 (5): 378-84; Nielsen et al., Protein Sci. 2003 May, 12 (5): 1007-17, Bioinformatics. 2004 Jun 12:20 (9): 1388 -97).
- Peptide synthesis Peptides were synthesized by Biosynthesis (Lewisville, Texas) according to standard solid phase synthesis and purified by reverse phase high performance liquid chromatography (HPLC). The purity (> 90%) and identity of the peptides were analyzed by analytical HPLC and mass spectrometry, respectively. The peptide was dissolved in dimethyl sulfoxide at 20 mg / ml and stored at -80 ° C.
- CTL-derived monocyte-derived dendritic cells were used as antigen presenting cells to induce cytotoxic T lymphocyte (CTL) responses to peptides presented on human leukocyte antigen (HLA).
- CTL cytotoxic T lymphocyte
- HLA human leukocyte antigen
- AIM-V medium Invitrogen
- AS heat-inactivated autoserum
- a population enriched for monocytes was cultured in the presence of 4 (R & D System). After 7 days of culture, cytokine-induced DCs were pulsed with 20 ⁇ g / ml of each synthetic peptide in AIM-V medium at 37 ° C. for 3 hours in the presence of 3 ⁇ g / ml ⁇ 2-microglobulin.
- the generated cells appeared to express DC related molecules such as CD80, CD83, CD86, and HLA class II on their cell surface (data not shown).
- DC related molecules such as CD80, CD83, CD86, and HLA class II on their cell surface (data not shown).
- These peptide-pulsed DCs were then inactivated by X-ray irradiation (20 Gy) and mixed with autologous CD8 + T cells obtained by positive selection using CD8 Positive Isolation Kit (Dynal) at a ratio of 1:20 . These cultures were seeded in 48-well plates (Corning). Each well was filled with 1.5 x 10 4 peptide-pulsed DC, 3 x 10 5 CD8 + T cells and 10 ng / ml IL-7 (R & D System) in 0.5 ml AIM-V / 2% AS medium. ) Included.
- IL-2 (CHIRON) was added to these cultures to a final concentration of 20 IU / ml.
- T cells were further stimulated with peptide-pulsed autologous DCs.
- DC was prepared each time by the same method as above.
- CTL proliferation procedure described by Riddell et al. (Walter EA et al., N Engl J Med 1995, 333 (16): 1038-44; Riddell SR et al., Nat Med 1996, 2 (2): 216-23) CTL were grown in culture using methods similar to those used. CTL was combined with 2 human B lymphoblastoid cell lines treated with 5 ⁇ 10 6 cells / flask mitomycin C and 40 ng / ml anti-CD3 antibody for a total of 25 ml of 5% AS-containing AIM-V Co-cultured in (AIM-V / 5% AS) medium. One day after the start of culture, 120 IU / ml IL-2 was added to the culture.
- CTL clones Dilution was carried out so that there were 0.3, 1, and 3 cells / well of CTL in a 96 round bottom microtiter plate (Nalge Nunc International). CTL in a total volume of 150 ⁇ l with 2 human B lymphoblastoid cell lines treated with 1 ⁇ 10 4 cells / well mitomycin C, 30 ng / ml anti-CD3 antibody, and 125 IU / ml IL-2 / Well in AIM-V / 5% AS medium. Ten days later, 50 ⁇ l / well of IL-2 was added to the medium so that the final concentration of IL-2 reached 125 IU / ml.
- IFN- ⁇ ELISPOT assay IFN- ⁇ enzyme-linked immunosorbent assay (ELISA) were performed. Specifically, peptide-pulsed C1R-A03 (1 ⁇ 10 4 cells / well) was prepared as a stimulator cell. Induced CTL, ie CTL lines and CTL clones, were used as responder cells. IFN- ⁇ ELISPOT assay and IFN- ⁇ ELISA were performed according to the manufacturer's procedure.
- the target gene and a target cell in which HLA-A * 0301 was forcibly expressed or a cDNA encoding an open reading frame of HLA-A * 0301 was amplified by PCR.
- the PCR amplification product was cloned into an expression vector.
- Lipofectamine 2000 (Invitrogen) according to the manufacturer's recommended procedure, transfect either the target gene and / or HLA-A * 0301 expression vector or both into the target gene and HLA negative cell line COS7 I did it.
- Table 3a and Table 3b show CDCA1-derived 9mer and 10mer peptides predicted to bind to HLA-A * 0301 in descending order of binding affinity. A total of 24 peptides that could have HLA-A * 0301 binding ability were selected and examined to determine the epitope peptides.
- CTL lines showed strong IFN- ⁇ production against target cells pulsed with the corresponding peptide compared to target cells that were not pulsed with the peptide.
- CTL clones were established by limiting dilution from CTL strains as described in the “Materials and Methods” section above, and IFN- ⁇ production from CTL clones against C1R-A03 pulsed with peptide was determined by IFN- ⁇ ELISA. It was measured.
- CDCA1-A03-9-219 (SEQ ID NO: 7) (a), CDCA1-A03-10-400 (SEQ ID NO: 38) (b) and CDCA1-A03-10-257 (SEQ ID NO: 47) (c) Strong IFN- ⁇ production was observed from CTL clones stimulated with (Fig. 11).
- CDCA1-A03-9-219 (SEQ ID NO: 7) and CDCA1-A03-10-400 (SEQ ID NO: 38) showed both CDCA1 and HLA-A * 0301 It showed strong CTL activity against expressing COS7 cells (FIG. 12). On the other hand, no significant specific CTL activity was detected relative to the control cells.
- CDCA1-A03-9-219 (SEQ ID NO: 7) and CDCA1-A03-10-400 (SEQ ID NO: 38) are peptides generated by the endogenous processing of CDCA1, and HLA-A * It is clearly demonstrated that it is presented on target cells with the 0301 molecule and recognized by CTL.
- CDCA1-A03-9-219 (SEQ ID NO: 7) and CDCA1-A03-10-400 (SEQ ID NO: 38) are suitable as cancer vaccines for patients with cancer that express CDCA1. It shows the possibility of being.
- CDCA1-A03-9-219 SEQ ID NO: 7
- CDCA1-A03-10-400 SEQ ID NO: 38
- CDCA1-A03-10-257 SEQ ID NO: 47
- CDCA1-A03-10-257 SEQ ID NO: 47
- Example 4 Preparation of emulsion formulation Peptide is dissolved in water for injection or sterile physiological saline to 1.0 to 10.0 mg / ml and collected in a syringe. This is connected by a syringe and a connector filled with an equivalent amount of IFA to water for injection or physiological saline, and stirred by alternately pressing the syringe plungers of the two connected syringes. After stirring for several minutes, the completion of the emulsion is evaluated by the drop test method.
- the drop test method can be performed by dropping one drop of a stirred sample on the water surface. If the sample dropped on the surface of the water does not immediately diffuse into the water, it is evaluated that the emulsion is complete.
- the finished emulsion can be administered to cancer patients by subcutaneous injection.
- Cancer patients to be administered include bladder cancer, breast cancer, cervical cancer, cholangiocellular cancer, chronic myelogenous leukemia (CML), esophageal cancer, stomach cancer, non-small cell lung cancer, lymphoma, osteosarcoma, prostate A patient suffering from cancer, kidney cancer, small cell lung cancer, head and neck cancer, soft tissue tumor, colon cancer or the like can be selected.
- Preparation of lyophilized preparation Dissolve the peptide in water for injection to 1.0 to 10.0 mg / ml, and sterilize by filtration. This is filled into a sterilized vial and a sterilized rubber stopper is half-capped. After the vial is lyophilized, a freeze-dried preparation is prepared by wrapping all stoppers and aluminum caps. In use, water for injection or sterile physiological saline is poured into the vial to re-dissolve the lyophilized powder. The redissolved solution in the vial is collected using a syringe, and connected to a syringe filled with the same amount of IFA as the collected redissolved solution by a connector.
- the finished emulsion can be administered to cancer patients by subcutaneous injection.
- Cancer patients to be administered include bladder cancer, breast cancer, cervical cancer, cholangiocellular cancer, chronic myelogenous leukemia (CML), esophageal cancer, stomach cancer, non-small cell lung cancer, lymphoma, osteosarcoma, prostate
- CML chronic myelogenous leukemia
- esophageal cancer stomach cancer, non-small cell lung cancer, lymphoma, osteosarcoma
- prostate A patient suffering from cancer, kidney cancer, small cell lung cancer, head and neck cancer, soft tissue tumor, colon cancer or the like can be selected.
- the present invention induces a powerful and specific anti-tumor immune response and thus may have applicability to a wide range of cancer types, a novel HLA-A11-restricted, HLA-A33-restricted, and HLA-derived from CDCA1 A03 restricted epitope peptides are provided.
- the peptides, compositions, APCs and CTLs of the present invention can be used for cancers that express CDCA1, such as bladder cancer, breast cancer, cervical cancer, cholangiocellular cancer, chronic myelogenous leukemia (CML), esophageal cancer. It can be used as a peptide vaccine for gastric cancer, non-small cell lung cancer, lymphoma, osteosarcoma, prostate cancer, kidney cancer, small cell lung cancer, head and neck cancer, soft tissue tumor, and colon cancer.
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Abstract
Description
本発明はまた、本発明のペプチドのいずれか1つをコードする単離されたポリヌクレオチドを提供する。これらのポリヌクレオチドは、本発明のペプチドと同様に、CTL誘導能を有するAPCを誘導するために用いることができ、またはCDCA1を発現するがん細胞に対する免疫応答を誘導するために対象に投与することができる。
本発明の態様を実施または試験するにあたって、本明細書に記載の方法および材料と類似のまたは同等の任意の方法および材料を用いることができるが、好ましい方法、装置、および材料をここに記載する。しかしながら、本発明の材料および方法について記載する前に、本明細書に記載の特定の大きさ、形状、寸法、材料、方法論、プロトコール等は慣例的な実験法および最適化に応じて変更可能であるため、本発明がこれらに限定されないことが理解されるべきである。本記載に使用する専門用語は特定の型または態様のみを説明する目的のためのものであり、添付の特許請求の範囲によってのみ限定される本発明の範囲を限定することは意図されないことも、また理解されるべきである。
本明細書で用いる「1つの(a)」、「1つの(an)」および「その(the)」という単語は、他に特記されない限り「少なくとも1つの」を意味する。
物質(例えば、ペプチド、抗体、ポリヌクレオチド等)に関して用いる「単離された」および「精製された」という用語は、該物質がそうでなければ天然源中に含まれ得る少なくとも1種の物質を実質的に含まないことを示す。したがって、単離または精製されたペプチドは、そのペプチドが由来する細胞もしくは組織源からの他の細胞材料、例えば糖質、脂質、および他の混入タンパク質を実質的に含まないペプチドを指す。またはペプチドが化学合成される場合には、単離または精製されたペプチドは前駆体物質もしくは他の化学物質を実質的に含まないペプチドを指す。「細胞材料を実質的に含まない」という用語は、それが単離された細胞または組換え産生された細胞の細胞成分から、ペプチドが分離されたペプチドの調製物を含む。したがって、細胞材料を実質的に含まないペプチドは、約30%、20%、10%、または5%、3%、2%または1%(乾燥重量ベース)未満の他の細胞材料を含有する、ペプチドの調製物を包含する。ペプチドを組換え産生する場合、単離または精製されたペプチドは、培養培地も実質的に含まず、培養培地を実質的に含まないペプチドは、培養培地をペプチド調製物の容量の約20%、10%、または5%、3%、2%または1%(乾燥重量ベース)未満で含有する、ペプチドの調製物を包含する。ペプチドを化学合成によって生成する場合、単離または精製されたペプチドは、前駆体物質および他の化学物質を実質的に含まず、前駆体物質および他の化学物質を実質的に含まないペプチドは、前駆体物質および他の化学物質をペプチド調製物の容量の約30%、20%、10%、5%、3%、2%または1%(乾燥重量ベース)未満で含有する、ペプチドの調製物を包含する。特定のペプチド調製物が単離または精製されたペプチドであることは、例えば、ドデシル硫酸ナトリウム(SDS)-ポリアクリルアミドゲル電気泳動およびゲルのクーマシーブリリアントブルー染色等の後の単一バンドの出現によって確認することができる。好ましい態様では、本発明のペプチドおよびポリヌクレオチドは単離または精製されている。
HLA-A11およびHLA-A33は、アジア人の中でよく見られるアリルであり(Sette A, Sidney J., Immunogenetics 1999, 50:201-12)、HLA-A03は白人の中でよく見られるアリルである(Cao et al., Hum Immunol. 2001;62(9):1009-30)。そのため、HLA-A11、HLA-A33またはHLA-A03によって拘束されるCDCA1由来のCTL誘導性ペプチドを提供することにより、多くのアジア人または白人に、CDCA1を発現するがんの有効な治療方法を提供することができる。よって、本発明は、HLA-A11、HLA-A33またはHLA-A03拘束性の様式でCTLを誘導し得るCDCA1由来のペプチドを提供する。
1)アラニン(A)、グリシン(G);
2)アスパラギン酸(D)、グルタミン酸(E);
3)アスパラギン(N)、グルタミン(Q);
4)アルギニン(R)、リジン(K);
5)イソロイシン(I)、ロイシン(L)、メチオニン(M)、バリン(V);
6)フェニルアラニン(F)、チロシン(Y)、トリプトファン(W);
7)セリン(S)、スレオニン(T);および
8)システイン(C)、メチオニン(M)(例えば、Creighton,Proteins 1984を参照されたい)。
(a)N末端から2番目のアミノ酸がスレオニン、バリン、イソロイシン、ロイシン、フェニルアラニンまたはチロシンで置換されている;
(b)N末端から3番目のアミノ酸がロイシン、フェニルアラニン、チロシン、イソロイシンまたはアラニンで置換されている;
(c)N末端から7番目のアミノ酸がロイシン、イソロイシン、チロシン、バリンまたはフェニルアラニンで置換されている;および
(d)C末端のアミノ酸がリジンまたはアルギニンで置換されている。
(a)N末端から2番目のアミノ酸がスレオニン、バリン、イソロイシン、ロイシン、フェニルアラニンまたはチロシンで置換されている;および
(b)C末端のアミノ酸がリジンまたはアルギニンで置換されている。
(a)N末端から1番目のアミノ酸がアスパラギン酸またはグルタミン酸で置換されている;
(b)N末端から2番目のアミノ酸がフェニルアラニン、チロシン、アラニン、イソロイシン、ロイシンまたはバリンで置換されている;および
(c)C末端のアミノ酸がアルギニンまたはリジンで置換されている。
(a)N末端から2番目のアミノ酸がフェニルアラニン、チロシン、アラニン、イソロイシン、ロイシンもしくはバリンで置換されている;および
(b)C末端のアミノ酸がアルギニンまたはリジンで置換されている。
(a)N末端から2番目のアミノ酸がロイシン、メチオニン、バリン、アラニン、イソロイシン、セリン、またはスレオニンで置換されている;および
(b)C末端のアミノ酸がアルギニン、リジン、チロシン、またはフェニルアラニンで置換されている。
(a)配列番号:3、5~7、9、10、12~14、17、19、21、30、35、38~40、45、53、56、58、27、60、28、67、69および47の中から選択されるアミノ酸配列からなる元のアミノ酸配列に対して、1個、2個、または数個のアミノ酸残基が置換、欠失、挿入、および/または付加されたアミノ酸配列からなる候補配列を作成する段階;
(b)(a)で作成した候補配列の中からCDCA1以外のいかなる公知のヒト遺伝子産物とも有意な相同性(配列同一性)を有さない候補配列を選択する段階;
(c)(b)で選択した候補配列からなるペプチドと、APCとを接触させる段階;
(d)(c)のAPCとCD8陽性T細胞とを接触させる段階;および
(e)元のアミノ酸配列からなるペプチドよりも同等かまたはより高いCTL誘導能を有するペプチドを選択する段階。
周知の技法を用いて、本発明のペプチドを調製することができる。例えば、組換えDNA技術または化学合成を用いて、本発明のペプチドを調製することができる。本発明のペプチドは、個々に、または2つもしくはそれ以上のペプチドを含むより長いポリペプチドとして、合成することができる。組換えDNA技術を用いて宿主細胞内で産生させた後、または化学合成した後、宿主細胞または合成反応物から、本発明のペプチドを単離することができる。すなわち、他の宿主細胞タンパク質およびそれらの断片、または他のいかなる化学物質も実質的に含まないように、本発明のペプチドを精製または単離することができる。
(i)Peptide Synthesis, Interscience, New York, 1966;
(ii)The Proteins, Vol. 2, Academic Press, New York, 1976;
(iii)「ペプチド合成」(日本語), 丸善, 1975;
(iv)「ペプチド合成の基礎と実験」(日本語), 丸善, 1985;
(v)「医薬品の開発」(日本語), 続第14巻(ペプチド合成), 広川書店, 1991;
(vi)WO99/67288;および
(vii)Barany G. & Merrifield R.B., Peptides Vol. 2, Solid Phase Peptide Synthesis, Academic Press, New York, 1980, 100-118。
本発明はまた、本発明のペプチドのいずれかをコードするポリヌクレオチドを提供する。これらには、天然CDCA1遺伝子(例えば、GenBankアクセッション番号NM_145697(配列番号:81)またはGenBankアクセッション番号NM_031423(配列番号:83))由来のポリヌクレオチド、およびその保存的に改変されたヌクレオチド配列を有するポリヌクレオチドが含まれる。本明細書において「保存的に改変されたヌクレオチド配列」という語句は、同一のまたは本質的に同一のアミノ酸配列をコードする配列を指す。遺伝暗号の縮重のため、数多くの機能的に同一の核酸が任意の特定のタンパク質をコードする。例えば、コドンGCA、GCC、GCG、およびGCUはすべて、アミノ酸のアラニンをコードする。したがって、あるコドンによってアラニンが指定される任意の位置において、コードされるポリペプチドを変化させることなく、該コドンを前記の対応するコドンのいずれかに変更することができる。そのような核酸の変異は「サイレント変異」であり、保存的に改変された変異の一種である。ペプチドをコードする本明細書中のあらゆる核酸配列は、該核酸のあらゆる可能なサイレント変異をも表す。核酸中の各コドン(通常メチオニンに対する唯一のコドンであるAUG、および通常トリプトファンに対する唯一のコドンであるTGGを除く)を改変して、機能的に同一の分子を得ることができることを、当業者は認識するであろう。したがって、ペプチドをコードする核酸の各サイレント変異は、開示した各配列において非明示的に記載されている。
本発明はさらに、本発明のペプチドとHLA抗原との間に形成された複合体を自身の表面上に提示する、エキソソームと称される細胞内小胞を提供する。エキソソームは、例えば特表平11-510507号およびWO99/03499に詳述されている方法を用いて調製することができ、治療および/または予防の対象となる患者から得られたAPCを用いて調製することができる。本発明のエキソソームは、本発明のペプチドと同様の様式で、ワクチンとして接種することができる。
本発明はまた、HLA抗原と本発明のペプチドとの間に形成される複合体を自身の表面上に提示するAPCを提供する。あるいは、本発明は、HLA抗原と本発明のペプチドとの間に形成された複合体をその細胞表面上に有するAPCを提供する。本発明のAPCは、単離されたAPCであり得る。細胞(APC、CTL等)に関して用いられる場合、「単離された」という用語は、該細胞が他の種類の細胞から分離されていることを指す。本発明のAPCは、治療および/または予防の対象となる患者に由来するAPCから誘導されたものであってもよく、かつ単独で、または本発明のペプチド、エキソソーム、もしくはCTLを含む他の薬物と併用して、ワクチンとして投与することができる。
(a)第1の対象からAPCを回収する段階、
(b)段階(a)のAPCをペプチドと接触させる段階、および
(c)段階(b)のAPCを第2の対象に投与する段階。
(a)HLA-A11(より好ましくはHLA-A*1101)、HLA-A33(より好ましくはHLA-A*3303)およびHLA-A03(より好ましくはHLA-A*0301)の中より選択される少なくとも1つのHLAを発現しているAPCを本発明のペプチドと接触させる段階;
(b)HLA-A11(より好ましくはHLA-A*1101)、HLA-A33(より好ましくはHLA-A*3303)およびHLA-A03(より好ましくはHLA-A*0301)の中より選択される少なくとも1つのHLAを発現しているAPCに、本発明のペプチドをコードするポリヌクレオチドを導入する段階。
本発明のペプチドによって誘導されたCTLは、インビボでCDCA1を発現するがん細胞を標的とする免疫応答を増強するため、本発明のペプチドと同様にワクチンとして用いることができる。したがって本発明は、本発明のペプチドによって誘導または活性化された、CTLを提供する。本発明のCTLは、本発明のペプチドを標的とするCTLであり、本発明のペプチドとHLA抗原との複合体に結合することができるCTLである。該複合体へのCTLの結合は、CTLの細胞表面上に存在するT細胞受容体(TCR)を介して行われる。本発明のCTLは、単離されたCTLであり得る。好ましいCTLは、ヒト由来の単離されたCTLである。また本発明のCTLはそれぞれ異なる本発明のペプチドを標的とするCTLの混合物であってもよい。
(a)CD8陽性T細胞を、HLA-A11(より好ましくはHLA-A*1101)、HLA-A33(より好ましくはHLA-A*3303)またはHLA-A03(より好ましくはHLA-A*0301)と本発明のペプチドとの複合体を自身の表面上に提示するAPCまたはエキソソームとインビトロで接触させる段階;
(b)CD8陽性T細胞に、細胞表面上にHLA-A11(より好ましくはHLA-A*1101)、HLA-A33(より好ましくはHLA-A*3303)またはHLA-A03(より好ましくはHLA-A*0301)により提示された本発明のペプチドに結合し得るTCRの各サブユニットをコードするポリヌクレオチドを導入する段階。
本発明はまた、細胞表面上にHLA抗原により提示された本発明のペプチドに結合し得るTCRの各サブユニットをコードするポリヌクレオチドを含む組成物、およびそれを使用する方法を提供する。該ポリヌクレオチドは、細胞表面上にHLA抗原により細胞表面上に提示された本発明のペプチドに結合し得るTCRを発現させることにより、CDCA1を発現するがん細胞に対する特異性をCD8陽性T細胞に付与する。当技術分野における公知の方法を用いることにより、本発明のペプチドで誘導されたCTLのTCRサブユニットとしてのα鎖およびβ鎖をコードするポリヌクレオチドを同定することができる(WO2007/032255、およびMorgan et al., J Immunol, 171, 3288 (2003))。例えば、TCRを分析するためにはPCR法が好ましい。分析のためのPCRプライマーは、例えば、次の5'側プライマーに、以下の3'側プライマーを組み合わせて増幅用プライマーセットとすることができるが、これらに限定されない。
5'側プライマー:
5'-Rプライマー(5'-gtctaccaggcattcgcttcat-3')(配列番号:77)
3'側プライマー:
TCRα鎖C領域に特異的:
3-TRa-Cプライマー(5'-tcagctggaccacagccgcagcgt-3')(配列番号:78)
TCRβ鎖C1領域に特異的:
3-TRb-C1プライマー(5'-tcagaaatcctttctcttgac-3')(配列番号:79)、または
TCRβ鎖C2領域に特異的:
3-TRβ-C2プライマー(5'-ctagcctctggaatcctttctctt-3')(配列番号:80)
同定されたポリヌクレオチドをCD8陽性T細胞に導入することによって形成されるTCRは、本発明のペプチドを提示する標的細胞と高い結合力で結合することができ、かつ、本発明のペプチドを提示する標的細胞の効率的な殺傷をインビボおよびインビトロで媒介する。
本発明はまた、以下の中から選択される少なくとも1つの有効成分を含む、組成物または薬学的組成物を提供する:
(a)本発明のペプチド;
(b)本発明のペプチドを発現可能な形態でコードするポリヌクレオチド;
(c)本発明のAPC;
(d)本発明のエキソソーム;
(e)本発明のCTL。
(a)本発明のペプチド;
(b)本発明のペプチドを発現可能な形態でコードするポリヌクレオチド;
(c)本発明のAPC;
(d)本発明のエキソソーム;
(e)本発明のCTL。
CDCA1の発現は、正常組織と比較して、がん細胞において、有意に上昇する。そのため、本発明のペプチドまたは該ペプチドをコードするポリヌクレオチドを、がんの治療および/もしくは予防、ならびに/または術後のその再発の予防に用いることができる。したがって本発明は、がんの治療および/もしくは予防、ならびに/または術後のその再発の予防のための薬学的組成物であって、本発明のペプチドまたはポリヌクレオチドの1種類または複数種を有効成分として含む組成物を提供する。あるいは、薬学的組成物として用いるために、本発明のペプチドを、エキソソームまたはAPCの表面上に提示させることができる。加えて、本発明のペプチドのいずれか1つを標的とする本発明のCTLもまた、本発明の薬学的組成物の有効成分として用いることができる。本発明の薬学的組成物は、治療的有効量または薬学的有効量の上記有効成分を含み得る。
本発明の薬学的組成物は、ヒトである対象または患者において、がんを治療および/もしくは予防するため、ならびに/または術後のその再発を予防するために用いることができる。本発明の薬学的組成物は、HLA-A11、HLA-A33およびHLA-A03の中より選択される少なくとも1つのHLAが陽性の対象に対して、好ましく使用することができる。また、本発明の薬学的組成物は、HLA-A11、HLA-A33およびHLA-A03の中より選択される少なくとも1つのHLAならびにCDCA1を発現するがんを治療および/もしくは予防するため、ならびに/または術後のその再発を予防するために、好ましく用いることができる。
(a)本発明のペプチド;
(b)本発明のペプチドを発現可能な形態でコードするポリヌクレオチド;
(c)本発明のペプチドを自身の表面上に提示するAPC;
(d)本発明のペプチドを自身の表面上に提示するエキソソーム;および
(e)本発明のCTL。
(a)本発明のペプチド;
(b)本発明のペプチドを発現可能な形態でコードするポリヌクレオチド;
(c)本発明のペプチドを自身の表面上に提示するAPC;
(d)本発明のペプチドを自身の表面上に提示するエキソソーム;および
(e)本発明のCTL。
(a)本発明のペプチド;
(b)本発明のペプチドを発現可能な形態でコードするポリヌクレオチド;
(c)本発明のペプチドを自身の表面上に提示するAPC;
(d)本発明のペプチドを自身の表面上に提示するエキソソーム;および
(e)本発明のCTL。
(a)本発明のペプチド;
(b)本発明のペプチドを発現可能な形態でコードするポリヌクレオチド;
(c)本発明のペプチドを自身の表面上に提示するAPC;
(d)本発明のペプチドを自身の表面上に提示するエキソソーム;および
(e)本発明のCTL。
(a)本発明のペプチド;
(b)本発明のペプチドを発現可能な形態でコードするポリヌクレオチド;
(c)本発明のペプチドを自身の表面上に提示するAPC;
(d)本発明のペプチドを自身の表面上に提示するエキソソーム;および
(e)本発明のCTL。
本発明のペプチドを含む薬学的組成物は、必要であれば、従来の製剤化法によって製剤化することができる。本発明の薬学的組成物は、本発明のペプチドに加えて、医薬品に通常用いられる担体、賦形剤等を特に制限なく必要に応じて含み得る。本発明の薬学的組成物に使用可能な担体の例としては、滅菌水(例えば、注射用水)、生理食塩水、リン酸緩衝液、リン酸緩衝生理食塩水、トリス緩衝生理食塩水、0.3%グリシン、培養液等が挙げられる。さらに、本発明の薬学的組成物は、必要に応じて、安定剤、懸濁液、保存剤、界面活性剤、溶解補助剤、pH調整剤、凝集抑制剤等を含み得る。本発明の薬学的組成物は、CDCA1を発現するがん細胞に対して特異的な免疫を誘導することができるため、がんの治療または予防の目的に用いることができる。
本発明の薬学的組成物はまた、本発明のペプチドを発現可能な形態でコードするポリヌクレオチドを含み得る。本明細書において、「発現可能な形態で」という語句は、ポリヌクレオチドが、細胞に導入された場合に、本発明のペプチドが発現されることを意味する。例示的な態様において、本発明のポリヌクレオチドの配列は、本発明のペプチドの発現に必要な調節エレメントを含む。本発明のポリヌクレオチドには、標的細胞のゲノムへの安定した挿入が達成されるために必要な配列を備えさせることができる(相同組換えカセットベクターの説明に関しては、例えばThomas KR & Capecchi MR, Cell 1987, 51: 503-12を参照されたい)。例えば、Wolff et al., Science 1990, 247: 1465-8;米国特許第5,580,859号;第5,589,466号;第5,804,566号;第5,739,118号;第5,736,524号;第5,679,647号;およびWO98/04720を参照されたい。DNAに基づく送達技術の例には、「naked DNA」、促進された(ブピバカイン、ポリマー、ペプチド媒介性)送達、カチオン性脂質複合体、および粒子媒介性(「遺伝子銃」)または圧力媒介性の送達が含まれる(例えば、米国特許第5,922,687号を参照されたい)。
本発明のペプチドおよびポリヌクレオチドを用いて、APCおよびCTLを誘導することができる。本発明のエキソソームおよびAPCを用いて、CTLを誘導することもできる。本発明のペプチド、ポリヌクレオチド、エキソソーム、およびAPCは、それらのCTL誘導能が阻害されない限り、任意の他の化合物と組み合わせて用いることができる。したがって、本発明のペプチド、ポリヌクレオチド、APCおよびエキソソームのいずれかを含む薬学的組成物を用いて本発明のCTLを誘導することができる。また、本発明のペプチドまたはポリヌクレオチドを含む薬学的組成物を用いて本発明のAPCを誘導することができる。
本発明は、本発明のペプチドまたはポリヌクレオチドを用いて、CTL誘導能を有するAPCを誘導する方法を提供する。
(a)対象からAPCを回収する段階;および
(b)段階(a)のAPCを本発明のペプチドと接触させる段階。
前記APCは特定の種類の細胞に限定されず、リンパ球によって認識されるように自身の細胞表面上にタンパク質性抗原を提示することが知られているDC、ランゲルハンス細胞、マクロファージ、B細胞、および活性化T細胞を用いることができる。DCはAPCの中で最も強力なCTL誘導能を有するため、好ましくはDCを用いることができる。本発明の任意のペプチドを単独で、または本発明の他のペプチドと共に用いることができる。また、本発明のペプチドと、他のCTL誘導性ペプチド(例えば、他のTAA由来のCTL誘導性ペプチド)とを組み合わせて用いることもできる。
(a)対象からAPCを回収する段階;および
(b)本発明のペプチドをコードするポリヌクレオチドを段階(a)のAPCに導入する段階。
段階(b)は、「VI.抗原提示細胞(APC)」の章に上述したように行うことができる。
(a)APCを本発明のペプチドで接触させる段階;
(b)本発明のペプチドをコードするポリヌクレオチドをAPCに導入する段階。
(a)APCを本発明のペプチドで接触させる段階;
(b)本発明のペプチドをコードするポリヌクレオチドをAPCに導入する段階。
本発明の方法によって誘導されたAPCは、CDCA1に特異的なCTL(すなわち本発明のCTL)を誘導することができる。
本発明はまた、本発明のペプチド、ポリヌクレオチド、エキソソーム、またはAPCを用いてCTLを誘導する方法を提供する。本発明はまた、本発明のペプチドとHLA抗原との複合体を認識し得るT細胞受容体(TCR)を形成し得るポリペプチド(すなわち、TCRサブユニット)をコードする1つまたは複数のポリヌクレオチドを使用してCTLを誘導する方法を提供する。好ましくは、CTLを誘導する方法は、以下の中より選択される少なくとも1つの段階を含む:
(a)CD8陽性T細胞を、HLA抗原と本発明のペプチドとの複合体を自身の表面上に提示する抗原提示細胞と接触させる段階;
(b)CD8陽性T細胞を、HLA抗原と本発明のペプチドとの複合体を自身の表面上に提示するエキソソームと接触させる段階;および
(c)本発明のペプチドとHLA抗原との複合体を認識することができるTCRを形成し得るポリペプチドをコードする1つまたは複数のポリヌクレオチドをCD8陽性T細胞に導入する段階。
(a)対象からAPCを回収する段階、
(b)段階(a)のAPCを本発明のペプチドと接触させる段階、および
(c)段階(b)のAPCをCD8陽性T細胞と共培養する段階。
誘導されたCTLは、その後対象に戻してもよい。
(a)CD8陽性T細胞を、HLA抗原と本発明のペプチドとの複合体を自身の表面上に提示するAPCと共培養する段階;
(b)CD8陽性T細胞を、HLA抗原と本発明のペプチドとの複合体を自身の表面上に提示するエキソソームと共培養する段階;および
(c)細胞表面上にHLA抗原により提示された本発明のペプチドに結合し得るTCRの各サブユニットをコードするポリヌクレオチドを含むベクターを、CD8陽性T細胞に導入する段階。
(a)本発明のペプチド;
(b)本発明のペプチドを発現可能な形態でコードするポリヌクレオチド;
(c)本発明のペプチドを自身の表面上に提示するAPC;および
(d)本発明のペプチドを自身の表面上に提示するエキソソーム。
(a)本発明のペプチド;
(b)本発明のペプチドを発現可能な形態でコードするポリヌクレオチド;
(c)本発明のペプチドを自身の表面上に提示するAPC;および
(d)本発明のペプチドを自身の表面上に提示するエキソソーム。
(a)本発明のペプチド;
(b)本発明のペプチドを発現可能な形態でコードするポリヌクレオチド;
(c)本発明のペプチドを自身の表面上に提示するAPC;および
(d)本発明のペプチドを自身の表面上に提示するエキソソーム。
(a)本発明のペプチド;
(b)本発明のペプチドを発現可能な形態でコードするポリヌクレオチド;
(c)本発明のペプチドを自身の表面上に提示するAPC;および
(d)本発明のペプチドを自身の表面上に提示するエキソソーム。
(a)本発明のペプチド;
(b)本発明のペプチドを発現可能な形態でコードするポリヌクレオチド;
(c)本発明のペプチドを自身の表面上に提示するAPC;および
(d)本発明のペプチドを自身の表面上に提示するエキソソーム。
さらに本発明は、CDCA1を発現するがんに対する免疫応答を誘導する方法を提供する。適用可能ながんには、膀胱がん、乳がん、子宮頸がん、胆管細胞がん、慢性骨髄性白血病(CML)、食道がん、胃がん、非小細胞肺がん、リンパ腫、骨肉腫、前立腺がん、腎がん、小細胞肺がん、頭頸部がん、軟部組織腫瘍、および大腸がんなどが含まれるが、これらに限定されない。また、がんは、HLA-A11、HLA-A33およびHLA-A03の中より選択される少なくとも1つのHLAを発現していることが好ましい。
(a)本発明のペプチド;
(b)本発明のペプチドを発現可能な形態でコードするポリヌクレオチド;
(c)本発明のペプチドを自身の表面上に提示するAPC;
(d)本発明のペプチドを自身の表面上に提示するエキソソーム;および
(e)本発明のCTL。
(a)本発明のペプチド;
(b)本発明のペプチドを発現可能な形態でコードするポリヌクレオチド;
(c)本発明のペプチドを自身の表面上に提示するAPC;
(d)本発明のペプチドを自身の表面上に提示するエキソソーム;および
(e)本発明のCTL。
(a)本発明のペプチド;
(b)本発明のペプチドを発現可能な形態でコードするポリヌクレオチド;
(c)本発明のペプチドを自身の表面上に提示するAPC;
(d)本発明のペプチドを自身の表面上に提示するエキソソーム;および
(e)本発明のCTL。
(a)本発明のペプチド;
(b)本発明のペプチドを発現可能な形態でコードするポリヌクレオチド;
(c)本発明のペプチドを自身の表面上に提示するAPC;
(d)本発明のペプチドを自身の表面上に提示するエキソソーム;および
(e)本発明のCTL。
(a)本発明のペプチド;
(b)本発明のペプチドを発現可能な形態でコードするポリヌクレオチド;
(c)本発明のペプチドを自身の表面上に提示するAPC;
(d)本発明のペプチドを自身の表面上に提示するエキソソーム;および
(e)本発明のCTL。
(a)本発明のペプチド;
(b)本発明のペプチドを発現可能な形態でコードするポリヌクレオチド;
(c)本発明のペプチドを自身の表面上に提示するAPC;
(d)本発明のペプチドを自身の表面上に提示するエキソソーム;および
(e)本発明のCTL。
(a)本発明のペプチド;
(b)本発明のペプチドを発現可能な形態でコードするポリヌクレオチド;
(c)本発明のペプチドを自身の表面上に提示するAPC;
(d)本発明のペプチドを自身の表面上に提示するエキソソーム;および
(e)本発明のCTL。
i)がんを有する対象の疾患部位から採取された生体試料中のCDCA1の発現レベルを測定する段階;
ii)i)で測定されたCDCA1の発現レベルに基づいて、CDCA1を発現するがんを有する対象を特定する段階;および
iii)上記の(a)~(e)からなる群より選択される少なくとも1つの成分を、正常対照と比較してCDCA1を過剰発現するがんを有する対象に投与する段階。
i)がんを有する対象の疾患部位から採取された生体試料中のCDCA1の発現レベルを測定する段階;
ii)i)で測定されたCDCA1の発現レベルに基づいて、CDCA1を発現するがんを有する対象を特定する段階;および
iii)ii)で特定された対象を、上記の(a)~(e)からなる群より選択される少なくとも1つの有効成分で治療され得る対象として特定または選択する段階。
生体試料中のCDCA1の発現レベルは公知の方法で測定することができ、例えば、CDCA1遺伝子の転写産物をプローブやPCR法により検出する方法(例えば、cDNAマイクロアレイ法、ノーザンブロット法、RT-PCR法など)、CDCA1遺伝子の翻訳産物を抗体等により検出する方法(例えば、ウェスタンブロット法、免疫染色法など)等を用いることができる。また、生体試料は血液試料であってもよく、この場合には、CDCA1またはその断片に対する抗体の血中レベルを測定し、該血中レベルに基づいて疾患部位におけるCDCA1の発現レベルを評価してもよい。CDCA1に対する抗体の血中レベルの測定は公知の方法を用いて行うことができ、例えば、CDCA1タンパク質や本発明のペプチドを抗原として用いた酵素免疫測定法(EIA)、酵素結合免疫吸着測定法(ELISA)、および放射免疫測定法(RIA)等を用いることができる。
本発明はさらに、本発明のペプチドに結合する抗体を提供する。好ましい抗体は本発明のペプチドに特異的に結合し、本発明のペプチドではないものには結合しない(または弱く結合する)。別の態様において、そのような抗体は、HLA分子との関連でペプチドを認識する抗体、すなわちペプチド-MHC複合体に結合する抗体を含み得る。抗体の結合特異性は、阻害試験で確認することができる。すなわち、分析する抗体と全長CDCA1ポリペプチドとの間の結合が、本発明のペプチドの存在下で阻害される場合、この抗体が本発明のペプチドに特異的に結合することが示される。本発明のペプチドに対する抗体は、疾患の診断および予後診断のアッセイ、ならびに本発明の薬学的組成物の投与対象の選択および本発明の薬学的組成物のモニタリングにおいて使用され得る。
例えば、本発明の抗体は、対象の血液試料(例えば血清試料)中に存在する本発明のペプチドを検出するために用いることもできる。あるいは、逆に、対象の血液試料(例えば血清試料)中に存在する本発明の抗体を、本発明のペプチドを用いて検出することもできる。対象の血液試料中において本発明のペプチドまたは本発明の抗体を測定した結果は、本発明の薬学的組成物の投与対象の選択、または本発明の薬学的組成物の効果のモニタリングに役立てることができる。そのほか、たとえばワクチンとして投与するペプチドに対する抗体を有する患者は、ワクチンに対する応答性が高い場合があることが報告されている。したがって、本発明のペプチドは、当該ペプチドをワクチンとして投与したときに、応答性の高い患者の抗体を指標とすることによって選択するためのイムノアッセイ用抗原として利用することができる。
本発明はまた、本発明のペプチドをコードするポリヌクレオチドを含むベクターおよび該ベクターが導入された宿主細胞を提供する。本発明のベクターは、宿主細胞中に本発明のポリヌクレオチドを保持するため、宿主細胞に本発明のペプチドを発現させるため、または遺伝子治療用に本発明のポリヌクレオチドを投与するために使用され得る。
[1]以下の群より選択されるアミノ酸配列を含む、細胞傷害性T細胞(CTL)誘導能を有する15アミノ酸未満のペプチド:
(a)配列番号:3、5~7、9、10、12~14、17、19、21、30、35、38~40、45、53、56、58、27、60、28、67、69および47からなる群より選択されるアミノ酸配列;および
(b)配列番号:3、5~7、9、10、12~14、17、19、21、30、35、38~40、45、53、56、58、27、60、28、67、69および47からなる群より選択されるアミノ酸配列に対して、1個、2個、または数個のアミノ酸が置換、欠失、挿入および/または付加されているアミノ酸配列。
[2]以下の(i)~(iii)からなる群より選択される、[1]に記載のペプチド:
(i)配列番号:3、5~7、9、10、12~14、17、19、21、30、35、38~40、45、53、56および58からなる群より選択されるアミノ酸配列に対して以下の(a)~(d)からなる群より選択される1個以上の置換がされたアミノ酸配列を含むペプチド:
(a)N末端から2番目のアミノ酸が、スレオニン、バリン、イソロイシン、ロイシン、フェニルアラニンおよびチロシンからなる群より選択されるアミノ酸に置換されている;
(b)N末端から3番目のアミノ酸が、ロイシン、フェニルアラニン、チロシン、イソロイシンおよびアラニンからなる群より選択されるアミノ酸に置換されている;
(c)N末端から7番目のアミノ酸が、ロイシン、イソロイシン、チロシン、バリンおよびフェニルアラニンからなる群より選択されるアミノ酸に置換されている;および
(d)C末端のアミノ酸が、リジンおよびアルギニンからなる群より選択されるアミノ酸に置換されている。
(ii)配列番号:27、3、60、28、5、67および69からなる群より選択されるアミノ酸配列に対して以下の(a)~(c)からなる群より選択される1個以上の置換がされたアミノ酸配列を含むペプチド:
(a)N末端から1番目のアミノ酸が、アスパラギン酸およびグルタミン酸からなる群より選択されるアミノ酸に置換されている;
(b)N末端から2番目のアミノ酸が、フェニルアラニン、チロシン、アラニン、イソロイシン、ロイシンおよびバリンからなる群より選択されるアミノ酸に置換されている;および
(c)C末端のアミノ酸が、アルギニンおよびリジンからなる群より選択されるアミノ酸に置換されている;
(iii)配列番号:7、38および47からなる群より選択されるアミノ酸配列に対して以下の(a)~(b)からなる群より選択される1個以上の置換がされたアミノ酸配列を含むペプチド:
(a)N末端から2番目のアミノ酸が、ロイシン、メチオニン、バリン、アラニン、イソロイシン、セリンおよびスレオニンからなる群より選択されるアミノ酸に置換されている;および
(b)C末端のアミノ酸が、アルギニン、リジン、チロシンおよびフェニルアラニンからなる群より選択されるアミノ酸に置換されている。
[3]配列番号:3、5~7、9、10、12~14、17、19、21、30、35、38~40、45、53、56、58、27、60、28、67、69および47からなる群より選択されるアミノ酸配列からなる、[1]に記載のペプチド。
[4][1]~[3]のいずれか一項に記載のペプチドをコードする、ポリヌクレオチド。
[5]薬学的に許容される担体と、以下の(a)~(e)からなる群より選択される少なくとも1つの成分とを含む組成物:
(a)[1]~[3]のいずれか一項に記載の1種類もしくは複数種のペプチド;
(b)[1]~[3]のいずれか一項に記載のペプチドを発現可能な形態でコードする1種類もしくは複数種のポリヌクレオチド;
(c)[1]~[3]のいずれか一項に記載のペプチドとHLA抗原との複合体を自身の細胞表面上に提示する抗原提示細胞(APC);
(d)[1]~[3]のいずれか一項に記載のペプチドとHLA抗原との複合体を自身の細胞表面上に提示するエキソソーム;および
(e)[1]~[3]のいずれか一項に記載のペプチドを標的とするCTL。
[6]前記成分が以下の(a)~(d)からなる群より選択される少なくとも1つの成分であり、CTLを誘導するための組成物である、[5]に記載の組成物:
(a)[1]~[3]のいずれか一項に記載の1種類もしくは複数種のペプチド;
(b)[1]~[3]のいずれか一項に記載のペプチドを発現可能な形態でコードする1種類もしくは複数種のポリヌクレオチド;
(c)[1]~[3]のいずれか一項に記載のペプチドとHLA抗原との複合体を自身の細胞表面上に提示する抗原提示細胞(APC);および
(d)[1]~[3]のいずれか一項に記載のペプチドとHLA抗原との複合体を自身の細胞表面上に提示するエキソソーム。
[7]薬学的組成物である、[5]に記載の組成物。
[8](i)がんの治療、(ii)がんの予防、および(iii)がんの術後の再発の予防からなる群より選択される1以上の用途のための薬学的組成物である、[7]に記載の組成物。
[9]がんに対する免疫応答を誘導するための、[7]に記載の組成物。
[10]がんが、膀胱がん、乳がん、子宮頸がん、胆管細胞がん、慢性骨髄性白血病(CML)、食道がん、胃がん、非小細胞肺がん、リンパ腫、骨肉腫、前立腺がん、腎がん、小細胞肺がん、頭頸部がん、軟部組織腫瘍、および大腸がんからなる群より選択される、[8]または[9]に記載の組成物。
[11]HLA-A11、HLA-A33およびHLA-A03からなる群より選択される少なくとも1つのHLAが陽性である対象への投与のために製剤化される、[5]~[10]のいずれか一項に記載の組成物。
[12]以下からなる群より選択される段階を含む、CTL誘導能を有するAPCを誘導する方法:
(a)APCを、[1]~[3]のいずれか一項に記載のペプチドとインビトロ、エクスビボ、またはインビボで接触させる段階、および
(b)[1]~[3]のいずれか一項に記載のペプチドをコードするポリヌクレオチドをAPCに導入する段階。
[13]以下の(a)~(c)からなる群より選択される段階を含む、CTLを誘導する方法:
(a)CD8陽性T細胞を、HLA抗原と[1]~[3]のいずれか一項に記載のペプチドとの複合体を自身の表面上に提示するAPCと共培養する段階、
(b)CD8陽性T細胞を、HLA抗原と[1]~[3]のいずれか一項に記載のペプチドとの複合体を自身の表面上に提示するエキソソームと共培養する段階、および
(c)細胞表面上にHLA抗原により提示された[1]~[3]のいずれか一項に記載のペプチドに結合し得るT細胞受容体(TCR)の各サブユニットをコードするポリヌクレオチドをCD8陽性T細胞に導入する段階。
[14]HLA抗原と[1]~[3]のいずれか一項に記載のペプチドとの複合体を自身の表面上に提示するAPC。
[15][12]に記載の方法によって誘導される、[14]に記載のAPC。
[16][1]~[3]のいずれか一項に記載のペプチドを標的とするCTL。
[17][13]に記載の方法によって誘導される、[16]に記載のCTL。
[18]以下の(a)~(e)からなる群より選択される少なくとも1つの成分を対象に投与する段階を含む、がんに対する免疫応答を誘導する方法:
(a)[1]~[3]のいずれか一項に記載の1種類もしくは複数種のペプチド;
(b)[1]~[3]のいずれか一項に記載のペプチドを発現可能な形態でコードする1種類もしくは複数種のポリヌクレオチド;
(c)[1]~[3]のいずれか一項に記載のペプチドとHLA抗原との複合体を自身の細胞表面上に提示する抗原提示細胞(APC);
(d)[1]~[3]のいずれか一項に記載のペプチドとHLA抗原との複合体を自身の細胞表面上に提示するエキソソーム;および
(e)[1]~[3]のいずれか一項に記載のペプチドを標的とするCTL。
[19]以下の(a)~(e)からなる群より選択される少なくとも1つの成分を対象に投与する段階を含む、がんを治療および/もしくは予防する、ならびに/または術後のその再発を予防する方法:
(a)[1]~[3]のいずれか一項に記載の1種類もしくは複数種のペプチド;
(b)[1]~[3]のいずれか一項に記載のペプチドを発現可能な形態でコードする1種類もしくは複数種のポリヌクレオチド;
(c)[1]~[3]のいずれか一項に記載のペプチドとHLA抗原との複合体を自身の細胞表面上に提示する抗原提示細胞(APC);
(d)[1]~[3]のいずれか一項に記載のペプチドとHLA抗原との複合体を自身の細胞表面上に提示するエキソソーム;および
(e)[1]~[3]のいずれか一項に記載のペプチドを標的とするCTL。
[20][1]~[3]のいずれか一項に記載のペプチドに結合する抗体。
[21]CTL誘導能を有するペプチドをスクリーニングする方法であって、以下の段階を含む方法:
(a)配列番号:3、5~7、9、10、12~14、17、19、21、30、35、38~40、45、53、56、58、27、60、28、67、69および47からなる群より選択されるアミノ酸配列からなる元のアミノ酸配列に対して、1個、2個、または数個のアミノ酸残基が置換、欠失、挿入、および/または付加されたアミノ酸配列からなる候補配列を作成する段階;
(b)(a)で作成した候補配列の中からCDCA1以外のいかなる公知のヒト遺伝子産物とも有意な相同性(配列同一性)を有さない候補配列を選択する段階;
(c)(b)で選択した候補配列からなるペプチドと、APCとを接触させる段階;
(d)(c)のAPCとCD8陽性T細胞とを接触させる段階;および
(e)元のアミノ酸配列からなるペプチドよりも同等かまたはより高いCTL誘導能を有するペプチドを選択する段階。
[22]がんに対する免疫応答を誘導するための組成物の製造における、以下の(a)~(e)からなる群より選択される少なくとも1つの有効成分の使用:
(a)[1]~[3]のいずれか一項に記載の1種類もしくは複数種のペプチド;
(b)[1]~[3]のいずれか一項に記載のペプチドを発現可能な形態でコードする1種類もしくは複数種のポリヌクレオチド;
(c)[1]~[3]のいずれか一項に記載のペプチドとHLA抗原との複合体を自身の細胞表面上に提示する抗原提示細胞(APC);
(d)[1]~[3]のいずれか一項に記載のペプチドとHLA抗原との複合体を自身の細胞表面上に提示するエキソソーム;および
(e)[1]~[3]のいずれか一項に記載のペプチドを標的とするCTL。
[23]がんの治療および/もしくは予防、ならびに/または術後のその再発の予防のための薬学的組成物の製造における、以下の(a)~(e)からなる群より選択される少なくとも1つの成分の使用:
(a)[1]~[3]のいずれか一項に記載の1種類もしくは複数種のペプチド;
(b)[1]~[3]のいずれか一項に記載のペプチドを発現可能な形態でコードする1種類もしくは複数種のポリヌクレオチド;
(c)[1]~[3]のいずれか一項に記載のペプチドとHLA抗原との複合体を自身の細胞表面上に提示する抗原提示細胞(APC);
(d)[1]~[3]のいずれか一項に記載のペプチドとHLA抗原との複合体を自身の細胞表面上に提示するエキソソーム;および
(e)[1]~[3]のいずれか一項に記載のペプチドを標的とするCTL。
[24]がんに対する免疫応答を誘導するための、以下の(a)~(e)からなる群より選択される少なくとも1つの成分の使用:
(a)[1]~[3]のいずれか一項に記載の1種類もしくは複数種のペプチド;
(b)[1]~[3]のいずれか一項に記載のペプチドを発現可能な形態でコードする1種類もしくは複数種のポリヌクレオチド;
(c)[1]~[3]のいずれか一項に記載のペプチドとHLA抗原との複合体を自身の細胞表面上に提示する抗原提示細胞(APC);
(d)[1]~[3]のいずれか一項に記載のペプチドとHLA抗原との複合体を自身の細胞表面上に提示するエキソソーム;および
(e)[1]~[3]のいずれか一項に記載のペプチドを標的とするCTL。
[25]がんを治療および/もしくは予防する、ならびに/または術後のその再発を予防するための、以下の(a)~(e)からなる群より選択される少なくとも1つの成分の使用:
(a)[1]~[3]のいずれか一項に記載の1種類もしくは複数種のペプチド;
(b)[1]~[3]のいずれか一項に記載のペプチドを発現可能な形態でコードする1種類もしくは複数種のポリヌクレオチド;
(c)[1]~[3]のいずれか一項に記載のペプチドとHLA抗原との複合体を自身の細胞表面上に提示する抗原提示細胞(APC);
(d)[1]~[3]のいずれか一項に記載のペプチドとHLA抗原との複合体を自身の細胞表面上に提示するエキソソーム;および
(e)[1]~[4]のいずれか一項に記載のペプチドを標的とするCTL。
[26]以下の(a)~(e)からなる群より選択される少なくとも1つの成分を対象に投与する段階を含む、CDCA1を発現する細胞に対する細胞傷害活性を誘導する方法:
(a)[1]~[3]のいずれか一項に記載の1種類もしくは複数種のペプチド;
(b)[1]~[3]のいずれか一項に記載のペプチドを発現可能な形態でコードする1種類もしくは複数種のポリヌクレオチド;
(c)[1]~[3]のいずれか一項に記載のペプチドとHLA抗原との複合体を自身の細胞表面上に提示する抗原提示細胞(APC);
(d)[1]~[3]のいずれか一項に記載のペプチドとHLA抗原との複合体を自身の細胞表面上に提示するエキソソーム;および
(e)[1]~[3]のいずれか一項に記載のペプチドを標的とするCTL。
[27][1]~[3]のいずれか一項に記載の1種類もしくは複数種のペプチドを含む凍結乾燥製剤。
[28][1]~[3]のいずれか一項に記載の1種類もしくは複数種のペプチドを水溶性の担体に溶解し、ろ過滅菌することを含む方法で調製された、薬学的組成物。
[29][1]~[3]のいずれか一項に記載の1種類もしくは複数種のペプチドと水溶性の担体とを含む水溶液であって、ろ過滅菌された水溶液。
[30][1]~[3]のいずれか一項に記載の1種類もしくは複数種のペプチド、水溶性の担体、および油性アジュバントを含むエマルション。
[31][5]~[11]のいずれか一項に記載の組成物が収容されている容器、およびアジュバントが収容されている容器を含むキット。
[32][1]~[3]のいずれか一項に記載のペプチドを含む凍結乾燥製剤が収納されている容器、アジュバントが収納されている容器、および凍結乾燥製剤のための再溶解液が収納されている容器を含むキット。
なお、本明細書において引用された全ての先行技術文献は、参照として本明細書に組み入れられる。
材料および方法
細胞株
HLA-AおよびHLA-B陰性ヒトBリンパ芽球様細胞株であるC1R、ならびにアフリカミドリザル腎細胞株であるCOS7は、ATCCから購入した。
HLA-A*1101を安定的に発現するC1R(C1R-A11)は、刺激細胞として使用された。HLA-A*1101のオープンリーディングフレームをコードするcDNAはPCRで増幅され、発現ベクターへクローニングされた。C1R細胞は発現ベクターで形質転換され、その後、G418(Invitrogen)を用いて2週間選択された。G418選択細胞はG418が添加された培養培地を含む96ウェルプレートのウェルへ蒔かれ、さらに30日培養された。外来性HLA-A*1101のC1R細胞における発現はフローサイトメトリー解析で確認された。
HLA-A*1101分子に結合するCDCA1由来の9merおよび10merのペプチドを、結合予測サーバー「NetMHC 3.2」(www.cbs.dtu.dk/services/NetMHC/) (Buus et al., Tissue Antigens. 2003 Nov, 62(5):378-84; Nielsen et al., Protein Sci. 2003 May, 12(5):1007-17, Bioinformatics. 2004 Jun 12:20(9):1388-97)を用いて予測した。
これらのペプチドは、Biosynthesis(Lewisville,Texas)により、標準的な固相合成法に従って合成し、逆相高速液体クロマトグラフィー(HPLC)によって精製した。該ペプチドの純度(>90%)および同一性は、それぞれ分析用HPLCおよび質量分析によって分析した。ペプチドはジメチルスルホキシドに20mg/mlで溶解し、-80℃で保存した。
単球由来の樹状細胞(DC)を抗原提示細胞として使用し、ヒト白血球抗原(HLA)上に提示されたペプチドに対する細胞傷害性Tリンパ球(CTL)応答を誘導した。他所に記載されているように、DCはインビトロで作製した(Nakahara S et al.,Cancer Res 2003,63(14):4112-8)。具体的には、Ficoll-Paque plus(Pharmacia)溶液によって健常なボランティア(HLA-A*1101陽性)から単離した末梢血単核細胞を、プラスチック製の組織培養ディッシュ(Becton Dickinson)へ付着させることによって分離し、それらを単球画分として濃縮した。2%の加熱不活化した自己血清(AS)を含むAIM-V培地(Invitrogen)中、1000 IU/mlの顆粒球マクロファージコロニー刺激因子(R&D System)および1000 IU/mlのインターロイキン(IL)-4(R&D System)の存在下で、単球が濃縮された集団を培養した。7日間の培養後、サイトカインで誘導したDCに、AIM-V培地中で37℃で3時間、3μg/mlのβ2-ミクログロブリンの存在下で20μg/mlの各合成ペプチドをパルスした。作製された細胞は、自身の細胞表面上にCD80、CD83、CD86、およびHLAクラスIIなどのDC関連分子を発現しているようであった(データは示さず)。次いで、ペプチドパルスしたこれらのDCをX線照射(20Gy)によって不活化し、CD8 Positive Isolation Kit(Dynal)を用いた陽性選択によって得られた自己CD8+T細胞と1:20の比率で混合した。これらの培養物を48ウェルプレート(Corning)中に播種した。各ウェルは、0.5mlのAIM-V/2%AS培地中に、1.5 x 104個のペプチドパルスしたDC、3 x 105個のCD8+T細胞および10ng/mlのIL-7(R&D System)を含むようにした。3日後、これらの培養物に、IL-2(CHIRON)を最終濃度20IU/mlまで添加した。7日目および14日目に、ペプチドパルスした自己DCでT細胞をさらに刺激した。DCは上記と同一の方法によって毎回調製した。21日目に、3回目のペプチド刺激後、ペプチドパルスしたC1R-A11細胞に対してCTLをヒト インターフェロン(IFN)-γ 酵素結合免疫スポット(ELISPOT)アッセイにて試験した(Tanaka H et al., Br J Cancer 2001, 84(1): 94-9; Umano Y et al., Br J Cancer 2001, 84(8): 1052-7; Uchida N et al., Clin Cancer Res 2004, 10(24): 8577-86; Suda T et al., Cancer Sci 2006, 97(5): 411-9; Watanabe T et al., Cancer Sci 2005, 96(8): 498-506)。
Riddellら(Walter EA et al., N Engl J Med 1995, 333(16): 1038-44; Riddell SR et al., Nat Med 1996, 2(2): 216-23)によって記載されている方法と類似の方法を使用して、CTLを培養下で増殖させた。CTLを、5 x 106個細胞/フラスコのマイトマイシンCによって処理された2種類のヒトBリンパ芽球様細胞株、および40ng/mlの抗CD3抗体とともに、総量25mlの5%AS含有AIM-V(AIM-V/5%AS)培地中で共培養した。培養開始1日後に、120IU/mlのIL-2を該培養物に添加した。5、8および11日目に、30IU/mlのIL-2を含む新鮮なAIM-V/5%AS培地を、該培養物に添加した(Tanaka H et al., Br J Cancer 2001, 84(1): 94-9; Umano Y et al., Br J Cancer 2001, 84(8): 1052-7, Uchida N et al., Clin Cancer Res 2004, 10(24): 8577-86; Suda T et al., Cancer Sci 2006, 97(5): 411-9, Watanabe T et al., Cancer Sci 2005, 96(8): 498-506)。
96丸底マイクロタイタープレート(Nalge Nunc International)においてCTL 0.3個、1個、および3個細胞/ウェルとなるように、希釈を行った。CTLを、1 x 104個細胞/ウェルのマイトマイシンCによって処理された2種類のヒトBリンパ芽球様細胞株、30ng/mlの抗CD3抗体、および125IU/mlのIL-2とともに、総量150μl/ウェルのAIM-V/5%AS培地中で培養した。10日後、50μl/ウェルのIL-2を、IL-2の最終濃度が125IU/mlに達するように該培地に添加した。14日目にCTL活性を試験し、上記と同一の方法を使用してCTLクローンを増殖させた(Uchida N et al., Clin Cancer Res 2004, 10(24): 8577-86; Suda T et al., Cancer Sci 2006, 97(5): 411-9; Watanabe T et al., Cancer Sci 2005, 96(8)): 498-506)。
特異的CTL活性を調べるために、IFN-γ ELISPOTアッセイおよびIFN-γ酵素結合免疫吸着アッセイ(ELISA)を実施した。具体的には、ペプチドパルスしたC1R‐A11(1 x 104個細胞/ウェル)を刺激細胞として調製した。誘導したCTL、すなわちCTL株およびCTLクローンを応答細胞として使用した。IFN-γ ELISPOTアッセイおよびIFN-γ ELISAは、製造業者の手順に従って実施した。
標的遺伝子またはHLA-A*1101のオープンリーディングフレームをコードするcDNAをPCRによって増幅した。PCR増幅産物を発現ベクターにクローニングした。製造業者の推奨する手順に従ってリポフェクタミン2000(Invitrogen)を使用して、標的遺伝子およびHLAの陰性細胞株であるCOS7に標的遺伝子の発現ベクターおよびHLA-A*1101の発現ベクターのいずれかまたは両方をトランスフェクトした。トランスフェクションから2日後、トランスフェクトした細胞をベルセン(Invitrogen)を用いて回収し、CTL活性アッセイのための標的細胞(5 x 104個細胞/ウェル)として使用した。
CDCA1由来のHLA-A * 1101結合ペプチドの予測
表1aおよび表1bは、HLA-A*1101への結合が予測されたCDCA1由来の9merペプチドおよび10merペプチドを結合親和性の高い順に示す。HLA-A*1101結合能を有する可能性のある合計58種のペプチドを、エピトープペプチドを決定するために選択し、調べた。
CDCA1由来のペプチドに対するCTLを、「材料および方法」に記載したプロトコールに従って作製した。IFN-γ ELISPOTアッセイによって、ペプチド特異的なCTL活性を測定した(図1)。CDCA1-A11-9-123(配列番号:3)を用いたウェル番号#7(a)、CDCA1-A11-9-105(配列番号:5)を用いたウェル番号#8(b)、CDCA1-A11-9-419(配列番号:6)を用いたウェル番号#1(c)、CDCA1-A11-9-219(配列番号:7)を用いたウェル番号#4(d)、CDCA1-A11-9-439(配列番号:9)を用いたウェル番号#2(e)、CDCA1-A11-9-343(配列番号:10)を用いたウェル番号#2(f)、CDCA1-A11-9-21(配列番号:12)を用いたウェル番号#8(g)、CDCA1-A11-9-157(配列番号:13)を用いたウェル番号#3(h)、CDCA1-A11-9-244(配列番号:14)を用いたウェル番号#7(i)、CDCA1-A11-9-213(配列番号:17)を用いたウェル番号#6(j)、CDCA1-A11-9-335(配列番号:19)を用いたウェル番号#7(k)、CDCA1-A11-9-25(配列番号:21)を用いたウェル番号#4(l)、CDCA1-A11-10-339(配列番号:30)を用いたウェル番号#3(m)、CDCA1-A11-10-452(配列番号:35)を用いたウェル番号#2(n)、CDCA1-A11-10-400(配列番号:38)を用いたウェル番号#7(o)、CDCA1-A11-10-289(配列番号:39)を用いたウェル番号#6(p)、CDCA1-A11-10-203(配列番号:40)を用いたウェル番号#5(q)、CDCA1-A11-10-156(配列番号:45)を用いたウェル番号#2(r)、CDCA1-A11-10-340(配列番号:53)を用いたウェル番号#2(s)、CDCA1-A11-10-106(配列番号:56)を用いたウェル番号#4(t)およびCDCA1-A11-10-358(配列番号:58)を用いたウェル番号#2(u)におけるCTLは、対照ウェルと比較して強力なIFN-γ産生を示した。一方、表1aおよび表1bに示される他のペプチドは、HLA-A*1101との結合活性を有する可能性があるにもかかわらず、それらのペプチドでの刺激によっては、特異的なCTL活性が測定されなかった。典型的な陰性データの例であるが、CDCA1-A11-10-391(配列番号:33)で刺激したCTLからは特異的IFN-γ産生が観察されなかった(v)。結果としてCDCA1に由来する21種のペプチドが、強力なCTLを誘導することができるペプチドとして選択されることが示された。
IFN-γ ELISPOTアッセイにてペプチド特異的CTL活性を示したCDCA1-A11-9-123(配列番号:3)を用いたウェル番号#7(a)、CDCA1-A11-9-105(配列番号:5)を用いたウェル番号#8(b)、CDCA1-A11-9-419(配列番号:6)を用いたウェル番号#1(c)、CDCA1-A11-9-219(配列番号:7)を用いたウェル番号#4(d)、CDCA1-A11-9-439(配列番号:9)を用いたウェル番号#4(e)、CDCA1-A11-9-157(配列番号:13)を用いたウェル番号#3(f)、CDCA1-A11-9-25(配列番号:21)を用いたウェル番号#4(g)、CDCA1-A11-10-339(配列番号:30)を用いたウェル番号#3(h)、CDCA1-A11-10-358(配列番号:58)を用いたウェル番号#2(i)におけるCTLを増殖させ、CTL株を樹立した。これらのCTL株のCTL活性をIFN-γ ELISAによって測定した(図2)。これらのCTL株は、ペプチドをパルスしなかった標的細胞と比較して、対応するペプチドをパルスした標的細胞に対して強力なIFN-γ産生を示した。さらに、上記の「材料および方法」の章に記載された通りCTL株から限界希釈によってCTLクローンを樹立し、ペプチドをパルスしたC1R-A11に対するCTLクローンからのIFN-γ産生をIFN-γ ELISAによって測定した。CDCA1-A11-9-105(配列番号:5)(a)、CDCA1-A11-9-419(配列番号:6)(b)およびCDCA1-A11-9-219(配列番号:7)(c)で刺激したCTLクローンから強力なIFN-γ産生が観察された(図3)。
CDCA1-A11-9-219(配列番号:7)に対して樹立されたCTLクローンを、CDCA1およびHLA-A*1101分子を発現する標的細胞を認識する能力に関して調べた。全長CDCA1およびHLA-A*1101遺伝子の両方をトランスフェクトしたCOS7細胞(CDCA1およびHLA-A*1101遺伝子を発現する標的細胞の特異的モデル)を標的細胞として調製した。全長CDCA1またはHLA-A*1101のいずれかをトランスフェクトしたCOS7細胞を対照として調製した。CDCA1-A11-9-219(配列番号:7)で刺激したCTLクローンが、CDCA1およびHLA-A*1101の両方を発現するCOS7細胞に対して強力なCTL活性を示した(図4)。一方、対照細胞に対して有意な特異的CTL活性は検出されなかった。これらのデータにより、CDCA1-A11-9-219(配列番号:7)が、CDCA1の内因的プロセシングにより生じるペプチドであり、かつHLA-A*1101分子とともに標的細胞上に提示されてCTLによって認識されることが明確に実証された。これらの結果は、CDCA1-A11-9-219(配列番号:7)が、CDCA1を発現するがんを有する患者に対するがんワクチンとして適している可能性を示している。
CDCA1-A11-9-123(配列番号:3)、CDCA1-A11-9-105(配列番号:5)、CDCA1-A11-9-419(配列番号:6)、CDCA1-A11-9-219(配列番号:7)、CDCA1-A11-9-439(配列番号:9)、CDCA1-A11-9-343(配列番号:10)、CDCA1-A11-9-21(配列番号:12)、CDCA1-A11-9-157(配列番号:13)、CDCA1-A11-9-244(配列番号:14)、CDCA1-A11-9-213(配列番号:17)、CDCA1-A11-9-335(配列番号:19)、CDCA1-A11-9-25(配列番号:21)、CDCA1-A11-10-339(配列番号:30)CDCA1-A11-10-452(配列番号:35)、CDCA1-A11-10-400(配列番号:38)、CDCA1-A11-10-289(配列番号:39)、CDCA1-A11-10-203(配列番号:40)、CDCA1-A11-10-156(配列番号:45)、CDCA1-A11-10-340(配列番号:53)、CDCA1-A11-10-106(配列番号:56)およびCDCA1-A11-10-358(配列番号:58)で刺激したCTLは、有意かつ特異的なCTLの活性を示した。これらの結果は、CDCA1-A11-9-123(配列番号:3)、CDCA1-A11-9-105(配列番号:5)、CDCA1-A11-9-419(配列番号:6)、CDCA1-A11-9-219(配列番号:7)、CDCA1-A11-9-439(配列番号:9)、CDCA1-A11-9-343(配列番号:10)、CDCA1-A11-9-21(配列番号:12)、CDCA1-A11-9-157(配列番号:13)、CDCA1-A11-9-244(配列番号:14)、CDCA1-A11-9-213(配列番号:17)、CDCA1-A11-9-335(配列番号:19)、CDCA1-A11-9-25(配列番号:21)CDCA1-A11-10-339(配列番号:30)、CDCA1-A11-10-452(配列番号:35)、CDCA1-A11-10-400(配列番号:38)、CDCA1-A11-10-289(配列番号:39)、CDCA1-A11-10-203(配列番号:40)、CDCA1-A11-10-156(配列番号:45)、CDCA1-A11-10-340(配列番号:53)、CDCA1-A11-10-106(配列番号:56)およびCDCA1-A11-10-358(配列番号:58)の配列が、ヒト免疫系を感作することが知られている他の分子に由来するペプチドと相同であるという事実に起因する可能性がある。この可能性を排除するために、BLASTアルゴリズム(blast.ncbi.nlm.nih.gov/Blast.cgi)を用いて、これらのペプチドの配列をクエリーとして相同性解析を行った。その結果、CDCA1-A11-9-123(配列番号:3)、CDCA1-A11-9-105(配列番号:5)、CDCA1-A11-9-419(配列番号:6)、CDCA1-A11-9-219(配列番号:7)、CDCA1-A11-9-439(配列番号:9)、CDCA1-A11-9-343(配列番号:10)、CDCA1-A11-9-21(配列番号:12)、CDCA1-A11-9-157(配列番号:13)、CDCA1-A11-9-244(配列番号:14)、CDCA1-A11-9-213(配列番号:17)、CDCA1-A11-9-335(配列番号:19)、CDCA1-A11-9-25(配列番号:21)CDCA1-A11-10-339(配列番号:30)、CDCA1-A11-10-452(配列番号:35)、CDCA1-A11-10-400(配列番号:38)、CDCA1-A11-10-289(配列番号:39)、CDCA1-A11-10-203(配列番号:40)、CDCA1-A11-10-156(配列番号:45)、CDCA1-A11-10-340(配列番号:53)、CDCA1-A11-10-106(配列番号:56)およびCDCA1-A11-10-358(配列番号:58)の配列に対して有意な相同性を有する配列は認められなかった。したがって、本発明者らの知る限りでは、これらのペプチドが、他の関連のない分子に対して意図しない免疫応答を引き起こす可能性はほとんどないと考えられる。結論として、CDCA1由来の新規HLA-A11拘束性エピトープペプチドが同定された。さらに、CDCA1由来のエピトープペプチドは、がん免疫療法に適用し得ることが示された。
材料および方法
細胞株
HLA-AおよびHLA-B陰性ヒトBリンパ芽球様細胞株であるC1R、ならびにアフリカミドリザル腎細胞株であるCOS7は、ATCCから購入した。
HLA-A*3303を安定的に発現するC1R(C1R-A33)は、刺激細胞として使用された。HLA-A*3303のオープンリーディングフレームをコードするcDNAはPCRで増幅され、発現ベクターへクローニングされた。C1R細胞は発現ベクターで形質転換され、その後、G418(Invitrogen)を用いて2週間選択された。G418選択細胞はG418が添加された培養培地を含む96ウェルプレートのウェルへ蒔かれ、さらに30日培養された。外来性HLA-A*3303のC1R細胞における発現はフローサイトメトリー解析で確認された。
HLA-A*3303分子に結合するCDCA1由来の9merおよび10merのペプチドを、結合予測サーバー「NetMHC pan2.8」(www.cbs.dtu.dk/services/NetMHCpan/) (Nielsen et al., PLoS One. 2007;29;2(8):e796; Hoof et al., Immunogenetics. 2009;61(1): 1-13)を用いて予測した。
ペプチドは、Biosynthesis(Lewisville,Texas)により、標準的な固相合成法に従って合成し、逆相高速液体クロマトグラフィー(HPLC)によって精製した。該ペプチドの純度(>90%)および同一性は、それぞれ分析用HPLCおよび質量分析によって分析した。ペプチドはジメチルスルホキシドに20mg/mlで溶解し、-80℃で保存した。
単球由来の樹状細胞(DC)を抗原提示細胞として使用し、ヒト白血球抗原(HLA)上に提示されたペプチドに対する細胞傷害性Tリンパ球(CTL)応答を誘導した。他所に記載されているように、DCはインビトロで作製した(Nakahara S et al.,Cancer Res 2003,63(14):4112-8)。具体的には、Ficoll-Paque plus (Pharmacia)溶液によって健常なボランティア(HLA-A*3303陽性)から単離した末梢血単核細胞を、プラスチック製の組織培養ディッシュ(Becton Dickinson)へ付着させることによって分離し、それらを単球画分として濃縮した。2%の加熱不活化した自己血清(AS)を含むAIM-V培地(Invitrogen)中、1000 IU/mlの顆粒球マクロファージコロニー刺激因子(R&D System)および1000 IU/mlのインターロイキン(IL)-4(R&D System)の存在下で、単球が濃縮された集団を培養した。7日間の培養後、サイトカインで誘導したDCに、AIM-V培地中で37℃で3時間、3μg/mlのβ2-ミクログロブリンの存在下で20μg/mlの各合成ペプチドをパルスした。作製された細胞は、自身の細胞表面上にCD80、CD83、CD86、およびHLAクラスIIなどのDC関連分子を発現しているようであった(データは示さず)。次いで、ペプチドパルスしたこれらのDCをX線照射(20Gy)によって不活化し、CD8 Positive Isolation Kit(Dynal)を用いた陽性選択によって得られた自己CD8+T細胞と1:20の比率で混合した。これらの培養物を48ウェルプレート(Corning)中に播種した。各ウェルは、0.5mlのAIM-V/2%AS培地中に、1.5 x 104個のペプチドパルスしたDC、3 x 105個のCD8+T細胞および10ng/mlのIL-7(R&D System)を含むようにした。3日後、これらの培養物に、IL-2(CHIRON)を最終濃度20IU/mlまで添加した。7日目および14日目に、ペプチドパルスした自己DCでT細胞をさらに刺激した。DCは上記と同一の方法によって毎回調製した。21日目に、3回目のペプチド刺激後、ペプチドパルスしたC1R-A33細胞に対してCTLをヒト インターフェロン(IFN)-γ 酵素結合免疫スポット(ELISPOT)アッセイにて試験した(Tanaka H et al., Br J Cancer 2001, 84(1): 94-9; Umano Y et al., Br J Cancer 2001, 84(8): 1052-7; Uchida N et al., Clin Cancer Res 2004, 10(24): 8577-86; Suda T et al., Cancer Sci 2006, 97(5): 411-9; Watanabe T et al., Cancer Sci 2005, 96(8): 498-506)。
Riddellら(Walter EA et al., N Engl J Med 1995, 333(16): 1038-44; Riddell SR et al., Nat Med 1996, 2(2): 216-23)によって記載されている方法と類似の方法を使用して、CTLを培養下で増殖させた。CTLを、5 x 106個細胞/フラスコのマイトマイシンCによって処理された2種類のヒトBリンパ芽球様細胞株、および40ng/mlの抗CD3抗体とともに総量25mlの5%AS含有AIM-V(AIM-V/5%AS)培地中で共培養した。培養開始1日後に、120IU/mlのIL-2を該培養物に添加した。5、8および11日目に、30IU/mlのIL-2を含む新鮮なAIM-V/5%AS培地を、該培養物に添加した(Tanaka H et al., Br J Cancer 2001, 84(1): 94-9; Umano Y et al., Br J Cancer 2001, 84(8): 1052-7, Uchida N et al., Clin Cancer Res 2004, 10(24): 8577-86; Suda T et al., Cancer Sci 2006, 97(5): 411-9, Watanabe T et al., Cancer Sci 2005, 96(8): 498-506)。
96丸底マイクロタイタープレート(Nalge Nunc International)においてCTL 0.3個、1個、および3個細胞/ウェルとなるように、希釈を行った。CTLを、1 x 104個細胞/ウェルのマイトマイシンCによって処理された2種類のヒトBリンパ芽球様細胞株、30ng/mlの抗CD3抗体、および125IU/mlのIL-2とともに、総量150μl/ウェルのAIM-V/5%AS培地中で培養した。10日後、50μl/ウェルのIL-2を、IL-2の最終濃度が125IU/mlに達するように該培地に添加した。14日目にCTL活性を試験し、上記と同一の方法を使用してCTLクローンを増殖させた(Uchida N et al., Clin Cancer Res 2004, 10(24): 8577-86; Suda T et al., Cancer Sci 2006, 97(5): 411-9; Watanabe T et al., Cancer Sci 2005, 96(8)): 498-506)。
特異的CTL活性を調べるために、IFN-γ ELISPOTアッセイおよびIFN-γ酵素結合免疫吸着アッセイ(ELISA)を実施した。具体的には、ペプチドパルスしたC1R‐A33(1 x 104個細胞/ウェル)を刺激細胞として調製した。誘導したCTL、すなわちCTL株およびCTLクローンを応答細胞として使用した。IFN-γ ELISPOTアッセイおよびIFN-γ ELISAは、製造業者の手順に従って実施した。
標的遺伝子またはHLA-A*3303のオープンリーディングフレームをコードするcDNAをPCRによって増幅した。PCR増幅産物を発現ベクターにクローニングした。製造業者の推奨する手順に従ってリポフェクタミン2000(Invitrogen)を使用して、標的遺伝子およびHLAの陰性細胞株であるCOS7に標的遺伝子の発現ベクターおよびHLA-A*3303の発現ベクターのいずれかまたは両方をトランスフェクトした。トランスフェクションから2日後、トランスフェクトした細胞をベルセン(Invitrogen)を用いて回収し、CTL活性アッセイのための標的細胞(5 x 104個細胞/ウェル)として使用した。
CDCA1由来のHLA-A * 3303結合ペプチドの予測
表2aおよび表2bは、HLA-A*3303への結合が予測されたCDCA1由来の9merペプチドおよび10merペプチドを結合親和性の高い順に示す。HLA-A*3303結合能を有する可能性のある合計32種のペプチドを、エピトープペプチドを決定するために選択し、調べた。
CDCA1由来のペプチドに対するCTLを、「材料および方法」に記載したプロトコールに従って作製した。IFN-γ ELISPOTアッセイによって、ペプチド特異的なCTL活性を測定した(図5)。CDCA1-A33-9-43(配列番号:27)を用いたウェル番号#2(a)、CDCA1-A33-9-123(配列番号:3)を用いたウェル番号#1(b)、CDCA1-A33-9-108(配列番号:60)を用いたウェル番号#3(c)、CDCA1-A33-9-261(配列番号:28)を用いたウェル番号#2(d)、CDCA1-A33-9-105(配列番号:5)を用いたウェル番号#6(e)、CDCA1-A33-10-10(配列番号:67)を用いたウェル番号#8(f)およびCDCA1-A33-10-260(配列番号:69)を用いたウェル番号#5(g)におけるCTLは、対象と比較して強力なIFN-γ産生を示した。一方、表2aおよび表2bに示される他のペプチドは、HLA-A*3303との結合活性を有する可能性があるにもかかわらず、それらのペプチドでの刺激によっては、特異的なCTL活性が測定されなかった。典型的な陰性データの例ではあるが、CDCA1-A33-10-122(配列番号:68)で刺激したCTLからは特異的IFN-γ産生が観察されなかった(h)。結果としてCDCA1に由来する7種のペプチドを、強力なCTLを誘導することができるペプチドとして選択した。
IFN-γ ELISPOTアッセイにおいてペプチド特異的CTL活性を示したCDCA1-A33-9-43(配列番号:27)を用いたウェル番号#2(a)、CDCA1-A33-9-123(配列番号:3)を用いたウェル番号#1(b)、CDCA1-A33-10-10(配列番号:67)を用いたウェル番号#8(c)およびCDCA1-A33-10-260(配列番号:69)を用いたウェル番号#5(d)におけるCTLを増殖させ、CTL株を樹立した。これらのCTL株のCTL活性をIFN-γ ELISAによって測定した(図6)。これらのCTL株は、ペプチドをパルスしなかった標的細胞と比較して、対応するペプチドをパルスした標的細胞に対して強力なIFN-γ産生を示した。さらに、上記の「材料および方法」の章に記載された通りCTL株から限界希釈によってCTLクローンを樹立し、ペプチドをパルスしたC1R-A33に対するCTLクローンからのIFN-γ産生をIFN-γ ELISAによって測定した。CDCA1-A33-9-43(配列番号:27)(a)、およびCDCA1-A33-9-123(配列番号:3)(b)で刺激したCTLクローンから強力なIFN-γ産生が観察された(図7)。
CDCA1-A33-9-43(配列番号:27)に対して樹立されたCTLクローンを、CDCA1およびHLA-A*3303分子を発現する標的細胞を認識する能力に関して調べた。全長CDCA1およびHLA-A*3303遺伝子の両方をトランスフェクトしたCOS7細胞(CDCA1およびHLA-A*3303遺伝子を発現する標的細胞の特異的モデル)を標的細胞として調製した。全長CDCA1またはHLA-A*3303のいずれかをトランスフェクトしたCOS7細胞を対照として調製した。CDCA1-A33-9-43(配列番号:27)で刺激したCTLクローンが、CDCA1およびHLA-A*3303の両方を発現するCOS7細胞に対して強力なCTL活性を示した(図8)。一方、対照細胞に対して有意な特異的CTL活性は検出されなかった。これらのデータにより、CDCA1-A33-9-43(配列番号:27)が、CDCA1の内因的プロセシングにより生じるペプチドであり、かつHLA-A*3303分子とともに標的細胞上に提示されてCTLによって認識されることが明確に実証される。これらの結果は、CDCA1-A33-9-43(配列番号:27)が、CDCA1を発現するがんを有する患者に対するがんワクチンとして適している可能性を示している。
CDCA1-A33-9-43(配列番号:27)、CDCA1-A33-9-123(配列番号:3)、CDCA1-A33-9-108(配列番号:60)、CDCA1-A33-9-261(配列番号:28)、CDCA1-A33-9-105(配列番号:5)、CDCA1-A33-10-10(配列番号:67)およびCDCA1-A33-10-260(配列番号:69)の配列が、ヒト免疫系を感作することが知られている他の分子に由来するペプチドと相同であるという事実に起因する可能性がある。この可能性を排除するために、BLASTアルゴリズム(blast.ncbi.nlm.nih.gov/Blast.cgi)を用いて、これらのペプチドの配列をクエリーとして相同性解析を行った。その結果、CDCA1-A33-9-43(配列番号:27)、CDCA1-A33-9-123(配列番号:3)、CDCA1-A33-9-108(配列番号:60)、CDCA1-A33-9-261(配列番号:28)、CDCA1-A33-9-105(配列番号:5)、CDCA1-A33-10-10(配列番号:67)およびCDCA1-A33-10-260(配列番号:69)の配列に対して有意な相同性を有する配列は認められなかった。したがって、本発明者らの知る限りでは、これらのペプチドが、他の関連のない分子に対して意図しない免疫応答を引き起こす可能性はほとんどないと考えられる。結論として、CDCA1由来の新規HLA-A33拘束性エピトープペプチドが同定された。さらに、CDCA1由来のエピトープペプチドはがん免疫療法に適用し得ることが示された。
材料および方法
細胞株
HLA-AおよびHLA-B陰性ヒトBリンパ芽球様細胞株であるC1R、ならびにアフリカミドリザル腎細胞株であるCOS7は、ATCCから購入した。
HLA-A*0301を安定的に発現するC1R(C1R-A03)は、刺激細胞として使用された。HLA-A*0301のオープンリーディングフレームをコードするcDNAはPCRで増幅され、発現ベクターへクローニングされた。C1R細胞は発現ベクターで形質転換され、その後、G418(Invitrogen)を用いて2週間選択された。G418選択細胞はG418が添加された培養培地を含む96ウェルプレートのウェルへ蒔かれ、さらに30日培養された。外来性HLA-A*0301のC1R細胞における発現はフローサイトメトリー解析で確認された。
HLA-A*0301分子に結合するCDCA1由来の9merおよび10merのペプチドを、結合予測サーバー「NetMHC 3.2」(www.cbs.dtu.dk/services/NetMHC/) (Buus et al., Tissue Antigens. 2003 Nov, 62(5):378-84; Nielsen et al., Protein Sci. 2003 May, 12(5):1007-17, Bioinformatics. 2004 Jun 12:20(9):1388-97)を用いて予測した。
ペプチドは、Biosynthesis(Lewisville,Texas)により、標準的な固相合成法に従って合成し、逆相高速液体クロマトグラフィー(HPLC)によって精製した。該ペプチドの純度(>90%)および同一性は、それぞれ分析用HPLCおよび質量分析によって分析した。ペプチドはジメチルスルホキシドに20mg/mlで溶解し、-80℃で保存した。
単球由来の樹状細胞(DC)を抗原提示細胞として使用し、ヒト白血球抗原(HLA)上に提示されたペプチドに対する細胞傷害性Tリンパ球(CTL)応答を誘導した。他所に記載されているように、DCはインビトロで作製した(Nakahara S et al.,Cancer Res 2003,63(14):4112-8)。具体的には、Ficoll-Paque plus (Pharmacia)溶液によって健常なボランティア(HLA-A*0301陽性)から単離した末梢血単核細胞を、プラスチック製の組織培養ディッシュ(Becton Dickinson)へ付着させることによって分離し、それらを単球画分として濃縮した。2%の加熱不活化した自己血清(AS)を含むAIM-V培地(Invitrogen)中、1000 IU/mlの顆粒球マクロファージコロニー刺激因子(R&D System)および1000 IU/mlのインターロイキン(IL)-4(R&D System)の存在下で、単球が濃縮された集団を培養した。7日間の培養後、サイトカインで誘導したDCに、AIM-V培地中で37℃で3時間、3μg/mlのβ2-ミクログロブリンの存在下で20μg/mlの各合成ペプチドをパルスした。作製された細胞は、自身の細胞表面上にCD80、CD83、CD86、およびHLAクラスIIなどのDC関連分子を発現しているようであった(データは示さず)。次いで、ペプチドパルスしたこれらのDCをX線照射(20Gy)によって不活化し、CD8 Positive Isolation Kit(Dynal)を用いた陽性選択によって得られた自己CD8+T細胞と1:20の比率で混合した。これらの培養物を48ウェルプレート(Corning)中に播種した。各ウェルは、0.5mlのAIM-V/2%AS培地中に、1.5 x 104個のペプチドパルスしたDC、3 x 105個のCD8+T細胞および10ng/mlのIL-7(R&D System)を含むようにした。3日後、これらの培養物に、IL-2(CHIRON)を最終濃度20IU/mlまで添加した。7日目および14日目に、ペプチドパルスした自己DCでT細胞をさらに刺激した。DCは上記と同一の方法によって毎回調製した。21日目に、3回目のペプチド刺激後、ペプチドパルスしたC1R-A03に対してCTLをヒト インターフェロン(IFN)-γ 酵素結合免疫スポット(ELISPOT)アッセイにて試験した(Tanaka H et al., Br J Cancer 2001, 84(1): 94-9; Umano Y et al., Br J Cancer 2001, 84(8): 1052-7; Uchida N et al., Clin Cancer Res 2004, 10(24): 8577-86; Suda T et al., Cancer Sci 2006, 97(5): 411-9; Watanabe T et al., Cancer Sci 2005, 96(8): 498-506)。
Riddellら(Walter EA et al., N Engl J Med 1995, 333(16): 1038-44; Riddell SR et al., Nat Med 1996, 2(2): 216-23)によって記載されている方法と類似の方法を使用して、CTLを培養下で増殖させた。CTLを、5 x 106個細胞/フラスコのマイトマイシンCによって処理された2種類のヒトBリンパ芽球様細胞株、および40ng/mlの抗CD3抗体とともに、総量25mlの5%AS含有AIM-V(AIM-V/5%AS)培地中で共培養した。培養開始1日後に、120IU/mlのIL-2を該培養物に添加した。5、8および11日目に、30IU/mlのIL-2を含む新鮮なAIM-V/5%AS培地を、該培養物に添加した(Tanaka H et al., Br J Cancer 2001, 84(1): 94-9; Umano Y et al., Br J Cancer 2001, 84(8): 1052-7, Uchida N et al., Clin Cancer Res 2004, 10(24): 8577-86; Suda T et al., Cancer Sci 2006, 97(5): 411-9, Watanabe T et al., Cancer Sci 2005, 96(8): 498-506)。
96丸底マイクロタイタープレート(Nalge Nunc International)においてCTL 0.3個、1個、および3個細胞/ウェルとなるように、希釈を行った。CTLを、1 x 104個細胞/ウェルのマイトマイシンCによって処理された2種類のヒトBリンパ芽球様細胞株、30ng/mlの抗CD3抗体、および125IU/mlのIL-2とともに、総量150μl/ウェルのAIM-V/5%AS培地中で培養した。10日後、50μl/ウェルのIL-2を、IL-2の最終濃度が125IU/mlに達するように該培地に添加した。14日目にCTL活性を試験し、上記と同一の方法を使用してCTLクローンを増殖させた(Uchida N et al., Clin Cancer Res 2004, 10(24): 8577-86; Suda T et al., Cancer Sci 2006, 97(5): 411-9; Watanabe T et al., Cancer Sci 2005, 96(8)): 498-506)。
特異的CTL活性を調べるために、IFN-γ ELISPOTアッセイおよびIFN-γ酵素結合免疫吸着アッセイ(ELISA)を実施した。具体的には、ペプチドパルスしたC1R‐A03(1 x 104個細胞/ウェル)を刺激細胞として調製した。誘導したCTL、すなわちCTL株およびCTLクローンを応答細胞として使用した。IFN-γ ELISPOTアッセイおよびIFN-γ ELISAは、製造業者の手順に従って実施した。
標的遺伝子またはHLA-A*0301のオープンリーディングフレームをコードするcDNAをPCRによって増幅した。PCR増幅産物を発現ベクターにクローニングした。製造業者の推奨する手順に従ってリポフェクタミン2000(Invitrogen)を使用して、標的遺伝子およびHLAの陰性細胞株であるCOS7に標的遺伝子の発現ベクターおよびHLA-A*0301の発現ベクターのいずれかまたは両方をトランスフェクトした。トランスフェクションから2日後、トランスフェクトした細胞をベルセン(Invitrogen)を用いて回収し、CTL活性アッセイのための標的細胞(5 x 104個細胞/ウェル)として使用した。
CDCA1由来のHLA-A * 0301結合ペプチドの予測
表3aおよび表3bは、HLA-A*0301への結合が予測されたCDCA1由来の9merペプチドおよび10merペプチドを結合親和性の高い順に示す。HLA-A*0301結合能を有する可能性のある合計24種のペプチドを、エピトープペプチドを決定するために選択し、調べた。
CDCA1由来のペプチドに対するCTLを、「材料および方法」に記載したプロトコールに従って作製した。IFN-γ ELISPOTアッセイによって、ペプチド特異的なCTL活性を測定した(図9)。CDCA1-A03-9-219(配列番号:7)を用いたウェル番号#3(a)、CDCA1-A03-10-400(配列番号:38)を用いたウェル番号#1(b)およびCDCA1-A03-10-257(配列番号:47)を用いたウェル番号#2(c)におけるCTLは、対照と比較して強力なIFN-γ産生を示した。一方、表3aおよび表3bに示される他のペプチドは、HLA-A*0301との結合活性を有する可能性があるにもかかわらず、それらのペプチドでの刺激によっては、特異的なCTL活性が測定されなかった。典型的な陰性データの例であるが、CDCA1-A03-9-343(配列番号:10)で刺激したCTLからは特異的IFN-γ産生が観察されなかった(d)。結果として、CDCA1に由来する3種のペプチドを、強力なCTLを誘導することができるペプチドとして選択した。
IFN-γ ELISPOTアッセイにてペプチド特異的CTL活性を示したCDCA1-A03-9-219(配列番号:7)を用いたウェル番号#3(a)、CDCA1-A03-10-400(配列番号:38)を用いたウェル番号#1(b)およびCDCA1-A03-10-257(配列番号:47)を用いたウェル番号#2(c)におけるCTLを増殖させ、CTL株を樹立した。これらのCTL株のCTL活性をIFN-γ ELISAによって測定した(図10)。これらのCTL株は、ペプチドをパルスしなかった標的細胞と比較して、対応するペプチドをパルスした標的細胞に対して強力なIFN-γ産生を示した。さらに、上記の「材料および方法」の章に記載された通りCTL株から限界希釈によってCTLクローンを樹立し、ペプチドをパルスしたC1R-A03に対するCTLクローンからのIFN-γ産生をIFN-γ ELISAによって測定した。CDCA1-A03-9-219(配列番号:7)(a)、CDCA1-A03-10-400(配列番号:38)(b)およびCDCA1-A03-10-257(配列番号:47)(c)で刺激したCTLクローンから強力なIFN-γ産生が観察された(図11)。
CDCA1-A03-9-219(配列番号:7)(a)およびCDCA1-A03-10-400(配列番号:38)(b)に対して樹立されたCTLクローンを、CDCA1およびHLA-A*0301分子を発現する標的細胞を認識する能力に関して調べた。全長CDCA1およびHLA-A*0301遺伝子の両方をトランスフェクトしたCOS7細胞(CDCA1およびHLA-A*0301遺伝子を発現する標的細胞の特異的モデル)を標的細胞として調製した。全長CDCA1またはHLA-A*0301のいずれかをトランスフェクトしたCOS7細胞を対照として調製した。CDCA1-A03-9-219(配列番号:7)(a)およびCDCA1-A03-10-400(配列番号:38)(b)で刺激したCTLクローンが、CDCA1およびHLA-A*0301の両方を発現するCOS7細胞に対して強力なCTL活性を示した(図12)。一方、対照細胞に対して有意な特異的CTL活性は検出されなかった。これらのデータにより、CDCA1-A03-9-219(配列番号:7)およびCDCA1-A03-10-400(配列番号:38)が、CDCA1の内因的プロセシングにより生じるペプチドであり、かつHLA-A*0301分子とともに標的細胞上に提示されてCTLによって認識されることが明確に実証される。これらの結果は、CDCA1-A03-9-219(配列番号:7)およびCDCA1-A03-10-400(配列番号:38)が、CDCA1を発現するがんを有する患者に対するがんワクチンとして適している可能性を示している。
CDCA1-A03-9-219(配列番号:7)、CDCA1-A03-10-400(配列番号:38)およびCDCA1-A03-10-257(配列番号:47)で刺激したCTLは、有意かつ特異的なCTL活性を示した。この結果は、CDCA1-A03-9-219(配列番号:7)、CDCA1-A03-10-400(配列番号:38)およびCDCA1-A03-10-257(配列番号:47)の配列が、ヒト免疫系を感作することが知られている他の分子に由来するペプチドと相同であるという事実に起因する可能性がある。この可能性を排除するために、BLASTアルゴリズム(blast.ncbi.nlm.nih.gov/Blast.cgi)を用いて、これらのペプチドの配列をクエリーとして相同性解析を行った。その結果、CDCA1-A03-9-219(配列番号:7)、CDCA1-A03-10-400(配列番号:38)およびCDCA1-A03-10-257(配列番号:47)の配列に対して有意な相同性を有する配列は認められなかった。したがって、本発明者らの知る限りでは、これらのペプチドが、他の関連のない分子に対して意図しない免疫応答を引き起こす可能性はほとんどないと考えられる。結論として、本発明者は、CDCA1由来の新規HLA-A03拘束性エピトープペプチドが同定された。さらに、CDCA1由来のエピトープペプチドは、がん免疫療法に適用し得ることが示された。
エマルション製剤の調製
ペプチドを注射用水または滅菌生理食塩水に1.0~10.0mg/mlとなるように溶解し、シリンジに採取する。これを注射用水または生理食塩水と等量のIFAを充填したシリンジとコネクタにより連結し、連結した2本のシリンジのシリンジプランジャを交互に押圧することによって撹拌する。数分間撹拌した後、ドロップテスト法により、エマルションの完成を評価する。ドロップテスト法は、撹拌したサンプル1滴を水面上に滴下することによって行うことができる。水面上に滴下したサンプルが直ちに水中に拡散しない場合にはエマルション完成と評価し、ただちに水中に拡散する場合にはエマルション未完成と評価する。エマルション未完成と評価された場合には、さらに撹拌を行ってエマルションを完成させる。完成したエマルションは、皮下注射により、がん患者に投与することができる。投与対象のがん患者としては、膀胱がん、乳がん、子宮頸がん、胆管細胞がん、慢性骨髄性白血病(CML)、食道がん、胃がん、非小細胞肺がん、リンパ腫、骨肉腫、前立腺がん、腎がん、小細胞肺がん、頭頸部がん、軟部組織腫瘍、または大腸がん等に罹患している患者を選択することができる。
ペプチドを注射用水に1.0~10.0mg/mlとなるように溶解し、ろ過滅菌を行う。これを滅菌バイアルに充填し、滅菌したゴム栓を半打栓する。このバイアルを凍結乾燥した後、全打栓およびアルミキャップの巻き締めを行うことにより、凍結乾燥製剤を作成する。使用の際には、バイアルに注射用水または滅菌生理食塩水を注入して凍結乾燥粉末を再溶解する。シリンジを用いてバイアル中の再溶解液を採取し、採取した再溶解液と等量のIFAを充填したシリンジとコネクタにより連結する。連結した2本のシリンジのシリンジプランジャを交互に押圧することによって撹拌する。数分間撹拌した後、ドロップテスト法により、エマルションの完成を評価する。完成したエマルションは、皮下注射により、がん患者に投与することができる。投与対象のがん患者としては、膀胱がん、乳がん、子宮頸がん、胆管細胞がん、慢性骨髄性白血病(CML)、食道がん、胃がん、非小細胞肺がん、リンパ腫、骨肉腫、前立腺がん、腎がん、小細胞肺がん、頭頸部がん、軟部組織腫瘍、または大腸がん等に罹患している患者を選択することができる。
Claims (23)
- 以下の群より選択されるアミノ酸配列を含む、細胞傷害性T細胞(CTL)誘導能を有する15アミノ酸未満のペプチド:
(a)配列番号:3、5~7、9、10、12~14、17、19、21、30、35、38~40、45、53、56、58、27、60、28、67、69および47からなる群より選択されるアミノ酸配列;および
(b)配列番号:3、5~7、9、10、12~14、17、19、21、30、35、38~40、45、53、56、58、27、60、28、67、69および47からなる群より選択されるアミノ酸配列に対して、1個、2個、または数個のアミノ酸が置換、欠失、挿入および/または付加されているアミノ酸配列。 - 以下の(i)~(iii)からなる群より選択される、請求項1に記載のペプチド:
(i)配列番号:3、5~7、9、10、12~14、17、19、21、30、35、38~40、45、53、56および58からなる群より選択されるアミノ酸配列に対して以下の(a)~(d)からなる群より選択される1個以上の置換がされた、アミノ酸配列を含むペプチド:
(a)N末端から2番目のアミノ酸が、スレオニン、バリン、イソロイシン、ロイシン、フェニルアラニンおよびチロシンからなる群より選択されるアミノ酸に置換されている;
(b)N末端から3番目のアミノ酸が、ロイシン、フェニルアラニン、チロシン、イソロイシンおよびアラニンからなる群より選択されるアミノ酸に置換されている;
(c)N末端から7番目のアミノ酸が、ロイシン、イソロイシン、チロシン、バリンおよびフェニルアラニンからなる群より選択されるアミノ酸に置換されている;および
(d)C末端のアミノ酸が、リジンおよびアルギニンからなる群より選択されるアミノ酸に置換されている;
(ii)配列番号:27、3、60、28、5、67および69からなる群より選択されるアミノ酸配列に対して以下の(a)~(c)からなる群より選択される1個以上の置換がされたアミノ酸配列を含むペプチド:
(a)N末端から1番目のアミノ酸が、アスパラギン酸およびグルタミン酸からなる群より選択されるアミノ酸に置換されている;
(b)N末端から2番目のアミノ酸が、フェニルアラニン、チロシン、アラニン、イソロイシン、ロイシンおよびバリンからなる群より選択されるアミノ酸に置換されている;および
(c)C末端のアミノ酸が、アルギニンおよびリジンからなる群より選択されるアミノ酸に置換されている;
(iii)配列番号:7、38および47からなる群より選択されるアミノ酸配列に対して以下の(a)~(b)からなる群より選択される1個以上の置換がされたアミノ酸配列を含むペプチド:
(a)N末端から2番目のアミノ酸が、ロイシン、メチオニン、バリン、アラニン、イソロイシン、セリンおよびスレオニンからなる群より選択されるアミノ酸に置換されている;および
(b)C末端のアミノ酸が、アルギニン、リジン、チロシンおよびフェニルアラニンからなる群より選択されるアミノ酸に置換されている。 - 配列番号:3、5~7、9、10、12~14、17、19、21、30、35、38~40、45、53、56、58、27、60、28、67、69および47からなる群より選択されるアミノ酸配列からなる、請求項1に記載のペプチド。
- 請求項1~3のいずれか一項に記載のペプチドをコードする、ポリヌクレオチド。
- 薬学的に許容される担体と、以下の(a)~(e)からなる群より選択される少なくとも1つの成分とを含む組成物:
(a)請求項1~3のいずれか一項に記載の1種類もしくは複数種のペプチド;
(b)請求項1~3のいずれか一項に記載のペプチドを発現可能な形態でコードする1種類もしくは複数種のポリヌクレオチド;
(c)請求項1~3のいずれか一項に記載のペプチドとHLA抗原との複合体を自身の細胞表面上に提示する抗原提示細胞(APC);
(d)請求項1~3のいずれか一項に記載のペプチドとHLA抗原との複合体を自身の細胞表面上に提示するエキソソーム;および
(e)請求項1~3のいずれか一項に記載のペプチドを標的とするCTL。 - 前記成分が以下の(a)~(d)からなる群より選択される少なくとも1つの成分であり、CTLを誘導するための組成物である、請求項5に記載の組成物:
(a)請求項1~3のいずれか一項に記載の1種類もしくは複数種のペプチド;
(b)請求項1~3のいずれか一項に記載のペプチドを発現可能な形態でコードする1種類もしくは複数種のポリヌクレオチド;
(c)請求項1~3のいずれか一項に記載のペプチドとHLA抗原との複合体を自身の細胞表面上に提示する抗原提示細胞(APC);および
(d)請求項1~3のいずれか一項に記載のペプチドとHLA抗原との複合体を自身の細胞表面上に提示するエキソソーム。 - 薬学的組成物である、請求項5に記載の組成物。
- (i)がんの治療、(ii)がんの予防、および(iii)がんの術後の再発の予防からなる群より選択される1以上の用途のための、請求項7に記載の組成物。
- がんに対する免疫応答を誘導するための、請求項7に記載の組成物。
- がんが、膀胱がん、乳がん、子宮頸がん、胆管細胞がん、慢性骨髄性白血病(CML)、食道がん、胃がん、非小細胞肺がん、リンパ腫、骨肉腫、前立腺がん、腎がん、小細胞肺がん、頭頸部がん、軟部組織腫瘍、および大腸がんからなる群より選択される、請求項8または9に記載の組成物。
- HLA-A11、HLA-A33およびHLA-A03からなる群より選択される少なくとも1つのHLAが陽性である対象への投与のために製剤化される、請求項5~10のいずれか一項に記載の組成物。
- 以下からなる群より選択される段階を含む、CTL誘導能を有するAPCを誘導する方法:
(a)APCを、請求項1~3のいずれか一項に記載のペプチドとインビトロ、エクスビボ、またはインビボで接触させる段階、および
(b)請求項1~3のいずれか一項に記載のペプチドをコードするポリヌクレオチドをAPCに導入する段階。 - 以下からなる群より選択される段階を含む、CTLを誘導する方法:
(a)CD8陽性T細胞を、HLA抗原と請求項1~3のいずれか一項に記載のペプチドとの複合体を自身の表面上に提示するAPCと共培養する段階、
(b)CD8陽性T細胞を、HLA抗原と請求項1~3のいずれか一項に記載のペプチドとの複合体を自身の表面上に提示するエキソソームと共培養する段階、および
(c)細胞表面上にHLA抗原により提示された請求項1~3のいずれか一項に記載のペプチドに結合し得るT細胞受容体(TCR)の各サブユニットをコードするポリヌクレオチドをCD8陽性T細胞に導入する段階。 - HLA抗原と請求項1~3のいずれか一項に記載のペプチドとの複合体を自身の表面上に提示するAPC。
- 請求項12に記載の方法によって誘導される、請求項14記載のAPC。
- 請求項1~3のいずれか一項に記載のペプチドを標的とするCTL。
- 請求項13に記載の方法によって誘導される、請求項16に記載のCTL。
- 以下の(a)~(e)からなる群より選択される少なくとも1つの成分を対象に投与する段階を含む、がんに対する免疫応答を誘導する方法:
(a)請求項1~3のいずれか一項に記載の1種類もしくは複数種のペプチド;
(b)請求項1~3のいずれか一項に記載のペプチドを発現可能な形態でコードする1種類もしくは複数種のポリヌクレオチド;
(c)請求項1~3のいずれか一項に記載のペプチドとHLA抗原との複合体を自身の細胞表面上に提示する抗原提示細胞(APC);
(d)請求項1~3のいずれか一項に記載のペプチドとHLA抗原との複合体を自身の細胞表面上に提示するエキソソーム;および
(e)請求項1~3のいずれか一項に記載のペプチドを標的とするCTL。 - 以下の(a)~(e)からなる群より選択される少なくとも1つの成分を対象に投与する段階を含む、がんを治療および/もしくは予防する、ならびに/または術後のその再発を予防する方法:
(a)請求項1~3のいずれか一項に記載の1種類もしくは複数種のペプチド;
(b)請求項1~3のいずれか一項に記載のペプチドを発現可能な形態でコードする1種類もしくは複数種のポリヌクレオチド;
(c)請求項1~3のいずれか一項に記載のペプチドとHLA抗原との複合体を自身の細胞表面上に提示する抗原提示細胞(APC);
(d)請求項1~3のいずれか一項に記載のペプチドとHLA抗原との複合体を自身の細胞表面上に提示するエキソソーム;および
(e)請求項1~3のいずれか一項に記載のペプチドを標的とするCTL。 - 請求項1~3のいずれか一項に記載のペプチドに結合する抗体。
- CTL誘導能を有するペプチドをスクリーニングする方法であって、以下の段階を含む方法:
(a)配列番号:3、5~7、9、10、12~14、17、19、21、30、35、38~40、45、53、56、58、27、60、28、67、69および47からなる群より選択されるアミノ酸配列からなる元のアミノ酸配列に対して、1個、2個、または数個のアミノ酸残基が置換、欠失、挿入、および/または付加されたアミノ酸配列からなる候補配列を作成する段階;
(b)(a)で作成した候補配列の中からCDCA1以外のいかなる公知のヒト遺伝子産物とも有意な相同性(配列同一性)を有さない候補配列を選択する段階;
(c)(b)で選択した候補配列からなるペプチドと、APCとを接触させる段階;
(d)(c)のAPCとCD8陽性T細胞とを接触させる段階;および
(e)元のアミノ酸配列からなるペプチドよりも同等かまたはより高いCTL誘導能を有するペプチドを選択する段階。 - 請求項1~3のいずれか一項に記載の1種類もしくは複数種のペプチド、水溶性の担体、および油性アジュバントを含むエマルション。
- 請求項5~11のいずれか一項に記載の組成物が収容されている容器、およびアジュバントが収容されている容器を含むキット。
Priority Applications (14)
Application Number | Priority Date | Filing Date | Title |
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KR1020177006015A KR20170040315A (ko) | 2014-08-04 | 2015-07-31 | Cdca1-유래 펩티드 및 이를 포함하는 백신 |
EP15830292.7A EP3178839B1 (en) | 2014-08-04 | 2015-07-31 | Cdca1-derived peptide and vaccine containing same |
MX2017001650A MX2017001650A (es) | 2014-08-04 | 2015-07-31 | Peptido derivado de cdca1 y vacuna que lo contiene. |
US15/500,906 US10676514B2 (en) | 2014-08-04 | 2015-07-31 | CDCA1-derived peptide and vaccine containing same |
CN201580052500.2A CN107074908B (zh) | 2014-08-04 | 2015-07-31 | Cdca1衍生的肽和含有它们的疫苗 |
JP2016540195A JP6700512B2 (ja) | 2014-08-04 | 2015-07-31 | Cdca1由来ペプチドおよびそれを含むワクチン |
EP23194566.8A EP4282883A3 (en) | 2014-08-04 | 2015-07-31 | Cdca1-derived peptide and vaccine containing same |
SG11201700839SA SG11201700839SA (en) | 2014-08-04 | 2015-07-31 | Cdca1-derived peptide and vaccine containing same |
RU2017106888A RU2699543C2 (ru) | 2014-08-04 | 2015-07-31 | Пептид, полученный из cdca1, и содержащая его вакцина |
AU2015300258A AU2015300258B2 (en) | 2014-08-04 | 2015-07-31 | CDCA1-derived peptide and vaccine containing same |
BR112017002212A BR112017002212A2 (pt) | 2014-08-04 | 2015-07-31 | peptídeo derivado de cdca1 e vacina contendo o mesmo |
CA2956132A CA2956132A1 (en) | 2014-08-04 | 2015-07-31 | Cdca1-derived peptide and vaccine containing same |
IL250215A IL250215B (en) | 2014-08-04 | 2017-01-22 | A peptide derived from cdca1 and a component containing it |
AU2020202681A AU2020202681B2 (en) | 2014-08-04 | 2020-04-22 | CDCA1-derived peptide and vaccine containing same |
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EP (2) | EP3178839B1 (ja) |
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CN (3) | CN107074908B (ja) |
AU (2) | AU2015300258B2 (ja) |
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CA (1) | CA2956132A1 (ja) |
IL (1) | IL250215B (ja) |
MX (1) | MX2017001650A (ja) |
RU (1) | RU2699543C2 (ja) |
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WO2020027239A1 (ja) | 2018-08-02 | 2020-02-06 | オンコセラピー・サイエンス株式会社 | Cdca1由来ペプチドおよびそれを含むワクチン |
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KR101943978B1 (ko) * | 2017-12-28 | 2019-01-30 | 주식회사 하이센스바이오 | 신규한 펩타이드 |
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Cited By (4)
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WO2020027239A1 (ja) | 2018-08-02 | 2020-02-06 | オンコセラピー・サイエンス株式会社 | Cdca1由来ペプチドおよびそれを含むワクチン |
KR20210040106A (ko) | 2018-08-02 | 2021-04-12 | 온코세라피 사이언스 가부시키가이샤 | Cdca1유래 펩타이드 및 이를 포함하는 백신 |
JPWO2020027239A1 (ja) * | 2018-08-02 | 2021-08-10 | オンコセラピー・サイエンス株式会社 | Cdca1由来ペプチドおよびそれを含むワクチン |
JP7448124B2 (ja) | 2018-08-02 | 2024-03-12 | オンコセラピー・サイエンス株式会社 | Cdca1由来ペプチドおよびそれを含むワクチン |
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