WO2005007694A1 - Peptide her2/neu et utilisation therapeutique de celui-ci - Google Patents

Peptide her2/neu et utilisation therapeutique de celui-ci Download PDF

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WO2005007694A1
WO2005007694A1 PCT/JP2004/010547 JP2004010547W WO2005007694A1 WO 2005007694 A1 WO2005007694 A1 WO 2005007694A1 JP 2004010547 W JP2004010547 W JP 2004010547W WO 2005007694 A1 WO2005007694 A1 WO 2005007694A1
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seq
peptide
her2
hla
neu
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PCT/JP2004/010547
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Japanese (ja)
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Kyogo Itoh
Shigeki Shichijo
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Green Peptide Co., Ltd.
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A61K39/001102—Receptors, cell surface antigens or cell surface determinants
    • A61K39/001103—Receptors for growth factors
    • A61K39/001106—Her-2/neu/ErbB2, Her-3/ErbB3 or Her 4/ErbB4
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/82—Translation products from oncogenes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the present invention relates to a HER2 / neu cancer antigen peptide. More specifically, the present invention relates to HLA-A24 or mono-A2-binding HER2 / neu peptide capable of inducing humoral immunity as well as cell-mediated immunity, and a cancer vaccine containing the peptide. I do. Background art
  • the immune system in the body plays an important role against cancer. This exemption
  • cytotoxic T cells act as powerful effectors and can provide long-term immunity in response to tumor antigens produced in tumor cells. It can.
  • Tumor antigens are decomposed in specific cells to form an antigen peptide consisting of 8 to 11 amino acids, which binds to the major histocompatibility antigen, human leukocyte antigen (HLA) molecule, and presents it on the tumor cell surface. Is done.
  • HLA human leukocyte antigen
  • the tight peptide sequence presented on the tumor cell surface by the HLA molecule is recognized by CTL (Townsend. A., et al., Annu. Rev. Immunol. 7, 601-624, 1989). ).
  • CTL recognizes the complex of the antigen peptide and HLA and damages tumor cells.
  • HLA is a cell membrane antigen roughly classified into a class I antigen and a class II antigen. It is class I antigen that is recognized by CTL together with the antigenic peptide, and is expressed on almost all nucleated cells. HLA class I antigens are further classified into HLA-A, B, C, etc., and there are polymorphisms in their genes. Among them, the HLA—A24 allele (a 1 e 1 e) is about 60% of Japanese (often 95% of the genotype is A2402), 20% of Caucasians, and 1 ⁇ % of Africans. The HLA-A2 allele is about 40 Japanese. /. The Chinese about 53. /.
  • HLA Molecules have been reported to play a critical role in antigen presentation of various tumor antigens (Bauer, MP, et al., In Histcompatibility Testing, ed.
  • TAAs tumor-associated antigens
  • MAGE melanoma antigen
  • HER 2 Human Epidermal Growth Factor Receptor 2
  • This HER2 is one of the human epidermal growth factor receptors, and its HER2 gene encodes a transmembrane tyrosine kinase receptor and is frequently overexpressed in many solid tumors, especially breast cancer.
  • Overexpression of HER2 induces oncogenic transformation and promotes cell growth and results in dysregulation.
  • a correlation between HER2 overexpression and prognosis has been reported in many clinical studies.
  • overexpression of HER2 in breast cancer has been recognized as an important indicator for predicting the prognosis of breast cancer as well as its therapeutic effect.
  • HER2 Z neu has high homology to epidermal growth factor receptor Is a 185-kilodanoleton transmembrane tyrosine kinase receptor
  • HER2Zneu is highly expressed in various epithelial tumors, particularly in colorectal cancer, not only in its primary site but also in metastatic regions (Tanaka, H., et al., Brit. J. Cancer ,
  • HER2 / neu is highly expressed in many malignant tumors, but not expressed in normal cells or expressed only at low levels, so it is considered as a prototype TAA, It can be a strong candidate for immunotherapy.
  • HER2 / neu-derived HLA-A24 binding peptides nine peptides (GP2, amino acid positions 654-662) consisting of the transmembrane membrane of the HER2 / neu protein are found in ovarian and breast cancers. CTLs that are widely expressed in both HER2Zneu-positive tumor cells and that recognize cancer-specifically were found.
  • GP2 is thought to be useful as a peptide vaccine for cancer immunotherapy (Peoples, GE, et al .: Proc.
  • HLA—A2 binding HER2 / neu peptide p 369-377 (p 369) is used as a cancer immunotherapy for HLA-2 / neu overexpressing metastatic breast, ovarian or colorectal HLA —
  • a Cases of use in patients with 2-positive cancer have also been reported (Zaks, TZ, et al., Cancer Res. 58, 4902-4908, Nov. 1, 1998). '
  • Helper T cells as well as CTLs play a decisive role in the antitumor immune response. It is thought that if an antigenic peptide capable of inducing both helper T cell and CTL responses can be identified and introduced as a vaccine, cancer immunotherapy can be effectively performed.
  • HER211eu peptides have been reported (Kobayashi, H., et al., Cancer Res. 60, 5228-5236, Sept. 15, 2000).
  • the amino acid sequence of the reported CTL epitope in the peptide is shown below (the number in parentheses indicates the starting position of the amino acid of CTL epitope):
  • HER1124 Tyr Val Ala Pro Leu Thr Cys Ser Pro;
  • HER765 (768) Tyr Val Met Ala Gly Val Gly Ser Pro;
  • HER883 The amino acid sequence of the reported CTL epitope in the peptide is shown below (the number in parentheses indicates the starting position of the amino acid of CTL epitope):
  • HER1124 Tyr Val Ala Pro Leu Thr Cys Ser Pro;
  • HER765 768) : Tyr Val Met Ala Gly Val Gly Ser Pro;
  • HER822 (
  • HER883 (886): lie Lys Trp Met Ala Leu Glu Ser lie; HER605 (608): Leu Ser Tyr Met Pro lie Trp Lys Phe; HER62 (65): Leu Pro Thr Asn Ala Ser Leu Ser Phe; HER648 (651): Leu Thr Ser lie lie Ser Ala Val Val.
  • HER2 is expressed at high levels in many cancers, including breast cancer, and may be a target in humoral and cellular immunity (zum Buschenfelde, CM, et al., Cancer Res. 62, 2244-2247, April 15, 2002).
  • Herceptin a humanized anti-HER2 / neu antibody, regresses tumors in breast cancer patients whose tumors overexpress the HER2 / neu antigen (Vogel, CL, et al., J. Clin. Oncol., 20, 719-726, 2002; Burstein, HJ, et al., J. Clin. Oncol.,. 21, 46-53, 2003).
  • CTL epitope peptides Some of the peptides identified as CTL epitope peptides have the ability to induce both cellular and humoral immune responses in phase I clinical trials (Mine, T ., Et al., Cancer Sci., 94: 548-56, 2003) and reported that a peptide capable of inducing both a cellular immune response and a humoral immune response has immunogenicity. (Noguchi, ⁇ ⁇ , et al., Prostate, 57:
  • the present invention provides a novel candidate for a peptide vaccine that is recognized by HLA-A24 or 1A2-restricted cytotoxic T cells (CTLs), induces CTL, and induces humoral immunity.
  • CTLs cytotoxic T cells
  • the aim is to identify useful HER2 / neu peptides and provide effective cancer immunotherapy to HER2 / neu positive cancer patients.
  • the present inventors have identified CTL epitope peptides that induce HER2Zneu-specific cellular and humoral immune responses. IgG to HLA-A24 or mono-A2-binding peptides was frequently detected in the sera of cancer patients tested. In addition, they have found that these peptides induce peptide-specific and tumor-reactive CTL activity in peripheral blood mononuclear cells (PBMC) of the patient, and thus completed the present invention.
  • PBMC peripheral blood mononuclear cells
  • HER 2 3 42- 35 o SEQ ID NO: 1
  • HER 2 485 - 493 SEQ ID NO: 2
  • HER 2 553 -56 i SEQ ID NO: 3
  • HER 2 907 - 915 SEQ ID NO: 4
  • HER 2 44 4-453 SEQ ID NO: 5
  • HER 2 466 _ 474 SEQ ID NO: 6
  • HER 2 484 - 4 93 SEQ ID NO: 7
  • HER 2 785 - 794 SEQ ID NO: 8
  • HER 2 851 One 85 9 (SEQ ID NO: 9);
  • nucleic acid sequence molecule which is an oligonucleotide encoding the polypeptide according to (1) or (2) or the antigen peptide according to claim 3, or a polynucleotide or a complementary chain thereof;
  • HER 2 55 3 _56 i SEQ ID NO: 3
  • HER 2 9 915 SEQ ID NO: 4 in which recognizes HER 2 / neu reactive cytotoxic complexes with antigens base peptide and HLA-A24 antigen Sex T cells;
  • HER 2 444 — 453 (SEQ ID NO: 5), HER 2 466 _ 474 (SEQ ID NO: 6), HER 2 484 493 (SEQ ID NO: 7), HER 2 7 794 (SEQ ID NO: 8), or HER 2 851 over 859 (SEQ ID NO: 9) antigenic peptide and HLA-A2 antigen and a conjugate that recognizes HER 2 / neu reactive cytotoxic T cells that are;
  • the present invention provides the nucleic acid sequence molecule of the present invention, and / or The present invention relates to a diagnostic method for diagnosing the peptide-related disease by analyzing the peptide or the polypeptide as a marker.
  • the HER2 / neu peptide of the present invention is recognized by humoral immunity as well as cellular immunity and induces them, so that it is useful as a cancer vaccine or for diagnosis of such peptide-related diseases. '' Brief description of the drawings
  • Figure 1 shows the detection of IgG in response to a HER27 ⁇ eu-derived peptide with an HLA-A24 binding motif in the sera of three breast cancer patients (Pt) and one healthy female (HD). Graph.
  • FIG. 3 is a graph showing MHC-restricted cell-damaging activity induced by stimulation of HLA-A24-binding peptide.
  • FIG. 4A is a graph showing the detection of IgG that reacts with a HER27 ⁇ eu-derived peptide having an HLA-A2 binding motif in the sera of six breast cancer patients (Pt).
  • FIG. 4B is a graph showing the peptide specificity of anti-peptide IgG by an absorption test.
  • FIG. 5A is a graph showing the cytotoxic activity of CTL induced by stimulation of an HLA-A2-binding peptide. SKOV 3-A2 and SKOV 3-nu11 were used as target cells.
  • FIG. 5B is a graph showing the cytotoxic activity of CTL induced by stimulation of HLA-A2 binding peptide.
  • FIG. 5C is a graph showing the cytotoxic activity of CTL induced by stimulation of HLA-A2 binding peptide.
  • 1-87 cell line (HLA-A2 positive) and QG59 (HLA-A2 negative) were used as target cells.
  • FIG. 5D is a graph showing the cytotoxic activity of CTL induced by stimulation of HLA-A2 binding peptide.
  • 1-87 cell lines HL A-A2 positive
  • QG59 HLA-A2 negative
  • PHA blast cells were used as target cells.
  • FIG. 6A shows CTL MHC induced by HLA-A2 binding peptide stimulation. Restrictive and CD8 + T cell dependent. T2 cells (HL A_A2 positive, ATCC deposit number: CRL—1992) were used as target cells.
  • FIG. 6B shows confirmation of CTL MHC restriction and CD8 + T cell dependence induced by stimulation of HLA-A2 binding peptide, and peptide-specific cytotoxic activity of CTL.
  • Figure 7 shows the identification of HER2 / neu protein in various cell lines by Western blot.
  • the antigenic peptide of the present invention is a HER2 / neu HLA-A24 or mono-A2-binding peptide that induces specific cytotoxic T cells and induces specific antibodies.
  • the number of amino acid residues is usually 8 to 11, preferably 9 to 11, and more preferably 9 or 10.
  • HER 2 342 _ 35 As an example, HER 2 342 _ 35.
  • HER 2 342 _ 350 refers to peptides corresponding to Amino acid positions 342-350 of the total amino acid sequence of HER 2 / neu.
  • HER2 342 — 35. is also described as “HER2 / neu 342” by the number of the starting position of the amino acid sequence. The entire amino acid sequence of HER2 / neu has been deposited with Gene Bank under accession number P 04626 (SEQ ID NO: 10).
  • the antigenic peptide of the present invention comprises the peptide represented by any one of the amino acid sequences of SEQ ID NOS: 1 to 9, preferably at least about 70%, more preferably at least about 80%, and still more preferably It may be a mutant peptide having a homology of more than about 90%. Peptides having such homology can be selected using at least the strength of recognition by HLA-A24 or HLA-A2-restricted CTL as an index.
  • the number of amino acids in these peptides is a number that can be bound to the HLA molecule and presented on the surface of the antigen-presenting cell, and is a number that is recognized by CTL and has the properties as an epitope peptide, and is at least about 8 Or more, preferably about 9 or more, and more preferably 9 to 10.
  • the polypeptide containing the antigenic peptide of the present invention usually has 8 to 50 amino acid residues, preferably 9 to 30 amino acids, and has 111 ⁇ -8-24 or: ⁇ 11 ⁇ 8_2 restricted restriction proteins. The selection is made by using the strength of recognition by 1 ⁇ as an index.
  • the mutant polypeptide of the present invention preferably has about 70% or more, more preferably about 80% or more, and still more preferably about 90% or more homology with the above-mentioned polypeptide, and has an HLA-A24 or HLA- A2 restricted Polypeptide selected based on the strength of recognition by CTL. '
  • Techniques for determining the homology of an amino acid sequence include, for example, a method for directly determining an amino acid sequence, and a method for determining a predicted nucleotide sequence and then determining the amino acid encoded by the amino acid sequence based on the nucleotide sequence.
  • a method for estimating the sequence or the like can be used.
  • Mutant peptides and mutant polypeptides may have one or several amino acids deleted, substituted, attached, inserted, or induced mutation in the amino acid sequence of the antigenic peptide or polypeptide of the present invention. And peptides having an amino acid sequence having the same.
  • peptide of the present invention can be modified to such an extent that the function is not significantly changed, for example, by modifying the constituent amino group or carboxyl group.
  • the peptide of the present invention is recognized by HLA-A24 or HLA-A2-restricted CTL, can activate the CTL, and has a function as a tumor antigen.
  • the peptide of the present invention can be produced by a known method in ordinary peptide chemistry (Peptide Synthesis (Maruzen) 1975; "Peptide Synthesis", Interscience, New York, 1996; ihe Proteins, ol. 2, Academic Press
  • the antigenic peptide of the present invention is selected from candidate peptides corresponding to a part of the amino acid sequence (SEQ ID NO: 10) of HER2Zneu and having an HLA-A24 or 1A2 binding motif by humoral immunity. Selection is based on the ability to recognize and induce peptide-specific CTL.
  • Peptide-specific IgG is measured by ELISA on serum samples using candidate peptides. Peptide-specific IgG was prepared by serially diluting a serum sample with 0.05% Tween 20-Block Ace (registered trademark, MEGMI LK, Japan), adding the diluted serum to a peptide-fixed plate, and using it as a secondary antibody. It can be detected using rabbit anti-human IgG ( ⁇ ) chain specificity.
  • peripheral blood mononuclear cells from HER2 / neu-positive breast cancer patients were cultured with candidate peptides, and their PBMC IFNs were compared to target cells pulsed with the corresponding peptides. — Measure ⁇ -producing ability.
  • the PBMCs showing the above-mentioned IFN- ⁇ production were further measured for their cytotoxicity against peptide-pulsed target cells using a standard 51- Cr release assay for 6 hours. And examine its ability to induce peptide-specific CTL.
  • the mutant peptide of the antigen peptide of the present invention, the polypeptide containing the antigen peptide of the present invention, and the mutant polypeptide thereof can be obtained by using the strength of recognition by HLA-A24 or HLA-A2-restricted CTL as an index. You can choose. Recognition by CTLs can be assessed by IFN- ⁇ production and cytotoxic 1 "production of CTLs specific to the peptide's underlying antigenic peptide against target cells pulsed with those peptides. it can.
  • the nucleic acid sequence molecule of the present invention is an oligonucleotide or a polynucleotide encoding the amino acid sequence of the peptide of the present invention or a complementary chain thereof, and is composed of at least about 24 or more bases.
  • Nucleic acid sequence molecules can be readily prepared according to typical genetic engineering techniques, as described in standard textbooks such as Molecular Cloning, 2nd ed., Cold Spring Harbor Laboratory Press (1989). it can.
  • the vector containing the nucleic acid sequence molecule of the present invention incorporates the nucleic acid sequence molecule of the present invention into a vector which is conventionally used for gene expression, and includes a plasmid vector and a virus vector.
  • viral vectors include retrovirus, adenovirus, adeno-associated virus, herpes virus, vaccinia virus, box virus, polio virus, and Sindbis virus.
  • HER2 / neu-reactive CTLs are induced by culturing PBMCs obtained from patients such as breast cancer and ovarian cancer together with antigen-presenting cells displaying CTL epitope peptides using the peptides or nucleic acid sequence molecules of the present invention. it can.
  • the induction of CTL to make sure as before mentioned in I FN- ⁇ production you Yopi standard 6 hours 51 C r release Atsu Si.
  • the induced HER2 / neu-reactive CTL can be used for measuring the efficacy of the peptide of the present invention and also for adoptive immunotherapy.
  • the peptide or nucleic acid sequence molecule of the present invention can be used as a CTL inducer, In addition, it can be said that it can be used for a method for inducing HER 2 Zn eu reactive CTL.
  • the antibody according to the present invention is capable of immunologically recognizing the peptide of the present invention, and can be prepared using the antigenic determinant of the peptide of the present invention.
  • the antigenic determinant is composed of at least 5, more preferably at least 8-10 amino acids. This amino acid sequence does not need to be completely homologous to the amino acid sequence shown in SEQ ID NOs: 1 to 9, but it is necessary that at least a peptide consisting of this amino acid sequence has recognizability by CTL. is there.
  • the antibody of the present invention comprises a peptide of the present invention or an antigenic determinant thereof, in the presence or absence of an adjuvant, alone or conjugated to a carrier, which is used to produce a humoral immune response and / or a cellular immunity against animals. It can be produced by inducing immunity such as response.
  • the carrier is not particularly limited as long as it does not cause harmful effects on the host itself.
  • cellulose, polymerized amino acids, albumin and the like can be used.
  • mice, rats, rabbits, goats, rabbits and the like are preferably used.
  • the obtained polyclonal antibody can be preferably obtained from serum by a known antibody recovery method such as immunoaffinity mouth chromatography.
  • a monoclonal antibody can be produced by collecting antibody-producing cells from an animal to which the above-mentioned immunization method has been applied and introducing the cells into perpetually grown cells known per se.
  • the polyclonal or monoclonal antibody thus obtained can be used as an antibody for purification, reagent, labeling marker, or the like.
  • composition according to the present invention can be prepared by using the peptide, nucleic acid sequence molecule, vector, or antibody of the present invention alone or in combination of two or more. '
  • the peptide of the present invention can be used as a so-called cancer actin for treating cancer.
  • Cancers that can be treated with the peptide of the present invention include breast cancer, ovarian cancer, etc., with breast cancer being particularly preferred.
  • a combination of peptides of the same class Since the CTL of a cancer patient is a population of cells that recognize multiple tumor antigens, it is better to use a combination of different tumor antigens as a cancer vaccine than to use a single peptide as a cancer vaccine. It is expected that higher effects can be obtained.
  • a tumor antigen is used in combination, a peptide selected from the peptides of the present invention may be used in combination.
  • the cancer vaccine according to the present invention may be used alone or in combination with a carrier in the presence or absence of an appropriate adjuvant to activate cell-mediated humoral immunity.
  • a carrier in the presence or absence of an appropriate adjuvant to activate cell-mediated humoral immunity.
  • the carrier to be used is not particularly limited as long as it does not have a harmful effect on the human body.
  • cellulose, polymerized amino acid, albumin and the like can be used.
  • the dosage form can be appropriately selected by applying peptide preparation means known per se. The dose varies depending on the recognizability of the CTL, but is generally 0.1 mg or less as the active substance. O OmgZ days / adult human, preferably 0.1 mg to 10 mg / day / adult human. It is given once every few days or months. ⁇
  • the pharmaceutical composition according to the present invention can be used by, for example, a method of directly carrying it into the body by being carried on a vector, or a method of collecting cells from human and introducing it outside the body.
  • Vectors that can be used include, for example, retrovirus, adenovirus, vaccinia virus, etc., but retroviruses are preferred. Of course, these viruses are replication defective.
  • the dose varies depending on the recognizability of the CTL, but is generally 0.1 ⁇ g-10 Omg / day / adult human, preferably 1 ⁇ g 5 Omg daily Adult human. It is administered once every few days or months.
  • PBMC peripheral blood mononuclear cells
  • C and serum were collected from 9 healthy female subjects (HD). All sera were stored at -80 ° C and PBMC were stored frozen at -196 ° C.
  • the human ovarian cancer cell line SKOV3 (HLA-A3 / 28) expressing HER2 / neu and its HLA-A24 transfectant SKQV3-A24 were kindly provided by Mie University (Okugawa et al., A novel human HER2-derived peptide homologous to the mouse Kd-restricted cytotoxic T lymphocytes in ovarian cancer patients and healthy individuals).
  • CIR-A2402 HLA-2402 transfectant cell line
  • HER 2 / neu derived a peptide HER 2 8 - 16, HER 2 6 3 one 7 1, HER 2 342 - 35 .
  • SEQ ID NO: 1 HER 2 44Q - 448 , HER 2 485 - 493 ( SEQ ID NO: 2), HER 2 553 -56 i ( SEQ ID NO: 3), HER2 9. 7 - 915 (SEQ ID NO: 4), HER 2 968 - 976 , HER 2 1022 - 1030, HE R 2 2 ⁇ 2 - 220 used in this experiment.
  • Serum peptide-specific IgG levels were measured by ELISA as previously reported (Ohkouchi, S., et al., Non-mutated tumor-rejection antigen peptides elict type-1 allergy in the majority of healthy individuals.Tissue Antigen, 59: 259-, 2002., Kawamoto N et al., IgG reactive to CTL-directed epitopes of self-antigens is either lacking or unbalanced in atopic dermatitis patients.Tissue Antigens, 61: 352-361, 2003 .) '
  • Serum or plasma samples are serially diluted with 0.05% Tween 20_ Block Ace (ME GM ILK, Japan) and diluted serum (100 ⁇ L / per bottle) is diluted with peptide (200 per per well). ⁇ g) The solid phase was added to a Nunc Cova 1 ink plate. Antibodies were detected using Egret anti-human IgG ( ⁇ -chain specific).
  • the sera from 14 healthy individuals were tested for the reactivity of HIV peptide as a negative control peptide by the above assay.
  • the mean SD value of the optical density by ELISA was 0.040 ⁇ 0.030.
  • the average + SD value (0.0770) was determined as the cutoff value.
  • a serum sample (diluted 1: 100 in 0.05% PBST x 100) was added to the plate well in a plate well. 2 hours standing was coupled with at phasing peptide (1 Ueru per 2 0 mu ⁇ / m 1 peptide solution in immobilized) 3 7. After this binding procedure was repeated three times, its activity was tested by ELISA.
  • PBMCs from eight HLA-A24-positive breast cancer patients and five HLA-A24-positive healthy subjects were used as subjects for CTL induction.
  • peptide-specific CTL As previously reported (Ito, M., S. Shichijo, Y. Miyagi, T. Kobayashi, N. Tsuda, A. Yamada, N. Saito, and K. Itoh. Identification of SART3- derived peptides capable of inducing HLA-A2 ⁇ restriced and tumor-specific CTLs in cancer patients with different HLA-A2 subtypes.Int.J. Cancer 88: 633, 2000.) Culture in 200 ⁇ L of IL-2 containing medium supplemented with each peptide (10 M) in a micro culture plate did. .
  • C1R-A24 HLA-A2402 cDNA
  • C1R plasma cell leukemia-derived mutant cell line
  • anti-HLA class I antibody respectively 20 ⁇ / ⁇ 1 (W6 / 32, I gG2 a), anti-HLA-A2 antibody (BB7.2, I g G 2 b ), anti-CD 8 antibodies ( Nu-Ts, IgG2a), anti-HLA class II antibodies (H-DR-1, IgG2a), and anti-CD4 antibodies (Nu-Th / i, IgGl) were used.
  • Anti-CD14 antibody JML-HI4, IgG2a was used as control.
  • Statistical analysis was performed using the Two-tailed Student 's-test.
  • IgG that responds to HER2 / neu-derived peptide having HLA-A24 binding motif is breast cancer patient (13 patients with HER2Zn eu positive tumor and 4 HER2 / neu negative patients) and healthy female (HD) It was examined whether it could be detected in the serum of eight patients. .
  • the cells were pulsed with the peptide and compared with the cells.If production of ⁇ ⁇ or more was observed in the cells pulsed with the corresponding peptide, it was judged that the induction of the specificity of the peptide was successful. As described in Implementation! ? Fold ⁇ , '' "
  • HER 2 342 one 350, HER 2 485 one 493, HER 2 5 53 one 561 and HER 2 907 one 915 peptide and previously reported CTL Epito Pupepuchido (HER2 8 - 16 and HER 2 63 - (71 peptides) were tested for their ability to induce CTL (CTL inducibility was performed for all 10 types; see Table 1).
  • PBMC from eight breast cancer patients (six HER2-positive tumor patients and two HER2-negative tumor patients) and five healthy female (HD) patients were treated with six peptides (purity 90% or less E ⁇ 2) 3 42- 350, HC ⁇ 2485-493 HLR ⁇ 5 53-'561 HE 29 0 7
  • HER 2 63 respectively HER 2 8 _ 16 of Rapi - 71 induced specific I FN- y production to the peptide.
  • HER 2 342 35 in 4, 5, 2, and 1 patients, respectively.
  • HE R 2 485 - 493, HER 2 553 - 561 and HER2 9. 7 - 915, induced specific I FN- gamma production to the peptide.
  • some of these peptides also induced such activity in a few healthy donors.
  • the levels of IFN- ⁇ produced by these P BMCs are significantly suppressed by anti-class I antibodies (W6 / 32) or anti-CD8 antibodies, but not by other antibodies. This suggests that these CTL activities are carried by HLA class I restricted CD8 + T cells. Furthermore, using 311 ⁇ 3_24 cells (HL A-A24 positive, HER2 / neu positive) as target cells, a 51 Cr release assay was performed, and significant levels of CTLs in PBMCs showing the above positive response. was confirmed to have been induced.
  • the HLA-A2-binding antigen peptide of HER2 / neu was identified in the same manner as in Example 1. .
  • HER 2 444 — 453 SEQ ID NO: 5
  • HER 2 466 — 474 SEQ ID NO: 6
  • HER 2 484 — 493 SEQ ID NO: 7
  • FIG. 4A shows representative results of six cases
  • FIG. 4B shows results of confirmation of peptide specificity
  • Table 3 shows the results of CTL induction for peptides detected in multiple sera.
  • HER2 / neu369 is a peptide reported as a common epitope recognized by CTL (Fisk et al., Entill cat ion of an immunodominant peptide of HER2 / neu
  • IgG that reacts with the CTL epitope peptide is often detected in the serum of cancer patients before vaccination and in the serum of healthy subjects.
  • serum anti-peptide antibody levels after vaccination indicate that patients with advanced lung cancer Had a strong correlation with the survival of the elderly.
  • IgG that reacts to these peptides is either deficient or unbalanced in the serum of patients with atopic disease.
  • Anti-peptide IgG when tested, does not react with the protein from which the peptide was derived, and directly inhibits cell growth of tumor cells in vivo, as well as being antibody dependent on tumor cells. Neither did it elicit sexual cytotoxic activity.
  • the anti-HER2Zneu peptide IgG in the present invention does not appear to play a role in direct action on tumor cells. Rather, these antibodies are thought to be involved in infiltrating immunocompetent cells at the tumor site through the induction of an inflammatory response around the tumor site.
  • the present invention is expected to open a new door to HER2 / neu peptide-based immunotherapy for HLA-A24 or A2-positive and HER2 / neu positive breast cancer patients.

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Abstract

L'invention concerne un peptide HER2/neu utile dans l'immunothérapie à base notamment d'un peptide destiné à des patientes atteintes du cancer du sein. Plus précisément, l'invention concerne un polypeptide comprenant un peptide antigène HER2342-350, HER2485-493, HER2553-561, HER2907-915, HER2444-453, HER2466-474, HER2484-493, HER2785-794 ou HER2851-859; un tel peptide antigène; des molécules d'une séquence d'acides nucléiques codant ceux-ci; et des compositions médicales renfermant ceux-ci.
PCT/JP2004/010547 2003-07-16 2004-07-16 Peptide her2/neu et utilisation therapeutique de celui-ci WO2005007694A1 (fr)

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WO2014080730A1 (fr) 2012-11-22 2014-05-30 株式会社糖鎖工学研究所 Lieur glycosylé, composé contenant un fragment de lieur glycosylé et un fragment de substance physiologiquement active ou son sel et procédés de production dudit composé ou de son sel
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JP2009508113A (ja) * 2005-09-08 2009-02-26 ヘンリー エム．ジャクソン ファウンデイション フォー ザ アドバンスメント オブ ミリタリー メディシン，インコーポレイティド 免疫源性ペプチドの標的特異的同定方法
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JP2014196341A (ja) * 2005-09-08 2014-10-16 ザ ヘンリー エム．ジャクソン ファウンデイション フォー ザ アドバンスメント オブ ミリタリー メディスン，インコーポレイティド 免疫源性ペプチドの標的特異的同定方法
US8883164B2 (en) 2005-09-08 2014-11-11 The Henry M. Jackson Foundation For The Advancement Of Military Medicine, Inc. Targeted identification of immunogenic peptides
US8945573B2 (en) 2005-09-08 2015-02-03 The Henry M. Jackson Foundation For The Advancement Of Military Medicine, Inc. Targeted identification of immunogenic peptides
US9050322B2 (en) 2005-09-08 2015-06-09 The Henry M. Jackson Foundation For The Advancement Of Military Medicine, Inc. Targeted identification of immunogenic peptides
US9720000B2 (en) 2005-09-08 2017-08-01 The Henry M. Jackson Foundation For The Advancement Of Military Medicine, Inc. Targeted identification of immunogenic peptides
JP2010501160A (ja) * 2006-08-11 2010-01-21 デンドレオン コーポレイション 無差別ＨＥＲ−２／ＮｅｕＣＤ４Ｔ細胞エピトープ
WO2014080730A1 (fr) 2012-11-22 2014-05-30 株式会社糖鎖工学研究所 Lieur glycosylé, composé contenant un fragment de lieur glycosylé et un fragment de substance physiologiquement active ou son sel et procédés de production dudit composé ou de son sel

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