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WO2012032763A1 - Peptides vangl1 et vaccins les contenant - Google Patents

Peptides vangl1 et vaccins les contenant Download PDF

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Publication number
WO2012032763A1
WO2012032763A1 PCT/JP2011/004984 JP2011004984W WO2012032763A1 WO 2012032763 A1 WO2012032763 A1 WO 2012032763A1 JP 2011004984 W JP2011004984 W JP 2011004984W WO 2012032763 A1 WO2012032763 A1 WO 2012032763A1
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Prior art keywords
peptide
peptides
vangl1
present
cancer
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Inventor
Yusuke Nakamura
Takuya Tsunoda
Ryuji Osawa
Sachiko Yoshimura
Tomohisa Watanabe
Gaku Nakayama
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Oncotherapy Science Inc
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Oncotherapy Science Inc
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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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4748—Tumour specific antigens; Tumour rejection antigen precursors [TRAP], e.g. MAGE

Definitions

  • VANGL1 Vang-like 1
  • peptides that bind to HLA antigen and include the VANGL1 sequence (SEQ ID NO: 69) or an immunologically active fragment thereof.
  • VANGL1 sequence SEQ ID NO: 69
  • These peptides are expected to have CTL inducibility and, thus, can be used to induce CTL in vitro, in vivo or ex vivo or to be administered to a subject for inducing immune responses against cancers, examples of which include, but are not limited to, bladder cancer, breast cancer, cervical cancer, cholangiocellular carcinoma, endometriosis, liver cancer, NSCLC, osteosarcoma, pancreatic cancer, SCLC and AML.
  • Preferred peptides are nonapeptides and decapeptides, and more preferably, nonapeptides and decapeptides having an amino acid sequence selected from among SEQ ID NOs: 1 and 3 to 67.
  • Peptides having an amino acid sequence selected from among SEQ ID NOs: 1, 12 and 21 show strong CTL inducibility and thus are particularly preferred.
  • one object of the present invention is to provide agents and/or compositions that include or incorporate any peptides or polynucleotides of the present invention for inducing CTL.
  • agents and/or compositions can be used for the treatment and/or prophylaxis of a primary cancer or a metastasis or post-operative recurrence thereof.
  • cancers contemplated by the present invention include, but are not limited to, bladder cancer, breast cancer, cervical cancer, cholangiocellular carcinoma, endometriosis, liver cancer, NSCLC, osteosarcoma, pancreatic cancer, SCLC and AML.
  • pharmaceutical compositions or agents that include or incorporate any peptides or polynucleotides of the present invention formulated the treatment and/or prophylaxis of a primary cancer or a metastasis or post-operative recurrence thereof.
  • the present pharmaceutical agents and/or compositions may include as active ingredients APCs or exosomes that present any of the present peptides.
  • CTLs obtained by such methods find use in the treatment and/or prevention of cancers, more particularly bladder cancer, breast cancer, cervical cancer, cholangiocellular carcinoma, endometriosis, liver cancer, NSCLC, osteosarcoma, pancreatic cancer, SCLC and AML. Therefore, it is yet another object of the present invention to provide CTLs obtained by the present methods.
  • the applicability of the present invention extends to any of a number of diseases relating to or arising from VANGL1 overexpression, examples of which include, but are not limited to, bladder cancer, breast cancer, cervical cancer, cholangiocellular carcinoma, endometriosis, liver cancer, NSCLC, osteosarcoma, pancreatic cancer, SCLC and AML.
  • Figure 1 is composed of a series of photographs, (a) - (d), depicting the results of IFN-gamma ELISPOT assay on CTLs that were induced with peptides derived from VANGL1.
  • Figure 2 is composed of a series of line graphs, (a) and (b), depicting the results of an IFN-gamma ELISA assay that, in turn demonstrates the IFN-gamma production of the CTL lines stimulated with VANGL1-A02-9-194 (SEQ ID NO:1) (a) and VANGL1-A02-9-484 (SEQ ID NO:21) (b).
  • the results demonstrate CTL lines established by stimulation with each peptide show potent IFN-gamma production as compared with the control.
  • “+” indicates the IFN-gamma production against target cells pulsed with the appropriate peptide
  • "-" indicates the IFN-gamma production against target cells not pulsed with any peptides.
  • Figure 4 is a line graph depicting specific CTL activity against target cells that exogenously express VANGL1 and HLA-A*0201.
  • COS7 cells transfected with HLA-A*0201 or the full length VANGL1 gene were prepared as the controls.
  • the CTL line established with VANGL1-A02-9-484 (SEQ ID NO:21) showed specific CTL activity against COS7 cells transfected with both VANGL1 and HLA-A*0201 (black lozenge).
  • no significant specific CTL activity was detected against target cells expressing either HLA-A*0201 (triangle) or VANGL1 (circle).
  • peptide having CTL inducibility wherein the peptide consists of the amino acid sequence of VANGL1 or an immunologically active fragment thereof.
  • CTL cytotoxic T lymphocyte
  • [7] The isolated peptide of any one of [1] to [6], wherein said peptide is nonapeptide or decapeptide.
  • compositions for the treatment and/or prophylaxis of cancer, and/or the prevention of a postoperative recurrence thereof wherein the composition comprises one or more of the peptide(s) of any one of [1] to [7], or one or more of the polynucleotide(s) of [8].
  • composition comprises one or more of the peptide(s) of any one of [1] to [7], or one or more of the polynucleotide(s) of [8].
  • [12]A method for inducing an antigen-presenting cell (APC) with CTL inducibility comprising the step of selected from the group consisting of: (a) contacting an APC with a peptide of any one of [1] to [7] in vitro, ex vivo or in vivo, and (b) introducing a polynucleotide encoding the peptide of any one of [1] to [7] into an APC.
  • APC antigen-presenting cell
  • a method for inducing CTL that comprises a step selected from the group consisting of: (a) co-culturing CD8 positive T cells with APCs that present on the surface a complex of an HLA antigen and the peptide of any one of [1] to [7], (b) co-culturing CD8 positive T cells with exosomes that present on the surface a complex of an HLA antigen and the peptide of any one of [1] to [7], and (c) introducing a gene that comprises a polynucleotide encoding a T cell receptor (TCR) subunit polypeptide bound to a peptide of any one of [1] to [7] into a T cell.
  • TCR T cell receptor
  • the present invention also provides following peptides and use thereof: [1]An isolated peptide having CTL inducibility, wherein the peptide consists of the amino acid sequence of VANGL1 or an immunologically active fragment thereof, or an isolated peptide having CTL inducibility, wherein the peptide comprises or consists of an amino acid sequence of an immunologically active fragment of the peptide consisting of the amino acid sequence of SEQ ID NO: 69, [2]The isolated peptide of [1], wherein the peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 and 3 to 67, [3]An isolated peptide of [1] or [2] in which one, two, or several amino acid(s) are substituted, inserted, deleted or added to yield a modified peptide that retains the CTL inducibility of the original peptide, [4]The isolated peptide of [3], wherein, in the context of HLA-A2, the peptide
  • an isolated or purified peptide refers to peptide that are substantially free of cellular material such as carbohydrate, lipid, or other contaminating proteins from the cell or tissue source from which the peptide is derived, or substantially free of chemical precursors or other chemicals when chemically synthesized.
  • substantially free of cellular material includes preparations of a peptide in which the peptide is separated from cellular components of the cells from which it is isolated or recombinantly produced.
  • a peptide that is substantially free of cellular material includes preparations of polypeptide having less than about 30%, 20%, 10%, or 5% (by dry weight) of heterologous protein (also referred to herein as a "contaminating protein").
  • heterologous protein also referred to herein as a "contaminating protein”
  • the peptide is recombinantly produced, it is also preferably substantially free of culture medium, which includes preparations of peptide with culture medium less than about 20%, 10%, or 5% of the volume of the peptide preparation.
  • amino acid refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that similarly function to the naturally occurring amino acids.
  • Amino acid may be either L-amino acids or D-amino acids.
  • Naturally occurring amino acids are those encoded by the genetic code, as well as those modified after translation in cells (e.g., hydroxyproline, gamma-carboxyglutamate, and O-phosphoserine).
  • amino acid analog refers to compounds that have the same basic chemical structure (an alpha carbon bound to a hydrogen, a carboxy group, an amino group, and an R group) as a naturally occurring amino acid but have a modified R group or modified backbones (e.g., homoserine, norleucine, methionine, sulfoxide, methionine methyl sulfonium).
  • modified R group or modified backbones e.g., homoserine, norleucine, methionine, sulfoxide, methionine methyl sulfonium.
  • amino acid mimetic refers to chemical compounds that have different structures but similar functions to general amino acids.
  • Amino acids may be referred to herein by their commonly known three letter symbols or the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission.
  • the terms "gene”, “polynucleotides”, “nucleotides” and “nucleic acids” are used interchangeably herein and, unless otherwise specifically indicated are similarly to the amino acids referred to by their commonly accepted single-letter codes.
  • compositions are used interchangeably herein to refer to a product that includes the specified ingredients in the specified amounts, as well as any product which results, directly or indirectly, from combination of the specified ingredients in the specified amounts.
  • Such term in relation to pharmaceutical composition is intended to encompass a product that includes the active ingredient(s), and the inert ingredient(s) that make up the carrier, as well as any product which results, directly or indirectly, from combination, complexation or aggregation of any two or more of the ingredients, or from dissociation of one or more of the ingredients, or from other types of reactions or interactions of one or more of the ingredients.
  • the pharmaceutical compositions of the present invention encompass any composition made by admixing a compound of the present invention and a pharmaceutically or physiologically acceptable carrier.
  • active ingredient refers to a substance in an agent or composition that is biologically or physiologically active.
  • active ingredient refers to a substance that shows an objective pharmacological effect.
  • active ingredients in the agents or compositions may lead to at least one biological or physiologically action on cancer cells and/or tissues directly or indirectly.
  • such action may include reducing or inhibiting cancer cell growth, damaging or killing cancer cells and/or tissues, and so on.
  • indirect effect of active ingredients is inductions of CTLs recognizing or killing cancer cells.
  • the "active ingredient” may also be referred to as "bulk", "drug substance” or "technical product”.
  • phrases "pharmaceutically acceptable carrier” or “physiologically acceptable carrier”, as used herein, means a pharmaceutically or physiologically acceptable material, composition, substance, compound or vehicle, including, but are not limited to, a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting the active ingredient from one organ, or portion of the body, to another organ, or portion of the body.
  • a pharmaceutical agent or compositions of the present invention find particular use as vaccines.
  • the phrase “vaccine” also referred to as an “immunogenic composition” refers to an agent or composition that has the function to improve, enhance and/or induce anti-tumor immunity upon inoculation into animals.
  • HLA-A2 refers to the HLA-A2 type containing the subtypes, examples of which include, but are not limited to, HLA-A*0201, HLA-A*0202, HLA-A*0203, HLA-A*0204, HLA-A*0205, HLA-A*0206, HLA-A*0207, HLA-A*0210, HLA-A*0211, HLA-A*0213, HLA-A*0216, HLA-A*0218, HLA-A*0219, HLA-A*0228 and HLA-A*0250.
  • kit is used in reference to a combination of reagents and other materials. It is contemplated herein that the kit may include microarray, chip, marker, and so on. It is not intended that the term “kit” be limited to a particular combination of reagents and/or materials.
  • prevention and prophylaxis can occur “at primary, secondary and tertiary prevention levels.” While primary prevention and prophylaxis avoid the development of a disease, secondary and tertiary levels of prevention and prophylaxis encompass activities aimed at the prevention and prophylaxis of the progression of a disease and the emergence of symptoms as well as reducing the negative impact of an already established disease by restoring function and reducing disease-related complications. Alternatively, prevention and prophylaxis can include a wide range of prophylactic therapies aimed at alleviating the severity of the particular disorder, e.g. reducing the proliferation and metastasis of tumors.
  • the treatment and/or prophylaxis of cancer and/or the prevention of postoperative recurrence thereof include any of the following steps, such as the surgical removal of cancer cells, the inhibition of the growth of cancerous cells, the involution or regression of a tumor, the induction of remission and suppression of occurrence of cancer, the tumor regression, and the reduction or inhibition of metastasis.
  • Effective treatment and/or the prophylaxis of cancer decreases mortality and improves the prognosis of individuals having cancer, decreases the levels of tumor markers in the blood, and alleviates detectable symptoms accompanying cancer.
  • reduction or improvement of symptoms constitutes effectively treating and/or the prophylaxis include 10%, 20%, 30% or more reduction, or stable disease.
  • the VANGL1 gene is over expressed in cancer cells such as bladder cancer, breast cancer, cervical cancer, cholangiocellular carcinoma, endometriosis, liver cancer, NSCLC, osteosarcoma, pancreatic cancer, SCLC and AML and not expressed in most normal organs, it is a good target for immunotherapy.
  • cancer cells such as bladder cancer, breast cancer, cervical cancer, cholangiocellular carcinoma, endometriosis, liver cancer, NSCLC, osteosarcoma, pancreatic cancer, SCLC and AML and not expressed in most normal organs, it is a good target for immunotherapy.
  • the present invention provides nonapeptides (peptides composed of nine amino acid residues) and decapeptides (peptides composed of ten amino acid residues) of CTL-recognized epitopes from VANGL1.
  • the present invention provides an isolated peptide which binds to an HLA antigen and induces cytotoxic T lymphocytes (CTL), wherein the peptide has the amino acid sequence set forth in SEQ ID NO: 69 or is an immunologically active fragment thereof. More specifically, in some embodiments, the present invention provides peptides having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 12 and 21.
  • the present invention encompasses peptides composed of any fragments derived from VANGL1 that are determined to bind with HLA antigens by such known programs. Furthermore, such peptides may include the peptide composed of the full length VANGL1 sequence.
  • the peptides of the present invention may be flanked with additional amino acid residues so long as the peptide retains its CTL inducibility.
  • the particular additional amino acid residues may be composed of any kind of amino acids so long as they do not impair the CTL inducibility of the original peptide.
  • the present invention encompasses peptides having a binding affinity for HLA antigens, in particular peptides derived from VANGL1.
  • Such peptides are, for example, less than about 40 amino acids, often less than about 20 amino acids, usually less than about 15 amino acids.
  • modified peptides i.e., peptides composed of an amino acid sequence modified by substituting, deleting, inserting, or adding one, two or several amino acid residues to an original reference sequence
  • modified peptides have been 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).
  • amino acid side chains examples include 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), and side chains having the following functional groups or characteristics in common: an aliphatic side-chain (G, A, V, L, I, P); a hydroxyl group containing side-chain (S, T, Y); a sulfur atom containing side-chain (C, M); a carboxylic acid and amide containing side-chain (D, N, E, Q); a base containing side-chain (R, K, H); and an aromatic containing side-chain (H, F, Y, W).
  • A, I, L, M, F, P, W, Y, V hydrophilic amino acids
  • R, D, N, C, E, Q amino acids
  • G, A, V, L, I, P a hydroxyl group containing side-chain
  • the following eight groups each contain amino acids that are conservative substitutions for one another: 1) Alanine (A), Glycine (G); 2) Aspartic acid (D), Glutamic acid (E); 3) Aspargine (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, e.g., Creighton, Proteins 1984).
  • Such conservatively modified peptides are also considered to be peptides of the present invention.
  • the peptide of the present invention is not restricted thereto and may include non-conservative modifications, so long as the resulting modified peptide retains the CTL inducibility of the original unmodified peptide.
  • the modified peptides should not exclude CTL inducible peptides of polymorphic variants, interspecies homologues, and alleles of VANGL1.
  • Amino acid residues may be inserted, substituted or added to the peptides of the present invention or, alternatively, amino acid residues may be deleted therefrom to achieve a higher binding affinity.
  • severe means 5 or fewer amino acids, for example, 4, or 3 or fewer.
  • the percentage of amino acids to be modified may be 20% or less, for example, 15% or less, even more preferably 10% or less, for example 1 to 5%.
  • peptides possessing high HLA-A2 binding affinity tend to have the second amino acid from the N-terminus substituted with phenylalanine, tyrosine, methionine, or tryptophan.
  • peptides in which the C-terminal amino acid is substituted with phenylalanine, leucine, isoleucine, tryptophan, or methionine can also be favorably used.
  • peptides having an amino acid sequence selected from among SEQ ID NOs: 1, 12 and 21 wherein the second amino acid from the N-terminus of the amino acid sequence of said SEQ ID NO is substituted with leucine or methionine, and peptides, and/or wherein the C-terminus of the amino acid sequence of said SEQ ID NO is substituted with valine or leucine are contemplated by the present invention.
  • peptides showing high HLA-A2 binding affinity it may be desirable to substitute the second amino acid from the N-terminus with leucine or methionine or the amino acid at the C-terminus with valine or leucine can.
  • peptides having amino acid sequences selected from among SEQ ID NOs: 1, 12, and 21 wherein the second amino acid from the N-terminus of the amino acid sequence is substituted with leucine or methionine, and/or wherein the C-terminus of the amino acid sequence is substituted with valine or leucine are encompassed by the present invention.
  • Substitutions may be introduced not only at the terminal amino acids but also at the position of potential T cell receptor (TCR) recognition of peptides.
  • TCR T cell receptor
  • a peptide with amino acid substitutions may be equal to or better than the original, for example CAP1, p53 (264-272), Her-2/neu (369-377) or gp100 (209-217) (Zaremba et al. Cancer Res. 57, 4570-4577, 1997, T. K. Hoffmann et al. J Immunol. (2002);168(3):1338-47., S. O. Dionne et al. Cancer Immunol immunother. (2003) 52: 199-206 and S. O. Dionne et al.
  • the present invention provides an isolated peptide of less than 14, 13, 12, 11, or 10 amino acids in length which has CTL inducibility and comprises the amino acid sequence selected from the group consisting of: (i) an amino acid sequence in which one, two or several amino acid(s) are modified in the amino acid sequence selected from the group consisting of SEQ ID NOs: 1 and 3 to 33, wherein the peptide binds an HLA antigen and induces cytotoxic T lymphocytes, and (ii) the amino acid sequence of (i), wherein the amino acid sequence has one or both of the following characteristics: (a) the second amino acid from the N-terminus of said SEQ ID NO is modified to leucine or methionine; and (b) the C-terminal amino acid of said SEQ ID NO is modified to valine or leucine.
  • the present invention also provides an isolated peptide of less than 15, 14, 13, 12, or 11 amino acids in length which has CTL inducibility and comprises the amino acid sequence selected from the group consisting of: (i') an amino acid sequence in which one , two or several amino acid(s) are modified in the amino acid selected from the group consisting of SEQ ID NOs: 34 to 67, wherein the peptide binds an HLA antigen and induces cytotoxic T lymphocytes, and (ii') the amino acid sequence of (i'), wherein the amino acid sequence has one or both of the following characteristics: (a) the second amino acid from the N-terminus of said SEQ ID NO is modified to leucine and methionine; and (b) the C-terminal amino acid of said SEQ ID NO is modified to valine and leucine.
  • These peptides are processed in APC to present a peptide of (i), (ii), (i'), and (ii') thereon, when these peptides are contacted with, or
  • CTL inducibility indicates the ability of the peptide to induce CTLs when presented on antigen-presenting cells (APCs). Further, “CTL inducibility” includes the ability of the peptide to induce CTL activation, CTL proliferation, promote CTL lysis of target cells, and to increase CTL IFN-gamma production.
  • Confirmation of CTL inducibility is accomplished by inducing APCs carrying human MHC antigens (for example, B-lymphocytes, macrophages, and dendritic cells (DCs)), or more specifically DCs derived from human peripheral blood mononuclear leukocytes, and after stimulation with the peptides, mixing with CD8-positive cells, and then measuring the IFN-gamma produced and released by CTL against the target cells.
  • human MHC antigens for example, B-lymphocytes, macrophages, and dendritic cells (DCs)
  • DCs dendritic cells
  • nonapeptides or decapeptides selected from among those peptides having an amino acid sequence as set forth in SEQ ID NOs: 1, 12 and 21 showed particularly high CTL inducibility as well as high binding affinity to an HLA antigen.
  • these peptides are exemplified as preferred embodiments of the present invention.
  • the preferred peptides of the present invention are composed of the amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 12 and 21.
  • non-VANGL1 tumor associated antigen peptides also can be used substantially simultaneously to increase immune response via HLA class I and/or class II. It is well established that cancer cells can express more than one tumor associated gene. It is within the scope of routine experimentation for one of ordinary skill in the art to determine whether a particular subject expresses additional tumor associated genes, and then include HLA class I and/or HLA class II binding peptides derived from expression products of such genes in VANGL1 compositions or vaccines.
  • polytopes i.e., groups of two or more potentially immunogenic or immune response stimulating peptides which can be joined together in various arrangements (e.g., concatenated, overlapping).
  • the polytope (or nucleic acid encoding the polytope) can be administered in a standard immunization protocol, e.g., to animals, to test the effectiveness of the polytope in stimulating, enhancing and/or provoking an immune response.
  • polytopes can be joined together directly or via the use of flanking sequences to form polytopes, and the use of polytopes as vaccines is well known in the art (see, e.g., Thomson et al., Proc. Natl. Acad. Sci USA 92(13):5845-5849, 1995; Gilbert et al., Nature Biotechnol. 15(12):1280-1284, 1997; Thomson et al., J Immunol. 157(2):822-826, 1996; Tarn et al., J Exp. Med. 171(l):299-306, 1990).
  • Polytopes containing various numbers and combinations of epitopes can be prepared and tested for recognition by CTLs and for efficacy in increasing an immune response.
  • the peptides of the present invention may be further linked to other substances, so long as they retain the CTL inducibility.
  • substances may include: peptides, lipids, sugar and sugar chains, acetyl groups, natural and synthetic polymers, etc.
  • the peptides may contain modifications such as glycosylation, side chain oxidation, or phosphorylation; so long as the modifications do not destroy the biological activity of the peptides as described herein. These kinds of modifications may be performed to confer additional functions (e.g., targeting function, and delivery function) or to stabilize the polypeptide.
  • polypeptides For example, to increase the in vivo stability of a polypeptide, it is known in the art to introduce D-amino acids, amino acid mimetics or unnatural amino acids; this concept may also be adopted for the present polypeptides.
  • the stability of a polypeptide may be assayed in a number of ways. For instance, peptidases and various biological media, such as human plasma and serum, can be used to test stability (see, e.g., Verhoef et al., Eur J Drug Metab Pharmacokin 1986, 11: 291-302).
  • the present invention also provides the method of screening for or selecting modified peptides having same or higher activity as compared to originals.
  • the method may include steps of: a: substituting, deleting or adding at least one amino acid residue of a peptide of the present invention, b: determining the activity of said peptide, and c: selecting the peptide having same or higher activity as compared to the original.
  • said activity may include MHC binding activity, APC or CTL inducibility and cytotoxic activity.
  • the peptides of the present invention may also be described as "VANGL1 peptide(s)" or "VANGL1 polypeptide(s)".
  • a peptide of the present invention may be obtained through chemical synthesis based on the selected amino acid sequence.
  • conventional peptide synthesis methods that may be adopted for the synthesis include: (i) Peptide Synthesis, Interscience, New York, 1966; (ii) The Proteins, Vol. 2, Academic Press, New York, 1976; (iii) Peptide Synthesis (in Japanese), Maruzen Co., 1975; (iv) Basics and Experiment of Peptide Synthesis (in Japanese), Maruzen Co., 1985; (v) Development of Pharmaceuticals (second volume) (in Japanese), Vol. 14 (peptide synthesis), Hirokawa, 1991; (vi) WO99/67288; and (vii) Barany G. & Merrifield R.B., Peptides Vol. 2, "Solid Phase Peptide Synthesis", Academic Press, New York, 1980, 100-118.
  • polynucleotide that encode any of the aforementioned peptides of the present invention. These include polynucleotides derived from the natural occurring VANGL1 gene (GenBank Accession No. AB057596, NM_001172411, NM_001172412 or NM_138959 (for example SEQ ID NO: 68)) and those having a conservatively modified nucleotide sequences thereof.
  • VANGL1 GenBank Accession No. AB057596, NM_001172411, NM_001172412 or NM_138959 (for example SEQ ID NO: 68)
  • conservatively modified nucleotide sequence refers to sequences which encode identical or essentially identical amino acid sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein.
  • each codon in a nucleic acid may be modified to yield a functionally identical molecule. 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 may be composed of DNA, RNA, and derivatives thereof.
  • a DNA molecule is suitably composed of bases such as the naturally occurring bases A, T, C, and G, and T is replaced by U in an RNA.
  • bases such as the naturally occurring bases A, T, C, and G, and T is replaced by U in an RNA.
  • non-naturally occurring bases be included in polynucleotides, as well.
  • the polynucleotide of the present invention may encode multiple peptides of the present invention with or without intervening amino acid sequences.
  • the intervening amino acid sequence may provide a cleavage site (e.g., enzyme recognition sequence) of the polynucleotide or the translated peptides.
  • the polynucleotide may include any additional sequences to the coding sequence encoding the peptide of the present invention.
  • the polynucleotide may be a recombinant polynucleotide that includes regulatory sequences required for the expression of the peptide or may be an expression vector (plasmid) with marker genes and such.
  • such recombinant polynucleotides may be prepared by the manipulation of polynucleotides through conventional recombinant techniques using, for example, polymerases and endonucleases.
  • a polynucleotide may be produced by insertion into an appropriate vector, which may be expressed when transfected into a competent cell.
  • a polynucleotide may be amplified using PCR techniques or expression in suitable hosts (see, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York, 1989).
  • a polynucleotide may be synthesized using the solid phase techniques, as described in Beaucage SL & Iyer RP, Tetrahedron 1992, 48: 2223-311; Matthes et al., EMBO J 1984, 3: 801-5.
  • Exosomes The present invention further provides intracellular vesicles called exosomes, which present complexes formed between the peptides of this invention and HLA antigens on their surface. Exosomes may be prepared, for example, using the methods detailed in Japanese Patent Application Kohyo Publications Nos. Hei 11-510507 and WO99/03499, and may be prepared using APCs obtained from patients who are subject to treatment and/or prevention. The exosomes of this invention may be inoculated as vaccines, similarly to the peptides of this invention.
  • HLA antigens included in the complexes must match that of the subject requiring treatment and/or prevention.
  • HLA-A2 particularly HLA-A*0201 and HLA-A*0206 is often appropriate.
  • A2 type that are highly expressed among the Japanese and Caucasian is favorable for obtaining effective results, and subtypes such as A*0201 and A*0206 find use.
  • the type of HLA antigen of the patient requiring treatment is investigated in advance, which enables appropriate selection of peptides having high levels of binding affinity to this antigen, or having CTL inducibility by antigen presentation.
  • the present invention also provides isolated APCs that present complexes formed between HLA antigens and the peptides of this invention on its surface.
  • the APCs may be derived from patients who are subject to treatment and/or prevention, and may be administered as vaccines by themselves or in combination with other drugs including the peptides of this invention, exosomes, or CTLs.
  • the APCs are not limited to a particular kind of cells and include dendritic cells (DCs), Langerhans cells, macrophages, B cells, and activated T cells, which are known to present proteinaceous antigens on their cell surface so as to be recognized by lymphocytes. Since DC is a representative APC having the strongest CTL inducing action among APCs, DCs find use as the APCs of the present invention.
  • the APCs of the present invention may be obtained by inducing DCs from peripheral blood monocytes and then contacting (stimulating) them with the peptides of this invention in vitro, ex vivo or in vivo.
  • the APCs of the present invention may be administered to a subject for inducing immune response against cancer in the subject by themselves or in combination with other drugs including the peptides, exosomes or CTLs of this invention.
  • the ex vivo administration may include steps of: a: collecting APCs from a first subject, b: contacting with the APCs of step a, with the peptide, and c: administering the APCs of step b to a second subject.
  • the first subject and the second subject may be the same individual, or may be different individuals.
  • the APCs obtained by step b may be administered as a vaccine for treating and/or preventing cancers, examples of which include, but are not limited to, bladder cancer, breast cancer, cervical cancer, cholangiocellular carcinoma, endometriosis, liver cancer, NSCLC, osteosarcoma, pancreatic cancer, SCLC and AML.
  • the present invention also provides a method or process for manufacturing a pharmaceutical composition for inducing APCs, wherein the method includes the step of admixing or formulating the peptide of the invention with a pharmaceutically acceptable carrier.
  • the APCs have a high level of CTL inducibility.
  • high level of CTL inducibility the high level is relative to the level of that by APC contacting with no peptide or peptides which may not induce the CTL.
  • Such APCs having a high level of CTL inducibility may be prepared by a method which includes the step of transferring a polynucleotide encoding the peptide of this invention to APCs in vitro as well as the method mentioned above.
  • the introduced genes may be in the form of DNAs or RNAs. Examples of methods for introduction include, without particular limitations, various methods conventionally performed in this field, such as lipofection, electroporation, and calcium phosphate method may be used.
  • the gene may be performed as described in Cancer Res 1996, 56: 5672-7; J Immunol 1998, 161: 5607-13; J Exp Med 1996, 184: 465-72; Published Japanese Translation of International Publication No. 2000-509281.
  • the gene undergoes transcription, translation, and such in the cell, and then the obtained protein is processed by MHC Class I or Class II, and proceeds through a presentation pathway to present partial peptides.
  • the APCs of the present invention are APCs that present complexes of HLA-A2 antigen and the peptide of the present invention on their surface.
  • CTLs Cytotoxic T lymphocytes
  • a CTL induced against any of the peptides of the present invention strengthens the immune response targeting cancer cells in vivo and thus may be used as vaccines similar to the peptides.
  • the present invention provides isolated CTLs that are specifically induced or activated by any of the present peptides.
  • Such CTLs may be obtained by (1) administering the peptide(s) of the present invention to a subject or (2) contacting (stimulating) subject-derived APCs, and CD8-positive cells, or peripheral blood mononuclear leukocytes in vitro with the peptide(s) of the present invention or (3) contacting CD8-positive cells or peripheral blood mononuclear leukocytes in vitro with the APCs or exosomes presenting a complex of an HLA antigen and the peptide on its surface or (4) introducing a gene that includes a polynucleotide encoding a T cell receptor (TCR) subunit binding to the peptide of this invention.
  • TCR T cell receptor
  • the CTLs of this invention may be derived from patients who are subject to treatment and/or prevention, and may be administered by themselves or in combination with other drugs including the peptides of this invention or exosomes for the purpose of regulating effects.
  • the obtained CTLs act specifically against target cells presenting the peptides of this invention, for example, the same peptides used for induction.
  • the target cells may be cells that endogenously express VANGL1, such as cancer cells, or cells that are transfected with the VANGL1 gene; and cells that present a peptide of this invention on the cell surface due to stimulation by the peptide may also serve as targets of activated CTL attack.
  • the CTLs of the present invention are CTLs that recognize cells presenting complexes of HLA-A2 antigen and the peptide of the present invention.
  • the phrase “recognize a cell” refers to binding a complex of HLA-A2 antigen and the peptide of the present invention on the cell surface via its TCR and showing specific cytotoxic activity against the cell.
  • specific cytotoxic activity refers to showing cytotoxic activity against the cell presenting a complex of HLA-A2 antigen and the peptide of the present invention but not other cells.
  • T cell receptor The present invention also provides a composition including nucleic acids encoding polypeptides that are capable of forming a subunit of a T cell receptor (TCR), and methods of using the same.
  • the TCR subunits have the ability to form TCRs that confer specificity to T cells against tumor cells presenting VANGL1.
  • the nucleic acids of alpha- and beta- chains as the TCR subunits of the CTL induced with one or more peptides of this invention may be identified (WO2007/032255 and Morgan et al., J Immunol, 171, 3288 (2003)).
  • the PCR method is preferred to analyze the TCR.
  • the PCR primers for the analysis can be, for example, 5'-R primers (5'-gtctaccaggcattcgcttcat-3') as 5' side primers (SEQ ID NO: 70) and 3-TRa-C primers (5'-tcagctggaccacagccgcagcgt-3') specific to TCR alpha chain C region (SEQ ID NO: 71), 3-TRb-C1 primers (5'-tcagaaatcctttctcttgac-3') specific to TCR beta chain C1 region (SEQ ID NO: 72) or 3-TRbeta-C2 primers (5'- ctagcctctggaatcctttctcttt-3') specific to TCR beta chain C2 region (SEQ ID NO: 73) as 3' side primers, but not limited.
  • the derivative TCRs may bind target cells displaying the VANGL1 peptide with high avidity, and optionally mediat
  • the nucleic acids encoding the TCR subunits may be incorporated into suitable vectors, e.g., retroviral vectors. These vectors are well known in the art.
  • the nucleic acids or the vectors including them usefully may be transferred into a T cell, for example, a T cell from a patient.
  • the present invention provides an off-the-shelf composition allowing rapid modification of a patient's own T cells (or those of another mammal) to rapidly and easily produce modified T cells having excellent cancer cell killing properties.
  • the specific TCR is a receptor capable of specifically recognizing a complex of a peptide of the present invention and HLA molecule, giving a T cell specific activity against the target cell when the TCR on the surface of the T cell.
  • a specific recognition of the above complex may be confirmed by any known methods, and preferred methods include, for example, tetramer analysis using HLA molecule and peptide of the present invention, and ELISPOT assay.
  • tetramer analysis using HLA molecule and peptide of the present invention and ELISPOT assay.
  • ELISPOT assay By performing the ELISPOT assay, it can be confirmed that a T cell expressing the TCR on the cell surface recognizes a cell by the TCR, and that the signal is transmitted intracellularly.
  • the confirmation that the above-mentioned complex can give a T cell cytotoxic activity when the complex exists on the T cell surface may also be carried out by a known method.
  • a preferred method includes, for example, the determination of cytotoxic activity against an HLA positive target cell, such as chromium release assay.
  • the present invention provides CTLs which are prepared by transduction with the nucleic acids encoding the TCR subunits polypeptides that bind to the VANGL1 peptide, e.g., SEQ ID NOs: 1, 12 and 21 in the context of HLA-A2.
  • the transduced CTLs are capable of homing to cancer cells in vivo, and may be expanded by well known culturing methods in vitro (e.g., Kawakami et al., J Immunol., 142, 3452-3461 (1989)).
  • the CTLs of the present invention may be used to form an immunogenic composition useful in treating or the prevention of cancer in a patient in need of therapy or protection (WO2006/031221).
  • cancer such as bladder cancer, breast cancer, cervical cancer, cholangiocellular carcinoma, endometriosis, liver cancer, NSCLC, osteosarcoma, pancreatic cancer, SCLC and AML compared with normal tissue
  • the peptides of or polynucleotides of the present invention may be used for treating and/or for the prophylaxis of cancer, and/or prevention of postoperative recurrence thereof.
  • the present invention provides a pharmaceutical composition or agent for the treatment and/or prophylaxis of cancer, and/or for prevention of postoperative recurrence thereof, such composition or agent including as an active ingredient one or more of the peptides, or polynucleotides of this invention as an active ingredient.
  • the present peptides may be expressed on the surface of any of the foregoing exosomes or cells, such as APCs for the use as pharmaceutical compositions or agents.
  • the aforementioned CTLs which target any of the peptides of the present invention may also be used as the active ingredient of the present pharmaceutical compositions or agents.
  • the pharmaceutical and compositions or agents of the present invention also find use as a vaccine.
  • the phrase "vaccine” also referred to as an "immunogenic composition” refers to a composition or agent that has the function to induce anti-tumor immunity upon inoculation into animals.
  • compositions of the present invention can be used to treat and/or prevent cancers, and/or prevention of postoperative recurrence thereof in subjects or patients including human and any other mammal including, but not limited to, mouse, rat, guinea-pig, rabbit, cat, dog, sheep, goat, pig, cattle, horse, monkey, baboon, and chimpanzee, particularly a commercially important animal or a domesticated animal.
  • the present invention also provides the use of an active ingredient in manufacturing a pharmaceutical composition or agent for treating cancer or tumor, said active ingredient selected from among: (a) a peptide of the present invention; (b) a nucleic acid encoding such a peptide as disclosed herein in an expressible form; (c) an APC or an exosome presenting a peptide of the present invention on its surface; and (d) a cytotoxic T cell of the present invention.
  • the present invention further provides an active ingredient for use in the treatment and/or prevention of cancers or tumors, said active ingredient selected from among: (a) a peptide of the present invention; (b) a nucleic acid encoding such a peptide as disclosed herein in an expressible form; (c) an APC or an exosome presenting a peptide of the present invention on its surface; and (d) a cytotoxic T cell of the present invention.
  • the present invention further provides a method or process for manufacturing a pharmaceutical composition or agent for treating or preventing cancer or tumor, wherein the method or process includes the step of formulating a pharmaceutically or physiologically acceptable carrier with an active ingredient selected from among: (a) a peptide of the present invention; (b) a nucleic acid encoding such a peptide as disclosed herein in an expressible form; (c) an APC or an exosome presenting a peptide of the present invention on its surface; and (d) a cytotoxic T cell of the present invention.
  • a pharmaceutically or physiologically acceptable carrier with an active ingredient selected from among: (a) a peptide of the present invention; (b) a nucleic acid encoding such a peptide as disclosed herein in an expressible form; (c) an APC or an exosome presenting a peptide of the present invention on its surface; and (d) a cytotoxic T cell of the present invention.
  • the present invention also provides a method or process for manufacturing a pharmaceutical composition or agent for treating or preventing cancer or tumor, wherein the method or process includes the steps of admixing an active ingredient with a pharmaceutically or physiologically acceptable carrier, wherein the active ingredient is selected from among: (a) a peptide of the present invention; (b) a nucleic acid encoding such a peptide as disclosed herein in an expressible form; (c) an APC or an exosome presenting a peptide of the present invention on its surface; and (d) a cytotoxic T cell of the present invention.
  • the present pharmaceutical compositions or agents find use as a vaccine.
  • the phrase "vaccine” also referred to as an immunogenic composition refers to a composition or agent that has the function to induce anti-tumor immunity upon inoculation into animals.
  • Cancers to be treated by the pharmaceutical compositions or agents of the present invention are not limited and include any cancer in which VANGL1 is involved (e.g., is over-expressed), including, but not limited to, bladder cancer, breast cancer, cervical cancer, cholangiocellular carcinoma, endometriosis, liver cancer, NSCLC, osteosarcoma, pancreatic cancer, SCLC and AML.
  • the present pharmaceutical compositions or agents may contain in addition to the aforementioned active ingredients, other peptides which have the ability to induce CTLs against cancerous cells, other polynucleotides encoding the other peptides, other cells that present the other peptides, or such.
  • the other peptides that have the ability to induce CTLs against cancerous cells are exemplified by cancer specific antigens (e.g., identified TAAs), but are not limited thereto.
  • the pharmaceutical compositions or agents of the present invention may optionally include other therapeutic substances as an active ingredient, so long as the substance does not inhibit the antitumoral effect of the active ingredient, e.g., any of the present peptides.
  • formulations may include anti-inflammatory compositions, pain killers, chemotherapeutics, and the like.
  • the medicaments of the present invention may also be administered sequentially or concurrently with the one or more other pharmacologic compositions.
  • the amounts of medicament and pharmacologic substance or composition depend, for example, on what type of pharmacologic substance(s) or composition(s) is/are used, the disease being treated, and the scheduling and routes of administration. It should be understood that in addition to the ingredients particularly mentioned herein, the pharmaceutical compositions or agents of this invention may include other compositions conventional in the art having regard to the type of formulation in question.
  • the present pharmaceutical compositions or agents may be included in articles of manufacture and kits containing materials useful for treating the pathological conditions of the disease to be treated, e.g., cancer.
  • the article of manufacture may include a container of any of the present pharmaceutical compositions or agents with a label. Suitable containers include bottles, vials, and test tubes. The containers may be formed from a variety of materials, such as glass or plastic.
  • the label on the container should indicate the composition or agent is used for treating or prevention of one or more conditions of the disease.
  • the label may also indicate directions for administration and so on.
  • kits including a pharmaceutical composition or agent of the present invention may optionally further include a second container housing a pharmaceutically-acceptable diluent. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use.
  • compositions or agent can, if desired, be presented in a pack or dispenser device which can contain one or more unit dosage forms containing the active ingredient.
  • the pack can, for example, include metal or plastic foil, such as a blister pack.
  • the pack or dispenser device can be accompanied by instructions for administration.
  • compositions containing the peptides as the active ingredient can be administered directly as a pharmaceutical composition or agent , or if necessary, may be formulated by conventional formulation methods.
  • carriers, excipients, and such that are ordinarily used for drugs can be included as appropriate without particular limitations. Examples of such carriers are sterilized water, physiological saline, phosphate buffer, culture fluid and such.
  • the pharmaceutical compositions or agents can contain as necessary, stabilizers, suspensions, preservatives, surfactants and such.
  • the pharmaceutical compositions of this invention can be used for anticancer purposes.
  • the peptides of this invention can be prepared in a combination, which includes two or more of peptides of the present invention, to induce CTL in vivo.
  • the peptides can be in a cocktail or can be conjugated to each other using standard techniques.
  • the peptides can be chemically linked or expressed as a single fusion polypeptide sequence that may have one or several amino acid as a linker (e.g., Lysine linker: K. S. Kawamura et al. J. Immunol. 2002, 168: 5709-5715).
  • the peptides in the combination can be the same or different.
  • the peptides are presented at a high density by the HLA antigens on APCs, then CTLs that specifically react toward the complex formed between the displayed peptide and the HLA antigen are induced.
  • APCs e.g., DCs
  • APCs are removed from subjects and then stimulated by the peptides of the present invention to obtain APCs that present any of the peptides of this invention on their cell surface.
  • compositions or agents for the treatment and/or prevention of cancer can also include an adjuvant so that cellular immunity will be established effectively, or they can be administered with other active ingredients, and they can be administered by formulation into granules.
  • An adjuvant refers to a compound that enhances the immune response against the protein when administered together (or successively) with the protein having immunological activity.
  • An adjuvant that can be applied includes those described in the literature (Clin Microbiol Rev 1994, 7: 277-89).
  • the peptides of the present invention may also be administered in the form of a pharmaceutically acceptable salt.
  • the salts include salts with an alkali metal, salts with a metal, salts with an organic base, salts with an organic acid and salts with an inorganic acid.
  • pharmaceutically acceptable salt refers to those salts which retain the biological effectiveness and properties of the compound and which are obtained by reaction with inorganic acids or bases such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid and the like.
  • the pharmaceutical compositions or agents of the present invention include a component that primes CTL.
  • Lipids have been identified as compositions capable of priming CTL in vivo against viral antigens.
  • acid residues can be attached to the epsilon- and alpha-amino groups of a lysine residue and then linked to a peptide of the present invention.
  • the lipidated peptide can then be administered either directly in a micelle or particle, incorporated into a liposome, or emulsified in an adjuvant.
  • lipid priming of CTL responses E.
  • coli lipoproteins such as tripalmitoyl-S-glycerylcysteinyl-seryl-serine (P3CSS) can be used to prime CTL when covalently attached to an appropriate peptide (see, e.g., Deres et al., Nature 1989, 342: 561-4).
  • P3CSS tripalmitoyl-S-glycerylcysteinyl-seryl-serine
  • the method of administration can be oral, intradermal, subcutaneous, intravenous injection, or such, and systemic administration or local administration to the vicinity of the targeted sites.
  • the administration can be performed by single administration or boosted by multiple administrations.
  • the dose of the peptides of this invention can be adjusted appropriately according to the disease to be treated, age of the patient, weight, method of administration, and such, and is ordinarily 0.001 mg to 1,000 mg, for example, 0.01 mg to 100 mg, for example, 0.1 mg to 10 mg, and can be administered once in a few days to few months.
  • One skilled in the art can appropriately select a suitable dose.
  • compositions containing polynucleotides as the active ingredient can also include nucleic acids encoding the peptides disclosed herein in an expressible form.
  • the phrase "in an expressible form” means that the polynucleotide, when introduced into a cell, will be expressed in vivo as a polypeptide that induces anti-tumor immunity.
  • the nucleic acid sequence of the polynucleotide of interest includes regulatory elements necessary for expression of the polynucleotide.
  • the polynucleotide(s) can be equipped so to achieve stable insertion into the genome of the target cell (see, e.g., Thomas KR & Capecchi MR, Cell 1987, 51: 503-12 for a description of homologous recombination cassette vectors). See, e.g., Wolff et al., Science 1990, 247: 1465-8; U.S. Patent Nos. 5,580,859; 5,589,466; 5,804,566; 5,739,118; 5,736,524; 5,679,647; and WO 98/04720.
  • DNA-based delivery technologies include "naked DNA”, facilitated (bupivacaine, polymers, peptide-mediated) delivery, cationic lipid complexes, and particle-mediated (“gene gun”) or pressure-mediated delivery (see, e.g., U.S. Patent No. 5,922,687).
  • the peptides of the present invention can also be expressed by viral or bacterial vectors.
  • expression vectors include attenuated viral hosts, such as vaccinia or fowlpox. This approach involves the use of vaccinia virus, e.g., as a vector to express nucleotide sequences that encode the peptide. Upon introduction into a host, the recombinant vaccinia virus expresses the immunogenic peptide, and thereby elicits an immune response.
  • Vaccinia vectors and methods useful in immunization protocols are described in, e.g., U.S. Patent No. 4,722,848. Another vector is BCG (Bacille Calmette Guerin).
  • BCG vectors are described in Stover et al., Nature 1991, 351: 456-60.
  • a wide variety of other vectors useful for therapeutic administration or immunization e.g., adeno and adeno-associated virus vectors, retroviral vectors, Salmonella typhi vectors, detoxified anthrax toxin vectors, and the like, will be apparent. See, e.g., Shata et al., Mol Med Today 2000, 6: 66-71; Shedlock et al., J Leukoc Biol 2000, 68: 793-806; Hipp et al., In Vivo 2000, 14: 571-85.
  • Delivery of a polynucleotide into a patient can be either direct, in which case the patient is directly exposed to a polynucleotide-carrying vector, or indirect, in which case, cells are first transformed with the polynucleotide of interest in vitro, then the cells are transplanted into the patient.
  • two approaches are known, respectively, as in vivo and ex vivo gene therapies.
  • CTL can be also induced by using them ex vivo, and after inducing CTL, the activated CTLs are returned to the subject.
  • the method can include steps of : a: collecting APCs from subject:, b: contacting with the APCs of step a, with the peptide:, and c: co-culturing the APCs of step b with CD8-positive cells.
  • the Tm is the temperature (under a defined ionic strength, pH and nucleic acid concentration) at which 50% of the probes complementary to their target sequence hybridize to the target sequence at equilibrium. Since the target sequences are generally present at excess, at Tm, 50% of the probes are occupied at equilibrium.
  • stringent conditions will be those in which the salt concentration is less than about 1.0 M sodium ion, typically about 0.01 to 1.0 M sodium ion (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30 degree Centigrade for short probes or primers (e.g., 10 to 50 nucleotides) and at least about 60 degree Centigrade for longer probes or primers. Stringent conditions may also be achieved with the addition of destabilizing substances, such as formamide.
  • a control level determined from a biological sample that is known to be non-cancerous is referred to as a "normal control level”.
  • the control level is determined from a cancerous biological sample, it is referred to as a "cancerous control level”.
  • Difference between a sample expression level and a control level can be normalized to the expression level of control nucleic acids, e.g., housekeeping genes, whose expression levels are known not to differ depending on the cancerous or non-cancerous state of the cell.
  • Exemplary control genes include, but are not limited to, beta-actin, glyceraldehyde 3 phosphate dehydrogenase, and ribosomal protein P1.
  • reagents suitable for detecting mRNA of the VANGL1 gene include nucleic acids that specifically bind to or identify the VANGL1 mRNA, such as oligonucleotides that have a complementary sequence to a portion of the VANGL1 mRNA. These kinds of oligonucleotides are exemplified by primers and probes that are specific to the VANGL1 mRNA. These kinds of oligonucleotides may be prepared based on methods well known in the art. If needed, the reagent for detecting the VANGL1 mRNA may be immobilized on a solid matrix. Moreover, more than one reagent for detecting the VANGL1 mRNA may be included in the kit.
  • the number of sites displaying a detectable signal provides a quantitative indication of the amount of VANGL1 mRNA present in the sample.
  • the detection sites may be configured in any suitably detectable shape and are typically in the shape of a bar or dot spanning the width of a test strip.
  • the kit of the present invention may further include a positive control sample or VANGL1 standard sample.
  • the positive control sample of the present invention may be prepared by collecting VANGL1 positive samples and then assaying their VANGL1 levels.
  • a purified VANGL1 protein or polynucleotide may be added to cells that do not express VANGL1 to form the positive sample or the VANGL1 standard sample.
  • purified VANGL1 may be a recombinant protein.
  • the VANGL1 level of the positive control sample is, for example, more than the cut off value.
  • the present invention further provides a diagnostic kit including, a protein or a partial protein thereof specifically recognized by the antibody of the present invention or the fragment thereof.
  • the partial peptide of the protein of the present invention include polypeptides composed of at least 8, preferably 15, and more preferably 20 contiguous amino acids in the amino acid sequence of the protein of the present invention.
  • Cancer can be diagnosed by detecting an antibody in a sample (e.g., blood, tissue) using a protein or a peptide (polypeptide) of the present invention.
  • the method for preparing the protein of the present invention and peptides are as described above.
  • a diagnostic kit of the present invention may include the peptide of the present invention and an HLA molecule binding thereto.
  • the method for detecting antigen specific CTLs using antigenic peptides and HLA molecules has already been established (for example, Altman JD et al., Science. 1996, 274(5284): 94-6).
  • the complex of the peptide of the present invention and the HLA molecule can be applied to the detection method to detect tumor antigen specific CTLs, thereby enabling earlier detection, recurrence and/or metastasis of cancer.
  • it can be employed for the selection of subjects applicable with the pharmaceuticals including the peptide of the present invention as an active ingredient, or the assessment of the treatment effect of the pharmaceuticals.
  • peptides of the present invention may be used in tetramer staining assays to assess peripheral blood mononuclear cells for the presence of antigen-specific CTLs following exposure to a tumor cell antigen or an immunogen.
  • the HLA tetrameric complex may be used to directly visualize antigen specific CTLs (see, e. g., Ogg et al., Science 279 : 2103-2106, 1998 ; and Altman et al, Science 174 : 94-96, 1996) and determine the frequency of the antigen-specific CTL population in a sample of peripheral blood mononuclear cells.
  • a tetramer reagent using a peptide of the invention may be generated as described below.
  • a peptide that binds to an HLA molecule is refolded in the presence of the corresponding HLA heavy chain and beta 2- microglobulin to generate a trimolecular complex.
  • carboxyl terminal of the heavy chain is biotinylated at a site that was previously engineered into the protein.
  • streptavidin is added to the complex to form tetramer composed of the trimolecular complex and streptavidin.
  • the tetramer can be used to stain antigen-specific cells.
  • the cells can then be identified, for example, by flow cytometry. Such an analysis may be used for diagnostic or prognostic purposes. Cells identified by the procedure can also be used for therapeutic purposes.
  • the present invention also provides reagents to evaluate immune recall responses (see, e. g., Bertoni etaL, J. Clin. Invest. 100 : 503-513, 1997 and Penna et aL, J Exp. Med. 174 : 1565-1570, 1991) including peptides of the present invention.
  • immune recall responses see, e. g., Bertoni etaL, J. Clin. Invest. 100 : 503-513, 1997 and Penna et aL, J Exp. Med. 174 : 1565-1570, 1991
  • patient PBMC samples from individuals with cancer to be treated are analyzed for the presence of antigen-specific CTLs using specific peptides.
  • a blood sample containing mononuclear cells can be evaluated by cultivating the PBMCs and stimulating the cells with a peptide of the invention. After an appropriate cultivation period, the expanded cell population can be analyzed, for example, for CTL activity.
  • the peptides of the invention may be also used to make antibodies, using techniques well known in the art (see, e. g. CURRENTPROTOCOLSINIMMUNOLOGY, Wiley/Greene, NY ; and Antibodies A Laboratory Manual, Harlow and Lane, Cold Spring Harbor Laboratory Press, 1989), which may be useful as reagents to diagnose or monitor cancer.
  • Such antibodies may include those that recognize a peptide in the context of an HLA molecule, i. e., antibodies that bind to a peptide-MHC complex.
  • VANGL1 in a biological sample can also be tested by standard PCR amplification protocols using VANGL1 primers.
  • An example of tumor expression is presented herein and further disclosure of exemplary conditions and primers for VANGL1 amplification can be found in WO2003/27322.
  • the diagnostic methods involve contacting a biological sample isolated from a subject with an agent specific for the VANGL1 HLA binding peptide to detect the presence of the VANGL1 HLA binding peptide in the biological sample.
  • contacting means placing the biological sample in sufficient proximity to the agent and under the appropriate conditions of, e. g., concentration, temperature, time, ionic strength, to allow the specific interaction between the agent and VANGL1 HLA binding peptide that are present in the biological sample.
  • the conditions for contacting the agent with the biological sample are conditions known by those of ordinary skill in the art to facilitate a specific interaction between a molecule and its cognate (e.
  • the diagnosis can be done, by a method which allows direct quantification of antigen- specific T cells by staining with Fluorescein-labelled HLA multimeric complexes (for example, Altman, J. D. et al., 1996, Science 274 : 94; Altman, J. D. et al., 1993, Proc. Natl. Acad. Sci. USA 90 : 10330 ;). Staining for intracellular lymphokines, and interferon-gamma release assays or ELISPOT assays also has been provided.
  • Tetramer staining, intracellular lymphokine staining and ELISPOT assays all appear to be at least 10-fold more sensitive than more conventional assays (Murali-Krishna, K. et al., 1998, Immunity 8 : 177; Lalvani, A. et al., 1997, J. Exp. Med. 186 : 859; Dunbar, P. R. et al., 1998, Curr. Biol. 8 : 413;). Pentamers (e.g., US 2004-209295A), dextramers (e.g., WO 02/072631), and streptamers (e.g., Nature medicine 6. 631-637 (2002)) may also be used.
  • Pentamers e.g., US 2004-209295A
  • dextramers e.g., WO 02/072631
  • streptamers e.g., Nature medicine 6. 631-637 (2002)
  • the present invention provides a method for diagnosing or evaluating an immunological response of a subject administered at least one of VANGL1 peptides of the present invention, the method including the steps of: (a) contacting an immunogen with immunocompetent cells under the condition suitable of induction of CTL specific to the immunogen; (b) detecting or determining induction level of the CTL induced in step (a); and (c) correlating the immunological response of the subject with the CTL induction level.
  • the immunogen is at least one of (a) a VANGL1 peptide selected from among the amino acid sequences of SEQ ID NOs: 1 and 3 to 67, peptides having such amino acid sequences, and peptides having in which such amino acid sequences have been modified with one, two or more amino acid substitution(s).
  • a VANGL1 peptide selected from among the amino acid sequences of SEQ ID NOs: 1 and 3 to 67, peptides having such amino acid sequences, and peptides having in which such amino acid sequences have been modified with one, two or more amino acid substitution(s).
  • immunocompetent cells may be cultured in vitro under the presence of immunogen(s) to induce immunogen specific CTL.
  • any stimulating factors may be added to the cell culture.
  • IL-2 is preferable stimulating factors for the CTL induction.
  • Antibodies The present invention provides antibodies that bind to the peptide of the present invention. Preferred antibodies specifically bind to the peptide of the present invention and will not bind (or will bind weakly) to non- peptide of the present invention. Alternatively, antibodies bind the peptide of the invention as well as the homologs thereof.
  • Antibodies against the peptide of the invention can find use in cancer diagnostic and prognostic assays, and imaging methodologies. Similarly, such antibodies can find use in the treatment, diagnosis, and/or prognosis of other cancers, to the extent VANGL1 is also expressed or over-expressed in cancer patient. Moreover, intracellularly expressed antibodies (e. g., single chain antibodies) are therapeutically useful in treating cancers in which the expression of VANGL1 is involved, examples of which include, but are not limited to, bladder cancer, breast cancer, cervical cancer, cholangiocellular carcinoma, endometriosis, liver cancer, NSCLC, osteosarcoma, pancreatic cancer, SCLC and AML.
  • the present invention also provides various immunological assay for the detection and/or quantification of VANGL1 protein (for example SEQ ID NO: 69) or fragments thereof including polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1 and 3 to 67.
  • Such assays can include one or more anti-VANGL1 antibodies capable of recognizing and binding a VANGL1 protein or fragments thereof, as appropriate.
  • anti-VANGL1 antibodies binding to VANGL-1 polypeptide preferably recognize a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1 and 3 to 67. A binding specificity of antibody can be confirmed with inhibition test.
  • immunological assays are performed within various immunological assay formats well known in the art, including but not limited to various types of radio-immunoassays, immuno-chromatgraph technique, enzyme-linked immunosorbent assays (ELISA), enzyme- linked immunofluorescent assays (ELIFA), and the like.
  • immunological but non-antibody assays of the invention also include T cell immunogenicity assays (inhibitory or stimulatory) as well as major histocompatibility complex (MHC) binding assays.
  • immunological imaging methods capable of detecting cancers expressing VANGL1 are also provided by the invention, including but not limited to radioscintigraphic imaging methods using labeled antibodies of the present invention. Such assays are clinically useful in the detection, monitoring, and prognosis of VANGL1 expressing cancers including, but not limited to, bladder cancer, breast cancer, cervical cancer, cholangiocellular carcinoma, endometriosis, liver cancer, NSCLC, osteosarcoma, pancreatic cancer, SCLC and AML.
  • the present invention provides an antibody that binds to the peptide of the invention.
  • the antibody of the invention can be used in any form, such as monoclonal or polyclonal antibodies, and includes antiserum obtained by immunizing an animal such as a rabbit with the peptide of the invention, all classes of polyclonal and monoclonal antibodies, human antibodies and humanized antibodies produced by genetic recombination.
  • a peptide of the invention used as an antigen to obtain an antibody may be derived from any animal species, but preferably is derived from a mammal such as a human, mouse, or rat, more preferably from a human.
  • a human-derived peptide may be obtained from the nucleotide or amino acid sequences disclosed herein.
  • antigens may be diluted and suspended in an appropriate amount of phosphate buffered saline (PBS), physiological saline, etc.
  • PBS phosphate buffered saline
  • the antigen suspension may be mixed with an appropriate amount of a standard adjuvant, such as Freund's complete adjuvant, made into emulsion and then administered to mammalian animals.
  • a standard adjuvant such as Freund's complete adjuvant
  • an appropriately amount of Freund's incomplete adjuvant every 4 to 21 days.
  • An appropriate carrier may also be used for immunization.
  • serum is examined by a standard method for an increase in the amount of desired antibodies.
  • Polyclonal antibodies against the peptides of the present invention may be prepared by collecting blood from the immunized mammal examined for the increase of desired antibodies in the serum, and by separating serum from the blood by any conventional method.
  • Polyclonal antibodies include serum containing the polyclonal antibodies, as well as the fraction containing the polyclonal antibodies may be isolated from the serum.
  • Immunoglobulin G or M can be prepared from a fraction which recognizes only the peptide of the present invention using, for example, an affinity column coupled with the peptide of the present invention, and further purifying this fraction using protein A or protein G column.
  • Resulting hybridomas obtained by the cell fusion may be selected by cultivating them in a standard selection medium, such as HAT medium (hypoxanthine, aminopterin and thymidine containing medium).
  • HAT medium hyperxanthine, aminopterin and thymidine containing medium.
  • the cell culture is typically continued in the HAT medium for several days to several weeks, the time being sufficient to allow all the other cells, with the exception of the desired hybridoma (non-fused cells), to die. Then, the standard limiting dilution is performed to screen and clone a hybridoma cell producing the desired antibody.
  • human lymphocytes such as those infected by EB virus may be immunized with a peptide, peptide expressing cells or their lysates in vitro. Then, the immunized lymphocytes are fused with human-derived myeloma cells that are capable of indefinitely dividing, such as U266, to yield a hybridoma producing a desired human antibody that is able to bind to the peptide can be obtained (Unexamined Published Japanese Patent Application No. (JP-A) Sho 63-17688).
  • the obtained hybridomas are subsequently transplanted into the abdominal cavity of a mouse and the ascites are extracted.
  • the obtained monoclonal antibodies can be purified by, for example, ammonium sulfate precipitation, a protein A or protein G column, DEAE ion exchange chromatography or an affinity column to which the peptide of the present invention is coupled.
  • the antibody of the present invention can be used not only for purification and detection of the peptide of the present invention, but also as a candidate for agonists and antagonists of the peptide of the present invention.
  • an immune cell such as an immunized lymphocyte
  • producing antibodies may be immortalized by an oncogene and used for preparing monoclonal antibodies.
  • Monoclonal antibodies thus obtained can be also recombinantly prepared using genetic engineering techniques (see, for example, Borrebaeck and Larrick, Therapeutic Monoclonal Antibodies, published in the United Kingdom by MacMillan Publishers LTD (1990)).
  • a DNA encoding an antibody may be cloned from an immune cell, such as a hybridoma or an immunized lymphocyte producing the antibody, inserted into an appropriate vector, and introduced into host cells to prepare a recombinant antibody.
  • the present invention also provides recombinant antibodies prepared as described above.
  • Antibodies obtained as above may be purified to homogeneity.
  • the separation and purification of the antibody can be performed according to separation and purification methods used for general proteins.
  • the antibody may be separated and isolated by the appropriately selected and combined use of column chromatographies, such as affinity chromatography, filter, ultrafiltration, salting-out, dialysis, SDS polyacrylamide gel electrophoresis and isoelectric focusing (Antibodies: A Laboratory Manual. Ed Harlow and David Lane, Cold Spring Harbor Laboratory (1988)), but are not limited thereto.
  • a protein A column and protein G column can be used as the affinity column.
  • Exemplary protein A columns to be used include, for example, Hyper D, POROS and Sepharose F.F. (Pharmacia).
  • Exemplary chromatography with the exception of affinity includes, for example, ion-exchange chromatography, hydrophobic chromatography, gel filtration, reverse-phase chromatography, adsorption chromatography and the like (Strategies for Protein Purification and Characterization: A Laboratory Course Manual. Ed Daniel R. Marshak et al., Cold Spring Harbor Laboratory Press (1996)).
  • the chromatographic procedures can be carried out by liquid-phase chromatography, such as HPLC and FPLC.
  • an enzyme substrate such as p-nitrophenyl phosphate
  • a fragment of the peptide such as a C-terminal or N-terminal fragment, may be used as the antigen to evaluate the binding activity of the antibody.
  • BIAcore (Pharmacia) may be used to evaluate the activity of the antibody according to the present invention.
  • the above methods allow for the detection or measurement of the peptide of the invention, by exposing the antibody of the invention to a sample assumed to contain the peptide of the invention, and detecting or measuring the immune complex formed by the antibody and the peptide. Because the method of detection or measurement of the peptide according to the invention can specifically detect or measure a peptide, the method may be useful in a variety of experiments in which the peptide is used.
  • the present invention also provides a vector and host cell into which a nucleotide encoding the peptide of the present invention is introduced.
  • a vector of the present invention is useful to keep a nucleotide, especially a DNA, of the present invention in host cell, to express the peptide of the present invention, or to administer the nucleotide of the present invention for gene therapy.
  • E. coli When E. coli is a host cell and the vector is amplified and produced in a large amount in E. coli (e.g., JM109, DH5 alpha, HB101 or XL1Blue), the vector should have "ori" to be amplified in E. coli and a marker gene for selecting transformed E. coli (e.g., a drug-resistance gene selected by a drug such as ampicillin, tetracycline, kanamycin, chloramphenicol or the like).
  • a marker gene for selecting transformed E. coli e.g., a drug-resistance gene selected by a drug such as ampicillin, tetracycline, kanamycin, chloramphenicol or the like.
  • M13-series vectors, pUC-series vectors, pBR322, pBluescript, pCR-Script, etc. can be used.
  • pGEM-T pDIRECT and pT7 can also be used for subcloning and extracting cDNA as well as the vectors described above.
  • an expression vector is especially useful.
  • an expression vector to be expressed in E. coli should have the above characteristics to be amplified in E. coli.
  • the vector should have a promoter, for example, lacZ promoter (Ward et al., Nature 341: 544-6 (1989); FASEB J 6: 2422-7 (1992)), araB promoter (Better et al., Science 240: 1041-3 (1988)), T7 promoter or the like, that can efficiently express the desired gene in E. coli.
  • a promoter for example, lacZ promoter (Ward et al., Nature 341: 544-6 (1989); FASEB J 6: 2422-7 (1992)), araB promoter (Better et al., Science 240: 1041-3 (1988)), T7 promoter or the like, that can efficiently express the desired gene in E. coli.
  • the host is preferably BL21 which expresses T7 RNA polymerase
  • the vector may also contain a signal sequence for peptide secretion.
  • An exemplary signal sequence that directs the peptide to be secreted to the periplasm of the E. coli is the pelB signal sequence (Lei et al., J Bacteriol 169: 4379 (1987)).
  • Means for introducing of the vectors into the target host cells include, for example, the calcium chloride method, and the electroporation method.
  • the vector In order to express the vector in animal cells, such as CHO, COS or NIH3T3 cells, the vector should have a promoter necessary for expression in such cells, for example, the SV40 promoter (Mulligan et al., Nature 277: 108 (1979)), the MMLV-LTR promoter, the EF1 alpha promoter (Mizushima et al., Nucleic Acids Res 18: 5322 (1990)), the CMV promoter and the like, and preferably a marker gene for selecting transformants (for example, a drug resistance gene selected by a drug (e.g., neomycin, G418)).
  • a promoter necessary for expression in such cells for example, the SV40 promoter (Mulligan et al., Nature 277: 108 (1979)), the MMLV-LTR promoter, the EF1 alpha promoter (Mizushima et al., Nucleic Acids Res 18: 5322 (1990)
  • DCs cytotoxic T lymphocyte
  • HLA human leukocyte antigen
  • DCs were generated in vitro as described elsewhere (Nakahara S et al., Cancer Res 2003 Jul 15, 63(14): 4112-8). Specifically, peripheral blood mononuclear cells isolated from a normal volunteer (HLA-A*0201 positive) by Ficoll-Plaque (Pharmacia) solution were separated by adherence to a plastic tissue culture dish (Becton Dickinson) so as to enrich them as the monocyte fraction.
  • the monocyte-enriched population was cultured in the presence of 1000 U/ml of granulocyte-macrophage colony-stimulating factor (R&D System) and 1000 U/ml of interleukin (IL)-4 (R&D System) in AIM-V Medium (Invitrogen) containing 2% heat-inactivated autologous serum (AS). After 7 days of culture, the cytokine-induced DCs were pulsed with 20 micro-g/ml of each of the synthesized peptides in the presence of 3 micro-g/ml of beta 2-microglobulin for 3 hr at 37 degrees C in AIM-V Medium.
  • R&D System granulocyte-macrophage colony-stimulating factor
  • IL-4 interleukin-4
  • AS heat-inactivated autologous serum
  • the generated cells appeared to express DC-associated molecules, such as CD80, CD83, CD86 and HLA class II, on their cell surfaces (data not shown).
  • DC-associated molecules such as CD80, CD83, CD86 and HLA class II
  • These peptide-pulsed DCs were then inactivated by X-irradiation (20 Gy) and mixing at a 1:20 ratio with autologous CD8+ T cells, obtained by positive selection with CD8 Positive Isolation Kit (Dynal). These cultures were set up in 48-well plates (Corning); each well contained 1.5 x 10 4 peptide-pulsed DCs, 3 x 10 5 CD8+ T cells and 10 ng/ml of IL-7 (R&D System) in 0.5 ml of AIM-V/2% AS medium.
  • CTL Expansion Procedure CTLs were expanded in culture using the method similar to the one described by Riddell et al. (Walter EA et al., N Engl J Med 1995 Oct 19, 333(16): 1038-44; Riddell SR et al., Nat Med 1996 Feb, 2(2): 216-23). A total of 5 x 10 4 CTLs were suspended in 25 ml of AIM-V/5% AS medium with 2 kinds of human B-lymphoblastoid cell lines, inactivated by Mitomycin C, in the presence of 40 ng/ml of anti-CD3 monoclonal antibody (Pharmingen). One day after initiating the cultures, 120 IU/ml of IL-2 were added to the cultures.
  • CTL clones The dilutions were made to have 0.3, 1, and 3 CTLs/well in 96 round-bottomed micro titer plate (Nalge Nunc International). CTLs were cultured with 1 X 10 4 cells/well of 2 kinds of human B-lymphoblastoid cell lines, 30ng/ml of anti-CD3 antibody, and 125 U/ml of IL-2 in a total of 150 micro-l/well of AIM-V Medium containing 5%AS. 50 micro-l /well of IL-2 were added to the medium 10 days later so to reach a final concentration of 125 U/ml IL-2.
  • CTL activity was tested on the 14th day, and CTL clones were expanded using the same method as described above (Uchida N et al., Clin Cancer Res 2004 Dec 15, 10(24): 8577-86; Suda T et al., Cancer Sci 2006 May, 97(5): 411-9; Watanabe T et al., Cancer Sci 2005 Aug, 96(8): 498-506).
  • the cDNA encoding an open reading frame of target genes or HLA-A*0201 was amplified by PCR.
  • the PCR-amplified product was cloned into a vector.
  • the plasmids were transfected into COS7, which is the target genes and HLA-A*0201-null cell line, using lipofectamine 2000 (Invitrogen) according to the manufacturer's recommended procedures. After 2 days from transfection, the transfected cells were harvested with versene (Invitrogen) and used as the target cells (5 X 10 4 cells/ well) for CTL activity assay.
  • Specific CTL activity against target cells expressing VANGL1 and HLA-A*0201 The established CTL lines and clones raised against each peptide were examined for the ability to recognize target cells that express VANGL1 and HLA-A*0201 molecule.
  • Specific CTL activity against COS7 cells which transfected with both the full length of VANGL1 and HLA-A*0201 gene was tested by using the CTL lines and clones raised by corresponding peptide as the stimulator cells.
  • COS7 cells transfected with either full length of VANGL1 or HLA-A*0201 were prepared as the controls.
  • VANGL1-A02-9-194 SEQ ID NO: 1
  • VANGL1-A02-9-235 SEQ ID NO: 12
  • VANGL1-A02-9-484 SEQ ID NO: 21
  • This result may be due to the fact that the sequence of VANGL1-A02-9-194 (SEQ ID NO:1), VANGL1-A02-9-235 (SEQ ID NO: 12) and VANGL1-A02-9-484 (SEQ ID NO: 21) are homologous to peptide derived from other molecules that are known to sensitize the human immune system.
  • the present invention provides new TAAs, particularly those derived from VANGL1 that induce potent and specific anti-tumor immune responses and have applicability to a wide array of cancer types.
  • TAAs are useful as peptide vaccines against diseases associated with VANGL1, e.g., cancer, more particularly, bladder cancer, breast cancer, cervical cancer, cholangiocellular carcinoma, endometriosis, liver cancer, NSCLC, osteosarcoma, pancreatic cancer, SCLC and AML.

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Abstract

Cette invention concerne des peptides isolés ou des fragments immunogènes dérivés de SEQ ID N°: 69 qui se lient à un antigène HLA et induisent des lymphocytes T cytotoxiques (CTL). Les peptides peuvent comprendre une des séquences d'acides aminés précitées avec ou sans substitution, délétion, ou addition d'une, de deux, ou de plusieurs séquences d'acides aminés. Cette invention concerne également des compositions pharmaceutiques contenant ces peptides. Les peptides selon l'invention s'avèrent utiles dans le traitement du cancer.
PCT/JP2011/004984 2010-09-07 2011-09-06 Peptides vangl1 et vaccins les contenant Ceased WO2012032763A1 (fr)

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Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003027322A2 (fr) * 2001-09-25 2003-04-03 Japan As Represented By The President Of The University Of Tokyo Genes et polypeptides associes aux carcinomes hepatocellulaires, et methode de detection de carcinomes hepatocellulaires
WO2004024766A1 (fr) * 2002-09-12 2004-03-25 Oncotherapy Science, Inc. Peptides kdr et vaccins les renfermant
WO2006090810A2 (fr) * 2005-02-25 2006-08-31 Oncotherapy Science, Inc. Vaccins a base de peptides pour cancers du poumon exprimant des polypeptides ttk, urlc10 ou koc1
WO2007013576A1 (fr) * 2005-07-27 2007-02-01 Oncotherapy Science, Inc. Gene tom34 lie au cancer du colon
WO2008047473A1 (fr) * 2006-10-17 2008-04-24 Oncotherapy Science, Inc. Vaccins peptidiques pour des cancers exprimant les polypeptides mphosph1 ou depdc1
WO2010100878A1 (fr) * 2009-03-04 2010-09-10 Oncotherapy Science, Inc. Peptides vangl1 et vaccins contenant ces peptides

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003027322A2 (fr) * 2001-09-25 2003-04-03 Japan As Represented By The President Of The University Of Tokyo Genes et polypeptides associes aux carcinomes hepatocellulaires, et methode de detection de carcinomes hepatocellulaires
WO2004024766A1 (fr) * 2002-09-12 2004-03-25 Oncotherapy Science, Inc. Peptides kdr et vaccins les renfermant
WO2006090810A2 (fr) * 2005-02-25 2006-08-31 Oncotherapy Science, Inc. Vaccins a base de peptides pour cancers du poumon exprimant des polypeptides ttk, urlc10 ou koc1
WO2007013576A1 (fr) * 2005-07-27 2007-02-01 Oncotherapy Science, Inc. Gene tom34 lie au cancer du colon
WO2008047473A1 (fr) * 2006-10-17 2008-04-24 Oncotherapy Science, Inc. Vaccins peptidiques pour des cancers exprimant les polypeptides mphosph1 ou depdc1
WO2010100878A1 (fr) * 2009-03-04 2010-09-10 Oncotherapy Science, Inc. Peptides vangl1 et vaccins contenant ces peptides

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