Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
  • C07K7/04—Linear peptides containing only normal peptide links
  • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/0005—Vertebrate antigens
  • A61K39/0011—Cancer antigens
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/10—Dispersions; Emulsions
  • A61K9/127—Synthetic bilayered vehicles, e.g. liposomes or liposomes with cholesterol as the only non-phosphatidyl surfactant
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
  • A61P37/02—Immunomodulators
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
  • A61P37/02—Immunomodulators
  • A61P37/04—Immunostimulants
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• • 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
  • C07K14/4713—Autoimmune diseases, e.g. Insulin-dependent diabetes mellitus, multiple sclerosis, rheumathoid arthritis, systemic lupus erythematosus; Autoantigens
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
  • A61K2039/55511—Organic adjuvants
  • A61K2039/55566—Emulsions, e.g. Freund's adjuvant, MF59
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies

Definitions

• the present invention relates to the field of biological science, more specifically to the field of cancer therapy.
• the present invention relates to Foxp3 peptides that are extremely effective as cancer vaccines, and drugs for treating and preventing tumors.
• TIL tumor- infiltrating lymphocytes
• PBL peripheral blood lymphocytes
• T-regs require forkhead transcription factor scurfin (Foxp3; SEQ ID NO 2) encoded by the Foxp3 gene (GenBank Accession No. NM_014009; SEQ ID NO 1), which controls their development and regulatory properties (Fontenot JD et al., Nat Immunol 4: 330-6, 2003, Hori S et al., Science 299: 1057-61, 2003, Khattri R et al., Nat Immunol 4: 304-6, 2003).
• Foxp3 forkhead transcription factor scurfin
• the present invention provides methods for regulating immunosuppression, which methods comprise the step of administering Foxp3 polypeptides of the invention.
• Anti-immunosuppression i.e., reversing or counteracting immunosuppression
• the present invention provides methods for inducing anti-immunosuppression, which methods comprise the step of administering the Foxp3 polypeptides, as well as pharmaceutical agents for regulating immunosuppression, comprising the Foxp3 polypeptides.
• the invention provides peptides having cytotoxic T cell indu- cibility, wherein the peptide comprises or consists of the amino acid sequence selected from the group of:
• the invention provides peptides having cytotoxic T cell indu- cibility, wherein the peptide comprises the amino acid sequence selected from the group of :
• the second amino acid from the N-terminus is phenylalanine, tyrosine, methionine, or tryptophan.
• the C-terminal amino acid is phenylalanine, leucine, isoleucine, tryptophan, or methionine.
• the second amino acid from the N-terminus is leucine or methionine.
• the C-terminal amino acid is valine or leucine.
• the substituted peptide comprises the amino acid sequence of SEQ ID NO: 95, 97 or 98
• the invention further provides compositions comprising Foxp3 peptides of the invention or polynucleotides encoding Foxp3 peptides of the invention, and a pharmaceutically acceptable carrier or excipient.
• the compositions are formulated to be administered as a vaccine.
• the invention provides compositions comprising a polynucleotide encoding a Foxp3 peptide of the invention.
• the compositions comprise a plurality (i.e., two or more) polynucleotides encoding a plurality of Foxp3 peptides of the invention.
• the compositions comprise a polynucleotide that encodes a plurality of Foxp3 peptides of the invention.
• compositions comprise another peptide which has the ability to induce cytotoxic T cells against cancerous cells or another polynucleotide encoding the other peptide.
• the invention provides an exosome that presents on its surface a complex comprising an HLA antigen and a Foxp3 peptide of the invention.
• the HLA antigen is selected from the group consisting of HLA- A24, HLA-A2402, HLA- A02 and HLA-A0201.
• the invention provides methods for treating cancer (e.g., reducing tumor cell growth, promoting tumor cell death) by administering to an individual a Foxp3 peptide or a polynucleotide encoding a Foxp3 peptide.
• the invention provides methods of inducing cytotoxic T cells by administering a Foxp3 peptide of the invention or a polynucleotide encoding the Foxp3 peptide.
• the invention provides an antigen-presenting cell, which comprises a complex formed between an HLA antigen and a Foxp3 peptide of the invention.
• the antigen presenting cell is isolated.
• the invention provides methods of regulating T-reg cells in a subject comprising administering to the subject a vaccine comprising a Foxp3 peptide of the invention or an immunologically active fragment of the peptide, or a poly- nucleotide encoding the peptide.
• the subject or patient can be a human.
• Figure 1 depicts photographs showing the results of IFN-gamma ELISPOT assay on CTLs that were induced with peptides derived from Foxp3.
• Figure IA the CTLs in well numbers #2 and 7 stimulated with Foxp3-A24-9-363 (SEQ ID NO 3),#1 and #6 with Foxp3-A24-9-366 (SEQ ID NO 7), #5 with Foxp3-A24-9-190 (SEQ ID NO 9), and #7 with Foxp3-A24- 10-87 (SEQ ID NO 67), and with Foxp3-A24- 10-60 (SEQ ID NO 74) showed potent IFN-gamma production compared with the control.
• FIG.2C In Figure 2C, the CTLs in well numbers #1, #2, #4, #5, #7, #9, #11 and #12 with Foxp3-A2-9-390 (SEQ ID NO 15), #5 and #11 with Foxp3-A2-9-304 (SEQ ID NO 19), #7 with Foxp3-A2-9-68 (SEQ ID NO 7) and #12 with Foxp3-A2-9-252 (SEQ ID NO 17) showed potent IFN-gamma production compared with the control.
• FIG.3A-D Figure 3 shows that the cells in the positive wells were expanded and performed IFN-gamma ELISA assay.
• Figure 4 shows specific CTL activity against the target cells endogenously expressing Foxp3 and HLA-A* 02or 24.
• CTL lines raised by Foxp3-A02-9-390 (SEQ ID NO: 15) and Foxp3-A02-9-252 (SEQ ID NO: 17) showed high specific CTL activity against 293T that transfected both Foxp3 and HLA- A02. On the other hand, it did not show significant specific CTL activity against controls.
• FIG 4C CTL lines raised by Foxp3-A02-9-252 (SEQ ID NO: 17) showed high specific CTL activity against 293T that transfected both Foxp3 and HLA-A24. On the other hand, it did not show significant specific CTL activity against controls.
• IFN-gamma ELISA assay was performed using CTL line induced with Foxp3-9-252-WT peptide as responder cells (IxIO 5 cells/well) and Foxp3-9-252-WT (-solid circle-), Foxp3-9-252-9V (-open circle-) or HIV- A02 (-solid triangle-) peptide pulsed T2 cells as stimulator cells (IxIO 4 cells/well). Peptide pulse of stimulator cells was conducted at 37 degree Celsius for 2 hours in each kind of peptide and peptide concentration.
• CTL line induced with Foxp3-9-252-9V peptide was used as responder cells.
• T2 cells incubated with Foxp3-9-252-9V (-solid circle-) or Foxp3-9-252-WT (-open circle-) peptide and without any peptide (-open square-) were used as stimulator cells in this assay (IxIO 4 cells/well).
• polypeptide polypeptide
• peptide protein
• protein protein
• amino acid polymers in which one or more amino acid residue is a modified residue, or a non-naturally occurring residue, such as an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers.
• amino acid refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that similarly functions to the naturally occurring 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 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 are referred to herein by their commonly known three letter symbols or the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission.
• Foxp3-A2-9-304 (SEQ ID NO 19), Foxp3-A24- 10-87 (SEQ ID NO 67) and Foxp3-A24- 10-60 (SEQ ID NO 74) .
• Foxp3-A24- 10-60 (SEQ ID NO 74) are epitope peptides restricted by HLA- A24 and HLA- A2. Since Foxp3 is expressed in most cancer patients and is associated with immunosuppression induced by immunosuppressive factors due to tumors, Foxp3 is a good target for immunotherapy to promote the clinical efficacy of antigen specific immunotherapy against cancer.
• the Foxp3 peptides of the present invention can be flanked with additional amino acid residues so long as the Foxp3 peptide retains its CTL inducibility.
• Such peptides with CTL inducibility can be less than about 40 amino acids, for example, less than about 20 amino acids, for example, less than about 15 amino acids.
• the amino acid sequence flanking the peptides consisting of the amino acid sequence selected from the group of SEQ ID NOs: 3-5, 7-9, 12, 15-19, 22, 24, 27-30, 37, 67 and 74 is not limited and can be composed of any kind of amino acids so long as it does not inhibit the CTL inducibility of the peptide.
• the present invention also provides peptides having CTL inducibility, which comprises the amino acid sequence selected from the group of SEQ ID NOs: 3-5, 7-9, 12, 15-19, 22, 24, 27-30, 37, 67 and 74.
• modified peptides i.e., peptides composed of an amino acid sequence modified by substituting 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 81: 5662-6, 1984; Zoller and Smith, Nucleic Acids Res 10: 6487-500, 1982; Dalbadie- McFarland et al., Proc Natl Acad Sci USA 79: 6409-13, 1982.
• the peptide having CTL inducibility of the present invention can be composed of the amino acids comprising the amino acid sequence of SEQ ID NOs: 3-5, 7-9, 12, 15-19, 22, 24, 27-30, 37, 67 or 74, wherein one or more amino acids are added and/or substituted.
• 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).
• the following eight groups each contain amino acids that are conservative substitutions for one another:
• 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 can include non-conservative modifications, so long as the peptide retains the CTL inducibility.
• the modified peptides do not exclude CTL inducible peptides of polymorphic variants, interspecies homologues, and alleles of Foxp3.
• the term “several” means 5 or less, or for example, 3 or less.
• the percentage of amino residues modified can be 20% or less, for example, 15% or 10% or less, for example, 1 to 5% of the entirety of the amino acids sequence of SEQ ID NOs: 3-5, 7-9, 12, 15-19, 22, 24, 27-30, 37, 67 and 74.
• Foxp3-A2-9-69 (SEQ ID NO 16), Foxp3-A2-9-252 (SEQ ID NO 17), Foxp3-A2- 10-359 (SEQ ID NO 22), Foxp3-A2- 10-263 (SEQ ID NO 24), Foxp3-A2- 10-94 (SEQ ID NO 27), Foxp3-A2- 10-233 (SEQ ID NO 28), Foxp3-A2-10-152 (SEQ ID NO 29), Foxp3-A2- 10-77 (SEQ ID NO 30), Foxp3-A2- 10-246 (SEQ ID NO 37), Foxp3-A2-9-68 (SEQ ID NO 18), Foxp3-A2-9-304 (SEQ ID NO 19), Foxp3-A24- 10-87 (SEQ ID NO 67) and
• Foxp3-A24- 10-60 (SEQ ID NO 74) showed that they do not have significant homology with peptides derived from any other known human gene products. This lowers the possibility of unknown or undesired immune responses when used for immunotherapy.
• the present peptides When used in immunotherapy, the present peptides will be presented on the surface of a cell or exosome as a complex with an HLA antigen. Therefore, peptides are selected with high binding affinity to the HLA antigen in addition to their CTL indu- cibility.
• the peptides can be modified by substitution, addition and such of the amino acid residues to achieve a higher binding affinity.
• peptides showing high HLA- A24 binding affinity can have their second amino acid from the N-terminus substituted with phenylalanine, tyrosine, methionine, or tryptophan, and peptides whose amino acid at the C-terminus is substituted with phenylalanine, leucine, isoleucine, tryptophan, or methionine also find use.
• peptides which second amino acid from the N-terminus is substituted with leucine or methionine, and in which the C-terminal amino acid is substituted with valine or leucine can be used as peptides with high HLA- A02 binding affinity.
• the peptide sequence is identical to a portion of the amino acid sequence of an endogenous or exogenous protein having a different function, side effects such as autoimmune disorders or allergic symptoms against specific substances may be induced. Therefore, homology searches can be performed using available databases to avoid, reduce or minimize situations in which the sequence of the peptide matches the amino acid sequence of another protein. When it becomes clear from the homology searches that there exists no other peptide with 1 or 2 amino acids difference to the objective peptide, the objective peptide can be modified in order to increase the binding affinity with HLA antigens, and/or increase the CTL inducibility without any danger of the side effects.
• CTL inducibility indicates the ability of the peptide to induce CTLs when presented on antigen-presenting cells.
• CTL inducibility includes the ability of the peptide to induce CTL activation, CTL proliferation, and to increase IFN-gamma production.
• Confirmation of CTL inducibility can be accomplished by inducing antigen- presenting cells carrying human MHC antigens (for example, B-lymphocytes, macrophages, and dendritic cells), or more specifically dendritic cells 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
• IFN-gamma produced and released by CTL can be examined by measuring IFN-gamma produced and released by CTL in the presence of antigen-presenting cells that carry immobilized peptides, and visualizing the inhibition zone on the media using anti-IFN-gamma monoclonal antibodies.
• the peptides of the present invention can be further linked to other substances, so long as they retain the CTL inducibility.
• Usable substances include: peptides, lipids, sugar and sugar chains, acetyl groups, natural and synthetic polymers, etc.
• the peptides can 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 can 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 particularly useful various D-amino acids, amino acid mimetics or unnatural amino acids; this concept can also be adopted for the present polypeptides.
• the stability of a polypeptide can be assayed in a number of ways. For instance, peptidases and various biological media, such as human plasma and serum, have been used to test stability (see, e.g., Verhoef et al., Eur J Drug Metab Pharmacokin 11: 291-302, 1986). [0053] III. Preparation of the peptides
• the present invention provides polynucleotides which encode any of the aforementioned peptides of the present invention. These include polynucleotides derived from the natural occurring Foxp3 gene and those having a conservatively modified nucleotide sequence thereof.
• the phrase "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. For instance, the codons GCA, GCC, GCG, and GCU all encode the amino acid alanine.
• nucleic acid variations are "silent variations," which are one species of conservatively modified variations.
• Every nucleic acid sequence herein which encodes a peptide also describes every possible silent variation of the nucleic acid.
• each codon in a nucleic acid except AUG, which is ordinarily the only codon for methionine, and TGG, which is ordinarily the only codon for tryptophan
• TGG which is ordinarily the only codon for tryptophan
• the polynucleotide of the present invention can be composed of DNA, RNA, and derivatives thereof.
• a DNA is suitably composed of bases such as A, T, C, and G. T is replaced by U in an RNA.
• the Foxp3 polynucleotides of the present invention can encode multiple Foxp3 peptides of the present invention with or without intervening amino acid sequences in between.
• the intervening amino acid sequence can provide a cleavage site (e.g., enzyme recognition sequence) of the polynucleotide or the translated peptides.
• the polynucleotides can include any additional sequences to the coding sequence encoding the peptide of the present invention.
• the polynucleotides can be recombinant polynucleotides that include regulatory sequences required for the expression of the peptide. In general, such recombinant polynucleotides can be prepared by the manipulation of polynucleotides through conventional recombinant techniques using, for example, polymerases and endonucleases.
• both recombinant and chemical synthesis techniques can be used to produce the polynucleotides of the present invention.
• the polynucleotides can be produced by insertion into an appropriate vector, which can be expressed when transfected into a competent cell.
• the polynucleotides can 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).
• the polynucleotides can be synthesized using the solid phase techniques, as described in Beaucage S. L. & Iyer R.P., Tetrahedron 48: 2223-311, 1992; Matthes et al., EMBO J 3: 801-5, 1984.
• the Foxp3 peptides or polynucleotides encoding the Foxp3 peptides of this invention can be used for regulating T-reg cells.
• the present invention provides a pharmaceutical agent for regulating T-reg cells, which comprise one or more of peptides of this invention, or polynucleotides encoding the peptides as an active ingredient.
• regulating T-reg cells indicates to modify the state of the T-reg cells in vivo, for example, by inhibiting proliferation of or suppressing the function of the T- reg cells.
• T-reg cell is thought to be one of the major players to suppress various types of immune response and "suppressing the function of the T-reg cells” herein means to decrease the ability of the T-reg cells to suppress an immune response.
• T- reg cells act in the periphery as called peripheral tolerance (Miescher S et al., J Immunol 136: 1899-907, 1986; Young RC et al., Am J Med 52: 63-72, 1972; Alexander JP et al., Cancer Res 53: 1380-7, 1997; Horiguchi S et al., Cancer Res 59: 2950-6, 1999; Kono K et al., Clin Cancer Res 11: 1825-8, 1996; Kiessling R et al., Cancer Immunol Immunother 48: 353-62, 1999; Fontenot JD et al., Nat Immunol 4: 330-6, 2003, Hori S et al., Science 299: 1057-61, 2003; Khattri R et al., Nat Immunol 4: 304-6, 2003).
• T-reg cells provide immunosuppressive effect, for example, in a cancer patient. Therefore, the Foxp3 peptides of the present invention, which are over- expressed in the T-reg cells, or polynucleotides encoding the Foxp3 peptides can be used as a pharmaceutical agent (e.g., vaccine) for treating cancer.
• a pharmaceutical agent e.g., vaccine
• the phrase "vaccine” (also referred to as an immunogenic composition) refers to a substance that has the function to induce anti-tumor immunity or immunity to regulate T-regs upon inoculation into animals.
• polypeptides comprising the amino acid sequence of SEQ ID NOs: 3-5, 7-9, 12, 15-19, 22, 24, 27-30, 37, 67 or 74 are HLA- A24 or HLA- A02 restricted epitope peptides that can induce potent and specific immune response against T-reg cells expressing Foxp3. Therefore, the present pharmaceutical agents are intended for the administration to subjects whose HLA antigen is either HLA- A24 or HLA- A02.
• Cancers to be treated by the pharmaceutical agents of the present invention are not limited and include all kinds of cancers wherein Foxp3 is expressed in the subject.
• Exemplified cancers include breast cancer, AML, bladder cancer, cervical, cholangio- cellular carcinoma, CML, colon and rectum, endometriosis, esophagus, gastric, gastric diffuse-type, liver, lung, lymphoma, neuroblastoma, osteosarcoma, ovarian, pancreatic cancer, prostate, renal carcinoma, small cell lung cancer, soft tissue tumor and testicular tumor.
• the pharmaceutical agents of the present invention can optionally include other therapeutic substances as an active ingredient, so long as the substance does not inhibit the T-reg cell regulating effect of the peptide of interest.
• formulations can include anti-inflammatory agents, pain killers, chemotherapeutics, and the like.
• the medicaments of the present invention can also be administered sequentially or concurrently with the one or more other pharmacologic agents. The amounts of medicament and pharmacologic agent depend, for example, on what type of pharmacologic agent(s) is/are used, the disease being treated, and the scheduling and routes of administration.
• antigen presenting cells that have immobilized the Foxp3 peptides of this invention on their cell surface are obtained by removing dendritic cells from the subjects, which are stimulated by the peptides of this invention, CTL is induced in the subjects by readministering the Foxp3 peptide-loaded dendritic cells to the subjects, and as a result, aggressiveness towards the target cells can be increased.
• the pharmaceutical agents of the invention can also comprise nucleic acids encoding the Foxp3 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 has induces anti-tumor immunity.
• the nucleic acid sequence of the polynucleotide of interest includes regulatory elements necessary for expression of the polynucleotide in a target cell.
• the polynucleotide(s) can be equipped to stably insert into the genome of the target cell (see, e.g., Thomas KR & Capecchi MR, Cell 51: 503-12, 1987 for a description of homologous recombination cassette vectors). See, e.g., Wolff et al., Science 247: 1465-8, 1990; 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 (bupivicaine, 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 present invention provides intracellular vesicles called exosomes, which present complexes formed between the peptides of this invention and HLA antigens on their surface.
• Exosomes can be prepared, for example by using the methods described in detail in Published Japanese Translation of International Publication Nos. Hei 11-510507 and 2000-512161, and can be prepared using antigen presenting cells obtained from subjects who are targets of treatment and/or prevention.
• the exosomes of this invention can be inoculated as vaccines, similarly to the peptides of this invention.
• the present invention also provides antigen-presenting cells (APCs) that present complexes formed between HLA antigens and the peptides of this invention on its surface.
• APCs antigen-presenting cells
• the APCs that are obtained by contacting the peptides of this invention, or the nucleotides encoding the peptides of this invention can be prepared from subjects who are the targets of treatment and/or prevention, and can be administered as vaccines by themselves or in combination with other drugs including the peptides of this invention, exosomes, or cytotoxic T cells.
• the CD8 + T cells having cytotoxic activity obtained by step d can be administered to the subject as a vaccine.
• the APCs to be mixed with the CD8 + T cells in above step c can also be prepared by transferring genes coding for the present peptides into the APCs as detailed above under the item of "V-(4) Antigen-presenting cells"; but are not limited thereto and any APC or exosome which effectively presents the present peptides to the T cells can be used for the present method.
• the protein when a certain protein induces any one of these immune responses upon inoculation into an animal, the protein is decided to have anti-immunosupression inducing effect.
• the induction of the anti-immunosupression by a protein can be detected by observing in vivo or in vitro response of the host immune system against the protein.
• a method for detecting the induction (i.e., activation) of cytotoxic T lymphocytes is well known. Specifically, it is known that a foreign substance that enters the living body is presented to T cells and B cells by the action of antigen presenting cells (APCs).
• APCs antigen presenting cells
• a method for evaluating the action to induce CTLs using dendritic cells (DCs) as APC is well known in the art.
• DCs dendritic cells
• a test peptide is initially contacted with DC and then this DC is contacted with T cells.
• Detection of T cells having cytotoxic effects against cells expressing (i.e., presenting on an HLA molecule) the peptide of interest after the contact with DC shows that the test peptide has an activity of inducing the cytotoxic T cells.
• Activity of CTL against T-regs can be detected, for example, using the lysis of 51 Cr-labeled tumor cells as the indicator.
• the method of evaluating the degree of T-regs damage using 3 H-thymidine uptake activity or LDH (lactose dehydrogenase)-release as the indicator is also well known and can be used in the present invention.
• peripheral blood mononuclear cells can also be used as the APC.
• the induction of CTL is reported to be enhanced by culturing PBMC in the presence of GM-CSF and IL-4.
• CTL has been shown to be induced by culturing PBMC in the presence of keyhole limpet hemocyanin (KLH) and IL-7.
• KLH keyhole limpet hemocyanin
• Anti-immunosuppression is induced by administering the vaccine of this invention, and the induction enables dissolution of immunosuppression.
• Such effects can be statistically significant. For example, in observation, at a significance level of 5% or less, wherein the regulating effect of a vaccine against T-regs is compared to a control without vaccine administration.
• Student's t-test, the Mann- Whitney litest or ANOVA can be used for statistical analyses.
• T-regs can be regulated (i.e., suppressed), for example, by the ex vivo method. More specifically, PBMCs of the subject receiving treatment or prevention are collected, the cells are contacted with the polypeptide ex vivo, and following the induction of APC or CTL, the cells can be administered to the subject.
• APC can be also induced by introducing a vector encoding the polypeptide into PBMCs ex vivo.
• APC or CTL induced in vitro can be cloned prior to administration. By cloning and growing cells having high activity of damaging target cells, cellular immunotherapy can be performed more effectively.
• APC and CTL isolated in this manner can be used for cellular immunotherapy not only against individuals from whom the cells are derived, but also against similar types of diseases in other individuals.
• A24LCL cells HLA-A24/24
• T2 cells HLA- A02/02
• human B-lymphoblastoid cells 293T and COS7 were purchased from ATCC.
• Monocyte-derived dendritic cells were used as antigen-presenting cells (APCs) to induce CTL responses against peptides presented on HLA.
• DCs were generated in vitro as described elsewhere (Horiguchi S. et al. Cancer Res. 59:2950-6). Specifically, peripheral blood mononuclear cells (PBMCs) isolated from normal volunteer (HLA- A*2402 and/or HLA-A*0201) with Ficoll-Plaque (Pharmacia) solution were separated by adherence to plastic tissue culture dish (Becton Dickinson) so as to enrich them for the monocyte fraction.
• the monocyte-enriched population was cultured in the presence of 1000 U/ml of GM-CSF (R&D System) and 1000 U/ml of IL-4 (R&D System) in AIM-V medium (Invitrogen) containing 2% heat-inactivated autologous serum (AS). After 7 days in the culture, the cytokine-generated DCs were pulsed with 20 mcg/ml of the synthesized peptides in the presence of 3 mcg/ml of beta2-microglobulin for 4 hrs at 20 degrees C in AIM-V medium.
• peptide - pulsed DCs were then inactivated with mitomycin C (MMC) (30mcg/ml for 30 mins) and mixed at a 1:20 ratio with autologous CD8 + T cells, obtained by positive selection with CD8 Positive Isolation Kit (Dynal).
• MMC mitomycin C
• CD8 Positive Isolation Kit CD8 Positive Isolation Kit
• These cultures were set up in 48-well plates (Corning); each well contained 1.5xlO 4 peptide-pulsed DCs, 3xlO 5 CD8 + T cells and 10 ng/ml of IL-7 (R&D System) in 0.5 ml of AIM-V/2% AS. Three days later, these cultures were supplemented with IL-2 (CHIRON) to a final concentration of 20 IU/ml.
• IL-2 CHIRON
• the T cells were further restimulated with the peptide-pulsed autologous DCs.
• the DCs were prepared each time through the same procedure described above. CTL activity was tested against peptide-pulsed A24LCL cells or T2 cells after the 3rd round of peptide stimulation on day 21.
• CTLs were expanded in culture using a similar method as reported by Riddell, et al. (Walter et al., N Engl J Med 333(16): 1038-44, 1995; Riddell et al., Nat Med 2(2): 216-23, 1996 Feb).
• a total of 5 x 10 4 CTLs were resuspended in 25 ml of AIM-V/5% AS with 2 kinds of human B-lymphoblastoid cell lines, inactivated with MMC, in the presence of 40 ng/ml of anti-CD3 monoclonal antibody (Pharmingen).
• 120 IU/ml of IL-2 were added to the cultures.
• the cultures were fed with fresh AIM-V/5% AS containing 30 IU/ml of IL-2 on days 5, 8 and 11.
• IFN-gamma ELISPOT assay and IFN-gamma ELISA were performed. Briefly, peptide-pulsed A24-LCL, T2 cell (1 x 10 4 /well) or the cells endogenously expressing Foxp3 and HLA molecule was prepared as a stimulator cells. Cultured CTL lines in 48 wells were used as a responder cells. IFN-gamma ELISPOT assay and IFN-gamma ELISA were performed under manufacture procedure.
• 4Tl cells (lxl ⁇ 5 per mouse) were injected s.c. into the right flank of BALB/c mice on day 0. Vaccination was done on days 3 and 10 using hFoxp3-252 (KLSAMQAHL: SEQ ID NO: 17) or mFoxp3-252 (KLGAMQAHL: SEQ ID NO: 88) IFA-conjugated peptides.
• IFN-gamma ELISA assay was performed to examine the affinity of substituted peptide to HLA-A2 molecule.
• CTLs induced with Foxp3-9-252-WT (KLSAMQAHL: SEQ ID NO: 17) peptide were used as responder cells and T2 cells were prepared as stimulator cells by incubation with Foxp3-9-252-WT, Foxp3-9-252-9V (KLSAMQAHV: SEQ ID NO: 95) and HIV- A02 (SLYNTYATL) peptide at 37 degree Celsius for 2 hours.
• Peptide pulse to T2 cells were performed with wide range concentration (10-10 4 mcg/ml) of each peptide.
• Start position indicates the number of amino acid from the N-terminus of Foxp3. Binding score is derived from "BIMAS” described in Materials and Methods. [Table 2]
• the established CTL clone raised against these peptides were examined for their ability to recognize the target cells endogenously expressing Foxp3 and HLA-A*24 or 02.
• Foxp3-9-252 peptide (SEQ ID NO 17) is identified as epitope peptide restricted with both HLA-A*2402 and HLA-A*0201.
• a single or a couple of amino acid(s) substitutions were selected to achieve higher binding affinity to HLA-A*2402 or HLA- A*0201 molecule than natural Foxp3-9-252 peptide; Foxp3-9-252-WT (KLSAMQAHL)(SEQ ID NO 17).
• Binding score of amino acid substitution in Foxp3-9-252 (SEQ ID NO 17) are derived from the BIMAS software.
• Table4 shows amino acid sequences and binding score to HLA-A*2402 and 0201 molecule of substituted peptide from Foxp3-9-252. Binding score of peptide are derived from the BIMAS software. Six or nine kinds of substitutions, a total of fifteen peptides, which predicted to have higher binding affinity to HLA- A24 or HLA- A2 molecule than wild type were synthesized (Table 4). [0131] [Table 4]
• FOXp3-A24-10-114 (SEQ ID NO 12), FOXp3-A2-9-390 (SEQ ID NO 15), FOXp3-A2-9-69 (SEQ ID NO 16), FOXp3-A2-9-252 (SEQ ID NO 17), FOXp3-A2- 10-359 (SEQ ID NO 22), FOXp3-A2- 10-263 (SEQ ID NO 24), FOXp3-A2- 10-94 (SEQ ID NO 27), FOXp3-A2- 10-233 (SEQ ID NO 28), FOXp3-A2-10-152 (SEQ ID NO 29), FOXp3-A2- 10-77 (SEQ ID NO 30), FOXp3-A2- 10-246 (SEQ ID NO 37), FOXp3-A2-9-68 (SEQ ID NO 18), FOXp3-A2-9-304 (SEQ ID NO 19) Foxp3-A24- 10-87 (SEQ ID NO 67) and

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