WO2022244341A1 - Peptide et composition médicinale - Google Patents

Peptide et composition médicinale Download PDF

Info

Publication number
WO2022244341A1
WO2022244341A1 PCT/JP2022/004920 JP2022004920W WO2022244341A1 WO 2022244341 A1 WO2022244341 A1 WO 2022244341A1 JP 2022004920 W JP2022004920 W JP 2022004920W WO 2022244341 A1 WO2022244341 A1 WO 2022244341A1
Authority
WO
WIPO (PCT)
Prior art keywords
peptide
amino acid
cell
cells
stap
Prior art date
Application number
PCT/JP2022/004920
Other languages
English (en)
Japanese (ja)
Inventor
正 松田
Original Assignee
国立大学法人北海道大学
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 国立大学法人北海道大学 filed Critical 国立大学法人北海道大学
Publication of WO2022244341A1 publication Critical patent/WO2022244341A1/fr

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/08—Peptides having 5 to 11 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • 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
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
    • 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

Definitions

  • the present invention relates to peptides and pharmaceutical compositions.
  • This application claims priority based on Japanese Patent Application No. 2021-83215 filed in Japan on May 17, 2021, the content of which is incorporated herein.
  • T cells which play a central role in the immune system
  • TCR T cell receptor
  • the kinase molecule LCK present in the TCR protein complex interacts with the CD3 ⁇ molecule, resulting in the immunoreceptor tyrosine-based activating motif ITAM (immunoreceptor tyrosine-based activating motif) in CD3 ⁇ .
  • ITAM immunoreceptor tyrosine-based activating motif
  • Non-Patent Document 1 When ITAM is phosphorylated, ZAP-70 is recruited and promotes tyrosine phosphorylation of a series of TCR signal downstream molecules, and through activation of transcription factors such as NF- ⁇ B and NFAT, T cell proliferation-inducing cytokine IL- 2 production and the subsequent proliferation and activation of T cells. Therefore, it has been reported that T cell activation through TCR signaling plays an important role in the immune response, and that its decreased function induces immunodeficiency, while its overabundance promotes the development of autoimmune diseases and allergies. (Non-Patent Document 1).
  • Steroids and cytotoxic drugs such as cyclophosphamide, azathioprine, and methotrexate are used as immunosuppressive agents for the treatment of autoimmune diseases and allergies, and cyclosporin A and tacrolimus, which target the TCR downstream molecule calcineurin, as specific immunosuppressive agents.
  • cytotoxic drugs such as cyclophosphamide, azathioprine, and methotrexate
  • cyclosporin A and tacrolimus which target the TCR downstream molecule calcineurin
  • STAP-2 (Signal transducing adaptor protein-2) is a PH domain with phospholipid-binding properties from the N-terminal side, an SH2-like domain that is a phosphorylated tyrosine-binding domain, and a proline-rich protein that shows binding properties to SH3 domain-containing proteins. It is an adapter protein with domains.
  • STAP-2 has the function of regulating IgE/Fc ⁇ RI signaling in mast cells responsible for allergic responses, enhancing LPS/TLR4 signaling in macrophages, and enhancing NF- by LMP1 in EB virus-infected B cells. It has been reported that it suppresses the activity of ⁇ B (Non-Patent Documents 2-4).
  • STAP-2 regulates T cell adhesion, cell migration induced by chemokines, activated cell death (AICD), and B cell hematopoietic stress (Non-Patent Documents 5-8).
  • STAP-2 interacts with the breast cancer-specific tyrosine kinase Brk (breast tumor kinase) and contributes to the activation of STAT3, and gene knockdown of STAP-2 suppresses the growth of breast cancer cells and prostate cancer. have been reported (Non-Patent Documents 9 to 11).
  • STAP-2 enhances BCR-ABL-dependent cell proliferation by enhancing the activity of BCR-ABL tyrosine kinase, which is the causative gene of chronic myelogenous leukemia (CML). It has also been reported that down suppresses tumorigenesis of human CML cancer cells (Non-Patent Documents 12 and 13).
  • An object of the present invention is to provide a peptide having immunosuppressive activity and a pharmaceutical composition containing the peptide as an active ingredient.
  • the present inventors have completed the present invention by finding that a peptide containing a partial amino acid sequence of STAP-2 binds to ITAM, suppresses the activity of STAP-2, and has immunosuppressive activity.
  • the present invention provides the following peptides and pharmaceutical compositions.
  • [1] comprising an amino acid sequence represented by Trp-Xaa 1 -Xaa 2 -Ile-Leu (SEQ ID NO: 1; where Xaa 1 and Xaa 2 represent arbitrary amino acid residues), and immunosuppressive activity Peptides with [2]
  • Xaa 2 is a Val residue or an Ala residue.
  • [4] The peptide according to any one of [1] to [3], which consists of an amino acid sequence with 20 or fewer residues.
  • [5] A peptide consisting of an amino acid sequence represented by any one of SEQ ID NOS: 2-4.
  • the pharmaceutical composition of [8], wherein the immune-related disease is an autoimmune disease or a disease caused by abnormal activation of the immune system.
  • the pharmaceutical composition of [7] for treating T lymphoma The pharmaceutical composition of [7] for treating T lymphoma.
  • the present invention can provide a peptide having immunosuppressive activity and a pharmaceutical composition containing the peptide as an active ingredient.
  • Example 2 the results of analyzing the binding of ITAM to GST-tagged STAP-2 (GST-STAP-2) protein C-terminal region-deficient protein (GST-STAP-2C) by pull-down and Western blotting were shown.
  • GST-tagged STAP-2 GST-tagged STAP-2
  • GST-STAP-2C protein C-terminal region-deficient protein
  • FIG. 10 shows the results of analysis of the binding of a peptide (GST-STAP-2 C 332-348) having the 332nd to 348th amino acid sequence of STAP-2 to ITAM by pull-down method and Western blotting method in Example 2. is.
  • FIG. 10 shows the results of treating Jurkat cells with Peptide #1 or Peptide #2 and measuring cell proliferation in Example 3.
  • FIG. 10 shows the results of treating Jurkat cells with peptide #1 or peptide #2 and measuring IL-2 production upon TCR signal activation in Example 3.
  • FIG. 10 shows the results of treating Jurkat cells with Peptide #A, Peptide #B, Peptide #A-WT, or Peptide #A-1A and measuring cell proliferation in Example 3.
  • Fig. 2 shows the results of treating Jurkat cells with Peptide #A, Peptide #B, Peptide #A-WT or Peptide #A-1A and measuring IL-2 production upon TCR signal activation in Example 3. is.
  • non-pep indicates the results when no peptide was added.
  • Example 3 treatment of Jurkat cells with Peptide #A-WT, Peptide #A-1A, Peptide #A-2A, Peptide #A-3A, Peptide #A-4A or Peptide #A-5A reduced cell proliferation. It is the figure which showed the measured result.
  • Example 4 Jurkat cells were treated with Peptide #A-WT, Peptide #W1, Peptide #W2 or Peptide #W3, TCR signal was activated, and IL-2 production was measured. be.
  • FIG. 10 shows the results of treating Jurkat cells with Peptide #A-WT, Peptide #W1, Peptide #W2 or Peptide #W3 and measuring cell proliferation in Example 4.
  • FIG. 10 shows the results of treating Jurkat cells with Peptide #A-WT, Peptide #W1, Peptide #W2 or Peptide #W3 and measuring cell proliferation in Example 4.
  • Example 4 Jurkat cells were treated with Peptide #A-WT, Peptide #TW, Peptide #FW or Peptide #HW, TCR signal was activated, and IL-2 production was measured. be.
  • FIG. 10 shows the results of treating Jurkat cells with Peptide #A-WT, Peptide #TW, Peptide #FW or Peptide #HW and measuring cell proliferation in Example 4.
  • FIG. 5 human T cell line MOLT4 cells, mouse T cell line EL-4, or mouse NKT cell line 2E10 cells were treated with peptide #A-WT or peptide #WT-3B, and cell proliferation was measured. It is a diagram.
  • FIG. 10 shows the results of treating human prostate cancer cell line DU145 cells with peptide #A-WT or peptide #WT-3B and measuring cell proliferation in Example 5.
  • FIG. 6 experimental autoimmune encephalomyelitis model mice were used, peptide #A-WT or peptide #WT-3B was administered, and the results of observing pathological condition scores are shown.
  • Group 1 indicates the 1% DMSO/PBS control administration group
  • Group 2 indicates the peptide #A-WT administration group
  • Group 3 indicates the peptide #WT-3B administration group.
  • the peptide according to the present invention is a peptide containing an amino acid sequence represented by Trp-Xaa 1 -Xaa 2 -Ile-Leu (SEQ ID NO: 1) and having immunosuppressive activity (hereinafter also referred to as an immunosuppressive peptide).
  • SEQ ID NO: 1 an amino acid sequence represented by Trp-Xaa 1 -Xaa 2 -Ile-Leu
  • immunosuppressive peptide hereinafter also referred to as an immunosuppressive peptide.
  • SEQ ID NO: 1 and Xaa 2 represent arbitrary amino acid residues.
  • Xaa 1 and Xaa 2 may be any amino acid residue as long as the immunosuppressive peptide according to the present invention has immunosuppressive activity.
  • Examples of Xaa 1 include Pro residues and Ala residues. be done.
  • Xaa 2 includes Val residue, Ala residue and the like.
  • Examples of the amino acid sequence represented by SEQ ID NO: 1 include Trp-Pro-Val-Ile-Leu (SEQ ID NO: 2), Trp-Pro-Ala-Ile-Leu (SEQ ID NO: 3), Trp-Ala-Val- Ile-Leu (SEQ ID NO: 4) and the like.
  • STAP-2 The amino acid sequence of STAP-2 is known, and the amino acid sequence of human STAP-2 (SEQ ID NO: 5) has been registered as NP_001013863.1 in the Reference Sequence Database of the National Center for Biotechnology Information (NCBI).
  • STAP-2 has, from the N-terminal side, a PH domain exhibiting phospholipid binding (regions 1 to 147 of SEQ ID NO: 5), an SH2-like domain (152 to 247 of SEQ ID NO: 5) that is a phosphorylated tyrosine binding region 247 to 403 of SEQ ID NO: 5) and a proline-rich region (region from 247 to 403 in SEQ ID NO: 5) that exhibits binding to SH3 domain-containing proteins.
  • amino acid sequence represented by SEQ ID NO: 2 corresponds to the 332nd to 336th partial amino acid sequence of SEQ ID NO: 5.
  • the immunosuppressive peptide according to the present invention preferably consists of an amino acid sequence with 20 residues or less.
  • the number of amino acid residues of the immunosuppressive peptide according to the present invention is 19 or less, 18 or less, 17 or less, 16 or less, 15 or less, 14 or less, 13 or less, 12 or less, 11 or less, or 10 or less.
  • the number of amino acid residues of the immunosuppressive peptide according to the present invention is 5 or more, and may be, for example, 6 or more, 7 or more, 8 or more, or 9 or more.
  • the immunosuppressive peptide according to the present invention can contain any amino acid sequence other than the amino acid sequence represented by SEQ ID NO:1.
  • the immunosuppressive peptide according to the present invention includes a partial amino acid sequence adjacent to the amino acid sequence represented by SEQ ID NO: 5 on the N-terminal side or the C-terminal side of the amino acid sequence represented by SEQ ID NO: 2. It can be additionally included on the N-terminal side or C-terminal side of the amino acid sequence represented by SEQ ID NO:1.
  • the amino acid sequence additionally comprising a partial amino acid sequence adjacent to the N-terminal side or C-terminal side of the amino acid sequence represented by SEQ ID NO: 2 includes, for example, SEQ ID NO: 332nd to 343rd partial amino acid sequences of SEQ ID NO: 5, 332nd to 348th partial amino acid sequences of SEQ ID NO: 5, and the like.
  • T cell activation is induced by the T cell receptor (TCR) expressed on the surface of T cells recognizing the MHC/antigen peptide complex.
  • TCR T cell receptor
  • the kinase molecule LCK present in the TCR protein complex interacts with the CD3 ⁇ molecule, resulting in the immunoreceptor tyrosine-based activating motif ITAM (ITAM) in CD3 ⁇ . ; hereinafter also referred to as ITAM) is induced.
  • ITAM immunoreceptor tyrosine-based activating motif ITAM
  • STAP-2 binds ITAM and promotes binding of LCK to CD3 ⁇ .
  • the immunosuppressive peptides of the present invention inhibit downstream signaling from LCK by competitively inhibiting binding of STAP-2 to ITAM and inhibiting binding of LCK to CD3 ⁇ .
  • LCK phosphorylation is suppressed by the immunosuppressive peptide of the present invention, tyrosine phosphorylation of a series of TCR signal downstream molecules is suppressed, and T cell proliferation is mediated by the activation of transcription factors such as NF- ⁇ B and NFAT.
  • transcription factors such as NF- ⁇ B and NFAT.
  • the production of the inducing cytokine IL-2 and the subsequent proliferation and activation of T cells are suppressed.
  • the immunosuppressive peptides according to the invention have immunosuppressive activity.
  • the immunosuppressive activity is not particularly limited as long as it suppresses the immune activity of T cells, and includes, for example, suppression of IL-2 production in T cells, suppression of T cell proliferation, suppression of T cell activation, and the like. . Since the immunosuppressive peptide according to the present invention has immunosuppressive activity, it is useful for treating immune-related diseases such as autoimmune diseases and diseases such as T-cell lymphoma.
  • autoimmune diseases include Graves' disease, rheumatoid arthritis, Hashimoto's thyroiditis, type 1 diabetes, systemic lupus erythematosus, vasculitis, Addison's disease, polymyositis, dermatomyositis, psoriasis, Sjögren's syndrome, systemic scleroderma, Glomerulonephritis etc. are mentioned.
  • cytokine release syndrome cytokine storm
  • COVID-19 chimeric antigen receptor-transfected T cell
  • immunosuppressive peptides according to the present invention can be used in the form of modified peptides to which other substances are added as long as they do not impair the immunosuppressive activity, and these modified peptides are also included in the present invention.
  • modified peptide is a peptide in which a membrane-permeable peptide is added to the end of the immunosuppressive peptide described above with or without a linker sequence. Therefore, the present invention further provides peptides in which a membrane-permeable peptide is added to the end of the immunosuppressive peptide described above, with or without a linker sequence.
  • Membrane-permeable peptides are peptides that have the ability to increase the cell-membrane permeability of bound substances. It is used as a tool for introducing drugs, nanoparticles, liposomes, etc. into cells.
  • any one selected from known CPPs can be used, examples of which include oligoarginine peptides, arginine-rich basic peptides derived from Tat protein of human immunodeficiency virus type 1 (TAT peptide, SEQ ID NO: 6), arginine-rich basic peptide from Rex protein of human T-cell leukemia virus type II (HTLV-II-Rex, SEQ ID NO: 7), arginine-rich basic peptide from flockhouse virus (FHV coat (35-49), SEQ ID NO: 8) and the like.
  • An oligoarginine peptide is a peptide consisting of multiple consecutive Arg residues. The number of Arg residues in the oligoarginine peptide may be about 4-16, preferably 6-12, more preferably 8-12.
  • CPPs may be added to either the N-terminus or the C-terminus of the immunosuppressive peptides of the present invention. CPPs may also be added directly to the N-terminus or C-terminus of the immunosuppressive peptides of the present invention, or via a linker sequence.
  • linker sequence As the linker sequence (linker peptide), one appropriately selected from linker sequences known to those skilled in the art that can connect two peptides without interfering with their respective functions can be used.
  • the number of amino acid residues in the linker sequence is preferably 1 to 10, such as 1 to 5, 1 to 4, 1 to 3, 1 to 2, 2 to 3, 2 to 4, 2 to 5 or 2 to 10.
  • the amino acid residue constituting the linker sequence is preferably an amino acid residue with a relatively small volume such as Gly, Ala, Ser, Pro, etc., particularly preferably Gly.
  • the linker sequence is a sequence of 1-5, 1-4, 1-3, 1-2, 2-3, 2-4, or 2-5 consecutive glycines. be.
  • a preferred example of the immunosuppressive peptide according to the present invention to which a membrane-permeable peptide is added is an immunosuppressive peptide to which a membrane-permeable peptide is added via a linker sequence consisting of 1 to 5 glycines.
  • modified peptides are immunosuppressive peptides in which one or more amino acid residues have been chemically modified with suitable substances.
  • Chemical modifications can include, for example, acylation, prenylation, acetylation, phosphorylation, glycosylation, PEGylation, and the like.
  • modified peptides are immunosuppressive peptides to which fluorescent or other labeling compounds have been added.
  • labeling compounds include fluorescent substances (e.g., FITC, rhodamine, etc.), metal particles (e.g., gold colloids, etc.), fluorescent microbeads (e.g., Luminex (registered trademark, Luminex), etc.), chromoproteins (e.g., phycoerythrin, phycocyanin, etc.).
  • radioactive isotopes eg, 3 H, 14 C, 32 P, 35 S, 125 I, 131 I, etc.
  • enzymes eg, peroxidase, alkaline phosphatase, etc.
  • biotin streptavidin, and the like.
  • modified peptide is an immunosuppressive peptide fused with a functional protein.
  • functional proteins include tag peptides such as His tag, GST tag, HA tag and FLAG tag, and fluorescent proteins such as GFP.
  • peptides are synthesized using amino acids modified with various protective groups as raw materials, for example, by organic chemical synthesis methods such as the Fmoc method (fluorenylmethyloxycarbonyl method) and the tBoc method (t-butyloxycarbonyl method).
  • organic chemical synthesis methods such as the Fmoc method (fluorenylmethyloxycarbonyl method) and the tBoc method (t-butyloxycarbonyl method).
  • Peptides are produced by genetic engineering methods, which involve introducing an expression vector containing a peptide-encoding nucleic acid into a suitable host cell for expression, or by translating a peptide-encoding nucleic acid in a cell-free protein synthesis system. It can also be produced by genetic engineering production methods. Each operation in genetic engineering production methods, including preparation of nucleic acids encoding peptides, types of host cells and methods of introducing genes, expression and purification of peptides, etc., includes experimental procedures that describe various genetic engineering operations in detail Based on the instructions in the manual, it can be carried out by methods known or known to those skilled in the art.
  • the invention provides nucleic acids encoding immunosuppressive peptides, expression vectors containing such nucleic acids, and transformed host cells.
  • the pharmaceutical composition according to the present invention contains the immunosuppressive peptide according to the present invention as an active ingredient.
  • the immunosuppressive peptides of the present invention competitively inhibit binding of STAP-2 to ITAM and inhibit downstream signaling from LCK by inhibiting binding to LCK and CD3 ⁇ .
  • LCK phosphorylation is suppressed by the immunosuppressive peptide of the present invention
  • tyrosine phosphorylation of a series of TCR signal downstream molecules is suppressed
  • T cell proliferation is mediated by the activation of transcription factors such as NF- ⁇ B and NFAT.
  • the production of the inducing cytokine IL-2 and the subsequent proliferation and activation of T cells are suppressed.
  • a pharmaceutical composition containing the immunosuppressive peptide of the present invention as an active ingredient is useful for treating immune-related diseases such as autoimmune diseases and diseases such as T-cell lymphoma.
  • immune-related diseases such as autoimmune diseases and diseases such as T-cell lymphoma.
  • autoimmune diseases include Graves' disease, rheumatoid arthritis, Hashimoto's thyroiditis, type 1 diabetes, systemic lupus erythematosus, vasculitis, Addison's disease, polymyositis, dermatomyositis, psoriasis, Sjögren's syndrome, systemic scleroderma, Glomerulonephritis etc. are mentioned.
  • the pharmaceutical composition according to the present invention is useful for treating exacerbation of novel coronavirus infection caused by abnormal activation of the immune system.
  • the pharmaceutical composition according to the present invention may contain other active ingredients as long as they do not impair the action of the immunosuppressive peptide of the present invention.
  • Other active ingredients include, for example, known immunosuppressants.
  • known immunosuppressants include steroids and cytotoxic agents such as cyclophosphamide, azathioprine and methotrexate, as well as specific immunosuppressants such as cyclosporine A and tacrolimus, which target the TCR downstream molecule calcineurin, and IL-2 receptors.
  • Examples include rapamycin, which targets mTOR downstream of the body, and tocilizumab, a therapeutic drug for cytokine release syndrome (cytokine storm), which is known as a side effect associated with chimeric antigen receptor gene-transfected T cell (CART) therapy.
  • rapamycin which targets mTOR downstream of the body
  • tocilizumab a therapeutic drug for cytokine release syndrome (cytokine storm)
  • CART chimeric antigen receptor gene-transfected T cell
  • administration route of the pharmaceutical composition according to the present invention is not particularly limited, and is appropriately determined according to target cells and tissues containing them.
  • administration routes of the pharmaceutical composition according to the present invention include oral administration, direct administration to joints, transdermal administration, intravenous administration, intraperitoneal administration, enema administration and the like.
  • the pharmaceutical composition according to the present invention can be prepared by conventional methods, such as oral solid formulations such as powders, granules, capsules, tablets, chewable formulations and sustained release formulations, oral liquid formulations such as solutions and syrups, and injections. It can be formulated into medicines, enemas, sprays, patches, ointments, and the like.
  • excipients, binders, lubricants, disintegrants, fluidizing agents, solvents, solubilizers, buffers, suspending agents, emulsifiers, tonicity agents , stabilizers, preservatives, antioxidants, flavoring agents, coloring agents and the like can be blended in a conventional manner.
  • the animal is not particularly limited, and may be a human or an animal other than a human, but a human is preferred.
  • Non-human animals include mammals such as cows, pigs, horses, sheep, goats, monkeys, dogs, cats, rabbits, mice, rats, hamsters and guinea pigs, and birds such as chickens, quails and ducks.
  • the pharmaceutical composition of the present invention contains an agent for promoting intracellular translocation of the peptides, such as a cationic lipid, non-covalently Substances used in protein transfection reagents, such as membrane-permeable peptides that form complexes with proteins and translocate into cells, Sendai virus-derived envelope (HVJ-E), and magnetic nanoparticles, are added to the pharmaceutical composition of the present invention. It may be incorporated into a product or administered together with a pharmaceutical composition according to the present invention.
  • an agent for promoting intracellular translocation of the peptides such as a cationic lipid, non-covalently
  • Substances used in protein transfection reagents such as membrane-permeable peptides that form complexes with proteins and translocate into cells, Sendai virus-derived envelope (HVJ-E), and magnetic nanoparticles, are added to the pharmaceutical composition of the present invention. It may be incorporated into a product or administered together with a pharmaceutical composition according to
  • Human T cell lines Jurkat, MOLT4
  • mouse T cell line EL-4
  • mouse NKT cell line (2E10) used in subsequent experiments were RPMI medium containing 10% fetal calf serum (FCS), derived from human fetuses.
  • FCS fetal calf serum
  • Renal epithelial cell line HEK293T and human prostate cancer cell line DU145 were each subcultured in DMEM medium containing 10% FCS under conditions of 37° C. and 5% CO 2 .
  • cell proliferation/survival was measured using a commercially available cell counting kit (product name: Cell Counting Kit-8, manufactured by Dojindo Laboratories). Specifically, various cell lines were replated into 96-well plates at a rate of 20,000 cells/well. Then, after culturing for a predetermined time, the number of surviving cells in each well was counted using the commercially available kit.
  • HEK293T cells were transfected with the STAP-2 expression vector gene by the calcium phosphate method.
  • This solution was added dropwise to 50% confluent HEK293T cells cultured in a 10 cm dish, cultured at 37°C, 5% CO2 for 36 hours, and then centrifuged at 6,000 rpm for 1 minute at 4°C.
  • the cell pellet after washing with PBS was gently treated with a cell lysate [50 mM Tris-HCl (pH 7.4), 0.15 M NaCl, 1% NP-40, 1 mM PMSF, 1 mM Na 3 VO 4 ] at 4° C. for 30 minutes.
  • the mixture was stirred at room temperature for solubilization, centrifuged at 4° C. and 15,000 rpm for 60 minutes, and the resulting supernatant was used as a total cell lysate sample.
  • Glutathione Sepharose Glutathione Sepharose (GSH pull-down assay) or Sepharose beads (pull-down assay using ITAM peptide) added with ITAM peptide (QQCQNQLYNELNLGRREEYDVLDKRRGR; SEQ ID NO: 9) were added to the cell lysate and gently stirred at 4°C for 1.5 hours. did. After the reaction, the reaction was treated in the same manner as in the immunoprecipitation method, a sample for Western blot analysis was prepared, and the protein that binds to the ITAM peptide was analyzed by Western blotting.
  • STAP-2 constitutively expressing Jurkat cells were established by introducing STAP-2 expression plasmid DNA into Jurkat cells by electroporation. Specifically, Jurkat cells were harvested and washed three times with RPMI (without FCS and antibiotics), and 1 ⁇ 10 7 cells were suspended in 500 ⁇ L of RPMI (without FCS and antibiotics). Pulses were applied at 300 V and 975 ⁇ F using Pulser II (manufactured by Bio-Rad) to introduce the plasmid DNA into the cells. The resulting cells were cultured in medium containing 1 mg/mL G418 for 2 weeks for drug selection, and the cells were cloned to establish a Jurkat cell line that constitutively expresses STAP-2.
  • TCR signal activation by anti-CD3 antibody and anti-CD28 antibody Activation of TCR signals by anti-CD3 antibody and anti-CD28 antibody was performed as follows. 5 ⁇ g/mL anti-CD3 antibody and 1.5 ⁇ g/mL anti-CD28 antibody for 3 ⁇ 10 6 cells/mL of STAP-2 constitutively expressing Jurkat cells (hereinafter abbreviated as Jurkat cells) obtained above. After adding to the final concentration, it was incubated at 37° C. for a certain period of time to activate the TCR signal.
  • each cell was treated with a cell lysate to prepare a total cell lysate sample.
  • the resulting cell lysate was subjected to SDS-PAGE to separate proteins and then transferred to a PVDF membrane. After reacting this transfer membrane with each primary antibody solution, it was reacted with an HRP-labeled secondary antibody, and a chemiluminescent reagent (product name: "Immobilon (registered trademark) Western Chemiluminescent HRP Substrate", manufactured by Millipore) was used. Chemiluminescence was performed and detected as protein bands.
  • Example 1 Synthesis of Peptides Peptides shown in Table 1 below were designed with reference to the amino acid sequence of STAP-2 (SEQ ID NO: 5). Each peptide consists of, in order from the N-terminal side, an oligoarginine sequence (octaarginine sequence) consisting of 8 Arg residues, a linker sequence consisting of 2 Gly residues, and a partial amino acid sequence of STAP-2 or a modified sequence thereof. have These chemically synthesized peptides (peptide purity of 95% or higher) were purchased from GL Biochem, and used in the experiments after being made into 10% DMSO solutions.
  • Example 2 Identification of ITAM-binding sequence in SATP-2 Various STAP-2 protein C-terminal regions (GST-STAP-2 C) to which a GST tag was added were prepared, and GST-STAP-2 C and ITAM were identified. Binding was analyzed by the pull-down method. The results are shown in FIG. The results in FIG. 1 show that the 244th to 348th amino acid sequences of STAP-2 bind to ITAM, and the 244th to 328th amino acid sequences of STAP-2 do not bind to ITAM. 2 (GST-STAP-2 C 329-348) was found to bind to ITAM. FIG.
  • FIG. 2 shows the results of analyzing the binding of a peptide having the 332nd to 348th amino acid sequence of STAP-2 (GST-STAP-2 C 332-348) to ITAM by a pull-down method and Western blotting using an anti-GST antibody. each shown. From the results in FIG. 2, it was found that the peptide having the 332nd to 348th amino acid sequence of STAP-2 binds to ITAM.
  • Example 3 Investigation of cell growth suppression and IL-2 production suppression activity in Jurat cells of a peptide to which a membrane-permeable peptide was added Jurkat cells were treated with peptide #1 or peptide #2 obtained in Example 1, Activation of the TCR signal was performed and IL-2 production and cell proliferation were measured. IL-2 was measured by ELISA method. Specifically, ELISA MAX (registered trademark) Standard Sets (manufactured by BioLegend) were used according to the attached protocol. The results are shown in FIGS. 3A and 3B. As shown in FIGS.
  • peptide #1 which is a peptide having an octaarginine sequence added to the 332nd to 348th amino acid sequences of STAP-2 at the N-terminus
  • peptide #1 A significant decrease in IL-2 production and cell proliferation was observed compared to untreated Jurkat cells.
  • peptide #2 which is a peptide in which leucine at position 336 and leucine at position 341 of peptide #1 were substituted with alanine. This result indicates that peptide #1 inhibited ITAM-mediated TCR signaling and suppressed IL-2 production and cell proliferation.
  • peptide #B which is a peptide added to the above-mentioned membrane-permeable peptide to the 337th to 348th amino acid sequence of STAP-2.
  • peptide #A-WT which is a peptide added to the membrane-permeable peptide to the amino acid sequence of 5 residues at positions 332 to 336 of STAP-2, also produced significant IL-2 similar to peptide #A. A decrease in cell growth and a decrease in cell proliferation were observed.
  • peptide #A-WT peptide #A-1A, peptide #A-2A, peptide #A-1A, peptide #A-2A, peptide Jurkat cells were treated with #A-3A, peptide #A-4A or peptide #A-5A for activation of TCR signaling and the effect on cell proliferation was measured.
  • the results are shown in FIG.
  • FIG. 5 in peptide #A-3A and peptide #A-4A, suppression of cell proliferation was observed in the same manner as peptide #A-WT, but peptide #A-1A, peptide #A-2A and No cell growth inhibition was observed with peptide #A-5A.
  • Example 4 Investigation of cell growth inhibitory activity and IL-2 production inhibitory activity in Jurat cells of peptides with no added membrane-permeable peptide and peptides with different membrane-permeable peptides Peptides shown in Table 2 below were synthesized. Cell growth inhibition and IL-2 production inhibition activity in Jurkat cells were evaluated by the same method as in 3 above.
  • Peptide #W1 consists of the 332nd to 336th amino acid sequences of STAP-2, and is a peptide obtained by removing the octaarginine sequence and the linker sequence from peptide #A-WT.
  • Peptide #W2 consists of the 344th to 348th amino acid sequence of STAP-2
  • peptide #W3 consists of the 339th to 345th amino acid sequence of STAP-2
  • Peptides #TW1, #FW1 and #HW1 are peptides in which the octaarginine sequence of peptide #A-WT is replaced with other membrane-permeable peptide sequence, respectively, TAT peptide, FHV coat (35-49 ) or HTLV-II-Rex, a linker sequence consisting of two Gly residues, and the 332nd to 336th amino acid sequences of STAP-2.
  • Peptide #W1 is a peptide consisting of the 332nd to 336th amino acid sequences of STAP-2, excluding the membrane permeable sequence from peptide #A-WT. No decrease and no decrease in cell proliferation was observed.
  • peptide #TW1, peptide #FW1, and peptide #HW1, in which different membrane-permeable peptides are added to peptide #W1, are all IL-like peptide #A-WT.
  • peptide #FW1 was observed to have a stronger reduction in IL-2 production and cell proliferation than peptide #A-WT.
  • Example 5 Examination of cell growth suppression and IL-2 production suppression activity against T cells other than Jurkat cells instead of Jurkat cells, human T cell line MOLT4 cells, which are human T cells other than Jurkat cells, By the same method as in Example 3, the effect of peptide #A-WT on cell proliferation of human T cells was examined. In addition, ITAM in the TCR protein complex of T cells contributes to T cell activation across animal species. The effect of WT on cell proliferation of mouse T cells was examined. As a negative control, the 332nd to 336th amino acid sequences of STAP-2 of peptide #A-WT were replaced with the 344th to 348th amino acid sequences of SPAP-2, consisting of the amino acid sequence represented by SEQ ID NO: 25.
  • Peptide #WT-3B was used. The results are shown in FIG. As shown in FIG. 10, cell growth suppression was observed in human T cell line MOLT4 cells upon treatment with peptide #A-WT. In contrast, the negative control peptide #WT-3B did not exhibit cell growth inhibitory activity. In addition, as shown in FIG. 10, peptide #A-WT also exhibited growth-inhibitory activity in mouse-derived cells, demonstrating that it acts across animal species.
  • Example 6 Examination of the effects of immunosuppressive peptides using experimental autoimmune encephalomyelitis model mice From the results of Example 5, it was found that peptide #A-WT acts across animal species. Therefore, it was speculated that immunosuppressive peptides could also act in mouse models. Therefore, an experimental autoimmune encephalomyelitis (EAE) mouse model, which is known to strongly depend on T cell activation, was used to examine the effects of the immunosuppressive peptides of the present invention. EAE is widely used as an autoimmune disease model in elucidating treatment methods for MS, since its pathology is similar to that of human multiple sclerosis (MS).
  • MS multiple sclerosis
  • EAE lymphocytes activated in peripheral tissues infiltrate the central nervous system, including the spinal cord and brain, and induce inflammation through the production of inflammatory cytokines, resulting in demyelination and limb paralysis. be done. Therefore, peptide #A-WT was administered to an EAE mouse model to verify its effect. Specifically, to EAE mice subcutaneously administered MOG antigen to the tail root, pertussis toxin was administered to the tail vein immediately after the administration of the antigen and 2 days after the administration of the antigen. - WT or peptide #WT-3B as a negative control was administered every other day into the tail vein, and the disease score was observed. The results are shown in FIG. As shown in FIG.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Organic Chemistry (AREA)
  • Immunology (AREA)
  • General Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Epidemiology (AREA)
  • Hematology (AREA)
  • Oncology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Transplantation (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Genetics & Genomics (AREA)
  • Molecular Biology (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Peptides Or Proteins (AREA)

Abstract

La présente invention concerne : un peptide ayant une séquence d'acides aminés (SEQ ID NO. 1) représentée par Trp-Xaa1-Xaa2-Ile-Leu (ici, Xaa1 et Xaa2 représentent chacun un résidu d'acide aminé défini arbitrairement) et ayant une activité immunosuppressive ; et une composition médicinale contenant ledit peptide en tant que principe actif.
PCT/JP2022/004920 2021-05-17 2022-02-08 Peptide et composition médicinale WO2022244341A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2021083215A JP2022176672A (ja) 2021-05-17 2021-05-17 ペプチド及び医薬用組成物
JP2021-083215 2021-05-17

Publications (1)

Publication Number Publication Date
WO2022244341A1 true WO2022244341A1 (fr) 2022-11-24

Family

ID=84140204

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2022/004920 WO2022244341A1 (fr) 2021-05-17 2022-02-08 Peptide et composition médicinale

Country Status (2)

Country Link
JP (1) JP2022176672A (fr)
WO (1) WO2022244341A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115651067A (zh) * 2022-11-24 2023-01-31 安徽农业大学 具有肝癌细胞毒性和α-葡萄糖苷酶抑制活性的环色-丙-缬-异亮-亮肽及制备方法

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH06261941A (ja) * 1993-01-18 1994-09-20 Kuraray Co Ltd 抗デオキシリボ核酸抗体の吸着剤
JPH11503309A (ja) * 1995-03-23 1999-03-26 イミュネックス・コーポレーション Ｉｌ−１７受容体
JP2006523226A (ja) * 2003-02-28 2006-10-12 ザ ジョンズ ホプキンス ユニバーシティ Ｔ細胞調節方法
US20070110760A1 (en) * 2005-01-14 2007-05-17 Monroe John G Methods and compositions targeting viral and cellular ITAM motifs, and use of same in identifying compounds with therapeutic activity
WO2009033788A2 (fr) * 2007-09-11 2009-03-19 Mondobiotech Laboratories Ag Utilisation d'un peptide comme agent thérapeutique
JP2020039315A (ja) * 2018-09-12 2020-03-19 国立大学法人北海道大学 移植片対宿主病モデル動物、及び移植片対宿主病の治療剤のスクリーニング方法

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH06261941A (ja) * 1993-01-18 1994-09-20 Kuraray Co Ltd 抗デオキシリボ核酸抗体の吸着剤
JPH11503309A (ja) * 1995-03-23 1999-03-26 イミュネックス・コーポレーション Ｉｌ−１７受容体
JP2006523226A (ja) * 2003-02-28 2006-10-12 ザ ジョンズ ホプキンス ユニバーシティ Ｔ細胞調節方法
US20070110760A1 (en) * 2005-01-14 2007-05-17 Monroe John G Methods and compositions targeting viral and cellular ITAM motifs, and use of same in identifying compounds with therapeutic activity
WO2009033788A2 (fr) * 2007-09-11 2009-03-19 Mondobiotech Laboratories Ag Utilisation d'un peptide comme agent thérapeutique
JP2020039315A (ja) * 2018-09-12 2020-03-19 国立大学法人北海道大学 移植片対宿主病モデル動物、及び移植片対宿主病の治療剤のスクリーニング方法

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115651067A (zh) * 2022-11-24 2023-01-31 安徽农业大学 具有肝癌细胞毒性和α-葡萄糖苷酶抑制活性的环色-丙-缬-异亮-亮肽及制备方法
CN115651067B (zh) * 2022-11-24 2024-04-30 安徽农业大学 具有肝癌细胞毒性和α-葡萄糖苷酶抑制活性的环色-丙-缬-异亮-亮肽及制备方法

Also Published As

Publication number Publication date
JP2022176672A (ja) 2022-11-30

Similar Documents

Publication Publication Date Title
US11834519B2 (en) Compositions and methods for modulating γ-c-cytokine activity
Fukui et al. Unc93B1 biases Toll-like receptor responses to nucleic acid in dendritic cells toward DNA-but against RNA-sensing
RU2435782C2 (ru) Hla-a*3303-рестриктированный пептид wt1 и содержащая его фармацевтическая композиция
AU2021203530B2 (en) Modulating gamma - c -cytokine activity
Moon et al. The Fos-related antigen 1–JUNB/activator protein 1 transcription complex, a downstream target of signal transducer and activator of transcription 3, induces T helper 17 differentiation and promotes experimental autoimmune arthritis
JP5936129B2 (ja) ＲＥＩＣ／Ｄｋｋ−３タンパク質の部分領域ポリペプチド
Arima et al. A putative silencer element in the IL-5 gene recognized by Bcl6
WO2022244341A1 (fr) Peptide et composition médicinale
US8722052B2 (en) Vaccinia virus protein A46 peptide and use thereof
US7923545B2 (en) Caterpiller gene family
Ding et al. Plectin regulates the signaling and trafficking of the HIV-1 co-receptor CXCR4 and plays a role in HIV-1 infection
JP5967448B2 (ja) ２型ｔｎｆ受容体の発現を誘導する合成ペプチド及びその利用
WO2014095977A1 (fr) Nouveau peptide pellino
EP1755649A1 (fr) Elimination selective de cellules cancereuses par induction d'acetyltransferase via des cytokines tnf-alpha et il-6
JP4283531B2 (ja) マスト細胞の細胞死誘発剤
JP4602772B2 (ja) Ｗｔ１遺伝子トランスジェニック動物
KR20200050715A (ko) Tlr 억제 펩타이드를 포함하는 루푸스 예방 또는 치료용 조성물
WO2003070946A1 (fr) Molecules s'associant au domaine terminal c dans une cellule receptrice
Monhasery Transcytosis of Interleukin (IL-) 11 and apical redirection of gp130 is mediated by IL-11-receptor and Identification of MAD2B as a novel regulatory protein of IL-6 classic signaling
Townsend Characterization of 6E3-p52: A novel cell surface molecule involved in T cell activation
Kullak-Ublick et al. Dietrich Keppler". Jörg König', Yunhai Cui", Anne T. Nies'

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 22804263

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 22804263

Country of ref document: EP

Kind code of ref document: A1