Peptides having ability to inhibit t cell activation and pharmaceutical compositions

Abstract

The present invention provides peptides that comprise an amino acid sequence that has 5–20 residues and includes an amino acid sequence that is represented by Xaa1-Xaa2-L-F-F (in which Xaa1 and Xaa2 are each independently any amino acid residue), the peptides having the ability to inhibit T cell activation, peptides obtained by adding a membrane-permeable peptide to a terminus of the abovementioned peptides via a linker sequence or not, nucleic acids that code for the peptides, and pharmaceutical compositions that include the peptides or the nucleic acids.

Description

Peptides and pharmaceutical compositions having T-cell activation inhibitory activity

[0001] The present invention relates to a peptide having the ability to suppress T cell activation, and a pharmaceutical composition containing the peptide.

[0002] The activation of T cells, which play a central role in the immune system, is induced by the recognition of antigens by T cell receptors (TCRs) located on the surface of T cells. TCRs form complexes with various proteins, including the CD3 protein group (γ, δ, ε, ζ). When the TCR complex binds to an antigen-presenting MHC molecule, the phosphorylation enzyme LCK present in the TCR complex is activated by phosphorylation. Subsequently, phosphorylation of downstream TCR signaling molecules (ITK, PLC-γ1, etc.) occurs, activating transcription factors such as NF-κB and NFAT, leading to IL-2 production and subsequent T cell proliferation and activation. T cell activation via TCR signaling plays a crucial role in the immune response, and its abnormal enhancement can lead to the development of autoimmune diseases and immune-related diseases such as allergies (e.g., Non-Patent Document 1). Many details of the regulatory mechanism of TCR signaling remain unclear, and elucidating it is expected to greatly contribute to understanding the pathogenesis of various immune-related diseases and developing new treatments.

[0003] Signal transducing adaptor protein-1 (STAP-1) is an adapter protein having a PH domain with a phospholipid-binding motif and an SH2 domain with a phosphorylated tyrosine-binding site at its N-terminus. STAP-1 is strongly expressed in immune and hematopoietic tissues such as the spleen, thymus, and bone marrow, suggesting its involvement in signal transduction by immune and hematopoietic cells. To date, STAP-1 has been reported to be involved in maintaining iNKT cell counts and suppressing the pathogenesis of autoimmune hepatitis (Non-Patent Literature 2), and to exacerbate the pathogenesis of chronic myeloid leukemia (Non-Patent Literature 3). It has also been reported to weakly bind to LCK, a core kinase of TCR-mediated signal transduction (Non-Patent Literature 4).

[0004] J. Lin et al., J Cell Sci. 114: 243-244, 2001.Ji. Kashiwakura et al., PLoS ONE 15(11): e0241440, 2020.J. Toda et al., Oncogene 39: 5601-5615, 2020.M. Masuhara et al., Biochem. Biophys. Res. Commun. 268: 697-703, 2000.

[0005] This invention provides a novel therapeutic method for immune-related diseases involving T cell activation.

[0006] The inventors of this invention have reported that STAP-1 enhances downstream signaling and promotes T cell activation by forming a complex with LCK, ITK, and PLC-γ1, and that STAP-1 deficiency suppresses antigen-induced airway inflammation in a bronchial asthma model, and also suppresses T cell infiltration into the spinal cord and inflammatory cytokine production in an experimental autoimmune encephalomyelitis model, thereby reducing the severity of the disease (K. Kagohashi et al., J Immunol (2024) 212 (6): 951-961). Furthermore, the inventors focused on STAP-1 inhibition as a novel therapeutic approach for immune-related diseases involving T cell activation, and found that peptides containing specific amino acid sequences have the ability to suppress STAP-1-mediated T cell activation.

[0007] This disclosure provides the following inventions: Item 1. A peptide comprising an amino acid sequence having 5 to 100 residues, for example 5 to 20 residues, including Xaa1-Xaa2-LFF (wherein Xaa1 and Xaa2 are any amino acid residues independently of each other), and having the ability to suppress T cell activation. Item 2. The peptide according to Item 1, comprising a partial sequence of the human STAP-1 amino acid sequence shown in SEQ ID NO: 9, the partial sequence including TTLFF (SEQ ID NO: 3), or an amino acid sequence having 80% or more sequence identity with the partial sequence. Item 3. The peptide according to Item 1 or 2, wherein at least one of Xaa1 or Xaa2 is threonine or alanine. Item 4. The peptide according to any one of Items 1 to 3, wherein at least one of Xaa1 and Xaa2 is threonine and the other is threonine or alanine. Item 5. A peptide according to any one of claims 1 to 4, comprising EYEHYWTELRGTTLFFYTD (SEQ ID NO: 7), a partial sequence of the amino acid sequence shown in SEQ ID NO: 7, including TTLFF (SEQ ID NO: 3), or an amino acid sequence having 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 7 or the partial sequence. Claim 6. A peptide according to any one of claims 1 to 5, comprising an amino acid sequence shown in any one of SEQ ID NOs: 1 to 8. Claim 7. A peptide having T cell activation inhibitory ability, wherein a functional peptide is attached to the terminus of a peptide defined in any one of claims 1 to 6, with or without the linker sequence. Claim 8. The peptide according to claim 7, wherein the functional peptide is a membrane-permeable peptide. Claim 9. A peptide according to any one of claims 1 to 8, comprising one or more chemically modified amino acid residues. Claim 10. A nucleic acid encoding a peptide defined in any one of claims 1 to 8. Claim 11. A pharmaceutical composition for the treatment or prevention of an immune-related disease involving T cell activation, comprising a peptide according to any one of claims 1 to 9, or the nucleic acid according to claim 10. Item 12. The pharmaceutical composition according to Item 11, wherein the immune-related disease involving T cell activation is an autoimmune disease or an allergic disease. Item 13. The pharmaceutical composition according to Item 11 or 12, wherein the immune-related disease involving T cell activation is a central nervous system autoimmune demyelinating disease.Item 14. A pharmaceutical composition according to any one of items 11 to 13, wherein the immune-related disease involving T cell activation is multiple sclerosis or neuromyelitis optica. Item 15. A pharmaceutical composition for the treatment or prevention of an autoimmune or allergic disease, comprising a peptide according to any one of items 1 to 9, or a nucleic acid according to item 10. Item 16. A pharmaceutical composition for the treatment or prevention of a central nervous system autoimmune demyelinating disease, comprising a peptide according to any one of items 1 to 9, or a nucleic acid according to item 10. Item 17. A pharmaceutical composition for the treatment or prevention of multiple sclerosis or neuromyelitis optica, comprising a peptide according to any one of items 1 to 9, or a nucleic acid according to item 10. Item 18. A pharmaceutical composition for the treatment of T-cell lymphoma, comprising a peptide according to any one of items 1 to 9, or a nucleic acid according to item 10.

[0008] According to the present invention, it is possible to provide a novel therapeutic method for immune-related diseases and T-cell lymphomas that involve T-cell activation.

[0009] This figure shows the correspondence between the peptides used in the examples and the PH domain of STAP-1 and its N-terminal amino acid sequence (amino acid sequences 1 to 121 of SEQ ID NO: 9). This graph shows the proliferation of EL-4 cells in the presence of each peptide. This graph shows the proliferation of EL-4 cells (top), Jurkat cells (middle), and human PBMCs (bottom) in the presence of peptide PH-3Ca (also referred to as iSP1 from Figure 3 onwards) or peptide PH-3Ca5A (also referred to as iCont from Figure 3 onwards). This graph shows the proliferation of Ramos cells (left) and HeLa cells (right) in the presence of iSP1 or iCont. This is a histogram of Jurkat cells treated with FITC-labeled iSP1 analyzed by flow cytometry. This graph shows IL-2 production in Jurkat cells stimulated with TCR (left) or PMA / ionomycin (right) in the presence of iSP1 or iCont. This graph shows IL-2 production in EL-4 cells stimulated via TCR in the presence of iSP1 or iCont. It also shows the production of IL-2 in mouse CD4 cells stimulated via TCR in the presence of iSP1 or iCont. + This graph shows T cell proliferation (left) and IL-2 production (right). This figure shows the regulation of TCR signaling by STAP-1 and its suppression by iSP1. The upper left is a conceptual diagram of the mechanism of TCR signaling regulation by STAP-1, the lower left is an immunoblotting using an extract of Jurkat cells stimulated with TCR in the presence of iSP1 or iCont, and the lower center and lower right are graphs showing the quantitative values ​​of each signaling molecule from the immunoblotting bands. This shows the protocol (upper panel) of a peptide administration test in experimentally autoimmune encephalomyelitis (EAE) mice and a graph (lower panel) showing the changes in clinical scores. This shows tissue staining images of the spinal cord of EAE mice administered peptide (H&E staining: upper left and lower left, Luxol Fast Blue staining: upper right and lower right) and graphs showing the area of ​​cell infiltration (left) and demyelination (right) calculated from these images. This graph shows the percentage (top and bottom left) and absolute number (top and bottom right) of Th1 and Th17 cells that infiltrated the central nervous system of EAE mice administered peptides.

[0010] The following descriptions may be based on representative embodiments or specific examples, but the present invention is not limited to such embodiments or specific examples. In this specification, numerical ranges represented using "~" or "-" mean a range that includes the numbers at both ends as the upper and lower limits, respectively, unless otherwise specified. The upper and lower limits of each numerical range exemplified in this specification can be combined in any way. In addition, amino acid residues are generally represented by a single letter.

[0011] [T cell activation inhibitory peptide] This disclosure provides a peptide (hereinafter also referred to as a T cell activation inhibitory peptide) that includes or consists of an amino acid sequence represented by Xaa1-Xaa2-LFF (wherein Xaa1 and Xaa2 are any amino acid residues independently of each other).

[0012] Xaa1 and Xaa2 may be any amino acid residues, independently of each other, as long as their peptides have the ability to suppress T cell activation. Xaa1 and Xaa2 may each be neutral amino acids such as serine, threonine, cysteine, asparagine, glutamine, tyrosine, glycine, alanine, proline, valine, leucine, isoleucine, methionine, phenylalanine, or tryptophan. Preferably, at least one of Xaa1 and Xaa2 is threonine or alanine, and examples of such amino acid sequences include Xaa1-TLFF (SEQ ID NO: 1) and T-Xaa2-LFF (SEQ ID NO: 2). More preferably, at least one of Xaa1 and Xaa2 is threonine and the other is either threonine or alanine.

[0013] The amino acid sequences represented by Xaa1-Xaa2-LFF include, for example, TTLFF (SEQ ID NO: 3), ATLFF (SEQ ID NO: 4), TALFF (SEQ ID NO: 5), and AALFF (SEQ ID NO: 6). TTLFF (SEQ ID NO: 3) is a partial sequence of the human STAP-1 amino acid sequence and corresponds to positions 52-56 of the human STAP-1 amino acid sequence (SEQ ID NO: 9, 295 amino acids in total) registered as Q9ULZ2 ​​in the UniProt database. The PH domain of STAP-1 corresponds to positions 25-121 of SEQ ID NO: 9, and the SH2 domain corresponds to positions 177-280.

[0014] The number of amino acid residues in the T cell activation inhibitory peptide is, for example, 5-100, 5-90, 5-80, 5-70, 5-60, 5-50, 5-40, 5-35, or 5-30, preferably 5-25, more preferably 5-20. The number of amino acid residues in the T cell activation inhibitory peptide may be 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20.

[0015] The T cell activation inhibitory peptide may contain any amino acid sequence other than the amino acid sequence represented by Xaa1-Xaa2-LFF. For example, the T cell activation inhibitory peptide may consist of a partial sequence of the amino acid sequence shown in SEQ ID NO: 9, which includes TTLFF (SEQ ID NO: 3). Alternatively, the T cell activation inhibitory peptide may consist of an amino acid sequence having 80% or more, preferably 85% or more, more preferably 90% or more, sequence identity with the partial sequence of the amino acid sequence shown in SEQ ID NO: 9, which includes TTLFF (SEQ ID NO: 3).

[0016] Preferred examples of T cell activation inhibitory peptides are a partial sequence of the amino acid sequence shown in SEQ ID NO: 7, including EYEHYWTELRGTTLFFYTD (SEQ ID NO: 7) and TTLFF (SEQ ID NO: 3), or a peptide consisting of an amino acid sequence having 80% or more, preferably 85% or more, more preferably 90% or more, sequence identity with the amino acid sequence shown in SEQ ID NO: 7 or its partial sequence. An example of a partial sequence of the amino acid sequence shown in SEQ ID NO: 7, including TTLFF (SEQ ID NO: 3), is TTLFFYTD (SEQ ID NO: 8).

[0017] Amino acid sequence identity refers to the percentage of identical amino acids relative to all overlapping amino acids in the optimal alignment calculated using an algorithm known in the art (preferably, the algorithm may consider introducing gaps into one or both sequences for optimal alignment). Identity can be calculated, for example, by aligning two amino acid sequences using NCBI BLAST-2 (National Center for Biotechnology Information Basic Local Alignment Search Tool) with default settings.

[0018] This disclosure also provides peptides (also referred to as modified peptides) in which a functional peptide is attached to the terminus of the T cell activation inhibitory peptide described above, either via or without a linker sequence. The functional peptide only needs to not impair the T cell activation inhibitory ability of the T cell activation inhibitory peptide described above, and one example of such is a cell-penetrating peptide. Cell-penetrating peptides are peptides that have the ability to increase the cell membrane permeability of the bound substance, and are used as tools for introducing various substances, such as proteins, peptides, high molecular weight drugs, nanoparticles, liposomes, etc., into cells.

[0019] Examples of membrane-permeable peptides that can be used in this disclosure include oligoarginine peptides, arginine-rich basic peptides derived from human immunodeficiency virus type 1 Tat protein (TAT peptide, SEQ ID NO: 10), arginine-rich basic peptides derived from human T-cell leukemia virus type 2 Rex protein (HTLV-II-Rex, SEQ ID NO: 11), and arginine-rich basic peptides derived from Flockhouse virus (FHV coat (35-49), SEQ ID NO: 12). Oligoarginine peptides are peptides consisting of multiple consecutive arginine residues. The number of arginine residues in an oligoarginine peptide may be about 4 to 16, preferably 6 to 12, and more preferably 8 to 12.

[0020] Other examples of functional peptides include tagged peptides such as His-tagged, GST-tagged, HA-tagged, and FLAG-tagged peptides.

[0021] The functional peptide may be attached to either the N-terminus or the C-terminus of the T cell activation inhibitory peptide described above. Furthermore, the functional peptide may be directly attached to the N-terminus or C-terminus of the T cell activation inhibitory peptide, or it may be attached via a linker sequence.

[0022] The linker sequence (linker peptide) that can be used in this disclosure is one that can link two peptides without interfering with each other's functions. The number of amino acid residues in the linker sequence is preferably 1 to 10, for example 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. Furthermore, the amino acid residues constituting the linker sequence are preferably relatively small amino acid residues such as glycine, alanine, serine, and proline, with glycine being particularly preferred. The linker sequence is, for example, a sequence of 1 to 5, 1 to 4, 1 to 3, 1 to 2, 2 to 3, 2 to 4, or 2 to 5 consecutive glycine residues.

[0023] A preferred example of the modified peptides of this disclosure is a peptide in which a membrane-permeable peptide is added to the N-terminus of a T cell activation inhibitory peptide without a linker sequence.

[0024] The T cell activation inhibitory peptides and modified peptides of this disclosure may contain one or more chemically modified amino acid residues. Examples of chemical modifications include modification of the amino group of an amino acid residue (biotination, myristoylation, palmitoylation, acetylation, maleimidation, methylation, malonylation, etc.), modification of the carboxyl group (amidation, esterification, etc.), modification of the thiol group (farnesylation, geranylation, methylation, palmitoylation, etc.), modification of the hydroxyl group (phosphorylation, sulfation, etc.), PEGylation, glycosylation, etc. These chemical modifications can be carried out by known methods.

[0025] The T cell activation inhibitory peptides and modified peptides of this disclosure include fluorescent substances (e.g., FITC, rhodamine, etc.), metal particles (e.g., gold colloid, etc.), radioisotopes (e.g., 3 H, 14 C, 32 P, 35 S, 125 I, 131 It may also be labeled with a labeled compound such as (I) etc.

[0026] The T cell activation inhibitory peptides and modified peptides disclosed herein possess T cell activation inhibitory activity and can be used as T cell activation inhibitors. T cell activation inhibitory activity is the ability to suppress T cell activation and can also be expressed as the ability to suppress TCR signaling or IL-2 production under TCR stimulation. Although not bound by theory, it is thought that the T cell activation inhibitory peptides and modified peptides competitively inhibit the binding of STAP-1 to LCK, thereby suppressing the phosphorylation of LCK and a series of downstream TCR signaling molecules, leading to the suppression of activation of transcription factors such as NF-κB and NFAT, and subsequently the suppression of IL-2 production and the subsequent suppression of T cell proliferation and activation.

[0027] In this disclosure, peptides can be produced using amino acids modified with various protecting groups as raw materials by organic chemical synthesis methods such as the Fmoc method (fluorenylmethyloxycarbonyl method) or the tBoc method (t-butyloxycarbonyl method).

[0028] Furthermore, peptides can be produced using nucleic acids encoding the peptide by genetic engineering methods. The nucleic acid may be DNA or mRNA, and if it is DNA, it may be in the form of an expression vector. The expression vector contains DNA encoding the peptide, operably ligated to a regulatory sequence such as a promoter. The expression vector includes a replication start site and a promoter, and may optionally include enhancers, transcription termination sequences (terminators), ribosome binding sites, polyadenylation signals, etc.

[0029] For example, peptides can be produced by introducing an expression vector containing DNA encoding the peptide into a suitable host cell, such as E. coli, insect cells, or animal cells, and expressing the peptide. Alternatively, the expression vector may be introduced into the T cells themselves, whose activation is to be suppressed. Genetic engineering methods, including the preparation of DNA and expression vectors, the type of host cell and the method of introducing the expression vector, peptide expression, and purification, are well known to those skilled in the art and can be carried out based on the instructions in an experimental operation manual that describes various methods in detail. This disclosure thus provides nucleic acids encoding the above-mentioned T cell activation suppressing peptide or modified peptide, and host cells transformed with said nucleic acids.

[0030] Peptides can also be produced, for example, by synthesizing proteins in a cell-free system using DNA or mRNA that encodes the peptide. Examples of cell-free protein synthesis systems include systems that utilize cell extracts from E. coli, wheat germ, yeast, rabbit reticulocytes, insect cells, and mammalian cultured cells, as well as reconstituted systems that are constructed by combining factors necessary for protein synthesis.

[0031] [Pharmaceutical Compositions] The Disclosure further provides pharmaceutical compositions containing the above-described T-cell activation inhibitory peptides or modified peptides, or nucleic acids encoding them, as active ingredients. The pharmaceutical compositions can be used for the treatment or prevention of immune-related diseases involving T-cell activation, or for the treatment or prevention of T-cell lymphoma. The pharmaceutical compositions can be used for the treatment or prevention of autoimmune or allergic diseases, such as central nervous system autoimmune demyelinating diseases like multiple sclerosis or neuromyelitis optica. As used herein, the term “treatment” encompasses all medically acceptable types of therapeutic interventions aimed at curing, temporarily relieving, or otherwise treating a disease or condition. The term “prevention” also encompasses all medically acceptable types of preventive interventions aimed at preventing or suppressing the onset or development of a disease. Therefore, the treatment or prevention of a disease in this Disclosure encompasses medically acceptable interventions for a variety of purposes, including improving symptoms associated with the disease, delaying or halting its progression, preventing its onset, or preventing its recurrence.

[0032] The pharmaceutical compositions of this disclosure contain an effective amount of the above-mentioned T-cell activation inhibitory peptide or modified peptide, or nucleic acids encoding them, for the treatment or prevention of immune-related diseases or T-cell lymphomas involving T-cell activation. The pharmaceutical compositions of this disclosure contain an effective amount of the above-mentioned T-cell activation inhibitory peptide or modified peptide, or nucleic acids encoding them, for the treatment or prevention of autoimmune or allergic diseases, such as central nervous system autoimmune demyelinating diseases like multiple sclerosis or neuromyelitis optica. These effective amounts can be appropriately determined depending on the method of use, the age, sex, weight, type and severity of the disease, and other factors.

[0033] Examples of diseases that can be treated or prevented by the pharmaceutical compositions of this disclosure include autoimmune diseases such as rheumatoid arthritis, psoriasis, systemic lupus erythematosus, Behçet's disease, Sjögren's syndrome, polymyositis, dermatomyositis, hyperthyroidism, hypothyroidism, autoimmune adrenal insufficiency, euerythrocytic anemia, central nervous system autoimmune demyelinating diseases (e.g., multiple sclerosis, neuromyelitis optica, etc.), autoimmune hepatitis, Crohn's disease, and ulcerative colitis; and allergic diseases such as bronchial asthma, allergic rhinitis, food allergy, allergic gastroenteritis, contact dermatitis, latex allergy, and drug allergy (drug hypersensitivity). These are included in immune-related diseases that involve T cell activation.

[0034] Examples of T-cell lymphomas that can be treated or prevented by the pharmaceutical compositions disclosed herein include peripheral T-cell lymphoma, cutaneous T-cell lymphoma (e.g., mycosis fungoides, Sézary syndrome, etc.), extranodal NK / T-cell lymphoma, and adult T-cell leukemia / lymphoma.

[0035] The pharmaceutical compositions of this disclosure can be used in combination with other agents for the treatment or prevention of immune-related diseases involving T cell activation, such as immunosuppressants, steroidal anti-inflammatory drugs, TNF inhibitors, IL-6 receptor inhibitors, IL-12 / 23 inhibitors, T cell costimulatory molecule inhibitors, etc. The pharmaceutical compositions of this disclosure can also be used in combination with other agents for the treatment or prevention of T-cell lymphoma, such as anticancer agents.

[0036] In addition to the active ingredient, the pharmaceutical composition of the present disclosure can contain pharmaceutically acceptable additives. Examples of pharmaceutically acceptable additives include excipients, binders, lubricants, solvents, disintegrants, solubilizers, suspending agents, emulsifiers, isotonic agents, stabilizers, preservatives, antioxidants, flavoring agents, coloring agents, buffering agents, flow promoters, and the like. Pharmaceutically acceptable additives are well known to those skilled in the art and can be appropriately selected and used within the scope of the ordinary implementation ability of those skilled in the art.

[0037] The pharmaceutical composition of the present disclosure is preferably a parenteral preparation, and examples thereof include injections, infusions, and the like. The administration route of the pharmaceutical composition is not particularly limited, but in the case of a parenteral preparation, for example, intravenous administration (preferably intravenous administration), subcutaneous administration, intramuscular administration, intraperitoneal administration, local administration to the target site, and the like can be mentioned.

[0038] The pharmaceutical composition of the present disclosure may contain a DDS material such as an agent for promoting the intracellular translocation of a peptide or nucleic acid, for example, a cationic lipid (such as lipofectamine), a membrane-permeable peptide that non-covalently forms a complex with a protein and translocates into cells (membrane-translocating peptides such as the above-mentioned oligoarginine peptide), Sendai virus-derived envelope (HVJ-E), magnetic nanoparticles, viral vectors, lipid membrane structures such as liposomes or micelles, or may be administered together with the pharmaceutical composition. The present disclosure thus provides a pharmaceutical composition in a form in which the above-mentioned T cell activation inhibitory peptide or modified peptide, or a nucleic acid encoding them, is carried or encapsulated in a DDS material.

[0039] The pharmaceutical composition of the present disclosure is administered to a subject who is at risk of developing or has developed the above-mentioned disease. The subject is a human or a non-human animal, and examples of non-human animals include mammals such as rodents including mice, rats, hamsters, guinea pigs, primates including chimpanzees, macaques, livestock including pigs, cows, goats, horses, sheep, and pet animals including dogs, cats. The pharmaceutical composition is preferably administered to humans.

[0040] [Therapeutic or preventive method] The present disclosure further provides a method for treating or preventing an immune-related disease or T-cell lymphoma in which T-cell activation is involved, or a subject at risk of developing or having developed such a disease, comprising administering to the subject a pharmaceutical composition containing the above-described T-cell activation inhibitory peptide or modified peptide, or a nucleic acid encoding the same. The present disclosure further provides a method for treating or preventing an autoimmune disease or allergic disease, such as a central nervous system autoimmune demyelinating disease such as multiple sclerosis or neuromyelitis optica, in a subject at risk of developing or having developed the disease, comprising administering to the subject a pharmaceutical composition containing the above-described T-cell activation inhibitory peptide or modified peptide, or a nucleic acid encoding the same.

[0041] In addition, the present disclosure provides the use of the above-described T-cell activation inhibitory peptide or modified peptide, or a nucleic acid encoding the same, in the manufacture of a pharmaceutical composition for the treatment or prevention of an immune-related disease or T-cell lymphoma in which T-cell activation is involved, and; the use of the above-described T-cell activation inhibitory peptide or modified peptide, or a nucleic acid encoding the same, for the treatment or prevention of the disease. The present disclosure provides the use of the above-described T-cell activation inhibitory peptide or modified peptide, or a nucleic acid encoding the same, in the manufacture of a pharmaceutical composition for the treatment or prevention of an autoimmune disease or allergic disease, such as a central nervous system autoimmune demyelinating disease such as multiple sclerosis or neuromyelitis optica, and; the use of the above-described T-cell activation inhibitory peptide or modified peptide, or a nucleic acid encoding the same, for the treatment or prevention of the disease.

[0042] The present invention will be described in more detail by the following examples, but the present invention is not limited thereto.

[0043] [Materials and Methods] Antibodies: Anti-human CD3 monoclonal antibody (clone OKT3) and anti-human CD28 monoclonal antibody (clone CD28.8) were purchased from eBioscience (San Diego, CA), anti-mouse CD3 monoclonal antibody (clone 145-2C11) was purchased from American Type Culture Collection (Manassas, VA), and anti-mouse CD28 monoclonal antibody (clone PV-1) was purchased from BioXCell (West Lebanon, NH). Anti-Myc antibody, anti-Flag antibody, anti-HA antibody, and anti-β-actin antibody were purchased from Sigma-Aldrich (St. Louis, MO). Anti-phospho-PLCg1 (Tyr783) antibody, anti-phopho-ZAP-70 (Tyr319), anti-i, anti-phospho-ERK (Thr202 / Tyr204) antibody, anti-phospho-LCK (Tyr394) antibody, and anti-ZAP-70 antibody were purchased from Cell Signaling Technology (Beverly, MA). Anti-PLCg1 antibody for Western blotting was purchased from R&D Systems (Minneapolis, MN), and anti-ITK antibody was purchased from Proteintech Group (Rosemont, IL). APC anti-mouse CD3e antibody (clone 145-2C11), PE conjugate anti-mouse CD4 monoclonal antibody (clone GK1.5), and other antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Human T-cell lymphoma cell line Jurkat, mouse T-cell lymphoma cell line EL-4, and human B-cell lymphoma cell line Ramos were subcultured in RPMI 1640 containing 10% fetal bovine serum (FCS), while human cervical cancer cell line HeLa was subcultured in DMEM containing 10% FCS under conditions of 37°C and 5% CO2. Human peripheral blood mononuclear cells (hPBMCs) from healthy individuals were purchased from Lonza. CD4 cells were derived from normal mice. + T cells were prepared from mouse spleen using the EasySep mouse CD4+ T cell isolation kit (STEMCELL Technologies, Vancouver, BC, Canada), and the cells were confirmed to be a preparation with over 95% CD3 + CD4 + cells by flow cytometry. - Statistical analysis Statistical analysis was performed using GraphPad Prism 6.02. The Mann-Whitney U test, unpaired Student t test, Tukey multiple comparison test, and Sidak multiple comparison test were employed. Outliers were identified by the ROUT method of GraphPad Prism. Data were considered significant at p < 0.05. Data were shown as mean ± SEM.

[0044] [Example 1] Referring to the PH domain of STAP-1 and the amino acid sequence on its N-terminal side, a peptide was designed by adding an oligoarginine sequence (octaarginine sequence) consisting of 8 arginine residues to the N-terminal of the amino acid sequences shown in Table 1. These chemically synthesized peptides (peptide purity of 95% or more) were purchased from GL Biochem and used in experiments as a 10% DMSO solution. For the positions of the amino acid sequences shown in Table 1 in the amino acid sequence of STAP-1, refer to FIG. 1.

[0045] [Example 2] (1) EL-4 cells were treated with the synthetic peptide of Example 1 at a final concentration of 10 μM or 50 μM for 30 minutes in an RPMI1640 environment without FCS, then replaced with RPMI1640 containing 10% FCS and seeded in a 96-well plate at a density of 2x10 4 cells / well. After culturing at 37°C for 48 hours, 10 μl of WST-8 reagent (Dojindo Laboratories, Kumamoto, Japan) was added, and the cells were incubated at 37°C in the dark for 2 hours, and then the absorbance at 450 nm was measured as an index of cell proliferation.

[0046] Figure 2 shows the cell proliferation with each synthetic peptide added, with cell proliferation without the addition of synthetic peptides set to 100. Peptides PH-3, PH-3C, and PH-3Ca, which contain the amino acid sequence TTLFF, and peptides PH-3Ca1A and PH-3Ca2A, in which one threonine residue of the amino acid sequence TTLFF is replaced with an alanine residue, suppressed EL-4 cell proliferation in a concentration-dependent manner. No signs of cytotoxicity were observed in EL-4 cells treated with these peptides.

[0047] Furthermore, the inhibition of EL-4 cell proliferation observed when peptides PH-1A and PH-4 were added was not observed with PH-1Aa, PH-1Ab, PH-1Ac, PH-4A, PH-4B, and PH-4C, which correspond to partial peptides of these amino acid sequences. Therefore, it was determined that this inhibition was not based on inhibition of PH domain binding and was excluded from consideration. In addition, the inhibition of EL-4 cell proliferation observed when peptides PH-1B, PH-2, and PH-2A were determined to be highly likely to be due to cytotoxicity based on observation of the cell's appearance and were therefore excluded from consideration.

[0048] (2) The growth inhibitory activity of peptide PH-3Ca (hereinafter also referred to as iSP1) and peptide PH-3Ca5A (hereinafter also referred to as iCont), which did not show activity as a comparison, against EL-4 cells, Jurkat cells, and hPBMCs was evaluated using the same method as in (1), except that the peptide addition concentration was changed. It was confirmed that iSP1 showed concentration-dependent growth inhibitory activity against human and mouse T-cell lymphoma cell lines as well as PBMCs derived from healthy individuals (Figure 3).

[0049] (3) The inhibitory activity of iSP1 and iCont on Ramos cells and HeLa cells was also evaluated using the same method as in (1). It was confirmed that neither iSP1 nor iCont affected the proliferation of B-cell lymphoma cells or cervical cancer cells (Figure 4).

[0050] [Example 3] (1) Jurkat cells were treated with FITC-labeled iSP1 for 30 minutes in an RPMI1640 environment without FCS, and then analyzed by flow cytometry. The proportion of FITC-positive cells increased upon addition of FITC-labeled iSP1 (Figure 5), confirming the intracellular translocation of iSP1.

[0051] (2) In addition, Jurkat cells, EL-4 cells, or CD4 cells derived from normal mice are collected in 96-well plates coated with 3 mg / ml anti-CD3 antibody solution and 1 mg / ml anti-CD28 antibody solution. + T cells (1x10 5 Cells were seeded (cells / well) and cultured at 37°C for 48 hours using RPMI 1640 containing 10% FCS supplemented with iSP1 or iCont. The culture supernatant was collected, and the IL-2 concentration was measured using an ELISA kit (BioLegend). As a comparison, the IL-2 concentration in the culture supernatant of cells that received nonspecific stimulation by adding Phorbol 12-myristate 13-acetate (PMA) and ionomycin instead of anti-CD3 / anti-CD28 TCR stimulation was measured. iSP1 suppressed IL-2 production in human and mouse T-cell lymphoma cell lines stimulated by TCR (Figures 6-8). Note that CD4 + Regarding T cells, cell proliferation was also evaluated using WST-8 reagent on the culture supernatant, and the cell proliferation inhibitory activity of iSP1 was confirmed, similar to Example 2 (Figure 8).

[0052] [Example 4] Jurkat cells were stimulated with TCR in the presence of iSP1 or iCont in the same manner as in Example 3(2), cultured for 48 hours, and then harvested. Cell lysates were prepared using lysis buffer (50 mM Tris-HCl [pH 7.4], 0.15 M NaCl, 1% Nonidet P-40, 1 mM PMSF, 1 mM Na3VO4, 1 mM NaF), boiled in SDS sample buffer, analyzed by SDS-PAGE, and transferred to polyvinylidene fluoride membranes. The membranes were blocked with 2% bovine serum albumin TBST or 5% skim milk TBST and incubated with primary antibodies. Proteins reacted with primary antibodies were visualized using HRP-labeled secondary antibodies (GE Healthcare Bio-Sciences, Uppsala, Sweden) and Immobilon Western Chemiluminescent HRP substrate (Millipore, Bedford, MA).

[0053] STAP-1 forms a complex with LCK and ITK, which are components of TCR signaling, thereby promoting the efficient phosphorylation of downstream molecules and enhancing T cell activation (see K. Kagohashi et al., J Immunol (2024) 212 (6): 951-961; also see the conceptual diagram in the upper left of Figure 9). The presence of iSP1 suppressed the phosphorylation of LCK, ZAP-70, and PLC-γ1 (Figure 9), indicating that iSP1 suppresses T cell signaling and thus inhibits T cell activation.

[0054] [Example 5] The therapeutic effect of iSP1 was evaluated as follows using experimental autoimmune encephalomyelitis (EAE) mice. EAE is a model of central nervous system autoimmune demyelinating disease that exhibits pathological conditions similar to multiple sclerosis and neuromyelitis optica. It is thought to develop when autoreactive T cells infiltrate the central nervous system, inducing inflammation and causing demyelination.

[0055] C57 / BL6 mice were subcutaneously immunized at the base of the tail with 100 mg of MOG (Sigma-Aldrich) emulsified with CFA (BD, Sparks, MD). At the time of immunization and two days later, mice were intravenously injected with 400 ng of pertussis toxin (List Biological Laboratories), and from 8 to 20 days after immunization, 40 μg of peptide iSP1 or iCont was administered intravenously every other day. The severity of EAE was monitored using a clinical score according to previously reported criteria (K. Saitoh et al., Biochem. Biophys. Rep. (2016) 8: 139-145). Formalin-fixed, paraffin-embedded sections of the spinal cord were stained with H&E or Luxol Fast Blue to analyze inflammation or demyelination. To analyze Th1 and Th17 cells in the central nervous system, brain and spinal cord cells were homogenized, resuspended in 30% Percoll, and the cell suspension was overlaid in 70% Percoll. After centrifugation, the 30-70% Percoll interface was collected and used as mononuclear cells for detecting Th1 and Th17 cells in the central nervous system of EAE mice. Mononuclear cells were stimulated with PMA (50 ng / ml) + ionomycin (1 mg / ml) in the presence of brefeldin (5 mg / ml) for 5 hours and then stained intracellularly.

[0056] Figure 10 shows the trial protocol and the progression of the EAE clinical score. The clinical score indicates the severity of symptoms, with higher numbers representing a normal state with no abnormal symptoms, and 5 representing death. iSP1 suppressed disease exacerbation compared to iCont. Furthermore, iSP1 suppressed cell infiltration and demyelination into the spinal cord (Figure 11), and suppressed the infiltration of Th1 and Th17 cells into the central nervous system (Figure 12). Therefore, it was considered that iSP1 suppressed demyelination by inhibiting T cell activation, thereby delaying the onset of EAE and suppressing disease exacerbation.

Claims

1. A peptide consisting of an amino acid sequence of 5 to 20 residues containing Xaa1-Xaa2-LFF (where Xaa1 and Xaa2 are any amino acid residues independently of each other), which has the ability to suppress T cell activation.

2. The peptide according to claim 1, comprising a partial sequence of the human STAP-1 amino acid sequence shown in Sequence ID No. 9, the partial sequence including TTLFF (Sequence ID No. 3), or an amino acid sequence having 80% or more sequence identity with the partial sequence.

3. The peptide according to claim 1, wherein at least one of Xaa1 or Xaa2 is threonine or alanine.

4. The peptide according to claim 1, wherein at least one of Xaa1 and Xaa2 is threonine and the other is threonine or alanine.

5. The peptide according to claim 1, comprising EYEHYWTELRGTTLFFYTD (SEQ ID NO: 7), a partial sequence of the amino acid sequence shown in SEQ ID NO: 7, the partial sequence including TTLFF (SEQ ID NO: 3), or an amino acid sequence having 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 7 or the partial sequence.

6. The peptide according to claim 1, comprising the amino acid sequence shown in any of SEQ ID NOs: 1 to 8.

7. A peptide having T cell activation inhibitory activity, wherein a functional peptide is attached to the terminus of the peptide defined in claim 1, either via or without a linker sequence.

8. The peptide according to claim 7, wherein the functional peptide is a membrane-permeable peptide.

9. The peptide according to claim 1 or 7, comprising one or more chemically modified amino acid residues.

10. A nucleic acid encoding the peptide as defined in claim 1 or 7.

11. A pharmaceutical composition for the treatment or prevention of an immune-related disease involving T cell activation, comprising the peptide described in claim 1 or 7, or a nucleic acid encoding the peptide defined in claim 1 or 7.

12. The pharmaceutical composition according to claim 11, wherein the immune-related disease involving T cell activation is an autoimmune disease or an allergic disease.

13. The pharmaceutical composition according to claim 11, wherein the immune-related disease involving T cell activation is a central nervous system autoimmune demyelinating disease.

14. A pharmaceutical composition for the treatment of T-cell lymphoma, comprising a peptide according to claim 1 or 7, or a nucleic acid encoding the peptide as defined in claim 1 or 7.

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