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WO2021208106A1 - 一种治疗自身免疫病的融合肽 - Google Patents

一种治疗自身免疫病的融合肽 Download PDF

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Publication number
WO2021208106A1
WO2021208106A1 PCT/CN2020/085447 CN2020085447W WO2021208106A1 WO 2021208106 A1 WO2021208106 A1 WO 2021208106A1 CN 2020085447 W CN2020085447 W CN 2020085447W WO 2021208106 A1 WO2021208106 A1 WO 2021208106A1
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Prior art keywords
syndrome
immune checkpoint
pdl1
autoimmune
peptide
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PCT/CN2020/085447
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English (en)
French (fr)
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魏化伟
杨承刚
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北京泽勤生物医药有限公司
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Priority to PCT/CN2020/085447 priority Critical patent/WO2021208106A1/zh
Priority to US17/996,451 priority patent/US20230227533A1/en
Publication of WO2021208106A1 publication Critical patent/WO2021208106A1/zh

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    • 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
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70596—Molecules with a "CD"-designation not provided for elsewhere
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • 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
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • 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
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/70521—CD28, CD152
    • 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
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/70532—B7 molecules, e.g. CD80, CD86
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/03—Fusion polypeptide containing a localisation/targetting motif containing a transmembrane segment

Definitions

  • the invention belongs to the field of biomedicine, and specifically relates to a fusion peptide for treating autoimmune diseases.
  • Autoimmune diseases are mainly diseases caused by the body's immune response to self-antigens, a large number of immune cells infiltrate, and the damage of self-tissues caused by abnormal immune tissue metabolism.
  • Autoimmune diseases can be divided into two major categories according to the scope of the diseased tissue.
  • the first category is organ-specific autoimmune diseases, which can affect multiple organs including the brain, thyroid, and stomach; the second category is non-organ-specific Autoimmune diseases can affect multiple tissues such as muscles, skin, and joints.
  • organ-specific autoimmune diseases which can affect multiple organs including the brain, thyroid, and stomach
  • non-organ-specific Autoimmune diseases can affect multiple tissues such as muscles, skin, and joints.
  • autoimmune diseases There are currently more than 100 known autoimmune diseases, affecting about 5% of the population. Common ones include systemic lupus erythematosus, rheumatoid arthritis, systemic vasculitis, and scleroderma.
  • the basic reaction of the life system to ensure life generally has a strictly controlled chemical environment.
  • the pH inside and outside the cell is generally relatively constant, and the pH outside the cell is pH 7.4.
  • the extracellular pH is generally between 6.2 and 7.0, resulting in an acidic extracellular microenvironment.
  • the low pH insertion peptide (pHLIP, pH low insertion peptide) derived from bacterial rhodopsin-derived transmembrane helix protein C happens to be a polypeptide carrier that can target this slightly acidic environment.
  • pHLIP is composed of flanking sequences and transmembrane (TM) sequences.
  • the flanking sequences are composed of protonated amino acid residues, and the TM sequence is composed of hydrophobic residues.
  • pHLIP does not form a single helix across the membrane in a neutral solution, but pHLIP can form a C-helix structure and insert into the cell membrane in an acidic solution and the presence of a double lipid layer.
  • pHLIP generally has three states. In healthy tissues with a pH of about 7.4, in a dissolved state (state I) or weakly bound to the membrane (state II), pHLIP is flushed from the membrane by normal perfusion and continues to circulate in the body. In inflammatory tissues with acidic pH, pHLIP spontaneously folds into a transmembrane spiral and inserts into the membrane (state III).
  • pHLIP is widely used in nuclear imaging, fluorescence-guided surgery, gene therapy and nanotechnology.
  • the use of pHLIP's acidic environment tropism to develop drugs that are beneficial to the treatment of autoimmune diseases is a hot research topic in the future.
  • the present invention is based on the following concept: connect the extracellular segment of PD-L1 to the N-terminal of pHLIP and display the extracellular segment of PD-L1 on the cell membrane of the lesion tissue through pHLIP, and use pHLIP to enhance PD-1/PD- of the lesion tissue L1 negative signal inhibits the immune response of effector T cells from the source, and plays a certain role in preventing the occurrence and development of autoimmune diseases.
  • One of the objectives of the present invention is to provide a fusion peptide formed by PD-L1 and a low pH insert peptide.
  • the second objective of the present invention is to provide a pharmaceutical composition for the treatment of autoimmune diseases formed by the above-mentioned fusion peptide.
  • the third objective of the present invention is to provide the application of PD-L1 in the preparation of the above-mentioned fusion peptide.
  • the third objective of the present invention is to provide the use of the above-mentioned fusion peptide.
  • the present invention provides a fusion peptide of a low pH insert peptide, the fusion peptide comprising a low pH insert peptide, an immune checkpoint ligand or a fragment thereof.
  • Immune checkpoints include PD-1, Lag-3, Tim-3, TIGIT, CTLA-4.
  • PD-1 ligands include PD-L1 and PD-L2.
  • Lag-3 ligands include fibrinogen-like protein 1, LSECtin.
  • Tim-3 ligands include galectin-9, phosphatidylserine, high mobility group protein B1, Ceacam-1.
  • TIGIT ligands include CD155 and CD122.
  • CTLA-4 ligands include CD86 (B7-2) and CD80 (B7-1).
  • the low pH insert peptide that can be used to construct the fusion peptide of the present invention includes the polypeptide shown in SEQ ID NO. 1 or a variant thereof.
  • WT polypeptide whose sequence is SEQ ID NO. 1 is abbreviated as WT in the present invention, and variants of WT include Var1-Var16.
  • WT ACEQNPIY WARYADWLFTTPLLLLDLALLVDADEGT (SEQ ID NO.1);
  • Var1 ACEDQNPY WARYADWLFTTPLLLLDLALLVDG (SEQ ID NO. 2);
  • Var2 ACEDQNPY WRAYADLFTPLTLLDLLALWDG (SEQ ID NO.3);
  • Var3 ACDDQNP WRAYLDLLFPTDTLLLDLLW (SEQ ID NO.4);
  • Var4 ACEEQNP WRAYLELLFPTETLLLELLW (SEQ ID NO.5);
  • Var5 ACDDQNP WARYLDWLFPTDTLLLDL (SEQ ID NO. 6);
  • Var6 CDNNNP WRAYLDLLFPTDTLLLDW (SEQ ID NO. 7);
  • Var7 ACEEQNP WARYLEWLFPTETLLLEL (SEQ ID NO. 8);
  • Var9 CEEQQP WRAYLELLFPTETLLLEW (SEQ ID NO. 10);
  • Var11 ACEEQNP WARYAEWLFPTTLLLLE (SEQ ID NO. 12);
  • Var12 ACEDQNP WARYADLLFPTTLAW (SEQ ID NO. 13);
  • Var13 ACEEQNP WARYAELLFPTTLAW (SEQ ID NO.14);
  • Var14 TEDAD VLLALDLLLLPTTFLWDAYRAWYPNQECA (SEQ ID NO.15);
  • Var16 CDDDDDNPNY WARYAPWLFTTPLLLLPGALLVEAEET (SEQ ID NO. 17).
  • the underlined part above represents the extracellular segment of the low pH insert peptide.
  • the PD-L1 protein or fragments thereof of the present invention is connected to the N-terminus of the low pH insert peptide through Linker.
  • the Linker sequence is GGGS.
  • the immune checkpoint used is PD-1, and its ligand is selected as PD-L1.
  • the PD-L1 fragment linked to the low pH insert peptide sequence is the extracellular segment of the PD-L1 protein.
  • sequence of the extracellular segment of the PD-L1 protein is shown in SEQ ID NO.18 or SEQ ID NO.19.
  • the amino acid sequence shown in SEQ ID NO. 18 or SEQ ID NO. 19 has undergone one or several amino acid residue substitutions and/or deletions and/or additions and is identical to that shown in SEQ ID NO. 18 or SEQ ID NO. 19
  • Polypeptides derived from the amino acid sequence shown in SEQ ID NO. 18 or SEQ ID NO. 19 with the same function also belong to the extracellular segment of the PD-L1 protein, that is, the extracellular segment of the PD-L1 protein of the present invention includes wild type And its variants.
  • the extracellular segment variant of PD-L1 protein has at least 80% homology (also called sequence identity) with the amino acid sequence shown in SEQ ID NO. 18 or SEQ ID NO. 19, and more preferably, it is identical to SEQ ID.
  • the amino acid sequence shown in NO. 18 or SEQ ID NO. 19 has at least about 90% to 95% homology, and often 96%, 97%, 98%, or 99% homology.
  • extracellular segment variants of PD-L1 protein also include non-conservative modifications to the amino acid sequence shown in SEQ ID NO.18 or SEQ ID NO.19, as long as the modified polypeptide still retains the biological activity of the binding ligand. Can.
  • the fusion peptide constructed using the extracellular segment of the PD-L1 protein and the low pH insert peptide is expressed as follows: PDL1-WT pHLIP, in the specific embodiment of the present invention, its amino acid sequence is shown in SEQ ID NO.20; PDL1-var3 In the specific embodiment of the present invention, its amino acid sequence is shown in SEQ ID NO. 21; PDL1-var7, in the specific embodiment of the present invention, its amino acid sequence is shown in SEQ ID NO. 22.
  • the present invention provides a pharmaceutical composition for the treatment of autoimmune diseases, the pharmaceutical composition comprising the aforementioned fusion peptide.
  • the pharmaceutical composition also includes a pharmaceutically acceptable carrier.
  • Examples of pharmaceutically acceptable carriers include, but are not limited to: water; water carriers such as but not limited to sodium chloride injection, Ringer injection, glucose injection, glucose and sodium chloride injection, and lactated Ringer injection Liquid; water-miscible carriers such as but not limited to ethanol, polyethylene glycol and polypropylene glycol; and non-aqueous carriers such as but not limited to corn oil, cottonseed oil, peanut oil, sesame oil, ethyl oleate, isopropyl myristate and Benzyl benzoate.
  • water carriers such as but not limited to sodium chloride injection, Ringer injection, glucose injection, glucose and sodium chloride injection, and lactated Ringer injection Liquid
  • water-miscible carriers such as but not limited to ethanol, polyethylene glycol and polypropylene glycol
  • non-aqueous carriers such as but not limited to corn oil, cottonseed oil, peanut oil, sesame oil, ethyl oleate, isopropyl myr
  • the pharmaceutical composition also includes other drugs for the treatment of autoimmune diseases.
  • composition of the present invention can be administered to human patients by any route, including but not limited to: intravenous, intradermal, transdermal, subcutaneous, intramuscular, inhalation (such as via aerosol), buccal (such as tongue) Bottom), topical (ie skin and mucosal surfaces, including airway surfaces), intrathecal, intraarticular, intrapleural, intracerebral, intraarterial, intraperitoneal, oral, intralymphatic, intranasal, rectal or vaginal administration, by Local catheter perfusion or direct injection into the lesion.
  • the composition of the present invention is administered by intravenous bolus injection or intravenous infusion within a given time (0.5-2 hours). Can be passed through a peristaltic pump, or in the form of a long-acting formulation
  • the pharmaceutical composition of the present invention is delivered, but as is understood in the art, the most suitable route in any given situation depends on factors such as the type, age, sex and general health of the subject, and the characteristics of the disease being treated And severity and/or characteristics of the particular composition (i.e. dosage, dosage form) administered.
  • the route of administration is a bolus or continuous infusion over a period of time, once a week or twice a week.
  • the route of administration is subcutaneous injection, optionally once or twice a week.
  • the pharmaceutical composition of the present invention is administered to outpatients.
  • the dosage of the pharmaceutical composition of the present invention is measured in units of mg/kg of the patient's body weight. In other embodiments, the dosage of the pharmaceutical composition is measured in units of mg/m2 of the patient's body surface area. In other embodiments, the dosage of the pharmaceutical composition is measured in units of mg/dose administered to the patient. Any dosage measurement method can be used in combination with the composition and method of the present invention, and the dosage unit can be converted by standard methods in the art.
  • the dosage can be selected according to a variety of factors, including the age, sex, type, and disease of the subject, the required degree of cell consumption, and the dosage can be determined by those skilled in the art.
  • the effective dose of the pharmaceutical composition of the present invention can be extrapolated from the dose-response curve of the in vitro detection system or animal model detection system.
  • the dose of initial treatment is usually higher and/or the frequency of dosing is higher compared to the maintenance regimen.
  • the present invention provides the use of immune checkpoint ligands or fragments thereof in the preparation of the aforementioned fusion peptides.
  • the present invention provides the application of the aforementioned fusion peptide in the preparation of drugs for the treatment of autoimmune diseases.
  • autoimmune disease refers to a subject's disease characterized by cell, tissue and/or organ damage caused by the subject's immune response to its own cells, tissues and/or organs.
  • exemplary autoimmune diseases include alopecia areata, ankylosing spondylitis, antiphospholipid syndrome, autoimmune Addison's disease, autoimmune diseases of the adrenal glands, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune oophoritis, and Orchitis, autoimmune thrombocytopenia, Behcet syndrome, bullous pemphigoid, cardiomyopathy, stomatitis, diarrheal dermatitis, chronic fatigue immune dysfunction syndrome, chronic inflammatory demyelinating polyneuropathy, colliculus Shier's syndrome, scar pemphigoid, CREST syndrome, cold agglutinin disease, Crohn's disease, discoid lupus, idiopathic mixed cryoglobulinemia, diabetes, eosinophilic muscle Meningit
  • the medicine includes the aforementioned pharmaceutical composition.
  • the medicine or pharmaceutical composition of the present invention can be used in combination with other medicines for treating autoimmune diseases.
  • NSAIDs non-limiting examples include but are not limited to: aspirin, diflunisal, diclofenac, etodolac, fenamic acid esters, fenoprofen, flurbiprofen, cloth Profen, pendomethacin, ketoprofen, methyl salicylate, nabumetone, naproxen, piperazine, phenylbutazone, piroxicam, sulindac and tolmetin); analgesia Drugs (non-limiting examples are acetaminophen, phenacetin and tramadol); CSI, including but not limited to: celecoxib and rofecoxib; glucocorticoids (preferably low-dose oral corticosteroids) Hormones, such as ⁇ 7.5 mg/d prednisone, or monthly pulsed high-dose glucocorticoids or intra-articular
  • drugs for the treatment of osteoarthritis include but are not limited to: analgesics (non-limiting examples are acetaminophen, with a dose up to 4000 mg/d; phenacetin; tramadol, with a daily dose ranging from 200 to 300 mg) ; NSAID (non-limiting examples include but are not limited to: aspirin, diflunisal, diclofenac, etodolac, fenamic acid esters, fenoprofen, flurbiprofen, ibuprofen, indomethacin, ketone Profen, methyl salicylate, nabumetone, naproxen, piperazine, phenylbutazone, piroxicam, sulindac and tolmetin.
  • analgesics non-limiting examples are acetaminophen, with a dose up to 4000 mg/d
  • phenacetin tramadol, with a daily dose ranging
  • NSAIDs Preferably low-dose NSAIDs, for example, 1200 mg/d ibuprofen Fen, 500mg/d naproxen.
  • Gastric protective agents such as misoprostol, famotidine or omeprazole are preferably used simultaneously with NSAID; non-acetylated salicylate includes but is not limited to salicylate
  • Cyclooxygenase (Cox)-2-specific inhibitors (CSI) include but are not limited to celecoxib and rofecoxib; long-acting glucocorticoid preparations; hyaluronic acid; capsaicin cream.
  • the present invention provides a labeling system, which includes the aforementioned fusion peptide.
  • the labeling system may also include Cyanine 5.5, Alexa Flour 750, Alexa Fluor 647, Alexa Flour 488, Alexa Flour 546, 64 Cu-DOTA, 68 Ga-DOTA, 18 FO-pyridine, 18 F-liposomes, liposomal Rhodamine, Nanogold, TAMRA.
  • the fusion peptide in the labeling system is inserted into the cell membrane under the action of the low pH insert peptide.
  • the PDL1 in the fusion peptide is positioned on the surface of the target cell, and the PDL1 ligand (such as PD-1) on the surface of the immune cell recognizes the PDL1 on the surface of the target cell. PDL1 ligand binds to PDL1, thereby inhibiting immune cell function.
  • the present invention provides the application of the aforementioned fusion peptide in the preparation of an autoimmune lesion tissue cell labeling system.
  • the marking system is the same as described above.
  • the present invention provides a treatment method for autoimmune diseases, the treatment method comprising administering the aforementioned fusion peptide or the aforementioned pharmaceutical composition of the present invention to a person in need.
  • the present invention provides a method for labeling an immune checkpoint ligand or a fragment thereof on the cell membrane of a lesion tissue, the method comprising linking the immune checkpoint ligand or a fragment thereof with a low pH insertion peptide Form a fusion peptide.
  • the method further includes introducing the fusion peptide into the lesion tissue and inserting it into the cell membrane of the lesion tissue.
  • the immune checkpoint includes the aforementioned immune checkpoint; the immune checkpoint ligand includes the aforementioned immune checkpoint ligand; and the immune checkpoint ligand fragment includes the aforementioned immune checkpoint ligand fragment.
  • the low pH insert peptides include the aforementioned low pH insert peptides.
  • the present invention provides the use of immune checkpoint ligands or fragments thereof in the preparation of drugs for the treatment of autoimmune diseases.
  • the immune checkpoint includes the aforementioned immune checkpoint; the immune checkpoint ligand includes the aforementioned immune checkpoint ligand; the immune checkpoint ligand fragment includes the aforementioned immune checkpoint ligand Fragment.
  • autoimmune diseases are defined as described above.
  • the medicine includes the aforementioned pharmaceutical composition.
  • the present invention provides the application of an immune checkpoint ligand or a fragment thereof in the preparation of a tissue cell labeling system for immune disease lesions.
  • the immune checkpoint includes the aforementioned immune checkpoint; the immune checkpoint ligand includes the aforementioned immune checkpoint ligand; the immune checkpoint ligand fragment includes the aforementioned immune checkpoint ligand Fragment.
  • the marking system includes the marking system described above.
  • the present invention provides the use of a low pH insert peptide in the preparation of the aforementioned fusion peptide.
  • the definition of the low pH insert peptide is the same as described above.
  • the present invention provides the use of low pH insert peptides in the preparation of drugs for treating autoimmune diseases.
  • the definition of the low pH insert peptide is the same as described above.
  • autoimmune diseases are defined as described above.
  • the medicine includes the aforementioned pharmaceutical composition.
  • the present invention provides the application of a low pH insert peptide in the preparation of a tissue cell labeling system for autoimmune disease lesions.
  • the definition of the low pH insert peptide is the same as described above.
  • autoimmune diseases are defined as described above.
  • the marking system includes the marking system described above.
  • sequences of the present invention are listed in sequence from the N-terminus to the C-terminus.
  • PDL1 and “PD-L1” can be used interchangeably.
  • autoimmune diseases use antibodies or antagonists to block the function of a certain cytokine (IL-6/TNF- ⁇ /IL-17), so as to achieve the purpose of alleviating the disease.
  • IL-6/TNF- ⁇ /IL-17 cytokine
  • the occurrence of autoimmune diseases is the result of multi-factor synergy.
  • This application uses pHLIP to enhance the PD-1/PD-L1 negative signal of the lesion tissue and suppress the immune response of effector T cells from the source. It is expected that the treatment of autoimmune diseases may have a certain curative effect.
  • This application uses pHLIP to accurately display PD-L1 at the lesion site, and the polypeptide has the characteristics of a short half-life, which can avoid the potential risk of long-term inhibition to the body.
  • Figure 1 shows the SDS-PAGE staining diagram of the synthetic peptide of the present invention, where A: PD-L1-WT pHLIP; B: PDL1-Ig; C: extracellular segment of PD-L1; D: PDL1-var3; E: PD-L1 -var7;
  • Figure 2 shows the ELISA results of the binding of PDL1-WT pHLIP to PD-1
  • Figure 3 shows the result of ELISA of the binding of PD-1 and PDL1 protein
  • Figure 4 shows the ELISA result diagram of the binding of PDL1-WT pHLIP and PDL1 antibody
  • Figure 5 shows the ELISA results of PDL1-var3 binding to PD-1
  • Figure 6 shows the ELISA results of PDL1-var3 binding to PDL1 antibody
  • Figure 7 shows the ELISA results of PDL1-var7 binding to PD-1
  • Figure 8 shows the ELISA result diagram of the binding of PDL1-var7 and PDL1 antibody
  • Figure 9 shows the fluorescence image of PDL1-WT pHLIP localization on HEK293 cells observed by confocal
  • Figure 10 shows the fluorescence image of PDL1-var3 localization on HEK293 cells observed by confocal
  • Figure 11 shows the fluorescence image of PDL1-var7 localization on HEK293 cells observed by confocal
  • Figure 12 shows photos of the soles of mice treated with PDL1-WT pHLIP
  • Figure 13 shows a photograph of the soles of mice treated with PDL1-var3
  • Figure 14 shows a photograph of the soles of mice treated with PDL1-var7
  • Figure 15 shows the results of CIA scores treated with PDL1-WT pHLIP
  • Figure 16 shows the result of PDL1-var3 processing CIA score
  • Figure 17 shows the result of PDL1-var7 processing CIA score
  • Figure 18 shows the results of serum cytokine detection after PDL1-WT pHLIP treatment
  • Figure 19 shows the results of serum cytokine detection after PDL1-var3 treatment
  • Figure 20 shows the results of the detection of serum cytokines treated with PDL1-var7
  • Figure 21 shows the results of PDL1-WT pHLIP processing palm heel thickness detection
  • Figure 22 shows the results of PDL1-var3 processing palm and heel thickness detection
  • Figure 23 shows the results of PDL1-var7 processing palm and heel thickness detection.
  • the wild-type pHLIP (amino acid sequence is shown in SEQ ID NO. 1) and its variants 3 and 7 are represented as WT pHLIP, var3 and var7, respectively, which are all synthesized by Hangzhou Zhongpi Biochemical Co., Ltd.
  • the extracellular segment of PDL1 (the amino acid sequence is shown in SEQ ID NO.18, and the nucleotide sequence is shown in SEQ ID NO.26), a fusion peptide prepared from the extracellular segment of PD-L1 and wild-type pHLIP, var3, var7
  • they are represented as PDL1-WT pHLIP (amino acid sequence is shown in SEQ ID NO. 20, and nucleotide sequence is shown in SEQ ID NO. 23), PDL1-var3 (amino acid sequence is shown in SEQ ID NO. 21).
  • the nucleotide sequence is shown in SEQ ID NO. 24
  • PDL1-var7 the amino acid sequence is shown in SEQ ID NO.
  • the PET28a vector was constructed using restriction sites Nde I and XhoI, and transformed into the recipient bacteria BL21 (DE3) after construction, and clones were selected for verification (sequencing and expression).
  • Solution A 50mM Tris, 2mM EDTA, pH 8.0, wash twice;
  • Solution B 50mM Tris, 2mM EDTA, 0.1% Triton, pH 8.0, wash once;
  • Solution C 20mM Tris, 1M urea, pH8.0, wash once.
  • Ni column purification equilibrate the chromatographic column with 0.3M NaCL, 10mM PBS, pH8.0, wash the impurities with an equilibration buffer containing 40mM imidazole after loading, and elute the target protein with an equilibration buffer containing 300mM imidazole.
  • the purity of the target protein is greater than 95%.
  • the purified peptide was subjected to SDS-PAGE, and the result of Coomassie brilliant blue staining is shown in Figure 1, indicating that the peptide was successfully expressed.
  • Biotin BioLegend
  • PDL1-WT pHLIP PBS (Gibco).
  • PDL1-WT pHLIP group WT pHLIP control group; BSA control group; BLANK group.
  • Biotin BioLegend
  • PD-L1 extracellular segment PBS (Gibco).
  • PDL1 extracellular segment group PDL1-WT pHLIP group; WT pHLIP control group; BSA control group; BLANK group.
  • Biotin-anti-PDL1 BioLegend
  • PDL1-WT pHLIP PBS (Gibco).
  • PDL1-WT pHLIP group WT pHLIP control group; BSA control group; BLANK group.
  • PDL1-var3 group PDL1-var3 group; var3 control group; BSA control group; BLANK group.
  • Biotin-anti-PDL1 BioLegend
  • PDL1-var3 PBS (Gibco).
  • PDL1-var3 group PDL1-var3 group; var3 control group; BSA control group; BLANK group.
  • PDL1-var7 group PDL1-var7 group; var7 control group; BSA control group; BLANK group.
  • Biotin-anti-PDL1 BioLegend
  • PDL1-var7 PBS (Gibco).
  • PDL1-var7 group PDL1-var7 group; var7 control group; BSA control group; BLANK group.
  • Human embryonic kidney cell line HEK293 (purchased from ATCC).
  • DMEM medium Gibco
  • fetal bovine serum Gibco
  • Tris-HCL Tris-HCL (1mol/mL)
  • PD-1 PDL1 extracellular segment
  • PDL1-WT pHLIP WT pHLIP
  • PDL1-var7 var7
  • PDL1-var3, var7 PE cross-linking kit
  • Collect HEK293 cells in log phase discard the culture medium, and wash twice with PBS. After digestion with appropriate amount of trypsin, the digestion was terminated with culture solution, transferred to a 10ml test tube, centrifuged at 1000rpm for 5min, the supernatant was aspirated, and 1ml of DMEM medium containing 10% fetal bovine serum was added to resuspend and mix. Aspirate 10 ⁇ L of cell suspension to a cell counting plate for counting, add a certain amount of cell suspension to a laser confocal culture dish, adjust the culture medium to 5*10 5 , 1mL cell system, and place it in an incubator for overnight culture.
  • the supernatant is aspirated and washed twice with pH 7.4 PBS buffer.
  • the extracellular segment of PDL1 with a final concentration of 180 ⁇ g/mL and WT pHLIP, or var3, or var7 with a final concentration of 20 ⁇ g/mL were added to the PBS buffer at pH 7.4 and 6.3.
  • Chicken type II collagen (#20012), complete Freund's adjuvant (#7001), incomplete Freund's adjuvant (#7002), all are Chondrex, USA, WT pHLIP, var3, var7 are synthesized by Hangzhou Zhongtide Biochemical Co., Ltd. , PDL1 extracellular segment, PDL1-WT pHLIP, PDL1-var3, PDL1-var7, PDL1-Ig are prepared by our company.
  • the CIA score results are shown in Figure 15-17.
  • the CIA score of the PDL1-pHLIP group, PDL1-var3 group, or PDL1-var7 group was significantly reduced.
  • the results of serum cytokine detection are shown in Figure 18-20.
  • the levels of TNF- ⁇ , IFN- ⁇ , IL-6, and IL-17A in the PDL1-pHLIP group, PDL1-var3 group, or PDL1-var7 group were significantly reduced.

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Abstract

PDL1-pHLIP、制备方法及其在治疗自身免疫病中的应用。由低pH插入肽与PDL1的胞外段连接而成融合肽。在酸性环境下，低pH插入肽可插入到病灶组织细胞膜上，利用低pH插入肽的上述性质，将与其连接的PDL1靶向定位在病灶部位，利用低pH插入肽增强病灶组织PD-1/PD-L1负性信号，从源头上抑制效应T细胞免疫反应，从而发挥防治自身免疫病的作用。

Description

一种治疗自身免疫病的融合肽 技术领域
本发明属于生物医药领域，具体涉及一种治疗自身免疫病的融合肽。
背景技术
自身免疫病主要是机体对自身抗原发生免疫反应，免疫细胞大量浸润，免疫组织代谢异常导致的自身组织损害所引起的疾病。
自身免疫病按病变组织涉及的范围可分为两大类，第一类是器官特异性自身免疫病，可累及包括脑、甲状腺、胃在内的多个器官；第二类为非器官特异性自身免疫病，可累及肌肉、皮肤、关节等多个组织。目前已知的自身免疫病就有100多种，约累及5％的人口，常见的有系统性红斑狼疮、类风湿关节炎、系统性血管炎、硬皮病等。
生命系统为保证生命的基本反应一般具有严格控制的化学环境.健康组织的正常细胞中，细胞内外pH一般相对恒定，细胞外pH为pH7.4，但在自身免疫病的病变部位，由于炎性免疫细胞的大量浸润和高代谢，导致局部乳酸浓度急剧升高，这种平衡会遭到破坏，细胞外pH一般在6.2-7.0之间，产生酸性的细胞外微环境。来源于细菌视紫红质的跨膜螺旋蛋白C的低pH插入肽(pHLIP，pH low insertion peptide)恰巧是一种可以靶向这种微酸环境的多肽载体。pHLIP由侧翼序列和跨膜(trans membrane,TM)序列组成，其中侧翼序列由质子化氨基酸残基构成，TM序列由疏水残基构成。pHLIP在中性的溶液中不形成跨膜的单螺旋，但在酸性溶液中且存在双脂层时pHLIP可以形成C-螺旋结构并插入到细胞膜上。
pHLIP一般有三种状态，在pH值为7.4左右的健康组织中，处于溶解状态(状态I)或弱结合在膜上(状态II)，pHLIP通过正常灌注从膜上冲洗，并继续在体内循环。在pH值为酸性的炎症组织中，pHLIP自发折叠成跨膜螺旋，插入膜上(状态III)。
目前pHLIP在核成像，荧光引导手术，基因治疗及纳米技术中的应用都是十分广泛的。利用pHLIP的酸性环境趋向性开发利于治疗自身免疫病的药物是未来的研究热点。
发明内容
本发明是基于以下构思完成的：将PD-L1胞外段与pHLIP的N端相连通过pHLIP将PD-L1胞外段展示在病变部位组织细胞膜上，利用pHLIP增强病灶组织PD-1/PD-L1负性信号，从源头上抑制效应T细胞免疫反应，对防治自身免疫病的发生、发展起一定作用。
本发明的目的之一在于提供一种PD-L1与低pH插入肽形成的融合肽。
本发明的目的之二在于提供上述融合肽形成的治疗自身免疫病的药物组合物。
本发明的目的之三在于提供PD-L1在制备上述融合肽中的应用。
本发明的目的之三在于提供上述融合肽的用途。
为了实现上述目的，本发明采用了如下技术方案：
根据本发明的一个方面，本发明提供了一种低pH插入肽的融合肽，所述融合肽包括低pH插入肽、免疫检查点配体或其片段。
免疫检查点包括PD-1、Lag-3、Tim-3、TIGIT、CTLA-4。
PD-1配体包括PD-L1、PD-L2。
Lag-3配体包括纤维蛋白原样蛋白1、LSECtin。
Tim-3配体包括半乳糖凝集素-9、磷脂酰丝氨酸、高迁移率族蛋白B1、Ceacam-1。
TIGIT配体包括CD155、CD122。
CTLA-4配体包括CD86(B7-2)、CD80(B7-1)。
可用于构建本发明融合肽的低pH插入肽包括序列为SEQ ID NO.1所示的多肽或其变体。
序列为SEQ ID NO.1所示的多肽在本发明中简称WT，WT的变体包括Var1-Var16。
WT及其变体的序列如下：
WT: ACEQNPIYWARYADWLFTTPLLLLDLALLVDADEGT(SEQ ID NO.1)；
Var1: ACEDQNPYWARYADWLFTTPLLLLDLALLVDG(SEQ ID NO.2)；
Var2: ACEDQNPYWRAYADLFTPLTLLDLLALWDG(SEQ ID NO.3)；
Var3: ACDDQNPWRAYLDLLFPTDTLLLDLLW(SEQ ID NO.4)；
Var4: ACEEQNPWRAYLELLFPTETLLLELLW(SEQ ID NO.5)；
Var5: ACDDQNPWARYLDWLFPTDTLLLDL(SEQ ID NO.6)；
Var6: CDNNNPWRAYLDLLFPTDTLLLDW(SEQ ID NO.7)；
Var7: ACEEQNPWARYLEWLFPTETLLLEL(SEQ ID NO.8)；
Var8: CEEQQPWAQYLELLFPTETLLLEW(SEQ ID NO.9)；
Var9: CEEQQPWRAYLELLFPTETLLLEW(SEQ ID NO.10)；
Var10: ACEDQNPWARYADWLFPTTLLLLD(SEQ ID NO.11)；
Var11: ACEEQNPWARYAEWLFPTTLLLLE(SEQ ID NO.12)；
Var12: ACEDQNPWARYADLLFPTTLAW(SEQ ID NO.13)；
Var13: ACEEQNPWARYAELLFPTTLAW(SEQ ID NO.14)；
Var14: TEDADVLLALDLLLLPTTFLWDAYRAWYPNQECA(SEQ ID NO.15)；
Var15: CDDDDDNPNYWARYANWLFTTPLLLLNGALLVEAEET(SEQ ID NO.16)；
Var16: CDDDDDNPNYWARYAPWLFTTPLLLLPGALLVEAEET(SEQ ID NO.17)。
上述划线部分表示低pH插入肽的胞外段。
WT、Var1-Var16肽的胞外段重复一次或多次之后形成的肽((胞外段)n+低pH插入肽，其中n＝1、2、3……)也属于本发明的低pH插入肽的范畴。
本发明的所述PD-L1蛋白或其片段通过Linker连接于所述低pH插入肽的N端。
所述Linker是本领域常规使用的，序列可以是(GGGS)m，也可以是(GGGGS)m，其中m＝自然数。
优选地，所述Linker序列是GGGS。
在本发明的一个具体实施方案中，所用免疫检查点是PD-1，其配体选择的是PD-L1。
在本发明的具体实施例中，与低pH插入肽序列连接的PD-L1片段是PD-L1蛋白的胞外段。
进一步，所述PD-L1蛋白的胞外段序列如SEQ ID NO.18或SEQ ID NO.19所示。SEQ ID NO.18或SEQ ID NO.19所示的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加且与SEQ ID NO.18或SEQ ID NO.19所示的序列具有相同功能的由SEQ ID NO.18或SEQ ID NO.19所示的氨基酸序列衍生的多肽也属于PD-L1蛋白胞外段，即本发明的PD-L1蛋白的胞外段包括野生型及其变体。
PD-L1蛋白的胞外段变体与SEQ ID NO.18或SEQ ID NO.19所示的氨基酸序列具有至少80％同源性(又称为序列同一性)，更优选地，与SEQ ID NO.18或SEQ ID NO.19所示的氨基酸序列至少约90％至95％的同源性，常为96％、97％、98％、99％同源性。
通常，已知的是，一个蛋白质或者多肽中一个或多个氨基酸的修饰不会影响蛋白质的功能。本领域技术人员会认可改变单个氨基酸或小百分比的氨基酸或对氨基酸序列的个别添加、缺失、插入、替换是保守修饰，其中蛋白质多肽的改变产生具有相似功能的蛋白质或多肽。提供功能相似的氨基酸的保守替换表是本领域公知的。
PD-L1蛋白的胞外段变体也包括对SEQ ID NO.18或SEQ ID NO.19所示的氨基酸序列的非保守修饰，只要经过修饰的多肽仍然能够保留结合配体的生物学 活性即可。
利用PD-L1蛋白的胞外段与低pH插入肽构建的融合肽表示如下：PDL1-WT pHLIP，在本发明的具体实施例中，其氨基酸序列如SEQ ID NO.20所示；PDL1-var3，在本发明的具体实施例中，其氨基酸序列如SEQ ID NO.21所示；PDL1-var7，在本发明的具体实施例中，其氨基酸序列如SEQ ID NO.22所示。
根据本发明的又一个方面，本发明提供了一种治疗自身免疫病的药物组合物，所述药物组合物包括前面所述的融合肽。
进一步，所述药物组合物还包括药学上可接受的载体。
药学上可接受的载体的实例包括但不限于：水；水载体，如但不限于氯化钠注射液、林格注射液、葡萄糖注射液、葡萄糖和氯化钠注射液以及乳酸化林格注射液；水混溶载体，如但不限于乙醇、聚乙二醇和聚丙二醇；以及非水载体如但不限于玉米油、棉籽油、花生油、芝麻油、油酸乙酯、肉豆蔻酸异丙酯和苯甲酸苄酯。
更进一步，所述药物组合物还包括其他用于治疗自身免疫病的药物。
可通过任何途径将本发明的药物组合物给予人患者，这些途径包括但不限于：静脉内、皮内、透皮、皮下、肌内、吸入(如通过气雾剂)、含服(如舌下)、局部(即皮肤和粘膜表面，包括气道表面)、鞘内、关节内、胸膜内、大脑内、动脉内、腹膜内、口服、淋巴内、鼻内、直肠或阴道给药，通过局部导管灌注或病损内直接注射。在一个实施方式中，通过在给定时间(0.5-2小时)内静脉内推注或静脉内输注给予本发明组合物。可通过蠕动泵，或以长效制剂形式
递送本发明的药物组合物，但如本领域所了解的那样，在任何给定情况下最合适的途径取决于以下因素，例如对象的种类、年龄、性别和总体健康状况，所治疗疾病的特性和严重程度和/或所给予的特定组合物(即剂量、剂型)的特性。在具体实施方式中，给药途径是在一段时间，每周一次或每周两次推注或连续输注。在其它特定实施方式中，给药途径是皮下注射，任选每周一次或两次。在一个实施方式中，对门诊患者给予本发明的的药物组合物。
在某些实施方式中，本发明的药物组合物的剂量以mg/kg患者体重为单位计 量。在其它实施方式中，药物组合物的剂量以mg/m2患者体表面积为单位计量。在其它实施方式中，药物组合物的剂量以mg/给予患者的剂量为单位计量。任何剂量衡量方法均可与本发明组合物和方法联用，可通过本领域标准方式转换剂量单位。
本领域技术人员应理解，可根据多种因素选择剂量，包括对象的年龄、性别、种类和病症，所需的细胞消耗程度，剂量可由本领域技术人员进行确定。例如，可由来自体外检测系统或动物模型检测系统的剂量反应曲线外推得到本发明的药物组合物的有效量。
本领域技术人员应理解，与维持方案相比，初始治疗的剂量通常较高和/或给药频率较高。
根据本发明的又一个方面，本发明提供了免疫检查点配体或其片段在制备前面所述的融合肽中的应用。
免疫检查点配体或其片段的限定同前面所述。
根据本发明的又一个方面，本发明提供了前面所述的融合肽在制备治疗自身免疫病的药物中的应用。
术语“自身免疫病”指特征是对象对其自身的细胞、组织和/或器官产生免疫反应而引起的细胞、组织和/或器官损伤的对象病症。示例性的自身免疫病包括斑秃、强直性脊柱炎、抗磷脂综合征、自身免疫性艾迪森病、肾上腺的自身免疫病、自身免疫性溶血性贫血、自身免疫肝炎、自身免疫性卵巢炎和睾丸炎、自身免疫性血小板减少、贝切特综合征、大疱型类天疱疮、心肌病、口炎性腹泻皮炎、慢性疲劳免疫功能障碍综合征、慢性炎性脱髓鞘多神经病、丘施二氏综合征、疤痕性类天疱疮、CREST综合征、冷凝集素病、克罗恩氏病、盘状狼疮、特发性混合型冷球蛋白血症、糖尿病、嗜酸细胞性筋膜炎、纤维肌痛-纤维肌炎、肾小球肾炎、格拉夫斯病、格-巴二氏综合征、桥本甲状腺炎、亨-舍二氏紫癜、特发性肺纤维化、特发性/自身免疫血小板减少性紫癜、IgA神经病、青少年关节炎、扁平苔藓、红斑狼疮、美尼尔综合征、混合型结缔组织病、多发性硬化、1型或免疫介导的糖尿病、重症肌无力、天疱疮相关疾病、恶性贫血、结节性多动脉炎、多软骨炎、多腺体综合征、风湿性多肌痛、多肌炎、皮肌炎、原发性无Y球蛋 白血症、原发性胆汁性肝硬化、牛皮癣、牛皮癣关节炎、雷氏现象、莱特尔综合征、类风湿性关节炎、结节病、硬皮病、斯耶格伦综合征、全身肌强直综合征、系统性红斑狼疮、斯维特综合征、斯提耳病、红斑狼疮、高安动脉炎、暂时性动脉炎/巨细胞动脉炎、溃疡性结肠炎、葡萄膜炎、血管炎、白癜风、韦格纳肉芽肿病。
进一步，所述药物包括前面所述的药物组合物。
本发明的药物或药物组合物可以与其他治疗自身免疫病的药物联合应用。
治疗类风湿性关节炎的常用药物包括NSAID(非限制性例子包括但不限于：阿司匹林、二氟尼柳、双氯芬酸、依托度酸、灭酸酯类、非诺洛芬、氟比洛芬、布洛芬、喷哚美辛、酮洛芬、甲基水杨酸酯、萘丁美酮、萘普生、哌拉嗪、保泰松、吡罗昔康、舒林酸和托美丁)；镇痛药(非限制性例子是对乙酰氨基酚、非那西丁和曲马多)；CSI，包括但不限于：塞来考昔和罗非考昔；糖皮质激素类(优选低剂量口服糖皮质激素、如<7.5mg/d泼尼松、或每月脉冲给予高剂量糖皮质激素类、或关节内糖皮质激素)；疾病调理性抗风湿药(DMARD)，包括但不限于：氨甲喋呤(优选给定间歇性低剂量、如7.5-30mg每周一次)、金化合物(如金盐)、D-青霉胺、抗疟药(如氯喹)和柳氮磺吡啶；TNF-α中和剂，包括但不限于：依那西普和英利昔单抗；免疫抑制剂和细胞毒剂(例子包括但不限于：硫唑嘌呤、来氟米特、环孢霉素和环磷酰胺)。
治疗骨关节炎的的常用药物包括但不限于：镇痛药(非限制性例子是对乙酰氨基酚，剂量高达4000mg/d；非那西丁；曲马多，每天剂量范围是200至300mg)；NSAID(非限制性例子包括但不限于：阿司匹林、二氟尼柳、双氯芬酸、依托度酸、灭酸酯类、非诺洛芬、氟比洛芬、布洛芬、吲哚美辛、酮洛芬、甲基水杨酸酯、萘丁美酮、萘普生、哌拉嗪、保泰松、吡罗昔康、舒林酸和托美丁。优选低剂量NSAID，例如，1200mg/d布洛芬，500mg/d萘普生。胃保护剂，如米索前列醇、法莫替丁或奥美拉唑优选与NSAID同时使用；非乙酰化的水杨酸酯包括但不限于双水杨酯；环加氧酶(Cox)-2-特异性抑制剂(CSI)包括但不限于塞来考昔和罗非考昔；长效糖皮质激素制剂；透明质酸；辣椒碱乳膏。
根据本发明的又一个方面，本发明提供了一种标记系统，所述标记系统包括前面所述的融合肽。
进一步，所述标记系统还可以包括Cyanine 5.5、Alexa Flour 750、Alexa Fluor647、Alexa Flour 488、Alexa Flour 546、 64Cu-DOTA、 68Ga-DOTA、 18F-O-pyridine、 18F-liposomes、liposomal Rhodamine、Nanogold、TAMRA。
标记系统中的融合肽在低pH插入肽的作用下插入到细胞膜上，融合肽中的PDL1在靶细胞表面定位，免疫细胞表面上的PDL1配体(如PD-1)识别靶细胞表面PDL1，PDL1配体与PDL1结合，从而抑制免疫细胞功能。
根据本发明的又一个方面，本发明提供了前面所述的融合肽在制备自身免疫病病灶组织细胞标记系统中的应用。
进一步，所述标记系统同前面所述。
根据本发明的又一个方面，本发明提供了一种自身免疫病的治疗方法，所述治疗方法包括给有需要者施用本发明前面所述的融合肽或前面所述的药物组合物。
根据本发明的又一个方面，本发明提供了一种在病灶组织细胞膜上标记免疫检查点配体或其片段的方法，所述方法包括将免疫检查点配体或其片段与低pH插入肽连接形成融合肽。
进一步，所述方法还包括将所述融合肽导入病灶组织并插入到病灶组织细胞膜上。
所述免疫检查点包括前面所述的免疫检查点；所述免疫检查点配体包括前面所述的免疫检查点配体；免疫检查点配体片段包括前面所述的免疫检查点配体片段。
所述低pH插入肽包括前面所述的低pH插入肽。
根据本发明的又一个方面，本发明提供了免疫检查点配体或其片段在制备治疗自身免疫病的药物中的应用。
进一步，所述免疫检查点包括前面所述的免疫检查点；所述免疫检查点配体包括前面所述的免疫检查点配体；免疫检查点配体片段包括前面所述的免疫检查点配体片段。
进一步，所述自身免疫病限定同前面所述。
进一步，所述药物包括前面所述的药物组合物。
根据本发明的又一个方面，本发明提供了免疫检查点配体或其片段在制备身免疫病病灶组织细胞标记系统中的应用。
进一步，所述免疫检查点包括前面所述的免疫检查点；所述免疫检查点配体包括前面所述的免疫检查点配体；免疫检查点配体片段包括前面所述的免疫检查点配体片段。
进一步，所述标记系统包括前面所述的标记系统。
根据本发明的又一个方面，本发明提供了低pH插入肽在制备前面所述的融合肽中的应用。
进一步，低pH插入肽限定同前面所述。
根据本发明的又一个方面，本发明提供了低pH插入肽在制备治疗自身免疫病的药物中的应用。
进一步，低pH插入肽限定同前面所述。
进一步，所述自身免疫病限定同前面所述。
进一步，所述药物包括前面所述的药物组合物。
根据本发明的又一个方面，本发明提供了低pH插入肽在制备自身免疫病病灶组织细胞标记系统中的应用。
进一步，低pH插入肽限定同前面所述。
进一步，所述自身免疫病限定同前面所述。
进一步，所述标记系统包括前面所述的标记系统。
本发明的序列都是从N端到C端依次列出。
本发明中“PDL1”和“PD-L1”可互换使用。
本发明的优点和有益效果如下：
目前，自身免疫病的临床和临床前研究多是利用抗体或拮抗剂阻断某一细胞因子(IL-6/TNF-α/IL-17)的功能，从而达到减轻疾病的目的。然而，自身免疫病的发生是多因子协同作用的结果。本申请利用pHLIP增强病灶组织 PD-1/PD-L1负性信号，从源头上抑制效应T细胞免疫反应，期望对自身免疫病的治疗或有一定的疗效。
本申请利用pHLIP准确的将PD-L1展示在病变部位，且多肽具有半衰期较短的特点，可以避免长期抑制对机体的潜在风险。
附图说明
图1显示本发明合成肽的SDS-PAGE染色图，其中A：PD-L1-WT pHLIP；B：PDL1-Ig；C：PD-L1胞外段；D：PDL1-var3；E：PD-L1-var7；
图2显示PDL1-WT pHLIP与PD-1结合的ELISA结果图；
图3显示PD-1与PDL1蛋白结合的ELISA结果图；
图4显示PDL1-WT pHLIP与PDL1抗体结合的ELISA结果图；
图5显示PDL1-var3与PD-1结合的ELISA结果图；
图6显示PDL1-var3与PDL1抗体结合的ELISA结果图；
图7显示PDL1-var7与PD-1结合的ELISA结果图；
图8显示PDL1-var7与PDL1抗体结合的ELISA结果图；
图9显示利用confocal观察HEK293细胞上PDL1-WT pHLIP定位的荧光图；
图10显示利用confocal观察HEK293细胞上PDL1-var3定位的荧光图；
图11显示利用confocal观察HEK293细胞上PDL1-var7定位的荧光图；
图12显示PDL1-WT pHLIP处理小鼠脚掌照片；
图13显示PDL1-var3处理小鼠脚掌照片；
图14显示PDL1-var7处理小鼠脚掌照片；
图15显示PDL1-WT pHLIP处理CIA评分结果图；
图16显示PDL1-var3处理CIA评分结果图；
图17显示PDL1-var7处理CIA评分结果图；
图18显示PDL1-WT pHLIP处理血清细胞因子检测结果图；
图19显示PDL1-var3处理血清细胞因子检测结果图；
图20显示PDL1-var7处理血清细胞因子检测结果图；
图21显示PDL1-WT pHLIP处理掌跟厚度检测结果图；
图22显示PDL1-var3处理掌跟厚度检测结果图；
图23显示PDL1-var7处理掌跟厚度检测结果图。
具体实施方式
以下实例被包括以示范本发明的优选实施方式。本领域技术人员应理解的是，后文实施例中公开的技术代表发明人发现的在本发明的实践中发挥良好功能的技术，因此可被认为构成其实践的优选实施方式。但是，根据本公开，本领域技术人员应理解，可在公开的具体实施方式中进行多种改变，并仍得到同样或类似的结果，而没有背离本发明的宗旨和范围。本发明不限于本文所述的具体实施方式的范围，该实施方式意为本发明各个方面的单独示例，并且功能上等同的方法和组分也在本发明的范围内。事实上，除本文所示和所述的改变外，本发明的多种改变将由于前文描述而对于本领域技术人员显而易见。这种改变意欲落入所附权利要求的范围内。
实施例1 肽合成
野生型pHLIP(氨基酸序列如SEQ ID NO.1所示)及其变体3和7分别表示成WT pHLIP、var3和var7，均由杭州中肽生化有限公司合成。
PDL1胞外段(氨基酸序列如SEQ ID NO.18所示，核苷酸序列如SEQ ID NO.26所示)，PD-L1胞外段与野生型pHLIP、var3、var7制备而成的融合肽在本发明中分别表示为PDL1-WT pHLIP(氨基酸序列如SEQ ID NO.20所示，核苷酸序列如SEQ ID NO.23所示)、PDL1-var3(氨基酸序列如SEQ ID NO.21所示，核苷酸序列如SEQ ID NO.24所示)、PDL1-var7(氨基酸序列如SEQ ID NO.22所示，核苷酸序列如SEQ ID NO.25所示)；PDL1-Ig(氨基酸序列如SEQ ID NO.27所示，核苷酸序列如SEQ ID NO.28所示)，以上肽均由公司自行合成。
1、表达设计
按大肠杆菌表达设计基因序列，5‘端用Nde I酶切位点，3’端加终止子用XhoI酶切位点
2、菌种构建
利用酶切位点Nde I和XhoI分别构建PET28a载体，构建后转化受体菌BL21(DE3)，挑取克隆验证(测序和表达)。
3、培养诱导
将验证正确的菌种培养(LB培养基)，OD值到0.6-0.8时诱导(IPTG 0.5mM)，4-6小时离心收菌。
4、提取纯化
(1)包涵体洗涤
A液：50mM Tris，2mM EDTA，pH8.0，洗2次；
B液：50mM Tris，2mM EDTA，0.1％Triton，pH8.0，洗1次；
C液：20mM Tris，1M尿素，pH8.0，洗1次。
(2)抽提：用8M尿素，5mMβ-Me，0.3M NaCL，20mM Tris，pH8.0抽提包涵体，抽提比例1：20。
(3)复性透析：目的蛋白补加10mMβ-Me，40℃还原15分钟，稀释到0.2mg/ml用10mM PBS，50μM CuCl 2，pH8.0透析样品，换3次透析液，离心收集上清。
(4)Ni柱纯化：用0.3M NaCL，10mM PBS，pH8.0平衡色谱柱，上样后用含40mM咪唑的平衡缓冲液洗杂，用含300mM咪唑的平衡缓冲液洗脱目的蛋白。目的蛋白纯度大于95％。
(5)脱盐脱盐到20mM PBS，0.1M NaCL中。
5、考马斯亮蓝染色结果
纯化肽进行SDS-PAGE，考马斯亮蓝染色结果如图1所示，表明肽成功表达。
实施例2 PDL1-WT pHLIP结合配体能力的研究
一、PD-1与PDL1-WT pHLIP结合ELISA实验
1、实验材料
试剂：biotin生物素(BioLegend)；PDL1-WT pHLIP；PBS(Gibco)。
2、仪器
洗板机；酶标仪。
3、实验分组
PDL1-WT pHLIP组；WT pHLIP对照组；BSA对照组；BLANK组。
4、实验方法
(1)梯度包被PDL1-WT pHLIP、WT pHLIP、BSA，起始浓度50μg/ml，4℃过夜。
(2)洗板机洗板3次，酪蛋白封闭37℃1h。
(3)加入(1μg/ml)biotin-PD-1，37℃1h。
(4)加入二抗，室温避光45min。
(5)显色液显色5min。
(6)硫酸终止，酶标仪450nm读数。
二、PD-1与PDL1胞外段结合ELISA实验
1、实验材料
试剂：biotin生物素(BioLegend)；PD-L1胞外段，PBS(Gibco)。
2、仪器
洗板机；酶标仪。
3、实验分组
PDL1胞外段组；PDL1-WT pHLIP组；WT pHLIP对照组；BSA对照组；BLANK组。
4、实验方法
(1)梯度包被PDL1胞外段、PDL1-WT pHLIP、WT pHLIP、BSA，起始浓度50μg/ml，4℃过夜。
(2)洗板机洗板3次，酪蛋白封闭37℃1h。
(3)加入(1μg/ml)biotin-PD-1，37℃1h。
(4)加二抗，室温避光45min。
(5)显色液显色5min。
(6)硫酸终止，酶标仪450nm读数。
三、biotin-PDL1抗体与PDL1-WT pHLIP结合实验
1、实验材料
试剂：biotin-anti-PDL1(BioLegend)；PDL1-WT pHLIP；PBS(Gibco)。
2、仪器
洗板机；酶标仪。
3、实验分组
PDL1-WT pHLIP组；WT pHLIP对照组；BSA对照组；BLANK组。
4、实验方法
(1)梯度包被PDL1-WT pHLIP；WT pHLIP；BSA，起始浓度50μg/ml，4℃过夜。
(2)洗板机洗板3次，酪蛋白封闭37℃1h。
(3)加入biotin-anti-PDL1(1μg/ml)，37℃1h。
(4)加二抗，室温避光45min。
(5)显色液显色5min。
(6)硫酸终止，酶标仪450nm读数。
四、结果
结果如图2-4所示，PDL1-WT pHLIP与PD-1结合的EC50＝2.058μg/ml，PD-1与PDL1胞外段结合的EC50＝1.213μg/ml，PDL1-WT pHLIP与PDL1抗体结合的EC50＝0.5066μg/ml。上述结果表明，PDL1-WT pHLIP与PD-1的结合能力与PDL1胞外段相当，显示连接pHLIP不影响PDL1与PD-1的结合。
实施例3 PDL1-var3结合配体能力的研究
一、PD-1与PDL1-var3结合ELISA实验
1、实验材料
试剂：biotin生物素(BioLegend)；PDL1-var3；PBS(Gibco)。
2、仪器
洗板机；酶标仪。
3、实验分组
PDL1-var3组；var3对照组；BSA对照组；BLANK组。
4、实验方法
(1)梯度包被PDL1-var3、var3、BSA，起始浓度50μg/ml，4℃过夜。
(2)洗板机洗板3次，酪蛋白封闭37℃1h。
(3)加入(1μg/ml)biotin-PD-1，37℃1h。
(4)加入二抗，室温避光45min。
(5)显色液显色5min。
(6)硫酸终止，酶标仪450nm读数。
二、biotin-PDL1抗体与PDL1-var3结合实验
1、实验材料
试剂：biotin-anti-PDL1(BioLegend)；PDL1-var3；PBS(Gibco)。
2、仪器
洗板机；酶标仪。
3、实验分组
PDL1-var3组；var3对照组；BSA对照组；BLANK组。
4、实验方法
(1)梯度包被PDL1-var3；var3；BSA，起始浓度50μg/ml，4℃过夜。
(2)洗板机洗板3次，酪蛋白封闭37℃1h。
(3)加入biotin-anti-PDL1(1μg/ml)，37℃1h。
(4)加二抗，室温避光45min。
(5)显色液显色5min。
(6)硫酸终止，酶标仪450nm读数。
四、结果
结果如图5和6所示，PDL1-var3与PD-1结合的EC50＝1.985μg/ml，PDL1-var3与PDL1抗体结合的EC50＝0.5018μg/ml。上述结果表明，PDL1-var3与PD-1的结合能力与PDL1胞外段相当，显示连接var3不影响PDL1与PD-1的结合。
实施例4 PDL1-var7结合配体能力的研究
一、PD-1与PDL1-var7结合ELISA实验
1、实验材料
试剂：biotin生物素(BioLegend)；PDL1-var7；PBS(Gibco)。
2、仪器
洗板机；酶标仪。
3、实验分组
PDL1-var7组；var7对照组；BSA对照组；BLANK组。
4、实验方法
(1)梯度包被PDL1-var7、var7、BSA，起始浓度50μg/ml，4℃过夜。
(2)洗板机洗板3次，酪蛋白封闭37℃1h。
(3)加入(1μg/ml)biotin-PD-1，37℃1h。
(4)加入二抗，室温避光45min。
(5)显色液显色5min。
(6)硫酸终止，酶标仪450nm读数。
二、biotin-PDL1抗体与PDL1-var7结合实验
1、实验材料
试剂：biotin-anti-PDL1(BioLegend)；PDL1-var7；PBS(Gibco)。
2、仪器
洗板机；酶标仪。
3、实验分组
PDL1-var7组；var7对照组；BSA对照组；BLANK组。
4、实验方法
(1)梯度包被PDL1-var7；var7；BSA，起始浓度50μg/ml，4℃过夜。
(2)洗板机洗板3次，酪蛋白封闭37℃1h。
(3)加入biotin-anti-PDL1(1μg/ml)，37℃1h。
(4)加二抗，室温避光45min。
(5)显色液显色5min。
(6)硫酸终止，酶标仪450nm读数。
四、结果
结果如图7和8所示，PDL1-var7与PD-1结合的EC50＝1.743μg/ml，PDL1-var7与PDL1抗体结合的EC50＝0.4406μg/ml。上述结果表明，PDL1-var7与PD-1的结合能力与PDL1胞外段相当，显示连接var7不影响PDL1与PD-1的结合。
实施例5 融合肽在细胞上的定位
1、细胞系
人胚胎肾细胞系HEK293(购自ATCC)。
2、试剂
DMEM培养基(Gibco)，胎牛血清(Gibco)，PBS(pH＝7.4)(Gibco)，Tris-HCL(1mol/mL)，PD-1、PDL1胞外段和PDL1-WT pHLIP，WT pHLIP，PDL1-var7、var7、PDL1-var3、var7、PE交联试剂盒(Expedeon公司)。
3、仪器
超净台，二氧化碳孵化箱(Thermo)，离心机(Thermo)，激光共聚焦细胞培养皿(Corning)，电子pH剂(赛多利斯)，光学显微镜(Nikon)，激光共聚焦显微镜(Zeiss LSM 880)
4、实验步骤
收集处于对数期的HEK293细胞，弃去培养液，用PBS洗两次。加入适量胰酶消化后用培养液终止消化，移入10ml试管中，1000rpm离心5min，吸去上清，加入1mL含10％胎牛血清的DMEM培养基重悬混匀。吸10μL细胞悬液到细胞计数板中计数，取一定量的细胞悬液加入激光共聚焦培养皿中，用培养基调整至5*10 5，1mL的细胞体系，放入孵箱中培养过夜。
待培养过后的HEK293细胞贴壁后，吸去上清，用pH7.4的PBS缓冲液洗涤两次。
使用1mol/mL的盐酸与pH7.4的PBS缓冲液制备培养液，将盐酸逐滴加入PBS中，最终滴定缓冲液的pH值至6.3(5mL PBS缓冲液+12μLTris-盐酸)。将PDL1-WT pHLIP，或PDL1-var3，或PDL1-var7分别加入pH为7.4和6.3的PBS缓冲液中充分混匀，按比例稀释肽至终浓度为200μg/mL。对照组在pH为7.4和6.3的PBS缓冲液中加入终浓度为180μg/mL的PDL1胞外段和终浓度为20μg/mL的WT pHLIP，或var3，或var7。
将培养皿中的PBS缓冲液吸去，分别加入上述混合液1mL，空白对照组中加入1mL未混合的pH分别为7.4和6.3的PBS缓冲液，放入37度细胞孵箱中孵育1小时。
孵育结束后吸去上清，用对应pH的PBS缓冲液洗两遍，加入1μg/mL PD-1-PE 1mL，4度避光孵育半小时。
孵育结束后，用对应pH的PBS洗两遍，后加入对应pH的PBS缓冲液。
将制备好的培养皿放在激光共聚焦显微镜下观察细胞膜表面荧光情况。
5、结果
结果见图9，在酸性环境中，PDL1-pHLIP能够很好的展示在细胞膜表面。上述结果表明，PDL1不影响其低pH插入肽插入细胞膜的特性，同时PDL1与低pH插入肽连接也不影响其构象。
结果见图10，在酸性环境中，PDL1-var3能够很好的展示在细胞膜表面。上述结果表明，PDL1不影响其低pH插入肽插入细胞膜的特性，同时PDL1与低pH插入肽连接也不影响其构象。
结果见图11，在酸性环境中，PDL1-var7能够很好的展示在细胞膜表面。上述结果表明，PDL1不影响其低pH插入肽插入细胞膜的特性，同时PDL1与低pH插入肽连接也不影响其构象。
实施例6 融合肽治疗自身免疫病的效果评价
1、实验材料
动物：DBA/1J雄性小鼠(7周龄)。
2、试剂
鸡II型胶原(#20012)，完全弗氏佐剂(#7001)，不完全弗氏佐剂(#7002)，均是Chondrex，USA，WT pHLIP、var3、var7由杭州中肽生化有限公司合成，PDL1胞外段、PDL1-WT pHLIP、PDL1-var3、PDL1-var7、PDL1-Ig由本公司自己制备。
3、仪器
组织匀浆机(IKA，H44)。
4、实验方法
抗原的乳化
将等体积的佐剂，加入5ml一次性针管中，打开组织匀浆机，边低速搅拌边滴入胶原溶液，胶原全部加入后，加大转速，直至形成油包水样的乳滴。此过程将针管冰浴，防止蛋白变性。
第一次免疫，将2mg/ml的胶原与等体积的完全弗氏佐剂(4mg/ml)混合，并进行乳化(形成油包水样)后，最终胶原的浓度为1mg/ml，每只小鼠注射100μl，即100μg。从尾根部2cm处插入针头，直到针尖插入位置距尾根部0.5cm处开始注射。
第二次免疫，将2mg/ml的胶原与等体积的不完全弗氏佐剂混合，并进行乳化，每只小鼠注射100μg。注射部位在尾根部3cm处，针尖插入皮下距离鼠尾根部1.5cm处。
第二次免疫后2周(此时已出现CIA典型症状)，分成4个组：(1)腹腔注射PDL1-WT pHLIP(或PDL1-var3，或PDL1-var7)，1mg/kg，隔天给药一次；(2)腹腔注射PDL1(1mg/kg)+WT pHLIP(0.2mg/kg)(或PDL1-var3，或PDL1-var7)，隔天给药一次；(3)腹腔注射PDL1-Ig，1mg/kg，隔天给药一次；(4)对照组腹腔注射PBS。每组10只小鼠，26天后，检测掌跟厚度和血清细胞因子含量。
3、结果
图12-14的小鼠脚掌照片显示PDL1-pHLIP组，PDL1-var3组，或PDL1-var7组小鼠脚掌正常，炎症显著缓解。
CIA评分结果见图15-17，PDL1-pHLIP组，PDL1-var3组，或PDL1-var7组CIA评分明显降低。
血清细胞因子检测结果见图18-20，PDL1-pHLIP组，PDL1-var3组，或PDL1-var7组TNF-α、IFN-γ、IL-6、IL-17A含量显著降低。
掌跟厚度检测结果见图21-23，PDL1-pHLIP组，PDL1-var3组，或PDL1-var7组小鼠掌跟厚度显著降低。
上述结果表明，本发明的融合肽治疗能够显著减轻关节炎的症状，显示对关节炎有很好的治疗作用。

Claims (37)

  1. 一种低pH插入肽的融合肽，其特征在于，所述融合肽包括低pH插入肽，免疫检查点配体或其片段。
  2. 根据权利要求1所述的融合肽，其特征在于，所述低pH插入肽序列包括SEQ ID NO.1-17所示的序列中任一个。
  3. 根据权利要求1所述的融合肽，其特征在于，所述低pH插入肽序列包括SEQ ID NO.1-17所示的序列中胞外段序列重复一次或多次形成的序列。
  4. 根据权利要求1-4中任一项所述的融合肽，其特征在于，免疫检查点包括PD-1、Lag-3、Tim-3、TIGIT、CTLA-4。
  5. 根据权利要求4所述的融合肽，其特征在于，免疫检查点是PD-1。
  6. 根据权利要求5所述的融合肽，其特征在于，PD-1配体包括PDL1、PDL2；PDL1片段包括PD-L1的胞外段。
  7. 根据权利要求6所述的融合肽，其特征在于，PDL1蛋白的胞外段是SEQ ID NO.18或SEQ ID NO.19所示的氨基酸形成的多肽或SEQ ID NO.18或SEQ ID NO.19所示的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加且与SEQ ID NO.18或SEQ ID NO.19所示的序列具有相同功能的由SEQ ID NO.18或SEQ ID NO.19所示的氨基酸序列衍生的多肽。
  8. 根据权利要求7所述的融合肽，其特征在于，所述PDL1蛋白或其片段通过Linker连接于所述低pH插入肽的N端。
  9. 根据权利要求8所述的融合肽，其特征在于，所述Linker序列是(GGGS)m或(GGGGS)m，其中m＝自然数。
  10. 根据权利要求9所述的融合肽，其特征在于，所述Linker序列是GGGS。
  11. 根据权利要求1-10中任一项所述的融合肽，其特征在于，所述融合肽的序列如SEQ ID NO.20-22所示。
  12. 一种治疗自身免疫病的药物组合物，其特征在于，所述药物组合物包括权利要求1-10中任一项所述的融合肽。
  13. 一种标记系统，其特征在于，所述标记系统包括权利要求1-10中任一项所述的融合肽。
  14. 一种自身免疫病治疗方法，其特征在于，所述方法包括给有需要者施用权利要求1-10中任一项所述的融合肽或权利要求12所述的药物组合物。
  15. 一种在病灶组织细胞膜上标记免疫检查点配体或其片段的方法，其特征在于，所述方法包括将免疫检查点配体或其片段与低pH插入肽连接形成融合肽，将所述融合肽导入病灶组织并插入到病灶组织细胞膜上。
  16. 根据权利要求15所述的方法，其特征在于，所述免疫检查点包括权利要求1-10中任一项所述的免疫检查点；所述免疫检查点配体包括权利要求1-10中任一项所述的免疫检查点配体；免疫检查点配体片段包括权利要求1-10中任一项所述的免疫检查点配体片段。
  17. 根据权利要求15所述的方法，其特征在于，所述低pH插入肽包括权利要求1-10中任一项所述的低pH插入肽。
  18. 权利要求1-10中任一项所述的融合肽在制备治疗自身免疫病的药物中的应用。
  19. 根据权利要求18所述的应用，其特征在于，所述自身疫病包括斑秃、强直性脊柱炎、抗磷脂综合征、自身免疫性艾迪森病、肾上腺的自身免疫病、自身免疫性溶血性贫血、自身免疫肝炎、自身免疫性卵巢炎和睾丸炎、自身免疫性血小板减少、贝切特综合征、大疱型类天疱疮、心肌病、口炎性腹泻皮炎、慢性疲劳免疫功能障碍综合征、慢性炎性脱髓鞘多神经病、丘施二氏综合征、疤痕性类天疱疮、CREST综合征、冷凝集素病、克罗恩氏病、盘状狼疮、特发性混合型冷球蛋白血症、糖尿病、嗜酸细胞性筋膜炎、纤维肌痛-纤维肌炎、肾小球肾炎、格拉夫斯病、格-巴二氏综合征、桥本甲状腺炎、亨-舍二氏紫癜、特发性肺纤维化、特发性/自身免疫血小板减少性紫癜、IgA神经病、青少年关节炎、扁平苔藓、红斑狼疮、美尼尔综合征、混合型结缔组织病、多发性硬化、1型或免疫介导的糖尿病、重症肌无力、天疱疮相关疾病、恶性贫血、结节性多动脉炎、多 软骨炎、多腺体综合征、风湿性多肌痛、多肌炎、皮肌炎、原发性无Y球蛋白血症、原发性胆汁性肝硬化、牛皮癣、牛皮癣关节炎、雷氏现象、莱特尔综合征、类风湿性关节炎、结节病、硬皮病、斯耶格伦综合征、全身肌强直综合征、系统性红斑狼疮、斯维特综合征、斯提耳病、红斑狼疮、高安动脉炎、暂时性动脉炎/巨细胞动脉炎、溃疡性结肠炎、葡萄膜炎、血管炎、白癜风、韦格纳肉芽肿病。
  20. 免疫检查点配体或其片段在制备权利要求1-10中任一项所述的融合肽中的应用。
  21. 根据权利要求20所述的应用，其特征在于，所述免疫检查点包括权利要求1-10中任一项所述的免疫检查点；所述免疫检查点配体包括权利要求1-10中任一项所述的免疫检查点配体；免疫检查点配体片段包括权利要求1-10中任一项所述的免疫检查点配体片段。
  22. 免疫检查点配体或其片段在制备治疗自身免疫病的药物中的应用。
  23. 根据权利要求22所述的应用，其特征在于，所述免疫检查点包括权利要求1-10中任一项所述的免疫检查点；所述免疫检查点配体包括权利要求1-10中任一项所述的免疫检查点配体；免疫检查点配体片段包括权利要求1-10中任一项所述的免疫检查点配体片段。
  24. 根据权利要求22所述的应用，其特征在于，所述自身疫病包括斑秃、强直性脊柱炎、抗磷脂综合征、自身免疫性艾迪森病、肾上腺的自身免疫病、自身免疫性溶血性贫血、自身免疫肝炎、自身免疫性卵巢炎和睾丸炎、自身免疫性血小板减少、贝切特综合征、大疱型类天疱疮、心肌病、口炎性腹泻皮炎、慢性疲劳免疫功能障碍综合征、慢性炎性脱髓鞘多神经病、丘施二氏综合征、疤痕性类天疱疮、CREST综合征、冷凝集素病、克罗恩氏病、盘状狼疮、特发性混合型冷球蛋白血症、糖尿病、嗜酸细胞性筋膜炎、纤维肌痛-纤维肌炎、肾小球肾炎、格拉夫斯病、格-巴二氏综合征、桥本甲状腺炎、亨-舍二氏紫癜、特发性肺纤维化、特发性/自身免疫血小板减少性紫癜、IgA神经病、青少年关节炎、扁平苔藓、红斑狼疮、美尼尔综合征、混合型结缔组织病、多发性硬化、1型或免疫介导的糖尿病、重症肌无力、天疱疮相关疾病、恶性贫血、结节性多动脉炎、多软骨炎、多腺体综合征、风湿性多肌痛、多肌炎、皮肌炎、原发性无Y球蛋白血症、原发性胆汁性肝硬化、牛皮癣、牛皮癣关节炎、雷氏现象、莱特尔综合征、 类风湿性关节炎、结节病、硬皮病、斯耶格伦综合征、全身肌强直综合征、系统性红斑狼疮、斯维特综合征、斯提耳病、红斑狼疮、高安动脉炎、暂时性动脉炎/巨细胞动脉炎、溃疡性结肠炎、葡萄膜炎、血管炎、白癜风、韦格纳肉芽肿病。
  25. 免疫检查点配体或其片段在制备身免疫病病灶组织细胞标记系统中的应用。
  26. 根据权利要求25所述的应用，其特征在于，所述标记系统包括权利要求13所述的标记系统。
  27. 根据权利要求25所述的应用，其特征在于，所述免疫检查点包括权利要求1-10中任一项所述的免疫检查点；所述免疫检查点配体包括权利要求1-10中任一项所述的免疫检查点配体；免疫检查点配体片段包括权利要求1-10中任一项所述的免疫检查点配体片段。
  28. 权利要求1-10中任一项所述的融合肽在制备自身免疫病病灶组织细胞标记系统中的应用。
  29. 根据权利要求28所述的应用，其特征在于，所述标记系统包括权利要求13所述的标记系统。
  30. 低pH插入肽在制备权利要求1-10中任一项所述的融合肽中的应用。
  31. 根据权利要求30所述的应用，其特征在于，所述低pH插入肽包括权利要求1-10中任一项所述的低pH插入肽。
  32. 低pH插入肽在制备治疗自身免疫病的药物中的应用。
  33. 根据权利要求32所述的应用，其特征在于，所述低pH插入肽包括权利要求1-10中任一项所述的低pH插入肽。
  34. 根据权利要求32所述的应用，其特征在于，所述自身疫病包括斑秃、强直性脊柱炎、抗磷脂综合征、自身免疫性艾迪森病、肾上腺的自身免疫病、自身免疫性溶血性贫血、自身免疫肝炎、自身免疫性卵巢炎和睾丸炎、自身免疫性血小板减少、贝切特综合征、大疱型类天疱疮、心肌病、口炎性腹泻皮炎、慢性疲劳免疫功能障碍综合征、慢性炎性脱髓鞘多神经病、丘施二氏综合征、疤痕性类天疱疮、CREST综合征、冷凝集素病、克罗恩氏病、盘状狼疮、特发性混合 型冷球蛋白血症、糖尿病、嗜酸细胞性筋膜炎、纤维肌痛-纤维肌炎、肾小球肾炎、格拉夫斯病、格-巴二氏综合征、桥本甲状腺炎、亨-舍二氏紫癜、特发性肺纤维化、特发性/自身免疫血小板减少性紫癜、IgA神经病、青少年关节炎、扁平苔藓、红斑狼疮、美尼尔综合征、混合型结缔组织病、多发性硬化、1型或免疫介导的糖尿病、重症肌无力、天疱疮相关疾病、恶性贫血、结节性多动脉炎、多软骨炎、多腺体综合征、风湿性多肌痛、多肌炎、皮肌炎、原发性无Y球蛋白血症、原发性胆汁性肝硬化、牛皮癣、牛皮癣关节炎、雷氏现象、莱特尔综合征、类风湿性关节炎、结节病、硬皮病、斯耶格伦综合征、全身肌强直综合征、系统性红斑狼疮、斯维特综合征、斯提耳病、红斑狼疮、高安动脉炎、暂时性动脉炎/巨细胞动脉炎、溃疡性结肠炎、葡萄膜炎、血管炎、白癜风、韦格纳肉芽肿病。
  35. 低pH插入肽在制备自身免疫病病灶组织细胞标记系统中的应用。
  36. 根据权利要求35所述的应用，其特征在于，所述低pH插入肽包括权利要求1-10中任一项所述的低pH插入肽。
  37. 根据权利要求35所述的应用，其特征在于，所述标记系统包括权利要求13所述的标记系统。
PCT/CN2020/085447 2020-04-18 2020-04-18 一种治疗自身免疫病的融合肽 WO2021208106A1 (zh)

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Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103917243A (zh) * 2011-10-17 2014-07-09 海莱乌医院 基于pd-l1的免疫疗法
WO2017220602A1 (en) * 2016-06-21 2017-12-28 Herlev Hospital Pdl1 peptides for use in cancer vaccines
CN109467607A (zh) * 2017-12-28 2019-03-15 北京泽勤生物医药有限公司 一种靶向肿瘤的酸性敏感融合肽及其应用
CN110713522A (zh) * 2018-11-30 2020-01-21 北京泽勤生物医药有限公司 低pH插入肽的胞外段作为抗原的应用
CN110760008A (zh) * 2018-11-30 2020-02-07 北京泽勤生物医药有限公司 低pH插入肽的融合蛋白、药物组合物及应用
CN110790829A (zh) * 2017-12-19 2020-02-14 北京泽勤生物医药有限公司 pHLIP胞外段作为抗原制备的抗体在制备抗肿瘤药物中的应用

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103917243A (zh) * 2011-10-17 2014-07-09 海莱乌医院 基于pd-l1的免疫疗法
WO2017220602A1 (en) * 2016-06-21 2017-12-28 Herlev Hospital Pdl1 peptides for use in cancer vaccines
CN110790829A (zh) * 2017-12-19 2020-02-14 北京泽勤生物医药有限公司 pHLIP胞外段作为抗原制备的抗体在制备抗肿瘤药物中的应用
CN109467607A (zh) * 2017-12-28 2019-03-15 北京泽勤生物医药有限公司 一种靶向肿瘤的酸性敏感融合肽及其应用
CN110713522A (zh) * 2018-11-30 2020-01-21 北京泽勤生物医药有限公司 低pH插入肽的胞外段作为抗原的应用
CN110760008A (zh) * 2018-11-30 2020-02-07 北京泽勤生物医药有限公司 低pH插入肽的融合蛋白、药物组合物及应用

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
JIA XUELI, ZHANG JIA ZHAO TING DU QING CAO DEYING XIANG BAI GENG GEXIA QI XIANRONG: "Research situation of pH low insertion peptides", ACTA PHARMACEUTICA SINICA, YAOXUE XUEBAO, CN, vol. 53, no. 3, 31 December 2018 (2018-12-31), CN , pages 375 - 382, XP055857238, ISSN: 0513-4870, DOI: 10.16438/j.0513-4870.2017-0830 *
WEI YUSHUANG; LIAO RUFANG; MAHMOOD ABDULRAHMAN AHMED; XU HAIBO; ZHOU QIBING: "pH-responsive pHLIP (pH low insertion peptide) nanoclusters of superparamagnetic iron oxide nanoparticles as a tumor-selective MRI contrast agent", ACTA BIOMATERIALIA, ELSEVIER, AMSTERDAM, NL, vol. 55, 29 March 2017 (2017-03-29), AMSTERDAM, NL, pages 194 - 203, XP085034272, ISSN: 1742-7061, DOI: 10.1016/j.actbio.2017.03.046 *

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