CN111454371A

CN111454371A - PD L1-pH L IP, preparation method and application thereof in treatment of autoimmune disease

Info

Publication number: CN111454371A
Application number: CN202010308370.7A
Authority: CN
Inventors: 魏化伟; 杨承刚
Original assignee: Beijing Zeqin Biomedical Co ltd
Current assignee: Beijing Zeqin Biomedical Co ltd
Priority date: 2020-04-18
Filing date: 2020-04-18
Publication date: 2020-07-28
Anticipated expiration: 2040-04-18
Also published as: CN111454371B

Abstract

The invention discloses PD L1-pH L IP, a preparation method and application thereof in treating autoimmune diseases.A fusion peptide of the invention is formed by connecting a low-pH insertion peptide with an extracellular segment of PD L1. under an acidic environment, the low-pH insertion peptide can be inserted into a cell membrane of focal tissue, the PD L1 connected with the low-pH insertion peptide is targeted and positioned at a focal part by utilizing the properties of the low-pH insertion peptide, and negative signals of the focal tissue PD-1/PD-L1 are enhanced by utilizing the low-pH insertion peptide, so that the immune reaction of an effector T cell is inhibited from the source, thereby playing a role in preventing and treating the autoimmune diseases.

Description

PD L1-pH L IP, preparation method and application thereof in treatment of autoimmune disease

Technical Field

The invention belongs to the field of biological medicines, and particularly relates to PD L1-pH L IP, a preparation method and application thereof in treating autoimmune diseases.

Background

The autoimmune disease is mainly caused by the fact that an organism has an immune response to self-antigen, immune cells infiltrate in large quantity, and self-tissue damage is caused by abnormal metabolism of immune tissues.

Autoimmune diseases can be divided into two major classes according to the range of pathological tissue involvement, the first class is organ-specific autoimmune diseases, which can involve multiple organs including brain, thyroid, stomach; the second category is non-organ specific autoimmune diseases, which can involve multiple tissues such as muscle, skin, joints, etc. At present, more than 100 autoimmune diseases are known, about 5% of the population is involved, and common systemic lupus erythematosus, rheumatoid arthritis, systemic vasculitis, scleroderma and the like are known.

The extracellular pH is typically between 6.2 and 7.0, creating an acidic extracellular microenvironment.A low pH insertion peptide derived from the transmembrane helix protein C of bacteriorhodopsin (pH L IP, pH low insertion peptide) happens to be a polypeptide carrier that can target this slightly acidic environment.pH L IP consists of flanking sequences consisting of protonated amino acid residues and Transmembrane (TM) sequences consisting of hydrophobic residues.pH L IP does not form a single helix in neutral solution, but pH L IP can form a C-helix structure and insert onto the cell membrane in the presence of a lipid bilayer.

There are generally three states of pH L IP, either in solution (state I) or weakly bound to the membrane (state II) in healthy tissue at pH around 7.4, pH L IP is washed from the membrane by normal perfusion and continues to circulate in the body in inflammatory tissue at acidic pH L IP spontaneously folds into a transmembrane helix and intercalates into the membrane (state III).

At present, the application of pH L IP in nuclear imaging, fluorescence-guided surgery, gene therapy and nanotechnology is very wide, and the development of a medicament beneficial to treating autoimmune diseases by utilizing the acidic environment trend of pH L IP is a future research hotspot.

Disclosure of Invention

The method is completed based on the following conception that the PD-L1 extracellular segment is connected with the N end of pH L IP, the PD-L1 extracellular segment is displayed on the tissue cell membrane of a lesion part through pH L IP, the negative signal of the lesion tissue PD-1/PD-L1 is enhanced by utilizing the pH L IP, the immune response of an effect T cell is inhibited from the source, and a certain effect is played on the prevention and the treatment of the occurrence and the development of the autoimmune disease.

One of the purposes of the invention is to provide a fusion peptide formed by PD-L1 and a low-pH insertion peptide.

The invention also aims to provide a pharmaceutical composition for treating autoimmune diseases, which is formed by the fusion peptide.

The invention also aims to provide application of PD-L1 in preparing the fusion peptide.

The third object of the present invention is to provide the use of the above fusion peptide.

In order to achieve the purpose, the invention adopts the following technical scheme:

according to one aspect of the present invention, there is provided a fusion peptide of a low pH insertion peptide, the fusion peptide comprising the low pH insertion peptide, an immune checkpoint ligand, or a fragment thereof.

Immune checkpoints include PD-1, L ag-3, Tim-3, TIGIT, CT L A-4.

The PD-1 ligand comprises PD-L1 and PD-L2.

L ag-3 ligands include fibrinogen-like protein 1, L SECtin.

The Tim-3 ligand comprises galectin-9, phosphatidylserine, high mobility group protein B1 and Ceacam-1.

TIGIT ligands include CD155, CD 122.

The CT L A-4 ligand includes CD86(B7-2) and CD80 (B7-1).

The low pH insertion peptide useful for constructing the fusion peptide of the present invention includes a polypeptide having the sequence shown in SEQ ID NO.1 or a variant thereof.

The polypeptide with the sequence shown as SEQ ID NO.1 is abbreviated as WT in the invention, and the variant of WT comprises Var1-Var 16.

The sequences of WT and its variants are as follows:

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)。

the underlined part indicates the extracellular domain of the low pH insertion peptide.

The peptides formed after one or more repetitions of the extracellular domain of the WT, Var1-Var16 peptide ((extracellular domain) n + low pH insertion peptide, where n ═ 1, 2, 3 … …) also fall within the scope of the low pH insertion peptides of the present invention.

The PD-L1 protein or fragment thereof of the invention is linked to the N-terminus of the low pH insertion peptide via L inker.

The L inker is used conventionally in the art, and the sequence can be (GGGS) m, or (GGGGS) m, wherein m is a natural number.

Preferably, the L inker sequence is GGGS.

In a particular embodiment of the invention, the immune checkpoint used is PD-1, the ligand of which is selected to be PD-L1.

In a specific embodiment of the invention, the fragment of PD-L1 linked to the low pH insertion peptide sequence is the extracellular domain of the PD-L1 protein.

Furthermore, the sequence of the extracellular segment of the PD-L1 protein is shown in SEQ ID NO.18 or SEQ ID NO.19, the polypeptide which is derived from the amino acid sequence shown in SEQ ID NO.18 or SEQ ID NO.19 and has the same function with the sequence shown in SEQ ID NO.18 or SEQ ID NO.19 after the substitution and/or deletion and/or addition of one or more amino acid residues of the amino acid sequence shown in SEQ ID NO.18 or SEQ ID NO.19 also belongs to the extracellular segment of the PD-L1 protein, namely the extracellular segment of the PD-L1 protein comprises a wild type and a variant thereof.

The extracellular domain variant of the 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, more preferably at least about 90% to 95% homology, often 96%, 97%, 98%, 99% homology with the amino acid sequence shown in SEQ ID NO.18 or SEQ ID NO. 19.

In general, it is known that modification of one or more amino acids in a protein or polypeptide does not affect the function of the protein. One skilled in the art will recognize that individual amino acid changes or small percentage amino acids or individual additions, deletions, insertions, substitutions to an amino acid sequence are conservative modifications, wherein a change in a protein polypeptide results in a protein or polypeptide with similar function. Conservative substitution tables providing functionally similar amino acids are well known in the art.

Extracellular domain variants of the 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 partner.

The fusion peptide constructed by using the extracellular segment of the PD-L1 protein and the low-pH insertion peptide is represented by PD L1-WT pH L IP, the amino acid sequence of which is shown as SEQ ID NO.20 in the specific embodiment of the invention, PD L1-var 3, the amino acid sequence of which is shown as SEQ ID NO.21 in the specific embodiment of the invention, and PD L1-var 7, the amino acid sequence of which is shown as SEQ ID NO.22 in the specific embodiment of the invention.

According to still another aspect of the present invention, there is provided a pharmaceutical composition for treating autoimmune diseases, comprising the fusion peptide as described above.

Further, the pharmaceutical composition also comprises a pharmaceutically acceptable carrier.

Examples of pharmaceutically acceptable carriers include, but are not limited to: water; aqueous vehicles such as, but not limited to, sodium chloride injection, ringer's injection, dextrose and sodium chloride injection, and lactated ringer's injection; water miscible carriers such as, but not limited to, ethanol, polyethylene glycol, and polypropylene glycol; and nonaqueous carriers such as, but not limited to, corn oil, cottonseed oil, peanut oil, sesame oil, ethyl oleate, isopropyl myristate, and benzyl benzoate.

Still further, the pharmaceutical composition may further comprise other drugs for treating autoimmune diseases.

The pharmaceutical compositions of the present invention may be administered to a human patient by any route, including but not limited to: intravenous, intradermal, transdermal, subcutaneous, intramuscular, inhalation (e.g., by aerosol), buccal (e.g., sublingual), topical (i.e., skin and mucosal surfaces, including airway surfaces), intrathecal, intraarticular, intrapleural, intracerebral, intraarterial, intraperitoneal, oral, intralymphatic, intranasal, rectal, or vaginal administration, by local catheter infusion, or direct injection into a lesion. In one embodiment, the compositions of the present invention are administered by intravenous bolus injection or intravenous infusion over a given time (0.5-2 hours). Can be administered by peristaltic pump, or in long acting form

The pharmaceutical compositions of the present invention are delivered, but as is understood in the art, the most suitable route in any given case will depend upon factors such as the species, age, sex and general health of the subject, the nature and severity of the condition being treated and/or the nature of the particular composition (i.e., dose, dosage form) being administered. In particular embodiments, the route of administration is bolus or continuous infusion over a period of time, once or twice weekly. In other particular embodiments, the route of administration is subcutaneous injection, optionally once or twice weekly. In one embodiment, the pharmaceutical composition of the invention is administered to an outpatient.

In certain embodiments, the dosage of the pharmaceutical composition of the invention is measured in mg/kg of patient body weight. In other embodiments, the dosage of the pharmaceutical composition is measured in mg/m2 of the body surface area of the patient. In other embodiments, the dosage of the pharmaceutical composition is measured in mg/dose administered to the patient. Any dose-measuring method can be used in conjunction with the compositions and methods of the present invention, and dosage units can be converted by means standard in the art.

It will be understood by those skilled in the art that the dosage may be selected based on a variety of factors, including the age, sex, species and condition of the subject, the degree of cell depletion desired, and the dosage may be determined by those skilled in the art. For example, an effective amount of a pharmaceutical composition of the invention can be extrapolated from a dose-response curve from an in vitro test system or an animal model test system.

It will be appreciated by those skilled in the art that the initial treatment dose will generally be higher and/or the frequency of administration will be higher compared to a maintenance regimen.

According to a further aspect of the invention there is provided the use of an immune checkpoint ligand or fragment thereof in the preparation of a fusion peptide as hereinbefore described.

The immune checkpoint ligand or fragment thereof is as defined above.

According to a further aspect of the present invention there is provided the use of a fusion peptide as hereinbefore described in the manufacture of a medicament for the treatment of an autoimmune disease.

The term "autoimmune disease" refers to a condition of a subject characterized by cellular, tissue and/or organ damage resulting from 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 gland, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune oophoritis and orchitis, autoimmune thrombocytopenia, Behcet's syndrome, bullous pemphigoid, cardiomyopathy, sprue diarrhea dermatitis, chronic fatigue immune dysfunction syndrome, chronic inflammatory demyelinating polyneuropathy, Cushing's syndrome, cicatricial pemphigoid, CREST syndrome, cold agglutinin disease, Crohn's disease, discoid lupus, idiopathic mixed cryoglobulinemia, diabetes, eosinophilic fasciitis, fibromyalgia-fibromyositis, glomerulonephritis, Graves ' disease, Guillain-Barre syndrome, Hashimoto's thyroiditis, Henschel-Schnei's purpura, Idiopathic pulmonary fibrosis, idiopathic/autoimmune thrombocytopenic purpura, IgA neuropathy, juvenile arthritis, lichen planus, lupus erythematosus, meniere's syndrome, mixed connective tissue disease, multiple sclerosis, type 1 or immune-mediated diabetes, myasthenia gravis, pemphigus-related diseases, pernicious anemia, polyarteritis nodosa, polychondritis, polyadenylic syndrome, polymyalgia rheumatica, polymyositis, dermatomyositis, primary aspergillosis, primary biliary cirrhosis, psoriasis, psoriatic arthritis, leiomyelia, reiter's syndrome, rheumatoid arthritis, sarcoidosis, scleroderma, sjogren's syndrome, systemic myotonic syndrome, systemic lupus erythematosus, svt's syndrome, stewart's disease, lupus erythematosus, takayasu's arteritis, giant cell arteritis, and the like, Ulcerative colitis, uveitis, vasculitis, vitiligo, and wegener granulomatosis.

Further, the medicament comprises the pharmaceutical composition as described above.

The medicament or the pharmaceutical composition of the invention can be used in combination with other medicaments for treating autoimmune diseases.

Common drugs for the treatment of rheumatoid arthritis include NSAIDs (non-limiting examples include, but are not limited to, aspirin, diflunisal, diclofenac, etodolac, fenamates, fenoprofen, flurbiprofen, ibuprofen, pentostatin, ketoprofen, methyl salicylate, nabumetone, naproxen, piperazines, phenylbutazone, piroxicam, sulindac, and tolmetin), analgesics (non-limiting examples are acetaminophen, phenacetin, and tramadol), CSI, including, but not limited to, celecoxib and rofecoxib, glucocorticoids (preferably low dose oral glucocorticoids, such as <7.5mg/D prednisone, or monthly pulsed administration of high dose glucocorticoids, or intra-articular glucocorticoids), conditioned antirheumatics (DMAEMA), including, but not limited to, methotrexate RD (preferably given intermittent low dose, such as 7.5-30mg once a week), gold compounds (such as gold salts), D-chloropenimine (such as penicillamine), and azapiroxicam, including, and sulfadoxoramide (non-immune inhibitors, including, without limitation: α).

Common drugs for treating osteoarthritis include, but are not limited to: analgesics (a non-limiting example being acetaminophen at doses up to 4000 mg/d; phenacetin; tramadol at daily doses ranging from 200 to 300 mg); NSAIDs (non-limiting examples include, but are not limited to, aspirin, diflunisal, diclofenac, etodolac, fenamates, fenoprofen, flurbiprofen, ibuprofen, indomethacin, ketoprofen, methyl salicylate, nabumetone, naproxen, perazine, phenylbutazone, piroxicam, sulindac, and tolmetin.Low dose NSAIDs are preferred, e.g., 1200mg/d ibuprofen, 500mg/d naproxen.gastroprotectants, such as misoprostol, famotidine, or omeprazole, are preferably used in combination with NSAIDs; non-acetylated salicylates include, but are not limited to, salsalate; cyclooxygenase (Cox) -2-specific inhibitors (CSI) include, but are not limited to, celecoxib and rofecoxib; glucocorticoid formulations; hyaluronic acid; capsaicin.

According to a further aspect of the invention there is provided a labelling system comprising a fusion peptide as hereinbefore described.

Further, the labeling system may further include Cyanine 5.5, Alexa Flour 750, Alexa Fluor647, Alexa Flour 488, Alexa Flour 546, Alexa Flur 546, and,⁶⁴Cu-DOTA、⁶⁸Ga-DOTA、¹⁸F-O-pyridine、¹⁸F-liposomes、liposomal Rhodamine、Nanogold、TAMRA。

The fusion peptide in the labeling system is inserted into a cell membrane under the action of the low-pH insertion peptide, PD L1 in the fusion peptide is positioned on the surface of a target cell, a PD L1 ligand (such as PD-1) on the surface of an immune cell recognizes PD L1 on the surface of the target cell, and a PD L1 ligand is combined with PD L1, so that the function of the immune cell is inhibited.

According to still another aspect of the present invention, there is provided the use of the fusion peptide as described above for preparing a focal tissue cell marking system for autoimmune diseases.

Further, the marking system is as described above.

According to a further aspect of the present invention there is provided a method of treatment of an autoimmune disease, the method comprising administering to a subject in need thereof a fusion peptide as hereinbefore described or a pharmaceutical composition as hereinbefore described.

According to yet another aspect of the present invention, there is provided a method of marking an immune checkpoint ligand or a fragment thereof on a cell membrane of focal tissue, the method comprising linking the immune checkpoint ligand or a fragment thereof to a low pH insertion peptide.

The immune checkpoint comprises the immune checkpoint described above; the immune checkpoint ligand comprises the immune checkpoint ligand described previously; the immune checkpoint ligand fragment includes the immune checkpoint ligand fragment described previously.

The low pH insertion peptide includes the low pH insertion peptide described above.

According to a further aspect of the invention there is provided the use of an immune checkpoint ligand, or a fragment thereof, in the manufacture of a medicament for the treatment of an autoimmune disease.

Further, the immune checkpoint comprises the immune checkpoint described above; the immune checkpoint ligand comprises the immune checkpoint ligand described previously; the immune checkpoint ligand fragment includes the immune checkpoint ligand fragment described previously.

Further, the autoimmune disease is defined as described above.

According to a further aspect of the present invention there is provided the use of an immune checkpoint ligand or fragment thereof in the preparation of a focal tissue cell marking system for an autoimmune disease.

Further, the marking system comprises the marking system described above.

According to a further aspect of the invention, there is provided the use of a low pH insertion peptide in the preparation of a fusion peptide as hereinbefore described.

Further, the low pH insertion peptide is defined as described above.

According to a further aspect of the invention there is provided the use of a low pH insertion peptide in the manufacture of a medicament for the treatment of an autoimmune disease.

Further, the low pH insertion peptide is defined as described above.

Further, the autoimmune disease is defined as described above.

According to still another aspect of the present invention, there is provided a use of a low pH insertion peptide for preparing a focal tissue cell marking system for autoimmune diseases.

Further, the low pH insertion peptide is defined as described above.

Further, the autoimmune disease is defined as described above.

Further, the marking system comprises the marking system described above.

The sequences of the invention are listed in order from the N-terminus to the C-terminus.

"PD L1" and "PD-L1" are used interchangeably herein.

The invention has the following advantages and beneficial effects:

at present, most of clinical and preclinical researches on autoimmune diseases are to block the function of a certain cytokine (I L-6/TNF- α/I L-17) by using an antibody or an antagonist so as to achieve the aim of relieving the diseases, however, the autoimmune diseases are generated as a result of the synergistic effect of multiple factors, pH L IP is used for enhancing the negative signal of focal tissue PD-1/PD-L1, effector T cell immune response is inhibited from the source, and the treatment or certain curative effect on the autoimmune diseases is expected.

The application utilizes pH L IP to accurately display PD-L1 at a pathological part, and the polypeptide has the characteristic of short half-life, so that the potential risk of inhibiting an organism for a long time can be avoided.

Drawings

FIG. 1 shows SDS-PAGE staining patterns of synthetic peptides of the invention, wherein A is PD-L1-WT pH L IP, B is PD L1-Ig, C is PD-L1 extracellular domain, D is PD L1-var 3, E is PD-L1-var 7;

FIG. 2 shows the results of the E L ISA results of PD L1-WT pH L IP binding to PD-1;

FIG. 3 shows a graph of the results of the E L ISA binding of PD-1 to PD L1 protein;

FIG. 4 shows the results of the E L ISA results of PD L1-WT pH L IP binding to PD L1 antibody;

FIG. 5 shows the results of the E L ISA results of PD L1-var 3 in combination with PD-1;

FIG. 6 shows the results of the E L ISA binding of PD L1-var 3 to the PD L1 antibody;

FIG. 7 shows the results of the E L ISA results for PD L1-var 7 in combination with PD-1;

FIG. 8 shows the results of the E L ISA binding of PD L1-var 7 to the PD L1 antibody;

FIG. 9 shows a fluorescence plot using confocal to observe the location of PD L1-WT pH L IP on HEK293 cells;

FIG. 10 shows a fluorescence map using confocal to observe the localization of PD L1-var 3 on HEK293 cells;

FIG. 11 shows a fluorescence map using confocal to observe the localization of PD L1-var 7 on HEK293 cells;

FIG. 12 shows photographs of PD L1-WT pH L IP treated mouse footpads;

FIG. 13 shows photographs of the palms of PD L1-var 3-treated mice;

FIG. 14 shows photographs of the palms of PD L1-var 7-treated mice;

FIG. 15 is a graph showing the results of CIA scoring by PD L1-WT pH L IP treatment;

FIG. 16 shows a graph of the results of PD L1-var 3 processing of CIA scores;

FIG. 17 shows a graph of the results of PD L1-var 7 processing of CIA scores;

FIG. 18 is a graph showing the results of detection of serum cytokines by PD L1-WT pH L IP treatment;

FIG. 19 is a graph showing the results of detection of cytokines by PD L1-var 3 treated serum;

FIG. 20 is a graph showing the results of detection of cytokines by PD L1-var 7 treated serum;

FIG. 21 is a graph showing the results of measurement of thickness of heels treated with PD L1-WT pH L IP;

FIG. 22 is a graph showing the results of the thickness measurements of the heels processed by PD L1-var 3;

FIG. 23 is a graph showing the results of the thickness measurements of the heels processed by PD L1-var 7.

Detailed Description

The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred embodiments for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention. The present invention is not to be limited in scope by the specific embodiments described herein, which are intended as single illustrations of individual aspects of the invention, and functionally equivalent methods and components are within the scope of the invention. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are intended to fall within the scope of the appended claims.

Example 1 peptide Synthesis

Wild-type pH L IP (amino acid sequence shown in SEQ ID NO. 1) and variants 3 and 7 thereof are denoted WTK L IP, var3 and var7, respectively, and are synthesized by Hangzhou peptide Biochemical Co., Ltd.

PD L1 extracellular segment (amino acid sequence is shown as SEQ ID NO.18, nucleotide sequence is shown as SEQ ID NO. 26), PD-L1 extracellular segment and fusion peptide prepared from wild type pH L IP, var3 and var7 are respectively expressed as PD L1-WTT L IP (amino acid sequence is shown as SEQ ID NO.20, nucleotide sequence is shown as SEQ ID NO. 23), PD L1-var 3 (amino acid sequence is shown as SEQ ID NO.21, nucleotide sequence is shown as SEQ ID NO. 24), PD L1-var 7 (amino acid sequence is shown as SEQ ID NO.22, nucleotide sequence is shown as SEQ ID NO. 25), PD L1-Ig (amino acid sequence is shown as SEQ ID NO.27, nucleotide sequence is shown as SEQ ID NO. 28), and the peptides are synthesized by the company.

1. Expression design

Designing gene sequence according to colibacillus expression, using Nde I enzyme cutting site at 5 'end, using XhoI enzyme cutting site at 3' end and adding terminator

2. Strain construction

PET28a vectors were constructed using the enzyme sites Nde I and XhoI, respectively, and the recipient bacterium B L21 (DE3) was transformed after construction, and clone verification (sequencing and expression) was selected.

3. Culture induction

Culturing the strain (L B culture medium), inducing (IPTG 0.5mM) when OD value is 0.6-0.8, and centrifuging for 4-6 hr to collect the strain.

4. Extracting and purifying

(1) Inclusion body washing

Solution A: 50mM Tris, 2mM EDTA, pH8.0, washing 2 times;

and B, liquid B: 50mM Tris, 2mM EDTA, 0.1% Triton, pH8.0, washing 1 time;

and C, liquid C: 20mM Tris, 1M urea, pH8.0, 1 wash.

(2) Extraction, namely extracting the inclusion bodies by 8M urea, 5mM β -Me, 0.3M NaC L and 20mM Tris with the pH of 8.0, wherein the extraction ratio is 1: 20.

(3) Renaturation dialysis, the target protein is supplemented with 10mM β -Me, reduced at 40 ℃ for 15 minutes, diluted to 0.2mg/ml and then treated with 10mMPBS，50μM CuCl₂The sample was dialyzed at pH8.0, the dialysate was changed 3 times, and the supernatant was collected by centrifugation.

(4) And (3) Ni column purification, namely, balancing a chromatographic column by using 0.3M NaC L, 10mM PBS and pH8.0, washing impurities by using an equilibrium buffer solution containing 40mM imidazole after loading, and eluting the target protein by using the equilibrium buffer solution containing 300mM imidazole, wherein the purity of the target protein is more than 95 percent.

(5) Desalted into 20mM PBS, 0.1M NaC L.

5. Coomassie brilliant blue staining results

The purified peptide was subjected to SDS-PAGE, and the result of Coomassie blue staining is shown in FIG. 1, indicating successful expression of the peptide.

Example 2 study of the ability of PD L1-WT pH L IP to bind to ligands

First, PD-1 and PD L1-WT pH L IP combined E L ISA experiment

1. Experimental Material

The reagent is biotin (Bio L egend), PD L1-WT pH L IP, PBS (Gibco).

2. Instrument for measuring the position of a moving object

Washing the plate machine; a microplate reader.

3. Experiment grouping

PD L1-WT pH L IP group, WT pH L IP control group, BSA control group, and B L ANK group.

4. Experimental methods

(1) Gradient coating PD L1-WT pH L IP, WT pH L IP, BSA, initial concentration 50. mu.g/ml, 4 ℃ overnight.

(2) The plate was washed 3 times with a plate washer and blocked with casein for 1h at 37 ℃.

(3) biotin-PD-1 (1. mu.g/ml) was added at 37 ℃ for 1 h.

(4) Adding secondary antibody, and keeping away from light for 45min at room temperature.

(5) Developing with developing solution for 5 min.

(6) Sulfuric acid was stopped and the microplate reader read 450 nm.

II, combining PD-1 and PD L1 extracellular domain with E L ISA experiment

1. Experimental Material

The reagent is biotin (Bio L egend), PD-L1 extracellular domain, PBS (Gibco).

2. Instrument for measuring the position of a moving object

Washing the plate machine; a microplate reader.

3. Experiment grouping

PD L1 extracellular domain group, PD L1-WT pH L IP group, WT pH L IP control group, BSA control group, and B L ANK group.

4. Experimental methods

(1) The gradient was coated over PD L1 extracellular domain, PD L1-WT pH L IP, WT pH L IP, BSA at an initial concentration of 50. mu.g/ml, overnight at 4 ℃.

(3) biotin-PD-1 (1. mu.g/ml) was added at 37 ℃ for 1 h.

(5) Developing with developing solution for 5 min.

(6) Sulfuric acid was stopped and the microplate reader read 450 nm.

Third, binding experiment of biotin-PD L1 antibody and PD L1-WT pH L IP

1. Experimental Material

Reagents biotin-anti-PD L1 (Bio L egend), PD L1-WT pH L IP, PBS (Gibco).

2. Instrument for measuring the position of a moving object

Washing the plate machine; a microplate reader.

3. Experiment grouping

4. Experimental methods

(1) The gradient was coated with PD L1-WT pH L IP, WT pH L IP, BSA at an initial concentration of 50. mu.g/ml, overnight at 4 ℃.

(3) biotin-anti-PD L1 (1. mu.g/ml) was added at 37 ℃ for 1 h.

(5) Developing with developing solution for 5 min.

(6) Sulfuric acid was stopped and the microplate reader read 450 nm.

Fourthly, the result

The results are shown in fig. 2-4, where PD L1-WT pH L IP binds to PD-1 with EC50 ═ 2.058 μ g/ml, PD-1 binds to PD L01 extracellular domain with EC50 ═ 1.213 μ g/ml, and PD L1-WT pH L IP binds to PD L1 antibody with EC50 ═ 0.5066 μ g/ml, the above results show that PD L1-WT pH L IP binds to PD-1 with comparable binding ability to PD L1 extracellular domain, showing that the binding of PD L1 to PD-1 is not affected by the link pH L IP.

Example 3 study of the ability of PD L1-var 3 to bind to ligands

First, the ISA experiment of combination of PD-1 and PD L1-var 3 with E L

1. Experimental Material

The reagent is biotin (Bio L egend), PD L1-var 3, PBS (Gibco).

2. Instrument for measuring the position of a moving object

Washing the plate machine; a microplate reader.

3. Experiment grouping

PD L1-var 3 group, var3 control group, BSA control group, B L ANK group.

4. Experimental methods

(1) The gradient was coated with PD L1-var 3, var3, BSA at an initial concentration of 50. mu.g/ml, overnight at 4 ℃.

(3) biotin-PD-1 (1. mu.g/ml) was added at 37 ℃ for 1 h.

(5) Developing with developing solution for 5 min.

(6) Sulfuric acid was stopped and the microplate reader read 450 nm.

Two, experiment for binding biotin-PD L1 antibody and PD L1-var 3

1. Experimental Material

The reagent is biotin-anti-PD L1 (Bio L egend), PD L1-var 3 and PBS (Gibco).

2. Instrument for measuring the position of a moving object

Washing the plate machine; a microplate reader.

3. Experiment grouping

PD L1-var 3 group, var3 control group, BSA control group, B L ANK group.

4. Experimental methods

(1) Gradient coating PD L1-var 3, var3, BSA, starting at 50. mu.g/ml, at 4 ℃ overnight.

(3) biotin-anti-PD L1 (1. mu.g/ml) was added at 37 ℃ for 1 h.

(5) Developing with developing solution for 5 min.

(6) Sulfuric acid was stopped and the microplate reader read 450 nm.

Fourthly, the result

Results as shown in fig. 5 and 6, EC50 of PD L1-var 3 bound to PD-1 was 1.985 μ g/ml, and EC50 of PD L1-var 3 bound to PD L1 antibody was 0.5018 μ g/ml-the above results indicate that PD L1-var 3 binds to PD-1 with comparable capacity to the extracellular domain of PD L1, showing that attaching var3 did not affect the binding of PD L1 to PD-1.

Example 4 study of the ability of PD L1-var 7 to bind to ligands

First, the ISA experiment of combination of PD-1 and PD L1-var 7 with E L

1. Experimental Material

The reagent is biotin (Bio L egend), PD L1-var 7, PBS (Gibco).

2. Instrument for measuring the position of a moving object

Washing the plate machine; a microplate reader.

3. Experiment grouping

PD L1-var 7 group, var7 control group, BSA control group, B L ANK group.

4. Experimental methods

(1) The gradient was coated with PD L1-var 7, var7, BSA at an initial concentration of 50. mu.g/ml, overnight at 4 ℃.

(3) biotin-PD-1 (1. mu.g/ml) was added at 37 ℃ for 1 h.

(5) Developing with developing solution for 5 min.

(6) Sulfuric acid was stopped and the microplate reader read 450 nm.

Two, experiment for binding biotin-PD L1 antibody and PD L1-var 7

1. Experimental Material

The reagent is biotin-anti-PD L1 (Bio L egend), PD L1-var 7 and PBS (Gibco).

2. Instrument for measuring the position of a moving object

Washing the plate machine; a microplate reader.

3. Experiment grouping

PD L1-var 7 group, var7 control group, BSA control group, B L ANK group.

4. Experimental methods

(1) Gradient coating PD L1-var 7, var7, BSA, starting at 50. mu.g/ml, at 4 ℃ overnight.

(3) biotin-anti-PD L1 (1. mu.g/ml) was added at 37 ℃ for 1 h.

(5) Developing with developing solution for 5 min.

(6) Sulfuric acid was stopped and the microplate reader read 450 nm.

Fourthly, the result

Results as shown in fig. 7 and 8, EC50 of PD L1-var 7 bound to PD-1 was 1.743 μ g/ml, and EC50 of PD L1-var 7 bound to PD L1 antibody was 0.4406 μ g/ml-the above results indicate that PD L1-var 7 binds to PD-1 with comparable ability to bind to extracellular domain of PD L1, showing that attaching var7 did not affect the binding of PD L1 to PD-1.

Example 5 localization of fusion peptides on cells

1. Cell lines

Human embryonic kidney cell line HEK293 (purchased from ATCC).

2. Reagent

DMEM medium (Gibco), fetal bovine serum (Gibco), PBS (pH 7.4) (Gibco), Tris-HC L (1mol/m L), PD-1, PD L1 extracellular and PD L1-WT pH L IP, WT pH L IP, PD L1-var 7, var7, PD L1-var 3, var7, PE cross-linking kit (Expedeon).

3. Instrument for measuring the position of a moving object

Clean bench, carbon dioxide incubator (Thermo), centrifuge (Thermo), confocal laser cell culture dish (Corning), electronic pH agent (Sidolisi), optical microscope (Nikon), confocal laser microscope (Zeiss L SM880)

4. Experimental procedure

Collecting HEK293 cells in logarithmic phase, discarding culture solution, washing with PBS twice, adding appropriate amount of pancreatin, terminating digestion with culture solution, transferring into 10ml test tube, centrifuging at 1000rpm for 5min, removing supernatant, and adding 1m LRe-suspending and mixing DMEM culture medium containing 10% fetal calf serum, sucking 10 mu L cell suspension into a cell counting plate for counting, adding a certain amount of cell suspension into a laser confocal culture dish, and adjusting the cell suspension to 5 x 10 by using the culture medium⁵1m L, in an incubator overnight.

After the cultured HEK293 cells were attached to the wall, the supernatant was aspirated and washed twice with PBS buffer of ph 7.4.

Preparing a culture solution by using 1mol/m L hydrochloric acid and PBS buffer solution with pH7.4, dropwise adding the hydrochloric acid into the PBS, finally titrating the pH value of the buffer solution to 6.3(5m L PBS buffer solution +12 mu L0 Tris-hydrochloric acid), adding PD L11-WT pH L IP, or PD L1-var 3, or PD L1-var 7 into the PBS buffer solution with pH7.4 and 6.3 respectively, fully mixing, diluting peptides according to the proportion to the final concentration of 200 mu g/m L, and adding PD L1 extracellular segment with the final concentration of 180 mu g/m L and pH L IP with the final concentration of 20 mu g/m L, or var3, or var7 into the PBS buffer solution with pH7.4 and 6.3.

The PBS buffer solution in the culture dish was aspirated, 1m L of the above mixture was added, 1m L of unblended PBS buffer solution with pH of 7.4 and 6.3 was added to the blank control, and the mixture was incubated in a 37 ℃ cell incubator for 1 hour.

After the incubation, the supernatant was aspirated, washed twice with PBS buffer at the corresponding pH, added with 1. mu.g/m L PD-1-PE 1m L, and incubated at 4 ℃ for half an hour in the dark.

After the incubation was completed, the cells were washed twice with PBS at the corresponding pH, and then PBS buffer at the corresponding pH was added.

And (3) placing the prepared culture dish under a laser confocal microscope to observe the fluorescence condition of the cell membrane surface.

5. Results

The results are shown in FIG. 9, and PD L1-pH L IP can be well displayed on the surface of cell membrane in acidic environment, the results show that PD L1 does not affect the property of inserting low pH insertion peptide into cell membrane, and the PD L1 is connected with the low pH insertion peptide and does not affect the conformation.

The results are shown in FIG. 10, and PD L1-var 3 can be well displayed on the surface of the cell membrane in an acidic environment, and the results show that PD L1 does not affect the insertion property of the low pH insertion peptide into the cell membrane, and the connection of PD L1 and the low pH insertion peptide does not affect the conformation.

The results are shown in FIG. 11, and PD L1-var 7 can be well displayed on the surface of the cell membrane in an acidic environment, and the results show that PD L1 does not affect the insertion property of the low pH insertion peptide into the cell membrane, and the connection of PD L1 and the low pH insertion peptide does not affect the conformation.

Example 6 evaluation of the Effect of fusion peptides on treating autoimmune diseases

1. Experimental Material

Animals: DBA/1J male mice (7 weeks old).

2. Reagent

Chicken type II collagen (#20012), complete freund adjuvant (#7001), incomplete freund adjuvant (#7002), Chondrex, USA, WT pH L IP, var3, var7 were synthesized by midwife peptide biochemistry ltd, hangzhou, PD L1 extracellular segment, PD L1-WTpH L IP, PD L1-var 3, PD L1-var 7, PD L1-Ig were prepared by the company itself.

3. Instrument for measuring the position of a moving object

Tissue homogenizer (IKA, H44).

4. Experimental methods

Emulsification of antigens

Adding adjuvant with the same volume into a 5ml disposable needle tube, opening a tissue homogenizer, dripping collagen solution while stirring at low speed, and increasing the rotation speed until forming emulsion drops of a water-in-oil sample. The process was performed with an ice bath on the syringe to prevent protein denaturation.

For the first immunization, 2mg/ml of collagen was mixed with an equal volume of complete Freund's adjuvant (4mg/ml) and emulsified (forming a water-in-oil sample) to a final collagen concentration of 1mg/ml, 100. mu.l per mouse, i.e., 100. mu.g. The needle was inserted 2cm from the base of the tail until the point of insertion was 0.5cm from the base of the tail.

For the second immunization, 2mg/ml of collagen was mixed with an equal volume of incomplete Freund's adjuvant and emulsified, and 100. mu.g of collagen was injected per mouse. The injection site was 3cm from the caudal root, and the needle tip was inserted subcutaneously 1.5cm from the caudal root.

2 weeks after the second immunization (at which the typical symptoms of CIA had appeared), the mice were divided into 4 groups, (1) intraperitoneally administered PD L1-WTKH L IP (or PD L1-var 3, or PD L1-var 7), 1mg/kg, once every other day, (2) intraperitoneally administered PD L1 (1mg/kg) + WTKH L IP (0.2mg/kg) (or PD L1-var 3, or PD L1-var 7), once every other day, (3) intraperitoneally administered PD L1-Ig, 1mg/kg, once every other day, (4) intraperitoneally administered PBS in a control group, 10 mice in each group, and the heel thickness and serum cytokine content were measured 26 days later.

3. Results

The photographs of the mouse soles of fig. 12-14 show that the mouse soles of PD L1-pH L IP group, PD L1-var 3 group, or PD L1-var 7 group are normal with significant relief of inflammation.

The CIA score results are shown in FIGS. 15-17, with significant reductions in CIA scores for PD L1-pH L IP group, PD L1-var 3 group, or PD L1-var 7 group.

The detection result of the serum cytokines is shown in the figure 18-20, and the contents of PD L1-pH L IP group, PD L1-var 3 group or PD L1-var 7 group TNF- α, IFN-gamma, I L-6 and I L-17A are obviously reduced.

The palm heel thickness test results are shown in fig. 21-23, and the palm heel thickness of mice in PD L1-pH L IP group, PD L1-var 3 group or PD L1-var 7 group is remarkably reduced.

The results show that the fusion peptide can remarkably relieve the symptoms of arthritis and has good treatment effect on the arthritis.

Sequence listing

<110> Beijing Zeqin biopharmaceutical Co., Ltd

<120> PD L1-pH L IP, preparation method and application thereof in treatment of autoimmune diseases

<160>28

<170>SIPOSequenceListing 1.0

<210>1

<211>36

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>1

Ala Cys Glu Gln Asn Pro Ile Tyr Trp Ala Arg Tyr Ala Asp Trp Leu

1 5 10 15

Phe Thr Thr Pro Leu Leu Leu Leu Asp Leu Ala Leu Leu Val Asp Ala

20 25 30

Asp Glu Gly Thr

35

<210>2

<211>32

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>2

Ala Cys Glu Asp Gln Asn Pro Tyr Trp Ala Arg Tyr Ala Asp Trp Leu

1 5 10 15

Phe Thr Thr Pro Leu Leu Leu Leu Asp Leu Ala Leu Leu Val Asp Gly

20 25 30

<210>3

<211>30

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>3

Ala Cys Glu Asp Gln Asn Pro Tyr Trp Arg Ala Tyr Ala Asp Leu Phe

1 5 10 15

Thr Pro Leu Thr Leu Leu Asp Leu Leu Ala Leu Trp Asp Gly

20 25 30

<210>4

<211>27

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>4

Ala Cys Asp Asp Gln Asn Pro Trp Arg Ala Tyr Leu Asp Leu Leu Phe

1 5 10 15

Pro Thr Asp Thr Leu Leu Leu Asp Leu Leu Trp

20 25

<210>5

<211>27

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>5

Ala Cys Glu Glu Gln Asn Pro Trp Arg Ala Tyr Leu Glu Leu Leu Phe

1 5 10 15

Pro Thr Glu Thr Leu Leu Leu Glu Leu Leu Trp

20 25

<210>6

<211>25

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>6

Ala Cys Asp Asp Gln Asn Pro Trp Ala Arg Tyr Leu Asp Trp Leu Phe

1 5 10 15

Pro Thr Asp Thr Leu Leu Leu Asp Leu

20 25

<210>7

<211>24

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>7

Cys Asp Asn Asn Asn Pro Trp Arg Ala Tyr Leu Asp Leu Leu Phe Pro

1 5 10 15

Thr Asp Thr Leu Leu Leu Asp Trp

20

<210>8

<211>25

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>8

Ala Cys Glu Glu Gln Asn Pro Trp Ala Arg Tyr Leu Glu Trp Leu Phe

1 5 10 15

Pro Thr Glu Thr Leu Leu Leu Glu Leu

20 25

<210>9

<211>24

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>9

Cys Glu Glu Gln Gln Pro Trp Ala Gln Tyr Leu Glu Leu Leu Phe Pro

1 5 10 15

Thr Glu Thr Leu Leu Leu Glu Trp

20

<210>10

<211>24

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>10

Cys Glu Glu Gln Gln Pro Trp Arg Ala Tyr Leu Glu Leu Leu Phe Pro

1 5 10 15

Thr Glu Thr Leu Leu Leu Glu Trp

20

<210>11

<211>24

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>11

Ala Cys Glu Asp Gln Asn Pro Trp Ala Arg Tyr Ala Asp Trp Leu Phe

1 5 10 15

Pro Thr Thr Leu Leu Leu Leu Asp

20

<210>12

<211>24

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>12

Ala Cys Glu Glu Gln Asn Pro Trp Ala Arg Tyr Ala Glu Trp Leu Phe

1 5 10 15

Pro Thr Thr Leu Leu Leu Leu Glu

20

<210>13

<211>22

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>13

Ala Cys Glu Asp Gln Asn Pro Trp Ala Arg Tyr Ala Asp Leu Leu Phe

1 5 10 15

Pro Thr Thr Leu Ala Trp

20

<210>14

<211>22

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>14

Ala Cys Glu Glu Gln Asn Pro Trp Ala Arg Tyr Ala Glu Leu Leu Phe

1 5 10 15

Pro Thr Thr Leu Ala Trp

20

<210>15

<211>34

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>15

Thr Glu Asp Ala Asp Val Leu Leu Ala Leu Asp Leu Leu Leu Leu Pro

1 5 10 15

Thr Thr Phe Leu Trp Asp Ala Tyr Arg Ala Trp Tyr Pro Asn Gln Glu

20 25 30

Cys Ala

<210>16

<211>37

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>16

Cys Asp Asp Asp Asp Asp Asn Pro Asn Tyr Trp Ala Arg Tyr Ala Asn

1 5 10 15

Trp Leu Phe Thr Thr Pro Leu Leu Leu Leu Asn Gly Ala Leu Leu Val

20 25 30

Glu Ala Glu Glu Thr

35

<210>17

<211>37

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>17

Cys Asp Asp Asp Asp Asp Asn Pro Asn Tyr Trp Ala Arg Tyr Ala Pro

1 5 10 15

Trp Leu Phe Thr Thr Pro Leu Leu Leu Leu Pro Gly Ala Leu Leu Val

20 25 30

Glu Ala Glu Glu Thr

35

<210>18

<211>220

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>18

Phe Thr Ile Thr Ala Pro Lys Asp Leu Tyr Val Val Glu Tyr Gly Ser

1 5 10 15

Asn Val Thr Met Glu Cys Arg Phe Pro Val Glu Arg Glu Leu Asp Leu

20 25 30

Leu Ala Leu Val Val Tyr Trp Glu Lys Glu Asp Glu Gln Val Ile Gln

35 40 45

Phe Val Ala Gly Glu Glu Asp Leu Lys Pro Gln His Ser Asn Phe Arg

50 55 60

Gly Arg Ala Ser Leu Pro Lys Asp Gln Leu Leu Lys Gly Asn Ala Ala

65 70 7580

Leu Gln Ile Thr Asp Val Lys Leu Gln Asp Ala Gly Val Tyr Cys Cys

85 90 95

Ile Ile Ser Tyr Gly Gly Ala Asp Tyr Lys Arg Ile Thr Leu Lys Val

100 105 110

Asn Ala Pro Tyr Arg Lys Ile Asn Gln Arg Ile Ser Val Asp Pro Ala

115 120 125

Thr Ser Glu His Glu Leu Ile Cys Gln Ala Glu Gly Tyr Pro Glu Ala

130 135 140

Glu Val Ile Trp Thr Asn Ser Asp His Gln Pro Val Ser Gly Lys Arg

145 150 155 160

Ser Val Thr Thr Ser Arg Thr Glu Gly Met Leu Leu Asn Val Thr Ser

165 170 175

Ser Leu Arg Val Asn Ala Thr Ala Asn Asp Val Phe Tyr Cys Thr Phe

180 185 190

Trp Arg Ser Gln Pro Gly Gln Asn His Thr Ala Glu Leu Ile Ile Pro

195 200 205

Glu Leu Pro Ala Thr His Pro Pro Gln Asn Arg Thr

210 215 220

<210>19

<211>261

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>19

Phe Thr Val Thr Val Pro Lys Asp Leu Tyr Val Val Glu Tyr Gly Ser

1 5 10 15

Asn Met Thr Ile Glu Cys Lys Phe Pro Val Glu Lys Gln Leu Asp Leu

20 25 30

Ala Ala Leu Ile Val Tyr Trp Glu Met Glu Asp Lys Asn Ile Ile Gln

35 40 45

Phe Val His Gly Glu Glu Asp Leu Lys Val Gln His Ser Ser Tyr Arg

50 55 60

Gln Arg Ala Arg Leu Leu Lys Asp Gln Leu Ser Leu Gly Asn Ala Ala

65 70 75 80

Leu Gln Ile Thr Asp Val Lys Leu Gln Asp Ala Gly Val Tyr Arg Cys

85 90 95

Met Ile Ser Tyr Gly Gly Ala Asp Tyr Lys Arg Ile Thr Val Lys Val

100 105 110

Asn Ala Pro Tyr Asn Lys Ile Asn Gln Arg Ile Leu Val Val Asp Pro

115 120 125

Val Thr Ser Glu His Glu Leu Thr Cys Gln Ala Glu Gly Tyr Pro Lys

130 135 140

Ala Glu Val Ile Trp Thr Ser Ser Asp His Gln Val Leu Ser Gly Lys

145 150 155 160

Thr Thr Thr Thr Asn Ser Lys Arg Glu Glu Lys Leu Phe Asn Val Thr

165 170 175

Ser Thr Leu Arg Ile Asn Thr Thr Thr Asn Glu Ile Phe Tyr Cys Thr

180 185 190

Phe Arg Arg Leu Asp Pro Glu Glu Asn His Thr Ala Glu Leu Val Ile

195 200 205

Pro Glu Leu Pro Leu Ala His Pro Pro Asn Glu Arg Thr Gly Gly Gly

210 215 220

Ser Ala Cys Glu Gln Asn Pro Ile Tyr Trp Ala Arg Tyr Ala Asp Trp

225 230 235 240

Leu Phe Thr Thr Pro Leu Leu Leu Leu Asp Leu Ala Leu Leu Val Asp

245 250 255

Ala Asp Glu Gly Thr

260

<210>20

<211>260

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>20

Phe Thr Ile Thr Ala Pro Lys Asp Leu Tyr Val Val Glu Tyr Gly Ser

1 5 10 15

Asn Val Thr Met Glu Cys Arg Phe Pro Val Glu Arg GluLeu Asp Leu

20 25 30

Leu Ala Leu Val Val Tyr Trp Glu Lys Glu Asp Glu Gln Val Ile Gln

35 40 45

Phe Val Ala Gly Glu Glu Asp Leu Lys Pro Gln His Ser Asn Phe Arg

50 55 60

Gly Arg Ala Ser Leu Pro Lys Asp Gln Leu Leu Lys Gly Asn Ala Ala

65 70 75 80

Leu Gln Ile Thr Asp Val Lys Leu Gln Asp Ala Gly Val Tyr Cys Cys

85 90 95

Ile Ile Ser Tyr Gly Gly Ala Asp Tyr Lys Arg Ile Thr Leu Lys Val

100 105 110

Asn Ala Pro Tyr Arg Lys Ile Asn Gln Arg Ile Ser Val Asp Pro Ala

115 120 125

Thr Ser Glu His Glu Leu Ile Cys Gln Ala Glu Gly Tyr Pro Glu Ala

130 135 140

Glu Val Ile Trp Thr Asn Ser Asp His Gln Pro Val Ser Gly Lys Arg

145 150 155 160

Ser Val Thr Thr Ser Arg Thr Glu Gly Met Leu Leu Asn Val Thr Ser

165 170 175

Ser Leu Arg Val Asn Ala Thr Ala Asn Asp Val Phe Tyr Cys Thr Phe

180 185 190

Trp Arg Ser Gln Pro Gly Gln Asn His Thr Ala Glu Leu Ile Ile Pro

195 200 205

Glu Leu Pro Ala Thr His Pro Pro Gln Asn Arg Thr Gly Gly Gly Ser

210 215 220

Ala Cys Glu Gln Asn Pro Ile Tyr Trp Ala Arg Tyr Ala Asp Trp Leu

225 230 235 240

Phe Thr Thr Pro Leu Leu Leu Leu Asp Leu Ala Leu Leu Val Asp Ala

245 250 255

Asp Glu Gly Thr

260

<210>21

<211>251

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>21

Phe Thr Ile Thr Ala Pro Lys Asp Leu Tyr Val Val Glu Tyr Gly Ser

1 5 10 15

Asn Val Thr Met Glu Cys Arg Phe Pro Val Glu Arg Glu Leu Asp Leu

20 25 30

Leu Ala Leu Val Val Tyr Trp Glu Lys Glu Asp Glu Gln Val Ile Gln

35 40 45

Phe Val Ala Gly Glu Glu Asp Leu Lys Pro Gln His Ser Asn Phe Arg

50 55 60

Gly Arg Ala Ser Leu Pro Lys Asp Gln Leu Leu Lys Gly Asn Ala Ala

65 70 75 80

Leu Gln Ile Thr Asp Val Lys Leu Gln Asp Ala Gly Val Tyr Cys Cys

85 90 95

Ile Ile Ser Tyr Gly Gly Ala Asp Tyr Lys Arg Ile Thr Leu Lys Val

100 105 110

Asn Ala Pro Tyr Arg Lys Ile Asn Gln Arg Ile Ser Val Asp Pro Ala

115 120 125

Thr Ser Glu His Glu Leu Ile Cys Gln Ala Glu Gly Tyr Pro Glu Ala

130 135 140

Glu Val Ile Trp Thr Asn Ser Asp His Gln Pro Val Ser Gly Lys Arg

145 150 155 160

Ser Val Thr Thr Ser Arg Thr Glu Gly Met Leu Leu Asn Val Thr Ser

165 170 175

Ser Leu Arg Val Asn Ala Thr Ala Asn Asp Val Phe Tyr Cys Thr Phe

180 185 190

Trp Arg Ser Gln Pro Gly Gln Asn His Thr Ala Glu Leu Ile Ile Pro

195 200 205

Glu Leu Pro Ala Thr His Pro Pro Gln Asn Arg Thr Gly Gly GlySer

210 215 220

Ala Cys Asp Asp Gln Asn Pro Trp Arg Ala Tyr Leu Asp Leu Leu Phe

225 230 235 240

Pro Thr Asp Thr Leu Leu Leu Asp Leu Leu Trp

245 250

<210>22

<211>249

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>22

Phe Thr Ile Thr Ala Pro Lys Asp Leu Tyr Val Val Glu Tyr Gly Ser

1 5 10 15

Asn Val Thr Met Glu Cys Arg Phe Pro Val Glu Arg Glu Leu Asp Leu

20 25 30

Leu Ala Leu Val Val Tyr Trp Glu Lys Glu Asp Glu Gln Val Ile Gln

35 40 45

Phe Val Ala Gly Glu Glu Asp Leu Lys Pro Gln His Ser Asn Phe Arg

50 55 60

Gly Arg Ala Ser Leu Pro Lys Asp Gln Leu Leu Lys Gly Asn Ala Ala

65 70 75 80

Leu Gln Ile Thr Asp Val Lys Leu Gln Asp Ala Gly Val Tyr Cys Cys

85 90 95

Ile Ile Ser Tyr Gly Gly Ala Asp Tyr Lys Arg Ile Thr Leu Lys Val

100 105 110

Asn Ala Pro Tyr Arg Lys Ile Asn Gln Arg Ile Ser Val Asp Pro Ala

115 120 125

Thr Ser Glu His Glu Leu Ile Cys Gln Ala Glu Gly Tyr Pro Glu Ala

130 135 140

Glu Val Ile Trp Thr Asn Ser Asp His Gln Pro Val Ser Gly Lys Arg

145 150 155 160

Ser Val Thr Thr Ser Arg Thr Glu Gly Met Leu Leu Asn Val Thr Ser

165 170 175

Ser Leu Arg Val Asn Ala Thr Ala Asn Asp Val Phe Tyr Cys Thr Phe

180 185 190

Trp Arg Ser Gln Pro Gly Gln Asn His Thr Ala Glu Leu Ile Ile Pro

195 200 205

Glu Leu Pro Ala Thr His Pro Pro Gln Asn Arg Thr Gly Gly Gly Ser

210 215 220

Ala Cys Glu Glu Gln Asn Pro Trp Ala Arg Tyr Leu Glu Trp Leu Phe

225 230 235 240

Pro Thr Glu Thr Leu Leu Leu Glu Leu

245

<210>23

<211>783

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>23

tttaccatta ccgcgccgaa agatctgtat gtggtggaat atggcagcaa cgtgaccatg 60

gaatgccgtt ttccggtgga acgtgaactg gatctgctgg cgctggtggt gtattgggaa 120

aaagaagatg aacaggtgat tcagtttgtg gcgggcgaag aagatctgaa accgcagcat 180

agcaactttc gtggccgtgc gagcctgccg aaagatcagc tgctgaaagg caacgcggcg 240

ctgcagatta ccgatgtgaa actgcaggat gcgggcgtgt attgctgcat tattagctat 300

ggcggcgcgg attataaacg tattaccctg aaagtgaacg cgccgtatcg taaaattaac 360

cagcgtatta gcgtggaccc ggcgaccagc gaacatgaac tgatttgcca ggcggaaggc 420

tatccggaag cggaagtgat ttggaccaac agcgatcatc agccggtgag cggcaaacgt 480

agcgtgacca ccagccgtac cgaaggcatg ctgctgaacg tgaccagcag cctgcgtgtg 540

aacgcgaccg cgaacgatgt gttttattgc accttttggc gtagccagcc gggccagaac 600

cataccgcgg aactgattat tccggaactg ccggcgaccc atccgccgca gaaccgtacc 660

ggcggcggca gcgcctgcga gcagaacccc atctactggg ccaggtacgc cgactggctg 720

ttcaccaccc ccctgctgct gctggacctg gccctgctgg tggacgccga cgagggcacc 780

taa 783

<210>24

<211>756

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>24

tttaccatta ccgcgccgaa agatctgtat gtggtggaat atggcagcaa cgtgaccatg 60

gaatgccgtt ttccggtgga acgtgaactg gatctgctgg cgctggtggt gtattgggaa 120

aaagaagatg aacaggtgat tcagtttgtg gcgggcgaag aagatctgaa accgcagcat 180

agcaactttc gtggccgtgc gagcctgccg aaagatcagc tgctgaaagg caacgcggcg 240

ctgcagatta ccgatgtgaa actgcaggat gcgggcgtgt attgctgcat tattagctat 300

ggcggcgcgg attataaacg tattaccctg aaagtgaacg cgccgtatcg taaaattaac 360

cagcgtatta gcgtggaccc ggcgaccagc gaacatgaac tgatttgcca ggcggaaggc 420

tatccggaag cggaagtgat ttggaccaac agcgatcatc agccggtgag cggcaaacgt 480

agcgtgacca ccagccgtac cgaaggcatg ctgctgaacg tgaccagcag cctgcgtgtg 540

aacgcgaccg cgaacgatgt gttttattgc accttttggc gtagccagcc gggccagaac 600

cataccgcgg aactgattat tccggaactg ccggcgaccc atccgccgca gaaccgtacc 660

ggcggcggca gcgcctgcga cgaccagaac ccctggaggg cctacctgga cctgctgttc 720

cccaccgaca ccctgctgct ggacctgctg tggtaa 756

<210>25

<211>750

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>25

tttaccatta ccgcgccgaa agatctgtat gtggtggaat atggcagcaa cgtgaccatg 60

gaatgccgtt ttccggtgga acgtgaactg gatctgctgg cgctggtggt gtattgggaa 120

aaagaagatg aacaggtgat tcagtttgtg gcgggcgaag aagatctgaa accgcagcat 180

agcaactttc gtggccgtgc gagcctgccg aaagatcagc tgctgaaagg caacgcggcg 240

ctgcagatta ccgatgtgaa actgcaggat gcgggcgtgt attgctgcat tattagctat 300

ggcggcgcgg attataaacg tattaccctg aaagtgaacg cgccgtatcg taaaattaac 360

cagcgtatta gcgtggaccc ggcgaccagc gaacatgaac tgatttgcca ggcggaaggc 420

tatccggaag cggaagtgat ttggaccaac agcgatcatc agccggtgag cggcaaacgt 480

agcgtgacca ccagccgtac cgaaggcatg ctgctgaacg tgaccagcag cctgcgtgtg 540

aacgcgaccg cgaacgatgt gttttattgc accttttggc gtagccagcc gggccagaac 600

cataccgcgg aactgattat tccggaactg ccggcgaccc atccgccgca gaaccgtacc 660

ggcggcggca gcgcctgcga ggagcagaac ccctgggcca ggtacctgga gtggctgttc 720

cccaccgaga ccctgctgct ggagctgtaa 750

<210>26

<211>660

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>26

tttaccatta ccgcgccgaa agatctgtat gtggtggaat atggcagcaa cgtgaccatg 60

gaatgccgtt ttccggtgga acgtgaactg gatctgctgg cgctggtggt gtattgggaa 120

aaagaagatg aacaggtgat tcagtttgtg gcgggcgaag aagatctgaa accgcagcat 180

agcaactttc gtggccgtgc gagcctgccg aaagatcagc tgctgaaagg caacgcggcg 240

ctgcagatta ccgatgtgaa actgcaggat gcgggcgtgt attgctgcat tattagctat 300

ggcggcgcgg attataaacgtattaccctg aaagtgaacg cgccgtatcg taaaattaac 360

cagcgtatta gcgtggaccc ggcgaccagc gaacatgaac tgatttgcca ggcggaaggc 420

tatccggaag cggaagtgat ttggaccaac agcgatcatc agccggtgag cggcaaacgt 480

agcgtgacca ccagccgtac cgaaggcatg ctgctgaacg tgaccagcag cctgcgtgtg 540

aacgcgaccg cgaacgatgt gttttattgc accttttggc gtagccagcc gggccagaac 600

cataccgcgg aactgattat tccggaactg ccggcgaccc atccgccgca gaaccgtacc 660

<210>27

<211>459

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>27

Phe Thr Ile Thr Ala Pro Lys Asp Leu Tyr Val Val Glu Tyr Gly Ser

1 5 10 15

Asn Val Thr Met Glu Cys Arg Phe Pro Val Glu Arg Glu Leu Asp Leu

20 25 30

Leu Ala Leu Val Val Tyr Trp Glu Lys Glu Asp Glu Gln Val Ile Gln

35 40 45

Phe Val Ala Gly Glu Glu Asp Leu Lys Pro Gln His Ser Asn Phe Arg

50 55 60

Gly Arg Ala Ser Leu Pro Lys Asp Gln Leu Leu Lys Gly Asn Ala Ala

65 70 75 80

Leu Gln Ile Thr Asp Val Lys Leu Gln Asp Ala Gly Val Tyr Cys Cys

85 90 95

Ile Ile Ser Tyr Gly Gly Ala Asp Tyr Lys Arg Ile Thr Leu Lys Val

100 105 110

Asn Ala Pro Tyr Arg Lys Ile Asn Gln Arg Ile Ser Val Asp Pro Ala

115 120 125

Thr Ser Glu His Glu Leu Ile Cys Gln Ala Glu Gly Tyr Pro Glu Ala

130 135 140

Glu Val Ile Trp Thr Asn Ser Asp His Gln Pro Val Ser Gly Lys Arg

145 150 155 160

Ser Val Thr Thr Ser Arg Thr Glu Gly Met Leu Leu Asn Val Thr Ser

165 170 175

Ser Leu Arg Val Asn Ala Thr Ala Asn Asp Val Phe Tyr Cys Thr Phe

180 185 190

Trp Arg Ser Gln Pro Gly Gln Asn His Thr Ala Glu Leu Ile Ile Pro

195 200 205

Glu Leu Pro Ala Thr His Pro Pro Gln Asn Arg Thr Gly Gly Gly Gly

210 215 220

Gly Ser Glu Pro Arg Gly Pro Thr Ile Lys Pro Cys Pro Pro Cys Lys

225 230 235 240

Cys Pro Ala Pro Asn Leu Leu Gly Gly Pro Ser Val Phe Ile Phe Pro

245 250 255

Pro Lys Ile Lys Asp Val Leu Met Ile Ser Leu Ser Pro Ile Val Thr

260 265 270

Cys Val Val Val Asp Val Ser Glu Asp Asp Pro Asp Val Gln Ile Ser

275 280 285

Trp Phe Val Asn Asn Val Glu Val His Thr Ala Gln Thr Gln Thr His

290 295 300

Arg Glu Asp Tyr Asn Ser Thr Leu Arg Val Val Ser Ala Leu Pro Ile

305 310 315 320

Gln His Gln Asp Trp Met Ser Gly Lys Glu Phe Lys Cys Lys Val Asn

325 330 335

Asn Lys Asp Leu Pro Ala Pro Ile Glu Arg Thr Ile Ser Lys Pro Lys

340 345 350

Gly Ser Val Arg Ala Pro Gln Val Tyr Val Leu Pro Pro Pro Glu Glu

355 360 365

Glu Met Thr Lys Lys Gln Val Thr Leu Thr Cys Met Val Thr Asp Phe

370 375 380

Met Pro Glu Asp Ile Tyr Val Glu Trp Thr Asn Asn Gly Lys Thr Glu

385 390 395 400

Leu Asn Tyr Lys Asn Thr Glu Pro Val Leu Asp Ser Asp Gly Ser Tyr

405 410 415

Phe Met Tyr Ser Lys Leu Arg Val Glu Lys Lys Asn Trp Val Glu Arg

420 425 430

Asn Ser Tyr Ser Cys Ser Val Val His Glu Gly Leu His Asn His His

435 440 445

Thr Thr Lys Ser Phe Ser Arg Thr Pro Gly Lys

450 455

<210>28

<211>1380

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>28

tttaccatta ccgcgccgaa agatctgtat gtggtggaat atggcagcaa cgtgaccatg 60

gaatgccgtt ttccggtgga acgtgaactg gatctgctgg cgctggtggt gtattgggaa 120

aaagaagatg aacaggtgat tcagtttgtg gcgggcgaag aagatctgaa accgcagcat 180

agcaactttc gtggccgtgc gagcctgccg aaagatcagc tgctgaaagg caacgcggcg 240

ctgcagatta ccgatgtgaa actgcaggat gcgggcgtgt attgctgcat tattagctat 300

ggcggcgcgg attataaacg tattaccctg aaagtgaacg cgccgtatcg taaaattaac 360

cagcgtatta gcgtggaccc ggcgaccagc gaacatgaac tgatttgcca ggcggaaggc 420

tatccggaag cggaagtgat ttggaccaac agcgatcatc agccggtgag cggcaaacgt 480

agcgtgacca ccagccgtac cgaaggcatg ctgctgaacg tgaccagcag cctgcgtgtg 540

aacgcgaccg cgaacgatgt gttttattgc accttttggc gtagccagcc gggccagaac 600

cataccgcgg aactgattat tccggaactg ccggcgaccc atccgccgca gaaccgtacc 660

ggcggcggcg gcggatccga gcccagaggg cccacaatca agccctgtcc tccatgcaaa 720

tgcccagcac ctaacctctt gggtggacca tccgtcttca tcttccctcc aaagatcaag 780

gatgtactca tgatctccct gagccccata gtcacatgtg tggtggtgga tgtgagcgag 840

gatgacccag atgtccagat cagctggttt gtgaacaacg tggaagtaca cacagctcag 900

acacaaaccc atagagagga ttacaacagt actctccggg tggtcagtgc cctccccatc 960

cagcaccagg actggatgag tggcaaggag ttcaaatgca aggtcaacaa caaagacctc 1020

ccagcgccca tcgagagaac catctcaaaa cccaaagggt cagtaagagc tccacaggta 1080

tatgtcttgc ctccaccaga agaagagatg actaagaaac aggtcactct gacctgcatg 1140

gtcacagact tcatgcctga agacatttac gtggagtgga ccaacaacgg gaaaacagag 1200

ctaaactaca agaacactga accagtcctg gactctgatg gttcttactt catgtacagc 1260

aagctgagag tggaaaagaa gaactgggtg gaaagaaata gctactcctg ttcagtggtc 1320

cacgagggtc tgcacaatca ccacacgact aagagcttct cccggactcc gggtaaataa 1380

Claims

1. A fusion peptide of a low pH insertion peptide, comprising a low pH insertion peptide, an immune checkpoint ligand, or a fragment thereof; preferably, the low pH insertion peptide sequence comprises any one of the sequences shown in SEQ ID No. 1-17.

2. The fusion peptide of claim 1, wherein the immune checkpoint comprises PD-1, L ag-3, Tim-3, TIGIT, CT L a-4.

3. The fusion peptide of claim 2, wherein the immune checkpoint is PD-1.

4. The fusion peptide of claim 3, wherein the PD-1 ligand comprises PD L1, PD L2, and the PD L1 fragment comprises the extracellular domain of PD-L1.

5. The fusion peptide of claim 4, wherein the extracellular domain of the PD L1 protein is a polypeptide formed by the amino acids shown in SEQ ID NO.18 or SEQ ID NO.19 or a polypeptide derived from the amino acid sequence shown in SEQ ID NO.18 or SEQ ID NO.19 by substitution and/or deletion and/or addition of one or more amino acid residues of the amino acid sequence shown in SEQ ID NO.18 or SEQ ID NO.19 and having the same function as the sequence shown in SEQ ID NO.18 or SEQ ID NO. 19.

6. The fusion peptide of claim 5, wherein the PD L1 protein or fragment thereof is linked to the N-terminus of the low pH insertion peptide via L inker, preferably wherein the L inker sequence is (GGGS) m or (GGGGS) m, wherein m is a natural number, and more preferably wherein the L inker sequence is GGGS.

7. The fusion peptide of any one of claims 1 to 6, wherein the sequence of the fusion peptide is as shown in SEQ ID No.20 to SEQ ID No. 22.

8. A pharmaceutical composition for treating an autoimmune disease, comprising the fusion peptide of any one of claims 1 to 7.

9. A labelling system comprising the fusion peptide of any one of claims 1 to 7.

10. An application, comprising the application of any one of:

(1) use of a fusion peptide according to any one of claims 1 to 7 in the manufacture of a medicament for the treatment of an autoimmune disease; preferably, the autoimmune disease comprises alopecia areata, ankylosing spondylitis, antiphospholipid syndrome, autoimmune addison's disease, autoimmune diseases of the adrenal gland, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune oophoritis and orchitis, autoimmune thrombocytopenia, Behcet's syndrome, bullous pemphigoid, cardiomyopathy, sprue diarrhea dermatitis, chronic fatigue immune dysfunction syndrome, chronic inflammatory demyelinating polyneuropathy, Cushing's syndrome, cicatricial pemphigoid, CREST syndrome, cold agglutinin disease, Crohn's disease, discoid lupus, idiopathic mixed cryoglobulinemia, diabetes, eosinophilic fasciitis, fibromyalgia-fibromyositis, glomerulonephritis, Graves 'disease, Guillain-Barre syndrome, Hashimoto's thyroiditis, Henschel-Schonlein purpura, Graves 'disease, Crohn's disease, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune oophoritis, idiopathic pulmonary fibrosis, idiopathic/autoimmune thrombocytopenic purpura, IgA neuropathy, juvenile arthritis, lichen planus, lupus erythematosus, meniere's syndrome, mixed connective tissue disease, multiple sclerosis, type 1 or immune-mediated diabetes, myasthenia gravis, pemphigus-related diseases, pernicious anemia, polyarteritis nodosa, polychondritis, polyadenylic syndrome, polymyalgia rheumatica, polymyositis, dermatomyositis, primary aspergillosis, primary biliary cirrhosis, psoriasis, psoriatic arthritis, leiomyelia, reiter's syndrome, rheumatoid arthritis, sarcoidosis, scleroderma, sjogren's syndrome, systemic myotonic syndrome, systemic lupus erythematosus, svt's syndrome, stewart's disease, lupus erythematosus, takayasu's arteritis, giant cell arteritis, and the like, Ulcerative colitis, uveitis, vasculitis, vitiligo, wegener granulomatosis;

(2) use of the fusion peptide of any one of claims 1 to 7 for the preparation of a focal tissue cell marking system for autoimmune diseases; preferably, the marking system comprises the marking system of claim 9;

(3) use of an immune checkpoint ligand or fragment thereof in the preparation of a fusion peptide according to any one of claims 1 to 7; preferably, the immune checkpoint comprises the immune checkpoint of any one of claims 1-7; an immune checkpoint ligand comprising the immune checkpoint of any one of claims 1-7; an immune checkpoint ligand fragment comprising the immune checkpoint ligand fragment of any one of claims 1-7;

(4) use of an immune checkpoint ligand or fragment thereof in the preparation of a medicament for the treatment of an autoimmune disease; preferably, the autoimmune disease comprises alopecia areata, ankylosing spondylitis, antiphospholipid syndrome, autoimmune addison's disease, autoimmune diseases of the adrenal gland, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune oophoritis and orchitis, autoimmune thrombocytopenia, Behcet's syndrome, bullous pemphigoid, cardiomyopathy, sprue diarrhea dermatitis, chronic fatigue immune dysfunction syndrome, chronic inflammatory demyelinating polyneuropathy, Cushing's syndrome, cicatricial pemphigoid, CREST syndrome, cold agglutinin disease, Crohn's disease, discoid lupus, idiopathic mixed cryoglobulinemia, diabetes, eosinophilic fasciitis, fibromyalgia-fibromyositis, glomerulonephritis, Graves 'disease, Guillain-Barre syndrome, Hashimoto's thyroiditis, Henschel-Schonlein purpura, Graves 'disease, Crohn's disease, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune oophoritis, idiopathic pulmonary fibrosis, idiopathic/autoimmune thrombocytopenic purpura, IgA neuropathy, juvenile arthritis, lichen planus, lupus erythematosus, meniere's syndrome, mixed connective tissue disease, multiple sclerosis, type 1 or immune-mediated diabetes, myasthenia gravis, pemphigus-related diseases, pernicious anemia, polyarteritis nodosa, polychondritis, polyadenylic syndrome, polymyalgia rheumatica, polymyositis, dermatomyositis, primary aspergillosis, primary biliary cirrhosis, psoriasis, psoriatic arthritis, leiomyelia, reiter's syndrome, rheumatoid arthritis, sarcoidosis, scleroderma, sjogren's syndrome, systemic myotonic syndrome, systemic lupus erythematosus, svt's syndrome, stewart's disease, lupus erythematosus, takayasu's arteritis, giant cell arteritis, and the like, Ulcerative colitis, uveitis, vasculitis, vitiligo, wegener granulomatosis;

(5) the application of the immune check point ligand or the fragment thereof in preparing a focal tissue cell marking system of autoimmune diseases; preferably, the marking system comprises the marking system of claim 9;

(6) use of a low pH insertion peptide for the preparation of a fusion peptide according to any one of claims 1 to 7; preferably, the low pH insertion peptide comprises the low pH insertion peptide of any one of claims 1-7;

(7) use of a low pH insertion peptide in the manufacture of a medicament for the treatment of an autoimmune disease; preferably, the low pH insertion peptide comprises the low pH insertion peptide of any one of claims 1-7; preferably, the autoimmune disease comprises alopecia areata, ankylosing spondylitis, antiphospholipid syndrome, autoimmune addison's disease, autoimmune disease of the adrenal gland, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune oophoritis and orchitis, autoimmune thrombocytopenia, Behcet's syndrome, bullous pemphigoid, cardiomyopathy, sprue diarrhea dermatitis, chronic fatigue immune dysfunction syndrome, chronic inflammatory demyelinating polyneuropathy, Cushing's syndrome, cicatricial pemphigoid, CREST syndrome, cold agglutinin disease, Crohn's disease, discoid lupus, idiopathic mixed cryoglobulinemia, diabetes, eosinophilic fasciitis, fibromyalgia-fibromyositis, glomerulonephritis, Graaff disease, Guillain-Barre syndrome, Hashimoto's thyroiditis, Henschel-Schnei purpura, Idiopathic pulmonary fibrosis, idiopathic/autoimmune thrombocytopenic purpura, IgA neuropathy, juvenile arthritis, lichen planus, lupus erythematosus, meniere's syndrome, mixed connective tissue disease, multiple sclerosis, type 1 or immune-mediated diabetes, myasthenia gravis, pemphigus-related diseases, pernicious anemia, polyarteritis nodosa, polychondritis, polyadenylic syndrome, polymyalgia rheumatica, polymyositis, dermatomyositis, primary aspergillosis, primary biliary cirrhosis, psoriasis, psoriatic arthritis, leiomyelia, reiter's syndrome, rheumatoid arthritis, sarcoidosis, scleroderma, sjogren's syndrome, systemic myotonic syndrome, systemic lupus erythematosus, svt's syndrome, stewart's disease, lupus erythematosus, takayasu's arteritis, giant cell arteritis, and the like, Ulcerative colitis, uveitis, vasculitis, vitiligo, wegener granulomatosis;

(8) the application of the low pH insertion peptide in preparing a lesion tissue cell marking system of the autoimmune disease; preferably, the low pH insertion peptide comprises the low pH insertion peptide of any one of claims 1-7; preferably, the marking system comprises the marking system of claim 9.