FUSION PEPTIDE FOR TREATING AUTOIMMUNE DISEASE

Abstract

PDL1-pHLIP, a preparation method of the PDL1-pHLIP, and an application of the PDL1-pHLIP in treatment of autoimmune diseases are provided. A fusion peptide is prepared by binding a pH low insertion peptide and an extracellular domain of PDL1. The pH low insertion peptide may be inserted onto a cell membrane of a focus tissue in an acid environment; the PDL1 bound to the pH low insertion peptide is subjected to targeting localization at the focus by utilizing the above properties of the pH low insertion peptide; a PD-1/PD-L1 negative signal of the focus tissue is enhanced by utilizing the pH low insertion peptide; and immune response of effector T cells is suppressed at the source, thereby achieving an effect of preventing and treating the autoimmune diseases.

Claims

1-37. (canceled)

38. A fusion peptide of a pH low insertion peptide, comprising the pH low insertion peptide, an immune checkpoint ligand or fragments thereof.

39. The fusion peptide according to claim 38, wherein the pH low insertion peptide comprises any of sequences shown as SEQ ID NO: 1-17.

40. The fusion peptide according to claim 38, wherein the pH low insertion peptide comprises a sequence formed by repeating an extracellular domain sequence once or for many times in the sequences shown as SEQ ID NO: 1-17.

41. The fusion peptide according to claim 38, wherein immune checkpoints comprise PD-1, Lag-3, Tim-3, TIGIT and CTLA-4.

42. The fusion peptide according to claim 41, wherein the immune checkpoint is the PD-1.

43. The fusion peptide according to claim 42, wherein the PD-1 ligand comprises PDL1 and PDL2; and the PDL1 fragment comprises an extracellular domain of the PD-L1.

44. The fusion peptide according to claim 43, wherein the extracellular domain of a PDL1 protein is a peptide formed by amino acids shown as SEQ ID NO: 18 or SEQ ID NO: 19, or a peptide derived from the amino acid sequence shown as SEQ ID NO: 18 or SEQ ID NO: 19 that is subjected to substitution and/or deletion and/or addition of one or several amino acid residues in the amino acid sequence shown as SEQ ID NO: 18 or SEQ ID NO: 19 and that has the same function as the sequence shown as SEQ ID NO: 18 or SEQ ID NO: 19.

45. The fusion peptide according to claim 44, wherein the PDL1 protein or a fragment thereof is bound to an N terminal of the pH low insertion peptide through Linker.

46. The fusion peptide according to claim 45, wherein a sequence of the Linker is (GGGS)m or (GGGGS)m, wherein m=natural number.

47. The fusion peptide according to claim 46, wherein the sequence of the Linker is GGGS.

48. The fusion peptide according to claim 38, wherein sequences of the fusion peptide are shown as SEQ ID NO: 20-22.

49. A pharmaceutical composition for treating autoimmune diseases, comprising the fusion peptide according to claim 38.

50. A marker system, comprising the fusion peptide according to claim 38.

51. A method for treating autoimmune diseases, comprising: applying the fusion peptide according to claim 38 to patients in need.

52. A method for treating autoimmune diseases, comprising: the pharmaceutical composition according to claim 49 to patients in need.

53. A method for marking an immune checkpoint ligand or fragments thereof on a focus tissue cell membrane, comprising: binding the immune checkpoint ligand or fragments thereof to a pH low insertion peptide to form a fusion peptide, introducing the fusion peptide into the focus tissue, and inserting the fusion peptide onto the focus tissue cell membrane; the fusion peptide is the fusion peptide according to claim 38.

54. The method according to claim 53, wherein the immune checkpoints, the immune checkpoint ligand, or the fragments of the immune checkpoint ligand comprise PD-1, Lag-3, Tim-3, TIGIT and CTLA-4.

55. The method according to claim 53, wherein the pH low insertion peptide comprises any of sequences shown as SEQ ID NO: 1-17.

56. The method according to claim 53, wherein the pH low insertion peptide comprises the pH low insertion peptide that comprises a sequence formed by repeating an extracellular domain sequence once or for many times in the sequences shown as SEQ ID NO: 1-17.

57. The application according to claim 52, wherein the autoimmune diseases comprise alopecia areata, ankylosing spondylitis, antiphospholipide 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, oral inflammatory diarrhea dermatitis, chronic fatigue immunologic inadequacy syndrome, chronic inflammatory demyelinating polyneuropathy, Chusch-Schotter's syndrome, cicatricial pemphigoid, CREST syndrome, cold hemagglutinin disease, Crohn's disease, discoid lupus erythematosus, idiopathic mixed cryoglobulinemia, diabetes, eosinophilic fasciitis, fibromyalgia-fibromyositis, glomerulonephritis, Graves' disease, Guillain-Barre syndrome, hashimoto thyroiditis, purpura Henoch-Schonlein, idiopathic pulmonary fibrosis, idiopathic/autoimmune thrombocytopenic purpura, IgA neuropathy, juvenile arthritis, lichen planus, lupus erythematosus, Meniere's syndrome, mixed connective tissue disease, disseminated sclerosis, type 1 or immune-mediated diabetes, myasthenia gravis, pemphigus-related diseases, pernicious anemia, polyarteritis nodosa, polychondritis, polyglandular syndrome, polymyalgia rheumatic, polymyositis, dermatomyositis, primary agammaglobulinemia, primary biliary cirrhosis, psoriasis, psoriatic arthritis, Raynaud phenomenon, Reiter syndrome, rheumatoid arthritis, nodule disease, scleroderma, Sjogren syndrome, stiff-man syndrome, systemic lupus erythematosus, Sweet's syndrome, Still's disease, lupus erythematosus, Takayasu's arteritis, transient arteritis/giant cell arteritis, ulcerative colitis, uveitis, vasculitis, leucoderma and Wegener's granulomatosis.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0080] FIG. 1 shows an SDS-PAGE chromosome map of a synthetic peptide in the present invention, wherein A: PD-L1-WTpHLIP; B: PDL1-Ig; C: extracellular domain of PD-L1; D: PDL1-var3; E: PD-L1-var7;

[0081] FIG. 2 shows an ELISA result map of binding of PDL1-WT pHLIP and PD-1;

[0082] FIG. 3 shows an ELISA result map of binding of PD-1 and a PDL1 protein;

[0083] FIG. 4 shows an ELISA result map of binding of PDL1-WT pHLIP and a PDL1 antibody;

[0084] FIG. 5 shows an ELISA result map of binding of PDL1-var3 and PD-1;

[0085] FIG. 6 shows an ELISA result map of binding of PDL1-var3 and a PDL1 antibody;

[0086] FIG. 7 shows an ELISA result map of binding of PDL1-var7 and PD-1;

[0087] FIG. 8 shows an ELISA result map of binding of PDL1-var7 and a PDL1 antibody;

[0088] FIG. 9 shows a fluorogram of PDL1-WT pHLIP localization on HEK293 cells observed by utilizing confocal;

[0089] FIG. 10 shows a fluorogram of PDL1-var3 localization on HEK293 cells observed by utilizing confocal;

[0090] FIG. 11 shows a fluorogram of PDL1-var7 localization on HEK293 cells observed by utilizing confocal;

[0091] FIG. 12 shows a photo of mouse soles treated with PDL1-WT pHLIP;

[0092] FIG. 13 shows a photo of mouse soles treated with PDL1-var3;

[0093] FIG. 14 shows a photo of mouse soles treated with PDL1-var7;

[0094] FIG. 15 shows a grading result map of CIA treated with PDL1-WT pHLIP;

[0095] FIG. 16 shows a grading result map of CIA treated with PDL1-var3;

[0096] FIG. 17 shows a grading result map of CIA treated with PDL1-var7;

[0097] FIG. 18 shows a detection result map of a serum cell factor treated with PDL1-WT pHLIP;

[0098] FIG. 19 shows a detection result map of a serum cell factor treated with PDL1-var3;

[0099] FIG. 20 shows a detection result map of a serum cell factor treated with PDL1-var7;

[0100] FIG. 21 shows a thickness detection result map of a heel treated with PDL1-WT pHLIP;

[0101] FIG. 22 shows a thickness detection result map of a heel treated with PDL1-var3; and

[0102] FIG. 23 shows a thickness detection result map of a heel treated with PDL1-var7.

DETAILED DESCRIPTION OF THE INVENTION

[0103] The following embodiments are included to illustrate preferred implementation modes of the present invention. Those skilled in the art should understand that, technologies disclosed in embodiments below represent technologies that are discovered by the inventor and that achieve excellent functions in practice of the present invention. Therefore, the embodiments below are considered as preferred implementation modes forming the practice of the present invention. However, according to the present disclosure, those skilled in the art should understand that, multiple changes may be conducted in specific implementation modes, and identical or similar results are still obtained without departing from the aim and scope of the present invention. The present invention is not limited to the scope of the specific implementation modes in the present invention. The implementation modes mean individual examples of various aspects in the present invention; and functionally identical methods and components also fall within the scope of the present invention. In fact, in addition to the changes in the present invention, multiple changes of the present invention are obvious to those skilled in the art due to the above description. These changes will fall within the scope of claims.

Embodiment 1

Peptide Synthesis

[0104] Wild-type pHLIP (having an amino acid sequence shown as SEQ ID NO: 1) and variants 3 and 7 thereof are respectively expressed as WTpHLIP, var3 and var7, and are all synthesized by Chinese Peptide Company.

[0105] An extracellular domain of PDL1 (having an amino acid sequence shown as SEQ ID NO: 18 and a nucleotide sequence shown as SEQ ID NO: 26), fusion peptides that are prepared from the extracellular domain of PD-L1 and wild types pHLIP, var3 and var7 and that are respectively expressed as PDL1-WTpHLIP (having an amino acid sequence shown as SEQ ID NO: 20 and a nucleotide sequence shown as SEQ ID NO: 23), PDL1-var3 (having an amino acid sequence shown as SEQ ID NO: 21 and a nucleotide sequence shown as SEQ ID NO: 24) and PDL1-var7 (having an amino acid sequence shown as SEQ ID NO: 22 and a nucleotide sequence shown as SEQ ID NO: 25), and PDL1-Ig (having an amino acid sequence shown as SEQ ID NO: 27 and a nucleotide sequence shown as SEQ ID NO: 28) are synthesized by the company.

1. Expression Design

[0106] According to an Escherichia coli expression design gene sequence, a restriction site Nde I was used at a terminal 5′, and a restriction site XhoI for a terminator was added at a terminal 3′.

2. Strain Construction

[0107] A vector PET28a was respectively constructed by utilizing the restriction sites Nde I and XhoI; receptor bacteria BL21 (DE3) were transformed after construction; and clone was picked for validation (sequencing and expression).

3. Culture and Induction

[0108] The strain that was validated accurate was cultured (by an LB medium) and then induced (IPTG0.5 mM) when a value OD was up to 0.6-0.8; and then the strain was centrifuged for 4-6 hours for recovery.

4. Extraction and Purification

[0109] (1) Inclusion body washing

[0110] Solution A: 50 mM Tris and 2 mM EDTA, pH of 8.0, washed twice;

[0111] Solution B: 50 mM Tris, 2 mM EDTA and 0.1% Triton, pH of 8.0, washed once;

[0112] Solution C: 20 mM Tris and 1M urea, pH of 8.0, washed once. [0113] (2) Extraction: the inclusion body was extracted with 8M urea, 5 mM β-Me, 0.3M NaCl and 20 mM Tris at a pH of 8.0, wherein an extraction ratio was 1:20. [0114] (3) Renaturation dialysis: 10 mM β-Me was added to a target protein; the protein was reduced at 40° C. for 15 minutes and diluted to 0.2 mg/ml with 10 mM PBS; the sample was dialyzed with 50 μM CuCl.sub.2 at a pH of 8.0; dialysate was changed for 3 times; and then the supernatant was collected after centrifugation. [0115] (4) Ni column purification: a chromatographic column was balanced with 0.3M NaCl and 10 mM PBS at a pH of 8.0; impurities were washed with equilibration buffer containing 40mM imidazole after loading; and the target protein was eluted with equilibration buffer containing 300mM imidazole. Purity of the target protein was higher than 95%. [0116] (5) The protein was desalinized in 20 mM PBS and 0.1M NaCl.

5. Coomassie Blue Staining Results

[0117] The purified peptide was subjected to SDS-PAGE. Coomassie blue staining results were shown as FIG. 1, which indicated that peptide expression was successful.

Embodiment 2

Study of Ability of PDL1-WT pHLIP Binding Ligand

[0118] I. ELISA Experiment of Binding of PD-1 and PDL1-WT pHLIP

1. Experimental Materials

[0119] Reagents: biotin (BioLegend), PDL1-WT pHLIP and PBS (Gibco).

2. Instruments

[0120] Microplate washer and microplate reader

3. Experimental Grouping

[0121] PDL1-WT pHLIP group; WTpHLIP control group; BSA control group and BLANK group

4. Experimental Method

[0122] (1) The PDL1-WT pHLIP, WT pHLIP and BSA were subjected to gradient envelope at an initial concentration of 50 μg/ml, and stayed overnight at 4° C.

[0123] (2) The microplate was washed by the microplate washer for 3 times; and casein sealing was conducted at 37° C. for 1 h.

[0124] (3) biotin-PD-1 (1 μg/ml) was added at 37° C. for 1 h.

[0125] (4) A second antibody was added for keeping in dark place at a room temperature for 45 min.

[0126] (5) A developing solution was added for developing for 5 min.

[0127] (6) Sulfuric acid termination was conducted, and reading was conducted by the microplate reader at 450 nm.

II. ELISA Experiment of Binding of PD-1 and Extracellular Domain of PDL1

1. Experimental Materials

[0128] Reagents: biotin (BioLegend), extracellular domain of PD-L1 and PBS (Gibco).

2. Instruments

[0129] Microplate washer and microplate reader

3. Experimental Grouping

[0130] PDL1 extracellular domain group, PDL1-WT pHLIP group; WTpHLIP control group; BSA control group and BLANK group

4. Experimental Method

[0131] (1) The PDL1 extracellular domain, PDL1-WT pHLIP, WT pHLIP and BSA were subjected to gradient envelope at an initial concentration of 50 μg/ml, and stayed overnight at 4° C.

[0132] (2) The microplate was washed by the microplate washer for 3 times; and casein sealing was conducted at 37° C. for 1 h.

[0133] (3) biotin-PD-1 (1 μg/ml) was added at 37° C. for 1 h.

[0134] (4) A second antibody was added for keeping in dark place at a room temperature for 45 min.

[0135] (5) A developing solution was added for developing for 5 min.

[0136] (6) Sulfuric acid termination was conducted, and reading was conducted by the microplate reader at 450 nm.

III. Experiment of Binding of Biotin-PDL1 Antibody and PDL1-WT pHLIP

1. Experimental Materials

[0137] Reagents: biotin-anti-PDL1 (BioLegend), PDL1-WT pHLIP and PBS (Gibco).

2. Instruments

[0138] Microplate washer and microplate reader

3. Experimental Grouping

[0139] PDL1-WT pHLIP group; WTpHLIP control group; BSA control group and BLANK group

4. Experimental Method

[0140] (1) The PDL1-WT pHLIP, WT pHLIP and BSA were subjected to gradient envelope at an initial concentration of 50 μg/ml, and stayed overnight at 4° C.

[0141] (2) The microplate was washed by the microplate washer for 3 times; and casein sealing was conducted at 37° C. for 1 h.

[0142] (3) biotin-anti-PDL1 (1 μg/ml) was added at 37° C. for 1 h.

[0143] (4) A second antibody was added for keeping in dark place at a room temperature for 45 min.

[0144] (5) A developing solution was added for developing for 5 min.

[0145] (6) Sulfuric acid termination was conducted, and reading was conducted by the microplate reader at 450 nm.

IV Results

[0146] The results were shown as FIGS. 2-4. The EC50 was equal to 2.058 μg/ml during binding of the PDL1-WT pHLIP and the PD-1; the EC50 was equal to 1.213 μg/ml during binding of the PD-1 and the extracellular domain of PDL1; and the EC50 was equal to 0.5066 μg/ml during binding of the PDL1-WT pHLIP and the PDL1 antibody. The above results showed that, the binding ability of the

[0147] PDL1-WT pHLIP and the PD-1 was equivalent to the extracellular domain of PDL1; and the binding of the PDL1 and PD-1 was not affected by linking of the pHLIP.

Embodiment 3

Study of Ability of PDL1-var3 Binding Ligand

[0148] I. ELISA Experiment of Binding of PD-1 and PDL1-var3

1. Experimental Materials

[0149] Reagents: biotin (BioLegend), PDL1-var3 and PBS (Gibco).

2. Instruments

[0150] Microplate washer and microplate reader

3. Experimental Grouping

[0151] PDL1-var3 group; var3 control group; BSA control group and BLANK group

4. Experimental Method

[0152] (1) The PDL1-var3, var3 and BSA were subjected to gradient envelope at an initial concentration of 50 μg/ml, and stayed overnight at 4° C.

[0153] (2) The microplate was washed by the microplate washer for 3 times; and casein sealing was conducted at 37° C. for 1 h.

[0154] (3) biotin-PD-1 (1 μg/ml) was added at 37° C. for 1 h.

[0155] (4) A second antibody was added for keeping in dark place at a room temperature for 45 min.

[0156] (5) A developing solution was added for developing for 5 min.

[0157] (6) Sulfuric acid termination was conducted, and reading was conducted by the microplate reader at 450 nm.

II. Experiment of Binding of Biotin-PDL1 and PDL1-var3

1. Experimental Materials

[0158] Reagents: biotin-anti-PDL1 (BioLegend), PDL1-var3 and PBS (Gibco).

2. Instruments

[0159] Microplate washer and microplate reader

3. Experimental Grouping

[0160] PDL1-var3 group; var3 control group; BSA control group and BLANK group

4. Experimental Method

[0161] (1) The PDL1-var3, var3 and BSA were subjected to gradient envelope at an initial concentration of 50 μg/ml, and stayed overnight at 4° C.

[0162] (2) The microplate was washed by the microplate washer for 3 times; and casein sealing was conducted at 37° C. for 1 h.

[0163] (3) biotin-anti-PDL1 (1 μg/ml) was added at 37° C. for 1 h.

[0164] (4) A second antibody was added for keeping in dark place at a room temperature for 45 min.

[0165] (5) A developing solution was added for developing for 5 min.

[0166] (6) Sulfuric acid termination was conducted, and reading was conducted by the microplate reader at 450 nm.

IV Results

[0167] The results were shown as FIGS. 5 and 6. The EC50 was equal to 1.985 μg/ml during binding of the PDL1-var3 and the PD-1; and the EC50 was equal to 0.5018 μg/ml during binding of the PDL1-var3 and the PDL1. The above results showed that, the binding ability of the PDL1-var3 and the PD-1 was equivalent to the extracellular domain of PDL1; and the binding of the PDL1 and PD-1 was not affected by linking of the var3.

Embodiment 4

Study of Ability of PDL1-var7 Binding Ligand

[0168] I. ELISA Experiment of Binding of PD-1 and PDL1-var7

1. Experimental Materials

[0169] Reagents: biotin (BioLegend), PDL1-var7 and PBS (Gibco).

2. Instruments

[0170] Microplate washer and microplate reader

3. Experimental Grouping

[0171] PDL1-var7 group; var7 control group; BSA control group and BLANK group

4. Experimental Method

[0172] (1) The PDL1-var7, var7 and BSA were subjected to gradient envelope at an initial concentration of 50 μg/ml, and stayed overnight at 4° C.

[0173] (2) The microplate was washed by the microplate washer for 3 times; and casein sealing was conducted at 37° C. for 1 h.

[0174] (3) biotin-PD-1 (1 μg/ml) was added at 37° C. for 1 h.

[0175] (4) A second antibody was added for keeping in dark place at a room temperature for 45 min.

[0176] (5) A developing solution was added for developing for 5 min.

[0177] (6) Sulfuric acid termination was conducted, and reading was conducted by the microplate reader at 450 nm.

II. Experiment of Binding of Biotin-PDL1 and PDL1-var7

1. Experimental Materials

[0178] Reagents: biotin-anti-PDL1 (BioLegend), PDL1-var7 and PBS (Gibco).

2. Instruments

[0179] Microplate washer and microplate reader

3. Experimental Grouping

[0180] PDL1-var7 group; var7 control group; BSA control group and BLANK group

4. Experimental Method

[0181] (1) The PDL1-var7, var7 and BSA were subjected to gradient envelope at an initial concentration of 50 μg/ml, and stayed overnight at 4° C.

[0182] (2) The microplate was washed by the microplate washer for 3 times; and casein sealing was conducted at 37° C. for 1 h.

[0183] (3) biotin-anti-PDL1 (1 μg/ml) was added at 37° C. for 1 h.

[0184] (4) A second antibody was added for keeping in dark place at a room temperature for 45 min.

[0185] (5) A developing solution was added for developing for 5 min.

[0186] (6) Sulfuric acid termination was conducted, and reading was conducted by the microplate reader at 450 nm.

IV Results

[0187] The results were shown as FIGS. 7 and 8. The EC50 was equal to 1.743 μg/ml during binding of the PDL1-var7 and the PD-1; and the EC50 was equal to 0.4406 μg/ml during binding of the PDL1-var7 and the PDL1. The above results showed that, the binding ability of the PDL1-var7 and the PD-1 was equivalent to the extracellular domain of PDL1; and the binding of the PDL1 and PD-1 was not affected by linking of the var7.

Embodiment 5

Localization of Fusion Peptide on Cells

1. Cell Line

[0188] Human embryonic kidney cell line HEK293 (purchased from ATCC).

2. Reagents DMEM medium (Gibco), fetal calf serum (Gibco), PBS (pH=7.4) (Gibco), Tris-HCL (1 mol/mL), PD-1, PDL1 extracellular domain, PDL1-WT pHLIP, PDL1-WT pHLIP, PDL1-var7, var7, PDL1-var3, var7 and PE crosslinking kits (Expedeon Company).

3. Instruments

[0189] Super clean bench; carbon dioxide incubator (Thermo); centrifuge (Thermo); laser confocal cell-culture dish (Corning); electronic pH indicator (Sartorius); optical microscope (Nikon); laser scanning confocal microscope (Zeiss LSM 880).

4. Experimental Steps

[0190] HEK293 cells in a logarithmic phase were collected; a culture solution was removed; and the cells were washed with PBS twice. An appropriate amount of pancreatin was added for digestion;

[0191] digestion was terminated with the culture solution; the solution was transferred into a 10 ml test tube; the solution was centrifuged at 1000 rpm for 5 min; the supernatant was removed; and 1 mL of DMEM medium containing 10% of fetal calf serum was added for resuspending and uniform mixing. 10 μL of cell suspension was sucked into a cell counting plate for counting; a certain amount of cell suspension was added into the laser confocal cell-culture dish and adjusted to 5*10.sup.5 with a medium; and 1 mL of cell system was cultured in the incubator overnight.

[0192] After attachment of the cultured HEK293 cells, the supernatant was removed; and the solution was washed twice with PBS buffer having a pH value of 7.4.

[0193] A culture solution was prepared from 1 mol/mL of hydrochloric acid and the PBS buffer having the pH value of 7.4; the hydrochloric acid was added into the PBS dropwise; and finally the pH value of the buffer was titrated to 6.3 (5 mL of PBS buffer +12 μL of Tris-hydrochloric acid). PDL1-WT pHLIP, or PDL1-var3 or PDL1-var7 was respectively added into PBS buffer having pH values of 7.4 and 6.3 to be fully and uniformly mixed; and the peptide was diluted to a final concentration of 200 μg/mL according to a ratio. In a control group, a PDL1 extracellular domain having a final concentration of 180 μg/mL and WT pHLIP or var3 or var7 having a final concentration of 20μg/mL were added into the PBS buffer having pH values of 7.4 and 6.3.

[0194] The PBS buffer in the culture dish was removed; 1 mL of the above mixed solution was respectively added; 1 mL of unmixed PBS buffer having respective pH values of 7.4 and 6.3 was added into a blank control group; and the cells were incubated in a cell incubator at 37° C. for 1 hour.

[0195] The supernatant was removed after completion of incubation; the solution was washed twice with the PBS buffer having corresponding pH values; 1 mL of 1 μg/mL PD-1-PE was added; and the cells were incubated in a dark place at 4° C. for half an hour.

[0196] The solution was washed twice with the PBS having corresponding pH values after completion of incubation; and then PBS buffer having corresponding pH values was added.

[0197] The prepared culture dish was placed under the laser scanning confocal microscope for observing the fluorescence situation on the surface of the cell membrane.

5. Results

[0198] The results were shown as FIG. 9. The PDL1-pHLIP can be well shown on the surface of the cell membrane in an acid environment. The above result showed that, the PDL1 did not affect the characteristic that the pH low insertion peptide was inserted into the cell membrane; meanwhile, conformation of the PDL1 was not affected during binding of the PDL1 and the pH low insertion peptide.

[0199] The results were shown as FIG. 10. The PDL1-var3 can be well shown on the surface of the cell membrane in an acid environment. The above result showed that, the PDL1 did not affect the characteristic that the pH low insertion peptide was inserted into the cell membrane; meanwhile, conformation of the PDL1 was not affected during binding of the PDL1 and the pH low insertion peptide.

[0200] The results were shown as FIG. 11. The PDL1-var7 can be well shown on the surface of the cell membrane in an acid environment. The above result showed that, the PDL1 did not affect the characteristic that the pH low insertion peptide was inserted into the cell membrane; meanwhile, conformation of the PDL1 was not affected during binding of the PDL1 and the pH low insertion peptide.

Embodiment 6

Effect Evaluation of Fusion Peptide for Treating Autoimmune Diseases

1. Experimental Materials

[0201] Animal: DBA/1J male mouse (at an age of 7 weeks)

2. Reagents

[0202] Chicken collagen II (#20012), complete Freund's adjuvant (#7001) and incomplete Freund's adjuvant (#7002) were all Chondrex, USA; WT pHLIP, var3 and var7 were synthesized by Chinese Peptide Company; and PDL1 extracellular domain, PDL1-WT pHLIP, PDL1-var3, PDL1-var7 and PDL1-Ig were prepared by the company.

3. Instruments

[0203] Tissue homogenizer (IKA, H44)

4. Experimental Method

Emulsification of Antigen

[0204] Adjuvants of the same volume were added into a 5 ml disposable needle tube; the tissue homogenizer was turned on; a collagen solution was dropped while low-speed stirring; and after the collagens were totally added, the rotation speed was increased until water-in-oil emulsion droplets were formed. The needle tube was placed in an ice bath in the process for preventing protein denaturation.

[0205] First immunization: 2 mg/ml of the collagen was mixed with the complete Freund's adjuvant (4 mg/ml) of the same volume; emulsification was conducted (forming a water-in-oil sample); the collagen had a final concentration of 1 mg/ml; and each mouse was injected with 100 μl of the sample, i.e., 100 μg. The needle was inserted from a position that was 2 cm away from the tail head; and injection started when a needle tip position was 0.5 cm away from the tail head.

[0206] Second immunization: 2 mg/ml of the collagen was mixed with the incomplete Freund's adjuvant of the same volume; emulsification was conducted; and each mouse was injected with 100 μg of the sample. The injection site was a position that was 3 cm away from the tail head; and a subcutaneous depth of the inserted needle tip was 1.5 cm away from the tail head of the mouse.

[0207] Within 2 weeks after the second immunization (then CIA classical symptoms appeared), the experiment was divided into 4 groups: (1) intraperitoneal injection of PDL1-WT pHLIP (or PDL1-var3, or PDL1-var7), 1 mg/kg, administration once every other day; (2) intraperitoneal injection of PDL1 (1 mg/kg)+WT pHLIP (0.2 mg/kg) (or PDL1-var3, or PDL1-var7), administration once every other day; (3) intraperitoneal injection of PDL1-Ig, 1 mg/kg, administration once every other day; and (4) intraperitoneal injection of PBS in the control group. Each group included 10 mice; and heel thicknesses and serum cell factor content were detected within 26 days.

3. Results

[0208] Mouse sole photos in FIGS. 12-14 showed that, the mice in the PDL1-pHLIP group, the PDL1-var3 group or the PDL1-var7 group had normal soles; and inflammation was significantly relieved. CIA grading results were shown as FIGS. 15-17; and CIA grades in the PDL1-pHLIP group, the PDL1-var3 group or the PDL1-var7 group were significantly decreased.

[0209] Serum cell factor detection results were shown as FIGS. 18-20; and content of TNF-α, IFN-γ, IL-6 and IL-17A in the PDL1-pHLIP group, the PDL1-var3 group or the PDL1-var7 group was significantly decreased.

[0210] Heel thickness detection results were shown as FIGS. 21-23; and the mouse heel thickness in the PDL1-pHLIP group, the PDL1-var3 group or the PDL1-var7 group was significantly decreased. The above results show that, the fusion peptide in the present invention can significantly alleviate symptoms of the arthritis, which indicates that the fusion peptide has an excellent effect of treating the arthritis.