Targeted immunotolerance vaccine, preparation method therefor, and use thereof

Abstract

Disclosed in the present invention are a targeted immunotolerance vaccine, a preparation method therefor, and use thereof. The present invention separately modifies a rheumatoid arthritis-related autoantigen peptide and CTLA4 Ig with DSPE-PEG (DP) to prepare a targeted formulation DP-antigen peptide and a targeted formulation DP-CTLA4, which are then mixed to obtain the vaccine. The vaccine described in the present invention, after intravenous injection, binds to albumin in vivo by means of DSPE, "hitchhiking" on albumin to target and enrich in inflammatory lesions, the spleen, the liver, and other tolerance-inducing sites, inhibiting T cell activation, and inducing anergy and apoptosis of rheumatoid arthritis autoantigen-specific T cells, thereby achieving immunotolerance treatment and prevention of rheumatoid arthritis.

Description

Targeted immune tolerance vaccine and preparation method and use thereof TECHNICAL FIELD

[0001] The present application relates to the field of pharmaceutical technology, in particular to a targeted immune tolerance vaccine and preparation method and use thereof. BACKGROUND

[0002] Rheumatoid arthritis (RA) is an autoimmune disease characterized by inflammatory joint synovial membrane and cartilage destruction caused by the human immune system misidentifying self-antigens and attacking normal tissues. Small molecule drugs and biological agents with immunosuppressive effects are commonly used to treat RA in clinical practice. However, due to poor targeting and short half-life of the drugs, the drug concentration at the lesion is low, the therapeutic effect is weak, and non-specific suppression of the immune system can easily cause systemic toxic side effects.

[0003] Immune tolerance therapy for remodeling specific tolerance to self-antigens has attracted much scientific interest in recent years and is one of the most advanced and potential methods for treating RA. Immune tolerance is the immune unresponsiveness of the body's immune system to specific antigens. Once the immune tolerance of the body to self-antigens is broken, autoimmune diseases will occur. Immune tolerance vaccines can induce the body to produce immune tolerance to the antigen by injecting relevant self-antigens and immunosuppressive agents. However, the current tolerance vaccines lack sufficient targeting to the lesion and the organ for inducing tolerance, and the therapeutic effect on RA needs to be improved. Chinese patent publication CN115590974A discloses a functionalized targeting preparation composed of an amphiphilic polymer and a protein biological agent and its application in the treatment of autoimmune diseases such as rheumatoid arthritis. The applicant has found that the therapeutic effect of the application on autoimmune diseases needs to be further improved, and the prevention of rheumatoid arthritis is still lacking. The present application is thus derived. SUMMARY

[0004] To solve at least one of the technical problems in the background art, the present application provides a targeted immune tolerance vaccine and a preparation method and use thereof.

[0005] The technical solution of the present application is as follows: One object of the present application is to provide a targeted immune tolerance vaccine composed of a first targeting preparation and a second targeting preparation, wherein the first targeting preparation comprises a first amphiphilic polymer and a disease-related antigen peptide, and the second targeting preparation comprises a second amphiphilic polymer and a protein biological agent.

[0006] The first amphiphilic polymer and the second amphiphilic polymer are both one of DP (DSPE-PEG) with a reactive functional group.

[0007] Preferably, the reaction functional group is maleimide, the first amphiphilic polymer is DP-MAL, the first targeting preparation is prepared by Michael addition reaction between the first amphiphilic polymer and a disease-related antigen peptide, and the disease-related antigen peptide has a cysteine added to the C-terminal during synthesis.

[0008] Preferably, the first amphiphilic polymer and the disease-related antigen peptide are reacted at a molar ratio of 1:(1-5).

[0009] Preferably, after the completion of the Michael addition reaction, the reaction solution is dialyzed against ultrapure water in a dialysis bag with a molecular weight cut-off of 3500 Da for 48 h, and vacuum freeze-drying is performed to obtain the first targeting preparation.

[0010] Preferably, the disease-related antigen peptide is a rheumatoid arthritis-related antigen peptide.

[0011] Preferably, the reaction functional group is N-hydroxysuccinimide (NHS), the second amphiphilic polymer is DP-NHS, the protein biological preparation is CTLA4 Ig, and the second targeting preparation is prepared by amide reaction between the second amphiphilic polymer and the protein biological preparation.

[0012] Preferably, the second amphiphilic polymer and the protein biological preparation are reacted at a molar ratio of 1:4.

[0013] Preferably, after the completion of the amide reaction, the reaction solution is purified by ultrafiltration.

[0014] Another object of the present application is to provide a preparation method of the above-mentioned targeted immunological tolerance vaccine (hereinafter referred to as DADC vaccine), comprising the following steps:

[0015] Synthesis of the first targeting preparation (hereinafter referred to as DP-antigen peptide or DP-antigen):

[0016] Adding a cysteine to the C-terminal of the disease-related antigen peptide;

[0017] Performing Michael addition reaction at a molar ratio of disease antigen peptide: DP-MAL of 1:(1-5);

[0018] Transferring the reaction solution to a dialysis bag and dialyzing against ultrapure water for 48 h, the dialysis bag having a molecular weight cut-off of 3500 Da, and vacuum freeze-drying to obtain the first targeting preparation;

[0019] Synthesis of the second targeting preparation (hereinafter referred to as DP-CTLA4):

[0020] Mixing at a molar ratio of CTLA4 Ig: DP-NHS of 1:4;

[0021] Add NaHCO3 to adjust pH to 8.5, and carry out amide reaction;

[0022] The reaction solution is purified by ultrafiltration method;

[0023] Preparation of the vaccine: the first and second targeting preparations are dissolved and mixed to obtain the vaccine, and the mixing ratio of the two is preferably 1:(1-5).

[0024] The present application also aims to provide the use of the above-mentioned targeting immunological tolerance vaccine in the preparation of a medicament for preventing and treating autoimmune diseases, especially in the preparation of a medicament for specifically preventing and treating rheumatoid arthritis. BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1 is a schematic diagram of the preparation and mechanism of action of the targeting DADC vaccine (A is a schematic diagram of the preparation of the DADC vaccine; B is a schematic diagram of the mechanism of action of the DADC vaccine);

[0026] Figure 2A is an agarose gel electrophoresis characterization of DP-antigen peptide with different reaction ratios (1:1 / 1:2 / 1:5); B-D are MALDI-TOF-MS characterizations of DP-antigen peptide (B is antigen peptide, C is DP-MAL, D is MALDI-TOF-MS result of DP-antigen peptide, and antigen peptide is OVA 323-339 -C);

[0027] Figure 3 is a SEC characterization of DP-antigen peptide (A is DMSO, B is antigen peptide, C is DP-MAL, D is SEC result of DP-antigen peptide, and antigen peptide is OVA 323-339 -C);

[0028] Figure 4 is a characterization of DP-CTLA4 (A is 1H NMR characterization; B is DLS characterization);

[0029] Figure 5 is the effect of DP modification on the biological activity of antigen peptide and CTLA4 Ig (A is an experimental flowchart; B is a representative flow cytometry histogram of CD4 + T cell surface CD69; C is CD69 mean fluorescence quantification analysis; D is a representative flow cytometry histogram of CD4 + T cell surface CD25; E is CD25 mean fluorescence quantification analysis; F is an experimental flowchart; G is a representative flow cytometry histogram of CD80 + CD86 + cells in CD 11c +Representative flow cytometry plots in cells; H is CD86 mean fluorescence quantification analysis; I is the fluorescence histogram of T cell surface CD25 staining; J is CD25 mean fluorescence quantification analysis. Data are expressed as mean ± SD (n = 3), analyzed using One-way ANOVA and Tukey post-hoc test; ns, no significant difference; ****, P < 0.0001);

[0030] Figure 6 is the characterization of DP-antigen peptide and DP-CTLA4 in vitro loading with albumin (A is the fluorescence imaging of DP-antigen peptide loading with albumin, B is the coomassie blue imaging; C is the fluorescence imaging of DP-CTLA4 loading with albumin, D is the coomassie blue imaging);

[0031] Figure 7 is the cytotoxicity detection of DP-antigen peptide and DP-CTLA4 (A is the cytotoxicity of DP-antigen peptide on BMDC; B is the cytotoxicity of DP-CTLA4 on BMDC);

[0032] Figure 8 is the in vivo distribution of DP-antigen peptide-Cy5 (A is the experimental flow chart; B is the fluorescence images of organs (He: Heart; Li: Liver; Sp: Spleen; Lu: Lung; Ki: Kidney; LN: Lymph Nodes) and paw (Lp: Left paw; Rp: Right paw) at different time points, C is the fluorescence intensity; antigen peptide is OVA 323-339 C, data are expressed as mean ± SD (n = 4), analyzed using Unpaired t test; ns, no significant difference; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001);

[0033] Figure 9 is the in vivo distribution of DP-CTLA4-Cy5 (A is the experimental flow chart; B is the fluorescence images of organs (He: Heart; Li: Liver; Sp: Spleen; Lu: Lung; Ki: Kidney; LN: Lymph Nodes) and paw (Lp: Left paw; Rp: Right paw) at different time points, C is the fluorescence intensity; data are expressed as mean ± SD (n = 4), analyzed using Unpaired t test; ns, no significant difference; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001);

[0034] Figure 10. DADC tolerizing vaccine significantly suppressed the proliferation of antigen-specific ACT cells in spleen, lymph node and peripheral blood (A, experimental scheme; Booster, activating vaccine DP-OVA323-339 and DP-CpG; B, representative flow cytometry plot of the percentage of ACT cells in CD3 + cells; C, number of ACT cells; D, CFSE fluorescence of ACT cells; E, number of ACT cells in lymph node; F, number of ACT cells in peripheral blood; the molar ratio of DP-antigen peptide to DP-CTLA4 used in this experiment was about 2:1; data were expressed as mean ± SD (Gl-G5, n=6; G6, n=5), analyzed using One-way ANOVA and Tukey post-hoc test; ns, no significant difference; **, P<0.01; ***, P<0.001; ****, P<0.0001);

[0035] Figure 11. The effective duration of DADC vaccine (A, experimental scheme; Booster, activating vaccine DP-gplOO25-33 and DP-CpG; B, percentage of ACT cells in CD8 + cells in spleen at different time points; C, number of ACT cells in spleen; D, percentage of ACT cells in CD8 + cells in lymph node at different time points; E, number of ACT cells in lymph node; the molar ratio of DP-antigen peptide to DP-CTLA4 used in this experiment was about 2:1; data were expressed as mean ± SD (n=6), analyzed using Two-way ANOVA and Tukey post-hoc test; ns, no significant difference; **, P<0.01; ***, P<0.001; ****, P<0.0001);

[0036] Figure 12. DADC vaccine induced antigen-specific immune tolerance (A, experimental scheme; Booster, activating vaccine DP-gplOO25-33 and DP-CpG; B, representative flow cytometry plot of the percentage of ACT cells in CD8 + cells in spleen; C, number of ACT cells in spleen; D, number of ACT cells in lymph node; E, concentration of IFN-γ, TNF-a and IL-6 in the culture medium after stimulating spleen cells with gplOO25-33 in vitro; the molar ratio of DP-antigen peptide to DP-CTLA4 used in this experiment was about 2:1; data were expressed as mean ± SD (n=6), analyzed using One-way ANOVA and Tukey post-hoc test; ns, no significant difference; *, P<0.05; **, P<0.01; ****, P<0.0001);

[0037] Figure 13 is the therapeutic evaluation of DADC vaccine on CIA mice (A, experimental flow chart; B, arthritis score; C, body weight change; D, right hind paw photo; E, paw thickness; F, maximum speed; G, running time; H, hind paw Micro-CT picture; arrow indicates bone erosion site; the molar ratio of DP-antigenic peptide to DP-CTLA4 used in this experiment is about 3:1; data are expressed as mean ± SD (n = 8), analyzed by Two-way ANOVA and Tukey's post-hoc test (B and C) or One-way ANOVA and Tukey's post-hoc test (E to G); ns, no significant difference; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001);

[0038] Figure 14 is the inflammation inhibition effect of DADC vaccine on CIA mice (A, experimental flow chart; B, the concentration of IL-6, IL-1β and TNF-α in serum; C, the concentration of IL-6, IL-1β and TNF-α in synovial membrane; D, the flow cytometry representative graph of CD80 expression in CD11c + cells; E, the quantitative analysis of CD80 + percentage in CD11c + cells; F, the concentration of IFN-γ in culture medium after stimulating spleen cells with Collagen259-272 in vitro for 48h; the molar ratio of DP-antigenic peptide to DP-CTLA4 used in this experiment is about 3:1; data are expressed as mean ± SD (n = 7), analyzed by One-way ANOVA and Tukey's post-hoc test; ns, no significant difference; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001);

[0039] Figure 15 is the prevention of in vivo proliferation of antigen-specific T cells by DADC vaccine (A, experimental flow chart, Inducer is inducer (DP-OVA323-339 and DP-CpG); B, representative flow cytometry of ACT cell percentage in CD3 + cells; C, the number of ACT cells in spleen; D, representative flow cytometry of CFSE fluorescence of ACT cells; E, the number of ACT cells in lymph node; the molar ratio of DP-antigenic peptide to DP-CTLA4 used in this experiment is about 2:1; data are expressed as mean ± SD (n = 6), analyzed by Brown-Forsythe ANOVA and Tukey's post-hoc test; ns, no significant difference; *, P < 0.05; **, P < 0.01; ***, P < 0.001);

[0040] Figure 16. DADC vaccine can induce antigen-specific prophylactic effect (A, experimental flow chart; Inducer, inducer (DP-OVA323-339 and DP-CpG); B, quantification of the percentage of ACT cells in CD4 + T cells; C, quantification of the percentage of ACT cells in the spleen; D, number of ACT cells in the spleen; E, quantification of the percentage of ACT cells in the lymph node; F, number of ACT cells in the lymph node; G, concentration of IFN-γ, TNF-α and IL-6 in the supernatant after in vitro stimulation of splenocytes with OVA323-339; the molar ratio of DP-antigen peptide to DP-CTLA4 used in this experiment was about 2:1; data are expressed as mean ± SD (n=6), analyzed using One-way ANOVA and Tukey post-hoc test; ns, no significant difference; *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001);

[0041] Figure 17. DADC vaccine prevents RA mice from developing disease (A, experimental flow chart; B, arthritis score; C, body weight change; D, paw thickness; E, maximum speed; F, running time; G, concentration of IL-6, IL-1β and TNF-α in serum; H, concentration of IL-6, IL-1β and TNF-α in synovium; the molar ratio of DP-antigen peptide to DP-CTLA4 used in this experiment was about 2:1; data are expressed as mean ± SD (n=7), analyzed using Two-way ANOVA and Tukey post-hoc test (B and C) or One-way ANOVA and Tukey post-hoc test (D to F); ns, no significant difference; *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001);

[0042] Figure 18. Analysis of ankle Micro-CT and tissue sections (A, Micro-CT image of hind paw; arrow indicates bone erosion part; B, H&E staining of section; C, Masson staining; D, Safranin O-fast green staining; E, synovitis score; F, synovial fibrosis area; G, cartilage thickness; the molar ratio of DP-antigen peptide to DP-CTLA4 used in this experiment was about 2:1; data are expressed as mean ± SD (n=7), analyzed using One-way ANOVA and Tukey post-hoc test; ns, no significant difference; *, P<0.05; ****, P<0.0001; scale bar, 100 μm). DETAILED DESCRIPTION

[0043] The above solutions are further described in the following detailed description in conjunction with the accompanying drawings and specific examples.

[0044] T cell activation requires dual signals - antigen stimulation signal and costimulation signal. Only antigen stimulation signal, lack of costimulation signal will lead to anergy or apoptosis of self-reactive T cells, inducing immune tolerance of the body. Abatacept (CTLA4 Ig) is a biological agent for the treatment of RA in clinic, which is a fusion protein connecting CTLA4 on the surface of T cells with the Fc part of IgG1 antibody. It can bind to the costimulatory signal CD80 / 86 on the surface of antigen presenting cells (APC), competitively block the binding of CD80 / 86 to CD28, inhibit T cell activation, and thus reduce the inflammatory response and disease activity caused by RA.

[0045] Albumin is the most abundant protein in blood, interstitial fluid and lymph fluid. Compared with other plasma proteins, the half-life of albumin in the body can be up to 3 weeks. Albumin has excellent targeting to inflammatory sites and lymphoid organs. For example, MTX-loaded albumin nanoparticles can be enriched at the RA lesion joints. In addition, using ligands that can bind to albumin to modify drug molecules can achieve drug loading on albumin in the body, thereby prolonging the plasma half-life and stability of the drug and reducing the degradation of the drug by enzymes. Distearoyl phosphatidyl ethanolamine (DSPE) can efficiently bind to albumin. The amphiphilic DSPE-polyethylene glycol (DSPE-PEG, DP) polymer modified with small molecules or polypeptides at the end can load albumin in the body through DSPE after subcutaneous administration, and use the lymph node backflow property of albumin to promote the lymph node enrichment of small molecules or polypeptides.

[0046] Based on the above theory, the present application respectively modifies RA related autoantigen peptide and CTLA4 Ig with DP to prepare DP-antigen and DP-CTLA4, and mixes to prepare DADC vaccine (A in FIG. 1). After intravenous injection, DADC vaccine binds to albumin in the body through DSPE, and "hitches a ride" on albumin to target and enrich to inflammatory lesions, spleen and liver and other tolerance induction sites. When dendritic cells (DCs) present RA autoantigens, CTLA4 Ig can bind to the costimulatory molecules CD80 / CD86 on the surface of DCs, competitively block the binding of CD80 / CD86 to T cell CD28, and inhibit T cell activation, thereby inducing anergy and apoptosis of RA autoantigen-specific T cells, achieving efficient and precise RA immune tolerance treatment and prevention (B in FIG. 1).

[0047] 1 Experimental method

[0048] 1.1 Materials

[0049] ​​​​​​​​​​Collagen type II, Freund's complete adjuvant and Freund's incomplete adjuvant were purchased from Chondrex, USA. DSPE-PEG2000-maleimide (DSPE-PEG 2000 MAL, DP-MAL), DSPE-PEG2000-activated ester (DSPE-PEG 2000 NHS, DP-NHS) were purchased from Shanghai Bingsuo Biotechnology Co., Ltd. Antigen peptides Collagen 259-272 C, OVA 323-339 C, OVA 257-264 C, gp100 25-33 C were purchased from Shanghai Jiersheng Biochemical Co., Ltd.

[0050] 1.2 Experimental animals

[0051] OT-2, Pmel-1 mice were purchased from Jackson Laboratory, USA, and C57BL / 6, BALB / c, DBA / 1 mice were purchased from Changzhou Cavens Experimental Animal Co., Ltd. The mice used were 6-8 weeks old, and the animal experiment operation met the relevant specification requirements of experimental animal welfare and the ethics of experimental animals of Suzhou University.

[0052] 1.3 Synthesis of DP-antigen peptides

[0053] Cysteine was added to the C-terminus of the synthesized antigen peptide, and DP-antigen peptide was prepared by Michael addition reaction of DP-MAL (structure, properties and preparation method refer to patent publication CN115590974A) with cysteine side chain-SH. The molar ratio of antigen peptide: DP-MAL was 1:1, 1:2 and 1:5, and the four-dimensional rotator was reacted for 24 h. The reaction solution was transferred to a dialysis bag (MWCO = 3500 Da), and ultra-pure water dialysis was performed for 48 h. Vacuum freeze-drying machine was freeze-dried to obtain DP-antigen peptide.

[0054] 1.4 Characterization of DP-antigen peptides

[0055] Agarose gel electrophoresis characterization: a certain amount of FITC-labeled antigen peptide was weighed to prepare a DP-antigen peptide-FITC sample. Then 0.8% agarose gel was prepared and placed in an electrophoresis tank, and the prepared sample was added. The voltage was adjusted to 100 V, and the electrophoresis time was 60 min. After electrophoresis, the gel sample was placed in a multifunctional biomolecular imager, and imaging was performed using the fluorescence of FITC.

[0056] MALDI-TOF-MS: Weigh an appropriate amount of α-cyano-4-hydroxycinnamic acid (CHCA) and dissolve it in TA30 (300 μL acetonitrile + 700 μL double-distilled water + 1 μL trifluoroacetic acid) to a concentration of 7 mg / mL to prepare the sample spotting matrix. Dilute the antigenic peptide and DP-antigen peptide with DEPC water to a concentration of 1 mg / mL to prepare the sample solution. Spot the sample using a sandwich method (matrix + sample + matrix). After the sample is completely dry, place it in the instrument for detection.

[0057] Size exclusion chromatography: The relative molecular weights of DP-antigen peptide and antigen peptide were determined using a size exclusion chromatograph. Liquid chromatography conditions: BioCore SEC-120 column (4.6 × 300 mm, 1.8 μm); detection wavelength 220 nm; flow rate 0.15 mL / min; injection volume 1 μL; mobile phase was an aqueous solution containing 20% ​​acetonitrile and 50 mM ammonium acetate.

[0058] 1.5 Effect of DP modification on the biological activity of antigenic peptides

[0059] (1) Extraction and culture of bone marrow-derived dendritic cells (BMDC): ① Select 6-8 week old C57BL / 6 mice and euthanize them; ② Dissect the mice and collect the tibia and femur; ③ Wash the bone marrow with PBS into centrifuge tubes and centrifuge at 350g for 5 min; ④ Discard the supernatant, add ACK lysis buffer to lyse the cells, add PBS to stop the reaction after 3 min, and centrifuge at 350g for 5 min; ⑤ Discard the supernatant, dilute the cells with medium containing 20 ng / mL GM-CSF, and culture at 1×10⁻⁶. 6 Seed cells at a density of 1 / mL into culture dishes and incubated in a cell culture incubator; ⑥ After 3 days, add one volume of medium containing 20 ng / mL GM-CSF to the cells and continue culturing; ⑦ On the 7th day, collect the cells for subsequent experiments.

[0060] (2)OT2 CD4 + T cell extraction: ① Extract spleens from OT2 mice, crush them on a moistened filter, and centrifuge at 700g for 5 min at 4℃; ② Discard the supernatant, add ACK lysis buffer to lyse red blood cells, add PBS to stop the reaction after 3 min, centrifuge at 700g for 5 min at 4℃ to obtain OT2 mouse spleen cells; ③ Follow EasySep... TM mouse CD4 + The instructions for the T-cell positive selection kit extract OT2 CD4. + T cells.

[0061] (3) Take BMDC cells that have been induced to mature and are in good growth condition, and use 2×10 5Live cells were seeded in U-bottom 96-well plates at a density of 1 x 105cells / well, and co-cultured with antigen peptides or DP-antigen peptides for 2 h. Then the original culture medium was removed by centrifugation, and the separated OT2 CD4 + T cells were added at a ratio of 1:1, and co-cultured for 48 h. The cells were collected for flow cytometry staining, and then detected by flow cytometry.

[0062] 1.6 Synthesis of DP-CTLA4

[0063] DP-CTLA4 was prepared by the method of amide reaction between DP-NHS (its structure, properties and preparation method refer to patent publication No. CN115590974A) and -NH2 of CTLA4 Ig. The molar ratio of CTLA4 Ig: DP-NHS was 1:4, 1 mol / L NaHCO3 was added to adjust the pH value to 8.5, and the four-dimensional rotator was reacted for 24 h. The reaction solution was purified by ultrafiltration. Nanodrop was used to detect the absorbance at A280, and the protein concentration was quantified for subsequent use.

[0064] 1.7 Characterization of DP-CTLA4

[0065] 1 H NMR: an appropriate amount of prepared DP-CTLA4 sample was freeze-dried using a vacuum freeze dryer, the freeze-dried sample was weighed and dissolved in 1.5 mL deuterium water (D2O), 2.5 μL DMSO was added as an internal standard, and the structure of the sample was characterized by nuclear magnetic resonance hydrogen spectrum (H NMR). 1 H NMR).

[0066] DLS: an appropriate amount of the above prepared DP-CTLA4 was taken, the sample concentration was adjusted, and the sample particle size was determined on a laser nanoparticle size instrument, and each group of samples was determined in parallel for 5 times.

[0067] 1.8 Effect of DP modification on the biological activity of CTLA4

[0068] The OT2 mouse spleen cells were extracted and prepared into a cell suspension, which was then divided into four groups, and OVA 323-339 peptide and CTLA4 Ig / DP-CTLA4 were added for co-culture. The groups were as follows: ① PBS group; ② OVA 323-339 peptide group; ③ OVA 323-339 peptide + CTLA4 Ig group; ④ OVA 323-339 peptide + DP-CTLA4 group. After 24 h, part of the cells were taken for flow cytometry staining to analyze the binding of CTLA4 Ig to DC surface CD80 / CD86. After 48 h, the remaining cells were taken for flow cytometry staining to analyze the T cell activation.

[0069] 1.9 DP-antigen peptide and DP-CTLA4 in vitro loading albumin

[0070] 1.9.1 DP-antigen peptide loading albumin

[0071] (1) A certain amount of FITC-labeled antigen peptide was weighed to prepare a DP-antigen peptide-FITC sample; and a molar ratio of DP-antigen peptide-FITC to albumin was 1:2, and a constant temperature shaking reaction was carried out at 37°C for 4h.

[0072] (2) 0.8% agarose gel was prepared and placed in an electrophoresis tank, and the prepared sample was added. The voltage was adjusted to 100V, and the electrophoresis time was 60min. After electrophoresis, the gel sample was placed in a multifunctional biomolecular imager, and imaging was performed using the fluorescence of FITC. After fluorescence imaging was completed, the gel sample was recovered, and coomassie brilliant blue staining solution was added and stained for 60min. Then the staining solution was poured out, and an appropriate amount of coomassie brilliant blue staining and decolorizing solution was added. After decolorization was completed, the gel sample was placed in a multifunctional biomolecular imager for development.

[0073] 1.9.2 DP-CTLA4 loading albumin

[0074] (1) FAM-NHS was weighed and dissolved in DMSO to 10mg / mL, and a molar ratio of CTLA4 Ig:FAM-NHS was 1:2 for feeding, and a four-dimensional rotator was reacted overnight to prepare CTLA4 Ig-FAM. Then, the CTLA4 Ig-FAM was ultrafiltrated until the supernatant was colorless. The protein concentration was quantified by Nanodrop, and part of the CTLA4 Ig-FAM sample was prepared as DP-CTLA4-FAM. A molar ratio of DP-CTLA4-FAM to albumin was 1:2, and BSA solution was added to the DP-CTLA4-FAM sample, and reacted at 37°C for 4h.

[0075] (2) The DP-CTLA4-FAM was characterized by agarose gel electrophoresis according to the method of 1.9.1 herein.

[0076] 1.10 Cytotoxicity of DP-antigen peptide and DP-CTLA4

[0077] (1) BMDC cells induced to mature and in good growth state were taken, and inoculated in a 96-well plate at a density of 2×10 5 Live cells / well, and set a cell-free group as a blank well, and cultured in a cell incubator for 12h. Then, different concentrations of DP-antigen peptide or DP-CTLA4 prepared in advance were added, and cultured for 48h.

[0078] (2) After the end of the culture, CCK-8 solution was added for 2h of co-incubation. After the positive wells turned color, the absorbance value (OD) of each well at 450nm was measured using an enzyme label meter, and the cell viability was calculated.

[0079] 1.11 In vivo biodistribution of DP-antigen peptide and DP-CTLA4

[0080] 1.11.1 In vivo distribution study of DP-antigen peptide

[0081] (1) Establishment of LPS acute inflammation model: 6-8 week old female BALB / c mice were injected with LPS in the left hind paw to induce inflammation. The concentration of LPS was 2.5mg / mL, and 20μL was injected subcutaneously for each mouse.

[0082] (2) After 2 days of modeling, the mice were grouped: ① antigen peptide-Cy5 group; ② DP-antigen peptide-Cy5 group; ③ PBS group. Intravenous administration was performed according to the grouping, and the dose was 20μg per mouse. The mice were euthanized at 6h, 12h, 24h and 48h after administration, and the heart, liver, spleen, lung, kidney, inguinal lymph node, axillary lymph node and hind paw were dissected for small animal imaging instrument (IVIS) monitoring (Ex=620nm, Em=680nm) to analyze the in vivo biodistribution and inflammation targeting. The antigen peptide was OVA 323-339 peptide.

[0083] 1.11.2 In vivo distribution study of DP-CTLA4

[0084] (1) LPS acute inflammation model was established according to the method of 1.11.1.

[0085] (2) After 2 days of modeling, the mice were grouped: ① CTLA4 Ig-Cy5 group; ② DP-CTLA4-Cy5 group; ③ PBS group. Intravenous administration was performed according to the grouping, and the dose was 20μg per mouse. The mice were euthanized at 12h, 24h and 48h after administration, and the heart, liver, spleen, lung, kidney, inguinal lymph node, axillary lymph node and hind paw were dissected for small animal imaging instrument (IVIS) monitoring (Ex=620nm, Em=680nm) to analyze the in vivo biodistribution and inflammation targeting.

[0086] 1.12 Inhibition of in vivo proliferation of antigen-specific adoptive T cells (ACT) by DADC vaccine

[0087] OT-2 mouse spleen cells were extracted, cultured for 3 days, and then ficoll-treated to obtain CD4 323-339 cells that could specifically recognize OVA +T cells, CFSE staining marker, each mouse tail vein injection of 10 million. 12 h after injection of T cells, the mice were grouped: ① PBS group; ② positive group; ③ DP-antigen peptide group; ④ DP-CTLA4 group; ⑤ DP-antigen peptide + DP-CTLA4 group; ⑥ DP-antigen peptide + rapamycin group. Except for 5 mice in the DP-antigen peptide + rapamycin group, 6 mice in each group were injected with drugs according to the grouping. The dosage of DP-CTLA4 was 60 μg per mouse, the dosage of DP-antigen peptide was 5 μg per mouse, and the dosage of rapamycin was 1 mg / kg. The antigen peptide used in this experiment was OVA 323-339 Peptide. Two days after injection of T cells, the mice were subcutaneously injected with DP-antigen peptide and DP-CpG. Except for the PBS group, the remaining groups were subcutaneously injected with a mixture of DP-antigen peptide and DP-CpG. The dosage of DP-antigen peptide was 5 μg per mouse, and the dosage of DP-CpG was 1.24 nmol per mouse. Four days after injection of T cells, the peripheral blood, axillary lymph nodes, inguinal lymph nodes, and spleen cells of the mice were taken for flow cytometry detection.

[0088] 1.13 Time-effect evaluation of DADC vaccine on ACT cells

[0089] Pmel-1 mouse spleen cells were extracted, cultured for 3 days, and then ficoll-treated to obtain CD8 25-33 T cells that could specifically recognize gp100 + peptide. Each mouse was injected with 1.5 million T cells via the tail vein. 12 h after adoptive T cells, the mice were grouped and administered, and the groups were as follows: ① positive group; ② DP-antigen peptide + DP-CTLA4 group, 18 mice in each group; the dosage of DP-CTLA4 was 60 μg per mouse, and the dosage of DP-antigen peptide was 5 μg per mouse. The antigen peptide used in this experiment was gp100 + peptide that could specifically activate Pmel CD8 25-33 T cells. Two, four, and eight days after adoptive T cells, 6 mice were subcutaneously injected with DP-antigen peptide and DP-CpG to immunize the mice. The dosage of DP-antigen peptide was 5 μg per mouse, and the dosage of DP-CpG was 1.24 nmol per mouse. Two days after immunization, the mice were euthanized, and the lymph nodes and spleen cells were dissected for flow cytometry detection.

[0090] 1.14 DADC vaccine specifically inhibits ACT cells

[0091] Pmel-1 CD8 + T cells were transferred to C57BL / 6 mice according to the method of 1.13, and 12 h after adoptive T cells, the mice were grouped and administered, and the groups were as follows: ① positive group; ② DP-gp100 25-33 peptide + DP-CTLA4 group; ③ DP-OVA 323-339Peptide + DP-CTLA4 group. 6 mice in each group; DP-CTLA4 dosage was 60 μg per mouse, and DP-antigen peptide dosage was 5 μg per mouse. On the 4th day after adoptive T cells, the mice were subcutaneously injected with DP-antigen peptide and DP-CpG to immunize the mice. DP-antigen peptide dosage was 5 μg per mouse, and DP-CpG dosage was 1.24 nmol per mouse. After 2 days of immunization, the mice were euthanized, and the axillary lymph nodes, inguinal lymph nodes, and spleen cells of the mice were dissected for flow cytometry detection. Part of the spleen cells were taken and stimulated with antigen peptide gp100 25-33 (10 μg / mL) for 48 h, and the concentrations of IFN-γ, TNF-α, and IL-6 in the supernatant were detected by ELISA.

[0092] 1.15 Therapeutic effect of DADC vaccine on RA mice

[0093] (1) Male DBA / 1 mice were immunized with bovine type II collagen at the tail root twice to induce arthritis, that is, the CIA model. On day 0, bovine type II collagen and CFA were mixed in a volume ratio of 1:1 to prepare a water-in-oil emulsion, and 100 μL of the emulsion was injected intradermally into the tail root of each mouse. On day 21, the emulsion was prepared in the same way for the second immunization induction (CFA was replaced by IFA).

[0094] (2) When the mice's paws appeared slightly red and swollen, the mice were grouped: ① model group, ② healthy group, ③ Collagen 259-272 peptide + CTLA4 Ig group, ④ DP-Collagen 259-272 peptide group, ⑤ DP-CTLA4 group, ⑥ DP-OVA 323-339 peptide + DP-CTLA4 (irrelevant antigen peptide) group, ⑦ DADC vaccine (DP-Collagen 259-272 + DP-CTLA4) group, and ⑧ DADC vaccine subcutaneous administration group, with 8 mice in each group.

[0095] (3) The mice were administered on days 27, 30, and 33, except that the ⑧ group was administered subcutaneously, and the other groups were administered intravenously; the antigen peptide and DP-antigen peptide were administered at a dose of 5 μg per mouse; CTLA4 Ig and DP-CTLA4 were administered at a dose of 40 μg per mouse. After the start of administration, the body weight and paw swelling score of the mice in each group were recorded. On day 41, the paw thickness of the mice in each group was measured with a vernier caliper, and the mice were subjected to a rotarod test.

[0096] (4) On day 41, the mice were anesthetized with isoflurane gas, and blood was taken from the retro-orbital plexus. According to the instructions of the ELISA kit, the concentrations of IL-6, IL-1β, and TNF-α in the serum were determined.

[0097] (5) Dissect the mouse ankle synovial tissue, use BCA kit to determine the protein concentration, and according to the instructions of the ELISA kit, determine the concentration of IL-6, IL-1β and TNF-α in the synovium.

[0098] (6) Dissect the mouse spleen cells, and perform flow detection. The remaining spleen cells are cultured for 48h after being stimulated by antigen peptide (Collagen 259-272 ) and the supernatant is collected for ELISA determination of IFN-γ secretion level.

[0099] (7) Dissect the mouse left hind paw, fix it in paraformaldehyde, and then perform Micro-CT imaging.

[0100] 1.16 DADC vaccine prevents in vivo proliferation of antigen-specific T cells

[0101] The mice were grouped 7 days before adoptive T cells: ① PBS group; ② positive group; ③ antigen peptide group; ④ DP-antigen peptide group; ⑤ antigen peptide+CTLA4 Ig group; ⑥ DP-antigen peptide+DP-CTLA4 group. According to the grouping, the tail vein was injected with drugs, the dosage of DP-CTLA4 was 60 μg per mouse, the dosage of DP-antigen peptide was 5 μg per mouse, and OVA + peptide was used as antigen peptide specific to activate OT2 CD4 323-339 T cells. On day 0, each mouse was given 10 million CFSE-labeled OT2 CD4 + T cells. On day 1, except for the PBS group, the rest of the groups were subcutaneously injected with the inducer DP-antigen peptide and DP-CpG to immunize the mice. The dosage of DP-antigen peptide was 5 μg per mouse, and the dosage of DP-CpG was 1.24 nmol per mouse. On day 4, the lymph nodes and spleen were dissected for flow detection.

[0102] 1.17 DADC vaccine induces antigen-specific preventive effect

[0103] The mice were grouped and administered 7 days before adoptive T cells: ① PBS group; ② positive group; ③ DP-CTLA4 group; ④ DP-OVA 257-264 peptide+DP-CTLA4 group; ⑤ DP-OVA 323-339 peptide+DP-CTLA4 group. The dosage of DP-CTLA4 was 60 μg per mouse, and the dosage of DP-antigen peptide was 5 μg per mouse. On day 0, each mouse was given 5 million CFSE-labeled OT2 CD4 + T cells. On day 1, except for the PBS group, the rest of the groups were subcutaneously injected with the inducer DP-OVA 323-339Peptide and DP-CpG immunize mice. The dose of DP-antigen peptide is 5 μg per mouse, and the dose of DP-CpG is 1.24 nmol per mouse. On day 4, dissect lymph nodes and spleen for flow cytometry. At the same time, take part of the spleen cells and add OVA 323-339 Peptide stimulation for 48 h, ELISA detection of IFN-γ, TNF-α and IL-6 concentration in supernatant.

[0104] 1.18 DADC vaccine effectively prevents RA in mice

[0105] (1) Randomly divide the non-modeled DBA / 1 mice into 7 groups: ① model group, ② healthy group, ③ Collagen 259-272 peptide group, ④ Collagen 259-272 peptide + CTLA4 Ig group, ⑤ Collagen 259-272 peptide + DP-CTLA4 group, ⑥ DADC vaccine (DP-Collagen 259-272 peptide + DP-CTLA4) group, and ⑦ DADC vaccine subcutaneous administration group, 7 mice in each group.

[0106] (2) Drug administration to mice 7 days before modeling, intravenous administration for the rest of the groups except for the ⑦ group which is subcutaneous administration; Collagen 259-272 peptide and DP-Collagen 259-272 peptide 5 μg per mouse; CTLA4 Ig and DP-CTLA4 60 μg per mouse. After seven days of drug administration, establish the CIA model, and record the body weight and RA score of mice after the second modeling is completed. On day 46, measure the paw thickness of mice in each group, and perform the rotating cage test on mice.

[0107] (3) On day 46, anesthetize mice with isoflurane gas, and take blood from the retro-orbital plexus. According to the instructions of the ELISA kit, measure the concentration of IL-6, IL-1β and TNF-α in serum.

[0108] (4) Dissect the synovial tissue of mouse ankle joints, use the BCA kit to measure the protein concentration, and according to the instructions of the ELISA kit, measure the concentration of IL-6, IL-1β and TNF-α in synovium.

[0109] (5) Dissect the mouse paw, fix it in paraformaldehyde, and then perform decalcification, paraffin embedding and sectioning operations to prepare ankle joint sections. Perform hematoxylin-eosin (H&E), safranin O-fast green and Masson trichrome (Masson) staining on ankle joint sections, and analyze after panoramic optical microscope scanning.

[0110] (6) Dissect the left hind paw of the mouse, fix it in paraformaldehyde, and then perform Micro-CT imaging.

[0111] 2 Experimental Results

[0112] 2.1 Successful synthesis of DP-antigen peptide

[0113] For DP-MAL, free antigenic peptides (in the form of antigenic peptide OVA) 323-339 (Taking C as an example) and different ratios of DP-antigen peptides were characterized by agarose gel electrophoresis. The fluorescence chromatograms show that, under the same electrophoresis conditions, the band migration rates of the reaction products at different ratios were significantly slower than those of the free antigen peptide, indicating that DP-antigen peptides at different reaction ratios could be successfully synthesized (Figure 2A). The DP-antigen peptide generated at a 1:2 reaction ratio had a slower band migration rate than that at a 1:1 ratio, indicating higher synthesis efficiency at this ratio. The band migration rate of the synthesized product at a 1:5 ratio was not significantly different from that at 1:2, so a 1:2 ratio was used in subsequent experiments.

[0114] For DP-MAL, free antigenic peptides (in the form of antigenic peptide OVA) 323-339 The antigen peptide (C-type) and DP-antigen peptide were characterized by MALDI-TOF-MS (Figure 2, B to D). The molecular weight of the free antigen peptide was 2382 Da, the average molecular weight of DP-MAL was 3031 Da, and the average molecular weight of the reaction product DP-antigen peptide increased to 5547 Da, proving that DP was successfully modified onto the antigen peptide.

[0115] When the antigenic peptide (in the form of antigenic peptide OVA) 323-339 Taking -C as an example, after reacting with DP-MAL to generate DP-antigen peptide, its molecular weight increases, and it can be characterized by size exclusion chromatography. The solvent DMSO shows a solvent peak at 25 min; the antigen peptide group, in addition to the solvent peak, shows a characteristic peak at around 21 min, which is the absorption peak of the peptide at 220 nm; the DP-MAL group shows no other characteristic peaks besides the solvent peak, indicating that DP-MAL has no absorption under these conditions; the DP-antigen peptide group, in addition to the solvent peak, shows an absorption peak at around 13 min, eluting 8 min earlier than the antigen peptide, proving the successful preparation of the DP-antigen peptide (Figure 3, A to D). The yield was calculated based on the area under the curve (AUC) of the DP-antigen peptide and the antigen peptide at 13 min and 21 min, respectively (Yield = Peak area after reaction (AUC1) / Total peak area (AUC1 + AUC2)). The results show that the reaction yield of the antigen peptide with DP-MAL is 95% (Figure 3, E).

[0116] 2.2 Successful synthesis of DP-CTLA4

[0117] The characteristic proton peak of internal reference DMSO (about 2.6 ppm) was found in the NMR spectrum of CTLA4 Ig. The NMR spectrum of DP-CTLA4 had two proton peaks, one was the proton peak of DMSO and the other was the proton peak of PEG (about 3.5 ppm) (A in FIG. 4). The hydration particle size of CTLA4 Ig and DP-CTLA4 was determined. The average particle size of unmodified CTLA4 Ig was 9.8 nm, and the average particle size increased to 12.5 nm after DP modification (B in FIG. 4), indicating that DP was modified to CTLA4 Ig.

[0118] 2.3 DP modification does not affect the biological activity of antigen peptide

[0119] To investigate whether DP modification affects the presentation of antigen peptide, BMDCs were extracted and incubated with antigen peptide for 2 h, and then OT2 CD4 + T cells were added for co-incubation (A in FIG. 5). Compared with the PBS group, OT2 CD4 323-339 T cells were activated by OVA 323-339 peptide and DP-OVA + peptide-treated BMDCs, which increased the expression of CD69 and CD25, surface activation index molecules of T cells (B-E in FIG. 5). There was no statistically significant difference in the expression of CD69 and CD25 of T cells in the OVA 323-339 peptide and DP-OVA 323-339 peptide groups, indicating that DP modification does not affect the ability of antigen peptide to be recognized by the corresponding T cell receptor (TCR). At the same time, a Collagen + peptide stimulation group was set, which could not be recognized by OT2 CD4 259-272 T cell TCR, and the expression of CD69 and CD25 on the surface of T cells in this group had no significant difference from that in the PBS group, indicating that the activation of T cells is antigen specific.

[0120] 2.4 DP modification does not affect the biological activity of CTLA4 Ig

[0121] The ability of CTLA4 Ig and DP-CTLA4 to bind to DC surface costimulatory molecules CD80 / CD86 in vitro was investigated by flow cytometry (F in FIG. 5). OVA 323-339In the peptide group (G2), the expression of CD80 / 86 on the DC surface was significantly increased compared to the PBS group. The staining fluorescence of CD80 / 86 on the DC surface in the CTLA4 Ig group (G3) and the DP-CTLA4 group (G4) was significantly lower than that in the G2 group, indicating that both CTLA4 Ig and DP-CTLA4 can competitively bind to co-stimulatory molecules on the DC surface and reduce the fluorescence intensity of fluid binding. Furthermore, there was no significant difference in the binding ability of CD80 / 86 between the G3 and G4 groups, indicating that DP modification does not affect the biological function of CTLA4 Ig (G to H in Figure 5). Simultaneously, the ability of CTLA4 Ig to inhibit T cell activation was evaluated by detecting the T cell surface activating protein CD25. OVA alone... 323-339 Peptideome, CD4 + The positive rate of CD25 on the surface of T cells was higher than 95%, while the expression of CD25 was significantly reduced in the CTLA4 Ig and DP-CTLA4 groups, indicating that both CTLA4 Ig and DP-CTLA4 can inhibit T cell activation. There was no statistically significant difference in the positive rate of CD25 on the surface of T cells between the G3 and G4 groups, proving that DP modification does not affect the ability of CTLA4 Ig to inhibit T cells (I to G in Figure 5).

[0122] 2.5 Both DP-antigen peptide and DP-CTLA4 can effectively carry albumin.

[0123] Compared to free antigenic peptides, the synthesized DP-antigen peptide has a larger molecular weight. After co-incubation with albumin, it can bind to albumin, further increasing its molecular weight. The fluorescence chromatogram shows that, under the same electrophoresis conditions, the band migration rate in lane 2 is significantly slower than in lane 1, proving the successful synthesis of the DP-antigen peptide (Figure 6A). The fluorescent bands in lanes 1 and 4 are in the same position, indicating that the free antigenic peptide lacks interaction with albumin. However, the band in lane 5 migrates significantly slower than in lane 2, indicating that the synthesized product can bind to albumin, resulting in slower migration. Coomassie Brilliant Blue staining of the sample showed no bands in lanes 1 and 2 because the peptide lacks a tertiary structure and cannot react with Coomassie Brilliant Blue (Figure 6B). Lane 3 contains albumin samples, which show a clear albumin band after Coomassie Brilliant Blue staining. The band position of lane 4 is consistent with that of lane 3, indicating that the antigen peptide failed to bind to albumin. Lane 5 shows a clear tail compared to lane 3, indicating that the DP-antigen peptide bound to albumin. Due to the range distribution of PEG molecular weight, the band distribution is relatively wide.

[0124] Similarly, the DP-CTLA4 with albumin was characterized by agarose gel electrophoresis. Under the same electrophoresis conditions, the band migration rate of lane 2 was significantly slower than that of lane 1, proving that DP-CTLA4 was successfully synthesized; the fluorescence band positions of lane 1 and lane 4 were consistent, indicating that free CTLA4 Ig lacked interaction with albumin; the band of lane 5 was significantly slower than that of lane 2, indicating that DP-CTLA4 successfully combined with albumin (Fig. 6C). The Coomassie blue results were consistent with the fluorescence results (Fig. 6D).

[0125] 2.6 DP-antigen peptide and DP-CTLA4 have no cytotoxicity

[0126] The cytotoxicity of DP-antigen peptide and DP-CTLA4 was detected by CCK-8 test method, and the experimental results are shown in Fig. 7. Even high concentrations of DP-antigen peptide and DP-CTLA4 had no significant effect on the growth activity of BMDC, indicating that DP-antigen peptide / DP-CTLA4 had no obvious cytotoxicity.

[0127] 2.7 In vivo biodistribution of DP-antigen peptide and DP-CTLA4

[0128] We injected LPS into the left paw of BALB / c mice to induce an acute inflammation model, and then injected Cy5 fluorescently labeled antigen peptide / DP-antigen peptide intravenously to explore its biodistribution and inflammation targeting (Fig. 8A). 6h after injection, the accumulation of free antigen peptide in the left and right hind paws was consistent, and the enrichment of DP-antigen peptide in the inflammatory paw was 2.3 times higher than that in the healthy paw; at the same time, the fluorescence intensity of DP-antigen peptide and free antigen peptide in the right hind paw without modeling was similar, but in the inflammatory paw, the fluorescence intensity of DP-antigen peptide was 1.9 times that of free antigen peptide, indicating that DP modification can significantly enhance the inflammation targeting of antigen peptide (Fig. 8B and C). Unmodified free antigen peptide and DP-antigen peptide had a certain degree of accumulation in the liver, spleen and kidney. The fluorescence intensity of DP-antigen peptide in the spleen and liver was higher than that of free antigen peptide, and the fluorescence intensity in the kidney was lower than that of free antigen peptide, proving that DP modification can enhance the accumulation of antigen peptide in the spleen and liver and reduce the excretion in the kidney. 12h, 24h and 48h after injection, DP modification also increased the accumulation of antigen peptide in the inflammatory paw, and the fluorescence intensity of the inflammatory paw of DP-antigen peptide group mice was 1.8 times, 1.8 times and 2.1 times that of free antigen peptide group, respectively.

[0129] Similarly, we investigated the biodistribution and inflammation targeting of DP-CTLA4 in LPS-induced acute inflammation model (Fig. 9A). The accumulation of CTLA4 Ig in the left and right hind paws was consistent at 12 h, 24 h and 48 h, and DP-CTLA4 was enriched in the inflamed paw by 1.7, 1.4 and 1.5 times compared to the healthy paw; meanwhile, the inflammation accumulation of DP-CTLA4 was 1.7, 1.5 and 1.4 times that of CTLA4 Ig (Fig. 9B and C). The accumulation of DP-modified DP-CTLA4 was enhanced in the spleen and liver, and reduced in the kidney.

[0130] 2.8 DADC vaccine can inhibit antigen-specific T cells in vivo

[0131] OT2 mice CD4 323-339 T cells that can specifically recognize antigen peptide OVA + were adoptively transferred into C57BL / 6 mice after CFSE staining, and then injected with DADC vaccine (DP-OVA 323-339 / DP-CTLA4). After 1.5 days of treatment, a booster (containing DP-OVA 323-339 and DP-CpG) was subcutaneously injected to promote the in vivo proliferation of injected antigen-specific T cells. The number of ACT cells in peripheral blood, spleen and lymph nodes was detected to evaluate the inhibitory effect of DADC tolerance vaccine on OVA 323-339 specific T cells (Fig. 10A).

[0132] When only T cells were transferred (G1), the proportion and number of ACT cells in CD3 + T cells in the spleen were low, and after injection of the booster, the number of ACT cells increased by 13.7 times (G2), proving that the booster can expand ACT cells (Fig. 10B and C). Compared with the G2 group, DP-OVA 323-339 alone (G3) actually promoted ACT proliferation, while DP-CTLA4 alone (G4) inhibited 67% of ACT proliferation. The DADC tolerance vaccine combining DP-OVA 323-339 and DP-CTLA4 (G5) can reverse the proliferation effect of DP-OVA 323-339 and inhibit 90% of ACT cell proliferation, about 1.3 times more effective than DP-CTLA4 alone. At the same time, the immunosuppressant rapamycin (G6) used in the dose could not inhibit the proliferation of ACT cells (Fig. 10C). Cell proliferation would cause CFSE fluorescence to decrease, and the average CFSE fluorescence intensity of ACT cells in the G5 group was significantly higher than that in the G2 group, and had no significant difference from the PBS group (Fig. 10D), indicating that intravenous injection of DADC vaccine can effectively inhibit the in vivo proliferation of ACT cells.

[0133] In the lymph nodes, the proliferation trend of ACT cells was consistent with that in the spleen. When only ACT cells were adoptively transferred, the number of ACT cells in the CD3 + T cells in the T cells, while after booster injection, the number of ACT cells increased by 37.8 times. Compared with the G2 group, DP-CTLA4 alone could inhibit 70% of ACT cell proliferation, DADC tolerance vaccine could inhibit 97% of ACT cell proliferation, and the effect of DP-CTLA4 was increased by about 1.4 times (Fig. 10E). In the peripheral blood, compared with the G2 group, DP-CTLA4 alone could inhibit 60% of ACT cell proliferation, DADC vaccine could inhibit 85% of ACT cell proliferation, and the effect of DP-CTLA4 was increased by about 1.4 times (Fig. 10F).

[0134] 2.9 Duration of the effect of DADC vaccine

[0135] We adoptively transferred Pmel-1 mouse CD8 25-33 T cells that could recognize the antigenic peptide gp100 + into C57BL / 6 mice, and then injected DADC vaccine containing gp100 25-33 (DP-gp100 25-33 / DP-CTLA4). ACT cells were activated by subcutaneous injection of booster (DP-gp100 25-33 / DP-CpG) at different times after treatment (1.5 days, 3.5 days, and 7.5 days) (Fig. 11A).

[0136] Compared with PBS, DADC vaccine could reduce 90% of ACT cells in the spleen 1.5 days after treatment; 87% of ACT cells could be reduced by DADC vaccine 3.5 days after treatment; 87% of ACT cell proliferation could still be inhibited by DADC vaccine 7.5 days after treatment (Fig. 11B and C). The inhibitory effect of DADC vaccine on ACT cells in the lymph nodes was similar to that in the spleen. Compared with PBS, DADC vaccine could reduce the number of antigen-specific ACT cells by at least 72% 1.5 days, 3.5 days, and 7.5 days after treatment (Fig. 11D and E).

[0137] 2.10 Antigen-specificity of immune tolerance induced by DADC vaccine

[0138] To investigate whether the immune tolerance induced by DADC vaccine was antigen-specific, we adoptively transferred Pmel-1 mouse CD8 25-33 T cells that could recognize the antigenic peptide gp100 + into C57BL / 6 mice, and then injected DADC vaccine containing gp100 25-33 (DP-gp100 25-33 / DP-CTLA4) or DADC vaccine containing irrelevant antigen OVA 323-339 (DP-OVA 323-339 / DP-CTLA4). After 3.5 days of treatment, a booster injection (DP-gplOO 25-33 / DP-CpG) was given to activate the ACT cells (A in Fig. 12).

[0139] Compared with the PBS group, the combination of DP-CTLA4 and irrelevant antigen peptide DP-OVA 323-339 (G3) reduced the proliferation of ACT cells by 49% (B and C in Fig. 12) due to the immunosuppressive effect of CTLA4 Ig, while the DADC tolerance vaccine combining DP-CTLA4 and relevant antigen peptide DP-gplOO 25-33 (G2) reduced the proliferation of ACT cells by 87% and improved the therapeutic effect by 1.8 times (B and C in Fig. 12). In the lymph nodes, the G3 group reduced the proliferation of ACT cells by 35%, while the antigen-specific tolerance vaccine G2 group reduced the number of ACT cells by 75%, with an inhibition effect 2.1 times that of the G3 group (D in Fig. 12).

[0140] The spleen cells of mice in each group were collected and stimulated with gplOO 25-33 peptide for 48 h, and the concentrations of IFN-γ, TNF-α and IL-6 in the culture medium were detected. As shown in E in Fig. 12, the Pmel-1 T cells in the PBS group were activated and secreted a large amount of inflammatory factors; compared with the PBS group, the antigen-specific tolerance vaccine reduced the secretion of IFN-γ by 62.9%, TNF-α by 49.5% and IL-6 by 67.5%, indicating that the antigen-specific T cells in the spleen were indeed reduced.

[0141] 2.11 Therapeutic effect of DADC vaccine on RA mice

[0142] The therapeutic effect of the DADC vaccine was evaluated in a collagen-induced mouse rheumatoid arthritis model (CIA), and the experimental procedure is shown in Figure 13A. The degree of paw swelling of each group of mice was scored, and the RA index of the model group mice was 13.6, and the RA index of the G4 group mice was 13.4, indicating that the antigen peptide alone cannot delay the progression of RA. The RA index of the G3 group mice was 9.9, indicating that intravenous administration of antigen peptide and CTLA4 Ig can alleviate the incidence of RA to some extent and reduce the degree of paw swelling in mice. The RA index of the G5 group mice was 7.6, indicating that intravenous administration of DP-CTLA4 has a certain therapeutic effect. The RA index of the G6 group mice was 7.1, similar to the G5 group, showing that the combination of irrelevant antigen peptide and DP-CTLA4 cannot improve the therapeutic effect (Figure 13B). However, the RA index of the G7 group mice was 4, which was significantly lower than that of the G5 group, indicating that the combination of disease-related antigen peptide and DP-CTLA4 can more effectively inhibit the development of the disease. At the same time, the RA index of the G8 group mice was 6.4, indicating that intravenous injection of the tolerance vaccine is better than subcutaneous injection (Figure 13B).

[0143] Weight loss is also an indicator of the progression of RA. Except for the G4 group, the weight loss of the mice in the other treatment groups was improved to some extent compared to the model group, indicating that each treatment group had a therapeutic effect. Among them, the G7 group had a more gradual decrease in mouse weight compared to the other treatment groups, indicating that intravenous administration of the tolerance vaccine was the most effective treatment for RA (Figure 13C).

[0144] On day 41, the right hind paws of each group of mice were photographed and recorded. The right hind paws of the model group and the G4 group mice were severely diseased, with all the joints including the ankle joint swollen; the right hind paws of the healthy group mice were normal in shape and had no swelling; the right hind paws of the mice in the remaining treatment groups were swollen to varying degrees, and the right hind paws of the G7 group mice had only one joint swollen, with the least degree of disease (Figure 13D). At the same time, the thickness of the four paws of each group of mice was measured using a vernier caliper and the sum was calculated. The paw thickness of the healthy group mice was 8.9 mm, that of the model group was 14.8 mm, and the paw thickness of the mice in the remaining administration groups was reduced to some extent compared to the model group; among them, the paw thickness of the G7 group mice was the lowest, only 10.2 mm (Figure 13E).

[0145] Motor ability and paw grasping ability in RA mice were inversely proportional to the severity of the disease. The severity of the disease was evaluated by recording the duration of exercise at a fixed rotation speed of 25 rpm and the maximum rotation speed that the mice could withstand in a rotating cage. On day 41, the mice were placed in a rotating cage for behavioral assessment. As the rotation speed of the cage gradually increased (up to a maximum of 70 rpm), the maximum rotation speed that healthy mice could withstand was 63 rpm, and that of the model group was 13.4 rpm. Except for the G4 group, whose maximum rotation speed (12.3 rpm) was lower than that of the model group, the maximum rotation speed that the mice in the other treatment groups could withstand was increased to some extent compared with the model group. Among them, the G7 group showed the least decline in paw grasping ability, and the maximum rotation speed that could withstand was significantly increased to 51.1 rpm (Figure 13, F). When the rotation speed of the cage was fixed at 25 rpm, the duration of exercise that healthy mice could withstand was 81.3 s, and that of the model group was 15.8 s. Among the treatment groups, the G7 group had the longest duration of exercise, approximately 63.6 s (Figure 13, G).

[0146] To assess bone erosion at the joints of mice in each group, we performed Micro-CT imaging on the paws of the mice. The three-dimensional images of the mouse paw bones showed that the paw surfaces of the healthy group were smooth and intact, with normal bone morphology, while the paw surfaces of the model group were rough, and bone erosion was observed in multiple joints. Except for group G4, all other groups showed improvement in the bone morphology and bone erosion of the mouse paws. Among them, group G7 showed the best treatment effect; the overall bone morphology of the paw bones in this group was normal, with no significant difference from the healthy group (H in Figure 13).

[0147] The concentrations of pro-inflammatory cytokines IL-6, TNF-α, and IL-1β in the serum and synovium of mice in each group were measured by ELISA. In serum, the model group mice showed the highest levels of secreted inflammatory factors. Except for group G4, the secretion of inflammatory factors in the serum of mice in all other treatment groups was reduced. Group G7 showed the strongest inhibitory effect on inflammatory factor secretion, inhibiting 81% of IL-6, 87% of IL-1β, and 93% of TNF-α secretion, respectively (Figure 14, B). Simultaneously, group G7 showed the highest inhibitory efficiency on inflammatory factors in the synovium of the mouse lesion site, inhibiting 89% of IL-6, 94% of IL-1β, and 85% of TNF-α secretion (Figure 14, C).

[0148] Flow cytometry was used to detect co-stimulatory molecules and CD4 on the surface of dendritic cells (DCs) in the spleen of mice. + The expression of IFN-γ on T cells was detected. The expression of CD80 on the surface of DCs in the model group mice was 33.6%, while that in the healthy group was 4.76%. Except for the G4 group, the expression level of CD80 on the surface of DCs in all other treatment groups was lower than that in the model group. The expression level of CD80 on the surface of DCs in the G7 group was the lowest, at only 8.10% (D and E in Figure 14).

[0149] Meanwhile, we took part of the spleen cells, added the collagen 259-272 peptide associated with RA onset, and stimulated the cells for 48 h. Then we detected the concentration of IFN-γ in the supernatant by ELISA. Except for the G4 group, the secretion level of IFN-γ was consistent with the model group, the rest of the treatment groups could significantly inhibit the secretion of inflammatory factor IFN-γ; the inhibition effect of the G7 group was the best, which could inhibit 80.5% of the abnormal secretion of IFN-γ (Fig. 14F).

[0150] 2.12 DADC can prevent the in vivo proliferation of antigen-specific T cells

[0151] To verify whether the DADC vaccine has the effect of preventing the proliferation of antigen-specific T cells, we inoculated the mice with the preventive vaccine 7 days before the adoptive T cells, and injected the OT2 mouse CD4 323-339 T cells that can specifically recognize the antigen peptide OVA + into the mice after CFSE staining. One day after injecting the T cells, we subcutaneously injected the inducer containing the antigen peptide and the immune-activating adjuvant (DP-OVA 323-339 / DP-CpG, Inducer) to promote the proliferation of the injected antigen-specific T cells (Fig. 15A). On the fourth day, we evaluated the preventive effect of the DADC vaccine by detecting the proliferation of ACT cells in the spleen and lymph nodes.

[0152] Compared with the G1 group injected with PBS, the injection of the inducer (G2) significantly increased the proportion of ACT cells in the spleen CD3 + T cells, and increased the number of ACT cells in the spleen by 9.2 times (Fig. 15B and C). Compared with the G2 group, the antigen peptide OVA 323-339 (G3) could reduce the proliferation of ACT cells by 57%, the DP-modified antigen peptide DP-OVA 323-339 (G4) could inhibit the proliferation of ACT cells by 70%, which was about 1.2 times the effect of the antigen peptide. The combination of OVA 323-339 and CTLA4 Ig (G5) could inhibit the proliferation of ACT cells by 84%, which was 1.5 times the effect of the G3 group, indicating that the immunosuppressant CTLA4 Ig and the antigen peptide could synergistically promote the body to produce immune tolerance. The DADC vaccine with both components DP-modified (DP-OVA 323-339 and DP-CTLA4, G6) could further reduce the proportion and number of ACT cells, and inhibit the proliferation of ACT cells by 99.9%, which had no statistical difference with the G1 group, completely offsetting the effect of the inducer. The proliferation of ACT cells would cause the CFSE fluorescence to decrease, and the CFSE fluorescence of the ACT cells in the DADC vaccine group was higher than that in the G2 group, which had no obvious difference with the PBS group, which proved that the DADC vaccine could effectively inhibit the proliferation of ACT cells (Fig. 15D).

[0153] The proliferation trend of ACT cells in lymph nodes was consistent with that in the spleen. Compared with PBS, the inducer increased the number of ACT cells in lymph nodes by 47.5 times (Fig. 15E). The DADC vaccine with two-component DP modification had the strongest effect on inhibiting ACT cell proliferation, which reduced the proliferation of 94% of ACT cells in lymph nodes.

[0154] 2.13 DADC vaccine induces antigen-specific preventive effect

[0155] To explore whether the preventive immune tolerance induced by the DADC vaccine is antigen-specific, we designed the DP-CTLA4 group without antigen peptides, the combination of OT2 CD4 + T cells that cannot recognize DP-OVA 257-264 and DP-CTLA4 groups, and the DADC vaccine group with the combination of OT2 CD4 + T cells that can recognize DP-OVA 323-339 and DP-CTLA4. Seven days before the adoptive T cells, the mice were administered, and then the OT2 mouse CD4 323-339 T cells that can specifically recognize the antigen peptide OVA + were injected into the mice after CFSE staining, subcutaneous injection of inducers (DP-OVA 323-339 / DP-CpG) to promote the proliferation of ACT cells, and then the proliferation of ACT cells in the spleen and lymph nodes of each group of mice was detected (Fig. 16A).

[0156] The proportion of ACT cells in the PBS group (G1) in the spleen CD4 + T cells was low, and the proportion of ACT cells increased to 5.2% after injection of the inducer. Although there was no statistical difference in the proportion of ACT cells between the DP-CTLA4 (G3) and the combination of antigen peptide DP-OVA 257-264 and DP-CTLA4 (G4) groups and the G2 group, it could reduce the number of ACT cells by about 45%, which was probably due to the broad-spectrum immunosuppressive effect of CTLA4 Ig (Fig. 16B-D). The DADC vaccine with the combination of antigen peptide DP-OVA 323-339 and DP-CTLA4 (G5) could reduce the proportion of ACT cells to 2.4% and reduce the number of ACT cells by 97%, which was more than 2 times higher than that of the G3 and G4 groups and had no statistical difference with the PBS group. The proliferation trend of ACT cells in the lymph nodes of each group was consistent with that in the spleen, and the DADC vaccine with the combination of antigen peptide DP-OVA 323-339 and DP-CTLA4 could reduce the proliferation of ACT cells by 89% (Fig. 16E and F).

[0157] The spleen cells of each group of mice were added with OVA323-339 Peptides, in vitro stimulation for 48 h, then detect the concentration of IFN-γ, TNF-α and IL-6 in the culture medium. After OVA 323-339 After peptide stimulation, ACT cells in G2 group were activated and secreted inflammatory factors, and the amount of pro-inflammatory cytokine secretion in G4 and G5 groups was slightly lower than that in G2 group, but the secretion amount of IFN-γ, TNF-α and IL-6 in DADC vaccine group was only 32.6%, 26.2 and 39.4% of that in G2 group, and had no statistical difference with PBS group (Fig. 16G). The results showed that the DADC vaccine containing the relevant antigen peptide could efficiently reduce the number of activated antigen-specific T cells in the spleen.

[0158] 2.14 DADC vaccine efficiently prevents RA in mice

[0159] The experimental process of DADC vaccine preventing CIA model mice from getting sick is shown in Fig. 17A. Compared with the model group, each administration group can prevent the onset of RA to varying degrees and reduce the swelling of the mouse paw. Among them, the RA index of the model group mice was 11.9; the RA index of the G4 group mice was 6.4; the RA index of the G5 group mice was 3.7, and DP-CTLA4 modified by DP had better effect on preventing the onset of RA than unmodified CTLA4 Ig; the RA index of the G6 group mice was 1.6, indicating that intravenous administration of DP double-modified DADC vaccine can prevent the joint swelling of RA mice to the highest degree; the average RA index of the G7 group mice was 7.6, indicating that subcutaneous administration can also prevent the onset of RA, but its effect is not as good as intravenous administration (Fig. 17B).

[0160] The weight loss of mice in each treatment group was improved to some extent compared with the model group, indicating that each administration group can prevent the onset of RA to some extent. The weight loss of mice in the G6 group was lower than that in other administration groups, indicating that intravenous administration of the tolerance vaccine has the best effect on preventing the onset of RA (Fig. 17C).

[0161] On day 46, the paw thickness (four) of each group of mice was measured using a vernier caliper and the total was calculated. The paw thickness of the healthy group mice was 9.1 mm, that of the model group was 13.5 mm, and the paw thickness of the mice in each administration group was reduced to some extent compared with the model group; among them, the paw thickness of the mice in the G6 group was the lowest, only 9.8 mm, which prevented 84% of the paw thickening compared with the model group (Fig. 17D).

[0162] On day 46, the mice were subjected to behavioral tests. When the rotation cage speed was gradually increased (up to 70 rpm), the maximum speed that healthy mice could withstand was 66.9 rpm, and that of the model group was 14.6 rpm. The maximum speed that mice in each drug group could withstand was increased to some extent compared with the model group. Among them, the grip (movement) ability of mice in the G6 group decreased to the lowest degree, and the maximum speed that the mice could withstand was significantly increased to 56.4 rpm (E in FIG. 17). When the rotation cage speed was fixed at 25 rpm, the movement time of healthy mice could last for 85.6 s, and that of the model group was 15.7 s. The movement time of mice in the G6 group was about 68.6 s (F in FIG. 17).

[0163] The concentrations of pro-inflammatory cytokines IL-6, TNF-a and IL-1β in the serum and synovium of mice in each group were determined by ELISA. In the serum, the model group mice secreted the highest level of inflammatory factors, and the secretion of inflammatory factors in the serum of mice in each drug group was reduced, indicating that each drug group could prevent the onset of RA. Among them, the G6 group secreted the least inflammatory factors IL-1β, IL-6 and TNF-a. Compared with the model group, the G6 group could inhibit 87.6% of IL-6, 92.2% of IL-1β and 97.7% of TNF-a secretion (G in FIG. 17). At the same time, the G6 group had the highest inhibition efficiency of inflammatory factors in the synovium of mice at the lesion site, which could inhibit 70.3% of IL-6, 92.7% of IL-1β and 88.4% of TNF-a (H in FIG. 17).

[0164] As can be seen from the three-dimensional imaging of the mouse paw bones, the surface of the paw of the healthy group mice was smooth and complete, and the bone morphology was normal. The surface of the paw of the model group mice was rough, and bone erosion occurred in multiple joints. Each drug group could improve the bone morphology and bone erosion of the mouse paw. The G6 group had the best prevention effect, and the bone morphology of the mouse paw in this group was generally normal, and only the joint part of the phalanx bone showed slight bone erosion (A in FIG. 18).

[0165] As can be seen from the H&E staining chart (B in FIG. 18), the synovial tissue of the model group mice appeared malignant hyperplasia, the lining cell layer was severely thickened, there were a large number of immune cell infiltrations around, and there was a pannus formation, and the joint was severely destroyed; the synovial tissue of the healthy group and the G6 group mice was normal in morphology, the lining cells were arranged in order, and there was no thickening phenomenon (2-3 layers), and there was no synovial stromal cell hyperplasia and inflammatory cell infiltration; the lining cell layer of the synovial tissue of the G4 group and the G7 group mice appeared moderate thickening (5-6 layers), and the synovial stromal cells were hyperplastic and accompanied by partial inflammatory cell infiltration; the lining cell layer of the synovial tissue of the G5 group mice appeared mild thickening (4-5 layers), and there were a small amount of inflammatory cell infiltrations around. At the same time, three trained members scored the synovial inflammation index of the synovial tissue of the mice in each group, and the synovial inflammation index score of the model group mice was 8.7, the healthy group was 0.3, the G6 group was 0.9, the G7 group was 5.4, the G3 group was 7.5, the G4 group was 3.9, and the G5 group was 2 (E in FIG. 18).

[0166] As can be seen from the Masson staining chart (C in FIG. 18), the synovium of the model group mice appeared severe fibrosis; the synovium of the healthy group and the G6 group mice was normal in structure and had no fibrosis; the synovium of the rest of the groups appeared fibrosis to varying degrees, but the fibrosis degree was lower than that of the model group. Image J software was used to quantitatively analyze the fibrosis degree of the synovium of the mice in each group, and the synovium fibrosis of the model group was 68.2%, the healthy group was 8.1%, the G6 group was 12.6%, the G7 group was 34.4%, the G3 group, the G4 group and the G5 group were 57.8%, 34.4% and 25.1% respectively (F in FIG. 18).

[0167] As can be seen from the Safranin O-fast green staining chart (D in FIG. 18), the articular cartilage of the model group and the G3 group mice was severely degenerated and had no normal cartilage morphology; the articular cartilage of the healthy group and the G6 group mice was normal in morphology; the articular cartilage of the G4 group and the G7 group mice appeared partial degeneration and was lightly colored; the articular cartilage structure of the G5 group mice was normal, but the cartilage thickness in some areas was reduced. Photoshop software was used to quantitatively analyze the articular cartilage thickness of the mice in each group, and the articular cartilage thickness of the model group was 25.7 μm, the healthy group was 54.3 μm, the G6 group was 48.7 μm, the G7 group was 30.8 μm, the G3 group, the G4 group and the G5 group were 28.0 μm, 36.9 μm and 41.8 μm respectively (G in FIG. 18).

[0168] The above only describes the preferred embodiments of the present application, and the protection scope of the present application is not limited to the above-mentioned embodiments only, and any technical solution falling within the concept of the present application belongs to the protection scope of the present application.

Claims

1. A targeted tolerance vaccine, characterized in that, The first targeting preparation and the second targeting preparation are mixed, wherein the first targeting preparation comprises a first amphiphilic polymer and a disease-related antigen peptide, and the second targeting preparation comprises a second amphiphilic polymer and a protein biological preparation; The first amphiphilic polymer and the second amphiphilic polymer are both one of DPs with a reactive functional group.

2. The targeted tolerance vaccine of claim 1, wherein, The reactive functional group is a maleimide, the first amphiphilic polymer is DP-MAL, the first targeting preparation is prepared by Michael addition reaction of the first amphiphilic polymer and the disease-related antigen peptide, and a cysteine is added to the C-terminal of the disease-related antigen peptide during synthesis.

3. The targeted tolerance vaccine of claim 2, wherein the antigen is a tumor antigen. The first amphiphilic polymer and the disease-related antigen peptide are reacted in a molar ratio of 1:(1-5).

4. The targeted tolerance vaccine according to claim 2 or 3, characterized in that, After the Michael addition reaction is completed, the reaction solution is dialyzed against ultrapure water in a dialysis bag with a molecular weight cut-off of 3500 Da for 48 h, and vacuum freeze-dried to obtain the first targeting preparation.

5. The targeted tolerance vaccine of claim 1, wherein the antigen is a tumor antigen. The disease-related antigen peptide is a rheumatoid arthritis-related antigen peptide.

6. The targeted tolerance vaccine of claim 1, wherein the antigen is a tumor antigen. The reactive functional group is N-hydroxysuccinimide, the second amphiphilic polymer is DP-NHS, and the protein biological preparation is CTLA4Ig, and the second targeting preparation is prepared by amide reaction of the second amphiphilic polymer and the protein biological preparation.

7. The targeted tolerance vaccine of claim 6, wherein the antigen is a tumor antigen. The second amphiphilic polymer and the protein biological preparation are reacted in a molar ratio of 1:

4.

8. The targeted tolerance vaccine of claim 6 or 7, wherein the antigen is a tumor antigen. The second targeting preparation is purified by ultrafiltration after the amide reaction is completed.

9. The method of producing a targeted tolerance vaccine according to any one of claims 1 to 8, wherein, The method comprises the following steps: Synthesis of the first targeting preparation: A cysteine is added to the C-terminal of the disease-related antigen peptide; Michael addition reaction is performed in a molar ratio of disease antigen peptide: DP-MAL of 1:(1-5); The reaction solution is transferred to a dialysis bag and dialyzed against ultrapure water for 48 h, the dialysis bag has a molecular weight cut-off of 3500 Da, and vacuum freeze-drying is performed to obtain the first targeting preparation; Synthesis of the second targeting preparation: CTLA4Ig: DP-NHS is mixed in a molar ratio of 1:4; NaHCO3 is added to adjust the pH value to 8.5, and amide reaction is performed; The reaction solution is purified by ultrafiltration; Preparation of the vaccine: the first targeting preparation and the second targeting preparation are dissolved and mixed to obtain the vaccine.

10. Use of the targeting immunotolerance vaccine according to any one of claims 1-8 or the targeting immunotolerance vaccine prepared by the preparation method of claim 9 in the preparation of a drug for specifically preventing and treating autoimmune diseases.

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