RA autoantigen epitope polypeptide and antigen-specific T cell vaccine prepared therefrom, preparation method and application thereof

Abstract

The present invention provides an RA autoantigen epitope polypeptide and an antigen-specific T cell vaccine prepared therefrom, a preparation method and an application thereof, and belongs to the technical field of immunotherapy. The present invention modifies the N-terminus and the C-terminus and transforms specific amino acids to make the RA autoantigen epitope polypeptide CII 1237‑1249 It has good stimulating effect and prolonged half-life. 1237‑1249 The peptide was used as an autoantigen epitope peptide for rheumatoid arthritis, combined with IL-7 and IL-15 as well as IL-2 and PHA stimulation, and then inactivated and flow-sorted antigen-specific CD4 + T cells are obtained to obtain an antigen-specific T cell vaccine that does not proliferate but retains immunogenicity. After the antigen-specific T cell vaccine is subcutaneously injected, it can effectively correct immune disorders and achieve the purpose of treating rheumatoid arthritis. The present invention provides a safe and reliable new method for the treatment of rheumatoid arthritis.

Description

Technical Field

[0001] The present invention belongs to the technical field of immunotherapy, and specifically relates to an RA autoantigen epitope polypeptide and an antigen-specific T cell vaccine prepared therefrom, a preparation method and an application thereof. Background Art

[0002] Rheumatoid arthritis (RA) is the most common clinically disabling autoimmune disease, with a prevalence of 0.28% to 0.41%, and a remission rate of only 8.6%. Without formal treatment, the disability rate is as high as 75%, making it one of the diseases with the highest disability rates in my country. The characteristics of rheumatoid arthritis include the occurrence of inflammatory synovial hyperplasia, chronic destructive arthritis, and the production of various autoantibodies (such as rheumatoid factor, anti-citrullinated antibodies, etc.), which lead to joint dysfunction and deformity, and can affect the heart, lungs, kidneys, nerves and other organs of the body. Currently, the treatment of RA is mainly through nonsteroidal anti-inflammatory drugs (NSAIDs) and disease-modifying antirheumatic drugs (DMARDs) combined with glucocorticoids for first-line treatment, but only 40% to 50% of patients respond to this treatment. Further combined with biological agents (such as TNF inhibitors and JAK inhibitors), about 70% of RA patients achieve clinical remission or are in a state of low disease activity. At present, we are still facing the severe situation of lack of specific treatments for RA. Developing safer and more effective drug treatments to achieve individualized and precise treatment of RA is an urgent problem to be solved in clinical practice.

[0003] Abnormal T cell activation driven by autoantigens is a key factor in the pathogenesis of RA. Under the influence of endogenous or exogenous factors, the body's reactivity to certain autoantigens is enhanced, promoting the specific recognition and binding of autoantigen peptides and HLA-DRB1 with TCR, activating pathogenic antigen-specific T cells, and thus mediating chronic inflammatory responses. In the inflamed joints of patients, through cell contact and release of soluble mediators, antigen-specific T cells stimulate macrophages and synovial fibroblasts to produce inflammatory mediators and proteases that destroy bone and cartilage, promoting joint inflammation. At the same time, antigen-specific T cells also promote B cells to produce a large number of rheumatoid factors and anti-cyclic citrullinated protein antibodies, causing synovial inflammatory responses, and then leading to pathological joint damage.

[0004] Drugs and technologies targeting T cells have been successful in clinical practice. T cell activation inhibition therapy represented by CTLA4 fusion protein Abatacept is currently a relatively mature solution. It binds to CD80 / CD86 on the surface of antigen-presenting cells to prevent the interaction between CD80 / CD86 and CD28 on the surface of T cells, thereby inhibiting autoantigen-induced T cell activation and weakening its downstream inflammatory response, thereby inhibiting osteoclastogenesis and exerting a therapeutic effect. In addition, there are currently a variety of T cell-related cytokine fusion proteins or monoclonal antibodies used clinically to treat rheumatoid arthritis. In addition, JAK1 / 2 / 3 inhibitors can simultaneously inhibit the functions of multiple inflammatory cytokines. Currently, the JAK inhibitor Tofacitinib has good benefits for patients in clinical applications. However, the above schemes all indiscriminately block T cell activation and downstream inflammation for treatment, which may lead to broad-spectrum immunosuppression, attack effector cells that play a protective function, and inevitably cause side effects such as infection, digestive tract, liver and kidney damage. Under this treatment situation, we urgently need to speed up the search for a safer and more effective treatment method to control the occurrence and development of the disease, correct the disordered immune system of RA patients, and achieve individualized and precise treatment for RA patients.

[0005] At present, "immune balance restoration" as a treatment goal has been valued by immunology experts and clinical researchers, including the use of therapeutic vaccines to achieve immune balance restoration. As a type of cell vaccine, T cell vaccines have been effective in a variety of immune diseases. T cell vaccines are similar to attenuated vaccines in that they inactivate pathogenic T cells to make them lose their pathogenicity, but retain the immune characteristics of pathogenic T cells in order to induce immune tolerance and exert therapeutic effects. T cell vaccines are a new direction of precision medicine in recent years. Unlike traditional vaccines that only work through humoral immunity, cell vaccines can mobilize both cellular immunity and humoral immunity. Compared with the currently popular CAR-T technology, CAR-T only targets the expression of a specific cell subset, while T cell vaccines can target a variety of autoreactive pathogenic cells, induce immune tolerance, and regulate the immune system to restore balance, which is crucial for patients with autoimmune diseases. In the early stage, the use of T cell vaccines in autoimmune diseases adopted non-targeted amplification, and did not separate the specific cell subsets, so the treatment effect was limited and the efficacy needs to be further improved.

[0006] In addition, immune cells usually have difficulty recognizing the entire antigen molecule, but recognize the specific partial sequence of the antigen macromolecule, namely the antigen epitope, through surface receptors. The antigen epitope is the main functional unit in the antigen molecule, which can effectively stimulate the body's cellular immunity and humoral immunity. At present, the antigen epitope of T cells is usually a linear epitope. To determine whether it is a T cell epitope, it is necessary to predict the affinity of the peptide to HLA and determine the activity by using T cell proliferation experiments, enzyme-linked immunosorbent assays, etc. The stimulation effect of autoantigen peptides corresponding to different epitopes varies greatly, affecting the activation and function of T cells. Due to the bystander tolerance effect, although there are multiple autoantigens involved in the pathogenesis of rheumatoid arthritis, selecting a key antigen to induce immune tolerance can regulate the immune system to restore balance. At present, there is no report on the use of autoantigen epitopes to stimulate T cell proliferation to produce antigen-specific T cells, and these cells are separated for the preparation of antigen-specific T cell vaccines. Summary of the invention

[0007] In view of this, the purpose of the present invention is to provide an RA rheumatoid arthritis autoantigen epitope polypeptide, which is modified to prolong its half-life and enhance the immune activation effect, so as to specifically stimulate the proliferation of peripheral antigen-specific CD4+ derived from RA samples, and enhance its reactivity to the autoantigen polypeptide after the combined treatment of IL-7 and IL-15, further promote its proliferation by using antigen presenting cells to bind to the antigen polypeptide, and finally use IL-2 and PHA to obtain the effect of massive proliferation of the target cells.

[0008] The purpose of the present invention is to provide an antigen-specific T cell vaccine, which can treat joint lesions caused by systemic immune system disorders by exerting the effects of immunosuppressive regulatory T cells.

[0009] The purpose of the present invention is also to provide a method for preparing an antigen-specific T cell vaccine, which has the characteristics of promoting the rapid proliferation of antigen-specific T cells and also performs separation treatment to obtain a high-purity antigen-specific T cell vaccine and then inactivates it to prepare an antigen-specific T cell vaccine.

[0010] The present invention also aims to provide an application of an antigen-specific T cell vaccine in the preparation of a rheumatoid arthritis drug.

[0011] The present invention provides a rheumatoid arthritis autoantigen epitope polypeptide, the amino acid sequence of the rheumatoid arthritis autoantigen epitope polypeptide is shown in SEQ ID NO: 1, the N-terminal glutamine of the rheumatoid arthritis autoantigen epitope polypeptide is replaced with D-glutamine, the N-terminus of the rheumatoid arthritis autoantigen epitope polypeptide is acetamidate-modified, and the C-terminus of the rheumatoid arthritis autoantigen epitope polypeptide is amide-modified.

[0012] The present invention provides an antigen-specific T cell vaccine, which is obtained by inactivating and separating spleen cells derived from rheumatoid arthritis samples after being stimulated with the rheumatoid arthritis autoantigen epitope polypeptide.

[0013] Preferably, the antigen-specific T cell vaccine is an inactivated antigen-specific CD4 + T cells.

[0014] The present invention provides a method for preparing the antigen-specific T cell vaccine, comprising the following steps:

[0015] The spleen cells or peripheral PBMC cells derived from rheumatoid arthritis samples were cultured in a culture medium containing the rheumatoid arthritis autoantigen epitope polypeptide for 7 days, co-cultured with antigen presenting cells that captured the rheumatoid arthritis autoantigen epitope polypeptide for 3 days, cultured with IL-7 and IL-15 on the 8th day of culture, IL-2 and PHA on the 13th day of culture, and collected and inactivated on the 16th day of culture to separate antigen-specific CD4 + T cells.

[0016] Preferably, the culture medium is based on RPIM1640 as the basic culture medium, and further comprises the following components: 10% FBS, 5mM HEPES, 2mM L-glutamine, 0.05mM mercaptoethanol, 100U / mL streptomycin and penicillin, 5-10ng / mL IL-15 and IL-7 by volume percentage;

[0017] In the culture medium, the concentration of the autoantigen is 5 to 50 μg / mL.

[0018] Preferably, the concentration of the spleen cells derived from the rheumatoid arthritis sample is (1-10)×10 7 / mL.

[0019] Preferably, the method for preparing the antigen presenting cells that capture autoantigens comprises co-incubating 5 to 50 μg / mL rheumatoid arthritis autoantigen epitope polypeptide solution with antigen presenting cells;

[0020] The concentration of the antigen presenting cells is (1-10)×10 6 / mL;

[0021] The antigen presenting cells include trophoblast cells or mature dendritic cells;

[0022] The method for preparing trophoblast cells comprises: performing red lysis treatment on spleen derived from rheumatoid arthritis samples to obtain spleen single cells, and then inactivating the cells to obtain trophoblast cells;

[0023] The method for preparing mature dendritic cells comprises: subjecting mouse bone marrow cells to erythroblastosis, differentiation and culture, and inducing maturation with LPS to obtain mature dendritic cells;

[0024] The reagent for differentiation culture is an aqueous solution containing 10-25 ng / mL GM-CSF and 5-10 ng / mL IL-4;

[0025] On the seventh day of culture, the ratio of cells to antigen-presenting cells that captured self-antigens was 1 to 5:1.

[0026] Preferably, the final concentration of IL-7 and IL-15 is 5 to 20 ng / mL;

[0027] The final concentration of IL-2 is 5 to 20 ng / mL;

[0028] The final concentration of the PHA is 1-10 μg / mL.

[0029] Preferably, the method of inactivating cells is treating with 20-50 μg / mL mitomycin for 30-60 min;

[0030] The isolated CD4 + The T cell method is to use commercial tetramer antibody staining to isolate antigen-specific CD4 + T cells.

[0031] The present invention provides the use of the antigen-specific T cell vaccine or the antigen-specific T cell vaccine obtained by the preparation method in preparing a drug for preventing and / or treating rheumatoid arthritis.

[0032] The present invention provides a modified rheumatoid arthritis autoantigen epitope polypeptide, the amino acid sequence of the rheumatoid arthritis autoantigen epitope polypeptide is shown in SEQ ID NO: 1, the N-terminal glutamine of the rheumatoid arthritis autoantigen epitope polypeptide is replaced with D-glutamine, the N-terminal of the rheumatoid arthritis autoantigen epitope polypeptide is acetamidate-modified, and the C-terminal of the rheumatoid arthritis autoantigen epitope polypeptide is amide-modified. The present invention screens multiple rheumatoid arthritis autoantigen epitope polypeptides, and compares the CD4 + The results of T cell combined with anti-CD28 stimulation showed that CII 1237-1249 The stimulation effect is better; the autoantigen peptide is modified to replace the N-terminal glutamine with the D-type, and the N-terminal acetylamide and C-terminal amidation are modified to extend the half-life of the peptide and improve the immune activation ability. Therefore, the modified CII is selected 1237-1249 Preparation of antigen-specific T cell vaccine as rheumatoid arthritis autoantigen epitope peptides.

[0033] The present invention provides an antigen-specific T cell vaccine, which is obtained by specifically amplifying spleen cells or peripheral PBMC cells derived from rheumatoid arthritis samples after stimulation with rheumatoid arthritis autoantigen epitope polypeptides, and then separating and inactivating. The antigen-specific T cell vaccine has high specificity for rheumatoid arthritis autoantigens and a fast proliferation rate. At the same time, the preventive and therapeutic effects of the antigen-specific T cell vaccine on rheumatoid arthritis were verified, and the results showed that the antigen-specific T cell vaccine achieves the purpose of preventing and treating rheumatoid arthritis by immunomodulating Tregs and upregulating the expression of related inhibitory molecules on the surface of Tregs cells. The present invention provides an antigen-specific T cell vaccine that can enhance the immune response in the body and enhance the body's therapeutic effect on rheumatoid arthritis. It has the characteristics of strong pertinence and lasting effect, and is safe to use without any toxic side effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 The results of the screening of autoantigen epitope polypeptides in Example 1 of the present invention, wherein A is the amino acid sequence of three antigen epitope polypeptides to be screened, B is the strength of binding to HLA predicted by the algorithm for several polypeptides in the polypeptide immune library, and the scoring results of the immunogenicity strength, (C) After obtaining PBMCs of RA patients, different concentrations of autoantigen polypeptides were used for stimulation, and CFSE dilution was used to detect cell proliferation after 72 hours; (D) Proliferation index SI = cell proliferation ratio of the group with polypeptide added to the stimulation group / cell proliferation ratio of the control group without polypeptide added, and the proliferation index was used to compare the proliferation capacity of multiple groups of autoantigen polypeptides with different concentrations; (E) After obtaining spleen cells from the collagen-induced mouse arthritis model, 10 μg / mL different polypeptides were used for stimulation to detect cell proliferation; (F) The proliferation index was used to compare the differences in proliferation capacity between the groups;

[0035] Figure 2 The half-life and immune activation effect of the modified autoantigen polypeptide used in Example 1 of the present invention; A. The strategy for modifying the CII polypeptide is shown, and the modified polypeptide is recorded as CII-mut; B. The left figure shows the change in the half-life of the CII prototype polypeptide, and the right figure shows the change in the half-life of the modified CII-mut polypeptide; C. After obtaining PBMC of RA patients, the modified CII-mut is used for stimulation, and CFSE dilution is used to detect cell proliferation after 72 hours; D. After obtaining PBMC of RA patients, the modified CII-mut is used for stimulation, and the levels of IFNγ, IL-17A and IL-6 in the culture supernatant are detected after 48 hours;

[0036] Figure 3The results of the effectiveness test of the antigen-specific T cell vaccine in Example 2 of the present invention are shown in Figure 2. A is the effect of the antigen-specific T cell vaccine on the immune cell subsets in the peripheral blood mononuclear cells (PBMCs) of RA patients; B is the effect of the antigen-specific T cell vaccine on the cytokines produced in the PBMCs of RA patients; C is the CD4 + The result of the ratio test of effector cells / memory cells of T cells, D is the result of the cell morphology test;

[0037] Figure 4 The results of cell subsets in rheumatoid arthritis mice after antigen-specific T cell vaccine treatment (A) and cytokine results in the supernatant (B) in Example 3;

[0038] Figure 5 Disease scoring results of rheumatoid arthritis mice after antigen-specific T cell vaccine treatment;

[0039] Figure 6 This is a micro-CT image of bone erosion and bone destruction in the joints of rheumatoid arthritis mice after antigen-specific T cell vaccine treatment in Example 4;

[0040] Figure 7 The changes of pathogenic cell subsets Th1 / Th17 in the spleen of rheumatoid arthritis mice after treatment with antigen-specific T cell vaccine in Example 4;

[0041] Figure 8 The results of up-regulating the proportion of Treg cells in the draining lymph nodes of rheumatoid arthritis mice after antigen-specific T cell vaccine treatment in Example 4;

[0042] Fig. 9 The changes in the proportion of Treg cells and the immune regulatory molecules in the spleen of CIA mice after treatment with the antigen-specific T cell vaccine in Example 5. DETAILED DESCRIPTION

[0043] The present invention provides a rheumatoid arthritis autoantigen epitope polypeptide, the amino acid sequence of the rheumatoid arthritis autoantigen epitope polypeptide is shown in SEQ ID NO: 1 (QYMRCitADQAAGGLR), the N-terminal glutamine of the rheumatoid arthritis autoantigen epitope polypeptide is replaced with D-glutamine, the N-terminus of the rheumatoid arthritis autoantigen epitope polypeptide is acetamidate-modified, and the C-terminus of the rheumatoid arthritis autoantigen epitope polypeptide is amide-modified.

[0044] The present invention uses a variety of autoantigen epitope polypeptides, such as Vimentin (65-77), Fibrinogen α (79-91), and Type II collagen, to prepare different concentrations to stimulate CD4 in peripheral PBMC of HLA-DR+RA patients.+ T cell proliferation and results showed that CII 1237-1249 The stimulation effect was better, and the stimulation effect was similar at 10μg / mL and 20μg / mL, so 10μg / mL was selected for subsequent use. + In T cells, combined with anti-CD28 stimulation, the results also showed that CII 1237-1249 The stimulation effect is better, so type II collagen polypeptide (QYMCitADQAAGGLR) is used as the antigen polypeptide for preparing antigen-specific T cell vaccines. The stability of the polypeptide in the subsequent culture process affects the vaccine preparation effect. The present invention uses two modification methods (acetamidation modification and amide modification) to modify the autoantigen to study the effect on the half-life of the polypeptide. The serum of RA patients and 200 μM polypeptide in equal volumes are co-incubated at 37°C, and the supernatant of the solution at multiple time points from 0 hours to 72 hours is taken. After methanol precipitation, it is detected by HPLC, and the half-life change is obtained by comparison with the standard. The results show that the unmodified CII 1237-1249 Half-life of autoantigen peptide 1 / 2 =28.66min, after transformation CII 1237-1249 The half-life of the autoantigen peptides was significantly prolonged, and about 75% of the peptides remained stable after 72 hours of incubation.

[0045] The present invention provides an antigen-specific T cell vaccine, which is obtained by specifically proliferating spleen cells or peripheral PBMC derived from rheumatoid arthritis samples after being stimulated by the rheumatoid arthritis autoantigen epitope polypeptide, and then being specifically separated and inactivated.

[0046] In the present invention, the antigen-specific T cell vaccine is preferably CD4 + The stimulation concentration of the rheumatoid arthritis autoantigen epitope polypeptide is preferably 5 to 50 μg / mL, more preferably 5 to 30 μg / mL, further preferably 8 to 20 μg / mL, and most preferably 10 μg / mL. The density of the spleen cells is preferably 1 to 10×10 7 / mL, more preferably 1 to 5×10 7 / mL, and the most preferred is 1×10 7 During the stimulation, the culture medium of the spleen cells is preferably based on RPIM1640 as the basic culture medium, and further comprises the following components: 10% FBS, 5mM HEPES, 2mM L-glutamine, 0.05mM mercaptoethanol, 100U / mL penicillin and streptomycin, 5-10ng / mL IL-15 and IL-7 by volume percentage.

[0047] The present invention provides a method for preparing the antigen-specific T cell vaccine, comprising the following steps:

[0048] The spleen cells or peripheral PBMC cells derived from rheumatoid arthritis samples were cultured for 7 days, co-cultured with antigen presenting cells that had captured the rheumatoid arthritis autoantigen epitope polypeptide for 3 days, and cultured with IL-7 and IL-15 on the 8th day of culture, IL-2 and PHA on the 13th day of culture, and collected and inactivated on the 16th day of culture. The antigen-specific CD4 + T cells.

[0049] In the present invention, the spleen cells of the model mice or the peripheral PBMC cells of the patient from which the rheumatoid arthritis sample is derived are used as the raw material to illustrate the preparation method. The specific preparation steps are as follows:

[0050] Obtain: The spleen and draining lymph node samples of mice with rheumatoid arthritis are crushed, subjected to red lysis treatment and neutralized with PBS, and spleen cell single cells are collected for culture. The red lysis treatment solution is 1× red lysis solution. The formula of the 1× red lysis solution is preferably 139.6mmol / LNH4Cl, 16.96mmol / LTris, and the pH value is adjusted to 7.2 with 1mol / L HCl. The culture temperature is preferably 37°C. The culture time is preferably 14 to 16 days, more preferably 16 days. The concentration of spleen cells derived from the rheumatoid arthritis sample is preferably (1 to 10)×10 7 / mL, more preferably 1 to 5×10 7 / mL, and the most preferred is 1×10 7 / mL.

[0051] In the present invention, the culture medium is preferably based on RPIM1640 as the basic culture medium, and also includes the following components: 10% FBS, 5mM HEPES, 2mM L-glutamine, 0.05mM mercaptoethanol, 100U / mL penicillin and streptomycin, 5-10ng / mL IL-15 and 5-10ng / mL IL-7 by volume. In the culture medium, the concentration of the autoantigen is preferably 2-50μg / mL, more preferably 5-30μg / mL, further preferably 8-20μg / mL, and most preferably 10μg / mL. In vitro innovative use of autoantigen epitope polypeptides to promote the proliferation of antigen-specific T cells after stimulation. The culture temperature is preferably 37°C. The culture time is preferably 14-16d, more preferably 16d.

[0052] In the present invention, the final concentration of IL-7 and IL-15 is preferably 5-10 ng / mL, more preferably 6-8 ng / mL, and most preferably 7 ng / mL. The final concentration of IL-2 is 5-20 ng / mL, more preferably 10-15 ng / mL. The final concentration of PHA is preferably 1-10 μg / mL, more preferably 2-8 μg / mL, and most preferably 5 μg / mL.

[0053] In the present invention, the method for inactivating cells is to treat with 20-50 μg / mL mitomycin for 30-60 min, more preferably, to treat with 30-40 μg / mL mitomycin for 30 min. + The T cell method is to use commercial tetramer antibody staining to isolate antigen-specific CD4 + T cells.

[0054] In the present invention, the method for preparing the antigen presenting cells for capturing autoantigens preferably comprises co-incubating 15-25 μg / mL rheumatoid arthritis autoantigen epitope polypeptide solution with the antigen presenting cells. The concentration of the antigen presenting cells is (1-10)×10 6 / mL. The antigen presenting cells include trophoblast cells or mature dendritic cells. The method for preparing the trophoblast cells preferably comprises: treating the spleen derived from the rheumatoid arthritis sample with red blood cells to obtain spleen single cells, inactivating the cells, and obtaining trophoblast cells. The method for preparing the mature dendritic cells,

[0055] In the present invention, mouse bone marrow cells are lysed and then cultured using GM-CSF and IL-4, and mature dendritic cells are obtained by inducing maturation with LPS.

[0056] In the present invention, the cells on the 7th day of culture and the antigen presenting cells that capture the autoantigen are preferably in a ratio of (1:1) to (5:1), more preferably 2:1. The role of the antigen presenting cells that capture the autoantigen epitope polypeptide is to present the captured autoantigen epitope polypeptide to T cells during the co-culture process, so that the autoantigen epitope polypeptide binds to the MHCII molecules on the surface of the T cells, further stimulates the T cell response, and produces specific T cells.

[0057] In the present invention, the final concentration of IL-7 and IL-15 is preferably 5-20 ng / mL, more preferably 5-10 ng / mL, and most preferably 10 ng / mL. The addition of IL-7 and IL-15 on the 8th day of culture is to increase the sensitivity of CD4+T cells to self-antigen peptides and facilitate specific expansion.

[0058] In the present invention, the final concentration of IL-2 is preferably 5-20 ng / mL, more preferably 8-15 ng / mL, and most preferably 10 ng / mL. The final concentration of PHA is preferably 1-10 μg / mL, more preferably 5-10 μg / mL, and most preferably 5 μg / mL. The addition of IL-2 and PHA in the last three days of culture is to activate and stimulate the antigen-specific T cells already present in the culture system, and then amplify them in large quantities to obtain a sufficient number of cells for subsequent vaccine preparation.

[0059] In the present invention, the method of inactivating cells is preferably to treat with 25 μg / mL mitomycin for half an hour. + The T cell method uses Stemcell CD4 + Separation is performed by separating magnetic beads. The immunomagnetic bead method for separating cells is based on the fact that cell surface antigens can bind to specific monoclonal antibodies connected to magnetic beads. In an external magnetic field, cells connected to magnetic beads through antibodies are adsorbed and retained in the magnetic field. Cells without such surface antigens cannot bind to specific monoclonal antibodies connected to magnetic beads and therefore have no magnetism and do not stay in the magnetic field, thereby allowing the cells to be separated. The separation of antigen-specific T cells utilizes tetramer technology combined with flow cytometry. TCR recognizes the MHC-antigen peptide complex on the surface of antigen-presenting cells or target cells. Based on signal-labeled streptavidin, four MHC molecule monomers are cross-linked. Four MHC-antigen peptide monomer molecules and fluorescent dyes are combined into an MHC tetramer, and in vitro analysis of antigen-specific T cells is successfully achieved by flow cytometry. In the process of preparing antigen-specific T cell vaccines of the present invention, magnetic bead sorting combined with tetramer technology is used to separate antigen-specific CD4 + T cells are treated with mitomycin in clinical tumor treatment instead of traditional radiation source irradiation to obtain antigen-specific T cell vaccines with better purity, and this treatment method is easier to promote in clinical practice; antigen-specific T cell vaccines with weak proliferation ability but retained immune activity are obtained, which can induce immune tolerance after autologous transfusion and correct immune disorders to achieve the treatment goal.

[0060] In the present invention, in order to detect the effectiveness of the antigen-specific T cell vaccine, the antigen-specific T cell vaccine prepared above was co-cultured with PBMC, and the changes in several types of cell subpopulations and cytokines were detected respectively. The results showed that Treg is the key cell for the antigen-specific T cell vaccine to exert its therapeutic effect, and at the same time effectively inhibits the level of the inflammatory cytokine IFNγ.

[0061] The present invention provides the antigen-specific T cell vaccine or the use of the antigen-specific T cell vaccine in preparing a drug for preventing and / or treating rheumatoid arthritis.

[0062] In the present invention, the collagen-induced arthritis mouse (CIA) model is a classic rheumatoid arthritis animal model, which is similar to the human rheumatoid arthritis phenotype, and the production of autoantibodies further induces systemic immune system disorders, leading to joint lesions. In the present embodiment, in order to verify the therapeutic effect of antigen-specific T cell vaccine on rheumatoid arthritis, the CIA mouse model was used as an experimental subject, and mice were immunized with antigen-specific T cell vaccine. The results showed that it can effectively reduce the swelling of mice and significantly reduce bone erosion and bone destruction; in addition, in terms of the impact on the immune system, after treatment, Th1 / Th17 decreased, Tfh and GCB decreased, and CD4 + IL10 + Th1 and Th17 are important pathogenic cell subsets in rheumatoid arthritis, which affect osteoclasts by secreting cytokines; Tfh and GCB affect the progression of the disease by affecting the production of autoantibodies (which is the main feature and pathogenesis of this autoimmune disease).

[0063] The following is a detailed description of an RA rheumatoid arthritis autoantigen epitope polypeptide provided by the present invention, an antigen-specific T cell vaccine prepared therefrom, a preparation method and applications thereof in conjunction with the examples, but they should not be construed as limiting the scope of protection of the present invention.

[0064] Example 1

[0065] A method for screening RA rheumatoid arthritis autoantigen epitope polypeptides

[0066] 1. Through the IEDB database (https: / / www.iedb.org / ), we combined multiple autoantigens in RA patients, including type II collagen, vimentin, Fibrinogen, etc., with multiple algorithms in the database, including MHC binding prediction, antigen presentation prediction, and immunogenicity prediction, to screen for key autoantigen T cell epitope peptides in RA. Citrulline modification in autoantigens of RA patients enhances the ability to bind to HLA through charge changes, thereby enhancing pathogenicity. Therefore, based on the combined score of multiple algorithms and the characteristics of citrulline modification in RA patients, we screened Figure 1 The autoantigenic peptides shown ( Figure 1 A and B).

[0067] 2. Synthesize the antigen polypeptide to be screened, the amino acid sequence is as follows:

[0068] Type II collagen(1237-1249): QYMRCitADQAAGGLR(SEQ ID NO:1);

[0069] Vimentin(65-77):SAVRACitSSVPGVR(SEQ ID NO:2);

[0070] Fibrinogen alpha (79-91): QDFTNRCitINKLKNS (SEQ ID NO: 3).

[0071] 3. The three selected antigen peptides were used to stimulate peripheral PBMC of HLA-DR+RA patients at different concentrations to detect CD4 + T cell proliferation. The specific method is as follows:

[0072] Select RA patients and extract two tubes of purple blood. Separate the blood with sterile PBMC separation solution on the table. Dilute the blood to 7 mL with PBS and gently spread the above on the top of the PBMC separation solution. Centrifuge at 1800 rpm for 20 min to absorb the white film in the middle layer. Granulocytes and red blood cells are at the bottom of the tube. After adding 5 mL of PBS for washing, continue centrifugation to obtain PBMC. 6 / mL PBMCs were stimulated, and the concentrations of the stimulating antigen polypeptides included the following three concentrations: 2.5μg / mL, 10μg / mL, and 20μg / mL.

[0073] Stimulation index (%) = ratio of proliferating cells in the autoantigen polypeptide / proliferating cells in the irrelevant peptide or no polypeptide control group × 100% Formula I.

[0074] The results showed that CII 1237-1249 The stimulation effect was better, and the stimulation effect was similar at 10μg / mL and 20μg / mL, so 10μg / mL was selected for subsequent use ( Figure 1 CD4 in collagen-induced arthritis mice + In T cells, antigen peptides were combined with anti-CD28 to stimulate cells, and the results also showed that CII 1237-1249 Better stimulation effect ( Figure 1 Therefore, subsequent studies mainly took type II collagen (QYMCitADQAAGGLR) as an example.

[0075] Modify autoantigen peptides and determine their half-life and immune activation effects ( Figure 2 )

[0076] The modification scheme is: the C and N termini of the autoantigen polypeptide are replaced with amide and acetamide caps respectively, and then the natural amino acids at the C and N termini are replaced with non-natural D-configuration amino acids. Figure 2A in the middle shows the schematic diagram before and after the transformation. The peptide was purified by solid phase and high performance liquid chromatography, and the purity of the peptide was determined by liquid chromatography-mass spectrometry (>95%). The peptide was dissolved in PBS and mixed with an equal volume of RA patient serum for incubation. Multiple time points were selected for the incubation time, and 3 replicate wells were prepared for each time point. Then 30 μL of supernatant was taken from each sample and added to 60 μL of methanol solution. After centrifugation, the supernatant was taken for HLPC-MS detection. The peak position was determined by comparing with the standard dissolved in acetonitrile. The peak area after different time points was used to judge the change in the half-life of the peptide in RA serum. Figure 2 Middle B shows: half-life of unmodified autoantigen peptide t 1 / 2 =28.66min, CII after two transformations 1237-1249 The half-life of the autoantigen peptides was significantly prolonged, and about 75% of the peptides remained stable after 72 hours of incubation.

[0077] Isolation of HLA-DR+ PBMC from patients with rheumatoid arthritis using modified CII 1237-1249 The autoantigen peptides were used for stimulation for 72 hours, and the proliferation of CD4+T cells was detected by CFSE dilution method. The formula I was used for detection. Figure 2 Zhong C found that the proliferation ability of CD4+T cells was enhanced after transformation. Figure 2 The expression of inflammatory cytokines in the supernatant of medium D was increased.

[0078] Example 2

[0079] A method for preparing antigen-specific T cell vaccine

[0080] 1. Experimental animals: Specific pathogen free (SPF) 6-8 week old DBA1 / J male mice were purchased from Beijing Huafukang Biotechnology Co., Ltd. and placed in the SPF environment of the animal laboratory of Peking University People's Hospital. The model was established after 7 days of continuous feeding.

[0081] 2. Modeling method: Dissolve bovine type II collagen (CII) in 0.1 mol glacial acetic acid, adjust the final concentration of CII to 4 mg / mL, and fully dissolve it at 4°C overnight in the dark. The next day, mix the dissolved CII with equal volumes of Freund's complete adjuvant to fully emulsify the two to obtain a collagen emulsion with a final concentration of 2 mg / mL. Primary immunization: Remove the hair from the mouse tail, and inject a total of 200 μg of collagen emulsion into the skin at multiple points, which is recorded as immunization day 0; booster immunization: On the 21st day of immunization, obtain CII solution in the same dissolution method, fully emulsify the CII solution with Freund's incomplete adjuvant, and inject a total of 100 μg of collagen emulsion into the mouse tail at multiple points. Five days after the booster immunization, the mice became ill one after another, reaching a peak on the 14th day, and CIA mice were obtained.

[0082] 3. Stimulation of autoantigen peptides and proliferation and culture of specific T cells

[0083] After CIA mice were euthanized by CO2, the abdominal cavity of the mice was exposed to obtain spleen tissue and draining lymph nodes, which were placed in a culture dish containing 1640 culture medium and placed on ice. The spleen was cut into pieces and ground and filtered through a 70 μm filter to obtain a single cell suspension. The spleen of one mouse was lysed with 3 mL of 1× lysate, neutralized with 3 volumes of PBS, and centrifuged at 1800 rpm for 5 min. After cell counting, the cells were divided into 1×10 7 / mL culture. 10μg / mL CII polypeptide was added to the culture system for subsequent antigen-specific T cell vaccine, while the group directly cultured without adding antigen polypeptide was used as a non-antigen-specific T cell vaccine for control. The composition of the culture medium was RPIM 1640 + 10% FBS + 5mM HEPES + 2mM L-glutamine + 0.05mM mercaptoethanol + 100U / mL penicillin and streptomycin + ng / mL IL-15 and IL-7. During the seven-day culture process, the color of the culture medium and the density of T cell vaccine proliferation were observed every other day;

[0084] Add antigen presenting cells on the 7th day of culture:

[0085] Antigen presenting cells are trophoblast cells or mature DC cells.

[0086] The preparation method of trophoblast cells is as follows: the spleen of CIA mice was removed, and the red blood cells were lysed to obtain a single cell suspension; 5×10 7 / mL of culture medium (based on RPIM1640, and also including the following components: 10% FBS, 5mM HEPES, 2mM L-glutamine, 0.05mM mercaptoethanol, 100U / mL streptomycin and penicillin by volume), treated with 50μg / mL mitomycin for 30min, and then washed three times to obtain trophoblast cells;

[0087] The preparation method of DC cells is as follows: obtain mouse bone marrow cells in advance, split them into red cells, and differentiate them into DC cells with 25ng / mL GM-CSF and 10ng / mL IL-4 for 7 days; obtain mature DCs by inducing them with 1μg / mL LPS for two days, and then treat them with 50μg / mL mitomycin for 30 minutes, and then wash them three times to obtain DC cells.

[0088] After 20 μg / mL CII peptide and antigen presenting cells were co-incubated in the incubator for 12 hours, T cell vaccine was added at a ratio of 2:1 for co-culture, and the culture density was 0.5×10 7 / mL;

[0089] On the 8th day of culture, IL-15 and IL-7 were added at a final concentration of 5-10 ng / mL.

[0090] On the 13th day of culture, IL-2 with a final concentration of 20 ng / mL and 5 μg / mL PHA were added to the culture system. The color of the culture medium and the cell density were observed every other day. At this stage, the cells will proliferate rapidly. If necessary, add an appropriate amount of culture medium. On the 16th day of culture, the cells were collected and treated with 25 μg / mL mitomycin for half an hour. After washing three times, the cells were cultured using Stemcell CD4 + Separation magnetic beads (Cat. No. 18952) were used to separate and obtain CD4 + T cells; resuspended in saline to 2×10 7 / mL of cell suspension to obtain antigen-specific T cell vaccine.

[0091] The flow chart of the whole process of the above cultivation is Figure 3 As shown in Figure A, the purity of the separation was verified by flow cytometry. Figure 3 B, and detect the CD4 + The ratio of effector cells to memory cells Figure 3 C, as well as the cell morphology, were tested Figure 3 Middle D.

[0092] Example 3

[0093] Experimental study on the effectiveness of antigen-specific T cell vaccines.

[0094] The antigen-specific T cell vaccine (CD4 + T cells) were counted and 4 × 10 5 / 400μL CD4 + T cells as TCV individual wells, 4 × 10 5 / 400μLPBMC as PBMC wells, and set up the following experimental groups at the same time, CD4 + The ratios of T cells to PBMC were 1:1, 1:2, and 1:4, respectively. + The T cell concentration was 4 × 10 5 / 400μL.

[0095] After 48 hours of culture, the following indicators were tested:

[0096] (1) Detect the changes of various T cell subsets in different treatment groups, including Th1 / Th2 / Th17 / Treg; before culturing for 48 hours, PMA and ionomycin were combined to stimulate the cells for 4 hours, and then membrane permeabilization staining (ebioscience, catalog number 88-8824-00) was performed. Treg was permeabilized by nuclear permeabilization staining (ebioscience, catalog number 00-5523-00). The staining antibodies are shown in Table 1.

[0097] Table 1 Antibodies for staining

[0098] marker brand Part Number PE-Cy7-CD4 Biolegend 300512 BV421-IL17A Biolegend 512321 FITC-IFNγ Invitrogen 4S.B3 PE-IL-4 Invitrogen 2247540 PE-Foxp3 Invitrogen 12-4777-4290 BV506-FVD Invitrogen 65-0866-14

[0099] (2) Detect the changes of inflammatory cytokines in the supernatant, mainly detecting IFNγ, IL-17, and IL-6. The kit was purchased from Xinbosheng Company and the kit instructions were referred to.

[0100] result

[0101] (1) Staining schemes for different immune cells:

[0102] Th1 (CD4 + IFNγ + ), Th2(CD4 + IL-4 + ), Th17(CD4 + IL - 17A + ),

[0103] Treg (CD4 + Foxp3 + ).

[0104] The results of detecting several types of cell subpopulations are shown in Figure 4 Middle A. Compared with the PBMC culture group alone, the proportion of Treg cells in the experimental group increased.

[0105] (2) The results of cytokine detection in the supernatant are shown in Figure 4 B. Compared with the PBMC group cultured alone, the IFNγ in the culture supernatant of the experimental group was significantly reduced.

[0106] Example 4

[0107] Animal experiment on the treatment of rheumatoid arthritis with antigen-specific T cell vaccine

[0108] A total of 60 CIA model mice were constructed according to the method of Example 2. After the fifth day of booster immunization, mice with consistent scores (1 to 3 points) were randomly divided into the following groups: a. saline control group; b. nonspecific T cell vaccine treatment group; c. antigen-specific T cell vaccine treatment group; 200 μl saline or 2×106 Cells were treated three times on days 1, 7, and 14 of the mice. The mice were sacrificed on day 21 of booster immunization, and the following indicators were tested:

[0109] (1) The joint swelling of mice was monitored and scored once a day. The scoring was performed independently by two experimenters at the same time, and the average score of the two was taken as the final score. The scoring joints included the four foot joints of the front and back limbs of the mice. The scoring criteria for each joint were as follows: 1 point for less than 2 swollen toes, 2 points for more than 2 swollen toes, 1 point for swollen soles, and 1 point for swollen ankles. Each joint was scored up to 4 points, and each mouse was scored up to 16 points.

[0110] Results Figure 5 The disease scores of mice were monitored and recorded, and the results showed that the T cell vaccine had an overall therapeutic effect compared with the saline group; the antigen-specific T cell vaccine obtained through antigen stimulation and amplification had a significantly better therapeutic effect than the non-specific T cell vaccine.

[0111] (2) Arthritis inflammation

[0112] Joint imaging and histopathological indicators for qualitative and quantitative evaluation of bone destruction

[0113] Small animal MicroCT was used to detect bone erosion and bone destruction in the joints of arthritic mice in the treatment group and the control group. Figure 6 As shown, the antigen-specific T cell vaccine significantly reduced bone erosion and bone destruction compared with the T cell vaccine without antigen stimulation and the saline group, showing a good therapeutic effect.

[0114] (3) Monitoring of immune subpopulations in mice in different treatment groups using flow cytometry

[0115] The lymph nodes of the model mice in the control group and the experimental group were placed in 1640 serum-containing culture medium, ground with a filter, and made into a single cell suspension, all of which were used for flow cytometry. The spleen was placed in 1640 serum-containing culture medium, ground with a filter, and made into a single cell suspension. Add 2 ml of red blood cell lysis buffer and place at room temperature for 7 minutes. Centrifuge at room temperature, 1600 rpm, for 5 minutes, and discard the supernatant. Wash twice with PBS buffer, keep the precipitate after centrifugation, count the cells after resuspending with PBS, and adjust the cell number to 2-3×10 6 / 100μl, set aside. The flow cytometry method is as follows: Flow cytometry is used to detect changes in immune cell subsets such as Th1 / 2 / 17 and Treg. The specific staining steps are as follows:

[0116] Th1 / 2 / 17: Th1 (CD4 + IFN-γ + ), Th2(CD4 + IL-4 +), Th17 (CD4 + IL-17A + )

[0117] 1) Stimulation: Add 1 ml of stimulation solution (1 μg / ml ionomycin, 10 μg / ml BFA, 50 ng / ml PMA, dissolved in 1640 serum-free and double-antibody-free medium) to each sample; incubate at 37°C in the dark for 5 h;

[0118] 2) Staining: Surface staining - add surface antigen staining marker (CD4-FITC, 1:50), incubate at 4°C, away from light, for 30 minutes. Permeabilize the membrane before intracellular staining, then add intracellular antigen staining marker (IL-17A-BV421, 1:50; IFN-γ-PE, 1:50: IL-4-APC, 1:50), incubate at 4°C, away from light, for 30 minutes.

[0119] 3) Detection: Wash, centrifuge, resuspend in 200 μL PBS buffer, and send to flow cytometer for detection.

[0120] Tfh:CD4 + CD44 + PD-1 + CXCR5 + Bcl-6 + Foxp3 - ;

[0121] Treg: CD4 + CD25 + Foxp3 + .

[0122] 1) Blocking: Add CD16 / CD32 (i.e., FcR blocking monoclonal antibody) to each sample at 1:200;

[0123] 2) Staining: add surface antigen staining marker (1:200 or 1:100), incubate at room temperature, away from light, on a shaker for 40 minutes; second layer of surface staining: add SA-BV711 (1:100), incubate at room temperature, away from light, on a shaker for 30 minutes; intracellular staining after membrane permeabilization: add intracellular antigen staining marker (1:50), incubate at 4°C, away from light for 30 minutes.

[0124] 3) Detection: Wash, centrifuge, resuspend in 200 μL PBS buffer, and detect on a detector.

[0125] Th1 and Th17 are important pathogenic cell subsets in rheumatoid arthritis, which affect osteoclasts by secreting cytokines; Tfh and GCB affect disease progression by affecting the production of autoantibodies. The experimental results showed that after treatment with antigen-specific T cell vaccines, Th1 / Th17 decreased and Treg increased ( Figure 7 , Figure 8 ). This shows that antigen-specific T cell vaccines can effectively treat rheumatoid arthritis by inhibiting pathogenic cell subsets.

[0126] Example 5

[0127] Treg is a key cell subset for antigen-specific T cell vaccines to exert therapeutic effects.

[0128] According to literature research, Treg is the main cell subpopulation affected in therapeutic cell vaccines, and this group of cells also plays an important role in RA. Therefore, in this example, arthritis mice were treated with antigen-specific T cell vaccines, and flow cytometry was used to analyze the number of Treg cells and the expression of related inhibitory molecules in the lymph nodes and spleen of the mice after treatment.

[0129] The results showed that after treatment with antigen-specific T cell vaccine, Treg cells increased significantly and upregulated the expression of cell surface-related inhibitory molecules ( Fig. 9 A and B).

[0130] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Use of an antigen-specific T cell vaccine in the preparation of a drug for preventing and / or treating rheumatoid arthritis, wherein the antigen-specific T cell vaccine is an antigen-specific CD4 T cell vaccine obtained by inactivating and separating spleen cells derived from rheumatoid arthritis samples after stimulation with rheumatoid arthritis autoantigen epitope polypeptides. + T cells; The amino acid sequence of the rheumatoid arthritis autoantigen epitope polypeptide is shown as QYMCitADQAAGGLR, the N-terminal glutamine of the rheumatoid arthritis autoantigen epitope polypeptide is replaced with D-glutamine, the N-terminus of the rheumatoid arthritis autoantigen epitope polypeptide is acetamidate-modified, and the C-terminus of the rheumatoid arthritis autoantigen epitope polypeptide is amide-modified; The antigen-specific T cell vaccine is obtained by treating with 20 to 50 μg / mL mitomycin for 30 to 60 minutes; The method for preparing the antigen-specific T cell vaccine comprises the following steps: The spleen cells derived from rheumatoid arthritis samples were cultured in a medium containing rheumatoid arthritis autoantigen epitope polypeptides for 7 days, co-cultured with antigen presenting cells that captured the rheumatoid arthritis autoantigen epitope polypeptides for 3 days, and cultured with IL-7 and IL-15 on the 8th day of culture, IL-2 and PHA were added on the 13th day of culture, and cells were collected on the 16th day of culture, inactivated, and antigen-specific CD4 + T cells.

2. The application according to claim 1, characterized in that: The culture medium is based on RPIM1640 and further includes the following components: 10% FBS, 5mM HEPES, 2mM L-glutamine, 0.05mM mercaptoethanol, 100U / mL streptomycin and penicillin, and 5-10ng / mL IL-15 and IL-7 by volume percentage; In the culture medium, the concentration of the autoantigen epitope polypeptide is 5-50 μg / mL.

3. The application according to claim 1, characterized in that: The concentration of spleen cells from the rheumatoid arthritis sample is (1-10)×10 7 / mL.

4. The use according to claim 1, characterized in that: The method for preparing the antigen presenting cells comprises co-incubating a 5-50 μg / mL rheumatoid arthritis autoantigen epitope polypeptide solution with the antigen presenting cells; The concentration of the antigen presenting cells is (1~10)×10 6 / mL; The antigen presenting cells include trophoblast cells or mature dendritic cells; The method for preparing trophoblast cells comprises: performing red lysis treatment on spleen derived from rheumatoid arthritis samples to obtain spleen single cells, and then inactivating the cells to obtain trophoblast cells; The method for preparing mature dendritic cells comprises: subjecting mouse bone marrow cells to erythroblastosis, differentiation and culture, and inducing maturation with LPS to obtain mature dendritic cells; The reagent for differentiation culture is an aqueous solution containing 10-25 ng / mL GM-CSF and 5-10 ng / mL IL-4; On the 7th day of culture, the ratio of cells to antigen-presenting cells that captured self-antigens was 1-5:

1.

5. The use according to claim 1, characterized in that: The final concentration of IL-7 and IL-15 is 5-20 ng / mL; The final concentration of IL-2 is 5-20 ng / mL; The final concentration of PHA is 1-10 μg / mL.

6. The use according to any one of claims 1 to 5, characterized in that: The method for inactivating cells is to treat with 20-50 μg / mL mitomycin for 30-60 minutes; The separation of specific CD4 + The T cell method is to use commercial tetramer antibody staining to isolate antigen-specific CD4 + T cells.

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