Antigen-specific B cell vaccines and their applications

Abstract

The present invention discloses an antigen-specific B cell vaccine and its application, the vaccine is prepared by B cells obtained after sorting of collagen-induced arthritis mouse spleen cells; the B cells obtained after sorting are total B cells, Plasma cells or pathogenic B cells; the preparation method is that the B cells obtained after sorting are cultured under the stimulation of anti-CD40, CpG, type II collagen (CII) and BAFF, and then irradiated by a cobalt source to obtain the vaccine. The present invention also discloses a human antigen-specific B cell vaccine, which is prepared by replacing the above-mentioned collagen-induced arthritis mouse spleen cells with peripheral blood cells of rheumatoid arthritis patients. Compared with traditional drugs, the B cell vaccine of the present invention has a smaller dosage, a lower frequency of administration, and a longer maintenance period; subcutaneous injection, simple and convenient; treatment of diseases by immunomodulation; can effectively control the condition, reduce bone erosion and joint destruction; autologous reinfusion, no immune tolerance problems; small side effects.

Description

[0001] This application is a divisional application with application number 201910891701.1, application date 2019.09.20, and the name of the invention: Application of B cell vaccine in the preparation of drugs for the treatment of rheumatoid arthritis. Technical Field

[0002] The present invention relates to the technical field of drugs for treating autoimmune diseases, and in particular to an antigen-specific B cell vaccine and application thereof in the preparation of drugs for treating rheumatoid arthritis. Background Art

[0003] Rheumatoid arthritis (RA) is a disease with irreversible bone erosion and a high disability rate. However, current treatment options still have certain limitations: lifelong medication is required, and most patients can only maintain remission but cannot be cured. Therefore, more specific and effective causal treatment options are urgently needed.

[0004] Currently, the most commonly used drugs for the treatment of RA are mainly divided into c-DMARDs and b-DMARDs; in addition, they also include newly developed small molecule ts-DMARDs, such as JAK inhibitors.

[0005] The onset of RA is closely related to the overactivation of B cells that produce a large number of autoantibodies. In view of the important role of B cells in the pathogenesis of RA, B lymphocyte targeted therapy targeting B lymphocyte surface molecules has been widely studied and applied in recent years.

[0006] B lymphocyte depletion therapy represented by CD20 monoclonal antibodies is a relatively mature research program and has achieved great success in clinical treatment, such as Rituximab, Ocrelizumab, Ofatumumab, etc. In addition, there are antibodies targeting BLyS and its receptors, such as belimumab, atacicept, BR3-Fc, etc., and monoclonal antibodies targeting CD22, such as Epratuzumab, etc.

[0007] In addition to B cell clearance, there are also neutralizing drugs targeting B cell growth factor (BAFF), including BAFF neutralizing antibodies (such as Belimumab), fusion proteins of BAFF receptors TACI or BR3 and immunoglobulin Fc segment.

[0008] Since RA is an autoimmune disease, immunologists have gradually realized that regulating the disordered immune system to restore its balance may have better therapeutic effects. The concept of T cell vaccine therapy was first proposed. T cell vaccine (TCV) refers to pathogenic T cells inactivated by chemical or physical methods. They lose their proliferation and pathogenicity, but maintain immunogenicity and immune activation characteristics. At present, there are precedents for the use of T cell vaccine regimens to treat autoimmune diseases such as RA both at home and abroad, showing good therapeutic effects and fewer side effects compared to drug treatment. In addition, other therapeutic vaccines based on inducing T cell tolerance have also been proposed, including TCR peptide vaccines, antigen peptide-MHC molecule complex vaccines, vaccines targeting MHC, peptide vaccines targeting cytokines (such as IL-17, TNF-α, IL-1β), complement peptide vaccines, etc.

[0009] Although therapeutic vaccines for RA are available, clinical options are still limited, and most vaccines are not satisfactory in terms of therapeutic effects. Summary of the invention

[0010] The specific B cells sorted from the donor are expanded in vitro and given antigen stimulation, then inactivated by irradiation so that they cannot proliferate but maintain immunogenicity. They are then re-infused into the recipient to achieve a therapeutic effect by inducing immune tolerance and correcting immune disorders. This vaccine is an antigen-specific B cell vaccine.

[0011] There are few types of existing RA therapeutic vaccines, with limited choices and poor effects. The onset of rheumatoid arthritis is closely related to the production of a large number of autoantibodies by B cells. In the past, more attention was paid to drug development using B cells as targets. The present invention changes the thinking. In view of the core role of B cells in the onset of RA, the development of therapeutic vaccines based on B cells has important application prospects. Therefore, B cell vaccines are developed, and it is further discovered that B cell vaccines can more directly regulate the immune system than T cell vaccines to achieve therapeutic effects. Based on the research results, the present invention provides the following technical solutions:

[0012] In a first aspect, the present invention provides an antigen-specific B cell vaccine, which is prepared by sorting B cells obtained from spleen cells of collagen-induced arthritis (CIA) mice; the B cells obtained after sorting are CD19 + Total B cells, Plasma cells (one embodiment such as CD19 - / + CD138 + ) or pathogenic B cells; the preparation method is to stimulate and culture the B cells obtained after sorting in the presence of anti-CD40 functional antibodies, CpG, type II collagen (CII) and BAFF, and then irradiate with a cobalt source to obtain a vaccine.

[0013] The mice selected are DBA / 1 male mice, which have a high model rate of about 80-90%. Moreover, compared with other arthritis models, the CIA model can cause joint damage and immune system disorders in mice, induce systemic lesions, and is closer to the disease phenotype of rheumatoid arthritis patients. Both chicken and bovine type II collagen (C II) can cause arthritis, and bovine collagen is preferred.

[0014] Optionally or preferably, in the above-mentioned B cell vaccine, the total dose of the cobalt source irradiation is 20-30 Gy, and the irradiation method is irradiation at an intensity of 1.5-2.0 Gy / min for 15 minutes. Preferably, the total dose is 30 Gy and the intensity is 2 Gy / min.

[0015] Optionally or preferably, in the above-mentioned B cell vaccine, the B cell is CD19 + For total B cells, the sorting method is: spleen cells are sorted by magnetic beads (for example, using the kit STEM CELL, catalog number 18954), and positive selection is used to obtain all CD19 + B cells.

[0016] Optionally or preferably, in the above-mentioned B cell vaccine, when the B cells are plasma cells, the sorting method is: spleen cells are surface stained with antibodies CD19-FITC and CD138-APC, and then the phenotype of CD19 is sorted by flow cytometry. - / + CD138 + Plasma cells.

[0017] Optionally or preferably, in the above-mentioned B cell vaccine, when the B cells are pathogenic B cells, the sorting method is: using flow sorting to enrich CD19 + Collagen-antibody + B cells, that is, obtaining pathogenic B cells.

[0018] In a second aspect, the present invention also provides a human antigen-specific B cell vaccine, which is prepared by using peripheral blood cells of rheumatoid arthritis patients to replace the spleen cells of collagen-induced arthritis mice described in any of the above schemes. Except for the replacement of the spleen cells used, the rest of the preparation method is the same as described in the above schemes, including total B cells (CD19 + ) vaccines, Plasma cells (CD19 + CD20 - CD27 + CD38 + ) vaccine, or pathogenic B cell (ACPA-specific Bcells) vaccine.

[0019] In a third aspect, the present invention also provides an application of an antigen-specific B cell vaccine for preparing a drug for treating rheumatoid arthritis, wherein the B cell vaccine is any of the B cell vaccines described above.

[0020] In a fourth aspect, the present invention also provides a drug for treating rheumatoid arthritis, wherein the drug component contains any of the above-mentioned B cell vaccines.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] The present invention creatively discovered that antigen-specific B cell vaccines can be used to treat rheumatoid arthritis RA, so that the immunotherapy of RA no longer relies solely on traditional drugs, T cell vaccines or B cell targeted therapy, providing a new treatment approach for clinical practice.

[0023] Compared with other drug treatments, the B cell vaccine described in the present invention has the following advantages: smaller dosage, lower administration frequency, and longer maintenance period; subcutaneous injection, simple and convenient; treatment of diseases through autologous immune regulation function; effective control of the disease, reduction of bone erosion and joint damage; autologous reinfusion, no immune tolerance problems; small side effects.

[0024] When CIA mice were treated, CD19 + The total B cell vaccine dose is 1×10 7 The dosage of plasma cell vaccine and pathogenic B cell vaccine is 1×10 5 , once every 5 days, for a total of 3 injections.

[0025] Literature reports that T cell vaccines can treat CIA, mostly as preventive vaccines, that is, vaccine intervention is given before the second immunization, and the therapeutic dose is a single 1×10 7 Compared with the prevention model, the treatment model can more accurately reflect the efficacy and has greater clinical prospects (reference: T Cell Vaccination Inhibits Th1 / Th17 / Tfh Frequencies and Production of Autoantibodies in Collagen-Induced Arthritis. Clinical and Developmental Immunology, 2013, 967301).

[0026] CD20 monoclonal antibody (Rituxan) exerts its therapeutic effect by eliminating B cells and is currently a commonly used drug for the treatment of RA. However, it cannot completely deplete B cells, especially plasma cells, and cannot eliminate tissue-resident B cells, nor can it specifically target pathogenic B cells, which has certain limitations. According to the pharmacokinetic attenuation, it requires a large number of multiple injections, and 20 μg is required per day (reference: B-cell inhibition by cross-linking CD79b is superior to B-cell depletion with anti-CD20 antibodies in treating murine collagen-induced arthritis. Eur J Immunol, 2015, 45(3): 705-15). BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 Design of vaccine preparation and experimental procedures in Examples 1-4.

[0028] Figure 2 These are the arthritis phenotype scoring results of the arthritis mice (normal saline group, B cell vaccine group, Non-B10 B cell vaccine group) in Example 5.

[0029] Figure 3 For the normal mice in Example 5 and micro CT imaging results of the paws of arthritic mice (saline group, B cell vaccine group, and Non-B10 B cell vaccine group).

[0030] Figure 4 For the normal mice in Example 5 HE staining of the paws and joint injury scoring results of arthritis mice (normal saline group, B cell vaccine group, and Non-B10 B cell vaccine group).

[0031] Figure 5 These are the results of flow cytometry detection of spleen immune cells in arthritis mice (normal saline group, B cell vaccine group, Non-B10 B cell vaccine group) in Example 5. DETAILED DESCRIPTION

[0032] Example 1CD19 + Preparation of total B cell vaccine

[0033] Experimental animals: Specific pathogen free (SPF) 6-8 week old DBA / 1 male mice were purchased from Beijing Huafukang Biotechnology Co., Ltd. and housed in the SPF environment of the animal laboratory of Peking University People's Hospital. Modeling experiments were performed after one week of adaptive feeding.

[0034] Modeling method: Bovine Type II collagen (C II) was dissolved in 100 mmol / L glacial acetic acid at 4°C overnight, mixed with complete Freund's adjuvant (CFA) at a volume ratio of 1:1, and fully emulsified. Two points were subcutaneously injected at the base of the mouse tail, each containing 100 μg of C II emulsion; on the 21st day after the first immunization, C II and incomplete Freund's adjuvant (IFA) were fully emulsified at a volume ratio of 1:1, and an emulsion containing 100 μg C II was subcutaneously injected at the base of the mouse tail to complete the induction of the collagen-induced arthritis (CIA) model. Six days after the second immunization, the mice gradually became ill, reaching a peak on the 14th day, and CIA mice were obtained.

[0035] (1) CIA mouse CO 2 After killing, the mice were dislocated by neck dislocation, and the abdomen was cut open with sterilized scissors. The spleen tissue was cut out, and the fat tissue around the spleen was trimmed and placed in a culture dish containing incomplete RPMI-1640 culture medium prepared in advance, and placed in an ice box for subsequent experiments.

[0036] (2) Take a clean culture dish and place a filter with a pore size of 100 μm on it. Add about 1 mL of incomplete RPMI-1640 culture medium to the filter to fully moisten the filter. Take out the spleen and place it on the filter. Grind it thoroughly with a grinding rod. Take a small amount of culture medium to rinse the filter and discard the filter.

[0037] (3) After repeatedly pipetting the liquid in the culture dish, transfer it to a 5 mL flow tube, centrifuge at 1800 rpm for 5 min, and discard the supernatant.

[0038] (4) Add 3 mL of physiological saline to resuspend the cells, filter using a flow tube with a cell strainer (BD, Catalog No. 352235), centrifuge at 1800 rpm for 5 min, and discard the supernatant.

[0039] (5) Add 3 mL of lysing solution to the flow tube, mix thoroughly by pipetting, and let stand at room temperature for 3 min (10×Fixative FreeFCM lysing solution was added with ddH 2 O to prepare 1× red lysis solution).

[0040] (6) Centrifuge at 1800 rpm for 5 min, discard the supernatant, add 3 mL of saline to resuspend the cell pellet, and centrifuge at 1800 rpm for 5 min, discard the supernatant to obtain the spleen cell pellet.

[0041] (7) Add 1 mL of physiological saline to resuspend the cell pellet to prepare a spleen cell suspension.

[0042] (8) Perform CD19 according to the instructions of CD19 positive selection kit II (Cat. No. 18954) from STEM CELL. + Magnetic bead sorting of total B cells.

[0043] (9) After sorting, complete RPMI-1640 medium (incomplete RPMI-1640 medium with 10% FBS and 1% penicillin and streptomycin) was added to the cells, mixed by pipetting, and plated. 3 μg / mL anti-CD40, 10 μg / mL CpG, 20 μg / mL CII, and 20 ng / mL BAFF were added and incubated in a 37°C incubator for 3 days.

[0044] (10) After 3 days of stimulation and culture, the cells were mixed by pipetting and collected in a 15 mL centrifuge tube. The tube was centrifuged at 1800 rpm for 5 min and the supernatant was discarded.

[0045] (11) Add 10 mL of normal saline and mix thoroughly by pipetting. Centrifuge at 1800 rpm for 5 min, then discard the supernatant and wash away the residual serum.

[0046] (12) Add 10 mL of incomplete RPMI-1640 medium and resuspend.

[0047] (13) The total dose was 30 Gy and the intensity was 2 Gy / min for 15 min, which caused the cells to lose their proliferation function but maintain their immunogenicity.

[0048] (14) Centrifuge at 1800 rpm for 5 min, discard the supernatant, add 10 mL of normal saline, mix by pipetting, and centrifuge at 1800 rpm for 5 min, discard the supernatant.

[0049] (15) Add physiological saline to resuspend the cells to a final concentration of 1×10 7 cell / 300μl, CD19 + Total B cell vaccines.

[0050] Example 2 Preparation of Non-B10 B cell vaccine

[0051] (1) Prepare spleen cell pellet according to the methods (1) to (6) in Example 1.

[0052] (2) Add 300 μL of saline to resuspend the spleen cell pellet, add 5 μL of surface antibodies CD19-FITC, CD5-APC, and CD1d-PE in a dark place, vortex and incubate at room temperature in a dark place for 30 min (vortex once every 15 min).

[0053] (3) Add 2 mL of physiological saline solution and mix thoroughly, then centrifuge at 1800 rpm for 5 min and discard the supernatant.

[0054] (4) Add 1 mL of sorting solution (PBS containing 2% FBS) to resuspend the cell pellet and then place it on a flow cytometer (BD FACS AriaII sorter) to sort out B10 cells (CD19 + CD5 + CD1d hi All B cells except CD19 + CD5 - / + CD1d low ), that is, Non-B10 B cells are obtained. 1.5-2.0*10 7 Non-B10 B cells (reference: A regulatory B cell subset with a unique CD1d hi CD5 + Phenotype controls T cell-dependent inflammatory responses, Immunity, 28(2008), pp.639-650).

[0055] The sorting conditions are: select a 75mm nozzle in purity mode, receive a 15mL centrifuge tube, the flow rate does not exceed 5 during sorting, and the loading speed does not exceed 15,000 events / s. According to the normal flow sorting process, as long as the flow rate is controlled reasonably according to efficiency and maintained below 5, it will not affect the state of the cells.

[0056] (5) The cells in the sorting collection tube were centrifuged at 1800 rpm for 10 min, resuspended in complete RPMI-1640 medium, and then subjected to subsequent culture stimulation and cobalt source irradiation in accordance with steps (9) to (14) in Example 1. Physiological saline was added and resuspended to a concentration of 1×10 7 cell / 300μL, and the Non-B10 B cell vaccine was prepared.

[0057] Example 3 Preparation of Plasma Cell Vaccine

[0058] (1) Prepare spleen cell pellet according to the methods (1) to (6) in Example 1.

[0059] (2) Add 300 μL of saline to resuspend the spleen cell pellet, and then add 5 μL of surface antibodies CD19-FITC and CD138-APC in a dark environment. Add enough antibodies to allow the cells to fully bind to the antibodies. Add 5-10 μL according to the number of cells in each tube.

[0060] (3) Using flow cytometry, sort cells with a phenotype of CD19 - / + CD138 + Plasma cells.

[0061] (4) Plasma cell vaccine was prepared in the same manner as steps (9) to (14) in Example 1. The concentration was 1×10 5 cell / 300μL.

[0062] Example 4 Preparation of pathogenic B cell vaccine

[0063] (1) Prepare spleen cell pellets according to the methods (1) to (6) in Example 1.

[0064] (2) Flow cytometry was used to separate all pathogenic B cells (CD19 + Collagen-antibody + B cells) are enriched, stimulated and cultured in vitro and irradiated with a cobalt source according to the treatment methods of steps (9) to (14) in Example 1, and finally prepared into a suspension of corresponding concentration with physiological saline to obtain a pathogenic B cell vaccine.

[0065] Example 5: Tests to verify the therapeutic effects of each vaccine

[0066] Experimental animals: Specific pathogen free (SPF) 6-8 week old DBA / 1 male mice were purchased from Beijing Huafukang Biotechnology Co., Ltd. and housed in the SPF environment of the animal laboratory of Peking University People's Hospital. Modeling experiments were performed after one week of adaptive feeding.

[0067] Modeling method: Bovine Type II collagen (C II) was dissolved in 100 mmol / L glacial acetic acid at 4°C overnight, mixed with complete Freund's adjuvant (CFA) at a volume ratio of 1:1, and fully emulsified. Subcutaneous injection was performed at two points at the base of the mouse tail, each containing 100 μg of C II emulsion; on the 21st day after the initial immunization, C II and incomplete Freund's adjuvant (IFA) were fully emulsified at a volume ratio of 1:1, and an emulsion containing 100 μg C II was subcutaneously injected at the base of the mouse tail to complete the induction of collagen-induced arthritis (CIA) model.

[0068] Grouping: A total of 40 CIA model mice were constructed. On the 5th day after the secondary immunization, 20 mice with similar scores were selected and randomly divided into the following three groups to ensure that the mean joint phenotype score of each group was consistent: The first group had a total of 6 mice, using the CD19 prepared in Example 1 + The second group, a total of 8 mice, was treated with the Non-B10 B cell vaccine prepared in Example 2; the third group, a control group, a total of 6 mice, was treated with normal saline.

[0069] Treatment methods: The three groups were treated with the same treatment methods: each mouse in each group was subcutaneously injected with 300 μL of saline or saline cell suspension, and the number of cells injected into the vaccine treatment group was 1×10 7 The treatment was given three times on days 5, 10 and 15 after the second immunization.

[0070] Final experiment: The mice in the vaccine treatment group and the control group were killed and dissected 20 days after the second immunization. The spleen tissue, paws, etc. were removed and the following evaluations were performed:

[0071] (1) Joint phenotype scoring: The joint swelling scores of mice were recorded starting from the first day after the second immunization. The scoring criteria are as follows:

[0072] 0 points: no joint redness or swelling;

[0073] 1 point: slight erythema or swelling of one toe;

[0074] 2 points: erythema and swelling of more than one toe;

[0075] 3 points: erythema and swelling of ankles or wrists;

[0076] 4 points: all erythema and swelling of toes and ankles or fingers and wrists, and the ankle or wrist cannot be bent;

[0077] The results of mouse joint swelling scores were as follows Figure 2As shown in the results, the vaccine group had a significantly lower score than the control group, the incidence of arthritis was significantly reduced, and the effect of the Non-B10 B cell vaccine group was better than that of the CD19 + Total B cells in the vaccine group.

[0078] (2) MicroCT: The ipsilateral paw was fixed in 4% paraformaldehyde and imaged using Tri-Modality FLEXTriump at 130μA and 80kVP. Three-dimensional reconstruction was performed to analyze the bone erosion of the mouse paw. The results are shown in Figure 3 As shown, it can be clearly seen that CD19 + Compared with the control group of normal saline, the bone erosion in the total B cell vaccine group and the Non-B10 B cell vaccine group was significantly weakened, the joints were more intact, and the treatment effect of the Non-B10 B cell vaccine group was more significant.

[0079] (3) HE staining: The ipsilateral mouse paw was fixed in 4% paraformaldehyde, embedded in paraffin, decalcified, stained with HE, and scanned with white light to observe the inflammatory infiltration site and joint injury score. The scoring criteria are as follows:

[0080] 0 points: normal;

[0081] 1 point: synovial hyperplasia and inflammatory cell infiltration;

[0082] 2 points: pannus formation and articular cartilage destruction;

[0083] 3 points: most articular cartilage is destroyed and subchondral bone is eroded;

[0084] 4 points: loss of joint integrity, ankylosis;

[0085] The rating results are as follows Figure 4 As shown in the results, it can be seen that compared with the control group, especially the Non-B10 B cell vaccine treatment group, most mice in the vaccine treatment group only progressed to synovial hyperplasia and inflammatory cell infiltration, without obvious articular cartilage destruction. However, most of the mice in the control group suffered from loss of joint integrity, and a small number suffered from cartilage destruction and erosion, and the disease progression was very serious.

[0086] (4) Flow cytometry of spleen immune cells: The B10, plasma, Th1, and Th17 subpopulations of the spleen of the three groups of mice were detected. It was found that the plasma cells in the vaccine treatment group were significantly reduced, B10 was significantly increased, and Th1 had a downward trend. From the perspective of immunological indicators, it was confirmed that the disease in the treatment group was alleviated (such as Figure 5 shown).

[0087] The above experimental results show that B cell vaccine can effectively control the condition of rheumatoid arthritis, reduce bone erosion and joint destruction, and has a good therapeutic effect.

[0088] The invention concept is described in detail using specific examples herein, and the description of the above embodiments is only used to help understand the core idea of ​​the invention. It should be pointed out that any obvious modification, equivalent substitution or other improvement made by a person of ordinary skill in the art without departing from the invention concept should be included in the protection scope of the present invention.

Claims

1. An antigen-specific B cell vaccine, characterized in that: Prepared from B cells obtained by sorting spleen cells from mice with collagen-induced arthritis; The B cells obtained after the sorting are CD19+ total B cells, and the sorting method is: spleen cells are sorted by magnetic beads to positively select all CD19+ B cells; The method for preparing the B cell vaccine is to stimulate and culture the B cells obtained after sorting in the presence of anti-CD40 functional antibodies, CpG, type II collagen and BAFF, and then irradiate them with a cobalt source to obtain the vaccine.

2. The B cell vaccine according to claim 1, characterized in that The total dose of the cobalt source irradiation is 20-30 Gy, and the irradiation method is irradiation at an intensity of 1.5-2.0 Gy / min for 15 min.

3. A human antigen-specific B cell vaccine, characterized in that: Prepared from B cells obtained by sorting peripheral blood cells from patients with rheumatoid arthritis; The B cells obtained after the sorting are CD19+ total B cells, and the sorting method is: peripheral blood cells are sorted by magnetic beads to positively select all CD19+ B cells; The method for preparing the B cell vaccine is to stimulate and culture the B cells obtained after sorting in the presence of anti-CD40 functional antibodies, CpG, type II collagen and BAFF, and then irradiate them with a cobalt source to obtain the vaccine.

4. Application of antigen-specific B cell vaccines, characterized in that: The antigen-specific B cell vaccine is used to prepare a drug for treating rheumatoid arthritis, and the B cell vaccine is the B cell vaccine according to claim 1 or 2.

5. A drug for treating rheumatoid arthritis, characterized in that: The medicinal component contains the B cell vaccine according to claim 1 or 2.

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