CN110041404A - Citrullinated antigens modified peptides and its application - Google Patents

Abstract

The present invention provides a citrullinated antigen-modified peptide and its application, and relates to the technical field of rheumatoid arthritis treatment. The citrullinated antigen-modified peptide has the amino acid sequence shown in SEQ ID No.1 or SEQ ID No.2. The citrullinated antigen-modified peptide specifically binds to HLA-DRB1 related to rheumatoid arthritis, and inhibits the binding of the antigenic peptide to HLA-DRB1, thereby inhibiting the activation of T cells and achieving the purpose of treating rheumatoid arthritis.

Description

Citrullinated antigen-modified peptide and its application

technical field

The invention relates to the technical field of rheumatoid arthritis treatment, in particular to citrullinated antigen-modified peptides and applications thereof.

Background technique

Rheumatoid arthritis is a systemic autoimmune disease characterized by chronic progressive joint disease. The lesions mainly accumulate in multiple joints in the whole body, showing multiple and symmetrical chronic synovitis, which in turn causes the destruction of cartilage and bone, and in severe cases can lead to joint deformity and even disability. Rheumatoid arthritis is one of the major diseases causing labor loss and disability.

The etiology of rheumatoid arthritis is not yet clear, and it is generally believed that genetic factors play an important role in the pathogenesis of rheumatoid arthritis. In recent years, studies on the pathogenesis of rheumatoid arthritis have shown that T cell activation driven by autoantigens is the basis for the pathogenesis and evolution of rheumatoid arthritis. Antigen peptides are recognized and presented by antigen-presenting cells in the body, and are then recognized by the T cell receptor (TCR) on the surface of antigen-specific T cells to form MHC-antigen peptide-TCR trimolecular complexes to activate antigen-specific T and B Cells, leading to increased production of inflammatory cytokines, immunoglobulins, etc., causing synovial inflammation, pannus formation, resulting in cartilage and bone destruction and other characteristic pathological changes in rheumatoid arthritis.

Currently, there are no drugs that can fundamentally control rheumatoid arthritis. Clinically, disease-modifying anti-rheumatic drugs and biological agents are mainly used to relieve symptoms, inhibit inflammation, delay joint damage and improve patient function. In recent years, the application of biological agents and small molecule targeted drugs has provided more options for the treatment of rheumatoid arthritis patients, and the remission rate has also been further improved. Nonetheless, some patients still do not respond well to treatment. Biologics and small-molecule targeted drugs are expensive and impose a huge economic burden on patients and society. Moreover, these drugs do not act on the initial link of the disease, and it is difficult to control the disease in a targeted manner. Moreover, there are many adverse reactions such as bone marrow suppression, liver function damage, and increased risk of infection, which cause many patients to be unable to tolerate the treatment of these drugs.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a citrullinated antigen-modified peptide and its application, wherein the citrullinated antigen-modified peptide specifically binds to HLA-DRB1 related to rheumatoid arthritis, and inhibits the binding of the antigenic peptide to HLA-DRB1, thereby The activation of T cells is inhibited to achieve the purpose of treating rheumatoid arthritis.

The present invention provides a citrullinated antigen-modified peptide, the citrullinated antigen-modified peptide has the amino acid sequence shown in SEQ ID No. 1 or SEQ ID No. 2.

Preferably, myristic acid is attached to the nitrogen terminus of the citrullinated antigen-modified peptide.

The present invention also provides the application of the citrullinated antigen-modified peptide described in the above technical solution in the preparation of a medicament for preventing and treating rheumatoid arthritis.

Preferably, the medicament further includes a pharmaceutically acceptable carrier or adjuvant for the citrullinated antigen-modified peptide.

Preferably, the dosage forms of the medicine include tablets, pills, capsules, syrups, powders or solutions.

The present invention also provides the application of the citrullinated antigen-modified peptide described in the above technical solution in the preparation of a drug for inhibiting the recognition of antigen by T cells.

The present invention also provides the application of the citrullinated antigen-modified peptide described in the above technical solution in the preparation of a medicine for inhibiting the autoimmune response caused by T cells' recognition of the antigen.

The present invention also provides the application of the citrullinated antigen-modified peptide described in the above technical solution in the preparation of a medicine for blocking the binding of T cells and antigen peptides.

The present invention also provides the application of the citrullinated antigen-modified peptide described in the above technical scheme in the preparation of a medicine for inhibiting the function of HLA-DRB1 molecules.

The present invention provides citrullinated antigen-modified peptides and applications thereof, the citrullinated antigen-modified peptides have the amino acid sequence shown in SEQ ID No. 1 or SEQ ID No. 2, and the citrullinated antigen-modified peptides are related to rheumatoid The specific binding of arthritis-related HLA-DRB1 inhibits the binding of antigenic peptides to HLA-DRB1, thereby inhibiting the activation of T cells and achieving the purpose of treating rheumatoid arthritis.

Description of drawings

Fig. 1 is the screening of citrullinated antigen-modified peptides;

Figure 2 is a further verification of the effects of aVP3 and aVP5 on the secretion of IL-6 from PBMCs of patients with rheumatoid arthritis;

Fig. 3 is the inhibitory effect of aVP3 and aVP5 nasal mucosa administration on experimental arthritis;

Figure 4 shows that aVP3 and aVP5 inhibit the development of experimental arthritis by inhibiting Tfh.

Detailed ways

The present invention provides a citrullinated antigen-modified peptide, the citrullinated antigen-modified peptide has the amino acid sequence shown in SEQ ID No.1 or SEQ ID No.2, and the specific sequence is as follows:

SEQ ID No. 1: SAVAL-Cit-SSVPGVR;

SEQ ID No. 2: SAVRL-Cit-RSVPGVR.

In the present invention, the design idea of the citrullinated antigen-modified peptide is:

1. The ability of the citrullinated antigen-modified peptide to interact with HLA is enhanced or unchanged, at least not significantly weakened;

2. The ability of citrullinated antigen-modified peptide to interact with TCR that can recognize HLA-DRB1*04:01 was significantly weakened.

The citrullinated antigen-modified peptide binds to HLA-DRB1*04:01 in vivo, but cannot cause activation of corresponding autoreactive T cells, thereby alleviating the symptoms of rheumatoid arthritis and even achieving the effect of curing.

In the present invention, the citrullinated antigen-modified peptide is synthesized by solid-phase synthesis and purified by high-performance liquid chromatography by China Peptide Biochemical Co., Ltd. and Beijing Saibaisheng Gene Technology Co., Ltd.

In the present invention, myristic acid is preferably connected to the nitrogen end of the citrullinated antigen-modified peptide, and the specific sequence is as follows:

SEQ ID No. 3: Myr-SAVAL-Cit-SSVPGVR;

SEQ ID No. 4: Myr-SAVRL-Cit-RSVPGVR.

In the present invention, myristic acid is connected to the nitrogen end of the citrullinated antigen-modified peptide, and the myristic acid not only increases the water solubility and stability of the polypeptide, but also promotes the transport and presentation of the polypeptide into cells.

The present invention also provides the application of the citrullinated antigen-modified peptide described in the above technical solution in the preparation of a medicament for preventing and treating rheumatoid arthritis.

In the present invention, the medicament preferably further comprises a pharmaceutically acceptable carrier or adjuvant for the citrullinated antigen-modified peptide. The present invention does not specifically limit the types of the pharmaceutical carriers and adjuvants, and conventional ones can be used.

In the present invention, the dosage form of the drug preferably includes tablets, pills, capsules, syrups, powders or solutions. The present invention does not specifically limit the preparation method of the dosage form of the above-mentioned medicine, and the conventional preparation method of the dosage form can be used. The present invention does not specifically limit the content of the citrullinated antigen-modified peptide in the dosage form, and the content of the drug in the conventional dosage form can be used.

The present invention also provides the application of the citrullinated antigen-modified peptide described in the above technical solution in the preparation of a medicine for inhibiting the expression of HLA-DRB1 molecules.

The present invention also provides the application of the citrullinated antigen-modified peptide described in the above technical solution in the preparation of a drug for inhibiting the recognition of antigen by T cells.

The present invention also provides the application of the citrullinated antigen-modified peptide described in the above technical solution in the preparation of a medicine for inhibiting the autoimmune response caused by T cells' recognition of the antigen.

The present invention also provides the application of the citrullinated antigen-modified peptide described in the above technical solution in the preparation of a medicine for blocking the binding of T cells and antigen peptides.

The present invention also provides the application of the citrullinated antigen-modified peptide described in the above technical scheme in the preparation of a medicine for inhibiting the function of HLA-DRB1 molecules.

The citrullinated antigen-modified peptides and their applications of the present invention will be described in further detail below with reference to specific examples. The technical solutions of the present invention include but are not limited to the following examples.

Example 1

Screening of citrullinated antigen-modified peptides, a total of 12 citrullinated antigen-modified peptides were designed, which are:

aVP1 (SEQ ID No. 6: SAVEL-Cit-SSVPGVR);

aVP2 (SEQ ID No. 7: SAVDL-Cit-SSVPGVR);

aVP3 (SEQ ID No. 3);

aVP4 (SEQ ID No. 8: SAVGL-Cit-SSVPGVR);

aVP5 (SEQ ID No. 4);

aVP6 (SEQ ID No. 9: SAVRL-Cit-FSVPGVR);

aVP7 (SEQ ID No. 10: SAVRL-Cit-SSVEGVR);

aVP8 (SEQ ID No. 11: SAVRL-Cit-SSVKGVR);

aVP9 (SEQ ID No. 12: SAVRL-Cit-SSVWGVR);

aVP10 (SEQ ID No. 13: SAVEL-Cit-WSVPGVR);

aVP11 (SEQ ID No. 14: SAVDL-Cit-SSVRGVR);

aVP12 (SEQ ID No. 15: SAVAL-Cit-KSVFGVR);

Prototype peptide (SEQ ID No. 16: SAVRL-Cit-SSVPGVR).

According to Figure 1, under the stimulation of the prototype peptide, the level of IL-6 produced by peripheral blood PBMCs of patients with rheumatoid arthritis increased. However, under the stimulation of aVP2, aVP3, aVP4, aVP5 and aVP6 allosteric peptides, the level of IL-6 decreased significantly, especially the aVP3 and aVP5 peptides.

Citrullinated antigen-modified peptides aVP3 (SEQ ID No. 3) and aVP5 (SEQ ID No. 4), while synthesizing vimentin 66-78 prototype peptides (with myristic acid attached to the nitrogen terminus of SEQ ID No. 16) (wildvimentinpeptide , wVP). The above-mentioned polypeptides were synthesized by China Peptide Biochemical Co., Ltd. and Beijing Saibaisheng Gene Technology Co., Ltd. by solid-phase synthesis and purified by high-performance liquid chromatography. In order to facilitate the transport of the peptide into the cell, myristic acid was connected to the N-terminus of both the citrullinated antigen-modified peptide and the prototype peptide, as shown in Table 1. The results of mass spectrometry showed that the sequence of the polypeptide was correct and the purity was over 95%.

Table 1 Synthetic polypeptides and their sequences

Among them, Myr is myristic acid, S is serine, A is alanine, V is valine, R is arginine, L is leucine, Cit is citrulline, P is proline, G is glycine .

Example 2

Hyporesponsiveness of citrullinated antigen-modified peptides in peripheral blood T cells of patients with rheumatoid arthritis.

In an embodiment of the present invention, the reactivity of peripheral blood T cells of patients with rheumatoid arthritis to two citrullinated antigen-modified peptides aVP3 and aVP5 was studied, and the inflammatory cytokine IL-6 stimulated by allosteric peptides was detected. produce level changes.

Starting with 4 mL of each patient's blood, the blood was added to 4 mL of Ficoll-Paque PLUS in a 15 mL centrifuge tube centrifuged at 1200 rpm for 30 minutes. The corresponding mononuclear cell (PBMC) layers were extracted. Subsequently, cells were washed twice with 15 mL of 1×PBS, and they were centrifuged at 1200 rpm after each wash. Finally, the cell pellet was resuspended in RPMI1640 containing 10% fetal bovine serum and penicillin (100 U/mL), streptomycin (100 μg/mL), 25 mM/L HEPES and 2 mM L-glutamine (all obtained from Gibco BRL) middle.

The obtained PBMCs were seeded in 96-well circular plates (Costar) at a number of ² x 105 cells/well. Subsequently, citrullinated antigen-modified peptides were added at a concentration of 30 μg/mL. 10 μg/ml phytohemagglutinin (PHA) was used as a positive control for cell stimulation, while RPMI1640 alone was used as a control for basal cell growth. The culture system of each well was 200 μL.

The cells were cultured for 120 hours, then the supernatant from each well was removed and the concentration of the cytokine IL-6 was determined by using a specific ELISA kit (Daktronics).

At the same time, 200 μl of the whole blood of the patient was taken, and the DNA was extracted by using a blood DNA extraction kit (Tiangen), and sent to Beijing Siercheng Biotechnology Co., Ltd. to detect its HLA-DRB1 typing.

IL-6 levels regulated by individual peptide fragments in PBMCs of RA patients are shown in FIG. 2 . As observed in Figure 2, both aVP3 and aVP5 peptides significantly reduced IL-6 levels compared to wVP peptides, especially in rheumatoid arthritis patients with shared epitopes. IL-6 is one of the crucial cytokines in the inflammatory process of rheumatoid arthritis, thus this peptide is a potential therapeutic candidate for the treatment of this disease.

Example 3

The therapeutic effects of aVP3 and aVP5 allosteric peptides were evaluated in an animal model of bovine collagen II (CII)-induced arthritis.

In this example, in addition to wVP, aVP3 and aVP5, an irrelevant peptide (iP) was added. The peptide fragment of the unrelated peptide is the reverse sequence of wVP Myr-RVGPVSS-Cit-LRVAS (SEQ ID No. 5).

The experimental arthritis model in the present invention adopts the internationally recognized collagen-induced arthritis (CIA) model. The experimental animals were inbred DBA/1 mice. The mice used in this experiment were from Beijing Huafukang Biotechnology Co., Ltd. Mice were kept in the Animal Experimental Center (SPF grade) of Peking University People's Hospital. All experimental mice were 6-8 week old male mice. All procedures involving animals in the experiments were carried out in accordance with the regulations for the management of laboratory animals in my country.

The bovine type II collagen was purchased from Sigma, dissolved in anhydrous glacial acetic acid to make a solution of 3 mg/ml, then emulsified with an equal volume of Freund's complete adjuvant (Sigma), and injected intradermally at the base of the tail of each mouse 100μl emulsion, the injection dose of CII is 150μg/only. After 21 days, 3 mg/ml bovine type II collagen prepared by dissolving anhydrous glacial acetic acid was emulsified with an equal volume of incomplete Freund's adjuvant (Sigma) in the same way, and 50 μl emulsification was intradermally injected into the tail root of each mouse. The injection dose of CII was 75 μg/only. In this experiment, the arthritis model of DBA/1 mice was successfully established.

The swelling of the ankle joints and interphalangeal joints of the mice was observed every day after the second immunization. Arthritis was assessed on a scale of 0 to 16 points. Each of the four paws was scored as 0-4 points, of which 1-2 toes were swollen as 1 point, 3 or more toes were swollen as 2 points, and the soles and ankles were swollen 1 point each . The joint scores for the affected joints were summed up to a maximum score of 16.

After the second immunization, the mice were observed daily for disease. The affected mice were randomly divided into 5 groups, with 8-10 mice in each group. Allosteric peptides, prototype peptides, irrelevant peptides and water nasal instillation were given respectively on the day of onset. The doses of allosteric peptides, prototype peptides and irrelevant peptides were 10 μl/time (polypeptide dissolved in water with a concentration of 2.5 mg/ml), once a day for 15 consecutive days. The control group was given 10 μl of nasal drop water each time. Mice were observed daily and the number of arthritic joints was scored for arthritis using the arthritis assessment method previously described.

28 days after the onset of the disease, the mice were sacrificed, the draining lymph nodes were taken, and the cells were gently ground into a cell suspension. After filtration, 1×10 ⁶ (100 μl) was added to the flow tube, and 0.1 μl of the flow antibody FVD-eFluor506 was added, and incubated at room temperature for 20 minutes in the dark. . Add 1 ml of PBS, and centrifuge at 1500 rpm for 5 minutes after mixing. After discarding the supernatant, add the following flow-through antibodies to each tube: CD4-eFluor4501 μl, CD8-APC-EF7801 μl, CD25-PE-Cy 71 μl, and incubate at 4 degrees for 30 minutes. Add 1 ml of PBS, centrifuge at 1500 rpm for 5 minutes after mixing, discard the supernatant, add 1 ml of permeabilization solution (concentrate: diluent = 1:3) to each tube, and treat at room temperature for 30 minutes in the dark. Add more than 2 times the volume of 1× permeabilization solution and centrifuge at 1500 rpm for 5 minutes. After discarding the supernatant, 102 μl of Foxp3-PE-eFluor6 was added, and incubated at room temperature for 1 hour in the dark. Add 1 ml of PBS, and centrifuge at 1500 rpm for 5 minutes after mixing. Discard the supernatant and add 200 ul of PBS to resuspend.

Another 1×10 ⁶ (100 μl) was added to the flow tube, 3 ul of purified rat anti-mouse CXCR5 antibody was added, and incubated at 4 degrees for 1 hour. Add 1 ml of PBS to each tube, centrifuge at 1500 rpm for 5 minutes after mixing, discard the supernatant, add 2 μl of biotin-linked anti-rat IgG, and incubate at 4 degrees for 30 minutes. Add PBS 1ml, mix well and centrifuge at 1500rpm for 5 minutes, discard the supernatant and add the following anti-mouse flow-through antibodies: SA-APC 1μl, CD3-PEcy 72μl, CD4-Percp Cy5.52μl, CD8-FITC 2μl, CD44-APC Cy72μl and PD-1-PE 2μl, incubated at 4 degrees for 30 minutes in the dark. Add 1 ml of PBS, and centrifuge at 1500 rpm for 5 minutes after mixing. Discard the supernatant and add 200 μl of PBS to each tube to resuspend.

The flow-through antibodies and permeabilizing fluid used in the experiments were from eBioscience.

On the machine, CD4+CD25+Foxp3+Treg and CD4+CD44+PD-1+CXCR5+Tfh cells were detected.

Arthritis was induced in 52 male DBA/1 mice with bovine CII, and the results showed that 45 mice had different degrees of joint swelling within 1 to 3 weeks after the second immunization. On the day of the onset of arthritis, mice were randomly divided into 5 groups, treated with modified peptide, prototype peptide, unrelated peptide and water nasal instillation, respectively, and the number of joints with arthritis was scored by the aforementioned arthritis assessment method.

The results showed that there was no significant difference in the onset time and pre-treatment arthritis scores of the mice in each group. The joint scores of the aVP5 peptide-treated group became stable after 1 week of treatment, while the joint swelling of mice in other groups, especially the unrelated peptide group, gradually increased (Figure 3). After the experiment, lymph node cell flow detection analysis showed that compared with the water group, the Tfh of the modified peptide group was decreased (Fig. 4). Tfh is related to the production of antibodies by B cells. These results suggest that intranasal administration of citrullinated antigen-modified peptides can attenuate autoimmune inflammation in CIA mice, reduce antibody-producing Tfh cells from helper B cells, and inhibit the degree of arthritis. The nasal mucosa administration of the polypeptide has a therapeutic effect on rheumatoid arthritis.

It can be seen from the above examples that the citrullinated antigen-modified peptide specifically binds to HLA-DRB1 related to rheumatoid arthritis, inhibits the binding of the antigenic peptide to HLA-DRB1, thereby inhibiting the activation of T cells and achieving therapeutic effects. The purpose of rheumatoid arthritis.

The above are only the preferred embodiments of the present invention. It should be pointed out that for those skilled in the art, without departing from the principles of the present invention, several improvements and modifications can be made. It should be regarded as the protection scope of the present invention.

sequence listing

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Beijing University

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Claims (9)

1. A citrullinated antigen-modified peptide, characterized in that the citrullinated antigen-modified peptide has the amino acid sequence shown in SEQ ID No. 1 or SEQ ID No. 2. 2 . The citrullinated antigen-modified peptide according to claim 1 , wherein myristic acid is connected to the nitrogen terminus of the citrullinated antigen-modified peptide. 3 . 3. The application of the citrullinated antigen-modified peptide of claim 1 or 2 in the preparation of a medicament for preventing and treating rheumatoid arthritis. 4. The use according to claim 3, wherein the medicine further comprises a pharmaceutically acceptable carrier or adjuvant for the citrullinated antigen-modified peptide. 5. The application according to claim 3 or 4, characterized in that, the dosage form of the medicine comprises tablet, pill, capsule, syrup, powder or solution. 6. The application of the citrullinated antigen-modified peptide of claim 1 or 2 in the preparation of a drug for inhibiting T cell recognition of an antigen. 7. Use of the citrullinated antigen-modified peptide of claim 1 or 2 in the preparation of a medicament for inhibiting the autoimmune response caused by T cells recognizing the antigen. 8. The application of the citrullinated antigen-modified peptide of claim 1 or 2 in the preparation of a medicine for blocking the binding of T cells and antigenic peptides. 9. Use of the citrullinated antigen-modified peptide of claim 1 or 2 in the preparation of a medicament for inhibiting the function of HLA-DRB1 molecules.