An optimized citrullinated collagen type ii polypeptide and uses thereof

Abstract

The application discloses citrullinated collagen type II polypeptide and application thereof. The amino acid sequence of the polypeptide is Ste-QYMCitADQAAGGLR (SEQ ID NO. 1), wherein the N terminal of glutamine Q in the sequence is connected with octadecanoic acid Ste, and the fourth position in the sequence is modified by citrullination. The citrullinated collagen type II polypeptide has a long drug half-life, can effectively treat rheumatoid arthritis, and has small side effects.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to an optimized citrullinated type II collagen polypeptide and its applications. Background Technology

[0002] Rheumatoid arthritis (RA) is a chronic, inflammatory autoimmune disease characterized by synovitis, pannus formation, autoantibody production (rheumatoid factor and anti-citrullinated protein antibodies), and destruction of articular cartilage and bone, ultimately leading to joint deformities and loss of function. RA also increases the risk of other diseases, such as cardiovascular diseases like myocardial infarction and stroke. Epidemiological studies show that the global prevalence of RA is 0.5-1.0%. In China, the prevalence of RA is 0.28%, with a total affected population of approximately 5 million. The male-to-female ratio is approximately 1:4, and the prevalence increases with age, especially in middle-aged and older women. The incidence of disability and functional limitation in RA patients is as high as 77.6%, and this increases with the duration of the disease, placing a heavy burden on patients' families and society.

[0003] Treatment for rheumatoid arthritis (RA) includes nonsteroidal anti-inflammatory drugs (NSAIDs), disease-modifying antirheumatic drugs (DMARDs), and glucocorticoids, all of which require lifelong medication. NSAIDs can relieve pain and reduce symptoms, but they cannot intervene in joint damage. Traditional DMARDs such as methotrexate can slow the progression of arthritis, but they usually have a slow onset of action, and their mechanisms of action are not yet fully understood. Biological DMARDs such as TNF-α inhibitors and targeted synthetic DMARDs such as JAK inhibitors are expensive and increase the risk of opportunistic infections such as tuberculosis. Glucocorticoids can rapidly improve symptoms and disease, but long-term use can cause side effects such as osteoporosis.

[0004] In recent years, antigen-specific immunotherapy has gradually gained attention in autoimmune diseases. Unlike standard treatments that induce systemic immunosuppression, antigen-specific therapy only reprograms or deletes self-reactive T cells or B cells, inducing immune tolerance to self-antigens without impairing systemic immunity and host defense.

[0005] In view of this, the present invention is hereby proposed. Summary of the Invention

[0006] The purpose of this invention is to address the technical problems of existing RA treatment drugs, such as significant side effects and short drug retention time, by providing a new and optimized citrullinated type II collagen polypeptide that treats RA by inducing immune tolerance.

[0007] The technical solution of this invention is described in detail below:

[0008] In a first aspect, the present invention provides an optimized citrullinated type II collagen polypeptide with the amino acid sequence Ste-QYMCitADQAAGGLR (SEQ ID NO.1), wherein the N-terminus of glutamine Q is linked to octadecanoic acid Ste, and the fourth position of the sequence is modified by citrullinated Cit.

[0009] Secondly, the present invention provides the application of the above-mentioned optimized citrullinated type II collagen polypeptide in the preparation of a drug for treating rheumatoid arthritis.

[0010] Optionally or preferably, the drug is a nasal drop preparation.

[0011] Thirdly, the present invention provides a medicament for treating rheumatoid arthritis, which contains the above-described optimized citrullinated type II collagen polypeptide.

[0012] Optionally or preferably, the drug is a nasal drop preparation.

[0013] Optionally or preferably, the nasal drops contain: optimized citrullinated type II collagen peptides, dimethyl sulfoxide (DMSO), and water.

[0014] Compared with the prior art, the present invention has the following beneficial effects:

[0015] 1. Long half-life. The optimized citrullinated type II collagen polypeptide provided by this invention, after modification with octadecanoic acid, greatly prolongs the drug residence time, with a half-life of more than 80 hours.

[0016] 2. Good immunogenicity. The optimized citrullinated type II collagen polypeptide provided by this invention can activate peripheral blood mononuclear cells (PBMCs) of rheumatoid arthritis patients or healthy individuals carrying the HLA-DRB1 risk gene for rheumatoid arthritis in vitro, promote their secretion of the pro-inflammatory cytokine IL-6, and has strong antigenicity.

[0017] 3. Less irritating. This invention utilizes optimized citrullinated type II collagen peptides to prepare nasal drops, which are less irritating to patients than injectable formulations and are more acceptable to patients.

[0018] 4. Excellent therapeutic effect. The optimized citrullinated type II collagen peptide provided by this invention can effectively treat RA. Animal experiments show that intranasal administration of the optimized citrullinated type II collagen peptide of this invention can significantly alleviate arthritis manifestations in RA model mice and reduce histopathological scores. Flow cytometry results show that intranasal administration of the optimized citrullinated type II collagen peptide of this invention can reduce the proportion of Th1 and Th17 cells and increase the proportion of regulatory B cells (Breg); reduce the levels of inflammatory cytokines IL-6 and TNF-α and increase the level of anti-inflammatory cytokine IL-10. BCR sequencing shows that this peptide can correct the abnormal imbalance of VJ and VJD combinations and CDR3.

[0019] 5. Minimal side effects. Histopathological examination shows that the optimized citrullinated type II collagen peptide provided by this invention has no significant side effects during the treatment of RA.

[0020] In summary, this invention provides an effective new strategy for the treatment of RA, which can induce antigen-specific immune tolerance without causing significant side effects. Attached Figure Description

[0021] Figure 1 The HPLC purification results of the optimized citrullinated type II collagen peptide Ste-citAg are shown in the figure.

[0022] Figure 2 Mass spectrometry identification of the optimized citrullinated type II collagen peptide Ste-citAg.

[0023] Figure 3 The degradation of unmodified citrullinated type II collagen peptides and optimized citrullinated type II collagen peptides modified with Ste in the plasma of RA patients.

[0024] Figure 4 Figure showing the effect of the optimized citrullinated type II collagen peptide Ste-citAg on the level of cytokines secreted by PBMCs in RA patients.

[0025] Figure 5 The results of the optimized citrullinated type II collagen peptide Ste-citAg in an animal model of rheumatoid arthritis (RA) are shown in (a) for arthritis scores in different groups after administration, (b) for histopathological scores in different groups 28 days after administration. In both figures, circles represent the Ste-IP control peptide administration group and squares represent the Ste-citAg administration group; (c) for joint tissue staining results and bone tissue scans in different groups 28 days after administration.

[0026] Figure 6The figure shows the results of lymphocyte subset analysis in arthritis mice after treatment with the optimized citrullinated type II collagen peptide Ste-citAg.

[0027] Figure 7 The figure shows the changes in the B cell receptor repertoire of arthritic mice after treatment with the optimized citrullinated type II collagen peptide Ste-citAg.

[0028] Figure 8 Figure 1 shows the experimental results of tissue damage and side effects in arthritic mice after treatment with the optimized citrullinated type II collagen peptide Ste-citAg. Detailed Implementation

[0029] To enable those skilled in the art to better understand the present application, the present application will be clearly and completely described below with reference to embodiments and accompanying drawings. Obviously, the described embodiments are only some embodiments of the present application, and not all embodiments. Based on the embodiments of the present application, all other embodiments obtained by those of ordinary skill in the art without creative effort should fall within the scope of protection of the present application. Unless otherwise specified, the instruments and reagents used in the embodiments are all from commercial channels.

[0030] Example 1: Optimized preparation and half-life of citrullinated type II collagen peptide Ste-citAg

[0031] The amino acid sequence of the optimized citrullinated type II collagen polypeptide is shown in SEQ ID NO.1: Ste-QYMCitADQAAGGLR (abbreviated as Ste-citAg), in which the N-terminus of glutamine Q is linked to octadecanoic acid Ste, and the 4th position of the sequence is modified by citrullinated Cit.

[0032] The above sequence was synthesized by Shanghai Nuoyou Biotechnology Co., Ltd. using the Fmoc solid-phase synthesis method, purified by high-performance liquid chromatography (HPLC), identified by mass spectrometry (MS), and endotoxin was removed. The results of HPLC purification and MS identification are as follows: Figure 1 and Figure 2 As shown.

[0033] Unmodified peptide (citAg) and octadecanoic acid-modified peptide (Ste-citAg) were dissolved separately in PBS at a concentration of 200 μM. Subsequently, the peptides were mixed with human serum (from Peking University People's Hospital) at a ratio of 50% (v / v) in a total volume of 300 μL in a 1.5 mL EP tube. The reaction tube was then placed in a shaker and incubated at 37 °C with continuous shaking at 200 rpm. At each specified time point, 30 μL of the reaction mixture was taken from each replicate and diluted with twice the volume of cold acetone. After a precipitation step at -20 °C for 15 min, serum proteins were removed by centrifugation at 14,000 g for 10 min (4 °C). The resulting supernatant was transferred to a new tube and diluted with an equal volume of ACN / H₂O at 50% (v / v). The solution was then filtered through a 0.2 μm filter and analyzed by HPLC. The experiments were performed in triplicate.

[0034] The results showed that the peptide without octadecanoic acid (Ste) modification ( Figure 3 The half-life of citAg in the plasma of RA patients is only 30 minutes; while the half-life of the peptide modified with octadecanoic acid (Ste) is much shorter. Figure 3 Ste-citAg has a half-life of more than 80 hours in the plasma of RA patients.

[0035] Experiments show that when glutamine Q is linked to octadecanoic acid (Ste) at its N-terminus in the above polypeptide sequence, the destructive effect of proteolytic enzymes on the polypeptide is greatly reduced, effectively slowing down the degradation rate of the polypeptide and facilitating its transport into the cell.

[0036] Example 2: Immunogenicity of optimized citrullinated type II collagen peptides

[0037] Experimental materials:

[0038] Ste-citAg sequence: Ste-QYMCitADQAAGGLR (citrullinated at position 4);

[0039] Ste-Ag sequence: Ste-QYMRADQAAGGLR (uncitrullinated);

[0040] Ste-IP sequence: Ste-RLGGAAQDARMYQ (irrelevant peptide, used as a control peptide);

[0041] PHA: Phytohemagglutinin (positive control).

[0042] Experimental methods:

[0043] We randomly recruited 12 RA patients and performed HLA-DRB1 genotyping on them. A total of 5 RA patients carrying the rheumatoid susceptibility gene were recruited, carrying the HLA-DRB1*01:01 gene (2 cases) and the 04:05 gene (3 cases), respectively.

[0044] 4 mL of EDTA-anticoagulated blood from RA patients was collected, and PBMCs (peripheral blood mononuclear cells) were obtained by density gradient centrifugation and seeded into 96-well plates (2*10). 5 Cells were cultured at 37°C and 5% CO2 for 5 days. The cell culture supernatant was collected and the levels of IL-6, IFN-γ and IL-17A were detected by enzyme-linked immunosorbent assay (ELISA).

[0045] Whole blood was collected and the HLA-DRB1 genotype was identified by Beijing Jingzhun Biotechnology Co., Ltd. to determine the SE epitope status.

[0046] Experimental results:

[0047] We co-cultured the different peptides mentioned above with PBMCs individually and detected the levels of cytokines in the cell culture supernatant using ELISA. The results are as follows: Figure 4 Compared with the Ste-IP control group, Ste-citAg can stimulate human peripheral blood mononuclear cells to secrete IL-6, IFN-γ and IL-17A, and the effect is stronger than that of Ste-Ag, which proves that Ste-citAg has better immunogenicity.

[0048] Example 3: Therapeutic effect of optimized citrullinated type II collagen peptides in an RA animal model

[0049] The internationally recognized collagen-induced arthritis (CIA) animal model was used, and the experimental animals were inbred DBA / 1 mice.

[0050] Bovine collagen type II (bCII) and complete Freund's adjuvant (CFA) / incomplete Freund's adjuvant (IFA) were co-emulsified and injected intradermally into mice on days 0 and 21 to induce arthritis. On day 7 after the onset of the disease, mice were randomly assigned to groups according to their scores and were given 10 μL of Ste-citAg or Ste-IP control peptide (concentration of 2.5 mg / mL) once daily via nasal drops for 3 weeks. The disease status and joint phenotype scores of the mice were observed and recorded: no joint redness and swelling was scored as 0 points; swelling of 1-2 toe joints was scored as 1 point, and more than 2 joints were scored as 2 points; swelling of the paw was scored as 1 point; swelling of the carpal joint was scored as 1 point; the maximum score per paw was 4 points, and the maximum score per mouse was 16 points.

[0051] Mice were euthanized after treatment, and their hind paws were collected and soaked in a tissue fixative solution containing 4% paraformaldehyde. Paraffin sections were prepared using EDTA for decalcification and then stained with hematoxylin and eosin (HE) to assess the presence and severity of synovial hyperplasia, inflammatory cell infiltration, and bone destruction in the joints. The scores were as follows: normal (0 points); synovial hyperplasia and inflammatory cell infiltration (1 point); pannus formation and articular cartilage destruction (2 points); extensive articular cartilage destruction and subchondral bone erosion (3 points); and loss of joint integrity and ankylosis (4 points).

[0052] We used a small animal CT (Computer tomography) system to image the affected joints of mice and observe the bone erosion.

[0053] Experimental results are as follows Figure 5 As shown, starting from day 3 of treatment, the arthritis scores of mice in the Ste-citAg group began to decrease significantly compared with the Ste-IP control peptide treatment group; at the end of treatment (day 28), the arthritis scores of mice in the Ste-citAg group were significantly lower than those in the control group (6.80±1.19 vs. 10.80±1.04; P<0.05). Pathological results of the toe and ankle joints showed that most mice in the Ste-IP group had pannus formation, articular cartilage destruction, partial loss of joint integrity, and joint ankylosis; the Ste-citAg treatment group only showed synovial hyperplasia and inflammatory cell infiltration, with less obvious articular cartilage destruction, and its pathological score (1.15±0.35) was significantly lower than that of the Ste-IP treatment group (2.35±0.24, P=0.0178). Micro CT scans revealed very severe bone erosion in the Ste-IP treatment group, while the bone erosion was significantly reduced in the Ste-citAg treatment group.

[0054] Example 4: Optimized citrullinated type II collagen peptides regulate the proportion of immune cell subsets and cytokine levels in CIA mice.

[0055] Implementation method:

[0056] Mice were euthanized after treatment, and spleens, draining lymph nodes, and mesenteric lymph nodes were harvested. Single-cell suspensions were obtained by grinding the cells using a filter screen, and changes in T and B cell subsets such as Th1, Th2, Th17, Treg, Tfh, and Breg were detected by flow cytometry.

[0057] Blood was collected from the eyes of anesthetized mice after treatment. After 2 hours, serum was collected by centrifugation at 3500 rpm / 10 min. The levels of cytokines such as IL-6, TNF-α, IL-17A, and IFN-γ were detected by ELISA.

[0058] Experimental results are as follows Figure 6 As shown, lymphocyte subset analysis revealed that, compared with the Ste-IP treatment group (i.e., the control group), the Ste-citAg group mice had decreased Th1 (control group: 1.58±0.15%, Ste-citAg group: 1.25±0.16%) and Th17 (control group: 1.35±0.12%, Ste-citAg group: 1.12±0.06%), and increased Th2 (control group: 0.33±0.03%, Ste-citAg group: 0.55±0.10%; P=0.05) and B10 (control group: 10.60±0.58%, Ste-citAg group: 12.73±0.55%; P=0.02).

[0059] The cytokine detection results are shown in Table 1 below. Compared with the control peptide Ste-IP, Ste-citAg significantly reduced the level of the pro-inflammatory cytokine TNF-α (Ste-IP group: 129.2±33.45 pg / mL, Ste-citAg group: 45.12±26.56 pg / mL); the levels of pro-inflammatory cytokines IL-6 and IL-17A were also reduced. The level of IL-4 was significantly increased (Ste-IP group: 1.44±0.26 pg / mL, Ste-citAg: 4.26±1.25 pg / mL), and the level of the anti-inflammatory cytokine IL-10 was also increased to some extent.

[0060] Table 1. Results of Cytokine Detection

[0061]

[0062]

[0063] Example 5: Optimized citrullinated type II collagen peptides restore B-cell receptor repertoire balance

[0064] Implementation method:

[0065] Mice were euthanized after treatment, and spleens were harvested to isolate lymphocytes. Total RNA was extracted, and endogenous DNA was removed using DNase. One-stranded cDNA was synthesized using six-base random primers, followed by the preparation of a two-stranded cDNA synthesis reaction system. Multiplex PCR amplification was performed using BCR heavy chain primers. After purification, the PCR products underwent end repair, A-tailing, and ligation with sequencing adapters, and were then subjected to PCR amplification. The constructed library was quality-checked using an Agilent 2100 Bioanalyzer and then sequenced using an Illumina HiSeq XTMTen.

[0066] Sequencing data were compared with the sequences of the V, J, and D regions of this species in the IMGT (http: / / www.imgt.org / ) database to comprehensively examine the expression of the V, J, and D regions, as well as the VJ and VDJ combinations, in each sample; the diversity of CDR3 amino acid clonal types and the similarity between samples were assessed; and significant differences in V genes, J genes, VJ combinations, VDJ combinations, and CDR3 amino acid clonal types among groups were analyzed. All sequencing and analysis were performed by Qingdao Ouyi Biomedical Technology Co., Ltd.

[0067] Experimental results are as follows Figure 6 The results showed that, compared with the non-model group, 6 VJ combinations were significantly elevated and 7 VJ combinations were significantly decreased in the Ste-IP control group. Compared with the Ste-IP control group, after Ste-citAg treatment, 6 VJ combinations were significantly elevated and 5 VJ combinations were significantly decreased. Among them, the IGHV1S21_IGHJ2, IGHV6-5_IGHJ2, and IGHV9-2-1_IGHJ2 combinations were elevated after model establishment and decreased after treatment, which may be involved in the process of Ste-citAg treatment for CIA.

[0068] Compared to the untreated group, the Ste-IP control group showed a significant increase in 34 VDJ combinations and a significant decrease in 146 VDJ combinations; compared to the Ste-IP control group, the Ste-citAg treatment group showed a significant increase in 71 VDJ combinations and a significant decrease in 39 VDJ combinations. Among them, the combinations of IGHV1S17_IGHJ4_IGHD3-1, IGHV1-75_IGHJ1_IGHD1-1, IGHV1-19_IGHJ3_IGHD2-12, IGHV5-9_IGHJ4_IGHD5-4, IGHV2-9-2_IGHJ2_IGHD2-9, IGHV1S35_IGHJ1_IGHD3-3, IGHV1S134_IGHJ4_IGHD3-2, IGHV1S47_IGHJ4_IGHD3-2, IGHV9-3-1_IGHJ4_IGHD2-9, and IGHV9-3_IGHJ4_IGHD2-9 increased after CIA modeling and decreased after treatment, which may be involved in the process of Ste-citAg treatment for CIA.

[0069] In addition, we also compared changes in CDR3. The results showed that the CARDNWGFAYW, CARAGTGYAMDYW, CARDAGGSYAMDYW, CAGDNWDW, and CARDEDFDYW fragments increased after modeling and decreased after treatment, which may be involved in the process of Ste-citAg treatment for CIA.

[0070] Example 6 shows that the optimized citrullinated type II collagen peptide treatment does not cause significant tissue damage or side effects.

[0071] Experimental methods:

[0072] After treatment, the mice's body weight and heart weight were measured, and the mice's heart, liver, spleen, lungs, kidneys, stomach, small intestine, pancreas, and brain were collected for routine lesion analysis.

[0073] Experimental results are as follows Figure 7 The results showed that, compared with the Ste-IP control group, there was no significant difference in body weight and heart weight in CIA mice after Ste-citAg treatment, and no obvious lesions or damage to any organs, with no significant differences between groups.

[0074] These results indicate that Ste-citAg can induce antigen-specific immune tolerance, induce regulatory B cells, reduce effector T cells, and restore immune repertoire balance, while having no obvious side effects, providing a new strategy for the treatment of rheumatoid arthritis.

[0075] This article uses specific examples to illustrate the inventive concept in detail. The description of the above embodiments is only for the purpose of helping to understand the core idea of ​​the present invention. It should be noted that any obvious modifications, equivalent substitutions or other improvements made by those skilled in the art without departing from the inventive concept should be included within the protection scope of the present invention.

Claims

1. Use of an optimized citrullinated collagen type II polypeptide for the preparation of a medicament for the treatment of rheumatoid arthritis, characterized in that, The amino acid sequence is Ste-QYMCitADQAAGGLR (SEQ ID NO. 1), wherein the N-terminus of glutamine Q in the sequence is connected with octadecanoic acid Ste, and the 4th position in the sequence is citrullinated; and the drug is a nasal drop preparation.

2. Use according to claim 1, characterized in that, The components of the nasal drop preparation are: optimized citrullinated type II collagen polypeptide, dimethyl sulfoxide (DMSO) and water.

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