Search

KR-20260068070-A - Citrullinated SETDB1 polypeptide and diagnosis of rheumatoid arthritis using the same

KR20260068070AKR 20260068070 AKR20260068070 AKR 20260068070AKR-20260068070-A

Abstract

A method for diagnosing rheumatoid arthritis based on the level of an anti-citrullinized SETDB1 antibody and a kit used in said method are provided. Additionally, a polypeptide for diagnosing the level of said anti-citrullinized SETDB1 antibody and its use in the diagnosis of rheumatoid arthritis are provided.

Inventors

  • 거, 셩샹
  • 시아, 닝샤오
  • 웡, 린
  • 천, 쥬안
  • 리우, 웨이
  • 덩, 원
  • 허우, 왕헝
  • 송, 리우웨이
  • 치아오, 산
  • 장, 준

Assignees

  • 시아먼 유니버시티
  • 베이징 완타이 바이오로지컬 파마시 엔터프라이즈 코포레이션 리미티드
  • 시아먼 이노디엑스 바이오테크 컴퍼니 리미티드

Dates

Publication Date
20260513
Application Date
20240808
Priority Date
20230809

Claims (20)

  1. As an isolated polypeptide or a variant thereof, The above polypeptide consists of at least 11 consecutive amino acid residues of the SETDB1 protein MBD domain; At least one of the above consecutive amino acid residues is an arginine residue, and at least one of the above arginine residues is citrullized; The above variant differs from the polypeptide derived therefrom only by the substitution of one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, or 9) amino acid residues (e.g., conservative or non-conservative substitution) and retains the biological function of the polypeptide derived therefrom (e.g., activity recognized and bound by an anti-citrullinated SETDB1 antibody), Isolated polypeptide or a variant thereof.
  2. In claim 1, the isolated polypeptide comprises at least one arginine residue among amino acid positions corresponding to the following positions of the SETDB1 protein MBD domain: 2, 16, 17, 21, 22, 23, 26, 43 and/or 50, and at least one of the arginine residues is citrullinated; Preferably, the isolated polypeptide comprises at least one arginine residue among amino acid positions corresponding to the following positions of the SETDB1 protein MBD domain: 16, 17, 21, 22, 23 and/or 26, isolated polypeptide or a variant thereof.
  3. In paragraph 1 or 2, The isolated polypeptide comprises at least an amino acid residue corresponding to positions 16 to 26 of the SETDB1 protein MBD domain; at least one of the consecutive amino acid residues at positions 16 to 26 is an arginine residue, and at least one of the arginine residues is citrullinated; Preferably, the isolated polypeptide comprises at least one arginine residue (e.g., 1, 2, 3, 4, 5, or all 6) at amino acid positions corresponding to the following positions of the SETDB1 protein MBD domain: 16, 17, 21, 22, 23, and/or 26; and at least one of the arginine residues (e.g., 1, 2, 3, 4, 5, or all 6) is citrullinated; Preferably, the isolated polypeptide comprises at least the sequence indicated by SEQ ID NO. 8; Preferably, the isolated polypeptide further comprises at least one arginine residue (e.g., 1, 2, or all 3) of amino acid positions corresponding to the following positions of the SETDB1 protein MBD domain: 2, 43, and/or 50, wherein at least one of the arginine residues (e.g., 1, 2, or all 3) is citrullinated, the isolated polypeptide or a variant thereof.
  4. In any one of claims 1 to 3, the isolated polypeptide comprises a sequence of amino acid residues selected from amino acid residues at the next position of the SETDB1 protein MBD domain: 5 to 27, 8 to 27, 12 to 27, 14 to 27, 16 to 27, 16 to 26, 1 to 27, or 5 to 32; at least one of the sequence of amino acid residues is an arginine residue, and at least one of the arginine residues is citrullinated; Preferably, the isolated polypeptide comprises at least one arginine residue (e.g., 1, 2, 3, 4, 5, or all 6) at an amino acid position corresponding to the following positions of the SETDB1 protein MBD domain: 16, 17, 21, 22, 23, and/or 26, wherein at least one of the arginine residues (e.g., 1, 2, 3, 4, 5, or all 6) is citrullinated; Preferably, the isolated polypeptide further comprises an arginine residue at an amino acid position corresponding to position 2 of the SETDB1 protein MBD domain, and the isolated polypeptide or a variant thereof, wherein the arginine residue can be optionally citrullinated.
  5. In any one of claims 1 to 4, the isolated polypeptide comprises 72 or fewer consecutive amino acid residues of the SETDB1 protein MBD domain (e.g., 70 or fewer, 65 or fewer, 60 or fewer, 55 or fewer, 50 or fewer, 45 or fewer, 40 or fewer, 35 or fewer, or 30 or fewer), isolated polypeptide or a variant thereof.
  6. In any one of claims 1 to 5, the variant does not include amino acid substitutions at amino acid positions corresponding to the following positions of the SETDB1 protein MBD domain: 16, 17, 21, 22, 23, and 26; Preferably, the variant is an isolated polypeptide or a variant thereof that does not further comprise amino acid substitutions at amino acid positions corresponding to the following positions of the SETDB1 protein MBD domain: 2, 43 and/or 50.
  7. In any one of claims 1 to 6, the isolated polypeptide comprises at least amino acid residues at positions 2 to 26 of the SETDB1 protein MBD domain; Preferably, the isolated polypeptide comprises arginine residues at amino acid positions corresponding to the following positions of the SETDB1 protein MBD domain: 2, 16, 17, 21, 22, 23, and 26, and at least one of the arginine residues (e.g., 1, 2, 3, 4, 5, 6, or all 7) is citrullinated; Preferably, an isolated polypeptide or a variant thereof in which all arginine residues at amino acid positions corresponding to the following positions of the SETDB1 protein MBD domain: 2, 16, 17, 21, 22, 23, and 26 are citrullinated.
  8. In any one of claims 1 to 7, the SETDB1 protein MBD domain is an isolated polypeptide or a variant thereof having the sequence indicated by SEQ ID NO. 1.
  9. In any one of claims 1 to 8, the isolated polypeptide has a sequence represented by any one of SEQ ID NOs 3 to 10; Preferably, the variant has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with respect to the sequence indicated by any one of SEQ ID NOs 3 to 10, and is an isolated polypeptide or a variant thereof having completely identical positions and numbers of citrullines.
  10. In any one of claims 1 to 9, the separated polypeptide or its variant has a modifier that can be attached to the surface of a stationary support or attached to the stationary support; Preferably, the modifier is biotin or avidin; Preferably, the stationary support is an isolated polypeptide or a variant thereof selected from magnetic beads or microtiter plates (e.g., microplates or ELISA plates).
  11. In any one of claims 1 to 9, the isolated polypeptide or its variant has a detectable label; Preferably, the detectable label is an isolated polypeptide or a variant thereof selected from an enzyme (e.g., horseradish peroxidase or alkaline phosphatase), a chemiluminescent reagent (e.g., acridinium ester compound), a fluorescent dye, or biotin.
  12. An isolated nucleic acid molecule comprising a nucleotide sequence encoding an isolated polypeptide or a variant thereof according to any one of claims 1 to 9.
  13. A vector containing isolated nucleic acid molecules according to paragraph 12.
  14. A host cell comprising an isolated nucleic acid molecule according to paragraph 12 or a vector according to paragraph 13.
  15. A kit comprising a separated polypeptide or a variant thereof according to any one of claims 1 to 9.
  16. In claim 15, the above kit comprises a capture reagent selected from a separated polypeptide or a variant thereof according to any one of claims 1 to 9; Preferably, the kit further includes instructions on a method of using the isolated polypeptide or a variant thereof as a capture reagent to measure the level of an antibody specific to citrullinated SETDB1 protein in a sample, and optionally includes instructions for determining whether a subject has rheumatoid arthritis; Preferably, the capture reagent is selected from the isolated polypeptide or a variant thereof according to claim 10; Preferably, the object is a kit that is a mammal such as a human.
  17. In Paragraph 16, The above kit further comprises a detection reagent, said detection reagent is selected from secondary antibodies having a detectable label; Preferably, the detectable label is selected from an enzyme (e.g., horseradish peroxidase or alkaline phosphatase), a chemiluminescent reagent (e.g., acridinium ester compound), a fluorescent dye, or biotin; Preferably, the secondary antibody is specific to the species from which the antibody to be detected originated (e.g., human); Preferably, the secondary antibody is an anti-immunoglobulin antibody; Preferably, the anti-immunoglobulin antibody is selected from anti-IgG antibody, anti-IgM antibody and anti-IgA antibody, in the kit.
  18. In claim 16, the kit further comprises a detection reagent, wherein the detection reagent is selected from the isolated polypeptide or a variant thereof according to claim 11.
  19. Use of a reagent capable of detecting antibodies specific to citrullinated SETDB1 protein in the manufacture of a kit for determining whether a subject has rheumatoid arthritis; Preferably, the reagent is a reagent capable of detecting antibodies specific to the citrullinated SETDB1 protein by immunological analysis; preferably, the immunological analysis is selected from enzyme immunoassay (e.g., ELISA), chemiluminescent immunoassay, fluorescence immunoassay, or radioimmunoassay; Preferably, the subject is a human; Preferably, the reagent is used to measure the level of antibodies specific to citrullinated SETDB1 protein in a sample derived from the subject; Preferably, the sample is a blood sample (e.g., whole blood, plasma, or serum) or a tissue fluid sample (e.g., synovial fluid).
  20. In claim 19, the reagent capable of detecting an antibody specific to the citrullinated SETDB1 protein comprises a capture reagent, said capture reagent is selected from isolated polypeptides or variants thereof according to any one of claims 1 to 7; Preferably, the capture reagent is selected from the isolated polypeptide or a variant thereof according to claim 10.

Description

Citrullinated SETDB1 polypeptide and diagnosis of rheumatoid arthritis using the same Cross-reference of related applications This application is based on Chinese application No. CN 202310997528.X (filed August 9, 2023) and claims priority thereof. The disclosure of said Chinese application is incorporated herein by reference in its entirety. Technology field The present invention relates to immunological detection, and more particularly to the field of immunological diagnosis. More specifically, the present invention provides a method for diagnosing rheumatoid arthritis based on the level of an anti-citrullinized SETDB1 antibody and a kit for use in said method. Furthermore, the present invention provides a citrullinized SETDB1 polypeptide used for said diagnosis and its use in the diagnosis of rheumatoid arthritis. Citrullination is a type of post-translational modification of proteins. It is a post-translational modification that occurs under the catalyzeance of Ca + -dependent peptidylarginine deiminase (PAD) (Wang, R., et al., Gut stem cell necroptosis by genome instability triggers bowel inflammation. Nature, 2020. 580(7803): p. 386-390.). Because citrullination fundamentally alters the structure and function of proteins, an abnormal increase in citrullination within the body can lead to abnormally elevated levels of autoantibodies against citrullinated residue epitopes, potentially resulting in disease. The association between citrullination and diseases such as rheumatoid arthritis, systemic lupus erythematosus, and tumors has been reported. Among these, citrullination has been the most frequently studied in rheumatoid arthritis. Rheumatoid arthritis is a systemic autoimmune disease of unknown etiology that affects 0.5–1.0% of adults worldwide. It generally affects the synovial membrane of the joints and commonly manifests as joint pain and swelling. In severe cases, it can lead to joint deformity and even disability (MCINNES I B, SCHETT G. The pathogenesis of rheumatoid arthritis [J]. N Engl J Med, 2011, 365(23): 2205-2219.). According to research, arthritis is one of 33 medical causes of movement-related loss in American adults (HOOTMAN J M, HELMICK C G, BRADY T J. A public health approach to addressing arthritis in older adults: the most common cause of disability [J]. Am J Public Health, 2012, 102(3): 426-433.). Therefore, rheumatoid arthritis can no longer be regarded merely as a common medical condition, but is also a public health issue requiring significant attention. Furthermore, some health economists have conducted studies on the economic burden caused by rheumatoid arthritis. As a result, it has been shown that reducing risk factors or intervening in the early stages of the disease can significantly reduce costs compared to subsequent hospitalization and surgery (LANES S F, LANZA L L, RADENSKY P W, et al. Resource utilization and cost of care for rheumatoid arthritis and osteoarthritis in a managed care setting: the importance of drug and surgery costs[J]. Arthritis Rheum, 1997, 40(8): 1475-1481.). In conclusion, "early detection, early diagnosis, and early treatment" are required to control the onset and progression of rheumatoid arthritis. Studies have shown that the formation of autoantibodies is one of the characteristics of rheumatoid arthritis that distinguishes it from other inflammatory arthritis, such as psoriatic arthritis, reactive arthritis, and osteoarthritis (KOURILOVITCH M, GALARZA-MALDONADO C, ORTIZ-PRADO E. Diagnosis and classification of rheumatoid arthritis[J]. J Autoimmun, 2014, 48-49(26-30).). Currently, the diagnosis of rheumatoid arthritis in laboratory tests relies primarily on rheumatoid factors (RFs) and anti-citrullinizing protein antibodies (ACPAs). Rheumatoid factors are produced in the body in response to stimulation by persistent infection with exogenous pathogens such as viruses and mycoplasma. Therefore, positive results can be detected in patients with other immune diseases, patients with chronic infections, and even 5% of healthy individuals, and it is generally used as an indicator for early disease screening. Among anti-citrullinated protein antibodies, anti-cyclic citrullinated peptide (CCP) antibodies have established themselves as the most widely used clinical diagnostic markers due to their high specificity. Studies show that while this marker has a specificity of up to 90%, its sensitivity is only about 70% (Wegner, N., Lundberg, K., Kinloch, A., Fisher, B., Malmstrφm, V., Feldmann, M., & Venables, P. J. (2010). Autoimmunity to specific citrullinated proteins gives the first clues to the etiology of rheumatoid arthritis. Immunological reviews, 233(1), 34-54. https://doi.org/10.1111/j.0105-2896.2009.00850.x). Due to the problem of low sensitivity, misdiagnosis in patients occurs frequently, and it is urgent to identify new diagnostic markers for rheumatoid arthritis. In addition, CCP is an artificially designed cyclic citrulline peptide that can o