Citrullinated setdb1 polypeptides and their use in the diagnosis of rheumatoid arthritis

Abstract

The present invention relates to the field of immunological detection, in particular immunological diagnostics. Specifically, the present invention provides methods for diagnosing rheumatoid arthritis based on the level of anti-citrullinated SETDB1 antibodies, as well as kits for use in said methods. The present invention also provides polypeptides of citrullinated SETDB1 for use in the aforementioned diagnosis and their use for diagnosing rheumatoid arthritis.

Description

Technical Field

[0001] This invention relates to immunological detection, particularly to the field of immunological diagnostics. Specifically, this invention provides a method for diagnosing rheumatoid arthritis based on anti-citrullinated SETDB1 antibody levels, and a kit for using said method. This invention also provides a citrullinated SETDB1 peptide for the above-mentioned diagnosis and its use in the diagnosis of rheumatoid arthritis. Background Technology

[0002] Citrullination is a type of post-translational modification of proteins. It is a post-translational modification that occurs under the catalysis of a Ca+-dependent peptidylarginine deiminase (PAD) (Wang, R., et al., Gut stem cell necroptosis by genome instability triggers bowelinflammation. Nature, 2020. 580(7803): p.386-390.). Because citrullination fundamentally alters protein structure and function, an abnormal increase in citrullination in the body can lead to an abnormally high level of autoantibodies against citrullinated residue epitopes, resulting in disease. Diseases such as rheumatoid arthritis, systemic lupus erythematosus, and tumors have all been reported to be related to citrullination.

[0003] Citrullination is the most frequently studied finding in rheumatoid arthritis. Rheumatoid arthritis is a systemic autoimmune disease of unknown etiology, affecting 0.5-1.0% of adults worldwide. It generally affects the synovium of joints and is often manifested as joint pain and swelling. In severe cases, it can lead to joint deformities and even disability (MCINNES IB, SCHETT G. The pathogenesis of rheumatoid arthritis[J]. N Engl J Med, 2011, 365(23):2205-2219.). Studies have found that two of the 33 medical causes of motor-related functional loss in American adults are caused by arthritis (HOOTMAN JM, HELMICK CG, BRADY T JA. 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 simply regarded as an ordinary medical disease, but rather as a public health issue that needs attention. In addition, some health economists have conducted relevant studies on the economic burden caused by rheumatoid arthritis. The results show that the costs incurred by reducing risk factors or intervening in the disease as early as possible are far lower than the costs incurred by later hospitalization and surgery (LANES SF, LANZA LL, RADENSKY PW, 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, controlling the occurrence and development of rheumatoid arthritis requires "early detection, early diagnosis, and early treatment".

[0004] Studies have shown that the formation of autoantibodies is one of the characteristics that distinguishes rheumatoid arthritis 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, in laboratory tests, the diagnosis of rheumatoid arthritis mainly relies on rheumatoid factor (RFs) and anti-citrullinated protein antibodies (ACPA). Rheumatoid factor is produced by the body due to the stimulation of persistent infection by foreign pathogens such as viruses and mycoplasma. Therefore, positive results can also be detected in other immune diseases, patients with chronic infections, and 5% of healthy individuals, and it is generally used as an indicator for early disease screening. Among anti-citrullinated protein antibodies, anti-cyclic citrullinated polypeptide (CCP) antibodies have become the most commonly used clinical diagnostic marker due to their high specificity. Studies have shown that this biomarker has a specificity of up to 90%, but a sensitivity of only about 70% (Wegner, N., Lundberg, K., Kinloch, A., Fisher, B.). V., Feldmann, M., & Venables, PJ (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 its low sensitivity, rheumatoid arthritis is often missed, so there is an urgent need to screen for new diagnostic biomarkers. Furthermore, CCP is an artificially designed cyclic citrullinated peptide, which can only be used for the clinical diagnosis of rheumatoid arthritis and is not very useful for exploring the disease mechanism.

[0005] In summary, citrullination is involved in the development and progression of rheumatoid arthritis (RA). Therefore, naturally occurring citrulline biomarkers in RA patients are expected to become high-performance biomarkers for diagnosis and mechanism exploration. Screening for naturally occurring high-level citrulline biomarkers in patients can not only solve the problem of the current lack of single clinical biomarkers, but also hopefully find key factors in the pathogenesis of RA. Summary of the Invention

[0006] Histone-lysine N-methyltransferase (SETDB1) belongs to the speckle repressor 39 (SUV39) protein family and is one of the lysine methyltransferases (PKMTs) containing the SET domain involved in epigenetic regulation. It enables the trimethylation of histone H3 at the ninth lysine residue (H3K9me3). Current research mainly suggests that SETDB1 is closely related to cancer. In many cancers, SETDB1 expression levels tend to increase, influencing cancer development and progression, such as colorectal cancer, liver cancer, gastric cancer, and lung cancer. SETDB1 can serve as a combination target for tumor therapy. For example, inhibiting SETDB1 can enhance the sensitivity of cetuximab in colorectal cancer, overcoming its resistance. In melanoma cells with high SETDB1 expression, treatment with SETDB1 inhibitors in combination with BAKR and MEK inhibitors can effectively kill cells resistant to targeted therapy.

[0007] The inventors of this application innovatively screened serum samples from rheumatoid arthritis (RA) patients for citrulline biomarkers by constructing chemical derivatives, and for the first time discovered a citrullinated SETDB1 epitope polypeptide. This epitope polypeptide exhibits high levels of autoantibodies in RA patients, demonstrating its diagnostic value in rheumatoid arthritis. Therefore, the inventors provide a RA diagnostic method based on the detection of anti-citrullinated SETDB1 antibody levels, as well as a citrullinated SETDB1 polypeptide for use in this method, which can effectively reduce the missed diagnosis rate of early-stage RA patients and may even outperform current clinical diagnostic biomarkers.

[0008] Isolated polypeptides or their variants

[0009] In a first aspect, the present invention provides an isolated polypeptide or a variant thereof, wherein the polypeptide consists of at least 11 consecutive amino acid residues of the MBD domain of the SETDB1 protein;

[0010] Wherein, at least one of the continuous amino acid residues is an arginine residue, and at least one of the arginine residues is citrullinated;

[0011] The variant differs from its source polypeptide only in 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; e.g., conservative substitution), and retains the biological function of its source polypeptide (e.g., the activity of being recognized and bound by anti-citrullinated SETDB1 antibody).

[0012] In this document, the biological functions of the polypeptides or variants thereof of the present invention include, but are not limited to, the activity of being recognized and bound as epitope peptides by anti-citrullinated antibodies (e.g., anti-citrullinated SETDB1 antibodies).

[0013] In this document, when referring to the amino acid position of the MBD domain of the SETDB1 protein, the sequence shown in SEQ ID NO:1 is used for description. For example, the statement "the second amino acid residue of the MBD domain of the SETDB1 protein" refers to the second amino acid residue and its corresponding position in the sequence shown in SEQ ID NO:1. The corresponding position refers to the position in the sequence to be compared that is identical to the specific amino acid position in SEQ ID NO:1 when the sequence to be compared is optimally aligned with SEQ ID NO:1 (i.e., to obtain the highest percentage of identity).

[0014] In some embodiments, all arginine residues in the continuous amino acid residues are citrullinated (i.e., citrulline).

[0015] In some embodiments, the isolated polypeptide has at least one (e.g., position 1, 2, 3, 4, 5, 6, 7, 8, or all 9) arginine residues at the following amino acid positions corresponding to the MBD domain of the SETDB1 protein: 2, 16, 17, 21, 22, 23, 26, 43, and / or 50; and at least one of the arginine residues (e.g., 1, 2, 3, 4, 5, 6, 7, 8, or all 9) is citrullinated.

[0016] In some embodiments, the isolated polypeptide contains at least amino acid residues 16-26 of the MBD domain of the SETDB1 protein; wherein at least one of the consecutive amino acid residues 16-26 is an arginine residue, and at least one of the arginine residues is citrullinated. In some embodiments, all the arginine residues in the consecutive amino acid residues 16-26 are citrullinated.

[0017] In some embodiments, the isolated polypeptide has at least one (e.g., position 1, 2, 3, 4, 5, or all 6) arginine residues at the following amino acid positions corresponding to the MBD domain of the SETDB1 protein: 16, 17, 21, 22, 23, and / or 26; and at least one (e.g., one, two, three, four, five, or all 6) of the arginine residues is citrullinated. In some embodiments, all of the arginine residues are citrullinated.

[0018] In some embodiments, the isolated polypeptide contains the sequence shown in SEQ ID NO:8 at positions 16-26 corresponding to the MBD domain of the SETDB1 protein.

[0019] In some embodiments, the isolated polypeptide further comprises at least one (e.g., one, two, or all three) arginine residues at the following amino acid positions corresponding to the MBD domain of the SETDB1 protein: 2, 43, and / or 50; and at least one (e.g., one, two, or all three) of the arginine residues is citrullinated.

[0020] In some embodiments, the isolated polypeptide consists of a series of amino acid residues selected from the following: amino acid residues at positions 5-27, 8-27, 12-27, 14-27, 16-27, 16-26, 1-27, or 5-32 of the MBD domain of the SETDB1 protein; wherein at least one of the series of amino acid residues is an arginine residue, and at least one of the arginine residues is citrullinated. In some embodiments, all the arginine residues in the series of amino acid residues are citrullinated.

[0021] In some embodiments, the isolated polypeptide has at least one (e.g., position 1, 2, 3, 4, 5, or all 6) arginine residues at the following amino acid positions corresponding to the MBD domain of the SETDB1 protein: 16, 17, 21, 22, 23, and / or 26; and at least one (e.g., one, two, three, four, five, or all 6) of the arginine residues is citrullinated.

[0022] In some embodiments, the isolated polypeptide further comprises an arginine residue at position 2 of the MBD domain of the SETDB1 protein, which is optionally citrullinated.

[0023] In some embodiments, the variants described in this invention have the exact same citrulline characteristics as the polypeptides from which they are derived, i.e., the same number and position of citrulline.

[0024] In some embodiments, the variant does not include amino acid substitutions at the following positions corresponding to the MBD domain of the SETDB1 protein, compared to its derived polypeptide: 16, 17, 21, 22, 23, and 26. That is, the variant is identical to its derived polypeptide at amino acid positions 16, 17, 21, 22, 23, and 26 of the MBD domain of the SETDB1 protein. In some embodiments, the variant further does not include amino acid substitutions at positions 2, 43, and / or 50 of the MBD domain of the SETDB1 protein. That is, the variant is further identical to its derived polypeptide at amino acid positions 2, 43, and / or 50 of the MBD domain of the SETDB1 protein.

[0025] In some embodiments, the variants of the present invention differ from the polypeptide from which they are derived only in the substitution of one, two, three, or four amino acid residues (e.g., conservative or non-conservative substitution; e.g., conservative substitution), and retain the biological function of the polypeptide from which it is derived (e.g., the activity of being recognized and bound by anti-citrullinated SETDB1 antibodies).

[0026] In some embodiments, the polypeptides and variants thereof of the present invention are arginine residues at amino acid positions 16, 17, 21, 22, 23 and 26 corresponding to the MBD domain of the SETDB1 protein, and at least one (e.g., one, two, three, four, five or all six) of the arginine residues are citrullinated.

[0027] In some embodiments, the polypeptides and variants of the present invention contain citrullinated arginine (i.e., citrulline) at amino acid positions 16, 17, 21, 22, 23 and 26 corresponding to the MBD domain of the SETDB1 protein.

[0028] In some embodiments, the above-mentioned polypeptide and its variants further have an arginine residue at position 2 of the MBD domain of the SETDB1 protein, which is optionally citrullinated.

[0029] In some embodiments, the above-described polypeptide and its variants further have an arginine residue at the amino acid position corresponding to position 43 of the MBD domain of the SETDB1 protein, which is optionally citrullinated.

[0030] In some embodiments, the above-described polypeptide and its variants further have an arginine residue at the amino acid position corresponding to position 50 of the MBD domain of the SETDB1 protein, which is optionally citrullinated.

[0031] In some embodiments, the isolated polypeptide comprises no more than 72 SETDB1 MBD domains (e.g., no more than 70, no more than 69, no more than 68, no more than 67, no more than 66, no more than 65, no more than 64, no more than 63, no more than 62, no more than 61, no more than 60, no more than 59, no more than 58, no more than 57, no more than 56, no more than 55, no more than 54, no more than 53, no more than 52, no more than 51, no more than 50, no more than 72, no more than 53, no more than 52, no more than 51, no more than 50, no more than 7 ... Composed of a continuous amino acid residue of more than 49, no more than 48, no more than 47, no more than 46, no more than 45, no more than 44, no more than 43, no more than 42, no more than 41, no more than 40, no more than 39, no more than 38, no more than 37, no more than 36, no more than 35, no more than 34, no more than 33, no more than 32, no more than 31, no more than 30, no more than 29, or no more than 28.

[0032] In some embodiments, the isolated polypeptide consists of 11 to 30 (e.g., 11 to 28) consecutive amino acid residues of the MBD domain of the SETDB1 protein. In some embodiments, the isolated polypeptide consists of 11, 12, 14, 16, 20, 23, 27, or 28 consecutive amino acid residues of the MBD domain of the SETDB1 protein.

[0033] In some embodiments, the polypeptides and variants thereof of the present invention contain at least amino acid residues 2-26 of the MBD domain of the SETDB1 protein.

[0034] In some embodiments, the isolated polypeptide has arginine residues at amino acid positions 2, 16, 17, 21, 22, 23, and 26 corresponding to the MBD domain of the SETDB1 protein, and at least one of the arginine residues (e.g., one, two, three, four, five, six, or all seven) is citrullinated.

[0035] In some embodiments, the isolated polypeptide is citrullinated at positions 2, 16, 17, 21, 22, 23 and 26 corresponding to the MBD domain of the SETDB1 protein.

[0036] In some embodiments, the variant does not include, compared to its derived polypeptide, amino acid substitutions at the following positions corresponding to the MBD domain of the SETDB1 protein: 2, 16, 17, 21, 22, 23, and 26. That is, the variant is identical to its derived polypeptide at the amino acid positions corresponding to 2, 16, 17, 21, 22, 23, and 26 of the MBD domain of the SETDB1 protein.

[0037] In some embodiments, the isolated polypeptide and its variants consist of no more than 50 (e.g., no more than 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, or 30) consecutive amino acid residues of the SETDB1 MBD domain.

[0038] In some embodiments, the isolated polypeptide and its variants consist of 25 to 30 consecutive amino acid residues of the MBD domain of the SETDB1 protein.

[0039] In some embodiments, the MBD domain of the SETDB1 protein has a sequence as shown in SEQ ID NO:1 or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with it.

[0040] In some embodiments, the isolated polypeptide has a sequence as shown in any one of SEQ ID NOs:3-10. In some embodiments, 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 the sequence shown in any one of SEQ ID NOs:3-10, and has exactly the same citrulline positions and quantities.

[0041] Derivatized peptides or their variants

[0042] The polypeptides or variants thereof of the present invention can be derivatized, for example, by linking other functional units, such as detectable markers, protein tags, solid supports, etc.

[0043] In one embodiment, the isolated polypeptide or its variant is attached to the surface of a solid support or has a modifying group that can be attached to the solid support. In some embodiments, the N-terminus of the isolated polypeptide or its variant is attached to the surface of the solid support or has a modifying group that can be attached to the solid support. In some embodiments, the modifying group is biotin or avidin. In some embodiments, the solid support is selected from magnetic beads or microtiter plates (e.g., microplates or ELISA plates).

[0044] In another embodiment, the isolated polypeptide or a variant thereof carries a detectable label. In some embodiments, the detectable label is selected from enzymes (e.g., horseradish peroxidase or alkaline phosphatase), chemiluminescent reagents (e.g., acridine esters), fluorescent dyes, or biotin.

[0045] Preparation of peptides

[0046] The polypeptides or variants thereof of the present invention are not limited by their manner of production; for example, they can be produced by genetic engineering methods (recombinant technology) or by chemical synthesis methods.

[0047] In another aspect, the present invention provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide or a variant thereof of the present invention.

[0048] In another aspect, the present invention also provides a vector comprising the isolated nucleic acid molecules as described above. The vector of the present invention can be a cloning vector or an expression vector. In a preferred embodiment, the vector of the present invention is, for example, a plasmid, a granulocyte, a bacteriophage, a Cosmid, etc.

[0049] In another aspect, the present invention also provides host cells comprising the isolated nucleic acid molecules or vectors of the present invention. Such host cells include, but are not limited to, prokaryotic cells such as Escherichia coli cells, and eukaryotic cells such as yeast cells, insect cells, plant cells, and animal cells (such as mammalian cells, such as mouse cells, human cells, etc.).

[0050] In another aspect, the present invention also provides a method for preparing the polypeptide or variant thereof of the present invention, comprising culturing the host cell of the present invention under conditions that allow expression of the polypeptide or variant thereof, and recovering the polypeptide or variant thereof from the cultured host cell culture.

[0051] Test kit

[0052] In a second aspect, the present invention provides a kit comprising a polypeptide selected from the isolated polypeptides or variants thereof described in the first aspect.

[0053] In some embodiments, the kit of the present invention includes a capture reagent selected from the isolated peptides or variants thereof described in the first aspect. In some embodiments, the kit further includes instructions for using the isolated peptides or variants thereof as capture reagents to detect antibodies specific to citrullinated SETDB1 protein in a sample, or instructions for using the isolated peptides or variants thereof as capture reagents to determine the level of antibodies specific to citrullinated SETDB1 protein in a sample from a subject, thereby determining whether the subject has rheumatoid arthritis.

[0054] In some implementations, the subject is a mammal, such as a human.

[0055] In some embodiments, the capture reagent is attached to the surface of a solid support. In some embodiments, the N-terminus of the capture reagent is attached to the solid support. Hereinafter, the solid support includes well-drained plates, test tubes, beads (e.g., latex particles), or films (e.g., nitrocellulose membranes) made of or coated with polymeric materials (e.g., polyvinyl chloride, polystyrene, polyacrylamide, or cellulose), or magnetic beads pre-coated with functional groups (e.g., amino, carboxyl, biotin, or avidin). In some embodiments, the solid support is selected from magnetic beads or microtiter plates (e.g., microplates or ELISA plates).

[0056] In other embodiments, the capture reagent has a modifying group that can be linked to a solid support. In some embodiments, the N-terminus of the capture reagent has a modifying group that can be linked to a solid support. In such embodiments, the kit may further comprise a coating reagent for coating the capture reagent onto the solid support, such as a coating buffer (e.g., carbonate buffer, phosphate buffer, Tris-HCl buffer, or borate buffer). Methods for coating proteins or peptides onto solid supports are well known in the art, such as physical adsorption, covalent coupling via aminated or carboxylated surfaces, or mediated binding via avidin-biotin systems, polylysine pre-coated surfaces, protein A or protein G pre-coated surfaces. In some embodiments, the modifying group is biotin or avidin, and the solid support surface has a corresponding linker group.

[0057] In some embodiments, the kit contains at least a solid-phase carrier coated with avidin or streptomycin, and the aforementioned capture reagent, in a separate container or in a separate compartment of a single container unit.

[0058] In some embodiments, the method for determining whether a subject has rheumatoid arthritis by measuring the level of anti-citrullinated SETDB1 antibodies in a sample is performed in an indirect manner. Measuring antibody levels in a sample indirectly is well known to those skilled in the art. In such assays, a "capture antigen" first forms an immune complex with antibodies in the sample, and the captured antibodies are then detected by a secondary antibody (e.g., an anti-immunoglobulin antibody).

[0059] Therefore, in some embodiments, the kit further comprises a detection reagent selected from secondary antibodies with a detectable label. In some embodiments, the detectable label is selected from enzymes (e.g., horseradish peroxidase or alkaline phosphatase), chemiluminescent reagents (e.g., acridine esters), fluorescent dyes, or biotin. In some exemplary embodiments, the detectable label is selected from enzymes (e.g., horseradish peroxidase or alkaline phosphatase).

[0060] In some implementations, the secondary antibody is specific to the species (e.g., human) from which the test antibody originates.

[0061] In some implementations, the secondary antibody is an anti-immunoglobulin antibody.

[0062] In some exemplary embodiments, the kit is used to detect anti-citrullinated SETDB1 protein IgG antibodies. In such embodiments, the anti-immunoglobulin antibody is selected from anti-IgG antibodies, such as anti-human IgG antibodies.

[0063] In some exemplary embodiments, the kit is used to detect anti-citrullinated SETDB1 protein IgM antibodies. In such embodiments, the anti-immunoglobulin antibody is selected from anti-IgM antibodies, such as anti-human IgM antibodies.

[0064] In some exemplary embodiments, the kit is used to detect anti-citrullinated SETDB1 protein IgA antibodies. In such embodiments, the anti-immunoglobulin antibody is selected from anti-IgA antibodies, such as anti-human IgA antibodies.

[0065] In other embodiments, the method for determining whether a subject has rheumatoid arthritis by measuring the level of anti-citrullinated SETDB1 antibodies in a sample is performed in a double-antigen sandwich configuration. Determining antibody levels in a sample using a double-antigen sandwich method is well known to those skilled in the art. In such assays, the "capture antigen" and "detect antigen" processes create a bridge between two specific antigens, which are typically identical, have the same core epitope, or exhibit immune cross-reactivity, allowing one antibody to bind to both antigens.

[0066] Therefore, in some embodiments, the kit further comprises a detection reagent selected from the isolated peptides or variants thereof described in the first aspect.

[0067] In some embodiments, the detection reagent carries a detectable label. In some embodiments, the detectable label is selected from enzymes (e.g., horseradish peroxidase or alkaline phosphatase), chemiluminescent reagents (e.g., acridine esters), fluorescent dyes, or biotin. In some exemplary embodiments, the detectable label is selected from enzymes (e.g., horseradish peroxidase or alkaline phosphatase).

[0068] In some embodiments, the polypeptide sequences contained in the detection reagent and the capture reagent are identical or substantially identical. In some embodiments, "substantially identical" means that the two polypeptide sequences contained in the detection reagent and the capture reagent contain the same core epitope, the core epitope comprising at least the 16-26th consecutive amino acid residues of the MBD domain of the SETDB1 protein, at least one of the consecutive amino acid residues being an arginine residue, and at least one of the arginine residues being citrullinated (preferably all of them being citrullinated). In some embodiments, the core epitope comprises at least the sequence shown in SEQ ID NO:8.

[0069] In some embodiments, the kit of the present invention may also contain one or more reagents or devices selected from: (i) a device for collecting or storing samples from a subject (e.g., a blood collection device); and (ii) other reagents required for performing the assay (e.g., buffer solutions, diluents, blocking solutions, and / or standards).

[0070] Detection uses and methods

[0071] In a third aspect, the present invention provides a method for detecting antibodies specific to citrullinated SETDB1 protein in a sample, comprising the following steps:

[0072] (1) The sample is contacted with a capture reagent to obtain an antigen-antibody immune complex; the capture reagent is selected from the isolated polypeptides or variants thereof described in the first aspect;

[0073] (2) Determine the amount of antigen-antibody immune complex obtained in step (1).

[0074] In some implementations, the method is performed in vitro.

[0075] In some implementations, the method is used for non-diagnostic purposes.

[0076] In some implementations, in step (2), the amount of the immune complex is determined by immunological detection.

[0077] In some embodiments, the immunological assay is selected from enzyme immunoassay (e.g., ELISA), chemiluminescent immunoassay, fluorescence immunoassay, or radioimmunoassay.

[0078] In some embodiments, the capture reagent is attached to the surface of a solid support.

[0079] In other embodiments, the capturing agent has a modifying group that can be linked to a solid support. In such embodiments, prior to step (1), the method further includes the step of coating the capturing agent onto the surface of the solid support.

[0080] In some embodiments, the N-terminus of the capturing reagent is attached to the surface of the solid support, or has a modifying group that can be attached to the solid support.

[0081] In some embodiments, the assay is performed in an indirect manner. Assessing antibody levels in a sample using indirect methods is well known to those skilled in the art. In such assays, a "capture antigen" first forms an immune complex with antibodies in the sample, and the captured antibodies are then detected by a secondary antibody (e.g., an anti-immunoglobulin antibody).

[0082] Therefore, in some embodiments, in step (2), a detection reagent is used to detect the amount of the immune complex, the detection reagent being selected from secondary antibodies with detectable labels.

[0083] In some embodiments, the detectable marker is selected from enzymes (e.g., horseradish peroxidase or alkaline phosphatase), chemiluminescent reagents (e.g., acridine esters), fluorescent dyes, or biotin.

[0084] In some implementations, the secondary antibody is specific to the species (e.g., human) from which the test antibody originates.

[0085] In some implementations, the secondary antibody is an anti-immunoglobulin antibody.

[0086] In some exemplary embodiments, the antibody being detected is an IgG antibody. In such embodiments, the anti-immunoglobulin antibody is selected from anti-IgG antibodies, such as anti-human IgG antibodies.

[0087] In some exemplary embodiments, the antibody being detected is an IgM antibody. In such embodiments, the anti-immunoglobulin antibody is selected from anti-IgM antibodies, such as anti-human IgM antibodies.

[0088] In some exemplary embodiments, the antibody being detected is an IgA antibody. In such embodiments, the anti-immunoglobulin antibody is selected from anti-IgA antibodies, such as anti-human IgA antibodies.

[0089] In other embodiments, the assay is performed in a double-antigen sandwich configuration. Assessing antibody levels in a sample using a double-antigen sandwich method is well known to those skilled in the art. In such assays, the "capture antigen" and "detect antigen" processes create a bridge between two specific antigens, which are typically identical, have the same core epitope, or exhibit immune cross-reactivity, allowing one antibody to bind to both antigens.

[0090] Therefore, in some embodiments, in step (2), a detection reagent is used to detect the amount of the immune complex, the detection reagent being selected from the isolated polypeptides or variants thereof described in the first aspect.

[0091] In some embodiments, the detection reagent is selected from the isolated polypeptide or variants thereof described in the first aspect, the isolated polypeptide or variant thereof bearing a detectable label. In some embodiments, the detectable label is selected from enzymes (e.g., horseradish peroxidase or alkaline phosphatase), chemiluminescent reagents (e.g., acridine esters), fluorescent dyes, or biotin. In some exemplary embodiments, the detectable label is selected from enzymes (e.g., horseradish peroxidase or alkaline phosphatase).

[0092] In some embodiments, the polypeptide sequences contained in the detection reagent and the capture reagent are identical or substantially identical. In some embodiments, "substantially identical" means that the two polypeptide sequences contained in the detection reagent and the capture reagent contain the same core epitope, the core epitope comprising at least the 16-26th consecutive amino acid residues of the MBD domain of the SETDB1 protein, at least one of the consecutive amino acid residues being an arginine residue, and at least one of the arginine residues being citrullinated (preferably all of them being citrullinated). In some embodiments, the core epitope comprises at least the sequence shown in SEQ ID NO:8.

[0093] In another aspect, the invention also relates to the use of the isolated polypeptide or variant thereof described in the first aspect in the preparation of a kit for detecting antibodies specific to citrullinated SETDB1 protein in a sample. In some embodiments, the kit detects antibodies specific to citrullinated SETDB1 protein in a sample by means of the method described in the third aspect.

[0094] Diagnostic uses and methods

[0095] In a fourth aspect, the present invention provides a method for determining whether a subject suffers from rheumatoid arthritis, comprising:

[0096] (1) Determine the level of antibodies specific to citrullinated SETDB1 protein in samples from the subjects; and,

[0097] (2) Compare the level with the reference value.

[0098] In some implementations, if the level is higher than a reference value, the subject is determined to have rheumatoid arthritis.

[0099] In some implementations, the method is performed in vitro.

[0100] In this article, the reference values ​​are derived from values ​​of subjects or healthy individuals who do not have rheumatoid arthritis (e.g., subjects without detectable disease and without a history of rheumatoid arthritis), or indicate the level of antibody specific to citrullinated SETDB1 protein in the corresponding samples of subjects or healthy individuals who do not have rheumatoid arthritis.

[0101] In some embodiments, the sample is a blood sample (e.g., whole blood, plasma, or serum) or a tissue fluid sample (e.g., synovial fluid).

[0102] In some implementations, the subject is a mammal, such as a human.

[0103] In some embodiments, the level of antibodies specific to citrullinated SETDB1 protein in the sample is determined by an immunoassay. In some embodiments, the immunoassay is selected from enzyme immunoassays (e.g., ELISA), chemiluminescent immunoassays, fluorescence immunoassays, or radioimmunoassays.

[0104] In some embodiments, the assay includes using the isolated polypeptide or a variant thereof described in the first aspect as a capture reagent.

[0105] In some implementations, step (1) includes the following steps:

[0106] (1a) A sample from the subject is contacted with a capture reagent to obtain an antigen-antibody immune complex; the capture reagent is selected from the isolated polypeptides or variants thereof described in the first aspect;

[0107] (1b) Determine the amount of antigen-antibody immune complex obtained in step (1a).

[0108] In some embodiments, in step (1b), the amount of the immune complex is determined by an immunological assay. In some embodiments, the immunological assay is selected from enzyme immunoassay (e.g., ELISA), chemiluminescent immunoassay, fluorescence immunoassay, or radioimmunoassay.

[0109] In some embodiments, the capture reagent is attached to the surface of a solid support.

[0110] In other embodiments, the capturing agent has a modifying group that can be linked to a solid support. In such embodiments, prior to step (1), the method further includes the step of coating the capturing agent onto the surface of the solid support.

[0111] In some embodiments, the N-terminus of the capturing reagent is attached to the surface of the solid support, or has a modifying group that can be attached to the solid support.

[0112] In some embodiments, the assay is performed in an indirect manner. Assessing antibody levels in a sample using indirect methods is well known to those skilled in the art. In such assays, a "capture antigen" first forms an immune complex with antibodies in the sample, and the captured antibodies are then detected by a secondary antibody (e.g., an anti-immunoglobulin antibody).

[0113] Therefore, in some embodiments, in step (1b), a detection reagent is used to detect the amount of the immune complex, the detection reagent being selected from secondary antibodies with detectable labels.

[0114] In some embodiments, the detectable marker is selected from enzymes (e.g., horseradish peroxidase or alkaline phosphatase), chemiluminescent reagents (e.g., acridine esters), fluorescent dyes, or biotin.

[0115] In some implementations, the secondary antibody is specific to the species (e.g., human) from which the test antibody originates.

[0116] In some implementations, the secondary antibody is an anti-immunoglobulin antibody.

[0117] In some exemplary embodiments, the antibody being detected is an IgG antibody. In such embodiments, the anti-immunoglobulin antibody is selected from anti-IgG antibodies, such as anti-human IgG antibodies.

[0118] In some exemplary embodiments, the antibody being detected is an IgM antibody. In such embodiments, the anti-immunoglobulin antibody is selected from anti-IgM antibodies, such as anti-human IgM antibodies.

[0119] In some exemplary embodiments, the antibody being detected is an IgA antibody. In such embodiments, the anti-immunoglobulin antibody is selected from anti-IgA antibodies, such as anti-human IgA antibodies.

[0120] In other embodiments, the assay is performed in a double-antigen sandwich configuration. Assessing antibody levels in a sample using a double-antigen sandwich method is well known to those skilled in the art. In such assays, the "capture antigen" and "detect antigen" processes create a bridge between two specific antigens, which are typically identical, have the same core epitope, or exhibit immune cross-reactivity, allowing one antibody to bind to both antigens.

[0121] Therefore, in some embodiments, in step (1b), a detection reagent is used to detect the amount of the immune complex, the detection reagent being selected from the isolated polypeptides or variants thereof described in the first aspect.

[0122] In some embodiments, the detection reagent is selected from the isolated polypeptide or variants thereof described in the first aspect, the isolated polypeptide or variant thereof bearing a detectable label. In some embodiments, the detectable label is selected from enzymes (e.g., horseradish peroxidase or alkaline phosphatase), chemiluminescent reagents (e.g., acridine esters), fluorescent dyes, or biotin. In some exemplary embodiments, the detectable label is selected from enzymes (e.g., horseradish peroxidase or alkaline phosphatase).

[0123] In some embodiments, the polypeptide sequences contained in the detection reagent and the capture reagent are identical or substantially identical. In some embodiments, "substantially identical" means that the two polypeptide sequences contained in the detection reagent and the capture reagent contain the same core epitope, the core epitope comprising at least the 16-26th consecutive amino acid residues of the MBD domain of the SETDB1 protein, at least one of the consecutive amino acid residues being an arginine residue, and at least one of the arginine residues being citrullinated (preferably all of them being citrullinated). In some embodiments, the core epitope comprises at least the sequence shown in SEQ ID NO:8.

[0124] In some embodiments, the method described in any of the above embodiments further includes: providing a sample from the subject prior to step (1).

[0125] In some embodiments, the method described in any of the above embodiments further includes, after step (2), administering a therapeutically effective dose of a treatment for rheumatoid arthritis (e.g., chemotherapy, radiotherapy, and / or immunotherapy) to the subject diagnosed with rheumatoid arthritis.

[0126] In some implementations, the treatment for rheumatoid arthritis is selected from surgical treatment, chemotherapy (such as hydroxychloroquine sulfate, sulfasalazine, leflunomide, methotrexate, prednisone, celecoxib, ibuprofen, diclofenac sodium, loxoprofen sodium), targeted therapy (such as tumor necrosis factor antagonists), immunotherapy (such as tocilizumab), and combination therapy (such as chemotherapy + surgery).

[0127] In another aspect, the present invention also relates to the use of reagents capable of determining antibodies specific to citrullinated SETDB1 protein in the preparation of a kit for determining whether a subject has rheumatoid arthritis.

[0128] In some embodiments, the reagent is capable of measuring the level of antibodies specific to citrullinated SETDB1 protein by an immunological assay. In some embodiments, the immunological assay is selected from enzyme immunoassays (e.g., ELISA), chemiluminescent immunoassays, fluorescence immunoassays, or radioimmunoassays.

[0129] In some implementations, the subject is a mammal, such as a human.

[0130] In some embodiments, the sample is a blood sample (e.g., whole blood, plasma, or serum) or a tissue fluid sample (e.g., synovial fluid).

[0131] In some embodiments, the kit determines whether a subject has rheumatoid arthritis by means of:

[0132] (1) Determine the level of antibodies specific to citrullinated SETDB1 protein in samples from the subjects; and,

[0133] (2) Compare the level with the reference value.

[0134] In some implementations, if the level is higher than a reference value, the subject is determined to have rheumatoid arthritis.

[0135] In some embodiments, the reagent capable of detecting antibodies specific to citrullinated SETDB1 protein comprises a capture reagent selected from the isolated polypeptides or variants thereof described in the first aspect.

[0136] In some implementations, step (1) above includes the following steps:

[0137] (1a) A sample from the subject is contacted with a capture reagent to obtain an antigen-antibody immune complex; the capture reagent is selected from the isolated polypeptides or variants thereof described in the first aspect;

[0138] (1b) Determine the amount of antigen-antibody immune complex obtained in step (1a).

[0139] In some embodiments, in step (1b), the amount of the immune complex is determined by an immunological assay. In some embodiments, the immunological assay is selected from enzyme immunoassay (e.g., ELISA), chemiluminescent immunoassay, fluorescence immunoassay, or radioimmunoassay.

[0140] In some embodiments, the capture reagent is attached to the surface of a solid support.

[0141] In other embodiments, the capturing agent has a modifying group that can be linked to a solid support. In such embodiments, prior to step (1) above, the method further includes the step of coating the capturing agent onto the surface of the solid support.

[0142] In some embodiments, the N-terminus of the capturing reagent is attached to the surface of the solid support, or has a modifying group that can be attached to the solid support.

[0143] In some embodiments, the reagent capable of detecting antibodies specific to citrullinated SETDB1 protein further comprises a detection reagent selected from secondary antibodies with a detectable label.

[0144] In some implementations, in step (1b) above, the detection reagent is used to detect the amount of the immune complex.

[0145] In some embodiments, the detectable marker is selected from enzymes (e.g., horseradish peroxidase or alkaline phosphatase), chemiluminescent reagents (e.g., acridine esters), fluorescent dyes, or biotin.

[0146] In some implementations, the secondary antibody is specific to the species (e.g., human) from which the test antibody originates.

[0147] In some implementations, the secondary antibody is an anti-immunoglobulin antibody.

[0148] In some exemplary embodiments, the antibody being detected is an IgG antibody. In such embodiments, the anti-immunoglobulin antibody is selected from anti-IgG antibodies, such as anti-human IgG antibodies.

[0149] In some exemplary embodiments, the antibody being detected is an IgM antibody. In such embodiments, the anti-immunoglobulin antibody is selected from anti-IgM antibodies, such as anti-human IgM antibodies.

[0150] In some exemplary embodiments, the antibody being detected is an IgA antibody. In such embodiments, the anti-immunoglobulin antibody is selected from anti-IgA antibodies, such as anti-human IgA antibodies.

[0151] In other embodiments, the reagent capable of detecting antibodies specific to citrullinated SETDB1 protein further comprises a detection reagent selected from the isolated polypeptides or variants thereof described in the first aspect.

[0152] In some implementations, in step (1b) above, the detection reagent is used to detect the amount of the immune complex.

[0153] In some embodiments, the detection reagent is selected from the isolated polypeptide or variants thereof described in the first aspect, the isolated polypeptide or variant thereof bearing a detectable label. In some embodiments, the detectable label is selected from enzymes (e.g., horseradish peroxidase or alkaline phosphatase), chemiluminescent reagents (e.g., acridine esters), fluorescent dyes, or biotin. In some exemplary embodiments, the detectable label is selected from enzymes (e.g., horseradish peroxidase or alkaline phosphatase).

[0154] In some embodiments, the polypeptide sequences contained in the detection reagent and the capture reagent are identical or substantially identical. In some embodiments, "substantially identical" means that the two polypeptide sequences contained in the detection reagent and the capture reagent contain the same core epitope, the core epitope comprising at least the 16-26th consecutive amino acid residues of the MBD domain of the SETDB1 protein, at least one of the consecutive amino acid residues being an arginine residue, and at least one of the arginine residues being citrullinated (preferably all of them being citrullinated). In some embodiments, the core epitope comprises at least the sequence shown in SEQ ID NO:8.

[0155] Terminology Definition

[0156] In this invention, unless otherwise stated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, to better understand this invention, definitions and explanations of relevant terms are provided below.

[0157] As used herein, the term "citrullination" refers to a form of post-translational modification (PTM) specifically, a chemical change catalyzed by peptidyl arginine deiminoase (PAD) to deimino the amino acid arginine (R) into citrulline (C), resulting in the release of the nitrogen moiety. In a citrullinated sequence, at least one arginine residue is replaced by a citrulline residue. Therefore, the expressions "arginine residue is citrullinated" and "arginine residue is replaced by a citrulline residue" have the same meaning.

[0158] As used herein, the terms “citrulline” and “cit” are used interchangeably and refer to 2-amino-5-(carbamoylamino)valerate, an α-amino acid with the following formula: H2NC(O)NH(CH2)3CH(NH2)CO2H.

[0159] As used in this article, the term "anti-cyclic citrullinated peptide (CCP) antibody" refers to an autoantibody that uses a synthetic cyclic citrullinated peptide (CCP) as an antigen, primarily of the IgG type. Anti-CCP antibodies are spontaneously secreted by B lymphocytes in patients with rheumatoid arthritis, while B lymphocytes in patients with other diseases and healthy individuals do not spontaneously secrete anti-CCP antibodies. Therefore, anti-CCP antibodies have high specificity for rheumatoid arthritis.

[0160] As used herein, the term “SETDB1” refers to a histone methyltransferase that methylates the lysine residue at position 9 of histone H3 (H3K9) (Loyola A, et al., EMBO Reports. 2009; 10(7):769-775; Gurard-Levin ZA, et al., Annu Rev Biochem. 2014; 83:487-517). The human SETDB1 gene is located on human chromosome 1q21. The sequence of SETDB1 is well known to those skilled in the art; for example, the human SETDB1 gene sequence can be found in the Ensembl database accession number: ENSG00000143379. In this document, “citrullinated SETDB1 protein” refers to a SETDB1 protein in which at least one arginine residue is replaced with a citrulline residue. Preferably, at least one arginine residue in positions 16-26 of the MBD domain of the citrullinated SETDB1 protein is citrullinated (preferably all arginine residues are citrullinated). Preferably, the citrullinated SETDB1 protein has the sequence shown in SEQ ID NO:8 at positions 16-26 of its MBD domain.

[0161] As used herein, the term "SETDB1 protein MBD (methyl-CpG-binding domain)" refers to the methylated CpG-binding domain contained at the N-terminus of the SETDB1 protein, located at positions 594-665. The sequence of the SETDB1 protein MBD domain is well known to those skilled in the art, and an exemplary sequence is provided in SEQ ID NO:1.

[0162] As used herein, when referring to the amino acid sequence of the MBD domain of the SETDB1 protein, the sequence shown in SEQ ID NO: 1 is used for description. For example, the expression "amino acid residues 16-26 of the MBD domain of the SETDB1 protein" refers to amino acid residues 16-26 of the polypeptide shown in SEQ ID NO: 1. However, those skilled in the art will understand that mutations or variations can be naturally generated or artificially introduced into the amino acid sequence of the MBD domain of the SETDB1 protein without affecting its biological function. Therefore, in this invention, the term "MBD domain of the SETDB1 protein" and similar expressions should include all such sequences, including, for example, the sequence shown in SEQ ID NO: 1 and its natural or artificial variants (e.g., variants having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity). Furthermore, when describing a sequence fragment of the MBD domain of the SETDB1 protein, it includes not only the sequence fragment of SEQ ID NO: 1 but also the corresponding sequence fragments in its natural or artificial variants. For example, the expression "amino acid residues 16-26 of the MBD domain of the SETDB1 protein" includes amino acid residues 16-26 of SEQ ID NO: 1, and the corresponding fragment in its variants (natural or artificial). According to the present invention, the expression "corresponding sequence fragment" or "corresponding fragment" refers to a fragment located at an equivalent position in the compared sequences when the sequences are optimally aligned, i.e., when the sequences are aligned to obtain the highest percentage of identity.

[0163] As used herein, the terms "specific binding" or "specific to..." refer to a non-random binding reaction between two molecules (i.e., a binding molecule and a target molecule), such as the reaction between an antibody and the antigen it targets. The binding affinity between two molecules can be described by the KD value. The KD value is the dissociation constant obtained by the ratio of kd (the dissociation rate of a specific binding molecule-target molecule interaction; also known as koff) to ka (the association rate of a specific binding molecule-target molecule interaction; also known as kon), or kd / ka expressed as molar concentration (M). The smaller the KD value, the stronger the binding between the two molecules and the higher the affinity. In some embodiments, an antibody that specifically binds to an antigen (or an antibody that is specific to an antigen) means that the antibody binds at a rate less than about 10... -5 M, for example, less than approximately 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M or 10 -10 The antigen is bound by an affinity of M or less (KD). The KD value can be determined by methods well known in the art, such as by surface plasmon resonance (SPR) in a BIACORE instrument.

[0164] As used herein, the term "immunological assay" refers to an assay that utilizes the specific interaction / binding affinity between an antigen and antibody, and is generally used to detect the presence or level of a specific antigen or antibody in a sample. Such immunological assays are well known to those skilled in the art and include, but are not limited to, enzyme immunoassays (EIA), chemiluminescent immunoassays (CLIA), radioimmunoassays (RIA), fluorescence immunoassays (FIA), Western blotting, immunoturbidimetry, surface plasmon resonance, etc. In some embodiments, the immunological assay is an enzyme immunoassay (EIA), such as an ELISA, Elispot assay, or CLEIA assay. For a detailed description of immunological assays, see, for example, Fundamental Immunology, Ch. 7, Paul W., ed., 2nd edition, Raven Press, NY (1989).

[0165] As used herein, the term "epitope" refers to a site on an antigen that is specifically bound by an immunoglobulin or antibody. "Epitope" is also referred to in the art as an "antigenic determinant." For example, an epitope typically comprises at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 consecutive or non-continuous amino acids in a distinctive spatial conformation, and can be "linear" or "conformal." In a linear epitope, all points of interaction between the protein and the interacting molecule (e.g., an antibody) are present linearly along the primary amino acid sequence of the protein. In a conformational epitope, points of interaction are present across protein amino acid residues that are separated from each other.

[0166] As used herein, the term "detectable label" can refer to any substance that can be detected by fluorescent, spectroscopic, photochemical, biochemical, immunological, electrical, optical, or chemical means. Particularly preferred are such labels that are suitable for immunological assays (e.g., enzyme-linked immunosorbent assays, radioimmunoassays, fluorescence immunoassays, chemiluminescent immunoassays, etc.). Such labels are well known in the art and include, but are not limited to, enzymes (e.g., horseradish peroxidase, alkaline phosphatase, β-galactosidase, urease, glucose oxidase, etc.) and radionuclides (e.g., 3 H, 125 I, 35 S, 14 C or 32P), fluorescent dyes (e.g., fluorescein isothiocyanate (FITC), fluorescein, tetramethylrhodamine isothiocyanate (TRITC), phycoerythrin (PE), Texas Red, rhodamine, quantum dots or cyanine dye derivatives (e.g., Cy7, Alexa 750)), luminescent materials (e.g., chemiluminescent materials, such as acridine esters), magnetic beads (e.g., The invention includes, but is not limited to, calorimetric markers such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads, and biotin for binding avidin (e.g., streptavidin) modified with the aforementioned markers. Patents teaching the use of these markers include, but are not limited to, U.S. Patents 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and 4,366,241 (all incorporated herein by reference). The markers covered in this invention can be detected by methods known in the art. For example, radioactive markers can be detected using photographic film or a scintillation calculator, and fluorescent markers can be detected using a photodetector to detect emitted light. Enzyme markers are generally detected by providing an enzyme with a substrate and detecting the reaction product produced by the enzyme's action on the substrate, and calorimetric markers are detected by simple, visually appealing colored markers. In some implementations, the detectable markers described above can be connected to the detection antibodies or antigens via connectors of varying lengths to reduce potential steric hindrance.

[0167] As used herein, the term "antibody" refers to an immunoglobulin molecule typically composed of two pairs of polypeptide chains (each pair consisting of one light chain (LC) and one heavy chain (HC)). Antibody light chains can be classified as κ (kappa) and λ (lambda) light chains. Heavy chains can be classified as μ, δ, γ, α, or ε, and antibody isotypes are defined as IgM, IgD, IgG, IgA, and IgE, respectively. Within both light and heavy chains, variable and constant regions are linked by a "J" region of approximately 12 or more amino acids, and the heavy chain also contains a "D" region of approximately 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of three domains (CH1, CH2, and CH3). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region consists of one domain, CL. Constant domains do not directly participate in antibody-antigen binding but exhibit various effector functions, such as mediating the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. The VH and VL regions can be further subdivided into highly degenerated regions (called complementarity-determining regions (CDRs)) interspersed with more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions (VH and VL) of each heavy / light chain pair form the antigen-binding sites.

[0168] As used herein, the terms "separated" or "isolated" refer to substances obtained artificially from their natural state. If a substance or component is found in nature as a "separated" entity, it may be due to an alteration of its natural environment, the separation of the substance from its natural environment, or both. For example, a certain unseparated polynucleotide or polypeptide may naturally exist in the body of a living animal, and a high-purity identical polynucleotide or polypeptide separated from this natural state is called a separated one. The terms "separated" or "isolated" do not exclude the presence of artificial or synthetic substances, nor do they exclude the presence of other impurities that do not affect the substance's activity.

[0169] As used herein, the term "vector" refers to a nucleic acid delivery vehicle into which polynucleotides can be inserted. When a vector enables the expression of a protein encoded by the inserted polynucleotide, it is called an expression vector. Vectors can be introduced into host cells through transformation, transduction, or transfection, allowing the genetic material elements they carry to be expressed in the host cells. Vectors are well-known to those skilled in the art and include, but are not limited to: plasmids; phage particles; Cos plasmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived artificial chromosomes (PAC); bacteriophages such as λ phage or M13 phage; and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retrotranscriptoviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomaviruses (such as SV40). A vector may contain multiple elements controlling expression, including but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, a vector may contain a replication initiation site.

[0170] As used herein, the term "host cell" refers to a cell that can be used to introduce a vector, including but not limited to prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells, or human cells.

[0171] As used herein, the term "identity" refers to the sequence matching between two polypeptides or two nucleic acids. Two compared sequences are identical at a position when the same base or amino acid monomeric subunit occupies the same location (e.g., a position in each of two DNA molecules is occupied by adenine, or a position in each of two polypeptides is occupied by lysine). The "percentage identity" between two sequences is a function of the number of matching positions shared by the two sequences divided by the number of positions compared × 100. For example, if six out of ten positions in two sequences match, then the two sequences have 60% identity. For example, the DNA sequences CTGACT and CAGGTT share 50% identity (three out of six positions match). Typically, two sequences are compared to produce the maximum identity. Such comparisons can be made using methods readily available, for example, computer programs such as the Align program (DNAstar, Inc.) Needleman et al. (1970) J. Mol. Biol. 48: 443-453. The percentage identity between two amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl Biosci., 4:11-17 (1988)) integrated into the ALIGN program (version 2.0), which uses a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. Alternatively, the percentage identity between two amino acid sequences can be determined using the Needleman and Wunsch algorithm (J MoIBiol. 48:444-453 (1970)) in the GAP program integrated into the GCG software package (available at www.gcg.com), which uses a Blossum 62 matrix or a PAM250 matrix, along with gap weights of 16, 14, 12, 10, 8, 6, or 4, and length weights of 1, 2, 3, 4, 5, or 6.

[0172] As used herein, the term "conservative substitution" means an amino acid substitution that does not adversely affect or alter the intended properties of a protein / peptide containing an amino acid sequence. For example, conservative substitutions can be introduced using standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include substitutions of amino acid residues with amino acid residues having similar side chains, such as substitutions with residues that are physically or functionally similar to the corresponding amino acid residues (e.g., having similar size, shape, charge, chemical properties, including the ability to form covalent or hydrogen bonds). Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid and glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, and tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, and methionine), β-branched side chains (e.g., threonine, valine, and isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, and histidine). Therefore, it is preferable to replace the corresponding amino acid residue with another amino acid residue from the same side chain family. Furthermore, amino acid residues can also be classified into categories defined by optional physical and functional properties. For example, residues containing alcohol groups (S and T), aliphatic residues (I, L, V and M), cycloalkenyl-related residues (F, H, W and Y), hydrophobic residues (A, C, F, G, H, I, L, M, R, T, V, W and Y), negatively charged residues (D and E), polar residues (C, D, E, H, K, N, Q, R, S and T), positively charged residues (H, K and R), small residues (A, C, D, G, N, P, S, T and V), very small residues (A, G and S), residues involved in corner formation (A, C, D, E, G, H, K, N, Q, R, S, P and T), and flexible residues (Q, T, K, S, G, P, D, E and R). Methods for identifying conserved amino acid substitutions are well known in the art (see, for example, Brummell et al., Biochem. 32:1180-1187 (1993); Kobayashi et al., Protein Eng. 12(10):879-884 (1999); and Burks et al., Proc. Natl Acad. Set USA 94:412-417 (1997), which are incorporated herein by reference).

[0173] The twenty common amino acids mentioned in this article are written in accordance with conventional usage. See, for example, Immunology-ASynthesis (2nd Edition, E.S. Golub and D.G. Ren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference. In this invention, the terms “polypeptide” and “protein” have the same meaning and are used interchangeably. Furthermore, in this invention, amino acids are generally represented by single-letter and three-letter abbreviations known in the art. For example, alanine can be represented by A or Ala.

[0174] As used herein, the term “subject” includes, but is not limited to, various animals, particularly mammals such as humans.

[0175] Beneficial effects

[0176] This invention provides a citrullinated SETDB1 peptide and a detection method based on anti-citrullinated SETDB1 antibody levels. The technical solution of this invention has high specificity and sensitivity, enabling early diagnosis of rheumatoid arthritis (RA). The technical solution of this invention is simple to operate; compared to disease diagnosis through imaging, a preliminary diagnosis can be made by detecting the level of anti-citrullinated SETDB1 antibodies in the patient's serum. The technical solution of this invention achieves a positive detection rate of up to 50% in individuals negative for anti-cyclic citrullinated peptide antibodies, significantly improving the detection rate and solving the problem of low sensitivity of diagnostic markers in existing technologies, leading to missed diagnoses of early RA patients. Compared to artificially designed anti-CCP antibodies, the technical solution of this invention is a naturally occurring protein in the patient's body, which is more conducive to discovering the pathogenesis of rheumatoid arthritis.

[0177] In summary, the citrullinated SETDB1 peptide and the detection method based on anti-citrullinated SETDB1 antibody levels provided by this invention offer an effective solution to the problem of single diagnostic biomarkers for RA and the ease with which false negatives occur in the prior art, and are also of great significance for the study of the pathogenesis of RA. Attached Figure Description

[0178] Figure 1A-1B Evaluation of antibody levels and diagnostic value of different citrulline short peptides in rheumatoid arthritis. Among them, Figure 1A The antibody levels of different citrulline peptides in rheumatoid arthritis; Figure 1B This study evaluates the diagnostic value of different citrulline short peptides in rheumatoid arthritis.

[0179] Figure 2Comparison of the diagnostic value of citrullinated SETDB1 short peptide (cit-4) and anti-citrullinated polypeptide antibody (CCP2).

[0180] Figure 3 : Study on the expression level of citrullinated short peptide (cit-4) in anti-citrullinated peptide antibody (CCP2) negative population.

[0181] Figures 4A-4B A study on the antibody levels and diagnostic value of citrullinated SETDB1 short peptide (cit-4) in patients with different autoimmune diseases. Figure 4A It refers to the antibody levels of cit-4 in patients with different autoimmune diseases; Figure 4B This is an assessment of the diagnostic value of cit-4 in patients with different autoimmune diseases.

[0182] Figures 5A-5B The detection results of SETDB1 citrulline peptides of different lengths in rheumatoid arthritis. Among them, Figure 5A The antibody levels of SETDB1 citrulline peptides of different lengths in rheumatoid arthritis; Figure 5B This study evaluates the diagnostic value of SETDB1 citrulline peptides of different lengths in rheumatoid arthritis.

[0183] Sequence information

[0184] Table 1: Information about the sequences involved in this invention is provided in the table below.

[0185]

[0186]

[0187] X represents citrulline. Detailed Implementation

[0188] The invention will now be described in the following non-limiting embodiments.

[0189] Those skilled in the art will understand that the embodiments are described by way of example only and are not intended to limit the scope of protection claimed in this application. Unless otherwise specified, the experimental methods in the embodiments are conventional methods. Where specific conditions are not specified in the embodiments, they are performed according to conventional conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0190] The ELISA reagents and consumables used in the following examples were donated by Beijing Wantai Biological Pharmacy Co., Ltd.; the detection antigens used were synthesized by Nanjing Genscript Biotech Co., Ltd.

[0191] Example 1: Study on antibody levels of citrullinated SETDB1 short peptide (cit-4) in rheumatoid arthritis

[0192] First, citrulline biomarkers were screened from the serum of rheumatoid arthritis patients using mass spectrometry, initially yielding short citrulline peptides including: cit-2 (SEQ ID NO:11), cit-3 (SEQ ID NO:12), cit-4 (SEQ ID NO:3), cit-5 (SEQ ID NO:13), cit-8 (SEQ ID NO:14), and cit-9 (SEQ ID NO:15). Subsequently, these short peptides were conjugated with biotin and chemically synthesized to provide biotin-labeled short peptides for further ELISA screening.

[0193] The expression level of autoantibodies against citrulline markers was detected using an indirect ELISA method. In short, a short peptide with biotin conjugated to its N-terminus was first coated onto a PABC plate at a concentration of 50 ng / well. After coating, serum from 24 rheumatoid arthritis (RA) patients and 24 healthy subjects (HC) were diluted 10-fold as primary antibodies and incubated for 30 minutes. Secondary antibodies were prepared using HRP-labeled goat anti-human IgG (GAH-HRP), and incubation was performed for 30 minutes. Finally, the results were read. The results are shown in Figure 1. Figure 1A The results showed that the level of CIT-4 autoantibodies was significantly elevated in rheumatoid arthritis, with statistical significance. ROC curve analysis of the test results also yielded the following results: Figure 1B As shown, cit-4 has high diagnostic value in rheumatoid arthritis, with an AUC value of 0.9462, which is significantly better than other short peptides.

[0194] Example 2: Study on the diagnostic value of citrullinated SETDB1 short peptide (cit-4) for rheumatoid arthritis

[0195] The expression levels of citrulline marker autoantibodies in patients with rheumatoid arthritis were detected using an indirect ELISA method (see Example 1 for details). Additionally, an anti-citrulline peptide antibody (CCP2) was used as a control. The anti-citrulline peptide antibody kit was purchased from Beijing Bell Biotechnology Co., Ltd., and the relevant tests were performed according to the kit instructions. Results are shown below. Figure 2 middle. Figure 2 The results showed that the AUC value of cit-4 was 0.9887, which was greater than that of CCP2 (AUC value was 0.9667), indicating that it had a higher diagnostic efficacy compared to CCP2.

[0196] Example 3: Study on the expression level of citrullinated short peptide (cit-4) in anti-citrullinated peptide antibody (CCP2) negative population

[0197] Cit-4 antibody levels were detected in a patient population that tested negative for CCP2 using an indirect ELISA method (see Example 1 for details). Results are shown below. Figure 3 middle. Figure 3 The results showed that the positive rate of cit-4 could reach 50% in the CCP2-negative patient population, indicating that cit-4 is more sensitive than CCP2.

[0198] Example 4: Diagnostic value of citrullinated SETDB1 short peptide (cit-4) in patients with different autoimmune diseases

[0199] The levels of citrullinated SETDB1 autoantibodies in the serum of patients with different autoimmune diseases (RA rheumatoid arthritis, SLE systemic lupus erythematosus, PSS primary Sjögren's syndrome, and SSc scleroderma) were detected using an indirect ELISA method (see Example 1 for details). The results are shown in Figure 4. Figure 4A The results showed that citrullinated SETDB1 was the most abundant autoantibody in rheumatoid arthritis, among autoimmune diseases. ROC curve analysis of the results also yielded the following results: Figure 4B As shown, among autoimmune diseases, citrullinated SETDB1 has the best diagnostic value for patients with rheumatoid arthritis.

[0200] Example 5: Expression levels of truncated SETDB1 variants of different lengths in the serum of patients with rheumatoid arthritis

[0201] Based on the citrullinated short peptide cit-4 (SEQ ID NO:3), truncated variants of citrullinated SETDB1 of different lengths (SEQ ID NOs:4-10) were further designed and synthesized, all containing SEQ ID NO:8 (i.e., amino acid residues 16-26 of the MBD domain). The expression levels of citrullinated marker autoantibodies in rheumatoid arthritis patients were detected using these truncated variants of SETDB1 (SEQ ID NOs:3-10) via indirect ELISA (see Example 1 for details). The results are shown in Figure 5. Figure 5A The results showed that citrullinated SETDB1 truncated peptides of different lengths significantly increased the expression level of autoantibodies in the serum of patients with rheumatoid arthritis. ROC curve analysis of the detection results also yielded the following results: Figure 5B As shown, citrullinated SETDB1 truncated peptides of different lengths can all be used for the diagnosis of rheumatoid arthritis.

[0202] Although specific embodiments of the invention have been described in detail, those skilled in the art will understand that various modifications and variations can be made to the details based on all the published teachings, and all such changes are within the scope of protection of the invention. The full scope of the invention is given by the appended claims and any equivalents thereof.

Claims

1. An isolated polypeptide, wherein, The sequence of the isolated polypeptide is any one of SEQ ID NOs: 3-10, where X represents citrulline.

2. The isolated polypeptide according to claim 1, wherein, The separated polypeptide is attached to the surface of a solid support or has modifying groups that can be attached to the solid support.

3. The isolated polypeptide according to claim 2, wherein, The modifying group is biotin or avidin.

4. The isolated polypeptide according to claim 2, wherein, The solid support is selected from magnetic beads or microtiter plates.

5. The isolated polypeptide according to claim 4, wherein, The microtiter plate is a microplate or an enzyme-labeled plate.

6. The isolated polypeptide according to claim 1, wherein, The isolated polypeptides are labeled with detectable markers.

7. The isolated polypeptide according to claim 6, wherein, The detectable marker is selected from enzymes, chemiluminescent reagents, or fluorescent dyes.

8. The isolated polypeptide according to claim 7, wherein, The enzyme is horseradish peroxidase or alkaline phosphatase.

9. The isolated polypeptide according to claim 7, wherein, The chemiluminescent reagent is an acridine ester compound.

10. An isolated nucleic acid molecule comprising a nucleotide sequence encoding the isolated polypeptide of claim 1.

11. A vector comprising the isolated nucleic acid molecule of claim 10.

12. A host cell comprising the isolated nucleic acid molecule of claim 10 or the vector of claim 11.

13. A kit comprising the isolated polypeptide of claim 1.

14. The kit of claim 13, comprising a capture reagent selected from the isolated polypeptides of claim 1.

15. The kit of claim 14, wherein, The kit also includes instructions for using the isolated peptide as a capture reagent to determine the level of antibodies specific to citrullinated SETDB1 protein in samples from subjects and to determine whether the subjects have rheumatoid arthritis.

16. The kit of claim 15, wherein, The capture reagent is selected from the isolated polypeptides according to any one of claims 2-5.

17. The kit of claim 15, wherein, The subjects were mammals.

18. The kit of claim 17, wherein, The mammal in question is a human.

19. The kit of claim 14, further comprising a detection reagent, wherein, The detection reagent is selected from secondary antibodies with detectable labels.

20. The kit of claim 19, wherein, The detectable marker is selected from enzymes, chemiluminescent reagents, or fluorescent dyes.

21. The kit of claim 20, wherein, The enzyme is horseradish peroxidase or alkaline phosphatase.

22. The kit of claim 20, wherein, The chemiluminescent reagent is an acridine ester compound.

23. The kit of claim 19, wherein, The secondary antibody is specific to the species from which the test antibody originates.

24. The kit of claim 23, wherein, The antibody to be tested was derived from a human.

25. The kit of claim 19, wherein, The secondary antibody is an anti-immunoglobulin antibody.

26. The kit of claim 25, wherein, The anti-immunoglobulin antibody is selected from anti-IgG antibody, anti-IgM antibody, and anti-IgA antibody.

27. The kit of claim 14, further comprising a detection reagent, wherein, The detection reagent is selected from the isolated polypeptides according to any one of claims 6-9.

28. The use of reagents capable of determining antibodies specific to citrullinated SETDB1 protein in the preparation of a kit for determining whether a subject has rheumatoid arthritis, wherein, The reagent capable of detecting antibodies specific to citrullinated SETDB1 protein comprises a capture reagent selected from the isolated polypeptides of claim 1.

29. The use as described in claim 28, wherein, The reagent can be used to detect antibodies specific to citrullinated SETDB1 protein by immunological assay.

30. The use as described in claim 29, wherein, The immunological assays are selected from enzyme immunoassay, chemiluminescent immunoassay, fluorescence immunoassay or radioimmunoassay.

31. The use as described in claim 30, wherein, The enzyme immunoassay method is ELISA.

32. The use as described in claim 28, wherein, The subjects were human.

33. The use as described in claim 28, wherein, The reagent is used to determine the level of antibodies specific to citrullinated SETDB1 protein in samples from the subject.

34. The use as described in claim 33, wherein, The sample is a blood sample or a tissue fluid sample.

35. The use as described in claim 34, wherein, The blood sample is whole blood, plasma, or serum.

36. The use as described in claim 34, wherein, The tissue fluid sample was synovial fluid from a joint.

37. The use as described in claim 28, wherein, The capture reagent is selected from the isolated polypeptides according to any one of claims 2-5.

38. The use as described in claim 37, wherein, The reagent capable of detecting antibodies specific to citrullinated SETDB1 protein further comprises a detection reagent selected from secondary antibodies with a detectable label.

39. The use as described in claim 38, wherein, The detectable marker is selected from enzymes, chemiluminescent reagents, or fluorescent dyes.

40. The use as described in claim 39, wherein, The enzyme is horseradish peroxidase or alkaline phosphatase.

41. The use as described in claim 39, wherein, The chemiluminescent reagent is an acridine ester compound.

42. The use as described in claim 38, wherein, The secondary antibody is specific to the species from which the test antibody originates.

43. The use as described in claim 42, wherein, The antibody to be tested was derived from a human.

44. The use as described in claim 38, wherein, The secondary antibody is an anti-immunoglobulin antibody.

45. The use as described in claim 44, wherein, The anti-immunoglobulin antibody is selected from anti-IgG antibody, anti-IgM antibody or anti-IgA antibody.

46. ​​The use as described in claim 45, wherein, The anti-immunoglobulin antibody is an anti-IgG antibody.

47. The use as described in claim 28, wherein, The reagent capable of detecting antibodies specific to citrullinated SETDB1 protein further comprises a detection reagent selected from the isolated polypeptides according to any one of claims 6-9.

48. Use of the isolated polypeptide according to any one of claims 1-9 in the preparation of a kit for detecting antibodies specific to citrullinated SETDB1 protein in a sample.

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