Methods and kits for detecting Anti-citrullinated CLEC12a autoantibodies in rheumatoid arthritis

Abstract

Described herein are methods and kits for diagnosing or determining a progression of rheumatoid arthritis (RA), comprising detecting or determining levels of autoantibodies specific to citrullinated-CLEC12A (C- type lectin domain family 12 member A).

Description

[0001] METHODS AND KITS FOR DETECTING ANTI-CITRULLINATED CLEC12A AUTOANTIBODIES IN RHEUMATOID ARTHRITIS

[0002] The present description relates to a method and kits for diagnosing or determining the progression of rheumatoid arthritis (RA), comprising determining levels of autoantibodies specific citrullinated- CLEC12A (C-type lectin domain family 12 member A).

[0003] The present description refers to a number of documents, the contents of which are herein incorporated by reference in their entirety.

[0004] BACKGROUND

[0005] Rheumatoid arthritis (RA) is a systemic autoimmune disease with a prevalence of 1% in the general population. While genetic and environmental factors contribute to the development of RA, autoantibodies play a key role in the chronic inflammation of diarthrodial joints that characterizes this type of arthritis. The most specific autoantibodies in RA recognize a diverse array of post-translationally modified proteins with citrullinated antigens being the first to be identified.

[0006] Citrullination involves the conversion of arginine to citrulline by protein arginine deiminase (PAD) enzymes. This post-transcriptional modification plays a role in several physiological processes such as the epigenetic control of transcription (Wang et al.), neutrophil extracellular trap formation and epidermal differentiation (Aghamdi et al.). Inflammation-induced citrullination generates antigens that in susceptible individuals cause immune tolerance breakdown resulting in autoantibody production.

[0007] Tolerance to citrullinated antigens is lost in approximately 70% of RA patients. Of these antigens, fibrinogen is the major citrullinated antigen in RA. Using several experimental approaches, many potential citrullinated antigens have been identified in RA. They are detected in circulation, synovial fluid and synovial tissue (Tilvawala et al.). The clinical relevance of anti -citrullinated protein antibodies (A CPA) is underscored by their diagnostic value and the ability to stratify RA patients into two distinct groups: those seropositive or seronegative for ACPA. Patients expressing ACPA have different genetic and environmental risk factors for RA, worse prognosis with more erosive joint disease and extra- articular manifestations as well as different responses to therapy than seronegative patients.

[0008] Overall, there is a need for new diagnostic and prognostic markers in RA.

[0009] SUMMARY

[0010] In some embodiments described herein, is a method for identifying a subject having rheumatoid arthritis (RA) by detecting whether autoantibodies specific to citrullinated-CLEC12A (C-type lectin domain family 12 member A) are present; wherein said method comprises: a) contacting a biological sample from a subject with citmllinated-CLEC12A to form a complex between autoantibodies and citrullinated-CLEC12A; b) detecting the complex between the autoantibodies and citrullinated-CLEC12A; c) optionally, measuring the levels of the autoantibodies specific to citrullinated-CLEC12A; and d) identifying the subject having RA when the autoantibodies specific to citrullinated-CLEC12A relative to a control is present.

[0011] In some embodiments described herein, is a method for the treatment of rheumatoid arthritis (RA) in an RA subject in need thereof, wherein said method comprises: a) identifying an RA subject by detecting whether autoantibodies specific to citrullinated- CLEC12A (C-type lectin domain family 12 member A) are present by a method which comprises: i. contacting a biological sample from a human subject suspected of suffering or suffering from RA with citrullinated-CLEC12A to form a complex between the autoantibodies and citrullinated-CLEC12A; ii. detecting the complex between the autoantibodies and citrullinated-CLEC12A; iii. optionally, measuring the levels of autoantibodies specific to citrullinated-CLEC12A; and iv. identifying the RA subject when the autoantibodies specific to citrullinated- CLEC12A relative to a control is present; and b) when the RA subject is identified, treating the RA subject with anti-RA therapy.

[0012] In some embodiments described herein, is a method for diagnosing or determining a progression of rheumatoid arthritis RA) in a subject (e.g., human subject suspected of suffering or suffering from RA), said method comprising: a) optionally, providing a biological sample from the subject; b) detecting autoantibody levels specific to citrullinated-CLEC12A (C-type lectin domain family 12 member A) in the sample; and c) diagnosing the subject as having RA or determining the progression of RA by observing significantly increased autoantibody levels of specific to citrullinated-CLEC12A as compared to a subject not having RA.

[0013] In some embodiments described herein, is a method for clinically assessing rheumatoid arthritis (RA) in a subject (e.g., human subject suspected of suffering or suffering from RA), said method comprising: a) quantifying the level of autoantibodies specific to citrullinated-CLEC12A (C-type lectin domain family 12 member A) in a sample from the subject; and b) comparing the level of autoantibodies specific to citmllinated-CLEC12A (C-type lectin domain family 12 member A) to a suitable reference value indicative of the presence, stage and / or progression of RA, thereby clinically assessing RA in the subject.

[0014] In some embodiments described herein, is an in vitro method for preparing a clinical human sample, said method comprising: a) optionally, identifying a human subject as having or suspected of having rheumatoid arthritis (RA); b) obtaining a sample comprising autoantibodies specific to citrullinated-CLEC12A (C-type lectin domain family 12 member A) from the human subject; and c) quantifying the level of the autoantibodies specific to citrullinated-CLEC12A in the sample or detecting autoantibodies specific to citrullinated-CLEC12A in the sample.

[0015] In some embodiments described herein, is an in vitro method for processing a clinical human sample, said method comprising: a) optionally, identifying a human subject clinically assessed as having or suspected of having rheumatoid arthritis (RA); b) obtaining a sample comprising autoantibodies specific to citrullinated-CLEC12A (C-type lectin domain family 12 member A) from the human subject; and c) quantifying the level of the autoantibodies specific to citrullinated-CLEC12A in the sample or detecting autoantibodies specific to citrullinated-CLEC12A in the sample.

[0016] In some embodiments described herein, is a method of detecting one or more autoantibodies specific to citrullinated-CLEC12A (C-type lectin domain family 12 member A) in a biological sample from a human subject having or suspected of having rheumatoid arthritis (RA); said method comprising: a) contacting the biological sample from the human subject with citrullinated-CLEC12A to form a complex between autoantibodies and citrullinated-CLEC12A; b) detecting the complex between the autoantibodies and citrullinated-CLEC12A; and c) optionally, measuring the levels of the autoantibodies specific to citrullinated-CLEC12A.

[0017] In some embodiments described herein, is a kit for use in diagnosing or determining the progression of rheumatoid arthritis (RA) in a subject, said kit comprising one or more reagents for detecting autoantibodies specific to citrullinated-CLEC12A (C-type lectin domain family 12 member A) in a biological sample from the subject.

[0018] In some embodiments described herein, is a method of producing a complex comprising citrullinated-CLEC12A (C-type lectin domain family 12 member A) and one or more autoantibodies specific to the citrullinated-CLEC12A; wherein said method comprises: a) contacting a biological sample from a subject comprising one or more autoantibodies, with citrullinated-CLEC12A, wherein the subject has or is suspected to have rheumatoid arthritis (RA); and b) detecting the complex between the autoantibodies and citrullinated-CLEC12A.

[0019] In some embodiments described herein, is a complex for use in the identification, prognosis, diagnosis, or determination of progression of rheumatoid arthritis (RA) in a subject, wherein the complex comprises or is formed by citrullinated-CLEC12A (C-type lectin domain family 12 member A) and autoantibodies specific to the citrullinated-CLEC12A.

[0020] In some embodiments described herein, is a complex for use as a research tool in the detection of autoantibodies specific to by citrullinated-CLEC12A (C-type lectin domain family 12 member A) in a biological sample, wherein the complex comprises citrullinated-CLEC12A and autoantibodies specific to citrullinated-CLEC 12A.

[0021] In some embodiments described herein, is a kit for use in the detection of autoantibodies specific to citrullinated-CLEC 12A (C-type lectin domain family 12 member A) in a biological sample, wherein the kit comprises citrullinated-CLEC 12A and reagents for the detection of the autoantibodies.

[0022] In some embodiments described herein, is a method of manufacturing the kit as defined herein, said method comprising: a) immobilizing the citrullinated-CLEC 12A; and b) providing reagents for the detection of autoantibodies specific to the citrullinated-CLEC 12A.

[0023] In some embodiments described herein, is a method for producing or modifying a test for detecting rheumatoid arthritis (RA), the method comprising adding or integrating into said test quantifying the level of autoantibodies specific to citrullinated-CLEC 12A (C-type lectin domain family 12 member A) in a biological sample or detecting autoantibodies specific to citrullinated-CLEC 12A in a biological sample.

[0024] In some embodiments described herein, is a recombinant CLEC12A (C-type lectin domain family 12 member A) polypeptide comprising an extracellular or ectodomain from a CLEC12a (e.g., SEQ ID NO: 1), wherein the recombinant CLEC12A is citrullinated.

[0025] In some embodiments described herein, is one or more peptides of the recombinant CLEC12A polypeptides as defined herein, wherein the one or more peptides comprise one or more citrullinated residues.

[0026] In some embodiments described herein, is a use of the recombinant CLEC12A polypeptide or the one or more peptides defined herein for the identification, prognosis, diagnosis, or determination of progression of rheumatoid arthritis (RA) in a subject.

[0027] In some embodiments described herein, is a use of the recombinant CLEC12A polypeptide or the one or more peptides defined herein in the method defined herein or in the kit defined herein. In some embodiments described herein, is a polynucleotide encoding the recombinant CLEC12A polypeptide or the one or more peptides as defined herein.

[0028] In some embodiments described herein, is a vector comprising the polynucleotide defined herein.

[0029] BRIEF DESCRIPTION OF THE DRAWINGS

[0030] In the appended drawings:

[0031] Fig. 1 shows the alignment of the ectoCLEC12A constructs, IL3ss-FLAG-ectoCLEC12A (SEQ ID NO: 2), IL3ss-FLAG-ectoCLEC12A No Arg (SEQ ID NO: 3), and ectoCLEC12A-K2 (SEQ ID NO: 4). IL3ss-FLAG-ectoCLEC12A (SEQ ID NO: 2): the extracellular domain of human CLEC12A (residues 76-262 corresponding toresidues 69-265 of SEQ ID NO: 1) fused at the N-terminus to the FLAG tag (residues 66-73) via the dipeptide linker / spacer “TR” (residues 74-75), and the IL-3 signal sequence (residues 1-26) and a biotinylation consensus sequence via the spacer ‘GARQ’. IL3ss-FLAG- ectoCLEC12A No Arg (SEQ ID NO: 3): the extracellular domain of human CLEC12A (residues 76-262 corresponding to residues 67-265 of SEQ ID NO: 1) fused at the N-terminus to the FLAG tag (residues 66-73) via the dipeptide linker / spacer “TK” (residues 74-75), and the IL-3 signal sequence (residues 1-26) via the linker / spacer of residues 27-65. EctoCLEC12A-K2 (SEQ ID NO: 4): the extracellular domain of humanCLEC12A (corresponding residues 69-265 of SEQ ID NO: 1) comprising the dipeptide linker / spacer “TK” (residues 1-2). Potential citrullination sites in all ectoClecl2A constructs are shaded.

[0032] Fig. 2A shows the CLEC12A-PAD2 (Protein-arginine deiminase type-2 enzyme) complex predicted by AlphaFold™-Multimer. CLEC12A (right) and PAD2 (left) complex and close-up of CLEC12A-PAD2 interaction is shown. CLEC12A has R185, R201, R204, R232 and R245 (with respect to SEQ ID NO: 1) in contact with the active site (D351, H471, D473 and C647) in the C-terminal catalytic domain of PAD2 (SEQ ID NO: 7). Fig. 2B shows the CLEC12A-PAD4 complex. The 3D homology model (105-253 amino acid) of CLEC12A (Uniprot ID: Q5QGZ9) was constructed by the modeling software Modeller™ (Webb et al.), using as templates from PDB database, 3g81_A (105-125 amino acid) and lyxk B (126-253 amino acid). Model quality was assessed by Ramachandran plot analysis through PROCHECK™ (Laskowski et al.). Structure images were generated using PyMOL™. CLEC12A (and PAD4 complex and close-up of CLEC12A-PAD4 interaction is shown. CLEC12A has R135 (with respect to SEQ ID NO: 1) in contact with the active site of PAD4.

[0033] Fig. 3A shows the carboxymethylated, citrullinated, deamidated, and oxidated residues in residues 27-262 of IL3ss-FLAG-ectoCLEC12A No Arg (SEQ ID NO: 3) by mass spectrometry before incubation with PAD2 in vitro. Fig. 3B shows the carboxymethylated, citrullinated, deamidated, and oxidated residues in residues 27-262 of IL3ss-FLAG-ectoCLEC12A No Arg (SEQ ID NO: 3) by mass spectrometry after incubation with PAD2 in vitro. Citrullinated arginines are represented in boxes. Fig. 4A shows anti-CitCLEC12A (citrullinated CLEC12A) antibody levels in rheumatoid arthritis patient sera. Sera from rheumatoid arthritis (RA) patients, n=68 (circles) and healthy controls (HC), n=36 (squares) were tested for IgG antibodies targeting CitCLEC12A using ELISA. Each symbol represents a study subject. The solid lines represent the median values with error bars for IQR (interquartile range). The dashed line represents the cut-off value for positivity (24. 1 RU / mL). Fig. 4B shows anti-CLEC12A and anti-CitCLEC12A (citrullinated CLEC12A) antibody levels in rheumatoid arthritis patient sera. Sera from rheumatoid arthritis (RA) patients, n=68 (circles) and healthy controls (HC), n=36 (squares) were tested for IgG antibodies targeting CLEC12A or CitCLEC12A using ELISA. Each symbol represents a study subject. ***p-value < 0.001; ****p-value < 0.0001 by the Mann-Whitney test.

[0034] Fig. 5A shows the expression of anti-citrullinated protein antibodies (ACPA) and anti- homocitrullinated protein antibodies (AHCPA) in anti-CitCLEC12A positive subjects. Fig. 5B shows the expression of anti-citrullinated protein antibodies (ACPA) and anti-homocitrullinated protein antibodies (AHCPA) in anti-CitCLEC12A negative subjects. Serum IgG anti-CCP2 (cyclic citrullinated peptide 2), anti-CitJED (citrullinated peptide), and anti-HomoCitJED (homocitrullinated peptide) antibodies were measured by ELISA. Values represented as % of subjects expressing none, 1 or more of these antibodies.

[0035] Fig. 6 shows the correlation between anti-CitCLEC12A antibody and anti-CCP2 antibody levels in RA patients. Sera from rheumatoid arthritis (RA) patients (n=68) were tested for IgG antibodies targeting CitCLEC12A and anti-CCP2 using ELISA. Each symbol represents a study subject; r=0.3189, p=0.0075 by Spearman test.

[0036] SEQUENCE LISTING

[0037] This application contains a Sequence Listing in computer readable form created November 18, 2025. The computer readable form is incorporated herein by reference.

[0038] Table 1: Sequence Listing DETAILED DESCRIPTION

[0039] General Definitions

[0040] Headings, and other identifiers, e.g., (a), (b), (i), (ii), etc., are presented merely for ease of reading the specification and claims. The use of headings or other identifiers in the specification or claims does not necessarily require the steps or elements be performed in alphabetical or numerical order or the order in which they are presented.

[0041] The use of the word “a” or “an” when used in conjunction with the term “comprising” in the claims and / or the specification may mean “one” but it is also consistent with the meaning of “one or more”, “at least one”, and “one or more than one”.

[0042] The term “about”, when used herein, indicates that a value includes the standard deviation of error for the device or method being employed in order to determine the value. In general, the terminology “about” is meant to designate a possible variation of up to 10%. Therefore, a variation of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10% of a value is included in the term “about”. Unless indicated otherwise, use of the term “about” before a range applies to both ends of the range.

[0043] As used in this specification and claim(s), the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

[0044] As used herein, “protein” or “polypeptide” or “peptide” means any peptide-linked chain of amino acids, which may or may not comprise any type of modification (e.g., chemical or post- translational modifications such as acetylation, carboxymethylation, deamidation, phosphorylation, citrullination, homocitrullination, glycosylation, oxidation, sulfatation, sumoylation, prenylation, ubiquitination, etc.).

[0045] As used herein, “autoantibody” refers to an antibody produced by the immune system of a subject and that is directed to a self-antigen. The term “autoantibody” and “antibody”, when referring to anti-citCLEC12A, can be used interchangeably herein.

[0046] As used herein, “subject” refers to a mammal, including but not limited to a human, mouse, rat, or rabbit. The subject may be a human patient, such as a patient having or suspected of having rheumatoid arthritis (RA). The subject may also be a “healthy”, “reference” (e.g., suitable reference), or “control” subject, which may not suffer from RA or does not present symptoms associated with RA and may be a healthy subject, which may be used as a control or reference in the determination of the presence, diagnosis, prognosis, classification, or progression of RA. Symptoms associated with RA may include but are not limited to joint pain, stiffness and swelling, decreased physical function, fatigue, anemia, fevers, nodules, difficulty breathing, cough, chest pain, eye redness and pain, visual loss, numbness, tingling, and skin ulcers. In some cases, the patient having or suspected of having rheumatoid arthritis (RA) is a patient at high risk of developing RA.

[0047] As used herein, “sample” or “biological sample” refers to any sample comprising or being tested for the presence of antibodies or autoantibodies. Samples can include but are not limited to cellular extracts, live or dead / dying cells, cell lysates, extracellular fluid, fluid harvested from the body of a subject, culture media, blood, bone marrow, plasma, serum, sputum, bronchoalveolar lavage (BAL), synovial fluid, biopsy, or any organ.

[0048] As used herein the expression “recombinant” refers to any polypeptide or polynucleotide that has a polypeptide or polynucleotide sequence that has been altered by human intervention to include a sequence that is not the same as that found in the native, progenitor, or host cell. Moreover, the polynucleotide sequence alteration can be chromosomal or extrachromosomal.

[0049] As used herein, the term “citrullination” or “citrullinated” (also referred to as deimination) refers to the conversion of an arginine amino acid to a citrulline. Citrullination may be catalyzed via a PAD (Protein-arginine deiminase or peptidylarginine deiminase) enzyme (e.g., PAD1, PAD2, PAD3, PAD4, PAD5, or PAD6) or an arginine deiminase (ADI). Citrullination may occur intracellularly (e.g., via post-translational modification) or extracellularly. As defined herein, a polypeptide is defined as being citrullinated if at least one arginine anywhere in the polypeptide, either in the native or recombinant polypeptide sequence, is citrullinated. In some aspects, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 arginines in the polypeptide may be citrullinated. In some aspects, at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% of the arginines in the polypeptide may be citrullinated. In some aspects, at least one of the arginines in the polypeptide near or in proximity to the active site of a PAD enzyme (e.g., PAD1, PAD2, PAD3, PAD4, PAD5, or PAD6) may be citrullinated.

[0050] The present invention relates to a novel autoantigen in rheumatoid arthritis, citrullinated CLEC12A (also referred to as myeloid inhibitory C-type lectin or MICL), and methods of diagnosis or prognosis of RA by quantifying or detecting autoantibodies specific to citrullinated CLEC12A. To date, citrullination of CLEC12A, or autoantibodies specific thereto, have not been previously described or reported in either healthy humans or in RA patients. Previously, native non-citrullinated CLEC12A expression was shown to correlate with rheumatoid factor levels in RA patients (Galligan et al.), and RA patients were previously shown to develop autoantibodies specific to non-citrullinated CLEC12A ( Redelinghuys et al.; Malamud, et al.). Furthermore, while older studies described an association between certain polymorphisms in and around the CLEC12A gene and RA (Michou et al.), more recent findings with larger cohorts suggest that there is in fact no clear association (Redelinghuys et al.; Okada et al.). In one aspect, there is described a method for identifying a subject having rheumatoid arthritis (RA) by detecting whether autoantibodies specific to citrullinated-CLEC12A (C-type lectin domain family 12 member A) are present. Said method may comprise contacting a biological sample from a subject with citrullinated-CLEC12A to form a complex between autoantibodies and citrullinated- CLEC12A; detecting the complex between the autoantibodies and citrullinated-CLEC12A; optionally, measuring the levels of the autoantibodies specific to citrullinated-CLEC12A; and identifying the subject having RA when the autoantibodies specific to citrullinated-CLEC12A relative to a control is present.

[0051] In another aspect, there is described a method for the treatment of rheumatoid arthritis (RA) in an RA subject in need thereof. Said method may comprise optionally identifying an RA subject by detecting whether autoantibodies specific to citrullinated-CLEC12A (C-type lectin domain family 12 member A) are present by a method which comprises contacting a biological sample from a human subject suspected of suffering or suffering from RA with citrullinated-CLEC12A to form a complex between the autoantibodies and citrullinated-CLEC12A; detecting the complex between the autoantibodies and citrullinated-CLEC12A; optionally, measuring the levels of autoantibodies specific to citrullinated- CLEC12A; and identifying the RA subject when the autoantibodies specific to citrullinated-CLEC12A relative to a control is present. Furthermore, when the RA subject is identified, treating the RA subject with anti-RA therapy.

[0052] Anti-RA therapy may include but is not limited to anti-inflammatory, or non-steroidal antiinflammatory drugs and disease modifying anti -rheumatic drugs (e.g., sulfasalazine), corticosteroids, corticosteroid-sparing agents, analgesics, antimalarial drugs (e.g., hydroxychloroquine), immunosuppressants (e.g., leflunomide, methotrexate, azathioprine, cyclophosphamide,), biologies (e.g., , rituximab, sarilumab, abatacept, anakinra, tocilizumab), , tumor necrosis factor (e.g., TNF-alpha) inhibitors (e.g., etanercept, adalimumab, infliximab, certolizumab pegol, golimumab), janus kinase inhibitors, , disease-modifying antirheumatic drugs, transplantation, and / or surgery.

[0053] In another aspect, there is described a method for diagnosing or determining / assessing a progression of rheumatoid arthritis RA) in a subject (e.g., human subject suspected of suffering or suffering from RA). Said method may comprise optionally, providing a biological sample from the subject; detecting autoantibody levels specific to citrullinated-CLEC12A (C-type lectin domain family 12 member A) in the sample; and diagnosing the subject as having RA or determining the progression of RA by observing significantly increased autoantibody levels of specific to citrullinated-CLEC12A as compared to a subject not having RA.

[0054] In another aspect, there is described a method for clinically assessing rheumatoid arthritis (RA) in a subject (e.g., human subject suspected of suffering or suffering from RA). Said method may comprise quantifying the level of autoantibodies specific to citrullinated-CLEC12A (C-type lectin domain family 12 member A) in a sample from the subject; and comparing the level of autoantibodies specific to citrullinated-CLEC12A (C-type lectin domain family 12 member A) to a suitable reference value indicative of the presence, stage and / or progression of RA, thereby clinically assessing RA in the subject.

[0055] In some aspects, clinical assessment comprises diagnosing RA in the subject, staging RA in the subject, predicting a response to treatment, and / or monitoring the progression of RA in the subject.

[0056] In another aspect, there is described an in vitro method for preparing a clinical human sample. The method may comprise optionally, identifying a human subject as having or suspected of having rheumatoid arthritis (RA); obtaining a sample comprising autoantibodies specific to citrullinated- CLEC12A (C-type lectin domain family 12 member A) from the human subject; and quantifying the level of the autoantibodies specific to citrullinated-CLEC12A in the sample or detecting autoantibodies specific to citrullinated-CLEC12A in the sample.

[0057] In another aspect, there is described an in vitro method for processing a clinical human sample. The method may comprise optionally, identifying a human subject clinically assessed as having or suspected of having rheumatoid arthritis (RA); obtaining a sample comprising autoantibodies specific to citrullinated-CLEC12A (C-type lectin domain family 12 member A) from the human subject; and quantifying the level of the autoantibodies specific to citrullinated-CLEC12A in the sample or detecting autoantibodies specific to citrullinated-CLEC12A in the sample.

[0058] In another aspect, there is described a method of detecting, screening, or panning one or more autoantibodies specific to citrullinated-CLEC12A (C-type lectin domain family 12 member A) in a biological sample from a human subject having or suspected of having rheumatoid arthritis (RA). The method may comprise contacting the biological sample from the human subject with citrullinated- CLEC12A to form a complex between autoantibodies and citrullinated-CLEC12A; detecting the complex between the autoantibodies and citrullinated-CLEC12A; and optionally, measuring the levels of the autoantibodies specific to citrullinated-CLEC12A.

[0059] In another aspect, there is described a method of producing a complex comprising citrullinated- CLEC12A (C-type lectin domain family 12 member A) and one or more autoantibodies specific to the citrullinated-CLEC12A. Said method may comprise contacting a biological sample from a subject comprising one or more autoantibodies, with citrullinated-CLEC12A, wherein the subject has or is suspected to have rheumatoid arthritis (RA); and detecting the complex between the autoantibodies and citrullinated-CLEC 12A.

[0060] In another aspect, there is described a method for producing or modifying a test for detecting rheumatoid arthritis (RA). The method may comprise adding or integrating into said test detecting and / or quantifying the level of autoantibodies specific to citrullinated-CLEC 12A (C-type lectin domain family 12 member A) in a biological sample or detecting autoantibodies specific to citrullinated-CLEC12A in the sample.

[0061] In another aspect, there is described a kit for use in diagnosing or determining the progression of rheumatoid arthritis (RA) in a subject. Said kit may comprise one or more reagents for detecting autoantibodies specific to citrullinated-CLEC12A (C-type lectin domain family 12 member A) in a biological sample from the subject. The kit may further include a plate (such as a microplate), tube, or any suitable support, upon which a protein (such as an autoantigen) is or may be immobilized. Reagents for the kits described herein may include but are not limited to reagents commonly used in an immunoassay, such as in an enzyme-linked immunosorbent assay (ELISA), or for Mass Spectrometry. For example, the reagents may include a detecting antibody for the detection of autoantibodies bound to an immobilized autoantigen. In some aspects, the kit may comprise instructions to perform the methods described herein.

[0062] In some aspects, the detection or quantification of one or more autoantibody / autoantibodies is determined using commonly known methods for the detection of antibodies, including but not limited to an immunoassay or Mass Spectrometry. Immunoassays may include an enzyme-linked immunosorbent assay (ELISA), such as a direct binding or sandwich ELISA.

[0063] In some aspects, the methods and kits described herein further comprise or are used in combination with detecting or identifying the levels of one or more autoantibodies known to be associated with the identification, diagnosis or progression of said RA. These autoantibodies may include but are not limited to anti -rheumatoid factor (RF), anti -cyclic citrullinated peptides (CCP1, CCP2, CCP3) antibodies, anti-citrullinated protein antibodies (ACPA), anti-carbamylated protein (anti-CarP) antibodies, antisynthetic citrullinated peptide (CitJED), and anti-homocitrullinated peptide (HomoCitJED).

[0064] In some aspects methods and kits described herein further comprise or are used in combination with determining whether one or more criteria selected from one or more known diagnostic or classification measures of RA are present. This may include but not limited to the American College of Rheumatology (ACR) classification criteria and European League Against Rheumatism (EULAR) classification criteria.

[0065] In some aspects described herein, the methods and kits described herein are for the detection or diagnosis of any stage of RA. In some aspects described herein, the methods and kits described herein are for the detection or diagnosis from early RA (early onset arthritis) to established RA to severe RA. In some aspects, determining the progression of RA comprises staging RA. These stages may include but are not limited to stage I (synovitis or early RA), stage II (pannus), stage III (fibrous ankylosis), and stage IV (bony ankylosis).

[0066] In another aspect, there is described a complex for use in the identification, diagnosis, prognosis, or determination of progression of rheumatoid arthritis (RA) in a subject, wherein the complex comprises or is formed by citrullinated-CLEC12A (C-type lectin domain family 12 member A) and autoantibodies specific to the citrullinated-CLEC12A. The complex can also be isolated. The complex can be an immunocomplex.

[0067] In another aspect, there is described a complex for use as a research tool in the detection of autoantibodies specific to by citrullinated-CLEC12A (C-type lectin domain family 12 member A) in a biological sample, wherein the complex comprises citrullinated-CLEC12A and autoantibodies specific to citrullinated-CLEC12A. The complex can also be isolated. The complex can be an immunocomplex.

[0068] In another aspect, there is described a method of manufacturing the kit as defined herein, said method comprising immobilizing the citrullinated-CLEC12A and providing reagents for the detection of autoantibodies specific to the citrullinated-CLEC12A.

[0069] In another aspect, there is described a recombinant CLEC12A (C-type lectin domain family 12 member A) protein or peptides thereof comprising an extracellular or ectodomain from a CLEC12a (e.g., SEQ ID NO: 1), wherein the recombinant CLEC12A is citrullinated. In some aspects, the recombinant citrullinated CLEC12A defined herein is used in the methods and kits defined herein. In some aspects, the recombinant CLEC12A is citrullinated by a PAD (Protein-arginine deiminase) enzyme (e.g., PAD1, PAD2, PAD3, PAD4, PAD5, or PAD6) or an ADI. In some aspects, one or more arginines residues corresponding to, homologous to, or positionally equivalent to the arginines such as R104, R119, R135, R136, R185, R201, R204, R217, R232, R245, and R263 of SEQ ID NO: 1, may be citrullinated. In some aspects, one or more arginines residues corresponding to, homologous to, or positionally equivalent to the arginines such as R87, R104, R119, R135, R136, R185, R201, R204, R217, R232, R245, and R263 of SEQ ID NO: 1, may be citrullinated. In some aspects, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 arginines in CLEC12A may be citrullinated. In some aspects, at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% of the arginines of CLEC12A may be citrullinated. In some aspects, at least one of the arginines of CLEC12A near or in proximity to the active site of a PAD enzyme (e.g., PAD1, PAD2, PAD3, PAD4, PAD5, or PAD6) may be citrullinated.

[0070] In some aspects, described herein is one or more peptides of the recombinant CLEC12A polypeptides as defined herein, wherein the one or more peptides comprise one or more citrullinated residues. The one or more peptides may comprise a citrullination on one or more arginines residues corresponding to, homologous to, or positionally equivalent to the arginines selected from the group consisting of R104, R119, R135, R136, R185, R201, R204, R217, R232, R245, and R263 of SEQ ID NO: 1

[0071] Generally, positionally equivalent mutations in two or more different CLEC12A genes or proteins (e.g., from the same or different organism, or different isoforms, homologs, analogs, orthologs, or paralogs) occur at homologous amino acid positions in the amino acid sequences of the CLEC12A. Hence, use herein of the term “positionally equivalent” or “homologous” to a given mutation, regardless of whether or not the particular function of the mutated amino acid is known. It is possible to identify positionally equivalent or homologous amino acid residues in the amino acid sequences of two or more different CLEC12A genes or proteins on the basis of sequence alignment and / or molecular modelling.

[0072] In some aspects, the extracellular or ectodomain of CLEC12A comprises an amino acid sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to residues 69-265 of SEQ ID NO: 1

[0073] In some aspects, the recombinant CLEC12A described herein may comprise a signal sequence (e.g., IL-3 signal sequence) and / or a tag (e.g., FLAG) to induce the secretion and to purify the protein, respectively. In some aspects, the recombinant CLEC12A comprises an amino acid sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 2.

[0074] In some aspects, there is described a use of the recombinant CLEC12A protein described herein or one or more peptides thereof (or as defined herein) for the identification, prognosis, diagnosis, or determination of progression of rheumatoid arthritis (RA) in a subject. In some aspects, the recombinant CLEC12A protein described herein or one or more peptides thereof (or as defined herein) is used in the methods and kits described herein.

[0075] In some aspects, there is described a polynucleotide encoding the recombinant CLEC12A protein described herein or one or more peptides thereof (or as defined herein). In some aspects, there is described a vector comprising the polynucleotide described herein.

[0076] In some aspects, there is described a method for screening for anti-citCLEC12A autoantibodies or for screening for different autoimmune diseases in a human subject. Said method may comprise administering the recombinant CLEC12A protein described herein or one or more peptides thereof (or as defined herein) in said patient, and / or detecting the presence / amount of anti-citCLEC12A autoantibodies in said subject (e.g., in a sample). In some aspects, the subject may or may not have RA. In some aspect, said method comprise further diagnosing or determining a progression an autoimmune disease (e.g., RA) by observing significantly increased autoantibody levels of specific to citrullinated-CLEC12A as compared to a subject not having an autoimmune disease (e.g., RA). In some aspects, there is described a use of the recombinant CLEC12A protein described herein or one or more peptides thereof (or as defined herein) in a screening assay or method for screening for anti-citCLEC12A autoantibodies or for screening for different autoimmune diseases in a human subject.

[0077] Other objects, advantages and features of the present description will become more apparent upon reading of the following non-restrictive description of specific embodiments thereof, given by way of example only with reference to the accompanying drawings. EXAMPLES

[0078] Example 1: Materials and Methods

[0079] Study Population

[0080] Sera samples were collected from healthy control subjects (HC), and from RA patients attending a tertiary care Rheumatology clinic at St. Joseph’s Health Care London (SJHC) (London ON, Canada). All RA patients (n=68) satisfied the American College of Rheumatology 2010 criteria for RA and were treated with disease modifying agents as per standard of care (Aletaha et al.). HC sera samples (n=36) were collected from individuals accompanying RA patients to their clinic visit, and from staff members at SJHC or Western University (London ON, Canada) (Lac et al.). HC related to the RA patients or that had a history of RA, other autoimmune disease or had inflammatory joint symptoms (swelling or morning stiffness) were excluded from the study. RA patients and healthy subjects were age- and sex-matched 2 to 1. Patient characteristics were collected by interview and medical record review using standardized case report forms at the time of blood sample collection. Data included participant demographics, smoking history, RA disease duration, swollen joint count and presence of significant morning joint stiffness (defined as > 30 min). This research study was approved by the Human Research Ethics Board of Western University (London, Ontario, Canada) (REB#105594).

[0081] Plasmid constructs

[0082] The wild type, CLEC12A coding sequence used for our constructs corresponds to the CLEC12A isoform 2 sequence (UniProt Q5QGZ9-2; SEQ ID NO: 1). A recombinant form of CLEC12A composed of its extracellular domain (ectoCLEC12A; residues 69-265 of SEQ ID NO: 1) fused to a FLAG tag via the dipeptide linker “TR” (residues 74-75) and further fused to an IL-3 signal sequence via the linker / spacer of residues 27-65 to make IL3ss-FLAG-ectoCLEC12A (SEQ ID NO: 2). The construct coding for this recombinant CLE12A-ecto was further modified by mutating the first arginine to lysine (residue 75) in the dipeptide linker / spacer, as well as the three arginines in the IL-3ss and first spacer / linker, in the IL3ss- FLAG-ectoCLEC12A No Arg sequence (SEQ ID NO: 3) using HiFI™ site-directed mutagenesis with the following primers forward; 5 -CAATAGCCACCAAATTAGCTCGTGAGCTATATAGC-3’ (SEQ ID NO: 5) and reverse primer 5 -GCTATATAGCTCACGAGCTAATTTGGTGGCTATTG-3’ (SEQ ID NO: 6). Finally, the ectoCLEC12A-K2 sequence (SEQ ID NO: 4) was made adding the dipeptide linker / spacer “TK” (residues 1-2) to the ectodomain (residues 69-265) of SEQ ID NO: 1. The alignment of the constructs, as well as potential sites for arginine citrullination, is shown in Fig. 1. Citrullination of CitCLEC12A

[0083] Human recombinant CLEC12A (IL3ss-FLAG-ectoCLEC12A No Arg construct; SEQ ID NO: 3) was citrullinated in vitro by incubating 7 units of peptidylarginine deiminase 2 (PAD2) (Sigma-Aldrich) with 0.25 mg of CitCLEC12A in 0.1 M Tris-HCL (pH 7.4), 10 mM CaC12 for 3 h at 50°C (Hill et al.). Citrullination was confirmed by mobility shift on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and mass spectrometry as described below.

[0084] Mass Spectrometry

[0085] Citrullinated and non-citrullinated CLEC12A were isolated from SDS-PAGE gels and submitted to the Western University Functional Proteomics Facility for digest with LysC and AspN (Thermofisher Scientific). Following enzymatic digest of gels according to manufacturer instructions for each enzyme, samples were analyzed as previously described using a FT / IT / CI Orbitrap™ Elite mass spectrometer (Waters). Data were analyzed using the MASCOT™ server with the National Center for Biotechnology Information database for citrullination (modification of arginine, R).

[0086] Antibody Assays

[0087] Sera were tested for the presence of antibodies to CLEC12A, citCLEC12A, cyclic citrullinated peptide 2 (CCP), another synthetic citrullinated peptide called CitJED and a homocitrullinated peptide called HomoCitJED (Lac et al.) using Enzyme-Linked ImmunoSorbant Assay (ELISA). All ELISA experiments were conducted at least in duplicate with the average value reported.

[0088] Methods for the CLEC12A and citCLEC12A ELISA were adapted from (Hill et al.). Briefly, MaxiSorp™ Nunc™ 96-well plates (Thermofisher Scientific) were coated overnight at 4°C with 10 pg / mL of protein / well or 1% BSA (background control) in carbonate coating buffer. Plates were then washed with PBS containing 0.05% Tween (PBST) and blocked with 0.1% BSA in PBS. After washing, sera samples diluted in 0. 1% BSA PBST were incubated in duplicate on the plate for 30 minutes before being washed away with PBST. Biotin-conjugated goat anti-human IgG secondary antibodies (Jackson) were diluted 1:5,000 in 0.1% BSA PBST containing streptavidin / horseradish peroxidase diluted 1:4,000 (Abeam) and incubated in the wells for 30 min before being washed with PBST. 100 pL of TMB substrate (Sigma- Aldrich) was incubated in each well for 10 minutes before the reaction was stopped with 50 pL of IM H2SO4. Optical Density (OD) at 655nm was subtracted from OD at 450nm using a microplate reader (BIORAD). Antibody reactivity to BSA (Sigma- Aldrich) was subtracted from each duplicate sample.

[0089] Antibodies to CLEC12A and citCLEC12A were quantified in Relative Units / mL (RU / mL) by normalization to a serial dilution of a reference serum sample (16 replicates, coefficient of variation 20%). The cut-off value for positive anti-CitClecl2A was 24. 1 RU / mL and were determined using the mean of HC sera + 2 standard deviations. Samples with OD values below the detection limit of the assay (<0.1 OD) were set to 0 RU / mL.

[0090] Anti-CCP IgG antibodies were detected using an ELISA kit from Euroimmun (Luebeck, Germany) and carried out according to the manufacturer’s instructions with the recommended positive cut-off of >25 RU / mL. Antibodies to CitJED and HomoCitJED were measured as previously described with positive cut-offs of 3.7 RU / mL and 25.3 RU / mL respectively (Lac et al.).

[0091] Three-dimensional modeling of CLEC12A based on the complex with PAD4

[0092] The 3D homology model (105-253 residues of SEQ ID NO: 1) of CLEC12A (Uniprot ID: Q5QGZ9-2) was constructed by the modeling software Modeller™ (Webb and Sali, 2014), using as templates from PDB database, 3g81_A (105-125 amino acid) and lyxk B (126-253 amino acid) based on the complex with PAD4. Model quality was assessed by Ramachandran plot analysis through PROCHECK™ (Laskowski et al., 1993). Structure images were generated using PyMOL™.

[0093] Prediction of the complex CLEC12A-PAD2

[0094] The sequences CLEC12A (UniProt: Q5QGZ9-2; SEQ ID NO: 1) and PAD2 HUMAN (UniProt: Q9Y2J8-1; SEQ ID NO: 7) were used in this work. The prediction of the complex CL12A-PAD2 was conducted by AlphaFold-Multimer™ (AF-M). AF-M is able to model complexes of globular proteins with accuracy comparable to experimental structures and superior to AlphaFold™ and docking methods (Evans et al., 2021). Structure images were generated using PyMOL.

[0095] Statistical analyses

[0096] Study participant characteristics were presented using descriptive statistics. Antibody levels were compared with the Student T-test or Mann-Whitney test depending on the normality of the data. Correlations between antibody levels were performed using the Spearman test. Categorical variables were compared using the Fisher exact test. A p<0.05 was considered significant. All statistical analyses were performed using GraphPad™ (version 9.2.0).

[0097] Example 2: Results

[0098] Citrullination of CLEC12A

[0099] To assess anti-citCLEC12A reactivity in RA patients and healthy donors, different constructs consisting of the extracellular domain of CLEC12A (SEQ ID NO: 1) were first produced. Fig. 1 shows the alignment of the ectoCLEC12A constructs, IL3ss-FLAG-ectoCLEC12A (SEQ ID NO: 2), and IL3ss-FLAG-ectoCLEC12A No Arg (SEQ ID NO: 3), and ectoCLEC12A-K2 (SEQ ID NO: 4). IL3ss-FLAG-ectoCLEC12A (SEQ ID NO: 2): the extracellular domain of human CLEC12A (residues 76-262 corresponding toresidues 69-265 of SEQ ID NO: 1) fused at the N-terminus to the FLAG tag (residues 66-73) via the dipeptide linker / spacer “TR” (residues 74-75), and the IL-3 signal sequence (residues 1-26) via the linker / spacer of residues 27-65. IL3ss-FLAG-ectoCLEC12A No Arg (SEQ ID NO: 3): the extracellular domain of human CLEC12A (residues 76-262 corresponding to residues 67-265 of SEQ ID NO: 1) fused at the N-terminus to the FLAG tag (residues 66-73) via the dipeptide linker / spacer “TK” (residues 74-75), and the IL-3 signal sequence (residues 1-26) via the linker / spacer of residues 27-65. EctoCLEC12A-K2 (SEQ ID NO: 4): the extracellular domain of human CLEC12A (corresponding residues 69-265 of SEQ ID NO: 1) comprising the dipeptide linker / spacer “TK” (residues 1-2). Potential citrullination sites in all ectoClecl2A constructs are shaded.

[0100] Fig. 2A shows the CLEC12A-PAD2 (Protein-arginine deiminase type-2 enzyme) complex predicted by AlphaFold™-Multimer. CLEC12A (right) and PAD2 (left) complex and close-up of CLEC12A-PAD2 interaction is shown. CLEC12A has R185, R201, R204, R232 and R245 (with respect to SEQ ID NO: 1) in contact with the active site (D351, H471, D473 and C647) in the C-terminal catalytic domain of PAD2 (SEQ ID NO: 7). Fig. 2B shows the CLEC12A-PAD4 complex. The 3D homology model (105-253 amino acid) of CLEC12A (Uniprot ID: Q5QGZ9) was constructed by the modeling software Modeller (Webb et al.), using as templates from PDB database, 3g81_A (105-125 amino acid) and lyxk B (126-253 amino acid). Model quality was assessed by Ramachandran plot analysis through PROCHECK (Laskowski et al.). Structure images were generated using PyMOL. CLEC12A (left) and PAD4 (right) complex and close-up of CLEC12A-PAD4 interaction is shown. CLEC12A has R135 (with respect to SEQ ID NO: 1) in contact with the active site of PAD4.

[0101] Fig. 3A shows the carboxymethylated, citrullinated, deamidated, and oxidated residues in residues 27-262 of IL3ss-FLAG-ectoCLEC12A No Arg (SEQ ID NO: 3) by mass spectrometry before incubation with PAD2 in vitro. Fig. 3B shows the carboxymethylated, citrullinated, deamidated, and oxidated residues in residues 27-262 of IL3ss-FLAG-ectoCLEC12A No Arg (SEQ ID NO: 3) by mass spectrometry after incubation with PAD2 in vitro. Citrullinated arginines are represented in boxes. As shown, mass spectrometry of CLEC12A-ecto confirmed that PAD2 citrullinates all arginines of CLEC12A-ecto. Notably, the predicted arginines R135, R185, R201, R204, R232 and R245 of CLEC12A from Figs. 2A and 2B were citrullinated by PAD2.

[0102] These data indicate that recombinant CLEC12A-ecto can be produced and citrullinated by PAD in vitro, an enzyme that CLEC12A is predicted to interact with from docking analysis. RA patients are seropositive for anti-citCLEC12A autoantibodies

[0103] Next, sera from RA patients and healthy controls were evaluated for the presence and levels of anti-CLEC12A and anti-citCLEC12A autoantibodies. The patient cohort screened for anti-citCLEC12A seropositivity was comprised of 68 RA patients with a median age of 59 (IQR 14) years and a disease duration of 4 (IQR14) years (Table 2). Most patients were women (79%) and 58% of patients had a history of smoking.

[0104] Table 2: Characteristics of RA patients

[0105] Fig. 4A and Table 2 show anti-CitCLEC12A (citmllinated CLEC12A) antibody levels in rheumatoid arthritis patient sera. Sera from rheumatoid arthritis (RA) patients, n=68 (circles) and healthy controls (HC), n=36 (squares) were tested for IgG antibodies targeting CitCLEC12A using ELISA. Each symbol represents a study subject. The solid lines represent the median values with error bars for IQR (interquartile range). The dashed line represents the cut-off value for positivity (24. 1 RU / mL). Each symbol represents a study subject. ***p-value < 0.001; ****p-value < 0.0001 by the Mann-Whitney test. As shown, forty percent (40%) (27 / 68) of RA patients were positive for anti-citCLEC12A autoantibodies (Table 2 and Fig. 4A). These data were confirmed in a validation cohort of 36 RA patients, whereby 47% of RA patients were seropositive for anti-citCLEC12A autoantibodies (data not shown). Furthermore, similar to the data in Fig. 4A and Table 2, significant levels of anti-citCLEC12A autoantibodies were observed in RA patients of the validation cohort as compared to healthy controls (data not shown).

[0106] Table 3 shows the correlation between these anti-citCLEC12A autoantibodies and the inflammation marker ESR (erythrocyte sedimentation rate; p = 0.0287). The correlation with inflammation is supported by a tendency of these autoantibodies to associate with MDGlobal (physician global assessment of disease severity; p = 0.0518) (Table 3). Thus, the presence of these anti- citCLEC12A autoantibodies is associated with inflammation in RA patients and a strong tendency to be associated with more severe disease as per the MDGlobal assessment.

[0107] Table 3: Correlation of anti-citCLEC12A autoantibodies with severe outcomes in RA

[0108] 1Based on Wilcoxon Mann Whitney Test.

[0109] *** <0.001; ** <0.01; * <0.05;T< 0.15

[0110] To evaluate whether these anti-CitCLEC12A autoantibodies are cross-reactive with uncitrullinated CLEC12A, sera from RA patients and healthy controls were assayed by ELISA using CLEC12A and citCLEC12A as the coating antigen. Fig. 4B shows anti-CLEC12A and anti-CitCLEC12A (citrullinated CLEC12A) antibody levels in rheumatoid arthritis patient sera. Sera from rheumatoid arthritis (RA) patients, n=68 (circles) and healthy controls (HC), n=36 (squares) were tested for IgG antibodies targeting CLEC12A or CitCLEC12A using ELISA. Each symbol represents a study subject. ***p-value < 0.001; ****p-value < 0.0001 by the Mann-Whitney test. As shown, there was a significant increase in anti-CitCLEC12A with respect to anti-CLEC12A, indicating that these autoantibodies are specific for citCLEC12A.

[0111] Together, these data indicate that citCLEC12A is a novel autoantigen in RA.

[0112] Seropositivity for anti-citCLEC12A autoantibodies correlates with age

[0113] To determine the clinical relevance of anti-CitCLEC12A autoantibodies, we determined whether they correlated with patient characteristics. In our discovery cohort, seropositive patients for anti- citCLEC12A were younger (p= 0.0058) than those that tested negative for anti-CitCLEC12A antibodies (p=0.0058) (Table 2). While RA patients positive for anti-citCLEC12A antibodies were more likely to be smokers and achieve remission and reported less joint stiffness, this did not reach statistical significance, likely due to low patient numbers in the analysis and limited information with respect to the patients in the analysis. Collectively, these data indicate that in established RA patients, anti-citCLEC12A positivity inversely correlates with age.

[0114] Seropositivity for anti-citCLEC12A autoantibodies and other citrullinated antigens partially overlap

[0115] The routine test utilized in the clinic setting that identifies autoantibodies to citrullinated antigens, CCPs, is not specific to one autoantigen, but is able to detect autoantibodies to many citrullinated proteins in 70% of patients (n >50 patients). Many generations of CCP tests are currently being utilized but none, especially on their own, can accurately diagnose RA or determine disease severity or progression (e.g., prognosis). For example, the CCP2 test is a standard of care test to assist and / or confirm a diagnosis of RA and is a component of RA classification criteria utilized in clinical trials, but alone, cannot accurately diagnose RA.

[0116] Since ACPA (Anti-citrullinated protein antibodies) exhibit cross-reactivity with several citrullinated antigens, we compared anti-citCLEC12A seropositivity to the presence of autoantibodies against known citrullinated autoantigens. Most patients positive for anti-citCLEC12A antibodies (70%) were seropositive for CCP, CitJED and HomoCitJED, a small proportion of patients (7%; see “None” referring to the proportion seropositive to other citrullinated antigens) were positive only for citCLEC12A antibodies (Fig. 5A). In contrast, only 30% of RA patients negative for citCLEC12A were seropositive for CCP, CitJED and HomoCitJED (Fig. 5B). We also observed a potential, but weak correlation between levels of anti-CitCLEC12A and anti-CCP2 (Fig. 6) indicative that CLEC12A seropositivity is likely nonoverlapping with that of other seropositivities in RA. There is thus, a partial overlap between citCLEC12A seropositivity and autoantibodies against other known citrullinated autoantigens. These data were confirmed in a validation cohort of 36 RA patients, whereby 8.3% of RA patients were seropositive for citCLECL12A but not any other citrullinated antigen, including CCPs (data not shown).

[0117] Taken together, these data indicate that anti-citCLEC12A autoantibodies may be used as diagnostic and / or prognostic markers for RA or early RA and / or markers for predicting the onset of RA, early RA, or severe outcomes in RA. These data further suggest that citCLEC12A reactivity may be distinct from the reactivity to other autoantigens, and therefore a potentially highly specific or sensitive marker for RA.

[0118] Example 3: Discussion

[0119] ACPA are a hallmark of RA and recognize a diverse array of antigens collectively known as the RA citrullinome. We report CLEC12A as a novel citrullinated autoantigen that has not previously been identified in RA patients. We also show that autoantibodies to citCLEC12A are present in a significantly higher proportion of younger RA patients.

[0120] Seropositivity for citCLEC12A in RA patients was determined using CLEC12A-ecto produced in a eukaryotic cell line and citrullinated with PAD2. This approach optimizes for the detection of autoantibodies that bind conformational-dependent, citrullinated epitopes. While several studies reported that citrullinated epitopes on small peptides can bind antibodies in RA patients, these specificities require confirmation with the native protein. ACPA seem to bind citrullinated histone, vimentin and fibrinogen in a conformation-dependent manner (Roman-Melendez). The use of the PAD2 enzyme to citrullinate CLEC12A-ecto is also relevant to RA as both PAD2 and PAD4 generate citrullinated epitopes that are immunogenic in RA. While it remains unknown whether CLEC12A-ecto is preferentially citrullinated by PAD2 or PAD4, our in silico, docking data indicate that CLEC12A-ecto interacts with PAD4. It is highly likely that both PAD enzymes citrullinate CLEC12A as it harbours 3 arginines within a PAD2 citrullination motif (RXXXXK; in Larman) and one arginine within a known PAD4 citrullination motif (RGXXXX) in its extracellular domain. We thus consider the citCLEC12A preparation used in this study is representative of citrullination events that may occur on this inhibitory receptor in RA.

[0121] Without wishing to be bound to a specific theory, the present inventors believe that the use of a natively folded CLEC12A preparation in our study may explain why anti-citCLEC12A antibodies have not yet been reported in the literature. The inventors believe that the immunogenicity of citCLEC12A may be conformation-dependent, which means that these autoantibodies would not be identified by high throughput screens of citrullinated peptides of the human proteome to screen for ACPA reactivities in RA sera. Moreover, most studies use serum to screen for ACPA and CLEC12A is a cell-surface receptor that we have not been able to detect in cell-free blood (unpublished data). In addition, some studies limit their analysis to known ACPA specificities.

[0122] We observed high levels of reactivity of RA sera to citCLEC12A. This observation supports the notion that citCLEC12A is a bona fide, RA autoantigen. Increased antibody binding suggests high avidity of an antibody towards its antigen, a criterion considered to confirm the specificity of ACPA in previous studies.

[0123] Similar to other ACPA ELISAs, a significant proportion of RA patients are seropositive for anti- citCLEC12A antibodies and exhibit cross-reactivity with other ACPA specificities. This cross-reactivity is partly explained by the recognition of arginine motifs shared by many proteins that can be citrullinated, defined in many cases by the presence of a glycine in the +1, +2 or +3 position after the arginine. More research is required, however, to identify the arginines that are predominantly citrullinated on CLEC12A in RA patients. Our in silico analysis indicates that the most likely arginine citrullinated by PAD4 is R135. The observation that anti-citCLEC12A antibodies are more prevalent in younger patients is clinically relevant as it indicates that these autoantibodies are highly likely characteristic of the earlier stages of RA. The presence of anti-citCLE12A antibodies during the early phase of RA would support the potential of these autoantibodies as prognostic biomarkers for RA. They may also be predictive of a response to treatment as reported for ACPA reactivity detected with commercial kits.

[0124] While many potential, citrullinated autoantigens have been identified in RA, the impact of these citrullination events on the function of these proteins remains incompletely understood. CLEC12A is an inhibitory receptor that dampens activation signaling pathways. Since citrullination can modify protein structure and function, this post-translational modification is a potential mechanism through which CLEC12A function is dysregulated in RA. In addition, binding of anti-citCLEC12A antibodies to the receptor may induce its internalization, diminishing its inhibitory activity and enhancing inflammation. While we did not observe an association between anti-citCLEC12A seropositivity and joint stiffness in this discovery cohort, a study in a larger validation cohort of RA patients in addition to mechanistic studies are needed to address this question.

[0125] Collectively, our observations indicate that citCLEC12A is a novel autoantigen in RA.

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[0139] Webb, B. et al. Comparative Protein Structure Modeling Using MODELLER. Curr Protoc Bioinformatics. 2016 Jun 20;54:5.6.1-5.6.37. doi: 10.1002 / cpbi.3.

[0140] In some aspects, embodiments of the present disclosure as described herein include the following items:

[0141] 1. A method for identifying a subject having rheumatoid arthritis (RA) by detecting whether autoantibodies specific to citrullinated-CLEC12A (C-type lectin domain family 12 member A) are present; wherein said method comprises: a) contacting a biological sample from a subject with citrullinated-CLEC12A to form a complex between autoantibodies and citrullinated-CLEC12A; b) detecting the complex between the autoantibodies and citrullinated-CLEC12A; c) optionally, measuring the levels of the autoantibodies specific to citrullinated-CLEC12A; and d) identifying the subject having RA when the autoantibodies specific to citrullinated-CLEC12A relative to a control is present. A method for the treatment of rheumatoid arthritis (RA) in an RA subject in need thereof, wherein said method comprises: a) identifying an RA subject by detecting whether autoantibodies specific to citrullinated- CLEC12A (C-type lectin domain family 12 member A) are present by a method which comprises: i. contacting a biological sample from a human subject suspected of suffering or suffering from RA with citrullinated-CLEC12A to form a complex between the autoantibodies and citrullinated-CLEC12A; ii. detecting the complex between the autoantibodies and citrullinated-CLEC12A; iii. optionally, measuring the levels of autoantibodies specific to citrullinated-CLEC12A; and iv. identifying the RA subject when the autoantibodies specific to citrullinated- CLEC12A relative to a control is present; and b) when the RA subject is identified, treating the RA subject with anti-RA therapy. A method for diagnosing or determining a progression of rheumatoid arthritis RA) in a subject (e.g., human subject suspected of suffering or suffering from RA), said method comprising: a) optionally, providing a biological sample from the subject; b) detecting autoantibody levels specific to citrullinated-CLEC12A (C-type lectin domain family 12 member A) in the sample; and c) diagnosing the subject as having RA or determining the progression of RA by observing significantly increased autoantibody levels of specific to citrullinated-CLEC12A as compared to a subject not having RA. A method for clinically assessing rheumatoid arthritis (RA) in a subject (e.g., human subject suspected of suffering or suffering from RA), said method comprising: a) quantifying the level of autoantibodies specific to citrullinated-CLEC12A (C-type lectin domain family 12 member A) in a sample from the subject; and b) comparing the level of autoantibodies specific to citrullinated-CLEC12A (C-type lectin domain family 12 member A) to a suitable reference value indicative of the presence, stage and / or progression of RA, thereby clinically assessing RA in the subject. The method of item 4, wherein said clinical assessment comprises diagnosing RA in the subject, staging RA in the subject, predicting a response to treatment, and / or monitoring the progression of RA in the subject. An in vitro method for preparing a clinical human sample, said method comprising: a) optionally, identifying a human subject as having or suspected of having rheumatoid arthritis (RA); b) obtaining a sample comprising autoantibodies specific to citrullinated-CLEC12A (C-type lectin domain family 12 member A) from the human subject; and c) quantifying the level of the autoantibodies specific to citrullinated-CLEC12A in the sample or detecting autoantibodies specific to citrullinated-CLEC12A in the sample. An in vitro method for processing a clinical human sample, said method comprising: a) optionally, identifying a human subject clinically assessed as having or suspected of having rheumatoid arthritis (RA); b) obtaining a sample comprising autoantibodies specific to citrullinated-CLEC12A (C-type lectin domain family 12 member A) from the human subject; and c) quantifying the level of the autoantibodies specific to citrullinated-CLEC12A in the sample or detecting autoantibodies specific to citrullinated-CLEC12A in the sample. A method of detecting one or more autoantibodies specific to citrullinated-CLEC12A (C-type lectin domain family 12 member A) in a biological sample from a human subject having or suspected of having rheumatoid arthritis (RA); said method comprising: a) contacting the biological sample from the human subject with citrullinated-CLEC12A to form a complex between autoantibodies and citrullinated-CLEC12A; b) detecting the complex between the autoantibodies and citrullinated-CLEC12A; and c) optionally, measuring the levels of the autoantibodies specific to citrullinated-CLEC12A. The method of any one of items 1 to 8, wherein the detecting or quantifying the level of the autoantibody / autoantibodies is determined using an immunoassay. The method of any one of items 1 to 9, wherein the subject is a human. The method of any one of items 1 to 10, wherein the sample is blood, serum, plasma, synovial fluid, sputum, bronchoalveolar lavage (BAL), live cells, dying cells, dead cells, or cell lysates. The method of any one of items 1 to 11, wherein the method further comprises a step of detecting or identifying the levels of one or more autoantibodies known to be associated with the identification, diagnosis or progression of said RA (e.g., anti-rheumatoid factor (RF), anti -cyclic citrullinated peptides (CCP1, CCP2, CCP3), anti-citrullinated protein antibodies (ACPA), anti-carbamylated protein (anti-CarP) antibodies, anti-synthetic citrullinated peptide (CitJED), and anti- homocitrullinated peptide (HomoCitJED)). The method of any one of items 1 to 12, wherein the method further comprises a step of determining whether one or more criteria selected from the group consisting of American College of Rheumatology (ACR) classification criteria and European League Against Rheumatism (EULAR) classification criteria are present. The method of any one of items 1 to 13, wherein the RA is early RA (early onset arthritis). A kit for use in diagnosing or determining the progression of rheumatoid arthritis (RA) in a subject, said kit comprising one or more reagents for detecting autoantibodies specific to citrullinated- CLEC12A (C-type lectin domain family 12 member A) in a biological sample from the subject. The kit of item 15, wherein the detecting of autoantibody levels is determined using an immunoassay. The kit of item 15 or 16, wherein the subject is a human. The kit of any one of items 15 to 17, wherein the sample is blood, serum, plasma, synovial fluid, sputum, bronchoalveolar lavage (BAL), live cells, dying cells, dead cells, or cell lysates. The kit for use of any one of items 15 to 18, further comprising reagents for detecting one or more autoantibodies known to be associated with the identification, diagnosis or progression of said RA (e.g., anti-rheumatoid factor (RF), anti-cyclic citrullinated peptides (CCP1, CCP2, CCP3), anti- citmllinated protein antibodies (ACPA), anti-carbamylated protein (anti-CarP) antibodies, antisynthetic citrullinated peptide (CitJED), and anti-homocitrullinated peptide (HomoCitJED)). The kit for use of any one of items 14 to 19, wherein the kit is used in combination with the detection of one or more autoantibodies known to be associated with the identification, diagnosis or progression of said RA (e.g., anti-rheumatoid factor (RF), anti-cyclic citrullinated peptides (CCP1, CCP2, CCP3), anti-citrullinated protein antibodies (ACPA), anti-carbamylated protein (anti-CarP) antibodies, antisynthetic citrullinated peptide (CitJED), and anti-homocitrullinated peptide (HomoCitJED)). The kit for use of any one of items 14 to 20, wherein the kit is used in combination with one or more criteria selected from one or more known diagnostic or classification measures of RA (e.g., American College of Rheumatology (ACR) classification criteria and European League Against Rheumatism (EULAR) classification criteria). A method of producing a complex comprising citrullinated-CLEC12A (C-type lectin domain family 12 member A) and one or more autoantibodies specific to the citrullinated-CLEC12A; wherein said method comprises: a) contacting a biological sample from a subject comprising one or more autoantibodies, with citrullinated-CLEC12A, wherein the subject has or is suspected to have rheumatoid arthritis (RA); and b) detecting the complex between the autoantibodies and citrullinated-CLEC12A. The method of item 22, further comprising one or more features as defined in any one of items 9 to 14. A complex for use in the identification, prognosis, diagnosis, or determination of progression of rheumatoid arthritis (RA) in a subject, wherein the complex comprises or is formed by citrullinated- CLEC12A (C-type lectin domain family 12 member A) and autoantibodies specific to the citrullinated-CLEC 12A. A complex for use as a research tool in the detection of autoantibodies specific to by citrullinated- CLEC12A (C-type lectin domain family 12 member A) in a biological sample, wherein the complex comprises citrullinated-CLEC 12A and autoantibodies specific to citrullinated-CLEC 12A. A kit for use in the detection of autoantibodies specific to citrullinated-CLEC 12A (C-type lectin domain family 12 member A) in a biological sample, wherein the kit comprises citrullinated- CLEC12A and reagents for the detection of the autoantibodies. A method of manufacturing the kit as defined in any one of items 15 to 21 and 26, said method comprising: a) immobilizing the citrullinated-CLEC 12A; and b) providing reagents for the detection of autoantibodies specific to the citrullinated-CLEC 12A. A method for producing or modifying a test for detecting rheumatoid arthritis (RA), the method comprising adding or integrating into said test quantifying the level of autoantibodies specific to citrullinated-CLEC 12A (C-type lectin domain family 12 member A) in a biological sample or detecting autoantibodies specific to citrullinated-CLEC 12A in a biological sample. The method of item 28, further comprising one or more features as defined in any one of items 9 to 14. A recombinant CLEC12A (C-type lectin domain family 12 member A) polypeptide comprising an extracellular or ectodomain from a CLEC12a (e.g., SEQ ID NO: 1), wherein the recombinant CLEC12A is citrullinated. The recombinant CLEC12A polypeptide of item 30, wherein the recombinant CLEC12A is citrullinated by a PAD (Protein-arginine deiminase) enzyme (e.g., PAD1, PAD2, PAD3, PAD4, PAD5, or PAD6). The recombinant CLEC12A polypeptide of item 30 or 31, wherein the recombinant CLEC12A is citrullinated on one or more arginines residues corresponding to, homologous to, or positionally equivalent to the arginines selected from the group consisting of R104, R119, R135, R136, R185, R201, R204, R217, R232, R245, and R263 of SEQ ID NO: 1. The recombinant CLEC12A polypeptide of any one of items 30 to 32, wherein the extracellular or ectodomain of CLEC12a comprises an amino acid sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to residues 67-265 of SEQ ID NO: 1. The recombinant CLEC12A polypeptide of any one of items 30 to 33, comprising an amino acid sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 2 One or more peptides of the recombinant CLEC12A polypeptides as defined in any one of items 30 to 34, wherein the one or more peptides comprise one or more citrullinated residues. The one or more peptides of item 35, comprising a citrullination on one or more arginines residues corresponding to, homologous to, or positionally equivalent to the arginines selected from the group consisting of R104, R119, R135, R136, R185, R201, R204, R217, R232, R245, and R263 of SEQ ID NO: 1. The recombinant CLEC12A polypeptide of any one of items 30 to 34 or the one or more peptides of item 35 or 36, for use in the identification, prognosis, diagnosis, or determination of progression of rheumatoid arthritis (RA) in a subject. The recombinant CLEC12A polypeptide of any one of items 30 to 34 or the one or more peptides of item 35 or 36, for use in the method of any one of items 1 to 14, 22, 23, and 27 to 29 or in the kit of any one of items 15 to 21. Use of the recombinant CLEC12A polypeptide of any one of items 30 to 34 or the one or more peptides of item 35 or 36 for the identification, prognosis, diagnosis, or determination of progression of rheumatoid arthritis (RA) in a subject. Use of the recombinant CLEC12A polypeptide of any one of items 30 to 34 or the one or more peptides of item 35 or 36 in the method of any one of items 1 to 14, 22, 23, and 27 to 29 or in the kit of any one of items 15 to 21. A polynucleotide encoding the recombinant CLEC12A polypeptide as defined in any one of items 30 to 34 or the one or more peptides of item 35 or 36. A vector comprising the polynucleotide of item 41.

Claims

CLAIMS:

1. A method for identifying a subject having rheumatoid arthritis (RA) by detecting whether autoantibodies specific to citrullinated-CLEC12A (C-type lectin domain family 12 member A) are present; wherein said method comprises: a) contacting a biological sample from a subject with citrullinated-CLEC12A to form a complex between autoantibodies and citrullinated-CLEC12A; b) detecting the complex between the autoantibodies and citrullinated-CLEC12A; c) optionally, measuring the levels of the autoantibodies specific to citrullinated-CLEC12A; and d) identifying the subject having RA when the autoantibodies specific to citrullinated-CLEC12A relative to a control is present.

2. A method for the treatment of rheumatoid arthritis (RA) in an RA subject in need thereof, wherein said method comprises: a) identifying an RA subject by detecting whether autoantibodies specific to citrullinated- CLEC12A (C-type lectin domain family 12 member A) are present by a method which comprises: i. contacting a biological sample from a human subject suspected of suffering or suffering from RA with citrullinated-CLEC12A to form a complex between the autoantibodies and citrullinated-CLEC12A; ii. detecting the complex between the autoantibodies and citrullinated-CLEC12A; iii. optionally, measuring the levels of autoantibodies specific to citrullinated-CLEC12A; and iv. identifying the RA subject when the autoantibodies specific to citrullinated- CLEC12A relative to a control is present; and b) when the RA subject is identified, treating the RA subject with anti-RA therapy.

3. A method for diagnosing or determining a progression of rheumatoid arthritis RA) in a subject (e.g., human subject suspected of suffering or suffering from RA), said method comprising: a) optionally, providing a biological sample from the subject; b) detecting autoantibody levels specific to citrullinated-CLEC12A (C-type lectin domain family 12 member A) in the sample; andc) diagnosing the subject as having RA or determining the progression of RA by observing significantly increased autoantibody levels of specific to citrullinated-CLEC12A as compared to a subject not having RA.

4. A method for clinically assessing rheumatoid arthritis (RA) in a subject (e.g., human subject suspected of suffering or suffering from RA), said method comprising: a) quantifying the level of autoantibodies specific to citrullinated-CLEC12A (C-type lectin domain family 12 member A) in a sample from the subject; and b) comparing the level of autoantibodies specific to citrullinated-CLEC12A (C-type lectin domain family 12 member A) to a suitable reference value indicative of the presence, stage and / or progression of RA, thereby clinically assessing RA in the subject.

5. The method of claim 4, wherein said clinical assessment comprises diagnosing RA in the subject, staging RA in the subject, predicting a response to treatment, and / or monitoring the progression of RA in the subject.

6. An in vitro method for preparing a clinical human sample, said method comprising: a) optionally, identifying a human subject as having or suspected of having rheumatoid arthritis (RA); b) obtaining a sample comprising autoantibodies specific to citrullinated-CLEC12A (C-type lectin domain family 12 member A) from the human subject; and c) quantifying the level of the autoantibodies specific to citrullinated-CLEC12A in the sample or detecting autoantibodies specific to citrullinated-CLEC12A in the sample.

7. An in vitro method for processing a clinical human sample, said method comprising: a) optionally, identifying a human subject clinically assessed as having or suspected of having rheumatoid arthritis (RA); b) obtaining a sample comprising autoantibodies specific to citrullinated-CLEC12A (C-type lectin domain family 12 member A) from the human subject; and c) quantifying the level of the autoantibodies specific to citrullinated-CLEC12A in the sample or detecting autoantibodies specific to citrullinated-CLEC12A in the sample.

8. A method of detecting one or more autoantibodies specific to citrullinated-CLEC12A (C-type lectin domain family 12 member A) in a biological sample from a human subject having or suspected of having rheumatoid arthritis (RA); said method comprising: a) contacting the biological sample from the human subject with citrullinated-CLEC12A to form a complex between autoantibodies and citrullinated-CLEC12A; b) detecting the complex between the autoantibodies and citrullinated-CLEC12A; and c) optionally, measuring the levels of the autoantibodies specific to citrullinated-CLEC12A.

9. The method of any one of claims 1 to 8, wherein the detecting or quantifying the level of the autoantibody / autoantibodies is determined using an immunoassay.

10. The method of any one of claims 1 to 9, wherein the subject is a human.

11. The method of any one of claims 1 to 10, wherein the sample is blood, serum, plasma, synovial fluid, sputum, bronchoalveolar lavage (BAL), live cells, dying cells, dead cells, or cell lysates.

12. The method of any one of claims 1 to 11, wherein the method further comprises a step of detecting or identifying the levels of one or more autoantibodies known to be associated with the identification, diagnosis or progression of said RA (e.g., anti-rheumatoid factor (RF), anti -cyclic citrullinated peptides (CCP1, CCP2, CCP3), anti-citrullinated protein antibodies (ACPA), anti-carbamylated protein (anti-CarP) antibodies, anti-synthetic citrullinated peptide (CitJED), and anti- homocitrullinated peptide (HomoCitJED)).

13. The method of any one of claims 1 to 12, wherein the method further comprises a step of determining whether one or more criteria selected from the group consisting of American College of Rheumatology (ACR) classification criteria and European League Against Rheumatism (EULAR) classification criteria are present.

14. The method of any one of claims 1 to 13, wherein the RA is early RA (early onset arthritis).

15. A kit for use in diagnosing or determining the progression of rheumatoid arthritis (RA) in a subject, said kit comprising one or more reagents for detecting autoantibodies specific to citrullinated- CLEC12A (C-type lectin domain family 12 member A) in a biological sample from the subject.

16. The kit of claim 15, wherein the detecting of autoantibody levels is determined using an immunoassay.

17. The kit of claim 15 or 16, wherein the subject is a human.

18. The kit of any one of claims 15 to 17, wherein the sample is blood, serum, plasma, synovial fluid, sputum, bronchoalveolar lavage (BAL), live cells, dying cells, dead cells, or cell lysates.

19. The kit for use of any one of claims 15 to 18, further comprising reagents for detecting one or more autoantibodies known to be associated with the identification, diagnosis or progression of said RA (e.g., anti-rheumatoid factor (RF), anti-cyclic citrullinated peptides (CCP1, CCP2, CCP3), anti- citmllinated protein antibodies (ACPA), anti-carbamylated protein (anti-CarP) antibodies, antisynthetic citrullinated peptide (CitJED), and anti-homocitrullinated peptide (HomoCitJED)).

20. The kit for use of any one of claims 14 to 19, wherein the kit is used in combination with the detection of one or more autoantibodies known to be associated with the identification, diagnosis or progression of said RA (e.g., anti-rheumatoid factor (RF), anti-cyclic citrullinated peptides (CCP1, CCP2, CCP3), anti-citrullinated protein antibodies (ACPA), anti-carbamylated protein (anti-CarP) antibodies, anti-synthetic citrullinated peptide (CitJED), and anti-homocitrullinated peptide (HomoCitJED)).

21. The kit for use of any one of claims 14 to 20, wherein the kit is used in combination with one or more criteria selected from one or more known diagnostic or classification measures of RA (e.g., American College of Rheumatology (ACR) classification criteria and European League Against Rheumatism (EULAR) classification criteria).

22. A method of producing a complex comprising citrullinated-CLEC12A (C-type lectin domain family 12 member A) and one or more autoantibodies specific to the citrullinated-CLEC12A; wherein said method comprises: a) contacting a biological sample from a subject comprising one or more autoantibodies, with citrullinated-CLEC12A, wherein the subject has or is suspected to have rheumatoid arthritis (RA); and b) detecting the complex between the autoantibodies and citrullinated-CLEC12A.

23. The method of claim 22, further comprising one or more features as defined in any one of claims 9 to 14.

24. A complex for use in the identification, prognosis, diagnosis, or determination of progression of rheumatoid arthritis (RA) in a subject, wherein the complex comprises or is formed by citrullinated- CLEC12A (C-type lectin domain family 12 member A) and autoantibodies specific to the citrullinated-CLEC 12A.

25. A complex for use as a research tool in the detection of autoantibodies specific to by citrullinated- CLEC12A (C-type lectin domain family 12 member A) in a biological sample, wherein the complex comprises citrullinated-CLEC 12A and autoantibodies specific to citrullinated-CLEC 12A.

26. A kit for use in the detection of autoantibodies specific to citrullinated-CLEC 12A (C-type lectin domain family 12 member A) in a biological sample, wherein the kit comprises citrullinated- CLEC12A and reagents for the detection of the autoantibodies.

27. A method of manufacturing the kit as defined in any one of claims 15 to 21 and 26, said method comprising: a) immobilizing the citrullinated-CLEC 12A; and b) providing reagents for the detection of autoantibodies specific to the citrullinated-CLEC 12A.

28. A method for producing or modifying a test for detecting rheumatoid arthritis (RA), the method comprising adding or integrating into said test quantifying the level of autoantibodies specific to citrullinated-CLEC 12A (C-type lectin domain family 12 member A) in a biological sample or detecting autoantibodies specific to citrullinated-CLEC 12A in a biological sample.

29. The method of claim 28, further comprising one or more features as defined in any one of claims 9 to 14.

30. A recombinant CLEC12A (C-type lectin domain family 12 member A) polypeptide comprising an extracellular or ectodomain from a CLEC12a (e.g., SEQ ID NO: 1), wherein the recombinant CLEC12A is citrullinated.

31. The recombinant CLEC12A polypeptide of claim 30, wherein the recombinant CLEC12A is citmllinated by a PAD (Protein-arginine deiminase) enzyme (e.g., PAD1, PAD2, PAD3, PAD4, PAD5, or PAD6).

32. The recombinant CLEC12A polypeptide of claim 30 or 31, wherein the recombinant CLEC12A is citmllinated on one or more arginines residues corresponding to, homologous to, or positionally equivalent to the arginines selected from the group consisting of R104, R119, R135, R136, R185, R201, R204, R217, R232, R245, and R263 of SEQ ID NO: 1.

33. The recombinant CLEC12A polypeptide of any one of claims 30 to 32, wherein the extracellular or ectodomain of CLEC12a comprises an amino acid sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to residues 67-265 of SEQ ID NO: 1.

34. The recombinant CLEC12A polypeptide of any one of claims 30 to 33, comprising an amino acid sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO:

235. One or more peptides of the recombinant CLEC12A polypeptides as defined in any one of claims 30 to 34, wherein the one or more peptides comprise one or more citmllinated residues.

36. The one or more peptides of claim 35, comprising a citmllination on one or more arginines residues corresponding to, homologous to, or positionally equivalent to the arginines selected from the group consisting of R104, R119, R135, R136, R185, R201, R204, R217, R232, R245, and R263 of SEQ ID NO: 1.

37. The recombinant CLEC12A polypeptide of any one of claims 30 to 34 or the one or more peptides of claim 35 or 36, for use in the identification, prognosis, diagnosis, or determination of progression of rheumatoid arthritis (RA) in a subject.

38. The recombinant CLEC12A polypeptide of any one of claims 30 to 34 or the one or more peptides of claim 35 or 36, for use in the method of any one of claims 1 to 14, 22, 23, and 27 to 29 or in the kit of any one of claims 15 to 21.

39. Use of the recombinant CLEC12A polypeptide of any one of claims 30 to 34 or the one or more peptides of claim 35 or 36 for the identification, prognosis, diagnosis, or determination of progression of rheumatoid arthritis (RA) in a subject.

40. Use of the recombinant CUEC12A polypeptide of any one of claims 30 to 34 or the one or more peptides of claim 35 or 36 in the method of any one of claims 1 to 14, 22, 23, and 27 to 29 or in the kit of any one of claims 15 to 21.

41. A polynucleotide encoding the recombinant CLEC12A polypeptide as defined in any one of claims 30 to 34 or the one or more peptides of claim 35 or 36.

42. A vector comprising the polynucleotide of claim 41.

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