WO2023275235A1 - Method and means for diagnosis of spondyloarthritis - Google Patents

Method and means for diagnosis of spondyloarthritis Download PDF

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Publication number
WO2023275235A1
WO2023275235A1 PCT/EP2022/068047 EP2022068047W WO2023275235A1 WO 2023275235 A1 WO2023275235 A1 WO 2023275235A1 EP 2022068047 W EP2022068047 W EP 2022068047W WO 2023275235 A1 WO2023275235 A1 WO 2023275235A1
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seq
antibody
peptide
variant
binding fragment
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PCT/EP2022/068047
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French (fr)
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Veerle Somers
Patrick VANDORMAEL
Dana QUADEN
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Universiteit Hasselt
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/564—Immunoassay; Biospecific binding assay; Materials therefor for pre-existing immune complex or autoimmune disease, i.e. systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, rheumatoid factors or complement components C1-C9
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/10—Musculoskeletal or connective tissue disorders

Definitions

  • the present invention in general relates to the field of antibody profiling in spondyloarthritis.
  • the invention provides methods and means for diagnosing spondyloarthritis, for evaluating the disease severity of spondyloarthritis and/or for evaluating the response of a subject diagnosed with spondyloarthritis to therapeutic treatment based on antibody profiling.
  • Axial spondyloarthritis is a rheumatic disease affecting between 0.5 and 1.4% of the population in Europe and North America, and is mainly characterized by chronic inflammation of spinal and sacroiliac joints. Diagnosis of axSpA is challenging since clinical manifestations, such as inflammatory back pain, peripheral arthritis and inflammatory bowel disease often overlap with other disorders. In the absence of diagnostic criteria, classification criteria developed by the Assessment in SpondyloArthritis international Society (ASAS) are often used for diagnosis in patients with suspect of axial or peripheral spondyloarthritis.
  • SpondyloArthritis international Society are often used for diagnosis in patients with suspect of axial or peripheral spondyloarthritis.
  • HLA human leukocyte antigen
  • CRP C-reactive protein
  • testing for antibodies to these 3 UH-axSpA-lgG peptides increased the post-test probability for axSpA from 79% to 91%, and could therefore be a valuable addition to the current diagnostic process (Quaden et al., Arthritis Rheumatol. 2020; 72(12): 2094-105).
  • one limitation to the use of antibodies against this panel of 3 UH-axSpA-lgG peptides is the limited sensitivity, leaving about 85% of axSpA patients seronegative for these novel markers.
  • the inventors of the present application identified a novel tool for the diagnosis of spondyloarthritis (SpA), in particular in axial spondyloarthritis (axSpA).
  • the invention further provides tools for evaluating the disease severity of spondyloarthritis and for the evaluation of therapy response in patients with spondyloarthritis, in particular axSpA, More specific, in the present invention novel antibody biomarkers were identified that can identify SpA patients and that can be used for the evaluation of the disease severity or of the therapeutic treatment in SpA patients.
  • the present invention provides an in vitro method for diagnosing spondyloarthritis in a subject, said method comprising: a) providing a biological sample from the subject, and b) determining the presence or quantity of antibodies against one or more peptides; in particular 2, 3, or 4 peptides selected from the list consisting of SEQ ID No: 1 , SEQ ID No: 2, SEQ ID No: 3, SEQ ID No: 4, SEQ ID No: 5, SEQ ID No: 6, and SEQ ID No: 7, or against an immune-reactive fragment or antibody-binding fragment or variant thereof; wherein the presence of or an increase in antibody levels against at least one of said peptides or immune-reactive fragments or antibody-binding fragments or variants thereof in said biological sample compared to a reference sample is indicative for the presence of spondyloarthritis.
  • the present invention provides an in vitro method for diagnosing spondyloarthritis in a subject, said method comprising: a) providing a biological sample from the subject, and b) determining the presence or quantity of antibodies against a peptide as shown in SEQ ID No: 2, or an immune- reactive fragment or antibody-binding fragment or variant thereof, against a peptide as shown in SEQ ID NO: 1 or against an antibody-binding fragment or variant thereof, and against a peptide as shown in SEQ ID NO: 7 or against an antibody-binding fragment or variant thereof; wherein the presence or an increase in antibody levels against at least one of said peptides or immune-reactive fragments or antibody-binding fragments or variants thereof in said biological sample compared to a reference sample is indicative for the presence of spondyloarthritis in the subject.
  • the presence or an increase in antibody levels against the peptide as shown in SEQ ID NO: 2 or against an immune- reactive fragment or against an antibody-binding fragment or variant thereof compared to the reference sample is indicative for the presence of spondyloarthritis in the subject.
  • the presence or increase in antibody levels against the peptide as shown in SEQ ID NO: 2 or against an immune-reactive fragment or against an antibody-binding fragment or variant thereof, against the peptide as shown in SEQ ID NO 1 or against an immune-reactive fragment or an antibody-binding fragment or variant thereof, and against the peptide as shown in SEQ ID NO: 7 or against an immune-reactive fragment or an antibody-binding fragment or variant thereof, in said sample compared to the reference sample is indicative for the presence of spondyloarthritis in the subject.
  • the method of the invention further comprises determining the presence or quantity of antibodies against one or more peptides; in particular 2, 3 or 4 peptides, as shown in SEQ ID No: 3, SEQ ID No: 4, SEQ ID No: 5, and SEQ ID No: 6, or against an immune-reactive fragment or antibody-binding fragment or variant thereof, wherein the presence or an increase in antibody levels against one or more of said peptides or against said immune-reactive fragments or antibody-binding fragments or variants thereof compared to the reference sample is indicative for the presence of spondyloarthritis in the subject.
  • the method further comprises determining the presence or quantity of antibodies against one or more peptides; in particular 2, 3 or 4 peptides, as shown in SEQ ID No: 8, SEQ ID No: 9, SEQ ID No: 10, SEQ ID No: 11 , SEQ ID No: 12, SEQ ID No: 13, SEQ ID No: 14, SEQ ID No: 15, SEQ ID No: 16, SEQ ID No: 17, SEQ ID No: 18 and SEQ ID No: 19, or against an immune-reactive fragment or antibody-binding fragment or variant thereof in the first and second biological sample, wherein the presence or an increase in antibody levels against one or more of said peptides or against said immune-reactive fragments or antibody-binding fragments or variants thereof in the biological sample compared to the reference sample is indicative for the presence of spondyloarthritis in the subject.
  • the present invention provides an in vitro method for evaluating the disease severity of spondyloarthritis in a subject, said method comprising: a) providing a biological sample from the subject, and b) determining the presence or quantity of antibodies against one or more peptides; in particular 2, 3, or 4 peptides selected from the list consisting of SEQ ID No: 1 , SEQ ID No: 2, SEQ ID No: 3, SEQ ID No: 4, SEQ ID No: 5, SEQ ID No: 6, and SEQ ID No: 7, or against an immune-reactive fragment or antibody-binding fragment or variant thereof; wherein the presence of or an increase in antibody levels against at least one of said peptides or immune-reactive fragments or antibody-binding fragments or variants thereof in said biological sample compared to a reference sample is indicative for the disease severity of spondyloarthritis.
  • the present invention provides an in vitro method for evaluating the disease severity of spondyloarthritis in a subject, said method comprising: a) providing a biological sample from the subject, and b) determining the presence or quantity of antibodies against a peptide as shown in SEQ ID No: 2, or an immune-reactive fragment or antibody-binding fragment or variant thereof, against a peptide as shown in SEQ ID NO: 1 or against an antibody-binding fragment or variant thereof, and against a peptide as shown in SEQ ID NO: 7 or against an antibody-binding fragment or variant thereof; wherein the presence or an increase in antibody levels against at least one of said peptides or immune- reactive fragments or antibody-binding fragments or variants thereof in said biological sample compared to a reference sample is indicative for the disease severity of spondyloarthritis in the subject.
  • the presence or an increase in antibody levels against the peptide as shown in SEQ ID NO: 2 or against an immune-reactive fragment or against an antibody-binding fragment or variant thereof compared to the reference sample is indicative forthe disease severity of spondyloarthritis in the subject.
  • the present invention provides an in vitro method for evaluating a therapeutic treatment of spondyloarthritis in a subject, said method comprising: a) providing a first biological sample from the subject obtained before the start of the therapeutic treatment, b) providing a second biological sample from the subject obtained after therapeutic treatment, and c) determining the presence or quantity of antibodies against one or more peptides; in particular 2, 3, or 4 peptides selected from the list consisting of SEQ ID No: 1 , SEQ ID No: 2, SEQ ID No: 3, SEQ ID No: 4, SEQ ID No: 5, SEQ ID No: 6, and SEQ ID No: 7, or against an immune-reactive fragment or antibody-binding fragment or variant thereof in the first and second biological sample; wherein the presence of or a difference in antibody levels against at least one of said peptides or immune-reactive fragments or antibody-binding fragments or variants thereof in the second biological sample compared to the first biological sample indicates that the subject is responding to the therapeutic treatment.
  • the present invention provides an in vitro method for evaluating a therapeutic treatment of spondyloarthritis in a subject, said method comprises a) providing a first biological sample from the subject obtained before the start of the therapeutic treatment, b) providing a second biological sample from the subject obtained after therapeutic treatment, and c) determining the presence or quantity of antibodies against a peptide as shown in SEQ ID No: 2 or against an antibody-binding fragment or variant thereof, against a peptide as shown in SEQ ID NO: 1 or against an antibody-binding fragment or variant thereof, and against a peptide as shown in SEQ ID NO: 7 or against an antibodybinding fragment or variant thereof in the first and second biological sample; wherein a deviation or no deviation in antibody levels against at least one of said peptides or antibody-binding fragments or variants thereof in the second biological sample compared to the first biological sample indicates that the subject is responding to the therapeutic treatment; preferably wherein a decrease in antibody levels against at least one of said peptides or antibody-bind
  • the presence or a deviation or no deviation in antibody levels against the peptide as shown in SEQ ID NO: 2 or against an immune-reactive fragment or against an antibody-binding fragment or variant thereof in the second biological sample compared to the first biological sample indicates that the subject is responding to the therapeutic treatment.
  • a decrease in antibody levels against the peptide as shown in SEQ ID NO: 2 or against an immune-reactive fragment or against an antibody-binding fragment or variant thereof in the second biological sample compared to the first biological sample indicates that the subject is responding to the therapeutic treatment.
  • the presence or a deviation or no deviation in antibody levels against the peptide as shown in SEQ ID NO: 2 or against an immune-reactive fragment or against an antibody-binding fragment or variant thereof, against the peptide as shown in SEQ ID NO 1 or against an immune-reactive fragment or an antibody-binding fragment or variant thereof, and against the peptide as shown in SEQ ID NO: 7 or against an immune-reactive fragment or an antibody-binding fragment or variant thereof, in the second biological sample compared to the first biological sample indicates that the subject is responding to the therapeutic treatment.
  • the method further comprises determining the presence or quantity of antibodies against one or more peptides; in particular 2, 3 or 4 peptides, as shown in SEQ ID No: 3, SEQ ID No: 4, SEQ ID No: 5, and SEQ ID No: 6, or against an immune-reactive fragment or antibody-binding fragment or variant thereof in the first and second biological sample, wherein the presence or a deviation or no deviation in antibody levels against one or more of said peptides or against said immune-reactive fragments or antibody-binding fragments or variants thereof in the second biological sample compared to the first biological sample indicates that the subject is responding to the therapeutic treatment.
  • the method further comprises determining the presence or quantity of antibodies against one or more peptides; in particular 2, 3 or 4 peptides, as shown in SEQ ID No: 8, SEQ ID No: 9, SEQ ID No: 10, SEQ ID No: 11 , SEQ ID No: 12, SEQ ID No: 13, SEQ ID No: 14, SEQ ID No: 15, SEQ ID No: 16, SEQ ID No: 17, SEQ ID No: 18 and SEQ ID No: 19, or against an immune-reactive fragment or antibody-binding fragment or variant thereof in the first and second biological sample, wherein the presence or a deviation or no deviation in antibody levels against one or more of said peptides or against said immune-reactive fragments or antibody-binding fragments or variants thereof in the second biological sample compared to the first biological sample indicates that the subject is responding to the therapeutic treatment.
  • the present invention provides a peptide as shown in any one of SEQ ID NOs 1- 7, or of SEQ ID NOs: 8-19, or an immunoreactive fragment or an antibody-binding fragment or variant thereof.
  • the present invention provides a peptide as shown in any one of SEQ ID NOs 1-7, or of SEQ ID NOs: 8-19.
  • the invention provides a peptide comprising a sequence represented by any of SEQ ID Nos: 1-7, by any one of Seq ID Nos: 8-19, or an immunoreactive fragment or antibody-binding fragment or variant thereof, in particular comprising at least 4; preferably at least 6, preferably at least 7, more preferably at least 9, consecutive amino acids derived from said sequences.
  • the present invention provides a peptide consisting essentially of a sequence represented by SEQ ID Nos: 1-7, of a sequence of represented by Seq ID Nos: 8-19, or an immunoreactive fragment thereof, in particular comprising at least 4, preferably at least 6, consecutive amino acids derived from SEQ ID Nos: 1-7, or from SEQ ID Nos: 8-19.
  • detection agent comprising one or more peptides, antibody-binding fragments or variants thereof as disclosed herein.
  • composition comprising one or more of the peptides or immunoreactive fragments or antibody-binding fragments or variants thereof or detection agents as described above.
  • the peptides, the detection agents or the composition according to the present invention are for use in the diagnosis of spondyloarthritis.
  • the peptides, the detection agents or the composition according to the present invention are for use for evaluating the therapy response in a subject diagnosed with spondyloarthritis.
  • a peptide, an immunoreactive fragment thereof, an antibody-binding fragment or variant thereof as disclosed herein, or of a detection agent as taught herein, or of a composition comprising a peptide, immunoreactive fragment thereof, antibody-binding fragment or variant thereof or a detecting agent as disclosed herein is provided for detecting the presence or quantity of specific antibodies against said peptide, immunoreactive fragment or antibody-binding fragment or variant thereof or against the peptide, immunoreactive fragment or antibody-binding fragment or variant thereof present in the composition, preferably wherein the presence or increase in antibody levels as compared to a reference sample is indicative for the diagnosis of spondyloarthritis.
  • a peptide, immunoreactive fragment thereof, antibody-binding fragment or variant thereof as disclosed herein, or of a detection agent as taught herein, of a composition comprising a peptide, immunoreactive fragment thereof, antibody-binding fragment or variant thereof as disclosed herein is provided for detecting the presence or quantity of specific antibodies against said peptide, antibody-binding fragment or variant, or against the peptide, antibody-binding fragment or variant present in the composition, in a first and second biological sample of the subject wherein the first biological sample is obtained before the start of therapeutic treatment and the second biological sample is obtained after the start of therapeutic treatment; preferably wherein a decrease in antibody levels in the second biological sample as compared to the first biological sample is indicative for a response to the therapeutic treatment.
  • the present invention also provides a diagnostic kit for performing the in vitro method according to any of the embodiments of the present invention, said kit comprising a peptide or immunoreactive fragment or antibody-binding fragment or variant thereof, or a detection agent as taught herein, or a composition according to the invention, and reagents for detecting antibody binding to said one or more peptides or fragments thereof.
  • the present invention provides a method for treatment of a subject with spondyloarthritis, said method comprising: identifying a subject as having or not having one or more peptides or antibody-binding fragments as taught herein in a biological sample from the subject, such as by any one of the in vitro methods as taught herein, and administering a therapeutic treatment against spondyloarthritis to the subject.
  • Figure 1 Added value of a combination of our antibody biomarker panels for the diagnosis of early axSpA patients.
  • antibodies against our panel of UH-axSpA-lgG.1/4/8 peptides (PCT/EP2020/076120 published as WO2021/053152 A1) detected 16% of early axSpA patients.
  • Additional testing for antibody reactivity against the novel UH-axSpA-lgG.101/102/109/110 peptides increased the sensitivity for detecting early axSpA patients from 16% to 32%.
  • Additional testing for antibodies against the novel UH-axSpA-lgA.1/3/10 peptides leads to a further increase in sensitivity, allowing to detect up to 50% of early axSpA patients in total.
  • one or more or “at least one”, such as one or more members or at least one member of a group of members, is clear per se, by means of further exemplification, the term encompasses inter alia a reference to any one of said members, or to any two or more of said members, such as, e.g., any >3, >4, >5, >6 or >7 etc. of said members, and up to all said members.
  • “one or more” or “at least one” may refer to 1 , 2, 3, 4, 5, 6, 7 or more.
  • the inventors identified a novel tool for the diagnosis of spondyloarthritis as well as for the evaluation ofthe disease severity of spondyloarthritis and evaluation ofthe therapy response in patients diagnosed with spondyloarthritis. More specific, an unbiased screening was performed to identify novel (auto)antibodies in early axSpA patients that provide a novel tool for early axSpA diagnosis. To this end, the inventors applied the antibody profiling technique serological antigen selection (SAS).
  • SAS serological antigen selection
  • a cDNA phage display library constructed from axSpA hip synovial tissue was screened for reactivity with immunoglobulin A (IgA) antibodies in plasma of early axSpA patients.
  • IgA immunoglobulin A
  • phage particles expressing axSpA synovial antigens and randomly formed peptides, originating from a newly constructed axSpA cDNA phage display library were screened for IgA antibody reactivity in plasma of early axSpA patients.
  • Antibody reactivity against novel Hasselt University (UH)- axSpA-lgA peptides was determined in early axSpA patients and healthy controls (HC).
  • the inventors also investigated whether antibodies against a combination of the identified UH-axSpA- IgG peptides (SEQ ID’s 8 - 19), and the UH-axSpA-lgA targets (SEQ ID’s 1-7), could have a promising biomarker potential for the early diagnosis of axSpA patients.
  • the present invention provides an in vitro method for diagnosing of spondyloarthritis, wherein the method comprises providing a biological sample from the subject and determining the presence or quantity of one or more antibodies against one or more peptides or against one or more immunoreactive fragments or antibody-binding fragments or variants of said one or more peptides in the biological sample. More specific, the antibody reactivity towards said immunoreactive fragments or antibody-binding fragments or variants is comparable to the antibody reactivity towards the corresponding peptide itself.
  • the presence of or a deviation, such as an increase, in antibody levels against at least one of said peptides or immunoreactive fragments or antibody-binding fragments or variants thereof compared to a reference sample is indicative for the presence of spondyloarthritis in the subject.
  • an in vitro method for evaluating the disease severity of spondyloarthritis in a subject comprises providing a biological sample from the subject and determining the presence or quantity of one or more antibodies against one or more peptides or against one or more immunoreactive fragments or antibody-binding fragments or variants of said one or more peptides in the biological sample. More specific, the antibody reactivity towards said immunoreactive fragments or antibody-binding fragments or variants is comparable to the antibody reactivity towards the corresponding peptide itself.
  • the presence of or a deviation, such as an increase, in antibody levels against at least one of said peptides or immunoreactive fragments or antibody-binding fragments or variants thereof compared to a reference sample is indicative for the disease severity of spondyloarthritis in the subject.
  • an in vitro method for evaluating a therapeutic treatment of spondyloarthritis in a subject comprises providing a first and a second biological sample wherein the first biological sample is obtained at the start of the therapeutic treatment and the second sample is obtained after the therapeutic treatment, and determining in said first and second biological sample the presence or quantity of one or more antibodies against one or more peptides or against one or more immunoreactive fragments or antibody-binding fragments or variants of said one or more peptides. More specific, the antibody reactivity towards said immunoreactive fragments or antibody-binding fragments or variants is comparable to the antibody reactivity towards the corresponding peptide itself.
  • a deviation or no deviation, in particular a deviation, such as an increase or a decrease, in antibody levels against said one or more peptides or immunoreactive fragments or antibody-binding fragments or variants thereof in the second biological sample compared to the first biological sample indicates that the subject is responding to the therapeutic treatment.
  • a decrease in antibody levels against said one or more peptides or immunoreactive fragments or antibody-binding fragments or variants thereof in the second biological sample compared to the first biological sample indicates that the subject is responding to the therapeutic treatment
  • the methods and uses as taught herein comprise determining in one or more biological samples of the subject the presence or quantity of antibodies against one or more peptides selected from the group consisting of a peptide as shown in any one of SEQ ID NOs: 1-19 or against an antibody-binding fragment or variant thereof. Sequences are displayed in Table 1 .
  • the methods and uses as taught herein comprise determining in one or more biological samples of a subject the presence or quantity of antibodies against peptides selected from the group consisting of a peptide as shown in SEQ ID NO: 1 or an antibody-binding fragment or variant thereof.
  • the methods and uses as taught herein comprise determining in one or more biological samples of a subject the presence or quantity of antibodies against peptides selected from the group consisting of a peptide as shown in SEQ ID NO: 2 or an antibody-binding fragment or variant thereof.
  • the methods and uses as taught herein comprise determining in one or more biological samples of a subject the presence or quantity of antibodies against peptides selected from the group consisting of a peptide as shown in SEQ ID NO: 3 or an antibody-binding fragment or variant thereof.
  • the methods and uses as taught herein comprise determining in one or more biological samples of a subject the presence or quantity of antibodies against peptides selected from the group consisting of a peptide as shown in SEQ ID NO: 4 or an antibody-binding fragment or variant thereof.
  • the methods and uses as taught herein comprise determining in one or more biological samples of a subject the presence or quantity of antibodies against peptides selected from the group consisting of a peptide as shown in SEQ ID NO: 5 or an antibody-binding fragment or variant thereof.
  • the methods and uses as taught herein comprise determining in one or more biological samples of a subject the presence or quantity of antibodies against peptides selected from the group consisting of a peptide as shown in SEQ ID NO: 6 or an antibody-binding fragment or variant thereof.
  • the methods and uses as taught herein comprise determining in one or more biological samples of a subject the presence or quantity of antibodies against peptides selected from the group consisting of, and a peptide as shown in SEQ ID NO: 7 or an antibody-binding fragment or variant thereof.
  • the methods and uses as taught herein comprise determining in one or more biological samples of a subject the presence or quantity of antibodies against any combination of peptides selected from the group consisting of SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7.
  • the methods and uses as taught herein comprise determining in one or more biological samples of a subject the presence or quantity of antibodies against a peptide as shown in SEQ ID NO: 2 or an antibody-binding fragment or variant thereof, against a peptide as shown in SEQ ID NO: 1 or an antibody-binding fragment or variant thereof, and against a peptide as shown in SEQ ID NO: 7 or an antibody-binding fragment or variant thereof.
  • the methods and uses as taught herein comprise determining in one or more biological samples of a subject the presence or quantity of antibodies against a peptide as shown in SEQ ID NO: 2 or an antibody-binding fragment or variant thereof, against a peptide as shown in SEQ ID NO: 1 or an antibody-binding fragment or variant thereof, against a peptide as shown in SEQ ID NO: 3 or an antibody-binding fragment or variant thereof, against a peptide as shown in SEQ ID NO: 4 or an antibody-binding fragment or variant thereof, against a peptide as shown in SEQ ID NO: 5 or an antibody-binding fragment or variant thereof, against a peptide as shown in SEQ ID NO: 6 or an antibody-binding fragment or variant thereof, and against a peptide as shown in SEQ ID NO: 7 or an antibody-binding fragment or variant thereof.
  • the in vitro method disclosed herein is for diagnosing spondyloarthritis in a subject, said method comprising: a) providing a biological sample from the subject, and b) determining the presence or quantity of antibody levels against a peptide as shown in SEQ ID NO: 2 or against an antibody-binding fragment or variant thereof, against a peptide as shown in SEQ ID NO: 1 or against an antibody-binding fragment or variant thereof, and against a peptide as shown in SEQ ID No: 7; or against an antibody-binding fragment or variant thereof, wherein the presence of or an increase in antibody levels against at least one of said peptides or antibody-binding fragments or variants thereof in said biological sample compared to a reference sample is indicative for the presence or diagnosis of spondyloarthritis in the subject.
  • the in vitro method disclosed herein is for diagnosing spondyloarthritis in a subject, said method comprising: a) providing a biological sample from the subject, and b) determining the presence or quantity of antibody levels against a peptide as shown in SEQ ID NO: 2 or against an antibody-binding fragment or variant thereof, against a peptide as shown in SEQ ID NO: 1 or against an antibody-binding fragment or variant thereof, and against a peptide as shown in SEQ ID No: 7; or against an antibody-binding fragment or variant thereof, wherein the presence of or an increase in antibody levels against the peptide as shown in SEQ ID NO: 2 or against an antibody-binding fragment or variant thereof in the biological sample compared to a reference sample is indicative for the presence or diagnosis of spondyloarthritis in the subject.
  • the in vitro method disclosed herein is for diagnosing spondyloarthritis in a subject, said method comprising: a) providing a biological sample from the subject, and b) determining the presence or quantity of antibody levels against a peptide as shown in SEQ ID NO: 2 or against an antibody-binding fragment or variant thereof, against a peptide as shown in SEQ ID NO: 1 or against an antibody-binding fragment or variant thereof, and against a peptide as shown in SEQ ID No: 7; or against an antibody-binding fragment or variant thereof, wherein the presence or increase in antibody levels against the peptide as shown in SEQ ID NO: 2 or against an antibody-binding fragment or variant thereof, against the peptide as shown in SEQ ID NO 1 or against an antibody-binding fragment or variant thereof, and against the peptide as shown in SEQ ID NO: 7 or against an antibody-binding fragment or variant thereof, in said sample compared to a reference sample is indicative for the presence of spondyloarthriti
  • the in vitro method disclosed herein is for diagnosing spondyloarthritis in a subject, said method comprising: a) providing a biological sample from the subject, and b) determining the presence or quantity of antibody levels against a peptide as shown in SEQ ID NO: 2 or against an antibody-binding fragment or variant thereof, wherein the presence of or an increase in antibody levels against the peptide as shown in SEQ ID NO: 2 or against an antibody-binding fragment or variant thereof in the biological sample compared to a reference sample is indicative for the presence or diagnosis of spondyloarthritis in the subject.
  • the in vitro method disclosed herein is for diagnosing spondyloarthritis in a subject, said method comprising: a) providing a biological sample from the subject, and b) determining the presence or quantity of antibody levels against a peptide as shown in SEQ ID NO: 1 or against an antibody-binding fragment or variant thereof, wherein the presence of or an increase in antibody levels against the peptide as shown in SEQ ID NO: 1 or against an antibody-binding fragment or variant thereof in the biological sample compared to a reference sample is indicative for the presence or diagnosis of spondyloarthritis in the subject.
  • the in vitro method disclosed herein is for diagnosing spondyloarthritis in a subject, said method comprising: a) providing a biological sample from the subject, and b) determining the presence or quantity of antibody levels against a peptide as shown in SEQ ID NO: 7 or against an antibody-binding fragment or variant thereof, wherein the presence of or an increase in antibody levels against the peptide as shown in SEQ ID NO: 7 or against an antibody-binding fragment or variant thereof in the biological sample compared to a reference sample is indicative for the presence or diagnosis of spondyloarthritis in the subject.
  • the in vitro method disclosed herein is for diagnosing spondyloarthritis in a subject, said method comprising: a) providing a biological sample from the subject, and b) determining the presence or quantity of antibody levels against a peptide as shown in SEQ ID NO: 2 or against an antibody-binding fragment or variant thereof, against a peptide as shown in SEQ ID NO: 1 or against an antibody-binding fragment or variant thereof, and against a peptide as shown in SEQ ID No: 7; or against an antibody-binding fragment or variant thereof, c) determining the presence or quantity of antibodies against one or more peptides as shown in SEQ ID NO: 3, SEQ ID No: 4, SEQ ID No: 5, and SEQ ID No: 6, or against an antibody-binding fragment or variant thereof, wherein the presence or increase in antibody levels against the peptide as shown in SEQ ID NO: 2 or against an antibody-binding fragment or variant thereof, against the peptide as shown in SEQ ID NO 1 or against an antibody
  • the in vitro method disclosed herein is for diagnosing spondyloarthritis in a subject, said method comprising: a) providing a biological sample from the subject, b) determining the presence or quantity of antibody levels against a peptide as shown in SEQ ID NO: 2 or against an antibody-binding fragment or variant thereof, against a peptide as shown in SEQ ID NO: 1 or against an antibody-binding fragment or variant thereof, and against a peptide as shown in SEQ ID No: 7; or against an antibody-binding fragment or variant thereof, and c) determining the presence or quantity of antibodies against one or more peptides as shown in SEQ ID NO: 8, SEQ ID No: 9, SEQ ID No: 10, SEQ ID No: 11 , SEQ ID No: 12, SEQ ID No: 13, SEQ ID No: 14, SEQ ID No: 15, SEQ ID No: 16, SEQ ID No: 17, SEQ ID No: 18 and SEQ ID No: 19, or against an antibody-binding fragment or variant thereof, wherein
  • the in vitro method disclosed herein is for evaluating the disease severity of spondyloarthritis in a subject, said method comprising: a) providing a biological sample from the subject, and b) determining the presence or quantity of antibody levels against a peptide as shown in SEQ ID NO: 2 or against an antibody-binding fragment or variant thereof, against a peptide as shown in SEQ ID NO: 1 or against an antibody-binding fragment or variant thereof, and against a peptide as shown in SEQ ID No: 7; or against an antibody-binding fragment or variant thereof, wherein the presence of or an increase in antibody levels against the peptide as shown in SEQ ID NO: 2 or against an antibodybinding fragment or variant thereof in the biological sample compared to a reference sample is indicative for the disease severity of spondyloarthritis in the subject.
  • the in vitro method disclosed herein is for evaluating the disease severity of spondyloarthritis in a subject, said method comprising: a) providing a biological sample from the subject, and b) determining the presence or quantity of antibody levels against a peptide as shown in SEQ ID NO: 2 or against an antibody-binding fragment or variant thereof, against a peptide as shown in SEQ ID NO: 1 or against an antibody-binding fragment or variant thereof, and against a peptide as shown in SEQ ID No: 7; or against an antibody-binding fragment or variant thereof, wherein the presence or increase in antibody levels against the peptide as shown in SEQ ID NO: 2 or against an antibody-binding fragment or variant thereof, against the peptide as shown in SEQ ID NO 1 or against an antibody-binding fragment or variant thereof, and against the peptide as shown in SEQ ID NO: 7 or against an antibodybinding fragment or variant thereof, in said sample compared to a reference sample is indicative for the disease severity of spondy
  • the in vitro method disclosed herein is for evaluating the disease severity of spondyloarthritis in a subject, said method comprising: a) providing a biological sample from the subject, and b) determining the presence or quantity of antibody levels against a peptide as shown in SEQ ID NO: 2 or against an antibody-binding fragment or variant thereof, wherein the presence of or an increase in antibody levels against the peptide as shown in SEQ ID NO: 2 or against an antibody-binding fragment or variant thereof in the biological sample compared to a reference sample is indicative for the disease severity of spondyloarthritis in the subject.
  • the in vitro method disclosed herein is for evaluating the disease severity of spondyloarthritis in a subject, said method comprising: a) providing a biological sample from the subject, and b) determining the presence or quantity of antibody levels against a peptide as shown in SEQ ID NO: 1 or against an antibody-binding fragment or variant thereof, wherein the presence of or an increase in antibody levels against the peptide as shown in SEQ ID NO: 1 or against an antibody-binding fragment or variant thereof in the biological sample compared to a reference sample is indicative for the disease severity of spondyloarthritis in the subject.
  • the in vitro method disclosed herein is for evaluating the disease severity of spondyloarthritis in a subject, said method comprising: a) providing a biological sample from the subject, and b) determining the presence or quantity of antibody levels against a peptide as shown in SEQ ID NO: 7 or against an antibody-binding fragment or variant thereof, wherein the presence of or an increase in antibody levels against the peptide as shown in SEQ ID NO: 7 or against an antibody-binding fragment or variant thereof in the biological sample compared to a reference sample is indicative for the disease severity of spondyloarthritis in the subject.
  • antibody levels against the peptide as shown in SEQ ID No: 1 , or an immunoreactive fragment or antibody-binding variant thereof comprising at least 4, preferably at least 6, even more preferably at least 8, consecutive amino acids derived therefrom are determined.
  • antibody levels against the peptide as shown in SEQ ID No: 2, or an immunoreactive fragment or antibody-binding variant thereof comprising at least 4, preferably at least 6, even more preferably at least 8, consecutive amino acids derived therefrom are determined.
  • antibody levels against the peptide as shown in SEQ ID No: 7, or an immunoreactive fragment or antibody-binding variant thereof comprising at least 4, preferably at least 6, even more preferably at least 8, consecutive amino acids derived therefrom are determined.
  • antibody levels against the peptide as shown in SEQ ID No: 2, or an immunoreactive fragment or antibody-binding variant thereof comprising at least 4, preferably at least 6, even more preferably at least 8, consecutive amino acids derived therefrom are determined, in combination with antibody levels against the peptide as shown in SEQ ID No 1 and/or 7, or a fragment comprising at least 4, preferably at least 6, even more preferably at least 8, consecutive amino acids derived therefrom.
  • antibody levels against the peptide as shown in SEQ ID NO: 1 , SEQ ID NO: 2 and SEQ ID NO: 7, or against an immunoreactive fragment or antibody-binding fragment thereof, preferably said fragment comprising at least 4, preferably at least 6, more preferably at least 8, consecutive amino acids derived thereof, are determined in combination with determining the antibody levels against one or more peptides as shown in SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, or against an immu noreactive fragment or antibody-binding fragment or variant thereof, preferably said fragment comprising at least 4, preferably at least 6, more preferably at least 8, consecutive amino acids derived therefrom.
  • antibody levels against the peptide as shown in SEQ ID No: 1 , SEQ ID No 2 and SEQ ID No 7, or against an immunoreactive fragment or antibody-binding fragment thereof; preferably said fragment comprising at least 4, preferably at least 6, more preferably at least 8, consecutive amino acids derived therefrom, are determined.
  • said antibody levels are combined with antibody levels against one or more peptides as shown in SEQ ID No 8, SEQ ID No 9, SEQ ID No 16 and SEQ ID No 17, or against an immunoreactive fragment or antibody-binding fragment or variant thereof, preferably said fragment comprising at least 4, preferably at least 6, more preferably at least 8, consecutive amino acids derived therefrom.
  • the present invention provides an in vitro method for diagnosing the presence of spondyloarthritis or for evaluating the disease severity of spondyloarthritis in a subject, said method comprising: a) providing a biological sample from the subject, and b) determining antibody levels against one or more peptides selected from the list comprising SEQ ID NO: 1 , SEQ ID No: 2 or SEQ ID NO: 7, or an immune-reactive fragment or antibody-binding fragment or variant thereof comprising at least 4; in particular at least 6, consecutive amino acids derived therefrom; wherein an increase in antibody levels against said one or more peptides or fragments thereof in said biological sample compared to a reference sample is indicative for the presence of spondyloarthritis.
  • an increase in antibody levels against the peptide of SEQ ID NO: 2 or against an immunoreactive fragment or antibody-binding fragment or variant thereof comprising at least 4, in particular at least 6, consecutive amino acids derived therefrom, in said biological sample compared to a reference sample is indicative for the presence of spondyloarthritis or for the disease severity of spondyloarthritis in the subject.
  • the present invention provides an in vitro method for diagnosing of spondyloarthritis or for evaluating the disease severity of spondyloarthritis, said method comprising: a) providing a biological sample from the subject, and b) determining antibody levels against peptides selected from the list comprising SEQ ID No: 1 , SEQ ID No: 2, and SEQ ID No: 7, or an immune-reactive fragment or antibody-binding variant thereof; wherein an increase in antibody levels against said peptides or fragments thereof in said biological sample compared to a reference sample is indicative for the diagnosis of spondyloarthritis in the subject.
  • peptides are selected from the list consisting of SEQ ID NO: 1 , SEQ ID No: 2 and SEQ ID NO: 7, or an immune-reactive fragment or antibody-binding fragment or variant thereof; in particular at least 6 consecutive amino acids derived from the original sequence of the peptides.
  • the in vitro method disclosed herein is for diagnosing spondyloarthritis or for evaluating the disease severity of spondyloarthritis in a subject, said method comprising: a) providing a biological sample from the subject, and b) determining the presence or quantity of antibody levels against a peptide as shown in SEQ ID NO: 2 or against an antibody-binding fragment or variant thereof, against a peptide as shown in SEQ ID NO: 1 or against an antibody-binding fragment or variant thereof, and against a peptide as shown in SEQ ID No: 7; or against an antibody-binding fragment or variant thereof, wherein the presence of or an increase in antibody levels against at least one of said peptides or antibody-binding fragments or variants thereof in said biological sample compared to a reference sample is indicative for the presence or diagnosis of spondyloarthritis or for the disease severity of spondyloarthritis in the subject.
  • the present invention further provides an in vitro method for evaluating a therapeutic treatment of spondyloarthritis in a subject, said method comprising: a) providing a first biological sample from the subject obtained before the start of the therapeutic treatment, b) providing a second biological sample from the subject obtained after therapeutic treatment, and c) determining the presence or quantity of antibodies against a peptide as shown in SEQ ID No: 2 or against an antibody-binding fragment or variant thereof, against a peptide as shown in SEQ ID NO: 1 or against an antibody-binding fragment or variant thereof, and against a peptide as shown in SEQ ID NO: 7 or against an antibody-binding fragment or variant thereof in the first and second biological sample; wherein a deviation or no deviation, in antibody levels against at least one of said peptides or antibody-binding fragments or variants thereof in the second biological sample compared to the first biological sample indicates that the subject is responding to the therapeutic treatment.
  • a deviation such as a decrease, in antibody levels against at least one
  • the present invention also provides an in vitro method for evaluating a therapeutic treatment of spondyloarthritis in a subject, said method comprising: a) providing a first biological sample from the subject obtained before the start of the therapeutic treatment, b) providing a second biological sample from the subject obtained after therapeutic treatment, and c) determining the presence or quantity of antibodies against a peptide as shown in SEQ ID No: 2 or against an antibody-binding fragment or variant thereof, against a peptide as shown in SEQ ID NO: 1 or against an antibody-binding fragment or variant thereof, and against a peptide as shown in SEQ ID NO: 7 or against an antibody-binding fragment or variant thereof in the first and second biological sample; wherein a deviation or no deviation, in antibody levels against the peptide as shown in SEQ ID NO: 2 or against an antibody-binding fragment or variant thereof in the second biological sample compared to the first biological sample indicates that the subject is responding to the therapeutic treatment.
  • a deviation such as a decrease, in antibody levels against at least one of said peptides or antibody-binding fragments or variants thereof in the second biological sample compared to the first biological sample indicates that the subject is responding to the therapeutic treatment.
  • a deviation or no deviation in antibody levels against the peptide as shown in SEQ ID NO: 2 or against an antibody-binding fragment or variant thereof, against the peptide as shown in SEQ ID NO 1 or against an antibody-binding fragment or variant thereof, and against the peptide as shown in SEQ ID NO: 7 or against an antibody-binding fragment or variant thereof, in the second biological sample compared to the first biological sample indicates that the subject responds to the therapeutic treatment.
  • a deviation such as a decrease, in antibody levels against at least one of said peptides or antibody-binding fragments or variants thereof in the second biological sample compared to the first biological sample indicates that the subject is responding to the therapeutic treatment.
  • the in vitro method disclosed herein is for evaluating a therapeutic treatment of spondyloarthritis in a subject, said method comprising: a) providing a first biological sample from the subject obtained before the start of the therapeutic treatment, b) providing a second biological sample from the subject obtained after therapeutic treatment, and c) determining the presence or quantity of antibody levels against a peptide as shown in SEQ ID NO: 2 or against an antibody-binding fragment or variant thereof, wherein a deviation or no deviation, in antibody levels against the peptide as shown in SEQ ID NO: 2 or against an antibody-binding fragment or variant thereof in the second biological sample compared to the first biological sample indicates that the subject responds to the therapeutic treatment.
  • a deviation such as a decrease, in antibody levels against at least one of said peptides or antibody-binding fragments or variants thereof in the second biological sample compared to the first biological sample indicates that the subject is responding to the therapeutic treatment.
  • the in vitro method disclosed herein is for evaluating a therapeutic treatment of spondyloarthritis in a subject, said method comprising: a) providing a first biological sample from the subject obtained before the start of the therapeutic treatment, b) providing a second biological sample from the subject obtained after therapeutic treatment, and c) determining the presence or quantity of antibody levels against a peptide as shown in SEQ ID NO: 1 or against an antibody-binding fragment or variant thereof, wherein a deviation or no deviation, in antibody levels against the peptide as shown in SEQ ID NO: 1 or against an antibody-binding fragment or variant thereof in the second biological sample compared to the first biological sample indicates that the subject responds to the therapeutic treatment.
  • a deviation such as a decrease, in antibody levels against at least one of said peptides or antibody-binding fragments or variants thereof in the second biological sample compared to the first biological sample indicates that the subject is responding to the therapeutic treatment.
  • the in vitro method disclosed herein is for evaluating a therapeutic treatment of spondyloarthritis in a subject, said method comprising: a) providing a first biological sample from the subject obtained before the start of the therapeutic treatment, b) providing a second biological sample from the subject obtained after therapeutic treatment, and c) determining the presence or quantity of antibody levels against a peptide as shown in SEQ ID NO: 7 or against an antibody-binding fragment or variant thereof, wherein a deviation or no deviation, in antibody levels against the peptide as shown in SEQ ID NO: 7 or against an antibody-binding fragment or variant thereof in the second biological sample compared to the first biological sample indicates that the subject responds to the therapeutic treatment.
  • a deviation such as a decrease, in antibody levels against at least one of said peptides or antibody-binding fragments or variants thereof in the second biological sample compared to the first biological sample indicates that the subject is responding to the therapeutic treatment.
  • the in vitro method disclosed herein is for evaluating a therapeutic treatment of spondyloarthritis in a subject, said method comprising: a) providing a first biological sample from the subject obtained before the start of the therapeutic treatment, b) providing a second biological sample from the subject obtained after therapeutic treatment, c) determining the presence or quantity of antibody levels against a peptide as shown in SEQ ID NO: 2 or against an antibody-binding fragment or variant thereof, against a peptide as shown in SEQ ID NO: 1 or against an antibody-binding fragment or variant thereof, and against a peptide as shown in SEQ ID No: 7; or against an antibody-binding fragment or variant thereof in the first and second biological sample, and d) determining the presence or quantity of antibodies against one or more peptides as shown in SEQ ID NO: 3, SEQ ID No: 4, SEQ ID No: 5, and SEQ ID No: 6, or against an antibody-binding fragment or variant thereof in the first and second biological sample, wherein a deviation or no
  • the in vitro method disclosed herein is for evaluating a therapeutic treatment of spondyloarthritis in a subject, said method comprising: a) providing a first biological sample from the subject obtained before the start of the therapeutic treatment, b) providing a second biological sample from the subject obtained after therapeutic treatment, c) determining the presence or quantity of antibody levels against a peptide as shown in SEQ ID NO: 2 or against an antibody-binding fragment or variant thereof, against a peptide as shown in SEQ ID NO: 1 or against an antibody-binding fragment or variant thereof, and against a peptide as shown in SEQ ID No: 7; or against an antibody-binding fragment or variant thereof, in the first and second biological sample, and d) determining the presence or quantity of antibodies against one or more peptides as shown in SEQ ID NO: 8, SEQ ID No: 9, SEQ ID No: 10, SEQ ID No: 11 , SEQ ID No: 12, SEQ ID No: 13, SEQ ID No: 14, SEQ ID No: 15,
  • a ’’deviation” of a first value from a second value or a “difference” between a first value and a second value may generally encompass any direction (e.g., increase: first value > second value; or decrease: first value ⁇ second value) and any extent of alteration.
  • a deviation or a difference may encompass a decrease in a first value by, without limitation, at least about 10% (about 0.9-fold or less), or by at least about 20% (about 0.8-fold or less), or by at least about 30% (about 0.7-fold or less), or by at least about 40% (about 0.6-fold or less), or by at least about 50% (about 0.5-fold or less), or by at least about 60% (about 0.4-fold or less), or by at least about 70% (about 0.3-fold or less), or by at least about 80% (about 0.2-fold or less), or by at least about 90% (about 0.1-fold or less), relative to a second value with which a comparison is being made.
  • a deviation or a difference may encompass an increase of a first value by, without limitation, at least about 10% (about 1 .1 -fold or more), or by at least about 20% (about 1 .2-fold or more), or by at least about 30% (about 1 .3-fold or more), or by at least about 40% (about 1 .4-fold or more), or by at least about 50% (about 1 .5-fold or more), or by at least about 60% (about 1 .6-fold or more), or by at least about 70% (about 1 .7-fold or more), or by at least about 80% (about 1 .8-fold or more), or by at least about 90% (about 1 .9-fold or more), or by at least about 100% (about 2-fold or more), or by at least about 150% (about 2.5-fold or more), or by at least about 200% (about 3-fold or more), or by at least about 500% (about 6-fold or more), or by at least about 700% (about 8-fold or more), or like, relative to a second
  • a deviation or a difference may refer to a statistically significant observed alteration.
  • a deviation or a difference may refer to an observed alteration which falls outside of error margins of reference values in a given population (as expressed, for example, by standard deviation or standard error, or by a predetermined multiple thereof, e.g., +1xSD or +2xSD or +3xSD, or +1xSE or +2xSE or+3xSE).
  • Deviation or a difference may also refer to a value falling outside of a reference range defined by values in a given population (for example, outside of a range which comprises >40%, > 50%, >60%, >70%, >75% or >80% or >85% or >90% or >95% or even >100% of values in said population).
  • a deviation or a difference may be concluded if an observed alteration is beyond a given threshold or cut-off.
  • threshold or cut-off may be selected as generally known in the art to provide for a chosen accuracy, sensitivity and/or specificity of the prediction methods, e.g., accuracy, sensitivity and/or specificity of at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 85%, or at least 90%, or at least 95%.
  • receiver-operating characteristic (ROC) curve analysis can be used to select an optimal threshold or cut-off value of the quantity of a given biomarker for clinical use of the present diagnostic tests, based on acceptable global accuracy, sensitivity and/or specificity, or related performance measures which are well-known per se, such as positive predictive value (PPV), negative predictive value (NPV), positive likelihood ratio (LR+), negative likelihood ratio (LR-), Youden index, or similar.
  • PPV positive predictive value
  • NPV negative predictive value
  • LR+ positive likelihood ratio
  • LR- negative likelihood ratio
  • Youden index or similar.
  • an optimal threshold or cut-off value may be selected for each individual biomarker as a local extremum of the receiver operating characteristic (ROC) curve, i.e. a point of local maximum distance to the diagonal line, as described in Robin X., PanelomiX: a threshold-based algorithm to create panels of biomarkers, 2013, Translational Proteomics, 1 (1):57-64.
  • ROC receiver operating characteristic
  • a relevant threshold or cut-off value can be obtained by correlating the sensitivity and specificity and the sensitivity/specificity for any threshold or cut-off value.
  • any combination of two or more peptides selected from the list consisting of SEQ ID No 1-7 can suitably be used within the context of the present invention; preferably peptides are selected from the list consisting of SEQ ID Nos 1 , 2 and 7.
  • the peptides of the present invention may be provided as such, however, also an immune-reactive fragment or antibody-binding fragment or variant thereof; typically comprising at least 4, at least 5, in particular at least 6, consecutive amino acids derived from the original sequence of the peptide are suitable for use in the method of the current invention.
  • peptides are selected from the list consisting of SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID No: 10, SEQ ID NO: 11 , SEQ ID NO: 12, SEQ ID No: 13, SEQ ID No: 14, SEQ ID NO: 15, SEQ ID No: 16, SEQ ID No: 17, SEQ ID No: 18 and SEQ ID NO: 19, or an immune-reactive fragment thereof; typically comprising at least 4 consecutive amino acids derived therefrom; in particular at least 6 consecutive amino acids derived therefrom.
  • peptides are selected from the list comprising SEQ ID NO: 8, SEQ ID No: 9, SEQ ID No:16 and SEQ ID NO: 17, or an immune-reactive fragment thereof; typically comprising at least 4 consecutive amino acids derived therefrom; in particular at least 6 consecutive amino acids derived therefrom.
  • antibody levels against the peptide comprising a sequence of SEQ ID No: 8, or an immune-reactive fragment thereof; typically comprising at least 4 consecutive amino acids derived therefrom; preferably at least 6 consecutive amino acids derived therefrom are determined.
  • said antibody levels are combined with antibody levels against the peptide comprising a sequence of SEQ ID No 1 , 2 and/or 7, or an immune-reactive fragment thereof; typically comprising at least 4 consecutive amino acids derived therefrom; preferably at least 6 consecutive amino acids derived therefrom.
  • antibody levels against the peptide comprising a sequence of SEQ ID No: 9, or an immune-reactive fragment thereof; typically comprising at least 4 consecutive amino acids derived therefrom; preferably at least 6 consecutive amino acids derived therefrom are determined.
  • said antibody levels are combined with antibody levels against thepeptide comprising a sequence of SEQ ID No 1 , 2 and/or 7, or an immune-reactive fragment thereof; typically comprising at least 4 consecutive amino acids derived therefrom; preferably at least 6 consecutive amino acids derived therefrom.
  • antibody levels against the peptide comprising a sequence of SEQ ID No: 16, or an immune-reactive fragment thereof; typically comprising at least 4 consecutive amino acids derived therefrom;preferably at least 6 consecutive amino acids derived therefrom are determined.
  • said antibody levels are combined with antibody levels against the peptide comprising a sequence of SEQ ID No 1 , 2 and/or 7, or an immune-reactive fragment thereof; typically comprising at least 4 consecutive amino acids derived therefrom; preferably at least 6 consecutive amino acids derived therefrom.
  • antibody levels against the peptide comprising a sequence of SEQ ID No: 17, or an immune-reactive fragment thereof; typically comprising at least 4 consecutive amino acids derived therefrom; preferably at least 6 consecutive amino acids derived therefrom are determined.
  • said antibody levels are combined with antibody levels against the peptide comprising a sequence of SEQ ID No 1 , 2 and/or 7, an immune-reactive fragment thereof; typically comprising at least 4 consecutive amino acids derived therefrom; preferably at least 6 consecutive amino acids derived therefrom.
  • antibody levels against the peptide comprising a sequence of SEQ ID No: 8, SEQ ID No: 9 , SEQ ID No: 16 and/or SEQ ID No: 17, or an immune-reactive fragment thereof; typically comprising at least 4 consecutive amino acids derived therefrom; preferably at least 6 consecutive amino acids derived therefrom are determined.
  • said antibody levels are combined with antibody levels against the peptide comprising a sequence of SEQ ID No 1 , 2 and/or 7, or an immune-reactive fragment thereof; typically comprising at least 4 consecutive amino acids derived therefrom; preferably at least 6 consecutive amino acids derived therefrom.
  • any combination of two or more peptides selected from the list comprising SEQ ID No 1-7, with any combination of two or more peptides selected from the list comprising SEQ ID No 8-19 can suitably be used within the context of the present invention; preferably peptides are selected from the list comprising SEQ ID Nos 1 , 2 and 7, in combination with peptides selected from the list comprising SEQ ID No 8-19; in particular with the list comprising SEQ ID Nos 8, 9, 16 and 17.
  • the peptides of the present invention may be provided as such, however, also immune-reactive fragment thereof; typically comprising at least 4, at least 5, in particular at least 6, consecutive amino acids derived therefrom are suitable for use in the method of the current invention.
  • a “full- length” peptide it may alternatively be replaced by an immune-reactive fragment, comprising at least 4; at least 5, or at least 6 consecutive amino acids derived therefrom.
  • the methods as disclosed herein are characterized in that the one or more antibodies are detected using a detection agent comprising one or more peptides selected from the group consisting of the peptide of SEQ ID NO: 1-19 or an antibody-binding fragment or variant thereof.
  • the methods as disclosed herein are characterized in that the one or more antibodies are detected using a detection agent composing one or more peptides selected from the group consisting of: the peptide of SEQ ID NO: 1 or an antibody-binding fragment or variant thereof, the peptide of SEQ ID NO: 2 or an antibody-binding fragment or variant thereof, the peptide of SEQ ID NO: 3 or an antibody-binding fragment or variant thereof, the peptide of SEQ ID NO: 4 or an antibody-binding fragment or variant thereof, the peptide of SEQ ID NO: 5 or an antibody-binding fragment or variant thereof, the peptide of SEQ ID NO: 6 or an antibody-binding fragment or variant thereof, the peptide of SEQ ID NO: 7 or an antibody-binding fragment or variant thereof, or combinations thereof.
  • a detection agent composing one or more peptides selected from the group consisting of: the peptide of SEQ ID NO: 1 or an antibody-binding fragment or variant thereof, the peptide of SEQ ID NO: 2
  • the methods as disclosed herein are characterized in that the one or more antibodies are detected using a detection agent composing one or more peptides selected from the group consisting of: the peptide of SEQ ID NO: 1 or an antibody-binding fragment or variant thereof, the peptide of SEQ ID NO: 2 or an antibody-binding fragment or variant thereof, the peptide of SEQ ID NO: 7 or an antibody-binding fragment or variant thereof, or combinations thereof.
  • antibodies can be detected using detection agent comprising a peptide of SEQ ID NO: 1 or an antibody-binding variant or fragment thereof.
  • antibodies can be detected using detection agent comprising a peptide of SEQ ID NO: 2 or an antibody-binding variant or fragment thereof.
  • antibodies can be detected using detection agent comprising a peptide of SEQ ID NO: 7 or an antibody-binding variant or fragment thereof.
  • antibodies can be detected using detection agent comprising a peptide of SEQ ID NO: 2 or an antibody-binding variant or fragment thereof, in combination with a peptide of SEQ ID NO: 1 or an antibody-binding variant or fragment thereof and/or , a peptide of SEQ ID NO: 7 or an antibody-binding variant or fragment thereof.
  • the present invention also provides peptides, such as isolated peptides, comprising a sequence represented by any of SEQ ID Nos: 1-19, or an immune-reactive fragment or antibody-binding fragment or variant thereof.
  • the present application provides a peptide selected from the group consisting of the peptide of SEQ ID NO: 1 , the peptide of SEQ ID NO: 2, the peptide of SEQ ID NO: 3, the peptide of SEQ ID NO: 4, the peptide of SEQ ID NO: 5, the peptide of SEQ ID NO: 6, the peptide of SEQ ID NO: 7, the peptide of SEQ ID NO: 8, the peptide of SEQ ID NO: 9, the peptide of SEQ ID NO: 10, the peptide of SEQ ID NO: 11 , the peptide of SEQ ID NO: 12, the peptide of SEQ ID NO: 13, the peptide of SEQ ID NO: 14, the peptide of SEQ ID NO: 15, the peptide of SEQ ID NO: 16, the peptide of SEQ ID NO: 17, the peptide of SEQ ID NO: 18, and the peptide of SEQ ID NO: 19.
  • the present application provides a peptide selected from the group consisting of the peptide of SEQ ID NO: 1 , the peptide of SEQ ID NO: 2, the peptide of SEQ ID NO: 3, the peptide of SEQ ID NO: 4, the peptide of SEQ ID NO: 5, the peptide of SEQ ID NO: 6, and the peptide of SEQ ID NO: 7.
  • the present application provides a peptide selected from the group consisting of the peptide of SEQ ID NO: 1 , the peptide of SEQ ID NO: 2, and the peptide of SEQ ID NO: 7.
  • the present application provides a peptide as shown in SEQ ID NO: 1. In some embodiments, the present application provides a peptide that comprises a sequence as shown in SEQ ID NO: 1.
  • the present application a peptide as shown in SEQ ID NO: 2. In some embodiments, the present application provides a peptide that comprises a sequence as shown in SEQ ID NO: 2.
  • the present application a peptide as shown in SEQ ID NO: 7. In some embodiments, the present application provides a peptide that comprises a sequence as shown in SEQ ID NO: 7.
  • immu noreactive fragments or antibody-binding fragments or variants of the peptides as disclosed herein are also provided.
  • a peptide that comprises at least 4; preferably at least 6, consecutive amino acids derived from SEQ ID No: 1-19.
  • the present invention provides peptides consisting essentially of a sequence represented by SEQ ID Nos: 1-19, or an immune-reactive fragment or antibody-binding fragment thereof; typically comprising at least 4, preferably at least 6, consecutive amino acids derived from SEQ ID Nos: 1-19.
  • a detection agent comprising one or more peptides, antibody-binding fragments or variants thereof as disclosed herein.
  • the detection agent may comprise one or more or a combination of multiple peptides or antibody-binding fragments or variants thereof as disclosed herein.
  • the detection agent as disclosed herein is a polypeptide.
  • a detection agent, preferably a polypeptide is provided that comprises one or more peptides, antibodybinding fragments or variants thereof as taught herein.
  • the detection agent is a composite polypeptide that comprises the amino acid sequence of one or more of the peptides that are disclosed herein.
  • a detection agent according to the invention may comprise the sequence of SEQ ID NO: 1 , SEQ ID NO: 2 or SEQ ID NO: 7.
  • detection agent that is a composite polypeptide may comprise the sequence of SEQ ID NO: 1 and the sequence of SEQ ID NO: 2, or the sequence of SEQ ID NO: 2 and the sequence of SEQ ID NO: 7, or the sequence of SEQ ID NO: 1 and the sequence of SEQ ID NO: 2 and the sequence of SEQ ID NO: 7.
  • composition comprising one or more peptides, or antibody-binding fragments or variants thereof or detection agents as disclosed herein is provided.
  • a composition comprising a peptide comprising or consisting essentially of the sequence of SEQ ID NO: 2 or antibody-binding fragment or variant thereof is provided. In a certain embodiment, a composition comprising a peptide comprising or consisting essentially of the sequence of SEQ ID NO: 1 or antibody-binding fragment or variant thereof is provided. In a certain embodiment, a composition comprising a peptide comprising or consisting essentially of the sequence of SEQ ID NO: 7 or antibody-binding fragment or variant thereof is provided.
  • a composition comprising a peptide comprising or consisting essentially of the sequence of SEQ ID NO: 2 or antibodybinding fragment or variant thereof, a peptide comprising or consisting essentially of the sequence of SEQ ID NO: 1 or antibody-binding fragment or variant thereof, and a peptide comprising or consisting essentially of the sequence of SEQ ID NO: 7 or antibody-binding fragment or variant thereof is provided.
  • a composition may be provided that comprises a peptide as shown in SEQ ID NO: 2, or an antibody-binding fragment thereof in combination with at least one other peptide as shown in SEQ ID NO: 1 or SEQ ID NO: 7 or an antibody-binding fragment thereof.
  • the composition may comprise at least one other peptide comprising or consisting essentially of a sequence represented by any one of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6, or an antibody-binding fragment or variant thereof.
  • the composition may comprise at least one other peptide comprising or consisting essentially of a sequence represented by any one of SEQ ID NO: 8-19, or an antibody-binding fragment or variant thereof.
  • the composition can also comprise one or more peptides of SEQ ID NO: 1 , 2 and 7 or an antibody-binding fragment thereof, in combination with one or more peptides of SEQ ID No: 3, 4, 5 and 6, or an antibody-binding fragment thereof and in combination with one or more peptides of SEQ ID NO; 8, 9, 10, 11 , 12, 13, 15, 16, 17, 18 and 19, or an antibody-binding fragment thereof.
  • a composition comprising the peptides of SEQ ID NO: 1 , 2 and or an antibody-binding fragment thereof and the peptides of SEQ ID NO: 8, 9, 16, and 17, or an antibody-binding fragment thereof can be provided.
  • antibodies are detected against one or more peptides as disclosed herein or against their antibody-binding fragments or variants thereof.
  • the peptides as taught herein are non-naturally occurring peptides or non-physiological peptides. More specific, although it was found that peptides show some homology to a number of human proteins that are expressed in human hip synovial tissue of spondyloarthritis patients, the peptides envisaged herein are non-naturally occurring peptides.
  • the peptides are formed by out-of-frame cDNA translation or the translation of non-coding sequences.
  • the methods and uses as taught herein can also be performed using their naturally occurring counterparts, i.e. proteins having regions that share homology with such peptides, i.e. proteins having regions that share at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity with the (non- naturally) occurring peptides of the present invention.
  • the peptides of the present invention were found to have homology with human proteins such as indicated in the table 2 and 3 below:
  • the antibodies are IgA, IgG and/or IgM antibodies. In certain embodiments, the antibodies are IgA and/or IgG antibodies. In some embodiments, the antibodies are IgA antibodies. In some embodiments, the antibodies are IgG antibodies. In some embodiments, the antibodies are autoantibodies, in particular IgA, IgG and/or IgM autoantibodies. In some embodiments, the antibodies are IgA autoantibodies and/or IgG autoantibodies. In some embodiments, the antibodies are IgA autoantibodies. In some embodiments, the antibodies are IgG autoantibodies. In some embodiments, the antibodies are thus antibodies produced by the subject’s immune system and are directed against an antigen of the subject’s own proteins.
  • Antibodies are normally produced in response to a foreign protein or substance within the body, typically a pathogen. Normally, the immune system is able to recognize and ignore the body’s own proteins and not overreact to non-threatening substances in the environment. Sometimes, however, the immune system cease to recognize one or more of the body’s normal constituents as “self, leading to production of autoantibodies. These autoantibodies can attack the subject’s own cells, tissues and/or organs, causing inflammation and damage.
  • antibodies can also be detected against immunoreactive fragments or antibodybinding fragments or antibody-binding variants of the peptides of SEQ ID Nos 1 to 19.
  • immunoreactive fragment, antibody-binding fragment and antibody-binding variant are synonyms and can be used interchangeably.
  • an immunoreactive fragment or an antibody-binding fragment or variant of a peptide refers to a functionally equivalent fragment or variant of the corresponding peptide.
  • antibody-binding fragments or variants are to be understood as any fragment or variant of the peptide of SEQ ID NO: 1 to 19 that show an antibody reactivity that is similar or comparable to or substantially the same as the antibody reactivity of the corresponding peptide.
  • Antibody-binding fragments or variants can also be referred herein as immunoreactive or immune reactive peptides of the corresponding peptides of SEQ ID NO: 1 to 19.
  • Immune reactive peptides or antibody-binding fragments are thus to be understood as any peptide that shows an antibody reactivity that is similar or comparable to or substantially the same as the antibody reactivity of their corresponding peptides.
  • the antibody-binding fragments, antibodybinding variants or immune reactive peptides are peptides that can be recognized by the same antibodies that recognized their corresponding peptides of SEQ ID Nos: 1 to 19 as disclosed herein.
  • the antibodies recognize the same epitope on the peptides and their corresponding immune reactive peptides or antibody-binding fragments or variants.
  • the immune reactive peptides or antibody-binding fragments as taught herein can be a fragment of their corresponding peptide as defined in SEQ ID Nos: 1 to 19, preferably as defined in SEQ ID NOs; 1 to 7, such as defined in SEQ ID NO: 1 , 2 or 7.
  • the immune reactive peptide or antibody-binding fragment can thus be a fragment of its corresponding peptides, such as for example a peptide of at least 4, preferably at least 5, even more preferably at least 6, amino acids of the sequence of the peptide as taught herein.
  • fragment as used throughout this specification with reference to a peptide, polypeptide, or protein generally denotes a portion of the peptide, polypeptide, or protein, such as typically an N- and/or C-terminally truncated form of the peptide, polypeptide, or protein.
  • a fragment may comprise at least about 30%, e.g., at least about 50% or at least about 70%, preferably at least about 80%, e.g., at least about 85%, more preferably at least about 90%, and yet more preferably at least about 95% or even about 99% of the amino acid sequence length of said peptide, polypeptide, or protein.
  • a fragment may include a sequence of > 4 consecutive amino acids, or > 5 consecutive amino acids, or > 6 consecutive amino acids, or > 7 consecutive amino acids, or > 8 consecutive amino acids, or > 9 consecutive amino acids, or > 10 consecutive amino acids, of the corresponding full-length peptide, polypeptide, or protein.
  • an antibody-binding variant of the corresponding peptide can be a peptide that does not necessarily display the same amino acid sequence as the corresponding peptide.
  • Discontinuous homology as used herein is to be understood as partial similarity or identity in the amino acid sequences between the antibody-binding variant and its corresponding peptide, such as for example wherein part of the amino acid sequence of the immune reactive peptide is similar or identical to the corresponding peptide as defined in SEQ ID NOs 1 to 19 but wherein another part of the amino acid sequence of the immune reactive peptide differs from the corresponding peptide as defined in SEQ ID NOs 1 to 19.
  • the antibody-binding variants are still functionally equivalent to the corresponding peptides of SEQ ID NOs: 1 to 19 and thus these antibody-binding variants show an antibody reactivity that is similar or comparable to or substantially the same as the antibody reactivity of the corresponding peptides of SEQ ID NOs 1 to 19.
  • the amino acid sequence of a variant of a peptide may be at least about 80% identical or at least about 85% identical, e.g., preferably at least about 90% identical or at least about 95% identical to the amino acid sequence of the peptide. Sequence identity between proteins or polypeptides may be determined using suitable algorithms for performing sequence alignments and determination of sequence identity as know per se.
  • BLAST Basic Local Alignment Search Tool
  • a variant of a peptide may comprise one or more amino acid additions, deletions, and/or substitutions compared with the corresponding peptide.
  • the amino acid sequence of a variant of a peptide may differ by 5 or less, 4 or less, 3 or less, or 2 or less, such as 1 or 2 or 3, amino acid additions, deletions, and/or substitutions compared to the amino acid sequence of the peptide. In certain embodiments, at least some and preferably all substitutions may be conservative amino acid substitution.
  • Conservative amino acid substitutions include substitutions within the following groups: valine, alanine and glycine; leucine, valine, and isoleucine; aspartic acid and glutamic acid; asparagine and glutamine; serine, cysteine, and threonine; lysine and arginine; and phenylalanine and tyrosine.
  • the nonpolar hydrophobic amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan and methionine.
  • the polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine and glutamine.
  • the positively charged (i.e., basic) amino acids include arginine, lysine and histidine.
  • the negatively charged (i.e., acidic) amino acids include aspartic acid and glutamic acid. Any substitution of one member of the above-mentioned polar, basic, or acidic groups by another member of the same group can be deemed a conservative substitution. By contrast, a non-conservative substitution is a substitution of one amino acid for another with dissimilar characteristics.
  • fragment or variant or “variant or fragment” of any peptide also encompasses fragments of variants of such peptide, and variants of fragments of such peptide.
  • fragments and variants of proteins, polypeptides or peptides refers to or encompasses fragments or variants of proteins, polypeptides or peptides, this in particular denotes such fragments and variants which are biologically active.
  • biologically active is interchangeable with terms such as “functionally active” or “functional”, denoting that the fragment and variant at least partly retains the biological activity or intended functionality of the respective or corresponding protein, polypeptide, or peptide.
  • fragments and variants of the peptides disclosed herein, such as their immune reactive peptides show a similar or comparable or substantially the same antibody reactivity as their corresponding peptides.
  • fragments and variants of the peptides disclosed herein, such as their immune reactive peptides interact with the same antibodies as their corresponding peptides.
  • the antibodies that bind to the antibody-binding fragments or variants or immune reactive peptides as disclosed herein are the same as the antibodies that bind to their corresponding peptides.
  • the antibody-binding fragments or variants or immune reactive peptides have a similar immune reactivity with their antibodies as compared to their corresponding peptides.
  • the antibody reactivity of the peptide and of the antibody-binding fragment or variant or immune reactive peptide thereof is the same and hence both the peptide and the antibodybinding fragment or variant or immune reactive peptide can interact with the same antibody.
  • the signal or measurement obtained using a given molar quantity of the fragment or variant may be at least 1%, preferably at least 10%, such as at least 20%, at least 30%, or at least 40%, or more preferably at least 50%, such as at least 60%, or at least 70%, or even more preferably at least 80%, or at least 90%, still more preferably at least 95%, including about 100% or even more than 100%, of the signal or measurement obtained using the same molar quantity of the corresponding peptide, when all other parameters and conditions of the technique or assay are the same.
  • Any suitable means for detecting antibody levels against one or more peptides of the present invention may be used.
  • Methods for detecting antibody/antigen or immune complexes are well known in the art.
  • the present invention may be modified by one skilled in the art to accommodate the various detection methods known in the art.
  • the particular detection method chosen by one skilled in the art depends on several factors, including the amount of biological sample available, the type of biological sample, the stability of the biological sample, the stability of the antigen (i.e. peptide), and the affinity between the antibody and the antigen (i.e. peptide).
  • the method of the current invention may include the use of an immunoassay, such as, enzyme-linked immunosorbent assays (ELISAs), immunofluorescent techniques, radioimmunological assays (RIA) and immunoblotting and/or line blot.
  • an immunoassay such as, enzyme-linked immunosorbent assays (ELISAs), immunofluorescent techniques, radioimmunological assays (RIA) and immunoblotting and/or line blot.
  • ELISA-based assays antigens (i.e. peptides) are bound to a support, and the biological sample is combined therewith. Subsequently antibodies in the sample are allowed to bind to the antigens/peptides bound on the support, thereby forming immune complexes. After the immune complexes have formed, excess biological sample may be removed and the array may be washed to remove nonspecifically bound antibodies.
  • the immune complexes may then be reacted with an appropriate enzyme-labeled anti-immunoglobulin. Then anti-immunoglobulin is allowed to react with the antibodies in the immune complexes. After an optional further wash, the enzyme substrate may be added. The enzyme linked to the anti-immunoglobulin catalyzes a reaction that converts the substrate into a product, which can then be detected and used to quantify the amount of antibody in the sample.
  • the peptides of the present invention are selected as such that no or only a marginal antibody reactivity is found in a reference sample in contrast to a significantly higher antibody reactivity in patients having spondyloarthritis or having a more severe disease status of spondyloarthritis.
  • a reference sample is preferably a sample from a healthy individual not suffering from spondyloarthritis, or from an individual suffering from lower back pain without spondyloarthritis; in particular from non-inflammatory lower back pain without spondyloarthritis.
  • a predetermined threshold based on healthy individuals may be set to compare samples from patients suspected of having spondyloarthritis.
  • the reference sample is a sample from a patient suffering from spondyloarthritis but with a known disease status, such as a disease status that is determined based on the BASDAI score.
  • antibody reactivity is determined by calculating the ratio of the optical density (OD) of the specific signal to OD of the background signal. For each peptide target, a cut-off for seropositivity was calculated as the mean of this ratio (OD (specific)/OD (background)) in the non-reactive samples plus 3*Standard Deviation.
  • a sample is considered positive when its ratio (OD (specific)/OD (background)) is higher than this cut-off.
  • OD (specific)/OD (background) the higher the general reactivity (OD (specific)/OD (background)) for the peptides of the present invention, in a particular patient, the more likely it is that a positive diagnosis of spondyloarthritis is made or that a more severe disease status of spondylarthritis is present.
  • antibodies are provided that specifically bind to peptides or their antibodybinding fragments, variants or immune reactive peptides as described herein above.
  • Methods for generating antibodies are well known in the art.
  • the peptides forming part of the compositions of the invention may be synthesized chemically or may be in a recombinant way. They may also be coupled to a soluble carrier after synthesis or after recombination production. If a carrier is used, the nature of such a carrier should be such that it has a molecular weight greater than 5000 and should not be recognized by antibodies.
  • a carrier can be a protein.
  • Proteins which are frequently used as carriers are keyhole limpet hemocyanin, bovine gamma globulin, bovine serum albumin, and poly-L-lysine. There are many well described techniques for coupling peptides to carriers. The linkage may occur at the N-terminus, C-terminus or at an internal site in the peptide. The peptide may also be derivatized for coupling. The peptides may also be synthesized directly on an oligo- lysine core in which both the alpha as well as the epsilon-amino groups of lysines are used as growth points for the polypeptides. The number of lysines comprising the core is preferably 3 or 7. Additionally, a cysteine may be included near or at the C-terminus of the complex to facilitate the formation of homo- or heterodimers.
  • the invention discloses in some aspects a process for detecting or quantifying antibodies related to the diagnosis of spondyloarthritis or to the disease severity of spondyloarthritis or to evaluate therapy response in spondyloarthritis in a biological sample of a subject, preferably a human, liable to contain them.
  • This process comprises contacting the biological sample with a composition according to the invention under conditions enabling an immunological reaction between said composition and the antibodies which are possibly present in the biological sample and the detection of the antigen/antibody complex which may be formed.
  • the detection can be carried out according to any classical process, for example an immune-enzymatic process according to the ELISA technique or immunofluorescent or radioimmunological (RIA) or the equivalent ones (e.g.
  • LINE blot or LINE assay can be used.
  • peptides labelled by an appropriate label of the enzymatic, fluorescent, biotin, radioactive type can be used.
  • Such a method for detecting antibodies related to spondyloarthritis comprises for instance the following steps: deposit of determined amounts of a composition comprising peptides according to the invention on a support (e.g. into wells of a titration microplate), introduction on said support (e.g. into wells) of increasing dilutions of the body fluid (e.g. blood plasma or serum) to be diagnosed, incubation of the support (e.g. microplate), repeated rinsing of the support (e.g.
  • microplate introduction on the support labelled antibodies which are specific for immunoglobulins present in the body fluid, the labelling of these antibodies being based on the activity of an enzyme which is selected from among the ones which are able to hydrolyse a substrate to a product which absorbs light at a given wave length, and detection by comparing a control standard of the amount of hydrolysed substrate.
  • the invention also relates to a process for detecting and identifying an antigen related to the diagnosis of spondyloarthritis or to the disease severity of spondyloarthritis or to the evaluation of therapy response in spondyloarthritis in a biological sample liable to contain it, this process comprising contacting the biological sample with an appropriate antibody of the invention (i.e. antibodies with a specificity for a peptide, antibody-binding fragment, variant or immune reactive peptide thereof of the composition) under conditions enabling an immunological reaction between said antibody and the antigens which are possibly present in the biological sample and the detection of the antigen/antibody complex which may be formed.
  • an appropriate antibody of the invention i.e. antibodies with a specificity for a peptide, antibody-binding fragment, variant or immune reactive peptide thereof of the composition
  • antibodies in particular auto-antibodies, which recognize the peptides of the invention or their immune reactive peptides, can be detected in a variety of ways.
  • One method of detection is further described in the examples and uses enzyme-linked immunosorbent assay (ELISA) of the polypeptides of the invention or their immune reactive peptides displayed by phages (e.g. phage-ELISA technology).
  • ELISA enzyme-linked immunosorbent assay
  • the detection in ELISA uses a peptide or mixture of peptides bound to a solid support.
  • this will be a microtiter plate but may in principle be any sort of insoluble solid phase (e.g. glass, nitrocellulose).
  • a suitable dilution or dilutions of for example blood or serum to be tested is brought into contact with the solid phase to which the polypeptide is bound.
  • a solution hybridization is carried out in which high affinity interactions occur (e.g. biotinylated polypeptides of the composition are pre-incubated with serum). The incubation is carried out for a time necessary to allow the binding reaction to occur. Subsequently, unbound components are removed by washing the solid phase.
  • immune complexes i.e. antibodies present in for example human serum binding to at least one peptide of the invention
  • antibodies which specifically bind to human immunoglobulins and which have been labelled with an enzyme, preferably but not limited to either horseradish peroxidase, alkaline phosphatase, or beta-galactosidase, which is capable of converting a colourless or nearly colourless substrate or co-substrate into a highly coloured product or a product capable of forming a coloured complex with a chromogen.
  • an enzyme preferably but not limited to either horseradish peroxidase, alkaline phosphatase, or beta-galactosidase, which is capable of converting a colourless or nearly colourless substrate or co-substrate into a highly coloured product or a product capable of forming a coloured complex with a chromogen.
  • a detection system may employ an enzyme which, in the presence of the proper substrate(s), emits light.
  • the amount of product formed is detected either visually, spectrophotometrically, electrochemically, fluorescently or luminometrically, and is compared to a similarly treated control.
  • the detection system may also employ radioactively labelled antibodies, in which cases the amount of immune complex is quantified by scintillation counting or gamma counting.
  • Other detection systems which may be used include those based on the use of protein A derived from Staphylococcus aureus Cowan strain I, protein G from group C Staphylococcus sp. (strain 26RP66), or systems which make use of the high affinity biotin-avidin or streptavidin binding reaction.
  • the peptides of the invention or their antibody-binding fragments, variants or immune reactive peptides may be either labelled or unlabelled. Labels which may be employed may be of any type, such as enzymatic, chemical, fluorescent, luminescent, or radioactive.
  • the peptides may be modified for binding to surfaces or solid phases, such as, for example microtiter plates, nylon membranes, glass or plastic beads, and chromatographic supports such as cellulose, silica, or agarose.
  • the methods by which peptides or their antibody-binding fragments, variants or immune reactive peptides can be attached or bound to solid support or surface are well known to those skilled in the art.
  • the peptides of the invention or their antibody-binding fragments, variants or immune reactive peptides can be prepared according to the classical techniques in the field of peptide synthesis.
  • the synthesis can be carried out in homogeneous solution or in solid phase.
  • the synthesis technique in homogeneous solution which can be used is the one described by Houbenweyl in the book titled “Methode der organische chemie” (Method of organic chemistry) edited by E. Wunsch, vol. 15-1 et II. THIEME, Stuttgart 1974.
  • the peptides of the invention can also be prepared in solid phase according the method described by Atherton & Shepard in their book titled “Solid phase peptide synthesis” (Ed.
  • antibodies raised to peptides of the invention or to their antibody-binding fragments or variants or immune reactive peptides (or carrier-bound peptides or immune reactive peptides) can also be used in conjunction with labelled or unlabelled peptides of the invention or their immune reactive peptides for the detection of (auto)antibodies present in serum by competition assay.
  • antibodies raised to peptides are attached to a solid support which may be, for example, a plastic bead or a plastic tube. The peptide is then mixed with suitable dilutions of the fluid (e.g. serum) to be tested and this mixture is subsequently brought into contact with the antibody bound to the solid support.
  • the solid support is washed and the amount of labelled or unlabelled peptide is quantified.
  • a reduction in the amount of label bound to the solid support is indicative of the presence of (auto)antibodies in the original sample, such as blood plasma or serum.
  • the peptide may also be bound to the solid support.
  • Labelled antibody may then be allowed to compete with (auto)antibody present in the sample (e.g. serum) under conditions in which the amount of peptide is limiting.
  • a reduction of the measured signal is indicative of the presence of (auto)antibodies in the sample tested.
  • a competition ELISA can be used in which samples (e.g. plasma or serum samples) are pre-incubated with increasing concentrations of one or more synthetic peptides corresponding to the sequences defined by SEQ ID No: 1-19, before use in a phage ELISA.
  • a test for giving evidence of the fact that one or more peptides present in a composition of the invention are recognized by antibodies present in for example blood or serum (for example auto-antibodies present in serum of axSpa patients) is an immunoblotting (or Western blotting) analysis.
  • peptides can be chemically synthesized or peptides (or the protein) can be produced via recombinant techniques.
  • nitrocellulose membranes e.g. Hybond C. (Amersham)
  • the free reactive functions which are present in some of the amino acids, which are part of the constitution of the peptides of the invention or their immune reactive peptides particularly the free carboxyl groups which are carried by the groups Glu and Asp or by the C-terminal amino acid on the one hand and/or the free NH2 groups carried by the N-terminal amino acid or by amino acids inside the peptidic chain, for instance Lys, on the other hand, can be modified in so far as this modification does not alter the above mentioned properties of the polypeptide.
  • the peptides which are thus modified are naturally part of the invention.
  • the above-mentioned carboxyl groups can be acylated or esterified. Other modifications are also part of the invention.
  • the amine or carboxyl functions or both of terminal amino acids can be themselves involved in the bond with other amino acids.
  • the N-terminal amino acid can be linked to the C-terminal amino acid of another peptide comprising from 1 to several amino acids.
  • any peptidic sequences resulting from the modification by substitution and/or by addition and/or by deletion of one or several amino acids of the polypeptides according to the invention are part of the invention in so far as this modification does not alter the above-mentioned properties of said polypeptides.
  • the peptides according to the invention can be glycosylated or not, particularly in some of their glycosylation sites of the type Asn-X-Ser or Asn-X-Thr, X representing any amino acid.
  • the peptide may be synthesized with an extra cysteine residue added. This extra cysteine residue is preferably added to the amino terminus and facilitates the coupling of the peptide to a carrier protein which is necessary to render the small peptide immunogenic. If the peptide is to be labelled for use in radioimmune assays, it may be advantageous to synthesize the protein with a tyrosine attached to either the amino or carboxyl terminus to facilitate iodination. This peptide possesses therefore the primary sequence of the peptide above-mentioned but with additional amino acids which do not appear in the primary sequence of the protein and whose sole function is to confer the desired chemical properties to the peptide.
  • amount refers to but are not limited to the absolute or relative amount of antibodies, peptides, polypeptides or any other value or parameter associated with the latter or which can derive therefrom.
  • values or parameters comprise signal intensity values obtained by direct or indirect measurement, for example, in an ELISA assay.
  • the readout may be a mean, average, median, or the variance or other statistically or mathematically-derived value associated with the measurement.
  • the absolute values obtained for the antibody levels under identical conditions will display a variability that is inherent in live biological systems and also reflects individual antibody quantity variability as well as the variability inherent between individuals.
  • a relative quantity of a marker, antibody, peptide, polypeptide, or protein in a sample may be advantageously expressed as an increase or decrease or as a fold-increase or fold-decrease relative to said another value, such as relative to a reference value as taught herein.
  • first and second parameters e.g., first and second quantities
  • a measurement method may produce quantifiable readouts (such as, e.g., signal intensities) for said first and second parameters, wherein said readouts are a function of the value of said parameters, and wherein said readouts may be directly compared to produce a relative value for the first parameter vs. the second parameter, without the actual need to first convert the readouts to absolute values of the respective parameters.
  • Reference to the activity of a protein, peptide, or polypeptide may generally encompass any one or more aspects of the biological activity of the protein, peptide, or polypeptide, such as without limitation any one or more aspects of its antibody reactivity, biochemical activity, enzymatic activity, signaling activity, interaction activity, ligand activity, and/or structural activity, e.g., within a cell, tissue, organ or an organism.
  • the type of a marker e.g., antibody, peptide, polypeptide, protein
  • the type of the tested object e.g., a cell, cell population, tissue, organ, or organism
  • the type of biological sample of a subject e.g., serum, plasma, whole blood, tissue biopsy
  • the quantity and/or activity of a marker may be measured directly in the tested object, or the tested object may be subjected to one or more processing steps aimed at achieving an adequate measurement of the marker.
  • peptide as used throughout this specification preferably refers to a peptide as used herein consisting essentially of 50 amino acids or less, e.g., 45 amino acids or less, preferably 40 amino acids or less, e.g., 35 amino acids or less, more preferably 30 amino acids or less, e.g., 25 or less, 20 or less, 15 or less, 10 or less or 5 or less amino acids.
  • polypeptide as used throughout this specification generally encompasses polymeric chains of amino acid residues linked by peptide bonds. Hence, especially when a protein is only composed of a single polypeptide chain, the terms “protein” and “polypeptide” may be used interchangeably herein to denote such a protein. The term is not limited to any minimum length of the polypeptide chain. The term may encompass naturally, recombinantly, semi-synthetically or synthetically produced polypeptides.
  • polypeptides that carry one or more co- or post-expression- type modifications of the polypeptide chain, such as, without limitation, glycosylation, acetylation, phosphorylation, sulfonation, methylation, ubiquitination, signal peptide removal, N-terminal Met removal, conversion of pro-enzymes or pre-hormones into active forms, etc.
  • the term further also includes polypeptide variants or mutants which carry amino acid sequence variations vis-a-vis a corresponding native polypeptide, such as, e.g., amino acid deletions, additions and/or substitutions.
  • the term contemplates both full-length polypeptides and polypeptide parts or fragments, e.g., naturally- occurring polypeptide parts that ensue from processing of such full-length polypeptides.
  • any marker, antibody, peptide, polypeptide, or protein corresponds to the marker, antibody, peptide, polypeptide, or protein commonly known under the respective designations in the art.
  • the terms encompass such markers, antibodies, peptides, polypeptides, or proteins of any organism where found, and particularly of animals, preferably warm-blooded animals, more preferably vertebrates, yet more preferably mammals, including humans and non-human mammals, still more preferably of humans.
  • markers, antibodies, peptides, polypeptides, or proteins may be human, i.e. , their primary sequence may be the same as a corresponding primary sequence of or present in naturally occurring human markers, peptides, polypeptides, proteins, or nucleic acids. In certain embodiments, markers, antibodies, peptides, polypeptides, or proteins are non-naturally occurring.
  • markers, antibodies, peptides, polypeptides, or proteins may be present in or isolated from samples of human subjects or may be obtained by other means (e.g., by recombinant expression, cell-free transcription or translation, or non-biological nucleic acid or peptide synthesis).
  • any marker, antibody, peptide, polypeptide, protein, or fragment thereof may generally also encompass modified forms of said marker, antibody, peptide, polypeptide, protein, or fragment thereof, such as bearing post-expression modifications including, for example, phosphorylation, glycosylation, lipidation, methylation, cysteinylation, sulphonation, glutathionylation, acetylation, biotinylation, oxidation of methionine to methionine sulphoxide or methionine sulphone, and the like.
  • any marker, antibody, peptide, polypeptide, or protein may encompass measuring the marker, antibody, peptide, polypeptide, or protein and/or measuring one or more fragments or variants thereof.
  • any marker, antibody, peptide, polypeptide, or protein and/or one or more fragments or variants thereof may be measured collectively, such that the measured quantity corresponds to the sum amounts of the collectively measured species.
  • any marker, antibody, peptide, polypeptide, or protein and/or one or more fragments or variants thereof may be measured each individually.
  • a marker, antibody, peptide, polypeptide, or protein is “detected” or “measured” in a sample when the presence or absence, quantity and/or activity of said marker, antibody, peptide, polypeptide, or protein is determined or measured in the sample, preferably substantially to the exclusion of other markers, antibodies, peptides, polypeptides, or proteins.
  • the present invention also provides a diagnostic kit for performing one of the in vitro methods according to the present invention, said kit comprising one or polypeptides or antibody-binding fragments thereof of the invention or one or more detection agents according to the invention, or a composition according to an embodiment of the invention; and reagents for detecting antibody binding to said one or more peptides or antibody-binding fragments or variants therefrom or detection agents.
  • Said reagents are for example reagents for making a medium appropriate for the immunological reaction to occur, reagents enabling the antigen/antibody complex which has been produced by the immunological reaction, said reagents possibly having a label, or being liable to be recognizable by a labelled reagent, more particularly in the case where the abovementioned polypeptide is not labelled.
  • the application provides the use of one or more peptides or antibody-binding fragments or variants thereof as taught herein, or the use of a detection agent as taught herein, or the use of a composition comprising one or more peptides or antibody-binding fragments or variants thereof as taught herein, for detecting the presence or quantity of specific antibodies against said peptide, antibody-binding fragment or variant, or against the detection agent, or against the peptide, antibodybinding fragment or variant present in the composition, in a biological sample of the subject; preferably wherein the presence or increase in antibody levels as compared to a reference sample is indicative for the diagnosis of spondyloarthritis.
  • the application provides the use of one or more peptides or antibody-binding fragments or variants thereof as taught herein, or the use of a detection agent as taught herein, or the use of a composition comprising one or more peptides or antibody-binding fragments or variants thereof as taught herein, for detecting the presence or quantity of specific antibodies against said peptide, antibody-binding fragment or variant, or against the detection agent, or against the peptide, antibodybinding fragment or variant present in the composition, in a biological sample of the subject; preferably wherein the presence or increase in antibody levels as compared to a reference sample is indicative for the disease severity of spondyloarthritis.
  • the application provides the use of one or more peptides or antibody-binding fragments or variants thereof as taught herein, or the use of a detection agent as taught herein, or the use of a composition comprising one or more peptides or antibody-binding fragments or variants thereof as taught herein, for detecting the presence or quantity of specific antibodies against said peptide, antibody-binding fragment or variant, or against said detection agent, or against the peptide, antibodybinding fragment or variant present in the composition, in a first and second biological sample of the subject wherein the first biological sample is obtained before the start of therapeutic treatment and the second biological sample is obtained after the start of therapeutic treatment; preferably wherein a decrease in antibody levels in the second biological sample as compared to the first biological sample is indicative for a response to the therapeutic treatment.
  • the invention provides the use of one or more peptides or detection agents or a composition of the invention for detecting the presence of specific antibodies to at least one of said peptides or detection agents or to a peptide present in said composition wherein said antibodies are present in a body fluid of a mammal, preferably a human.
  • said use of a composition is an “in vitro” use of a composition. The latter implies a diagnostic method with no direct interaction with the patient.
  • the methods and means of the present invention are in particular suitable for diagnosing the presence of spondyloarthritis.
  • diagnosis or “diagnosis” is meant to include “predicting” or “detecting” the presence of spondyloarthritis.
  • Spondyloarthritis includes axial spondyloarthritis and peripheral spondyloarthritis; preferably selected from ankylosing spondylitis, non-radiographic axial spondyloarthritis, undifferentiated spondyloarthritis, juvenile spondyloarthritis, psoriatic arthritis.
  • antibodies against one or more of the peptides of the present invention are absent in healthy individuals, hence, in that instance, the mere detection or presence of an antibody response against said peptides or fragments thereof is already suitable for determining the diagnosis spondyloarthritis in said patient.
  • the methods according to the different embodiments of the present invention can be combined with any other method available for the diagnosis of spondyloarthritis or for evaluating the disease severity of spondyloarthritis. Said other methods can be selected from physical examination, imaging technology and/or laboratory tests for human leukocyte antigen (HLA)-B27 and C-reactive protein (CRP).
  • While the methods and means of the present invention are highly useful in the diagnosis of spondyloarthritis, they are also very suitable for use in determining the disease severity of spondyloarthritis or for evaluating therapeutic treatment in patients diagnosed with spondyloarthritis.
  • disease severity or the response to therapy can be evaluated by using the BASDAI score, which ranges from 0 (no disease activity) to 10 (very active disease) and a cutoff value of a BASDAI score of 4 is frequently used to define active disease.
  • the invention also relates to a process for detecting and identifying antigen of spondyloarthritis in a body fluid liable to contain them, this process comprising contacting the biological sample with an appropriate antibody of the invention (i.e. antibodies with a specificity for a polypeptide of the composition) under conditions enabling an immunological reaction between said antibody and the antigens of spondyloarthritis which are possibly present in the biological sample and the detection of the antigen/antibody complex which may be formed.
  • an appropriate antibody of the invention i.e. antibodies with a specificity for a polypeptide of the composition
  • one or more peptides, or the immune reactive peptides or antibody-binding fragments or variants thereof or detection agents as taught herein are provided for use in the diagnosis of spondyloarthritis or for use in the evaluation of disease severity or therapy response in spondyloarthritis, in particular wherein said one or more peptides comprise an epitope having a sequence selected from any one of SEQ ID No: 1 to 7 or an immune-reactive fragment or antibodybinding fragment or variant thereof; typically comprising at least 4, in particular at least 5, even more in particular at least 6, consecutive amino acids derived therefrom.
  • said one or more peptides or the immune reactive peptides or antibody-binding fragments or variants thereof or detection agents comprise an epitope having a sequence selected from any one of SEQ ID No: 1 , SEQ ID No: 2 or SEQ ID No: 7, or an immune-reactive fragment thereof; typically comprising at least 4, in particular at least 5, even more in particular at least 6, consecutive amino acids derived therefrom.
  • one or more peptides, or the immune reactive peptides or antibody-binding variants or fragments thereof or detection agents as taught herein are provided for use in the diagnosis of spondyloarthritis or for use in the evaluation of disease severity or therapy response in spondyloarthritis, in particular wherein said one or more peptides comprise an epitope having a sequence selected from any one of SEQ ID No: 8 to 19 or an immune-reactive fragment or antibodybinding fragment or variant thereof; typically comprising at least 4, in particular at least 5, even more in particular at least 6, consecutive amino acids derived therefrom.
  • said one or more polypeptides or the immune reactive peptides or antibody-binding fragments or variants thereof comprise an epitope having a sequence selected from any one of SEQ ID No: 8, SEQ ID No: 9, SEQ ID No:16 or SEQ ID No: 17, an immune-reactive fragment or antibody-binding fragment or variant thereof; typically comprising at least 4, in particular at least 5, even more in particular at least 6, consecutive amino acids derived therefrom.
  • the biological sample may be any type of sample suitable for the determination of antibody levels against said one or more peptides of the invention, and is in particular a blood sample, a tissue sample or a body fluid sample.
  • the sample is selected from the list comprising hair, skin, nails, saliva, synovial liquid, blood serum, blood plasma, urine, tears, bone marrow fluid, cerebrospinal fluid, lymphatic fluid, amniotic fluid, nipple aspiration fluid, and the like; more preferably selected from blood serum or blood plasma.
  • the sample is a blood sample, preferably a blood serum sample or a blood plasma sample.
  • the antibody level in the biological sample can be compared to the antibody level of a reference sample, wherein the antibody level in the one or more reference samples is considered as the reference value.
  • the reference sample is a biological sample obtained from a healthy subject, i.e. a subject that is not diagnosed with spondyloarthritis.
  • the reference sample is a biological sample obtained from a patient diagnosed with spondyloarthritis but that does not show any antibody reactivity against the antibodies that are determined in the present invention.
  • the reference sample is a biological sample obtained from a patient diagnosed with spondyloarthritis and with a known disease severity status.
  • the antibody level in two or more biological samples obtained from the subject can be compared.
  • a first biological sample is obtained before the start of the treatment of spondyloarthritis and a second or even a third or fourth biological sample is obtained after the treatment of spondyloarthritis, preferably after a particular time of treating the subject diagnosed with spondyloarthritis, such as after 3 months, after 6 months, after 12 months, after 24 months or after 36 months.
  • the terms “subject”, “individual” or “patient” are used interchangeably throughout this specification, and typically and preferably denote humans, but may also encompass reference to nonhuman animals, preferably warm-blooded animals, even more preferably mammals, such as, e.g. nonhuman primates, rodents, canines, felines, equines, ovines, porcines, and the like.
  • non-human animals includes all vertebrates, e.g., mammals, such as non-human primates (particularly higher primates), sheep, dog, rodent (e.g., mouse or rat), guinea pig, goat, pig, cat, rabbits, cows, and nonmammals such as chicken, amphibians, reptiles, etc.
  • the subject is a mammal.
  • the subject is a non-human mammal.
  • the subject is an experimental animal or animal substitute as a disease model. The term does not denote a particular age or sex.
  • Suitable subjects may include subjects that are suspected of having spondyloarthritis, such as subjects that are presenting with symptoms of back pain (slow-onset back pain with stiffness for at least three months or longer, intense pain in the mornings or after periods of inactivity, and it may begin to ease during the day with stretching and exercise), fatigue, joint pain, heel pain, sleep problems, and/or abdominal pain.
  • back pain slow-onset back pain with stiffness for at least three months or longer, intense pain in the mornings or after periods of inactivity, and it may begin to ease during the day with stretching and exercise
  • fatigue joint pain
  • heel pain sleep problems
  • abdominal pain abdominal pain
  • the subject did not yet receive any treatment with an anti-inflammatory and/or anti-rheumatic drug to treat spondyloarthritis. In certain embodiments, the subject did not receive any treatment with an anti-inflammatory and/or anti-rheumatic drug for a period of at least 3 months, preferably at least 6 months.
  • the subject is diagnosed with spondyloarthritis and is selected to receive treatment with an anti-inflammatory and/or anti-rheumatic drug, for example drugs selected from nonsteroidal anti-inflammatory drugs (NSAIDs), conventional disease-modifying antirheumatic drugs (cDMARDs) and biological DMARDs such as tumor necrosis factor-alpha (TNF-alpha) inhibitors, agents targeting Interleukin (IL)-17A and Janus kinase (JAK) inhibitors
  • NSAIDs nonsteroidal anti-inflammatory drugs
  • cDMARDs conventional disease-modifying antirheumatic drugs
  • TNF-alpha tumor necrosis factor-alpha
  • IL-17A Interleukin-17A
  • JK Janus kinase
  • a method for treatment of a subject with spondyloarthritis comprises identifying a subject as having or not having one or more antibodies as taught herein in a biological sample from the subject (such as by a method comprising determining the presence or quantity of the one or more antibodies in the sample), and administering an anti-spondyloarthritic drug to the subject.
  • the method comprises comparing the antibody levels in the biological sample of the subject with the antibody levels in one or more reference samples.
  • the subject when antibodies as disclosed herein are detected as compared to the reference sample, the subject is diagnosed with spondyloarthritis and the subject is treated with an anti-inflammatory or anti-rheumatc drug, in particular with a drug selected from nonsteroidal anti-inflammatory drugs (NSAIDs), conventional diseasemodifying anti-rheumatic drugs (cDMARDs, for example sulfasalazine) and biological DMARDs such as tumor necrosis factor-alpha (TNF-alpha) inhibitors.
  • NSAIDs nonsteroidal anti-inflammatory drugs
  • cDMARDs conventional diseasemodifying anti-rheumatic drugs
  • TNF-alpha tumor necrosis factor-alpha
  • the subject is first diagnosed with spondyloarthritis using any one of the in vitro methods, peptides, detection agents, compositions or diagnostic kits of the present invention, followed by selection of the appropriate therapy and treatment of the subject with said therapy against spondyloarthritis.
  • the method of treatment comprises the in vitro method for diagnosing the presence of spondyloarthritis in a subject, said method being any method as described herein above, followed by treatment of the subject diagnosed with spondyloarthritis with a therapy selected from nonsteroidal anti-inflammatory drugs (NSAIDs), conventional disease-modifying anti-rheumatic drugs (cDMARDs, for example sulfasalazine) and biological DMARDs such as tumor necrosis factor-alpha (TNF-alpha) inhibitors.
  • NSAIDs nonsteroidal anti-inflammatory drugs
  • cDMARDs conventional disease-modifying anti-rheumatic drugs
  • TNF-alpha tumor necrosis factor-alpha
  • the methods of treatment as taught herein may specifically relate to prophylactic and/or therapeutic treatment of spondyloarthritis.
  • said method relates to a therapeutic treatment of spondyloarthritis.
  • the therapeutic agent administered to the subject can be a therapeutic agent known to be effective against spondyloarthritis, as disclosed herein above.
  • the terms “therapy” or “treatment” refer to interventions, such as pharmacological interventions, that result in the alleviation or measurable lessening of one or more symptoms or measurable markers of a pathological condition such as a disease or disorder.
  • the terms encompass primary treatments as well as neo-adjuvant treatments, adjuvant treatments and adjunctive therapies.
  • the terms “therapy” or “treatment” broadly refer to interventions that result in the alleviation or measurable lessening of one or more symptoms or measurable markers of spondyloarthritis. Measurable lessening includes any clinically significant decline in a measurable marker or symptom.
  • the terms encompass both curative treatments and treatments directed to reduce symptoms and/or slow progression of the disease.
  • the terms encompass both the therapeutic treatment of an already developed pathological condition, as well as prophylactic or preventative measures, wherein the aim is to prevent or lessen the chances of incidence of a pathological condition.
  • the terms may relate to therapeutic treatments.
  • the terms may relate to preventative treatments. Treatment of a chronic pathological condition during the period of remission may also be deemed to constitute a therapeutic treatment.
  • the term may encompass ex vivo or in vivo treatments.
  • responsiveness or “susceptibility” or “sensitivity” may be used interchangeably herein and refer to the quality that predisposes a subject having spondyloarthritis to be sensitive or reactive to a particular therapeutic treatment.
  • a subject is “responsive” or “susceptible” or “sensitive” (which terms may be used interchangeably) to treatment with a particular therapeutic agent if the subject will have benefit from the treatment.
  • a subject is non-responsive to a particular treatment with a therapeutic agent if there is no effect of the treatment.
  • a subject is poorly responding to a particular treatment with a therapeutic agent if there is only minimal effect of the treatment.
  • the disease severity status of spondyloarthritis or the response to treatment is based on standard criteria known in the art.
  • Disease activity measurements can for example be based on the Bath Ankylosing Spondylitis Disease Activity Index (BASDAI), the Bath Ankylosing Spondylitis Functional Index (BASFI), erythrocyte sedimentation rate (ESR), and/or C-reactive protein (CRP) levels.
  • BASDAI Bath Ankylosing Spondylitis Disease Activity Index
  • BASFI Bath Ankylosing Spondylitis Functional Index
  • ESR erythrocyte sedimentation rate
  • CRP C-reactive protein
  • the presence of HLA-B27 can be linked to disease severity.
  • the disease activity is based on the BASDAI score.
  • axSpA or RA Patients with axSpA or RA were diagnosed by their treating rheumatologist and classified according to the ASAS classification criteria (Rudwaleit et a!., Ann Rheum Dis 2009, 68(6): 770-6; Rudwaleit et al., Ann Rheum Dis 2009, 68(6): 777-83) or the 1987 American College of Rheumatology criteria for RA (Arnett et al., Arthritis Rheum 1988; 31 (3): 315-24), respectively. All axSpA and RA patients were considered as early patients as plasma samples were collected within a maximum time of 5 years after diagnosis. The HC did not show signs of inflammation, rheumatic and/or joint related complaints or a positive family history of rheumatic and/or autoimmune diseases.
  • the SAS procedure uses plasma pools of axSpA patients and HC to identify novel axSpA-related antibodies.
  • a human RA cDNA phage display library and a human axSpA cDNA phage display library were used to screen for the presence of respectively IgG or IgA antibodies in pooled plasma of early axSpA patients.
  • This early axSpA SAS pool had a mean age of 40.6 ⁇ 11.8 years, a mean disease duration of 1 .4 ⁇ 0.5 years, 5 (50%) were male and 8 (80%) were HLA- B27 positive.
  • a HC SAS pool consisting of 10 HC plasma samples (mean age of 40.6 ⁇ 12.3 years and 5 (50%) were male) that were age- and gender- matched to the axSpA SAS pool, was used in the SAS procedure to perform counter selection. After the identification of novel antibodies and their corresponding targets by the SAS procedure, antibody reactivity against phage displayed peptides was tested in the early axSpA SAS pool and HC SAS pool, using phage enzyme linked immunosorbent assays (ELISA), in order to select the best peptide targets.
  • ELISA phage enzyme linked immunosorbent assays
  • Antibody reactivity against the 12 selected UH-axSpA-lgG peptides was determined and validated in additional individual plasma samples of 80 early axSpA patients, 46 HC and 46 RA patients, using phage ELISA.
  • Antibody reactivity against 7 UH-axSpA-lgA peptide targets was also determined by phage ELISA in individual plasma samples of 79 early axSpA patients and 92 HC.
  • the HC (n 92) had a mean age of 44.2 ⁇ 11.9 years and 52 (57%) were male.
  • a human axSpA cDNA phage display library was constructed according to the procedure as described in Vandormael et al. (Immunol Res, 2017; 65(1): 307-25). In brief, total RNA was extracted from hip synovial tissue of 3 axSpA patients and messenger RNA (mRNA) was converted into double-stranded cDNA, using the Maxima H-minus double-stranded cDNA synthesis kit (Thermo Fisher Scientific, Belgium). Patient-specific adaptors were ligated to the cDNA, which was then size-fractionated by agarose gel electrophoresis.
  • mRNA messenger RNA
  • cDNA molecules ranging from 200 to 10,000 base pairs were directionally cloned into the pSPVI-A/B/C phagemid vectors and transformed in TG1 E.coli cells via electroporation.
  • Phage particles from this resulting cDNA phage display library were produced by infecting TG1 E.coli cells with M13K07 helper phage and purifying via PEG/NaCI precipitation.
  • Phage particles from the human RA cDNA phage display library were screened for reactivity with IgG antibodies present in plasma of early axSpA patients by the previously described SAS procedure (Palmers et al., J Neuroinflammation, 2016, 13(1): 243; Somers et al., J Autoimmun, 2011 , 36(1): 33- 46; Somers et al., Ann NY Acad Sci, 2009, 1173: 92-102; Somers et al., J Immunol, 2008, 180(6): 3957- 63) with some minor modifications.
  • an immunotube (Nunc) was coated with 10 pg/mL rabbit anti-human IgG (Dako) diluted in coating buffer (0.1 M sodium hydrogen carbonate, pH 9.6), rotating overnight at 4°C.
  • coating buffer 0.1 M sodium hydrogen carbonate, pH 9.6
  • Approximately 4 x 10 13 phage particles from the RA cDNA phage display library were pre-incubated with the axSpA SAS pool, each plasma sample at a 1 :200 final dilution, in 2% skimmed milk powder in phosphate buffered saline (MPBS) for 1 .5 hours (hrs) at room temperature (RT) to allow formation of IgG antibody-phage complexes.
  • MPBS phosphate buffered saline
  • the antibody-phage pre-incubation mixture was added and incubated for 2 hrs and 30 minutes (min) at RT to capture antibody-phage complexes.
  • Non-bound antibodies and phage particles were washed away, while bound antibody-phage complexes were eluted by 100 mM triethylamine, pH 12.0 (Sigma- Aldrich) and neutralized using 1 M Tris-HCI, pH 7.4 (Sigma-Aldrich).
  • Eluted phage particles were amplified by infecting TG1 E.
  • coli bacterial cells which were then grown overnight at 30°C on 2xTY- ampicillin (100 pg/mL), 2% glucose agar plates. Resulting bacterial colonies were harvested and used as input for consecutive SAS selection rounds.
  • Four consecutive SAS selection rounds were performed using the early axSpA SAS plasma pool.
  • the fourth SAS selection round was immediately followed by one round of counter selection with the HC SAS pool, with each HC plasma sample at a 1 :200 final dilution, which was performed to prevent the isolation of common antibodies present in the general population.
  • phage particles not bound by IgG antibodies in the HC SAS pool were amplified and characterized.
  • the fusion regions between the M13 gene VI and cDNA inserts of selected phage clones were analyzed.
  • the cDNA inserts of randomly selected bacterial colonies were amplified using PCR primers flanking the cDNA inserts. Initial characterization of these clones was performed by DNA fingerprinting of these PCR products using restriction enzymes BstNI (Roche Diagnostics) and CviQI (New England Biolabs). Clones that showed a unique restriction pattern were identified by DNA sequencing of the fusion between M13 gene VI and the cDNA inserts. Both nucleotide and amino acid sequences of identified cDNA inserts were compared with human sequences using the BLAST tool of NCBI.
  • Novel IgA antibodies and their corresponding antigenic targets were identified by the SAS procedure.
  • phage particles displaying axSpA synovial antigens and randomly formed peptides, originating from the newly human axSpA cDNA phage display library were screened for IgA antibody reactivity in the early axSpA SAS pool using the previously described SAS procedure, with some minor modifications.
  • a Nunc immunotube was coated with 10 pg/mL of rabbit anti-human IgA (Dako), diluted in coating buffer (0.1 M sodium hydrogen carbonate, pH 9.6), rotating overnight at 4°C.
  • the immunotube was blocked with 2% skimmed milk powder in phosphate buffered saline (MPBS) for 2 hours (hrs) at room temperature (RT). Meanwhile, approximately 4 x 10 13 phage particles from the human axSpA cDNA phage display library were pre-incubated with the early axSpA SAS pool diluted in 2% MPBS (each plasma sample at a 1 :200 final dilution) for 1 .5 hrs at RT to allow the formation of IgA antibody- phage complexes. The antibody-phage pre-incubation mixture was added to the blocked immunotube and incubated for 2 hrs and 30 minutes (min) at RT to capture the antibody-phage complexes.
  • MPBS phosphate buffered saline
  • the nonbound antibodies and phage particles were removed by extensive washing, whereas the bound antibody-phage complexes were eluted by 100 mM triethylamine, pH 12.0 (Sigma-Aldrich) and neutralized using 1 M Tris-HCI, pH 7.4 (Sigma-Aldrich). Eluted phage particles were then amplified by infecting TG1 E. coli bacterial cells, which were grown overnight on 2xTY- ampicillin (100 pg/mL), 2% glucose agar plates. The resulting bacterial colonies were harvested and used as input for consecutive SAS selection rounds.
  • Antibody reactivity against peptides displayed on phage particles was determined by phage ELISA in pooled and individual plasma samples as described previously (Palmers et al., J Neuroinflammation, 2016, 13(1): 243), with some minor modifications.
  • Microlon high binding microplates (Greiner Bio-One) were coated respectively with 5 pg/mL anti-M13 monoclonal antibody (GE Healthcare) or 4.0 pg/mL anti-M13 mouse monoclonal antibody (clone MM05T, Sino Biological) diluted in coating buffer (0.2 M sodium carbonate bicarbonate buffer, pH 9.6) overnight at 4°C to determine the presence of UH-axSpA-lgG or UH-axSpA-lgA antibodies. Coated plates were blocked with 5% MPBS for 2 hrs at 37°C while shaking.
  • the plates were then incubated with 7 x 10 11 colony forming units (cfu)/mL of phage particles expressing the antigen of interest (specific phage) or phage particles without peptide (empty phage) for 1 hr at 37°C and 30 min at RT.
  • Bound phage particles were incubated with pooled or individual plasma samples diluted 1 : 100 in 5% MPBS, followed by incubation with respectively anti-human IgG-Fc antibody coupled with horseradish peroxidase (Bethyl laboratories) diluted 1 :50,000 in 5% MPBS or with cross-absorbed goat anti-human IgA-Fc conjugated with horse radish peroxidase (Bethyl Laboratories) diluted 1 :2,500 in 5% MPBS for 1 hr at RT.
  • Antibody reactivity against each phage-displayed peptide is expressed by the ratio of the optical density (OD) signal of each phage-displayed peptide over the OD signal of the phage without peptide (OD(specific phage)/OD(empty phage)).
  • a cut-off for seropositivity was calculated as the mean of the antibody reactivity in the non-reactive samples plus 3 x standard deviation. Samples with a mean ratio above this cut-off value were considered antibody-positive. Within each experiment, samples were tested in duplicate and experiments were performed independently for at least two times. Average values of experimental repeats had a coefficient of variation (% CV) lower than 20%.
  • Antibody reactivity against individual phage displayed antigens or panels of antigens was compared between axSpA patients and controls using the Fisher’s exact test.
  • Clinical characteristics were compared between antibody-positive and antibody-negative axSpA patients, in which continuous clinical characteristics were compared by Student’s t-tests and categorical characteristics were compared using Fisher’s exact tests.
  • LR+ positive likelihood ratio
  • Presence of antibodies against each of these 173 phage displayed peptides was first determined by phage ELISA in 6 additional plasma pools, each pool consisting of 10 early axSpA patients, and in 3 additional plasma pools, consisting of 10 HC each.
  • IgA antibodies against against 7 of these 173 peptides were more frequently present in the 6 early axSpA plasma pools as compared to the 3 HC plasma pools.
  • These 7 peptides were annotated UH-axSpA-lgA.1 till UH-axSpA-lgA.10 (University Hasselt-axSpA-lgA. isotype target number).
  • Nucleotide and amino acid sequences of the 7 selected UH-axSpA-lgA peptides were compared with human and microbial sequences using the custom DNAnalyzer program, which correspond to fragments of known proteins and novel linear peptides (see Table 4).
  • UH-axSpA-lgA.1 to UH-axSpA-lgA.9 had a length between 10 and 54 amino acids and showed only partial homology to different human proteins as these peptides resulted from the out-of-frame cDNA translation or the translation of non-coding cDNA sequences.
  • the DNA sequence of UH-axSpA-lgA.10 corresponded with an in-frame fusion to the 3’-coding region of the human Histone deacetylase 3 (HDAC3) gene.
  • HDAC3 variant showed 100% homology with the canonical HDAC3 sequence (NM_003883.3), resulting in the expression of a peptide with 100% homology to the last 22 amino acids of the C-terminal part of human HDAC3, whereas the total human HDAC3 protein has a size of 428 amino acids.
  • the peptide sequences of phage-displayed peptides UH-axSpA-lgA.1 , UH-axSpA-lgA.3, UH- axSpA-lgA.6 to UH-axSpA-lgA.10 also showed partial homology to proteins from several microbial species, including Escherichia (E.) coli, Klebsiella (K.) pneumoniae, Saccharomyces (S.) cerevisiae and Yersinia (Y.) pestis (Table 4).
  • Salmonella enterica (A0A426WQ81)
  • Multidrug resistance protein MdtA, mdtA, E. coli (B1LNW7)
  • Multidrug resistance protein MdtA, mdtA E. coli (B1LNW7)
  • MACROD2 MACROD2 (A1Z1Q3)
  • peptide sequences only showed partial homology to human proteins involved in several biological processes, including protein biosynthesis (Ribosome- releasing factor 2 (GFM2)), transcription regulation (SAGA-associated factor 29 (SGF29)), metabolic processes (Cysteine sulfinic acid decarboxylase (CSAD)), motility and muscle contraction (Titin (TTN)), transport (Bestrophin-1 (BEST1)), cell adhesion (Platelet glycoprotein 4 (CD36)), DNA regulation (DNA-dependent protein kinase catalytic subunit (PRKDC)) and to proteins with an yet unknown function (Coiled-coil domain- containing protein 171 (CCDC171)).
  • GMM2 protein biosynthesis
  • SGF29 transcription regulation
  • TTN motility and muscle contraction
  • BEST1 Bestrophin-1
  • CD36 Cell adhesion
  • PRKDC DNA regulation
  • CCDC171 CCDC171
  • the peptide sequences expressed by UH-axSpA-lgG.104, UH-axSpA-lgG.105 and UH-axSpA.lgG.108 encoded C-terminal portions of known human proteins: intraflagellar transport protein 43 (IFT43), target of Myb protein 1 (TOM1) and DNA polymerase subunit gamma-1 (POLG). Since the TAG stop codon of UH-axSpA.lgG.108 is an amber stop codon, and thus translated to glutamine (Q) in the TG1 E.coli bacteria, a 102 nucleotide stretch of the 3’UTR of POLG is also translated and expressed as the final 34 amino acids on this phage clone.
  • IFT43 intraflagellar transport protein 43
  • TOM1 target of Myb protein 1
  • POLG DNA polymerase subunit gamma-1
  • the antibody positive early axSpA patients did not show any significant difference in age, gender, HLA-B27 status, disease duration, treatment, Bath Ankylosing Spondylitis Disease Activity Index (BASDAI), Bath Ankylosing Spondylitis Functional Index (BASFI), erythrocyte sedimentation rate (ESR), CRP and presence of extra-articular manifestations, as compared to early axSpA patients lacking these antibodies (Table 8).
  • BASDAI Bath Ankylosing Spondylitis Disease Activity Index
  • BASFI Bath Ankylosing Spondylitis Functional Index
  • ESR erythrocyte sedimentation rate
  • CRP erythrocyte sedimentation rate
  • NSAID use (n, %) 15/21 (71.4) 39/55 (70.9) 1.000 cDMARD use (n, %) 6/21 (28.6) 24/55 (43.64) 0.2977 bDMARD use (n, %) 5/21 (23.8) 13/55 (23.6) 1.000
  • HLA Human Leukocyte Antigen
  • NSAID Non-Steroidal Anti-Inflammatory Drug
  • cDMARD conventional Disease- Modifying Anti-Rheumatic Drug
  • bDMARD biological Disease-Modifying Anti-Rheumatic Drug
  • BASDAI Bath Ankylosing Spondylitis Disease Activity Index
  • BASFI Bath Ankylosing Spondylitis Functional Index
  • ESR Erythrocyte Sedimentation Rate
  • CRP C-Reactive Protein Presence of antibodies against 12 novel UH-axSpA-igG peptides in axSpA patients and controls
  • Presence of antibodies against the 12 selected UH-axSpA-lgG peptides was determined in individual plasma samples of early axSpA patients, patients with RA, and HC from the UH cohort, using phage ELISA. Antibody reactivity against these individual UH-axSpA-lgG peptides was present in 1% (1/80) up to 21 % (17/80) of early axSpA patients, whereas presence of these antibodies ranged from 0% (0/46) to 15% (7/46) in HC and from 0% (0/46) to 20% (9/46) in RA patients (Table 9).
  • Table 10 Antibody reactivity against individual UH-axSpA-lgG peptides and a panel of 4 peptides in early axSpA patients compared to HC
  • NSAID use ( n , %) 9/15 (60.0) 23/60 (38.3) 0.1531 cDMARD use (n, %) 9/15 (60.0) 21/60 (35) 0.0876 bDMARD use (n, %) 0/15 (0) 18/63 (28.6) 0.0167
  • HLA-B27 Human Leukocyte Antigen-B27
  • NSAID Non-Steroidal Anti-Inflammatory Drug
  • cDMARD conventional Disease-Modifying Anti-Rheumatic Drug
  • bDMARD biological Disease-Modifying Anti-Rheumatic Drug
  • BASDAI Bath Ankylosing Spondylitis Disease Activity Index
  • BASFI Bath Ankylosing Spondylitis Functional Index
  • ESR Erythrocyte Sedimentation Rate
  • CRP C-Reactive Protein.
  • Patient 2 and 5 were treated with a biological Disease-Modifying Anti-Rheumatic Drug (DMARD), whereas patient 3 and 4 were treated with a conventional DMARD.
  • DMARD biological Disease-Modifying Anti-Rheumatic Drug

Abstract

The present invention in general relates to the field of antibody profiling in spondyloarthritis. In particular, the inventors found that antibody levels against selected peptides are raised in spondyloarthritis patients, and herein provide a diagnostic method and kit comprising such peptides for use in the diagnosis of spondyloarthritis.

Description

METHOD AND MEANS FOR DIAGNOSIS OF SPONDYLOARTHRITIS
FIELD OF THE INVENTION
The present invention in general relates to the field of antibody profiling in spondyloarthritis. In particular, the invention provides methods and means for diagnosing spondyloarthritis, for evaluating the disease severity of spondyloarthritis and/or for evaluating the response of a subject diagnosed with spondyloarthritis to therapeutic treatment based on antibody profiling.
BACKGROUND TO THE INVENTION
Axial spondyloarthritis (axSpA) is a rheumatic disease affecting between 0.5 and 1.4% of the population in Europe and North America, and is mainly characterized by chronic inflammation of spinal and sacroiliac joints. Diagnosis of axSpA is challenging since clinical manifestations, such as inflammatory back pain, peripheral arthritis and inflammatory bowel disease often overlap with other disorders. In the absence of diagnostic criteria, classification criteria developed by the Assessment in SpondyloArthritis international Society (ASAS) are often used for diagnosis in patients with suspect of axial or peripheral spondyloarthritis. These criteria combine physical examination, presence of sacroiliitis on imaging and serological tests for human leukocyte antigen (HLA)-B27 and C-reactive protein (CRP) (Rudwaleit et al., Ann Rheum Dis 2009; 68(6): 770-6; Rudwaleit et al., Ann Rheum Dis 2009, 68(6): 777-83). However, these criteria are not specific enough to distinguish axSpA patients from persons with non-specific chronic low back pain (LBP) at an early disease phase (de Vlam, Brest Parte Res Clin Rheumatol, 2010, 24(5); 671-82; de Winter et al., Arthritis Rheumatol, 2018, 70(7): 1042-8; Ez-Zaitouni et al., Rheumatology (Oxford), 2018: 57(7): 1173-9). Therefore, for many patients, axSpA diagnosis may be challenging, and is often delayed by several years after the occurrence of first clinical symptoms, which poses a problem for early treatment initiation. This underscores the importance of the discovery of new objective biomarkers to improve early axSpA diagnosis. Emerging evidence indicates a possible role of the humoral immune response in the pathophysiology of axSpA, and several antibodies against various antigens have already been reported (Quaden et al., Autoimmune Rev, 2016, 15(8): 820-32). Nevertheless, the clinical relevance of these antibodies and their targets must be studied and validated more extensively in different cohorts to establish the true accuracy of these potential diagnostic biomarkers for axSpA. Most of these antibodies are of the immunoglobulin (lg)G isotype, which is not surprising since IgG antibodies are the most common and abundant antibodies within the human blood, making them very useful as diagnostic biomarker. Our research group has recently described IgG antibody reactivity against a panel of 3 novel peptides (called UH-axSpA-lgG.1 , UH-axSpA-lgG.4 and UH-axSpA-lgG.8) in 14.2% (22/155) of early axSpA patients from two cohorts, in 8.4% (8/95) of healthy controls (HC), and in only 5% (4/75) of persons with low back pain (LBP), resulting in a specificity of 95%, and a positive likelihood ratio (LR+) of 2.7. Combined with a positive result for the laboratory tests HLA-B27 and CRP, testing for antibodies to these 3 UH-axSpA-lgG peptides increased the post-test probability for axSpA from 79% to 91%, and could therefore be a valuable addition to the current diagnostic process (Quaden et al., Arthritis Rheumatol. 2020; 72(12): 2094-105). Still, one limitation to the use of antibodies against this panel of 3 UH-axSpA-lgG peptides, is the limited sensitivity, leaving about 85% of axSpA patients seronegative for these novel markers.
SUMMARY OF THE INVENTION
As corroborated in the experimental section, the inventors of the present application identified a novel tool for the diagnosis of spondyloarthritis (SpA), in particular in axial spondyloarthritis (axSpA). The invention further provides tools for evaluating the disease severity of spondyloarthritis and for the evaluation of therapy response in patients with spondyloarthritis, in particular axSpA, More specific, in the present invention novel antibody biomarkers were identified that can identify SpA patients and that can be used for the evaluation of the disease severity or of the therapeutic treatment in SpA patients.
In a first aspect, the present invention provides an in vitro method for diagnosing spondyloarthritis in a subject, said method comprising: a) providing a biological sample from the subject, and b) determining the presence or quantity of antibodies against one or more peptides; in particular 2, 3, or 4 peptides selected from the list consisting of SEQ ID No: 1 , SEQ ID No: 2, SEQ ID No: 3, SEQ ID No: 4, SEQ ID No: 5, SEQ ID No: 6, and SEQ ID No: 7, or against an immune-reactive fragment or antibody-binding fragment or variant thereof; wherein the presence of or an increase in antibody levels against at least one of said peptides or immune-reactive fragments or antibody-binding fragments or variants thereof in said biological sample compared to a reference sample is indicative for the presence of spondyloarthritis.
In another aspect, the present invention provides an in vitro method for diagnosing spondyloarthritis in a subject, said method comprising: a) providing a biological sample from the subject, and b) determining the presence or quantity of antibodies against a peptide as shown in SEQ ID No: 2, or an immune- reactive fragment or antibody-binding fragment or variant thereof, against a peptide as shown in SEQ ID NO: 1 or against an antibody-binding fragment or variant thereof, and against a peptide as shown in SEQ ID NO: 7 or against an antibody-binding fragment or variant thereof; wherein the presence or an increase in antibody levels against at least one of said peptides or immune-reactive fragments or antibody-binding fragments or variants thereof in said biological sample compared to a reference sample is indicative for the presence of spondyloarthritis in the subject. In some embodiments, the presence or an increase in antibody levels against the peptide as shown in SEQ ID NO: 2 or against an immune- reactive fragment or against an antibody-binding fragment or variant thereof compared to the reference sample is indicative for the presence of spondyloarthritis in the subject.
In some embodiments, the presence or increase in antibody levels against the peptide as shown in SEQ ID NO: 2 or against an immune-reactive fragment or against an antibody-binding fragment or variant thereof, against the peptide as shown in SEQ ID NO 1 or against an immune-reactive fragment or an antibody-binding fragment or variant thereof, and against the peptide as shown in SEQ ID NO: 7 or against an immune-reactive fragment or an antibody-binding fragment or variant thereof, in said sample compared to the reference sample is indicative for the presence of spondyloarthritis in the subject.
In some embodiments, the method of the invention further comprises determining the presence or quantity of antibodies against one or more peptides; in particular 2, 3 or 4 peptides, as shown in SEQ ID No: 3, SEQ ID No: 4, SEQ ID No: 5, and SEQ ID No: 6, or against an immune-reactive fragment or antibody-binding fragment or variant thereof, wherein the presence or an increase in antibody levels against one or more of said peptides or against said immune-reactive fragments or antibody-binding fragments or variants thereof compared to the reference sample is indicative for the presence of spondyloarthritis in the subject.
In some embodiments, the method further comprises determining the presence or quantity of antibodies against one or more peptides; in particular 2, 3 or 4 peptides, as shown in SEQ ID No: 8, SEQ ID No: 9, SEQ ID No: 10, SEQ ID No: 11 , SEQ ID No: 12, SEQ ID No: 13, SEQ ID No: 14, SEQ ID No: 15, SEQ ID No: 16, SEQ ID No: 17, SEQ ID No: 18 and SEQ ID No: 19, or against an immune-reactive fragment or antibody-binding fragment or variant thereof in the first and second biological sample, wherein the presence or an increase in antibody levels against one or more of said peptides or against said immune-reactive fragments or antibody-binding fragments or variants thereof in the biological sample compared to the reference sample is indicative for the presence of spondyloarthritis in the subject.
In a related aspect, the present invention provides an in vitro method for evaluating the disease severity of spondyloarthritis in a subject, said method comprising: a) providing a biological sample from the subject, and b) determining the presence or quantity of antibodies against one or more peptides; in particular 2, 3, or 4 peptides selected from the list consisting of SEQ ID No: 1 , SEQ ID No: 2, SEQ ID No: 3, SEQ ID No: 4, SEQ ID No: 5, SEQ ID No: 6, and SEQ ID No: 7, or against an immune-reactive fragment or antibody-binding fragment or variant thereof; wherein the presence of or an increase in antibody levels against at least one of said peptides or immune-reactive fragments or antibody-binding fragments or variants thereof in said biological sample compared to a reference sample is indicative for the disease severity of spondyloarthritis.
In another related aspect, the present invention provides an in vitro method for evaluating the disease severity of spondyloarthritis in a subject, said method comprising: a) providing a biological sample from the subject, and b) determining the presence or quantity of antibodies against a peptide as shown in SEQ ID No: 2, or an immune-reactive fragment or antibody-binding fragment or variant thereof, against a peptide as shown in SEQ ID NO: 1 or against an antibody-binding fragment or variant thereof, and against a peptide as shown in SEQ ID NO: 7 or against an antibody-binding fragment or variant thereof; wherein the presence or an increase in antibody levels against at least one of said peptides or immune- reactive fragments or antibody-binding fragments or variants thereof in said biological sample compared to a reference sample is indicative for the disease severity of spondyloarthritis in the subject. In some embodiments, the presence or an increase in antibody levels against the peptide as shown in SEQ ID NO: 2 or against an immune-reactive fragment or against an antibody-binding fragment or variant thereof compared to the reference sample is indicative forthe disease severity of spondyloarthritis in the subject.
In another aspect, the present invention provides an in vitro method for evaluating a therapeutic treatment of spondyloarthritis in a subject, said method comprising: a) providing a first biological sample from the subject obtained before the start of the therapeutic treatment, b) providing a second biological sample from the subject obtained after therapeutic treatment, and c) determining the presence or quantity of antibodies against one or more peptides; in particular 2, 3, or 4 peptides selected from the list consisting of SEQ ID No: 1 , SEQ ID No: 2, SEQ ID No: 3, SEQ ID No: 4, SEQ ID No: 5, SEQ ID No: 6, and SEQ ID No: 7, or against an immune-reactive fragment or antibody-binding fragment or variant thereof in the first and second biological sample; wherein the presence of or a difference in antibody levels against at least one of said peptides or immune-reactive fragments or antibody-binding fragments or variants thereof in the second biological sample compared to the first biological sample indicates that the subject is responding to the therapeutic treatment.
In another aspect, the present invention provides an in vitro method for evaluating a therapeutic treatment of spondyloarthritis in a subject, said method comprises a) providing a first biological sample from the subject obtained before the start of the therapeutic treatment, b) providing a second biological sample from the subject obtained after therapeutic treatment, and c) determining the presence or quantity of antibodies against a peptide as shown in SEQ ID No: 2 or against an antibody-binding fragment or variant thereof, against a peptide as shown in SEQ ID NO: 1 or against an antibody-binding fragment or variant thereof, and against a peptide as shown in SEQ ID NO: 7 or against an antibodybinding fragment or variant thereof in the first and second biological sample; wherein a deviation or no deviation in antibody levels against at least one of said peptides or antibody-binding fragments or variants thereof in the second biological sample compared to the first biological sample indicates that the subject is responding to the therapeutic treatment; preferably wherein a decrease in antibody levels against at least one of said peptides or antibody-binding fragments or variants thereof in the second biological sample compared to the first biological sample indicates that the subject is responding to the therapeutic treatment.
In some embodiments, the presence or a deviation or no deviation in antibody levels against the peptide as shown in SEQ ID NO: 2 or against an immune-reactive fragment or against an antibody-binding fragment or variant thereof in the second biological sample compared to the first biological sample indicates that the subject is responding to the therapeutic treatment. Preferably, a decrease in antibody levels against the peptide as shown in SEQ ID NO: 2 or against an immune-reactive fragment or against an antibody-binding fragment or variant thereof in the second biological sample compared to the first biological sample indicates that the subject is responding to the therapeutic treatment.
In some embodiments, the presence or a deviation or no deviation in antibody levels against the peptide as shown in SEQ ID NO: 2 or against an immune-reactive fragment or against an antibody-binding fragment or variant thereof, against the peptide as shown in SEQ ID NO 1 or against an immune-reactive fragment or an antibody-binding fragment or variant thereof, and against the peptide as shown in SEQ ID NO: 7 or against an immune-reactive fragment or an antibody-binding fragment or variant thereof, in the second biological sample compared to the first biological sample indicates that the subject is responding to the therapeutic treatment.
In some embodiments, the method further comprises determining the presence or quantity of antibodies against one or more peptides; in particular 2, 3 or 4 peptides, as shown in SEQ ID No: 3, SEQ ID No: 4, SEQ ID No: 5, and SEQ ID No: 6, or against an immune-reactive fragment or antibody-binding fragment or variant thereof in the first and second biological sample, wherein the presence or a deviation or no deviation in antibody levels against one or more of said peptides or against said immune-reactive fragments or antibody-binding fragments or variants thereof in the second biological sample compared to the first biological sample indicates that the subject is responding to the therapeutic treatment.
In some embodiments, the method further comprises determining the presence or quantity of antibodies against one or more peptides; in particular 2, 3 or 4 peptides, as shown in SEQ ID No: 8, SEQ ID No: 9, SEQ ID No: 10, SEQ ID No: 11 , SEQ ID No: 12, SEQ ID No: 13, SEQ ID No: 14, SEQ ID No: 15, SEQ ID No: 16, SEQ ID No: 17, SEQ ID No: 18 and SEQ ID No: 19, or against an immune-reactive fragment or antibody-binding fragment or variant thereof in the first and second biological sample, wherein the presence or a deviation or no deviation in antibody levels against one or more of said peptides or against said immune-reactive fragments or antibody-binding fragments or variants thereof in the second biological sample compared to the first biological sample indicates that the subject is responding to the therapeutic treatment.
In still another aspect, the present invention provides a peptide as shown in any one of SEQ ID NOs 1- 7, or of SEQ ID NOs: 8-19, or an immunoreactive fragment or an antibody-binding fragment or variant thereof.
In another aspect, the present invention provides a peptide as shown in any one of SEQ ID NOs 1-7, or of SEQ ID NOs: 8-19.
In another aspect, the invention provides a peptide comprising a sequence represented by any of SEQ ID Nos: 1-7, by any one of Seq ID Nos: 8-19, or an immunoreactive fragment or antibody-binding fragment or variant thereof, in particular comprising at least 4; preferably at least 6, preferably at least 7, more preferably at least 9, consecutive amino acids derived from said sequences.
In another aspect, the present invention provides a peptide consisting essentially of a sequence represented by SEQ ID Nos: 1-7, of a sequence of represented by Seq ID Nos: 8-19, or an immunoreactive fragment thereof, in particular comprising at least 4, preferably at least 6, consecutive amino acids derived from SEQ ID Nos: 1-7, or from SEQ ID Nos: 8-19. Thus, a peptide selected from the list comprising SEQ ID No: 1 , SEQ ID No: 2, SEQ ID No: 3, SEQ ID No: 4, SEQ ID No: 5, SEQ ID No: 6, SEQ ID No: 7, SEQ ID No: 8, SEQ ID No: 9, SEQ ID No: 10, SEQ ID No: 11 , SEQ ID No: 12, SEQ ID No: 13, SEQ ID No: 14, SEQ ID No: 15, SEQ ID No: 16, SEQ ID No: 17, SEQ ID No: 18, SEQ ID No: 19; or an immunoreactive fragment or antibody-binding fragment or variant thereof, in particular comprising at least 4, in particular at least 5, even more in particular at least 6, consecutive amino acids derived therefrom, is provided.
Also provided is a detection agent comprising one or more peptides, antibody-binding fragments or variants thereof as disclosed herein.
In a related aspect, a composition is provided comprising one or more of the peptides or immunoreactive fragments or antibody-binding fragments or variants thereof or detection agents as described above.
In a related aspect, the peptides, the detection agents or the composition according to the present invention are for use in the diagnosis of spondyloarthritis.
In another aspect, the peptides, the detection agents or the composition according to the present invention are for use for evaluating the therapy response in a subject diagnosed with spondyloarthritis.
In another aspect, the use of a peptide, an immunoreactive fragment thereof, an antibody-binding fragment or variant thereof as disclosed herein, or of a detection agent as taught herein, or of a composition comprising a peptide, immunoreactive fragment thereof, antibody-binding fragment or variant thereof or a detecting agent as disclosed herein, is provided for detecting the presence or quantity of specific antibodies against said peptide, immunoreactive fragment or antibody-binding fragment or variant thereof or against the peptide, immunoreactive fragment or antibody-binding fragment or variant thereof present in the composition, preferably wherein the presence or increase in antibody levels as compared to a reference sample is indicative for the diagnosis of spondyloarthritis.
In a related aspect, the use of a peptide, immunoreactive fragment thereof, antibody-binding fragment or variant thereof as disclosed herein, or of a detection agent as taught herein, of a composition comprising a peptide, immunoreactive fragment thereof, antibody-binding fragment or variant thereof as disclosed herein, is provided for detecting the presence or quantity of specific antibodies against said peptide, antibody-binding fragment or variant, or against the peptide, antibody-binding fragment or variant present in the composition, in a first and second biological sample of the subject wherein the first biological sample is obtained before the start of therapeutic treatment and the second biological sample is obtained after the start of therapeutic treatment; preferably wherein a decrease in antibody levels in the second biological sample as compared to the first biological sample is indicative for a response to the therapeutic treatment.
The present invention also provides a diagnostic kit for performing the in vitro method according to any of the embodiments of the present invention, said kit comprising a peptide or immunoreactive fragment or antibody-binding fragment or variant thereof, or a detection agent as taught herein, or a composition according to the invention, and reagents for detecting antibody binding to said one or more peptides or fragments thereof.
In a final aspect, the present invention provides a method for treatment of a subject with spondyloarthritis, said method comprising: identifying a subject as having or not having one or more peptides or antibody-binding fragments as taught herein in a biological sample from the subject, such as by any one of the in vitro methods as taught herein, and administering a therapeutic treatment against spondyloarthritis to the subject.
The above and further aspects and preferred embodiments of the invention as described in the following sections and the appended claims. The subject-matter of the appended claims is hereby specifically incorporated in this specification. BRIEF DESCRIPTION OF THE DRAWINGS
With specific reference now to the figures, it is stressed that the particulars shown are by way of example and for purposes of illustrative discussion of the different embodiments of the present invention only. They are presented in the cause of providing what is believed to be the most useful and readily description of the principles and conceptual aspects of the invention. In this regard, no attempt is made to show structural details of the invention in more detail than is necessary for a fundamental understanding of the invention. The description taken with the drawings making apparent to those skilled in the art how the several forms of the invention may be embodied in practice.
Figure 1. Added value of a combination of our antibody biomarker panels for the diagnosis of early axSpA patients. In the early axSpA patients that were tested for antibodies against each of the three UH-axSpA peptide panels (n=62), antibodies against our panel of UH-axSpA-lgG.1/4/8 peptides (PCT/EP2020/076120 published as WO2021/053152 A1) detected 16% of early axSpA patients. Additional testing for antibody reactivity against the novel UH-axSpA-lgG.101/102/109/110 peptides, increased the sensitivity for detecting early axSpA patients from 16% to 32%. Additional testing for antibodies against the novel UH-axSpA-lgA.1/3/10 peptides, leads to a further increase in sensitivity, allowing to detect up to 50% of early axSpA patients in total.
Figure 2. Presence of antibodies to UH-axSpA-lgA.3 is indicative for therapy response. A. The presence of antibodies to UH-axSpA-lgA.3 in axSpA patients (n=6) was evaluated at the time of diagnosis of the disease (timepoint 1) and after 12 months (timepoint 2), wherein the patients were treated with a biological or conventional Disease-Modifying Anti-Rheumatic Drug (DMARD). B. In these patients the clinical disease severity was assessed at both time point 1 and 2 by using the Bath Ankylosing Disease Activity Index (BASDAI).
DETAILED DESCRIPTION OF THE INVENTION
As used herein, the singular forms “a”, “an”, and “the” include both singular and plural referents unless the context clearly dictates otherwise.
The terms “comprising”, “comprises” and “comprised of as used herein are synonymous with “including”, “includes” or “containing”, “contains”, and are inclusive or open-ended and do not exclude additional, non-recited members, elements or method steps. The terms also encompass “consisting of and “consisting essentially of, which enjoy well-established meanings in patent terminology.
The recitation of numerical ranges by endpoints includes all numbers and fractions subsumed within the respective ranges, as well as the recited endpoints.
The terms “about” or “approximately” as used herein when referring to a measurable value such as a parameter, an amount, a temporal duration, and the like, are meant to encompass variations of and from the specified value, such as variations of +/-10% or less, preferably +/-5% or less, more preferably +/- 1% or less, and still more preferably +/-0.1% or less of and from the specified value, insofar such variations are appropriate to perform in the disclosed invention. It is to be understood that the value to which the modifier “about” refers is itself also specifically, and preferably, disclosed. Furthermore, the terms first, second, third and the like in the description and in the claims, are used for distinguishing between similar elements and not necessarily for describing a sequential or chronological order, unless specified. It is to be understood that the terms so used are interchangeable under appropriate circumstances and that the embodiments of the invention described herein are capable of operation in other sequences than described or illustrated herein.
Whereas the terms “one or more” or “at least one”, such as one or more members or at least one member of a group of members, is clear per se, by means of further exemplification, the term encompasses inter alia a reference to any one of said members, or to any two or more of said members, such as, e.g., any >3, >4, >5, >6 or >7 etc. of said members, and up to all said members. In another example, “one or more” or “at least one” may refer to 1 , 2, 3, 4, 5, 6, 7 or more.
The discussion of the background to the invention herein is included to explain the context of the invention. This is not to be taken as an admission that any of the material referred to was published, known, or part of the common general knowledge in any country as of the priority date of any of the claims.
Throughout this disclosure, various publications, patents and published patent specifications are referenced by an identifying citation. All documents cited in the present specification are hereby incorporated by reference in their entirety. In particular, the teachings or sections of such documents herein specifically referred to are incorporated by reference.
Unless otherwise defined, all terms used in disclosing the invention, including technical and scientific terms, have the meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. By means of further guidance, term definitions are included to better appreciate the teaching of the invention. When specific terms are defined in connection with a particular aspect of the invention or a particular embodiment of the invention, such connotation is meant to apply throughout this specification, i.e., also in the context of other aspects or embodiments of the invention, unless otherwise defined.
In the following passages, different aspects or embodiments of the invention are defined in more detail. Each aspect or embodiment so defined may be combined with any other aspect(s) or embodiment(s) unless clearly indicated to the contrary. In particular, any feature indicated as being preferred or advantageous may be combined with any other feature or features indicated as being preferred or advantageous.
Reference throughout this specification to “one embodiment”, “an embodiment” means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, appearances of the phrases “in one embodiment” or “in an embodiment” in various places throughout this specification are not necessarily all referring to the same embodiment, but may. Furthermore, the particular features, structures or characteristics may be combined in any suitable manner, as would be apparent to a person skilled in the art from this disclosure, in one or more embodiments. Furthermore, while some embodiments described herein include some but not other features included in other embodiments, combinations of features of different embodiments are meant to be within the scope of the invention, and form different embodiments, as would be understood by those in the art. For example, in the appended claims, any of the claimed embodiments can be used in any combination.
As corroborated in the experimental section, which illustrates certain representative embodiments ofthe invention, the inventors identified a novel tool for the diagnosis of spondyloarthritis as well as for the evaluation ofthe disease severity of spondyloarthritis and evaluation ofthe therapy response in patients diagnosed with spondyloarthritis. More specific, an unbiased screening was performed to identify novel (auto)antibodies in early axSpA patients that provide a novel tool for early axSpA diagnosis. To this end, the inventors applied the antibody profiling technique serological antigen selection (SAS). In this approach, a cDNA phage display library constructed from axSpA hip synovial tissue was screened for reactivity with immunoglobulin A (IgA) antibodies in plasma of early axSpA patients. In particular, in this SAS procedure, phage particles expressing axSpA synovial antigens and randomly formed peptides, originating from a newly constructed axSpA cDNA phage display library, were screened for IgA antibody reactivity in plasma of early axSpA patients. Antibody reactivity against novel Hasselt University (UH)- axSpA-lgA peptides was determined in early axSpA patients and healthy controls (HC). Furthermore, the inventors also investigated whether antibodies against a combination of the identified UH-axSpA- IgG peptides (SEQ ID’s 8 - 19), and the UH-axSpA-lgA targets (SEQ ID’s 1-7), could have a promising biomarker potential for the early diagnosis of axSpA patients.
Accordingly, in a first aspect, the present invention provides an in vitro method for diagnosing of spondyloarthritis, wherein the method comprises providing a biological sample from the subject and determining the presence or quantity of one or more antibodies against one or more peptides or against one or more immunoreactive fragments or antibody-binding fragments or variants of said one or more peptides in the biological sample. More specific, the antibody reactivity towards said immunoreactive fragments or antibody-binding fragments or variants is comparable to the antibody reactivity towards the corresponding peptide itself. The presence of or a deviation, such as an increase, in antibody levels against at least one of said peptides or immunoreactive fragments or antibody-binding fragments or variants thereof compared to a reference sample is indicative for the presence of spondyloarthritis in the subject.
In a related aspect, an in vitro method for evaluating the disease severity of spondyloarthritis in a subject is provided. Said comprises providing a biological sample from the subject and determining the presence or quantity of one or more antibodies against one or more peptides or against one or more immunoreactive fragments or antibody-binding fragments or variants of said one or more peptides in the biological sample. More specific, the antibody reactivity towards said immunoreactive fragments or antibody-binding fragments or variants is comparable to the antibody reactivity towards the corresponding peptide itself. The presence of or a deviation, such as an increase, in antibody levels against at least one of said peptides or immunoreactive fragments or antibody-binding fragments or variants thereof compared to a reference sample is indicative for the disease severity of spondyloarthritis in the subject.
In a related aspect, an in vitro method for evaluating a therapeutic treatment of spondyloarthritis in a subject is provided. Said method comprises providing a first and a second biological sample wherein the first biological sample is obtained at the start of the therapeutic treatment and the second sample is obtained after the therapeutic treatment, and determining in said first and second biological sample the presence or quantity of one or more antibodies against one or more peptides or against one or more immunoreactive fragments or antibody-binding fragments or variants of said one or more peptides. More specific, the antibody reactivity towards said immunoreactive fragments or antibody-binding fragments or variants is comparable to the antibody reactivity towards the corresponding peptide itself. A deviation or no deviation, in particular a deviation, such as an increase or a decrease, in antibody levels against said one or more peptides or immunoreactive fragments or antibody-binding fragments or variants thereof in the second biological sample compared to the first biological sample indicates that the subject is responding to the therapeutic treatment. Preferably a decrease in antibody levels against said one or more peptides or immunoreactive fragments or antibody-binding fragments or variants thereof in the second biological sample compared to the first biological sample indicates that the subject is responding to the therapeutic treatment
In certain embodiments, the methods and uses as taught herein comprise determining in one or more biological samples of the subject the presence or quantity of antibodies against one or more peptides selected from the group consisting of a peptide as shown in any one of SEQ ID NOs: 1-19 or against an antibody-binding fragment or variant thereof. Sequences are displayed in Table 1 .
Table 1. Overview of amino acid sequences of the peptides according to the invention and their corresponding SEQ ID NO.
Figure imgf000012_0001
Figure imgf000013_0001
In certain embodiments, the methods and uses as taught herein comprise determining in one or more biological samples of a subject the presence or quantity of antibodies against peptides selected from the group consisting of a peptide as shown in SEQ ID NO: 1 or an antibody-binding fragment or variant thereof.
In certain embodiments, the methods and uses as taught herein comprise determining in one or more biological samples of a subject the presence or quantity of antibodies against peptides selected from the group consisting of a peptide as shown in SEQ ID NO: 2 or an antibody-binding fragment or variant thereof.
In certain embodiments, the methods and uses as taught herein comprise determining in one or more biological samples of a subject the presence or quantity of antibodies against peptides selected from the group consisting of a peptide as shown in SEQ ID NO: 3 or an antibody-binding fragment or variant thereof.
In certain embodiments, the methods and uses as taught herein comprise determining in one or more biological samples of a subject the presence or quantity of antibodies against peptides selected from the group consisting of a peptide as shown in SEQ ID NO: 4 or an antibody-binding fragment or variant thereof.
In certain embodiments, the methods and uses as taught herein comprise determining in one or more biological samples of a subject the presence or quantity of antibodies against peptides selected from the group consisting of a peptide as shown in SEQ ID NO: 5 or an antibody-binding fragment or variant thereof.
In certain embodiments, the methods and uses as taught herein comprise determining in one or more biological samples of a subject the presence or quantity of antibodies against peptides selected from the group consisting of a peptide as shown in SEQ ID NO: 6 or an antibody-binding fragment or variant thereof.
In certain embodiments, the methods and uses as taught herein comprise determining in one or more biological samples of a subject the presence or quantity of antibodies against peptides selected from the group consisting of, and a peptide as shown in SEQ ID NO: 7 or an antibody-binding fragment or variant thereof.
In certain embodiments, the methods and uses as taught herein comprise determining in one or more biological samples of a subject the presence or quantity of antibodies against any combination of peptides selected from the group consisting of SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7.
In certain embodiments, the methods and uses as taught herein comprise determining in one or more biological samples of a subject the presence or quantity of antibodies against a peptide as shown in SEQ ID NO: 2 or an antibody-binding fragment or variant thereof, against a peptide as shown in SEQ ID NO: 1 or an antibody-binding fragment or variant thereof, and against a peptide as shown in SEQ ID NO: 7 or an antibody-binding fragment or variant thereof. In certain embodiments, the methods and uses as taught herein comprise determining in one or more biological samples of a subject the presence or quantity of antibodies against a peptide as shown in SEQ ID NO: 2 or an antibody-binding fragment or variant thereof, against a peptide as shown in SEQ ID NO: 1 or an antibody-binding fragment or variant thereof, against a peptide as shown in SEQ ID NO: 3 or an antibody-binding fragment or variant thereof, against a peptide as shown in SEQ ID NO: 4 or an antibody-binding fragment or variant thereof, against a peptide as shown in SEQ ID NO: 5 or an antibody-binding fragment or variant thereof, against a peptide as shown in SEQ ID NO: 6 or an antibody-binding fragment or variant thereof, and against a peptide as shown in SEQ ID NO: 7 or an antibody-binding fragment or variant thereof.
In certain embodiments, the in vitro method disclosed herein is for diagnosing spondyloarthritis in a subject, said method comprising: a) providing a biological sample from the subject, and b) determining the presence or quantity of antibody levels against a peptide as shown in SEQ ID NO: 2 or against an antibody-binding fragment or variant thereof, against a peptide as shown in SEQ ID NO: 1 or against an antibody-binding fragment or variant thereof, and against a peptide as shown in SEQ ID No: 7; or against an antibody-binding fragment or variant thereof, wherein the presence of or an increase in antibody levels against at least one of said peptides or antibody-binding fragments or variants thereof in said biological sample compared to a reference sample is indicative for the presence or diagnosis of spondyloarthritis in the subject.
In certain embodiments, the in vitro method disclosed herein is for diagnosing spondyloarthritis in a subject, said method comprising: a) providing a biological sample from the subject, and b) determining the presence or quantity of antibody levels against a peptide as shown in SEQ ID NO: 2 or against an antibody-binding fragment or variant thereof, against a peptide as shown in SEQ ID NO: 1 or against an antibody-binding fragment or variant thereof, and against a peptide as shown in SEQ ID No: 7; or against an antibody-binding fragment or variant thereof, wherein the presence of or an increase in antibody levels against the peptide as shown in SEQ ID NO: 2 or against an antibody-binding fragment or variant thereof in the biological sample compared to a reference sample is indicative for the presence or diagnosis of spondyloarthritis in the subject.
In certain embodiments, the in vitro method disclosed herein is for diagnosing spondyloarthritis in a subject, said method comprising: a) providing a biological sample from the subject, and b) determining the presence or quantity of antibody levels against a peptide as shown in SEQ ID NO: 2 or against an antibody-binding fragment or variant thereof, against a peptide as shown in SEQ ID NO: 1 or against an antibody-binding fragment or variant thereof, and against a peptide as shown in SEQ ID No: 7; or against an antibody-binding fragment or variant thereof, wherein the presence or increase in antibody levels against the peptide as shown in SEQ ID NO: 2 or against an antibody-binding fragment or variant thereof, against the peptide as shown in SEQ ID NO 1 or against an antibody-binding fragment or variant thereof, and against the peptide as shown in SEQ ID NO: 7 or against an antibody-binding fragment or variant thereof, in said sample compared to a reference sample is indicative for the presence of spondyloarthritis in the subject. In certain embodiments, the in vitro method disclosed herein is for diagnosing spondyloarthritis in a subject, said method comprising: a) providing a biological sample from the subject, and b) determining the presence or quantity of antibody levels against a peptide as shown in SEQ ID NO: 2 or against an antibody-binding fragment or variant thereof, wherein the presence of or an increase in antibody levels against the peptide as shown in SEQ ID NO: 2 or against an antibody-binding fragment or variant thereof in the biological sample compared to a reference sample is indicative for the presence or diagnosis of spondyloarthritis in the subject.
In certain embodiments, the in vitro method disclosed herein is for diagnosing spondyloarthritis in a subject, said method comprising: a) providing a biological sample from the subject, and b) determining the presence or quantity of antibody levels against a peptide as shown in SEQ ID NO: 1 or against an antibody-binding fragment or variant thereof, wherein the presence of or an increase in antibody levels against the peptide as shown in SEQ ID NO: 1 or against an antibody-binding fragment or variant thereof in the biological sample compared to a reference sample is indicative for the presence or diagnosis of spondyloarthritis in the subject.
In certain embodiments, the in vitro method disclosed herein is for diagnosing spondyloarthritis in a subject, said method comprising: a) providing a biological sample from the subject, and b) determining the presence or quantity of antibody levels against a peptide as shown in SEQ ID NO: 7 or against an antibody-binding fragment or variant thereof, wherein the presence of or an increase in antibody levels against the peptide as shown in SEQ ID NO: 7 or against an antibody-binding fragment or variant thereof in the biological sample compared to a reference sample is indicative for the presence or diagnosis of spondyloarthritis in the subject.
In certain embodiments, the in vitro method disclosed herein is for diagnosing spondyloarthritis in a subject, said method comprising: a) providing a biological sample from the subject, and b) determining the presence or quantity of antibody levels against a peptide as shown in SEQ ID NO: 2 or against an antibody-binding fragment or variant thereof, against a peptide as shown in SEQ ID NO: 1 or against an antibody-binding fragment or variant thereof, and against a peptide as shown in SEQ ID No: 7; or against an antibody-binding fragment or variant thereof, c) determining the presence or quantity of antibodies against one or more peptides as shown in SEQ ID NO: 3, SEQ ID No: 4, SEQ ID No: 5, and SEQ ID No: 6, or against an antibody-binding fragment or variant thereof, wherein the presence or increase in antibody levels against the peptide as shown in SEQ ID NO: 2 or against an antibody-binding fragment or variant thereof, against the peptide as shown in SEQ ID NO 1 or against an antibody-binding fragment or variant thereof, against the peptide as shown in SEQ ID NO: 7 or against an antibody-binding fragment or variant thereof, and against one or more peptides as shown in SEQ ID NO: 3, SEQ ID No: 4, SEQ ID No: 5, and SEQ ID No: 6, or against an antibody-binding fragment or variant thereof, in said sample compared to a reference sample is indicative for the presence or diagnosis of spondyloarthritis in the subject.
In certain embodiments, the in vitro method disclosed herein is for diagnosing spondyloarthritis in a subject, said method comprising: a) providing a biological sample from the subject, b) determining the presence or quantity of antibody levels against a peptide as shown in SEQ ID NO: 2 or against an antibody-binding fragment or variant thereof, against a peptide as shown in SEQ ID NO: 1 or against an antibody-binding fragment or variant thereof, and against a peptide as shown in SEQ ID No: 7; or against an antibody-binding fragment or variant thereof, and c) determining the presence or quantity of antibodies against one or more peptides as shown in SEQ ID NO: 8, SEQ ID No: 9, SEQ ID No: 10, SEQ ID No: 11 , SEQ ID No: 12, SEQ ID No: 13, SEQ ID No: 14, SEQ ID No: 15, SEQ ID No: 16, SEQ ID No: 17, SEQ ID No: 18 and SEQ ID No: 19, or against an antibody-binding fragment or variant thereof, wherein the presence or increase in antibody levels against the peptide as shown in SEQ ID NO: 2 or against an antibody-binding fragment or variant thereof, against the peptide as shown in SEQ ID NO 1 or against an antibody-binding fragment or variant thereof, against the peptide as shown in SEQ ID NO: 7 or against an antibody-binding fragment or variant thereof, and against one or more peptides as shown in SEQ ID NO: 8, SEQ ID No: 9, SEQ ID No: 10, SEQ ID No: 11 , SEQ ID No: 12, SEQ ID No: 13, SEQ ID No: 14, SEQ ID No: 15, SEQ ID No: 16, SEQ ID No: 17, SEQ ID No: 18 and SEQ ID No: 19, or against an antibody-binding fragment or variant thereof, in said sample compared to a reference sample is indicative for the presence or diagnosis of spondyloarthritis in the subject.
In certain embodiments, the in vitro method disclosed herein is for evaluating the disease severity of spondyloarthritis in a subject, said method comprising: a) providing a biological sample from the subject, and b) determining the presence or quantity of antibody levels against a peptide as shown in SEQ ID NO: 2 or against an antibody-binding fragment or variant thereof, against a peptide as shown in SEQ ID NO: 1 or against an antibody-binding fragment or variant thereof, and against a peptide as shown in SEQ ID No: 7; or against an antibody-binding fragment or variant thereof, wherein the presence of or an increase in antibody levels against the peptide as shown in SEQ ID NO: 2 or against an antibodybinding fragment or variant thereof in the biological sample compared to a reference sample is indicative for the disease severity of spondyloarthritis in the subject.
In certain embodiments, the in vitro method disclosed herein is for evaluating the disease severity of spondyloarthritis in a subject, said method comprising: a) providing a biological sample from the subject, and b) determining the presence or quantity of antibody levels against a peptide as shown in SEQ ID NO: 2 or against an antibody-binding fragment or variant thereof, against a peptide as shown in SEQ ID NO: 1 or against an antibody-binding fragment or variant thereof, and against a peptide as shown in SEQ ID No: 7; or against an antibody-binding fragment or variant thereof, wherein the presence or increase in antibody levels against the peptide as shown in SEQ ID NO: 2 or against an antibody-binding fragment or variant thereof, against the peptide as shown in SEQ ID NO 1 or against an antibody-binding fragment or variant thereof, and against the peptide as shown in SEQ ID NO: 7 or against an antibodybinding fragment or variant thereof, in said sample compared to a reference sample is indicative for the disease severity of spondyloarthritis in the subject.
In certain embodiments, the in vitro method disclosed herein is for evaluating the disease severity of spondyloarthritis in a subject, said method comprising: a) providing a biological sample from the subject, and b) determining the presence or quantity of antibody levels against a peptide as shown in SEQ ID NO: 2 or against an antibody-binding fragment or variant thereof, wherein the presence of or an increase in antibody levels against the peptide as shown in SEQ ID NO: 2 or against an antibody-binding fragment or variant thereof in the biological sample compared to a reference sample is indicative for the disease severity of spondyloarthritis in the subject.
In certain embodiments, the in vitro method disclosed herein is for evaluating the disease severity of spondyloarthritis in a subject, said method comprising: a) providing a biological sample from the subject, and b) determining the presence or quantity of antibody levels against a peptide as shown in SEQ ID NO: 1 or against an antibody-binding fragment or variant thereof, wherein the presence of or an increase in antibody levels against the peptide as shown in SEQ ID NO: 1 or against an antibody-binding fragment or variant thereof in the biological sample compared to a reference sample is indicative for the disease severity of spondyloarthritis in the subject.
In certain embodiments, the in vitro method disclosed herein is for evaluating the disease severity of spondyloarthritis in a subject, said method comprising: a) providing a biological sample from the subject, and b) determining the presence or quantity of antibody levels against a peptide as shown in SEQ ID NO: 7 or against an antibody-binding fragment or variant thereof, wherein the presence of or an increase in antibody levels against the peptide as shown in SEQ ID NO: 7 or against an antibody-binding fragment or variant thereof in the biological sample compared to a reference sample is indicative for the disease severity of spondyloarthritis in the subject.
In certain embodiments, antibody levels against the peptide as shown in SEQ ID No: 1 , or an immunoreactive fragment or antibody-binding variant thereof comprising at least 4, preferably at least 6, even more preferably at least 8, consecutive amino acids derived therefrom are determined. In certain embodiments, antibody levels against the peptide as shown in SEQ ID No: 2, or an immunoreactive fragment or antibody-binding variant thereof comprising at least 4, preferably at least 6, even more preferably at least 8, consecutive amino acids derived therefrom are determined. In certain embodiments, antibody levels against the peptide as shown in SEQ ID No: 7, or an immunoreactive fragment or antibody-binding variant thereof comprising at least 4, preferably at least 6, even more preferably at least 8, consecutive amino acids derived therefrom are determined.
In certain embodiments, antibody levels against the peptide as shown in SEQ ID No: 2, or an immunoreactive fragment or antibody-binding variant thereof comprising at least 4, preferably at least 6, even more preferably at least 8, consecutive amino acids derived therefrom are determined, in combination with antibody levels against the peptide as shown in SEQ ID No 1 and/or 7, or a fragment comprising at least 4, preferably at least 6, even more preferably at least 8, consecutive amino acids derived therefrom.
In certain embodiments, antibody levels against the peptide as shown in SEQ ID NO: 1 , SEQ ID NO: 2 and SEQ ID NO: 7, or against an immunoreactive fragment or antibody-binding fragment thereof, preferably said fragment comprising at least 4, preferably at least 6, more preferably at least 8, consecutive amino acids derived thereof, are determined in combination with determining the antibody levels against one or more peptides as shown in SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, or against an immu noreactive fragment or antibody-binding fragment or variant thereof, preferably said fragment comprising at least 4, preferably at least 6, more preferably at least 8, consecutive amino acids derived therefrom.
In certain embodiments, antibody levels against the peptide as shown in SEQ ID No: 1 , SEQ ID No 2 and SEQ ID No 7, or against an immunoreactive fragment or antibody-binding fragment thereof; preferably said fragment comprising at least 4, preferably at least 6, more preferably at least 8, consecutive amino acids derived therefrom, are determined. In certain embodiments, said antibody levels are combined with antibody levels against one or more peptides as shown in SEQ ID No 8, SEQ ID No 9, SEQ ID No 16 and SEQ ID No 17, or against an immunoreactive fragment or antibody-binding fragment or variant thereof, preferably said fragment comprising at least 4, preferably at least 6, more preferably at least 8, consecutive amino acids derived therefrom.
In certain embodiments, the present invention provides an in vitro method for diagnosing the presence of spondyloarthritis or for evaluating the disease severity of spondyloarthritis in a subject, said method comprising: a) providing a biological sample from the subject, and b) determining antibody levels against one or more peptides selected from the list comprising SEQ ID NO: 1 , SEQ ID No: 2 or SEQ ID NO: 7, or an immune-reactive fragment or antibody-binding fragment or variant thereof comprising at least 4; in particular at least 6, consecutive amino acids derived therefrom; wherein an increase in antibody levels against said one or more peptides or fragments thereof in said biological sample compared to a reference sample is indicative for the presence of spondyloarthritis. In particular, an increase in antibody levels against the peptide of SEQ ID NO: 2 or against an immunoreactive fragment or antibody-binding fragment or variant thereof comprising at least 4, in particular at least 6, consecutive amino acids derived therefrom, in said biological sample compared to a reference sample is indicative for the presence of spondyloarthritis or for the disease severity of spondyloarthritis in the subject.
In some embodiments, the present invention provides an in vitro method for diagnosing of spondyloarthritis or for evaluating the disease severity of spondyloarthritis, said method comprising: a) providing a biological sample from the subject, and b) determining antibody levels against peptides selected from the list comprising SEQ ID No: 1 , SEQ ID No: 2, and SEQ ID No: 7, or an immune-reactive fragment or antibody-binding variant thereof; wherein an increase in antibody levels against said peptides or fragments thereof in said biological sample compared to a reference sample is indicative for the diagnosis of spondyloarthritis in the subject. In some embodiments, peptides are selected from the list consisting of SEQ ID NO: 1 , SEQ ID No: 2 and SEQ ID NO: 7, or an immune-reactive fragment or antibody-binding fragment or variant thereof; in particular at least 6 consecutive amino acids derived from the original sequence of the peptides.
In certain embodiments, the in vitro method disclosed herein is for diagnosing spondyloarthritis or for evaluating the disease severity of spondyloarthritis in a subject, said method comprising: a) providing a biological sample from the subject, and b) determining the presence or quantity of antibody levels against a peptide as shown in SEQ ID NO: 2 or against an antibody-binding fragment or variant thereof, against a peptide as shown in SEQ ID NO: 1 or against an antibody-binding fragment or variant thereof, and against a peptide as shown in SEQ ID No: 7; or against an antibody-binding fragment or variant thereof, wherein the presence of or an increase in antibody levels against at least one of said peptides or antibody-binding fragments or variants thereof in said biological sample compared to a reference sample is indicative for the presence or diagnosis of spondyloarthritis or for the disease severity of spondyloarthritis in the subject.
The present invention further provides an in vitro method for evaluating a therapeutic treatment of spondyloarthritis in a subject, said method comprising: a) providing a first biological sample from the subject obtained before the start of the therapeutic treatment, b) providing a second biological sample from the subject obtained after therapeutic treatment, and c) determining the presence or quantity of antibodies against a peptide as shown in SEQ ID No: 2 or against an antibody-binding fragment or variant thereof, against a peptide as shown in SEQ ID NO: 1 or against an antibody-binding fragment or variant thereof, and against a peptide as shown in SEQ ID NO: 7 or against an antibody-binding fragment or variant thereof in the first and second biological sample; wherein a deviation or no deviation, in antibody levels against at least one of said peptides or antibody-binding fragments or variants thereof in the second biological sample compared to the first biological sample indicates that the subject is responding to the therapeutic treatment. Preferably, wherein a deviation, such as a decrease, in antibody levels against at least one of said peptides or antibody-binding fragments or variants thereof in the second biological sample compared to the first biological sample indicates that the subject is responding to the therapeutic treatment.
The present invention also provides an in vitro method for evaluating a therapeutic treatment of spondyloarthritis in a subject, said method comprising: a) providing a first biological sample from the subject obtained before the start of the therapeutic treatment, b) providing a second biological sample from the subject obtained after therapeutic treatment, and c) determining the presence or quantity of antibodies against a peptide as shown in SEQ ID No: 2 or against an antibody-binding fragment or variant thereof, against a peptide as shown in SEQ ID NO: 1 or against an antibody-binding fragment or variant thereof, and against a peptide as shown in SEQ ID NO: 7 or against an antibody-binding fragment or variant thereof in the first and second biological sample; wherein a deviation or no deviation, in antibody levels against the peptide as shown in SEQ ID NO: 2 or against an antibody-binding fragment or variant thereof in the second biological sample compared to the first biological sample indicates that the subject is responding to the therapeutic treatment. Preferably, wherein a deviation, such as a decrease, in antibody levels against at least one of said peptides or antibody-binding fragments or variants thereof in the second biological sample compared to the first biological sample indicates that the subject is responding to the therapeutic treatment. In certain embodiments, a deviation or no deviation in antibody levels against the peptide as shown in SEQ ID NO: 2 or against an antibody-binding fragment or variant thereof, against the peptide as shown in SEQ ID NO 1 or against an antibody-binding fragment or variant thereof, and against the peptide as shown in SEQ ID NO: 7 or against an antibody-binding fragment or variant thereof, in the second biological sample compared to the first biological sample indicates that the subject responds to the therapeutic treatment. Preferably, wherein a deviation, such as a decrease, in antibody levels against at least one of said peptides or antibody-binding fragments or variants thereof in the second biological sample compared to the first biological sample indicates that the subject is responding to the therapeutic treatment.
In certain embodiments, the in vitro method disclosed herein is for evaluating a therapeutic treatment of spondyloarthritis in a subject, said method comprising: a) providing a first biological sample from the subject obtained before the start of the therapeutic treatment, b) providing a second biological sample from the subject obtained after therapeutic treatment, and c) determining the presence or quantity of antibody levels against a peptide as shown in SEQ ID NO: 2 or against an antibody-binding fragment or variant thereof, wherein a deviation or no deviation, in antibody levels against the peptide as shown in SEQ ID NO: 2 or against an antibody-binding fragment or variant thereof in the second biological sample compared to the first biological sample indicates that the subject responds to the therapeutic treatment. Preferably, wherein a deviation, such as a decrease, in antibody levels against at least one of said peptides or antibody-binding fragments or variants thereof in the second biological sample compared to the first biological sample indicates that the subject is responding to the therapeutic treatment.
In certain embodiments, the in vitro method disclosed herein is for evaluating a therapeutic treatment of spondyloarthritis in a subject, said method comprising: a) providing a first biological sample from the subject obtained before the start of the therapeutic treatment, b) providing a second biological sample from the subject obtained after therapeutic treatment, and c) determining the presence or quantity of antibody levels against a peptide as shown in SEQ ID NO: 1 or against an antibody-binding fragment or variant thereof, wherein a deviation or no deviation, in antibody levels against the peptide as shown in SEQ ID NO: 1 or against an antibody-binding fragment or variant thereof in the second biological sample compared to the first biological sample indicates that the subject responds to the therapeutic treatment. Preferably, wherein a deviation, such as a decrease, in antibody levels against at least one of said peptides or antibody-binding fragments or variants thereof in the second biological sample compared to the first biological sample indicates that the subject is responding to the therapeutic treatment.
In certain embodiments, the in vitro method disclosed herein is for evaluating a therapeutic treatment of spondyloarthritis in a subject, said method comprising: a) providing a first biological sample from the subject obtained before the start of the therapeutic treatment, b) providing a second biological sample from the subject obtained after therapeutic treatment, and c) determining the presence or quantity of antibody levels against a peptide as shown in SEQ ID NO: 7 or against an antibody-binding fragment or variant thereof, wherein a deviation or no deviation, in antibody levels against the peptide as shown in SEQ ID NO: 7 or against an antibody-binding fragment or variant thereof in the second biological sample compared to the first biological sample indicates that the subject responds to the therapeutic treatment. Preferably, wherein a deviation, such as a decrease, in antibody levels against at least one of said peptides or antibody-binding fragments or variants thereof in the second biological sample compared to the first biological sample indicates that the subject is responding to the therapeutic treatment.
In certain embodiments, the in vitro method disclosed herein is for evaluating a therapeutic treatment of spondyloarthritis in a subject, said method comprising: a) providing a first biological sample from the subject obtained before the start of the therapeutic treatment, b) providing a second biological sample from the subject obtained after therapeutic treatment, c) determining the presence or quantity of antibody levels against a peptide as shown in SEQ ID NO: 2 or against an antibody-binding fragment or variant thereof, against a peptide as shown in SEQ ID NO: 1 or against an antibody-binding fragment or variant thereof, and against a peptide as shown in SEQ ID No: 7; or against an antibody-binding fragment or variant thereof in the first and second biological sample, and d) determining the presence or quantity of antibodies against one or more peptides as shown in SEQ ID NO: 3, SEQ ID No: 4, SEQ ID No: 5, and SEQ ID No: 6, or against an antibody-binding fragment or variant thereof in the first and second biological sample, wherein a deviation or no deviation, in antibody levels against the peptide as shown in SEQ ID NO: 2 or against an antibody-binding fragment or variant thereof, against the peptide as shown in SEQ ID NO 1 or against an antibody-binding fragment or variant thereof, against the peptide as shown in SEQ ID NO: 7 or against an antibody-binding fragment or variant thereof, and against one or more peptides as shown in SEQ ID NO: 3, SEQ ID No: 4, SEQ ID No: 5, and SEQ ID No: 6, or against an antibody-binding fragment or variant thereof, in the second biological sample compared to the first biological sample indicates that the subject responds to the therapeutic treatment. Preferably, wherein a deviation, such as a decrease, in antibody levels against at least one of said peptides or antibody-binding fragments or variants thereof in the second biological sample compared to the first biological sample indicates that the subject is responding to the therapeutic treatment.
In certain embodiments, the in vitro method disclosed herein is for evaluating a therapeutic treatment of spondyloarthritis in a subject, said method comprising: a) providing a first biological sample from the subject obtained before the start of the therapeutic treatment, b) providing a second biological sample from the subject obtained after therapeutic treatment, c) determining the presence or quantity of antibody levels against a peptide as shown in SEQ ID NO: 2 or against an antibody-binding fragment or variant thereof, against a peptide as shown in SEQ ID NO: 1 or against an antibody-binding fragment or variant thereof, and against a peptide as shown in SEQ ID No: 7; or against an antibody-binding fragment or variant thereof, in the first and second biological sample, and d) determining the presence or quantity of antibodies against one or more peptides as shown in SEQ ID NO: 8, SEQ ID No: 9, SEQ ID No: 10, SEQ ID No: 11 , SEQ ID No: 12, SEQ ID No: 13, SEQ ID No: 14, SEQ ID No: 15, SEQ ID No: 16, SEQ ID No: 17, SEQ ID No: 18 and SEQ ID No: 19, or against an antibody-binding fragment or variant thereof, in the first and second biological sample, wherein a deviation or no deviation, in antibody levels against the peptide as shown in SEQ ID NO: 2 or against an antibody-binding fragment or variant thereof, against the peptide as shown in SEQ ID NO 1 or against an antibody-binding fragment or variant thereof, against the peptide as shown in SEQ ID NO: 7 or against an antibody-binding fragment or variant thereof, and against one or more peptides as shown in SEQ ID NO: 8, SEQ ID No: 9, SEQ ID No: 10, SEQ ID No: 11 , SEQ ID No: 12, SEQ ID No: 13, SEQ ID No: 14, SEQ ID No: 15, SEQ ID No: 16, SEQ ID No: 17, SEQ ID No: 18 and SEQ ID No: 19, or against an antibody-binding fragment or variant thereof, in said sample compared to a reference sample indicates that the subject responds to the therapeutic treatment. Preferably, wherein a deviation, such as a decrease, in antibody levels against at least one of said peptides or antibody-binding fragments or variants thereof in the second biological sample compared to the first biological sample indicates that the subject is responding to the therapeutic treatment.
As used herein, a ’’deviation” of a first value from a second value or a “difference” between a first value and a second value may generally encompass any direction (e.g., increase: first value > second value; or decrease: first value < second value) and any extent of alteration.
For example, a deviation or a difference may encompass a decrease in a first value by, without limitation, at least about 10% (about 0.9-fold or less), or by at least about 20% (about 0.8-fold or less), or by at least about 30% (about 0.7-fold or less), or by at least about 40% (about 0.6-fold or less), or by at least about 50% (about 0.5-fold or less), or by at least about 60% (about 0.4-fold or less), or by at least about 70% (about 0.3-fold or less), or by at least about 80% (about 0.2-fold or less), or by at least about 90% (about 0.1-fold or less), relative to a second value with which a comparison is being made.
For example, a deviation or a difference may encompass an increase of a first value by, without limitation, at least about 10% (about 1 .1 -fold or more), or by at least about 20% (about 1 .2-fold or more), or by at least about 30% (about 1 .3-fold or more), or by at least about 40% (about 1 .4-fold or more), or by at least about 50% (about 1 .5-fold or more), or by at least about 60% (about 1 .6-fold or more), or by at least about 70% (about 1 .7-fold or more), or by at least about 80% (about 1 .8-fold or more), or by at least about 90% (about 1 .9-fold or more), or by at least about 100% (about 2-fold or more), or by at least about 150% (about 2.5-fold or more), or by at least about 200% (about 3-fold or more), or by at least about 500% (about 6-fold or more), or by at least about 700% (about 8-fold or more), or like, relative to a second value with which a comparison is being made.
Preferably, a deviation or a difference may refer to a statistically significant observed alteration. For example, a deviation or a difference may refer to an observed alteration which falls outside of error margins of reference values in a given population (as expressed, for example, by standard deviation or standard error, or by a predetermined multiple thereof, e.g., +1xSD or +2xSD or +3xSD, or +1xSE or +2xSE or+3xSE). Deviation or a difference may also refer to a value falling outside of a reference range defined by values in a given population (for example, outside of a range which comprises >40%, > 50%, >60%, >70%, >75% or >80% or >85% or >90% or >95% or even >100% of values in said population).
In a further embodiment, a deviation or a difference, such as an increase or a decrease, may be concluded if an observed alteration is beyond a given threshold or cut-off. Such threshold or cut-off may be selected as generally known in the art to provide for a chosen accuracy, sensitivity and/or specificity of the prediction methods, e.g., accuracy, sensitivity and/or specificity of at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 85%, or at least 90%, or at least 95%.
For example, receiver-operating characteristic (ROC) curve analysis can be used to select an optimal threshold or cut-off value of the quantity of a given biomarker for clinical use of the present diagnostic tests, based on acceptable global accuracy, sensitivity and/or specificity, or related performance measures which are well-known per se, such as positive predictive value (PPV), negative predictive value (NPV), positive likelihood ratio (LR+), negative likelihood ratio (LR-), Youden index, or similar.
For example, an optimal threshold or cut-off value may be selected for each individual biomarker as a local extremum of the receiver operating characteristic (ROC) curve, i.e. a point of local maximum distance to the diagonal line, as described in Robin X., PanelomiX: a threshold-based algorithm to create panels of biomarkers, 2013, Translational Proteomics, 1 (1):57-64.
The person skilled in the art will understand that it is not relevant to give an exact threshold or cut-off value. A relevant threshold or cut-off value can be obtained by correlating the sensitivity and specificity and the sensitivity/specificity for any threshold or cut-off value.
It is to the diagnostic engineers to determine which level of positive predictive value/negative predictive value/sensitivity/specificity is desirable and how much loss in positive or negative predictive value is tolerable. The chosen threshold or cut-off level could be dependent on other diagnostic parameters used in combination with the present method by the diagnostic engineers.
In certain embodiments of the invention, any combination of two or more peptides selected from the list consisting of SEQ ID No 1-7, can suitably be used within the context of the present invention; preferably peptides are selected from the list consisting of SEQ ID Nos 1 , 2 and 7. The peptides of the present invention may be provided as such, however, also an immune-reactive fragment or antibody-binding fragment or variant thereof; typically comprising at least 4, at least 5, in particular at least 6, consecutive amino acids derived from the original sequence of the peptide are suitable for use in the method of the current invention. Hence, at any mentioning of a “full-length” peptide, it may alternatively be replaced by an immune-reactive fragment or an antibody-binding fragment or variant, comprising at least 4; at least 5, or at least 6 consecutive amino acids derived therefrom. In some embodiments, peptides are selected from the list consisting of SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID No: 10, SEQ ID NO: 11 , SEQ ID NO: 12, SEQ ID No: 13, SEQ ID No: 14, SEQ ID NO: 15, SEQ ID No: 16, SEQ ID No: 17, SEQ ID No: 18 and SEQ ID NO: 19, or an immune-reactive fragment thereof; typically comprising at least 4 consecutive amino acids derived therefrom; in particular at least 6 consecutive amino acids derived therefrom.
In some embodiments, peptides are selected from the list comprising SEQ ID NO: 8, SEQ ID No: 9, SEQ ID No:16 and SEQ ID NO: 17, or an immune-reactive fragment thereof; typically comprising at least 4 consecutive amino acids derived therefrom; in particular at least 6 consecutive amino acids derived therefrom.
In some embodiments, antibody levels against the peptide comprising a sequence of SEQ ID No: 8, or an immune-reactive fragment thereof; typically comprising at least 4 consecutive amino acids derived therefrom; preferably at least 6 consecutive amino acids derived therefrom are determined. In some embodiments, said antibody levels are combined with antibody levels against the peptide comprising a sequence of SEQ ID No 1 , 2 and/or 7, or an immune-reactive fragment thereof; typically comprising at least 4 consecutive amino acids derived therefrom; preferably at least 6 consecutive amino acids derived therefrom.
In some embodiments, antibody levels against the peptide comprising a sequence of SEQ ID No: 9, or an immune-reactive fragment thereof; typically comprising at least 4 consecutive amino acids derived therefrom; preferably at least 6 consecutive amino acids derived therefrom are determined. In some embodiments, said antibody levels are combined with antibody levels against thepeptide comprising a sequence of SEQ ID No 1 , 2 and/or 7, or an immune-reactive fragment thereof; typically comprising at least 4 consecutive amino acids derived therefrom; preferably at least 6 consecutive amino acids derived therefrom.
In some embodiments, antibody levels against the peptide comprising a sequence of SEQ ID No: 16, or an immune-reactive fragment thereof; typically comprising at least 4 consecutive amino acids derived therefrom;preferably at least 6 consecutive amino acids derived therefrom are determined. In some embodiments, said antibody levels are combined with antibody levels against the peptide comprising a sequence of SEQ ID No 1 , 2 and/or 7, or an immune-reactive fragment thereof; typically comprising at least 4 consecutive amino acids derived therefrom; preferably at least 6 consecutive amino acids derived therefrom.
In some embodiments, antibody levels against the peptide comprising a sequence of SEQ ID No: 17, or an immune-reactive fragment thereof; typically comprising at least 4 consecutive amino acids derived therefrom; preferably at least 6 consecutive amino acids derived therefrom are determined. In some embodiments, said antibody levels are combined with antibody levels against the peptide comprising a sequence of SEQ ID No 1 , 2 and/or 7, an immune-reactive fragment thereof; typically comprising at least 4 consecutive amino acids derived therefrom; preferably at least 6 consecutive amino acids derived therefrom.
In some embodiments, antibody levels against the peptide comprising a sequence of SEQ ID No: 8, SEQ ID No: 9 , SEQ ID No: 16 and/or SEQ ID No: 17, or an immune-reactive fragment thereof; typically comprising at least 4 consecutive amino acids derived therefrom; preferably at least 6 consecutive amino acids derived therefrom are determined. In some embodiments, said antibody levels are combined with antibody levels against the peptide comprising a sequence of SEQ ID No 1 , 2 and/or 7, or an immune-reactive fragment thereof; typically comprising at least 4 consecutive amino acids derived therefrom; preferably at least 6 consecutive amino acids derived therefrom.
In certain embodiments, any combination of two or more peptides selected from the list comprising SEQ ID No 1-7, with any combination of two or more peptides selected from the list comprising SEQ ID No 8-19 can suitably be used within the context of the present invention; preferably peptides are selected from the list comprising SEQ ID Nos 1 , 2 and 7, in combination with peptides selected from the list comprising SEQ ID No 8-19; in particular with the list comprising SEQ ID Nos 8, 9, 16 and 17. The peptides of the present invention may be provided as such, however, also immune-reactive fragment thereof; typically comprising at least 4, at least 5, in particular at least 6, consecutive amino acids derived therefrom are suitable for use in the method of the current invention. Hence, at any mentioning of a “full- length” peptide, it may alternatively be replaced by an immune-reactive fragment, comprising at least 4; at least 5, or at least 6 consecutive amino acids derived therefrom.
In some embodiments, the methods as disclosed herein are characterized in that the one or more antibodies are detected using a detection agent comprising one or more peptides selected from the group consisting of the peptide of SEQ ID NO: 1-19 or an antibody-binding fragment or variant thereof.
In some embodiments, the methods as disclosed herein are characterized in that the one or more antibodies are detected using a detection agent composing one or more peptides selected from the group consisting of: the peptide of SEQ ID NO: 1 or an antibody-binding fragment or variant thereof, the peptide of SEQ ID NO: 2 or an antibody-binding fragment or variant thereof, the peptide of SEQ ID NO: 3 or an antibody-binding fragment or variant thereof, the peptide of SEQ ID NO: 4 or an antibody-binding fragment or variant thereof, the peptide of SEQ ID NO: 5 or an antibody-binding fragment or variant thereof, the peptide of SEQ ID NO: 6 or an antibody-binding fragment or variant thereof, the peptide of SEQ ID NO: 7 or an antibody-binding fragment or variant thereof, or combinations thereof.
In some embodiments, the methods as disclosed herein are characterized in that the one or more antibodies are detected using a detection agent composing one or more peptides selected from the group consisting of: the peptide of SEQ ID NO: 1 or an antibody-binding fragment or variant thereof, the peptide of SEQ ID NO: 2 or an antibody-binding fragment or variant thereof, the peptide of SEQ ID NO: 7 or an antibody-binding fragment or variant thereof, or combinations thereof.
For example, antibodies can be detected using detection agent comprising a peptide of SEQ ID NO: 1 or an antibody-binding variant or fragment thereof. Or, antibodies can be detected using detection agent comprising a peptide of SEQ ID NO: 2 or an antibody-binding variant or fragment thereof. Or, antibodies can be detected using detection agent comprising a peptide of SEQ ID NO: 7 or an antibody-binding variant or fragment thereof. Or, antibodies can be detected using detection agent comprising a peptide of SEQ ID NO: 2 or an antibody-binding variant or fragment thereof, in combination with a peptide of SEQ ID NO: 1 or an antibody-binding variant or fragment thereof and/or , a peptide of SEQ ID NO: 7 or an antibody-binding variant or fragment thereof.
The present invention also provides peptides, such as isolated peptides, comprising a sequence represented by any of SEQ ID Nos: 1-19, or an immune-reactive fragment or antibody-binding fragment or variant thereof.
In some embodiments, the present application provides a peptide selected from the group consisting of the peptide of SEQ ID NO: 1 , the peptide of SEQ ID NO: 2, the peptide of SEQ ID NO: 3, the peptide of SEQ ID NO: 4, the peptide of SEQ ID NO: 5, the peptide of SEQ ID NO: 6, the peptide of SEQ ID NO: 7, the peptide of SEQ ID NO: 8, the peptide of SEQ ID NO: 9, the peptide of SEQ ID NO: 10, the peptide of SEQ ID NO: 11 , the peptide of SEQ ID NO: 12, the peptide of SEQ ID NO: 13, the peptide of SEQ ID NO: 14, the peptide of SEQ ID NO: 15, the peptide of SEQ ID NO: 16, the peptide of SEQ ID NO: 17, the peptide of SEQ ID NO: 18, and the peptide of SEQ ID NO: 19.
In some embodiments, the present application provides a peptide selected from the group consisting of the peptide of SEQ ID NO: 1 , the peptide of SEQ ID NO: 2, the peptide of SEQ ID NO: 3, the peptide of SEQ ID NO: 4, the peptide of SEQ ID NO: 5, the peptide of SEQ ID NO: 6, and the peptide of SEQ ID NO: 7.
In some embodiments, the present application provides a peptide selected from the group consisting of the peptide of SEQ ID NO: 1 , the peptide of SEQ ID NO: 2, and the peptide of SEQ ID NO: 7.
In some embodiments, the present application provides a peptide as shown in SEQ ID NO: 1. In some embodiments, the present application provides a peptide that comprises a sequence as shown in SEQ ID NO: 1.
In some embodiments, the present application a peptide as shown in SEQ ID NO: 2. In some embodiments, the present application provides a peptide that comprises a sequence as shown in SEQ ID NO: 2.
In some embodiments, the present application a peptide as shown in SEQ ID NO: 7. In some embodiments, the present application provides a peptide that comprises a sequence as shown in SEQ ID NO: 7.
Also provided are immu noreactive fragments or antibody-binding fragments or variants of the peptides as disclosed herein.
In some embodiments, a peptide is provided that comprises at least 4; preferably at least 6, consecutive amino acids derived from SEQ ID No: 1-19. In some embodiments, the present invention provides peptides consisting essentially of a sequence represented by SEQ ID Nos: 1-19, or an immune-reactive fragment or antibody-binding fragment thereof; typically comprising at least 4, preferably at least 6, consecutive amino acids derived from SEQ ID Nos: 1-19.
Further provided is a detection agent comprising one or more peptides, antibody-binding fragments or variants thereof as disclosed herein. The detection agent may comprise one or more or a combination of multiple peptides or antibody-binding fragments or variants thereof as disclosed herein. In some embodiments, the detection agent as disclosed herein is a polypeptide. In some embodiments, a detection agent, preferably a polypeptide, is provided that comprises one or more peptides, antibodybinding fragments or variants thereof as taught herein. In some embodiments, the detection agent is a composite polypeptide that comprises the amino acid sequence of one or more of the peptides that are disclosed herein. For example, a detection agent according to the invention may comprise the sequence of SEQ ID NO: 1 , SEQ ID NO: 2 or SEQ ID NO: 7. Or, detection agent that is a composite polypeptide may comprise the sequence of SEQ ID NO: 1 and the sequence of SEQ ID NO: 2, or the sequence of SEQ ID NO: 2 and the sequence of SEQ ID NO: 7, or the sequence of SEQ ID NO: 1 and the sequence of SEQ ID NO: 2 and the sequence of SEQ ID NO: 7.
In a related aspect, a composition comprising one or more peptides, or antibody-binding fragments or variants thereof or detection agents as disclosed herein is provided.
In a certain embodiment, a composition comprising a peptide comprising or consisting essentially of the sequence of SEQ ID NO: 2 or antibody-binding fragment or variant thereof is provided. In a certain embodiment, a composition comprising a peptide comprising or consisting essentially of the sequence of SEQ ID NO: 1 or antibody-binding fragment or variant thereof is provided. In a certain embodiment, a composition comprising a peptide comprising or consisting essentially of the sequence of SEQ ID NO: 7 or antibody-binding fragment or variant thereof is provided. In a certain embodiment, a composition comprising a peptide comprising or consisting essentially of the sequence of SEQ ID NO: 2 or antibodybinding fragment or variant thereof, a peptide comprising or consisting essentially of the sequence of SEQ ID NO: 1 or antibody-binding fragment or variant thereof, and a peptide comprising or consisting essentially of the sequence of SEQ ID NO: 7 or antibody-binding fragment or variant thereof is provided. For example, a composition may be provided that comprises a peptide as shown in SEQ ID NO: 2, or an antibody-binding fragment thereof in combination with at least one other peptide as shown in SEQ ID NO: 1 or SEQ ID NO: 7 or an antibody-binding fragment thereof.
In further embodiments, the composition may comprise at least one other peptide comprising or consisting essentially of a sequence represented by any one of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6, or an antibody-binding fragment or variant thereof. In further embodiments, the composition may comprise at least one other peptide comprising or consisting essentially of a sequence represented by any one of SEQ ID NO: 8-19, or an antibody-binding fragment or variant thereof.
In some embodiments, the composition can also comprise one or more peptides of SEQ ID NO: 1 , 2 and 7 or an antibody-binding fragment thereof, in combination with one or more peptides of SEQ ID No: 3, 4, 5 and 6, or an antibody-binding fragment thereof and in combination with one or more peptides of SEQ ID NO; 8, 9, 10, 11 , 12, 13, 15, 16, 17, 18 and 19, or an antibody-binding fragment thereof. For example, a composition comprising the peptides of SEQ ID NO: 1 , 2 and or an antibody-binding fragment thereof and the peptides of SEQ ID NO: 8, 9, 16, and 17, or an antibody-binding fragment thereof can be provided.
With the present invention, antibodies are detected against one or more peptides as disclosed herein or against their antibody-binding fragments or variants thereof. In certain embodiments, the peptides as taught herein are non-naturally occurring peptides or non-physiological peptides. More specific, although it was found that peptides show some homology to a number of human proteins that are expressed in human hip synovial tissue of spondyloarthritis patients, the peptides envisaged herein are non-naturally occurring peptides. In some embodiments, the peptides are formed by out-of-frame cDNA translation or the translation of non-coding sequences.
As for the peptides with SEQ-ID’s 1-7, that are positive for IgA antibodies, and for the peptides with SEQ-ID’s 8-19, that are positive for IgG antibodies, the methods and uses as taught herein can also be performed using their naturally occurring counterparts, i.e. proteins having regions that share homology with such peptides, i.e. proteins having regions that share at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity with the (non- naturally) occurring peptides of the present invention. In that respect, the peptides of the present invention were found to have homology with human proteins such as indicated in the table 2 and 3 below:
Table 2. Peptides of SEQ ID NOs 1 -7 and their corresponding homology with human proteins.
Figure imgf000029_0001
a Peptide sequence of the translated cDNA insert, with the aa between parenthesis representing the transition between the M13 phage gene VI and the cDNA insert..
Q Amber stop codon, which is translated into glutamine by the bacterial strain *stop codon
Table 3. Peptides of SEQ ID Nos 8 to 19 and their corresponding homology with human proteins.
Figure imgf000030_0001
Figure imgf000031_0001
a Peptide sequence of the translated cDNA insert, with the aa between parenthesis representing the transition between the M13 phage gene VI and the cDNA insert..
Q Amber stop codon, which is translated into glutamine by the bacterial strain *stop codon
In certain embodiments, the antibodies are IgA, IgG and/or IgM antibodies. In certain embodiments, the antibodies are IgA and/or IgG antibodies. In some embodiments, the antibodies are IgA antibodies. In some embodiments, the antibodies are IgG antibodies. In some embodiments, the antibodies are autoantibodies, in particular IgA, IgG and/or IgM autoantibodies. In some embodiments, the antibodies are IgA autoantibodies and/or IgG autoantibodies. In some embodiments, the antibodies are IgA autoantibodies. In some embodiments, the antibodies are IgG autoantibodies. In some embodiments, the antibodies are thus antibodies produced by the subject’s immune system and are directed against an antigen of the subject’s own proteins. Antibodies are normally produced in response to a foreign protein or substance within the body, typically a pathogen. Normally, the immune system is able to recognize and ignore the body’s own proteins and not overreact to non-threatening substances in the environment. Sometimes, however, the immune system cease to recognize one or more of the body’s normal constituents as “self, leading to production of autoantibodies. These autoantibodies can attack the subject’s own cells, tissues and/or organs, causing inflammation and damage.
As taught herein, antibodies can also be detected against immunoreactive fragments or antibodybinding fragments or antibody-binding variants of the peptides of SEQ ID Nos 1 to 19. As used herein, the terms immunoreactive fragment, antibody-binding fragment and antibody-binding variant are synonyms and can be used interchangeably. As used herein, an immunoreactive fragment or an antibody-binding fragment or variant of a peptide refers to a functionally equivalent fragment or variant of the corresponding peptide. In the context of the present invention, antibody-binding fragments or variants are to be understood as any fragment or variant of the peptide of SEQ ID NO: 1 to 19 that show an antibody reactivity that is similar or comparable to or substantially the same as the antibody reactivity of the corresponding peptide. Antibody-binding fragments or variants can also be referred herein as immunoreactive or immune reactive peptides of the corresponding peptides of SEQ ID NO: 1 to 19. Immune reactive peptides or antibody-binding fragments are thus to be understood as any peptide that shows an antibody reactivity that is similar or comparable to or substantially the same as the antibody reactivity of their corresponding peptides. In other words, the antibody-binding fragments, antibodybinding variants or immune reactive peptides are peptides that can be recognized by the same antibodies that recognized their corresponding peptides of SEQ ID Nos: 1 to 19 as disclosed herein. In some embodiments, the antibodies recognize the same epitope on the peptides and their corresponding immune reactive peptides or antibody-binding fragments or variants.
In some embodiments, the immune reactive peptides or antibody-binding fragments as taught herein can be a fragment of their corresponding peptide as defined in SEQ ID Nos: 1 to 19, preferably as defined in SEQ ID NOs; 1 to 7, such as defined in SEQ ID NO: 1 , 2 or 7. In such embodiments, the immune reactive peptide or antibody-binding fragment can thus be a fragment of its corresponding peptides, such as for example a peptide of at least 4, preferably at least 5, even more preferably at least 6, amino acids of the sequence of the peptide as taught herein. The inventors found that peptides or their corresponding antibody-binding fragments or immunoreactive peptides, such as some fragments of the peptides, are also immune reactive with the same antibodies. The term “fragment” as used throughout this specification with reference to a peptide, polypeptide, or protein generally denotes a portion of the peptide, polypeptide, or protein, such as typically an N- and/or C-terminally truncated form of the peptide, polypeptide, or protein. Preferably, a fragment may comprise at least about 30%, e.g., at least about 50% or at least about 70%, preferably at least about 80%, e.g., at least about 85%, more preferably at least about 90%, and yet more preferably at least about 95% or even about 99% of the amino acid sequence length of said peptide, polypeptide, or protein. For example, insofar not exceeding the length of the full-length peptide, polypeptide, or protein, a fragment may include a sequence of > 4 consecutive amino acids, or > 5 consecutive amino acids, or > 6 consecutive amino acids, or > 7 consecutive amino acids, or > 8 consecutive amino acids, or > 9 consecutive amino acids, or > 10 consecutive amino acids, of the corresponding full-length peptide, polypeptide, or protein.
As used herein, an antibody-binding variant of the corresponding peptide can be a peptide that does not necessarily display the same amino acid sequence as the corresponding peptide. For example, there can be discontinuous homology between the antibody-binding variant and its corresponding peptide. Discontinuous homology as used herein is to be understood as partial similarity or identity in the amino acid sequences between the antibody-binding variant and its corresponding peptide, such as for example wherein part of the amino acid sequence of the immune reactive peptide is similar or identical to the corresponding peptide as defined in SEQ ID NOs 1 to 19 but wherein another part of the amino acid sequence of the immune reactive peptide differs from the corresponding peptide as defined in SEQ ID NOs 1 to 19. The antibody-binding variants, also considered herein as immune reactive peptides, are still functionally equivalent to the corresponding peptides of SEQ ID NOs: 1 to 19 and thus these antibody-binding variants show an antibody reactivity that is similar or comparable to or substantially the same as the antibody reactivity of the corresponding peptides of SEQ ID NOs 1 to 19. By means of an example and without limitation, the amino acid sequence of a variant of a peptide may be at least about 80% identical or at least about 85% identical, e.g., preferably at least about 90% identical or at least about 95% identical to the amino acid sequence of the peptide. Sequence identity between proteins or polypeptides may be determined using suitable algorithms for performing sequence alignments and determination of sequence identity as know per se. Exemplary but non-limiting algorithms include those based on the Basic Local Alignment Search Tool (BLAST) originally described by Altschul et al. 1990 (J Mol Biol 215: 403-10), such as the “Blast 2 sequences” algorithm described by Tatusova and Madden 1999 (FEMS Microbiol Lett 174: 247-250), for example using the published default settings or other suitable settings (such as, e.g., for the BLASTP algorithm: matrix = Blosum62 (Henikoff et al., 1992, Proc. Natl. Acad. Sci., 89:10915-10919), cost to open a gap = 11 , cost to extend a gap = 1 , expectation value = 10.0, word size = 3). A variant of a peptide may comprise one or more amino acid additions, deletions, and/or substitutions compared with the corresponding peptide. By means of an example, the amino acid sequence of a variant of a peptide may differ by 5 or less, 4 or less, 3 or less, or 2 or less, such as 1 or 2 or 3, amino acid additions, deletions, and/or substitutions compared to the amino acid sequence of the peptide. In certain embodiments, at least some and preferably all substitutions may be conservative amino acid substitution. Conservative amino acid substitutions include substitutions within the following groups: valine, alanine and glycine; leucine, valine, and isoleucine; aspartic acid and glutamic acid; asparagine and glutamine; serine, cysteine, and threonine; lysine and arginine; and phenylalanine and tyrosine. The nonpolar hydrophobic amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan and methionine. The polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine and glutamine. The positively charged (i.e., basic) amino acids include arginine, lysine and histidine. The negatively charged (i.e., acidic) amino acids include aspartic acid and glutamic acid. Any substitution of one member of the above-mentioned polar, basic, or acidic groups by another member of the same group can be deemed a conservative substitution. By contrast, a non-conservative substitution is a substitution of one amino acid for another with dissimilar characteristics.
Reference to “fragment or variant” or “variant or fragment” of any peptide also encompasses fragments of variants of such peptide, and variants of fragments of such peptide.
Where the present specification refers to or encompasses fragments or variants of proteins, polypeptides or peptides, this in particular denotes such fragments and variants which are biologically active. The term “biologically active” is interchangeable with terms such as “functionally active” or “functional”, denoting that the fragment and variant at least partly retains the biological activity or intended functionality of the respective or corresponding protein, polypeptide, or peptide. In particular, fragments and variants of the peptides disclosed herein, such as their immune reactive peptides, show a similar or comparable or substantially the same antibody reactivity as their corresponding peptides. In further particular embodiments, fragments and variants of the peptides disclosed herein, such as their immune reactive peptides, interact with the same antibodies as their corresponding peptides.
In some embodiments, the antibodies that bind to the antibody-binding fragments or variants or immune reactive peptides as disclosed herein are the same as the antibodies that bind to their corresponding peptides.
In certain embodiments, the antibody-binding fragments or variants or immune reactive peptides have a similar immune reactivity with their antibodies as compared to their corresponding peptides. Thus, in certain embodiments, the antibody reactivity of the peptide and of the antibody-binding fragment or variant or immune reactive peptide thereof is the same and hence both the peptide and the antibodybinding fragment or variant or immune reactive peptide can interact with the same antibody.
By means of further examples and not limitation, where binding of a given antibody to a peptide as disclosed herein or to a fragment or variant of that peptide is detectable by a technique which provides a quantifiable signal, optionally expressed as a quantitative measurement, the signal or measurement obtained using a given molar quantity of the fragment or variant may be at least 1%, preferably at least 10%, such as at least 20%, at least 30%, or at least 40%, or more preferably at least 50%, such as at least 60%, or at least 70%, or even more preferably at least 80%, or at least 90%, still more preferably at least 95%, including about 100% or even more than 100%, of the signal or measurement obtained using the same molar quantity of the corresponding peptide, when all other parameters and conditions of the technique or assay are the same. Any suitable means for detecting antibody levels against one or more peptides of the present invention may be used. Methods for detecting antibody/antigen or immune complexes are well known in the art. The present invention may be modified by one skilled in the art to accommodate the various detection methods known in the art. The particular detection method chosen by one skilled in the art depends on several factors, including the amount of biological sample available, the type of biological sample, the stability of the biological sample, the stability of the antigen (i.e. peptide), and the affinity between the antibody and the antigen (i.e. peptide). For example, the method of the current invention may include the use of an immunoassay, such as, enzyme-linked immunosorbent assays (ELISAs), immunofluorescent techniques, radioimmunological assays (RIA) and immunoblotting and/or line blot. For example, in ELISA-based assays, antigens (i.e. peptides) are bound to a support, and the biological sample is combined therewith. Subsequently antibodies in the sample are allowed to bind to the antigens/peptides bound on the support, thereby forming immune complexes. After the immune complexes have formed, excess biological sample may be removed and the array may be washed to remove nonspecifically bound antibodies. The immune complexes may then be reacted with an appropriate enzyme-labeled anti-immunoglobulin. Then anti-immunoglobulin is allowed to react with the antibodies in the immune complexes. After an optional further wash, the enzyme substrate may be added. The enzyme linked to the anti-immunoglobulin catalyzes a reaction that converts the substrate into a product, which can then be detected and used to quantify the amount of antibody in the sample.
The peptides of the present invention, including their immunoreactive peptides or antibody-binding fragments, are selected as such that no or only a marginal antibody reactivity is found in a reference sample in contrast to a significantly higher antibody reactivity in patients having spondyloarthritis or having a more severe disease status of spondyloarthritis. A reference sample is preferably a sample from a healthy individual not suffering from spondyloarthritis, or from an individual suffering from lower back pain without spondyloarthritis; in particular from non-inflammatory lower back pain without spondyloarthritis. On the other hand, a predetermined threshold based on healthy individuals may be set to compare samples from patients suspected of having spondyloarthritis. In some embodiments, the reference sample is a sample from a patient suffering from spondyloarthritis but with a known disease status, such as a disease status that is determined based on the BASDAI score. In a particular embodiment, antibody reactivity is determined by calculating the ratio of the optical density (OD) of the specific signal to OD of the background signal. For each peptide target, a cut-off for seropositivity was calculated as the mean of this ratio (OD (specific)/OD (background)) in the non-reactive samples plus 3*Standard Deviation. A sample is considered positive when its ratio (OD (specific)/OD (background)) is higher than this cut-off. In particular the higher the general reactivity (OD (specific)/OD (background)) for the peptides of the present invention, in a particular patient, the more likely it is that a positive diagnosis of spondyloarthritis is made or that a more severe disease status of spondylarthritis is present.
In certain embodiments, antibodies are provided that specifically bind to peptides or their antibodybinding fragments, variants or immune reactive peptides as described herein above. Methods for generating antibodies are well known in the art. For the purpose of generation of antibodies, the peptides forming part of the compositions of the invention may be synthesized chemically or may be in a recombinant way. They may also be coupled to a soluble carrier after synthesis or after recombination production. If a carrier is used, the nature of such a carrier should be such that it has a molecular weight greater than 5000 and should not be recognized by antibodies. Such a carrier can be a protein. Proteins which are frequently used as carriers are keyhole limpet hemocyanin, bovine gamma globulin, bovine serum albumin, and poly-L-lysine. There are many well described techniques for coupling peptides to carriers. The linkage may occur at the N-terminus, C-terminus or at an internal site in the peptide. The peptide may also be derivatized for coupling. The peptides may also be synthesized directly on an oligo- lysine core in which both the alpha as well as the epsilon-amino groups of lysines are used as growth points for the polypeptides. The number of lysines comprising the core is preferably 3 or 7. Additionally, a cysteine may be included near or at the C-terminus of the complex to facilitate the formation of homo- or heterodimers.
In general terms, the invention discloses in some aspects a process for detecting or quantifying antibodies related to the diagnosis of spondyloarthritis or to the disease severity of spondyloarthritis or to evaluate therapy response in spondyloarthritis in a biological sample of a subject, preferably a human, liable to contain them. This process comprises contacting the biological sample with a composition according to the invention under conditions enabling an immunological reaction between said composition and the antibodies which are possibly present in the biological sample and the detection of the antigen/antibody complex which may be formed. The detection can be carried out according to any classical process, for example an immune-enzymatic process according to the ELISA technique or immunofluorescent or radioimmunological (RIA) or the equivalent ones (e.g. LINE blot or LINE assay) can be used. Thus in some embodiments of the invention peptides labelled by an appropriate label of the enzymatic, fluorescent, biotin, radioactive type, can be used. Such a method for detecting antibodies related to spondyloarthritis comprises for instance the following steps: deposit of determined amounts of a composition comprising peptides according to the invention on a support (e.g. into wells of a titration microplate), introduction on said support (e.g. into wells) of increasing dilutions of the body fluid (e.g. blood plasma or serum) to be diagnosed, incubation of the support (e.g. microplate), repeated rinsing of the support (e.g. microplate), introduction on the support labelled antibodies which are specific for immunoglobulins present in the body fluid, the labelling of these antibodies being based on the activity of an enzyme which is selected from among the ones which are able to hydrolyse a substrate to a product which absorbs light at a given wave length, and detection by comparing a control standard of the amount of hydrolysed substrate.
In yet another aspect, the invention also relates to a process for detecting and identifying an antigen related to the diagnosis of spondyloarthritis or to the disease severity of spondyloarthritis or to the evaluation of therapy response in spondyloarthritis in a biological sample liable to contain it, this process comprising contacting the biological sample with an appropriate antibody of the invention (i.e. antibodies with a specificity for a peptide, antibody-binding fragment, variant or immune reactive peptide thereof of the composition) under conditions enabling an immunological reaction between said antibody and the antigens which are possibly present in the biological sample and the detection of the antigen/antibody complex which may be formed. Thus antibodies, in particular auto-antibodies, which recognize the peptides of the invention or their immune reactive peptides, can be detected in a variety of ways. One method of detection is further described in the examples and uses enzyme-linked immunosorbent assay (ELISA) of the polypeptides of the invention or their immune reactive peptides displayed by phages (e.g. phage-ELISA technology). The latter technology is fully described in Quaden et al., (Arthritis Rheumatology, 2020, 72(12)), wherein paragraph “Detection of antibodies to 9 novel UH-axSpA peptides” on page 2097 is herein specifically incorporated. In other ways, the detection in ELISA uses a peptide or mixture of peptides bound to a solid support. In some cases, this will be a microtiter plate but may in principle be any sort of insoluble solid phase (e.g. glass, nitrocellulose). In one embodiment a suitable dilution or dilutions of for example blood or serum to be tested is brought into contact with the solid phase to which the polypeptide is bound. In another embodiment “a solution hybridization” is carried out in which high affinity interactions occur (e.g. biotinylated polypeptides of the composition are pre-incubated with serum). The incubation is carried out for a time necessary to allow the binding reaction to occur. Subsequently, unbound components are removed by washing the solid phase. The detection of immune complexes (i.e. antibodies present in for example human serum binding to at least one peptide of the invention) is achieved using antibodies which specifically bind to human immunoglobulins, and which have been labelled with an enzyme, preferably but not limited to either horseradish peroxidase, alkaline phosphatase, or beta-galactosidase, which is capable of converting a colourless or nearly colourless substrate or co-substrate into a highly coloured product or a product capable of forming a coloured complex with a chromogen. Alternatively, a detection system may employ an enzyme which, in the presence of the proper substrate(s), emits light. The amount of product formed is detected either visually, spectrophotometrically, electrochemically, fluorescently or luminometrically, and is compared to a similarly treated control. The detection system may also employ radioactively labelled antibodies, in which cases the amount of immune complex is quantified by scintillation counting or gamma counting. Other detection systems which may be used include those based on the use of protein A derived from Staphylococcus aureus Cowan strain I, protein G from group C Staphylococcus sp. (strain 26RP66), or systems which make use of the high affinity biotin-avidin or streptavidin binding reaction.
The peptides of the invention or their antibody-binding fragments, variants or immune reactive peptides may be either labelled or unlabelled. Labels which may be employed may be of any type, such as enzymatic, chemical, fluorescent, luminescent, or radioactive. In addition, the peptides may be modified for binding to surfaces or solid phases, such as, for example microtiter plates, nylon membranes, glass or plastic beads, and chromatographic supports such as cellulose, silica, or agarose. The methods by which peptides or their antibody-binding fragments, variants or immune reactive peptides can be attached or bound to solid support or surface are well known to those skilled in the art.
The peptides of the invention or their antibody-binding fragments, variants or immune reactive peptides can be prepared according to the classical techniques in the field of peptide synthesis. The synthesis can be carried out in homogeneous solution or in solid phase. For instance, the synthesis technique in homogeneous solution which can be used is the one described by Houbenweyl in the book titled “Methode der organische chemie” (Method of organic chemistry) edited by E. Wunsch, vol. 15-1 et II. THIEME, Stuttgart 1974. The peptides of the invention can also be prepared in solid phase according the method described by Atherton & Shepard in their book titled “Solid phase peptide synthesis” (Ed. IRL Press, Oxford, NY, Tokyo, 1989). Synthesis protocols in the art generally employ the use of t- butyloxycarbonyl- or 9-fluorenylmethoxy-carbonyl-protected activated amino acids. The procedures for carrying out the syntheses, the types of side-chain protection, and the cleavage methods are amply described in, for example, Stewart and Young, Solid Phase Peptide Synthesis, 2nd Edition, Pierce Chemical Company, 1984; and Atherton and Sheppard, Solid Phase Peptide Synthesis, IRL Press 1989.
In certain embodiments, antibodies raised to peptides of the invention or to their antibody-binding fragments or variants or immune reactive peptides (or carrier-bound peptides or immune reactive peptides) can also be used in conjunction with labelled or unlabelled peptides of the invention or their immune reactive peptides for the detection of (auto)antibodies present in serum by competition assay. For example, antibodies raised to peptides are attached to a solid support which may be, for example, a plastic bead or a plastic tube. The peptide is then mixed with suitable dilutions of the fluid (e.g. serum) to be tested and this mixture is subsequently brought into contact with the antibody bound to the solid support. After a suitable incubation period, the solid support is washed and the amount of labelled or unlabelled peptide is quantified. A reduction in the amount of label bound to the solid support is indicative of the presence of (auto)antibodies in the original sample, such as blood plasma or serum. By the same token, the peptide may also be bound to the solid support. Labelled antibody may then be allowed to compete with (auto)antibody present in the sample (e.g. serum) under conditions in which the amount of peptide is limiting. As in the previous example, a reduction of the measured signal is indicative of the presence of (auto)antibodies in the sample tested.
In another embodiment, a competition ELISA can be used in which samples (e.g. plasma or serum samples) are pre-incubated with increasing concentrations of one or more synthetic peptides corresponding to the sequences defined by SEQ ID No: 1-19, before use in a phage ELISA.
In some embodiments, a test for giving evidence of the fact that one or more peptides present in a composition of the invention are recognized by antibodies present in for example blood or serum (for example auto-antibodies present in serum of axSpa patients) is an immunoblotting (or Western blotting) analysis. In the latter case peptides can be chemically synthesized or peptides (or the protein) can be produced via recombinant techniques. In short, after sodium dodecyl sulfate-polyacrylamide gel electrophoresis, peptides of the invention are blotted onto nitrocellulose membranes (e.g. Hybond C. (Amersham)) as described by Towbin H. et al., 1979, "Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications", Proc. Natl. Acad. Sci. USA 76:4350-4354.
It goes without saying that the free reactive functions which are present in some of the amino acids, which are part of the constitution of the peptides of the invention or their immune reactive peptides, particularly the free carboxyl groups which are carried by the groups Glu and Asp or by the C-terminal amino acid on the one hand and/or the free NH2 groups carried by the N-terminal amino acid or by amino acids inside the peptidic chain, for instance Lys, on the other hand, can be modified in so far as this modification does not alter the above mentioned properties of the polypeptide. The peptides which are thus modified are naturally part of the invention. The above-mentioned carboxyl groups can be acylated or esterified. Other modifications are also part of the invention. Particularly, the amine or carboxyl functions or both of terminal amino acids can be themselves involved in the bond with other amino acids. For instance, the N-terminal amino acid can be linked to the C-terminal amino acid of another peptide comprising from 1 to several amino acids. Furthermore, any peptidic sequences resulting from the modification by substitution and/or by addition and/or by deletion of one or several amino acids of the polypeptides according to the invention are part of the invention in so far as this modification does not alter the above-mentioned properties of said polypeptides. The peptides according to the invention can be glycosylated or not, particularly in some of their glycosylation sites of the type Asn-X-Ser or Asn-X-Thr, X representing any amino acid.
Variations of these peptides are also possible depending on its intended use. For example, if the peptide is to be used to raise antisera, the peptide may be synthesized with an extra cysteine residue added. This extra cysteine residue is preferably added to the amino terminus and facilitates the coupling of the peptide to a carrier protein which is necessary to render the small peptide immunogenic. If the peptide is to be labelled for use in radioimmune assays, it may be advantageous to synthesize the protein with a tyrosine attached to either the amino or carboxyl terminus to facilitate iodination. This peptide possesses therefore the primary sequence of the peptide above-mentioned but with additional amino acids which do not appear in the primary sequence of the protein and whose sole function is to confer the desired chemical properties to the peptide.
The terms “amount”, “quantity” or “level” are synonymous and as used herein refer to but are not limited to the absolute or relative amount of antibodies, peptides, polypeptides or any other value or parameter associated with the latter or which can derive therefrom. Such values or parameters comprise signal intensity values obtained by direct or indirect measurement, for example, in an ELISA assay. The readout may be a mean, average, median, or the variance or other statistically or mathematically-derived value associated with the measurement. The absolute values obtained for the antibody levels under identical conditions will display a variability that is inherent in live biological systems and also reflects individual antibody quantity variability as well as the variability inherent between individuals.
A relative quantity of a marker, antibody, peptide, polypeptide, or protein in a sample may be advantageously expressed as an increase or decrease or as a fold-increase or fold-decrease relative to said another value, such as relative to a reference value as taught herein. Performing a relative comparison between first and second parameters (e.g., first and second quantities) may but need not require determining first the absolute values of said first and second parameters. For examples, a measurement method may produce quantifiable readouts (such as, e.g., signal intensities) for said first and second parameters, wherein said readouts are a function of the value of said parameters, and wherein said readouts may be directly compared to produce a relative value for the first parameter vs. the second parameter, without the actual need to first convert the readouts to absolute values of the respective parameters.
Reference to the activity of a protein, peptide, or polypeptide may generally encompass any one or more aspects of the biological activity of the protein, peptide, or polypeptide, such as without limitation any one or more aspects of its antibody reactivity, biochemical activity, enzymatic activity, signaling activity, interaction activity, ligand activity, and/or structural activity, e.g., within a cell, tissue, organ or an organism.
Depending on factors that can be evaluated and decided on by a skilled person, such as inter alia the type of a marker (e.g., antibody, peptide, polypeptide, protein), the type of the tested object (e.g., a cell, cell population, tissue, organ, or organism), the type of biological sample of a subject, (e.g., serum, plasma, whole blood, tissue biopsy), the expected abundance of the marker in the tested object, the type, robustness, sensitivity and/or specificity of the detection method used to detect the marker, etc., the quantity and/or activity of a marker may be measured directly in the tested object, or the tested object may be subjected to one or more processing steps aimed at achieving an adequate measurement of the marker.
The term “peptide” as used throughout this specification preferably refers to a peptide as used herein consisting essentially of 50 amino acids or less, e.g., 45 amino acids or less, preferably 40 amino acids or less, e.g., 35 amino acids or less, more preferably 30 amino acids or less, e.g., 25 or less, 20 or less, 15 or less, 10 or less or 5 or less amino acids.
The term “polypeptide” as used throughout this specification generally encompasses polymeric chains of amino acid residues linked by peptide bonds. Hence, especially when a protein is only composed of a single polypeptide chain, the terms “protein” and “polypeptide” may be used interchangeably herein to denote such a protein. The term is not limited to any minimum length of the polypeptide chain. The term may encompass naturally, recombinantly, semi-synthetically or synthetically produced polypeptides. The term also encompasses polypeptides that carry one or more co- or post-expression- type modifications of the polypeptide chain, such as, without limitation, glycosylation, acetylation, phosphorylation, sulfonation, methylation, ubiquitination, signal peptide removal, N-terminal Met removal, conversion of pro-enzymes or pre-hormones into active forms, etc. The term further also includes polypeptide variants or mutants which carry amino acid sequence variations vis-a-vis a corresponding native polypeptide, such as, e.g., amino acid deletions, additions and/or substitutions. The term contemplates both full-length polypeptides and polypeptide parts or fragments, e.g., naturally- occurring polypeptide parts that ensue from processing of such full-length polypeptides.
The reference to any marker, antibody, peptide, polypeptide, or protein corresponds to the marker, antibody, peptide, polypeptide, or protein commonly known under the respective designations in the art. The terms encompass such markers, antibodies, peptides, polypeptides, or proteins of any organism where found, and particularly of animals, preferably warm-blooded animals, more preferably vertebrates, yet more preferably mammals, including humans and non-human mammals, still more preferably of humans.
In certain embodiments, markers, antibodies, peptides, polypeptides, or proteins may be human, i.e. , their primary sequence may be the same as a corresponding primary sequence of or present in naturally occurring human markers, peptides, polypeptides, proteins, or nucleic acids. In certain embodiments, markers, antibodies, peptides, polypeptides, or proteins are non-naturally occurring.
The qualifier “human” in this connection relates to the primary sequence of the respective markers, antibodies, peptides, polypeptides, or proteins, rather than to their origin or source. For example, such markers, antibodies, peptides, polypeptides, or proteins, may be present in or isolated from samples of human subjects or may be obtained by other means (e.g., by recombinant expression, cell-free transcription or translation, or non-biological nucleic acid or peptide synthesis).
Unless otherwise apparent from the context, reference herein to any marker, antibody, peptide, polypeptide, protein, or fragment thereof may generally also encompass modified forms of said marker, antibody, peptide, polypeptide, protein, or fragment thereof, such as bearing post-expression modifications including, for example, phosphorylation, glycosylation, lipidation, methylation, cysteinylation, sulphonation, glutathionylation, acetylation, biotinylation, oxidation of methionine to methionine sulphoxide or methionine sulphone, and the like.
Fragments or variants of any marker, antibody, peptide, polypeptide, or protein are also envisaged herein. Hence, for example, referring herein to measuring (or measuring the quantity of) any one marker, antibody, peptide, polypeptide, or protein may encompass measuring the marker, antibody, peptide, polypeptide, or protein and/or measuring one or more fragments or variants thereof. For example, any marker, antibody, peptide, polypeptide, or protein and/or one or more fragments or variants thereof may be measured collectively, such that the measured quantity corresponds to the sum amounts of the collectively measured species. In another example, any marker, antibody, peptide, polypeptide, or protein and/or one or more fragments or variants thereof may be measured each individually.
As used herein, a marker, antibody, peptide, polypeptide, or protein is “detected” or “measured” in a sample when the presence or absence, quantity and/or activity of said marker, antibody, peptide, polypeptide, or protein is determined or measured in the sample, preferably substantially to the exclusion of other markers, antibodies, peptides, polypeptides, or proteins.
The present invention also provides a diagnostic kit for performing one of the in vitro methods according to the present invention, said kit comprising one or polypeptides or antibody-binding fragments thereof of the invention or one or more detection agents according to the invention, or a composition according to an embodiment of the invention; and reagents for detecting antibody binding to said one or more peptides or antibody-binding fragments or variants therefrom or detection agents. Said reagents are for example reagents for making a medium appropriate for the immunological reaction to occur, reagents enabling the antigen/antibody complex which has been produced by the immunological reaction, said reagents possibly having a label, or being liable to be recognizable by a labelled reagent, more particularly in the case where the abovementioned polypeptide is not labelled.
In a further related aspect, the application provides the use of one or more peptides or antibody-binding fragments or variants thereof as taught herein, or the use of a detection agent as taught herein, or the use of a composition comprising one or more peptides or antibody-binding fragments or variants thereof as taught herein, for detecting the presence or quantity of specific antibodies against said peptide, antibody-binding fragment or variant, or against the detection agent, or against the peptide, antibodybinding fragment or variant present in the composition, in a biological sample of the subject; preferably wherein the presence or increase in antibody levels as compared to a reference sample is indicative for the diagnosis of spondyloarthritis.
In a further related aspect, the application provides the use of one or more peptides or antibody-binding fragments or variants thereof as taught herein, or the use of a detection agent as taught herein, or the use of a composition comprising one or more peptides or antibody-binding fragments or variants thereof as taught herein, for detecting the presence or quantity of specific antibodies against said peptide, antibody-binding fragment or variant, or against the detection agent, or against the peptide, antibodybinding fragment or variant present in the composition, in a biological sample of the subject; preferably wherein the presence or increase in antibody levels as compared to a reference sample is indicative for the disease severity of spondyloarthritis.
In a further related aspect, the application provides the use of one or more peptides or antibody-binding fragments or variants thereof as taught herein, or the use of a detection agent as taught herein, or the use of a composition comprising one or more peptides or antibody-binding fragments or variants thereof as taught herein, for detecting the presence or quantity of specific antibodies against said peptide, antibody-binding fragment or variant, or against said detection agent, or against the peptide, antibodybinding fragment or variant present in the composition, in a first and second biological sample of the subject wherein the first biological sample is obtained before the start of therapeutic treatment and the second biological sample is obtained after the start of therapeutic treatment; preferably wherein a decrease in antibody levels in the second biological sample as compared to the first biological sample is indicative for a response to the therapeutic treatment.
Thus the invention provides the use of one or more peptides or detection agents or a composition of the invention for detecting the presence of specific antibodies to at least one of said peptides or detection agents or to a peptide present in said composition wherein said antibodies are present in a body fluid of a mammal, preferably a human. In particular embodiments, said use of a composition is an “in vitro” use of a composition. The latter implies a diagnostic method with no direct interaction with the patient.
The methods and means of the present invention are in particular suitable for diagnosing the presence of spondyloarthritis. The term “diagnosing” or “diagnosis” is meant to include “predicting” or “detecting” the presence of spondyloarthritis. Spondyloarthritis includes axial spondyloarthritis and peripheral spondyloarthritis; preferably selected from ankylosing spondylitis, non-radiographic axial spondyloarthritis, undifferentiated spondyloarthritis, juvenile spondyloarthritis, psoriatic arthritis.
In some instances, antibodies against one or more of the peptides of the present invention are absent in healthy individuals, hence, in that instance, the mere detection or presence of an antibody response against said peptides or fragments thereof is already suitable for determining the diagnosis spondyloarthritis in said patient. In some other instances, the methods according to the different embodiments of the present invention can be combined with any other method available for the diagnosis of spondyloarthritis or for evaluating the disease severity of spondyloarthritis. Said other methods can be selected from physical examination, imaging technology and/or laboratory tests for human leukocyte antigen (HLA)-B27 and C-reactive protein (CRP).
While the methods and means of the present invention are highly useful in the diagnosis of spondyloarthritis, they are also very suitable for use in determining the disease severity of spondyloarthritis or for evaluating therapeutic treatment in patients diagnosed with spondyloarthritis. In patients with spondyloarthritis, disease severity or the response to therapy can be evaluated by using the BASDAI score, which ranges from 0 (no disease activity) to 10 (very active disease) and a cutoff value of a BASDAI score of 4 is frequently used to define active disease.
In yet another aspect, the invention also relates to a process for detecting and identifying antigen of spondyloarthritis in a body fluid liable to contain them, this process comprising contacting the biological sample with an appropriate antibody of the invention (i.e. antibodies with a specificity for a polypeptide of the composition) under conditions enabling an immunological reaction between said antibody and the antigens of spondyloarthritis which are possibly present in the biological sample and the detection of the antigen/antibody complex which may be formed.
In certain embodiments, one or more peptides, or the immune reactive peptides or antibody-binding fragments or variants thereof or detection agents as taught herein, are provided for use in the diagnosis of spondyloarthritis or for use in the evaluation of disease severity or therapy response in spondyloarthritis, in particular wherein said one or more peptides comprise an epitope having a sequence selected from any one of SEQ ID No: 1 to 7 or an immune-reactive fragment or antibodybinding fragment or variant thereof; typically comprising at least 4, in particular at least 5, even more in particular at least 6, consecutive amino acids derived therefrom. In certain embodiments, said one or more peptides or the immune reactive peptides or antibody-binding fragments or variants thereof or detection agents comprise an epitope having a sequence selected from any one of SEQ ID No: 1 , SEQ ID No: 2 or SEQ ID No: 7, or an immune-reactive fragment thereof; typically comprising at least 4, in particular at least 5, even more in particular at least 6, consecutive amino acids derived therefrom.
In a further embodiment, one or more peptides, or the immune reactive peptides or antibody-binding variants or fragments thereof or detection agents as taught herein are provided for use in the diagnosis of spondyloarthritis or for use in the evaluation of disease severity or therapy response in spondyloarthritis, in particular wherein said one or more peptides comprise an epitope having a sequence selected from any one of SEQ ID No: 8 to 19 or an immune-reactive fragment or antibodybinding fragment or variant thereof; typically comprising at least 4, in particular at least 5, even more in particular at least 6, consecutive amino acids derived therefrom. In certain embodiments, said one or more polypeptides or the immune reactive peptides or antibody-binding fragments or variants thereof comprise an epitope having a sequence selected from any one of SEQ ID No: 8, SEQ ID No: 9, SEQ ID No:16 or SEQ ID No: 17, an immune-reactive fragment or antibody-binding fragment or variant thereof; typically comprising at least 4, in particular at least 5, even more in particular at least 6, consecutive amino acids derived therefrom.
In the context of the present invention, the biological sample, sometimes also referred to as “sample”, may be any type of sample suitable for the determination of antibody levels against said one or more peptides of the invention, and is in particular a blood sample, a tissue sample or a body fluid sample. In some embodiments, the sample is selected from the list comprising hair, skin, nails, saliva, synovial liquid, blood serum, blood plasma, urine, tears, bone marrow fluid, cerebrospinal fluid, lymphatic fluid, amniotic fluid, nipple aspiration fluid, and the like; more preferably selected from blood serum or blood plasma. In preferred embodiments, the sample is a blood sample, preferably a blood serum sample or a blood plasma sample.
In some embodiments, the antibody level in the biological sample can be compared to the antibody level of a reference sample, wherein the antibody level in the one or more reference samples is considered as the reference value. In some embodiments, the reference sample is a biological sample obtained from a healthy subject, i.e. a subject that is not diagnosed with spondyloarthritis. In some embodiments, the reference sample is a biological sample obtained from a patient diagnosed with spondyloarthritis but that does not show any antibody reactivity against the antibodies that are determined in the present invention. In some embodiments, the reference sample is a biological sample obtained from a patient diagnosed with spondyloarthritis and with a known disease severity status.
In some embodiments, the antibody level in two or more biological samples obtained from the subject can be compared. In some embodiments, a first biological sample is obtained before the start of the treatment of spondyloarthritis and a second or even a third or fourth biological sample is obtained after the treatment of spondyloarthritis, preferably after a particular time of treating the subject diagnosed with spondyloarthritis, such as after 3 months, after 6 months, after 12 months, after 24 months or after 36 months.
As used herein, the terms “subject”, “individual” or “patient” are used interchangeably throughout this specification, and typically and preferably denote humans, but may also encompass reference to nonhuman animals, preferably warm-blooded animals, even more preferably mammals, such as, e.g. nonhuman primates, rodents, canines, felines, equines, ovines, porcines, and the like. The term “non-human animals” includes all vertebrates, e.g., mammals, such as non-human primates (particularly higher primates), sheep, dog, rodent (e.g., mouse or rat), guinea pig, goat, pig, cat, rabbits, cows, and nonmammals such as chicken, amphibians, reptiles, etc. In certain embodiments, the subject is a mammal. In some embodiments, the subject is a non-human mammal. In some preferred embodiments of the methods and uses as taught herein, the subject is a human subject. In other embodiments, the subject is an experimental animal or animal substitute as a disease model. The term does not denote a particular age or sex.
Suitable subjects may include subjects that are suspected of having spondyloarthritis, such as subjects that are presenting with symptoms of back pain (slow-onset back pain with stiffness for at least three months or longer, intense pain in the mornings or after periods of inactivity, and it may begin to ease during the day with stretching and exercise), fatigue, joint pain, heel pain, sleep problems, and/or abdominal pain.
In certain embodiments, the subject did not yet receive any treatment with an anti-inflammatory and/or anti-rheumatic drug to treat spondyloarthritis. In certain embodiments, the subject did not receive any treatment with an anti-inflammatory and/or anti-rheumatic drug for a period of at least 3 months, preferably at least 6 months. In some embodiments, the subject is diagnosed with spondyloarthritis and is selected to receive treatment with an anti-inflammatory and/or anti-rheumatic drug, for example drugs selected from nonsteroidal anti-inflammatory drugs (NSAIDs), conventional disease-modifying antirheumatic drugs (cDMARDs) and biological DMARDs such as tumor necrosis factor-alpha (TNF-alpha) inhibitors, agents targeting Interleukin (IL)-17A and Janus kinase (JAK) inhibitors
In a final aspect, a method for treatment of a subject with spondyloarthritis is provided that comprises identifying a subject as having or not having one or more antibodies as taught herein in a biological sample from the subject (such as by a method comprising determining the presence or quantity of the one or more antibodies in the sample), and administering an anti-spondyloarthritic drug to the subject. Particularly, the method comprises comparing the antibody levels in the biological sample of the subject with the antibody levels in one or more reference samples. In certain embodiments, when antibodies as disclosed herein are detected as compared to the reference sample, the subject is diagnosed with spondyloarthritis and the subject is treated with an anti-inflammatory or anti-rheumatc drug, in particular with a drug selected from nonsteroidal anti-inflammatory drugs (NSAIDs), conventional diseasemodifying anti-rheumatic drugs (cDMARDs, for example sulfasalazine) and biological DMARDs such as tumor necrosis factor-alpha (TNF-alpha) inhibitors.
In certain embodiments, the subject is first diagnosed with spondyloarthritis using any one of the in vitro methods, peptides, detection agents, compositions or diagnostic kits of the present invention, followed by selection of the appropriate therapy and treatment of the subject with said therapy against spondyloarthritis. For example, the method of treatment comprises the in vitro method for diagnosing the presence of spondyloarthritis in a subject, said method being any method as described herein above, followed by treatment of the subject diagnosed with spondyloarthritis with a therapy selected from nonsteroidal anti-inflammatory drugs (NSAIDs), conventional disease-modifying anti-rheumatic drugs (cDMARDs, for example sulfasalazine) and biological DMARDs such as tumor necrosis factor-alpha (TNF-alpha) inhibitors.
The methods of treatment as taught herein may specifically relate to prophylactic and/or therapeutic treatment of spondyloarthritis. In particular embodiments, said method relates to a therapeutic treatment of spondyloarthritis. The therapeutic agent administered to the subject can be a therapeutic agent known to be effective against spondyloarthritis, as disclosed herein above.
As used throughout this specification, the terms “therapy” or “treatment” refer to interventions, such as pharmacological interventions, that result in the alleviation or measurable lessening of one or more symptoms or measurable markers of a pathological condition such as a disease or disorder. The terms encompass primary treatments as well as neo-adjuvant treatments, adjuvant treatments and adjunctive therapies. The terms “therapy” or “treatment” broadly refer to interventions that result in the alleviation or measurable lessening of one or more symptoms or measurable markers of spondyloarthritis. Measurable lessening includes any clinically significant decline in a measurable marker or symptom. Generally, the terms encompass both curative treatments and treatments directed to reduce symptoms and/or slow progression of the disease. The terms encompass both the therapeutic treatment of an already developed pathological condition, as well as prophylactic or preventative measures, wherein the aim is to prevent or lessen the chances of incidence of a pathological condition. In certain embodiments, the terms may relate to therapeutic treatments. In certain other embodiments, the terms may relate to preventative treatments. Treatment of a chronic pathological condition during the period of remission may also be deemed to constitute a therapeutic treatment. The term may encompass ex vivo or in vivo treatments.
The terms “responsiveness” or “susceptibility” or “sensitivity” may be used interchangeably herein and refer to the quality that predisposes a subject having spondyloarthritis to be sensitive or reactive to a particular therapeutic treatment. A subject is “responsive” or “susceptible” or “sensitive” (which terms may be used interchangeably) to treatment with a particular therapeutic agent if the subject will have benefit from the treatment. A subject is non-responsive to a particular treatment with a therapeutic agent if there is no effect of the treatment. A subject is poorly responding to a particular treatment with a therapeutic agent if there is only minimal effect of the treatment.
In spondyloarthrhtis, the disease severity status of spondyloarthritis or the response to treatment is based on standard criteria known in the art. Disease activity measurements can for example be based on the Bath Ankylosing Spondylitis Disease Activity Index (BASDAI), the Bath Ankylosing Spondylitis Functional Index (BASFI), erythrocyte sedimentation rate (ESR), and/or C-reactive protein (CRP) levels. Also the presence of HLA-B27 can be linked to disease severity. In a preferred embodiment, the disease activity is based on the BASDAI score.
It is apparent that there have been provided in accordance with the invention products, methods and uses, that provide substantial advantages as set forth above. While the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications, and variations will be apparent to those skilled in the art in light of the foregoing description. Accordingly, it is intended to embrace all such alternatives, modifications, and variants as follows in the spirit and broad scope of the appended claims.
The above aspects and embodiments are further supported by the following non-limiting examples. Also other characteristics and advantages of the invention will appear in the following examples and the figures illustrating the invention. EXAMPLES
Materials and Methods Patients and controls
Plasma of early axial spondyloarthritis (axSpA) patients (n=98) and rheumatoid arthritis (RA) patients (n=46) was collected at the ReumaClinic in Genk, whereas plasma of healthy controls (HC; n=125) was collected in Hasselt and Genk (UH cohort). Plasma of 10 axSpA patients and 10 HC was used for screening in the SAS procedure, while the remaining samples were used for validation of antibody reactivity using phage ELISA.
Patients with axSpA or RA were diagnosed by their treating rheumatologist and classified according to the ASAS classification criteria (Rudwaleit et a!., Ann Rheum Dis 2009, 68(6): 770-6; Rudwaleit et al., Ann Rheum Dis 2009, 68(6): 777-83) or the 1987 American College of Rheumatology criteria for RA (Arnett et al., Arthritis Rheum 1988; 31 (3): 315-24), respectively. All axSpA and RA patients were considered as early patients as plasma samples were collected within a maximum time of 5 years after diagnosis. The HC did not show signs of inflammation, rheumatic and/or joint related complaints or a positive family history of rheumatic and/or autoimmune diseases.
The SAS procedure uses plasma pools of axSpA patients and HC to identify novel axSpA-related antibodies. In this study, a human RA cDNA phage display library and a human axSpA cDNA phage display library were used to screen for the presence of respectively IgG or IgA antibodies in pooled plasma of early axSpA patients. Plasma of 10 early axSpA patients, which did not receive biological therapy, was pooled into an early axSpA SAS pool. This early axSpA SAS pool had a mean age of 40.6 ± 11.8 years, a mean disease duration of 1 .4 ± 0.5 years, 5 (50%) were male and 8 (80%) were HLA- B27 positive. A HC SAS pool, consisting of 10 HC plasma samples (mean age of 40.6 ± 12.3 years and 5 (50%) were male) that were age- and gender- matched to the axSpA SAS pool, was used in the SAS procedure to perform counter selection. After the identification of novel antibodies and their corresponding targets by the SAS procedure, antibody reactivity against phage displayed peptides was tested in the early axSpA SAS pool and HC SAS pool, using phage enzyme linked immunosorbent assays (ELISA), in order to select the best peptide targets.
Antibody reactivity against the 12 selected UH-axSpA-lgG peptides was determined and validated in additional individual plasma samples of 80 early axSpA patients, 46 HC and 46 RA patients, using phage ELISA. The axSpA patients (n=80) had a mean age of 44.2 ± 12.4 years, a mean disease duration of 2.9 ± 1 .3 years, 36 (45%) were male and 34 (43%) were HLA-B27 positive. The HC (n=46) had a mean age of 43.7 ± 18.2 years and 22 (48%) were male. Patients with RA (n=46) had a mean age of 47.1 ± 11.1 years, a mean disease duration of 3.1 ± 1.2 years and 13 (28%) were male.
Antibody reactivity against 7 UH-axSpA-lgA peptide targets was also determined by phage ELISA in individual plasma samples of 79 early axSpA patients and 92 HC. The early axSpA patients (n=79) had a mean age of 43.2 ± 12.5 years, a mean disease duration of 3.0 ± 1.6 years, 45 (57%) were male and 45 (57%) were HLA-B27 positive. The HC (n=92) had a mean age of 44.2 ± 11.9 years and 52 (57%) were male.
This study was conducted in accordance with the Helsinki Declaration and was approved by the local Ethical Committees of Jessa Hospital, Hasselt University and Ziekenhuis Oost-Limburg (ZOL) (B243201422699). Plasma samples of axSpA patients and controls were previously collected under different studies, which were ethically approved by the Ethical Committees of University Hospitals Leuven, Jessa Hospital and Hasselt University (B322201215165 and B243201422699) after providing written informed consent. All human biological materials used for this study were stored in the University Biobank Limburg (UBiLim).
Construction of human axSpA cDNA phage display library
A human axSpA cDNA phage display library was constructed according to the procedure as described in Vandormael et al. (Immunol Res, 2017; 65(1): 307-25). In brief, total RNA was extracted from hip synovial tissue of 3 axSpA patients and messenger RNA (mRNA) was converted into double-stranded cDNA, using the Maxima H-minus double-stranded cDNA synthesis kit (Thermo Fisher Scientific, Belgium). Patient-specific adaptors were ligated to the cDNA, which was then size-fractionated by agarose gel electrophoresis. Subsequently, cDNA molecules ranging from 200 to 10,000 base pairs were directionally cloned into the pSPVI-A/B/C phagemid vectors and transformed in TG1 E.coli cells via electroporation. Phage particles from this resulting cDNA phage display library were produced by infecting TG1 E.coli cells with M13K07 helper phage and purifying via PEG/NaCI precipitation.
Identification of novel antibodies by SAS
Phage particles from the human RA cDNA phage display library were screened for reactivity with IgG antibodies present in plasma of early axSpA patients by the previously described SAS procedure (Palmers et al., J Neuroinflammation, 2016, 13(1): 243; Somers et al., J Autoimmun, 2011 , 36(1): 33- 46; Somers et al., Ann NY Acad Sci, 2009, 1173: 92-102; Somers et al., J Immunol, 2008, 180(6): 3957- 63) with some minor modifications. In brief, an immunotube (Nunc) was coated with 10 pg/mL rabbit anti-human IgG (Dako) diluted in coating buffer (0.1 M sodium hydrogen carbonate, pH 9.6), rotating overnight at 4°C. Approximately 4 x 1013 phage particles from the RA cDNA phage display library were pre-incubated with the axSpA SAS pool, each plasma sample at a 1 :200 final dilution, in 2% skimmed milk powder in phosphate buffered saline (MPBS) for 1 .5 hours (hrs) at room temperature (RT) to allow formation of IgG antibody-phage complexes. After blocking the immunotube with 2% MPBS for 2 hrs at RT, the antibody-phage pre-incubation mixture was added and incubated for 2 hrs and 30 minutes (min) at RT to capture antibody-phage complexes. Non-bound antibodies and phage particles were washed away, while bound antibody-phage complexes were eluted by 100 mM triethylamine, pH 12.0 (Sigma- Aldrich) and neutralized using 1 M Tris-HCI, pH 7.4 (Sigma-Aldrich). Eluted phage particles were amplified by infecting TG1 E. coli bacterial cells, which were then grown overnight at 30°C on 2xTY- ampicillin (100 pg/mL), 2% glucose agar plates. Resulting bacterial colonies were harvested and used as input for consecutive SAS selection rounds. Four consecutive SAS selection rounds were performed using the early axSpA SAS plasma pool. The fourth SAS selection round was immediately followed by one round of counter selection with the HC SAS pool, with each HC plasma sample at a 1 :200 final dilution, which was performed to prevent the isolation of common antibodies present in the general population. After this round, phage particles not bound by IgG antibodies in the HC SAS pool were amplified and characterized. In order to identify the antigen targets expressed on the surface of selected phage particles, the fusion regions between the M13 gene VI and cDNA inserts of selected phage clones were analyzed. The cDNA inserts of randomly selected bacterial colonies were amplified using PCR primers flanking the cDNA inserts. Initial characterization of these clones was performed by DNA fingerprinting of these PCR products using restriction enzymes BstNI (Roche Diagnostics) and CviQI (New England Biolabs). Clones that showed a unique restriction pattern were identified by DNA sequencing of the fusion between M13 gene VI and the cDNA inserts. Both nucleotide and amino acid sequences of identified cDNA inserts were compared with human sequences using the BLAST tool of NCBI.
Novel IgA antibodies and their corresponding antigenic targets were identified by the SAS procedure. In this approach, phage particles displaying axSpA synovial antigens and randomly formed peptides, originating from the newly human axSpA cDNA phage display library, were screened for IgA antibody reactivity in the early axSpA SAS pool using the previously described SAS procedure, with some minor modifications. A Nunc immunotube was coated with 10 pg/mL of rabbit anti-human IgA (Dako), diluted in coating buffer (0.1 M sodium hydrogen carbonate, pH 9.6), rotating overnight at 4°C. The immunotube was blocked with 2% skimmed milk powder in phosphate buffered saline (MPBS) for 2 hours (hrs) at room temperature (RT). Meanwhile, approximately 4 x 1013 phage particles from the human axSpA cDNA phage display library were pre-incubated with the early axSpA SAS pool diluted in 2% MPBS (each plasma sample at a 1 :200 final dilution) for 1 .5 hrs at RT to allow the formation of IgA antibody- phage complexes. The antibody-phage pre-incubation mixture was added to the blocked immunotube and incubated for 2 hrs and 30 minutes (min) at RT to capture the antibody-phage complexes. The nonbound antibodies and phage particles were removed by extensive washing, whereas the bound antibody-phage complexes were eluted by 100 mM triethylamine, pH 12.0 (Sigma-Aldrich) and neutralized using 1 M Tris-HCI, pH 7.4 (Sigma-Aldrich). Eluted phage particles were then amplified by infecting TG1 E. coli bacterial cells, which were grown overnight on 2xTY- ampicillin (100 pg/mL), 2% glucose agar plates. The resulting bacterial colonies were harvested and used as input for consecutive SAS selection rounds. After four positive SAS selection rounds, one round of counter selection with the HC SAS pool diluted in 2% MPBS, each plasma sample at a 1 :200 final dilution, was performed to prevent the isolation of common antibodies. After this counter selection, phage particles not bound by IgA antibodies in the HC SAS pool were amplified and characterized. In parallel, a second SAS screening was performed using largely the same approach, except that here two positive SAS selection rounds were followed by two alternating rounds of positive and counter selection, and each plasma sample of the early axSpA SAS plasma pool and HC SAS plasma pool was used at a 1 :2,000 final dilution.
From both SAS approaches, cDNA inserts of a random selection of phage particles were identified by sequencing their fusion to the M13 gene VI. The custom-made DNAnalyzer software, an Anaconda Python based multiprocessing program using Biopython, allowed automation of the comparison of resulting nucleotide and amino acid sequences to human and microbial sequences with the BLAST tool of NCBI. Phage ELISA
Antibody reactivity against peptides displayed on phage particles was determined by phage ELISA in pooled and individual plasma samples as described previously (Palmers et al., J Neuroinflammation, 2016, 13(1): 243), with some minor modifications.
In brief, half area 96 well Microlon high binding microplates (Greiner Bio-One) were coated respectively with 5 pg/mL anti-M13 monoclonal antibody (GE Healthcare) or 4.0 pg/mL anti-M13 mouse monoclonal antibody (clone MM05T, Sino Biological) diluted in coating buffer (0.2 M sodium carbonate bicarbonate buffer, pH 9.6) overnight at 4°C to determine the presence of UH-axSpA-lgG or UH-axSpA-lgA antibodies. Coated plates were blocked with 5% MPBS for 2 hrs at 37°C while shaking. The plates were then incubated with 7 x 1011 colony forming units (cfu)/mL of phage particles expressing the antigen of interest (specific phage) or phage particles without peptide (empty phage) for 1 hr at 37°C and 30 min at RT. Bound phage particles were incubated with pooled or individual plasma samples diluted 1 : 100 in 5% MPBS, followed by incubation with respectively anti-human IgG-Fc antibody coupled with horseradish peroxidase (Bethyl laboratories) diluted 1 :50,000 in 5% MPBS or with cross-absorbed goat anti-human IgA-Fc conjugated with horse radish peroxidase (Bethyl Laboratories) diluted 1 :2,500 in 5% MPBS for 1 hr at RT.
Antibody reactivity against each phage-displayed peptide is expressed by the ratio of the optical density (OD) signal of each phage-displayed peptide over the OD signal of the phage without peptide (OD(specific phage)/OD(empty phage)).
For each peptide target, a cut-off for seropositivity was calculated as the mean of the antibody reactivity in the non-reactive samples plus 3 x standard deviation. Samples with a mean ratio above this cut-off value were considered antibody-positive. Within each experiment, samples were tested in duplicate and experiments were performed independently for at least two times. Average values of experimental repeats had a coefficient of variation (% CV) lower than 20%.
Statistical analysis
Statistical analyses were performed using SAS JMP Pro version 14.2 and a P value of <0.05 was considered as statistically significant.
Antibody reactivity against individual phage displayed antigens or panels of antigens was compared between axSpA patients and controls using the Fisher’s exact test. Clinical characteristics were compared between antibody-positive and antibody-negative axSpA patients, in which continuous clinical characteristics were compared by Student’s t-tests and categorical characteristics were compared using Fisher’s exact tests.
For individual phage displayed antigens or a panel of antigens, also the positive likelihood ratio (LR+) was calculated based on the presence of antibody reactivity in axSpA patients compared to HC.
Results
Identification of novel IgA antibodies in early axSpA patients
Screening phage particles, expressing synovial antigens and randomly-formed peptides from the human axSpA cDNA phage display library, for antibody reactivity in the early axSpA SAS plasma pool, resulted in the identification of novel IgA antibodies targeting 173 different peptides. Of these 173 different phage- displayed peptides, 97 peptides resulted from the first SAS approach using the plasma pools at a 200x final dilution, whereas 76 peptides resulted from the second SAS approach using the plasma pools in a 2,000x final dilution.
Presence of antibodies against each of these 173 phage displayed peptides was first determined by phage ELISA in 6 additional plasma pools, each pool consisting of 10 early axSpA patients, and in 3 additional plasma pools, consisting of 10 HC each. As a result, IgA antibodies against against 7 of these 173 peptides were more frequently present in the 6 early axSpA plasma pools as compared to the 3 HC plasma pools. These 7 peptides were annotated UH-axSpA-lgA.1 till UH-axSpA-lgA.10 (University Hasselt-axSpA-lgA. isotype target number).
Identity of peptides targeted by novel IgA antibodies in axSpA patients
Nucleotide and amino acid sequences of the 7 selected UH-axSpA-lgA peptides were compared with human and microbial sequences using the custom DNAnalyzer program, which correspond to fragments of known proteins and novel linear peptides (see Table 4).
Peptide sequences displayed by UH-axSpA-lgA.1 to UH-axSpA-lgA.9 had a length between 10 and 54 amino acids and showed only partial homology to different human proteins as these peptides resulted from the out-of-frame cDNA translation or the translation of non-coding cDNA sequences. The DNA sequence of UH-axSpA-lgA.10 corresponded with an in-frame fusion to the 3’-coding region of the human Histone deacetylase 3 (HDAC3) gene. Our isolated HDAC3 variant showed 100% homology with the canonical HDAC3 sequence (NM_003883.3), resulting in the expression of a peptide with 100% homology to the last 22 amino acids of the C-terminal part of human HDAC3, whereas the total human HDAC3 protein has a size of 428 amino acids.
Moreover, the peptide sequences of phage-displayed peptides UH-axSpA-lgA.1 , UH-axSpA-lgA.3, UH- axSpA-lgA.6 to UH-axSpA-lgA.10 also showed partial homology to proteins from several microbial species, including Escherichia (E.) coli, Klebsiella (K.) pneumoniae, Saccharomyces (S.) cerevisiae and Yersinia (Y.) pestis (Table 4).
Table 4. Identity of 7 novel antigens targeted by IgA antibody responses in early axSpA patients
Antibody cDNA identity Fusion Peptide sequence of Size Homology on amino acid level (UniProt targets (NCBI Accession type3, in cDNA insert0 (aa)d Accession NO.)
No.) frame15
UH-axSpA- Phosphoribosyl mRNA, (A) G ETWPGAARR RQTT 19 Human proteins lgA.1 transferase domain coding, No GEAS* 8/10 (80%) Kinesin light chain 3, KLC3 (Q6P597) containing 1 9/13 (69%) Prothymosin alpha, PTMA (P06454)
(NM_001282786.1) 10/14 (71%) Capping protein, Arp2/3 and myosin-l linker protein 2, CARMIL2 (Q6F5E8)
Microbial proteins
11/23 (48%) DNA ligase B, ligB, Klebsiella (K.) pneumoniae (B5XTF0)
9/13 (69%) 60S ribosomal protein L37-A, RPL37A , Saccharomyces (S.) cerevisiae (P49166)
7/11 (64%) UPF0102 protein YraN, yra/V,
Salmonella enterica (A0A426WQ81)
UH-axSpA- 28S ribosomal N3 Ribosomal (A)GKANDQRSWGRNDL 53 Human proteins lgA.3 (NR_146154.1) RNA NLFSNFKWVRSPARWR 17/32 (53%) Coilin, COIL (P38432)
GAGRGMRVPSGPLLVS 17/27 (62%) Scaffold attachment factor B2, SAFB2
RTGAAG* (Q14151)
9/12 (75%) Enoyl-(acyl-carrier-protein) reductase mitochondrial, MECR (Q9BV79)
Microbial proteins
14/28 (50%) Hydrogenase-1 large chain, hyaB, E. coli (P0ACD8)
20/47 (43%) Multidrug resistance protein MdtA, mdtA, E. coli (B1LNW7)
9/14 (64%) Uncharacterized protein YciO, yciO, E. coli (P0AFR4)
UH-axSpA- Zinc finger CCCH- mRNA, (R)PAVADSGDGGKGDIT 26 Human proteins lgA.6 type containing 3 coding, No AADPPTAGSD* 14/29 (48%) Ryanodine receptor 1 , RYR1
(NM_015117.2) (P21817)
16/31 (51%) Uncharacterized protein C1orf167, C1orf167 (Q5SNV9)
12/24 (50%) 3-oxoacyl-acyl-carrier-protein synthase, OXSM ( Q9NWU1)
Microbial proteins
9/15 (60%) L-lactate dehydrogenase, IldD,
Yersinia (Y.) pestis , (A4TKI4)
11/25 (44%) Phthiocerol/phenolphthiocerol synthesis polyketide synthase type I PpsB, ppsB, Mycobacterium tuberculosis (Q7TXL9)
12/21 (57%) 30S ribosomal protein S3, rpsC, Mycobacterium sp. (A1 UBP2)
UH-axSpA- kinesin family mRNA, (T)RERDSDYE* 9 Human proteins lgA.7 member 2A 3’UTR 7/8 (87%) SH2B adapter protein 3, SH2B3
(N _004520.5) (Q9UQQ2)
6/6 (100%) Zinc finger protein 516, ZNF516 (Q92618)
6/7 (85%) Probable global transcription activator SNF2L2, SMARCA2 (P51531)
Microbial proteins
5/6 (83%) ATP-dependent RNA helicase MSS116, MSS116 , S. cerevisiae (P 15424)
6/8 (75%) E3 ubiquitin-protein ligase HEL2, HEL2 , S. cerevisiae (Q05580)
5/8 (63%) PH085 cyclin-5, PCL5 , S. cerevisiae (P38794)
UH-axSpA- Ubiquitin- Intron, nc (V)KHSLHEIFNTKPANGL 18 Human proteins lgA.8 conjugating enzyme S* 8/9 (88%) Multimerin-1 precursor, MMRN1
E2D 3 (UBE2D3) (Q13201) genome 6/6 (100%) N-lysine methyltransferase SETD6,
(AF213884S1) SETD6 (Q8TBK2)
9/14 (64%) Phosphatidylinositol 3,4,5- trisphosphate 5-phosphatase 1 , INPP5D (Q92835)
Microbial proteins
10/14 (71%) Ubiquitin carboxyl-terminal hydrolase 11 , RPN11, S. cerevisiae (P43588)
8/13 (62%) Putative tyrosine-protein kinase in cps region, NA, K. pneumoniae (Q48452)
9/21 (43%) Carboxylic acid reductase, car,
Mycobacterium marinum (B2FIN69)
UH-axSpA- RNA 28S ribosomal Ribosomal (G)KANDQRSWGRNDLN 52 Human proteins lgA.9 4 (NR_145822.1) RNA LFSNFKWVRSPARWRG 17/32 (53%) Coilin, COIL (P38432) AGRGMRVPSGPLLVSRT 17/27 (62%) Scaffold attachment factor B2, SAFB2 GAAG* (Q14151)
9/12 (75%) Enoyl-(acyl-carrier-protein) reductase mitochondrial, MECR (Q9BV79)
Microbial proteins
14/28 (50%) Hydrogenase-1 large chain, hyaB , E coli (P0ACD8)
20/47 (43%) Multidrug resistance protein MdtA, mdtA E. coli (B1LNW7)
9/14 (64%) Uncharacterized protein YciO, yciO , E coli (P0AFR4)
UFI-axSpA- Flistone deacetylase mRNA, (P)PEAPNEFYDGDFIDN 23 Human proteins
IgA.10 3 (NM_003883.3) coding, Yes DKESDVEI* 22/22 (100%) Histone deacetylase 3, HD AC 3
(015379)
12/17 (70%) ADP-ribose glycohydrolase
MACROD2, MACROD2 (A1Z1Q3)
10/15 (66%) Neuroendocrine convertase 1, PCSK1 (P29120)
Microbial proteins
10/14 (71%) PH085 cyclin-8, PCL8 , S. cerevisiae (Q08966)
9/3 (69%) Bud site selection protein 14, BUD14 , S. cerevisiae (P27637)
8/10 (80%) Uncharacterized protein YjgL, yjgL, E. coli (P39336) a Origin of the cDNA insert of the phage-displayed target b In-frame fusion of the cDNA coding region with the M13 gene VI: Yes/No. Translation of in-frame fusion results in expression of (part of) a human protein, whereas out-of-frame fusion results in a fusion construct with a random peptide sequence.
0 Peptide sequence of the translated cDNA insert, with the first amino acid between parenthesis representing the transition between the M13 phagemid vector and the cDNA insert. d Size of translated cDNA insert in amino acids
Q Amber stop codon, which is translated into glutamine by the bacterial strain * stop codon mRNA, messenger RNA; nc, non-coding; UTR, untranslated region
Identification of novel IgG antibodies in early axSpA patients
Screening phage particles, expressing protein and peptide antigens from the RA cDNA phage display library, for IgG antibody reactivity in the early axSpA SAS pool, resulted in the identification of novel antibodies directed against 12 novel UH-axSpA-lgG peptides (Table 5). Nine of the 12 selected peptide sequences (UH-axSpA-lgG.101 to UH-axSpA-lgG.103, UH-axSpA- lgG.106, UH-axSpA.lgG.107 and UH-axSpA-lgG.109 to UH-axSpA-lgG.112) resulted from out-of-frame cDNA translation, or translation of non-coding cDNA regions (e.g. 3’untranslated region (UTR)). As a result, these peptide sequences only showed partial homology to human proteins involved in several biological processes, including protein biosynthesis (Ribosome- releasing factor 2 (GFM2)), transcription regulation (SAGA-associated factor 29 (SGF29)), metabolic processes (Cysteine sulfinic acid decarboxylase (CSAD)), motility and muscle contraction (Titin (TTN)), transport (Bestrophin-1 (BEST1)), cell adhesion (Platelet glycoprotein 4 (CD36)), DNA regulation (DNA-dependent protein kinase catalytic subunit (PRKDC)) and to proteins with an yet unknown function (Coiled-coil domain- containing protein 171 (CCDC171)). The peptide sequences expressed by UH-axSpA-lgG.104, UH-axSpA-lgG.105 and UH-axSpA.lgG.108 encoded C-terminal portions of known human proteins: intraflagellar transport protein 43 (IFT43), target of Myb protein 1 (TOM1) and DNA polymerase subunit gamma-1 (POLG). Since the TAG stop codon of UH-axSpA.lgG.108 is an amber stop codon, and thus translated to glutamine (Q) in the TG1 E.coli bacteria, a 102 nucleotide stretch of the 3’UTR of POLG is also translated and expressed as the final 34 amino acids on this phage clone.
Table 5. Identity of 12 UH-AXSPA-IGG PEPTIDES targeted by novel antibodies in early axSpA patients
Figure imgf000056_0001
Figure imgf000057_0001
a Region of the cDNA insert fused to phage gene VI b In-frame fusion of the cDNA coding region with the M13 gene VI: Yes/ No. Translation of in-frame fusion results in expression of (part of) a human protein, whereas out-of-frame fusion results in a fusion construct with a random peptide sequence.
0 Peptide sequence of the translated cDNA insert, with the aa between parenthesis representing the transition between the M13 phage gene VI and the cDNA insert. d Size of translated cDNA insert in amino acids
Q Amber stop codon, which is translated into glutamine by the bacterial strain * Stop codon aa, amino acids; UTR, Untranslated region; mRNA, messenger RNA.
Antibody reactivity against 7 novel UH-axSpA-igA peptides
The presence of IgA antibodies against the 7 UH-axSpA-lgA peptides was determined in individual plasma samples of early axSpA patients (n= 79) and HC (n= 92) from the UH cohort. Antibody reactivity against individual UH-axSpA-lgA peptides was present in 3% (2/79) to 22% (17/79) of early axSpA patients and in 3% (3/92) to 21% (19/92) of HC (Table 4.). The highest antibody reactivity in the samples of the early axSpA patients was seen for UH-axSpA-lgA.8 (22%) and UH-axSpA-lgA.3 (13%). The lowest reactivity in the HC samples was seen for UH-axspA-lgA.10 (3%), UH-axSpA-lgA.1 (4%) and UH-axSpA-lgA.3 (5%), see Table 6.
Table 6. Antibody reactivity against individual peptide targets UH-axSpA-lgA.1 - 10 in early axSpA patients and HC from the UH cohort
Antibody targets Early axSpA HC
(n=79) (n=92)
UH-axSpA-lgA.1 5/79 (6%) 4/92 (4%)
UH-axSpA-lgA.3 10/79 (13%) 5/92 (5%)
UH-axSpA-lgA.6 2/79 (3%) 5/92 (5%)
UH-axSpA-lgA.7 6/79 (8%) 12/92 (13%)
UH-axSpA-lgA.8 17/79 (22%) 19/92 (21%)
UH-axSpA-lgA.9 7/79 (9%) 6/92 (7%)
UH-axSpA-lgA.10 6/79 (8%) 3/92 (3%)
Antibody reactivity against a panel of 3 UH-axSpA-igA peptides in early axSpA patients and HC from the UH cohort
We also investigated whether antibodies against a combination of specific UH-axSpA-lgA peptides could increase sensitivity in early axSpA patients from the UH cohort, while maintaining sufficient specificity. We combined the 3 UH-axSpA-lgA peptides whose antibody reactivity showed the highest LR+ into a panel: UH-axSpA-lgA.1 , UH-axSpA-lgA.3 and UH-axSpA-lgA.10. Antibody reactivity targeting this panel of UH-axSpA-lgA peptides was found in 27% (21/79) of early axSpA patients, and in only 12% (11/92) of HC (p=0.0185 ). As a result, this panel of 3 UH-axSpA-lgA peptides showed a specificity of 88%, and a LR+ of 2.2, see also Table 7.
Additionally, we explored whether antibody reactivity targeting this panel of 3 UH-axSpA-lgA peptides was linked to a certain subset of early axSpA patients. To this end, the clinical disease characteristics, were compared between early axSpA patients with (antibody positive) or without (antibody negative) IgA antibodies against this panel of peptides. Within this UH cohort, the antibody positive early axSpA patients did not show any significant difference in age, gender, HLA-B27 status, disease duration, treatment, Bath Ankylosing Spondylitis Disease Activity Index (BASDAI), Bath Ankylosing Spondylitis Functional Index (BASFI), erythrocyte sedimentation rate (ESR), CRP and presence of extra-articular manifestations, as compared to early axSpA patients lacking these antibodies (Table 8). Table 7. Antibody reactivity against UH-axSpA-lgA.1, UH-axSpA-lgA.3 and UH-axSpA-lgA.10 and a panel of these 3 UH-axSpA-lgA peptides in early axSpA patients and HC from the UH cohort
Figure imgf000059_0001
LR+, Positive Likelihood Ratio; Spec, Specificity; Sens, Sensitivity.
Table 8. Clinical characteristics of early axSpA patients antibody positive or antibody negative against a panel of 3 UH-axSpA-lgA peptides
Antibody positive Antibody
Olinical characteristics (n=2f) negative (n=55) P value
Age (mean, SD) 42.8 (13.1) 43.4 (12.6) 0.8576
Male ( n , %) 12/21 (57.1) 31/55 (56.4) 1.000
HLA-B27 positive ( n , %) 13/21 (61.9) 30/52 (57.7) 0.7977
Disease duration in years1 2.6 (1.4) 2.9 (1.3) 0.3981 (mean , SD)
No medication useb (n, %) 4/21 (19.0) 7/55 (12.7) 0.4841
NSAID use (n, %) 15/21 (71.4) 39/55 (70.9) 1.000 cDMARD use (n, %) 6/21 (28.6) 24/55 (43.64) 0.2977 bDMARD use (n, %) 5/21 (23.8) 13/55 (23.6) 1.000
BASDAI (mean, SD) 5.1 (1.9) 4.6 (2.2) 0.3750
BASFI (mean, SD) 5.0 (2.2) 4.0 (2.7) 0.1807
ESR, mm/h (mean, SD) 13.2 (12.2) 10.8 (16.2) 0.5198
CRP, mg/L (mean, SD) 6.8 (9.5) 5.6 (7.6) 0.6238
Extra-articular manifestations0 5 /21 (23.8) 10/55 (18.2) 0.7479
(n, %) a Disease duration, time between diagnosis and blood sampling b No medication use at the time of blood sampling 0 Including uveitis, inflammatory bowel disease and psoriasis
HLA, Human Leukocyte Antigen; NSAID, Non-Steroidal Anti-Inflammatory Drug; cDMARD, conventional Disease- Modifying Anti-Rheumatic Drug; bDMARD, biological Disease-Modifying Anti-Rheumatic Drug; BASDAI, Bath Ankylosing Spondylitis Disease Activity Index; BASFI, Bath Ankylosing Spondylitis Functional Index; ESR, Erythrocyte Sedimentation Rate; CRP, C-Reactive Protein Presence of antibodies against 12 novel UH-axSpA-igG peptides in axSpA patients and controls
Presence of antibodies against the 12 selected UH-axSpA-lgG peptides was determined in individual plasma samples of early axSpA patients, patients with RA, and HC from the UH cohort, using phage ELISA. Antibody reactivity against these individual UH-axSpA-lgG peptides was present in 1% (1/80) up to 21 % (17/80) of early axSpA patients, whereas presence of these antibodies ranged from 0% (0/46) to 15% (7/46) in HC and from 0% (0/46) to 20% (9/46) in RA patients (Table 9). The highest antibody reactivity in the samples of the early axSpA patients was seen for UH-axSpA-lgG.107 (21%) and UH- axSpA-lgG.112 (19%). The lowest reactivity in the HC samples was seen for UH-axspA-lgG.101 (0%), UH-axSpA-lgG.106 (0%) and UH-axSpA-lg.110 (0%).
Table 9. Antibody reactivity against individual UH-axSpA-lgG.101-112 peptide targets in early axSpA patients and controls from the UH cohort
Antibody targets Early axSpA HC RA
(n=80) (n=46) (n=46)
UH-axSpA-lgG.101 2/80 (3%) 0/46 (0%) 0/46 (0%) UH-axSpA-lgG.102 6/80 (8%) 1/46 (2%) 0/46 (0%) UH-axSpA-lgG.103 8/80 (10%) 3/46 (7%) 9/46 (20%) UH-axSpA-lgG.104 4/80 (5%) 2/46 (4%) 2/46 (4%) UH-axSpA-lgG.105 4/80 (5%) 2/46 (4%) 2/46 (4%) UH-axSpA-lgG.106 1/80 (1%) 0/46 (0%) 0/46 (0%) UH-axSpA-lgG.107 17/80 (21%) 7/46 (15%) 7/46 (15%) UH-axSpA-lgG.108 2/80 (3%) 3/46 (7%) 2/46 (4%) UH-axSpA-lgG.109 7/80 (9%) 1/46 (2%) 1/46 (2%) UH-axSpA-lgG.110 3/80 (4%) 0/46 (0%) 0/46 (0%) UH-axSpA-lgG.111 1/80 (1%) 0/46 (0%) 0/46 (0%) UH-axSpA-lgG.112 15/80 (19%) 4/46 (9%) 6/46 (13%)
Antibody reactivity against a panel of 4 UH-axSpA-lgG peptide targets in early axSpA patients and HC
From the 12 UH-axSpA.lgG peptides, we selected the 4 peptides whose antibody reactivity showed the highest LR+, and combined them into a panel: UH-axSpA-lgG.101 , UH-axSpA-lgG.102, UH-axSpA- lgG.109 and UH-axSpA-lgG.110. Antibodies against at least one of these 4 peptides were present in 21% (17/80) of early axSpA patients, compared to only 4% (2/46) of HC (p=0.010 ), resulting in a specificity of 96% and a LR+ of4.9 (Table 10). Antibodies against this panel of 4 UH-axSpA-lgG peptides were also able to discriminate axSpA patients from patients with RA, as they were present in only 2% (1/46) of RA patients (p=0.0028). Table 10. Antibody reactivity against individual UH-axSpA-lgG peptides and a panel of 4 peptides in early axSpA patients compared to HC
Figure imgf000061_0001
In addition, clinical and disease characteristics were compared between early axSpA patients that tested positive for antibodies against the panel of 4 peptides, UH-axSpA-lgG.101 , UH-axSpA-lgG.102, UH- axSpA-lgG.109 and UH-axSpA-lgG.110, and axSpA patients lacking these antibodies (antibody negative), see Table 11. Patients which were positive for antibodies against this peptide panel showed a trend towards a younger age (39.8 vs 45.4 years, p= 0.069), lower prevalence of males (23.5 vs 50.8%, p=0.057), increased NSAID use (60 vs 38%, p=0.153), increased cDMARD use (60 vs 35%, p=0.088), but showed a significantly decreased bDMARD use (0 vs 29%, p=0.017). In addition, no significant differences in HLA-B27 status, disease duration, Bath Ankylosing Spondylitis Disease Activity Index (BASDAI), Bath Ankylosing Spondylitis Functional Index (BASFI), erythrocyte sedimentation rate (ESR) and CRP could be detected between early axSpA patients with and without antibody reactivity against the panel of UH-axSpA-lgG.101/102/109/110 peptides.
Table 11. Comparison of clinical characteristics between early axSpA patients that were positive or negative for antibodies against the panel of 4 peptides targets UH-axSpA-lgG.101 , UH-axSpA- lgG.102, UH-axSpA-lgG.109 and UH-axSpA-lgG.110
Clinical characteristics Antibody Antibody P value positive negative (n=63) (n=17)
Age (mean, SD) 39.8 (10.31) 45.4 (12.7) 0.0688
Male (n, %) 4/17 (23.5) 32/63 (50.8) 0.0568
HLA-B27 positive (n, %) 6/15 (40.0) 29/58 (49.2) 0.5747
Disease duration in years3 2.9 (1.4) 2.8 (1.3) 0.8639 (mean , SD)
No medication useb ( n , %) 3/15 (20.0) 14/60 (23.3) 1.000
NSAID use ( n , %) 9/15 (60.0) 23/60 (38.3) 0.1531 cDMARD use (n, %) 9/15 (60.0) 21/60 (35) 0.0876 bDMARD use (n, %) 0/15 (0) 18/63 (28.6) 0.0167
BASDAI (mean, SD) 4.9 (2.5) 5.2 (2.2) 0.7196
BASFI (mean, SD) 4.6 (2.5) 4.5 (2.6) 0.9660
ESR, mm/h (mean, SD) 10.1 (9.8) 10.4 (15.0) 0.9289
CRP, mg/L (mean, SD) 6.05 (7.4) 4.9 (7.0) 0.5943
3 Disease duration, time between diagnosis and blood sampling b No medication use at the time of blood sampling
HLA-B27, Human Leukocyte Antigen-B27; NSAID, Non-Steroidal Anti-Inflammatory Drug; cDMARD, conventional Disease-Modifying Anti-Rheumatic Drug; bDMARD, biological Disease-Modifying Anti-Rheumatic Drug; BASDAI, Bath Ankylosing Spondylitis Disease Activity Index; BASFI, Bath Ankylosing Spondylitis Functional Index; ESR, Erythrocyte Sedimentation Rate; CRP, C-Reactive Protein.
Added value of testing for the panel of UH-axSpA-lgG.101/102/109/110 peptides and the panel of UH-axSpA-lgA.1/3/10 peptides
Our previous work already led to the identification of 3 peptides, UH-axSpA-lgG.1 , UH-axSpA-lgG.4 and UH-axSpA-lgG.8 , whose antibody reactivity was able to identify 14% of early axSpA patients (PCT/EP2020/076120). Next, we explored whether antibodies against the novel panels of UH-axSpA- lgG.101/102/109/110 peptides and UH-axSpA-lgA.1/3/10 peptides might provide a higher biomarker potential, by independently identifying additional axSpA patients, which were not identified by each of the other peptide panels.
To this end, we looked in axSpA patients samples that were tested for antibodies against multiple peptide panels, in order to investigate whether antibody seropositivity against each of these peptide panels would be independent from each other, in these shared patient samples. The percentage of seropositivity for antibodies against the UH-axSpA-lgG.101/102/109/110 panel was very similar in axSpA patients that were either seropositive (20%) or seronegative (19%) for antibodies against the UH- axSpA-lgG.1/4/8 panel (p=1.000), and also in axSpA patients that were either seropositive (17%) or seronegative (20%) for antibodies against the UH-axSpA-lgA.1/3/10 panel (p=1.000) (Table 12). Table 12. Presence of antibodies against the UH-axSpA-lgG.101/102/109/110 panel in shared early axSpA patients which are positive or negative for antibodies against the UH-axSpA- lgG.1/4/8 or the UH-axSpA-lgA.1/3/10 panels.
Figure imgf000063_0001
Similarly, seropositivity for antibodies against the UH-axSpA-lgA.1/3/10 panel was equally high in axSpA patients that were either seropositive (31%) or seronegative (26%) for antibodies against the UH- axSpA-lgG.1/4/8 panel (p=0.737), and also in in axSpA patients that were either seropositive (25%) or seronegative (30%) for antibodies against UH-axSpA-lgG.101/102/109/110 panel (p=1.000) (Table 13). These results strongly support the fact that antibody reactivity of these three different antibody biomarker panels is identifying totally independent parts of the axSpA patient population, and therefore, each panel has its own additional added value.
Table 13. Presence of antibodies against the UH-axSpA-lgA.1/3/10 panel in shared early axSpA patients which are positive or negative for antibodies against the UH-axSpA-lgG.1/4/8 or the UH- axSpA-lgG.101/102/109/110 panels.
Figure imgf000063_0002
Since the antibody reactivities against each of these 3 different peptide panels act independently, they can each contribute to the identification of different subsets of axSpA patients. From the HC cohorts tested for antibodies against the 3 different peptide panels, only 5 HC samples were shared over all panels. However, an expected overall specificity of 77% could be calculated from the antibody reactivities of each panel obtained with its HC cohort (8.5% for UH-axSpA-lgG.1/4/8, 4.3% for UH- axSpA-lgG.101/102/109/110 and 12% for UH-axSpA-lgA.1/3/10). On the other hand, 62 axSpA patients were shared for antibody testing against all 3 panels, allowing us to more accurately calculate the added value from each of these panels. Of the shared axSpA patients, 16% (10/62) were positive for our previously described antibodies against the UH-axSpA-lgG.1/4/8 peptides. Testing for antibodies against the 4 novel UH-axSpA-lgG.101/102/109/110 peptides, identifies an additional 19% (10/52) of the axSpA patients not detected by the UH-axSpA-lgG.1/4/8 panel, resulting in a positive antibody test for 32% (20/62) of early axSpA patients in total (Fig.1). Addition of testing for antibodies against the 3 novel UH-axSpA-lgA.1/3/10 peptides, identifies an additional 26% (11/42) of the axSpA patients not detected by the combined IgG peptide panels, resulting in a positive antibody test for 50% (31/62) of early axSpA patients in total, see also Figure 1 . Therefore, adding a test for antibodies against our novel UH-axSpA-lgG.101/102/109/110 and UH-axSpA-lgA.1/3/10 peptides, to our previous panel of UH- axSpA-lgG.1/4/8 peptides, increases the sensitivity from 16% to 50%, with a specificity of 77%, thus allowing to identify about half of early axSpA patients using this combination of 3 antibody biomarker panels.
Presence of antibodies to UH-asSpA-lgA.3 is indicative for therapy response
In a next step, we investigated whether the presence of antibodies to UH-axSpA-lgA.3 is indicative for therapy response using the phage ELISA. Therefore, we assessed changes in serology in a longitudinal setting and evaluated whether alterations in antibody status were related to clinical disease activity, indicated by the Bath Ankylosing Spondylitis Disease Activity Index (BASDAI). More precisely, antibody reactivity was determined after a 12-months’ follow-up period in samples from patients, who tested positive for IgA antibodies against UH-axSpA-lgA.3 (n=6) at baseline and they were treated with a biological or conventional Disease-Modifying Anti-Rheumatic Drug (DMARD) (TNFalpha antibody, TNF inhibitor or sulfasalazine).
For antibody reactivity towards UH-axSpA-lgGA3, a change from positive (above cut-off 1 .3) to negative status (below cut-off 1 .3) was observed for axSpA patients 2, 3, 4 and 5 (Fig. 2A) which was associated with a small decrease in disease activity, as indicated by the BASDAI score (Fig. 2B). Patient 2 and 5 were treated with a biological Disease-Modifying Anti-Rheumatic Drug (DMARD), whereas patient 3 and 4 were treated with a conventional DMARD.

Claims

1. An in vitro method for diagnosing spondyloarthritis in a subject, said method comprising: a) providing a biological sample from the subject, and b) determining the presence or quantity of
- antibodies against a peptide as shown in SEQ ID No: 2 or against an antibody-binding fragment or variant thereof,
- against a peptide as shown in SEQ ID NO: 1 or against an antibody-binding fragment or variant thereof, and
- against a peptide as shown in SEQ ID NO: 7 or against an antibody-binding fragment or variant thereof; wherein the presence of or an increase in antibody levels against at least one of said peptides or antibody-binding fragments or variants thereof in said biological sample compared to a reference sample is indicative for the presence of spondyloarthritis in the subject.
2. The in vitro method of claim 1 , wherein the presence or increase in antibody levels against the peptide as shown in SEQ ID NO: 2 or against an antibody-binding fragment or variant thereof in said biological sample compared to a reference sample is indicative for the presence of spondyloarthritis in the subject.
3. The in vitro method of claim 1 or 2, wherein the presence or increase in antibody levels
- against the peptide as shown in SEQ ID NO: 2 or against an antibody-binding fragment or variant thereof,
- against the peptide as shown in SEQ ID NO 1 or against an antibody-binding fragment or variant thereof, and
- against the peptide as shown in SEQ ID NO: 7 or against an antibody-binding fragment or variant thereof, in said sample compared to a reference sample is indicative for the presence of spondyloarthritis in the subject.
4. The in vitro method of any one of claims 1 to 3, further comprising determining the presence or quantity of antibodies against one or more peptides as shown in SEQ ID No: 3, SEQ ID No: 4, SEQ ID No: 5, and SEQ ID No: 6; or against an antibody-binding fragment or variant thereof, wherein the presence or increase in antibody levels against one or more of said peptides or against antibody-binding fragments or variants thereof in said biological sample compared to the reference sample is indicative for the presence of spondyloarthritis in the subject.
5. The in vitro method according to any one of claims 1 to 4, further comprising determining the presence or quantity of antibodies against one or more peptides as shown in SEQ ID No: 8, SEQ ID No: 9, SEQ ID No: 10, SEQ ID No: 11 , SEQ ID No: 12, SEQ ID No: 13, SEQ ID No: 14, SEQ ID No: 15, SEQ ID No: 16, SEQ ID No: 17, SEQ ID No: 18 and SEQ ID No: 19, or against an antibody-binding fragment or variant thereof, wherein the presence or increase in antibody levels against one or more of said peptides or against antibody-binding fragments or variants thereof in said biological sample compared to the reference sample is indicative for the presence of spondyloarthritis in the subject.
6. An in vitro method for evaluating the disease severity of spondyloarthritis in a subject, said method comprising: a) providing a biological sample from the subject, and b) determining the presence or quantity of antibodies against a peptide as peptide as shown in SEQ ID No: 2 or against an antibody-binding fragment or variant thereof, against; against a peptide as shown in SEQ ID NO: 1 or against an antibody-binding fragment or variant thereof, and against a peptide as shown in SEQ ID NO: 7 or against an antibody-binding fragment or variant thereof; wherein the presence or an increase in antibody levels against at least one of said peptides or antibodybinding fragments or variants thereof in said biological sample compared to a reference sample is indicative for the disease severity of spondyloarthritis in the subject.
7. The in vitro method of claim 6, wherein the presence or increase in antibody levels against the peptide as shown in SEQ ID NO: 2 or against an antibody-binding fragment or variant thereof in said biological sample compared to a reference sample is indicative for the disease severity of spondyloarthritis in the subject.
8. The in vitro method of claim 6 or 7, wherein the presence or increase in antibody levels against the peptide as shown in SEQ ID NO: 2 or against an antibody-binding fragment or variant thereof, against the peptide as shown in SEQ ID NO 1 or against an antibody-binding fragment or variant thereof, and against the peptide as shown in SEQ ID NO: 7 or against an antibody-binding fragment or variant thereof, in said sample compared to a reference sample is indicative for the disease severity of spondyloarthritis in the subject.
9. The in vitro method of any one of claims 6 to 8, further comprising determining the presence or quantity of antibodies against one or more peptides as shown in SEQ ID No: 3, SEQ ID No: 4, SEQ ID No: 5, and SEQ ID No: 6; or against an antibody-binding fragment or variant thereof, wherein the presence or increase in antibody levels against one or more of said peptides or against antibody-binding fragments or variants thereof in said biological sample compared to the reference sample is indicative for the disease severity of spondyloarthritis in the subject.
10. The in vitro method according to any one of claims 6 to 9, further comprising determining the presence or quantity of antibodies against one or more peptides as shown in SEQ ID No: 8, SEQ ID No: 9, SEQ ID No: 10, SEQ ID No: 11 , SEQ ID No: 12, SEQ ID No: 13, SEQ ID No: 14, SEQ ID No: 15, SEQ ID No: 16, SEQ ID No: 17, SEQ ID No: 18 and SEQ ID No: 19, or against an antibody-binding fragment or variant thereof, wherein the presence or increase in antibody levels against one or more of said peptides or against antibody-binding fragments or variants thereof in said biological sample compared to the reference sample is indicative forthe disease severity of spondyloarthritis in the subject.
11 . An in vitro method for evaluating a therapeutic treatment of spondyloarthritis in a subject, said method comprising: a) providing a first biological sample from the subject obtained before the start of the therapeutic treatment, b) providing a second biological sample from the subject obtained after the therapeutic treatment, and c) determining the presence or quantity of antibodies against a peptide as shown in SEQ ID No: 2 or against an antibody-binding fragment or variant thereof, against a peptide as shown in SEQ ID NO: 1 or against an antibody-binding fragment or variant thereof, and against a peptide as shown in SEQ ID NO: 7 or against an antibody-binding fragment or variant thereof in the first and second biological sample; wherein a deviation or no deviation in antibody levels against at least one of said peptides or antibodybinding fragments or variants thereof in the second biological sample compared to the first biological sample indicates that the subject is responding to the therapeutic treatment.
12. The in vitro method of claim 11 , wherein a deviation in antibody levels against the peptide as shown in SEQ ID NO: 2 or against an antibody-binding fragment or variant thereof in the second biological sample compared to the first biological sample indicates that the subject is responding to the therapeutic treatment.
13. The in vitro method of claim 11 or 12, wherein a deviation in antibody levels against the peptide as shown in SEQ ID NO: 2 or an antibody-binding fragment or variant thereof, against the peptide as shown in SEQ ID NO 1 or an antibody-binding fragment or variant thereof, and against the peptide as shown in SEQ ID NO: 7 or an antibody-binding fragment or variant thereof, in the second biological sample compared to the first biological sample indicates that the subject is responding to the therapeutic treatment.
14. The in vitro method of any one of claims 11 to 13, further comprising determining the presence or quantity of antibodies against one or more peptides as shown in SEQ ID No: 3, SEQ ID No: 4, SEQ ID No: 5, and SEQ ID No: 6; or against an antibody-binding fragment or variant thereof, in the first and second biological sample, wherein a deviation in antibody levels against one or more of said peptides or against antibody-binding fragments or variants thereof in the second biological sample compared to the first biological sample indicates that the subject is responding to the therapeutic treatment.
15. The in vitro method according to any one of claims 11 to 14, further comprising determining the presence or quantity of antibodies against one or more peptides as shown in SEQ ID No: 8, SEQ ID No: 9, SEQ ID No: 10, SEQ ID No: 11 , SEQ ID No: 12, SEQ ID No: 13, SEQ ID No: 14, SEQ ID No: 15, SEQ ID No: 16, SEQ ID No: 17, SEQ ID No: 18 and SEQ ID No: 19, or against an antibody-binding fragment or variant thereof, in the first and second biological sample, wherein a deviation in antibody levels against one or more of said peptides or against their antibody-binding fragments or variants thereof in the second biological sample compared to the first biological sample indicates that the subject is responding to the therapeutic treatment.
16. The in vitro method according to any one of the claims 1 to 15, wherein the antibody reactivity towards the antibody-binding fragment or variant of the peptide is comparable to the antibody reactivity towards the peptide itself.
17. The in vitro method according to any one of the claims 1 to 16, wherein the one or more antibodies are detected using a peptide selected from the group consisting of the peptide of any one of SEQ ID NOs: 1 to 19; preferably of any one of SEQ ID NOs: 1 , 2 or 7, or an antibody-binding fragment or variant thereof, and combinations thereof.
18. The in vitro method according to any one of the claims 1 to 16 wherein the one or more antibodies are detected using a detection agent comprising one or more peptides selected from the group consisting of the peptide of any one of SEQ ID NOs: 1 to 19; preferably of any one of SEQ ID NOs: 1 , 2 or 7, or an antibody-binding fragment or variant thereof, and combinations thereof; preferably wherein the detection agent is a polypeptide.
19. The in vitro method according to any one of claims 1 to 18, wherein the biological sample is a blood sample, a tissue sample or a body fluid sample.
20. The in vitro method according to any one of claims 1 to 19, wherein the biological sample is a blood sample, preferably a blood serum sample or a blood plasma sample.
21. The in vitro method according to any one of claims 1 to 20 wherein spondyloarthritis is axial spondyloarthritis or peripheral spondyloarthritis; preferably selected from ankylosing spondylitis, nonradiographic axial spondyloarthritis, undifferentiated spondyloarthritis, juvenile spondyloarthritis.
22. Use of the in vitro method according to any one of the preceding claims to tailor treatment to the patient’s individual needs or to evaluate the therapeutic treatment of spondyloarthritis in a patient suffering therefrom.
23. A peptide, antibody-binding fragment or variant selected from the group consisting of:
- the peptide of SEQ ID NO: 2, or an antibody-binding fragment or variant thereof;
- the peptide of SEQ ID NO: 1 , or an antibody-binding fragment or variant thereof; and
- the peptide of SEQ ID NO: 7, or an antibody-binding fragment or variant thereof; and
- combinations thereof.
24. A peptide of SEQ ID NO: 2 or an antibody-binding fragment or variant thereof.
25. A peptide as shown in SEQ ID NO: 2.
26. A detection agent comprising the peptide, antibody-binding fragment or variant thereof according to any one of claims 23 to 25.
27. A detection agent comprising the peptide, antibody-binding fragment or variant thereof according to claim 24 or 25.
28. A composition comprising one or more peptides, fragments or variants according to any one of claims 23 to 25 or one or more detection agents according to claim 26 or 27.
29. A composition comprising the peptide according to claim 24 or 25 or the detection agent according to claim 27.
30. The composition according to claim 29 further comprising at least one other peptide as shown in SEQ ID NO: 1 or SEQ ID NO: 7, or an antibody-binding fragment or variant thereof.
31 . The composition according to claim 29 or 30 further comprising at least one other peptide as shown in SEQ ID No: 3, SEQ ID No: 4, SEQ ID No: 5, and SEQ ID No: 6; or an antibody-binding fragment or variant thereof.
32. The composition according to any one of claims 29 to 31 , further comprising at least one other peptide as shown in SEQ ID No: 8, SEQ ID No: 9, SEQ ID No: 10, and SEQ ID No: 11 , SEQ ID No: 12, SEQ ID No: 13, SEQ ID No: 14, and SEQ ID No: 15, SEQ ID No: 16, SEQ ID No: 17, SEQ ID No: 18, or SEQ ID No: 19, or an antibody-binding fragment or variant thereof.
33. Use of the peptide, antibody-binding fragment or variant according to any one of claims 23 to 25, or of the detection agent according to claim 26 or 27, or of the composition according to any one of the claims 28 to 32, for detecting the presence or quantity of specific antibodies against said peptide, antibody-binding fragment or variant, against the detection agent, or against the peptide, antibodybinding fragment or variant or detection agent present in the composition, in a biological sample of the subject; preferably wherein the presence or increase in antibody levels as compared to a reference sample is indicative for the diagnosis of spondyloarthritis or for the disease severity of spondyloarthritis.
34. Use of the peptide, antibody-binding fragment or variant according to any one claims 23 to 25, or of the detection agent according to claim 26 or 27, or of the composition according to any one of the claims 28 to 32, for detecting the presence or quantity of specific antibodies against said peptide, antibodybinding fragment or variant, or against the detection agent, or against the peptide, antibody-binding fragment or variant or detection agent present in the composition, in a first and second biological sample of the subject wherein the first biological sample is obtained before the start of therapeutic treatment and the second biological sample is obtained after the start of therapeutic treatment; preferably wherein a deviation in antibody levels in the second biological sample as compared to the first biological sample is indicative for a response to the therapeutic treatment.
35. The peptide or antibody-binding fragment or variant according to any one of claims 23 to 25, or the detection agent according to claim 26 or 27, or the composition according to any one of claims 28 to 32, for use in the diagnosis of spondyloarthritis in the subject, or for evaluating the disease severity of spondyloarthritis in the subject or for evaluating the therapy response in a subject diagnosed with spondyloarthritis.
36. A diagnostic kit comprising one or more peptides or antibody-binding fragments or variants of any one of claims 23 to 25, or one or more detection agents according to claim 26 or 27, or a composition according to any one of claims 28 to 32, and reagents for detecting antibody binding to said one or more peptides, antibody-binding fragments or variants, or detection agents.
37. A diagnostic kit comprising the peptide or antibody-binding fragment or variant thereof of claim 24 or 25 or the detection agent of claim 27, or the composition according to claim 29, and reagents for detecting antibody binding to said peptide, antibody-binding fragment or variant thereof, to said detection agent.
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