WO2025012482A1

WO2025012482A1 - Method and means for prediction and evaluation of therapy response in rheumatoid arthritis

Info

Publication number: WO2025012482A1
Application number: PCT/EP2024/069997
Authority: WO
Inventors: Patrick Verschueren; Veerle Somers; Patrick VANDORMAEL
Original assignee: Katholieke Universiteit Leuven; Hasselt Universiteit
Current assignee: Katholieke Universiteit Leuven; Hasselt Universiteit
Priority date: 2023-07-13
Filing date: 2024-07-15
Publication date: 2025-01-16
Anticipated expiration: 2026-01-13

Abstract

The application discloses methods for predicting the response of a subject diagnosed with rheumatoid arthritis to treatment with an anti-rheumatic drug or for evaluating the response of subject diagnosed with rheumatoid arthritis to treatment with an anti-rheumatic drug. Said methods include detecting antibodies against one or more peptides, antibody-binding fragments, variants or immune reactive peptides thereof in a sample of the subject. Also provided are peptides, antibody-binding fragments, variants or immune reactive peptides, compositions comprising said peptides, antibody-binding fragments, variants or immune reactive peptides thereof and the use of peptides, antibody-binding fragments, variants or immune reactive peptides thereof and compositions.

Description

METHOD AND MEANS FOR PREDICTION AND EVALUATION OF THERAPY RESPONSE IN RHEUMATOID ARTHRITIS

FIELD OF THE INVENTION

The present invention generally relates to the field of antibody profiling in rheumatoid arthritis. More specific, the invention relates to methods and means for predicting and/or evaluating the response of a subject diagnosed with rheumatoid arthritis to treatment with an anti-rheumatic drug based on antibody profiling.

BACKGROUND OF THE INVENTION

Rheumatoid arthritis (RA) is an inflammatory autoimmune disease that affects multiple synovial joints and which, if left untreated leads to destruction of cartilage and the underlying bone. In the last two decades, a paradigm shift in RA disease management has been highly successful in driving back disease activity, resulting in reduced damage and disability for an increasing number of patients. This has been achieved by focusing on early recognition and diagnosis of patients, by the application of an early intensive treatment with the use of the treat-to-target strategy, aiming to reach remission or at least low disease activity (LDA), and the availability of many types of novel therapies and their use in treatment escalation.

In its 2019 update of recommendations for the management of RA, the European League Against Rheumatism (EULAR) endorsed the use of classical synthetic disease modifying drugs (csDMARDs), such as methotrexate (MTX), in combination with glucocorticoid (GC) bridging as a first-line treatment [Smolen et al. (2020) Ann Rheum Dis 79, 685-699], The Care in early RA trial (CareRA) has shown that such a combination therapy is effective at inducing DAS28CRP remission in about 70% of patients after two years and was found to be well tolerated and cost effective [Pazmino etal. (2020) Ann Rheum Dis 79, 556-565; Stouten etal. (2019) Rheumatol. 58, 2284- 2294; Verschueren et al. (2015) Ann Rheum Dis 74, 806-812], This strategy is also effective on the long term as more than half of patients did not have to intensify their DMARD treatment after 5 years [Stouten et al. (2021) Ann Rheum Dis 80, 965-973],

However, in case of insufficient response to this first-line treatment and in the presence of poor prognostic factors, the EULAR recommendations advise escalation to biological (b)DMARDs, which are directed against cytokine signalling, B or T lymphocytes, or to targeted synthetic (ts)DMARDs, which target Janus kinases.

If the applied therapy is not successful in effectively suppressing inflammation, patients experience a prolonged period of high disease activity, which leads to decreased functional capacity and progression of structural joint damage. It is generally recognized that there is an urgent need for novel biomarkers that allow immediate administration of the most appropriate drug to individual patients, which could in some cases require accelerated access to b/tsDMARDs.

SUMMARY

As corroborated in the experimental section, the inventors of the present application identified a novel tool to stratify rheumatoid arthritis (RA) patients in responders and non-responders to intensive conventional synthetic first-line therapy and predict their response before this therapy is initiated. More specific, in the present invention novel antibody biomarkers were identified that can identify RA patients that fail to reach remission or low disease activity after first-line combination therapy.

In an aspect of the invention, an in vitro method is provided for predicting the response of a subject diagnosed with rheumatoid arthritis to treatment with an anti-rheumatic drug. Said method comprises detecting in a biological sample of the subject one or more antibodies against one or more peptides selected from the group consisting of a peptide as shown in SEQ ID NO: 1 or an antibody-binding fragment or variant thereof, a peptide as shown in SEQ ID NO: 2 or an antibody-binding fragment or variant thereof, a peptide as shown in SEQ ID NO: 3 or an antibody-binding fragment or variant thereof, a peptide as shown in SEQ ID NO: 4 or an antibody-binding fragment or variant thereof, a peptide as shown in SEQ ID NO: 5 or an antibody-binding fragment or variant thereof, a peptide as shown in SEQ ID NO: 6 or an antibody-binding fragment or variant thereof, a peptide as shown in SEQ ID NO: 7 or an antibody-binding fragment or variant thereof, and a peptide as shown in SEQ ID NO: 8 or an antibody-binding fragment or variant thereof; preferably selected from the group consisting of a peptide as shown in SEQ ID NO: 1 or an antibody-binding fragment or variant thereof, a peptide as shown in SEQ ID NO: 2 or an antibody-binding fragment or variant thereof, and a peptide as shown in SEQ ID NO: 3 or an antibody-binding fragment or variant thereof.

A related aspect of the invention provides an in vitro method for evaluating the response of a subject diagnosed with rheumatoid arthritis to treatment with an anti-rheumatic drug. Said method comprises detecting in a biological sample of the subject one or more antibodies against one or more peptides selected from the group consisting of a peptide as shown in SEQ ID NO: 1 or an antibody-binding fragment or variant thereof, a peptide as shown in SEQ ID NO: 2 or an antibody-binding fragment or variant thereof, a peptide as shown in SEQ ID NO: 3 or an antibody-binding fragment or variant thereof, a peptide as shown in SEQ ID NO: 4 or an antibody-binding fragment or variant thereof, a peptide as shown in SEQ ID NO: 5 or an antibody-binding fragment or variant thereof, a peptide as shown in SEQ ID NO: 6 or an antibody-binding fragment or variant thereof, a peptide as shown in SEQ ID NO: 7 or an antibody-binding fragment or variant thereof, or a peptide as shown in SEQ ID NO: 8 or an antibody-binding fragment thereof; preferably selected from the group consisting of a peptide as shown in SEQ ID NO: 1 or an antibody-binding fragment or variant thereof, a peptide as shown in SEQ ID NO: 2 or an antibody-binding fragment or variant thereof, and a peptide as shown in SEQ ID NO: 3 or an antibody-binding fragment or variant thereof.

In some embodiments, the in vitro method comprises detecting in a biological sample of the subject antibodies against a peptide as shown in SEQ ID NO: 1 or an antibodybinding fragment or variant thereof, a peptide as shown in SEQ ID NO: 2 or an antibodybinding fragment or variant thereof, and a peptide as shown in SEQ ID NO: 3 or an antibody-binding fragment or variant thereof.

In some embodiments, the in vitro methods further comprise detecting in a biological sample of the subject antibodies against one or more peptides selected from the group consisting of: a peptide as shown in SEQ ID NO: 4 or an antibody-binding fragment or variant thereof; and a peptide as shown in SEQ ID NO: 5 or an antibody-binding fragment or variant thereof.

In some embodiments, the biological sample is a body fluid sample or a tissue sample, preferably the biological sample is a serum sample or a plasma sample.

A related aspect provides a peptide, an antibody-binding fragment or variant thereof that is selected from the group consisting of the peptide of SEQ ID NO: 1 or an antibodybinding 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, and the peptide of SEQ ID NO: 8 or an antibody-binding fragment or variant thereof.

In a related aspect, a peptide, antibody-binding fragment or variant thereof is provided that is selected from the group consisting of: 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. In another related aspect, a peptide, antibody-binding fragment or variant thereof is provided that is selected from the group consisting of: 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.

Also provided is a detection agent comprising one or more peptides, antibody-binding fragments or variants thereof as disclosed herein.

Further provided is a composition comprising one or more peptides, antibody-binding fragments or variants thereof or detection agents as disclosed herein.

In a related aspect, the peptides or antibody-binding fragments or variants thereof as disclosed herein or the detection agent or the composition as disclosed herein are for use in the prediction of the response of a subject diagnosed with rheumatoid arthritis to treatment with an anti-rheumatic drug.

In a related aspect, the peptides or antibody-binding fragments or variants thereof as disclosed herein or the detection agent or the composition as disclosed herein are for evaluating the response of a subject diagnosed with rheumatoid arthritis to treatment with an anti-rheumatic drug.

The present application also provides the use of a peptide or antibody-binding fragment or variant as disclosed herein, or a detection agent or a composition as disclosed herein for detecting the presence or quantity of specific antibodies to the peptide, antibodybinding fragment or variant thereof, or against the detection agent or against the peptide, antibody-binding fragment or variant thereof, or detection agent present in the composition, in a biological sample of a subject. In some embodiments, the detection or quantification of at least one specific antibody is indicative for a non-response or impaired response to treatment with an anti-rheumatic drug in a subject diagnosed with rheumatoid arthritis.

In a further related aspect, a diagnostic kit is provided comprising one or more peptides or antibody-binding fragments or variants thereof, one or more detection agents, or a composition as disclosed herein, and reagents for detecting antibody binding to said one or more peptides, antibody-binding fragments or variants or detection agents.

The above and further aspects and preferred embodiments of the invention are described in the following sections and in the appended claims. The subject-matter of appended claims is hereby specifically incorporated in this specification.

BRIEF DESCRIPTION OF DRAWINGS Figure 1. Competition ELISA to determine the antigen sequence recognised by anti-UH-RA.305/318/329 antibodies. Competition ELISA was performed using synthetic peptides to determine the antigen sequence recognized by anti-UH- RA.305/318/329 antibodies. For each antigen, two reactive serum samples were preincubated with increasing concentrations (0-30 pg/ml) of synthetic peptide (sequences in Table 3), and subsequently added to the respective antigen-expressing phage and to empty phage in a regular phage ELISA. Results are expressed as the ratio of antigenexpressing phage OD (optical density) over empty phage OD. Full length (FL) peptides had a minimal length of 10 amino acids. As UH-RA.305-FL and UH-RA.329-FL contained amino acids from the translated cloning adapter at their N-terminal side, epitope mapping using competition ELISA was used to investigate N-terminal truncation with one amino acid (-epi. A), with two amino acids (-epi.B), or to investigate the contribution of translated cloning adapter itself (-epi.CA). As a negative control, sample preincubation with a control peptide was carried out in parallel.

Figure 2. Competition ELISA using synthetic peptides to determine the minimal antigen sequence recognized by anti-UH-RA.314 antibodies. A reactive serum sample was pre-incubated with 30 pg/ml of synthetic peptide (sequences in Table 3), and subsequently added to UH-RA.314-expressing phage and to empty phage in a regular phage ELISA. Results are expressed as the ratio of antigen-expressing phage OD over empty phage OD. Full length (FL) peptides had a minimal length of 10 amino acids. As UH-RA.314-FL contained amino acids from the translated cloning adapter at its N-terminal side, epitope mapping using competition ELISA was used to investigate stepwise N-terminal truncation with between one (-epi. A) and six amino acids (-epi.F), or to investigate the contribution of translated cloning adapter itself (-epi.CA). As a negative control, sample pre-incubation with a control peptide was carried out in parallel.

Figure 3. Competition ELISA using synthetic peptides to determine the antigen sequence recognized by anti-UH-RA.107/ 108 antibodies. For each antigen, a reactive serum sample was pre-incubated with increasing concentrations (0-30 pg/ml) of synthetic peptide (sequences in Table 3), and subsequently added to the respective antigen-expressing phage in a regular phage ELISA. Results are expressed as the antigen-expressing phage OD signal. Full length (FL) peptides had a minimal length of 12 amino acids. As a negative control, sample pre-incubation with a control peptide was carried out in parallel. DESCRIPTION OF EMBODIMENTS

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 of the invention, the inventors identified a novel tool to stratify rheumatoid arthritis (RA) patients in responders and non-responders to intensive conventional synthetic first-line therapy and to even predict their response before this therapy is initiated. More specific, in the present invention novel antibody biomarkers that can identify RA patients that fail to reach remission or low disease activity after first-line RA therapy were identified. Identification of the antibody biomarkers was performed by screening of cDNA phage display libraries expressing RA synovial antigens for antibody reactivity in baseline sera from participants of a clinical trial failing to reach early remission (rem-) after intensive therapy with classical synthetic disease modifying drugs (csDMARDs) that are used as a standard care in the treatment of RA. Identified antibody biomarkers were further validated using ELISA, and correlations with remission or low disease activity over a two-year follow-up period were determined, according to the Disease Activity Score (DAS) based on 28 joints together with the level of C-reactive protein (DAS28CRP) or together with the erythrocyte sedimentation rate (DAS28ESR), and the clinical/simplified disease activity index (CDAI/SDAI).

Accordingly, an aspect relates to an in vitro method for predicting the response of a subject diagnosed with rheumatoid arthritis to treatment with an anti-rheumatic drug, wherein the method comprises detecting in a biological sample of the subject one or more antibodies against one or more peptides or against one or more antibody-binding fragments or variants of said one or more peptides. More specific, the antibody reactivity towards said antibody-binding fragments or variants is comparable to the antibody reactivity towards the corresponding peptide itself.

In a related aspect, an in vitro method for evaluating the response of a subject diagnosed with rheumatoid arthritis to treatment with an anti-rheumatic drug is provided. Said method comprises detecting in a biological sample of the subject one or more antibodies against one or more peptides or against one or more antibody-binding fragments or variants of said one or more peptides. More specific, the antibody reactivity towards said antibody-binding fragments or variants of the peptides is comparable to the antibody reactivity towards the peptide itself.

In certain embodiments, the methods and uses as taught herein comprise determining in a biological sample of a subject the presence or quantity of one or more antibodies against one or more peptides selected from the group consisting of a peptide as shown in SEQ ID NO: 1 or an antibody-binding fragment or variant thereof, a peptide as shown in SEQ ID NO: 2 or an antibody-binding fragment or variant thereof, a peptide as shown in SEQ ID NO: 3 or an antibody-binding fragment or variant thereof, a peptide as shown in SEQ ID NO: 4 or an antibody-binding fragment or variant thereof, a peptide as shown in SEQ ID NO: 5 or an antibody-binding fragment or variant thereof, a peptide as shown in SEQ ID NO: 6 or an antibody-binding fragment or variant thereof, a peptide as shown in SEQ ID NO: 7 or an antibody-binding fragment or variant thereof, and a peptide as shown in SEQ ID NO: 8 or 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.

In some embodiments, the methods and uses as taught herein comprise determining in a biological sample of a subject the presence or quantity of one or more antibodies against one or more peptides selected from the group consisting of a peptide as shown in SEQ ID NO: 1 or an antibody-binding fragment or variant thereof, a peptide as shown in SEQ ID NO: 2 or an antibody-binding fragment or variant thereof, a peptide as shown in SEQ ID NO: 3 or an antibody-binding fragment or variant thereof, a peptide as shown in SEQ ID NO: 4 or an antibody-binding fragment or variant thereof and a peptide as shown in SEQ ID NO: 5 or an antibody-binding fragment or variant thereof.

In some embodiments, the methods or uses as taught herein comprise determining in a biological sample of a subject the presence or quantity of one or more antibodies against one or more peptides selected from the group consisting of a peptide as shown in SEQ ID NO: 1 or an antibody-binding fragment or variant thereof, a peptide as shown in SEQ ID NO: 2 or an antibody-binding fragment or variant thereof and a peptide as shown in SEQ ID NO: 3 or an antibody-binding fragment or variant thereof.

In certain embodiments, the methods or uses as disclosed herein comprise determining the presence or quantity of antibodies against a peptide as shown in SEQ ID NO: 1, or against an antibody-binding fragment or variant thereof. In certain embodiments, the methods or uses as disclosed herein comprise 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. In certain embodiments, the methods or uses as disclosed herein comprise determining the presence or quantity of antibodies against a peptide as shown in SEQ ID NO: 3, or against an antibody-binding fragment or variant thereof. In certain embodiments, the methods or uses as disclosed herein comprise determining the presence or quantity of antibodies against a peptide as shown in SEQ ID NO: 4, or against an antibody-binding fragment or variant thereof. In certain embodiments, the methods or uses as disclosed herein comprise determining the presence or quantity of antibodies against a peptide as shown in SEQ ID NO: 5, or against an antibody-binding fragment or variant thereof. In certain embodiments, the methods or uses as disclosed herein comprise determining the presence or quantity of antibodies against a peptide as shown in SEQ ID NO: 6, or against an antibody-binding fragment or variant thereof. In certain embodiments, the methods or uses as disclosed herein comprise determining the presence or quantity of antibodies against a peptide as shown in SEQ ID NO: 7, or against an antibody-binding fragment or variant thereof. In certain embodiments, the methods or uses as disclosed herein comprise determining the presence or quantity of antibodies against a peptide as shown in SEQ ID NO: 8, or against an antibody-binding fragment or variant thereof.

In some embodiments, the methods or uses as disclosed herein comprise determining the presence or quantity of antibodies against a peptide as shown in SEQ ID NO: 1 or an antibody-binding fragment or variant thereof, a peptide as shown in SEQ ID NO: 2 or an antibody-binding fragment or variant thereof, and a peptide as shown in SEQ ID NO: 3 or an antibody-binding fragment or variant thereof.

In certain embodiments, the methods or uses as disclosed herein may further comprise determining the presence or quantity of antibodies against one or more peptides selected from the group consisting of a peptide as shown in SEQ ID NO: 4 or an antibody-binding fragment or variant thereof, and a peptide as shown in SEQ ID NO: 5 or an antibody-binding fragment or variant thereof. For example, the presence or quantity of antibodies 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: 4 and/or SEQ ID NO: 5 or an antibody-binding fragment or variant thereof can be determined. Or, the presence or quantity of antibodies against peptides as shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3 or an antibody-binding fragment or variant thereof and against a peptide as shown in SEQ ID NO: 4 and/or SEQ ID NO: 5 or an antibody-binding fragment or variant thereof can be determined.

In certain embodiments, the methods and uses as disclosed herein may further comprise determining the presence or quantity of antibodies against one or more peptides selected from the group consisting of a peptide as shown in SEQ ID NO: 6 or an antibody-binding fragment or variant thereof, a peptide as shown in SEQ ID NO: 7 or an antibody-binding fragment or variant thereof, and a peptide as shown in SEQ ID NO: 8 or an antibody-binding fragment or variant thereof. For example, the presence or quantity of antibodies against a peptide as shown in SEQ ID NO: 1 or an antibodybinding fragment or variant thereof and against a peptide as shown in SEQ ID NO: 6 and/or SEQ ID NO: 7 and/or SEQ ID NO: 8 or an antibody-binding fragment or variant thereof can be determined. Or, the presence or quantity of antibodies against peptides as shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3 or an antibody-binding fragment or variant thereof and against a peptide as shown in SEQ ID NO: 6 and/or SEQ ID NO: 7 and/or SEQ ID NO: 8 or an antibody-binding fragment or variant thereof can be determined.

In some embodiments, the methods and uses as disclosed herein are characterized in that the one or more antibodies are detected using a peptide 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: 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, and the peptide of SEQ ID NO: 8 or an antibody-binding fragment or variant thereof, or combinations thereof.

In some embodiments, the methods and uses as disclosed herein are characterized in that the one or more antibodies are detected using a peptide 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, or combinations thereof.

In some embodiments, the methods and uses as disclosed herein are characterized in that the one or more antibodies are detected using a peptide 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, or combinations thereof.

For example, antibodies are detected using a peptide of SEQ ID NO: 1 or an antibodybinding fragment or variant thereof. Or, antibodies are detected using a peptide of SEQ ID NO: 2 or an antibody-binding fragment or variant thereof. Or, antibodies are detected using a peptide of SEQ ID NO: 3 or an antibody-binding fragment or variant thereof. In some instances, antibodies are detected using a peptide of SEQ ID NO: 1, a peptide of SEQ ID NO: 2 and a peptide of SEQ ID NO: 3, or any antibody-binding fragment or variant of said peptides.

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 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: 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, and the peptide of SEQ ID NO: 8 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 comprising 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, and the peptide of SEQ ID NO: 5 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 comprising 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, and the peptide of SEQ ID NO: 3 or an antibody-binding fragment or variant thereof, or combinations thereof.

For example, antibodies are detected using a detection agent comprising a peptide of SEQ ID NO: 1 or an antibody-binding fragment or variant thereof. Or, antibodies are detected using a detection agent comprising a peptide of SEQ ID NO: 2 or an antibodybinding fragment or variant thereof. Or, antibodies are detected using a detection agent comprising a peptide of SEQ ID NO: 3 or an antibody-binding fragment or variant thereof. In some embodiments, antibodies are detected using a detection agent comprising a peptide of SEQ ID NO: 1 or an antibody-binding fragment or variant thereof, a detection agent comprising a peptide of SEQ ID NO: 2 or an antibody-binding fragment or variant thereof and a detection agent comprising a peptide of SEQ ID NO: 3, or any antibody-binding fragment or variant thereof.

In some embodiments, the detection agent may comprise 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.

Thus, in certain embodiments antibodies are detected against one or more peptides of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3 or against antibody-binding fragments or variants of each of said peptides, and against one or more peptides comprising a sequence of SEQ ID NO: 4 or SEQ ID NO: 5 or against antibody-binding fragments or variants of said peptides. For example, antibodies are detected against a peptide comprising a sequence of SEQ ID NO: 1 or against an antibody-binding fragment or variant thereof, whether or not in combination with the detection of antibodies against one or more peptides comprising a sequence of SEQ ID NO: 4 or SEQ ID NO: 5, or against antibody-binding fragments or variants thereof. Or, antibodies are detected against a peptide comprising a sequence of SEQ ID NO: 1, a peptide comprising a sequence of SEQ ID NO: 2, and a peptide comprising a sequence of SEQ ID NO: 3 or against antibody-binding fragments or variants of said peptides, and, optionally, a peptide comprising a sequence of SEQ ID NO: 4, or against an antibody-binding fragment or variant of said peptide. Or, antibodies are detected against a peptide comprising a sequence of SEQ ID NO: 1 or against an antibody-binding fragment or variant thereof, a peptide comprising a sequence of SEQ ID NO: 2 or against an antibody-binding fragment or variant thereof, and a peptide comprising a sequence of SEQ ID NO: 3 or against an antibody-binding fragment or variant thereof, and, optionally, against a peptide comprising a sequence of SEQ ID NO: 5 or against an antibody-binding fragment or variant of said peptide. Or, antibodies are detected against a peptide comprising a sequence of SEQ ID NO: 1 or an antibody-binding fragment or variant thereof, a peptide comprising a sequence of SEQ ID NO: 2 or an antibody-binding fragment or variant thereof, and a peptide comprising a sequence of SEQ ID NO: 3 or an antibody-binding fragment or variant thereof, and, optionally, a peptide comprising a sequence of SEQ ID NO: 4 and a peptide comprising a sequence of SEQ ID NO: 5, or against antibody-binding fragments or variants of said peptides.

In some embodiments antibodies can be detected against one or more peptides comprising a sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3 or against antibody-binding fragments or variants of said peptides, and against one or more peptides comprising a sequence of SEQ ID NO: 4 or SEQ ID NO: 5 or against antibody-binding fragments or variants of said peptides, and against one or more peptides comprising a sequence of SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO: 8, or against antibody-binding fragments or variants of said peptides.

In a further aspect, 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, and the peptide of SEQ ID NO: 8.

In another aspect, 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, and the peptide of SEQ ID NO: 5.

In related aspect, 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: 3.

Also provided are antibody-binding fragments or antibody-binding variants of the peptides as taught herein.

In some embodiments, a peptide is provided that comprises a combination of sequences that are selected from 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 and SEQ ID NO: 8. In some embodiments, a peptide is provided that comprises a combination of sequences that are selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5. In some embodiments, a peptide is provided that comprises a combination of sequences that are selected from SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3. For example, a peptide is provided that comprises the sequences of SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3.

Thus in some embodiments, a peptide comprising or consisting essentially of SEQ ID NO: 1 is provided, or an antibody-binding fragment or variant thereof. In some embodiments, a peptide comprising or consisting essentially of SEQ ID NO: 2 is provided, or an antibody-binding fragment or variant thereof. In some embodiments, a peptide comprising or consisting essentially of SEQ ID NO: 3 is provided, or an antibodybinding fragment or variant thereof. In some embodiments, a peptide comprising or consisting essentially of SEQ ID NO: 4 is provided, or an antibody-binding fragment or variant thereof. In some embodiments, a polypeptide comprising or consisting essentially of SEQ ID NO: 5 is provided, or an antibody-binding fragment or variant thereof. In some embodiments, a peptide comprising or consisting essentially of SEQ ID NO: 6 is provided, or an antibody-binding fragment or variant thereof. In some embodiments, a peptide comprising or consisting essentially of SEQ ID NO: 7 is provided, or an antibody-binding fragment or variant thereof. In some embodiments, a polypeptide comprising or consisting essentially of SEQ ID NO: 8 is provided, or an antibody-binding fragment or variant thereof.

In certain embodiments, a detection agent, preferably a polypeptide, is provided that comprises one or more peptides, antibody-binding fragments or variants as taught herein. In some embodiments, the polypeptide is a composite polypeptide that comprises the amino acid sequence of two or more of the peptides that are disclosed herein. For example, such 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: 1, SEQ ID NO: 2 and SEQ ID NO: 3.

In a related aspect, a composition comprising one or more of the peptides or antibodybinding 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: 1 or antibody-binding fragment or variant thereof is provided. In further embodiments, said composition further comprises at least one other peptide comprising or consisting essentially of a sequence represented by any one of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5, or an antibodybinding fragment or variant thereof. In certain embodiments, the composition as disclosed herein comprises a peptide comprising or consisting essentially of the sequence of SEQ ID NO: 1, a peptide comprising or consisting essentially of the sequence of SEQ ID NO: 2, and a peptide comprising or consisting essentially of the sequence of SEQ ID NO: 3, or one or more antibody-binding fragments or variants thereof In certain embodiments, the composition as disclosed herein comprises a peptide comprising or consisting essentially of the sequence of SEQ ID NO: 1, a peptide comprising or consisting essentially of the sequence of SEQ ID NO: 2, a peptide comprising or consisting essentially of the sequence of SEQ ID NO: 3, a peptide comprising or consisting essentially of the sequence of SEQ ID NO: 4, and a peptide comprising or consisting essentially of the sequence of SEQ ID NO: 5, or one or more antibody-binding fragments or variants thereof.

In an aspect of the invention, the peptides or the antibody-binding fragments or variant thereof, the detection agents or the compositions as taught herein are for use in the prediction of the response of a subject diagnosed with rheumatoid arthritis to treatment with an anti-rheumatic drug.

In a related aspect of the invention, the peptides or the antibody-binding fragments or variants thereof, the detection agents or the compositions as taught herein are for evaluating the response of a subject diagnosed with rheumatoid arthritis to treatment with an anti-rheumatic drug.

In a further related aspect, the application provides the use of one or more peptides or antibody-binding fragments thereof as taught herein, or the use of one or more detection agents 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, against the detection agent, or against the peptide, antibodybinding fragment or variant or detection agent present in the composition, in a biological sample of a subject diagnosed with rheumatoid arthritis. In a preferred embodiment, the detection or quantification of at least one specific antibody is indicative for a nonresponse or an impaired response to treatment with an anti-rheumatic drug in a subject diagnosed with rheumatoid arthritis. In particular embodiments, said use of a peptide, antibody-binding fragment or variant thereof or composition is an "in vitro" use of said peptide, antibody-binding fragment or variant thereof or composition. The latter implies a diagnostic method with no direct interaction with the patient.

In another aspect, the application provides an antibody that specifically binds to or reacts with a peptide or antibody-binding fragment or variant thereof as described herein.

With the present invention, antibodies are detected against one or more peptides as disclosed herein or against their antibody-binding fragments or variant 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 the peptides show some homology to a number of human proteins that are expressed in human synovial tissue of rheumatoid arthritis patients, the peptides as 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.

In certain embodiments, the antibodies are IgA, IgG and/or IgM antibodies. In certain embodiments, the antibodies are autoantibodies, in particular IgA, IgG and/or IgM 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, which is in an infectious organism. 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 ceases to recognise 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 antibody-binding fragments or antibody-binding variants of the peptides of SEQ ID NOs 1 to 8. As used herein, an antibody-binding fragment or an antibody-binding 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 NOs: 1 to 8 that shows 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 immune reactive peptides of the corresponding peptide of SEQ ID Nos: 1 to 8. Immune reactive peptides of a particular peptide are thus to be understood as any peptide that show 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, antibody-binding variants or immune reactive peptides are peptides that can be recognized by the same antibodies that recognize their corresponding peptides of SEQ ID Nos: 1 to 8 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 can be a fragment of their corresponding peptide as defined in SEQ ID NOs 1 to 8. In such embodiments, the immune reactive peptides are then referred to as antibody-binding fragments of the corresponding peptide. The immune reactive peptide or antibody-binding fragment can thus be a fragment of its corresponding peptide, such as for example a fragment 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 immune reactive peptides, such as some fragments of the peptides, for example as displayed in Table 3, are both immune reactive with the same antibodies (see Figure 2).

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 is 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 antibodybinding 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 8 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 8. 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 8 and thus these antibodybinding 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 8. 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 nonlimiting 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 antibody-binding 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 or detection agents 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 non- specifically bound antibodies. The immune complexes may then be reacted with an appropriate enzyme-labelled anti-immunoglobulin. After an optional further wash, the enzyme substrate may be added. The enzyme linked to the anti-immunoglobulin catalyses 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 the peptides, their antibody-binding fragments, variants or immune reactive peptides, are selected as such that no or only a marginal antibody reactivity is found in reference samples from patients having rheumatoid arthritis that do show a response to an anti-rheumatic drug, in contrast to a higher antibody reactivity in patients having rheumatoid arthritis that fail to show a response to this anti-rheumatic drug. A predetermined cut-off for antibody positivity can be determined based on non-reactive samples from healthy individuals or from patients diagnosed with rheumatoid arthritis but showing no reactivity. In a particular embodiment, antibody reactivity for a sample is determined by calculating the ratio of the optical density (OD) of the specific signal for a sample to the OD of the background signal for that sample. For example, for each peptide target, a cut-off for seropositivity can be calculated as five times the standard deviation (SD) above the mean ratio (AVG + 5SD) of all non-reactive samples, represented by the lowest subgroup from a changepoint analysis of all rheumatoid arthritis and healthy control samples. A sample is considered positive when its ratio (OD (specific)/OD (background)) is higher than this cut-off. In particular the higher the antibody reactivity (OD (specific)/OD (background)) for the peptides of the present invention, in a particular patient, the more likely it is that a non-response or poor response to an anti-rheumatic drug will be observed.

In certain embodiments, antibodies are provided that specifically bind to peptides or their antibody-binding 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 therapy response prediction in rheumatoid arthritis 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 rheumatoid arthritis 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 therapy response prediction in rheumatoid arthritis 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. (2020) Arthritis Rheumatology 72, 2094- 2105, 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 26R.P66), 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. IR.L 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, 2^nd Edition, Pierce Chemical Company, 1984; and Atherton and Sheppard, Solid Phase Peptide Synthesis, IR.L 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: 1-8, 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 RA 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 etal. (1979) 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 GIu 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 adds 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.

In an aspect of the invention, a diagnostic kit is provided for performing one of the in vitro methods according to the present invention. In certain embodiments, said kit comprises one or more peptides as taught herein, or one or more immune reactive peptides thereof as taught herein, 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 or immune reactive peptides thereof. 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 peptide is not labelled.

The present invention thus provides methods, polypeptides, or compositions to predict the response to therapy in a subject diagnosed with rheumatoid arthritis. Rheumatoid arthritis (RA) is a chronic inflammatory systemic autoimmune disease of unknown aetiology. It is characterized by symmetrical synovitis leading to cartilage damage and joint destruction if left untreated and it may be complicated by many extra-articular symptoms. RA is considered as an autoimmune disease, given the presence of autoantibodies. The diagnosis of RA can be made in a patient with inflammatory arthritis based on the number and distribution of involved joints (typically a symmetric in five or more small joints), the presence of rheumatoid factor (RF) and/or anti-citrullinated peptide/protein antibody (APCA), disease duration of more than six weeks, and elevated C-reactive protein (CRP) levels or erythrocyte sedimentation rate (ESR), in the absence of evidence of other diseases with similar clinical features. RA may also be diagnosed in patients without all of the classic findings of the disease as summarized in the European Alliance of Associations for Rheumatology (EULAR) and American College of Rheumatology (ACR) classification for RA, developed in 2010. This includes for instance patients with seronegative RA, those with clinically quiescent disease, and those with recent onset of RA. Such patients can be diagnosed by a rheumatologist based on findings or clinical features that are generally consistent with those described in the 2010 EULAR/ ACR classification criteria, even if they achieve a score which is insufficient (<6) for formal classification as RA.

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 non-human animals, preferably warm-blooded animals, even more preferably mammals, such as, e.g. non-human 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 non-mammals 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 diagnosed with rheumatoid arthritis. A diagnosis of rheumatoid arthritis can be made on the basis of typical clinical signs and symptoms known in the art, such as summarized in the EULAR/ACR classification criteria for RA as disclosed herein above.

In some embodiments, the subject is suspected to have RA.

In certain embodiments, the subject did not yet receive any treatment with an antirheumatic drug. In certain embodiments, the subject did not receive any treatment with an anti-rheumatic drug for a period of at least 3 months, preferably at least 6 months. In some embodiments, the subject is diagnosed with RA and is selected to receive treatment with an anti-rheumatic drug, such as for example a classical synthetic disease-modifying anti-rheumatic drug (csDMARD), a biological disease-modifying antirheumatic drug (bDMARD), or a targeted synthetic disease-modifying anti-rheumatic drug (tsDMARD), or combinations thereof. In preferred embodiments, the subject is diagnosed with RA and is selected to receive treatment with a csDMARD or combinations of csDMARDs. In some embodiments, the subject is diagnosed with RA and is selected to receive treatment with a csDMARD or combinations of csDMARDs in combination with glucocorticoids.

In certain embodiments, the antibody levels against the peptide(s) or against antibodybinding fragments, variants or immune reactive peptide(s) thereof as disclosed herein are determined in a biological sample of the subject and are compared to a reference value. Said reference value is determined based on the antibody levels against the peptide(s) or against antibody-binding fragments, variants or immune reactive peptide(s) thereof in one or more reference samples, and the presence or an increase in antibody levels in the biological sample as compared to the reference value is indicative for a non-response or an impaired response to treatment with an antirheumatic drug in the subject. As evidenced by the experimental data enclosed herein, the inventors found that the presence or increase in the level of one or more antibodies in a patient diagnosed with RA is indicative for a non-response or an impaired or poor response to treatment with an anti-rheumatic drug. Based on the detected levels of one or more antibodies, it can thus be predicted whether a subject will not or only poorly respond to treatment with an anti-rheumatic drug. In certain embodiments, the methods and uses as disclosed herein can thus be used to decide to start the treatment with a particular anti-rheumatic drug or combination of anti-rheumatic drugs or not.

In certain embodiments, the antibody level of the subject diagnosed with RA is thus compared to a reference value. The reference value can be determined based on antibody levels in a single reference sample, or based on antibody levels in a combination of multiple reference samples. In some embodiments, the reference sample is a biological sample obtained from a healthy subject, i.e. a subject that is not diagnosed with RA. In some embodiments, the reference sample is obtained from a patient having rheumatoid arthritis but wherein no or only a marginal antibody reactivity is found. Thus in some embodiments, the reference value is determined based on the antibody levels observed in one or more reference samples from healthy individuals. In some embodiments, the reference value is determined based on the antibody levels observed in one or more reference samples from individuals that are diagnosed with RA but wherein no or only a marginal antibody reactivity is found and that show a response to an anti-rheumatic drug. The reference value or pre-determined cut-off for antibody positivity can thus be determined based on non-reactive samples from healthy individuals or from patients diagnosed with rheumatoid arthritis but showing no antibody reactivity.

In some embodiments, identification of the presence or increase in the antibody level in the biological sample of the subject as compared to the reference value or to the antibody level in one or more reference samples, indicates that the subject will not or only poorly respond to treatment with an anti-rheumatic drug.

In certain embodiments, the presence or increase in the level of antibodies against the one or more peptides or against immune reactive peptides as disclosed herein can be used to evaluate the response of a subject diagnosed with RA to treatment with an antirheumatic drug. In some embodiments, the antibody level in the subject can be determined at a time point before the start of the treatment with an anti-rheumatic drug and at one or more time points after the start of the treatment with the antirheumatic drug, followed by comparing the antibody levels detected at the different time points after the start of the treatment with the antibody levels detected in the sample obtained before the start of the treatment. In some embodiments, the sample obtained before the start of the treatment is a reference sample of the subject and the antibody level detected in said reference sample are considered as a reference value, and the presence or increase in the antibody level in the samples obtained at the one or more time points after the start of the treatment is evaluated and compared to said reference value. An increase or presence of antibody levels in the samples obtained at the later time points as compared to said reference value is indicative for a nonresponse or impaired response of the subject to treatment with an anti-rheumatic drug. In some other embodiments, when the reference sample obtained at the start of the treatment shows a high antibody reactivity, a decrease or even absence of antibody levels in one or more subsequent samples obtained after the start of treatment with an anti-rheumatic drug as compared to the reference value of said reference sample obtained before the start of the treatment, is indicative for response of the subject to treatment with the anti-rheumatic drug.

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 rheumatoid arthritis. 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 "predicting", "prediction or "predictive" as used herein refer to an advance declaration, indication or foretelling of the response to treatment with an anti-rheumatic drug. For example, a prediction of a non-response or poor response to treatment with an anti-rheumatic drug in a subject may indicate that the subject will not respond or only poorly respond to treatment with the anti-rheumatic drug.

The terms "responsiveness" or "susceptibility" or "sensitivity" may be used interchangeably herein and refer to the quality that predisposes a subject having RA 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 RA, the response to treatment with an anti-rheumatic drug is based on standard criteria known in the art. Disease activity measurements after treatment with an antirheumatic drug allow to assess response to treatment following the treat-to-target strategy, aiming to reach the target of remission or at least low disease activity (LDA) in 6 months after treatment initiation [Smolen et al. (2016) Ann Rheum Dis 75, 3-15; Smolen et al. (2020) cited above]. In case this target is not reached, treatment should be adjusted, switching to a different csDMARD, or escalating to a bDMARD or tsDMARD. In some embodiments, the Disease Activity Score based on 28 joints with C-reactive protein (DAS28CRP) or the DAS28 with erythrocyte sedimentation rate (DAS28ESR) criteria are used to evaluate the response to treatment with an anti-rheumatic drug. The DAS28 is an index consisting of a 28 tender joint count, a 28 swollen joint count, a patient general health assessment on a visual analogue scale (range 0-100), and an acute phase reactant in the form of the erythrocyte sedimentation rate (ESR) or C- reactive protein (CRP) [Fransen et at. (2004 Rheumatology 43, 1252-1255],

In certain embodiments, the DAS28CRP or DAS28ESR criteria are used to evaluate the response to treatment with an anti-rheumatic drug. In certain embodiments, the DAS28CRP or DAS28ESR is determined in the subject before the start of the treatment and after a particular time period of treatment, for example after 4 weeks, 8 weeks, 12 weeks, 16 weeks, 24 weeks, or even more. For example, a subject with DAS28CRP/DAS28ESR>5.1 after treatment with the anti-rheumatic drug for a particular time period, for example after 16 weeks of treatment, is considered to have so-called high disease activity and not to respond to the treatment; a subject with 3.2<DAS28CRP/DAS28ESR<5.1 after treatment with the anti-rheumatic drug for a particular time period, for example after 16 weeks of treatment, is considered to have so-called moderate disease activity and to only poorly respond to the treatment; a subject with 2.6<DAS28CRP/DAS28ESR<3.2 after treatment with the anti-rheumatic drug for a particular time period, for example after 16 weeks of treatment, is considered to have so-called low disease activity (LDA) and a subject with DAS28CRP/DAS28ESR<2.61 after treatment with the anti-rheumatic drug for a particular time period, for example after 16 weeks of treatment, is considered to have reached disease remission and to respond well to the treatment. As used herein, subjects with a DAS28CRP/DAS28ESR>5.1, with a 3.2<DAS28CRP/DAS28ESR<5.1 or with 2.6<DAS28CRP/DAS28ESR<3.2 after treatment with the anti-rheumatic drug for a particular time period, for example after 16 weeks of treatment, are thus considered not to show remission, whereas subjects with a DAS28CRP/DAS28ESR<2.61 after treatment with the anti-rheumatic drug for a particular time period, for example after 16 weeks of treatment, are considered to show remission. Likewise, subjects with a DAS28CRP/DAS28ESR>5.1, or with a 3.2<DAS28CRP/DAS28ESR<5.1 after treatment with the anti-rheumatic drug for a particular time period, for example after 16 weeks of treatment, are considered not to show LDA, whereas subjects with 2.6<DAS28CRP/DAS28ESR<3.2 or with DAS28CRP<2.6 after treatment with the antirheumatic drug for a particular time period, for example after 16 weeks of treatment, are considered to show LDA.

In some other embodiments, the clinical disease activity index (CDAI) or the simplified disease activity index (SDAI) criteria are used to evaluate the response to treatment with an anti-rheumatic drug. The CDAI is an index consisting of a 28 tender joint count, a 28 swollen joint count, a general health assessment on a visual analogue scale (range 0-10) by the patient and by the medical doctor [Aletaha et al. (2005) Arthritis Res Ther 7, R796-R806], while the SDAI additionally includes an acute phase reactant in the form of C-reactive protein (CRP) [Smolen et al. (2003) Rheumatology 42, 244-257], In certain embodiments, the CDAI or SDAI is determined in the subject before the start of the treatment and after a particular time period of treatment, for example after 4 weeks, 8 weeks, 12 weeks, 16 weeks, 24 weeks, or even more. For example, a subject with CDAI>22 or SDAI>26 after treatment with the anti-rheumatic drug for a particular time period, for example after 16 weeks of treatment, is considered to have so-called high disease activity and not to respond to the treatment; a subject with 10<CDAI<22 or 11<SDAI<26 after treatment with the anti-rheumatic drug for a particular time period, for example after 16 weeks of treatment, is considered to have so-called moderate disease activity and to only poorly respond to the treatment; a subject with 2.8<CDAI< 10 or 3.3<SDAI< 11 after treatment with the anti-rheumatic drug for a particular time period, for example after 16 weeks of treatment, is considered to have so-called low disease activity (LDA) and; and a subject with CDAI <2.8 or SDAI <3.3 after treatment with the anti-rheumatic drug for a particular time period, for example after 16 weeks of treatment, is considered to have reached disease remission and to respond well to the treatment.

In certain embodiments, response to therapy is measured using the American College of Rheumatology (ACR) therapy response criteria. The ACR response criteria are a composite of score comprising clinical (swollen joint count, tender joint count, physician and patient response assessment, and healthy assessment questionnaire), and laboratory (acute phase response) parameters. The level of improvement is reported as an ACR20 (20%), ACR50 (50%), or ACR70 (70%) response, which indicates percent change (improvement) from the baseline score. Responses in RA may also be assessed using the Disease Activity Score (DAS)-based European League Against Rheumatism (EULAR) response criteria, which consider both the degree of improvement and the patient's current situation. These EULAR criteria classify individual patients as non-, moderate or good responders to a particular type of therapy, dependent on the extent of change and the level of disease activity reached. The EULAR response criteria have been shown comparable in validity to the ACR response criteria in clinical trials.

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 body fluid sample or a tissue sample; preferably 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 biological sample is a serum sample or a 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 RA. In some embodiments, the reference sample is a biological sample obtained from a patient diagnosed with RA but that does not show any antibody reactivity against the antibodies that are determined in the present invention. In some other embodiments, the reference sample is a sample of the subject that is obtained before the start of the treatment with an anti-rheumatic drug.

As disclosed herein, the present invention provides methods and tools to predict the response of a subject to treatment with an anti-rheumatic drug or the evaluate the response of a subject to treatment with an anti-rheumatic drug. As taught herein, an anti-rheumatic drug is considered as any agent or combination of agents that is known to ameliorate or improve symptoms of RA. General classes of drugs commonly used in the treatment of autoimmune diseases, such as RA, include corticosteroids and disease modifying drugs. Corticosteroids have a short onset of action, but many disease modifying drugs take several weeks or months to demonstrate a clinical effect. These agents include methotrexate, leflunomide (AravaTM), etanercept (EnbrelTM), infliximab (RemicadeTM), adalimumab (HumiraTM), anakinra (KineretTM), rituximab (RituxanTM), CTLA-4-Ig (abatacept), and the like.

Corticosterioids, e.g. prednisone, methylpredisone, prednisolone, solumedrol, etc., have both anti-inflammatory and immunoregulatory activity. They can be given systemically or can be injected locally. Corticosteroids are useful in early disease as temporary adjunctive therapy while waiting for disease modifying agents to exert their effects. Corticosteroids are also useful as chronic adjunctive therapy in patients with severe disease that is not well controlled with NSAIDs and other agents.

Disease modifying anti-rheumatoid drugs (DMARDS) have been shown to alter the disease course and improve radiography outcomes. It will be understood by the skilled person that these drugs can also be used in the treatment of other autoimmune diseases. DMARDs have an effect upon rheumatoid arthritis that is different and more delayed in onset than NSAIDs or corticosteroids.

Methotrexate (MTX) has become a popular first-line classical synthetic DMARD (csDMARD) agent in RA and other autoimmune diseases because of its early onset of action (4-6 weeks), good efficacy, favourable toxicity profile, ease of administration, and relative low cost. Methotrexate is the only conventional DMARD agent in which the majority of patients continue on therapy after 5 years. Methotrexate is effective in reducing the signs and symptoms of RA, as well as slowing or halting radiographic damage. Although the immunosuppressive and cytotoxic effects of MTX are due to the inhibition of difhydrofolate reductase, the anti-inflammatory effects in RA appear to be related at least in part to interruption of adenosine and TNF pathways. The onset of action of MTX is 4 to 6 weeks, with approximately 70% of patients showing some response.

Antimalarials such as hydroxychloroquine and chloroquine are rapidly absorbed, relatively safe, well-tolerated and often effective agents for treatment of RA, particularly for mild to moderate RA disease.

Sulfasalazine is another effective csDMARD for the treatment of RA, although its mechanism of action in RA is unknown. Like the other csDMARDs, it has been shown not only to reduce the signs and symptoms of RA but also to slow or halt radiographic progression. Sulfasalazine is a good alternative to methotrexate for patients with liver disease.

In current clinical practice, the first-line treatment for RA is treatment with one or more csDMARDs, such as MTX or sulfasalazine, often combined with glucocorticoids. However, in case of insufficient response to this first-line treatment with csDMARDs and in the presence of poor prognostic factors, the EULAR recommendations advise escalation to biological (b)DMARDs, which are directed against cytokine signalling, B or T lymphocytes, or to targeted synthetic (ts)DMARDs, which target Janus kinases. Examples of bDMARDS are, but are not limited to, etanercept (Enbrel™), infliximab (Remicade™), adalimumab (Humira™), anakinra (Kineret™), rituximab (Rituxan™), and abatacept (Orencia™). Examples of tsDMARDs are, but are not limited to, ofacitinib (Xeljanz™), baricitinib (Olumiant™), filgotinib (Jyseleca™), upadacitinib (Rinvoq™) .

In certain embodiments, the anti-rheumatic drug is selected from a csDMARD, a bDMARD, a tsDMARD or combinations thereof. In preferred embodiments, the antirheumatic drug is at least a csDMARD or at least a combination of several csDMARDs. In some embodiments, the anti-rheumatic drug is a csDMARD or a combination of several csDMARDs. For example, the anti-rheumatic drug is selected from MTX or sulfasalazine or combinations thereof, or the anti-rheumatic drug is MTX or sulfasalazine in combination with glucocorticoids.

The present application further provides a method of treatment comprising: 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 antirheumatic drug to the subject. Particularly, the method comprises comparing the antibody levels in the biological sample of the subject with a reference value, such as the antibody levels of one or more reference samples. In certain embodiments, when antibodies as disclosed herein are not detected or when no increase in the presence of said antibodies is detected as compared to the reference value, the subject is treated with an anti-rheumatic drug, in particular with a drug selected from csDMARD, bDMARD or tDMARD, or combinations thereof, even more preferably with a csDMARD or combinations of csDMARDs. In certain embodiments, when antibodies as disclosed herein are detected or when an increase of said antibodies is observed as compared the reference value, the subject is not treated with a csDMARD. In said embodiments, the subject is treated with a bDMARD, a tDMARD or combinations thereof.

The methods of treatment as taught herein may specifically relate to prophylactic and/or therapeutic treatment of RA. In particular embodiments, said method relates to a therapeutic treatment of RA. The therapeutic agent administered to the subject can be a therapeutic agent known to be effective against RA, as disclosed herein above.

It is apparent that there have been provided in accordance with the invention products, methods, and uses, that provide for 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 variations 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.

EXAMPLES METHODS Patients and controls

This study included patients with early RA from the CareRA trial, recruited at 13 rheumatology centres in Belgium [Verschueren et al. (2015) cited above], and healthy controls (HC), recruited at Hasselt University (Hasselt, Belgium). This study was approved by local ethics committees of Hasselt University and University Hospitals Leuven. Written informed consent was obtained from all participants. Samples were cryopreserved at the biobank from the University hospitals Leuven and the University Biobank Limburg.

Baseline serum samples, taken before the initiation of first-line csDMARD combination therapies, were obtained from 219 RA participants of the CareRA trial [Stouten et al., (2019) cited above; Verschueren et al. (2015) cited above; Verschueren et al. (2017) Ann Rheum Dis 76, 511-520], Disease activity measures investigated in the current study include remission (rem) and low disease activity (LDA) according to the DAS28CRP, DAS28ESR, SDAI and CDAI indices at fixed time points during the first two years of follow-up, sustained remission and LDA over the first two years, and therapy response according to the American College of Rheumatology (ACR20/50/70) criteria. DAS28CRP and DAS28ESR are the Disease Activity Score-28 for RA respectively in combination with CRP (C-reactive protein) or ESR (erythrocyte sedimentation rate) and represent a disease activity score based on 28 clinical markers in combination with respectively CRP levels or the ESR in a subject [Fransen et al. (2004) cited above], SDAI is the Simplified Disease Activity Index and CDAI is the Clinical Disease Activity Index as described in Aletha et al. (2005) cited above; Smolen et al., (2003) cited above. Details on demographics, clinical parameters, treatments used and responses of the entire CareRA study were published previously [Stouten et at. (2019) cited above; Verschueren et al. (2015) cited above; Verschueren et al. (2017) cited above]. Details on demographic and clinical parameters at baseline and week 16 of the 219 patients used in this study are presented in Table 2.

Baseline samples from 40 patients (Table 2) were used to screen for novel antibodies that correlate with failure to reach early disease remission using serological antigen selection (SAS) (13-15). Of these, the week (w)16 DAS28CRP rem- group consisted of 20 patients with moderate (3.2<DAS28CRP<5.1) or high (DAS28CRP>5.1) disease activity at baseline, who did not reach DAS28CRP remission (DAS28CRP<2.6) nor LDA (2.6<DAS28CRP<3.2) at week 16, and who showed the smallest improvement in DAS28CRP over the first 16 weeks among our study population. The wl6 DAS28CRP rem+ group consisted of 20 patients with high DAS28CRP disease activity at baseline, who reached DAS28CRP remission at weeks 16 and 52, and who showed the highest improvement in DAS28CRP over the first 16 weeks.

Antibody reactivity towards individual identified University Hasselt (UH)-RA antigens was determined by phage enzyme-linked immunosorbent assays (phage ELISA) in 4 serum pools of wl6 DAS28CRP rem- patients (10 patients/pool, median (IQR) baseline DAS28CRP 5.4 (5.0-6.3), median (IQR) week 16 DAS28CRP 3.8 (3.5-4.3)) and 5 pools of wl6 DAS28CRP rem+ patients (10 patients/pool, median (IQR) baseline DAS28CRP 5.8 (5.4-6.2), median (IQR) week 16 DAS28CRP 1.7 (1.4-2.0)), in order to select the antigens with specific reactivity against wl6 DAS28CRP rem- samples.

Antibody reactivity against 6 selected UH-RA antigens was validated using phage ELISA in 179 individual baseline samples from participants of the CareRA trial (table 2), and in 86 age- and gender-matched HC (mean (SD) age 51.8 (10.6) years, 56 (65%) were female). These RA samples consisted of 137 high-risk patients (based on presence of erosions, rheumatoid factor (RF) and/or anti-citrullinated protein antibody (ACPA), and baseline DAS28CRP [Verschueren et al. (2015) cited above], who received COBRA Classic (MTX/sulfasalazine/step-down GC, n=38), COBRA Slim (MTX/step-down GC, n=50) or COBRA Avant-Garde (MTX/leflunomide/step-down GC, n=49), and 42 low-risk patients, who received COBRA Slim (n = 19) or MTX monotherapy (n=23). After 16 weeks, at total of 66.5% (119/179) of these patients had achieved DAS28CRP remission.

Table 2. Demographic and clinical features of RA patients from the CareRA cohort that were used for SAS screening (n=40) and validation of antibody reactivity against identified UH-RA antigens n=179).

*Missing values for 1 patient. + Missing values for 2 to 4 patients. ACPA, anti-citrullinated protein antibodies; BL, baseline; CDAI, Clinical Disease Activity Index; CRP, C reactive protein; DAS28CRP, Disease Activity Score in 28 joints with C reactive protein; DAS28ESR, Disease Activity Score in 28 joints with erythrocyte sedimentation rate; ESR, erythrocyte sedimentation rate; IQR, interquartile range; RA, rheumatoid arthritis; rem-, patient not reaching disease remission; rem+, patient reaching disease remission; RF, Rheumatoid factor; SAS, serological antigen selection; SD, standard deviation; SDAI, Simplified Disease Activity Index; UH, University Hasselt; W, week. Identification of novel antigens using serological antigen selection (SAS)

Serological antigen selection (SAS) is a screening procedure that uses cDNA phage display to identify antigenic targets of novel antibodies of interest. In this study, two RA synovial cDNA phage display libraries expressing antigens from RA hip [Somers et al. (2009) Ann N Y Acad Sci 1173, 92-102] and RA knee synovia [Vandormael et at., (2017) Immunol Res 65, 307-325] were used. To specifically isolate novel antibodies associated with failure to reach early disease remission, these phage particles were used in rounds of positive selection to isolate complexes between antigens and antibodies in pooled sera of wl6 DAS28CRP rem- RA patients (n=20), and negative selection to remove complexes between antigens and antibodies in pooled sera of wl6 DAS28CRP rem-i- RA patients (n=20).

Serum antibodies with reactivity against phage or bacterial proteins were removed by first pre-absorbing sera to cyanogen-bromide sepharose beads (Merck, United States) coupled with phage and bacterial protein extracts. In a first negative round of the SAS procedure, equal amounts (~10el2 cfu/ml) of the two RA cDNA phage display libraries originating from RA hip and RA knee synovia were pooled, and pre-incubated with pooled wl6 DAS28CRP rem-i- sera in phosphate buffered saline (PBS) pH7.4 with 2% (w/v) skimmed milk powder (MPBS), and incubated for one hour rotating at room temperature (RT). Protein G agarose beads (Genscript, United States) were blocked with phage extract (100 pg/ml) in 2% MPBS for 1 hour at RT and washed with PBS. The negative pre-incubation mixture was transferred to the protein G beads, incubated 30 minutes rotating at RT, and the unbound phage particles were used immediately for positive selection. During positive selection, phage particles were pre-incubated with pooled wl6 DAS28CRP rem- sera in 2% MPBS, for one hour rotating at RT. The positive pre-incubation mixture was transferred to protein G beads, and incubated 30 minutes rotating at RT. Beads were washed 10 times with PBS with 0.1% Tween-20 and 5 times with PBS. The bound phage-antibody complexes were eluted with lOOmM triethylamine, neutralised with IM Tris-HCI pH7.4, and amplified by infecting TGI bacteria and plating on 2xTY plates with lOOpg/ml ampicillin and 2% glucose (2xTYAG). The output of this first SAS round was used as input for three additional SAS rounds, each consisting of two consecutive negative selection steps, followed by one positive selection step. After the final SAS round, colonies were randomly picked and stored in liquid 2xTYAG medium with 10% glycerol at -80°C. The DNA sequence of the M13 geneVI-cDNA fusion was characterised by Sanger sequencing, and the amino acid sequence of the corresponding displayed antigen was determined using DNAnalyzer software [Quaden et al. (2020) cited above]. Amino acid homology of the antigen sequences with human synovial proteins was determined using the RefSeq Select proteins database on NCBI and the blastp algorithm, sorted by E value. Expression of the homologous proteins in human synovial lymphoid, myeloid or fibroid cells was checked in the online database from Lewis et a!. (2019) Cell Rep 28, 2455-2470.

Detection of antibodies to UH-RA antigens

Antibody reactivity against antigens displayed on phage was measured by phage ELISA in pooled or individual serum samples as described in [Quaden et al. (2020) cited above]. In brief, half area 96-well MicroIon high-binding microplates (Greiner, Belgium) were coated overnight at 4°C with 3.5 pg/ml anti-M13 mouse monoclonal antibody (clone MM05T, Sino Biological, China) diluted in coating buffer (0.1 M sodium carbonate bicarbonate buffer, pH9.6). After washing (3 times using PBS pH7.4 with 0.1% Tween- 20, 1 time using PBS, shaking at RT), plates were blocked with 5% MPBS for two hours, shaking at 37°C. After washing, diluted antigen-expressing phage particles (7.0xl0ell cfu/ml in 5% MPBS) were added, and incubated for one hour standing at 37°C, followed by 30 minutes shaking at RT. After washing, plates were incubated with diluted serum samples (1/100 in 5% MPBS) for one hour standing at 37°C, and 30 minutes shaking at RT. After washing, plates were incubated with cross-adsorbed goat anti-human IgG- Fc, conjugated with horseradish peroxidase (Bethyl, United States) diluted 1/10000 in 5% MPBS, for one hour shaking at RT. Finally, after washing, plates were coloured with 3,3',5,5'-tetramethylbenzidine for ten minutes in the dark, stopped using 1.8 M H2SO4, and absorbance was read at 450nm.

All samples were tested in duplicate and experiments were performed independently at least twice. To correct for non-specific reactivity, samples were also tested on empty phage without displayed antigen. Samples with an optical density (OD) signal for the empty phage higher than 0.5 were excluded. Results are expressed as the average ratio of antigen-expressing phage OD over empty phage OD. The coefficient of variation for duplicate ODs, and for ratios of experimental repeats was lower than 20%. A cut-off for antibody-positivity was determined via changepoint analysis in RStudio using the Pruned Exact Linear Time algorithm. This algorithm allows to divide a series of ascending values in subgroups based on statistical changepoints. The cut-off for antibody-positivity was set at five times the standard deviation (SD) above the mean ratio (AVG + 5SD) of all non-reactive samples (represented by the lowest subgroup from the changepoint analysis). For the comparison of antibody reactivity between groups of RA patients, the positive likelihood ratio (LR+) consists of the ratio of the antibody reactivity percentage of the (sustained) remission negative or therapy response negative group, over the (sustained) remission positive or therapy response positive group (%sust rem-/%sust rem+; %rem-/%rem+; %resp-/%resp+). Competition ELISA

To confirm the phage-displayed peptide as the actual target of the observed antibody reactivity, competition ELISA was performed, as described in Quaden et al., (2020) cited above. Briefly, serum samples were pre-incubated with increasing concentrations (0-30 pg/ml) of synthetic peptide (> 85% purity, GL Biochem, China) (Table 3), for one hour shaking at RT. The pre-incubated samples were used in a regular phage ELISA and added to the respective antigen-expressing phage and to empty phage. Results are expressed as the ratio of antigen-expressing phage OD over empty phage OD. Initially, the synthetic peptides used in the competition ELISA had a minimal length of 10 amino acids. Therefore, for UH-RA.305, the peptide sequence was elongated with 4 N-terminal amino acids originating from the translated cloning adaptor (UH-RA.305-FL (full length)), for UH-RA.318, the antigenic peptide sequence following the cloning adaptor was used (UH-RA.318-FL), and for UH-RA.329, the peptide sequence was elongated with 8 N-terminal amino acids originating from the translated cloning adaptor (UH- RA.329-FL). Epitope mapping using competition ELISA could further narrow down the exact antigen sequence anti-UH-RA antibodies bound to. To this end, UH-RA.305-FL, UH-RA.314-FL, and UH-RA.329-FL were N-terminally truncated by 1 (-epi. A), 2 (-epi.B), or more (-epi.C to -epi.F) amino acids from the translated cloning adapter. In addition, peptides consisting of only the translated cloning adapter (-epi.CA) were used. UH- RA.107 and UH-RA.108 were elongated with respectively 7 and 3 N-terminal amino acids from the translated cloning adaptor (UH-RA.107-FL and UH-RA.108-FL). As a negative control, sample pre-incubation with a control peptide was carried out in parallel.

Table 3. Synthetic peptides used in competition ELISA.

* Amino acid sequence of synthetic peptide used in competition ELISA. For the UH-RA peptides, the amino acids indicated in bold originate from the translated cloning adapter, the regular amino acids originate from the translated cDNA insert. aa, amino acid; epi. A, epitope mapping truncating 1 N-terminai aa; epi.B, epitope mapping truncating 2 N-terminal aa; epi.CA, epitope mapping using translated cloning adaptor; FL, full length peptide; RA, rheumatoid arthritis; UH, University Hasselt.

Statistical analysis

Data analysis was performed using MP®, Version 15.2.0. Antibody-positivity for particular UH-RA antigens or antigen panels was compared between groups by applying Fisher's exact test. To test the probability that UH-RA antibody positivity is greater in the (sust)rem- than in the (sust)rem+ group, the one-tailed test was used. To test the probability that UH-RA antibody positivity is different in RA patients than healthy controls, the two-tailed test was used. A p value <0.05 was considered to be statistically significant.

RESULTS

Identification of novel antibody biomarkers that correlate with failure to achieve early DAS28CRP remission

We previously constructed two cDNA phage display libraries originating from hip [Somers et al. (2009) cited above] and knee [Vandormael et al. (2017) cited above] synovia from 4 RA patients. These libraries contain a highly diverse mixture of phage particles, each carrying on their surface a protein or protein fragment expressed in these target tissues, but also non-physiological peptides, formed by out-of-frame cDNA translation or the translation of non-coding sequences can be displayed. Combined, these libraries contained 1,2 x 10⁷ different antigens, which were used to specifically isolate novel antibodies associated with failure to reach early disease remission (rem-) after first-line therapy. To this end, these phage particles were used in rounds of positive selection to isolate complexes between antigens and antibodies in pooled sera of wl6 DAS28CRP rem- RA patients (n=20), and negative selection to remove complexes between antigens and antibodies in pooled sera of wl6 DAS28CRP rem+ RA patients (n=20).

Using this screening, 41 novel University Hasselt (UH)-RA antigens (UH-RA.301 to UH- RA.341) were identified. Initial antibody reactivity testing against these 41 individual antigens in serum pools of wl6 DAS28CRP rem- (4 pools of 10 patients each) and wl6 DAS28CRP rem-i- (5 pools of 10 patients each) patients, showed increased antibody reactivity in rem- pools for 6 antigens (results not shown). These antigens consist of short, non-physiological peptides, between 5 and 37 amino acids in length, which show homology to several human proteins (Table 4) that are expressed in human synovial tissue [Lewis et al. (2019) cited above].

Baseline antibody reactivity against UH-RA antigens in RA and HC

Antibody reactivity against each of the 6 UH-RA antigens was validated in baseline samples of 179 RA patients of the CareRA cohort, and in 86 age- and gender-matched healthy controls (HC). Anti-UH-RA antibody reactivity was found in 1.7% to 11.3% of RA patients and in 1.2% to 8.1% of HC (Table 5).

Baseline antibody reactivity against UH-RA antigens and early DAS28CRP remission

We examined whether baseline antibody reactivity towards the 6 UH-RA antigens correlated with failure to reach DAS28CRP remission after 8 or 16 weeks of first-line treatment. The largest difference in antibody reactivity between patients not reaching DAS28CRP remission at these time points, and those that did, was seen for UH-RA.305, UH-RA.318 and UH-RA.329 (Table 6).

Epitope mapping using competition ELISA with synthetic peptides could define the epitope sequences recognized by anti-UH-RA.305, UH-RA.314 and anti-UH-RA.329 antibodies, to the antigen sequences indicated in table 3 (Figure 1 A and C, and Figure 2). The required epitope recognized by the anti-UH-RA.305 and anti-UH-RA.329 antibodies had a size of 10 aa, while anti-UH-RA.314 antibodies could recognize an epitope as short as 4 aa. For the longer UH-RA.318 antigen, competition ELISA could show that the epitope recognized by anti-UH-RA.318 antibodies is part of the UH-RA antigen sequence indicated in Table 4 (Figure 1 B).

Baseline antibody reactivity against 3 UH-RA antigens and early remission and low disease activity

A panel of 3 antigens (UH-RA.305, UH-RA.318 and UH-RA.329) could be composed, whose combined antibody reactivity was 77 to 122% higher in baseline samples of RA patients that failed to reach DAS28CRP (p=0.007), DAS28ESR (p=0.007), SDAI (p=0.055) and CDAI (p=0.074) remission at week 8, compared to RA patients that did reach these respective outcome criteria (Table 7a). Moreover, antibody reactivity against these 3 antigens was respectively 109 and 120% higher in baseline samples of RA patients that had moderate to high disease activity at week 8 according to the SDAI (p=0.018) and CDAI (p=0.01) outcome criteria, compared to RA patients with low disease activity (Table 7a).

When looking at the contribution of the individual antigens of the UH-RA.305/318/329 panel, the strongest correlation with failure to reach early disease remission came from the antibody reactivity against the UH-RA.305 antigen. Baseline anti-UH-RA.305 antibody reactivity was about threefold higher in patients that did not reach remission at week 8 according to the DAS28CRP (p=0.035), DAS28ESR (p=0.039), SDAI (p=0.089) and CDAI (p=0.104) criteria (Table 7b).

In addition, baseline antibody reactivity against the panel of UH-RA.305/318/329 antigens was approximately twofold higher in patients failing to reach an ACR70 response at 16 weeks (p=0.030), 52 weeks (p=0.028) and 104 weeks (p=0.070) after first-line treatment initiation, and a similar trend was seen for the ACR20 and ACR50 responses, albeit not significant (Table 8).

Baseline antibody reactivity against 3 UH-RA antigens and early remission and LDA in RF/ACPA seronegative RA

In RA patients who are seronegative for rheumatoid factor (RF) and anti-citrullinated protein antibodies (ACPA), the difference in anti-UH-RA.305/318/329 antibody reactivity between RA patients that failed to reach different remission criteria and those that did, was much more pronounced (Table 9). At baseline, anti-UH-RA.305/318/329 antibody reactivity identified up to 45% of RF/ACPA seronegative patients that failed to reach DAS28CRP remission at week 8, compared to 6% in patients that did (p=0.007), resulting in a positive likelihood ratio (LR+) of 7.73. Likewise, baseline anti-UH- RA.305/318/329 antibodies in RF/ACPA seronegative patients, could distinguish between those patients who reached LDA at week 8 or not, according the DAS28CRP (p=0.049), SDAI (p=0.005) or CDAI (p=0.017) criteria (Table 9).

Baseline antibodies against 3 UH-RA antigens and remission during first year of first-line therapy

As baseline anti-UH-RA antibody reactivity was increased in patients failing to reach early remission, we also investigated the correlation between baseline antibody reactivity and remission at later time points during the first two years. Baseline anti- UH-RA.305/318/329 antibody reactivity was higher in patients failing to reach DAS28CRP remission at multiple time points during the first year of therapy (Table 10). In RF/ACPA seronegative RA patients, this effect was again more pronounced, with a larger LR+ (Table 10). For the DAS28ESR, SDAI and CDAI remission criteria, baseline antibody reactivity against the UH-RA.305/318/329 antigens was generally also higher in patients not reaching remission at different time points during the first year, albeit not significantly (results not shown). Finally, baseline antibody reactivity against the UH-RA.305/318/329 antigens showed no clear correlation with remission status at time points of the second year of therapy, for none of the remission indices studied (results not shown).

Baseline antibodies against 3 UH-RA antigens and sustained remission and LDA

In a single patient, disease activity can vary during follow up, sometimes switching above or below the thresholds for remission or LDA. For example, of the 110 patients that reached DAS28CRP remission at week 8 (Table 7), only 59 (54%) showed sustained remission at each time point between week 8 and 52 (Table 11a). Likewise, of the 136 RA patients who reached DAS28CRP LDA at week 8 (Table 7), 86 (68%) showed sustained LDA during this period (Table 11a). Therefore, we investigated whether baseline anti-UH-RA.305/318/329 antibody reactivity correlated with sustained remission, or with sustained LDA, over a longer period of time. For each of the remission criteria studied, baseline antibody reactivity against the UH-RA.305/318/329 antigens was significantly higher in RA patients that did not maintain sustained disease remission, or sustained LDA between week 8 and week 52, and was able to identify about 25% of sustained rem- patients, and about 30% of sustained LDA- patients during this period (Table 11a). Among these patients, the largest difference in antibody reactivity was seen between patients that did or did not reach sustained remission for the more stringent SDAI and CDAI remission criteria, with LR+ of resp. 6.62 and 6.32. In RF/ACPA seronegative patients, the difference in antibody positivity between patients that did, or did not reach sustained disease remission or LDA, was much more outspoken than in the total RA population, and was shown to be significantly higher in patients not reaching sustained LDA for the SDAI and CDAI criteria (Table 11a).

When looking at the contribution of the individual antigens of the UH-RA.305/318/329 panel, the strongest correlation with sustained disease remission during the first year, came from the antibody reactivity against the UH-RA.305 antigen. Baseline anti-UH- RA.305 antibody reactivity was four- to sixfold higher in patients that did not maintain sustained remission during the first year according to the DAS28CRP (p=0.030), DAS28ESR (p=0.087), SDAI (p=0.085) and CDAI (p=0.094) criteria (Table lib). In addition, baseline anti-UH-RA.305 antibody reactivity was three- to fourfold higher in patients who failed to reach sustained LDA according to the DAS28CRP (0.033), DAS28ESR (p=0.033) and SDAI (p=0.015) criteria, compared to patient that did reach these outcome measures (Table lib).

When looking at the correlation between baseline anti-UH-RA.305/318/329 reactivity and sustained remission or LDA over a two-year period, antibody reactivity was still significantly higher in patients failing to reach sustained LDA, according to each of the studied disease activity indices (Table 12).

Identification of novel antibody biomarkers for prediction of remission or LDA (2)

In addition, we performed a parallel screening using only the cDNA phage display library originating from knee synovia from 3 RA patients, containing 7.5 x 10⁵ different synovial antigens or non-physiological peptides [Vandormael et al. (2017) cited above]. This library was used in rounds of positive selection to isolate complexes between antigens and antibodies in pooled sera of wl6 DAS28CRP rem- RA patients (n = 6), and negative selection to remove complexes between antigens and antibodies in pooled sera of age- and gender-matched healthy controls (n=6). Using this screening, 50 novel University Hasselt (UH)-RA antigens were identified. Initial antibody reactivity testing against these 50 individual antigens in a serum pool of 6 wl6 DAS28CRP rem- patients, and a pool of 8 wl6 DAS28CRP rem+ patients, showed increased antibody reactivity in the rem- pool for 21 antigens (called UH-RA.101 to UH-RA.121) (results not shown). Further antibody reactivity testing in 42 individual wl6 DAS28CRP rem- patients and 44 wl6 DAS28CRP rem-i- patients showed increased antibody reactivity in rem- samples for 2 antigens, UH-RA.107 and UH-RA.108 (Table 13 and 14). Competition ELISA could show that the epitope recognized by anti-UH-RA.107/108 antibodies is part of the UH-RA antigen sequence indicated in Table 13 (Figure 3).

Combined antibody reactivity against the UH-RA.107 and UH-RA.108 antigens was between 3.8- and 5.1-fold higher in baseline samples of RA patients that failed to reach DAS28CRP (p = 0.096), DAS28ESR (p = 0.182), SDAI (p = 0.093) and CDAI (p = 0.099) remission at week 16, compared to RA patients that did reach these respective outcome criteria (Table 15).

Baseline anti-UH-RA.107/108 antibody reactivity was higher in patients failing to reach DAS28CRP, DAS28ESR, SDAI or CDAI remission at multiple time points during the first year of therapy, albeit not significant (Table 16 and results not shown).

For each of the remission criteria studied (DAS28CRP, DAS28ESR, SDAI and CDAI), baseline antibody reactivity against the UH-RA.107/108 antigens was higher in RA patients that did not maintain sustained disease remission, or sustained LDA between week 8 and week 52, and was able to identify about 10% of sustained rem- or LDA- patients during this period (Table 17). Among these patients, the largest difference in antibody reactivity was seen between patients that did or did not reach sustained remission for the DAS28ESR. remission criterium, and for the more stringent SDAI and CDAI remission criteria.

Table 4. Sequence, origin and homology of 6 novel UH-RA antigens

Table 5. Baseline anti-UH-RA reactivity in RA patients and healthy controls.

* Number and percentage of anti-UH-RA positive (baseline) samples from RA patients and healthy controls.

Ab, antibody; BL, baseline; HC, healthy control; LR+, positive likelihood ratio; RA, rheumatoid arthritis; UH, University Hasselt.

Table 6. Baseline anti-UH-RA reactivity according to DAS28CRP remission at week 8 and 16.

* Number and percentage of anti-UH-RA positive baseline samples from patients that did (rem+) or did not (rem-) reach DAS28CRP remission at week 8 or 16.

Ab, antibody; BL, baseline; DAS28CRP, Disease Activity Score in 28 joints with C reactive protein; LR+, positive likelihood ratio; RA, rheumatoid arthritis; rem-, patient not reaching disease remission; rem+, patient reaching disease remission; UH, University Hasselt; W, week.

Table 7a. Baseline anti-UH-RA.305/318/329 reactivity according to remission and low disease activity at week 8.

Table 7b. Baseline anti-UH-RA.305 reactivity according to remission and low disease activity at week 8.

Table 8. Baseline anti-UH-RA.305/318/329 reactivity according to ACR therapy response at week 16. 52 and 104.

* Number and percentage of anti-UH-RA.305/318/329 positive baseline samples from patients that did (resp+) or did not (resp-) reach ACR20, ACR50 or ACR70 response at weeks 16 to 104. ACR, American College of Rheumatology response criteria;

BL, baseline; LR+, positive likelihood ratio; RA, rheumatoid arthritis; resp-, patient not reaching therapy response criterium; resp+, patient reaching therapy response criterium; UH, University Hasselt, W, week.

Table 9. Baseline anti-UH-RA.305/318/329 reactivity in RA/ACPA seronegative RA according to remission and low disease activity at week 8.

* Number and percentage of anti-UH-RA.305/318/329 positive baseline samples from RF/ACPA seronegative RA patients that did (rem+) or did not (rem-) reach different remission criteria at week 8. § Number and percentage of anti-UH-RA.305/318/329 positive baseline samples from RF/ACPA seronegative RA patients that did (LDA+) or did not (LDA-) reach low disease activity according to different remission criteria at week 8. ACPA, anti-citrullinated protein antibodies; BL, baseline; CDAI, Clinical Disease Activity Index; DAS28CRP, Disease Activity Score in 28 joints with C reactive protein; DAS28ESR, Disease Activity Score in 28 joints with erythrocyte sedimentation rate; LDA-, patient not reaching low disease activity; LDA+, patient reaching low disease activity; LR+, positive likelihood ratio; RA, rheumatoid arthritis; rem-, patient not reaching disease remission; rem+, patient reaching disease remission; RF, Rheumatoid factor; SDAI, Simplified Disease Activity Index; UH, University Hasselt; W, week.

Table 10. Baseline anti-UH-RA.305/318/329 reactivity according to DAS28CRP remission at weeks 8 to 52.

* Number and percentage of anti-UH-RA.305/318/329 positive baseline samples from RA patients that did (rem+) or did not (rem-) reach DAS28CRP remission at weeks 8 to 52. § Number and percentage of anti-UH-RA.305/318/329 positive baseline samples from RF/ACPA seronegative RA patients that did (rem+) or did not (rem-) reach DAS28CRP remission at weeks 8 to 52. ACPA, anti-citrullinated protein antibodies; BL, baseline; DAS28CRP, Disease Activity Score in 28 joints with C reactive protein; LR+, positive likelihood ratio; rem-, patient not reaching disease remission; rem+, patient reaching disease remission; RA, rheumatoid arthritis; RF, Rheumatoid factor; UH, University Hasselt; Wx, week x.

Table 11a. Baseline anti-UH-RA.305/318/329 reactivity according to sustained disease remission and LDA from week 8 to 52.

* Number and percentage of anti-UH-RA.305/318/329 positive baseline samples from patients that did (sust rem+) or did not (sust rem-) reach sustained remission from week 8 to 52. § Number and percentage of anti-UH-RA.305/318/329 positive baseline samples from patients that did (sust LDA+) or did not (sust LDA-) reach sustained low disease activity from week 8 to 52. ACPA, anti-citrullinated protein antibodies; BL, baseline; CDAI, Clinical Disease Activity Index; DAS28CRP, Disease Activity Score in 28 joints with C reactive protein; DAS28ESR, Disease Activity Score in 28 joints with erythrocyte sedimentation rate; LR+, positive likelihood ratio; RA, rheumatoid arthritis; RF, Rheumatoid factor; SDAI, Simplified Disease Activity Index; sust LDA-, patient not reaching sustained low disease activity; sust LDA+, patient reaching sustained low disease activity; sust rem-, patient not reaching sustained disease remission; sust rem+, patient reaching sustained disease remission; UH, University Hasse It.

Table lib. Baseline anti-UH-RA.305 reactivity according to sustained disease remission and LDA from week 8 to 52.

Table 12. Baseline anti-UH-RA.305/318/329 reactivity according to sustained disease remission from week 8 to 104.

* Number and percentage of anti-UH-RA.305/318/329 positive baseline samples from patients that did (sust rem+) or did not (sust rem-) reach sustained remission from week 8 to 104. § Number and percentage of anti-UH-RA.305/318/329 positive baseline samples from patients that did (sust LDA+) or did not (sust LDA-) reach sustained low disease activity from week 8 to 104. ACPA, anti-citrullinated protein antibodies; BL, baseline; CDAI, Clinical Disease Activity Index; DAS28CRP, Disease Activity Score in 28 joints with C reactive protein; DAS28ESR, Disease Activity Score in 28 joints with erythrocyte sedimentation rate; LR+, positive likelihood ratio; RA, rheumatoid arthritis; RF, Rheumatoid factor; SDAI, Simplified Disease Activity Index; sust LDA-, patient not reaching sustained low disease activity; sust LDA+, patient reaching sustained low disease activity; sust rem-, patient not reaching sustained disease remission; sust rem+, patient reaching sustained disease remission; UH, University Hasselt.

Table 13. Sequence, origin and homology of 2 novel UH-RA antigens.

* Sequence of the antigen as expressed on the phage surface. Aa before the bracketed aa come from translation of the cDNA cloning adaptor, while aa behind the bracketed aa originate from the translated cDNA insert. + Size of the antigen is expressed as the number of aa. t The fusion location indicates the region in the RNA where the cDNA was fused to M13 gene VI. § Type of fusion of the cDNA coding region with M13 gene VI. "No" indicates the cDNA coding region is not in frame with M13 gene VI, resulting in out-of-frame protein expression of the cDNA insert. 1) Human synovial proteins with amino acid homology to antigen sequence, with amount and percentage of identical amino acids indicated. Top 3 hits using RefSeq Select proteins database on NCBI using the blastp algorithm, which are sorted by E value, and which are expressed in human synovial lymphoid, myeloid or fibroid cells according to online database from Lewis et al. cited above. 3'UTR, 3' untranslated region; 5'UTR, 5' untranslated region; aa, amino acid; cDNA complementary DNA; mRNA, messenger RNA; N/A, not applicable; NCBI, National Center for Biotechnology Information; nr., number; RA, rheumatoid arthritis; UH, University Hasselt.

Table 14. Baseline anti-UH-RA reactivity according to DAS28CRP remission at week 8 and 16.

* Number and percentage of anti-UH-RA positive baseline samples from patients that did (rem+) or did not (rem-) reach DAS28CRP remission at week 8 or 16. Ab, antibody; BL, baseline; DAS28CRP, Disease Activity Score in 28 joints with C reactive protein; LR+, positive likelihood ratio; RA, rheumatoid arthritis; rem-, patient not reaching disease remission; rem+, patient reaching disease remission; UH, University Hasselt; W, week.

Table 15. Baseline anti-UH-RA.107/108 reactivity according to remission and low disease activity at week 16.

* Number and percentage of anti-UH-RA.305/318/329 positive baseline samples from patients that did (rem+) or did not (rem-) reach different remission criteria at week 16. § Number and percentage of anti-UH-RA.305/318/329 positive baseline samples from patients that did (LDA+) or did not (LDA-) reach low disease activity according to different remission criteria at week. BL, baseline; CDAI, Clinical Disease Activity Index; DAS28CRP, Disease Activity Score in 28 joints with C reactive protein; DAS28ESR, Disease Activity Score in 28 joints with erythrocyte sedimentation rate; LDA-, patient not reaching low disease activity; LDA+, patient reaching low disease activity; LR+, positive likelihood ratio; RA, rheumatoid arthritis; rem-, patient not reaching disease remission; rem+, patient reaching disease remission; SDAI, Simplified Disease Activity Index; UH, University Hasselt; W, week.

Table 16. Baseline anti-UH-RA.107/108 reactivity according to DAS28CRP remission between week 8 and 52.

Table 17. Baseline anti-UH-RA.107/108 reactivity according to sustained disease remission and low-disease activity from week 8 to 52.

* Number and percentage of anti-UH-RA.305/318/329 positive baseline samples from patients that did (sust rem+) or did not (sust rem-) reach sustained remission from week 8 to 52. § Number and percentage of anti-UH-RA.305/318/329 positive baseline samples from patients that did (sust LDA+) or did not (sust LDA-) reach sustained low disease activity from week 8 to 52. ACPA, anti-citrullinated protein antibodies; BL, baseline; CDAI, Clinical Disease Activity Index; DAS28CRP, Disease Activity Score in 28 joints with C reactive protein; DAS28ESR, Disease Activity Score in 28 joints with erythrocyte sedimentation rate; LR+, positive likelihood ratio; RA, rheumatoid arthritis; RF, Rheumatoid factor; SDAI, Simplified Disease Activity Index; sust LDA-, patient not reaching sustained low disease activity; sust LDA+, patient reaching sustained low disease activity; sust rem-, patient not reaching sustained disease remission; sust rem+, patient reaching sustained disease remission; UH, University Hasselt.

Claims

1. An in vitro method for predicting or evaluating the response of a subject diagnosed with rheumatoid arthritis to treatment with an anti-rheumatic drug, said method comprising detecting in a biological sample of the subject one or more antibodies against one or more peptides selected from the group consisting of:

- a peptide as shown in SEQ ID NO: 1 or an antibody-binding fragment or variant thereof,

- a peptide as shown in SEQ ID NO: 2 or an antibody-binding fragment or variant thereof, and

- a peptide as shown in SEQ ID NO: 3 or an antibody-binding fragment or variant thereof.

2. The method according to claim 1 wherein the antibody reactivity towards the antibody-binding fragment or variant of the peptide is comparable to the antibody reactivity towards the peptide itself.

3. The method according to claim 1 or 2, wherein the one or more antibodies are detected using a peptide 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; and

- combinations thereof.

4. The method according to claim 1 or 2, 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 SEQ ID NO: 3 or an antibody-binding fragment or variant thereof; and - combinations thereof.

5. The method according to claim 4, wherein the detection agent is a polypeptide.

6. The method according to any one of claims 1 to 5, comprising detecting in a biological sample of the subject antibodies against:

- a peptide as shown in SEQ ID NO: 1 or an antibody-binding fragment or variant thereof;

- a peptide as shown in SEQ ID NO: 2 or an antibody-binding fragment or variant thereof; and

7. The method according to any one of claims 1 to 6, further comprising detecting in a biological sample of the subject one or more antibodies against one or more peptides selected from the group consisting of:

- a peptide as shown in SEQ ID NO: 4 or an antibody-binding fragment or variant thereof; and

- a peptide as shown in SEQ ID NO: 5, or an antibody-binding fragment or variant thereof.

8. The method according to any one of claims 1 to 7 wherein the presence or an increase in antibody levels against the one or more peptide(s) in the biological sample compared to a reference value is indicative for a non-response or an impaired response to treatment with an anti-rheumatic drug in the subject.

9. The method according to any one of claims 1 to 8 wherein the biological sample is a body fluid sample or a tissue sample.

10. The method according to claim 9, wherein the sample is a serum sample or a plasma sample.

11. The method according to any one of claims 1 to 10, wherein the anti-rheumatic drug is selected from a classical synthetic disease-modifying anti-rheumatic drug (csDMARD), a biological disease-modifying anti-rheumatic drug (bDMARD), a targeted synthetic disease-modifying anti-rheumatic drug (tsDMARD), or combinations thereof,

12. The method according to claim 11, wherein the anti-rheumatic drug is at least a csDMARD or combinations thereof.

13. The method according to claim 11 or 12, wherein the anti-rheumatic drug is methotrexate or methotrexate in combination with glucocorticoids.

14. A peptide, antibody-binding fragment or variant 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; and

- combinations thereof.

15. A detection agent comprising one or more peptides, antibody-binding fragments or variants according to claim 14; preferably wherein the detection agent is a polypeptide.

16. A composition comprising one or more peptides, fragments or variant according to claim 14 or one or more detection agents according to claim 15.

17. Use of a peptide, antibody-binding fragment or variant according to claim 14, or of a detection agent according to claim 15, or of a composition according to claim 16 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, antibody-binding fragment or variant or detection agent present in the composition, in a biological sample of a subject

18 The use according to claim 17, wherein the detection or quantification of at least one specific antibody is indicative for a non-response or impaired response to treatment with an anti-rheumatic drug in a subject diagnosed with rheumatoid arthritis.

19. The use of claim 17 wherein the peptide has a sequence of SEQ ID NO: 1 or an antibody-binding fragment or variant thereof, wherein the detection agent comprises a peptide of SEQ ID NO: 1 or an antibody-binding fragment or variant thereof, or wherein the composition comprises a peptide of SEQ ID NO: 1 or an antibody-binding fragment or variant thereof or a detection agent comprising a peptide of SEQ ID NO: 1 or an antibody-binding fragment or variant thereof.

20. A diagnostic kit comprising one or more peptides or antibody-binding fragments or variants of claim 14, one or more detection agents according to claim 15, or a composition according to claim 16, and reagents for detecting antibody binding to said one or more peptides, antibody-binding fragments or variants, or detection agents.