WO2013185181A1 - Diagnostic method for diseases characterised by ro/la autoimmunity - Google Patents

Abstract

A method of detecting autoantibodies associated with Ro/La autoimmunity comprising detecting, in a serum sample or other sample comprising immunoglobulin G (IgG), autoantibodies to a Ro and/or La antigen comprising a variable (V)-region molecular signature comprising an unusual or common heavy (H) and light (L)-chain pairing and/or at least one mutation in one or more of said H- and L-chains and/or an adjacent region. The method enables the detection of autoantibodies associated with one or more autoimmune disease or disorder characterised by Ro/La autoimmunity such as Sjögren's syndrome (SS), systemic lupus erythematosus (SLE), neonatal lupus syndrome, other systemic rheumatic diseases, polymyositis, progressive systemic sclerosis, primary biliary cirrhosis and congenital heart block (CHB).

Description

DIAGNOSTIC METHOD FOR DISEASES CHARACTERISED BY Ro/La AUTOIMMUNITY

PRIORITY DOCUMENT

[0001] The present application claims priority from:

United States Provisional Patent Application No. 61 659922 titled "Diagnostic method for autoimmune diseases" and filed on 14 June 2012.

The content of this application is hereby incorporated by reference in its entirety.

TECHNICAL FIELD

[0002] The present invention relates to a method of detecting autoantibodies associated with Ro/La autoimmunity. In a particular application, the method may be used for diagnosis of one or more autoimmune disease or disorder characterised by Ro/La autoimmunity such as Sjogren's syndrome, systemic lupus erythematosus and congenital heart block.

BACKGROUND

[0003] Sjogren's syndrome (SS) and lupus (systemic lupus erythematosus; SLE) are two of the most common autoimmune diseases with, respectively, up to 4 million and 1.5 million patients in the United States alone^{1, 2}.

[0004] In Sjogren's syndrome, cell-mediated auto-immunity causes apoptosis of epithelial cells of the exocrine glands (which produce tears and saliva) leading to characteristic dry eyes (xerostomia) and dry mouth (keratoconjunctivitis sicca). The disease is, however, "systemic" and accordingly affects many other organs and tissues including the gastrointestinal (GI) tract, kidneys, lungs, liver, pancreas, blood vessels and the peripheral and central nervous system (PNS/CNS). About nine out of every ten patients are women and, while typical onset occurs in individuals in their late forties, the syndrome does occur in all age groups in both women and men. It can also exist as a disease in its own right (primary Sjogren's syndrome) or may otherwise follow the onset of an associated autoimmune disease such as SLE, rheumatoid arthritis, scleroderma and primary biliary cirrhosis (secondary Sjogren's syndrome).

[0005] In systemic lupus erythematosus, auto-immunity leads to the attack of organs and tissues (eg heart, joints, skin, kidneys, lungs, liver and blood vessels) resulting in inflammation and tissue damage. Like SS, SLE is vastly more prevalent in women than men. It is also more common in individuals of a non-European descent. Typically, in adult women, onset of the disease occurs between 15 and 35 years of age. In children, the onset of SLE generally occurs between 3 and 15 years of age. [0006] For both SS and SLE, it is recognised that early diagnosis and proper treatment are critically important in reducing patient symptoms and improving patient quality of life. However„unfortunately, both have proven to be very difficult to diagnose, in part due to the range and unpredictability of symptoms and their similarity to that of other diseases and conditions. As a consequence, SS can often be overlooked or misdiagnosed and, on average, it presently takes nearly seven years for a patient to receive a diagnosis of Sj6gren's syndrome after initial presentation¹. For both diseases, the diagnosis may be based, in part, upon serologic testing of anti-extractable nuclear antigen (ENA) and anti-nuclear antibody (ANA); particularly, anti-Ro60 (SS A), anti-Ro52 and/or anti-La (SSB) antibody.

[0007] Whilst most research on Ro60 and the linked Ro52 (also termed TR1M21) and La autoantigens has focussed on fine mapping of their epitopes and apotopes^{5, 6}, little is known about the clonality and B- cell receptor repertoires of the autoantibodies themselves. In part, this is due to the fact that animal models are of limited value in understanding B cell repertoire selection in Ro La autoimmunity, because spontaneous anti-Ro La autoantibodies are not produced in standard mouse models of lupus. In addition, genomic studies of single cells have likewise been unable to provide a comprehensive snapshot of the B- cell receptor repertoire of systemic autoantibodies since these techniques involve a selection bias and are not easily applicable to the study of large patient populations.

[0008] While Burnet's Forbidden Clone hypothesis⁷ suggests that autoantibodies should be clonal, single cell approaches are unsuitable for identifying shared (ie "public") autoreactive B-cell clonotypes, an important consideration in diseases such as SS and SLE in which molecular characterisation of clonotypic autoantibodies may lead to therapeutic strategies aimed at removing autoreactive B-cell clones. On the other hand, direct protein sequencing of clonotypic autoantibodies from human serum samples, offers distinct advantages over conventional genomic approaches in terms of analysing the autoantibody repertoire in human disease⁸ and, to this end, the present applicants have now identified such an approach which enables the determination of the actual protein sequence (rather than the predicted translated sequence) of the variable (V)-region of autoantibodies from multiple patient samples, which in turn, enables the identification of characteristic VH/V_l families and/or common or "public" mutations. Thus, the present invention is directed at the identification of autoantibody

"molecular signatures" of autoimmune diseases such as SS and SLE, and the uses of such molecular signatures in diagnostic methods.

SUMMARY

[0009] The present invention provides a method of detecting autoantibodies associated with Ro/La autoimmunity, said method comprising the steps of;

(i) providing a serum sample or other sample comprising immunoglobulin G (IgG), and (ii) detecting the presence of autoantibodies to a Ro and/or La antigen comprising a variable (V)- region molecular signature comprising an unusual or common heavy (H) and light (L)-chain pairing and/or at least one mutation in one or more of said H- and L-chains and or an adjacent region.

[0010] The method enables the detection of autoantibodies associated with one or more autoimmune disease or disorder characterised by Ro/La autoimmunity such as, for example, Sj6gren's syndrome (and, particularly, primary SS), systemic lupus erythematosus (SLE), neonatal lupus syndrome and congenital heart block (CHB). The step of detecting the autoantibodies is preferably conducted using a targeted mass spectroscopy (MS) approach to directly sequence at least part of the V-region(s) of the autoantibodies comprising the molecular signature. _^

BRIEF DESCRIPTION OF FIGURES

[001 1] Figure 1 shows the results of direct sequencing of anti-Ro60peg IgG, revealing a public clonotype with a distinctive molecular signature: (A) L-chain V-region sequences are aligned with germline V_K3-20 and K joining family J2. A common arginine (R) mutation was found in the L-chain CDR3 region with a G-to-R substitution at position 91 or an S-to-R substitution at the adjacent position 92; (B) H-chain V-region sequences are aligned with germline V_H3-23 and H joining family J5. Shared mutational patterns were observed in the H-chain CDR1 with an S-to-D substitution at position 30 or 31 or an S-to-N substitution at position 30. Dots indicate amino acids matching with germline sequence, germline CDRs are Underlined, amino acids which are divergent from the germline are indicated as text, with common mutations shown in grey. Spaces indicate areas of incomplete sequence. Patient codes are given as pSSl to SS7;

[0012] Figure 2 provides examples of targeted MS spectra of surrogate peptides from serum: (A) pepl ; (B) pep5; (C) pep6; and (D) pep7. The precursor ion error mass tolerance was set to 10 ppm with the fragment ion error mass tolerance 0.8 Da;

[0013] Figure 3 shows the compiled results of the direct sequencing of anti-LaA IgG from seven primary SS patients, revealing public variable (V)-region public (shared) mutations: (A) H-chain V- region sequences aligned with germline V_H3-43 and V_H3-30; (B) L-chain V-region sequences aligned with germline V_K3-20 and V_K3-15; and (C) L-chain J-regions aligned with J2 and J4 germline sequence and H-chain J-regions aligned with J2. Public mutations divergent from the germline sequence are indicated and coded according to the prevalence of the mutation in the primary SS patient cohort analysed. Dots indicate amino acids matching to the germline sequence, and CDRs are boxed; and

[0014] Figure 4 shows the compiled results of the direct sequencing of anti-Ro52 IgG from eight primary SS patients, revealing clonal restriction and public variable (V)-region mutations: (A) L-chain V-region sequences aligned with germline V_K3-20 and K joining families Jl and J5; and (B) H-chain V- region sequences aligned with germline V_H3-23 and V_H3-7, and with the H joining peptides J2 and J6*2. Public mutations divergent from the germline are indicated and coded according to prevalence. Again, dots indicate amino acids matching with germline sequence, germline CD s are boxed, and double underlining indicates the surrogate peptides used in the targeted mass spectroscopy (MS).

DETAILED DESCRIPTION

[0015] The application of mass spectroscopy (MS) to clinical diagnostics remains in its infancy; to date this technology has not been considered as a tool for detection of specific autoantibodies in biological samples from patients with autoimmune diseases. Instead, solid-phase ELISA and/or immunoblot (lineblot) assays are currently the most widely used methods for detecting serum autoantibodies against extractable nuclear antigen (ENA) family members such as Ro60, Ro52 and La, although these have been improved through semi-automation and have largely replaced older techniques such as immunodiffusion. However, immunoassays are typically expensive; time-consuming; require careful checks and balances in the diagnostic setting because of quality issues relating to antigen stability and antibod conjugates; are often difficult to interpret when results lie in the indeterminate cut-off zone; and, additionally, provide only an indirect measure of circulating autoantibody via multiple detection steps. The present invention relates to a novel diagnostic method for direct detection of autoantibodies in patient samples that utilises V-region domain molecular signatures of public autoantibody clonotypes identified via, for example, a targeted MS surrogate V-region peptide approach. While not wishing to be bound by theory, the success of the method relies, at least in part, on the present applicants' recognition that in human systemic autoimmune disease, the production of pathogenic autoantibodies is driven by restricted sets of public B-cell clonotypes which undergo clonal succession.

[0016] In a first aspect, the present invention provides a method of detecting autoantibodies associated with Ro La autoimmunity, said method comprising the steps of;

(i) providing a serum sample or other sample comprising immunoglobulin G (IgG), and

(ii) detecting the presence of autoantibodies to a Ro and or La antigen comprising a variable (V)- region domain molecular signature comprising an unusual or common heavy (H) and light (L)- chain pairing and/or at least one mutation in one or more of said H- and L-chains and/or an adjacent region.

[0017] The method enables the detection of autoantibodies associated with Ro/La autoimmunity; that is, autoantibodies directed against a Ro antigen (eg Ro60 and Ro52) or a La antigen. As such, the method enables the detection of autoantibodies associated with one or more autoimmune disease or disorder characterised by Ro La autoimmunity such as, for example, SjSgren's syndrome (and, particularly, primary SS), systemic lupus erythematosus (SLE), neonatal lupus syndrome, other systemic rheumatic diseases, polymyositis, progressive systemic sclerosis, primary biliary cirrhosis and congenital heart block (CHB)(nb. it has been reported that anti-Ro60 autoantibodies initiate tissue damage in CHB and drive the production of type I interferons in systemic autoimmunity¹⁷' ^IS) such that the autoantibodies represent one or more biomarkers of the autoimmune disease or disorder.

Accordingly, in one embodiment, the method of the present invention is used for the diagnosis of an autoimmune disease or disorder selected from the group consisting of SS, SLE, neonatal lupus syndrome and CHB. In another embodiment, the method of the present invention is used for the diagnosis of an autoimmune disease or disorder selected from the group consisting of systemic rheumatic diseases, polymyositis, progressive systemic sclerosis and primary biliary cirrhosis.

[0018] Accordingly, the method of the present invention may be used for the diagnosis of an autoimmune disease or disorder associated with Ro/La autoimmunity such as those mentioned in the preceding paragraph. It is to be understood that where the method is used for such a diagnostic purpose, the method may involve detecting autoantibodies to more than one autoantigen. For example, the method may involve detecting autoantibodies to Ro60 and La for the diagnosis of primary SS, SLE, neonatal lupus syndrome or other autoimmune disease or disorder associated with Ro La autoimmunity (nb. it is known that Ro60 and La are physically linked as protein components of a ribonucleoprotein complex where they bind to non-coding cytoplasmic (Y) RNAs, and this is thought to explain the recurrence of anti-Ro60 and anti-La autoantibodies as linked autoantibody sets via intermolecular epitope spreading³). Moreover, it has been found that autoantibodies to Ro52 are strongly linked to anti- Ro60 La responses in primary SS and SLE, so the method of the invention may also involve detecting autoantibodies to Ro60, La and Ro52 or autoantibodies to La and Ro 52 for the diagnosis of primary SS, SLE or other autoimmune disease or disorder associated with Ro/La autoimmunity. On the other hand, it is known that anti-Ro52 autoantibodies may occur as an isolated species in patients with polymyositis, systemic sclerosis, primary biliary cirrhosis and primary SS²⁰. Thus the method may involve detecting autoantibodies to Ro52 only.

[0019] In addition, when used for the diagnosis of an autoimmune disease or disorder associated with Ro/La autoimmunity, it will be understood by those skilled in the art that the method of the invention may be used in combination with an independent analysis of one or more other biomarkers or potential biomarkers of the autoimmune disease or disorder and/or other tests such as, for example, for primary SS: the detection of rheumatoid factor (RF) in blood, Schirmer's test (for deficient lacrimal function), non-stimulated whole saliva collection test and other criteria included in the revised Classification Criteria for Sjogren's Syndrome²¹. [0020] The method of the present invention is preferably conducted with a serum sample (eg whole serum), however other sample types comprising IgG (eg polyclonal IgG) may also be suitable such as a sample of whole blood or plasma. Alternatively, the sample may be a sample that has been enriched for IgG or even the desired autoantibody component. For example, an enriched IgG sample may be prepared from a serum sample by any of the standard IgG purification processes well known to those skilled in the art (eg melon gel IgG purification). Additionally or alternatively, a sample may be enriched for the desired autoantibody component by, for example, passing the sample through an appropriate affinity column, and thereafter eluting the column-bound IgG. Thus, for example, to enrich a sample for anti-Ro60 autoantibodies, a serum sample can be subjected to affinity column purification with a column comprising Ro60peg fragment (amino acids 193-236 including an immunodominant apical peg-like epitope) to bind anti-Ro60 autoantibodies. Similarly, to enrich a sample for anti-Ro52 autoantibodies, a serum sample can be subjected to affinity column purification with a column comprising a protein p200 fragment (comprising amino acids 200-239¹⁹) to bind anti-Ro52

autoantibodies. To enrich a serum sample for anti-La autoantibodies, serum can be subjected to affinity column purification with a column comprising LaA (an immunodominant region comprising amino acids 1-107²⁶).

)

[0021] The step of detecting the presence of anti-Ro or anti-La autoantibodies involves determining the presence of a V-region domain molecular signature characteristic of these autoantibodies. Such a molecular signature preferably comprises, for example, a V-region with a V_K3-20-encoded L-chain paired with a V_H3-23-encoded H-chain, a V_H3-7-encoded H-chain or a V_H3-43-encoded H-chain, and/or at least one mutation in one or more of said H- and L-chains. The mutation(s) may be within one or more complementarity-determining region (CDR) region. For example, in one embodiment, the mutation(s) may be within one or more CDR within the V_H3-23-encoded, Vn3-7-encoded H-chain or V_H3 -43 -encoded H-chain and/or V_K3-20-encoded L-chain, but may additionally or alternatively be located within the adjacent framework (FR) and/or Joining (J)-segment regions (eg within 1-30 amino acids of the C-terminal amino acids of the H- and/or L-chain, depending upon the presence of any Diversity (D)-region). In another embodiment, mutation(s) may be within one or more CDR within the V_H3-30-encoded H-chain and or V_K3-15-encoded L-chain, but may additionally or alternatively be located within the adjacent FR and/or J-segment regions (eg within 1-30 amino acids of the C-terminal amino acids of the H- and/or L-chain, depending upon the presence of any D-region).

[0022] As used herein, the term "V-region domain" is to be understood as referring to an antibody domain comprising the Variable (V)-region of the H- and/or L-chain and any Diversity (D)- and Joining (J)-regions.

[0023] Accordingly, the step of detecting the presence of anti-Ro or anti-La autoantibodies may, in one embodiment, comprise simply determining the presence of IgG with a V-region with a V_K3-20-encoded L-chain paired with a V_H3-23-encoded, V_H3-7 -encoded or V_H3 -43 -encoded H-chain. However, in a preferred embodiment, the step of detecting the presence of anti-Ro or anti-La autoantibodies may comprise determining the presence of one or more mutation in one or more H- and L-chains of the V- region domain. Of course, the present invention also contemplates a step of detecting the presence of anti-Ro or anti-La autoantibodies that comprises determining both the presence of IgG with a V-region with a V_K3-20-encoded L-chain paired with a V_H3 -23 -encoded, V_H3-7-encoded or V_H3-43-encoded H- chain, and one or more mutation in one or more H- and L-chains of the V-region domain.

[0024] The one or more mutation that may be detected in accordance with the invention will preferably represent a mutation(s) that is commonly found in samples from unrelated subjects (eg unrelated primary SS patients); for example, a mutation that is found in the autoantibodies present in samples from more than 50% of relevant patients or a subset of relevant patients. Such mutations may be regarded as "public". They may characterise useful subsets of subjects (eg SLE patients at risk of having CHB babies). The mutation may be an amino acid substitution, deletion or addition. Where two or more mutations are to be detected, the mutations may be any combination of an amino acid substitution, deletion and addition, or all may be amino acid substitutions, deletions or additions.

[0025] In one embodiment of the method of the present invention, wherein the method is conducted to detect autoantibodies against Ro52, the method may involve detecting one or more mutation in CDRl , CDRl and/or CDR3 of the V_K3-20-encoded L-chain and/or one or more mutation in CDRl and/or CDR2 of the V_H3-23-encoded or V_H3-7-encoded H-chain, but more preferably involves detecting at least one sequence motif selected from the group consisting of LSCAASGFTFR (SEQ ID NO: 1), LSCAASGFTF.fi. (SEQ ID NO: 2), LSCAASGFR (SEQ ID NO: 3), LSCAASGF (SEQ ID NO: 4), RLVESGGGLVQPGGSLR (SEQ ID NO: 5), ALVESGGGLVQPGGSLR (SEQ ID NO: 6),

LLLYGASNR (SEQ ID NO: 7), and ASQSVSSNLAWYQQK (SEQ ID NO: 8), or at least one of the mutations found therein (ie as indicated in italics, although for the motif of SEQ ID NO: 8 there is also a deletion mutation after residue N).

[0026] In another embodiment of the method of present invention, wherein the method is conducted to detect autoantibodies against Ro60, the method may involve detecting one or more mutation in CDR3 of the V_K3-20-encoded L-chain and/or one or more mutation in CDR2 of the Vn3-23-encoded H-chain, but more preferably involves detecting at least one sequence motif selected from the group consisting of LEPEDFAVYYCQQYR (SEQ ID NO: 9), LEPEDFAVYYCQQYGR (SEQ ID NO: 10) and

LSCAASGFTF£>SYAMSWVR (SEQ ID NO: 11), or at least one of the mutations found therein (ie as indicated in italics).

[0027] In yet another embodiment of the method of present invention, wherein the method is conducted to detect autoantibodies against La, the method may involve detecting one or more mutation in CDRl of the V_K3-20-encoded L-chain and/or one or more mutation in the FR1 of the V_H3-30-encoded H-chain, but more preferably involves detecting at least one sequence motif selected from the group consisting of ASQSVSNYLAWYQQK (SEQ ID NO: 12) or ASQSVSNNLAWYQQK (SEQ ID NO: 13), or at least one of the mutations found therein (ie as indicated in italics).

[0028] Preferably, the step of detecting the presence of anti-Ro or anti-La autoantibodies is conducted by direct sequencing of the autoantibodies or, preferably, a part(s) thereof (eg the full-length V-region or CDRs therein).

[0029] Particularly suitable direct sequencing processes are those utilising mass spectroscopy (MS) as are well known to those skilled in the art. In the examples described hereinafter, direct sequencing of autoantibodies is achieved with high-resolution orbitrap mass spectrometry utilising IgG fragments generated by trypsin digestion. The potential mass accuracy offered by an orbitrap mass spectrometer (<10 ppm) allows for the highly specific selection of a desired "target" autoantibody peptide fragment(s) (eg fragments making up the full length V-region or a CDR therein) referred to as the target precursor ion mass; this, combined with the sensitivity of the ion trap (eg a detection rate of > 7 scans per second), allows for the subsequent fragmentation and sequencing of the target peptide fragment(s). Such a targeted MS approach to direct sequencing may utilise a reference or "surrogate" peptide (typically labelled with a stable isotope such as ²H, ^I3C or ^,5Na provided on, preferably, a C-terminus amino acid (eg lysine- or arginine-labelled) corresponding to a desired autoantibody peptide fragment (eg a surrogate V-region peptide) so as to enable the identification of the target precursor ion mass (ie mass to charge ratio (m/z)). Once that target precursor ion mass/charge is identified, the orbitrap may be operated to isolate the target peptide fragment based upon m/z for MS-based direct sequencing.

[0030] As mentioned above, in the examples described hereinafter, direct sequencing of autoantibodies is achieved with high-resolution orbitrap mass spectrometry. Those skilled in the art will, however, recognise that direct sequencing could be otherwise conducted using, for example, a triple quadrupole mass spectrometer operated with an ion trap.

[0031] In some embodiments of the method of the present invention, wherein targeted MS is used to directly sequence an autoantibody fragment(s), the method may be conducted in a manner whereby a V- region peptide fragment is isolated (for direct sequencing) having a suitable mass to charge ratio (m/z) in the range of, for example, 300 to 2000, more preferably 450 to 1000, such as, for example, a V-region domain peptide fragment consisting of the amino acid sequence: LSCAASGFTFR (SEQ ID NO: 1) (m/z 608.79), LSCAASGFTFK (SEQ ID NO: 2) (m/z 594.79), LSCAASGFR (SEQ ID NO: 3) (m/z 484.73), LSCAASGFK (SEQ ID NO: 4) (m/z 470.73), LVESGGGLVQPGGSLR (SEQ ID NO: 5) (m/z 763.42), LLLYGASNR (SEQ ID NO: 7) (m/z 503.78), ASQSVSSNLAWYQQK (SEQ ID NO: 8) (m/z 848.92), LEPEDFAVYYCQQYR (SEQ ED NO: 9), LEPEDFAVYYCQQYGR (SEQ ID NO: 10), and ASQSVSNYLAWYQQK (SEQ ID NO: 12) (m/z 443.23) or ASQSVSNNLAWYQQK (SEQ ID NO: 13) (m/z 430.97).

[0032] It will be understood by those skilled in the art that when targeted MS is used in the method of the present invention, the targeted MS may be conducted in a multiplexed manner. That is, the method of the present invention has the capacity for multiplex antibody detection in a sample (eg a test sample of serum) by simultaneously using multiple surrogate peptides.

[0033] In a second aspect, the present invention provides a peptide comprising or consisting of an amino acid sequence selected from the group consisting of: LSCAASGFTFR (SEQ ID NO: 1), LSCAASGFTFK (SEQ ID NO: 2), LSCAASGFR (SEQ ID NO: 3), LSCAASGFK (SEQ ID NO: 4), LVESGGGLVQPGGSLR (SEQ ID NO: 5), LLLYGASNR (SEQ ID NO: 7), ASQSVSSNLAWYQQK (SEQ ID NO: 8), LEPEDFAVYYCQQYR (SEQ ID NO: 9), LEPEDFAVYYCQQYGR (SEQ ID NO: 10), ASQSVSNYLAWYQQK (SEQ ID NO: 12) and ASQSVSNNLAWYQQK (SEQ ID NO: 13).

[0034] Where the peptide comprises one of the said amino acid sequences, it is to be understood that the peptide will be of no more than about 20 amino acids in length. The additional amino acids may represent tag or linker sequences (eg to link the peptide to a protein carrier molecule or solid support surface) or one or more amino acids found adjacent to the particular amino acid sequence within the antibody V-region from which it is derived.

[0035] The peptide may be provided in a substantially isolated form.

[0036] The peptide may be produced by recombinant techniques or protein synthesis methods well known to those skilled in the art.

[0037] The peptide may be used as a positive control for the method of the present invention.

[0038] The peptide may be labelled with a stable isotope (eg ²H, ¹³C and ¹⁵N) or other detectable label including, but not limited to, radioisotopes, haptens such as, for example, biotin, and fluorescent labels such as fluorescein derivatives (eg FITC) and rhodamine derivatives (eg TAMRA)).

[0039] When labelled with a stable isotope, the peptide may be used in a method for the mass spectroscopic quantification of autoantibodies associated with Ro La. Suitable MS-based methods for quantification of a peptide in a sample are known to those skilled in the art⁹. [0040] The peptide may also be used in the production of monoclonal and polyclonal antibodies (eg anti-idiotypic antibodies) by any of the antibody production methods well known to those skilled in the art.

[0041] Thus, in a third aspect, the present invention provides a monoclonal or polyclonal antibody, preferably in an isolated form, that specifically binds to a peptide comprising or consisting of an amino acid sequence selected from the group consisting of: LSCAASGFTFR (SEQ ID NO: 1),

LSCAASGFTFK (SEQ ID NO: 2), LSCAASGFR (SEQ ID NO: 3), LSCAASGFK (SEQ ID NO: 4), LVESGGGLVQPGGSLR (SEQ ID NO: 5), LLLYGASNR (SEQ ID NO: 7), ASQSVSSNLAWYQQK (SEQ ID NO: 8), LEPEDFAVYYCQQYR (SEQ ID NO: 9), LEPEDFAVYYCQQYGR (SEQ ID NO: 10), ASQSVSNYLAWYQQK (SEQ ID NO: 12) and ASQSVSNNLAWYQQK (SEQ ID NO: 13).

[0042] The invention is hereinafter described by way of the following, non-limiting examples.

EXAMPLES

Example 1 Identification of molecular signatures on anti-Ro60 autoantibodies

[0043] The Ro60 autoantigen is a major target of humoral autoimmunity in primary SS and SLE. However, while considerable effort has been focused on Ro60 epitope recognition and, more recently, on the mapping of Ro60 apotopes, little is known about the molecular characteristics of the anti-Ro60 autoantibodies themselves. A direct way of analysing the clonality and immunoglobulin variable (IGV) gene usage of humoral anti-Ro60 autoimmunity is to study the proteome of the circulating

autoantibodies. However, this has been hampered by the marked diversity and polyclonality of established humoral anti-Ro60 responses, which have generally undergone marked intra- and inter- molecular spreading to the linked Ro52 and La/SSB proteins by the onset of symptoms^{4, 11}. Thus by the time of clinical presentation, individual clonotypes are likely to be obscured by polyclonal anti-Ro/La autoantibody reactivity. The present applicants reasoned that an initial positive selection of complex anti-Ro/La polyclonal sera on a structurally defined Ro60 epitope might narrow the clonotypic diversity of established anti-Ro60 responses sufficiently for direct sequencing of purified autoantibodies.

Materials and Methods

[0044] Patient samples

[0045] Serum samples were collected from seven patients with primary SS with anti-Ro La autoantibodies who were positive for anti-Ro60peg on a maltose binding protein (MBP)-Ro60peg fusion protein ELISA¹². Control samples included samples from two patients with primary SS with anti-Ro/La autoantibodies without anti-Ro60peg antibodies, one asymptomatic donor with anti-Ro La

autoantibodies with anti-Ro60 peg antibodies, one asymptomatic mother of a baby with CHB with anti- R06O peg antibodies, and four healthy donors.

[0046] Preparation and specificity analysis of affinity-purified anti-Ro60 IgG

[0047] Affinity purification columns were made using cyanogen bromide (CnBr)-activated Sepharose 4B beads (GE Healthcare, Chalfont St Giles, Bucks, United Kingdom) coupled with the protein antigen/epitope (soluble recombinant MBP-R06O aa 82-244 fragment that includes the Ro60peg epitope) used for affinity purification. Five ml of serum from each patient or control subject was then passed over the column and, after overnight washing with phosphate buffered saline (PBS), bound IgG fraction was eluted with 0.1M glycine, 0.5M sodium chloride, pH 2.3. Eluted IgG fractions were then neutralised in 1M Tris HCL, pH 8.0, dialysed against PBS, pH 7.4, and then concentrated to 50 μg/ml with an Amicon concentrator (Millipore Corporation, Billerica, MA, United States of America).

[0048] Preparation of samples for mass spectrometry (MS)

[0049] Peptide samples were prepared for MS analysis by:

(i) In-solution digestions of affinity-purified IgG

Two or more affinity-purified IgGs from each serum sample were precipitated using a 2D Clean Up Kit (Bio-Rad Laboratories, Inc., Hercules, CA, United States of America) according to manufacturer's instructions and resuspended in 20 μΐ of lOOmM ammonium bicarbonate, pH 8.0. The IgG was then reduced by the addition of DTT to 5 mM with incubation at 100 °C for 5 min and alkylated by the addition of iodoacetamide to 20 mM. Samples were then digested with 100 ng of Trypsin Gold

(Promega Corporation, Madison, WI, United States of America) followed by incubation for 16 h at 37 °C;

or

(ii) In-gel digestions of affinity-purified IgG

Gel plugs were excised, washed 5 times with 100 mM ammonium bicarbonate and shrunk with acetonitrile. The gel plugs were then dried in a 37 °C oven and 10 μΐ of Trypsin Gold (10 ng μΐ, Promega) added followed by incubation on ice for 15 minutes. Excess trypsin was removed and 10 μΐ of lOOmM ammonium bicarbonate was added. Samples were incubated for 16 h at 37 °C. The digested solutions were then removed from each sample and placed in MS vials. [0050] MS analysis

[0051] Peptides were analysed using a LTQ Thermo Orbitrap XL linear ion trap mass spectrometer fitted with a nanospray source (Thermo Electron Corporation, Waltham, MA, United States of

America).

[0052] The digested peptides were applied to either a 300 μπι i.d. x 5 mm C_)gPepMap 100 NanoViper trap column (Dionex, Sunnyvale, CA, United States of America) or a 100 um i.d. x 20 mm 100 A precolumn (Dionex), and separated on a 75 mm x 150 mm C_]8 5 μπι 100 A column (Nikkyo Technos Co., Ltd, Tokyo, Japan), using a Dionex Ultimate 3000 HPLC (Dionex). A 55 min gradient from 2% acetonitrile to 45% acetonitrile containing 0.1% formic acid at a flow rate of 200 nl/min was run, followed by a step to 77% acetonitrile for 9 min. The mass spectrometer was operated in positive ion mode with one Fourier transform MS scan of mass/charge (m/z) 300-2000 at 60,000 resolution followed by high energy collision-induced dissociation fragmentation and Fourier transform MS product ion scans of the 6 most intense ions. A dynamic exclusion of 30 sec with 10 ppm low and high mass width relative to the reference mass with an exclusion list of 500 and collision energy of 35%. Only multiply charged ions were selected for MS/MS.

[0053] Protein sequence data analysis

[0054] Database searches were carried out with Proteome Discoverer version 1.2 (Thermo Electron Corporation) using the Sequest algorithm against a combined database of IMGT (ImMunoGeneTics information system®) sequences¹⁰, containing human H- and L-chains and the Uniprot 2010-06 database. The database search parameters were: a maximum of 2 missed trypsin cleavages, cross- correlation scores (Xcorr) of matches were greater than 1.5, 2.0, 2.25, 2.5 and 2.75 for charge states 1, 2, 3, 4 and 5 peptide ions respectively; peptide probability was high and 2 or more unique peptides were sequenced for each protein. The mass tolerance for peptide identification of precursor ions was 10 ppm and 0.01 Da for product ions. Searches were carried out with the following variable modifications; the oxidation of methionine, phosphorylation of serine, threonine and tyrosine, and carboxymethylation of cysteine. De novo sequencing was performed on raw data files with Peaks Studio version 5.2

(Bioinformatics Solutions Inc., Waterloo, ON, Canada). Sequences in Peaks were searched against the combined MGT/ Uniprot 2010-06 database. Data refinement was carried out on the raw data files with the following parameters: scans were merged with a retention time of 1 min, a precursor m z error tolerance of 10 ppm and a minimum charge state of 2, scans were filtered a precursor mass of between 350 and 5000 Da and a quality value of greater than 0.65. De novo sequencing in Peaks was carried out using the de novo module with the parent ion error tolerance set to 10 ppm and the variable

modifications of the oxidation of methionine, phosphorylation of serine, threonine and tyrosine, deamidation of asparagine and glutamine, and the carboxymethylation of cysteine. The SPIDER search tool was used to identify mutations or variations from the germline IMGT sequences by searching against the combined IMGT Uniprot 2010-06 database with the following parameters: a homology match query type, a mass error tolerance of 0.01 Da and the previously described variable modifications. Spectra was analysed in the inChorus search tool which combined the Peaks Protein ID, X! Tandem and OMSSA search tools with the following parameters: a parent and fragment ion error tolerance of 10 ppm and 0.01 Da respectively, up to 3 missed trypsin cleavages and the previously described variable modifications.

Results

[0055] IgGs for Ro60 peg were obtained and specificity confirmed by analysing starting, flow-through and eluted column fractions on Ro52/Ro60/La ELISAs and Ro60 subfragment ELISAs (data not shown). In addition, immunoblots of overlapping Ro60 subfragments confirmed that reactivity was restricted to the Ro60peg determinant (data not shown). In control experiments, IgGs were not detectable after passing normal human sera (n=4) or sera from primary SS patients with anti-Ro/La who were negative for anti-Ro60peg (n=2) over the MBP-R06O aa 82-244 column; or after passing anti- Ro60peg-positive sera (n=2) over a sham MBP column.

[0056] Sequencing of constant (C)-regions confirmed a kappa-restricted monotypic population of the IgGl isotype. No lambda L-chain sequence was detected. Combined database-driven and de novo amino acid sequencing of the variable (V)-regions of H- and L-chains was performed and achieved a near full- length V-region sequence in a single run of less than 50 ng of affinity-purified human IgG in the multiple patient samples. In agreement with recent reports of de novo immunoglobulin sequencing, reliable H-CDR3 sequence was not obtained because of the highly random nature and variable sequence quality through this region¹³' ¹⁴. Accordingly, analysis of the Ro60peg-specific IgG V-region sequences revealed a single V_K3-20-encoded L-chain in each patient that was further specified by arginine substitutions in the H-CDR3 at either position 91 (3 primary SS patients) or 92 (4 primary SS patients) of the CDR3s (Figure 1 A). Faint adjacent L-chain 2D-GE gel spots near the dominant pi 6.8 species were excised as gel plugs, sequenced and shown to be V_K3-20-encoded, in keeping with L-chain isoforms of monoclonal antibodies commonly observed on 2D-GE. Similarly, mass spectrometric sequencing of individual Ro60peg-specific IgGs revealed a single VH3 -23 -encoded H-chain in all patients, thereby confirming by direct protein sequencing that the clonotypic autoantibody identified on 2D-GE is a monoclonal autoantibody with a unique H L pairing signature. H-chains from different patients contained shared mutations within the H-CDR1 with either aspartic acid substitutions at positions 30 or 31 (4 primary SS patients) or an asparagine substitution at position 30 (2 primary SS patients) (Figure IB). The sequence at position 31 of subject 7 (pSS 7) was unattainable due to trypsin cleavage at the adjacent amino acid site. Apparent random mutations were noted in the L-chain CDR1 and H-chain CDR2 of individual patients. As with L-chains, sequencing of closely spaced H-chain gel plugs confirmed V_H3 -23 -encoded H-chain isoforms that are assumed to arise from post-translational modifications such as N-glycosylation and/or deamidation. Since flow-through fractions were depleted of anti-Ro60peg binding activity (data not shown), it was apparent that all of the anti-Ro60peg reactivity is accounted for by the V_H3-23/V_K3-20 clonotype. Subsequent studies on serum samples collected retrospectively from four of the primary SS patients also indicated that the anti-Ro60peg immune response is long-lived with high-titre humoral responses persisting over many years. Moreover, relative autoantibody affinities as assessed by KSCN elution of bound anti-Ro60peg IgGs remains remarkably constant over years, consistent with a stable phenotype (data not shown).

Discussion

[0057] A key finding of the¹ study described in this example, is that a subset of unrelated primary SS patients, defined by an immune response against an identical determinant, express a public Ro60peg- reactive B cell clonotype specified by a H-chain derived from the V_H3-23 gene segment paired with a L- chain derived from the V_K3-20 gene segment. An implied corollary is that primary SS patients who express this B cell clonotype share a common pathway of autoimmunity to the Ro60peg determinant. To the best of the present applicants' knowledge, this specific H-/L-chain pairing has not been reported previously for human autoantibodies. VH3-23 is commonly employed by human autoantibodies and over-represented in the normal B-cell repertoire¹⁵, and is also used preferentially in some MALT lymphomas. V 3-20, on the other hand, is expressed in B-cell clones of type II mixed

cryoglobulinaemia, an autoimmune disorder that shares a risk for lymphoma in primary SS¹⁶. The identification of common ("public") mutations within the L-chain CDR1 and H-chain CDR2 of patient anti-Ro60peg autoantibodies allows for the recognition of molecular signatures that can serve as biomarkers, such as the motif LEPEDF A V YYCQQYR (SEQ ID NO: 10), LEPEDFAVYYCQQYGR (SEQ ED NO: 1 1) and LSCAASGFTFDSYAMSWVR (SEQ ID NO: 12). Knowledge of these motifs enables the identification of surrogate V-region peptides for use in targeted MS so as to enable the identification of a target precursor ion mass (ie for use in a MS-based method of the present invention). Suitable examples may include the peptides: LEPEDF A VYYCQQYR (SEQ ID NO: 10) and

LEPEDF AVYYCQQYG ? (SEQ ID NO: 1 1 ).

Example 2 Identification of molecular signatures on anti-Ro52 autoantibodies

Materials and Methods

[0058] Patient samples

[0059] Serum samples were collected from eight patients diagnosed with primary SS. [0060] Preparation and specificity analysis of affinity-purified anti-Ro52 IgG

[0061] Affinity purification columns were made using cyanogen bromide (CnBr)-activated Sepharose 4B beads (GE Healthcare) coupled with the protein antigen/epitope (p200 spanning amino acids 200- 239¹⁹) used for affinity purification. Serum from each patient was then passed over the column and, after overnight washing with phosphate buffered saline (PBS), bound IgG fraction was eluted with 0.1M glycine, 0.5M sodium chloride, pH 2.3. Eluted IgG fractions were then neutralised in 1M Tris HCL, pH 8.0, dialysed against PBS, pH 7.4, and concentrated to 50 μ /τη\ with an Amicon concentrator (Millipore Corporation).

[0062] Preparation of samples for mass spectrometry (MS)

[0063] Peptide samples were prepared for MS analysis by:

(i) In-solution digestions of affinity-purified IgG

Affinity-purified IgG samples were precipitated and resuspended in 20 μΐ of lOOmM ammonium bicarbonate, pH 8.0. The IgG was then reduced by the addition of DTT to 5 raM and alkylated by the addition of iodoacetamide to 20 mM. Subsequently, the samples were incubated with 1 μΐ of Trypsin Gold 100 ng μΐ (Promega) overnight at 37 °C;

or

(ii) In-solution digestions of serum

Serum samples were diluted 1 in 20 with lOOmM ammonium bicarbonate, pH 8.0, reduced by the addition of DTT to 5 mM and alkylated by the addition of iodoacetamide to 20 mM. Samples were then incubated with 1 μΐ of Trypsin Gold 100 ng μΐ (Promega) overnight at 37 °C. Prior to MS analysis, the digested serum samples were further diluted 1 in 60 with ultra-pure water.

[0064] Alternative preparation of samples for mass spectroscopy (MS)

[0065] An alternative approach may utilise immunoassay plates, wherein plate wells are coated in a suitable epitope/antigen (in this case, for example, recombinant Ro52 protein lacking the IgG Fc-binding domain), typically at 10 μg/ml in coating buffer. Following incubation of the plate (eg for 1 hr at 37°C), washing in PBS, blocking with a suitable protein, and further washing in PBS, the plate wells may be incubated with an aliquot of patient serum (typically, diluted in PBS) for 2 hrs at 37°C. The plate wells may then be washed three times with 20 mM ammonium bicarbonate, and 30 μΐ of Trypsin Gold (Promega) solution (at 0.5 ng/μΐ in 20 mM ammonium bicarbonate) added to each well, followed by overnight incubation at 37°C. Appropriate wells are then pooled (ie capture is performed in multiple wells) and the final sample boiled down to a volume of approximately 20 μΐ. [0066] MS analysis

[0067] Peptide samples were analysed using a LTQ Thermo Orbitrap XL linear ion trap mass spectrometer fitted with a nanospray source (Thermo Electron Corporation).

[0068] The peptides were applied to a 100 μπι i.d. x 2 cm PepMapl 00 NanoViper trap column (Dionex) and separated on a 100 um i.d. x 150 mm CI 8 5 μηι 100 A column (Nikkyo Technos) using a Dionex Ultimate 3000 HPLC (Dionex), and thereafter eluted with a 55 min gradient from 2% acetonitrile to 45% acetonitrile containing 0.1% formic acid at a flow rate of 200 nl/min, followed by a step to 77% acetonitrile for 12 min. The mass spectrometer was operated in positive ion mode with one Fourier transform (FT) MS scan of mass/charge (m/z) 300-2000 at 60,000 resolution followed by collision induced dissociation (CID) fragmentation and FT MS product ion scans of the 6 most intense ions. The instrument method contained a dynamic exclusion of 25 seconds with an exclusion list of 500, 10 ppm low and high mass width relative to the reference mass and collision energy of 35%. Only multiply charged ions were selected for MS/MS.

[0069] For targeted MS, the methodology was as described above except that the m/z of the target peptides is added to a parent mass list and the 6 most intense ions from the parent list are detected in the linear ion trap. De novo sequencing for the targeted MS was performed as previously described⁸ except with a fragment ion tolerance of 0.8 Da.

[0070] Protein sequence data analysis

[0071] Peaks Studio v5.3 software (Bioinformatics Solutions) was used for all MS analysis. Scores for all peptides were given as a -lOlgP value, with a minimum score of at least 8 (as recommended by Peaks). Data refinement was carried out on the raw data files with the following parameters: scans were merged with a retention time of 30 seconds and a precursor m z tolerance of <10 ppm for the merged scans, precursor charge state minimum of 2, maximum of 5, scan quality value > 0.65, with data processing (peak centroiding, charge deconvolution, deistotope). De novo sequencing of the input spectrum was performed using the Peaks algorithm with precursor and fragment ion error tolerances of 10 ppm and 0.02 Da respectively, trypsin as the specified digestion enzyme, and the variable modifications of oxidation of methionine, carbamidomethylation of cysteine, and deamidation of asparagine and glutamine. The Peaks module was used to perform searches against a combined database of IMGT sequences¹⁰ (containing human H- and L-chains) and the Uniprot 2010-06 database under the parameters described above. Further, the SPIDER module was used to identify variations from the germline IMGT sequences using a homology match query type under the parameters described above. Results

[0072] To determine the clonality of humoral anti-Ro52 responses, Ro52-specific IgGs were first purified from eight patients with primary SS by passing sera with mixed anti-Ro52 Ro60/La specificities over a Ro52 affinity column comprising a recombinant Ro52 protein lacking the IgG Fc-binding domain, in order to avoid non-specific binding of IgG. The specificities of the eluted IgGs were confirmed by Ro52 Ro60 La ELISAs as being monospecific for Ro52.

[0073] De novo amino acid sequencing of the affinity-purified anti-Ro52 IgGs was performed to obtain near full-length autoantibody variable (V)-region sequences, thereby enabling the identification of identifying common ("public") V-region mutations and surrogate peptide fragments (eg incorporating one or more public mutation) in a targeted MS approach to the detection of R052 autoantibodies.

Analysis of the V-region sequences identified public Ro52-reactive B cell clonotypes characterised by a V_K3-20-encoded L-chain paired with either a V_H3-23- or V_H3-7-encoded H-chain. Sequencing of Joining (J) regions revealed a JK 1 -, JK5-, JH2- and JH6-encoded region.

[0074] The anti-Ro52 autoantibody proteome was characterised further by both random and shared amino acid mutations (ie substitutions) in the V-regions of the H- and L-chains. These mutations were particularly evident in the complementarity-determining regions (CDRs), consistent with multiple rounds of selection of intraclonal variants by the continuing exposure to self-antigen. The prevalence of the public mutations in the V_K3-20-J -encoded L-chains and structurally-related V_H3-23 and V_H3-7 Hi- chains were tabulated by proteomic heat maps (see Figure 4).

[0075] To determine whether the expressed anti-Ro52 autoantibody proteome observed in primary SS patients was shared by anti-Ro52 responses in other systemic autoimmune diseases, affinity-purified Ro52 autoantibodies from individual patients with SLE, polymyositis and progressive systemic sclerosis, were also sequenced (data not shown). Irrespective of the disease phenotype and presence of accompanying autoantibodies, each Ro52-specific autoantibody comprised the same public Ro52- reactive B cell clonotypes characterised by a V_K3-20-encoded L-chain paired with either a V_H3-23- or VH3-7 -encoded H-chain. This common molecular signature of Ro52 autoantibodies therefore appears to be a general feature of systemic humoral autoimmunity.

[0076] On the basis of the prevalence of the common V-region mutations, seven public V-region surrogate peptides were selected for targeted MS detection. These were designated as follows: pepl , amino acids 20-31 common to both V_H3-7 and V_H3-23 (ie LSCAASGFTFR; SEQ ID NO: 1); pep2, amino acids 20-31 common to both V_H3-7 and V_H3-23 (ie LSCAASGFTFK: SEQ ID NO: 2); pep3, amino acids 20-29 common to both V_H3-7 and V_H3-23 (ie LSCAASGFR: SEQ ID NO: 3); pep4, amino acids 20-29 common to both V_H3-7 and V_H3-23 (ie LSCAASGFK: SEQ ID NO: 4); pep5, spanning amino acids 4-16 of V_H3-7 (ie LVESGGGLVQPGGSLR: SEQ ID NO: 5); pep6, amino acids 45-53 of V_K3-20 (ie LLLYGASNR; SEQ ID NO: 6); and pep7, amino acids 25-39 of V_K3-20 (ie

ASQSVSSNLAWYQQK: SEQ ID NO: 7). The surrogate peptides were detected in at least 50% of the patients' Ro52-specific IgGs via de novo sequencing; this increased significantly using the more sensitive targeted mass spectrometry (MS).

[0077] Those skilled in the art will understand that leucine and isoleucine residues are essentially itidistinguishable by the MS-based direct sequencing method employed in this example as they share the same ion mass. Hence, leucine residues in the pepl-pep7 sequences may be interchangeable with isoleucine, and vice versa. Thus, for example, an alternative surrogate peptide to pep6 is LLIYGASNR (SEQ ED NO: 14).

Discussion

[0078] As with Ro60peg clonotypes, the V_H- and V_L-regions of the anti-Ro52 IgGs were specified by unique molecular signatures with shared and random mutations, consistent with an active immune response with antigen-driven somatic mutation. The Ro52pepl peptide was selected for testing as the basis of a targeted MS autoantibody detection assay, since this molecular signature is common (ie "public") to all anti-Ro52-positive patients (primary SS and SLE) tested to date. It contains a unique serine to arginine (S-to-R) substitution at amino acid position 3Q that is not present in IMTG or Uniprot 2010-06 databases. Using purified anti-Ro52 IgG as a substrate, the lower limit of detection of the Ro52VHpepl peptide in targeted MS that gave good quality spectra was -50 fg IgG, compared with ~0.5 ng IgG for a parallel, reference anti-Ro52 line blot ELISA (Euroimmun Medizinische

Labordiagnostika AG, Lubeck, GermanyX-Aata not shown). This difference represents a 10,000-fold increase in sensitivity of detection. The results obtained by the two detection methods (using polyclonal IgGs from subjects that were either positive (n=6) or negative (n=6) for anti-Ro52 IgG antibodies) were, however, concordant, thereby providing preliminary validation of the use of targeted MS for a target V- region peptide fragment as an alternative autoantibody platform to ELISA. This can be seen from the representative spectra shown in Figure 3; where (A) shows the mass spectra of Ro52pepl, m/z 608.79, specific to anti-Ro52 autoantibodies, (B) provides a base peak chromatogram of trypsin-digested polyclonal IgG displaying the complexity of the peptide mixture, (C) shows an extracted ion chromatogram indicating that non-targeted de novo sequencing of polyclonal IgG is not sufficiently sensitive to detect peptides specific to anti-Ro52 autoantibodies such as Ro52pepl, this being in contrast to the extracted ion chromatogram of (D) obtained with targeted MS of peptides specific to anti-Ro52 autoantibodies (eg Ro52pepl) which allows for their detection and sequencing in polyclonal IgG. Accordingly, the identification of a V-region molecular signature for Ro52 autoantibodies comprising a V_K3-20 L-chain paired with a V_H3-23 or V_H3-7 H-chain and/or one or more V-region amino acid mutation, enables the use of Ro52 autoantibodies present in patient serum samples as a biomarker for one or more autoimmune disease or disorder characterised by Ro52 autoimmunity.

Example 3 Identification of molecular signatures on anti-La autoantibodies

[0079] High-titre IgG autoantibodies to the 48 kDa La/SSB protein are a serological hallmark of primary SS but are also associated with SLE and neonatal lupus syndrome. La is physically linked with the 60 kDa Ro/SSA (Ro60) protein in ribonucleoprotein (RNP) complexes that are thought to drive combined anti-Ro60/La humoral responses via T cell-dependent intermolecular determinant spreading". Epitope mapping of the La autoantigen in patients with primary SS has revealed three immunodominant regions: LaA (aa 1-107); LaC (aa 1 1 1-242); and LaL2/3 (aa 346-408)²⁶. Autoantibodies directed against the conserved winged-helix LaA determinant²⁵ are of particular significance because they occur in —100% of precipitin-positive sera and appear to arise early in the anti-La response²⁶; are present at the highest concentration (mg/ml range) of any determinant²⁷; bind the analagous LaA apotope on the surface of apoptotic cells where they form IgG-immune complexes²⁸'²⁹; react with a conserved discontinuous epitope³⁰; and are present in sera from -80% of mothers of children with CHB²⁹. While an early study reported restricted heterogeneity and kappa L-chain oligoclonality of humoral responses against recombinant fragments encompassing LaC and LaL2/3³¹, nothing is known at a molecular level about the variable (V) gene usage and V-region mutational status of spontaneous Human anti-La autoantibodies in primary SS patients.

Materials and Methods

[0080] Patient samples

[0081] Serum samples were collected from seven primary SS patients with anti-Ro/La autoantibodies who were positive for anti-LaA antibody on a glutathione-S-transferase (GST)-LaA fusion protein enzyme-linked immunosorbent assay (ELISA). Patient characteristics and serological findings are shown in Table 1. Control samples included samples from two anti-Ro La positive primary SS patients who were negative for anti-LaA antibody, one asymptomatic donor with anti-Ro/La autoantibodies with anti-LaA antibody and four healthy donors. None of the patients had evidence of malignant B cell disorders nor were any being treated with steroids or other immunosuppressive drugs. [0082] Table 1 Characteristics of patients with primary SS

Autoantibodies were measured by enzyme-linked immunosorbent assay, with optical density (OD) <0.2 considered normal;† total IgG and rheumatoid factor (RF) were measured by nephelometry.

[0083] Preparation and specificity of affinity purified anti-LaA autoantibodies

[0084] CNBr-activated Sepharose 4B beads (Pharmacia Corporation, Uppsala, Sweden) were coupled with the soluble recombinant GST-LaA fragment (aa 1-107) in accordance with manufacturer's instructions. Five mL of serum from each patient or control subject was passed over the column and washed overnight with phosphate buffered saline (PBS, pH 7.4). The bound IgG fraction was then eluted with 0.1M glycine in 0.5M NaCl, pH 2.3 and neutralised in 1M Tris HC1, pH 8.0. The resulting fraction was dialysed against PBS overnight at 4°C and then concentrated in an Amicon concentrator (Millipore) to a final volume of ~500uL.

[0085] The activity and specificity of the affinity purified IgGs was determined by testing the starting serum (1 :500 dilution), flow-through fraction (volume normalised to the starting serum) and the eluted bound fraction (1 :50 dilution) on two ELISA sets. The first tested the specificity of the affinity purified IgG on various recombinant La fragments and native La. The various La fragments were expressed as GST fusion protein constructs prepared from the pGEX vector (New England BioLabs Inc, Ipswich, MA, United States of America). The plates (Maxisorp™; Nunc A S, Roskilde, Denmark) were coated with 5ug mL of native La (Arotec Diagnostics Limited, Wellington, New Zealand), GST-LaA, GST- LaC (aa 111-242), and GST control in 0.03M carbonate buffer (pH 8.2). Non-specific sites were blocked with 3% skim milk powder and duplicate wells were incubated with the different fractions. Bound IgG was detected with anti-human IgG conjugated with alkaline phosphatase as described previously¹². The second ELISA set tested the affinity-purified anti-LaA sample against native Ro60 (Arotec Diagnostics), full length Ro52 expressed in a maltose binding protein (MBP) fusion construct from the pMAL vector (New England Biolabs) and MBP control. ^ [0086] Two-dimensional gel electrophoresis (2-DE)

[0087] Affinity-purified anti-LaA samples were precipitated using a 2-D Clean Up Kit according to the manufacturer's instructions (Bio-Rad Laboratories Inc, Hercules, CA, United States of America) and were analysed using 2-DE as previously described⁸.

[0088] Preparation of samples for mass spectrometry (MS)

[0089] Samples were prepared for MS from either in-solution digests of affinity-purified IgGs or from H- and L-chain or intact immunoglobulin gel plugs excised from 2-DE gels in the manner described in Example 1 and 2 above.

[0090] MS analysis

[0091] Analysis of peptides was carried out in an LTQ Thermo Orbitrap XL linear ion trap mass spectrometer fitted with a nanospray source (Thermo Electron Corporation).

[0092] Protein sequence data analysis ,

[0093] Database searches were first carried out with Proteome Discoverer version 1.2 (Thermo Electron Corporation) using the Sequest algorithm against two combined databases; the IMGT and Uniprot 2010-06 databases. The database search parameters were as described in Example 1 ; peptide probability was high and 2 or more unique peptides were sequenced for each protein. The mass tolerance for peptide identification was lOppm for precursor ions and 0.01 Da for product ions. Searches were carried out with the following modifications: the oxidation of methionine; phosphorylation of serine, threonine and tyrosine; and carboxymethylation of cysteine. De novo sequencing was performed on raw data files with Peaks Studio version 5.3 (Bioinformatics Solutions). As there is a large degree of sequence homology between the various Ig gene families, the following strict set of rules were followed to minimise erroneous assignments of families: all spectra were manually inspected for quality; peptide lists were generated from the Peaks software program; a minimum of two framework (FR) peptides were matched back to the germline sequence; and sequence homology had to be >80% when the sequences were Ig Blasted²² against the IMGT database. Further data refinement parameters were set in the Peaks software program; scans were merged with a retention time of 1 minute, a precursor m/z error tolerance of lOppm and a minimum charge state of 2. Scans were filtered for a precursor mass of between 350 and 5000 Da and a quality value of greater than 0.65. Mutational analysis and deviations from the germline IMGT sequence were carried out using the SPIDER search tool by searching against the combined IMGT Uniprot 2010-06 databases with the following parameters: a homology match query type; a mass error tolerance of 0.01 Da; and the previously described variable modifications. Results

[0094] Autoantibodies specific for either an immunodominant determinant or the intact autoantigen, in this case the LaA epitope located at the NH2-terminus of La protein, were obtained and confirmed as specific for LaA by analysing starting serum, flow-through and eluted fractions on Ro52 Ro60/La ELISAs (data not shown). In ontrol experiments, no eluted IgGs were detected after normal human sera (n = 4) or serum from primary SS patients without anti-LaA antibody (n=2) were passed over the GST- LaA column. Further, no IgGs were detected in column eluates after anti-LaA-positive sera (n=3) were passed over a sham GST column.

[0095] The clonality of the affinity-purified anti-LaA samples was assessed using high-resolution 2- DE. Under reduced conditions, anti-LaA IgG resolved into several overlapping H-chain species migrating at 55kD with a range of isoelectric points (pi) from 7 to 8.5. These spots are likely to represent charge variants due to post-translational modifications of oligoclonal species such as glycosylation, as has been observed previously for mouse monoclonal antibodies and a clonotypic anti- Ro60 autoantibody^{8, 23}' ²⁴. L-chains, evident at ~25kD, resolved into four equally spaced spots ranging in pi from 5.2 to 6.8; identified as V_K3-15 and V_K3-20. The complete H-chain area was gel excised and found to comprise two dominant V gene families, namely V_H3-43 and V_H3-30. In order to determine actual H- and L-chain pairing, non-reduced 2-DE gels of anti-LaA IgG were performed and revealed biclonal species of similar staining intensity migrating to 150kD; one comprising a V_H3-30/V_K3-l 5 pairing and the second comprising a V_H3-43/V_K3-20 pairing as identified by MS.

[0096] In order to confirm both the clonality and H- and L-chain pairings of the anti-LaA IgGs, and determine the extent of IgV-region somatic hypermutation, in-solution trypsin digests of purified anti- LaA IgGs were performed followed by combined database and de novo Orbitrap MS sequencing. This confirmed the presence of the two IgGl H-chain species, V_H3-30 and V_H3-43, and two kappa L-chains encoded by V_K3-20 and V_K3-15. JK2 and JK4 regions were also identified from the in-solution digests together with a single heavy chain JH2, indicating that the V_H3-30 and V_H3-43 clonotypes utilise a single J-region. In addition, extensive amino acid mutations (ie substitutions) within the V-region (including some within the J-region of the V-region) were identified using the SPIDER analysis module. Public (common) mutations that are shared among patients are tabulated as a proteomic heat map shown in Figure 3. These are present in both FR and CDRs of each clonotypes. Most notably, there is a serine to asparagine substitution at position 31 in the CDR1 region of the V_K3-20-encoded L-chain present in all of the primary SS patients, as was an arginine to lysine substitution at position 16 in the FR1 region of the V_H3-30-encoded H-chain. Discussion

[0097] The study described in this example has determined the secreted autoantibody proteome specific for an epitope on La protein that is expressed as an apotope on the surface of apoptotic cells and available for binding of maternal anti-La in CHB. In particular, two dominant public IgGl kappa clonotypes were identified in unrelated patients with primary SS: one specified by a H-chain derived from the V_H3-30 gene segment paired with a V_K3-15 L-chain; and the second comprising a V_H3-43/V_K3- 20 pairing. This second clonotype is particularly interesting as it indicates that the Ro La autoantibody proteome is characterised by the shared expression of Ig L- and H-heavy chain V and J gene segments across the responses. Thus, from the results obtained in Examples 1 and 2, it is apparent that the anti- Ro52 autoantibody proteome has the same VH3-23 V_k3-20 molecular signature as the anti-Ro60peg clonotype, and now, from the results obtained in this study, it is clear that V_K3-20 is shared by clonotypes that encompass the entire Ro52 Ro60/La response, with V_K3-20 combined with KJ2 for anti-LaA Ro60peg clonotypes. This sharing of germline-encoded structures indicates a recombinatorial bias in the generation of these linked autoreactive species.

[0098] As was also found with the anti-Ro60 and anti-Ro52 autoantibody clonotypes, the anti-LaA IgGs included public (common) somatic mutations indicating that they are generated through independent antigen-driven clonal selection events. This allows for the recognition of unique amino acid sequences that represent molecular signatures (or biomarkers) for diseases and disorders such as primary SS, SLE, neonatal lupus syndrome and CHB, such as the motif ASQSVSNYLAWYQQK (SEQ ID NO: 12) or ASQSVSNNLAWYQQK (SEQ ID NO: 13). In turn, this allows the identification of surrogate V- region peptides for use in a diagnostic method employing a targeted MS approach.

Example 4 Anti-Ro52 autoantibody detection assay for primary SS using V-region domain peptide targeted MS

[0099] The suitability of the seven surrogate peptides pepl-pep7 in a targeted MS approach to the detection of Ro52 autoantibodies in unfractionated serum from patients diagnosed with primary SS was tested.

Materials and Methods [00100] Patient samples

[00101] Serum samples were collected from 17 patients diagnosed with primary SS. [00102] Preparation of samples for mass spectrometry (MS)

[00103] 10 μΐ samples of unfractionated patient serum were diluted 1 in 20 with lOOmM ammonium bicarbonate, pH 8.0, reduced by the addition of DTT to 5 mM and alkylated by the addition of iodoacetamide to 20 mM. Samples were then incubated with 1 μΐ of Trypsin Gold 100 ng/ μΐ (Promega) overnight at 37 °C. Prior to MS analysis, the digested serum samples were further diluted 1 in 60 with ultra-pure water.

[00104] MS analysis

[00105] About 1 ng of the digested serum was subjected to separation by nanoflow HPLC and sequencing using a Thermo Orbitrap XL linear ion trap with a methodology integrating a precursor mass list including the mass of each of pepl-pep7 (ie m/z values, respectively, of 608.79, 594.79, 484.73, 470.73, 763.42, 503.78 and 848.92). Where the precursor ion of interest (ie corresponding to one of the surrogate peptides) is found, the ion was isolated and measured in the FT cell. Fragmentation and subsequent sequencing of the isolated target peptide(s) was performed in the ion trap, increasing the sensitivity of detection. Positive detection of the target peptide(s) was determined by the presence of the precursor ion mass with a mass error tolerance of 10 ppm, product ion mass error tolerance of 0.8 Da, the intensity of the matched peaks, enzyme specificity and the de novo sequence (examples of MS/MS spectra of the surrogate Ro52 peptides are shown at Figure 2).

Results

[00106] The results are shown in Table 2. A sample was considered to be "positive" when > two anti- Ro52 target peptides were detected. 15 of 17 sera from the primary SS patients were positive compared with just 1 of 14 samples from matched controls. Targeted MS detection of anti-Ro52 in serum had both high sensitivity (87.5%) and specificity (92.9%) compared with detection by Ro52 ELISA (P = 0.0001). It was notable that some clonotypic species are detected by the targeted MS assay in normal subjects, but their serum concentrations are below the threshold of detection by conventional ELISA.

Discussion

[00107] The results indicated that the detection of anti-Ro52 autoantibodies in unfractionated human sera, as the basis of a new diagnostic method, is feasible through the use, for example, of a targeted MS approach. [00109] Table 2 Targeted MS of Ro52 autoantibody surrogate peptides in serum

pSS - primary Sjogren's syndrome; C - healthy control; + = target peptide detected; and

- = target peptide not detected [00110] Throughout the specification and the claims that follow, unless the context requires otherwise, the words "comprise" and "include" and variations such as "comprising" and "including" will be understood to imply the inclusion of a stated integer or group of integers, but not the exclusion of any other integer or group of integers.

[001 11] The reference to any prior art in this specification is not, and should not be taken as, an acknowledgement of any form of suggestion that such prior art forms part of the common general knowledge.

[00112] It will be appreciated by those skilled in the art that the invention is not restricted in its use to the particular application described. Neither is the present invention restricted in its preferred embodiment with regard to the particular elements and/or features described or depicted herein. It will be appreciated that the invention is not limited to the embodiment or embodiments disclosed, but is capable of numerous rearrangements, modifications and substitutions without departing from the scope of the invention as set forth and defined by the following claims.

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Claims

1. A method of detecting autoantibodies associated with Ro La autoimmunity, said method

comprising the steps of;

(i) providing a serum sample or other sample comprising immunoglobulin G (IgG), and

(ii) detecting the presence of autoantibodies to a Ro and/or La antigen comprising a variable (V)-region molecular signature comprising an unusual or common heavy (H) and light (L)-chain pairing and/or at least one mutation in one or more of said H- and L-chains and/or an adjacent region.

2. The method of claim 1, wherein the molecular signature comprises a V-region with a V_K3-20- encoded L-chain paired with a V_H3 -23 -encoded heavy (H)-chain, V_H3-7-encoded heavy (H)- chain or V_H3-43 -encoded heavy (H)-chain and/or at least one mutation in one or more of said H- and L-chains and/or an adjacent region.

3. The method of claim 1 or 2, when used for the diagnosis of an autoimmune disease or disorder selected from the group consisting of Sjogren's syndrome (SS), systemic lupus erythematosus (SLE), other systemic rheumatic diseases, polymyositis, progressive systemic sclerosis, primary biliary cirrhosis and congenital heart block (CHB).

4. The method of claim 1 or 2, when used for the diagnosis of an autoimmune disease or disorder selected from the group consisting of Sjogren's syndrome (SS), systemic lupus erythematosus (SLE), neonatal lupus syndrome and congenital heart block (CHB).

5. The method of claim 1 or 2, wherein step (i) consists of providing a serum sample.

6. The method of claim 1 or 2, wherein step (ii) comprises detecting the presence of autoantibodies against Ro52.

7. The method of claim 1 or 2, wherein step (ii) comprises detecting the presence of autoantibodies against Ro60.

8. The method of claim 1 or 2, wherein step (ii) comprises detecting the presence of autoantibodies against La.

9. The method of claim 1 or 2, wherein the at least one mutation in one or more H- and L-chain CDR region.

10. The method of claim 2, wherein the method is conducted to detect autoantibodies against Ro52, and the method comprises detecting one or more mutation in CDR1, CDR2 and/or CDR3 of the V_K3-20-encoded L-chain and/or one or more mutation in CDR1 and/or CDR2 of the VH3-23- encoded or VH3-7 H-chain.

1 1. The method of claim 10, wherein step (ii) comprises detecting at least one motif selected from the group consisting of LSCAASGFTFR (SEQ ID NO: 1), LSCAASGFTFK (SEQ ID NO: 2), LSCAASGFR (SEQ ID NO: 3), LSCAASGFK (SEQ ID NO: 4), RLVESGGGLVQPGGSLR (SEQ ID NO: 5), KLVESGGGLVQPGGSLR (SEQ ID NO: 6), LLLYGASNR (SEQ ID NO: 7), and ASQSVSSNLAWYQQK (SEQ ID NO: 8), or at least one of the mutations found therein.

12. The method of claim 2, wherein the method is conducted to detect autoantibodies against Ro60, and the method comprises detecting one or more mutation in GDR3 of the V_K3-20-encoded L- chain and/or one or more mutation in CDR2 of the VH3 -23 -encoded H-chain.

13. The method of claim 12, wherein step (ii) comprises detecting at least one motif selected from the group consisting of LEPEDFAVYYCQQYR (SEQ ID NO: 9), LEPEDFAVYYCQQYGR (SEQ ID NO: 10) and LSCAASGFTFDSYAMSWVR (SEQ ID NO: 11), or at least one of the mutations found therein.

14. The method of claim 2, wherein the method is conducted to detect autoantibodies against La, and the method comprises detecting one or more mutation in CDR1 of the V_K3-20-encoded L- chain and/or one or more mutation in FR1 of the V_H3-30-encoded H-chain.

15. The method of claim 14, wherein step (ii) comprises detecting at least one motif selected from the group consisting of ASQSVSNYLAWYQQK (SEQ ID NO: 12) or

ASQSVSNNLAWYQQK (SEQ ID NO: 13), or at least one of the mutations found therein.

16. The method of claim 1 or 2, wherein step (ii) comprises direct sequencing of autoantibodies or a part(s) thereof.

17. The method of claim 10, wherein step (ii) comprises a targeted mass spectroscopy (MS)

approach to directly sequence for the presence of a V-region peptide fragment comprising or consisting of the amino acid sequence: LSCAASGFTFR (SEQ ID NO: 1), LSCAASGFTFK (SEQ ID NO: 2), LSCAASGFR (SEQ ID NO: 3), LSCAASGFK (SEQ ID NO: 4), LVESGGGLVQPGGSLR (SEQ ID NO: 5), LLLYGASNR (SEQ ID NO: 7) or ASQSVSSNLAWYQQK (SEQ ID NO: 8).

18. The method of claim 12, wherein step (ii) comprises a targeted mass spectroscopy (MS)

approach to directly sequence for the presence of a V-region peptide fragment comprising or consisting of the amino acid sequence: LEPEDFAVYYCQQYR (SEQ ID NO: 9) or

LEPEDFAVYYCQQYGR (SEQ ID NO: 10).

19. The method of claim 14, wherein step (ii) comprises a targeted mass spectroscopy (MS)

approach to directly sequence for the presence of a V-region peptide fragment comprising or consisting of the amino acid sequence: ASQSVSNYLAWYQQK (SEQ ID NO: 12) or ASQSVSNNLAWYQQK (SEQ ID NO: 13).

i

20. A peptide comprising or consisting of an amino acid sequence selected from the group

consisting of: LSCAASGFTFR (SEQ ED NO: 1), LSCAASGFTFK (SEQ ID NO: 2),

LSCAASGFR (SEQ ID NO: 3), LSCAASGFK (SEQ ID NO: 4), LVESGGGLVQPGGSLR (SEQ ID NO: 5), LLLYGASNR (SEQ ID NO: 7), ASQSVSSNLAWYQQK (SEQ ID NO: 8), LEPEDFAVYYCQQYR (SEQ ED NO: 9), LEPEDFAVYYCQQYGR (SEQ ID NO: 10), ASQSVSNYLAWYQQK (SEQ ID NO: 1 1) and ASQSVSNNLAWYQQK (SEQ ID NO: 12).

21. A monoclonal or polyclonal antibody that specifically binds to a peptide comprising or

consisting of an amino acid sequence selected from the group consisting of: LSCAASGFTFR (SEQ ID NO: 1), LSCAASGFTFK (SEQ ID NO: 2), LSCAASGFR (SEQ ED NO: 3), LSCAASGFK (SEQ ID NO: 4), LVESGGGLVQPGGSLR (SEQ ID NO: 5), LLLYGASNR (SEQ ID NO: 7), ASQSVSSNLAWYQQK (SEQ ID NO: 8), LEPEDFAVYYCQQYR (SEQ ID NO: 9), LEPEDFAVYYCQQYGR (SEQ ID NO: 10), ASQSVSNYLAWYQQK (SEQ ID NO:

12) and ASQSVSNNLAWYQQK (SEQ ED NO: 13).