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Title:
DIAGNOSIS AND THERAPY OF ANKYLOSING SPONDYLITIS
Document Type and Number:
WIPO Patent Application WO/2018/024732
Kind Code:
A1
Abstract:
The present invention relates to synthetic or recombinant peptides able to bind ankylosing spondylitis auto-antibodies, their use in a diagnostic method of such disease and to relative support, kit and antibody.

Inventors:
PUCCETTI, Antonio (Ospedale Pediatrico Bambino Gesù, Piazza S. Onofrio 4, Rome, 00165, IT)
LUNARDI, Claudio (Ospedale Pediatrico Bambino Gesù, Piazza S. Onofrio 4, Rome, 00165, IT)
MORETTA, Lorenzo (Ospedale Pediatrico Bambino Gesù, Piazza S. Onofrio 4, Rome, 00165, IT)
Application Number:
EP2017/069451
Publication Date:
February 08, 2018
Filing Date:
August 01, 2017
Export Citation:
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Assignee:
OSPEDALE PEDIATRICO BAMBINO GESU' (Piazza S. Onofrio 4, Rome, 00165, IT)
International Classes:
G01N33/564; C07K7/08; C07K16/18; G01N33/543; G01N33/68
Attorney, Agent or Firm:
CAPASSO, Olga et al. (De Simone and Partners S.p.A, Via Vincenzo Bellini 20, Rome, 00198, IT)
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Claims:
CLAIMS

1. A synthetic or recombinant peptide able to bind ankylosing spondylitis auto-antibodies selected from the group consisting of:

c) a peptide comprising the amino acid sequence

NIGYIGPVPERALGLGI (SEQ ID NO. 1) and/or

d) a peptide comprising the amino acid sequence

RIGHVGARPSRH (SEQ ID NO. 2)

2. The synthetic or recombinant peptide according to claim 1 being at least 18 amino acid long, preferably at least

20 amino acid long, preferably at least 25 aminoacid long .

3. The synthetic or recombinant peptide according to claim 2 essentially consisting of any of SEQ ID No. 1 or 2. 4. The synthetic or recombinant peptide according to claim

1 essentially consisting of AARGNIGYIG PVPERALGLG IAADK (SEQ ID NO. 3) .

5. The synthetic or recombinant peptide according to any one of previous claim in linear or MAP form.

6. The synthetic or recombinant peptide according to any one of previous claim wherein the peptide further comprises a functional group.

7. The synthetic or recombinant peptide according to claim

6, wherein the functional group is selected from the group consisting of: biotin, succinimide, thiol, azide, alkyne, cyclic alkyne, aldehyde, thiazolidine, alkene, cyclic alkene, maleimide, phosphine, tiol- pyridyl disulfide, oxime, aminoxy, tetrazole, nitrone, thioester, acilidrazide, epoxide.

8. The synthetic or recombinant peptide according to any one of previous claim wherein said peptide is immobilized on a solid support.

9. A solid support comprising at least one synthetic or recombinant peptide as defined in claims 1 to 8.

10. The solid support according to claim 9 wherein said support is coated with streptavidin or avidin.

11. The solid support according to claim 9 wherein said support is coated with a polymer bearing a functional group .

12. The solid support according to claim 11 in which the functional group is selected from the group consisting of: succinimide, thiol, azide, alkyne, cyclic alkyne, aldehyde, thiazolidine, alkene, cyclic alkene, maleimide, phosphine, thiol-pyridyl disulfide, oxime , aminoxy, tetrazole, nitrone, thioester, acilidrazide, epoxide .

13. The solid support according to any claim from 9 to

12 consisting of a material selected from the group consisting of: gold, silicon, glass, paper, ITO (Indium Tin Oxide) , plastic and / or thermoplastic material, porous support.

14. The solid support according to claim 13 wherein the porous support is selected from the group consisting of: nitrocellulose, PVDF, Teflon, alumina, silicon.

15. The solid support according to claim 13, wherein the plastic material is selected from the group consisting of: polydimethylsiloxane (PDMS) , polypropylene, polycarbonate, polymethylmethacrylate, Teflon, COC, polystyrene, parylene.

16. The solid support according to any claim from 9 to

15 wherein said support is in the form of a chip, array, microarray, plate, slide, membrane, well or sphere.

17. The solid support according to any claim from 9 to

16 wherein at least one peptide is immobilized on said support, preferably in the form of a spot.

18. A method for detecting ankylosing spondylitis auto¬ antibodies in a patient, said method comprising:

c) obtaining a biological fluid isolated from the patient; and

d) detecting whether ankylosing spondylitis autoantibodies are present in the biological sample by contacting the biological sample with at least one synthetic or recombinant peptide according to any of claim 1 to 8 and detecting binding between ankylosing spondylitis auto-antibodies and the at least one synthetic or recombinant peptide.

19. A method for the diagnosis or for monitoring the progression of ankylosing spondylitis or for identifying or monitoring a therapy for ankylosing spondylitis characterized in detecting ankylosing spondylitis auto¬ antibodies in a biological sample isolated from a subject by means of binding to at least one synthetic or recombinant peptide according to any one of claim 1 to 8.

20. A kit for the diagnosis or for monitoring the progression of ankylosing spondylitis or for identifying or monitoring a therapy for ankylosing spondylitis comprising at least one synthetic or recombinant peptide according to any one of claim 1 to 8.

21. A nucleic acid molecule encoding for any one of the synthetic or recombinant peptide or fragment thereof according to any one of claim 1 to 8.

22. An antibody or a recombinant or synthetic derivative thereof able to recognize and bind to at least one synthetic or recombinant peptide according to claims 1 to 8.

23. The antibody or a recombinant or synthetic derivative thereof according to claim 22 for use in a diagnostic method of ankylosing spondylitis and/or for monitoring the progression of ankylosing spondylitis and/or for identifying or monitoring a therapy of ankylosing spondylitis.

Description:
DIAGNOSIS AND THERAPY OF ANKYLOSING SPONDYLITIS

TECHNICAL FIELD

The present invention relates to synthetic or recombinant peptides able to bind ankylosing spondylitis auto-antibodies, their use in a diagnostic method of such disease and to relative support, kit and antibody.

BACKGROUND ART

Ankylosing spondylitis (AS) is one of the most important forms of chronic inflammatory arthritis and represents the "prototype" of a group of interrelated diseases known as spondyloarthritides (SpA) . The disease affects primarily the spine, the sacroiliac joints and the peripheral entheses and j oints [1,2] .

The diagnosis of AS is based on clinical examination and on the exclusion of other types of seronegative arthritis. Medical history, physical examination, blood tests, and imaging of the joints may be used for diagnostic purposes. There is no definitive diagnostic test for AS and therefore the identification of a biochemical or immunological test with combined high sensitivity and specificity for the diagnosis and the follow-up of patients with AS is an important goal in medicine.

The presence of autoantibodies, which are typically detectable in many rheumatic diseases, is not commonly considered to be a feature of AS. Antibodies directed against leukocytes [ 3 ] , neutrophils [4], and some collagen proteins have been reported [5] . More recently Wright C. et al . [6] have shown the presence of multiple autoantibodies directed against proteins expressed in connective and skeletal tissue in about 40% of patients with AS by using a nucleic acid programmable protein array. Nevertheless autoantibodies typically associated with AS have not been identified so far. Another aspect of AS that needs to be clarified is the relationship between Klebsiella pneumoniae and the ethiopatogenesis of the disease.

A large body of evidence based on genetic, microbiological, molecular and immunological studies suggests that Klebsiella pneumoniae is the main microbial agent implicated in the etiopathogenesis of AS, as a triggering and/or a perpetuating factor [7] . However the precise role played by Klebsiella pneumoniae in the disease has not been clarified yet.

There is still the need for identifying a potential serologic marker that may help in the diagnosis and/or in the monitoring of disease activity of patients with AS. SUMMARY OF THE INVENTION

Ankylosing spondilitis (AS) is a chronic inflammatory arthritis of unknown cause. Its autoimmune origin has been suggested but never proven. Several reports have implicated Klebsiella pneumoniae as a triggering or perpetuating factor in AS, however its role in disease pathogenesis is still debated. Moreover, despite extensive investigation, a biomarker for AS has not been identified yet. To clarify these issues, the inventors screened a random peptide library with pooled IgGs obtained from 40 patients with AS.

A peptide (AS peptide) selected with the library, was recognized by serum IgGs from 170 of 200 (85%) patients with AS, but not by serum specimens from 100 healthy controls. Interestingly the AS peptide shows sequence similarity with several molecules expressed at the fibrocartilaginous sites that are primarily involved in AS inflammatory process. Moreover the peptide is highly homologous to a Klebsiella pneumoniae dipeptidase (DPP) protein. Antibodies affinity purified against the AS peptide recognize the autoantigens and the DPP protein. Moreover serum IgG antibodies against the Klebsiella DPP 121 -145 peptide epitope were detected in 190 of 200 patients with AS (95%), in 3 of 200 patients with rheumatoid arthritis (1.5%) and only in 1 of 100 (1%) patients with psoriatic arthritis. Such reactivity was not detected in control healthy donors. The present results show that antibodies directed against an epitope of a Klebsiella pneumoniae-derived protein are present nearly in all the patients with AS. In the absence of serological biomarkers for AS such antibodies may represent a useful tool in the diagnosis and prognosis of the disease.

Then, the present invention provides a synthetic or recombinant peptide able to bind ankylosing spondylitis auto- antibodies selected from the group consisting of:

a) a peptide comprising the amino acid sequence NIGYIGPVPERALGLGI (SEQ ID NO. 1) and/or

b) a peptide comprising the amino acid sequence RIGHVGARPSRH (SEQ ID NO. 2)

Preferably the synthetic or recombinant peptide is at least 18 amino acid long, preferably at least 20 amino acid long, preferably at least 25 amino acid long.

Preferably the synthetic or recombinant peptide essentially consists of any of SEQ ID No. 1 or 2.

Preferably the synthetic or recombinant peptide essentially consisting of AARGNIGYIG PVPERALGLG IAADK (SEQ ID NO. 3) . Preferably the synthetic or recombinant peptide is in linear or MAP form.

Preferably the synthetic or recombinant peptide further comprises a functional group.

Preferably the functional group is selected from the group consisting of: biotin, succinimide, thiol, azide, alkyne, cyclic alkyne, aldehyde, thiazolidine, alkene, cyclic alkene, maleimide, phosphine, tiol- pyridyl disulfide, oxime, aminoxy, tetrazole, nitrone, thioester, acilidrazide, epoxide .

Preferably the synthetic or recombinant peptide is immobilized on a solid support.

The present invention also provides a solid support comprising at least one synthetic or recombinant peptide as defined above.

Preferably said support is coated with streptavidin or avidin.

More preferably said support is coated with a polymer bearing a functional group.

Preferably the functional group is selected from the group consisting of: succinimide, thiol, azide, alkyne, cyclic alkyne, aldehyde, thiazolidine, alkene, cyclic alkene, maleimide, phosphine, thiol-pyridyl disulfide, oxime , aminoxy, tetrazole, nitrone, thioester, acilidrazide, epoxide .

Preferably the support consists of a material selected from the group consisting of: gold, silicon, glass, paper, ITO (Indium Tin Oxide), plastic and / or thermoplastic material, porous support.

Preferably the porous support is selected from the group consisting of: nitrocellulose, PVDF, Teflon, alumina, silicon.

Preferably the plastic material is selected from the group consisting of: polydimethylsiloxane (PDMS) , polypropylene, polycarbonate, polymethylmethacrylate, Teflon, COC, polystyrene, parylene.

Preferably the support is in the form of a chip, array, microarray, plate, slide, membrane, well or sphere.

Preferably at least one peptide is immobilized on said support, preferably in the form of a spot. The present invention also provides a method for detecting ankylosing spondylitis auto-antibodies in a patient, said method comprising:

a) obtaining a biological fluid isolated from the patient; and

b) detecting whether ankylosing spondylitis autoantibodies are present in the biological sample by contacting the biological sample with at least one synthetic or recombinant peptide as defined above and detecting binding between ankylosing spondylitis auto-antibodies and the at least one synthetic or recombinant peptide.

The present invention also provides a method for the diagnosis or for monitoring the progression of ankylosing spondylitis or for identifying or monitoring a therapy for ankylosing spondylitis characterized in detecting ankylosing spondylitis auto-antibodies in a biological sample isolated from a subject by means of binding to at least one synthetic or recombinant peptide as defined above.

The present invention also provides a kit for the diagnosis or for monitoring the progression of ankylosing spondylitis or for identifying or monitoring a therapy for ankylosing spondylitis comprising at least one synthetic or recombinant peptide as defined above.

The present invention also provides a nucleic acid molecule encoding for any one of the synthetic or recombinant peptide or fragment thereof as defined above.

The present invention also provides an antibody or a recombinant or synthetic derivative thereof able to recognize and bind to at least one synthetic or recombinant peptide as defined above.

Preferably the antibody or a recombinant or synthetic derivative thereof is for use in a diagnostic method of ankylosing spondylitis and/or for monitoring the progression of ankylosing spondylitis and/or for identifying or monitoring a therapy of ankylosing spondylitis.

The invention will be illustrated by means of non-limiting examples in reference to the following figures.

Fig 1. Peptides used in the study and sequence homologies. Sequence homology between the AS peptide and self-proteins (colons indicate identity and asterisks indicate conservative substitutions) .

Fig 2. Binding of anti-peptide antibodies to autoantigens .

Binding of affinity purified anti-AS peptide antibodies to collagen type I (A) , collagen type II (B) , fibronectin (C) , PER peptide (D) , ADM peptide (E) . Black continous line: antibodies affinity purified against the AS peptide from the sera of patients affected by AS. Dotted line: antibodies affinity purified against an irrelevant control peptide. A, B and C: ELISA assay. D and E: DELFIA assay. X axis: increasing antibody concentration by two fold ranging from 1.25 microgram/ml to 10 microgram/ml . Y axis: Optical Density values obtained at 405 nm wavelength for ELISA assay and IgG international units for DELFIA assay. F: western blot analysis of the binding of anti-peptide antibodies to ASAP1. Lane 1: antibodies affinity purified against an irrelevant control peptide, lane 2: antibodies affinity purified against the AS peptide, lane 3: commercially available monoclonal antibody directed against ASAP1.

Fig 3. Anti- peptide antibodies bind Klebsiella pneumoniae- derived proteins. (A) Sequence homology between the AS peptide and the Klebsiella pneumoniae-derived proteins: sugar transporter (SET) protein, Xaa-Pro dipeptidase (DPP) protein and L-fucose isomerase (LFI) protein.

Binding of affinity purified anti-AS peptide antibodies (black continuous line) to the SET (B) and LFI (C) peptide in DELFIA. Binding of affinity purified anti-AS peptide antibodies to recombinant DPP protein in ELISA (D) . Dotted line: antibodies affinity purified against an irrelevant control peptide. X axis: increasing antibody concentration by two fold ranging from 1.25 microgram/ml to 10 microgram/ml . Y axis: IgG international units for DELFIA assay and Optical Density values obtained at 405 nm wavelength for ELISA assay. Fig 4. Antibodies directed against the Klebsiella pneumoniae- derived DPP121-145 peptide in the sera of patients with inflammatory arthritides . (A) The results of an assay of europium-labeled antihuman IgG antibodies are shown; each circle represents a measurement for one patient, and the dashed horizontal line indicates the cut-off value of 32,250 international units.

ROC curves showing sensitivity and specificity of the assay of IgG antibodies against the Klebsiella pneumoniae-derived DPP 121 -145 peptide for differentiating between AS sera and 1 healthy donors (B) , between AS samples and RA (C) and between AS sera and PsA samples (D) .

DETAILED DESCRIPTION OF THE INVENTION

MATERIAL AND METHODS

Patients

Between January 2005 and November 2013, the inventors obtained serum samples from patients and healthy controls. All serum samples were stored at -20°C. Blood samples were obtained after all the subjects provided written informed consent, and the local ethics committee of the Azienda Ospedaliera Universitaria of Verona, Verona, Italy approved the study. The inventors studied a cohort of 200 patients (165 males and 35 females, mean age: 47114 years) affected by AS, attending the Unit of Autoimmune Diseases at the University Hospital of Verona and the Rheumatology Department of Lucania, San Carlo Hospital of Potenza and Madonna delle Grazie Hospital of Matera. The diagnosis of AS was assessed following the modified Criteria of New York [12] . The clinical features of the 200 patients were the following: sacroiliitis or spondylitis was present in all the subjects, peripheral joint arthritis was present in 63/200 patients. Eye involvement was present in 57/200 subjects, and cardiac involvement in 4/200. The HLA B27 allele was detected in 189/200 patients.

A cohort of 100 patients (64 males and 36 females, mean age: 57114 years) affected by psoriatic arthritis (PsA) was also studied. All patients fulfilled the Classification criteria for Psoriatic Arthritis (CASPAR) criteria for classification of PsA [13] . Two hundred patients with rheumatoid arthritis (RA) were also included in the study. RA patients met the American College of Rheumatology classification criteria for RA [14] . All the patients were enrolled consecutively irrespective of disease activity and treatment.

One hundred age- and sex-matched healthy subjects served as control group.

All the investigations have been conducted according to the principles expressed in the Helsinki declaration.

Peptide Library

A random dodecamer peptide library that expresses peptides on the surface of Escherichia coli was screened with pooled immunoglobulins purified from the serum samples from the 40 patients with AS, according to the manufacturer's instructions (FliTrx Panning Kit, Invitrogen) [8-11]. After five rounds of biopanning experiments, the enriched library was grown, and single colonies were induced to express the fusion peptides with tryptophan. Bacteria were lysed in sample buffer and tested by means of Western blotting with the pooled immunoglobulin fraction from the patients with ankylosing spondylitis in order to check for positive clones. DNA was extracted from positive clones and sequenced. A set of 15 of the 27 peptides obtained from the last biopanning round was synthetized and used in a dissociation-enhanced lanthanide fluorescence immunoassay (DELFIA) to test serum specimens from individual patients.

Peptide Synthesis

All the synthetic peptides, including AS peptide (RIGHVGARPSRH, SEQ ID NO. 2), the Klebsiella pneumoniae- derived proline dipeptidase (DPP) peptide (IGYIGPVPER, SEQ ID NO. 4) and DPP 12 i-i 4 5 peptide (AARGNIGYIG PVPERALGLG IAADK, SEQ ID NO. 3), the perlecan (PER) peptide (VGTRPSNH, SEQ ID NO. 5), the ADAMTSL3 /punctin 2 (ADM) peptide (GHLGARIQR, SEQ ID NO. 6), the Arf GAP With SH3 Domain, Ankyrin Repeat And PH Domain (ASAP1) peptide (IGHIEGQPSR, SEQ ID NO. 7) and the irrelevant control peptide (VTLPKDSDVELP, SEQ ID NO. 8), were manually synthesized by means of the standard method of solid-phase peptide synthesis; this method uses the 9- fluorenylmethoxycarbonyl strategy with minor modifications [15] .

Further, the inventors synthetized shorter peptides than the Peptide DPP 12 i-i 4 5 (SEQ ID No. 3) NH2- AARGNIGYIG

PVPERALGLG IAADK- COOH by deleting two amino acid residues at the time, one at the NH2-terminal and the other one at the COOH-terminal . In particular the 17 aa: NIGYIGPVPERALGLGI (SEQ ID No. 1) was synthetized.

Affinity Purification of Anti-Peptide Antibodies

The synthetic peptides (5 mg peptide per gram of dried Sepharose powder) were coupled to Sepharose 4B (Pharmacia, Uppsala, Sweden) according to the manufacturer's instructions. Sera diluted in PBS were applied to the columns. The columns were washed with PBS. Bound Ig were eluted with 0.1 M glycine (pH 2.5) and dialyzed against PBS. The purity of the preparations was assessed by SDS-PAGE followed by silver staining.

Assessment of Antibody Binding

a) DELFIA assay

The DELFIA is a time-resolved fluorescence method that can be used to study antibody binding to solid-phase proteins or peptides. The peptides were used at a concentration of 20 μg per milliliter in phosphate-buffered saline to coat DELFIA plates ( PerkinElmer, Waltham, Ma, USA) . Plates were then blocked for 1 hour with a blocking reagent (PerkinElmer) . Serum samples were diluted in a 1:50 solution in phosphate- buffered saline plus 1% bovine serum albumin (Sigma) and incubated on the plates overnight at 4 to 8°C. Plates were then washed 10 times with washing buffer (PerkinElmer) . Bound antibodies were detected with europium-labeled anti-human IgG antiserum (1:500 in diluting buffer, PerkinElmer). Plates were read on a Victor3 instrument (PerkinElmer) , and the data were analyzed with software supplied with the DELFIA instrument. Absorbance values higher than the mean (+3 SD) for each serum dilution in the control group were considered to be positive [10] .

b) ELISA assay

The ELISA assay for the binding of antibodies to collagen type I and II was performed using commercially available kits (CHONDREX Inc, Redmond, WA, USA) .

In the Elisa assay for the detection of anti-fibronectin antibodies plates (Immulon 2HB Thermo Scientific, Illkirch, France) were coated with 10 microgram/ml of human fibronectin (SIGMA, St. Louis, MO, USA), tested antibodies were diluted in PBS 1% BSA and incubated overnignt at 4°C. Plates were washed 3 times with PBS 1% Tween and one time with PBS alone. Alkaline-phosphatase labeled anti-human IgG antibodies were purchased from Sigma. IgG antibodies to Klebsiella pneumoniae were detected by ELISA using a bacterial extract adsorbed on the solid phase as elsewhere described in detail [16] . For the binding to recombinant Klebsiella pneumoniae proline dipeptidase (MyBioSource, San Diego, CA) plates were coated with 20 microgram/ml of recombinant protein in PBS. The test was then carried on as described above. The binding to the other two Klebsiella pneumoniae-derived proteins was assessed using the two synthetic peptides (LFI and SET peptide) using DELFIA as described above.

c) Western Blotting

The antibody binding to the ASAPl protein was assessed using the recombinant ASAPl, (Arf GAP With SH3 Domain, Ankyrin Repeat And PH Domain protein) (OriGene Inc, Rockville, MD, USA) by means of an immunoblot assay. The blots were probed with primary antibodies (human anti-peptide antibodies or mouse control monoclonal antibody) followed by either peroxidase-linked anti-human immunoglobulin antibodies or mouse IgG antibodies (Sigma) (10 μg per milliliter) . A mouse monoclonal antibody directed against ASAPl was purchased from OriGene and used as positive control. The blots were developed using a chemiluminescence kit by Thermo Scientific

Statistical Analysis

The inventors evaluated the sensitivity and specificity of the test with the use of receiver-operating-characteristic (ROC) curve analysis, estimating the area under the curve (AUC) with 95% confidence intervals. Statistical analysis was carried out using SPSS software, version 20 (SPSS) . RESULTS

Peptide Library

The inventors screened a peptide library with pooled immunoglobulins derived from a panel of 40 patients with AS. A set of 13 peptides, out of the 26 peptides obtained from the last biopanning step, was synthetized and used to screen the individual patients ' sera in a DELFIA assay employing the solid phase peptide. The inventors identified a peptide (AS peptide: RIGHVGARPSRH SEQ ID No. 2) that was recognized by IgG of serum samples from 33 of 40 patients with AS (83%) (absorbance (mean ± s.d.) : 41.900 ± 12.822 for a serum dilution of 1:100) but not by serum IgG from healthy controls. The results were validated in another cohort of 160 patients with AS, which were not used for the library screening. Anti-AS peptide antibodies were detected in 137/160 patients; by combining the two cohorts of patients the inventors observed that 170/200 (85%) patients' sera contained IgG antibodies against the AS peptide. These data indicate that this peptide sequence (AS peptide) contains an epitope recognized by the sera of most patients with AS.

Autoantigen Targets in Ankylosing Spondilitis

The inventors next compared the AS peptide with human proteins in a protein data bank (Swiss-Prot database of known human sequences) , using BLASTP software from the NCBI BLAST network service, and the inventors found that the AS peptide shared homology with different proteins highly expressed at the fibrocartilaginous sites which are primarily affected in the course of AS. These autoantigens include type I and II collagens (in particular collagen alpha-1 XXIV[17], collagen alpha-1 XXI [18], collagen alpha-2 XI, particularly abundant at cartilaginous sites [19]), the heparan sulfate proteoglycan 2, also known as perlecan, two glycoproteins particularly represented in the extracellular matrix, fibronectin and ADAMTSL3/punctin 2 [20,21], and a protein involved in cytoskeleton remodeling, ASAP1 (ArfGAP with SH3 domain, ankyrin repeat and PH domain 1 actin) [22] (Fig 1) . Affinity-purified antibodies against the AS peptide isolated from the sera of 10 patients with AS specifically recognized these molecules (Fig 2A-2F) . Indeed anti-AS peptide antibodies bound collagen type I (Fig 2A) , collagen type II (Fig 2B) and fibronectin (Fig 2C) in ELI SA .

Since the recombinant version of human perlecan and ADAMTSL3/punctin 2 molecules were not available, the crossreaction of anti-AS peptide antibodies with these proteins was assessed with a DELFIA assay in which the synthetic peptides (PER and ADM peptides, see above) had been used as solid phase antigens to coat the DELFIA plates (Fig 2D and 2E) . The binding of anti-AS peptide antibodies to ASAP1 was assessed using a cell line (HEK293T) overexpressing the molecule. Antibodies directed against the AS peptide specifically recognized the ASAP1 molecule; on the contrary affinity purified antibodies directed against an irrelevant control peptide did not react with the ASAP1 protein (Fig 2F) . Altogether these results show that sera of patients with AS contain IgG antibodies able to crossreact with several proteins expressed at the fibrocartilaginous sites, that are preferential targets of inflammatory aggression in AS.

Klebsiella pneumoniae and Ankylosing Spondilitis

Since Klebsiella pneumoniae has been associated with the pathogenesis of AS [7], the inventors decided to compare the AS peptide sequence with known microbial protein sequences in a protein data bank (Swiss-Prot database) , using BLASTP software from the Basic Local Alignment Search Tool (BLAST) network service of the National Center for Biotechnology Information (NCBI) . The inventors found that the AS peptide shares homology with 3 Klebsiella pneumoniae-derived proteins (Fig 3A) . Antibodies purified against the AS peptide bound the sugar trasporter (SET) peptide (Fig 3B) and the L-fucose peptide (Fig 3C) in DELFIA assay and recognized the recombinant dipeptidase (DPP) protein (Fig 3D) . The inventors next used the 3 bacterial peptides in DELFIA to test a panel of 50 sera from AS patients. Antibodies directed against the Klebsiella pneumoniae DPP peptide were detected in 36/50 (72%) serum samples, while antibodies against the Klebsiella pneumonia SET and against the Klebsiella pneumoniae L-fucose isomerase were detected at lower frequency (37% and 25% of the patients respectively) . Based on these results the inventors decided to focus their attention on the Klebsiella Pneumoniae derived DPP protein. The inventors therefore used the recombinant version of the DPP molecule to test a panel of 100 sera from AS patients using a DELFIA assay. Serum IgG antibodies against the Klebsiella-derived DPP protein were detected in 88 of 100 patients with AS, but not in control healthy donors. On the other hand since serum IgG antibodies against the Klebsiella- derived peptide (IGYIGPVPER, SEQ ID NO. 4) were detected only in 70 of 100 patients with AS, the inventors reasoned that the Klebsiella-derived DPP protein could contain other crucial epitopes for the anti-Klebsiella pneumoniae antibody response .

The inventors therefore decided to precisely map the reactivity of AS serum antibodies directed against the Klebsiella Pneumoniae-derived proline dipeptidase protein using 25 aminoacid-long synthetic peptides spanning the entire DPP sequence:

Klebsiella Pneumonia DPP- protein sequence (Primary

(citable) accession number: A6TGM5 UniprotKB/NCBI accession number : WP_004181656 (SEQ ID NO. 9)

1 meslaalykn hivtlqertr dvlarfqmda llihsgelvn vflddhpypf kvnpqfkawv

61 pvtqvpncwl Ivdgvnkpkl wfylpvdywh nveplptsfw teeidvialp kadgigsqlp 121 aargmgyig pvperalglg laadkmpkg vidylhyyra

yktdyelacm reaqksavng

181 hraayeafqs gmsefdinqa yltatghrdt dvpysnival

nehasvlhyt kldhrapaem

241 rsflldagae yngyaadltr twaahgdndf ahlikdvnde

qlalistmka gtsyidyhiq

301 fhqriakllr khqlvtdmse eamvendltg pfmphgighp

lglqvhdvag fmqddtgthl

361 aapskypylr ctriieprmv ltiepgiyfi esllapwreg

pfskhfnwqk idamkpfggi

421 riednvvihe nsienmtrdl kla

The inventors found that 190 out of 200 patients with AS had serum IgG antibodies directed against the peptide DPP 1 2 1 -145 (AARGNIGYIG PVPERALGLG IAADK, SEQ ID NO. 3) .

Anti-peptide antibodies were also investigated in two other inflammatory arthritides, RA and PsA, these antibodies were present in 3 of 200 patients with RA (1.5%) and only in 1 of 100 (1%) patients with PsA (Fig 4A) .

The sensitivity and specificity of the quantitative analysis of the assay in discriminating AS from healthy donors, with a cutoff value of 32,250 international units, were 95% and 100%, respectively. The AUC in the ROC analysis was 0.99 (95% confidence interval [CI] 0.9824 to 0.9990; P<0.0001) (Fig 4B) .

The ROC curves obtained by comparing AS serum specimens with RA and PsA samples are shown in Fig 4C and 4D. These data show that antibodies directed against a peptide epitope of the Klebsiella pneumoniae-derived protein DPP are typically present in the sera of patients with AS.

Further, the inventors synthetized shorther peptides than the Peptide DPP 12 i-i 4 5 (SEQ ID No. 3) NH2- AARGNIGYIG

PVPERALGLG IAADK- COOH by deleting two amino acid residues at the time, one at the NH2-terminal and the other one at the COOH-terminal . It was found that the 17 aa : (NIGYIGPVPERALGLGI, SEQ ID NO. 1) maintains the binding properties of the 25 aa peptide. The peptide was recognized by AS patients sera with the same frequency as the peptide DPP 1 2 1 -145 Further length reduction of the peptide induced a reduced binding.

DISCUSSION

The inventors report here on a serologic marker that is present in nearly all patients with AS.

In clinical practice, the diagnosis of AS is based on clinical history, physical examination and imaging and so far there are no biomarkers available to help in the diagnostic process. Moreover, despite extensive investigation, autoantibodies typically associated with AS and present in the patients' sera at high frequency have not been identified so far, leading to the hypothesis that AS may be an autoinflammatory rather than an autoimmune disease. Indeed autoantibodies directed against either collagen or neutrophils or nuclear antigens [3-5] have been detected in a few cases, and also in a recent report, which has employed a proteomic approach [6] antibodies directed against self antigens could be found only in 44% of the sera studied.

By using a peptide library approach the inventors could identify a peptide, the AS peptide, which is specifically recognized by 85% of the sera of patients with AS, but is not detectable in the sera of healthy controls.

The peptide shared similarity with different proteins present within the fibrocartilaginous sites which are primarily affected in the course of AS. These autoantigens include type I and II collagens (in particular collagen alpha-1 XXIV [17], collagen alpha-1 XXI [18], Collagen alpha-2 XI, particularly abundant at cartilagineous sites [19]), the heparan sulfate proteoglycan 2, also known as perlecan, two glycoproteins particularly represented in the extracellular matrix, fibronectin and ADAMTSL3/punctin 2 [20,21], and a protein involved in cytoskeleton remodeling, ASAP1 (ArfGAP with SH3 domain, ankyrin repeat and PH domain 1 actin) [22] .

A possible role for Klebsiella pneumoniae in the pathogenesis of AS has been reported by several groups (reviewed in ref. 7) . In this study, the inventors found that the AS peptide, which the inventors identified by screening a peptide library with sera from patients with AS, shares similarity with an amino acid sequence of the DPP protein of Klebsiella pneumoniae . Interestingly antibodies directed against the DPP 121 - 145 peptide were detectable even at higher frequency in AS serum samples when compared to the frequency of antibodies directed against the AS peptide (95"6 VGTSUS 85"6 ) . Noteworthy the an -Klebsiella pneumoniae-derived peptide antibodies are nearly absent in sera of patients affected by PsA and RA and are not detected in healthy donors.

It was also found that the 17 aa (NIGYIGPVPERALGLGI ) maintains the binding properties of the 25 aa peptide.

Although these results do not clarify the role played by Klebsiella pneumoniae in the pathogenesis of the disease, the ability of purified anti-AS peptide antibodies to recognize both autoantigens that are abundant in the entheses and amino acid sequences of Klebsiella pneumoniae-derived proteins suggest a possible link through a molecular mimicry mechanism. In conclusion, the inventors describe here the identification of an antibody that the inventors detected in the majority of the patients with AS; in the absence of other disease biomarkers this antibody may represents an interesting tool for the diagnosis of the disease.

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