WO2011045745A1

WO2011045745A1 - Antibody associated with autoimmune pancreatitis and uses thereof

Info

Publication number: WO2011045745A1
Application number: PCT/IB2010/054615
Authority: WO
Inventors: Antonio Puccetti; Claudio Lunardi; Luca Frulloni
Original assignee: Università Degli Studi Di Verona; Istituto G. Gaslini
Priority date: 2009-10-13
Filing date: 2010-10-12
Publication date: 2011-04-21
Also published as: EP2488865A1

Abstract

The present invention relates a method for detecting antibodies specific for a peptide newly identified as a marker of autoimmune pancreatitis.

Description

Antibody associated with Autoimmune Pancreatitis and uses thereof FIELD OF THE INVENTION

The present invention relates a method for detecting antibodies specific for a peptide newly identified as a marker of autoimmune pancreatitis. In particular, the method allows to discriminate between a subject affected by autoimmune pancreatitis from a subject affected by pancreatic adenocarcinoma or other autoimmune diseases such as systemic sclerosis or rheumatoid arthritis.

BACKGROUND ART

Autoimmune pancreatitis (AIP) is characterized by an inflammatory process in which prominent lymphocyte infiltration with associated fibrosis of the pancreas leads to organ dysfunction. AIP is an inflammatory disease of the pancreas with unique clinical and histological features.¹' ² Instrumentally, the involvement of pancreas in AIP may be diffuse or focal.¹' ³

Clinically, AIP may mimic pancreatic carcinoma and the differential diagnosis may be difficult. Several diagnostic criteria have been proposed,^4"6 but their real usefulness is still under debate. Serological markers of the disease include non organ specific autoantibodies (ANA)⁷ and organ specific autoantibodies, such as anti-carbonic anhydrase,^7"9 lactoferrin⁷ and anti-pancreatic secretory trypsin inhibitor.¹⁰ These organ specific autoantibodies have been found in AIP, but none of them has provided a satisfactory specificity and sensitivity. IgG4 has been proposed as a possible diagnostic marker,¹¹ but later studies have not confirmed this previous report.¹²' ¹³ Therefore, the identification of a serological marker for AIP is a major goal in clinical research.

The cause of the disease is still unknown. The autoimmune origin for AIP has been suggested but never proven and little is known about its pathogenesis.

The present invention identifies a serological marker able to discriminate AIP from pancreatic adenocarcinoma. The authors have used a molecular biology approach that has already been successfully applied to other autoimmune diseases.^14"17

DESCRIPTION OF THE INVENTION

To identify pathogenetically relevant autoantigen targets, the authors screened a random peptide library with pooled IgG immunoglobulins obtained from 20 patients with AIP. Peptide specific antibodies were detected in patients' sera. Among the identified peptides, peptide AIP i_₇ (SKDER F, SEQ ID NO. 2) was recognized by 18/20 AIP patients' sera, and by 4/40 sera of patients with pancreatic cancer (PC) but not by sera of normal donors. The peptide showed homology with an amino acid sequence of plasminogen-binding protein (PBP) of Helicobacter pylori (NCBI (national center accession number 025249) and with human UBR2 (NCBI accession number Q8IWV8), a protein ubiquitination enzyme highly expressed in acinar cells of the pancreas. Antibodies against the PBP peptide were detected in 19/20 patients (95%) with AIP and in 4/40 patients (10%) with PC. Such reactivity was not detected in patients with alcoholic chronic pancreatitis and intraductal papillary-mucinous neoplasm. The results were validated in another series of patients with AIP and PC: 14/15 patients (93%) with AIP and 1/70 (1,4%) tested positive for the presence of anti-PBP peptide antibodies. Combining the training and the validation sets the test was positive in 33/35 AIP patients (94%) and 5/110 PC patients (4,5%).

The authors have, thus, identified an antibody that was detectable in most patients with AIP but was also detected in some patients with pancreatic cancer.

It is therefore an object of the present invention a method for detecting antibodies specific for a peptide comprising the sequence X₁KX_{2 3}RR ₄ (SEQ ID No. 8) wherein Xi is S or A, X₂ is D or E, X3 is E or Q and X₄ is F, Y or Q in a biological sample comprising the steps of:

-reacting under proper conditions said biological sample with a reagent that specifically recognizes and binds the antibodies to produce a complex;

-detecting the complex.

Preferably, the peptide comprises the sequence selected from the group of: SKDERRF (SEQ ID NO. 2), AKEERRY (SEQ ID NO. 4) or AKEQRRQ (SEQ ID NO. 5).

Still preferably, the peptide consists of the sequence selected from the group of: SKDERRF (SEQ ID NO. 2), AKEERRY (SEQ ID NO. 4) or AKEQRRQ (SEQ ID NO. 5). Yet preferably, the peptide consists of AKEERRYLRDER, SEQ ID NO. 7.

In a preferred embodiment, the reagent that specifically recognizes and binds the antibodies is a peptide comprising the sequence X₁KX₂X₃RRX₄ (SEQ ID No. 8) wherein Xi is S or A, X₂ is D or E, X₃ is E or Q and X₄ is F, Y or Q.

Preferably the reagent that specifically recognizes and binds the antibodies is a peptide comprising the sequence selected from the group of: SKDERRF (SEQ ID NO. 2), AKEERRY (SEQ ID NO. 4) or AKEQRRQ (SEQ ID NO. 5). Still preferably the reagent that specifically recognizes and binds the antibodies is a peptide consisting of a sequence selected from the group of: SKDERRF (SEQ ID NO. 2), AKEER Y (SEQ ID NO. 4) or AKEQR Q (SEQ ID NO. 5).

Yet preferably the reagent that specifically recognizes and binds the antibodies is a peptide consisting of AKEER YLRDER, SEQ ID NO. 7.

In a preferred embodiment the biological sample is obtained from a subject suspected of autoimmune pancreatitis or from a subject under immunotherapy.

Preferably the detection of the complex allows to discriminate between a subject affected by autoimmune pancreatitis and a subject affected by pancreatic carcinoma.

It is a further object of the invention a method for the diagnosis and/or prognosis of autoimmune pancreatitis in a subject from which a sample is obtained comprising detecting antibodies specific for a peptide as disclosed above in the sample.

It is a further object of the invention a kit for the diagnosis and/or prognosis of autoimmune pancreatitis and/or for detecting antibodies specific for a peptide as disclosed above in a biological sample comprising at least:

- a reagent that specifically recognizes and binds the antibodies, said reagent being a peptide comprising the sequence X₁KX_{2 3}RR ₄ (SEQ ID No. 8) wherein Xi is S or A, X₂ is D or E, X₃ is E or Q and X₄ is F, Y or Q; and

- detecting means.

Preferably the reagent that specifically recognizes and binds the antibodies is a peptide comprising the sequence selected from the group of: SKDERRF (SEQ ID NO. 2), AKEERRY (SEQ ID NO. 4) or AKEQRRQ (SEQ ID NO. 5).

Still preferably the reagent that specifically recognizes and binds the antibodies is a peptide consisting of a sequence selected from the group of: SKDERRF (SEQ ID NO. 2), AKEERRY (SEQ ID NO. 4) or AKEQRRQ (SEQ ID NO. 5).

Yet preferably the reagent that specifically recognizes and binds the antibodies is a peptide consisting of AKEERRYLRDER, SEQ ID NO. 7.

The invention will be now illustrated in reference to the following non limiting figures and examples.

Figure 1. Sera of AIP patients recognized the PBP peptide, a. Training set: the different diseases studied are represented on the x axis: autoimmune pancreatitis (AIP); alcoholic chronic pancreatitis (ACP); pancreatic adenocarcinoma (PC); intraductal papillary-mucinous neoplasm (IPMN); systemic sclerosis (SSc) and rheumatoid arthritis (PvA). On the y axis the europium counts are reported, b. Validation set: autoimmune pancreatitis (AIP) and pancreatic cancer (PC) on the x axis; europium counts: y axis. The horizontal line represents the cut-off value.

Figure 2. Receiver Operating Characteristic (ROC) curve for IgG antibodies against PBP peptide comparing patients suffering from AIP and pancreatic cancer. X axis=False positive rate (specificity), Y axis=False negative rate (sensitivity) with an AUC in the ROC analysis of 0.988 (95% CI, 0.963 to 1.000; PO.001).

Figure 3. AIP serum IgG antibodies bind the Helicobacter pylori derived protein in Western blot. A Helicobacter pylori bacterial lysate was probed with: normal pooled human serum IgG (lane 1), rabbit antiserum raised against the peptide of the Helicobacter pylori plasminogen-binding protein (lane 2), antibodies affinity purified against the peptide of the the Helicobacter pylori plasminogen-binding protein from sera of patients with AIP (lane 3), sera from five different patients with AIP (lanes 4-8), antibodies affinity purified against an irrelevant control peptide from sera of patients with AIP (lane 9), sera of two healthy donors (lane 10 - 11). A peroxidase-labelled anti-human IgG antibody and an anti- rabbit IgG antibody (lane 2) were used for detection.

Figure 4. Inhibition experiments confirm cross-reactivity between anti-Helicobacter plasminogen-binding protein and anti-UBR2 peptide antibodies. Panel A: serum from an AIP patient, at a dilution that achieved 50% of the maximal binding to the Helicobacter plasminogen-binding protein coated plate (1 :400), was preincubated with increasing concentrations of either purified H. pylori protein (HP PROT) or PBP peptide (HP PEP) or UBR peptide (UBR2 PEP) or an irrelevant peptide (IRR PEP) for 1 hour at 37 C. The mixture was then transferred to the H.pylori derived protein coated ELISA plate. The remainder of the assay was then carried out as a direct ELISA test. The binding to the protein is inhibited by the Helicobacter protein, the PBP peptide and by the UBR2 peptide, but not by the irrelevant control peptide. Similar results were obtained in 5 other samples tested. The UBR2 peptide was used instead of the protein, since UBR2 expression in the cell is too low to purify an adequate amount of protein to perform this type of experiment. Moreover, the recombinant human UBR2 protein commercially available is only a part of the protein and does not comprise the homologous sequence of interest. Panel B: serum from an AIP patient, at a dilution that achieved 50% of the maximal binding to a UBR2 peptide (UBR2 PEP) coated plate (1 :800), was preincubated with increasing concentrations of either UBR2 peptide or purified H. pylori plasminogen-binding protein (HP PROT) or PBP peptide (HP PEP) or an irrelevant peptide (IR PEP) for 1 hour at 37 C. The mixture was then transferred to a UBR2 peptide coated ELISA plate. The remainder of the assay was then carried out as a direct ELISA test. The binding to UBR2 peptide is inhibited by the homologous peptide, by the Helicobacter protein, by the PBP peptide, but not by an irrelevant control peptide. Similar results were obtained in 4 other samples tested. X axis: inhibitor concentration (microg/mL), Y axis: percent of inhibition.

EXAMPLES METHODS

Patients

Serum samples were obtained from patients and healthy controls. All serum samples were stored at -20 C. Blood samples were obtained from all the subjects after written informed consent and the local ethical committee approved the study.

The characteristics of the patients included in the study are summarized in Table 1.

Table 1. Characteristics of the patients included in the study

HP+ serology (%) = Positive serology for Helycobacter Pylori

The authors studied twenty patients affected by focal autoimmune pancreatitis (AIP). The diagnosis of AIP was made on the basis of histology on pancreatic specimens in 2 patients who had undergone surgery. In 18 patients who had not had surgery, diagnosis was made based on the presence of 3 of the 4 following diagnostic criteria: 1) fine needle aspiration cytology (6 patients) or histology (7 patients), 2) association with other autoimmune diseases (10 patients); 3) typical radiological findings (CT or MR) (18 patients); 4) clinical, laboratory and instrumental response to steroid therapy (18 patients).³ Sera from AIP patients were collected within 1 month of the clinical onset of the disease and before steroid treatment. Ten patients had extra-pancreatic manifestations (5 ulcerative colitis, 3 sclerosing cholangitis, 2 interstitial nephritis, 1 retroperitoneal fibrosis).

Sera were collected within one month of symptom onset from 40 patients who had histologically confirmed pancreatic adenocarcinoma (PC). Furthermore, sera from 21 patients with alcoholic chronic pancreatitis (ACP) and from 18 patients with intraductal papillary-mucinous neoplasm (IPMN) were analysed. Finally sera from patients with two unrelated autoimmune diseases, systemic sclerosis (SSc) and rheumatoid arthritis (RA) were studied.

Forty age and sex matched healthy donors recruited among university and hospital workers without history of alcohol intake or of autoimmunity and with normal abdomen ultrasound, were also included in the analysis.

To validate the results obtained, the authors analysed the sera of an additional 15 patients with AIP and 70 patients with histologically confirmed PC. In these AIP patients, the diagnosis was made on the basis of pancreatic specimens in 1 patient. In the 14 non operated patients, diagnosis was made in the presence of: 1) fine needle aspiration cytology (4 patients) or histology (6 patients), 2) association with other autoimmune diseases (6 patients); 3) typical radiological findings (CT or MR) (14 patients); 4) clinical, laboratory and instrumental response to steroid therapy (14 patients).

Ten of the 15 AIP patients were affected by the diffuse and 5 by the focal form of the disease. Extrapancreatic manifestations were observed in 6 patients (4 ulcerative colitis, 1 sclerosing cholangitis and 1 retroperitoneal fibrosis).

All the patients with AIP and PC were tested for IgG4 levels. Moreover all the patients included in the study were tested for the presence of anti-Helicobacter pylori (H. pylori) antibodies.

IgG4 and Άη -Helicobacter pylori antibodies detection Serum IgG4 levels were determined using a commercially available immuno- nephelometric kit (Dade Behring, Marburg, Germany). The upper normal limit of serum IgG4 levels was considered 135 mg/dl, as reported by Hamano et al.¹¹

IgG antibodies to H. pylori were detected using a commercially available kit (Enzygnost H. Pylori, Siemens, Deerfield, IL, USA).

Library

The FliTrx random dodecamer peptide library, which displays peptides on the surface of Escherichia coli, was purchased from Invitrogen (Carlsbad, California, United States) and screened with pooled Immunoglobulins (Igs) purified from the sera of the 20 patients with AIP, according to manufacturer's instructions (FliTrx Panning Kit, Invitrogen). After five rounds of biopanning experiments, the enriched library was grown and single colonies induced with tryptophan to express the fusion peptides. Bacteria were lysed in sample buffer and tested in western blot with the pooled Ig fraction from autoimmune pancreatitis to check for positive clones. DNA was extracted from positive clones and sequenced. A set of 15 peptides, out of the 30 peptides obtained from the last biopanning round, was synthetized and used in a dissociation-enhanced lanthanide fluorescent immunoassay (DELFIA) assay to test individual patients' sera.

Peptide Synthesis

All the synthetic peptides, including the AIP peptide (SKDERRFEQPRV, SEQ ID No. 1), the AIPi_7 peptide (SKDERRF, SEQ ID No. 2), the AIP₆_i₂ peptide (RFEQPRV, SEQ ID No. 3), the PBP peptide (AKEERRY, SEQ ID No. 4), the UBR2 peptide (AKEQRRQ, SEQ ID No. 5) and the irrelevant control peptide (VTLPKDSDVELP, SEQ ID No. 6), were manually synthesized using the standard method of solid-phase peptide synthesis, which follows the 9-fluorenylmethoxycarbonyl (FMOC) strategy with minor modifications.¹⁸

DELFIA

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/ml in phosphate buffered saline (PBS) to coat DELFIA plates (Perkin Elmer, Boston MA). Plates were then blocked for 1 h with a blocking reagent (Perkin Elmer). Serum samples were diluted 1 :50 in bovine serum albumin (BSA) 1% (Sigma, Saint Louis MO) and incubated on the plates overnight at 4-8 °C. Plates were then washed ten times with washing buffer (Perkin Elmer). Bound antibodies were detected with a europium-labelled anti-human IgG antiserum (1 :500 in diluting buffer) (Perkin Elmer). Plates were read on a Victor 3 instrument (Perkin Elmer) and the data analyzed with a software supplied with the DELFIA instrument. Absorbance values higher than the mean + 3 SD of each serum dilution of the control group were considered positive.

Rabbit Antiserum Production

Polyclonal antibodies against a peptide corresponding to H. pylori plasminogen-binding protein (PBP), amino acid residues 298 to 309 (AKEEPv YLRDER, SEQ ID No. 7) were generated in New Zealand White rabbits using standard techniques.¹⁵

Western blot

A H. pylori extract (Virion, Ruschlikon, Switzerland) was used to detect the H. pylori PBP in immunoblot. The extract was enriched for the PBP by affinity chromatography using a Sepharose column coupled to a rabbit anti-PBP peptide antibody (see above). Blots were probed with primary antibodies followed by either peroxidase-linked anti-human Igs antibodies or anti-rabbit IgG antibodies (purchased from Sigma). HELA cell lysates were prepared using a commercially available kit (Nuclear Extract kit, Active Motif, Carlsbad, CA, USA). Cell lysates were immunoprecipitated with mouse polyclonal anti-human recombinant UBR2 (aa 1-439) antibodies (Abnova, Walnut, CA, USA) cross-linked to sepharose. Eluted proteins were resolved by SDS-PAGE and transferred to nitrocellulose membrane (Amersham Bioscience, Piscataway, NJ, USA). Blots were incubated with either mouse anti-UBR2 antibodies, purified human antibodies (10 μg/ml) or sera (1 :50 in diluting buffer). The Renaissance Chemiluminescence Kit (NEN, Boston, MA, USA) was used for detection.

Statistical analysis

The sensitivity and specificity of the test was evaluated using Receiver Operating Characteristic (ROC) curve analysis, estimating the area under the curve (AUC) with 95% CI. Statistical analysis was carried out using SPSS 13 software (SPSS Inc, Chicago, 111, USA). RESULTS

Peptide Library

The authors have screened a peptide library with pooled Igs derived from a panel of 20 patients with AIP, in order to identify only those peptides possibly relevant to the pathogenesis of the disease. The authors identified a peptide (named AIP peptide or AIP peptidei_i₂: SKDERRFEQPRV, SEQ ID NO. 1) recognized by serum IgGs of 17 out of 20 (85%) AIP patients but not by healthy donors, using a DELFIA assay. However 22 out of 40 (55%) patients' sera with pancreatic cancer and 10 out of 20 (50%) with ACP reacted with this peptide. In order to better map the Ig reactivity towards this epitope and to better discriminate the considered pancreatic diseases, the authors decided to synthetize two peptides named AIPi_₇ peptide (SKDERRF, SEQ ID NO. 2) and AIP₆_i₂ peptide (RFEQPRV, SEQ ID NO. 3) (Table 2A).

Table 2. Peptides used in the study and sequence homologies

A) list of the synthetic peptides used in the present invention

B) sequences homologies between the AIPi_₇ peptide and the Helicobacter pylori plasminogen-binding protein and

C) sequences homologies between the Helicobacter pylori plasminogen-binding protein and human E3 ubiquitin-protein ligase UBR2.

( : ) indicates identity; (*) indicates conservative substitutions.

The AIP₆_i₂ peptide was recognized by 4/20 (20%>) patients with AIP and 20/40 patients (50%) with PC.

The AIPi_₇ peptide was recognized by 18/20 AIP patients (90%) and by 4/40 (10%) patients with PC. These data demonstrate that the AIPi_₇ peptide sequence contains an epitope recognized by nearly all the sera of patients with AIP.

Helicobacter pylori and AIP

Since H. pylori infection has been associated with the pathogenesis of AIP,^19"21 the authors decided to compare the AIPi_₇ peptide sequence with known bacterial protein sequences in a protein data bank (Swiss-Prot database) using the BLASTP via the NCBI BLAST network service. The authors found that the peptide AIPi_₇ shared a high degree of homology (4 identities and 3 conservative substitutions) with the PBP encoded by H. pylori (Table 2B).

The authors then synthesized the bacterial peptide called PBP peptide (AKEERRY, SEQ ID NO. 4) and used it to test the panel of patients 'sera.

The authors found that 19 out of 20 (95%) patients with AIP had serum IgG antibodies against the peptide. Four (age: 48, 64, 72, 79 yrs) out of 40 (10%>) patients with PC recognized this sequence. Such reactivity was not detected in healthy donors and in patients affected by the other pancreatic diseases studied (Figure 1 A).

Serum antibodies against the identified peptide were not detected also in the two systemic autoimmune diseases considered, RA and SSc (Figure 1A).

The sensitivity and specificity of the quantitative analysis of the present invention's assay, with a cut off of 32.000 europium count, were 95% and 96.6%>, respectively (p<0.0001). The area under the curve (AUC) in the ROC analysis was 0,988 (CI 95%: 0.970-1.000). By comparing only patients suffering from AIP and pancreatic cancer, the sensitivity and specificity were 95% and 90% respectively, with AUC in the ROC analysis of 0.973 (CI: 95%: 0.937- 1.000)(p<0.0001) (Figure 2).

To validate these data, a second series of patients was analyzed for the presence of anti- PBP peptide antibodies, using the same europium count cut-off value. Fourteen out of 15 AIP patients (93%) and 1 (age 60 yrs) out of 70 PC patients (1.4%) resulted positive (Figure IB), with AUC in the ROC analysis of 0.988 (CI%: 0963- 1.000) (p<0.0001). Combining the training and the validation sets, 33 out of 35 AIP patients (94%) and 5 out of 110 PC patients (4.5%) were positive for antibodies, with a sensitivity of 94%, and a specificity of 95%.

IgG4 resulted positive in 19/35 AIP patients (54%), with a cut-off of 135 mg/dL ( i.e. if the concentration of IgG4 is > 135 mg/dL, then the sample is positive)¹¹. The 2 patients negative for the anti-PBP antibodies, were IgG4 positive, whereas all the remaining 16 IgG4 negative patients were positive for anti-peptide antibodies. Eleven of the 110 patients with PC (10%) tested positive for IgG4.

The results of the H. pylori testing are reported in Table 1. Patients with AIP have an higher frequency of antibodies against H. Pylori.

The ELISA results obtained with the PBP peptide were confirmed by western blot analysis using an H. pylori derived protein extract (Fig. 3); 32 out of the 35 AIP sera showed reactivity against a protein band corresponding to the PBP. Representative examples are shown in Figure 3. Antibodies directed against the H. pylori PBP were not detected in healthy donors and in patients with IPMN and ACP and were present in 4 out of 110 patients with pancreatic cancer.

Autoantigen targets in AIP

As AIP is characterized by chronic pancreatic tissue damage, the authors next compared the PBP peptide with human pancreatic proteins in a protein data bank (Swiss-Prot database of known human sequences) using the BLASTP via the NCBI BLAST network service, and found that the peptide is homologous (5 identities and 1 conservative substitution) to E3 ubiquitin-protein ligase (UBR2), AKEQRRQ (SEQ ID NO. 5) a component of the N-end rule pathway, highly expressed in acinar cells of the pancreas (Table 2C). Antibodies against the PBP peptide affinity purified from AIP patients' sera recognized the UBR2 protein in western blot (data not shown). Reactivity against such protein was also present in 29/35 sera of patients with AIP, but not in sera of patients affected by ACP. Moreover only 2/40 patients with pancreatic cancer bound UBR2 in western blot. IgG antibodies against the UBR2 peptide were detectable in 22/35 patients with AIP. Such reactivity was not present in sera of patients with other pancreatic diseases. Finally the binding of AIP serum antibodies to the purified H. pylori PBP was inhibited by the protein itself, the PBP peptide, by the UBR2 peptide, but not by an irrelevant control peptide. Similarly the binding of AIP serum antibodies to UBR2 peptide was inhibited by the homologous peptide, by the H. pylori protein, the PBP peptide, but not by the irrelevant peptide (Fig. 4).

Discussion The authors report a serological marker that is present in most patients with AIP. In clinical practice the differential diagnosis between AIP and PC is difficult. It has been reported that up to 10% of the patients who undergo pancreatic resection for a suspected pancreatic cancer have a final diagnosis of pancreatitis.²² The criteria for the diagnosis of AIP are not yet completely defined and therefore the identification of serological markers is relevant in clinical practice.

The present assay was able to discriminate between AIP and pancreatic cancer in the majority of the cases. Of note, 4,5% of patients with pancreatic cancer tested positive on the invention's assay. The mean age of patients with AIP is lower than that of patients with PC, however the authors do not believe that such difference influences the test. Indeed the test resulted negative in other groups of patients with a mean age similar to that of the AIP group.

High IgG4 levels are helpful in the diagnosis of AIP although the level chosen and the frequency reported are variable in different studies.^11"13 Therefore the ability of IgG4 to discriminate AIP from pancreatic cancer is still under debate. In the patients included in this study, the authors found the presence of high IgG4 levels in 54% of the patients with AIP and in 10% of patients with PC.

The anti-PBP peptide antibody the authors describe here was not positive in normal healthy donors or in patients with autoimmune diseases unrelated with AIP, such as SSc and RA.

19 21 23 24

A role for H. pylori infection in the pathogenesis of AIP has been suggested. ' ' In this study, the authors show that the peptide AIPi_₇, identified by peptide library screening of AIP sera, shares homology with an amino acid sequence of PBP of H. pylori. Of note, the PBP peptide is also homologous to the human protein UBR2, highly expressed in the pancreatic acinar cells. Therefore, it seems likely that the pancreatic acinar cells may be the target of the autoimmune aggression in AIP.

In conclusion the authors describe the identification of a novel antibody present in the majority of AIP patients. References

1. Finkelberg DL, et al, N Engl J Med 2006;355:2670-6.

2. Zamboni G, et al, Virchows Arch 2004.

3. Manfredi R, et al, Radiology 2008;247:435-43. 4. Chari ST. et al, J Gastroenterol 2007;42 Suppl 18:39-41.

5. Kim MH, Kwon S. J Gastroenterol 2007;42 Suppl 18:42-9.

6. Okazaki K, et al., J Gastroenterol 2007;42 Suppl 18:32-8.

7. Okazaki K, et al, Gastroenterology 2000;118:573-81.

8. Kino-Ohsaki J, et al., Gastroenterology 1996;110: 1579-86.

9. Frulloni L, et al, Pancreas 2000;20:382-8.

10. Asada M, et al., Pancreas 2006;33:20-6.

11. Hamano H, et al, N Engl J Med 2001 ;344:732-8.

12. Choi EK,et al, Pancreas 2007;35: 156-61.

13. Ghazale A, et al, Am J Gastroenterol 2007;102: 1646-53.

14. Zanoni G, et al., PLoS Med 2006;3:e358.

15. Lunardi C, et al., Nat Med 2000;6: 1 183-6.

16. Lunardi C, et al, Lancet 2002;360:915-21.

17. Puccetti A, et al, Clin Exp Allergy 2005;35: 1599-607.

18. Wellings DA, Atherton E. Methods Enzymol 1997;289:44-67.

19. Kountouras J, et al., J Cell Mol Med 2005;9: 196-207.

20. Guarneri F, et al, J Cell Mol Med 2005;9:741-4.

21. Kountouras J, et al., Pancreas 2005;30:192-3.

22. Wolfson D, et al., Pancreas 2005;31 :203- 17.

23. Kountouras J, et al., Gastroenterology 2007;133:368-9.

24. Rieder G, et al, World J Gastroenterol 2007; 13: 3939-3947.

Claims

1. A method for detecting antibodies specific for a peptide comprising the sequence X1KX2X3RRX4 (SEQ ID No. 8) wherein Xi is S or A, X₂ is D or E, X₃ is E or Q and X₄ is F, Y or Q in a biological sample comprising the steps of:

-detecting the complex.

2. The method of claim 1 wherein the antibodies are specific for a peptide comprising the sequence selected from the group of: SKDERRF (SEQ ID NO. 2), AKEERRY (SEQ ID

NO. 4) or AKEQRRQ (SEQ ID NO. 5).

3. The method of claim 2 wherein the antibodies are specific for a peptide consisting of the sequence selected from the group of: SKDERRF (SEQ ID NO. 2), AKEERRY (SEQ ID NO. 4) or AKEQRRQ (SEQ ID NO. 5).

4. The method of claim 2 wherein the antibodies are specific for a peptide consisting of AKEERRYLRDER, SEQ ID NO. 7.

5. The method of any one of previous claims wherein the reagent that specifically recognizes and binds the antibodies is a peptide comprising the sequence X1KX2X3RRX4 (SEQ ID No. 8) wherein Xi is S or A, X₂ is D or E, X₃ is E or Q and X₄ is F, Y or Q.

6. The method of any of claims 1 to 4 wherein the reagent that specifically recognizes and binds the antibodies is a peptide comprising the sequence selected from the group of: SKDERRF (SEQ ID NO. 2), AKEERRY (SEQ ID NO. 4) or AKEQRRQ (SEQ ID NO. 5).

7. The method of any of claims 1 to 4 wherein the reagent that specifically recognizes and binds the antibodies is a peptide consisting of a sequence selected from the group of: SKDERRF (SEQ ID NO. 2), AKEERRY (SEQ ID NO. 4) or AKEQRRQ (SEQ ID NO. 5).

8. The method of any of claims 1 to 4 wherein the reagent that specifically recognizes and binds the antibodies is a peptide consisting of AKEERRYLRDER, SEQ ID NO. 7.

9. The method of any one of previous claim wherein the biological sample is obtained from a subject suspected of autoimmune pancreatitis.

10. The method of claim 9 wherein the detection of the complex allows to discriminate between a subject affected by autoimmune pancreatitis and a subject affected by pancreatic carcinoma.

11. The method of any one of previous claim wherein the biological sample is obtained from a subject under immunotherapy.

12. A method for the diagnosis and/or prognosis of autoimmune pancreatitis in a subject from which a sample is obtained comprising detecting antibodies specific for a peptide as disclosed in any of claims 1 to 4 in the sample.

13. A kit for the diagnosis and/or prognosis of autoimmune pancreatitis and/or for detecting antibodies specific for a peptide as disclosed in any of claims 1 to 4 in a biological sample comprising at least:

- a reagent that specifically recognizes and binds the antibodies, said reagent being a peptide comprising the sequence X₁KX₂X₃RRX₄ (SEQ ID No. 8) wherein Xi is S or A, X₂ is D or E, X₃ is E or Q and X₄ is F, Y or Q; and

- detecting means.

14. The kit of claim 13 wherein the reagent that specifically recognizes and binds the antibodies is a peptide comprising the sequence selected from the group of: SKDERRF (SEQ ID NO. 2), AKEERRY (SEQ ID NO. 4) or AKEQRRQ (SEQ ID NO. 5).

15. The kit of claim 13 wherein the reagent that specifically recognizes and binds the antibodies is a peptide consisting of a sequence selected from the group of: SKDERRF (SEQ ID NO. 2), AKEERRY (SEQ ID NO. 4) or AKEQRRQ (SEQ ID NO. 5).

16. The kit of claim 13 wherein the reagent that specifically recognizes and binds the antibodies is a peptide consisting of AKEERRYLRDER, SEQ ID NO. 7.