WO2004087747A2

WO2004087747A2 - Citrullinated synthetic peptides and uses thereof

Info

Publication number: WO2004087747A2
Application number: PCT/IB2004/001056
Authority: WO
Inventors: Paola Migliorini
Original assignee: Universita' Di Pisa
Priority date: 2003-03-31
Filing date: 2004-03-29
Publication date: 2004-10-14
Also published as: ITTO20030246A1; WO2004087747A3

Abstract

Novel citrullinated linear synthetic peptides and multiple antigen peptides (MAP) comprising them, which are useful as antigens in assays for the detection of antibodies and T cells specific to citrullinated antigens, are described, the antibodies and T cells being characteristic of auto-immune diseases such as rheumatoid arthritis and multiple sclerosis.

Description

Novel citruUinated synthetic peptides and uses thereof

The present invention relates to novel citruUinated synthetic peptides and their use in the diagnosis and treatment of auto-immune diseases, particularly rheumatoid arthritis and multiple sclerosis.

In auto-immune diseases, the immune system responds to the organism's own antigens, producing antibodies and autoreactive T cells which cause damage to tissues and organs .

The mechanisms which cause this autoaggression are only partially explained. A possible mechanism is the modification of endogenous antigens by means of infective agents or of enzymatic reactions which occur at atypical sites or in uncontrolled manner. As a result of these modifications, the endogenous antigens are no longer recognized as "self" by the organism's immune system, which attacks them. One of these enzymatic reactions is the deimination of proteins, that is, the transformation of the amino-acid arginine into citrulline, which renders the proteins thus modified more susceptible to the action of proteases and thus accelerates their catabolism (Pritzker L.B. et al . , Biochemistry, 2000, 39 (18) : 5374-81) . This mechanism, which generally has a protective significance, may in some cases cause more extensive damage . The presence of deiminated proteins has in fact been demonstrated in areas of demyelination in multiple sclerosis (Wood D.D. et al . , Ann. Neurol. 1996, 40(l):18-24) and in the synovial membrane in rheumatoid arthritis (Masson-Bessiere C. et al., J. Immunol. 2001 Mar. 15; 166 (6) :4177-84 ; Jonca N. et al . , Arthritis Rheum. 2001, 4 (10) : 2255-62) , and hence at the sites where the lesions which are typical of these two auto-immune diseases occur. Moreover, both in rheumatoid arthritis and in multiple sclerosis, antibodies have been found which are capable of reaching with deiminated proteins and peptides and which are therefore potentially capable of contributing to the extension of the lesions. In rheumatoid arthritis in particular it has been demonstrated that the presence of these antibodies against deiminated peptides and proteins has a precise diagnostic and prognostic significance (Forslin K. et al . , Scand. J. Rheumatol . 2001, 30 (4) :221-4 ; Forslin K. et al . , Scand. J. Rheumatol. 2000, 29 (5) : 320-2 ; Schellekens G.A. et al . , Arthritis Rheum. 2000 Jan. 43 (1) : 155-163) . These antibodies are in fact present precociously and characterize the most aggressive forms of disease .

Since, in the early stages of arthritis, it is extremely difficult to make a diagnosis on an exclusively clinical basis, diagnostic tests which enable antibodies correlated with this disease to be detected are of great diagnostic use. One of these antibodies is the rheumatoid factor, which, however, is also present in other forms of arthritis as well as in various other diseases and in healthy elderly patients; moreover, it is present in the more advanced stages of rheumatoid arthritis but only rarely at the start . The above-mentioned antibodies that are directed against deiminated proteins and peptides are, on the contrary, found in approximately half of patients suffering from rheumatoid arthritis but not in patients suffering from other forms of arthritis. Moreover, these antibodies are present at the start of the disease, particularly in cases characterized by more serious joint damage (Nogueira . et al . , Ann. Rheum. Dis. 2001 Sep. 60 (9) :882-7; Vincent C. et al . , Arthritis Rheum. 2002 Aug. 46 (8) ) :2051-8) . In the light of these characteristics, tests for the detection of antibodies against deiminated peptides and proteins seem particularly desirable in the context of the diagnosis of rheumatoid arthritis.

Two methods of detecting antibodies against deiminated peptides and proteins (hereinafter also referred to an "anti- citrullinated-peptide antibodies") are currently available commercially.

The first method is an immunological assay based on the use, as antigens, of cyclic peptides bound to a solid phase, for example, a polystyrene plate. However, in this method, the preparation of the solid phase is particularly complex since the antigens used require a covalent attachment to the plate. Moreover, the synthesis of a cyclic peptide is much more complex, from the chemical point of view, than the synthesis of a linear peptide.

The other commercially-available method uses a deiminated protein - filaggrin - in an immunoblot assay which, as is known, is a complex and not very quantitative technique. Moreover, the method of obtaining the antigen is also quite complex in this case. On the one hand, it is in fact possible to use natural filaggrin which is already partially deiminated but which requires lengthy and complex extraction and purification procedures; alternatively, it is possible to use recombinant filaggrin which, however, must be purified from transfected bacteria and then subjected to deimination in vi tro with the enzyme peptide arginine deiminase. From the point of view of its structure and configuration, however, a recombinant protein often has characteristics which are different from those of the natural protein and which may give it a different antigenic reactivity. The object of the present invention is therefore to provide an antigen for the detection of anti-citrullinated-peptide antibodies which is easy to prepare and which permits the development of an assay that is simple to perform, versatile, and easily adaptable to automated systems.

This object is achieved by citruUinated linear synthetic peptides consisting of the following amino-acid sequence from the N-terminal to the C-terminal:

GPAGPX_ιGGGX₂GX₃GX₄GX₅GX₆GHNDGG,

wherein G is a glycine residue, P is a proline residue, A is an alanine residue, H is a histidine residue, N is an asparagine residue, D is an aspartic acid residue and X_x, X₂, X₃, X₄, X _; and X₆ are selected independently from an arginine residue and a citrulline residue, provided that at least one of Xi, X₂, X₃, X , X5. and X₆ is a citrulline residue.

The sequence of citruUinated peptides of the invention corresponds to the sequence from position 35 to position 58 of the protein EBNA I of the Epstein-Barr virus, in which at least one arginine residue is replaced by a citrulline residue .

It has been found that the above-described citruUinated peptides are specifically capable of binding anti- citrullinated-peptide antibodies which are typically present in the blood of patients suffering from rheumatoid arthritis and other auto-immune diseases. They are therefore suitable or use in an immunological assay method for the detect ion and/or quantification of anti-citrullinated-pept de antibodies in a sample of biological fluid suspected to contain such antibodies.

The citruUinated peptides of the invention also have chemical and structural characteristics which render t_hem particularly advantageous in comparison with the above- mentioned known antigens. In the first place, since they are linear, they are easy to synthesize by known methods for the chemical synthesis of peptides (Atherton E. et al., Bioorg. Chem. 1979, 8, 351). Moreover, these peptides are highly soluble and have a good capacity to bind to the solid phases that are commonly used for performing immunological assays (for example, polystyrene plates) simply by adsorption; this capacity is good enough to render the assay extremely versatile and easily adaptable to automated systems.

A preferred embodiment of the citruUinated peptides of the invention is that in which each of X_if X₂, X₃, X₄. Xs, and X₆ is a citrulline residue. A citruUinated peptide as described above in which all of the arginine residues are replaced by citrulline residues in fact provides the best results in an immunological assay for the detection of anti- citrullinated-peptide antibodies .

Antigenically effective peptide fragments derived from the above-described citruUinated peptides also fall within the scope of the invention. The expression "antigenically effective" is intended to indicate that the peptide fragments retain the same antigenic properties as the citruUinated peptides from which they are derived, that is, the capacity to bind anti-citrullinated-peptide antibodies specifically. Preferably, the sequence of these antigenically effective peptide fragments is constituted by at least 10 consecutive amino-acids of the above-described sequence of the linear citruUinated peptides and includes at least three GX repetitions in which at least one X is a citrulline residue.

According to another aspect of the invention, the above- described citruUinated peptides are in the form of multiple antigen peptides (MAP) .

As is known from the scientific literature, for example, Tam J.P. Proc. Natl. Acad. Sci. U.S.A. 1988, 85 (15) : 5409-13 , the structure of multiple antigen peptides is characterized by an immunologically inert amino-acid core, generally a radially branched core of lysine or of lysine and alanine, to which a plurality of identical copies of the antigen sequence of interest, generally 4 or 8 copies, are bound. The antigen sequences of interest are constructed on the amino-acid core by conventional methods for chemical synthesis on a solid phase.

The expression "immunologically inert core" means that, when the MAPs are used as immunogens, they do not induce the production of antibodies directed against the central amino- acid core.

MAPs have some characteristics which render them particularly advantageous for use as antigens in immunological assays : in the first place, they are extremely soluble, which leads to a good capacity for adsorption on the solid phase; moreover the antigen peptide sequences bound to the amino-acid core are exposed to a greater extent, which facilitates their recognition by the antibodies. A multiple antigen peptide (MAP) comprising a plurality of identical copies of a citruUinated linear synthetic peptide or of an antigenically effective fragment derived therefrom as described above, bound to an immunologically inert amino-acid core, therefore also falls within the scope of the present invention.

Preferably, the multiple antigen peptide comprises four identical copies of the citruUinated linear synthetic peptide or of the antigenically effective fragment derived therefrom.

Even more preferably, the multiple antigen peptide has the structure shown in Figure 1, in which K is a lysine residue and G, P, H, N, D, X_lf X₂, X₃, X , X5, and X₆ are as defined above. Even more preferably, each of X_if X₂, X₃, X₄, X₅, and X₆ in the structure of Figure 1 is a citrulline residue.

As indicated above, the citruUinated peptides of the invention, both in linear form and in MAP form, are particularly suitable for use an antigens in an immunological assay for detecting the presence and/or measuring the levels of anti-citrullinated-peptide antibodies in a sample of a biological fluid.

The sample of biological fluid may, for example, be a serum sample of an individual suspected of being affected by rheumatoid arthritis .

In order to perform the assay, the citruUinated peptide used as the antigen is coated to a solid substrate, for example, a micro-titration plate. One of the advantages of the citruUinated peptides of the invention is that they can be bound to the substrate simply by adsorption. The adsorption of the peptide on the substrate can be achieved, for example, by incubating a solution of the peptide in a suitable buffer, for example, PBS (phosphate buffered saline) with the solid substrate. In a first step of the assay method, the sample of biological fluid to be analyzed is placed in contact and incubated with the citruUinated peptide adsorbed on the solid substrate. Any anti-citrullinated-peptide antibodies that are possibly present in the sample are thus specifically bound by the peptide adsorbed on the solid substrate, producing a citruUinated peptide/anti-citrullinated-peptide antibody complex.

In a second step of the method, the sample of biological fluid is separated from the solid substrate so as to eliminate non-bound materials, for example, by washing.

In a third step of the method, an indicator antibody capable of binding any anti-citrullinated-peptide antibodies that are present on the substrate in the form of a citruUinated peptide/anti-citrullinated peptide antibody complex is added to the solid substrate, thus producing a citruUinated peptide/anti-citrullinated-peptide antibody/indicator antibody complex. The indicator antibody may, for example, be a human anti-IgG immunoglobulin.

Finally, in a fourth step of the method, the presence of the citruUinated peptide/anti-citrullinated peptide antibody/ indicator antibody complex on the solid substrate is detected, the presence of said complex on the solid substrate being indicative of the presence of anti-citrullinated- peptide antibodies in the sample of biological fluid.

In a preferred embodiment, the above-described method is an ELISA immunological assay in which the indicator antibody is conjugated to an enzyme.

In this embodiment, the presence of the citruUinated peptide/anti-citrullinated-peptide antibody/indicator antibody complex on the solid substrate is determined by detecting the activity of the enzyme conjugated to the indicator antibody.

Enzymes suitable for this purpose are, for example, alkaline phosphatase, radish peroxidase, and beta-galactosidase, i.e., enzymes capable of being detected with the use of a chromogenic substrate. A chromogenic substrate is a substance which, as a result of the reaction with the enzyme, gives rise to a coloured product which can thus be detected spectrophotometrically.

In another preferred embodiment, the solid substrate is a micro-titration plate, for example, of the type commonly used for performing ELISA immunological assays. The micro- titration plate is preferably a polystyrene plate.

The present invention also includes a kit for the detection of anti-citrullinated-peptide antibodies in a sample of biological fluid, comprising a peptide as defined above or an antigenically effective fragment derived therefrom and at least one further reagent. Preferably, the further reagent is an indicator antibody capable of binding the anti- citrullinated-peptide antibodies. Preferably, the indicator antibody is a human anti-IgG immunoglobulin conjugated to an enzyme capable of reacting with a chromogenic substrate.

The inventors have also found that the citruUinated peptides of the invention are capable of specifically recognizing not only anti-citrullinated-peptide antibodies as described above, but also T cells specific to citruUinated peptides. T cells specific to citruUinated peptides are characteristic of some auto-immune diseases in which there is a cell- mediated immune response to deiminated (citruUinated) antigens. For example, in multiple sclerosis, the existence of T cells specific to the basic protein of deiminated myelin (MBP) has been demonstrated (Tranquill L.R. et al . , 2000, Mult.Scler. 6, 220-5) .

The citruUinated synthetic peptides of the invention, both in linear form and in MAP form, can therefore be used as antigens for the detection of T cells specific to citruUinated antigens.

The type of assay commonly used to detect antigen-specific T cells is the proliferation test in which peripheral blood leukocytes of a patient to be examined are grown together with graduated doses of the antigen, in this case a citruUinated synthetic peptide as described above. If the patient has previously encountered citruUinated peptides and has a sufficient number of T cells specific thereto, proliferation will be achieved in the presence of this antigen.

Naturally, in the test, it is necessary to introduce a substance capable of inducing proliferation of all of the T cells (a mitogen) as a positive control, as well as some antigens against which the patient is assumed not to have been immunized, as a negative control.

The citruUinated peptides of the invention, particularly in MAP form, are optimal antigens for this type of assay. It is in fact known that the polymeric structure of MAPs facilitates their processing and presentation to T cells. The antigen-specific T cells can then be morphologically and functionally characterized e.g. they can be analyzed by cytofluorometry for the expression of surface molecules and the production of cytokines. Finally, the citruUinated peptides of the invention, both in linear form and in MAP form, can also be used as modulators of the immune response in auto-immune diseases, for example, multiple sclerosis or rheumatoid arthritis.

It is fact known that synthetic peptides have the ability to bind to MHC molecules with greater affinity than natural peptides in a system in which the synthetic peptide can act as agonist of the natural peptide, but stimulating only a few subsets of T cells, or as antagonist, inhibiting the maturation of T cells or their function. Peptides capable of competing with natural peptides and of inhibiting the autoimmune response have been described in animal models of diabetes (von Herrath M.G. et al . , 1998, J. Immunol. 161, 5087) . In animal models of rheumatoid arthritis, immunization with peptides of collagen II induces non- pathogenic IL4-producing T cells and therefore has a "protective" effect on the disease (Myers L.K. et al . , Arthritis Rheum. 2002, 46, 12, 3369) . In animal models of systemic lupus, the administration of a phosphorylated synthetic peptide derived from an auto-antigen slows the development of nephritis and extends the survival of the animals (Monnneaux F. et al . , 2003, Eur. J. Immunol. 33, 2, 287) .

A similar immunomodulating function of synthetic peptides is already used for therapeutic purposes in multiple sclerosis in which one of the treatments used is glatiramer acetate.

On the basis of this knowledge, it can therefore be predicted that the citruUinated synthetic peptides of the invention can be used as immunomodulators in rheumatoid arthritis, in multiple sclerosis, or in other auto-immune diseases in which the existence of a cell-mediated immune response to deiminated antigens has been demonstrated. The following example, which relates to the use of the citruUinated peptides of the invention as antigens in an ELISA assay for the determination of anti-citrullinated- peptide antibodies, is provided by way of illustration and is not intended in any way to limit the scope of the present invention as defined in the appended claims.

ELISA assay for the determination of anti-citrullinated- peptide antibodies

The antigen used was a multiple antigen peptide (MAP) having the structure shown in Figure 1, in which all of the arginine residues were replaced by citrulline.

This peptide was diluted to a concentration of 5 microgrammes/ml in PBS (phosphate buffered saline) and loaded into the wells of a polystyrene micro-titration plate (50 μl/well) . The plate was left overnight at +4°C to permit interaction between peptide and plastics (it may, however be incubated at 37°C for 1-2 hours with the same result) . Upon completion of the coating period, the wells containing the antigen, plus an equal number of wells which were used as controls, were treated for 1 hour at room temperature (RT) with 3% bovine serum albumin (BSA) in PBS. The patients' serum samples (diluted 1:200 in a buffer constituted by 1% BSA, 0.05% TWEEN X-100 in PBS) were then loaded onto the plate (50 μl/well) and left to incubate for 3 hours at RT. After the incubation period, one washing was performed with 1% PBS Tween X-100 and two washings were performed with PBS (150 μl/well) . An anti human-IgG antibody conjugated to the enzyme alkaline phosphatase, diluted 1:3000 in 1% PBS BSA, 0.05% TWEEN X-100, was used to show that the antigen/antibody reaction had taken place; the antibody (50 μl/well) was incubated for 3 hours at RT with stirring. Upon completion of the incubation, after three washings as described above, the alkaline phosphatase substrate p-nitrophenyl phosphate was added to the wells which, in the presence of the enzyme, produced a yellow product which was measurable by spectrophotometric techniques at a wavelength of 405 nm, and the quantity of which was proportional to the quantity of antibodies bound. The results of the test were expressed as the percentage of positiveness, calculated by dividing the absorbance of each serum sample by the absorbance of a positive serum sample the value of which was set arbitrarily at 100.

Serum samples of 270 patients suffering from rheumatoid arthritis (RA) , 29 patients suffering from psoriatic arthritis, 31 patients suffering from mixed cryoglobulinaemia, 30 patients suffering from systemic lupus erythematosus, 26 patients suffering from ankylosing spondylitis and 45 patients suffering from systemic sclerosis were tested by this method.

By considering each result that was greater than the 99th percentile of the control group (represented by 77 healthy transfusion-centre donors) to be positive, anti- citrullinated-peptide antibodies were found in 40% of the serum samples of patients with RA, in 3.8 % of the serum samples of patients with ankylosing spondylitis, in 3.4% of the serum samples of patients with psoriatic arthritis, and in 3.2% of the serum samples of patients with mixed cryoglobulinaemia; no patient with systemic lupus erythematosus or systemic sclerosis was positive.

Claims

1. A citruUinated linear synthetic peptide constituted by the amino-acid sequence:

GPAGPXιGGGX₂GX₃GX₄GX₅GX₆GHNDGG,

in which G is a glycine residue, P is a proline residue, A is an alanine residue, H is a histidine residue, N is an asparagine residue, D is an aspartic acid residue and Xi, X₂, X₃, X, X₅, and X₆ are selected independently from an arginine residue and a citrulline residue, provided that at least one of Xi, X₂, X₃, X₄, X₅, and X₆ is a citrulline residue, and antigenically effective fragments derived therefrom.

2. A citruUinated linear synthetic peptide according to Claim 1 in which each of Xi, X₂, X₃, X₄, X₅, and X₆ is a citrulline residue.

3. A citruUinated multiple antigen peptide (MAP) comprising a plurality of copies of a citruUinated linear synthetic peptide according to Claim 1 or Claim 2 , or of an antigenically effective fragment derived therefrom, bound to an immunologically inert amino-acid core.

4. A multiple antigen peptide (MAP) according to Claim 3, comprising four copies of a citruUinated linear synthetic peptide according to Claim 1 or Claim 2, or of an antigenically effective fragment derived therefrom.

5. A multiple antigen peptide (MAP) according to Claim 4, having the structure shown in Figure 1, in which K is a lysine residue and G, P, H, N, D, X_lf X₂, X₃, X₄, X₅, and Xβ are as defined in Claim 1.

6. A multiple antigen peptide (MAP) according to Claim 5 in which each of Xi, X₂, X₃, X₄, X₅, and X₆ is a citrull ne residue.

7. Use of a citruUinated peptide according to any one of Claims 1 to 6 as an antigen in an immunological assay for the detection of anti-citrullinated-peptide antibodies.

8. A immunological assay method for the detection of anti- citrullinated-peptide antibodies in a sample of a biological fluid, comprising the steps of:

(a) contacting and incubating said sample of biological fluid with a citruUinated peptide according to any one of Claims 1 to 6 adsorbed on a solid substrate,

(b) separating the sample of biological fluid from the solid substrate,

(c) adding to said solid substrate an indicator antibody capable of binding anti-citrullinated-peptide antibodies, and

(d) detecting the presence on said solid substrate of a citruUinated peptide/anti-citrullinated-peptide antibody/ indicator antibody complex, the presence of the complex on the solid substrate being indicative of the presence of anti- citrullinated-peptide antibodies in the sample of biological fluid.

9. A method according to Claim 8 in which the indicator antibody is conjugated to an enzyme.

10. A method according to Claim 9 in which the enzyme is selected from the group consisting of alkaline phosphatase, radish peroxidase, beta-galactosidase.

11. A method according to any one of Claims 8 to 10 which is an ELISA immunological assay.

12. A method according to any one of Claims 8 to 11 in which the solid substrate is a micro-titration plate.

13. A method according to Claim 12 in which the mic_ro- titration plate is a polystyrene plate.

14. A method according to any one of Claims 8 to 13 in which the biological fluid is serum.

15. A method according to any one of Claims 8 to 14 in which the sample of biological fluid is a sample of a biological fluid of an individual suspected of being ₍ affected by rheumatoid arthritis.

16. Use of a citruUinated peptide according to any one of Claims 1 to 6 as an antigen in a proliferation test for the detection of T cells specific to citruUinated antigens.

17. A citruUinated peptide according to any one of Claims 1 to 6 as a modulator of the immune response in auto-immune diseases.

18. A citruUinated peptide according to Claim 17, in which the auto-immune disease is rheumatoid arthritis or multiple sclerosis.

19. A kit for the detection of anti-citrullinated-peptide antibodies in a sample of a biological fluid, comprising a peptide according to any one of Claims 1 to 6 or an antigen fragment derived therefrom, and at least one further reagent.

20. A kit according to Claim 19 in which the further reagent is an indicator antibody capable of binding anti- citrullinated- peptide antibodies.