WO2009019657A2

WO2009019657A2 - Means for diagnosis and treatment of rheumatoid arthritis

Info

Publication number: WO2009019657A2
Application number: PCT/IB2008/053152
Authority: WO
Inventors: Gianfranco Ferraccioli; Francesco Ria
Original assignee: Universita' Cattolica Del Sacro Cuore
Priority date: 2007-08-06
Filing date: 2008-08-06
Publication date: 2009-02-12
Also published as: WO2009019657A3; ITRM20070429A1

Abstract

The present invention relates to oligonucleotide pairs (primers) allowing amplification of CDR3-Vbeta- Jbeta regions of TCR to rheumatoid arthritis, a method for diagnosis and/or monitoring of said disease using said oligonucleotides, a diagnostic kit for diagnosis and/or monitoring of rheumatoid arthritis comprising said oligonucleotides, nucleotide sequences amplified from said oligonucleotides as rheumatoid arthritis markers, amino acid sequences encoded by said nucleotide sequences, having such amino acid sequences as rheumatoid arthritis markers and the peptides themselves, together with one or more immunostimulant adjuvants and pharmacologically acceptable excipients, as polypeptide vaccines against rheumatoid arthritis.

Description

MEANS FOR DIAGNOSIS AND TREATMENT OF RHEUMATOID ARTHRITIS

DESCRIPTION

STATE OF THE PRIOR ART

Rheumatoid arthritis (RA) is an autoimmune chronic inflammatory disease characterized by chronic and often progressive arthritis, which up to some years ago led to loss of working activity 50% of patients at +10 years from onset of symptom's. Today the evolution of the disease is less dramatic, both due to an earlier diagnosis of the same and the new therapies currently available.

However, it remains a disease with a high social impact .

In the literature there is a general consensus in deeming that among the autoantigens determining the triggering of said autoimmune disease, type II collagen

(Coll-2) be one of those more involved. This is so because a loss of tolerance toward the sequestered autoantigen collagen has been demonstrated, by detection of IgG-type autoantibodies anti-Coll2. Moreover, it has been demonstrated that Coll-2 is able to stimulate self- reactive T cells within the context of Class II major histocompatibility complex HLA-DR4-DRl, and that the self-reactive clones are present in the synovial fluid, can persist over time, and produce cytokines capable of keeping tissue inflammation constantly active, whereas other cytokines such as TNFa reduce T cell reactivity toward the autoantigen (autoAg) .

All evidences published to date, tested on transgenic mice, demonstrate that T cell response in the presence of DR4-DR1 exhibits a high affinity in particular for the central peptide of Coll-2 (sequence 263-270) (ROSLONIEC EF, WHITTINGTON KB, ZALLER DM, KANG AH: "HLA-DRl (DRB1*0101) and DR4 (DRBl*0401) use the same anchor residues for binding an immunodominant peptide derived from human type II collagen". J. Immunol. 2002, 168: 253-9) . Therefore, self-reactivity appears within the scope of a well-outlined genetic context _r i.e. the presence of

HLA DR4+ or DRl+ haplotype (DRbetal or shared epitope) which is just that associated to RA in all populations examined.-

Given these premises, it should be possible to demonstrate the presence of T cells specific for this epitope in RA patients, above all in the early stages of the disease. However, to date no evidence has been reported about the possibility of identifying in clinical practice the self-reactive T-cell populations (comprised of T-cell clones, called T clonotypes) , i.e. the cells actually responsible for the disease, by a specific test for clonotype identification.

Evidently, the identification of such clonotypes would allow diagnosing the disease and monitoring its course simply and accurately.

To date, clonotype presence has been researched by studying, first under cytofluorimetry and then by PCR technique, the restriction of V (variable) beta chains of the TCR (T-cell receptor) .

The most variable region, CDR3 (i.e., 3^rd complementary determinant region) of TCR alpha ^' and beta chains, is that interacting directly with the antigenic peptide (pAg) . If expanded T clones were to exist, using the same receptor in different patients, in theory these clones should be identifiable by the restricted use of given "public" beta chains, and for the presence of very similar and therefore "public" sequences of the TCR.

However, to date cytofluorimetry results have been generally concordant in demonstrating oligoclonal restrictions, but very discordant in demonstrating the presence of reproducible restrictions. In fact, in the literature there are publications demonstrating restrictions for specific beta chains of

• the variable region, Vbetal4 (PAILLARD X, WEST SG, LAFFERTY JA: Evidence for the effect of a superantigen in RA. Science 1991, 153: 325-9), Vbetal7, Vbetal4, Vbeta3 (HOWELL MD, DIVELEY JP, LUNDEEN K : Limited T cell receptor beta chain heterogeneity among interleukin-2 receptor positive synovial T cells suggests a role for superantigen in rheumatoid arthritis. Proc Natl Acad Sci U S A 1991, 88: 10921-5), or Vbeta7 (SOTTINI A, IMBERTI L, GORLA R, CATTANEO R, PRIMI D: Restricted expression of TCR V beta but not V alpha genes in rheumatoid arthritis. Eur. J.Immunol. 1991, 21: 461-6).

The use of the more sensitive PCR, using specific primers for the various beta chains of the TCR, has certainly allowed to refine the approach to clonotype identification, yet results have been very contradictory as different clonotype families have been documented in the synovial fluid but not in the peripheral blood of the patients studied (JENKINS RN, NIKAEIN A, ZIMMERMANN A, MEEK K, LIPSKY PE: TCR V beta chain bias in rheumatoid arthritis. J.Clin. Invest . 1993, 92: 2688-701) . On the other hand it is known that, e.g., after a viral infection only less than the 1% of cytotoxic T lymphocytes expands; therefore, oligoclone demonstration requires a much more sensitive method.

A further step was accomplished with the introduction of the spectratyping or immunoscopy technique, in which multiple PCRs are run, by using multiple primers for the 23 beta chains of TCR, and this resulted in approximately 230 fractions and about 8 different lengths of the Vbeta region of CDR3. In undifferentiated cells, the information encoding this chain is subdivided into three segments: V (variable) segment, D (diversity) segment and J (joining) segment. C (constant) segment follows.

In the locus encoding the TCR beta chain there are about 26 V segments, 2 D segments and 13 J segments. When the cell differentiates, a V segment, a D segment and a J segment are joined by means of a site-specific recombination process. This essentially consists in a programmed deletion of the DNA sandwiched between the three segments, which then is discarded. In the case of the beta chain, there occur some variations in the sequence at the V-D and D-J junctions, adding a variability factor to the process. The CDR3 region is determined during V-D-J recombination. Joining of the V- D-J rearrangement to the C segment then occurs by RNA splicing, after transcription. It has been demonstrated that amplification of J region genes, besides of genes encoding the Vbeta region, involved in the V-J-beta recombination process, increases about 10-fold sensitivity in clonotype identification.

The main problem in the identification of self- reactive clonotypes has always been represented by the low number of self-reactive cells involved. If cytotoxic T-cells clonally expanding after a viral infection are estimated to be of the order of 1% of the CD8, in the scope of autoimmune diseases it is estimated that self- reactive CD4 cells be of the order of 1-2 log less. Therefore, to demonstrate their presence it is necessary on the one hand to surmise the Ag or Ags involved, and on the other hand to have available a technique allowing to identify restricted low-number populations. To date, Vbeta chain cytofluorimetry or mere spectratyping have been unable to identify any clonal population in peripheral blood.

SUMMARY OF THE INVENTION In the present invention a set of primers were selected in the Vbeta chain and the Jbeta chain of the CDR3 of the TCR receptor, which identify, by PCR, clonotypes of RA-associated T cells having specific amino acid sequences in the CDR3 region of the receptor. Such primers allow to diagnose both the disease and the relapse of the latter, even 2 to 3 months before symptoms occur, as well as identify gene sequences, amplified by said primers, encoding a group of localized peptides, each one in the CDR3 region of TCR, recognizing as antigen the region 261-273 of Coll-2 within the context of HLA-DR4 molecule and that are present only in individuals suffering from RA. Besides being RA markers, such peptides are also useful, together with immunostimulant adjuvants, in the formulation of polypeptide vaccines against RA, as they stimulate an immune response against T-cells bearing the TCR having said sequences in the CD3 region. Moreover, the primers selected in the present invention allow RA diagnosis and/or monitoring from T cells present in peripheral blood. Given the surprising earliness with which the clonotypes identified by said primers appear in peripheral blood, the primers of the present invention allow an extremely early and even presymptomatic diagnosis of disease presence and activity.

Therefore, object of the invention are oligonucleotide pairs (primers) allowing amplification of CDR3-Vbeta-Jbeta regions of TCR associated to rheumatoid arthritis, a method for diagnosis and/or monitoring of said disease using said oligonucleotides, a diagnostic kit for diagnosis and/or monitoring of rheumatoid arthritis comprising said oligonucleotides, nucleotide sequences amplified from , said oligonucleotides as rheumatoid arthritis markers, amino acid sequences encoded by said nucleotide sequences, peptides having such amino acid sequences as rheumatoid arthritis markers and the peptides themselves, together with one or more immunostimulant adjuvants and pharmacologically acceptable excipients, as polypeptide vaccines against rheumatoid arthritis.

DETAILED DESCRIPTION OF THE FIGURES Fig. 1: Fig. 1 shows examples of immunoscopy- spectratyping of the CDR3-Vbeta-Jbeta region of TCR, rearranged for Vbeta4 and Jbeta2.1, in the absence of Ag and in the presence of autoAg Coll-2. As it can be seen, there is a normal (Gaussian) distribution of T-cell populations of a normal subject.

Fig. 2: Fig. 2 shows that when instead it is assessed the rearrangement of the CDR3 region for Vbetall-Jbeta2.2, without Ag the polyclonal Gaussian distribution persists and, after autoAg stimulation, a clear peak appears, identifying a clone of self-reactive T cells in an RA patient, yet not in a normal subject.

Fig. 3: Fig. 3 shows that only some of the TCR rearrangements (for Vbetal-Jbeta2.6 ) demonstrate to be absent in peripheral blood, to appear after autoAg stimulation in peripheral blood, but above all to be already expanded in synovial fluid. Some of these clonotypes, identified during disease activity, disappeared post-remission, and had reappeared prior to relapse in 70% of examined patients.

Fig. 4: Fig. 4 shows that identification of key sequences of TCR enabled to design and compute the putative TCR structure binding the autoAg Coll.2. This structure enabled to design the peptides to be utilized in vaccination, and therefore render the TCR tolerant and prevent T-cell activation of the self-reactive clonotype.

DETAILED DESCRIPTION OF THE INVENTION According to the present invention, RA can be identified by the presence of PCR amplificates starting from cDNA of T cells present in peripheral blood and, optionally, also from cDNA of T cells present in synovial fluid, by oligonucleotide pairs having SEQ ID 1 and 39, 12 and 35, 15 and 36, 18 and 33,' and 18 and 38. In fact, said nucleotide pairs were selected as characterized in that they amplify sequences of cDNA coming from peripheral blood and/or synovial fluid T cells associated to rheumatoid arthritis.

Amplificates that can be' obtained by said RA- associated primers, and their length in bp are summarized in Table I reported below: TABLE I :

Pairs of primers in the CDR3-Vbeta-Jbeta region of the TCR, indicating, in the presence of amplificate from cDNA of T cells of peripheral blood as reported below, the presence of RA.

Therefore, according to the present invention, the presence of an amplificate from the cDNA of peripheral blood T cells, having at 5' the sequence of a forward

(fw) primer indicated in the above-reported square and at 3' the sequence of the corresponding reverse (rev) primer indicated in the same Table, indicates that the CDR encoded by the mRNA corresponding to said cDNA will react against coll2 to which the T cells on which it arises cause the autoimmune response of Rheumatoid Arthritis. In short, the presence of such amplificates indicates RA presence in the examined individual.

Advantageously, the primers selected in the present invention allow identification of RA-associated T-cell clonotypes that are present in peripheral blood, though in low concentrations, and that, following suitable medical care, disappear from peripheral blood, to reappear therein in case of relapse even two or three months prior to the relapse itself.

Therefore, the oligonucleotide pairs of the present invention are particularly indicated, both for a diagnosis of the disease in suspicious cases and for monitoring patients having the disease and properly treated in order to prevent the same disease relapses, as well as for monitoring subjects at risk (e.g., relatives of the ill) . Therefore, the extreme earliness with which the clonotypes identified by the oligonucleotide pairs arise in peripheral blood allows a precocity of diagnosis that even precedes the appearance of symptoms, making the therapeutic intervention particularly effective and timely.

The oligonucleotide sequences of the present invention amplify plural variable-length cDNA sequences reported in Table I, all associated to the disease.

Such amplificates can of course be sequenced by standard techniques or even with automatic sequencers, like, e.g., SEQ ID No 41 and 43, which are amplified by the pairs of primers having SEQ ID No 1 and 39 and that have been sequenced with said standard methods.

Given the primers, the amplification conditions and the length of the amplified sequences associated to the disease, to a technician skilled in the art it is absolutely evident how to sequence the amplificates obtained, which therefore are considered as described in the present description. Sequences 41 and 43, given by way of example, are to be construed as a mere example of the feasibility of such a sequencing. It should also be specified that to a technician skilled in the art are available not only the required technologies, kits, protocols for sequencing of short amplificates, but also specialized services to which the amplificate is provided and that take care of the sequencing. Therefore, what is described herein cannot be deemed insufficient to obtain without special efforts by a technician skilled in the art the sequences of the amplificates reported in table I, which are obtained with the oligonucleotides indicated in the present invention. The nucleotide sequence indicates also the amino acid sequence, the reading frame being easily deducible, as said oligonucleotides all amplify also a region upstream of the CDR3 common to all TCRs, represented by the amino acid sequence CAS or GAR. The reading frames of the primers of the invention are anyhow indicated in the sequence listing and in the following table:

Table II Reading frames of the forward primers of the invention

However, by using a program processing the three possible amino acid sequences starting from the nucleotide sequence, the right one will be that having the CAS or the GAR upstream of the CRD3 in frame.

An example of this are SEQ ID No 42 and 44, whose only difference lies in a small number of amino acids located at position 114-118 of the CDR3 of the TCR and in which the CAS sequence is at position 26-28 of both peptides . Accordingly, having provided the oligonucleotide pairs, and since the present description provides all indications necessary to optimally obtain the amplificates of the invention and the reading frames of the forward primers, that indicate from which base (1, 2, or 3) of said primer there starts the reading module yielding the right peptide, also the amplified nucleotide sequences and the deriving amino acid sequences are deemed to be sufficiently described.

The method of diagnosis of the invention provides, synthetically: purification of T cells from peripheral blood, culture of said cells in the presence of antigen Coll-2 261-273 and, in parallel, of a control in which said .antigen is absent, RNA extraction from said T cells, reverse transcription and then cDNA synthesis, a PCR with the forward primers of the invention and a reverse primer downstream of those indicated in Table I (in Cbeta region) so as to increase the number of amplificates comprising the region of interest, a PCR RUN-OFF reaction with the reverse primers indicated in Table I labeled; the amplificates thus obtained are read by (capillary or other) electrophoresis apparatus intended for DNA fragment analysis, such as, e.g., the Genetic Analyzer, and yielded patterns are overlapped and normalized, and there are identified as expanded in an antigen-specific manner those peaks exhibiting an at least 2-fold increase in the area of the antigen-stimulated sample with respect to that cultivated without antigen.

According to the present invention, the method for detection of RA presence or monitoring the course of already diagnosed RA may be carried out by starting from peripheral blood samples, and optionally, in parallel, from synovial fluid samples. In case of peripheral blood, mononucleated cells present in those samples are isolated according to standard methods known to a technician skilled in the art, and placed in culture in a suitable medium (e.g., RPMI 1640) according to standard techniques known to a technician skilled in the art, for a time period ranging from about 48 to 96 hours.

In case instead of synovial fluid, RNA extraction is carried out without prior cell culturing steps. RNA can be extracted according to standard ^• procedures known to a technician skilled in the art. In a non-limiting example of the process, synovial fluid may be directly centrifuged at about 1200-1500 rpm for 10 min, and the pellet thus obtained is then resuspended in buffered saline for a washing step and again centrifuged at the same speed and for a similar time. The pellet thus obtained could be further washed as indicated above, or directly used for RNA extraction according to standard techniques known to a person skilled in the art. In an embodiment of the invention, peripheral blood T cells, and, optionally, synovial fluid T cells are first purified on Percoll gradient and placed in 96-well plates (Costar Corp., Cambridge, MA) in the amount of 5xlO⁵ cells/well. A well of peripheral blood T cells will be placed in culture in the presence of concentrations ranging from about 10 μg/ml to about 30 μ/ml of peptide Coll.2 261-273, whereas another well will be placed in a parallel culture in the absence of said peptide. As culture medium, RPMI 1640 (Gibco BRL Life Technologies, Basel, Switzerland) may be used, supplemented with 2μM L- Glutamine, 50 μM 2-ME, 50 rαg/ml gentamicin (Sigma- Aldrich, St. Louis, MO, USA), and 1% human AB serum.

Synovial fluid T cells need no stimulation with antigen Coll. 2 261-273, as this cell population is already enriched in the clonotypes identified by the markers of the invention in case of RA presence, whereas for peripheral blood T cells it is preferable to perform stimulation with the indicated antigen, since clonotypes identified by the marker of the invention, though present also in peripheral blood in case of RA presence, are anyhow less represented and a previous culture with said antigen increases their percentage, allowing a more certain diagnosis.

Upon carrying out the cell culture as indicated, RNA extraction and preparation of the cDNA to be analyzed could be performed. Then, total RNA is extracted from said T cells and subjected to an RT PCR cycle for cDNA preparation.

Of course, mRNA for RT PCR can also be extracted. RNA extraction can be performed by using laboratory protocols known to the technician skilled in the art, or even by using commercial kits. RT PCR can be performed by using oligo-dT primers with standard methods or, in this case as well, by using commercial kits suitable for the purpose .

The cDNA will now be subjected to amplification cycles by PCR utilizing the pairs of primers indicated in Table 1, i.e., SEQ ID No 1 and SEQ ID No 39, SEQ ID No 12 and SEQ ID No35, SEQ ID No 15 and SEQ ID No 36, SEQ ID No 18 and SEQ ID No 33, SEQ ID No 18 and SEQ ID No 38.

The primers of the invention have been isolated and selected among many, as combining two very important properties; in fact, they allow to detect the sequences of interest when present and can be used under the same PCR conditions, thereby allowing use of a single all- comprising (dNTPs, MgCl₂, Taq and Buffer for PCR) PCR reaction mixture and of a single .reverse primer that can be distributed in plural reaction wells in which there will be placed the sample and a different forward primer for each well.

In the method of the invention a first amplification step is suggested, by using not the Jbeta primers indicated in Table I as reverse primers, but a common primer, downstream of said Jbeta primers, allowing to amplify a more common, and therefore more represented TCR region, and therefore exponentially increasing the sequences comprising also the region of interest. This step, envisaging an intermediate amplification step, serves to increase the (usually small) number of copies of sequences that can be amplified with the reverse Jbeta primers of the invention (see Table I) , thereby making more effective the hereto-described method for diagnosis. In fact, an increase in these sequences yields a higher number of amplificates for diagnosis and makes the result more "readable".

However, nothing would prevent one from carrying out the method of the invention by amplifying directly with the primers indicated in Table I; it is just that, for the diagnostic purposes of. the invention, the intermediate step with the primer having SEQ ID No 27, or with an analogous primer selected in the same region, is deemed to improve the yield of the diagnostic method, making it more reliable and more effective. For convenience's sake, the cDNA could be directly suspended into the reaction mixture and the different forward primers distributed in the different wells together with the reaction mixture thus prepared.

In the embodiment comprising the diagnostic kit of the invention, the forward primers could be aliquoted separately in ready-to-use or master concentrations, or said primers could be already present in a dehydrated form and in the right amounts, in labeled reaction wells provided with the same kit. The reaction buffer could be provided in a concentration suitable for its storage (e.g., 10X) and it could be already mixed with the right concentration of MgCl₂, or, to avoid problems of precipitation of said salt, the salt could be provided in a separate container. The single reverse primer could be provided in a ready-to-use concentration or in a master concentration to be diluted at the performing of the PCR reactions. Therefore, according to the present invention, the cDNA is amplified by multiple parallel PCR reactions (for convenience's sake they can of course be carried out at different times, yet the use of a multiple reaction on multiple media such as plural-well microtiters is clearly advantageous) .

According to the invention, as forward primers there could be used the primers denominated Vb in Table I, and therefore the primers having SEQ ID No 1, 12, 15, 18. Such primers are particularly advantageous, as they allow to identify the sequences of interest, have the same amplification conditions and can be used, in different wells, in a single multiplex PCR reaction. Moreover, for such primers there may be used- a single reverse primer selected in the present invention and having SEQ ID No

27.

Then, the first PCR reaction of the method of the invention can be performed by placing the cDNA to be assayed in as many wells as are the primers having SEQ ID No 1, 12, 15 and 18, together with the reaction mixture comprising dNTPs, Taq polymerase, MgCl₂, reverse primer having SEQ ID No 27 and H₂O as needed.

Therefore, the reaction mixture according to the invention could comprise reactants in the following concentrations:

H₂O q.s. to 50μl

PCR Buffer 1OX IX

MgCl₂ final concentration (f.c.) 2mM DNTPs f.c. 25OuM Fw primer f.c. 0.4 uM

Rev. primer f.c. 0.4 uM

Taq polymerase 2.5U cDNA ≤lμg

Evidently, the concentrations of the various components could be changed by following notions known to the technician skilled in the art on the matter of PCR (e.g., higher MgCl₂, lower annealing temperature). For the forward primers of the present invention and the reverse primer of SEQ ID No 27, the following PCR protocol is suitable:

PCR protocol Step I Denaturation 94⁰C 2 min

Step II Denaturation 94⁰C 30 sec

Step III Annealing 55°C 30 sec

Step IV Elongation 72°C 60 sec

II to IV repeated for 39 cycles Step V Stop 72°C 5 min

Amplificates obtained by the above-indicated PCR will now be subjected to a RUN-OFF reaction by PCR, in which the reverse primers indicated in Table I, labeled with fluorochromes, will be amplified with the products of the respective forward primers.

Accordingly, the cDNA amplified with the fw primer of SEQ ID 1 will be amplified with the rev. primer of SEQ ID No 39, that obtained with the fw primer of SEQ ID No 12 with the rev. primer of SEQ ID No 35, that obtained with the fw primer of SEQ ID No 15 with the rev. primer of SEQ ID No 36, and that obtained with the fw primer of SEQ ID No 18 will be amplified in two different reactions, with the rev. primer of SEQ ID No 33 and with that of SEQ ID No 38. The reverse primers, having SEQ ID No 33, 35, 36, 38 and 39, could be labeled with any suitable fluorochrome known to a technician skilled in the art, such as, e.g., 6-FAM, VIC, NED, ROX, TAM, TAMRA, TxRED, Cy5, Cy3. Therefore, ' the reaction mixture will comprise H₂O q.s. to 50μl

Buffer 1OX IX

MgCl₂ f.c. 3.2mM

DNTPs ^' f.c. 80OuM

Labeled Jβ primer f.c. 0.04uM Taq polymerase 2.5ϋ

Vbeta-Cbeta PCR product about 2μl

The conditions of the Run-off reaction with the primers of the invention are as follows: Run Off Reaction

Step I Denaturation 92⁰C 5 min Step II Denaturation 92°C 70 sec Step III Annealing 60⁰C 60 sec

Step IV Elongation 72°C 90 sec

II to IV repeated 13 times

Step V Stop 72°C 10 min

The cycle number of the reaction depends on how much amplificate is to be obtained. In the present invention it was observed that a cycle number ranging from 10 to 15 suffices to yield an amount of amplificate allowing a reliable diagnosis.

Run off reaction products are fractionated on an electrophoresis apparatus suitable for DNA fragment analysis (e.g., Genetic Analyzer in the various series of the company Applied Biosystems) . Obtained patterns are compared by suitable software programs (e.g., Gene Scan or Gene Mapper) . Then, results obtained from samples cultivated in the absence or in the presence of the antigen are graphically overlapped and modified, when required, by introduction of multiplication factors making overlappable the height and area of the majority

(78/10) of the peaks composing the Gaussian of CDR3 distribution (normalization) .

If among the T cells bearing the receptors characterized by recombination of a Vbeta-Jbeta pair there are some cells specific for peptide Coll-2 261-273, in the sample obtained from stimulated cultured cells it will be seen that one peak, or more seldom two peaks, will be of greater size with respect to the same peaks obtained in the control sample. Such an expansion is significant if the area of the peak obtained from the sample cultivated in the presence of the peptide is >2- fold that of the same peak obtained from the sample cultivated in the absence of antigen post-normalization. Therefore, the method of the invention will signal the presence of RA, when products of the Run-Off reaction of the samples stimulated with peptide Coll2 261-273 will yield peaks exhibiting an at least 2-fold area increase with respect to that cultivated without antigen, by overlapping and normalizing the obtained patterns.

The method of the invention is particularly advantageous as it allows, for the first time, to:

- early identify arthritic patients as carriers of rheumatoid autoimmune disease, who might greatly benefit from an immediate active therapy on T cells, therefore bringing these patients into remission over short times; identify in already diagnosed RA patients the disease still active from the autoimmune standpoint and, in cases into remission, those who still have the self- reactive clones and therefore that which might be a residual autoimmune disease, alike a residual disease in the hemato-oncological field;

- periodically monitor the disease and identify its reappearance from two to three months prior to clinical and biological relapse;

- study self-reactive T clones to identify any T- cell co-activation molecules, or molecules responsible for transmission of the self-activation signal, and advance an optional signal suppression with specific or nonspecific inhibition.

The kit according to the ^' present invention could contain merely aliquots of the primers indicated in Table I and of the primer having SEQ ID No 27, or it could contain also the reaction buffer 10X, the MgCl₂, the Taq polymerase; moreover, the kit ^' of the invention could comprise plural-well PCR plates, prepared beforehand and already containing the dehydrated pairs of primers required for carrying out the method of the invention, or the reaction mixture could already comprise the primer having SEQ ID No 27; furthermore, it could comprise the reactants required for RT PCR, those for RNA purification, be it total or mRNA, and the reverse primers for RUN-OFF reaction, i.e. the primers having SEQ ID No 33, 35, 36, 38, 39, could be labeled beforehand with a fluorochrome selected, e.g., among those indicated above, and ready-to-use. In the kit there could also be present other forward primers beside those already indicated comprised in the group of primers having SEQ ID No 1-26, and other reverse primers beside those already indicated comprised in the group SEQ ID No 28-40. Advantageously, these primers could be used to identify further RA-associated clonotypes by the method of the invention.

The invention also comprises the nucleotide sequences encoded by the amplification fragments delimited by the primers indicated in Table I, i.e., the fragments amplified by the following pairs of primers: fw primer of SEQ ID 1 rev. primer of SEQ ID No 39; length of said nucleotide sequences: 135 bp, fw primer of SEQ ID No 12 rev. primer of SEQ ID No 35; length of said nucleotide sequences: 135, 138 and 141 bp, fw primer of SEQ ID No 15 rev. primer of SEQ ID No 36; length of said nucleotide sequences: 199 and 202 bp, fw primer of SEQ ID No 18 rev. primer of SEQ ID No 33; length of said nucleotide sequences: 83, 86 and 89 bp e fw primer of SEQ ID No 18 primer rev of SEQ ID No 38; length of said nucleotide sequences: 83, 89 and 92 bp. These sequences are easily found by a technician skilled in the art; having the amplificate, the sequencing is obvious and can be carried out even by automatic means.

For instance, the amplification fw primer of SEQ ID 1 rev. primer of SEQ ID No 39 yielded two nucleotide sequences (SEQ ID No 41 and 43) and the respective amino acid sequences (SEQ ID No 42 and 44) . Said nucleotide sequences in turn encode, as exemplified above, amino acid sequences that are part of the TCR CDR3, and for which therefore the reading frame of the nucleotide sequences obtained is known and indicated in Table II. In the present invention the above-described peptides are of course them also RA markers, as representing just the TCR portion that reacts with Coll2 causing the autoimmune reaction, and have a length sufficient to be used, together with one or more immunostimulant adjuvants, like, e.g., Alum, Isco, immunomodulating cytokines and pharmacologically acceptable excipients.

Therefore, in one of its embodiments the invention comprises a vaccine comprising the peptides indicated above, or those encoded by the nucleotide sequences of the amplification fragments obtained with the primers of the invention, having the length indicated in Table I, and the reading frame indicated in Table II, one or more immunostimulant adjuvants and pharmacologically acceptable excipients. The vaccine of the invention may be manufactured by use of DNA sequences encoding the peptides of the invention, in plasmids intended for development of naked DNA vaccines, which optionally can contain also sequences encoding immunomodulating cytokines, like for instance, yet not exclusively, IL-2, IL-12, TNFalpha, GM-CSF, etc. More specifically, the peptides could be those encoded by the DNA fragments obtained by amplification according to the invention, such as, e.g., those delimited upstream by the fw primer of SEQ ID 1 and downstream by the rev. primer of SEQ ID No 39, said fragments having a length of 135pb and a reading frame starting from the^' base at position 3 of the primer having SEQ ID No 1, fw primer of SEQ ID No 12 rev. primer of SEQ ID No 35, said fragments having a length of 135, of 138 and of 141 bp and a reading frame starting from the base at position 2 of the primer having SEQ ID No 12, fw primer of SEQ ID No 15 rev. primer of SEQ ID No 36, said fragments having a length of 199 and of 202 bp and a reading frame starting from the base in position 1 of the primer having SEQ ID No 15, fw primer of SEQ ID No 18 rev. primer of SEQ ID No 33, said fragments having a length of 83, 86, 89 bp and a reading frame starting from the base at position 2 of the primer having SEQ ID No 18, and fw primer of SEQ ID No 18 rev primer of SEQ ID No 38, said fragments having a length of 83, 89 and 92 bp and a reading frame starting from the base at position 2 of the primer having SEQ ID No 18; like, e.g., the peptides having the amino acid sequences SEQ ID No 42 and 44, an immunostimulating adjuvant, and pharmacologically acceptable excipients.

The invention further comprises a therapeutic method for treatment of RA, comprising the administration of the vaccine of the invention in therapeutically effective doses to patients in need thereof.

EXAMPLES :

1. T~spec±fic proliferation post-challenge with Coll.2-peptide 261-273.

Peripheral blood and/or synovial fluid T cells were first purified in Percoll gradient and placed in 96-plate wells (Costar Corp., Cambridge, MA) in the amount of 5xlO⁵ cells/well in the presence of increasing concentrations of peptide Coll.2, sequences 250-264, 261-

273 and 289-303. The culture medium, RPMI 1640 (Gibco BRL

Life Technologies, Basel, Switzerland) , was supplemented with 2μM L-Glutamine, 50 μM 2-ME, 50 mg/ml gentamicin

(Sigma-Aldrich, St. Louis, MO, USA), and 1% human AB serum.

At +72 h, Ag-specific reaction of T cells was assessed by measuring [³H] thymidine incorporation, and this was the method that allowed to identify the TCR sequence of self-reactive T cells expanding with the AutoAg.

2. TCR repertoire analysis.

TCR repertoire analysis that allowed to identify the amino acid sequences of the invention was carried out by using a modification of a protocol described in RIA F, GALLARD A, GABAGLIA CR, GUERY JC, SERCARZ EE. Selection of similar naive T cell repertoires but induction of distinct T cell responses by native and modified antigen. J Immunol 2004; 172 : 3447-53.

In particular, peripheral blood and synovial fluid cells were cultivated in the presence or in the absence of the peptide for 3 days in RPMI 1640 as described. Total RNA was extracted from cell suspension by using RNeasy Mini Kit (Qiagen GmbH, Hilden, Germany) following standard procedures. TCR cDNA, tel quel (without stimulation) and after Ag stimulation was first amplified with two specific primers (i.e., pairs of nucleotide sequences of about 20 bases, obtained by chemical synthesis, complementary to lengths of the target DNA. For the carrying out of the invention and the identification of the specific primers selected in the present invention, there were used in the first PCR reaction of the method all of the Vb primers indicated in the sequence listing from SEQ ID No 1 to SEQ ID No 26 in 26 different wells, and as reverse primer the primer of SEQ ID No 27. PCR reaction was performed as indicated in the description, producing an exponential increase of the number of original DNA copies. Subsequently, to an aliquot of the product of each PCR reaction, in distinct wells for each new reverse primer used, fluorescent reverse primers having SEQ ID No 28-40 were added to bind each of the joining segments used for the RUN reaction. Then, chain reaction products were fractionated by using the Genetic Analyzer in accordance with nucleotide length. If there are no clonotype expansions, a Gaussian- type distribution of CDR3 length is observed. On the other hand, if there is clonal expansion, a perturbation in the Gaussian distribution is observed. The specific Vbeta-C-beta and Jbeta primer sequences were derived from IMGT database (http: //imgt . cines . fr) , following nomenclature .

Run-off reaction products were analyzed by Applied

Biosystems 3100 Prims, employing a gene scanning software

(Gene-scan 2.0 software - Applied Byosistems, Foster City, CA, USA) . Results are further reported as rate stimulation index RSI (rate stimulation index ^" = normalized peak area obtained from Ag-stimulated cells/ peak area of unstimulated cells) .

3. Clonotype sequence identification. Following the hereto-described protocol, it was possible to recognize two peptide sequences on the CDR3 region of the beta chain, encoding a Vbetal-Jbeta2.6 rearrangement, which proved able to identify specific clonotypes, that reappear after a relapse of the disease, disappear after remission and, above all, are found in peripheral blood and concomitantly in the articular environment (synovial fluid)^'.

They are encoded by the DNA sequences delimited by the following pairs of primers: fw primer of SEQ ID 1 rev. primer of SEQ ID No 39, fw primer of SEQ ID No 12 rev. primer of SEQ ID No 35, fw primer of SEQ ID No 15 rev. primer of SEQ ID No 36, fw primer of SEQ ID No 18 rev. primer of SEQ ID No 33 and fw primer of SEQ ID No 18 rev. primer of SEQ ID No 38. For instance, the amplification fw primer of SEQ ID 1 rev. primer of SEQ ID No 39 yielded two nucleotide sequences (SEQ ID No 41 and 43) and the respective amino acid sequences (SEQ ID No 42 and 44) .

Claims

- 27 - CLAIMS

1. Oligonucleotide pairs having SEQ ID 1 and 39, 12 and 35, 15 and 36, 18 and 33, and 18 and 38, characterized in that they amplify sequences of cDNA from peripheral blood and/or synovial fluid T cells associated to rheumatoid arthritis (RA) .

2. Nucleotide sequences amplified by the oligonucleotide pairs according to claim 1 as rheumatoid arthritis markers, characterized by the following length in base pairs (bp) : forward primer of SEQ ID 1, having a reading frame starting from base at position 3 and reverse primer of

SEQ ID No 39, said nucleotide sequence having a length of

135 bp, forward primer of SEQ ID No 12, having a reading frame starting from base at position 2 and reverse primer of SEQ ID No 35, said nucleotide sequence having a length di 135, 138 or 141 bp, forward primer of SEQ ID No 15, having a reading frame starting from base at position 1 and reverse primer of SEQ ID No 36, said nucleotide sequence having a length of 199 or 202 bp, forward primer of SEQ ID No 18, having a reading frame starting from base at position 2 and reverse primer of SEQ ID No 33, said nucleotide sequence having a length of 83, 86 or 89 bp, and forward primer of SEQ ID No 18, having a reading frame starting from base at position 2 and reverse primer of SEQ ID No 38, said nucleotide sequence _. having a length of 83, 89 or 92 bp.

3. Amino acid sequences encoded by the sequences according to claim 2 as rheumatoid arthritis markers.

4. A Method for diagnosis and/or monitoring of the course of rheumatoid arthritis, comprising the following steps: a. T cells present in samples of peripheral blood are isolated and placed in parallel cultures in the - 28 - presence and in the absence of antigen Coll2 261-273 for a time period ranging from 48 to 96 hours; b. total or messenger RNA of said cells is extracted; c. said RNA is subjected to an RT PCR cycle with oligo-dT primers and cDNA is generated; d. said cDNA is subjected to parallel PCR reactions, using for each reaction a forward primer selected from the group comprising the primers having SEQ ID No 1, 12, 15 and 18 so as to have a PCR reaction for each primer and, as reverse primer of each reaction, the primer having SEQ ID No 27; e. each amplificate obtained at d. is subjected to a RUN-OFF reaction by PCR with a fluorochrome-labeled nested reverse primer, the amplificate obtained with the forward primer having SEQ ID No 1. is amplified with the labeled primer having SEQ ID No 39, the amplificate obtained with the forward primer having SEQ ID No 12. is amplified with the labeled primer having SEQ ID No 35, the amplificate obtained with the forward primer having SEQ ID No 15. is amplified with the labeled primer having SEQ ID No 36, the amplificate obtained with the forward primer having SEQ ID No 18. is amplified with the labeled primer having SEQ ID No 33 and other amplificate obtained with the forward primer having SEQ ID No 1. is amplified with the labeled primer having SEQ ID No 38; f. the products obtained at e. are analyzed by electrophoretic apparatus suitable for DNA fragment analysis . g. the data obtained at f. are overlapped and normalized, and there are identified as expanded in an antigen-specific manner those peaks exhibiting an at least 2-fold increase in the area of the ^' antigen- stimulated sample with respect to that cultivated without antigen.

5. The method according to claim 4, wherein said peptide Coll2 261-273 is administered to said cells in - 29 - culture at a concentration ranging from 10 to 30 μg/ml.

6. A method for identification of T-cell clonotypes associated to rheumatoid arthritis comprising all of the steps according to any one of the claims 4 or 5, wherein at d. there are used further forward primers selected from the group o.f primers having SEQ ID No 1-26, and wherein at f. there are used further reverse primers labeled for each further amplificate obtained, selected from- the group comprising the primers having SEQ ID No 28-40.

7. A kit for early diagnosis of the presence or relapse of RA, comprising aliquots of oligonucleotides having SEQ ID No 1, 12, 15, 18, 27, 33, 35, 36, 38 and 39 wherein said oligonucleotides having SEQ ID No 33, 35, 36, 38 and 39 are optionally labeled with fluorochromes .

8. Kit for early diagnosis of the presence or relapse of RA, comprising plural-well plates comprising in each well one of the oligonucleotides having SEQ ID No 1, 12, 15, 18 and in each well the oligonucleotide having SEQ ID No 27 in concentrations suitable for performing a PCR reaction; aliquots of PCR Buffer, of dATP, dCTP, dGTP and dTTP optionally reunited in a single container, aliquots of MgCl₂, aliquots of Taq Polymerase, and aliquots of oligonucleotides having SEQ ID No 33, 35, 36, 38 and 39 optionally labeled with fluorochromes .

9. Kit according to claim 7 or 8, further comprising oligo-dT, inverse transcriptase, buffer and dNTP for reverse transcription.

10. A vaccine against rheumatoid arthritis, comprising one or more peptides according to claim 3, an immunostimulant adjuvant and pharmaceutically acceptable excipients.

11. A therapeutic method for treatment of rheumatoid • arthritis, comprising the administration of the vaccine according to claim 10 to patients in need thereof.