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Principal investigators:
Dr Raimon Sanmartí Sala
Hospital Clínic i Provincial de Barcelona
Dr Isabel Haro Villar
Institut d'Investigacions Químiques i Ambientals de Barcelona
Dr Alejandro Balsa Criado
Spanish Rheumatology Society. Madrid
Duration: 3 years


1. Summary

This is a co-ordinated multicentre project with the main aim of
developing a method of enzyme-immunoassay using citrullinated
synthetic peptides derived from human fibrin domains, which could
be used as a diagnostic test for rheumatoid arthritis (RA) and thus to
study its prognostic value.
The project consists of the following subprojects:
Subproject 1 (Dr Isabel Haro). Basic project with the aim of
chemically synthesising peptides derived from domains of the alpha
and beta chains of fibrin, in which the amino acid arginine is replaced
by the amino acid citrulline. These citrullinated peptides will act as an
antigenic substrate to detect by enzyme-immunoassay (ELISA)
antibodies present in the serum of patients with rheumatoid arthritis.
The ELISAs that use these citrullinated fibrin-derived peptides (widely
present in the synovial membrane of patients with RA) might be more
sensitive than those used up to now (derived from filaggrin and other
proteins) and might have greater prognostic ability.
In this project peptide sequences were selected from the primary
structure of the fibrin, using classic algorithms for prediction of
antigenicity and/or epitope mapping of the proteins and later
chemically determined citrullinated analogues were obtained
(following solid phase peptide synthesis methodologies) in linear,
cyclical and/or ramified form. Later, different ELISA were developed
for the detection of autoantibodies in the serum, using the
citrullinated peptides obtained. Covalent binding and direct adsorption
were measured, after hydrophobic derivation, of the peptide
sequences in the microtitre plates. Finally, a study was made of the
sensitivity and specificity of these ELISAs for diagnosis of RA and the
results were compared with the commercial first and second
generation ELISAs that use cyclic citrullinated peptides derived from
the structure of human filaggrin (anti-CCP1) and other peptide
sequences (anti-CCP2).

Subproject 2 (Dr Raimon Sanmartí). This project studied the
prognostic value of the new ELISAs developed in the first subproject,
which use synthetic citrullinated peptides derived from human fibrin,
in patients with early RA. The studied cohort was of patients with RA
with less than two years’ evolution, who were followed up with a
monitoring protocol for two years. The measure of bad prognosis was
considered to be the degree of articular destruction (evaluated by
radiography) at the end of the follow-up. The studied factors (clinical,
genetic and laboratory) were those associated with a greater articular
destruction, including the values of antibodies against human fibrin
citrullinated peptides. Other associations of these antibodies were
studied and their prognostic value was compared with the anti-CCP2.

Subproject 3 (Dr Alejandro Balsa). This is a multicentre project of
the Spanish Rheumatology Society with 34 participating centres
throughout Spain, which analysed the factors associated with the
development of RA in a prospective cohort of patients with recently-
onset arthritis who were followed up for 5 years. The role of the
antibodies against citrullinated peptides (CCP2 and against human
fibrin) in the future diagnosis of rheumatoid arthritis was found.

2. Results

Subproject 1. It was shown that the synthetic citrullinated peptides
related with fibrin are recognised by the serum of patients with RA.
Different citrullinated regions of the alpha and beta fibrin chains were
satisfactorily synthesised. Certain regions of these chains (alpha
(peptides p18, p19, p22) and beta (peptide p38)) showed very high
levels of sensitivity for detection of autoantibodies present in the
serum of patients with RA. Later, the antigenic capacity of the linear
peptides was shown to improve with cycling of the sequences. Finally,
it was shown that the combination in the same molecule of peptides
derived from filaggrin (cyclic peptide cfc1cyc) and from the alpha
chain of fibrin (chimeric fibrin/filaggrin (fb/f) peptides) increased the
sensitivity of the ELISAs and made it possible to identify antibodies in
the serum of patients with RA that were not detected by the
commercial ELISA tests (CCP1, CCP2).
An analysis of the sensitivity/specificity balance of the new ELISA
tests using the chimeric peptides fb/f, in different populations (RA,
blood donors and other rheumatic pathologies) showed the high
sensitivity of these ELISA for detecting autoantibodies in RA patients
(72-78%). In the specificity study of the different autoantibodies in
other pathologies where autoimmune phenomena are observed
(systemic Lupus erythematosus, chronic C virus hepatitis) or that
present are clinically similar to RA (psoriatic arthritis), it was shown
that the antichimeric antibodies fb/f are very specific, similarly to the
commercial anti-CCP2. This specificity is 97-98%, except for the SLE,
where it is somewhat lower (91-92%), as a result of the presence of
some false positives.

Subproject 2. A group of 188 patients with recently onset RA was
analysed and followed up for 2 for, for the prognostic value of the
antibodies against chimeric fibrin/filaggrin citrullinated synthetic
peptides p18, p19 and p22. Some 82% of the patients had antibodies
against one of the chimeric citrullinated peptides (against 74% with
the commercial test CCP2). The presence of these autoantibodies at
the time of inclusion (baseline values) was associated with greater
articular destruction, especially those aimed against the chimeric fb/f
p18, even higher than the commercial test CCP2. A very good
correlation was shown between the results of the three ELISAs that
use chimeric peptides and the commercial test CCP2, although there
were discrepancies in a large number of patients (around 14%).
However, an association was observed between the presence of these
autoantibodies, especially the chimeric fb/f p18, and a certain
genotype HLA (HLA-DRB04).

Subproject 3. A cohort of 171 patients with onset arthritis was
analysed with follow-up over an average of 3.6 years, for the
discrimination value of the anti-CCP2 and the fibrin/filaggrin chimeric
antibodies, for later development of RA. The large majority of the
patients (94%) were diagnosed with RA during the follow-up. Both
the anti-CCP2 and the antichimeric fb/f p18 (in phase of final
analysis) have been associated with later development of RA in this

3. Relevance and possible clinical implications of the final
results obtained

The antibodies against citrullinated peptides are the most specific
serological of RA. The final results obtained in this research project
made it possible to define chimeric peptides that contain domains of
two different proteins (filaggrin and fibrin) that are recognised by
autoantibodies present in the serum of patients with RA with a very
good specificity/sensitivity balance. We can conclude that the
citrullinated synthetic peptides show a clear interest in the
development of new improved tests for diagnosing RA. There is more
strength in the idea that epitopes of the alpha and beta chains of
human fibrin, present in rheumatoid synovial could be the main
antigens that trigger the immunological response to RA, with its
aetiopathogenic implications. As a result, there is a foreseeable great
clinical involvement of the fibrin-filaggrin chimeric peptides, as these
improve the sensitivity of detection of the autoantibodies present in
RA patients.
The sensitivity/specificity analysis of these autoantibodies has shown
that they have high sensitivity and specificity for diagnosis of RA.
Using a population of 322 patients with RA and 307 controls (Blood
Bank) and maintaining a specificity of 98%, the sensitivity of the
three fibrin/filaggrin (fb/f) antichimeric antibodies (p18, p19 and p22)
is 72-78%, no lower than that observed with the commercial anti-
CCP2 (74%). Interestingly, although the correlation between the
different autoantibodies is very narrow, there is a considerable
percentage of discrepancies among them (around 14%), which
clearly shows that the determination of more than one antibody can
increase the sensitivity (up to 81%) with practically no loss in
specificity (96-97%).
In the specificity study of the different autoantibodies in other
pathologies where autoimmune phenomena are observed (systemic
lupus erythematosus, chronic hepatitis with C virus) or that are
clinically similar to RA (psoriatic arthritis), it has been shown that the
fb/f antichimeric antibodies are very specific, similarly to the
commercial anti-CCP2. This specificity is 97-98%, except for the SLE,
where it is somewhat lower (91-92%), as a result of the presence of
some false positives.
An analysis of the prognostic value of those autoantibodies showed
that in a cohort of patients with early RA, their presence was
associated with a greater degree of articular destruction. The patients
with these autoantibodies (in particular with the fb/f antichimeric
antibody p18) present a greater degree radiological progression of
the disease after two years of follow-up, identifying those patients
with a more serious
In conclusion, these fibrin/filaggrin antichimeric antibodies have high
sensitivity for RA diagnosis and could be present in patients in whom
anti-CCP2 (commercial test, used in clinical practice) has been unable
to identify it. For this reason, the joint identification of these
antichimeric autoantibodies (in particular fb/f p18) increases the
diagnostic sensitivity for rheumatoid arthritis, as well as providing
additional information about the prognosis. The new ELISA tests that
use citrullinated synthetic peptides where domains of human fibrin
are included could be among the tests with a best
sensitivity/specificity balance for diagnosis of RA and be very useful in
determining its prognosis.
4. Publications

M.L. Pérez, M.J. Gómara, D. Kasi, A. Alonso, O. Viñas, G. Ercilla, R.
Sanmartí and I. Haro
Synthesis of overlapping fibrin citrullinated peptides and its use for
diagnosing rheumatoid arthritis s
Chem. Biol. Drug Des. 68, 194-200 (2006)

M.L. Pérez, M.J. Gómara, G. Ercilla, R. Sanmartí, I. Haro
Antibodies to citrullinated human fibrinogen synthetic peptides in
diagnosing rheumatoid arthritis.
J. Med. Chem. 50, 3573-3584 (2007)

Vázquez I, Graell E, Gratacós J, Cañete J.D, Viñas O, Ercilla M.G,
Gómez A, Hernández M.V, Rodríguez-Cros J.R, Larrosa M. and
Sanmartí R.
Prognostic markers of clinical remission in early rheumatoid arthritis
after two years of Dmards in a clinical setting.
Clin Exp Rheumatol 2007; 25: 232-8

Sanmartí R, *Gómez-Centeno A, **Ercilla G, *Larrosa M, **Viñas O,
Vazquez I, Gómez-Puerta J-A, *Gratacós J, Salvador G and Cañete
Prognostic factors of radiographic progression in early rheumatoid
arthritis. a two year prospective study after a structured therapeutic
strategy using dmards and very low doses of glucocorticoids.
Clinical Rheumatol 2007; 26: 1111-8.

I. Haro, M.J. Gómara, M.L. Pérez, R. Sanmartí
“Polipéptido quimérico fibrina-filagrina citrulinado capaz de detectar
los anticuerpos generados en la artritis reumatoide”
Application No.: P200701167
Holding body: CSIC-Fundació Clínic
Country: Spain
Year: application presented 30.04.2007