Anti-IL-6 vaccine composition

Abstract

The present invention relates to a pharmaceutical composition comprising, by way of active ingredient, at least one polypeptide comprising, or constituted by a sequence constituted by at least 8 contiguous amino acids and from at the most 30 contiguous amino acids chosen from within the interleukin-6 sequence and from at the most 30 contiguous amino acids chosen from within the complete IL-6 sequence.

Claims

1. A method for reducing symptoms of an arthritic disease, comprising administering to an individual in need thereof a therapeutically effective amount of at least one cyclized polypeptide with a length equal to 25 amino acids or less, wherein the at least one cyclized polypeptide comprises, or consists of, a sequence selected from the group consisting of SEQ ID NO: 2, 3, 17, 18 and 19, and a variant sequence thereof having at least 90% sequence identity with the sequence of SEQ ID NO: 2, 3, 17, 18 or 19 and elicits a protective anti-human IL-6 immune reaction.

2. The method of claim 1, wherein the arthritic disease is selected from the group consisting of rheumatoid arthritis, juvenile rheumatoid arthritis, psoriatic arthritis, arthrosis, refractory rheumatoid arthritis, chronic non-rheumatoid arthritis and ankylosing spondylitis.

3. The method of claim 1, wherein the cyclized polypeptide elicits anti-human IL-6 antibodies.

4. The method of claim 1, wherein the cyclized polypeptide is bound to a carrier macromolecule.

5. The method of claim 1, wherein the cyclized polypeptide comprises, or consists of, a sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:17, and a variant thereof having at least 90% sequence identity with the sequence of SEQ ID NO:2 or SEQ ID NO:17.

Description

DESCRIPTION OF THE FIGURES

(1) FIG. 1 represents the quantity of anti-mIL-6 antibodies present in serum diluted to 1/500.sup.th from OF1 mice immunized with the peptides mP1, mP2, mP3, mP4, mP5 and mP6 measured by ELISA (y-axis, optical density (OD) at 450 nm). The horizontal line at 0.3 OD represents the significance threshold.

(2) FIG. 2 represents the clinical score or disease activity index (DAI, y-axis) of untreated C57BL/6 mice (water) or mice treated with Dextran Sodium Sulfate (DSS) and vaccinated with the peptides mP1, mP2, mP3, mP4 and mP6, or with control vaccines without peptide and comprising buffer (PBS) or KLH carrier protein.

(3) FIG. 3 represents the histological colitis score (y-axis) of untreated C57BL/6 mice (control) or mice treated with DSS and vaccinated with the peptides mP1, mP2, mP3, mP4 and mP6, or with control vaccines without peptide and comprising buffer (PBS) or KLH carrier protein.

(4) FIG. 4 represents the length of the colon (y-axis, in cm) of untreated C57BL/6 mice (control) or mice treated with DSS and vaccinated with the peptides mP1, mP2, mP3, mP4 and mP6, or with control vaccines without peptide and comprising buffer (PBS) or KLH carrier protein.

(5) FIG. 5 represents the quantity of anti-hIL-6 antibodies present in serum diluted to 1/500.sup.th from OF1 mice immunized with the peptides hP1, hP2, hP3, hP4 and hP6 measured by ELISA (y-axis, optical density (OD) at 450 nm). The horizontal line at 0.3 OD represents the significance threshold.

(6) FIG. 6 represents the quantity of anti-hIL-6 antibodies present in the serum diluted to 1/500.sup.th from OF1 mice immunized with the peptides hP1, hP2, hP3, hP4 and hP6 measured by ELISA (y-axis, optical density (OD) at 450 nm). The horizontal line at 0.3 OD represents the significance threshold.

(7) FIG. 7 represents the anti-hIL-6 antibody titer (vertical axis) of sera from monkeys immunized against hP2 as a function of time (horizontal axis, in days).

(8) FIG. 8 represents the delayed-type hypersensitivity (DTH) clinical score (vertical axis, arbitrary units) of hP2-immunized monkeys and of control KLH-immunized monkeys.

(9) FIG. 9 represents the anti-mIL-6 antibody titer (vertical axis) of mice immunized against mP2 or KLH and having received injections of bleomycin (Bleo) or of NaCl.

(10) FIG. 10 represents hydroxyproline (OH proline) concentration (vertical axis, in g/ml) in skin biopsies of mice immunized with mP2 or KLH or having received an anti-IL-6 receptor antibody (anti IL-6R) and injected with either bleomycin (bleo) or NaCl.

(11) FIG. 11 represents dermal thickening (vertical axis, in arbitrary units) from histological sections of mice immunized with mP2 or KLH or having received an anti-IL-6 receptor antibody (anti IL-6R) and injected with either bleomycin (bleo) or NaCl.

EXAMPLES

Example 1

Recognition of the Whole Murine IL-6 (mIL-6) Cytokine and Cross-Reactivity Against the Human IL-6 (hIL-6) Cytokine by Serums from Mice Immunized with Peptides Derived from mIL-6

(12) Six peptides derived from murine IL-6 were chemically synthesized and coupled to a carrier protein, KLH (Keyhole Limpet Hemocyanin), five using the coupling agent bisdiazonium-benzidine (BDB) and one with glutaraldehyde. These peptides were then cyclized by the formation of disulfide bonds between cysteines (added or already present).

(13) For each peptide, Oncins France 1 mice (OF1, Charles River Laboratories, L'Arbresle, France) free from specific pathogenic organisms were immunized by intra-muscular route with 100 g of peptides derived from murine IL-6 mP1, mP2, mP3, mP4, mP5, and mP6 (see Table 1) in complete Freund's adjuvant (CFA) at the start of the experiment (D0) (n=4 per peptide).

(14) TABLE-US-00004 TABLE1 PeptidesderivedfrommIL-6(underlined)usedfortheimmunization Peptide IL-6region Sequence SEQIDNO mP1 61-75 BDB-Y-E.sub.61IVEMRKELCNGNSD.sub.75C.sub.+Amide 29 mP2 76-92 .sub.Acetyl+C.sub.76MNNDDALAENNLKLPE.sub.92C-Y-BDB 30 mP3 99-109 Gluta-C.sub.99YQTGYNQEIC.sub.109.sub.+Amide 26 mP4 125-40 BDB-Y-CE.sub.125FMKNNLKDNKKDKAR.sub.140C.sub.+Amide 32 mP5 154-168 .sub.Acetyl+CN.sub.154QEVKDLHKIVLPTP.sub.168C-Y-BDB 34 mP6 176-188 .sub.Acetyl+D.sub.176KCESQKEWLRTK.sub.188C-Y-BDB 33 The amino acids are annotated on the basis of Swissprot sequence P08505 (mIL-6)

(15) Incomplete Freund's adjuvant (IFA) boosters are then given every 15 days, on D15, D30 and D45. The sera from mice on D54 are tested by ELISA with a 1/500.sup.th dilution of the serums on plates covered with murine IL-6 or human IL-6 in order to evaluate their cross-reactivity.

(16) It is observed that all of the peptides tested with the exception of the peptide mP5 give rise to antibodies recognizing the murine cytokine (FIG. 1). Moreover, apart from the antibodies directed against the peptide mP2, the antibodies produced using the murine peptides show only a weak cross-reactivity with the human cytokine.

Example 2

Neutralization of the Biological Activity of the Murine IL-6 by Purified Antibodies Based on Serum from Rabbits Immunized with the Peptides Derived from Murine IL-6

(17) The neutralizing ability of purified IgGs based on serum from rabbits immunized with the peptides mP1, mP2, mP3, mP4, mP5 and mP6 respectively, was tested in a murine IL-6 neutralization test. This test is based on the fact that murine B9 hybridomas proliferate in a dose-dependent manner in response to murine IL-6 (Brakenhoff et al. (1987) J. Immunol. 139:4116-21).

(18) Description of the Experiment Carried Out:

(19) Plating (D0): seed a flat-bottom 96-well plate, treated for cell culture with 5000 cells per well in 50 L of neutralization medium. (RPMI 1640 without phenol red, 5% v/v Foetal Calf Serum (FCS), 2 mM L-Glutamine, 100 U/mL Penicillin/Streptomycin, 50 m of -Mercaptoethanol)

(20) In parallel a so-called pre-incubation plate is produced with the desired concentration of murine IL-6, samples and commercial anti-mIL-6 antibodies used as positive control, all in a final volume of 100 L of neutralization medium.

(21) The pre-incubation plate is left at 37 C. for 2 hours. 50 L per well is then transferred to the corresponding wells of the plate containing the cells, then the latter is incubated with 5% CO.sub.2 at 37 C. for 96 hours.

(22) Development (D4): Add 50 L of a solution of XTT (for 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide)-0.5% electron coupling reagent to each well. Leave to incubate at 37 C. for 7 hours. Reading of the optical density on a spectrophotometer set at 450 nm.

(23) It is observed that IgGs from the rabbits immunized with the peptides mP1, mP2, mP3, mP4 and mP6 neutralize the biological activity of murine IL-6 in a dose-dependent manner. On the other hand, IgGs from the rabbit immunized with the peptide mP5 do not neutralize the activity of murine IL-6.

Example 3

Clinical and Histological Protection after Vaccination Against a Peptide Derived from Murine IL-6 in a Murine Model of Chronic Colitis Induced by DSS

(24) Chronic colitis induced by Dextran Sodium Sulfate (DSS) is a well-known model of chronic inflammatory disease reproducing the main characteristics of the human diseases (Alex et al. (2009) Inflamm. Bowel Dis. 15:341-352).

(25) Briefly, groups of 6 C57BL/6 mice (Charles River Laboratories, L'Arbresle, France) were formed. The mice were immunized 4 times (once with CFA and three times with IFA) every 15 days with different vaccine preparations. The mice were respectively immunized with PBS (DSS/PBS group), 50 g per mouse of KLH (DSS/KLH group), and 100 g per mouse of peptide mP1, mP2, mP3, mP4, mP5 and mP6 coupled with KLH (DSS/mIL-6 peptide group). A control H.sub.2O group was also included. Four days after the first booster, colitis was induced by absorption of Dextran Sodium Sulfate (DSS) in water (2%) over 7 days followed by 7 days of water. Three DSS/water cycles were thus carried out, after which the mice received water until the end of the protocol. The clinical signs of the disease appear approximately on the fifth day after the start of treatment with DSS (D26 of the protocol). The development of the disease is measured from the start of the first DSS cycle up to the end of the protocol by a disease activity index (DAI) including weight loss, the presence of blood in the stools and the texture of the stools. The clinical scores are determined as follows: for the texture of the stools a score of 0 (hard stools) to 4 (diarrhoea), for the presence of blood in the stools a score of 0 (absence) to 4 (macroscopic blood), and weight loss a score of 0 (<1%) to 4 (>20%). Each mouse is thus assigned a clinical score with a maximum score of 12.

(26) It is observed that the mice immunized with the peptides mP1, mP2, mP3, mP4 and mP6 have a significantly reduced disease activity index compared with the control groups (FIG. 2)

(27) Then, on D67, the mice are killed by cervical dislocation. The histological sections from colon distal biopsies are scored blind, according to the severity of the inflammation (0: absence, 1: slight, 2: moderate, 3: severe), the extent of the inflammation (0: absence, 1: mucosal, 2: mucosal and sub-mucosal, 3: transmural), crypt damage (0: absence, 1: one-third damaged, 2: 2/3 damaged, 3: only the surface of the epithelium intact, 4: total destruction of the crypts and epithelium). Each mouse is thus assigned a histological score with a maximum score of 10.

(28) It is observed that the mice immunized with the peptides mP1, mP2, mP3, mP4 and mP6 have a significantly reduced histological score compared with the control mice treated with PBS or KLH alone (FIG. 3).

(29) The absorption of DSS significantly reduces the length of the colon. Interestingly, the mice immunized with the peptides mP1, mP2, mP3, mP4 and mP6 exhibit a significantly less severe reduction of the colon than the control mice (FIG. 4). On the other hand, immunizations with KLH have no effect on the reduction in the length of the colon induced by DSS.

Example 4

Recognition of the Whole hIL-6 Cytokine by the Antibodies from Mice Immunized with Human IL-6 Peptides

(30) Similarly to Example 1, five peptides derived from human IL-6, hP1, hP2, hP3, hP4 and hP6 were coupled with KLH, four using the coupling agent BDB and one with glutaraldehyde (see Table 2). These peptides were cyclized using two cysteines (added or already present).

(31) For each peptide, OF1 mice free from specific pathogenic organisms (n=4 per peptide) were immunized by intra-muscular route with 100 g of peptides derived from human IL-6 in CFA on D0. IFA boosters are then given every 15 days, on D15, D30 and D45. The serum is recovered after the boosters on D35 and D54 and tested by ELISA with a 1/500.sup.th dilution of the serums on plates covered with human IL-6 (FIG. 5).

(32) TABLE-US-00005 TABLE2 PeptidesderivedfromhIL-6(underlined)usedfortheimmunization Peptide IL-6region Sequence SEQIDNO hP1 63-78 BDB-Y-G.sub.63ISALRKETCNKSNMC.sub.78.sub.+Amide 16 hP2 78-94 .sub.Acetyl+C.sub.78ESSKEALAENNLNLPK.sub.94C-Y-BDB 17 hP3 101-111 Gluta-C.sub.10FQSGFNEETC.sub.111.sub.+Amide 3 hP4 127-141 BDB-Y-CE.sub.127FLQNRFESSEEQAR.sub.141C.sub.+Amide 18 hP6 177-189 .sub.Acetyl+T.sub.177KCQAQNQWLQDM.sub.189C-Y-BDB 19 The amino acids are annotated on the basis of Swissprot sequence P05231 (hIL-6)

Example 5

Neutralization of the Biological Activity of Human IL-6 by Purified Antibodies Based on Serum from Rabbits Immunized with Peptides Derived from Human IL-6

(33) New Zealand rabbits free from specific pathogenic organisms (n=1 per peptide) were immunized with 100 g of peptides hP1, hP2, hP3, hP4 and hP6 derived from human IL-6 by five immunizations with CFA/IFA on D0, D14, D28, D56 and J70. The neutralizing ability of the purified IgGs, derived from the serum of each rabbit, was tested in a human IL-6 neutralization test. This test is carried out on HEK293 cells (Human Embryonic Kidney cells) transfected with the gene encoding the human IL-6 receptor and the secreted embryonic alkaline phosphatase (SEAP) reporter gene (Supplier: Invivogen). The latter is then secreted in a dose-dependent manner in response to the human IL-6.

(34) Briefly, the inventors proceeded as follows:

(35) Plating (D0): seed a flat-bottom 96-well plate, treated for cell culture with 50,000 cells per well in 100 L of neutralization medium. (DMEM 4.5 g/L glucose, 10% v/v FCS, 2 mM L-Glutamine, 100 U/mL Penicillin/Streptomycin, 100 g/mL of Normocin).

(36) In parallel a so-called pre-incubation plate is prepared with the desired concentration of human IL-6, rabbit antibodies and commercial anti-hIL-6 antibodies used as a positive control, all in a final volume of 200 L of neutralization medium.

(37) The pre-incubation plate is left at 37 C. for 2 hours. 100 L per well is then transferred to the corresponding wells of the plate containing the cells, then the latter is incubated with 5% CO.sub.2 at 37 C. for 24 hours.

(38) Development (D1): Fill a flat-bottom 96-well plate with 180 L per well of QUANTI-Blue (InVivoGen, CA, USA). Add 20 L of supernatant from the plate treated with IL-6. Incubate with 5% CO.sub.2 at 37 C. for 3 hours. Determine the quantity of secreted alkaline phosphatase using a spectrophotometer set at 620 nm.

(39) It is observed that the IgGs from the rabbits immunized with the peptides hP1, hP2, hP3, hP4 and hP6 derived from human IL-6 neutralize the biological activity of the human IL-6 in a dose-dependent manner.

Example 6

Production of Monoclonal Antibodies Targeting the Peptides hP2 and hP6, Recognizing the Human hIL-6 Cytokine and Neutralizing its Biological Activity

(40) 1) Production

(41) 2 groups of three SWISS mice are immunized against the 2 peptides hP2 and hP6 of human IL-6 (6 mice in all) with the following protocol: an initial immunization with 100 g of peptide followed by 3 boosters on D20, D40, D60. The response titre is measured for each mouse and, for each peptide, the mouse exhibiting the best titre is used for the production of monoclonal antibodies.

(42) For this, the hybridoma technique is used. Spleen B lymphocytes are purified, then fused with transformed myeloma cells in order to obtain immortal lines. These fused cells, called hybridomas, are maintained in selective medium, in the presence of aminopterin and hypoxanthine, so that the non-fused cells die: the myeloma cells because they have no thymidine kinase, and the B lymphocytes because they are not transformed. Only the fused cells can survive and the limiting dilution technique is then used to isolate clones producing only one species of antibody. At this stage, it is possible to test the antibodies produced in the supernatant by these clones by means of in vitro tests: ELISA against the recombinant cytokine and cytokine biological activity neutralization tests.

(43) 2) Screening

(44) In a first step, for each peptide, 6 limiting-dilution plates are prepared. The dilutions are verified in each well by inverted-microscope analysis. A first screening is carried out by putting the supernatants into 12-well by 12-well groups and testing these groups by ELISA against the recombinant cytokine. The wells, the contents of which exhibit a positive signal, are tested individually: for the positive wells, a second limiting dilution is carried out on a plate.

(45) For the second screening, the wells are also monitored by inverted microscopy. The supernatants are put into 8-well by 8-well groups, tested by ELISA, and for the positive pools, the responses of the individual wells are again analyzed by ELISA.

(46) After the second screening step, it can reasonably be considered that the hybridomas present in the well originate from the same clone. These hybridomas are then cultured in bulk in order to produce larger quantities of antibodies.

(47) Three hybridomas are selected for each peptide. For this, these cells are injected into the abdomens of mice where they proliferate in the form of ascites. The antibodies are then recovered by collecting the ascitic fluid from the peritoneal cavity and they are purified by using protein G affinity chromatography.

(48) 3) Neutralization of the Biological Activity of Human IL-6 by the Purified Anti-hIL-6 Monoclonal Antibodies

(49) On the basis of the human IL-6 biological activity neutralization test described in Example 5, the neutralizing ability of the monoclonal antibodies is evaluated at variable dilutions in order to study the dose-dependency of the human IL-6 biological activity neutralizing properties.

Example 7

Recognition of the Whole hIL-6 Cytokine by Antibodies Derived from the Serum from Mice Immunized with Variants of the Peptides hP1, hP2, hP3, hP4 and hP6

(50) Five variants hP1, hP2, hP3, hP4 and hP6, derived from hP1, hP2, hP3, hP4, and hP6 respectively (see Table 3) were coupled to KLH, four using the coupling agent BDB and one with glutaraldehyde. These peptides were cyclized using two cysteines situated at the ends (added or already present in the native sequence).

(51) TABLE-US-00006 TABLE3 PeptidesderivedfromhIL-6usedfortheimmunization Peptide Sequence SEQIDNO %identity hP1 BDB-Y-GISALRKETCNKSNM-C.sub.+Amide 16 81.25% hP1 BDB-Y-GISAVRKDTCNKSQM-C.sub.+Amide 20 hP2 CESSKEALAENNLNLPK-C-Y-BDB 17 84.21% hP2 CESSKDAIAENQLNLPK-C-Y-BDB 21 hP3 Gluta-CFQSGFNEETC.sub.+Amide 3 81.81% hP3 Gluta-CFNSGFNEDTC.sub.+Amide 10 hP4 BDB-Y-C-EFLQNRFESSEEQAR-C.sub.+Amide 18 82.35% hP4 BDB-Y-C-EFLQNRFDSSDENAR-C.sub.+Amide 22 hP4 BDB-Y-C-EFLQNRFESSEEQAR.sub.+Amide 18 76.47% hP4 BDB-Y-C-DFLQNRFDSSDENAR.sub.+Amide 57 hP6 .sub.Acetyl+TKCQAQNQWLQDM-C-Y-BDB 19 80.00% hP6 .sub.Acetyl+TKCQANQQWLQEM-C-Y-BDB 23 The variant parts are underlined

(52) For each peptide, OF1 mice free from specific pathogenic organisms (n=4 per peptide) are immunized by intra-muscular route with 100 g of variants hP1, hP2, hP3, hP4 and hP6 in CFA on D0. IFA boosters are then given every 15 days, on D15, D30 and D45. The serums from mice are tested on D54 by ELISA with a 1/500.sup.th dilution on plates covered with human IL-6.

(53) It is observed that the peptide variants generate antibodies recognizing the human cytokine by ELISA (FIG. 6).

(54) Finally, segments larger than 30 amino acids, containing hP1, hP2, hP3, hP4 and hP6 are also tested. These segments are also capable of generating human cytokine neutralizing antibodies.

Example 8

Human IL-6 Recognition (hIL-6) by Cynomolgus Monkey Sera, Immunized with a Peptide Derived from hIL-6

(55) Four Cynomolgus monkeys were immunized intra-muscularly at day 0 with 150 g of the cyclized hP2 peptide derived from hIL-6 previously described coupled to the protein carrier KLH, and then emulsified with the ISA51 adjuvant (Seppic).

(56) Boosts were performed at d15, d30, d45 with an additional boost at d76. Blood samples were collected every two weeks until the end of the experiment (d120). These sera were tested in ELISA to evaluate the production of anti hIL-6 antibodies. A control group of four monkeys immunized with KLH only was used.

(57) All the monkeys immunized with hP2, developed antibodies against hIL-6 from d30 with a kinetics showing a peak (in average at day 60) and a decrease of the antibody titers by the end of the experiment (FIG. 7).

(58) The four control monkeys immunized with KLH only did not develop antibodies against hIL-6.

(59) During this experiment, the inventors also evaluated the ability of the immunization against the peptide hP2 to modulate a delayed hypersensitivity against tetanus toxoid (TTx).

(60) Briefly, the TTx vaccine (Sanofi-Pasteur, solution of 0.5 ml comprising 40 IU of Tetanus Toxoid and 0.6 mg of aluminium hydroxide) was administered intramuscularly at d59 in the thigh. Then an intradermic challenge was performed at d90 in two injection sites in the back of the monkey.

(61) Clinical features of the cutaneous reaction were evaluated once a day for each animal before the challenge and then 24 h, 48 h and 72 h after the challenge. Each injection site was monitored for the incidence, grade and duration of three parameters (erythema, thickening, nodules) and a cumulative clinical score of delayed-type hypersensitivity (DTH) comprising these three parameters was established for each group.

(62) Monkeys immunized with peptide hP2 showed a marked attenuation of the DTH response compared to the KLH group (FIG. 8), thus showing that the anti-IL6 antibodies induced by the immune reaction against the IL6 peptide immunogen are clinically effective.

Example 9

Clinical and Histological Protection after Vaccination with a Peptide Derived from Murine IL-6 in a Murine Model of Bleomycin Induced Systemic Sclerosis

(63) The murine model of bleomycin-induced systemic sclerosis is a well-known model of chronic inflammatory disease with main features being dermal thickness together with skin and pulmonary fibrosis due to excessive collagen production (Adamson & Bowden (1974) Am. J. Pathol. 77:185-197)

(64) Briefly, three groups of DBA/2 mice (Janvier, France) immunized intramuscularly four times (once in complete Freund's adjuvant (CFA), and three times in incomplete Freund's adjuvant (IFA)) every 15 days with different vaccine preparations: 8 mice were immunized with 100 g of the above-described cyclized mP2 peptide coupled to KLH (mP2-Bleo group) and were injected subcutaneously in the back with bleomycin 6 mice were immunized with 100 g of mP2 (mP2-Nacl group) and were injected subcutaneously in the back with NaCl (Negative control group) 8 mice were group immunized with 200 g of KLH alone (KLH-Bleo group) and were injected subcutaneously in the back with bleomycin. (Positive control group)

(65) The inventors also used an additional control group composed of mice injected intraperitoneally with 100 L (1 mg/mouse) of murine monoclonal anti IL-6 receptor antibody once a week (n=6).

(66) The subcutaneous injections of 100 L of bleomycin (0.5 mg/mL) were performed three days after the third boost and then every other day during three weeks in order to induce systemic sclerosis.

(67) At day 54, the mice were killed. Sera samples were collected and the antibody production against mIL-6 was quantified by ELISA. Furthermore, skin biopsies were performed to evaluate the OH-proline production, indicative of collagen production, and histological sections from the back were also performed to evaluate dermal thickness.

(68) All mice developed antibodies against mIL-6 after immunization against mP2 while the mice immunized against KLH only did not (FIG. 9).

(69) The collagen content was measured in the skin biopsy from each mouse by assaying hydroxyproline (OH-proline) content. OH-proline represents about 13% of collagen amino acids and is measured to evaluate collagen production. Briefly, an acid hydrolysis is performed on two biopsy punch, then OH-proline oxidation is realised which leads to pyloric derivatives with pink coloration as described by Woessner (1961) Arch. Biochem. Biophys. 93:440-447. The absorbance is read at 560 nm.

(70) The negative control group immunized with mP2 and injected subcutaneously with NaCl did not develop skin fibrosis whereas the positive control group, mice immunized with KLH only and injected with bleomycin, developed fibrosis. Furthermore the group immunized with mP2 and injected subcutaneously with bleomycin was protected from the development of fibrosis with the same efficacy as the group receiving the anti IL-6 receptor antibody (FIG. 10).

(71) Dermal thickness from histological sections was then measured in a blind way by two independent experts.

(72) Mice immunized against mP2 had a lower dermal thickening than mice immunized with KLH only, and it was identical with the dermal thickness observed for the group receiving anti mIL-6 receptor (FIG. 11).

(73) In conclusion, immunization against the mP2 peptide protected mice from fibrosis and dermal thickening in the murine model of bleomycin-induced systemic sclerosis.