The present application claims the benefit of US provisional application No. 62/275,903 filed on January 7, 2016 which is incorporated herein by reference.

Field of the invention

The present invention relates to method of treatment of self-protein-related diseases, in particular IL-6 related diseases, in an individual. Technical background

lnterleukin-6 (IL-6) is a key messenger of immune responses and inflammation processes. It is produced by a wide variety of cell types such as lymphocytes, macrophages, neutrophils or fibroblasts. IL-6 is the major regulator of acute phase protein synthesis in response to injury. It is also involved in the proliferation and differentiation of cytotoxic T cells by inducing the production of IL-2 and its receptor.

The overproduction of IL-6 has been associated with many autoimmune diseases or cancers. IL-6 serum concentration is increased in sera of rheumatoid arthritis (RA), systemic sclerosis (SSc) and multiple myeloma patients and is correlated with disease severity.

Several studies have shown a protective effect of anti-IL-6 receptor (IL-6R) blockade strategies in mouse models of autoimmune diseases. In humans, the pathological effects of IL-6 overproduction are specifically inhibited by tocilizumab, a commercialized monoclonal antibody directed against IL-6R, approved in 2009 for the treatment of rheumatoid arthritis, and in 201 1 for the treatment of systemic juvenile idiopathic arthritis.

Anti-cytokine biologies (monoclonal antibodies and soluble receptors) have successfully been used to treat chronic inflammatory diseases since the late 90's. These biologies present however several drawbacks including primary and secondary resistances, repeated injections and thus high compliance required from patients, side effects, and prohibitive costs for patients and healthcare systems (up to $20,000 per patient per year).

In order to overcome these disadvantages and to widen the therapeutic offer for the patients, new anti-cytokine targeted strategies are needed.

As an alternative and innovative strategy, the present inventors have developed anti- cytokine peptide-based active immunization, where cytokine-derived peptides linked to a carrier protein are used as immunogens to induce anti-cytokine autoantibodies production. Thus, the present inventors have recently demonstrated the efficacy of anti-IL-6 active immunization in the bleomycin mouse model of SSc, as described in Desallais, L. et al. Targeting IL-6 by both passive or active immunization strategies prevents bleomycin-induced skin fibrosis. Arthritis Res Ther 16, R1 57 (2014) and International Application WO2013/021 284 which is incorporated herein by reference.

Summary of the invention

The present inventors have now made the surprising finding that the efficacy of the treatment by anti-IL-6 active immunization could be improved by performing repeated administrations of the IL-6-derived peptide throughout the treatment.

Accordingly, the present invention relates to a method for treatment of self-protein- related diseases, or for vaccinating or immunizing against self-protein related diseases, in an individual during a treatment period, comprising performing several administrations of an effective amount of a peptide derived from the self-protein to the individual, wherein the administrations are distributed all along the treatment period.

The present invention also relates to a peptide derived from a self-protein for use in the treatment of self-protein-related diseases, or in the vaccination or immunization against self-protein related diseases, in an individual during a treatment period, wherein several administrations of the peptide derived from the self-protein are performed and the administrations are distributed all along the treatment period.

The present invention also relates to the use of a peptide derived from a self-protein for the manufacture of a medicament for the treatment of self-protein-related diseases, or for the vaccination or immunization against self-protein related diseases, in an individual during a treatment period, wherein several administrations of the peptide derived from the self- protein are performed and the administrations are distributed all along the treatment period.

The present invention also relates to a method for eliciting an immune response against a self-protein in an individual during a treatment period, comprising performing several administrations of an effective amount of a peptide derived from the self-protein to the individual, wherein the administrations are distributed all along the treatment period.

The present invention also relates to a peptide derived from a self-protein for use for eliciting an immune response against the self-protein in an individual during a treatment period, wherein several administrations of the peptide derived from the self-protein are performed and the administrations are distributed all along the treatment period.

The present invention also relates to the use of a peptide derived from a self-protein for the manufacture of a medicament for eliciting an immune response against the self- protein in an individual during a treatment period, wherein several administrations of the peptide derived from the self-protein are performed and the administrations are distributed all along the treatment period.

The present invention also relates to a method for treating an inflammatory disorder, or for vaccinating or immunizing against an inflammatory disorder, in an individual during a treatment period, comprising performing several administrations of an effective amount of a peptide derived from a pro-inflammatory protein to the individual, wherein the administrations are distributed all along the treatment period.

The present invention also relates to a peptide derived from a pro-inflammatory protein for use in the treatment of inflammatory disorders, or for vaccinating or immunizing against an inflammatory disorder, in an individual during a treatment period, wherein several administrations of the peptide derived from the self-protein are performed and the administrations are distributed all along the treatment period.

The present invention also relates to the use of a peptide derived from a pro- inflammatory protein for the manufacture of a medicament for the treatment of inflammatory disorders, or for vaccinating or immunizing against an inflammatory disorder in an individual, during a treatment period, wherein several administrations of the peptide derived from the self-protein are performed and the administrations are distributed all along the treatment period.

Description of the figures

Figure 1 : Study design. The monkeys received one priming (d1 ) and 4 boosters (d15, d30, d45, d76). The DTH reaction was evaluated on day 90, 30 days after the priming against TTx (day 59).

Figure 2: (a) Evolution of body weight (kg) in the hlS200-immunized group of monkeys, (b) Rectal temperature at week 7. (c) Rectal temperature at week 1 1 . BT: before treatment, T+6h: 6 hours after treatment, T+24h: 24 hours after treatment. Figure 3: Kinetics of the anti-IL-6 antibody production in the cynomolgus monkeys immunized against the hlS200 peptide immunogen. The control groups did not exhibit any antibodies against IL-6 detected by ELISA and are thus not shown.

Figure 4: Neutralizing capacity of the antibodies purified from sera of the immunized monkeys at d-8, d59, d90, d1 05 and d120. The four h lS200-immunized monkeys (in black) exhibit a significant IL-6 neutralization capacity higher than the background, while the neutralizing capacity of the control monkeys (in grey) is always below background value (threshold of 20 % marked by the dotted line). Only one control group (KLH) is shown for clarity of the figure (the values of the other control group are also all below background value). Figure 5: Cumulative inflammatory score of the local reactions of the immunized monkeys, based on erythema, dermal thickening, and nodules at the sites challenged with 0.05 ml of TTx vaccine. Figure 6: (a) IL-6 levels in the serum of monkeys were measured by ELISA. Data are presented as mean ± SD for each group at each time point. The mean level is always slightly higher in the hlS200-immunized group at each timepoint. (b) Analysis by the Mann-Whitney test shows that the mean IL-6 concentration over time is significantly higher in hlS200- immunized monkeys compared to the two control groups.

Detailed description of the invention

As intended herein, the word "comprising" is synonymous to "include", "contain" or "comprehend". When an object is said to comprise one or several features, this means that other features than those mentionned can be comprised in the object. Conversely, the expression "constituted of" is synonymous to "consisting of". When an object is said to consist of one or several features, this means that no other features than those mentionned are comprised in the object.

Preferably, the treatment period according to the invention comprises, or is constituted of, a priming period and a booster period.

Preferably, the administrations according to the invention are substantially evenly distributed all along the treatment period.

Preferably, the treatment period according to the invention is of at least 1 , 2, 3, 6, 1 2, 18, 24, or 36 months. Preferably, the treatment period is of a least 5, 1 0, 15, 20 or 30 years. Preferably, the treatment period is a lifelong treatment. Preferably, the treatment period lasts for the duration of the disease.

Preferably, consecutive administrations according to the invention are separated from one another by at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 or 12 week(s) all along the treatment period.

Preferably, consecutive administrations according to the invention are separated from one another by at least 1 , 2, or 3 week(s) during the priming period.

Preferably, consecutive administrations according to the invention are separated from one another by at least 4, 5, 6, 7, 8, 9, 10, 1 1 or 12 week(s) during the booster period.

Preferably, consecutive administrations are separated from one another by at most, or less than, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 or 12 week(s) all along the treatment period.

Prefarably, consecutive administrations are separated from one another by at most 1 ,

2 or 3 week(s) during the priming period. Preferably, consecutive administrations according to the invention are separated from one another by at most, or less than, 4, 5, 6, 7, 8, 9, 10, 1 1 or 1 2 week(s) during the booster period.

Preferably, the priming period is of at most, or less than 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 or 1 2 weeks.

Preferably, 2, 3, 4, 5 or 6 administration are performed during the priming period. More preferably, the treatment period comprises, or consists of:

A priming period during which 3, 4 or 5 administrations according to the invention are performed, wherein consecutive administrations are separated from one another by 1 to 3 weeks or by about 1 , 2 or 3 weeks, followed by:

- A booster period, preferably of at least one year, during which consecutive administrations are separated from one another by 1 month to less than 3 months, or by about 1 , 1 .2, 1 .4, 1 .6, 1 .8, 2, 2.2, 2.4, 2.6 or 2.8 months.

Preferably, the administrations according to the invention are selected from the group consisting of intramuscular administration, intravenous administration, subcutaneous administration, intradermic administration, transcutaneous administration, topical administration, intraperitoneal administration, oral administration, sublingual administration and nasal administration.

As intended herein a "self-protein" relates to a protein encoded by the genome of the individual. Accordingly, the self-protein according to the invention is not a viral, bacterial or parasitic protein.

Preferably, the self-protein according to the invention is a pro-inflammatory protein. More preferably the self-protein according to the invention is a pro-inflammatory cytokine. Most preferably, the self-protein according to the invention is IL-6.

As intended herein a "self-protein-related disease" relates to any disease of which an aetiologic agent and/or an symptom-causative agent is a self-protein.

Preferably, the self-protein-related diseases according to the invention are inflammatory diseases.

The self-protein-related diseases or the inflammatory diseases or disorders according to the invention are preferably selected from the group consisting of:

- chronic inflammatory diseases of the intestine, such as Crohn's disease and haemorrhagic rectocolitis or ulcerous colitis,

- arthritic diseases, in particular rheumatoid arthritis, juvenile rheumatoid arthritis, psoriatic arthritis, arthrosis, refractory rheumatoid arthritis, chronic non-rheumatoid arthritis, or ankylosing spondylitis,

- chronic or IL-6-related inflammatory bone diseases, in particular a bone resorption disorder or osteoporosis, - chronic or IL-6-related inflammatory diseases associated with an infection, such as septic shock, endotoxin shock, septicaemia, HCV hepatitis, malaria, meningitis, AIDS or HIV infections,

- chronic or IL-6-related inflammatory diseases of the cardiovascular system, such as atherosclerosis, ischaemia-reperfusion lesions, coronary diseases, and vasculitis, such as

Behget's disease or Wegener's granulomatosis,

- auto-immune diseases, such as scleroderma, in particular systemic scleroderma (also known as systemic sclerosis), lupus erythematosus, in particular disseminated lupus erythematosus, multiple sclerosis, or psoriasis,

- diseases linked to a graft, such as graft-versus-host reactions and graft rejections and traumas,

- allergies, in particular allergic asthma and skin disorders due to delayed hypersensitivity reactions,

- immune deficiencies, such as common variable immunodeficiency (CVID),

- chronic or IL-6-related inflammatory diseases of the respiratory system, in particular respiratory distress syndrome or pulmonary fibrosis,

- cancers having a chronic or IL-6-related inflammatory component, such as plasmacytoma, colorectal cancer, recurrent ovarian cancer, lymphoproliferative syndrome, multiple myeloma, in particular refractory multiple myeloma, or myeloproliferative syndrome, - diabetes, in particular juvenile diabetes,

- amyloidosis, in particular Alzheimer's disease,

- uveitis, in particular in its chronic recurrent form,

- cachexia, and

- endometriosis.

In particular, the self-protein-related diseases or the inflammatory diseases or disorders according to the invention are more preferably selected from the group consisting of rheumatoid arthritis, chronic inflammatory diseases of the intestine, such as Crohn's disease and haemorrhagic rectocolitis, lupus erythematosus, psoriasis, multiple myeloma, and colorectal cancer.

Preferably, the peptide is linked to a carrier protein. More preferably, the carrier protein is Keyhole Limpet Hemocyanin (KLH), hepatitis B surface antigen (HBsAg), bovine serum albumin (BSA), diphtheria toxoid or toxin (DT), tetanus toxoid or toxin (TT), or a viruslike particle (VLP).

Preferably, the peptide according to the invention is linked to a carrier protein acconrd to the invention by a coupling agent or a cross-linking agent, in particular bis-diazo-benzidine (BDB) or succinimidyl-6-(3-maleimidopropionamido)-hexanoate (SMPH). Preferably, the peptide according to the invention is Acetyl- CESSKEALAENNLNLPKCY (SEQ ID NO: 1 ).

Preferably, the peptide according to the invention is cyclized by the formation of an intramolecular disulphide bond between the two cysteine (C) residues.

Preferably, the peptide according to the invention is administered in an amount or at a dose of from 0.5 to 1 00 micrograms per kg of the individual (i.e. μg kg), more preferably 1 to 10 μg kg. Preferably also, the peptide according to the invention is administered in an unitary amount or at a unit dose of from 50 μg to 1 ,000 μg. As intended herein the dose or amount of the peptide relate to the total weight of the peptide optionnally associated the carrier protein if present.

Moreover, the peptide according to the invention can be combined with an adjuvant. The adjuvant can be of any type suited to enhancing the immune response, in particular the antibody response, of an individual, animal or human, to the administration of a peptide. It can thus be complete or incomplete Freund's adjuvant, ISA51 or ISA720, alum or calcium phosphate for example, ISA51 or ISA720 being preferred. The adjuvant can be combined with the peptide according to the invention by producing a 1 /2 up to a 1 /10 mixture, in particular a 1 /1 mixture, by volume of a solution of adjuvant and a solution comprising the polypeptide.

Preferably, the individual according to the invention is a mammal, more preferably a primate, and most preferably a human. Preferably also, the individual according to the invention is afflicted with an inflammatory disease.

The invention will now be further illustrated by the following non-limiting Figures and Example.

Example

In the present example, the inventors have tested the hlS200-coupled peptide in cynomolgus monkeys as, in this species, this peptide sequence is identical to the human one. Therefore, the inventors evaluated the ability of the vaccine to induce a strong anti- cytokine antibody response and active immunization efficacy in a delayed-type hypersensitivity (DTH) model (Bouchez, C. et al. Development of a Delayed-Type Hypersensitivity (DTH) Model in the Cynomolgus Monkey. J Toxicol Pathol 25, 183-188 (2012)).

Methods

Molecular design, peptide synthesis and coupling

The hlS200 peptide was designed using the human IL-6/IL-6Roc/gp130 structure (PDB ID: 1 P9M). This peptide was chosen in loops exposed to the protein surface, which correspond to a region involved in the interaction between the cytokine IL-6 and its receptor IL-6R0C.

The hlS200 peptide sequence is: (SEQ ID NO:

1 )-

The sequence in bold (CESSKEALAENNLNLPK) (SEQ ID NO: 2) is strictly identical between human and cynomolgus monkey IL-6. Peptides were synthesized as previously described in Desallais et al. (2014) op. cit. by Polypeptide Laboratories (Strasbourg, France). They were produced in a cyclized form by the formation of intramolecular disulphide bonds between cysteine residues and coupled with bis-diazobenzidine (BDB) to Keyhole Limpet Hemocyanin (KLH).

Animals

Twelve cynomolgus monkeys (Macaca fascicularis) of adult age (between 4.5 and 5.5 years old) were separated into three groups: the first group received KLH only, the second group received a control peptide coupled to KLH and the third group was immunized with the hlS200 peptide coupled to KLH. Individual data of the animals are presented in Table 1 below: Animal Gender Age (years) Starting weight (kg) Treatment

W62003 Male 5.2 3.97

W62004 Male 5.5 4.1 5

Control peptide

W62053 Female 5.1 3.39

W62054 Female 5.3 2.81

W62009 Male 5.3 5.9

W62059 Female 5.1 2.47

KLH

W62060 Female 5 3.1 2

W62061 Female 4.9 2.74

W6201 0 Male 5.4 4.6

W62062 Female 5 3.1 6

hlS200 peptide

W62063 Female 5.1 3.34

W62064 Female 5 4.1 2

Table 1 : Individual data of the monkeys.

Before inclusion in the experiment, each monkey received a complete health check including clinical examination, tuberculin testing, treatment against parasites and coprology tests. During the experiments, animals were housed in groups in dedicated pens at CIT (Centre International de Toxicologie) animal facilities (Evreux, France). The environment conditions were continuously recorded (temperature 22 ± 3°C and relative humidity 50 ± 30 %). The daily diet during the study consisted of commercial food pellets (Dietex France, SDS, Saint Gratien, France). In addition, a fruit supplement was given daily to each animal. Drinking water was provided ad libitum. The monkey W62010, from the hlS200-immunized group, received three sessions of non-steroidal anti-inflammatory drug (NSAID) and antibiotic drug administration (Finadyne® and Suramox® from days 9, 44 and 1 05) following a recurrent dental abscess (which started on day 9 and was not related to the immunization). Ethics

All study protocols and experimental procedures were approved by The CIT Ethical Committee and were carried out in accordance with the approved guidelines. Immunizations with the hlS200 peptide

The study design is presented in Fig. 1 .

Three groups of four monkeys were immunized five times intramuscularly into the right thigh, on days 1 , 15, 30, 45 and 76. The first group, which received the control peptide (150 μg/monkey) conjugated to KLH (300 μg/monkey according to the manufacturer of the conjugate, Polypeptide Laboratories) and the second group, which received KLH only (Pierce, IL, USA), were used as negative controls. The third group of four monkeys was immunized with the hlS200 peptide (150 μg/monkey) conjugated to KLH (300 μg/monkey according to the manufacturer of the conjugate, Polypeptide Laboratories). Each treatment was emulsified in Montanide ISA 51 VG (Seppic, France). All monkeys received an injection of 0.3 ml on every immunization day.

Clinical examinations

Each animal was checked for mortality or sign of morbidity at least twice a day during the study and clinical signs of systemic toxicity were checked. Body weights were also recorded once a week and rectal temperatures were measured before immunization, 6h and 24h after immunization on days 45 and 76.

Dosage of anti-IL-6 and anti-Tetanus toxoid antibodies by ELISA

Blood samples were taken from the monkeys in pre-dose (day -8), before the first immunization and then every two weeks until the end of the study (total of 9 samples). The IgG response against human IL-6 and Tetanus toxoid (TTx) was measured by ELISA as previously described in Desallais et al. (2014) op. cit. Briefly, 50 ng of human IL-6 (R&D Systems, Lille, France) or 50 ng of TTx (List Biological Laboratories, Campbell, USA) were adsorbed on microtitration plates (Nunc Maxisorp) overnight. After a saturation step, sera from immunized animals were serially diluted and added in coated wells. After wash, an incubation with polyclonal anti-monkey IgGs coupled to horseradish peroxidase (HRP) was performed. Plates were revealed with tetramethylbenzidine (TMB) and the reaction was stopped with 1 M sulfuric acid before reading on a spectrophotometer (Multiskan Ex, Thermo Scientific) at 450 nm. ELISA titers were expressed as those serum dilutions that lead to half- maximal OD450 (titer ₅ o).

Purification of immunoglobulins from monkey sera

Serum immunoglobulins were purified to avoid any non-specific serum interference in the IL-6 neutralization assay (see below). The total IgG fractions were purified from the sera of immunized monkeys using a protein A and protein G affinity chromatography (Biotem, Apprieu, France). In brief, 3 ml of each sera were diluted five times in PBS 1 X sodium EDTA 5 mM and injected on the column. After washes, elution of bound IgGs was accomplished with ammonium acetate 500 mM pH3 and neutralized with Tris 1 M pH 8,8. IgG fractions were then concentrated by centrifugation followed by two dialysis of two hours in PBS at 4°C. Final volume of purified IgGs was 3 ml.

IL-6 neutralization assay

This neutralization assay is based on the inhibition of the binding of IL-6 to its receptors, in an ELISA-derived assay. Microtitration plates were coated with 300 ng/ml of recombinant human gp130 Fc chimera protein (R&D Systems, Lille, France) in PBS overnight at 4°C. hlL-6 at 1 ,3 ng/ml and hlL-6Fta at 200 ng/ml (R&D Systems, Lille, France) were preincubated with the antibodies purified from the sera of immunized animals (at a dilution of 1 :3) for two hours at 37°C. After a saturation step with PBS containing 2% bovine serum albumin, 100 μΙ of the mix were added to the coated plates and incubated overnight at 4°C. The plates were then incubated with 200 ng/ml of h lL-6 biotinylated antibody (R&D Systems, Lille, France) for two hours at 37°C. After wash, plates were incubated with avidin- HRP (1 :500) for 30 minutes at 37°C and were revealed with 100 μΙ of TMB solution. The reaction was stopped with 50 μΙ of 1 M sulfuric acid solution. Absorbance was measured at 450 nm. Induction and evaluation of the DTH response in monkeys

The Tetanus vaccine (Sanofi Pasteur, France) was administered by intramuscular injection into the left thigh of the monkey on day 59, at a dose-volume of 0.5 mL per animal. A challenge was performed by intradermal injection on day 90 on two sites of the back of each animal with 0.05 mL of Tetanus vaccine. Animals were slightly anesthetized before the injections. Skin inflammatory reactions at the challenge sites were recorded once daily for each animal before administration, 24h, 48h and 72h after challenge. Three parameters were evaluated at each challenge site: erythema, dermal thickening and nodules. A score based on the incidence of the three-recorded parameters was used, with a 0 if the parameter was absent of the two challenged sites and a 1 if the parameter was present in at least one of the challenged sites. At each time point, the score for each animal was thus comprised between 0 and 3. The final score given to each animal corresponded to the sum of the scores obtained at 24h, 48h, or 72h. The score for each group was the sum of the scores of the group animals (max = 36). Additionally, the size of the nodules was also measured using a caliper on day 93. Serum levels of IL-6

Serum levels of IL-6 were measured in all monkeys, by using U-CyTech sandwich old world monkey IL-6 ELISA kits with a detection limit of 5 pg/ml (U-CyTech Biosciences, Utrecht, The Netherlands).

Statistics

A Fisher's exact test was used to compare the scores of the inflammatory response in the various groups of monkey. Mann-Whitney tests were used to compare IL-6 levels between the different groups. P values < 0.05 were considered significant.

Results

No adverse event observed during the monkey study

During the whole study (4 months), monkeys were monitored for potential side effect reactions. No unscheduled death and no clinical signs of systemic toxicity were recorded during the study. Some minor local reactions (nodules) were observed at the injection sites in some animals from all groups. They were considered to be part of the immunologic reaction following the immunization. No significant weight loss was observed in the hlS200- immunized group during the study (Fig. 2a) nor in the control groups. Moreover, no significant increase in the rectal temperature was recorded in any group, following injections with the peptide immunogens in week 7 (fourth injection) and week 1 1 (last injection) (Fig. 2b & 2c).

Production of anti-IL-6 antibodies in monkeys

A high production of antibodies recognizing human I L-6 was measured in all four hlS200-immunized monkeys (Fig. 3), showing that the self-tolerance was overcome. However, antibodies titers decreased at the end of the study without further boosts. No anti- human IL-6 (hlL-6) antibodies were found in the control groups.

Presence of anti-IL-6 neutralizing antibodies.

The IL-6 neutralization capacity of the antibodies induced by the immunization of monkeys was assessed by measuring the inhibition of binding of IL-6 to its receptors IL- 6Ra/gp130. In order to avoid any non-specific interference of serum, the IgG antibodies of the immunized monkeys were purified. Neutralizing antibody responses were observed in all hlS200-immunized monkeys with a maximum neutralizing capacity on d59 which decreased over time. No neutralization was observed for the control groups (Fig. 4). The neutralizing capacity in the h lS200-immunized monkeys along time followed a similar evolution as the anti-IL-6 antibody titersso. Significant decrease of the DTH response clinical signs following immunization against the hlS200-coupled peptide

Following the Tetanus toxoid challenge performed on day 90, all animals developed a DTH skin reaction at injection sites. In spite of the small size of the groups, the cumulative inflammatory score (erythema, dermal thickening, and nodules) for the hlS200-immunized group was significantly lower (P=0.014) compared to control groups immunized against a control peptide or against KLH (Fig. 5). In control groups, erythema grade and duration were increased compared to hlS200-immunized group as well as dermal thickening duration. Furthermore, the nodules observed in the hlS200-immunized group were too small to be measured compared to controls (Table 2). No correlation was observed between anti-IL-6 titers and the observed reduction in the DTH response among the four hlS200-immunized monkeys.

Table 2 : Local skin reactions at challenge sites injected with 0.05 ml of Tetanus vaccine (mean for 2 sites: total of 8 sites for 4 animals/group), (a): Grade of erythema and dermal thickening during the DTH evaluation (day 90 up to day 93), 1 : very slight; 2: well-defined, (b) : Mean duration (days) regarding DTH evaluation, (c) : Mean size of nodules (mm), nm: non measurable.

The TTx antibody response was also evaluated and was very similar in the three groups. The reduced clinical scores observed in the test group is thus likely not due to a decreased priming against TTx. Mean serum IL-6 levels increase after active immunization

The mean serum IL-6 concentration at each time point (e.g. test samples vs. control samples at day 15, or at day 30, etc.) showed no significant difference between the test group and the control groups. However, it can be observed that the mean serum IL-6 concentration for all time points was higher from day 15 to day 120 in the test group than in the two other groups (Fig. 6a). Using a global statistical approach over time (Mann-Whitney test, see methods), the inventors have found that the mean IL-6 concentration was in fact significantly higher in the hlS200-immunized group compared to the control peptide group (P=0.0001 ) and compared to the KLH control group (P=0.05) (Fig. 6b).

Discussion

In the present study, the inventors evaluated the safety, immunogenicity and efficacy of anti-IL-6 active immunization in cynomolgus monkeys. Cynomolgus monkeys were of particular interest because the sequence of the hlS200 peptide (derived from IL-6) is 100 % identical between human and cynomolgus monkey IL-6.

The inventors observed that the IL-6 peptide was well tolerated, highly immunogenic in monkeys, and capable of eliciting anti-IL-6 neutralizing antibodies. This antibody production was reversible and regular booster vaccines are required to sustain the antibody levels, which constitutes a safety feature.

The inventors also confirmed that anti-IL-6 active immunization had in vivo effects since it was able to modulate the inflammatory reactions following a DTH response induced by TTx. Indeed, the results showed a significant 57.9 % decrease of the inflammatory reactions in the IL-6-immunized group compared to controls. The inventors did not observe a direct correlation between anti-IL-6 titers and the reduction of the clinical score following TTx challenge within the IL-6 immunized group. Furthermore, the inventors have shown that the response to TTx was very similar in the hlS200-immunized group and in the control groups indicating that the decrease of DTH clinical scores in hlS200 immunized monkeys could not be induced by a decreased priming against TTx, but by the effective neutralization of IL-6 at day 90 due to the immunizations.

The inventors also observed that the mean serum IL-6 concentration at each time point showed indeed no significant difference between the test group and the control groups nor did the levels of C-reactive protein (CRP). However, the mean IL-6 serum concentration for all time points was always higher from day 15 to day 120 in the test group than in the two other groups. Using a global statistical approach over time, the inventors have found that the mean IL-6 concentration was in fact significantly higher in the hlS200-immunized group compared to the control peptide group (P=0.0001 ) and compared to the KLH control group (P=0.05). Such an increase of IL-6 serum concentration was observed with the anti-IL-6 monoclonal antibody Sirukumab and was explained by immune complexes. The increase of IL-6 concentration was also observed in patients treated with Tocilizumab, suggesting that this increase is well tolerated in patients treated by anti-IL-6 biologies. The CRP concentration was low in the three monkey groups of the study since they were healthy monkeys with no disease. Since the CRP values were low in all groups to begin with, it could not be reduced more by the vaccine in the h lS200-immunized group. A decrease of CRP levels has been observed in patients treated by anti-IL-6 biologies because they initially had a chronic inflammatory disease with high levels of CRP.

The present results confirm that active anti-cytokine immunization is an efficient alternative to anti-cytokine monoclonal antibodies. Compared to the infusion of monoclonal antibodies, the advantages of the anti-cytokine vaccines are manifolds: no anti-idiotypic response meaning less secondary resistance, decrease of the costs considering the small quantities of required immunogen, simplicity of production, and simplicity of administration to the patients. A few examples of vaccines based on whole-cytokine immunogens targeting pro-inflammatory cytokines have been described in mice. However, there are several advantages for the use of peptide immunogens over whole-cytokine immunogens: simplicity of production, better characterization of the immunogenic epitopes with less risk of T-cell immunity, cross-reactivity with other proteins or facilitating antibodies. In addition to IL-6, a few examples of successful active immunization against cytokine-derived peptides have been described in mice models of inflammatory diseases with peptide immunogens from TNFoc, IL-1 β, and IL-23p19.