IMMUNOGENIC PRODUCT COMPRISING IL-4 AND/OR IL-13 FOR TREATING DISORDERS ASSOCIATED WITH ABERRANT IL-4 AND/OR IL 13 EXPRESSION OR ACTIVITY

Abstract

An immunogenic product including a cytokine conjugated with a carrier protein, wherein the cytokine is selected from the group including IL-4, IL-13 and mixtures thereof, and wherein the carrier protein is CRM.sub.197. Further, a method for manufacturing the immunogenic product. Also, the therapeutic use of the immunogenic product for treating an inflammatory disorder associated with aberrant IL-4 and/or IL-13 expression or activity.

Claims

1-16. (canceled)

17. An immunogenic product comprising at least one cytokine conjugated with a carrier protein, wherein the at least one cytokine is selected from the group comprising IL-4, IL-13 and mixtures thereof, and wherein the carrier protein is CRM.sub.197.

18. The immunogenic product according to claim 17, wherein the at least one cytokine is IL-4.

19. The immunogenic product according to claim 17, wherein the at least one cytokine is IL-13.

20. The immunogenic product according to claim 17, comprising CRM.sub.197 coupled with both IL-4 and IL-13.

21. A composition comprising at least one immunogenic product comprising at least one cytokine conjugated with a carrier protein, wherein the at least one cytokine is selected from the group comprising IL-4, IL-13 and mixtures thereof, and wherein the carrier protein is CRM.sub.197.

22. The composition according to claim 21, comprising a mixture of at least two immunogenic products comprising at least one cytokine conjugated with a carrier protein, wherein the at least one cytokine is selected from the group comprising IL-4, IL-13 and mixtures thereof, and wherein the carrier protein is CRM.sub.197.

23. The composition according to claim 21, comprising a mixture of an immunogenic product comprising IL-4 and CRM.sub.197 with an immunogenic product comprising IL-13 and CRM.sub.197.

24. The composition according to claim 21, comprising a mixture of an immunogenic product comprising IL-4 and CRM.sub.197 with an immunogenic product comprising IL-13 and CRM.sub.197 at a weight ratio ranging from about 10:1 to about 1:10.

25. The composition according to claim 21, further comprising at least one pharmaceutically acceptable excipient and/or at least one adjuvant.

26. The composition according to claim 21, being an emulsion.

27. A method for producing an immunogenic product according to claim 17, the method comprising steps of: a) contacting the at least one cytokine with a heterobifunctional crosslinker containing a NHS-ester; b) contacting the carrier protein with a heterobifunctional crosslinker containing a NHS-ester, to generate a carrier-heterobifunctional crosslinker complex; c) contacting the heterobifunctional crosslinker-cytokine complex obtained at step (a) with the carrier heterobifunctional crosslinker complex obtained at step (b).

28. The method for producing an immunogenic product according to claim 27, wherein the a heterobifunctional crosslinker containing a NHS-ester of step a) is N-[-maleimidobutyryloxy]-succinimide ester (sGMBS) and wherein the heterobifunctional crosslinker containing a NHS-ester of step b) is N-succinimidyl-S-acetylthioacetate (SATA).

29. A method of treating an inflammatory disorder in a subject in need thereof or for inducing desensitization of a subject allergic to a specific antigen, comprising administering to said subject a therapeutically effective amount of an immunogenic product according to claim 17.

30. The method according to claim 29, wherein said inflammatory disorder is associated with aberrant IL-4 and/or IL-13 expression or activity.

31. The method according to claim 29, wherein the inflammatory disorder is selected from the group comprising asthma, allergic conditions, atopic disorders, bullous pemphigoid, respiratory disorders, nasal polyposis and other conditions involving airway inflammation; inflammatory and/or autoimmune disorders or conditions, gastrointestinal disorders or conditions; systemic lupus erythematosus, liver disorders or conditions, scleroderma; fibrotic diseases or disorders, scleroderma; solid tumors or cancers.

32. The method according to claim 29, wherein the inflammatory disorder is selected from the group consisting of allergic asthma, non-allergic asthma, food allergies, venom allergy, allergy to animals, drug allergy, hyper IgE syndrome, allergic rhinitis, allergic conjunctivitis, allergic enterogastritis, atopic dermatitis, urticaria, eczema, chronic obstructive pulmonary disease (COPD), eosinophilia, fibrosis, excess mucus production, systemic sclerosis (SSc), inflammatory bowel diseases (IBD), eosinophilic esophagitis (EE), eosinophilic-mediated gastrointestinal disease, ulcerative colitis, Crohn's disease, cirrhosis, hepatocellular carcinoma, fibrosis of the liver, leukemia, glioblastoma, lymphoma, mastocytosis.

33. The method according to claim 29, wherein the inflammatory disorder is selected from the group consisting of chronic idiopathic urticaria, chronic spontaneous urticaria, cystic fibrosis, pulmonary fibrosis, fibrosis caused by a hepatitis B virus, fibrosis caused by a hepatitis C virus, B cell chronic lymphocytic leukaemia, Hodgkin's lymphoma.

34. The method according to claim 29, wherein the inflammatory disorder is selected from asthma, atopic dermatitis, chronic obstructive pulmonary disease (COPD), pulmonary fibrosis, food allergy, nasal polyposis and eosinophilic esophagitis.

35. The method according to claim 29, wherein the inflammatory disorder is allergy, asthma, or atopic dermatitis.

36. The method according to claim 129, wherein the immunogenic product or the composition induces desensitization toward an allergen.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0335] FIG. 1 is a scheme of the chemical conjugation of sGMBS-modified IL-4 or IL-13 and SATA-labeled carrier protein through thiol-maleimide addition for the manufacturing of the immunogenic products of the present invention.

[0336] FIG. 2 is a combination of graphs showing the antigenicity of muIL-4 and muIL-13 immunogenic products. Capture was performed with anti-CRM.sub.197 antibody (A, C) or anti-KLH antibody (B, D) and detection with biotinylated polyclonal anti-muIL-4 antibody (A, B) or biotinylated polyclonal anti-muIL-13 antibody (C, D). Represented OD values are OD means of duplicates.

[0337] FIG. 3 is a study scheme of immunizations in Balb/c mice. Four immunizations at days 0, 7, 28 and 49 of immunogenic product (with CRM.sub.197 or KLH as carrier protein), unconjugated muIL-4 or muIL-13+CRM.sub.197 and CRM.sub.197 alone and PBS, all emulsified in squalene adjuvant and i.m. injected in 10 mice per group except for control group only 5 mice. Blood samples were performed before dosing and at day 39 and day 120.

[0338] FIG. 4 is a combination of graphs showing anti-muIL-4 (A), anti muIL-13 (B), anti-CRM.sub.197(C) and anti-KLH (D) antibody titers in mice sera. Ten mice per group were used in immunogenic products groups and 5 mice in control groups. Bars represent median.

[0339] FIG. 5 is a graph showing the muIL-4 neutralizing capacities in mice sera. Ten mice per group were used in immunogenic products groups and 5 mice in control groups. Bars represent median.

[0340] FIG. 6 is a graph showing muIL-13 neutralizing capacities in mice sera. Ten mice per group were used in immunogenic products groups and 5 mice in control groups. Bars represent median.

[0341] FIG. 7 is a study scheme of immunizations and asthma sensitization in Balb/c mice.

[0342] FIG. 8 is a combination of graphs showing anti-muIL-4 (A) and anti-muIL-13 (B) antibodies titers in mice sera. Twelve mice per group. Bars represent median.

[0343] FIG. 9 is a combination of graphs showing muIL-4 (A) and muIL-13 (B) neutralizing capacities in mice sera. Twelve mice per group. Bars represent median.

[0344] FIG. 10 is a graph showing an airway hyperresponsiveness to inhaled methacholine. Enhanced pause (Penh) values were measured by non-invasive whole body plethysmography. Data were obtained using 7-8 mice per group for HDM sensitized/challenged mice and n=16 for PBS controls. * or ** or ***: P<0.5 or 0.1 or 0.001 vs. CRM197-HDM group, using unpaired Mann-Whitney U test.

[0345] FIG. 11 is a combination of graphs showing the circulating IgE levels in serum collected before vaccination (A) and 24 hours after the last challenge with HDM or PBS (B). Data were obtained using 7-8 mice per group for HDM sensitized/challenged mice and n=16 for PBS controls. ***: P<0.001 vs. CRM197-HDM group, using unpaired Mann-Whitney U test.

[0346] FIG. 12 is a combination of graphs showing the levels of CD45+ cells (A) and eosinophils (B) in BAL collected 24 h after the last challenge with HDM. Data were obtained using 7-8 mice per group for HDM sensitized/challenged mice and n=16 for PBS controls. * or ** or ***: P<0.5 or 0.1 or 0.001 vs. CRM.sub.197-HDM group, using unpaired Mann-Whitney U test.

[0347] FIG. 13 is a combination of graphs showing the antigenicity of IL-4 immunogenic product (A), IL-13 immunogenic product (B) and Combo immunogenic product (A and B). Capture was performed with anti-CRM.sub.197 antibody and detection with biotinylated polyclonal anti-huIL-4 antibody (A) or biotinylated polyclonal anti-huIL-13 antibody (B). Represented OD values are OD means of duplicates.

[0348] FIG. 14 is a study scheme of immunization in Balb/c mice.

[0349] FIG. 15 is a combination of graphs showing the anti-IL-4 (A) and anti-IL-13 (B) antibody titers in mice sera. Ten mice per group. Bars represent median.

[0350] FIG. 16 is a combination of graphs showing the anti-IL-4 (A) and anti-IL-13 (B) neutralizing capacities in mice sera. Ten mice per group. Bars represent median.

EXAMPLES

[0351] The present invention is further illustrated by the following examples.

[0352] The present invention relates to an immunogenic product using CRM.sub.197 as a carrier protein. The properties of the immunogenic product of the invention are illustrated by the following examples. In addition, the product of the invention was compared to immunogenic product made with KLH to distinguish the present invention from previous art and to show its superiority to previous art.

[0353] CRM.sub.197 is a non-toxic form of diphtheria toxin without toxic activity due to a single base substitution, in its toxin domain, from glycine to glutamate in position 52 (Uchida et al. 1973 J Biol Chem). As an alternative, Keyhole Limpet Hemocyanin (KLH), a copper-containing protein that is found in arthropods and mollusca (Swaminathan et al. 2014), was also tested and compared to CRM.sub.197.

[0354] The immunogenic products of the invention were produced using the manufacturing process developed below.

[0355] A thiol-maleimide conjugation is employed for the preparation of IL-4 and IL-13 immunogenic products. Sulfhydryl moieties were introduced on the carrier protein CRM.sub.197 with SATA and subsequent hydroxylamine deprotection, while the cytokine muIL-4 or muIL-13 were derivatized by sGMBS, a maleimide-containing agent. Both SATA and sGMBS are heterobifunctional crosslinkers containing a NHS-ester, which reacts with primary amines (such as -amino groups of lysine residues and protein N-termini). An overview of the immunogenic product synthesis via thiol-maleimide conjugation is provided in FIG. 1.

Example 1: Preparation of the Murine IL-4 and IL-13 Immunogenic Products of the Invention with the Two Carrier Proteins

[0356] a) Carrier Protein Functionalization

[0357] CRM.sub.197 or KLH was diluted in the modification buffer comprising 70 mM sodium phosphate buffer, 150 mM NaCl, 5 mM EDTA (pH 7.2). SATA was diluted in DMSO to reach a 100 mM concentration. Then, SATA was added to CRM.sub.197 or KLH and after 30 minutes of incubation on a nutator at room temperature, SATA in excess was removed using Zeba desalting spin column, according to the manufacturer's instructions.

[0358] Subsequently, hydroxylamine was diluted in the same buffer at 500 mM. Then, CRM.sub.197-SATA or KLH-SATA was incubated with the hydroxylamine solution at a 50 mM final concentration, during 2 hours on a nutator at room temperature. Finally, the mixture was desalted and reagents in excess were removed using Zeba desalting spin column.

[0359] b) muIL-4 and muIL-13 Functionalization

[0360] muIL-4 or muIL-13 was dissolved in modification buffer (70 mM sodium phosphate buffer, 150 mM NaCl, 5 mM EDTA, pH 7.2). sGMBS was diluted in modification buffer at 10 mM. Then, sGMBS was added to muIL-4 or muIL-13 and after one hour of incubation on a nutator at room temperature, sGMBS in excess was removed using Zeba desalting spin column.

[0361] c) Conjugation

[0362] After CRM.sub.197, KLH, muIL-4 and muIL-13 functionalization, protein contents of each preparation were determined by Bradford assay.

[0363] Functionalized CRM.sub.197 or functionalized KLH was added to functionalized muIL-4 or functionalized muIL-13 at a molar ratio of 1:2 (carrier: muIL-4 or carrier: muIL-13) and 1:20 respectively. The ratio 1:20 for KLH was chosen based on the difference of molecular weight between KLH and CRM.sub.197 (CRM.sub.19758 kDa vs KLH subunit used in this manufacturing 400 kDa) and based on previous experience in making IFN immunogenic product, a vaccine currently evaluated in a phase IIb clinical trial in lupus patients (NCT02665364) allowing to mix similar quantity of cytokine and carrier in all manufacturings.

[0364] Individual immunogenic product preparations were incubated overnight at 4 C., on a nutator. The resulting immunogenic products were then concentrated using Amicon (3 kDa-cut-off membrane), 0.22 m-filtered and kept at 4 C.

[0365] d) Control (Unconjugated Cytokines and CRM.sub.197)

[0366] As controls, two mixtures (called unconjugated cytokines and CRM.sub.197) were prepared without proteins functionalization: [0367] muIL-4 and CRM.sub.197 were mixed at a molar ratio of 1:2 (CRM.sub.197:cytokine). The mix was prepared at 700 g/mL, without using any coupling reagent. [0368] muIL-13 and CRM.sub.197 were mixed at a molar ratio of 1:2 (CRM.sub.197:cytokine). The mix was prepared at 700 g/mL. without using any coupling reagent.

[0369] Both mixtures were 0.22 m-filtered and stored at 4 C.

[0370] e) Immunogenic Product Quantifications

[0371] Concentrations of muIL-4 immunogenic product and muIL-13 immunogenic product were determined by Coomassie Plus (Bradford) Protein Assay according to the manufacturer instructions.

Example 2: Antigenicity of the Murine Products

[0372] A sandwich ELISA was performed to evaluate the cytokine coupling to the carrier protein and also to evaluate whether epitopes are preserved during the manufacturing process.

[0373] Briefly, capture antibody (anti-carrier protein antibody) was coated in 96-well plates. After a blocking step with 2% (w/v) casein in PBS, immunogenic product samples were added and 2-fold serially diluted. After 90 minutes of incubation at 37 C., bound immunogenic products were detected using biotinylated anti-muIL-4 antibody (polyclonal goat IgG anti-muIL-4) or biotinylated anti-muIL-13 antibody (polyclonal goat IgG anti-muIL-13) and then revealed with streptavidin-HRP and OPD substrate. The enzymatic reaction was stopped with sulfuric acid and optical density (OD) was read at 490 nm. Results are shown in FIG. 2.

[0374] This test confirmed that the immunogenic products of the invention comprises muIL-4 or muIL-13 coupled to CRM.sub.197 or KLH. Furthermore, these results confirm that the immunogenic products are antigenic (i.e. recognized by anti-muIL-4 or anti-muIL-13 antibodies).

Example 3: Immunogenicity of the Immunogenic Products

[0375] Immunogenic products were administered in mice as an emulsion with a squalene-based adjuvant. Immunogenic products were diluted with PBS to the desired concentration and dilutions were mixed with an equal volume of adjuvant.

[0376] Mice Immunization Protocol

[0377] Each Balb/c mouse received four intramuscular (i.m.) injections of immunogenic products (with CRM.sub.197 or KLH) or controls such as PBS, the unconjugated cytokines with CRM.sub.197 or CRM.sub.197 alone, all emulsified (1:1) with a squalene-based adjuvant. Injections were performed on days 0, 7, 28 and 49 as detailed in Table 1 and FIG. 3. First immunization was performed at 7 weeks of age.

TABLE-US-00009 TABLE 1 Dose schedule of administrations Number of mice Dose/injection Article per group Day (g) Adjuvant muIL-4 10 0/7/28/49 30/30/10/10 Squalene immunogenic oil-in-water product (CRM.sub.197) adjuvant muIL-4 10 0/7/28/49 30/30/10/10 immunogenic product (KLH) muIL-13 10 0/7/28/49 30/30/10/10 immunogenic product (CRM.sub.197) muIL-13 10 0/7/28/49 30/30/10/10 immunogenic product (KLH) Unconjugated 5 0/7/28/49 30/30/10/10 muIL-4 + CRM197 Unconjugated 5 0/7/28/49 30/30/10/10 muIL-13 + CRM.sub.197 CRM.sub.197 5 0/7/28/49 30/30/10/10 DPBS 5 0/7/28/49

[0378] Blood collections were performed before dosing and at days 39, 60 and 120. Serum samples were prepared after coagulation at room temperature and centrifugation to remove the clot. Mice were sacrificed by lethal anesthesia at day 120.

[0379] Determination of Anti-Cytokines and Anti-Carrier Proteins Antibodies Titers by ELISA

[0380] Serum samples of immunized mice were assessed for the presence of anti-cytokines antibodies and anti-carrier protein antibodies by ELISA.

[0381] Briefly, muIL-4, muIL-13, CRM.sub.197 or KLH were coated in 96-well plates. After blocking with casein, serum samples were added, and two-fold serially diluted. After incubation at 37 C., bound antibodies were detected with HRP-conjugated anti-mouse IgG and plates were revealed using OPD substrate. The reaction was stopped with sulfuric acid and then absorbance at 490 nm was recorded.

[0382] Positive controls used for anti-muIL-4 antibody, anti-muIL-13 antibody, anti-KLH antibody and anti-CRM.sub.197 antibody titers were respectively, the rat monoclonal anti-muIL-4 IgG1 antibody, the mouse monoclonal anti-muIL13 antibody, pool of sera collected from mice immunized with KLH and the anti-diphtheria toxin A mouse monoclonal IgG1.

[0383] Samples were analyzed starting at dilution 500 dil.sup.1 up to 256,000 dil.sup.1, except for pre-immune sera analyzed only at 500 dil.sup.1.

[0384] Anti-muIL-4, anti-muIL-13, anti-CRM.sub.197 and anti-KLH titers were expressed as serum dilutions leading to half maximal OD.

[0385] Results are presented in FIG. 4. In all groups treated with immunogenic products as well as for mice injected with adjuvanted DPBS or carrier protein or unconjugated preparations, no anti-muIL-4, no anti-muIL-13 no anti-KLH and no anti-CRM.sub.197 were detected before dosing. In addition, no anti-cytokine or anti-carrier antibodies were detected at any timepoint in mice receiving adjuvanted DPBS.

[0386] Anti-CRM.sub.197 titers were detected in all groups treated with CRM.sub.197 control at days 39, 60 and 120 as well as in group treated with unconjugated muIL-4+CRM.sub.197 or with unconjugated muIL-13+CRM.sub.197. Anti-muIL-4, anti-muIL-13 and anti-CRM.sub.197 titers were detected in all groups treated with the immunogenic products made with CRM.sub.197 at days 39, 60 and 120. Anti-muIL-4, anti-muIL-13 and anti-KLH titers were detected but not in all mice in all groups treated with immunogenic products made with KLH at days 39, 60 and 120. Of note, the level of anti-IL-4 antibodies were higher in mice immunized with immunogenic products made with CRM.sub.197 than with the immunogenic products made with KLH.

[0387] muIL-4 Neutralization Bioassay

[0388] The antibodies induced by administrations of muIL-4 immunogenic products were further assessed for their anti-muIL-4 neutralizing capacities in a proliferative assay using CTLL-2 cells adapted from Soman et al, 2009. Briefly, CTLL-2 cells were grown in presence of IL-2 at 10 ng/mL final with RPMI supplemented with 2 mM glutamine, 1 mM sodium pyruvate, 1 mM HEPES, 100 units/mL penicillin, 100 g/mL streptomycin and 10% (v/v) FBS. For neutralization bioassays, IL-2 was replaced by muIL-4. Therefore, potential neutralizing muIL-4 antibodies induced after immunogenic products injections will prevent CTLL-2 growth.

[0389] Serum samples were added at 1/200 final and positive control polyclonal anti-muIL-4 antibody at 1 g/mL final, and were 2-fold serially diluted in 25 L per well RPMI+10% (v/v) FBS in culture plates. muIL-4 was then added at 2 ng/mL final to serum samples and incubated for 1 hour at room temperature. Then 20,000 CTLL-2 cells were added to pre-incubated samples (serum or positive control plus muIL-4). Plates were incubated for 48 h at 37 C., 5% CO.sub.2 in a humidified incubator. Cell viability was quantified by MTS/PMS assay. Forty microliters per well of MTS/PMS were added and after 4 h at 37 C., 5% CO.sub.2 in a humidified incubator, OD was read at 490 nm.

[0390] NC.sub.50 results were expressed as the serum dilution factor (dil.sup.1) neutralizing 50% of muIL-4 activity in presence of serum. The NC.sub.50 is determined by interpolating the serum dilution resulting in a 50% of IL-4 activity on the abscissa axis. Of note, a mouse is considered as a responder in this experiment if NC.sub.50200 dil.sup.1.

[0391] None of the mouse exhibited anti-muIL-4 neutralizing antibodies before dosing (FIG. 5 and Table 2). In the control groups, no anti-muIL-4 antibodies having a neutralizing capacity were detected at any time point. For mice immunized with immunogenic product made with CRM.sub.197, antibodies with neutralizing capacities against muIL-4 were detectable in 9 out of 10 mice. In contrast, in mice immunized with the immunogenic product made with KLH the level of anti-IL-4 neutralizing antibodies were lower at any time point than the one observed when CRM.sub.197 was the carrier and fewer sera exhibited neutralizing antibodies (at best time point only 6 sera exhibited neutralizing capacity (at day 120)). These results demonstrated that treatment with muIL-4 immunogenic product made with CRM.sub.197 was more immunogenic than the conjugated vaccine made with KLH.

TABLE-US-00010 TABLE 2 NC.sub.50 responders towards IL-4 muIL-4 immunogenic muIL-4 immunogenic muIL-4 + CRM.sub.197 product (CRM.sub.197) product (KLH) (unconjugated) D 0 D 39 D 60 D 120 D 0 D 39 D 60 D 120 D 0 D 39 D 60 D 120 Responders 0/10 5/10 7/10 9/10 0/10 2/10 4/10 6/10 0/5 0/5 0/5 0/5 (NC50 > 200 dil-1) CRM.sub.197 PBS D 0 D 39 D 60 D 120 D 0 D 39 D 60 D 120 Responders 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 (NC50 > 200 dil-1)
muIL-13 Neutralization Bioassay

[0392] Antibodies induced by administrations of muIL-13 immunogenic product were assessed for their neutralizing capacities by muIL-13 neutralization bioassay before dosing and at days 39, 60 and 120.

[0393] The neutralizing capacities of anti-muIL-13 antibodies were evaluated using a HEK-Blue IL-4/IL-13 reporter gene bioassay (InvivoGen #hkb-il413) by monitoring the activation of the STAT6 pathway. In response to this activation, this cell line produces secreted embryonic alkaline phosphatase (SEAP) which can be quantified using QUANTI-Blue (at =625 nm). Therefore, potential neutralizing muIL-13 antibodies induced after immunogenic products injections will prevent STAT6 pathway activation and can be evaluated.

[0394] Briefly, HEK-Blue IL-4/IL-13 cells were plated in an assay medium composed of DMEM GlutaMAX supplemented with 10% (v/v) FBS, 10 mM HEPES, 50 U/mL penicillin and 50 g/mL streptomycin. Then a mix of muIL-13 (at 2 ng/mL final concentration) and two-fold serially diluted serum sample starting at 1/100 final or control antibody two fold serially diluted from 1 g/mL final (polyclonal goat anti-muIL-13 antibody) were added to 40,000 HEK-Blue IL-4/IL-13 cells. Plates were incubated 24 h at 37 C., in a 5% CO.sub.2 humidified incubator. Then, in new flat-bottom plates, 10 L per well of cell supernatant were added to 90 L per well of QUANTI-Blue and after 1 h at 37 C., in a 5% CO.sub.2 humidified incubator, absorbances were read at 625 nm.

[0395] NC.sub.50 results were expressed as the serum dilution factor (dil.sup.1) neutralizing 50% of muIL-13 activity in presence of serum. The NC.sub.50 is determined by interpolating the serum dilution resulting in a 50% of muIL-13 activity on the abscissa axis. Of note, a mouse is considered as a responder in this experiment if NC.sub.50100 dil.sup.1.

[0396] No muIL-13 neutralizing antibodies were detected before dosing in all groups as well as in control groups at any time point (FIG. 6 and Table 3). Mice treated with the immunogenic product made with CRM.sub.197 produced antibodies with detectable neutralizing capacities against muIL-13 (FIG. 6 and Table 3). In mice immunized with immunogenic product made with KLH, no mice sera exhibited neutralizing antibodies against muIL-13. Of note, even if one mouse exhibited anti-muIL-13 antibodies as assessed by ELISA in the unconjugated muIL-13 and CRM.sub.197 group (FIG. 4-B), these antibodies were not neutralizing antibodies. These results demonstrated that treatment with the immunogenic product made with CRM.sub.197 is more immunogenic than the one made with KLH.

TABLE-US-00011 TABLE 3 NC.sub.50 responders towards IL-13 muIL-13 immunogenic muIL-13 immunogenic muIL-13 + CRM.sub.197 product (CRM.sub.197) product (KLH) (unconjugated) D 0 D 39 D 60 D 120 D 0 D 39 D 60 D 120 D 0 D 39 D 60 D 120 Responders 0/10 5/10 4/10 5/10 0/10 0/10 0/10 0/10 0/5 0/5 0/5 0/5 (NC50 > 100 dil-1) CRM.sub.197 PBS D 0 D 39 D 60 D 120 D 0 D 39 D 60 D 120 Responders 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 (NC50 > 100 dil-1)

[0397] As expected, no neutralizing anti-muIL-4 and anti-muIL-13 antibodies were detected in the CRM.sub.197 and DPBS control groups at any time point. In the control groups where the cytokines and CRM.sub.197 were mixed without chemical functionalization (unconjugated), immunization did not elicit anti-muIL-4 and anti-muIL-13 neutralizing antibodies, highlighting that conjugation between cytokine and carrier protein is mandatory to break B cell self-tolerance against cytokines. Moreover, for mice immunized with the immunogenic product made with KLH no anti-IL-13 neutralizing antibodies was observed while for mice immunized with immunogenic product made with CRM.sub.197, antibodies with neutralizing capacities against muIL-13 were detectable in 5 out of 10 mice. These results demonstrated that treatment with muIL-13 immunogenic product made with CRM.sub.197 was more immunogenic than the conjugated vaccine made with KLH.

Example 4: Residual Activities of Immunogenic Products

[0398] Residual Activity of muIL-4 Immunogenic Products

[0399] The residual activities of muIL-4 immunogenic products were assessed as described below (adapted from Soman et al, 2009).

[0400] Briefly, CTLL-2 cells are grown with IL-2. Culture medium was constituted of RPMIc medium supplemented with IL-2 at 10 ng/mL final and 10% (v/v) FBS.

[0401] For residual activity bioassays, IL-2 was replaced by muIL-4. Immunogenic products (with CRM.sub.197 or KLH) of the invention and muIL-4 control were two-fold serially diluted in RPMI +10% (v/v) FBS in 96-well plates beginning at 1000 ng/mL until 4 ng/mL for immunogenic products and 10 ng/mL until 0.04 ng/mL final for muIL-4. As a positive control, six wells with muIL-4 at 10 ng/mL were added and used as the maximum cell proliferation control. These samples are added to 20,000 CTLL-2 cells per well and plates were incubated for 48 h at 37 C., 5% CO.sub.2 in a humidified incubator. Cell proliferation was quantified by MTS/PMS assay. Forty microliters per well of MTS/PMS were added and after 4 h at 37 C., 5% CO.sub.2 in a humidified incubator, plates were read at 490 nm.

[0402] The effective dose 50 (ED.sub.50) value corresponds to the amount of immunogenic product or cytokine resulting in 50% of maximum cell signal. The value is determined by interpolating the 50% of maximum cell signal onto the abscissa axis using the y=ax+b formula from the curve passing by the dilution points surrounding the 50% inflexion point.

[0403] The inactivation factor was calculated by dividing the ED.sub.50 of the tested immunogenic product by the mean ED.sub.50 of the muIL-4 control standard curves.

TABLE-US-00012 TABLE 4 ED.sub.50 and inactivation factors muIL-4 control ED.sub.50 ED.sub.50 Inactivation Articles [ng .Math. mL.sup.1] [ng .Math. mL.sup.1] factor muIL-4 620.3 1.67 370 immunogenic product (CRM.sub.197) muIL-4 21.5 1.03 21 immunogenic product (KLH)

[0404] As shown in Table 4, muIL-4 residual activity was more reduced in immunogenic product made with CRM.sub.197 compared to the one prepared with KLH (much higher inactivation factor for muIL-4 immunogenic product prepared with CRM.sub.197).

[0405] Residual activity of muIL-13 immunogenic products The residual activity of muIL-13 immunogenic products was monitored using HEK-Blue IL-4/IL-13 reporter cell line as described below.

[0406] Briefly, muIL-13 immunogenic products and muIL-13 control were two-fold serially diluted in assay medium (DMEM, 10% (v/v) FBS, 10 mM HEPES buffer, 50 U/mL penicillin, 50 g/mL streptomycin) in round bottom plates beginning at 250 ng/mL and 10 ng/mL final, respectively. These mixes were added to 40,000 HEK-Blue IL-4/IL-13 cells per well. Plates were incubated 24 h at 37 C., in a 5% CO.sub.2humidified incubator. Then, in new flat bottom plates, 10 L per well of culture supernatant were added to 90 L per well of QUANTI-Blue and after 1 h at 37 C., in a 5% CO.sub.2humidified incubator, ODs were read at 625 nm.

[0407] The effective dose 50 (ED.sub.50) value, corresponding to the amount of the immunogenic product (or IL-13) resulting in 50% of maximum signal recorded for the considered samples, is determined by interpolating the ODmax/2 values to the corresponding sample concentrations using a four parameter logistic (4PL) nonlinear regression from the whole dilution points.

[0408] The inactivation factors were calculated by dividing the ED.sub.50 of the muIL-13 immunogenic products by the corresponding ED.sub.50 of the muIL-13 control standard curves.

TABLE-US-00013 TABLE 5 ED.sub.50 and inactivation factors muIL-13 control ED.sub.50 ED.sub.50 Inactivation Articles [ng .Math. mL.sup.1] [ng .Math. mL.sup.1] factor muIL-13 737 0.31 2369 immunogenic product (CRM.sub.197) muIL-13 297 0.41 723 immunogenic product (KLH)

[0409] As shown in Table 5, muIL-13 residual activity was more reduced in immunogenic product made with CRM.sub.197 compared to the one prepared with KLH (much higher inactivation factor for muIL-13 immunogenic product prepared with CRM.sub.197).

[0410] Intensity of the immune response against anti-IL-4 and anti-IL-13 was higher when the immunogenic product was made with CRM.sub.197 compared the one made with KLH as a carrier.

[0411] In addition, cytokines residual activities were more reduced in immunogenic product made with CRM.sub.197 compared to those prepared with KLH. From these results, we decided to perform a proof of concept with the products of the invention made with CRM.sub.197 as a carrier protein.

[0412] In addition, in allergy, both IL-4 and IL-13 are involved, so we decided to introduce a group of mice that would receive an immunogenic product comprising both IL-4 and IL-13 conjugates with CRM.sub.197. From now on, all the immunogenic products will be manufactured with CRM.sub.197 as a carrier protein and their mixture will refer to Combo immunogenic product.

Example 5: Efficacy of the Immunogenic Product of the Invention in an Allergy Mouse Model

[0413] Among all allergies, allergic asthma and food allergy are major public health problems now each affecting more than 300 million people worldwide. Allergies are considered to result from a breakdown of tolerance, resulting in type 2 immune responses characterized by the production of TH2 cytokines such as IL-4 and IL-13, high levels of IgE antibodies, and infiltration and expansion of immune cells (in particular mast cells, basophils, eosinophils and T cells) within the inflamed tissue.

[0414] House dust mite (HDM) is a major source of allergen that affects more than 50% of allergic patients (Meyer et al. 1994). In mice, repeated intranasal challenges with HDM reproduced key features of human chronic asthma including airway hyperresponsiveness (AHR), airflow obstruction, airway wall remodeling, mucus production and inflammatory responses in the lungs characterized by high levels of eosinophils.

[0415] Preparation of muIL-4/IL-13 Immunogenic Products

[0416] a) CRM.sub.197Functionalization

[0417] CRM.sub.197 was diluted in the modification buffer comprising 70 mM sodium phosphate buffer, 150 mM NaCl, 5 mM EDTA (pH 7.2). SATA was diluted in DMSO to reach a 100 mM concentration. Then, SATA was added to CRM.sub.197 and after 30 minutes of incubation on a nutator at room temperature, SATA in excess was removed using Zeba desaltingspin column, according to the manufacturer's instructions.

[0418] Subsequently, hydroxylamine was diluted in the same buffer at 500 mM. Then, CRM.sub.197-SATA was incubated with the hydroxylamine solution at a 50 mM final concentration, during 2 hours on a nutator at room temperature. Finally, the mixture was desalted and reagents in excess were removed using Zeba desalting spin column.

[0419] b) muIL-4 and muIL-13 Functionalization

[0420] muIL-4 was dissolved in modification buffer (70 mM sodium phosphate buffer, 150 mM NaCl, 5 mM EDTA, pH 7.2). sGMBS was diluted in modification buffer at 10 mM. Then, sGMBS was added to muIL-4 and after one hour of incubation on a nutator at room temperature, sGMBS in excess was removed using Zeba desalting spin column.

[0421] muIL-13 was dissolved in modification buffer (70 mM sodium phosphate buffer, 150 mM NaCl, 5 mM EDTA, pH 7.2). sGMBS was diluted in modification buffer at 10 mM. Then, sGMBS was added to muIL-13 and after one hour of incubation on a nutator at room temperature, sGMBS in excess was removed using Zeba desalting spin column.

[0422] c) Conjugation

[0423] After CRM.sub.197, muIL-4 and muIL-13 functionalization, protein contents of each preparation were determined by Bradford assay.

[0424] Functionalized CRM.sub.197 was added to functionalized muIL-4 or functionalized muIL-13 at a molar ratio of 1:2 (carrier:muIL-4) and 1:4 (or carrier:muIL-13). Individual immunogenic product preparations were incubated overnight at 4 C., on a nutator. The resulting immunogenic products were then concentrated using Amicon (3 kDa-cut-off membrane), 0.22 m-filtered and kept at 4 C.

[0425] d) Immunogenic Product Quantifications

[0426] Concentrations of muIL-4 immunogenic product and muIL-13 immunogenic product were determined by Coomassie Plus (Bradford) Protein Assay according to the manufacturer instructions.

[0427] muCombo Immunogenic Product Preparation

[0428] Independently synthesized muIL-4 immunogenic product and muIL-13 immunogenic product were mixed together in a 1-1 weight ratio after the concentration and 0.22 m sterile-filtration steps and resulting muCombo immunogenic product was stored at 4 C.

[0429] Mice Immunization, Allergic Asthma Protocol and Blood Sampling

[0430] Each Balb/c mouse received four intramuscular (i.m.) injections of immunogenic products alone or muCombo immunogenic product or CRM.sub.197 alone, as control, all emulsified with squalene-based adjuvant on days 0, 7, 28 and 55 as detailed in Table 6 and FIG. 7. First immunization was performed on mice at 7 weeks of age.

TABLE-US-00014 TABLE 6 Dose schedule of administrations Number of mice Dose Article per group Days (g) Adjuvant muIL-4 12 0/7/28/55 30/30/10/10 Squalene immunogenic oil-in-water product adjuvant muIL-13 12 0/7/28/55 30/30/10/10 immunogenic product muCombo 12 0/7/28/55 30/30/10/10 immunogenic product CRM.sub.197 12 0/7/28/55 15/15/5/5

[0431] Thirty-nine days after first immunogenic product immunization, chronic airway inflammation was induced in mice by three intra nasal injections of 100 g HDM from Dermatophagoides farinae (purchased from Greer) on days 39, 43 and 46. Starting on day 50, mice were challenged by intranasal injections with 25 g HDM twice weekly (total of 9 challenges).

[0432] Blood collections were performed seven days before dosing, at days 39, 64 and 24 hours after the last HDM challenge (mice sacrifice). Serum samples were prepared as previously described and were stored at 20 C. until analysis.

[0433] In this study were evaluated: [0434] Immunogenicity of the injected products by ELISA and bioassay, [0435] Airway hyperresponsiveness (AHR) measured by whole body plethysmography, [0436] Biological markers: circulating IgE levels in serum by ELISA, airway inflammation in lung and bronchoalveolar lavage (BAL) by FACS analysis and airway histology and inflammatory cell infiltration.

[0437] a) Immunogenicity of the Injected Products by ELISA and Bioassay

[0438] Antibody titers of anti-muIL-4 and anti-muIL-13 were measured by ELISA (as described above) from mice sera collected in the four treated groups.

[0439] No cytokine antibody titers were detected before dosing in all groups and in CRM.sub.197 receiving group at all time points. Anti-muIL-4 antibody titers were detected in all mice at all time points after muIL-4 immunogenic product and muCombo immunogenic product immunization (FIG. 8-A). Higher levels were observed in mice receiving muIL-4 immunogenic product compared to muCombo immunogenic product. Anti-muIL-13 antibody titers were detected in mice starting at D39 and with 100% responding mice at D65 in muIL-13 immunogenic product group (FIG. 8-B). In muCombo immunogenic product group, anti-muIL-13 antibodies were also detected as soon as D39 and with 9 out of 12 responding mice at D65. Overall, higher antibody titers were observed in sera after immunization with muIL-4 and muIL-13 immunogenic products than with muCombo immunogenic product, as expected since mice in the muCombo group received half the individual immunogenic product dose (FIG. 14).

[0440] The neutralization activity of each immunogenic products was assessed in vitro using two different bioassays as described above. Of note, a mouse is considered as a responder in this experiment if NC.sub.50 is 200 dil.sup.1 for IL-4 and 100 dil.sup.1 for IL-13.

[0441] High neutralizing capacities against muIL-4 were observed in mice immunized with muIL-4 immunogenic product and muCombo immunogenic product (FIG. 9-A and Table 7), n=11 out of 12 or n=9 out of 12 at least one time point, respectively.

TABLE-US-00015 TABLE 7 NC.sub.50 responders towards IL-4. muIL-4 immunogenic product muCombo immunogenic product CRM.sub.197 D 0 D 39 D 65 D 0 D 39 D 65 D 0 D 39 D 65 Responders 0/12 10/12 11/12 0/12 7/12 9/12 0/12 0/12 0/12 (NC50 > 200 dil-1)

[0442] Interestingly, one year after immunization with muIL-4 or muCombo immunogenic product, anti-IL-4 antibodies were still detected in mice (data not shown).

[0443] Neutralizing capacities against muIL-13 in mice immunized with muIL-13 immunogenic product and muCombo immunogenic product were also induced with n=10 out of 12 or n=7 out of 12 at least one time point, respectively (FIG. 9-B and Table 8).

TABLE-US-00016 TABLE 8 NC.sub.50 responders towards IL-13 muIL-13 immunogenic product muCombo immunogenic product CRM.sub.197 D 0 D 39 D 65 D 0 D 39 D 65 D 0 D 39 D 65 Responders 0/12 8/12 10/12 0/12 3/12 7/12 0/12 0/12 0/12 (NC50 > 100 dil-1)

[0444] Interestingly, one year after immunization with muIL-13 or muCombo immunogenic product, anti-IL-13 antibodies were still detected in mice (data not shown).

[0445] Of note, co-injection of muIL-4 and/or muIL-13 and CRM.sub.197 without prior chemical coupling did not elicit anti-IL-4 and anti-IL-13 antibodies, highlighting that conjugation between cytokine and carrier protein is mandatory to break B cell self-tolerance against cytokines.

[0446] Different classes (including in particular IgG1, IgG2a, IgG2b, IgG3, IgA, IgE and IgM) of anti-IL-4 and anti-IL-13 antibodies were produced after immunization, mostly IgG1 (data not shown).

[0447] b) Airway Hyperresponsiveness (AHR) Measured by Whole Body Plethysmography

[0448] Twenty-four hours after the last challenge, AHR was evaluated by whole body plethysmography. Responses to methacholine, a bronchoconstrictor agent, were measured in conscious mice.

[0449] Different doses of methacholine were administered by aerosol: 0 mg/mL, 3.5 mg/mL, 7 mg/mL and 14 mg/mL. Enhanced pause (Penh) has been used to evaluate changes in pulmonary function (Hamelmann et al., 1997). This measurement conceptualized the phase shift between the thoracic flow and the nasal flow curves: increased phase shift correlated with increased respiratory system resistance. Penh is calculated by the formula Penh=(Te/RT-1)PEF/PIF, where Te is expiratory time, RT the relaxation time, PEF the peak expiratory flow, and PIF is peak inspiratory flow. Penh values were recorded during 5 minutes after each methacholine challenge and the maximum value during the period is reported in the graphic.

[0450] A high bronchoconstriction in mice sensitized with HDM in CRM.sub.197 control group was observed (FIG. 10). The bronchoconstriction was partially inhibited with muIL-13 immunogenic product and muIL-4 immunogenic product while it was almost abrogated in mice immunized with muCombo immunogenic product showing the high protective effect of the products of the invention. Indeed, the level of residual bronchoconstriction observed in the muCombo group was similar to the one observed in HDM-unsensitized mice (circle in FIG. 10).

[0451] These results were confirmed using invasive airway response measurements. Indeed, HDM-treated control mice exhibited significantly increased changes in lung resistance and elastance upon methacholine challenge, as compared to the PBS-treated control group while these two features were partially reduced in mice treated with muIL-4, muIL-13 or muCombo immunogenic product (data not shown).

[0452] Altogether, these results indicate that AHR can be blocked upon dual vaccination against IL-4 and IL-13.

[0453] c) Biological Markers

[0454] Circulating IgE Levels in Serum by ELISA

[0455] Total circulating IgE levels were measured by ELISA following manufacturer's instructions (Mouse IgE ELISA Quantitation Set, Bethyl Labs E90-115) before immunogenic product vaccination and 24 hours after the last HDM challenge (FIG. 11).

[0456] No circulating IgE were detected before vaccination. Twenty-four hours after the last challenge with HDM, total circulating IgE were induced due to HDM sensitization (FIG. 11-B). The levels were reduced in all the immunogenic products receiving groups, demonstrating the protection effect of the products of the invention compared to CRM.sub.197 immunized mice. Of note, similar results were obtained when only HDM-specific IgE were measured by ELISA (data not shown).

[0457] HDM-treated mice also present elevated levels of HDM-specific IgG antibodies. However, these levels were not affected by treatment with muIL-4 and/or muIL-13 immunogenic product (data not shown).

[0458] Airway Inflammation in Lung and Bronchoalveolar Lavage (BAL)

[0459] A detailed time-course analysis of cellular changes in lung and bronchoalveolar lavage (BAL) of mice sensitized with HDM or PBS was carried out.

[0460] Inflammatory cells from BAL were analyzed by FACS using the following antibodies (Table 9): CD45-FITC, Ly6G-PE, CD11b-VG, SiglecF-PECy7, B220-APC and CD3-APC according to manufacturer's instructions.

TABLE-US-00017 Antibody Provider - Catalog number (Clone) Ly6G-PE BD Pharmingen - 561104 (Clone 1A8, Rat IgG.sub.2a, ) CD3-APC BD Pharmingen - 561826 (Clone 145-2C11, Armenian Hamster IgG1, ) CD45-FITC Miltenyi 130-110-658 (Clone REA737, recombinant human IgG1) SiglecF-PECy7 Miltenyi 130-112-334 (Clone REA798, recombinant human IgG1) CD11b-VG Miltenyi 130-110-559 (Clone REA713, recombinant human IgG1) B220-APC Miltenyi 130-102-259 (Clone RA3-6B2, rat IgG2a )

[0461] In lung of control mice, chronic intranasal exposure to HDM resulted in significant increases in the numbers of CD45.sup.+ cells infiltration of hematopoietic origin mainly constituted of eosinophils (CD45+, Ly6G, CD11b+, SiglecF+), as compared to PBS-treated animals (data not shown). Interestingly, immunogenic products vaccination prevented eosinophils infiltration among CD45.sup.+ infiltrating cells, especially in the muCombo immunogenic product group that exhibited a statistically significant decrease.

[0462] In bronchoalveolar lavage, HDM sensitization resulted in an inflammatory response characterized by CD45+ cells infiltration of hematopoietic origin mainly constituted of eosinophils (CD45+, Ly6G, CD11b+, SiglecF+) (FIG. 12). Interestingly, immunogenic products vaccination prevented eosinophils infiltration among CD45.sup.+ infiltrating cells, especially in the muCombo immunogenic product group that exhibited a statistically significant decrease.

[0463] Interestingly, vaccination with immunogenic products of the invention had no effect on the level of eosinophils in the blood (data not shown), indicating that the reduced airway eosinophilia observed in vaccinated mice is a consequence of reduced eosinophil recruitment to the lungs rather than systemic effects on the numbers of eosinophils or eosinophil progenitors.

[0464] Airway Histology

[0465] The effects of sensitization and vaccination on airway was examined by histological analyses. Briefly, left lung was excised post mortem, fixed with 4% PFA for 24 h at room temperature, and preserved in 70% ethanol. Longitudinal sections were done and stained with: [0466] Hematoxylin and Eosin (H&E) for assessment of leukocyte infiltration, [0467] Toluidine Blue for quantification of mast cell numbers, or [0468] Periodic acid Schiff (PAS) staining for assessment of goblet cells hyperplasia and mucus production.

[0469] Globally, these lung histological analyses confirm that vaccination with muIL-4, muIL-13 or muCombo immunogenic product in HDM-treated animals significantly reduced the numbers of leucocytes and intraepithelial mast cells, as compared to non-vaccinated HDM-treated animals (data not shown).

[0470] PAS histological analyses also confirm that vaccination with muIL-4, muIL-13 or muCombo immunogenic product in HDM-treated animals significantly reduced the goblet cell hyperplasia/mucus secretion, as compared to non-vaccinated HDM-treated animals (data not shown). These results support that the immunogenic products (in particular muIL-13 and muCombo immunogenic products) may control mucus hypersecretion induced by HDM sensitization.

[0471] In conclusion, we have demonstrated that the products of the invention made with CRM.sub.197 were immunogenic and induced anti-cytokine neutralizing antibodies whether injected alone or in combination (muCombo immunogenic product). The presence of anti-cytokine neutralizing antibodies was associated with a reduction of HDM-induced AHR, measured by Penh value following methacholine inhalation. In addition, these anti-cytokine neutralizing antibodies were able to limit circulating IgE levels as well as mast cells number in lung and eosinophils infiltration in airways.

[0472] Consequently, these results demonstrate that the immunogenic products of the invention are capable to break B cell tolerance against IL-4 and IL-13 and suggest that these immunogenic products may represent promising new therapeutic strategies in the treatment of asthma and/or allergy, and in particular in the treatment of allergic asthma.

[0473] Interestingly, individual immunogenic products were active but superior beneficial effects on asthma and/or allergy symptoms (and in particular on allergic asthma symptoms) and biological markers were observed when the immunogenic products were combined (muCombo immunogenic product).

Example 6: Preparation of the Human Immunogenic Products of the Invention

[0474] The preparation of human IL-4 immunogenic product, IL-13 immunogenic product and Combo immunogenic product was carried out following the same manufacturing process as described above, using the human cytokines IL-4 and IL-13 instead of the murine cytokines.

[0475] Preparation of IL-4 and IL-13 Immunogenic Product

[0476] a) CRM.sub.197 Functionalization

[0477] CRM.sub.197 functionalization was performed as described for the murine product.

[0478] b) IL-4 and IL-13 Functionalization

[0479] IL-4 or IL-13 dissolved in the buffer (70 mM sodium phosphate buffer, 150 mM NaCl, 5 mM EDTA, pH 7.2) were reacted with sGMBS previously dissolved at a 10 mM concentration in buffer. After one hour of reaction at room temperature with mild agitation, excess sGMBS was removed by Zeba desalting column.

[0480] c) Conjugation

[0481] After CRM.sub.197, IL-4 and IL-13 functionalization, protein contents of each preparation were determined by Bradford assay. Functionalized CRM.sub.197 was added to functionalized IL-4 or functionalized IL-13 at a molar ratio of 1:4 (carrier:IL-4 or carrier:IL-13). Individual immunogenic product preparations were incubated overnight at 4 C. on a nutator. The conjugates were submitted to desalting column to remove potential impurities, such as remaining hydroxylamine, from previous steps. Resulting immunogenic product was concentrated and filtered through a 0.22 m filter and stored at 4 C.

[0482] Preparation of Combo Immunogenic Product

[0483] Preparation of Combo immunogenic product can be performed using two different ways: by incubating both modified cytokines concomitantly with the modified carrier protein (simultaneous synthesis) or by mixing two independently synthesized IL-4 and IL-13 immunogenic products (mix preparation).

[0484] Combo immunogenic product preparation via simultaneous synthesis

[0485] a) CRM.sub.197Functionalization

[0486] CRM.sub.197 functionalization was performed as described for the murine product.

[0487] b) IL-4 and IL-13 Functionalization

[0488] IL-4 or IL-13 functionalization was performed as described for individual immunogenic product preparation.

[0489] c) Conjugation

[0490] After CRM.sub.197, IL-4 and IL-13 functionalization, protein contents of each preparation were determined by Bradford assay. All three modified proteins in phosphate buffer, 150 mM NaCl, 5 mM EDTA, pH 7.2 were mixed at a 2-2-1 molar ratio IL-4-IL-13-CRM.sub.197. The reaction was carried out at 4 C. overnight. The conjugate was submitted to desalting column to remove potential impurities, such as remaining hydroxylamine, from previous steps. Resulting immunogenic product was concentrated and filtered through a 0.22 m filter and stored at 4 C.

[0491] Combo Immunogenic Product Preparation Via Mix Preparation

[0492] Independently synthesized IL-4 and IL-13 immunogenic products were mixed together in a 1-1 weight ratio after the concentration and 0.22 m sterile-filtration steps. Combo immunogenic product was stored at 4 C. until used.

Example 7: Antigenicity of the Human Product

[0493] A sandwich ELISA was performed to evaluate the cytokine coupling to the carrier protein and also evaluating whether epitopes are preserved during the manufacturing process. The protocol used was the same as described hereabove using biotinylated anti-human IL-4 and IL-13 antibodies.

[0494] Results are shown in FIG. 13. This test confirmed that the immunogenic product of the invention comprises IL-4 or IL-13 coupled to CRM.sub.197. Furthermore, these results confirm that the immunogenic product is antigenic (i.e., recognized by anti-IL-4 or anti-IL-13 antibody), as well as in Combo immunogenic product.

Example 8: Immunogenicity of the Human Product

[0495] Mice Immunization Protocol

[0496] Each Balb/c mouse (10 mice per group) received four intramuscular (i.m.) injections of immunogenic product emulsified with squalene-based adjuvant on days 0, 7, 28 and 49 all performed at a 4 g dose, as detailed in FIG. 14. First immunization was performed at 7 weeks of age. Blood collections were carried out before dosing, and at days 38, 59, 90 and 120. Serum samples were prepared after coagulation at room temperature and centrifugation to remove the clot.

[0497] Determination of Anti-IL4 and Anti-IL-13 Antibodies Titers by ELISA

[0498] Anti-cytokines antibody titers were measured by ELISA from mice sera collected in each group.

[0499] The determination of each antibody titers in sera after immunization with immunogenic products or combo-immunogenic product were assessed as described above by coating human IL-4 and IL-13 on the 96-well plates.

[0500] No cytokine antibody titers were detected before dosing in all groups. Anti-IL-4 antibody titers were detected in all mice at all time points after IL-4 immunogenic product and muCombo immunogenic product immunizations (FIG. 15-A). Similar levels were observed in mice receiving IL-4 immunogenic product and Combo immunogenic product, indicating that we might have reached the plateau of the immune response. Anti-IL-13 antibody titers were detected in all mice at all time points after immunization in IL-13 immunogenic product group (FIG. 15-B). In Combo immunogenic product group, anti-IL-13 antibodies were also detected as soon as D38 and in 8 out of 10 mice at D59. Higher anti-IL-13 antibody titers were observed in sera after immunization with IL-13 immunogenic product than with Combo immunogenic product, since mice in the Combo group received half the individual immunogenic product dose.

[0501] Determination of Neutralizing Capacities after Immunizations

[0502] Anti-IL-4 and anti-IL-13 neutralizing capacities were assessed using HEK-Blue IL-4/IL-13 reporter cell line as described above. Of note, in this experiment, a mouse is considered as a responder if NC.sub.50 is >200 dil.sup.1 for IL-4 and >100 dil.sup.1 for IL-13.

[0503] High neutralizing capacities against IL-4 were observed in groups immunized with IL-4 immunogenic product and Combo immunogenic product with 100% of responding mice at all time points after immunization (FIG. 16-A and Table 10).

TABLE-US-00018 TABLE 10 NC.sub.50 responders towards IL-4 IL-4 immunogenic product Combo immunogenic product D 0 D 38 D 59 D 90 D 120 D 0 D 38 D 59 D 90 D 120 Responders 0/10 10/10 10/10 10/10 10/10 0/10 10/10 10/10 10/10 10/10 (NC50 > 200 dil-1)

[0504] Antibodies with neutralizing capacities against IL-13 were also induced in mice immunized with IL-13 immunogenic product and Combo immunogenic product with 100% of responding mice at D59 (FIG. 16-B and Table 11).

TABLE-US-00019 TABLE 11 NC.sub.50 responders towards IL-13 IL-13 immunogenic product Combo immunogenic product D 0 D 38 D 59 D 90 D 120 D 0 D 38 D 59 D 90 D 120 Responders 0/10 9/10 10/10 9/10 9/10 0/10 9/10 10/10 10/10 10/10 (NC50 > 200 dil-1)

[0505] Collectively, products of the invention were capable of inducing high anti-cytokines antibody titers with strong neutralizing capacities.

[0506] Residual Activity of Human Immunogenic Products

[0507] The residual activities of human IL-4 immunogenic product, IL-13 immunogenic product and Combo immunogenic product were monitored using HEK-Blue IL-4/IL-13 reporter cell line as described above for the residual activity of IL-4 and IL-13.

TABLE-US-00020 TABLE 12 ED.sub.50 and inactivation factors towards IL-4 IL-4 control ED.sub.50 ED.sub.50 Inactivation Articles [ng .Math. mL.sup.1] [ng .Math. mL.sup.1] factor IL-4 231 0.043 5287 immunogenic product Combo 382 0.043 8765 immunogenic product

TABLE-US-00021 TABLE 13 ED.sub.50 and inactivation factors towards IL-13 IL-13 control ED.sub.50 ED.sub.50 Inactivation Articles [ng .Math. mL.sup.1] [ng .Math. mL.sup.1] factor IL-13 444 0.258 1848 immunogenic product Combo 268 0.258 1116 immunogenic product

[0508] As shown in Table 12 and Table 13, inactivation factors towards IL-4 or IL-13 in the human immunogenic products were all greater than 1000 (and up to 8765), indicating that cytokines residual activities in the immunogenic products were reduced by a minimum of three orders of magnitude compared to native cytokines.

[0509] These highly reduced residual activities are an important element supporting the safety profile of the products of the invention.

Example 9: Therapeutic Efficacy of the Immunogenic Product of the Invention in a Mouse Model of Food Allergy Model

[0510] Food allergy is a major health issue in westernized countries with increasing prevalence over the last decades and a lack of curative treatment. Several major food staples, including milk, peanut, soy and wheat can induce food allergic reactions. Food allergies reflect a failure of oral tolerance of innocuous food allergens, resulting in the development of a dysregulated Th2-immune response, the secretion of Th2 cytokines (mostly IL-4 and IL-13), the secretion of allergen specific-IgE and the recruitment of effector cells to the gastrointestinal (GI) tract.

[0511] The therapeutic efficacy of the immunogenic product of the invention in food allergy was investigated using IL-4raF709 mutant mice. IL-4raF709 mice carry a gain-of-function mutation in the IL-4 receptor (IL-4R) alpha chain that disrupts the binding of the Src homology domain 2 containing protein tyrosine phosphatase 1 (SHP-1) to the receptor subunit and leads to augmented signal transducer and activator of transcription 6 (STAT6) activation by IL-4 and IL-13. These mice exhibit enhanced Th2 cell responses and IgE production. Thus, this mutation is prototypic of a number of human IL-4Ralpha polymorphisms that promote receptor signaling and are associated with atopy.

[0512] Preparation of muIL-4/IL-13 and muCombo Immunogenic Products

[0513] muIL-4, muIL-13 and muCombo immunogenic products are synthetized as described herein above.

[0514] Mice Immunization, Food Allergy Model, Anaphylaxis Response, Histological Analyses and Blood Sampling

[0515] Each IL-4raF709 mutant mice were immunized with muIL-4, muIL-13 or muCombo immunogenic products or CRM.sub.197, as a control, all emulsified with squalene-based adjuvant on days 0, 7, 28 and then boosted.

[0516] In parallel, mice were sensitized by oral gavage with either PBS or with 23 mg of Peanut (PE) butter, corresponding to 5 mg of peanut protein, suspended in 250 l of 0.1 M sodium bicarbonate (pH 8.0). Mice were challenged with an enteral bolus of 450 mg peanut butter (100 mg protein), diluted in water (Burton et al., 2014).

[0517] Just after the challenge, core body temperature was followed during one hour as well as the onset of diarrhea.

[0518] A clinical score was also determined: 0, no clinical symptom; 1, repetitive mouth/ear scratching and ear canal digging with hind legs; 2, decreased activity, self-isolation, puffiness around eyes and/or mouth; 3, periods of motionless for more than one minute, lying prone on stomach; 4, no response to whisker stimuli, reduced or no response to prodding; 5, tremor convulsion or death.

[0519] Sections of intestinal tissues were collected for histological analyses (e.g., mast cell quantification).

[0520] Blood was also collected to study the level of inflammation (e.g., mast cell degranulation product), antibody levels (e.g., anti-cytokines titers, neutralizing capacities, allergen-specific-IgG and -IgE and total IgE).

Example 10: Therapeutic Efficacy of the Immunogenic Product of the Invention in a Mouse Model of Atopic Dermatitis

[0521] Atopic dermatitis (AD) is a chronic or chronically relapsing, pruritic inflammatory skin disease. The incidence of AD has dramatically increased for the past three decades in industrialized countries. Immunological abnormalities of AD are generally characterized by sensitization with various allergens (e.g., foods, aeroallergens, microbes, and autoallergens), high serum IgE levels, and skin lesions with apoptotic keratinocytes and infiltration with immune cells that secrete Th2 cytokines such as IL-4, IL-5 and IL-13.

[0522] The therapeutic efficacy of the immunogenic products of the invention in AD was investigated by using an animal model in which repeated epicutaneous application of a house dust mite (HDM) extract and staphylococcal enterotoxin B induces eczematous skin lesions.

[0523] Preparation of muIL-4/IL-13 and muCombo Immunogenic Products

[0524] muIL-4, muIL-13 and muCombo immunogenic products are synthetized as described herein above.

[0525] Mice Immunization, Atopic Dermatitis Induction, Clinical Severity Measurement, Histological Analyses and Blood Sampling

[0526] Mice were immunized with muIL-4, muIL-13 or muCombo immunogenic products or CRM.sub.197, as a control, all emulsified with squalene-based adjuvant.

[0527] In parallel, atopic dermatitis was induced as previously described in Ando et al. (J Invest Dermatol. 2013 December; 133(12):2695-2705). Briefly, solutions of 500 ng of Staphylococcal enterotoxin (SEB) and 10 g of Dermatophagoides farinae extract (Der f is a house dust mite, HDM) were applied on a gauze pad placed on the shaved area. This portion of the back skin was occluded with a Tegaderm Transparent Dressing using bandages. Three days later, the dressings were replaced with a new one. After an additional 4 days had passed, the dressings were removed and the mice were kept without treatment for the next week. The one-week Der f/SEB treatment was repeated two more times, thus mice were the subjected to three cycles of such treatment.

[0528] Clinical severity was scored by an investigator who did not know the identities of mice 2 days after removing the dressings in the last cycle. Clinical scores were based on the severity (0, no symptoms; 1, mild; 2, intermediate; 3, severe) of four possible symptoms (redness, bleeding, eruption, and scaling). Maximum possible score is 12.

[0529] Mice were euthanized immediately after scoring, back skin specimens corresponding to the treated areas were obtained for histological analyses of epidermal thickness and eosinophilia. Blood was also collected to study the level of inflammation and antibody levels (e.g., anti-cytokines titers, neutralizing capacities, allergen-specific-IgG and -IgE and total IgE).

Example 11: Therapeutic Efficacy of the Immunogenic Product of the Invention in a Mouse Model of Chronic Obstructive Pulmonary Disease (COPD)

[0530] COPD is characterized by the progressive airflow limitation commonly associated with exaggerated inflammatory responses to inhaled irritants, which leads to the chronic obstructive bronchitis and the destruction of lung parenchyma, termed emphysema.

[0531] Several animal models of COPD have been established to elucidate possible mechanisms underlying the initiation and progression of COPD. Here, a protease-induced model was used to assess the therapeutic efficacy of the immunogenic product of the invention in COPD.

[0532] Preparation of muIL-4/IL-13 and muCombo Immunogenic Products

[0533] muIL-4, muIL-13 and muCombo immunogenic products are synthetized as described herein above.

[0534] Mice Immunization, Atopic Dermatitis Induction, Leukocyte Quantification and Histological Analyses

[0535] Mice were immunized with muIL-4, muIL-13 or muCombo immunogenic products or CRM.sub.197, as a control, all emulsified with squalene-based adjuvant.

[0536] In parallel, intranasal instillation of elastolytic enzymes, such as porcine pancreatic elastase was used to trigger COPD-like symptoms such as, for example, emphysema formation. Briefly, BALB/c mice were intranasally treated with 0.6 U porcine pancreatic elastase in 30 L of PBS (Shibata et al. Proc Natl Acad Sci USA. 2018 Dec 18; 115(51):13057-13062).

[0537] In a first experiment, mice were euthanized on day 5 for assessment of leukocytes: Number of leukocytes (e.g., monocytes, macrophages, neutrophils, T cells, B cells and eosinophils) was quantified by flow cytometry in bronchoalveolar lavage (BAL) fluids and single cell suspensions of lung cells. An additional assessment of leukocyte infiltration in the lungs was performed by histological examinations of lung tissues previously stained with hematoxylin and eosin (H&E). Blood was also collected to study the level of inflammation and antibody levels (e.g., anti-cytokines titers, neutralizing capacities).

[0538] In a second experiment, mice were sacrificed on day 21 after elastase treatment for assessment of lung emphysema by histology in H&E-stained lung tissue sections, using mean linear intercept (MLI) as an indicator of lung airspace enlargement. Blood was also collected to study the level of inflammation and antibody levels (e.g., anti-cytokines titers, neutralizing capacities, allergen-specific-IgG and -IgE and total IgE).

Example 12: Therapeutic Efficacy of the Immunogenic Product of the Invention in a Mouse Model of Pulmonary Fibrosis

[0539] Pulmonary fibrosis represents a broad spectrum of diseases that are characterized by different degrees of lung inflammation, excessive proliferation of lung fibroblasts, and increased lung collagen content.

[0540] The therapeutic efficacy of the immunogenic product of the invention in pulmonary fibrosis was investigated using an animal model in which bleomycin (BLM) administration in lungs leads to secretion of proinflammatory cytokines and chemokines, recruitment of leukocytes, increased collagen production, remodeling, and fibrosis lung inflammation.

[0541] Preparation of muIL-4/IL-13 and muCombo Immunogenic Products

[0542] muIL-4, muIL-13 and muCombo immunogenic products are synthetized as described herein above.

[0543] Mice immunization, pulmonary fibrosis induction, leukocyte quantification and histological analyses

[0544] Mice were immunized with muIL-4, muIL-13 or muCombo immunogenic products or CRM.sub.197, as a control, all emulsified with squalene-based adjuvant.

[0545] In parallel, pulmonary fibrosis was induced in C57BL/6 mice as described previously by Reber et al. (J Immunol 2014 Feb 15; 192(4):1847-54). Briefly, pulmonary fibrosis was induced by intranasal (i.n.) administration of BLM hydrochloride (0.1 mg in 25 l PBS; 12.5 l/nostril).

[0546] Body weight was monitored 5 times per week until the end of the experiment and mice were euthanized 7 days or 14 days after BLM treatment for assessment of lung fibrosis.

[0547] Number of leukocytes (e.g., monocytes, macrophages, neutrophils, T cells, B cells and eosinophils) was quantified by flow cytometry in bronchoalveolar lavage (BAL) fluids and single cell suspensions of lung cells.

[0548] Histological examinations of lung tissues stained with hematoxylin and eosin (for assessment of leukocyte infiltration), masson trichrome (for assessment of fibrosis), or toluidine blue (for assessment of mast cell numbers) were performed.

[0549] Blood was also collected to study the level of inflammation and antibody levels (e.g., anti-cytokines titers, neutralizing capacities).