RU2809548C2 - Immunogenic product containing il-4 and/or il-13 for treatment of disorders associated with aberrant expression or activity of il-4 and/or il-13 - Google Patents

Abstract

FIELD: pharmaceuticals; medicine.

SUBSTANCE: object 1 is an immunogenic product for treating an inflammatory disorder, comprising at least one cytokine conjugated to a carrier protein, wherein at least one cytokine is IL-13 or a mammalian variant of IL-13, wherein this variant exhibits at least 80% identity with the mammalian IL-13 from which it is derived, the carrier protein being CRM₁₉₇. Object 2 is a composition for the treatment of an inflammatory disorder containing at least one immunogenic product. Object 3 is a method of producing an immunogenic product using a heterobifunctional cross-linking agent containing an NHS ester. Objects 4 and 5 are the use of an immunogenic product or composition for treating an inflammatory disorder associated with aberrant expression or activity of IL-13, and for inducing desensitization of a subject allergic to a specific antigen.

EFFECT: effectiveness of the immunogenic product and the production of neutralizing antibodies upon its administration.

16 cl, 16 dwg, 13 tbl, 12 ex

Description

TECHNICAL FIELD

The present invention relates to an immunogenic product and its use for the treatment of disorders associated with aberrant expression or activity of IL-4 and/or IL-13, in particular asthma, atopic dermatitis and allergic disorders.

BACKGROUND OF THE ART

Allergic disorders are complex diseases that result from interactions between multiple genetic and environmental factors. The increase in allergies observed in recent decades is largely attributed to environmental changes occurring during the same period. Among all allergies, the major public health problems are allergic asthma, allergic rhinitis and various types of food allergies, each of which currently affects at least 300 million people worldwide. Moreover, it is estimated that half of the world's population will be affected by allergic disease by 2050. In terms of annual mortality, there are nearly 300,000 allergy-related deaths worldwide due to preventable asthma, food allergy or anaphylaxis. Thus, the increasing incidence of allergic diseases has become an important health issue worldwide, resulting in a significant socioeconomic burden, and for which there is still no effective long-term therapy.

The pathogenesis of allergic disorders occurs as a result of the effects of allergens on the immune system. Such exposures are thought to be responsible for the breakdown of tolerance, which leads to type 2 immune responses characterized by the production of T helper type 2 (Th2) cytokines such as interleukin 4 (IL-4) and interleukin 13 (IL-13), high levels antibodies such as immunoglobulin E (IgE), as well as the infiltration and spread of immune cells into inflamed tissue. Mast cells, basophils and eosinophils are primarily involved in the release of cytoplasmic granules containing pre-produced inflammatory mediators such as histamine. When exposed to an allergen, the allergen is recognized by IgE and binds to receptors on the surface of mast cells, basophils and eosinophils, which stimulates the degranulation of these cells and, consequently, the appearance of clinical symptoms. It should be noted that for these reasons, clinical diagnoses of various types of allergies are mainly based on measurements of allergen-specific IgE.

It is interesting to note that the cytokines IL-4 and IL-13 play key roles in the pathogenesis of allergic disorders. Both cytokines have long been associated with the pathogenesis of allergic disorders and are therapeutically important cytokines based on their biological functions. These two cytokines have a similar structure and share a common receptor subunit (IL-4Rα). However, despite their many similarities, IL-4 and IL-13 are also believed to have some nonredundant functions in allergy.

IL-4 is a pleiotropic cytokine that has been implicated in the development of allergy (Gour N. & Wills-Karp M., 2015), as elevated serum and bronchoalveolar lavage levels of IL-4 are observed in patients with asthma. It is believed that IL-4 specifically acts in the early phase of allergy development. The critical role of IL-4 lies in its multiple allergy-promoting effects, such as induction of IgE production, positive regulation of IgE receptor expression, and differentiation of naive type 0 helper T cells (Th0) into Th2 lymphocytes.

Type 2 immune cells play a key role in the allergic process by controlling humoral immunity and the switching of B cells to the IgE class in antibody responses. Thus, Th2 cells mediate the production of Ig (eg, IgE, IgG) and produce various cytokines as well as IL-4 and IL-13.

In contrast, IL-13 is more involved in the effector and late phases of allergic reactions (Gour N. & Wills-Karp M., 2015). IL-13 has been shown to be sufficient to induce the major manifestations of allergic diseases, including, but not limited to, airway hyperresponsiveness, mucus production, airway smooth muscle changes, and subepithelial fibrosis.

Given the range of cells involved in asthma that are known to be affected by IL-4 and IL-13, and the pathogenic functions associated with these interleukins, neutralization of one or both cytokines is a reliable approach to the treatment of allergic inflammatory disorders. Thus, since IL-4 and IL-13 represent promising therapeutic targets for the treatment of various types of allergies, there is a clear need to improve existing strategies to block these molecules in order to achieve long-term therapeutic effects.

Recently, new therapies have been developed to treat different types of allergies. These treatments, based on passive immunization, specifically target pathogenic factors involved in allergies. For example, the art has described the use of recombinant antibodies directed to IL-4 and IL-13 or their receptors. However, the use of recombinant antibodies is limited by high cost, the need for repeated injections, and the potential risks of anti-drug antibodies (ADA) or other adverse reactions.

In the present application, the present inventor has proposed a new immunogenic product based on the combination of a cytokine selected from IL-4 and IL-13 with CRM ₁₉₇ . The new immunogenic product is of particular interest in the treatment of inflammatory disorders such as, in particular, asthma, atopic dermatitis and allergic disorders.

BRIEF DESCRIPTION OF THE INVENTION

The present invention relates to an immunogenic product containing at least one cytokine conjugated to a carrier protein, wherein said at least one cytokine is selected from the group consisting of IL-4, IL-13 and mixtures thereof, and wherein said carrier protein represents CRM ₁₉₇ .

According to one embodiment of the present invention, the at least one cytokine is IL-4.

According to one embodiment of the present invention, the at least one cytokine is IL-13.

According to one embodiment of the present invention, the immunogenic product of the present invention contains CRM ₁₉₇ associated with both IL-4 and IL-13.

The present invention also relates to a composition containing at least one immunogenic product described herein above.

According to one embodiment of the present invention, the composition contains a mixture of at least two immunogenic products described above in this application.

According to one embodiment of the present invention, the composition contains a mixture of an immunogenic product containing IL-4 and CRM ₁₉₇ with an immunogenic product containing IL-13 and CRM ₁₉₇ .

According to one embodiment of the present invention, the composition contains a mixture of an immunogenic product containing IL-4 and CRM ₁₉₇ with an immunogenic product containing IL-13 and CRM ₁₉₇ in a weight ratio ranging from about 10:1 to about 1:10.

According to one embodiment of the present invention, the composition further comprises at least one pharmaceutically acceptable excipient and/or at least one adjuvant.

According to one embodiment of the present invention, the composition is an emulsion.

The present invention also relates to a method for producing the immunogenic product described in this application, said method comprising the steps of:

a) contacting at least one cytokine with a heterobifunctional cross-linker containing an NHS ester, preferably an N-[γ-maleimidobutyryloxy]-succinimide (sGMBS) ester;

b) contacting the carrier protein with a heterobifunctional cross-linker containing an NHS ester, preferably N-succinimidyl-S-acetylthioacetate (SATA), to produce a carrier-SATA complex;

c) bringing the sGMBS-cytokine complex obtained in step (a) into contact with the carrier-SATA complex obtained in step (b).

The present invention also relates to an immunogenic product described herein, or a composition described herein, for treating an inflammatory disorder.

According to one embodiment of the present invention, the inflammatory disorder is a disorder associated with aberrant expression or activity of IL-4 and/or IL-13.

According to one embodiment of the present invention, the inflammatory disorder is selected from the group consisting of asthma (both allergic and non-allergic), allergic conditions (such as, for example, various types of food allergies, poison allergies, animal allergies, drug allergies, hyper -IgE, allergic rhinitis, allergic conjunctivitis and allergic enterogastritis), atopic disorders (such as, for example, atopic dermatitis, urticaria (including chronic idiopathic urticaria and chronic spontaneous urticaria), eczema), bullous pemphigoid, respiratory disorders (such as allergic and non-allergic asthma, chronic obstructive pulmonary disease (COPD), nasal polyposis and other conditions involving airway inflammation (such as, for example, eosinophilia, fibrosis and excess mucus production, including cystic fibrosis and pulmonary fibrosis, systemic sclerosis (SSc)); inflammatory and/or autoimmune disorders or conditions, gastrointestinal disorders or conditions (such as, for example, inflammatory bowel disease (IBD) and eosinophilic esophagitis (EE), as well as eosinophil-mediated gastrointestinal disease, ulcerative colitis and Crohn's disease) ; systemic lupus erythematosus, liver disorders or conditions (such as, for example, cirrhosis and hepatocellular carcinoma), scleroderma; fibrotic diseases or disorders (such as, for example, liver fibrosis (such as, for example, fibrosis caused by hepatitis B and/or C virus)), scleroderma; solid tumors or various types of cancer, such as leukemia (such as, for example, chronic B-cell lymphocytic leukemia), glioblastoma, lymphoma (such as, for example, Hodgkin's lymphoma) and mastocytosis.

In one embodiment of the present invention, the inflammatory disease is selected from the group consisting of asthma (eg, allergic asthma), atopic dermatitis, chronic obstructive pulmonary disease (COPD), pulmonary fibrosis, food allergy, nasal polyposis, and eosinophilic esophagitis.

In one embodiment of the present invention, the inflammatory disease is selected from the group consisting of asthma (eg, allergic asthma), atopic dermatitis, chronic obstructive pulmonary disease (COPD), pulmonary fibrosis, and food allergy.

According to one embodiment of the present invention, the inflammatory disease is an allergy, asthma or atopic dermatitis.

According to one embodiment of the present invention, the immunogenic product or composition described herein induces desensitization of an allergic subject to an allergen.

The present invention also provides an immunogenic product or composition as described herein for inducing desensitization of a subject having an allergy to a specific antigen, wherein said immunogenic product or composition and said specific antigen must be administered to the subject having an allergy.

The present invention also provides a method for inducing desensitization of a subject allergic to a specific antigen, the method comprising administering to the subject an immunogenic product or composition described herein and said specific antigen.

The present invention also provides a method of increasing the effectiveness and/or decreasing the duration of desensitization of a subject allergic to a specific allergen, wherein the subject is administered an immunogenic product or composition described herein and then treated by desensitization.

DEFINITIONS

In the present invention, the following terms have the following meanings:

-As used herein, the term “about”, when referring to a measurable value such as quantity, length of time period, etc., is intended to include variations of ±20%, or in some cases ±10%, or in some cases ±5 %, or in some cases ±1%, or in some cases ±0.1% of the specified value, since such deviations are suitable for performing the disclosed methods.

-As used herein, “adjuvant” is a substance that enhances the immunogenicity of the immunogenic product of the present invention. Adjuvants are often administered to stimulate an immune response and are well known to those skilled in the art.

-As used herein, the term “carrier protein molecule” refers to a protein or peptide of at least 15, 30 or 50 amino acids in length which, when partially covalently associated with at least one cytokine selected from IL-4; IL-13 and mixtures thereof, to obtain heterocomplexes, ensures the presentation of a large number of antigens of the specified at least one cytokine to B lymphocytes.

-As used herein, the term “immune response” refers to the action of, for example, lymphocytes, antigen presenting cells, phagocytic cells and macromolecules produced by the above cells or the liver (including antibodies, cytokines and complement).

-As used herein, the term “immunogenic product” refers to at least one cytokine associated with a carrier protein that induces an immune response in a subject, preferably a mammal, to which said immunogenic product is administered, including a humoral immune response, i.e., production of antibodies , which neutralize properties, such as, for example, the biological activity of an endogenous cytokine.

-As used herein, the term antibody that “inhibits the biological activity” or “neutralizes the biological activity” of at least one cytokine selected from IL-4, IL-13, or mixtures thereof is intended to mean an antibody that inhibits the activity of said cytokine at at least 10%, 20%, 30%, 40%, 50%, 60%, 70% or 80% or more compared to the level of cytokine activity in the absence of antibody, for example, using a functional assay such as those described in Examples.

-As used herein, the term “pharmaceutically acceptable excipient” refers to an excipient that does not cause an undesirable, allergic or other adverse reaction when administered to a mammal, preferably a human. It includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents and absorption retarding agents, and the like. A pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid excipient, diluent, encapsulating material or formulation excipient of any type. For human administration, drugs must meet standards of sterility, pyrogenicity, general safety and purity as required by regulatory authorities such as the US Food and Drug Administration (FDA) or the European Medicines Agency (EMA).

-As used herein, the term “recombinant protein” refers to a protein (for example, a cytokine or CRM ₁₉₇ carrier protein) that is produced using recombinant DNA technology, such as, for example, a protein (for example, a cytokine or CRM ₁₉₇ carrier protein). expressed in prokaryotic cells (using a bacteriophage or plasmid expression system) or in eukaryotic cells (such as, for example, a yeast, insect or mammalian expression system). The term should also be interpreted to mean a protein (e.g., cytokine or CRM ₁₉₇ carrier protein) that has been produced by synthesizing a DNA molecule encoding a protein (e.g., cytokine or CRM ₁₉₇ carrier protein) and whose DNA molecule expresses the protein ( e.g., a cytokine or CRM ₁₉₇ carrier protein) or an amino acid sequence defining a protein (e.g., a cytokine or CRM ₁₉₇ carrier protein), wherein said DNA or amino acid sequence was produced using recombinant DNA or amino acid sequence technology that is available and well known in this field of technology.

-As used herein, the term “subject” is intended to include living organisms in which an immune response can be elicited (eg, mammals, particularly humans, primates, dogs, cats, horses, sheep, etc.). Preferably the subject is a human. According to one embodiment of the present invention, the subject may be a "patient", that is, a warm-blooded animal, preferably a human, who is awaiting or receiving medical care, or has been/is/will be the subject of a medical procedure or is being monitored for development of the target disease or condition, such as, for example, an inflammatory disorder. In one embodiment of the present invention, the subject is an adult (eg, a subject over 18 years of age). In another embodiment of the present invention, the subject is a child (eg, a subject under 18 years of age). According to one embodiment of the present invention, the subject is male. According to another embodiment of the present invention, the subject is female. According to one embodiment of the present invention, the subject is affected, preferably diagnosed with an inflammatory disorder. According to one embodiment of the present invention, the subject is at risk of developing an inflammatory disorder. Examples of risk factors include, but are not limited to, genetic predisposition or a family history of inflammatory disorders.

-As used herein, the term “therapeutically effective amount” refers to an amount of an immunogenic product described herein that is effective to achieve a particular biological result. Thus, the terms “therapeutically effective amount” mean a level or amount of an immunogenic product that is intended, without causing significant adverse or unwanted side effects to the target, to (1) delay or prevent the onset of the target disease or condition; (2) slowing or stopping the progression, exacerbation or worsening of one or more symptoms of the target disease or condition; (3) improving symptoms of the target disease or condition; (4) reducing the severity or incidence of the target disease or condition; or (5) cure the target disease or condition. A therapeutically effective amount may be administered prior to the onset of the target disease or condition for prophylactic or prophylactic effect. Alternatively or additionally, a therapeutically effective amount may be administered after the onset of the target disease or condition for therapeutic effect.

-As used herein, the term “treating” or “treating” refers to both therapeutic treatment and prophylactic or preventive measures; the goal being to prevent or slow down (reduce) the target disease or condition. Those in need of treatment include those who already have the condition, as well as those who are likely to have the condition, or those in whom the condition should be prevented. A subject is successfully "treated" for a disease or condition if, after receiving a therapeutic amount of an immunogenic product described herein, the subject shows an observable and/or measurable improvement in one or more of the following: a decrease in the number of pathogenic cells; reducing the percentage of all cells that are pathogenic; relief to some extent of one or more symptoms associated with a particular condition; reducing morbidity and mortality and/or improving problematic aspects of quality of life. The above parameters for assessing successful treatment and improvement can be easily measured using routine procedures known to the physician.

DETAILED DESCRIPTION

The present invention relates to an immunogenic product containing at least one cytokine conjugated to a carrier protein, wherein said at least one cytokine is selected from the group consisting of IL-4, IL-13 and mixtures thereof, and wherein said carrier protein represents CRM ₁₉₇ .

In the present application, the Applicants have demonstrated that the immunogenic product of the present invention provides advantages over the same immunogenic product containing KLH instead of CRM ₁₉₇ , particularly in terms of immunogenicity.

CRM ₁₉₇ is a non-toxic diphtheria toxin mutant having the sequence SEQ ID NO: 1, with no toxic activity due to a single base substitution (a mutational substitution of glycine for a glutamic acid residue at position 52).

SEQ ID NO: 1

GADDVVDSSKSFVMENFSSYHGTKPGYVDSIQKGIQKPKSGTQGNYDDDWKEFYSTDNKYDAAGYSVDNENPLSGKAGGVVKVTYPGLTKVLALKVDNAETIKKELGLSLTEPLMEQVGTEEFIKRFGDGASRVVLSLPFAEGSSSVEYINNWEQAKALSVELEINFETRGKRGQDAMYEYMAQACAGNR VRRSVGSSLSCINLDWDVIRDKTKTKIESLKEHGPIKNKMSESPNKTVSEEKAKQYLEEFHQTALEHPELSELKTVTGTNPVFAGANYAAWAVNVAQVIDSETADNLEKTTAALSILPGIGSVMGIADGAVHHNTEEIVAQSIALSSLMVAQAIPLVGELVDIGFAAYNFVESIINLFQVVHNSYNRPAYSPGHKTQPFLHDGY AVSWNTVEDSIIRTGFQGESGHDIKITAENTPLPIAGVLLPTIPGKLDVNKSKTHISVNGRKIRMRCRRAIDGDVTFCRPKSPVYVGNGVHANLHVAFHRSSSEKIHSNEISSDSIGVLGYQKTVDHTKVNSKLSLFFEIKS

According to one embodiment of the present invention, CRM ₁₉₇ can be produced using conventional methods known in the art, in autologous (C. diphtheriae) or heterologous systems (E. coli and P. fluorescens), as described by Hickey in 2018 (Hickey et al. 2018). For example, recombinant CRM ₁₉₇ can be produced by culturing cells containing an expression vector containing the CRM ₁₉₇ gene, collecting inclusion bodies, and purifying CRM ₁₉₇ . CRM ₁₉₇ can also be extracted from a culture of Corynebacterium diphtheriae from a bacterial strain purchased from ATCC (ATCC39255). In one embodiment of the present invention, CRM ₁₉₇ is commercially available and can be purchased, for example, from Reagent Proteins (San Diego, California, USA).

In one embodiment, the immunogenic product of the present invention comprises a CRM ₁₉₇ variant, wherein said variant exhibits at least about 70%, 75, 80, 85, 90, 95% or more identity with SEQ ID NO: 1. In one embodiment implementation of the present invention, the specified variant of CRM ₁₉₇ contains a mutational replacement of glycine with a glutamic acid residue at position 52 and, thus, is non-toxic.

The term "identity" or "identical", when used in reference to the sequence relationship between two or more nucleic acid sequences or two or more polypeptides, refers to the degree of sequence relatedness between nucleic acid sequences or polypeptides, determined by the number of matches between the strands of two or more nucleic acids. residues or amino acid residues, respectively. "Identity" measures the percentage of identical matches between the smaller of two or more gap-aligned sequences (if any) established by a particular mathematical model or computer program (i.e., "algorithms"). The identity of related nucleic acid or polypeptide sequences can be easily calculated using known methods. Such methods include, but are not limited to, those described in Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part 1, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M. Stockton Press, New York, 1991; and Carillo et al., SIAM J. Applied Math. 48, 1073 (1988). Preferred methods for determining identity are designed to ensure maximum agreement between the test sequences. Methods for determining identity are described in publicly available computer programs. Preferred computer program-based methods for determining identity between two sequences include ClustalO (Sievers F., et al 2011), the GCG software package including GAP (Devereux et al., Nucl. Acid. Res. \2, 387 (1984); Genetics Computer Group, University of Wisconsin, Madison, Wis.), BLASTP, BLASTN and FASTA (Altschul et al., J. MoI. Biol. 215, 403-410 (1990)). The BLASTX program is publicly available from the National Center for Biotechnology Information (NCBI) and other sources (BLAST Manual, Altschul et al. NCB/NLM/NIH, Bethesda, MD, USA. 20894; Altschul et al., supra). The well-known Smith-Waterman algorithm can also be used to determine identity.

According to one embodiment of the present invention, the CRM ₁₉₇ is a full-size CRM ₁₉₇ .

In one embodiment, the immunogenic product of the present invention comprises a CRM ₁₉₇ fragment, such as, for example, a fragment containing at least about 50, 100, 150, 200, 250, 300, 350, 400, 450, or 500 amino acids (preferably contiguous amino acids) from SEQ ID NO: 1.

According to one embodiment of the present invention, the at least one cytokine is IL-4.

According to one embodiment of the present invention, IL-4 is recombinant. Recombinant IL-4 can be produced using conventional methods known in the art using a nucleic acid sequence encoding IL-4. For example, recombinant IL-4 can be produced by culturing cells containing an expression vector containing the IL-4 gene, collecting inclusion bodies, and purifying the IL-4 cytokine. Recombinant IL-4 is available commercially and can be purchased, for example, from PeproTech (Rocky Hill, NJ, USA).

In one embodiment of the present invention, IL-4 is derived from a mammal.

In one embodiment, the IL-4 is a mammalian IL-4 variant, wherein the variant exhibits at least about 70%, 75%, 80%, 85%, 90%, 95% or more identity with the mammalian IL-4 from which it is derived. is happening.

According to one embodiment of the present invention, the IL-4 is full-length IL-4.

According to another embodiment of the present invention, the at least one cytokine is an IL-4 fragment, such as, for example, an IL-4 fragment containing at least about 50, 55, 60, 65, 70, 75, 80, 85, 90 , 95, 100, 105, 110, 115, 120, or 125 amino acids (preferably contiguous amino acids) of IL-4 from which it is derived.

According to one embodiment of the present invention, the fragment contains at least one specific epitope of IL-4.

In one embodiment of the present invention, the IL-4 is human IL-4, preferably recombinant human IL-4. Human IL-4 has the sequence SEQ ID NO: 2 (UniProt ID: P05112-1).

SEQ ID NO: 2

HKCDITLQEIIKTLNSLTEQKTLCTELTVTDIFAASKNTTEKETFCRAATVLRQFYSHHEKDTRCLGATAQQFHRHKQLIRFLKRLDRNLWGLAGLNSCPVKEANQSTLENFLERLKTIMREKYSKCSS

In one embodiment of the present invention, IL-4 is a variant of SEQ ID NO: 2, wherein the variant exhibits at least about 70%, 75, 80, 85, 90, 95% or more identity with SEQ ID NO: 2.

In one embodiment of the present invention, the IL-4 is full-length human IL-4.

According to another embodiment of the present invention, the at least one cytokine is a human IL-4 fragment, such as, for example, a human IL-4 fragment containing at least about 50, 55, 60, 65, 70, 75, 80, 85 , 90, 95, 100, 105, 110, 115, 120 or 125 amino acids (preferably contiguous amino acids) SEQ ID NO: 2.

According to one embodiment of the present invention, the fragment contains at least one specific epitope of human IL-4.

According to one embodiment of the present invention, the specified fragment contains or consists of the following sequence: AQQFHRHKQLIRFLKRLDRNLW (SEQ ID NO: 3).

In one embodiment of the present invention, the IL-4 is murine IL-4, preferably recombinant murine IL-4. Mouse IL-4 has the sequence SEQ ID NO: 4 (UniProt ID: P07750-1).

SEQ ID NO: 4

HIHGCDKNHLREIIGILNEVTGEGTPCTEMDVPNVLTATKNTTESELVCRASKVLRIFYLKHGKTPCLKKNSSVLMELQRLFRAFRCLDSSISCTMNESKSTSLKDFLESLKSIMQMDYS

In one embodiment of the present invention, IL-4 is a variant of SEQ ID NO: 4, wherein the variant exhibits at least about 70%, 75, 80, 85, 90, 95% or more identity with SEQ ID NO: 4.

In one embodiment of the present invention, the IL-4 is full-length murine IL-4.

In another embodiment of the present invention, the at least one cytokine is a murine IL-4 fragment, such as, for example, a murine IL-4 fragment containing at least about 50, 55, 60, 65, 70, 75, 80, 85 , 90, 95, 100, 105, 110 or 115 amino acids (preferably contiguous amino acids) SEQ ID NO: 4.

According to one embodiment of the present invention, the fragment contains at least one specific murine IL-4 epitope.

In one embodiment of the present invention, the IL-4 is canine IL-4, preferably recombinant canine IL-4. Canine IL-4 has the sequence SEQ ID NO: 5 (UniProt ID: O77762-1).

SEQ ID NO: 5

HNFNITIKEIIKMLNILTARNDSCMELTVKDVFTAPKNTSDKEIFCRAATVLRQIYTHNCSNRYLRGLYRNLSSMANKTCSMNEIKKSTLKDFLERLKVIMQKKYYRH

In one embodiment of the present invention, IL-4 is a variant of SEQ ID NO: 5, wherein the variant exhibits at least about 70%, 75, 80, 85, 90, 95% or more identity with SEQ ID NO: 5.

In one embodiment of the present invention, the IL-4 is full-length canine IL-4.

In another embodiment of the present invention, the at least one cytokine is a canine IL-4 fragment, such as, for example, a canine IL-4 fragment containing at least about 50, 55, 60, 65, 70, 75, 80, 85 , 90, 95, 100 or 105 amino acids (preferably contiguous amino acids) SEQ ID NO: 5.

According to one embodiment of the present invention, the fragment contains at least one specific canine IL-4 epitope.

In one embodiment of the present invention, the immunogenic product of the present invention contains IL-4 bound to CRM ₁₉₇ in an IL-4:CRM ₁₉₇ molar ratio ranging from about 16:1 to about 1:2, preferably from about 8:1 to about 2:1, more preferably about 4:1.

According to one embodiment of the present invention, the immunogenic product of the present invention contains IL-4 associated with CRM ₁₉₇ and is recognized by antibodies to IL-4.

The fact that an immunogenic product contains IL-4 bound to CRM ₁₉₇ and is recognized by anti-IL-4 antibodies can be verified using conventional methods known in the art. An example of such methods is a sandwich ELISA directed against a cytokine/carrier protein using, for example, a biotin-labeled detection antibody, a streptavidin-HRP signal amplification system, and an o-phenylenediamine hydrochloride (OPD) hydrochloride substrate solution.

In one embodiment of the present invention, ^{the IL-4} TEST A described herein can be used to test that the immunogenic product of the present invention contains IL-4 bound to CRM ₁₉₇ and is recognized by anti-IL-4 antibodies. TEST A ^IL‐4 is an ELISA‐based test directed against IL‐4/CRM ₁₉₇ .

TEST A ^IL-4 is carried out as follows:

-The plate is coated with a capture antibody directed to CRM ₁₉₇ , such as, for example, antibodies to diphtheria toxin from Abcam (AB53828) or Bio-Rad (3710-0956, 3710-0150 or 3710-0100),

-Block the plate with a blocking buffer (such as, for example, 2% casein (w/v) in phosphate-buffered saline (PBS, for example) for approximately 90 minutes at approximately 37°C,

-Incubate the plate for approximately 90 minutes at approximately 37°C with a 2-fold serial dilution of the immunogenic product, starting at 250 ng/ml, or with negative controls such as, for example, IL-4 and CRM ₁₉₇ .

- Incubate the plate for approximately 90 minutes at approximately 37°C with a biotinylated detection antibody directed against IL-4, such as, for example, biotinylated anti-IL-4 antibodies from Abcam (AB84278), R&D systems (BAF204) or PeproTech ( 500-P24BT), or biotinylated murine anti-IL-4 antibodies from Southern Biotech (10204-08), R&D systems (BAF404) or PeproTech (500-P54BT),

-Incubate the plate with streptavidin-HRP for approximately 30 minutes at approximately 37°C and develop the complex using OPD substrate solution for approximately 30 minutes,

-After stopping the enzymatic reaction, the intensity of the resulting color is determined using spectrophotometric methods at 490 nm.

According to one embodiment of the present invention, if the optical density of wells containing the immunogenic product of the present invention at about 25 ng per well is at least about 3 times, preferably at least about 5 times, and more preferably at least about 10 times the optical density of the wells containing the negative control, one skilled in the art can conclude that the immunogenic product of the present invention (i) is recognized by antibodies to IL-4, and (ii) contains IL-4 bound to CRM ₁₉₇ .

In one embodiment of the present invention, the immunogenic product of the present invention contains IL-4 bound to CRM ₁₉₇ and is highly inactivated, meaning that the immunogenic product exhibits less than about 10% of the initial IL-4 activity, preferably less than about 5%, and preferably less than about 1% of the initial IL-4 activity in the ^IL-4 TEST B condition mentioned herein below. TEST B ^IL-4 is a colorimetric T cell proliferation assay or reporter gene bioassay using a cell line expressing IL-4Rα that can be functionally activated by IL-4 contained in the immunogenic product of the present invention (i.e., which can bind IL-4 contained in the immunogenic product of the present invention and become activated after this binding).

In one embodiment, the immunogenic product of the present invention comprises murine IL-4 and TEST B ^IL-4 is a colorimetric T cell proliferation assay performed using CTLL-2 cells (from ECACC cell lines provided by Sigma-Aldrich, reference 93042610-1VL) according to the following method:

-CTLL-2 cells are grown in the presence of IL-2 at a final concentration of 10 ng/ml with RPMI supplemented with 2 mM glutamine, 1 mM sodium pyruvate, 1 mM HEPES, 100 U/ml penicillin, 100 μg/ml streptomycin (full RMPI or RPMIc) and 10% (v/v) fetal bovine serum (FBS),

-The immunogenic product of the present invention (IL-4/CRM ₁₉₇ ) and the muIL-4 control are serially diluted twofold in RPMIc + 10% (v/v) FBS in 96-well plates, starting at 1000 ng/ml to the final concentration 4 ng/ml for the immunogenic product and from 10 ng/ml to a final concentration of 0.04 ng/ml for muIL-4,

-Six wells of muIL-4 at 10 ng/ml were added as a positive control and used as a control for maximum cell proliferation,

-These samples are added to 20,000 CTLL-2 cells per well, and the plates are incubated for approximately 48 hours at approximately 37°C in a humidified 5% CO ₂ incubator.

-At the end of culture, cell viability is assessed using methods well known in the art. One example of such methods is mentioned below: 40 µl/well MTS/PMS solution (Promega, Madison, WI, USA) (where MTS stands for 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl) -2-(4-sulfophenyl)-2H-tetrazolium internal salt; and PMS stands for phenazine methosulfate) is added to the wells and the plate is incubated for an additional 4 hours at 37°C, 5% CO ₂ according to the manufacturer's instructions. The plate is then read at 490 nm on a spectrophotometer.

In another embodiment of the present invention, the immunogenic product of the present invention contains human IL-4 and ^{the IL-4} TEST B is a reporter gene assay that is performed using the HEK-Blue™ IL-4/IL-13 cell line purchased from InvivoGen (San Diego, California, USA). In these cells, stimulation with IL-4 or IL-13 activates the JAK/STAT6 pathway with subsequent SEAP production. The biological activity of IL-4 contained in the immunogenic product of the present invention can then be assessed by assessing the levels of SEAP in the supernatant.

According to this embodiment, TEST B ^IL-4 includes the following steps:

-HEK-Blue™ IL-4/IL-13 cells are cultured in assay media consisting of DMEM GlutaMAX™ supplemented with 10% (v/v) FBS, 10 mM HEPES, 50 U/mL penicillin and 50 μg /ml streptomycin,

-The immunogenic product (IL-4/CRM ₁₉₇ ) and IL-4 control are serially diluted twofold in assay medium to a final concentration of 8000 and 1 ng/ml, respectively.

-These dilution samples are then transferred to pre-seeded plates containing 40,000 HEK-Blue™ IL-4/IL-13 cells per well. The plates are incubated for approximately 24 hours at approximately 37°C in a humidified 5% CO ₂ incubator.

-At the end of culture, the activation pathway is assessed using methods well known in the art. One example of such methods is mentioned below: 90 μl/well of QUANTI-Blue™ solution (purchased from InvivoGen) is added to 10 μl/well of cell supernatant. The plate is then incubated for approximately 1 hour at approximately 37°C in a humidified 5% CO ₂ incubator. The plate is then read at 625 nm on a spectrophotometer.

An effective dose value of 50 (ED ₅₀ ) corresponds to the amount of immunogenic product or cytokine resulting in 50% of the maximum cell signal. This value is determined by interpolating 50% of the cell's maximum signal onto the x-axis using the formula y = ax + b based on a curve passing through the dilution points surrounding the 50% inflection point.

In TEST B ^IL-4, the inactivation coefficient is calculated by dividing the _ED50 of the immunogenic product tested by the average _ED50 value of the IL-4 control standard curves. A result with an inactivation coefficient of >100 means that for the same amount of protein, the IL-4 activity in the immunogenic product corresponds to less than 1% of the activity of native IL-4. According to one embodiment of the present invention, an inactivation ratio greater than about 2, 2.5, 3.33, 5 or 10, preferably about 20, more preferably about 100, indicates that the immunogenic product is highly inactivated. In one embodiment of the present invention, a residual activity of less than about 50%, 40%, 30%, 20%, or 10%, preferably less than about 5%, and more preferably less than about 1% of the activity of native IL-4 indicates that the immunogenic product strongly inactivated.

According to one embodiment of the present invention, the immunogenic product of the present invention contains IL-4 bound to CRM ₁₉₇ and is immunogenic, meaning that the immunogenic product is capable of inducing antibodies to IL-4 in vivo under ^IL-4 TEST C conditions. According to one embodiment of the present invention, the immunogenic product of the present invention is capable of inducing polyclonal antibodies to IL-4 in vivo, for example, under ^IL-4 TEST C conditions.

TEST C ^IL-4 is carried out in accordance with the following method:

Determined amounts of total proteins (as determined, for example, by the Bradford protein assay) of the immunogenic product are administered by injection into mice (over 3 weeks of age) three to four times over 120 days. In one embodiment of the present invention, ^{the IL-4} TEST C is a heterologous system, for example, an immunogenic product containing non-mouse IL-4 is administered by injection to a mouse, and the test includes administering a dose of total proteins ranging from about 0.3 up to 30 mcg. According to another embodiment of the present invention, TEST C ^IL-4 is a homologous system, i.e., an immunogenic product containing murine IL-4 is administered by injection to a mouse, and the test includes administering a dose of total proteins ranging from about 5 to about 30 μg . Serum samples are obtained prior to immunization (preimmunization serum sample) and between day 39 and day 120 (test serum sample). IL-4 ELISA is performed as explained below.

In general, a 96-well plate is coated with 1 μg/ml IL-4 used to prepare the immunogenic product and incubated overnight at a temperature ranging from about 2°C to about 8°C. The plate is then blocked with blocking buffer for approximately 90 minutes at approximately 37°C. 100 μl of pre-immunization sample and serum samples (pre-immunization and test) are added to the wells in a two-fold serial dilution, such as, for example, starting with 500 dilutions. ^-1 to 256000 rec. ^-1 . Finally, a labeled anti-mouse immunoglobulin secondary antibody (such as, for example, an HRP-conjugated antibody) is added to the wells, and the ELISA is developed using any colorimetric means known in the art, such as, for example, OPD substrate solution.

According to one embodiment of the present invention, if the optical density of the wells (490 nm) containing the test serum sample is at least about 1.5 times, preferably at least about 2 times, the optical density of the wells containing the serum sample before immunization, an immunogenic product is considered immunogenic, meaning that it induced antibodies to IL-4 in vivo.

In this test, titers were defined as the dilution of serum that achieves 50% of the ODmax minus the OD of the corresponding sample before immunization in the assay. This method of calculation is much more rigorous than the well-known seroconversion titers, but provides a more reliable analysis and fewer false-positive results. Titers were expressed as serum dilution factors ( ⁻¹ dil).

According to another embodiment of the present invention, in TEST C ^IL-4, the titer value is ≥250 rec. ^-1 , preferably ≥500 rec. ^-1 indicates that the immunogenic product of the present invention produces anti-IL-4 binding antibodies.

According to one embodiment of the present invention, the immunogenic product of the present invention contains IL-4 bound to CRM ₁₉₇ and is capable of neutralizing the activity of IL-4 under the conditions of ^{the IL-4} TEST D mentioned below herein. In accordance with the present invention, TEST D ^IL-4 is performed to evaluate the neutralizing ability of serum obtained from mice immunized with an immunogenic product. This assessment can be assessed using a colorimetric T cell proliferation assay for a murine product or a reporter gene bioassay for a human product using HEK-Blue™ IL-4/IL-13 cells. In these cells, stimulation with IL-4 or IL-13 activates the JAK/STAT6 pathway with subsequent SEAP production. Neutralizing antibodies to IL-4 induced by immunization with immunogenic products can then be assessed by assessing SEAP levels in the supernatant.

In one embodiment, the immunogenic product of the present invention comprises murine IL-4 and TEST D ^IL-4 is a colorimetric T cell proliferation assay that is performed using CTLL-2 cells according to the following method:

-CTLL-2 cells are grown in the presence of IL-2 at a final concentration of 10 ng/ml with RPMIc and 10% (v/v) FBS,

- Serum samples are added to the culture plates at a final ratio of 1/200 and a positive control polyclonal antibody to IL-4 at a final concentration of 1 μg/ml and serially diluted twofold in 25 μl per well of RPMIc + 10% (v/v) FBS .

-MuIL-4 is then added to the serum and control samples at a final concentration of 2 ng/ml and incubated for 1 hour at room temperature.

-Then 20,000 CTLL-2 cells per well are added to the pre-incubated samples. The plates are then incubated for approximately 48 hours at approximately 37°C in a humidified 5% CO ₂ incubator.

-At the end of culture, cell viability is assessed using methods well known in the art. One example of such methods is mentioned below: 40 μl/well of MTS/PMS solution is added to the wells and the plate is incubated for an additional 4 hours at 37°C, 5% CO ₂ . The plate is then read at 490 nm on a spectrophotometer.

According to another embodiment of the present invention, the immunogenic product of the present invention contains human IL-4, and TEST D ^IL-4 is a reporter gene bioassay that is performed using HEK-Blue™ IL-4/IL-13 cells, and includes the following steps :

-HEK-Blue™ IL-4/IL-13 cells are plated in assay media consisting of DMEM GlutaMAX™ supplemented with 10% (v/v) FBS, 10 mM HEPES, 50 U/mL penicillin and 50 μg /ml streptomycin.

-Serum samples, control antibody (polyclonal goat anti-IL-4 antibody) were diluted in assay medium at a final concentration of 1/200 and 1 μg/ml, respectively. Serum samples or control antibody were serially diluted twofold in 96-well round-bottom plates in the presence of IL-4 at a final concentration of 0.25 ng/ml and incubated for one hour at room temperature.

-These mixtures were then added to pre-seeded plates containing 40,000 HEK-Blue™ IL-4/IL-13 cells per well. The plates were incubated for approximately 24 hours at approximately 37°C in a humidified 5% CO ₂ incubator.

-At the end of culture, the activation pathway is assessed using methods well known in the art. One example of such methods is mentioned below: 90 μl/well of QUANTI-Blue™ solution is added to 10 μl/well of cell supernatant. The plates are then incubated for 1 hour at 37°C in a humidified 5% CO ₂ incubator. The plates are then read at 625 nm on a spectrophotometer.

HC ₅₀ results were expressed as the serum dilution factor ( ⁻¹ dil) neutralizing 50% of muIL-4 or IL-4 activity. HC ₅₀ is determined by interpolating the serum dilution resulting in 50% of IL-4 activity on the x-axis.

In TEST D ^IL-4, the NS value _{is 50} ≥100 rec. ^-1 , preferably ≥200 rec. ^-1 indicates that the immunogenic product of the present invention produces neutralizing antibodies against IL-4. According to one embodiment of the present invention, the anti-IL-4 neutralizing antibodies induced by administration of the immunogenic product of the present invention are polyclonal.

According to one embodiment of the present invention, the at least one cytokine is IL-13.

According to one embodiment of the present invention, IL-13 is recombinant. Recombinant IL-13 can be produced using conventional methods known in the art using a nucleic acid sequence encoding IL-13. For example, recombinant IL-13 can be produced by culturing cells containing an expression vector containing the IL-13 gene, collecting inclusion bodies, and purifying the IL-13 cytokine. Recombinant IL-13 is commercially available and can be purchased, for example, from PeproTech (Rocky Hill, NJ, USA).

In one embodiment of the present invention, IL-13 is derived from a mammal.

In one embodiment of the present invention, the IL-13 is a mammalian IL-13 variant, wherein the variant exhibits at least about 70%, 75, 80, 85, 90, 95% or more identity with the mammalian IL-13 from which it is derived. is happening.

According to one embodiment of the present invention, the IL-13 is full-length IL-13.

According to another embodiment of the present invention, the at least one cytokine is an IL-13 fragment, such as, for example, an IL-13 fragment containing at least about 50, 55, 60, 65, 70, 75, 80, 85, 90 , 95, 100, 105, 110, 115 or 120 amino acids (preferably contiguous amino acids) of IL-13 from which it is derived.

According to one embodiment of the present invention, the fragment contains at least one specific epitope of IL-13.

In one embodiment of the present invention, the IL-13 is human IL-13, preferably recombinant human IL-13. Human IL-13 has the sequence SEQ ID NO: 6 (UniProt ID: P35225-1).

SEQ ID NO: 6

LTCLGGFASPGPVPPSTALRELIEELVNITQNQKAPLCNGSMVWSINLTAGMYCAALESLINVSGCSAIEKTQRMLSGFCPHKVSAGQFSSLHVRDTKIEVAQFVKDLLLHLKKLFREGRFN

In one embodiment of the present invention, IL-13 is a variant of SEQ ID NO: 6, wherein the variant exhibits at least about 70%, 75, 80, 85, 90, 95% or more identity with SEQ ID NO: 6.

In one embodiment of the present invention, the IL-13 is full-length human IL-13.

According to another embodiment of the present invention, the at least one cytokine is a fragment of human IL-13, such as, for example, a fragment of human IL-13 containing at least about 50, 55, 60, 65, 70, 75, 80, 85 , 90, 95, 100, 105, 110, 115 or 120 amino acids (preferably contiguous amino acids) SEQ ID NO: 6.

According to one embodiment of the present invention, the fragment contains at least one specific epitope of human IL-13.

In one embodiment of the present invention, the IL-13 is murine IL-13, preferably recombinant murine IL-13. Mouse IL-13 contains the sequence SEQ ID NO: 7 (UniProt ID: P20109-1).

SEQ ID NO: 7

PVPRSVSLPLTLKELIEELSNITQDQTPLCNGSMVWSVDLAAGGFCVALDSLTNISNCNAIYRTQRILHGLCNRKAPTTVSSLPDTKIEVAHFITKLLSYTKQLFRHGPF

According to one embodiment of the present invention, IL-13 is a variant of SEQ ID NO: 7, wherein the variant exhibits at least about 70%, 75, 80, 85, 90, 95% or more identity with SEQ ID NO: 7.

In one embodiment of the present invention, the IL-13 is full-length murine IL-13.

In another embodiment of the present invention, the at least one cytokine is a murine IL-13 fragment, such as, for example, a murine IL-13 fragment containing at least about 50, 55, 60, 65, 70, 75, 80, 85 , 90, 95, 100 or 105 amino acids (preferably contiguous amino acids) SEQ ID NO: 7.

According to one embodiment of the present invention, the fragment contains at least one specific murine IL-13 epitope.

In one embodiment of the present invention, the IL-13 is canine IL-13, preferably recombinant canine IL-13. Canine IL-13 contains the sequence SEQ ID NO: 8 (UniProt ID: Q9N0W9-1).

SEQ ID NO: 8

SPSPVTPSPTLKELIEELVNITQNQASLCNGSMVWSVNLTAGMYCAALESLINVSDCSAIQRTQRMLKALCSQKPAAGQISSERSRDTKIEVIQLVKNLLTYVRGVYRHGNFR

In one embodiment of the present invention, IL-13 is a variant of SEQ ID NO: 8, wherein the variant exhibits at least about 70%, 75, 80, 85, 90, 95% or more identity with SEQ ID NO: 8.

In one embodiment of the present invention, the IL-13 is full-length canine IL-13.

In another embodiment of the present invention, the at least one cytokine is a canine IL-13 fragment, such as, for example, a canine IL-13 fragment containing at least about 50, 55, 60, 65, 70, 75, 80, 85 , 90, 95, 100, 105 or 110 amino acids (preferably contiguous amino acids) SEQ ID NO: 8.

According to one embodiment of the present invention, the fragment contains at least one specific canine IL-13 epitope.

In one embodiment, the immunogenic product of the present invention contains IL-13 bound to CRM ₁₉₇ in an IL-13:CRM ₁₉₇ molar ratio ranging from about 16:1 to about 1:2, preferably from about 8:1 to about 2:1, more preferably about 4:1.

According to one embodiment of the present invention, the immunogenic product contains IL-13 associated with CRM ₁₉₇ and is recognized by antibodies to IL-13.

The fact that the immunogenic product contains IL-13 associated with CRM ₁₉₇ and is recognized by antibodies to IL-13 can be verified using conventional methods known in the art. An example of such methods is a sandwich ELISA directed against a cytokine/carrier protein using, for example, a biotin-labeled detection antibody, a streptavidin-HRP signal amplification system, and/or an OPD substrate solution.

In one embodiment, ^{the IL-13} TEST A described herein can be used to test that the immunogenic product of the present invention contains IL-13 bound to CRM ₁₉₇ and is recognized by anti-IL-13 antibodies. TEST A ^IL-13 is an ELISA-based test directed against IL-13/CRM ₁₉₇ .

TEST A ^IL-13 is carried out as follows:

-The plate is coated with a capture antibody directed to CRM ₁₉₇ , such as, for example, antibodies to diphtheria toxin from Abcam (AB53828) or Bio-Rad (3710-0956, 3710-0150 or 3710-0100),

-The plate is blocked with blocking buffer (such as 2% casein (w/v) in PBS, for example) for approximately 90 minutes at approximately 37°C,

-The plate is incubated for approximately 90 minutes at approximately 37°C with a 2-fold serial dilution of the immunogenic product, starting at 250 ng/ml, or with negative controls such as, for example, IL-13 and CRM ₁₉₇ .

-The plate is incubated for approximately 90 minutes at approximately 37°C with a biotinylated detection antibody directed to IL-13, such as, for example, Biotinylated Anti-IL-13 Antibody from SouthernBiotech (10126-08), PeproTech (500-P13BT) or R&D systems (BAF213), or biotinylated mouse anti-IL-13 antibodies from Bio-Rad (AAM34B), PeproTech (500-P178BT) or R&D systems (BAF413),

-The plate is incubated with streptavidin-HRP for approximately 30 minutes at approximately 37°C, and the complex is developed using OPD substrate solution for approximately 30 minutes.

-After stopping the enzymatic reaction, the intensity of the resulting color is determined by spectrophotometric methods at 490 nm.

According to one embodiment of the present invention, if the optical density of wells containing the immunogenic product of the present invention at 25 ng per well is at least about 3 times, preferably at least about 5 times, and more preferably at least about 10 Based on the optical density of the wells containing the negative control, one skilled in the art can conclude that the immunogenic product of the present invention (i) is recognized by antibodies to IL-13 and (ii) contains IL-13 bound to CRM ₁₉₇ .

In another embodiment of the present invention, the immunogenic product of the present invention contains IL-13 bound to CRM ₁₉₇ and is highly inactivated, meaning that the immunogenic product exhibits less than about 15% residual activity in the ^IL-13 TEST B condition referred to herein. less than, preferably less than about 10% residual activity and more preferably less than about 5% residual activity. TEST B ^IL-13 is a reporter gene bioassay using HEK-Blue™ IL-4/IL-13 cells. In these cells, stimulation with IL-4 or IL-13 activates the JAK/STAT6 pathway with subsequent SEAP production. The biological activity of IL-13 contained in the immunogenic product of the present invention can then be assessed by assessing the levels of SEAP in the supernatant.

In one embodiment of the present invention, TEST B ^IL-13 uses the HEK-Blue™ IL-4/IL-13 cell line purchased from InvivoGen. In the presence of mouse or human IL-13, the HEK-Blue™ IL-4/IL-13 cell line STAT6 pathway is activated and produces SEAP, which can be quantified using methods well known in the art.

TEST B ^IL-13 includes the following steps:

-HEK-Blue™ IL-4/IL-13 cells are seeded in assay media consisting of DMEM GlutaMAX™ supplemented with 10% (v/v) FBS, 10 mM HEPES, 50 U/mL penicillin and 50 μg /ml streptomycin,

-Human immunogenic product (IL-13/CRM ₁₉₇ ) and IL-13 control are serially diluted twofold in assay media to a final concentration of 8000 and 10 ng/ml, respectively.

-The murine immunogenic product (muIL-13/CRM ₁₉₇ ) and muIL-13 control are serially diluted twofold in assay medium to a final concentration of 250 and 10 ng/ml, respectively.

-Samples are then transferred to pre-seeded plates containing 40,000 HEK-Blue™ IL-4/IL-13 cells per well. The plates are incubated for approximately 24 hours at approximately 37°C in a humidified 5% CO ₂ incubator.

-At the end of culture, the activation pathway is assessed using methods well known in the art. One example of such methods is mentioned below: 90 μl/well of QUANTI-Blue™ solution (purchased from InvivoGen) is added to 10 μl/well of cell supernatant. The plates are then incubated for approximately 1 hour at approximately 37°C in a humidified 5% CO ₂ incubator. The plates are then read at 625 nm on a spectrophotometer.

The effective dose 50 (ED ₅₀ ) value corresponding to the amount of immunogenic product (or IL-13) that results in 50% of the maximum signal recorded for the samples in question is determined by interpolating the ODmax/2 values to the corresponding sample concentrations using a four-parameter logistic ( 4PL) non-linear regression based on all dilution points.

In TEST B ^IL-13, the inactivation ratio is calculated by dividing the _ED50 of the test immunogenic product of the present invention by the corresponding _ED50 value of the IL-13 control standard curves. A result with an inactivation coefficient >20 means that for the same amount of protein, the IL-13 activity in the immunogenic product corresponds to less than 5% of the activity of native IL-13. According to one embodiment of the present invention, an inactivation ratio greater than about 2, 2.5, 3.33, 5 or 10, preferably about 20, indicates that the immunogenic product is highly inactivated. According to one embodiment of the present invention, a residual activity of less than about 50%, 40%, 30%, 20%, or 10%, preferably less than about 5% of the activity of native IL-13, indicates that the immunogenic product is highly inactivated.

According to one embodiment of the present invention, the immunogenic product of the present invention contains IL-13 bound to CRM ₁₉₇ and is immunogenic, meaning that the immunogenic product is capable of inducing antibodies to IL-13 in vivo under ^IL-13 TEST C conditions. According to one embodiment of the present invention, the immunogenic product of the present invention is capable of inducing polyclonal antibodies to IL-13 in vivo, for example, under ^IL-13 TEST C conditions.

TEST C ^IL-13 is carried out in accordance with the following method:

Determined amounts of total proteins (as determined, for example, by the Bradford protein assay) of the immunogenic product are administered by injection into mice (over 3 weeks of age) at least three times over 120 days. In one embodiment of the present invention, ^{the IL-13} TEST C is a heterologous system, for example, an immunogenic product containing non-mouse IL-13 is administered by injection into the mouse, and the test includes administering a dose of total proteins ranging from about 0.3 up to 10 mcg. According to another embodiment of the present invention, TEST C ^IL-13 is a homologous system, i.e., an immunogenic product containing murine IL-13 is administered by injection to a mouse, and the test includes administering a dose of total proteins ranging from about 5 to about 30 μg . Serum samples are obtained prior to immunization (pre-immunization serum sample) and between day 39 and day 120 (test serum sample). IL-13 ELISA is performed as explained below.

In general, a 96-well plate is coated with 1 μg/ml IL-13 used to prepare the immunogenic product and incubated overnight at a temperature ranging from about 2°C to about 8°C. The plate is then blocked with blocking buffer for approximately 90 minutes at approximately 37°C. 100 μl of pre-immunization sample and serum samples (pre-immunization and test) are added to the wells in a two-fold serial dilution, such as, for example, starting with 500 dilutions. ^-1 to 256000 rec. ^-1 . Finally, a labeled secondary anti-mouse immunoglobulin antibody (such as, for example, an HRP-conjugated antibody) is added to the wells, and the ELISA is developed using any colorimetric means known in the art, such as, for example, OPD substrate solution.

According to one embodiment of the present invention, if the optical density of the wells containing the test serum sample is at least about 1.5 times, preferably at least about 2 times, the optical density of the wells containing the serum sample before immunization, the immunogenic product is considered immunogenic , meaning that it induced antibodies to IL-13 in vivo.

In this test, titers were defined as the dilution of serum that achieves 50% of the ODmax minus the OD of the corresponding sample before immunization in the assay. This method of calculation is much more rigorous than the well-known seroconversion titers, but provides a more reliable analysis and fewer false-positive results. Titers were expressed as serum dilution factors ( ⁻¹ dil).

According to another embodiment of the present invention, in TEST C ^IL-13, the titer value is ≥250 rec. ^-1 , preferably ≥500 rec. ^-1 indicates that the immunogenic product of the present invention produces anti-IL-13 binding antibodies.

According to one embodiment of the present invention, the immunogenic product of the present invention contains IL-13 bound to CRM ₁₉₇ and is capable of neutralizing the activity of IL-13 under the conditions of ^{the IL-13} TEST D mentioned below herein. In accordance with the present invention, TEST D ^IL-13 is performed to evaluate the neutralizing ability of serum obtained from mice immunized with an immunogenic product using the HEK-Blue™ IL-4/IL-13 reporter cell line. In these cells, stimulation with IL-4 or IL-13 activates the JAK/STAT6 pathway with subsequent SEAP production. Neutralizing antibodies to IL-13 induced by immunization with immunogenic products can then be assessed by assessing SEAP levels in the supernatant.

TEST D ^IL-13 is performed using HEK-Blue™ IL-4/IL-13 cells according to the following method:

In the presence of bioactive IL-13, the STAT6 pathway of the HEK-Blue™ IL-4/IL-13 cell line is activated and produces SEAP, which can be quantified using methods well known in the art. This analysis is carried out according to the following method:

-HEK-Blue™ IL-4/IL-13 cells are cultured in assay media consisting of DMEM GlutaMAX™ supplemented with 10% (v/v) FBS, 10 mM HEPES, 50 U/mL penicillin and 50 μg /ml streptomycin.

-Serum samples obtained after administration of the human immunogenic product and a control antibody (polyclonal goat anti-IL-13 antibody, such as AF-213-NA) were diluted in assay medium to a final concentration of 1/100 and 4 μg/ ml, respectively, and added to 2 ng/ml IL-13.

-Serum samples obtained after administration of the murine immunogenic product and a control antibody (polyclonal goat anti-muIL-13 antibody such as AF-413-NA) were diluted in assay media to a final concentration of 1/100 and 1 μg/ml, respectively , and added to 2 ng/ml muIL-13.

-These mixtures were then incubated for 1 hour at room temperature before being added to 40,000 HEK-Blue™ IL 4/IL-13 cells per well. The plates were incubated for approximately 24 hours at approximately 37°C in a humidified 5% CO ₂ incubator.

-At the end of culture, the activation pathway is assessed using methods well known in the art. One example of such methods is mentioned below: 90 μl/well of QUANTI-Blue™ solution is added to 10 μl/well of cell supernatant. The plates are then incubated for 1 hour at 37°C in a humidified 5% CO ₂ incubator. The plates are then read at 625 nm on a spectrophotometer.

HC ₅₀ results were expressed as the serum dilution factor ( ⁻¹ dil) neutralizing 50% of muIL-13 or IL-13 activity. HC ₅₀ is determined by interpolating the serum dilution resulting in 50% of IL-13 activity on the x-axis.

In TEST D ^IL-13, the NS ₅₀ value is ≥50, preferably the NS ₅₀ value is ≥100 rec. ^-1 indicates that the immunogenic product of the present invention produces neutralizing antibodies against IL-13. According to one embodiment of the present invention, the anti-IL-13 neutralizing antibodies induced by administration of the immunogenic product of the present invention are polyclonal.

According to one embodiment of the present invention, the immunogenic product of the present invention contains IL-4 and IL-13.

In one embodiment of the present invention, IL-4, IL-13, or both are recombinant.

In one embodiment of the present invention, both IL-4 and IL-13 are derived from the same mammal. In one embodiment of the present invention, IL-4, IL-13, or both are human.

In one embodiment, the IL-4 is a variant of human IL-4, wherein the variant exhibits at least about 70%, 75, 80, 85, 90, 95% or more identity with human IL-4. In one embodiment, the IL-13 is a variant of human IL-13, wherein the variant exhibits at least about 70%, 75, 80, 85, 90, 95% or more identity with human IL-13.

In one embodiment of the present invention, IL-4, IL-13, or both are full-length.

According to one embodiment of the present invention, IL-4 is a fragment of full-length IL-4. According to one embodiment of the present invention, IL-13 is a fragment of full-length IL-13.

According to one embodiment of the present invention, the immunogenic product of the present invention contains IL-4 and IL-13, which are both associated with CRM ₁₉₇ .

In one embodiment of the present invention, the molar ratio of cytokines (i.e., IL-4 and IL-13):CRM ₁₉₇ ranges from about 16:1 to about 1:2, preferably from about 8:1 to about 2:1 , more preferably about 4:1.

According to one embodiment of the present invention, the molar ratio of IL-4:CRM ₁₉₇ ranges from about 8:1 to about 1:2, preferably from about 4:1 to about 1:1, more preferably about 2:1.

According to one embodiment of the present invention, the molar ratio of IL-13:CRM ₁₉₇ ranges from about 8:1 to about 1:2, preferably from about 4:1 to about 1:1, more preferably about 2:1.

According to one embodiment of the present invention, the molar ratio of IL-4:IL-13 ranges from about 5:1 to about 1:5, preferably from about 2:1 to about 1:2, more preferably about 1:1.

According to one embodiment of the present invention, the immunogenic product of the present invention contains IL-4 and IL-13 associated with CRM ₁₉₇ and is recognized by anti-IL-4 antibodies and anti-IL-13 antibodies.

The fact that the immunogenic product contains IL-4 and IL-13 associated with CRM ₁₉₇ and is recognized by anti-IL-4 antibodies and anti-IL-13 antibodies can be verified using conventional methods known in the art. An example of such methods is a sandwich ELISA directed against a cytokine/carrier protein using, for example, a biotin-labeled detection antibody, a streptavidin-HRP signal amplification system, and/or an OPD substrate solution.

In one embodiment of the present invention, recognition of an immunogenic product by anti-IL-4 antibodies and anti-IL-13 antibodies can be tested using TESTS A ( ^IL-4 TEST A and ^IL-13 TEST A) described herein.

In one embodiment of the present invention, the immunogenic product of the present invention contains IL-4 and IL-13 bound to CRM ₁₉₇ and is highly inactivated, meaning that the immunogenic product exhibits less than about 10% of the initial IL-4 activity, preferably less than about 10% of the initial IL-4 activity. 5% and preferably less than about 1% of the initial IL-4 activity and less than about 15% of the initial IL-13 activity, preferably less than about 10% and preferably less than about 5% of the initial IL-13 activity in the TESTS B condition (TEST B ^IL-4 and TEST B ^IL13 ) mentioned herein below.

According to one embodiment of the present invention, the immunogenic product contains IL-4 and IL-13 associated with CRM ₁₉₇ and is immunogenic, meaning that the immunogenic product is capable of (i) inducing antibodies to IL-4 in vivo under TEST C ^{IL- 4} and (i) induce antibodies to IL-13 in vivo under TEST C ^IL-13 conditions. According to one embodiment of the present invention, the immunogenic product of the present invention is capable of inducing polyclonal antibodies to IL-4 in vivo, for example, under TEST C ^IL-4 conditions, and antibodies to IL-13 in vivo, such as, for example, under TEST C conditions ^IL-13 .

According to one embodiment of the present invention, the immunogenic product of the present invention contains IL-4 and IL-13 bound to CRM ₁₉₇ and is capable of (i) neutralizing the activity of IL-4 in the ^IL-4 TEST D condition referred to herein, and (ii) neutralizing IL-13 activity in the ^IL-13 TEST D condition mentioned herein below. According to one embodiment of the present invention, the neutralizing antibodies against IL-4 and IL-13 induced by administration of the immunogenic product of the present invention are polyclonal.

The present invention also relates to a method for producing an immunogenic product containing at least one cytokine selected from IL-4, IL-13 and mixtures thereof, associated with a carrier protein, preferably CRM ₁₉₇ , which method includes the following steps:

a) contacting at least one cytokine with a heterobifunctional cross-linking agent containing an NHS ester, preferably an N-[γ-maleimidobutyryloxy]-succinimide (sGMBS) ester;

b) contacting the carrier protein with a heterobifunctional cross-linker containing an NHS ester, preferably N-succinimidyl-S-acetylthioacetate (SATA), to form a carrier-SATA complex;

c) bringing the sGMBS-cytokine complex obtained in step (a) into contact with the carrier-SATA complex obtained in step (b).

According to one embodiment of the present invention, in step a), the reaction buffer is in a liquid, preferably aqueous, solution.

According to one embodiment of the present invention, in step a), the reaction buffer has a pH ranging from about 6 to about 8, preferably ranging from about 6.5 to about 7.5, more preferably about pH=7.2.

According to one embodiment of the present invention, in step a), the cytokine is present in the solution at a concentration ranging from about 0.1 to about 10 mg/ml, preferably from about 0.5 to about 5 mg/ml, more preferably about 1 mg /ml.

According to one embodiment of the present invention, in step a), a heterobifunctional cross-linker containing an NHS ester, preferably sGMBS, is prepared in the reaction buffer at a concentration ranging from 1 mM to 100 mM, preferably from 5 mM to 50 mM and more preferably 10 mm.

According to one embodiment of the present invention, in step a), IL-4 and a heterobifunctional NHS ester-containing cross-linker, preferably sGMBS, are mixed in a molar ratio of IL-4:heterobifunctional NHS ester-containing cross-linker, preferably sGMBS, varying from about 1:120 to about 1:1, preferably from about 1:50 to about 1:10.

According to one embodiment of the present invention, in step a), IL-13 and a heterobifunctional cross-linker containing an NHS ester, preferably sGMBS, are mixed in a molar ratio of IL-13:heterobifunctional cross-linker containing an NHS ester, preferably sGMBS, varying from about 1:120 to about 1:1, preferably from about 1:50 to about 1:10.

According to one embodiment of the present invention, in step a), at least one cytokine is incubated with a heterobifunctional cross-linker containing an NHS ester, preferably sGMBS, for a period ranging from about 30 minutes to about 120 minutes, preferably from about 45 to about 90 minutes and more preferably for at least 60 minutes.

According to one embodiment of the present invention, in step a), the step of contacting the at least one cytokine with a heterobifunctional cross-linker containing an NHS ester, preferably sGMBS, is performed at a temperature ranging from about 15°C to about 35°C, preferably from about 18°C to about 27°C.

According to one embodiment of the present invention, after step a), small compounds having a molecular weight of less than about 10 kDa, less than about 5 kDa, or less than about 3 kDa that are present in the reaction mixture are removed. These small compounds comprise primarily excess heterobifunctional cross-linker containing NHS ester (and by-products associated with NHS ester hydrolysis), preferably sGMBS, and excess molecules that have not reacted. Such removal can be accomplished using methods well known in the art.

According to one embodiment of the present invention, at the end of step a), the protein content is determined using the Bradford assay or any method well known in the art.

According to one embodiment of the present invention, in step b), the reaction buffer is in a liquid, preferably aqueous, solution.

According to one embodiment of the present invention, in step b), the reaction buffer has a pH ranging from about 6 to about 8, preferably ranging from about 6.5 to about 7.5, more preferably about pH=7.2.

According to one embodiment of the present invention, in step b), CRM ₁₉₇ is present in solution at a concentration ranging from about 0.2 to about 20 mg/ml, preferably from about 1 to about 10 mg/ml, more preferably about 2 mg/ml. ml.

According to one embodiment of the present invention, in step b), a heterobifunctional cross-linker containing an NHS ester, preferably SATA, is present in solution, preferably in DMSO, at a concentration ranging from 20 mM to about 500 mM, preferably from about 50 mM to about 200 mM and more preferably at a concentration of about 100 mM.

According to one embodiment of the present invention, in step b), CRM ₁₉₇ and a heterobifunctional cross-linker containing an NHS ester, preferably SATA, are mixed in a molar ratio of carrier:heterobifunctional cross-linker containing an NHS ester, preferably SATA, ranging from about 1: 320 to approximately 1:10.

According to one embodiment of the present invention, in step b), CRM ₁₉₇ is incubated with a heterobifunctional crosslinker containing an NHS ester, preferably SATA, for a period of time ranging from about 10 minutes to about 60 minutes, preferably from about 15 minutes to about 45 minutes and most preferably within 30 minutes.

According to one embodiment of the present invention, contacting step b) is performed at a temperature ranging from about 15°C to about 35°C, preferably from about 18°C to about 27°C.

According to one embodiment of the present invention, after step b), small compounds having a molecular weight of less than about 10 kDa, less than about 5 kDa, or less than about 3 kDa that are present in the reaction mixture are removed. These small compounds comprise primarily excess heterobifunctional cross-linker containing NHS ester (and by-products associated with NHS ester hydrolysis), preferably SATA, DMSO, and excess molecules that have not reacted. Such removal can be accomplished using methods well known in the art.

According to one embodiment of the present invention, after step b), the complexes between CRM ₁₉₇ and a heterobifunctional crosslinker containing an NHS ester, preferably SATA, are deprotected to convert the protected group (a heterobifunctional crosslinker containing an NHS ester, preferably SATA) to the functional group. In one embodiment of the present invention, said deprotection step is carried out after the step of removing small compounds having a molecular weight of less than about 10 kDa, less than about 5 kDa, or less than about 3 kDa that are present in the reaction mixture.

Examples of methods for deprotecting a molecule are well known in the art and include, but are not limited to, the use of hydroxylamine, the use of methoxylamine, or the use of a base (such as, for example, NaOH, KOH, K ₂ CO ₃ , MeONa, NH ₃ in methanol).

In one embodiment of the present invention, the deprotection step comprises adding a hydroxylamine solution to the reaction mixture, preferably at a final concentration ranging from about 10 mM to about 500 mM, preferably from about 20 mM to about 100 mM, more preferably about 50 mM.

In one embodiment of the present invention, the hydroxylamine solution is incubated with the reaction mixture for a period of time ranging from about 60 minutes to about 180 minutes, preferably from about 90 minutes to about 150 minutes, and more preferably for 120 minutes.

According to one embodiment of the present invention, a hydroxylamine solution is added at 50 mM over 120 minutes.

According to one embodiment of the present invention, the incubation of the hydroxylamine solution with the reaction mixture is performed at a temperature ranging from about 15°C to about 35°C, preferably from about 18°C to about 27°C.

According to one embodiment of the present invention, after the deprotection step, small compounds having a molecular weight of less than about 10 kDa, 5 kDa or 3 kDa that are present in the reaction mixture are removed. These small compounds include mainly excess hydroxylamine and possible residual SATA from the previous step. Such removal can be accomplished using methods well known in the art.

According to one embodiment of the present invention, at the end of step b), the protein content is determined using the Bradford assay or any method well known in the art.

Then, in step c) of the method according to the present invention, the end product of step a) is brought into contact with the end product of step b) thereby obtaining an immunogenic product according to the present invention.

According to one embodiment of the present invention, in step c), the end product of step a) containing IL-4, and the end product of step b) containing CRM ₁₉₇ are brought into contact in a molar ratio of IL-4:CRM ₁₉₇ varying from about 16:1 to about 1:2, preferably about 8:1 to about 2:1, more preferably about 4:1.

According to one embodiment of the present invention, in step c), the end product of step a) containing IL-13, and the end product of step b) containing CRM ₁₉₇ are contacted at a ratio of IL-13:CRM ₁₉₇ ranging from about 16 :1 to about 1:2, preferably from about 8:1 to about 2:1, more preferably about 4:1.

In another embodiment of step c), the end product of step a) containing IL-4, the end product of step a) containing IL-13, and the end product of step b) are contacted. In one embodiment of the present invention, said contacting step is conducted at a cytokine (i.e., IL-4 and IL-13):CRM ₁₉₇ molar ratio ranging from about 16:1 to about 1:2, preferably from about 8: 1 to about 2:1, more preferably about 4:1. In one embodiment of the present invention, said contacting step is carried out at an IL-4:CRM ₁₉₇ molar ratio ranging from about 8:1 to about 1:2, preferably about 4:1 to about 1:1, more preferably about 2 :1. In one embodiment of the present invention, said contacting step is carried out at a molar ratio of IL-13:CRM ₁₉₇ ranging from about 8:1 to about 1:2, preferably from about 4:1 to about 1:1, more preferably about 2 :1. In one embodiment of the present invention, said contacting step is carried out at a molar ratio of IL-4:IL-13 ranging from about 5:1 to about 1:5, preferably from about 2:1 to about 1:2, more preferably about 1:1.

According to one embodiment of the present invention, in step c), the reaction buffer is in a liquid, preferably aqueous, solution.

According to one embodiment of the present invention, in step c), the reaction buffer has a pH ranging from about 6 to about 8, preferably ranging from about 6.5 to about 7.5, more preferably about pH=7.2.

In one embodiment of step c), the contacting step is carried out for a period of time ranging from about 2 hours to about 26 hours, preferably from about 10 to 18 hours, more preferably from about 12 to about 18 hours.

According to one embodiment of the present invention, incubation step c) is carried out at a temperature ranging from about 2°C to 10°C, preferably from about 3°C to about 7°C, and more preferably at about 4°C.

According to one embodiment of the present invention, after step c), small compounds having a molecular weight of less than about 100 kDa, less than about 50 kDa, less than about 25 kDa, less than about 10 kDa, less than about 5 kDa, or less than about 3 kDa, which present in the reaction mixture. These small compounds include mostly excess molecules that have not reacted. Such removal can be accomplished using methods well known in the art.

According to one embodiment of the present invention, the immunogenic product obtained in step c) is concentrated. Concentration of the immunogenic product can be accomplished by one skilled in the art using any technique known in the art, such as, for example, a centrifugal ultrafiltration method, which may optionally be combined with sterile filtration.

According to one embodiment of the present invention, the immunogenic product obtained in step c), and optionally concentrated, is lyophilized.

The present invention also relates to an immunogenic product that can be obtained using the method according to the present invention.

The present invention also relates to a composition containing, essentially consisting of or consisting of at least one immunogenic product described herein above. According to one embodiment of the present invention, said composition may be referred to as an immunogenic composition.

The present invention also relates to a pharmaceutical composition containing, essentially consisting of or consisting of at least one immunogenic product described above and at least one pharmaceutically acceptable excipient.

Pharmaceutically acceptable excipients that can be used in the pharmaceutical composition of the present invention include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins such as, for example, human serum albumin, buffers such as, for example, , phosphates, glycine, sorbic acid, potassium sorbate, mixtures of partial glycerides of saturated vegetable aliphatic acids, water, salts or electrolytes, such as, for example, protamine sulfate, disodium hydrogen phosphate, monopotassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silicon dioxide , magnesium trisilicate, polyvinylpyrrolidone, cellulose-based substances (eg sodium carboxymethylcellulose), polyethylene glycol, polyacrylates, waxes, polyethylene-polyoxypropylene block polymers, polyethylene glycol and lanolin.

The present invention also relates to a medicinal product containing, essentially consisting of or consisting of at least one immunogenic product described herein above.

As used herein, the term “consisting essentially of,” when applied to a composition, pharmaceutical composition, or drug, means that at least one immunogenic product of the present invention is the sole therapeutic or biologically active agent in said composition, pharmaceutical composition or medicine.

According to one embodiment of the present invention, the composition, pharmaceutical composition or drug according to the present invention contains or essentially consists of an immunogenic product containing IL-4 associated with CRM ₁₉₇ .

According to one embodiment of the present invention, the composition, pharmaceutical composition or drug according to the present invention contains or essentially consists of an immunogenic product containing IL-13 associated with CRM ₁₉₇ .

According to one embodiment of the present invention, the composition, pharmaceutical composition or drug according to the present invention contains or essentially consists of an immunogenic product containing CRM ₁₉₇ associated with both IL-4 and IL-13.

According to one embodiment of the present invention, the composition, pharmaceutical composition or drug according to the present invention contains or essentially consists of a mixture of an immunogenic product containing IL-4 associated with CRM ₁₉₇ and an immunogenic product containing IL-13 associated with CRM ₁₉₇ . in a weight ratio ranging from about 10:1 to about 1:10, preferably in a weight ratio ranging from about 4:1 to 1:4.

According to one embodiment of the present invention, the composition, pharmaceutical composition or drug according to the present invention is a vaccine composition. According to one embodiment of the present invention, the vaccine composition of the present invention contains at least one adjuvant.

The present invention also relates to a composition, pharmaceutical composition, drug or vaccine according to the present invention, wherein said composition, pharmaceutical composition, drug or vaccine contains an adjuvant.

According to one embodiment of the present invention, the composition, pharmaceutical composition, drug or vaccine according to the present invention thus contains one or more adjuvants.

Suitable adjuvants that can be used in the present invention include, but are not limited to:

(1) aluminum salts (alum), such as, for example, aluminum hydroxide, aluminum phosphate, aluminum sulfate, etc.;

(2) oil-in-water emulsion formulations (with or without other specific immunostimulating agents, such as, for example, muramyl peptides (defined below) or bacterial cell wall components), such as, for example, squalene-based emulsions (e.g. squalene-based oil-in-water emulsions) or squalane-based emulsions such as e.g.

(a) MF59 (an oil-in-water squalene adjuvant described in PCT Publication No. WO 90/14837) containing 5% squalene, 0.5% Tween 80 and 0.5% Span 85 (optionally containing various amounts of MTP-PE (see below, but optional)) made into submicron particles using a microfluidizer such as the Model 110Y Microfluidizer (Microfluidics, Newton, MA, USA),

(b) SAF containing 10% squalene, 0.4% Tween 80, 5% pluronic capped polymer L121 and thr-MDP (see below), either microfluidized to produce a submicron emulsion or shaken to produce a larger particle size emulsion , And

(c) Ribi™ adjuvant system (RAS), (Corixa, Hamilton, Montana, USA), containing 2% squalene, 0.2% Tween 80 and one or more bacterial cell wall components from the group consisting of 3-O-deacylated monophosphoryl lipid A (MPL™), described in US Pat. No. 4,912,094 (Corixa), trehalose dimycolate (TDM) and cell wall skeleton (CWS), preferably MPL+CWS (Detox™);

(d) a squalane-based adjuvant containing, but not limited to, the following composition: squalane 3.9%, w/v, sorbitan trioleate (0.47%, w/v) and polyoxyethylene (80), sorbitan monooleate (0.47%, w/v), dispersed in citrate buffer;

(3) water-in-oil emulsion formulations, such as, for example, ISA-51 or a squalene-based water-in-oil adjuvant (for example, ISA-720); Oily adjuvants suitable for use in water-in-oil emulsions may include mineral oils and/or metabolizable oils. Mineral oils can be selected from Bayol ^® , Marcol ^® and Drakeol, including Drakeol ^® 6VR (SEPPIC, France). Metabolizable oils may be selected from SP oil (described herein below), Emulsigen (MPV Laboratories, Ralston, New Zealand), Montanide 264,266,26 (Seppic SA, Paris, France), as well as vegetable oils, animal oils such as fish oils squalane and squalene, and tocopherol and its derivatives.

(4) saponin adjuvants such as Quil A or STIMULON™ can be used QS-21 (Antigenics, Framingham, MA, USA) (US Patent No. 5,057,540), or particles derived therefrom, such as ISCOM (immune stimulating complexes);

(5) bacterial lipopolysaccharides, synthetic analogs of lipid A, such as aminoalkylglucosamine phosphate compounds (AGP), or derivatives or analogs thereof, which are available from Corixa and which are described in US Pat. No. 6,113,918; one of the AGPs is 2-[(R)-3-tetradecanoyloxytetradecanoylamino]ethyl 2-deoxy-4-O-phosphono-3-Oi[(R)-3-tetradecanoyloxytetradecanoyl]-2-[(R)-3-tetradecanoyloxytetradecanoyl ]-b-D-glucopyranoside, also known as 529 (formerly known as RC529), which is manufactured in aqueous form or as a stable emulsion, synthetic polynucleotides such as oligonucleotides containing CpG motif(s) (U.S. Patent No. 6,207,646);

(6) cytokines such as interleukins (e.g. IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12, IL-15, IL-18, etc. .), interferons (eg, interferon gamma), granulocyte-macrophage colony-stimulating factor (GM-CSF), macrophage colony-stimulating factor (M-CSF), tumor necrosis factor (TNF), co-stimulatory molecules B7-1 and B7-2, etc. d.;

(7) detoxified mutants of a bacterial ADP-ribosylating toxin, such as cholera toxin (CT), either in wild type or in a mutant form, for example in which glutamic acid at amino acid position 29 is replaced by another amino acid, preferably histidine, according to published by international patent application WO 00/18434 (see also WO 02/098368 and WO 02/098369), pertussis toxin (PT) or heat labile E. coli toxin (LT), in particular LT-K63, LT-R72, CT-S109, PT-K9/G129 (see, for example, WO 93/13302 and WO92/19265); And

(8) other substances that act as immunostimulating agents to enhance the effectiveness of the composition. Muramyl peptides include, but are not limited to, N-acetylmuramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetylnormuramyl-L-alanine-2-(1'-2'-dipalmitoyl-sn-glycero-3 -hydroxyphosphoryloxy)-ethylamine (MTP-PE), etc.

The adjuvant used may depend, in part, on the recipient organism. Moreover, the amount of adjuvant to be administered will depend on the type and size of the animal.

According to one embodiment of the present invention, the composition, pharmaceutical composition, drug or vaccine composition of the present invention is (or contains) an emulsion further containing one or more surfactants and optionally at least one adjuvant described herein above. According to one embodiment of the present invention, the emulsion is a water-in-oil emulsion or an oil-in-water emulsion.

Examples of surfactants that can be used in the present invention are well known in the art and include, but are not limited to, mannide monooleate such as ^Montanide® 80, commercially available from Arlacel (SEPPIC, France), Tween 20, Tween 80, span 85, triton X-100.

According to one embodiment of the present invention, the composition, pharmaceutical composition, drug, vaccine composition of the present invention contains a therapeutically effective amount of at least one immunogenic product of the present invention.

According to one embodiment of the present invention and for storage purposes, the immunogenic product or composition, pharmaceutical composition, drug, vaccine composition or emulsion according to the present invention is lyophilized.

According to one embodiment of the present invention, the composition, pharmaceutical composition, drug, vaccine composition or emulsion according to the present invention can thus be provided in freeze-dried (lyophilized) form. According to this embodiment, the immunogenic product of the present invention is combined with one or more lyophilization auxiliaries. Various lyophilization auxiliaries are well known to one skilled in the art and include, but are not limited to, sugars such as lactose and mannitol.

According to one embodiment of the present invention, the composition, pharmaceutical composition, drug, vaccine composition or emulsion of the present invention may be mixed with stabilizers, for example, to protect proteins prone to degradation from degradation, to increase the shelf life of an immunogenic product, or to improve the efficiency of freeze-drying . Suitable stabilizers include, but are not limited to, SPGA, carbohydrates (such as sorbitol, mannitol, trehalose, starch, sucrose, dextran or glucose), proteins (such as, for example, albumin or casein, or breakdown products thereof), mixtures of amino acids, such as, for example, lysine or glycine, and buffers, such as, for example, alkali metal phosphates.

In one embodiment of the present invention, the immunogenic product, composition, pharmaceutical composition, vaccine composition, or emulsion of the present invention may be administered by injection, topically (eg, by transdermal delivery), rectally, nasally, or vaginally.

According to one embodiment of the present invention, the immunogenic product, composition, pharmaceutical composition, drug, vaccine composition or emulsion according to the present invention is in an adapted form for injection. Thus, according to one embodiment of the present invention, the immunogenic product, composition, pharmaceutical composition, drug or vaccine composition of the present invention must be injected into a subject by intramuscular, intraperitoneal or subcutaneous injection.

Examples of forms suitable for injection use include, but are not limited to, sterile solutions or dispersions and sterile powders for the preparation of sterile injectable solutions or dispersions for immediate administration. Prevention of microbial contamination can be achieved by adding preservatives to the composition, such as, for example, various antibacterial and antifungal agents (eg, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like). In one embodiment of the present invention, it may be advantageous to include isotonic agents, such as sugars or sodium chloride, to reduce pain during injection. According to one embodiment of the present invention, prolonged absorption of injectable compositions can be achieved through the use of absorption delaying agents in the compositions, for example, aluminum monostearate and gelatin.

According to one embodiment of the present invention, the lyophilized vaccine composition of the present invention is dissolved in water for injection and mixed gently; then add the immunoadjuvant described in this application above; The mixture is carefully mixed and added to a suitable syringe. Thus, the present invention also relates to a medical device, including a syringe, filled or pre-filled with a vaccine composition according to the present invention.

According to one embodiment of the present invention, the immunogenic product, composition, pharmaceutical composition, drug or vaccine composition of the present invention is in an adapted form for topical administration. Examples of forms adapted for topical administration include, but are not limited to, polymer patch or controlled release patch, and the like.

According to another embodiment of the present invention, the immunogenic product, composition, pharmaceutical composition, drug, vaccine composition or emulsion according to the present invention is in an adapted form for rectal administration. Examples of forms adapted for rectal administration include, but are not limited to, suppository, microenemas, enemas, gel, rectal foam, cream, ointment, etc.

The present invention also relates to a medical device, which is a syringe filled or pre-filled with a composition, pharmaceutical composition, drug or vaccine composition according to the present invention.

According to one embodiment of the present invention, the syringe is a two-chamber syringe, in which one chamber contains a solution with an immunogenic product according to the present invention, and the other chamber contains an adjuvant.

The present invention also relates to a medical device containing a vial pre-filled with an immunogenic product according to the present invention or a composition, pharmaceutical composition, drug or vaccine composition according to the present invention.

The present invention also relates to an immunogenic product, composition, pharmaceutical composition, drug or vaccine composition according to the present invention for treating an inflammatory disorder in a subject.

Thus, the present invention also provides a method for treating an inflammatory disorder in a subject, comprising administering to the subject an immunogenic product, composition, pharmaceutical composition, drug, vaccine composition or emulsion according to the present invention.

The present invention also provides a method of inducing an immune response against IL-4, IL-13, or both in a subject, comprising administering to the subject an immunogenic product, composition, pharmaceutical composition, drug or vaccine composition according to the present invention.

The present invention also provides a method of inducing in a subject the production of antibodies that inhibit the biological activity or neutralize the biological activity of IL-4, IL-13, or both, comprising administering to the subject an immunogenic product, composition, pharmaceutical composition, drug or vaccine composition according to the present invention . In one embodiment of the present invention, the antibodies are polyclonal antibodies.

According to one embodiment of the present invention, the subject is affected, preferably diagnosed with an inflammatory disorder, in particular, a disorder associated with aberrant expression or activity of IL-4 and/or IL-13.

According to one embodiment of the present invention, the subject is a human. Preferably, according to this embodiment, at least one cytokine contained in the immunogenic product of the present invention is human.

In one embodiment of the present invention, the subject is a non-human mammal (such as, for example, a pet). Preferably, in accordance with this embodiment, at least one cytokine contained in the immunogenic product of the present invention is derived from said non-human mammal.

According to one embodiment of the present invention, the inflammatory disorder is a disorder associated with aberrant expression or activity of IL-4 and/or IL-13.

Examples of an inflammatory disorder include, but are not limited to, asthma (both allergic and non-allergic), allergic conditions (such as, for example, various types of food allergies, poison allergies, cat allergies, drug allergies, hyper-IgE syndrome, allergic rhinitis, allergic conjunctivitis and allergic enterogastritis), atopic disorders (such as, for example, atopic dermatitis, urticaria (including chronic idiopathic urticaria and chronic spontaneous urticaria), eczema), bullous pemphigoid, respiratory disorders (such as allergic and non-allergic asthma, chronic obstructive pulmonary disease (COPD), nasal polyposis and other conditions involving airway inflammation (such as, for example, eosinophilia, fibrosis and excess mucus production, including cystic fibrosis and pulmonary fibrosis, systemic sclerosis (SSc)); inflammatory and/or autoimmune disorders or conditions, gastrointestinal disorders or conditions (such as, for example, inflammatory bowel diseases (IBD) and eosinophilic esophagitis (EE), as well as eosinophil-mediated gastrointestinal diseases, ulcerative colitis, Crohn's disease and systemic lupus erythematosus); systemic lupus erythematosus, liver disorders or conditions (such as, for example, cirrhosis and hepatocellular carcinoma), scleroderma; fibrotic diseases or disorders (such as, for example, liver fibrosis (such as, for example, fibrosis caused by hepatitis B and/or C virus)), scleroderma; solid tumors or various types of cancer, such as leukemia (such as, for example, chronic B-cell lymphocytic leukemia), glioblastoma, lymphoma (such as, for example, Hodgkin's lymphoma) and mastocytosis.

In one embodiment of the present invention, the inflammatory disorder is selected from the group consisting of asthma (eg, allergic asthma), atopic dermatitis, chronic obstructive pulmonary disease (COPD), pulmonary fibrosis, food allergy, nasal polyposis, and eosinophilic esophagitis.

In one embodiment of the present invention, the inflammatory disorder is selected from the group consisting of asthma (eg, allergic asthma), atopic dermatitis, chronic obstructive pulmonary disease (COPD), pulmonary fibrosis, and food allergy.

According to one embodiment of the present invention, the inflammatory disorder is an allergy, asthma or atopic dermatitis.

According to one embodiment of the present invention, the inflammatory disorder is allergic asthma.

According to one embodiment of the present invention, the inflammatory disorder is a solid tumor. According to one embodiment of the present invention, the method of the present invention is intended to prevent metastases from a solid tumor.

The present invention also provides a method for inducing desensitization of a subject allergic to a specific antigen, the method comprising administering to the subject an immunogenic product, composition, pharmaceutical composition, drug or vaccine composition according to the present invention and said allergen.

As used herein, the term “desensitization,” also known as allergen immunotherapy, desensitization or hyposensitization, or allergy vaccination, refers to the drug treatment of various types of environmental allergies, such as allergic asthma. Such treatment involves exposing people to increasing amounts of the allergen to reduce the immune system's response in the presence of the allergen.

Examples of allergens include, but are not limited to, inhaled allergens, ingested allergens, and contact allergens.

Examples of inhaled allergens include, but are not limited to, allergens from Astigmata (eg, Acarus siro (barn mite, Aca s 13), Blomia tropicalis (mite, Blo t), Dermatophagoides farinae (American house dust mite, Der f), Dermatophagoides microceras (house dust mite, Der m), Dermatophagoides pteronyssinus (European house dust mite, Der p), Euroglyphus maynei (house dust mite, Eur m), Glycyphagus domesticus (barn mite, Gly d 2), Lepidoglyphus destructor (barn mite, Lep d), Tyrophagus putrescentiae (barn mite, Tyr p); Blattaria (e.g. Blattella germanica (German cockroach, Bla g), Periplaneta americana (American cockroach, Per a); Coleoptera (e.g. Harmonia axyridis (Asian ladybug, Har a )), Diptera (e.g. Aedes aegypti (yellow fever mosquito, Aed a)), Chironomus kiiensis (flying mosquito, Chi k), Chironomus thummi thummi (flying mosquito, Chi t), Forcipomyia taiwana (biting midge, For t) , Glossina morsitans (savannah tsetse fly, Glo m), Hemidiptera: Triatoma protracta (California cone bug, Tria p), Hymenoptera (e.g. Apis cerana (eastern honey bee, Api c), Apis dorsata (giant honey bee, Api d), Apis mellifera (honeybee, Api m)), Bombus pennsylvanicus (bumblebee, Bom p), Bombus terrestris (bumblebee, Bom t), Dolichovespula arenaria (yellow hornet, Dol a), Dolichovespula maculata (spotted wasp, Dol m ), Myrmecia pilosula (Australian bulldog ant, Myr p), Polistes annularis (wasp, Pol a), Polistes dominulus (Mediterranean paper wasp, Pol d), Polistes exclamans (wasp, Pol e), Polistes fuscatus (wasp, Pol f ), Polistes gallicus (wasp, Pol g), Polistes metricus (wasp, Pol m), Polybia paulista (wasp, Pol p), Polybia scutellaris (wasp, Pol s), Solenopsis geminata (tropical fire ant, Sol g), Solenopsis invicta (introduced red fire ant, Sol i), Solenopsis richteri (black fire ant, Sol r), Solenopsis saevissima (Brazilian fire ant, Sol s), Vespa crabro (European hornet, Vesp c), Vespa mandarinia (Asian giant hornet, Vesp m), Vespula fiavopilosa (vesp f), Vespula germanica (vesp g), Vespula maculifrons (vesp m), Vespula pensylvanica (vesp p), Vespula squamosa (vesp, Vesp s), Vespula vidua (wasp, Vesp vi), Vespula vulgaris (lambug wasp, Vesp v), Ixodida (e.g. Argas reflexus (pigeon mite, Arg r), Lepidoptera (e.g. Bombyx niori (silkworm, Bomb n) ), Plodia interpunctella (Indian meal moth, Plo i), Thaumetopoea pityocampa (pine silkworm, Tha p), Thysanura (e.g. Lepisma saccharina (common silverfish, Lep s)), Siphonaptera (e.g. Ctenocephalides felis felis (cat flea, Cte f)), Carnivora (e.g. Canis familiaris (dog, Can f)), Felis domesticus (cat, Fel d); Lagomorpha (e.g. Oryctolagus cuniculus (rabbit, Ory c)), Perissodactlyla: Equus caballus (domestic horse, Equ c), Pleuronectiformes (e.g. Lepidorhombus whiffiagonis (megrim, citaricht, gallo, Lep w)), Rodentia (e.g. Cavia porcellus (guinea pig, Cav p)), Mus musculus (mouse, Mus m), Rattus norvegius (rat, Rat n)); Coniferales: Chamaecyparis obtusa (Japanese cypress, Cha o), Cupressus arizonica (cypress, Cup a), Cryptomeria japonica (sugi, Cry j), Cupressus sempervirens (common cypress, Cup s), Juniperus ashei (Mexican juniper, Jun a), Juniperus oxycedrus (red juniper, Jun o), Juniperus sabinoides (Mexican juniper, Jun s), Juniperus virginiana (Virginian juniper, Jun v); Gentianales (eg Catharanthus roseus (pink periwinkle, Cat r)); Poales (e.g. Anthoxanthum odoratum (scented spikelet, Ant o 1)), Cynodon dactylon (sweetroot, Cyn d 1, Cyn d 7, Cyn d 12, Cyn d 15, Cyn d 22w, Cyn d 23, Cyn d 24) , Dactylis glomerata (common hedgehog, Dae g 1, Dae g 2, Dae g 3, Dae g 4, Dae g 5), Festuca pratensis (meadow fescue, Fes p 4), Holcus lanatus (woolly broom, Hol l 1, Hol l 5), Hordeum vulgare (barley, Hor v 1, Hor v 5, Hor v 12, Hor v 15, Hor v 16, Hor v 17, Hor v 21), Lolium perenne (perennial ryegrass, Lol p 1, Lol p 2, Lol p 3, Lol p 4, Lol p 5, Lol p 11), Oryza sativa (rice, Ory s 1, Ory s 12), Paspalum notarum (buckwheat, Pas n 1), Phalaris aquatica (water canary , Pha a 1, Pha a 5), Phleum pratense (timothy, Phl p 1, Phl p 2, Phl p 4, Phl p 5, Phl p 6, Phl p 7, Phl p 11, Phl p 12, Phl p 13 ), Poa pratensis (meadow bluegrass, Poa p 1, Poa p 5), Secale cereale (rye, Sec c 1, Sec c 20), Sorghum halepense (Aleppo sorghum, Sor h 1), Triticum aestivum (wheat, Tri a 12 , Tri a 14, Tri a 185, Tri a 19, Tri a 25, Tri a 26, Tri a 27, Tri a 28, Tri a 29, Tri a 30), Zea mays (maize, Zea m 1, Zea m 12 , Zea m 14, Zea m 25), Fagales: Alnus glutinosa (alder, Aln g 1, Aln g 4), Betula verrucosa (silver birch, Bet v 1, Bet v 2, Bet v 3, Bet v 4, Bet v 5, Bet v 6, Bet v 7), Carpinus betuhxs (hornbeam, Car b 1); Lamiales (e.g. Fraxinus excelsior (common ash, Fra e l)), Ligustrum vulgare (common privet, Lig v), Syringa vulgaris (common lilac, Syr v); Malpighiales (for example, Hevea brasiliensis (latex), Hev b 1, Hev b 2, Hev b 3, Hev b 4, Hev b 5, Hev b 6, Hev b 7, Hev b 8, Hev b 9, Hev b 10, Hev b 11, Hev b 12, Hev b 13)); Proteales (e.g. Platanus acerifolia (Pla a 1, Pla a 2, Pla a 3), Platanus orientalis (Pla or 1, Pla or 2, Pla or 3)).

In one embodiment, the inhaled allergen is selected from the group consisting of or consisting of Acarus siro (barn mite, Aca s 13), Dermatophagoides farinae (American house dust mite, Der f), Dermatophagoides microceras (house dust mite, Der m), Dermatophagoides pteronyssinus (European house dust mite, Der p), Euroglyphus maynei (house dust mite, Eur m), Glycyphagus domesticus (barn mite, Gly d 2), Polistes annularis (wasp, Pol a), Polistes dominulus (Mediterranean paper wasp, Pol d ), Polistes exclamans (wasp, Pol e), Polistes fuscatus (wasp, Pol f), Polistes gallicus (wasp, Pol g), Polistes metricus (wasp, Pol m), Polybia paulista (wasp, Pol p), Polybia scutellaris ( wasp, Pol s), Felis domesticus (cat, Fel d), Poales and Betula verrucosa (silver birch, Bet v 1, Bet v 2, Bet v 3, Bet v 4, Bet v 5, Bet v 6, Bet v 7 ).

Examples of ingested allergens include, but are not limited to, allergens from Ascomycota fungi such as, for example, Dothideales (e.g., Alternaria alternata (Alt a), Cladosporium cladosporioides (Cla c), Cladosporium herbarum (Cla h), Curvularia lunata (Cur l), - Eurotiales: Aspergillus flavus (Asp fl), Aspergillus fumigatus (Asp f), Aspergillus niger (Asp n), Aspergillus oryzae (Asp o), Penicillium brevicompactum (Pen b), Penicillium chrysogenum (Pen ch) , Penicillium citrinum (Pen c), Penicillium oxalicum (Pen o), Hypocreales (e.g. Fusarium culmorum (Fus c)); Onygenales (e.g. Trichophyton rubrum (Tri r), Trichophyton tonsurans (Tri t)), Saccharomycetales: Candida albicans (yeast, Cand a), Candida boidinii (yeast, Cand b); Tuberculariales (e.g. Epicoccum purpurascens (Epi p)), allergens from basidiomycota such as e.g. Hymenomycetes (e.g. Coprinus comatus (white dung beetle) , Cop c)), Psilocybe cubensis (hallucinogenic mushroom, Psi c), Urediniomycetes (e.g. Rhodotorula mucilaginosa (yeast, Rho m)); Ustilaginomycetes (for example, Malassezia furfur (infective agent pityriasis versicolor, Mala f), Malassezia sympodialis (Mala s)); antibiotics (such as, for example, penicillins, cephalosporins, aminosides, quinolones, macrolides, tetracycline, sulfonamides); medicines (such as, for example, acetylsalicylic acid, vaccines, morphines and derivatives); vitamins, such as vitamin K1; and food allergens (such as, for example, milk, egg, peanut, tree nut (walnut, cashew, etc.), fish, shellfish, soy, wheat, as well as carrot, apple, pear, avocado, apricot , peach).

According to one embodiment of the present invention, the ingested allergen is a food allergen.

In one embodiment of the present invention, the food allergen is selected from the group consisting of or consisting of milk, egg, peanut, tree nut (walnut, cashew, etc.), fish, shellfish, soy, wheat, and carrot allergen, apple, pear, avocado, apricot, peach.

Examples of contact allergens include, but are not limited to, heavy metals (such as, for example, nickel, chromium, gold), latex, haptens such as, for example, halothane, hydralazine.

In one embodiment of the present invention, the allergen is selected from the group consisting of or consisting of Acarus siro (barn mite, Aca s 13), Dermatophagoides farinae (American house dust mite, Der f), Dermatophagoides microceras (house dust mite, Der m), Dermatophagoides pteronyssinus (European house dust mite, Der p), Euroglyphus maynei (house dust mite, Eur m), Glycyphagus domesticus (barn mite, Gly d 2), Polistes annularis (wasp, Pol a), Polistes dominulus (Mediterranean paper wasp, Pol d), Polistes exclamans (wasp, Pol e), Polistes fuscatus (wasp, Pol f), Polistes gallicus (wasp, Pol g), Polistes metricus (wasp, Pol m), Polybia paulista (wasp, Pol p), Polybia scutellaris (wasp, Pol s), Felis domesticus (cat, Fel d), Poales and Betula verrucosa (silver birch, Bet v 1, Bet v 2, Bet v 3, Bet v 4, Bet v 5, Bet v 6, Bet v 7) and food allergens.

The present invention also provides a method for increasing the effectiveness and/or reducing the duration of desensitization of a subject allergic to a specific allergen, wherein the subject is treated by desensitization and further administered an immunogenic product, composition, pharmaceutical composition, drug or vaccine composition according to the present invention.

According to one embodiment of the present invention, in the methods of the present invention, the subject is first administered an immunogenic product, composition, pharmaceutical composition, drug or vaccine composition according to the present invention, and then the allergen.

According to one embodiment of the present invention, in the methods of the present invention, the subject is administered first an allergen and then an immunogenic product, composition, pharmaceutical composition, drug or vaccine composition according to the present invention.

According to another embodiment of the present invention, in the method of the present invention, the subject receives a combined administration of an immunogenic product, composition, pharmaceutical composition, drug or vaccine composition of the present invention and an allergen.

The present invention also relates to a composition, pharmaceutical composition, drug or vaccine as described herein above, wherein said composition, pharmaceutical composition, drug or vaccine also contains at least one allergen.

In one embodiment of the present invention, a therapeutically effective amount of at least one immunogenic product of the present invention is or is to be administered to a subject. According to one embodiment of the present invention, the therapeutically effective amount corresponds to the amount of total proteins determined using the Bradford protein assay, well known in the art.

According to one embodiment of the present invention, the amount of the immunogenic product administered to a subject induces an immunoprotective response without significant undesirable effects.

According to one embodiment of the present invention, the amount of the immunogenic product administered to a subject induces desensitization to the allergen without significant adverse effects.

Optimal amounts of components for the immunogenic product of the present invention can be determined through standard studies involving observation of appropriate immune responses in subjects. After initial vaccination, subjects may receive one or more booster immunizations at appropriate intervals.

According to one embodiment of the present invention, the treatment consists of a single dose or multiple doses over a period of time.

According to one embodiment of the present invention, the subject to be treated is administered at least twice a month with a therapeutically effective amount of the immunogenic product described hereinabove.

According to another embodiment of the present invention, the subject to be treated is administered twice within 1 month with a therapeutically effective amount of the immunogenic product of the present invention. In this embodiment, the subject may be administered once on day 0 and a second time between day 7 and day 28. In one embodiment of the present invention, the subject may be administered once on day 0 and a second time on day 28.

According to another embodiment of the present invention, the subject to be treated is administered three times within 1 month with a therapeutically effective amount of the immunogenic product of the present invention. In this embodiment, the subject to be treated may be administered once on day 0, a second time between day 7 and day 14, and a third time between day 21 and day 28. In one embodiment of the present invention, the subject is administered once on day 0 , the second time on day 7 and the third time on day 28.

According to another embodiment of the present invention, the subject to be treated may be further administered once every three months with a therapeutically effective amount of the immunogenic product of the present invention.

According to one embodiment of the present invention, a therapeutically effective amount of the immunogenic product of the present invention can be administered to the subject to be treated three times within one month, as described herein above, and then additionally once every three months.

In another embodiment of the present invention, a therapeutically effective amount of the immunogenic product described hereinabove may be further administered to the subject to be treated when the amount of anti-IL-4 antibody is undetectable in a serum sample obtained from the subject.

In another embodiment of the present invention, a therapeutically effective amount of the immunogenic product described hereinabove may be further administered to the subject being treated when the amount of anti-IL-13 antibody is undetectable in a serum sample obtained from the subject.

In another embodiment of the present invention, a therapeutically effective amount of the immunogenic product described hereinabove may be further administered to the subject to be treated when the amount of anti-IL-4 and anti-IL-13 antibodies is undetectable in a serum sample obtained from the subject.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a diagram of the chemical conjugation of sGMBS-modified IL-4 or IL-13 and a SATA-tagged carrier protein by addition of a thiol-maleimide to produce the immunogenic products of the present invention.

Figure 2 is a set of graphs showing the antigenicity of muIL-4 and muIL-13 immunogenic products. Capture was performed using anti-CRM ₁₉₇ antibody (A, C) or anti-KLH antibody (B, D), and detection was performed using biotinylated polyclonal anti-muIL-4 antibody (A, B) or biotinylated polyclonal anti-muIL-13 antibody (C , D). The OD values shown are the average OD values of two replicates.

Figure 3 is a study immunization schedule in Balb/c mice. Four immunizations on days 0, 7, 28, and 49 of the immunogenic product (with CRM ₁₉₇ or KLH as carrier protein), unconjugated muIL-4 or muIL-13 + CRM ₁₉₇ , and CRM ₁₉₇ and PBS alone, all emulsified in an adjuvant-based squalene and administered by intramuscular (IM) injection to 10 mice per group, with the exception of a control group of only 5 mice. Blood samples were taken before dosing and on day 39 and day 120.

Figure 4 is a set of graphs showing antibody titers to muIL-4 (A), muIL-13 (B), CRM ₁₉₇ (C), and KLH (D) in mouse sera. Ten mice per group were used in the immunogenic product groups and 5 mice were used in the control groups. The bars represent the median.

Figure 5 is a graph showing muIL-4 neutralizing ability in mouse sera. Ten mice per group were used in the immunogenic product groups and 5 mice were used in the control groups. The bars represent the median.

Figure 6 is a graph showing muIL-13 neutralizing ability in mouse sera. Ten mice per group were used in the immunogenic product groups and 5 mice were used in the control groups. The bars represent the median.

Figure 7 is a study schedule of immunizations and asthma sensitization in Balb/c mice.

Figure 8 is a set of graphs showing titers of anti-muIL-4 antibodies (A) and anti-muIL-13 antibodies (B) in mouse sera. Twelve mice per group. The bars represent the median.

Figure 9 is a set of graphs showing muIL-4 (A) and muIL-13 (B) neutralizing capacity in mouse sera. Twelve mice per group. The bars represent the median.

Figure 10 is a graph showing airway hypersensitivity to inhaled methacholine. PenH (enhanced pause) index values were measured using noninvasive whole-body plethysmography. Data were obtained using 7-8 mice per group for HDM-sensitized/stimulated mice and n = 16 for PBS controls. * or ** or ***: P < 0.5 or 0.1 or 0.001 compared with the CRM197-HDM group using the unpaired Mann-Whitney U test.

Figure 11 is a set of graphs showing 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/stimulated mice and n = 16 for PBS controls. ***: P < 0.001 compared with CRM ₁₉₇ -HDM group, using unpaired Mann-Whitney U test.

Figure 12 is a set of graphs showing the levels of CD45 ⁺ cells (A) and eosinophils (B) in BAL fluid collected 24 hours after the last HDM stimulation. Data were obtained using 7-8 mice per group for HDM-sensitized/stimulated mice and n = 16 for PBS controls. * or ** or ***: P < 0.5 or 0.1 or 0.001 compared with the CRM ₁₉₇ -HDM group, using the unpaired Mann-Whitney U test.

Figure 13 is a set of graphs showing the antigenicity of the IL-4 immunogenic product (A), the IL-13 immunogenic product (B), and the Combo immunogenic product (A and B). Capture was performed using anti-CRM ₁₉₇ antibody, and detection was performed using biotinylated polyclonal anti-huIL-4 antibody (A) or biotinylated polyclonal anti-huIL-13 antibody (B). The OD values shown are the average OD values of two replicates.

Figure 14 is a study immunization schedule in Balb/c mice.

Figure 15 is a set of graphs showing titers of anti-IL-4 antibodies (A) and anti-IL-13 antibodies (B) in mouse sera. Ten mice per group. The bars represent the median.

Figure 16 is a set of graphs showing the neutralizing ability of anti-IL-4 antibodies (A) and anti-IL-13 antibodies (B) in mouse sera. Ten mice per group. The bars represent the median.

EXAMPLES

The present invention is further illustrated by the following examples.

The present invention relates to an immunogenic product using CRM ₁₉₇ as a carrier protein. The properties of the immunogenic product according to the present invention are illustrated by the following examples. In addition, the product of the present invention was compared with an immunogenic product prepared with KLH to distinguish the present invention from the prior art and to show its superiority over the prior art.

CRM ₁₉₇ is a nontoxic form of diphtheria toxin that lacks toxic activity due to a single base substitution in its toxin domain from a glycine to a glutamic acid residue at position 52 (Uchida et al. 1973 J Biol Chem). As an alternative, snail limpet hemocyanin (KLH), a copper-containing protein found in arthropods and molluscs (Swaminathan et al. 2014), was also tested and compared with CRM ₁₉₇ .

The immunogenic products of the present invention were prepared using a manufacturing method, the development of which is described below.

Thiol-maleimide conjugation is used to produce the immunogenic products IL-4 and IL-13. Sulfhydryl groups were introduced onto the CRM ₁₉₇ carrier protein using SATA and subsequent deprotection with hydroxylamine, while the cytokine muIL-4 or muIL-13 was derivatized using sGMBS, a maleimide-containing agent. Both SATA and sGMBS are heterobifunctional cross-linkers containing an NHS ester that reacts with primary amines (such as the ε-amino groups of lysine residues and protein N-termini). An overview of immunogenic product synthesis via thiol-maleimide conjugation is presented in Figure 1.

Example 1. Preparation of murine immunogenic products IL-4 and IL-13 according to the present invention with two carrier proteins

a) Functionalization of the carrier protein

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

Hydroxylamine was then diluted in the same buffer to 500 mM. CRM ₁₉₇ -SATA or KLH-SATA was then incubated with hydroxylamine solution at a final concentration of 50 mM for 2 hours on a rocking shaker at room temperature. Finally, the mixture was desalted and excess reagents were removed using a Zeba™ desalting spin column.

b) Functionalization of muIL-4 and muIL-13

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 to 10 mM. sGMBS was then added to muIL-4 or muIL-13 and after one hour of incubation on a shaker at room temperature, excess sGMBS was removed using a Zeba™ desalting spin column.

c) Conjugation

After functionalization of CRM ₁₉₇ , KLH, muIL-4 and muIL-13, the protein content of each drug was determined using the Bradford assay.

Functionalized CRM ₁₉₇ or functionalized KLH was added to functionalized muIL-4 or functionalized muIL-13 in a molar ratio of 1:2 (vehicle:muIL-4 or vehicle:muIL-13) and 1:20, respectively. The 1:20 ratio for KLH was chosen based on the difference in molecular weight between KLH and CRM ₁₉₇ (CRM ₁₉₇ ~58 kDa compared to the KLH subunit used in this manufacture, ~400 kDa) and based on previous experience with the immunogenic IFN product, vaccine that is currently being evaluated in a phase IIb clinical trial in lupus patients (NCT02665364), which allows mixing of similar amounts of cytokine and vehicle in all manufacturing methods.

Preparations of individual immunogenic products were incubated overnight at 4°C on a rocking shaker. The resulting immunogenic products were then concentrated using Amicon (3 kDa cut-off membrane), filtered through a 0.22 μm pore size filter and stored at 4°C.

d) Control (unconjugated cytokines and CRM ₁₉₇ )

As controls, two mixtures (called unconjugated cytokines and CRM ₁₉₇ ) were prepared without protein functionalization:

-muIL-4 and CRM ₁₉₇ were mixed in a molar ratio of 1:2 (CRM ₁₉₇ :cytokine). The mixture was prepared at a rate of 700 μg/ml without the use of any binding reagent.

-muIL-13 and CRM ₁₉₇ were mixed in a molar ratio of 1:2 (CRM ₁₉₇ :cytokine). The mixture was prepared at a rate of 700 μg/ml without the use of any binding reagent.

Both mixtures were filtered through a 0.22 μm filter and stored at 4°C.

e) Quantitative assessments of immunogenic products

Concentrations of muIL-4 immunogenic product and muIL-13 immunogenic product were determined using the Coomassie Plus protein assay (Bradford) according to the manufacturer's instructions.

Example 2: Antigenicity of Mouse Products

A sandwich ELISA was performed to assess the binding of the cytokine to the carrier protein, as well as to assess the retention of epitopes during the manufacturing method.

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

This test confirmed that the immunogenic products of the present invention contain muIL-4 or muIL-13 associated with CRM ₁₉₇ or KLH. In addition, these results confirm that the immunogenic products are antigenic (ie, recognized by anti-muIL-4 antibodies or anti-muIL-13 antibodies).

Example 3: Immunogenicity of immunogenic products

In mice, immunogenic products were administered as an emulsion with a squalene-based adjuvant. Immunogenic products were diluted in PBS to the target concentration, and the dilutions were mixed with an equal volume of adjuvant.

Mice immunization protocol

Each Balb/c mouse received four intramuscular (IM) injections of immunogenic products (with CRM ₁₉₇ or KLH) or controls such as PBS, unconjugated cytokines with CRM ₁₉₇ , or CRM ₁₉₇ alone, all emulsified (1:1) with adjuvant on based on squalene. Injections were performed on days 0, 7, 28 and 49, as detailed in Table 1 and Figure 3. The first immunization was performed at 7 weeks of age.

Table 1. Dose schedule

Product Number of mice per group Day Dose/injection (mcg) Adjuvant Immunogenic product muIL-4 (CRM ₁₉₇ ) 10 0/7/28/49 30/30/10/10 Squalene-based oil-in-water adjuvant Immunogenic product muIL-4 (KLH) 10 0/7/28/49 30/30/10/10 Immunogenic product muIL-13 (CRM ₁₉₇ ) 10 0/7/28/49 30/30/10/10 Immunogenic product muIL-13 (KLH) 10 0/7/28/49 30/30/10/10 Unconjugated muIL-4 + CRM ₁₉₇ 5 0/7/28/49 30/30/10/10 Unconjugated muIL-13 + CRM ₁₉₇ 5 0/7/28/49 30/30/10/10 CRM ₁₉₇ 5 0/7/28/49 30/30/10/10 DFSB 5 0/7/28/49 -

Blood sample collection was performed before dosing and on days 39, 60, and 120. Serum samples were prepared after coagulation at room temperature and centrifugation to remove clot. Mice were killed by lethal anesthesia on day 120.

Determination of titers of antibodies to cytokines and antibodies to carrier proteins using ELISA

Serum samples from immunized mice were assessed for the presence of cytokine antibodies and carrier protein antibodies using ELISA.

In general, 96-well plates were coated with muIL-4, muIL-13, CRM ₁₉₇ , or KLH. After blocking with casein, serum samples were added and serially diluted twofold. After incubation at 37°C, bound antibodies were detected using HRP-conjugated anti-mouse IgG, and plates were developed using OPD substrate. The reaction was stopped with sulfuric acid, and then the absorbance was recorded at 490 nm.

Positive controls used to determine the titers of anti-muIL-4 antibody and anti-muIL-13 antibody, anti-KLH antibody and anti-CRM ₁₉₇ antibody, respectively, were monoclonal rat IgG1 anti-muIL-4 antibody, monoclonal mouse anti-muIL13 antibody, pooled sera, collected from mice immunized with KLH and monoclonal mouse IgG1 to diphtheria toxin A.

Samples were analyzed starting from a dilution of 500 dilutions. ^-1 to 256000 rec. ^-1 , with the exception of sera before immunization, which were analyzed only at 500 dilutions. ^-1 .

Titers of anti-muIL-4 antibodies, anti-muIL-13 antibodies, anti-CRM ₁₉₇ antibodies, and anti-KLH antibodies were expressed as serum dilutions resulting in half the maximum OD.

The results are presented in Figure 4. In all groups treated with immunogenic products, as well as in mice injected with DPSB adjuvanted or carrier protein, or unconjugated drugs, no anti-muIL-4 antibodies, no anti-muIL-4 antibodies were detected before dosing. 13, antibodies to KLH and antibodies to CRM ₁₉₇ . In addition, mice treated with adjuvanted DPSB did not exhibit cytokine or vehicle antibodies at any time point.

Antibody titers to CRM ₁₉₇ were detected in all CRM ₁₉₇ control treated groups on days 39, 60 and 120, as well as in the unconjugated muIL-4 + CRM ₁₉₇ or unconjugated muIL-13 + CRM ₁₉₇ treated group. Titers of anti-muIL-4 antibodies, anti-muIL-13 antibodies and anti-CRM ₁₉₇ antibodies were detected in all groups treated with immunogenic products made with CRM ₁₉₇ on days 39, 60 and 120. Titers of anti-muIL-4 antibodies, anti-muIL-1 antibodies 13 and antibodies to KLH were detected, but not in all mice in all groups, treated with immunogenic products made with KLH on days 39, 60 and 120. Of note, the level of antibodies to IL-4 was higher in mice immunized with immunogenic products made with CRM ₁₉₇ than immunogenic products made with KLH.

MuIL-4 Neutralization Bioassay

Antibodies induced by administration of muIL-4 immunogenic products were further assessed for their neutralizing ability against muIL-4 in a proliferative assay using CTLL-2 cells, adapted from Soman et al, 2009. In general, CTLL-2 cells were grown in the presence of IL-2 at a final concentration of 10 ng/ml with RPMI supplemented with 2 mM glutamine, 1 mM sodium pyruvate, 1 mM HEPES, 100 U/ml penicillin, 100 μg/ml streptomycin, and 10% (v/v) FBS . For IL-2 neutralization bioassays, muIL-4 was replaced. Thus, potential muIL-4 neutralizing antibodies induced following injections of immunogenic products would prevent CTLL-2 propagation.

Culture plates were supplemented with serum samples at a final ratio of 1/200 and anti-muIL-4 polyclonal antibody as a positive control at a final concentration of 1 μg/ml and serially diluted twofold in 25 μl per well of RPMI + 10% (v/v) FBS. MuIL-4 was then added to the serum samples at a final concentration of 2 ng/ml and incubated for 1 hour at room temperature. 20,000 CTLL-2 cells were then added to preincubated samples (serum or positive control plus muIL-4). The plates were incubated for 48 hours at 37°C in a humidified incubator with 5% CO ₂ . Cell viability was quantified using MTS/PMS assay. Forty microliters of MTS/PMS per well was added and OD was read at 490 nm after 4 hours at 37°C in a humidified 5% CO ₂ incubator.

HC ₅₀ results were expressed as the serum dilution factor ( ⁻¹ dil) neutralizing 50% of muIL-4 activity in the presence of serum. HC ₅₀ is determined by interpolating the serum dilution resulting in 50% of IL-4 activity on the x-axis. It should be noted that a mouse is considered to have a response in this experiment if the HC ₅₀ is ≥200 rec. ^-1 .

None of the mice had neutralizing antibodies to muIL-4 before dosing (Figure 5 and Table 2). No neutralizing muIL-4 antibodies were detected in the control groups at any time point. For mice immunized with an immunogenic product formulated with CRM ₁₉₇ , muIL-4 neutralizing antibodies were detected in 9 of 10 mice. In contrast, in mice immunized with the immunogenic product formulated with KLH, the level of neutralizing antibodies against IL-4 was lower at any time point than that observed when the vehicle was CRM ₁₉₇ , and fewer sera showed neutralizing antibodies (at best point in time, only 6 sera showed neutralizing ability (on day 120)). These results demonstrated that treatment with the immunogenic muIL-4 product made with CRM ₁₉₇ was more immunogenic than the conjugate vaccine made with KLH.

Table 2. NS ₅₀ mice with IL-4 response

Immunogenic product muIL-4 (CRM ₁₉₇ ) Immunogenic product muIL-4 (KLH) muIL-4 + CRM ₁₉₇ (unconjugated) D0 D39 D60 D120 D0 D39 D60 D120 D0 D39 D60 D120 Those who have the answer
(NS ₅₀ >200 reconnaissance ^-1 ) 0/10 5/10 7/10 9/10 0/10 2/10 4/10 6/10 0/5 0/5 0/5 0/5 CRM ₁₉₇ FSB D0 D39 D60 D120 D0 D39 D60 D120 Having an answer (NS ₅₀ >200 reconnaissance ^-1 ) 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5

MuIL-13 Neutralization Bioassay

The neutralizing capacity of antibodies induced by administration of the muIL-13 immunogenic product was assessed using the muIL-13 Neutralization Bioassay before dosing and on days 39, 60 and 120.

The neutralizing ability of anti-muIL-13 antibodies was assessed using the HEK-Blue™ IL-4/IL-13 reporter gene bioassay (InvivoGen, cat. no. 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 λ = 625 nm). Thus, potential muIL-13 neutralizing antibodies induced following injections of immunogenic products would prevent activation of the STAT6 pathway and could be evaluated.

In general, HEK-Blue™ IL-4/IL-13 cells were plated in assay media consisting of DMEM GlutaMAX™ supplemented with 10% (v/v) FBS, 10 mM HEPES, 50 U/ml penicillin and 50 μg/ml streptomycin. Then a mixture of muIL-13 (at a final concentration of 2 ng/ml) and a serum sample, 2-fold serially diluted, starting with a final ratio of 1/100, or a control antibody, 2-fold serially diluted, starting with a final concentration of 1 μg/ml (polyclonal goat antibody to muIL-13) was added to 40,000 HEK-Blue™ IL-4/IL-13 cells. The plates were incubated for 24 h at 37°C in a humidified incubator with 5% CO ₂ . Then, in new flat bottom plates, 10 µl per well of cell supernatant was added to 90 µl per well of QUANTI-Blue™ and after 1 hour at 37°C in a humidified incubator with 5% CO ₂ the absorbance was read at 625 nm.

HC ₅₀ results were expressed as the serum dilution factor ( ⁻¹ dil) neutralizing 50% of muIL-13 activity in the presence of serum. HC ₅₀ is determined by interpolating the serum dilution resulting in 50% of muIL-13 activity onto the x-axis. It should be noted that in this experiment, a mouse is considered to have a response if the HC ₅₀ is ≥100 rec. ^-1 .

Neutralizing muIL-13 antibodies were not detected pre-dose in all groups or in control groups at any time point (Figure 6 and Table 3). Mice treated with the immunogenic product made with CRM ₁₉₇ produced antibodies with detectable neutralizing ability against muIL-13 (Figure 6 and Table 3). In mice immunized with the immunogenic product made with KLH, mouse sera did not show neutralizing antibodies to muIL-13. It should be noted that even if one mouse showed antibodies to muIL-13, as assessed by ELISA in the group receiving unconjugated muIL-13 and CRM ₁₉₇ (Figure 4-B), these antibodies were not neutralizing antibodies. These results demonstrated that treatment with an immunogenic product made with CRM ₁₉₇ is more immunogenic than treatment with an immunogenic product made with KLH.

Table 3. NS ₅₀ mice with IL-13 response

Immunogenic product muIL-13 (CRM ₁₉₇ ) Immunogenic product muIL-13 (KLH) muIL-13 + CRM ₁₉₇ (unconjugated) D0 D39 D60 D120 D0 D39 D60 D120 D0 D39 D60 D120 Those who have the answer
(NS ₅₀ >100 reconnaissance ^-1 ) 0/10 5/10 4/10 5/10 0/10 0/10 0/10 0/10 0/5 0/5 0/5 0/5 CRM ₁₉₇ FSB D0 D39 D60 D120 D0 D39 D60 D120 Those who have the answer
(NS ₅₀ >100 reconnaissance ^-1 ) 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5

As expected, neutralizing antibodies to muIL-4 and muIL-13 were not detected in the CRM ₁₉₇ and DFSB control groups at any time point. In control groups in which cytokines and CRM ₁₉₇ were mixed without chemical functionalization (unconjugated), immunization did not elicit the production of neutralizing antibodies to muIL-4 or muIL-13, highlighting that conjugation between a cytokine and a carrier protein is required to disrupt its own B cell tolerance against cytokines. In addition, no neutralizing antibodies to IL-13 were observed in mice immunized with an immunogenic product made with KLH, whereas in mice immunized with an immunogenic product made with CRM ₁₉₇ , neutralizing antibodies against muIL-13 were detected in 5 out of 10 mice. These results demonstrated that treatment with the muIL-13 immunogenic product made with CRM ₁₉₇ was more immunogenic than the conjugate vaccine made with KLH.

Example 4: Residual activity of immunogenic products

Residual activity of muIL-4 immunogenic products

Residual activity of muIL-4 immunogenic products was assessed as described below (adapted from Soman et al, 2009).

In general terms, CTLL-2 cells are grown with IL-2. The culture medium consisted of RPMIc medium supplemented with IL-2 at a final concentration of 10 ng/ml and 10% (v/v) FBS.

For residual activity bioassays, IL-2 was replaced by muIL-4. The immunogenic products (with CRM ₁₉₇ or KLH) of the present invention and the muIL-4 control were serially diluted twofold in RPMI + 10% (v/v) FBS in 96-well plates, starting at 1000 ng/ml to a final concentration of 4 ng /ml for immunogenic products and from 10 ng/ml to a final concentration of 0.04 ng/ml for muIL-4. As a positive control, six wells of muIL-4 at 10 ng/ml were added and used as a control for maximum cell proliferation. These samples were added to 20,000 CTLL-2 cells per well, and the plates were incubated for 48 hours at 37°C in a humidified 5% CO ₂ incubator. Cell proliferation was quantified using MTS/PMS assay. Forty microliters of MTS/PMS per well was added and after 4 hours at 37°C in a humidified 5% CO ₂ incubator, the plates were read at 490 nm.

The 50% effective dose ( _ED50 ) value corresponds to the amount of immunogenic product or cytokine that results in 50% of the maximum cell signal. The value is determined by interpolating 50% of the cell's maximum signal onto the x-axis using the formula y = ax + b based on a curve passing through the dilution points surrounding the 50% inflection point.

The inactivation coefficient was calculated by dividing the _ED50 of the immunogenic product tested by the average _ED50 value of the muIL-4 control standard curves.

Table 4. ED ₅₀ and inactivation coefficients

Products ED ₅₀ [ng.ml ^-1 ] Control muIL-4 ED ₅₀ [ng.ml ^-1 ] Inactivation coefficient Immunogenic product muIL-4 (CRM ₁₉₇ ) 620.3 1.67 370 Immunogenic product muIL-4 (KLH) 21.5 1.03 21

As shown in Table 4, residual muIL-4 activity was reduced to a greater extent in the immunogenic product prepared with CRM ₁₉₇ compared to the product prepared with KLH (significantly higher inactivation rate for the muIL-4 immunogenic product prepared with CRM ₁₉₇ ).

Residual activity of muIL-13 immunogenic products

Residual activity of muIL-13 immunogenic products was observed using the HEK-Blue™ IL-4/IL-13 reporter cell line as described below.

In general, muIL-13 immunogenic products and muIL-13 control were serially diluted twofold in assay medium (DMEM, 10% (v/v) FBS, 10 mM HEPES buffer, 50 U/ml penicillin, 50 μg/ ml streptomycin) in round-bottomed plates, starting at a final concentration of 250 ng/ml and 10 ng/ml, respectively. These mixtures were added to 40,000 HEK-Blue™ IL-4/IL-13 cells per well. The plates were incubated for 24 h at 37°C in a humidified incubator with 5% CO ₂ . Then, in new flat bottom plates, 10 µl per well of culture supernatant was added to 90 µl per well of QUANTI-Blue™ and after 1 hour at 37°C in a humidified incubator with 5% CO ₂ the OD was read at 625 nm.

The 50% effective dose ( _ED50 ) value corresponding to the amount of immunogenic product (or IL-13) that results in 50% of the maximum signal recorded for the samples in question is determined by interpolating the ODmax/2 values to the corresponding sample concentrations using four-parameter logistic (4PL) nonlinear regression based on all dilution points.

Inactivation coefficients were calculated by dividing the ED ₅₀ muIL-13 immunogenic products by the corresponding ED ₅₀ value of the muIL-13 control standard curves.

Table 5. ED ₅₀ and inactivation coefficients

Products ED ₅₀ [ng.ml ^-1 ] Control of muIL-13
ED ₅₀ [ng.ml ^-1 ] Inactivation coefficient Immunogenic product muIL-13 (CRM ₁₉₇ ) 737 0.31 2369 Immunogenic product muIL-13 (KLH) 297 0.41 723

As shown in Table 5, residual muIL-13 activity was reduced to a greater extent in the immunogenic product prepared with CRM ₁₉₇ compared to the product prepared with KLH (significantly higher inactivation rate for the muIL-13 immunogenic product prepared with CRM ₁₉₇ ).

The intensity of the immune response against IL-4 and IL-13 was higher if the immunogenic product was formulated with CRM ₁₉₇ compared to the product formulated with KLH as a carrier. In addition, residual cytokine activity was reduced to a greater extent in the immunogenic product prepared with CRM ₁₉₇ compared to the product prepared with KLH. Based on these results, we decided to obtain a proof of concept using the products of the present invention made with CRM ₁₉₇ as the carrier protein. Additionally, both IL‐4 and IL‐13 are involved in allergies, so we decided to introduce a group of mice that would receive an immunogenic product containing both IL‐4 and IL‐13 conjugates to CRM ₁₉₇ . From now on, all immunogenic products will be manufactured using CRM ₁₉₇ as the carrier protein, and the mixture will be called Combo immunogenic product.

Example 5: Efficacy of the immunogenic product of the present invention in a mouse model of allergy

Among all allergies, the major public health problems are allergic asthma and food allergies, each of which affects more than 300 million people worldwide. Various types of allergies are thought to result from a breakdown of tolerance, leading to type 2 immune responses characterized by the production of TH2 cytokines such as IL-4 and IL-13, high levels of IgE antibodies, and the infiltration and proliferation of immune cells (particularly , mast cells, basophils, eosinophils and T cells) in inflamed tissue.

House dust mite (HDM) is a major source of allergen, affecting more than 50% of allergy patients (Meyer et al. 1994). In mice, repeated intranasal stimulation using HDM recapitulated key features of human chronic asthma, including airway hyperresponsiveness (AHR), airway obstruction, airway wall remodeling, mucus production, and inflammatory responses in the lungs characterized by high levels of eosinophils.

Preparation of muIL-4/IL-13 immunogenic products

a) Functionalization of CRM ₁₉₇

CRM ₁₉₇ was diluted in modification buffer containing 70 mM sodium phosphate buffer, 150 mM NaCl, 5 mM EDTA (pH=7.2). SATA was diluted in DMSO to achieve a concentration of 100 mM. SATA was then added to CRM ₁₉₇ , and after 30 minutes of incubation on a shaker at room temperature, excess SATA was removed using a Zeba™ desalting spin column according to the manufacturer's instructions.

Hydroxylamine was then diluted in the same buffer to 500 mM. CRM ₁₉₇ -SATA was then incubated with hydroxylamine solution at a final concentration of 50 mM for 2 hours on a rocking shaker at room temperature. Finally, the mixture was desalted and excess reagents were removed using a Zeba™ desalting spin column.

b) Functionalization of muIL-4 and muIL-13

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 to 10 mM. sGMBS was then added to muIL-4 and after one hour of incubation on a shaker at room temperature, excess sGMBS was removed using a Zeba™ desalting spin column.

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 to 10 mM. sGMBS was then added to muIL-13 and after one hour of incubation on a shaker at room temperature, excess sGMBS was removed using a Zeba™ desalting spin column.

c) Conjugation

After functionalization of CRM ₁₉₇ , muIL-4, and muIL-13, the protein content of each drug was determined using the Bradford assay.

Functionalized CRM ₁₉₇ was added to functionalized muIL-4 or functionalized muIL-13 in a molar ratio of 1:2 (vehicle:muIL-4) and 1:4 (or vehicle:muIL-13). Preparations of individual immunogenic products were incubated overnight at 4°C on a rocking shaker. The resulting immunogenic products were then concentrated using Amicon ^® (3 kDa cut-off membrane), filtered through a 0.22 μm pore size filter and stored at 4°C.

d) Quantitative assessments of immunogenic products

Concentrations of muIL-4 immunogenic product and muIL-13 immunogenic product were determined using the Coomassie Plus protein assay (Bradford) according to the manufacturer's instructions.

MuCombo immunogenic product

The independently synthesized muIL-4 immunogenic product and the muIL-13 immunogenic product were mixed in a mass ratio of 1-1 after concentration and sterile filtration steps through a 0.22 μm pore size filter, and the resulting muCombo immunogenic product was stored at 4°C.

Mice immunization, allergic asthma protocol and blood collection

Each Balb/c mouse received four intramuscular (IM) injections of immunogenic products alone or muCombo immunogenic product or CRM ₁₉₇ alone as a control, all emulsified with a squalene-based adjuvant on days 0, 7, 28, and 55, as detailed in Table 6 and Figure 7. The first immunization was performed on mice at 7 weeks of age.

Table 6. Dose schedule

Product Number of mice per group Days Dose (mcg) Adjuvant Immunogenic product muIL-4 12 0/7/28/55 30/30/10/10 Squalene-based oil-in-water adjuvant Immunogenic product muIL-13 12 0/7/28/55 30/30/10/10 Immunogenic product muCombo 12 0/7/28/55 30/30/10/10 CRM ₁₉₇ 12 0/7/28/55 15/15/5/5

Thirty-nine days after the first immunization with the immunogenic product, mice were induced to develop chronic airway inflammation using three intranasal injections of 100 μg HDM from Dermatophagoides farinae (purchased from Greer) on days 39, 43, and 46. Beginning on day 50, mice were challenged with intranasal injections of 25 mcg HDM twice a week (9 stimulations in total).

Blood samples were collected seven days before dosing, days 39, 64, and 24 hours after the last HDM stimulation (mouse sacrifice). Serum samples were prepared as previously described and stored at −20°C until analysis.

This study assessed:

- Immunogenicity of injected products, measured using ELISA and bioassay,

- Airway hyperresponsiveness (AHR), measured using whole body plethysmography,

- Biological markers: serum circulating IgE levels by ELISA, airway inflammation in the lung and bronchoalveolar lavage (BAL) by FACS analysis, and airway histology and inflammatory cell infiltration.

a) Immunogenicity of injected products measured by ELISA and bioassay

Anti-muIL-4 and anti-muIL-13 antibody titers were measured by ELISA (as described above) in mouse sera collected from the four treated groups.

Cytokine antibody titers were undetectable before dosing in all groups and in the CRM ₁₉₇ group at all time points. Anti-muIL-4 antibody titers were detected in all mice at all time points after immunization with muIL-4 immunogenic product and muCombo immunogenic product (Figure 8-A). Higher levels were observed in mice treated with the muIL-4 immunogenic product compared to the muCombo immunogenic product. Anti-muIL-13 antibody titers were detected in mice starting at D39 and in mice with a 100% response to D65 in the muIL-13 immunogenic product group (Figure 8-B). In the muCombo immunogenic group, muIL-13 antibodies were also detected as early as D39 and in 9 of 12 mice responding to D65. Overall, higher antibody titers were observed in sera following immunization with the muIL-4 and muIL-13 immunogenic products than with the muCombo immunogenic product, as expected since mice in the muCombo group received half the dose of the individual immunogenic product (Figure 14).

The neutralizing activity of each immunogenic product was assessed in vitro using the two different bioassays described above. It should be noted that a mouse is considered to have responded in this experiment if the HC ₅₀ is ≥200 rec. ^-1 for IL-4 and ≥100 rec. ^-1 for IL-13.

High neutralizing capacity against muIL-4 was observed in mice immunized with muIL-4 immunogenic product and muCombo immunogenic product (Figure 9-A and Table 7), n=11 of 12 or n=9 of 12 at at least one time point, respectively.

Table 7. NS ₅₀ mice with IL-4 response

Immunogenic product muIL-4 Immunogenic product muCombo CRM ₁₉₇ D0 D39 D65 D0 D39 D65 D0 D39 D65 Having an answer (NS ₅₀ >200 reconnaissance ^-1 ) 0/12 10/12 11/12 0/12 7/12 9/12 0/12 0/12 0/12

It is interesting to note that one year after immunization with muIL-4 or muCombo immunogenic product, mice still showed antibodies to IL-4 (data not shown).

Induction of neutralizing activity against muIL-13 was also observed in mice immunized with the muIL-13 immunogenic product and the muCombo immunogenic product, with n=10 of 12 or n=7 of 12 at at least one time point, respectively (Figure 9-B and Table 8).

Table 8. NS ₅₀ mice with IL-13 response

Immunogenic product muIL-13 Immunogenic product muCombo CRM ₁₉₇ D0 D39 D65 D0 D39 D65 D0 D39 D65 Those who have the answer
(NS ₅₀ >100 reconnaissance ^-1 ) 0/12 8/12 10/12 0/12 3/12 7/12 0/12 0/12 0/12

It is interesting to note that one year after immunization with muIL-13 or muCombo immunogenic product, mice still showed antibodies to IL-13 (data not shown).

It should be noted that co-injection of muIL-4 and/or muIL-13 and CRM ₁₉₇ without prior chemical coupling did not induce the production of anti-IL-4 antibodies or anti-IL-13 antibodies, highlighting that conjugation between the cytokine and the carrier protein is required to break the intrinsic tolerance of B cells against cytokines.

Following immunization, various classes (including, but not limited to, IgG1, IgG2a, IgG2b, IgG3, IgA, IgE, and IgM) of anti-IL-4 antibodies and anti-IL-13 antibodies, mainly IgG1, were produced (data not shown).

b) Airway hyperresponsiveness (AHR) measured using whole body plethysmography

Twenty-four hours after the last stimulation, AHR was assessed using whole-body plethysmography. Responses to methacholine, a bronchoconstrictor, were measured in conscious mice.

Various doses of methacholine were administered via aerosol: 0 mg/ml, 3.5 mg/ml, 7 mg/ml and 14 mg/ml. The PenH index (Penh) was used to assess changes in pulmonary function (Hamelmann et al., 1997). This measurement provided insight into the phase shift between the thoracic flow and nasal flow curves: increased phase shift correlated with increased respiratory system resistance. PenH is calculated using the formula Penh = (Te/RT-1)×PEF/PIF, where Te is the expiratory time, RT is the relaxation time, PEF is the maximum expiratory flow, and PIF is the maximum inspiratory flow. PenH values were recorded for 5 min after each methacholine stimulation, and the maximum value during the period was plotted.

Severe bronchospasm was observed in HDM-sensitized mice in the CRM ₁₉₇ control group (Figure 10). Bronchospasm was partially inhibited by the muIL-13 immunogenic product and the muIL-4 immunogenic product, while it almost disappeared in mice immunized with the muCombo immunogenic product, indicating a high protective effect of the products of the present invention. Indeed, the level of residual bronchospasm observed in the muCombo group was similar to that observed in HDM non-sensitized mice (circle in Figure 10).

These results were confirmed using invasive measurements of airway response. Indeed, HDM-treated control mice exhibited significantly greater changes in lung resistance and elastance when stimulated with methacholine compared to PBS-treated controls, while mice treated with the immunogenic product muIL-4, muIL-13, or muCombo two traits were partially reduced (data not shown).

Taken together, these results suggest that AHR can be blocked by dual vaccination against IL-4 and IL-13.

c) Biological markers

Serum circulating IgE levels measured by ELISA

Total circulating IgE levels were measured by ELISA according to the manufacturer's instructions (Quantitative Mouse IgE ELISA Kit, Bethyl Labs E90-115) before vaccination with the immunogenic product and 24 hours after the last HDM challenge (Figure 11).

No circulating IgE was detected before vaccination. Twenty-four hours after the last HDM stimulation, there was an induction of total circulating IgE due to HDM sensitization (Figure 11-B). Levels were reduced in all groups receiving immunogenic products, demonstrating the protective effect of the products of the present invention compared to mice immunized with CRM ₁₉₇ . It should be noted that similar results were obtained when HDM-specific IgE was measured only by ELISA (data not shown).

Increased levels of HDM-specific IgG antibodies were also observed in HDM-treated mice. However, treatment with the muIL-4 and/or muIL-13 immunogenic product did not affect these levels (data not shown).

Airway inflammation in the lung and bronchoalveolar lavage (BAL)

A thorough temporal analysis of cellular changes in the lung and bronchoalveolar lavage fluid (BAL) of mice sensitized with HDM or PBS was performed.

Inflammatory cells from BAL fluid 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 the manufacturer's instructions.

Antibody Supplier - catalog number (clone) Ly6G-PE BD Pharmingen - 561104 (clone 1A8, rat IgG _2a , κ) CD3-APC BD Pharmingen - 561826 (clone 145-2C11, gray hamster IgG1, κ) 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 κ)

In the lungs of control mice, chronic intranasal exposure to HDM resulted in a significant increase in the number of infiltrating CD45 ⁺ cells of hematopoietic origin, mainly consisting of eosinophils (CD45 ⁺ , Ly6G ^- , CD11b ⁺ , SiglecF ⁺ ), compared with PBS-treated animals (data not shown ). Interestingly, vaccination with immunogenic products prevented eosinophil infiltration among infiltrating CD45 ⁺ cells, particularly in the muCombo immunogenic product group, which showed a statistically significant reduction.

In bronchoalveolar lavage fluid, HDM sensitization led to an inflammatory response characterized by infiltration of CD45 ⁺ cells of hematopoietic origin, mainly consisting of eosinophils (CD45 ⁺ , ^Ly6G- , CD11b ⁺ , SiglecF ⁺ ) (Figure 12). Interestingly, vaccination with immunogenic products prevented eosinophil infiltration among infiltrating CD45 ⁺ cells, particularly in the muCombo immunogenic product group, which showed a statistically significant reduction.

It is interesting to note that vaccination with the immunogenic products of the present invention had no effect on blood eosinophil levels (data not shown), indicating that the reduction in airway eosinophilia observed in vaccinated mice is a consequence of reduced eosinophil recruitment to the lungs rather than systemic eosinophils. effects on the number of eosinophils or eosinophil precursors.

Histology of the respiratory tract

The effects of sensitization and vaccination on the respiratory tract were examined using histological analyses. In general, the left lung was excised postmortem, fixed in 4% PFA for 24 h at room temperature, and preserved in 70% ethanol. Longitudinal sections were made and stained:

-hematoxylin and eosin (H&E) to assess leukocyte infiltration,

-toluidine blue to quantify the number of mast cells, or

-stained with periodic acid-Schiff (PAS) to evaluate goblet cell hyperplasia and mucus production.

Overall, these histological analyzes of the lungs confirm that in HDM-treated animals, vaccination with the immunogenic product muIL-4, muIL-13, or muCombo significantly reduced the number of leukocytes and intraepithelial mast cells, compared with unvaccinated HDM-treated animals (data not shown).

PAS histological analyzes also confirmed that in HDM-treated animals, vaccination with the immunogenic product muIL-4, muIL-13, or muCombo significantly reduced goblet cell hyperplasia/mucus production compared with unvaccinated HDM-treated animals (data not shown). These results confirm that immunogenic products (in particular, muIL-13 and muCombo immunogenic products) can control mucus hypersecretion induced by HDM sensitization.

In conclusion, we demonstrated that the products of the present invention formulated with CRM ₁₉₇ were immunogenic and induced cytokine neutralizing antibodies, whether administered by injection alone or in combination (muCombo immunogenic product). The presence of cytokine-neutralizing antibodies was associated with a decrease in HDM-induced AHR measured by Penh value after inhalation of methacholine. In addition, cytokine neutralizing antibodies were able to limit circulating IgE levels as well as mast cell numbers in the lung and eosinophil infiltration in the airways.

Therefore, these results demonstrate that the immunogenic products of the present invention are capable of breaking 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.

It is interesting to note that individual immunogenic products had active but superior beneficial effects on asthma and/or allergy symptoms (and in particular on allergic asthma symptoms) and biological markers were observed when combining immunogenic products (muCombo immunogenic product).

Example 6: Preparation of human immunogenic products according to the present invention

The preparation of the human IL-4 immunogenic product, the IL-13 immunogenic product and the Combo immunogenic product was carried out in accordance with the same manufacturing method as described above, using human IL-4 and IL-13 cytokines instead of mouse cytokines.

Preparation of immunogenic product IL-4 and IL-13

a) Functionalization of CRM ₁₉₇

Functionalization of CRM ₁₉₇ was performed as described for the mouse product.

b) Functionalization of IL-4 and IL-13

IL-4 or IL-13 dissolved in buffer (70 mM sodium phosphate buffer, 150 mM NaCl, 5 mM EDTA, pH=7.2) reacted with sGMBS previously dissolved in buffer at a concentration of 10 mM. After one hour of reaction at room temperature with gentle stirring, excess sGMBS was removed using a Zeba desalting column.

c) Conjugation

After functionalization of CRM ₁₉₇ , IL-4, and IL-13, the protein content of each drug was determined using the Bradford assay. Functionalized CRM ₁₉₇ was added to functionalized muIL-4 or functionalized IL-13 in a molar ratio of 1:4 (vehicle:IL-4 or vehicle:IL-13). Preparations of individual immunogenic products were incubated overnight at 4°C on a rocking shaker. The conjugates were added to a desalting column to remove possible impurities, such as remaining hydroxylamine, from previous steps. The resulting immunogenic product was concentrated, filtered through a 0.22 μm filter and stored at 4°C.

Preparation of the immunogenic Combo product

Preparation of the Combo immunogenic product can be accomplished using two different methods: by incubating both modified cytokines simultaneously with the modified carrier protein (simultaneous synthesis) or by mixing two independently synthesized immunogenic products IL-4 and IL-13 (mixed preparation).

Preparation of the immunogenic Combo product by simultaneous synthesis

a) Functionalization of CRM ₁₉₇

Functionalization of CRM ₁₉₇ was performed as described for the mouse product.

b) Functionalization of IL-4 and IL-13

Functionalization of IL-4 or IL-13 was performed as described for the preparation of the individual immunogenic product.

c) Conjugation

After functionalization of CRM ₁₉₇ , IL-4, and IL-13, the protein content of each drug was determined using the Bradford assay. All three modified proteins in phosphate buffer, 150 mM NaCl, 5 mM EDTA, pH = 7.2 were mixed in a molar ratio of 2-2-1 IL-4-IL-13-CRM ₁₉₇ . The reaction was carried out at 4°C overnight. The conjugate was added to a desalting column to remove possible impurities, such as remaining hydroxylamine, from previous steps. The resulting immunogenic product was concentrated, filtered through a 0.22 μm filter and stored at 4°C.

Preparation of an immunogenic Combo product by preparing a mixture

The independently synthesized immunogenic products IL-4 and IL-13 were mixed in a 1-1 weight ratio after concentration and sterile filtration steps through a 0.22 μm pore size filter. Combo immunogenic product was stored at 4°C until use.

Example 7: Antigenicity of a human product

A sandwich ELISA was performed to assess the binding of the cytokine to the carrier protein, as well as to assess the retention of epitopes during the manufacturing method. The protocol used was the same as described herein above, using biotinylated anti-human IL-4 antibodies and anti-human IL-13 antibodies.

The results are shown in Figure 13. This test confirmed that the immunogenic product of the present invention contains IL-4 or IL-13 associated with CRM ₁₉₇ . Additionally, these results confirm that the immunogenic product is antigenic (ie, recognized by anti-IL-4 antibody or anti-IL-13 antibody) as well as in the Combo immunogenic product.

Example 8: Immunogenicity of Human Product

Mice immunization protocol

Each Balb/c mouse (10 mice per group) received four intramuscular (IM) injections of the immunogenic product emulsified with a squalene-based adjuvant on days 0, 7, 28, and 49, all injections at a dose of 4 μg, as detailed described in Figure 14. The first immunization was performed at 7 weeks of age. Blood samples were collected before dosing and on days 38, 59, 90, and 120. Serum samples were prepared after coagulation at room temperature and centrifugation to remove clot.

Determination of titers of antibodies to IL4 and antibodies to IL-13 using ELISA

Cytokine antibody titers were measured by ELISA on mouse sera collected from each group.

Determination of titers of each antibody in sera after immunization with immunogenic products or immunogenic Combo product was assessed as described above by coating 96-well plates with human IL-4 and IL-13.

Anticytokine antibody titers were undetectable before dosing in all groups. Anti-IL-4 antibody titers were detected in all mice at all time points after immunization with the IL-4 immunogenic product and the muCombo immunogenic product (Figure 15-A). Similar levels were observed in mice treated with the IL-4 immunogenic product and the Combo immunogenic product, indicating that we may have reached a plateau in the immune response. Anti-IL-13 antibody titers were detected in all mice at all time points after immunization in the IL-13 immunogenic product group (Figure 15-B). In the Combo immunogenic product group, antibodies to IL-13 were also detected as early as D38 and in 8 out of 10 mice at D59. Higher titers of anti-IL-13 antibodies were observed in sera following immunization with the IL-13 immunogenic product compared with the Combo immunogenic product because mice in the Combo group received half the dose of the individual immunogenic product.

Determination of neutralizing ability after immunizations

Anti-IL-4 and anti-IL-13 neutralizing capacity was assessed using the HEK-Blue™ IL-4/IL-13 reporter cell line as described above. It should be noted that in this experiment, a mouse was considered to have a response if the HC ₅₀ was ≥200 rec. ^-1 for IL-4 and ≥100 rec. ^-1 for IL-13.

High neutralizing capacity against IL-4 was observed in the groups immunized with the IL-4 immunogenic product and the Combo immunogenic product, with 100% of mice having a response at all time points after immunization (Figure 16-A and Table 10).

Table 10. NS ₅₀ mice with IL-4 response

Immunogenic product IL-4 Immunogenic product Combo D0 D38 D59 D90 D120 D0 D38 D59 D90 D120 Having an answer (NS ₅₀ >200 reconnaissance ^-1 ) 0/10 10/10 10/10 10/10 10/10 0/10 10/10 10/10 10/10 10/10

Antibodies with neutralizing ability against IL-13 were also induced in mice immunized with the IL-13 immunogenic product and the Combo immunogenic product, with 100% of the mice responding to D59 (Figure 16-B and Table 11).

Table 11. NS ₅₀ mice with IL-13 response

Immunogenic product IL-13 Immunogenic product Combo D0 D38 D59 D90 D120 D0 D38 D59 D90 D120 Having an answer (NS ₅₀ >200 reconnaissance ^-1 ) 0/10 9/10 10/10 9/10 9/10 0/10 9/10 10/10 10/10 10/10

Collectively, the products of the present invention were capable of inducing high titers of antibodies against cytokines with strong neutralizing ability.

Residual activity of human immunogenic products

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

Table 12. ED ₅₀ and inactivation coefficients for IL-4

Products ED ₅₀ [ng.ml ^-1 ] Control of IL-4
ED ₅₀ [ng.ml ^-1 ] Inactivation coefficient Immunogenic product IL-4 231 0.043 5287 Immunogenic product Combo 382 0.043 8765

Table 13. ED ₅₀ and inactivation coefficients for IL-13

Products ED ₅₀ [ng.ml ^-1 ] Control of IL-13
ED ₅₀ [ng.ml ^-1 ] Inactivation coefficient Immunogenic product IL-13 444 0.258 1848 Immunogenic product Combo 268 0.258 1116

As shown in Table 12 and Table 13, all inactivation factors for IL-4 or IL-13 in human immunogenic products were greater than 1000 (and up to 8765), indicating that the residual cytokine activity in the immunogenic products was reduced by at least three order of magnitude compared to native cytokines.

The greatly reduced residual activity is an important element supporting the safety profile of the products of the present invention.

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

Food allergy is a major health problem in Western countries, with its prevalence increasing in recent decades and no definitive treatment available. Some staple foods, including milk, peanuts, soy and wheat, can induce food allergic reactions. Various types of food allergies represent a violation of oral tolerance to harmless food allergens, which leads to the development of a dysregulated Th2 immune response, secretion of Th2 cytokines (mainly IL-4 and IL-13), secretion of allergen-specific IgE and recruitment of effector cells into the gastrointestinal tract. - intestinal tract (GIT).

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

Preparation of immunogenic products muIL-4/IL-13 and muCombo

The immunogenic products muIL-4, muIL-13 and muCombo are synthesized as described above herein.

Mice immunization, food allergy model, anaphylactic response, histological analyzes and blood sampling

Each IL-4raF709 mutant mouse was immunized with the immunogenic products muIL-4, muIL-13 or muCombo or CRM ₁₉₇ , as a control, all emulsified with a squalene-based adjuvant, on days 0, 7, 28, and then stimulated.

Simultaneously, mice were sensitized by gavage of either PBS or 23 mg peanut (PE) oil, corresponding to 5 mg peanut protein suspended in 250 μl of 0.1 M sodium bicarbonate (pH = 8.0). Mice were stimulated using an enteral bolus of 450 mg peanut butter (100 mg protein) diluted in water (Burton et al., 2014).

Immediately after stimulation, core body temperature was monitored for one hour, as well as the onset of diarrhea.

A clinical score was also determined: 0, absence of clinical symptom; 1, repetitive scratching of the mouth/ear and digging of the ear canal with the hind paws; 2, decreased activity, self-isolation, swelling around the eyes and/or mouth; 3, periods of immobility of more than one minute, lying on the stomach; 4, lack of response to whisker stimuli, decreased or absent response to pushes; 5, tremors, convulsions or death.

Intestinal tissue sections were collected for histological analyzes (eg, mast cell quantification).

Blood was also collected to examine levels of inflammation (eg, mast cell degranulation product), antibody levels (eg, cytokine antibody titers, neutralizing capacity, allergen-specific IgG and IgE, and total IgE).

Example 10: Therapeutic efficacy of the immunogenic product of the present invention in a mouse model of atopic dermatitis

Atopic dermatitis (AD) is a chronic or chronically relapsing pruritic inflammatory skin disease. The incidence of AD has increased dramatically over the past three decades in industrialized countries. The immunological pathologies of AD are typically characterized by sensitization to various allergens (eg, food, aeroallergens, microbes, and autoallergens), high serum levels of IgE, and skin lesions with apoptotic keratinocytes and infiltration of immune cells that secrete Th2 cytokines such as IL-4, IL-5 and IL-13.

The therapeutic efficacy of the immunogenic products of the present invention in AD was studied using an animal model in which repeated cutaneous application of house dust mite (HDM) extract and staphylococcal enterotoxin B induces eczematous skin lesions.

Preparation of immunogenic products muIL-4/IL-13 and muCombo

The immunogenic products muIL-4, muIL-13 and muCombo were synthesized as described above herein.

Immunization of mice, induction of atopic dermatitis, measurement of clinical severity, histological analyzes and blood collection

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

Simultaneously, atopic dermatitis was induced as previously described by Ando et al. (J Invest Dermatol. 2013 Dec;133(12):2695-2705). In general, solutions of 500 ng staphylococcal enterotoxin (SEB) and 10 μg Dermatophagoides farinae extract (Der f is house dust mite, HDM) were applied to a gauze pad placed on the shaved area. This portion of the back skin was covered with a clear Tegaderm™ dressing using bandages. After three days, the dressing was replaced with a new one. After an additional 4 days, the bandages were removed and the mice were left untreated for the next week. Treatment with Der f/SEB for one week was repeated two more times, so the mice were subjected to three cycles of this treatment.

Clinical severity was assessed by an investigator blinded to mouse identifiers 2 days after dressing removal in the final cycle. Clinical scores were based on the severity (0, no symptoms; 1, mild; 2, moderate; 3, severe) of four possible symptoms (redness, bleeding, rash, and peeling). The maximum possible score is 12.

Mice were sacrificed immediately after scoring, and dorsal skin samples corresponding to the treated areas were obtained for histological analyzes of epidermal thickness and eosinophilia. Blood was also collected to examine inflammation levels and antibody levels (eg, cytokine antibody titers, neutralizing capacity, allergen-specific IgG and IgE, and total IgE).

Example 11: Therapeutic efficacy of the immunogenic product of the present invention in a mouse model of chronic obstructive pulmonary disease (COPD)

COPD is characterized by progressive airflow limitation, usually associated with increased inflammatory responses to inhaled irritants, leading to chronic obstructive bronchitis and destruction of the lung parenchyma called emphysema.

Several animal models of COPD have been developed to elucidate possible mechanisms underlying the onset and progression of COPD. In the present application, a protease-induced model was used to evaluate the therapeutic efficacy of the immunogenic product of the present invention in COPD.

Preparation of immunogenic products muIL-4/IL-13 and muCombo

The immunogenic products muIL-4, muIL-13 and muCombo are synthesized as described above herein.

Immunization of mice, induction of atopic dermatitis, leukocyte quantification and histological analyzes

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

At the same time, intranasal instillation of elastolytic enzymes, such as porcine pancreatic elastase, was used to trigger COPD-like symptoms, such as the formation of emphysema. In general terms, BALB/c mice were treated intranasally with 0.6 U. porcine pancreatic elastase in 30 µl PBS (Shibata et al. Proc Natl Acad Sci USA. 2018 Dec 18;115(51):13057-13062).

In the first experiment, mice were sacrificed on day 5 to assess leukocytes: leukocyte counts (eg, monocytes, macrophages, neutrophils, T cells, B cells, and eosinophils) were quantified by flow cytometry in bronchoalveolar lavage (BAL) fluids and single cell suspensions lungs. Additional assessment of leukocyte infiltration in the lungs was performed by histological examination of lung tissues prestained with hematoxylin and eosin (H&E). Blood was also collected to examine levels of inflammation and antibody levels (eg, cytokine antibody titers, neutralizing capacity).

In the second experiment, mice were sacrificed on day 21 after elastase treatment for histological assessment of emphysema in H&E-stained lung tissue sections, using the midline segment (MLI) as an indicator of lung airspace expansion. Blood was also collected to examine inflammation levels and antibody levels (eg, cytokine antibody titers, neutralizing capacity, allergen-specific IgG and IgE, and total IgE).

Example 12: Therapeutic efficacy of the immunogenic product of the present invention in a mouse model of pulmonary fibrosis

Pulmonary fibrosis is a broad spectrum of diseases that is characterized by varying degrees of lung inflammation, excessive proliferation of lung fibroblasts, and increased collagen content in the lungs.

The therapeutic efficacy of the immunogenic product of the present invention in pulmonary fibrosis was studied using an animal model in which administration of bleomycin (BLM) to the lungs results in the secretion of pro-inflammatory cytokines and chemokines, recruitment of leukocytes, increased collagen production, remodeling and fibrotic inflammation of the lungs.

Preparation of immunogenic products muIL-4/IL-13 and muCombo

The immunogenic products muIL-4, muIL-13 and muCombo are synthesized as described above herein.

Immunization of mice, induction of pulmonary fibrosis, quantification of leukocytes and histological analyzes

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

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

Body weight was monitored 5 times per week until the end of the experiment, and mice were sacrificed 7 or 14 days after BLM treatment to assess pulmonary fibrosis.

Leukocyte counts (eg, monocytes, macrophages, neutrophils, T lymphocytes, B cells, and eosinophils) were quantified by flow cytometry in bronchoalveolar lavage (BAL) fluids and single cell lung suspensions.

Histological examinations of lung tissues stained with hematoxylin and eosin (to assess leukocyte infiltration), Masson's trichrome (to assess fibrosis) or toluidine blue (to assess the number of mast cells) were performed.

Blood was also collected to examine levels of inflammation and antibody levels (eg, cytokine antibody titers, neutralizing capacity).

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List of sequences

<110> NEOVACS

INSERM (Institut National de la Santé et de la Recherche

Medicine)

INSTITUT PASTEUR

Grouard-Vogel Géraldine

Conde Garcia Eva

Bertrand Romain

Caillot Noémie

Reber Laurent

Bruhns Pierre

Serra Vincent

<120> Immunogenic product containing IL-4 and/or IL-13

for the treatment of disorders associated with aberrant expression

or the activity of IL-4 and/or IL-13

<130>CV-1058/PCT/2

<160> 8

<170> BiSSAP 1.3.6

<210> 1

<211> 535

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CRM197

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Asp Trp Lys Glu Phe Tyr Ser Thr Asp Asn Lys Tyr Asp Ala Ala Gly

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Val Lys Val Thr Tyr Pro Gly Leu Thr Lys Val Leu Ala Leu Lys Val

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Asp Asn Ala Glu Thr Ile Lys Lys Glu Leu Gly Leu Ser Leu Thr Glu

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Pro Leu Met Glu Gln Val Gly Thr Glu Glu Phe Ile Lys Arg Phe Gly

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Asp Gly Ala Ser Arg Val Val Leu Ser Leu Pro Phe Ala Glu Gly Ser

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Ser Ser Val Glu Tyr Ile Asn Asn Trp Glu Gln Ala Lys Ala Leu Ser

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Val Glu Leu Glu Ile Asn Phe Glu Thr Arg Gly Lys Arg Gly Gln Asp

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Arg Ser Val Gly Ser Ser Leu Ser Cys Ile Asn Leu Asp Trp Asp Val

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Ile Arg Asp Lys Thr Lys Thr Lys Ile Glu Ser Leu Lys Glu His Gly

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Pro Ile Lys Asn Lys Met Ser Glu Ser Pro Asn Lys Thr Val Ser Glu

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Glu Lys Ala Lys Gln Tyr Leu Glu Glu Phe His Gln Thr Ala Leu Glu

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His Pro Glu Leu Ser Glu Leu Lys Thr Val Thr Gly Thr Asn Pro Val

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Phe Ala Gly Ala Asn Tyr Ala Ala Trp Ala Val Asn Val Ala Gln Val

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Ile Asp Ser Glu Thr Ala Asp Asn Leu Glu Lys Thr Thr Ala Ala Leu

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Ser Ile Leu Pro Gly Ile Gly Ser Val Met Gly Ile Ala Asp Gly Ala

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Val His His Asn Thr Glu Glu Ile Val Ala Gln Ser Ile Ala Leu Ser

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Ser Leu Met Val Ala Gln Ala Ile Pro Leu Val Gly Glu Leu Val Asp

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Ile Gly Phe Ala Ala Tyr Asn Phe Val Glu Ser Ile Ile Asn Leu Phe

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Gln Val Val His Asn Ser Tyr Asn Arg Pro Ala Tyr Ser Pro Gly His

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Asp Ile Lys Ile Thr Ala Glu Asn Thr Pro Leu Pro Ile Ala Gly Val

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Gly Tyr Gln Lys Thr Val Asp His Thr Lys Val Asn Ser Lys Leu Ser

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<210> 2

<211> 129

<212> PROTEIN

<213> Homo sapiens

<220>

<223> Human IL-4

<400> 2

His Lys Cys Asp Ile Thr Leu Gln Glu Ile Ile Lys Thr Leu Asn Ser

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Cys Leu Gly Ala Thr Ala Gln Gln Phe His Arg His Lys Gln Leu Ile

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Arg Phe Leu Lys Arg Leu Asp Arg Asn Leu Trp Gly Leu Ala Gly Leu

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Ser

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<212> PROTEIN

<213> Homo sapiens

<220>

<223> Human IL-4 fragment

<400> 3

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<210> 4

<211> 120

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35 40 45

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Thr Pro Cys Leu Lys Lys Asn Ser Ser Val Leu Met Glu Leu Gln Arg

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Leu Phe Arg Ala Phe Arg Cys Leu Asp Ser Ser Ile Ser Cys Thr Met

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<220>

<223> IL-4 dogs

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<210> 6

<211> 122

<212> PROTEIN

<213> Homo sapiens

<220>

<223> Human IL-13

<400> 6

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<210> 8

<211> 113

<212> PROTEIN

<213> Canis

<220>

<223> Canine IL-13

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50 55 60

Arg Met Leu Lys Ala Leu Cys Ser Gln Lys Pro Ala Ala Gly Gln Ile

65 70 75 80

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85 90 95

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100 105 110

Arg

<---

Claims (25)

1. An immunogenic product for the treatment of an inflammatory disorder, comprising at least one cytokine conjugated to a carrier protein, wherein said at least one cytokine is IL-13 or a mammalian IL-13 variant, wherein said variant exhibits at least 80% identity with IL-13 of the mammal from which it is derived, wherein said carrier protein is CRM ₁₉₇ , and wherein said immunogenic product is produced by a process comprising the following steps: (a) contacting IL-13 or a variant thereof with a heterobifunctional cross-linker containing an NHS ester to produce an NHS-IL-13 ester complex; (b) contacting CRM ₁₉₇ with a heterobifunctional cross-linker containing an NHS ester to produce a CRM ₁₉₇ -NHS ester complex; (c) contacting said heterobifunctional cross-linker containing the NHS-IL-13 ester complex obtained in step (a) with the CRM ₁₉₇ -NHS ester complex obtained in step (b). 2. An immunogenic product according to claim 1, wherein said immunogenic product further comprises IL-4, wherein IL-4 and IL-13 are both linked to CRM ₁₉₇ . 3. A composition for the treatment of an inflammatory disorder, comprising at least one immunogenic product according to claim 1 or 2 and additionally containing at least one pharmaceutically acceptable excipient and/or at least one adjuvant. 4. The composition according to claim 3, wherein said composition further comprises an immunogenic product containing IL-4 and CRM ₁₉₇ . 5. The composition according to claim 4, wherein said composition contains a mixture of an immunogenic product containing IL-4 and CRM ₁₉₇ with an immunogenic product containing IL-13 and CRM ₁₉₇ in a mass ratio ranging from 10:1 to 1:10. 6. Composition according to any one of paragraphs. 3-5, which is an emulsion. 7. A method for producing an immunogenic product according to claim 1 or 2, wherein said method includes the steps of: a) contacting IL-13 or a variant thereof, wherein said variant exhibits at least 80% identity with the mammalian IL-13 from which it is derived, with a heterobifunctional cross-linker containing an NHS ester to produce an NHS ester complex -IL-13; b) contacting CRM ₁₉₇ with a heterobifunctional cross-linker containing an NHS ester to produce a CRM ₁₉₇ -NHS ester complex; c) contacting said heterobifunctional crosslinker containing the NHS-IL-13 ester complex obtained in step (a) with said CRM ₁₉₇ -NHS ester complex obtained in step (b). 8. The method of obtaining an immunogenic product according to claim 7, characterized in that said NHS ester in step (a) is N-[γ-maleimidobutyryloxy]-succinimide (GMBS). 9. The method of obtaining an immunogenic product according to claim 7, characterized in that said NHS ester in step (b) is N-succinimidyl-S-acetylthioacetate (SATA). 10. A method for obtaining an immunogenic product according to claim 7, including the following stages: a) contacting said IL-13 or a variant thereof with GMBS to produce a GMBS-IL-13 complex; b) contacting CRM ₁₉₇ with N-succinimidyl-S-acetylthioacetate (SATA) to produce a CRM ₁₉₇ -SATA complex; c) bringing said GMBS-IL-13 complex obtained in step (a) into contact with the CRM ₁₉₇ -SATA complex obtained in step (b). 11. Use of an immunogenic product according to claim 1 or 2 or a composition according to any one of claims. 3-6 for the treatment of an inflammatory disorder that is associated with aberrant expression or activity of IL-13. 12. The use of claim 11, wherein said inflammatory disorder is selected from the group consisting of asthma, allergic conditions, atopic disorders, bullous pemphigoid, respiratory disorders, nasal polyposis and other conditions involving inflammation of the respiratory tract; 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 various types of cancer, glioblastoma, lymphoma and mastocytosis. 13. Use according to claim 11 or 12, characterized in that said inflammatory disorder is selected from the group consisting of allergic asthma, non-allergic asthma, food allergies, poison allergy, animal allergy, drug allergy, hyper-IgE syndrome, allergic rhinitis, allergic conjunctivitis, allergic enterogastritis, atopic dermatitis, urticaria, eczema, chronic obstructive pulmonary disease (COPD), eosinophilia, fibrosis and excess mucus production, including cystic fibrosis and pulmonary fibrosis, systemic sclerosis (SSc), inflammatory bowel disease (IBD), eosinophilic esophagitis (EE), eosinophil-mediated gastrointestinal disease, ulcerative colitis, Crohn's disease, cirrhosis, hepatocellular carcinoma, liver fibrosis, leukemia and Hodgkin's lymphoma. 14. Application according to any one of paragraphs. 11-13, characterized in that said inflammatory disorder is selected from the group consisting of chronic idiopathic urticaria, chronic spontaneous urticaria, fibrosis caused by hepatitis B and/or C virus, and chronic B-cell lymphocytic leukemia. 15. Application according to any one of paragraphs. 11-14, characterized in that said inflammatory disorder is selected from asthma, atopic dermatitis, chronic obstructive pulmonary disease (COPD), pulmonary fibrosis, food allergy, nasal polyposis and eosinophilic esophagitis, wherein preferably said inflammatory disorder is an allergy, asthma or atopic dermatitis. 16. Use of an immunogenic product according to claim 1 or 2 or a composition according to any one of claims. 3-6 for inducing desensitization of a subject allergic to a specific antigen, wherein said immunogenic product or composition and said specific antigen are intended for administration to said subject having an allergy.