WO2008066443A1

WO2008066443A1 - Il-33 vaccine for the treatment of inflammatory conditions with a strong th2 component

Info

Publication number: WO2008066443A1
Application number: PCT/SE2007/001036
Authority: WO
Inventors: Lars Hellman
Original assignee: Theravac Pharmaceuticals Ab
Priority date: 2006-11-28
Filing date: 2007-11-26
Publication date: 2008-06-05
Also published as: SE0602549L; SE532250C2

Abstract

Methods designed to alleviate the symptoms or prevent the induction of various inflammatory conditions by targeting the cytokine IL-33. A vaccine comprising IL-33 or a fragment thereof is administered to a mammal in need thereof.

Description

IL-33 vaccine for the treatment of inflammatory conditions with a strong TH2 component

The present invention relates to methods designed to alleviate the symptoms or prevent the induction of various inflammatory conditions by targeting the cytokine IL-33. Although the invention generally relates to a vaccine for use in a mammal, preferred embodiments thereof relates to vaccines for the use in human, dog, cat or horse, the invention will be described below generally, and with reference to such vaccines for human, feline, equine or canine use.

Background of the invention

During the past few decades several diseases caused by the malfunction of the immune system have become major challenges of modern day medicine. One such area is allergic diseases. Allergies have in man become almost epidemic during the past 20-30 years and atopic, or IgE-mediated allergies, are the dominating form. Allergies are also a major problem for many domestic animals like dogs, cats and horses. However, the involvement of IgE is here less well documented. Common types of atopic allergies include in man, fur allergies, hay fever, dust mite allergies, insect venom allergies, extrinsic asthma and many types of food allergies. In addition, many of our domestic animals suffer from allergies directed against similar allergens. Allergies have been estimated to affect almost 30% of the human population.

Allergic diseases are caused by malfunctions in our immune system. Several of the cytokines regulating normal immune responses against various pathogens appear also to be directly involved in the allergic disease processes. Cytokines, or growth and differentiation factors of importance for the regulation of our immune system may thereby serve as potential targets for intervention.

One particular difficult problem in veterinary medicine is severe atopic dermatitis in dogs. Almost 50% of all visits to the veterinarians are due to skin problems where atopic dermatitis is the dominating factor. In human medicine a particularly difficult condition is instead severe asthma. For the most severe cases non of the existing treatment regiments show sufficient clinical effect. In the situation with severe atopic dermatitis in dogs many of these dogs have to be removed. We have here a great unmet medical need.

We have for many years been working on a potential treatment strategy against IgE mediated allergies in man and domestic animals, vaccination against IgE [1-4J. However in cases where IgE levels are exceptionally high as in dogs L5 |, this strategy has its clear limitations. These limitations are primarily due to strong tolerizing effects induced by the high concentrations of circulating IgE. In addition, it is difficult to obtain good clinical effects when large amounts of the target molecule have to be removed. We therefore saw it as almost impossible to reach our goal in dogs by vaccinating against IgE [5J. New innovative strategies had to be developed. We here present one potential solution to the problem, vaccination against one important cytokines regulating humoral immunity IL-33.

IL-33 is an IL-I like cytokine that shows strong TH2 potentiating effects. In vivo administration of this cytokine induces the expression of IL-4, IL-5 and IL-13, and leads to severe pathological changes in mucosal organs like the lung and digestive tract [6J. Intraperitoneal administration of IL-33 also result in increased numbers of eosinophils and lymphocytes and elevated levels of IgE and IgA [6J. IL-33 mediates its effects through the IL-I orphan receptor ST2. This receptor has been shown to act as a negative regulator of Toll like receptor and IL-I receptor signaling [7J. The ST2 receptor is expressed primarily on mast cells and on TH2 cells and has in earlier studies also been linked to TH2 type effector functions [8-13J. Interestingly, administration of antagonistic antibodies against ST2 or a soluble ST2 receptor have inhibitory effects on TH2 associated allergic airway inflammation and promotes THl responses [9, 13J.

The Prior Art

Patent applications describing the use of monoclonal antibodies or soluble receptors for blocking the activity of IL-33 has been filed. These strategies for targeting IL-33 is dependent on injections of highly purified recombinant protein every two to four weeks possibly for the rest of the life of the patient. A vaccine, as described in this application, could here serve as a major improvement over prior art due to that it rely on injections of recombinant protein in a much smaller scale, maybe as little as 1 / 10 000 compared to the amount needed for treatment with monoclonals or soluble receptor. A vaccines most likely also needs to be administrated one to four times a year as compared to the much more frequent administrations of monoclonals or soluble receptor as described above.

Object of the Invention

The object of the invention is to provide a convenient and cost effective method to treat various inflammatory conditions caused by excessive activation by IL-33. Treatment with a vaccine with a fusion protein consisting of IL-33 (or parts of IL-33) and a foreign carrier protein reduces the levels of free IL-33 and thereby reduces the symptoms caused by excessive release of IL-33.

Summary of the Invention

The above object is achieved according to the invention by a vaccine, which is characterized by containing a protein having the entire amino acid sequence or IL-33 from the species to be vaccinated or a segment larger than 5 amino acids of said amino acid sequence, in its original or multimerized form. The protein may optionally be coupled to one or more heterologous carrier proteins and by optionally containing an adjuvant.

Brief description of the figures

Figure 1 shows the nucleotide and the corresponding amino acid sequence of human IL-33.

Description of the invention

Anti-IL-33 antibodies are produced in the host by active immunization, so called vaccination. By injecting a modified IL-33 molecule into the host the immune system of the host produces a polyclonal antibody response directed against its own IL-33 thereby down regulating the effects of its potentially excessive IL-33 production. It is of major importance to modify the antigen so that the immune system of the host recognize the modified self-protein as a non-self protein. This can be achieved by covalent coupling of non-self amino acid regions to IL-33 or a selected region of IL- 33 from the species to be treated. The peptides within the non-self region then attract and activate non-tolerized T cells, which give help for the potentially auto-reactive B cells.

There are at least four possible strategies to do this modification of the self-protein. One method is to produce a fusion protein between a non-self protein, and the entire or a selected fragment of more than 5 amino acids of self IL-33 in a prokaryotic or eukaryotic expression system. The open reading frame of IL-33, as exemplified by canine and human IL-33 in figure 1, is then first being cloned into a bacterial, fungal or eukaryotic fusion protein vector. This fusion protein construct is then transfected into a mammalian or prokaryotic host for production of the desired fusion protein. The fusion partner can here be any non-self protein of any size from 10 amino acids to several hundred kD. However, it is usually favorable to use a fusion partner of approximately the same size as the self-protein.

Alternatively, an immunodominant peptide can be inserted into the IL-33 structure giving rise to a fusion protein with self-IL-33 sequences on both sides of the foreign peptide.

As a third alternative, a non-modified IL-33 can be produced in a mammalian or prokaryotic host or host cell line and then covalently attached to a carrier protein by chemical coupling.

The fourth alternative, which in our mind less favorable, is to produce selected regions of the IL-33 sequence as synthetic peptides and then to couple these peptides to a foreign carrier molecule by chemical coupling. This fourth alternative usually results, after injection into the patient, in antibody responses that show low binding activity against the native properly folded protein and thereby in lower clinical effect.

Following production the vaccine antigen is then purified and tested for pyrogen content and potential content of other contaminants. In order to obtain sufficiently strong immune response against the self-epitopes the vaccine antigen is then (optionally) mixed with an adjuvant before injection into the patient. After administration in the patient the vaccine induces an immune response against the vaccine antigen. Due to the presence of self-epitopes in the vaccine antigen this protein also induces an antibody response against the target molecule, here IL-33, thereby reducing the levels of this protein in the patient.

References

[ 1 1 Hellman L. Profound reduction in allergen sensitivity following treatment with a novel allergy vaccine. Eur J Immunol 1994;24(2):415-20.

[21 Hellman L. Is vaccination against IgE possible? Adv Exp Med Biol 1996;409:337-42.

[31 Hellman L, Carlsson M. Allergy vaccines: A review of developments. Clin Immunotherapeutics 1995;?:?

[4] Vernersson M, Ledin A, Johansson J, Hellman L. Generation of therapeutic antibody responses against IgE through vaccination. Faseb J 2002; 16(8): 875 -7. [5J Ledin A, Bergvall K, Hillbertz NS, Hansson H, Andersson G, Hedhammar A, et al. Generation of therapeutic antibody responses against IgE in dogs, an animal species with exceptionally high plasma IgE levels. Vaccine 2006;24(l):66-74. [6] Schmitz J, Owyang A, Oldham E, Song Y, Murphy E, McClanahan TK, et al. IL-33, an interleukin-1-like cytokine that signals via the IL-I receptor-related protein ST2 and induces T helper type 2-associated cytokines. Immunity 2005;23(5):479-90. [71 Brint EK, Xu D, Liu H, Dunne A, McKenzie AN, O'Neill LA, et al. ST2 is an inhibitor of interleukin 1 receptor and Toll-like receptor 4 signaling and maintains endotoxin tolerance. Nat Immunol 2004;5(4):373-9.

L81 Coyle AJ, Lloyd C, Tian J, Nguyen T, Erikkson C, Wang L, et al. Crucial role of the interleukin 1 receptor family member T1/ST2 in T helper cell type 2-mediated lung mucosal immune responses. J Exp Med 1999;190(7):895-902. |9| Lohning M, Stroehmann A, Coyle AJ, Grogan JL, Lin S, Gutierrez-Ramos JC, et al. T1/ST2 is preferentially expressed on murine Th2 cells, independent of interleukin 4, interleukin 5, and interleukin 10, and important for Th2 effector function. Proc Natl Acad Sci U S A 1998;95(12):6930-5. l lθ| Lohning M, Grogan JL, Coyle AJ, Yazdanbakhsh M, Meisel C, Gutierrez- Ramos JC, et al. T1/ST2 expression is enhanced on CD4+ T cells from schistosome egg-induced granulomas: analysis of Th cell cytokine coexpression ex vivo. J Immunol 1999;162(7):3882-9.

[1 11 Moritz DR, Rodewald HR, Gheyselinck J, Klemenz R. The IL-I receptor- related Tl antigen is expressed on immature and mature mast cells and on fetal blood mast cell progenitors. J Immunol 1998;161(9):4866-74.

| 12| Townsend MJ, Fallon PG, Matthews DJ, Jolin HE, McKenzie AN. T1/ST2- deficient mice demonstrate the importance of T1/ST2 in developing primary T helper cell type 2 responses. J Exp Med 2000;191(6): 1069-76.

113 J Xu D, Chan WL, Leung BP, Huang F, Wheeler R, Piedrafita D, et al. Selective expression of a stable cell surface molecule on type 2 but not type 1 helper T cells. J Exp Med 1998;187(5):787-94.

Claims

1. Vaccine comprising IL-33 or at least one fragment thereof and pharmaceutically acceptable adjuvants.

2. Vaccine according to claim 1, wherein at least one fragment of IL-33 in its original or multimerized form is coupled to a carrier molecule.

3. Vaccine according to claim 1 or 2, wherein the IL-33 is human, canine, feline or equine IL-33.

4. An IL-33 vaccine for use in medicine.

5. A process for the preparation of a vaccine according to claim 1 characterized by cloning of the cDNA, or a genomic sequence of IL-33 or a region encoding more than 5 amino acids thereof, ligating the same into a suitable vector, transforming the vector into an eukaryotic or prokaryotic host cell for the production of a fusion protein, containing the entire IL-33 sequence or a region thereof its original or in an mutated or multimerized form, and purifying and optionally mixing the obtained fusion protein with a suitable adjuvant.

6. The use of a fusion protein consisting of the entire or parts of IL-33 from the species to be treated and a foreign carrier protein for production of a vaccine for medical use.