SE532250C2

SE532250C2 - New formulations of IL-33 for the treatment of inflammatory conditions with a strong TH2 component by vaccination

Info

Publication number: SE532250C2
Application number: SE0602549A
Authority: SE
Inventors: Lars Hellman
Original assignee: Theravac Pharmaceuticals Ab
Priority date: 2006-11-28
Filing date: 2006-11-28
Publication date: 2009-11-24
Also published as: WO2008066443A1; SE0602549L

Description

532 250 IL-33 är en IL-1 liknande tillväxtfaktor som uppvisar kraftiga TH2-förstärkande effekter. In vivo administrering av denna tillväxtfaktor inducerar uttryck av lL-4, lL-S and IL-13, och leder till svåra patologiska förändringar i mukosala organ som lunga och tarmkanal [6]. Intraperitoneal administrering av IL-33 resulterar också i ökat antal eosinofiler och lymfocyter och ökade nivåer av IgE och IgA [6]. IL-33 får sin effekt genom att binda till receptorn ST2. Denna receptor har visats negativt reglera Toll- liknande receptorers och IL-1 receptorns signalering (71. ST2 receptorn är uttryckt främst på mast celler och på TH2 celler, och har även i tidigare studier visat sig kopplad till effektorfunktioner av TH2-typ [8-131. Vad som är intressant är att administrering av antagonistiska antikroppar mot ST2 eller med lösli g ST2 receptor så erhålls en inhibitorisk effekt på TH2 associerad allergisk luftvägsinflammation och THl svar förstärks [9, 131. 532 250 IL-33 is an IL-1-like growth factor that exhibits potent TH2-enhancing effects. In vivo administration of this growth factor induces the expression of IL-4, IL-S and IL-13, and leads to severe pathological changes in mucosal organs such as the lung and intestinal tract [6]. Intraperitoneal administration of IL-33 also results in increased eosinophils and lymphocytes and increased levels of IgE and IgA [6]. IL-33 gets its effect by binding to the receptor ST2. This receptor has been shown to negatively regulate the signaling of Toll-like receptors and the IL-1 receptor (71. The ST2 receptor is expressed mainly on mast cells and on TH2 cells, and has also in previous studies been shown to be linked to TH2-type effector functions [8- 131. What is interesting is that administration of antagonistic antibodies to ST2 or with soluble ST2 receptor obtains an inhibitory effect on TH2-associated allergic airway inflammation and enhances TH1 response [9, 131.

Yad som tidigare var känt inom fältet "Prior Art".Yad formerly known in the field "Prior Art".

Patentansökningar som beskriver användningen av monoklonala antikroppar och lösliga receptorer för blockering av IL-33s aktivitet har sänts in för erhållande av patent. Dessa strategier för att målsöka lL-33 är baserade på injektionen av högrenade rekombinanta proteiner var annan till var fjärde vecka under resten av patientens liv.Patent applications describing the use of monoclonal antibodies and soluble receptors to block IL-33 activity have been filed to obtain patents. These strategies for targeting IL-33 are based on the injection of highly purified recombinant proteins every two to four weeks for the rest of the patient's life.

Ett vaccin som beskrivs i denna patentansökan kan här komma att bli en avsevärd förbättring över tidigare känd teknik (prior art) p.g.a. att den bygger på injektion av rekombinanta proteiner i mycket mindre mängd, kanske så lite som en 10 000 del av vad som behövs vid behandling med monoklonala antikroppar eller lösliga receptorer.A vaccine described in this patent application may here be a significant improvement over prior art (prior art) due to that it is based on the injection of recombinant proteins in much smaller amounts, perhaps as little as 10,000 part of what is needed for treatment with monoclonal antibodies or soluble receptors.

Ett vaccin kommer troligtvis även att behöva administreras 2-4 gånger om året i jämförelse med de mycket mer frekventa administreringarna av monoklonaler och lösliga receptorer som beskrivits här ovan. i U ggﬁnningens mål Målet med uppfinningen är att kunna erbjuda en lätt och kostnadseffektiv metod för behandling av olika inflammatoriska sjukdomar orsakade av överdriven IL-33- medierad aktivering. Vaccination med ett fusionsprotein bestående av lL-33 (eller delar av IL-33) och ett främmande bärarprotein för att reducera nivåerna av fritt IL-33 och därmed mildra symptomen som orsakats av förhöjd frisättning av IL-33.A vaccine will probably also need to be administered 2-4 times a year compared to the much more frequent administrations of monoclonals and soluble receptors described above. Objectives of the Invention The object of the invention is to be able to offer an easy and cost-effective method for the treatment of various inflammatory diseases caused by excessive IL-33-mediated activation. Vaccination with a fusion protein consisting of IL-33 (or parts of IL-33) and a foreign carrier protein to reduce the levels of free IL-33 and thereby alleviate the symptoms caused by increased release of IL-33.

Sammanfattning av uppfinningen Målet som beskrivits ovan uppnås enligt uppfinningen med hjälp av ett vaccin som karaktäriseras av att det innehåller hela aminosyrasekvensen eller segment större än 5 aminosyror av IL-33 i dess ursprungliga eller multimeriserade form. Proteinet kan kopplas till ett eller ﬂera heterologa bärarproteinet och även innehålla ett adjuvans.Summary of the Invention The object described above is achieved according to the invention by means of a vaccine characterized in that it contains the entire amino acid sequence or segments larger than 5 amino acids of IL-33 in its original or multimerized form. The protein may be linked to one or more of the heterologous carrier proteins and may also contain an adjuvant.

Kgrtfatgtd beskﬁvning av ﬁgygerna Figur 1 visar nukieotid och den motsvarande aminosyra sekvensen för människans IL-33. 532 250 Beskrivning av uppfinningen Anti-IL-33 antikroppar produceras i patienten genom aktiv immunisering, vilket även kallas vaccination. Genom att injicera en modifierad IL-33 molekyl så börjar patientens immunsystem att producera ett polyklonalt antikroppssvar riktad mot dess eget IL~33 och reducerar därmed effektema av en för kraftig IL-33-produktion. Det är av största vikt att modifiera anti genet så att patientens immunsystem uppfattar vaccinantigenet som ett främmande protein. Detta kan åstadkommas genom kovalent koppling av ett främmande (icke själv) protein eller del av protein till hela eller delar av IL-33 från den art som skall behandlas. Peptidregioner inom det främmande, icke kroppsegna proteinet drar då till sig och aktiverar icke toleriserade T celler som kan ge hjälp till potentiellt autoreaktiva B-celler.Brief Description of the Eyes Figure 1 shows nucleotide and the corresponding amino acid sequence of human IL-33. Description of the Invention Anti-IL-33 antibodies are produced in the patient by active immunization, which is also called vaccination. By injecting a modified IL-33 molecule, the patient's immune system begins to produce a polyclonal antibody response directed against its own IL-33, thereby reducing the effects of excessive IL-33 production. It is of utmost importance to modify the antigen gene so that the patient's immune system perceives the vaccine antigen as a foreign protein. This can be accomplished by covalently coupling a foreign (non-self) protein or portion of protein to all or part of IL-33 of the species to be treated. Peptide regions within the foreign, non-native protein then attract and activate intolerant T cells that may aid potentially autoreactive B cells.

Det finns minst fyra olika sätt att göra denna modiﬁering av självproteinet. En metod är att producera vaccinantigenet som ett fusionsprotein mellan ett främmande protein och hela eller en utvald del på mer än 5 aminosyror i längd av själv- lL-33 i ett prokaryot eller eukaryot expressions system. Den öppna läsramen för IL-33, t.ex. humant IL-33 som kan ses i figur 1 klonas först ini bakteriell, svamp eller eukaryot fusionsproteinvektor. Denna vektorkonstruktion transfekteras sedan in i en eukaryot eller prokaryot värdcell (beroende på vald vektor) för produktion av det önskade proteinet. Denna fusionspartner kan här vara vilket för kroppen främmande protein som helst av en storlek från 10 aminosyror till flera hundra kD. Det är dock vanligen bäst att använda en fusionspartner av liknande storlek som självproteinet.There are at least four different ways to do this modification of the self-protein. One method is to produce the vaccine antigen as a fusion protein between a foreign protein and a whole or a selected portion of more than 5 amino acids in length of self-LL-33 in a prokaryotic or eukaryotic expression system. The open reading frame for IL-33, e.g. human IL-33 as seen in Figure 1 is first cloned into bacterial, fungal or eukaryotic fusion protein vector. This vector construct is then transfected into a eukaryotic or prokaryotic host cell (depending on the selected vector) to produce the desired protein. This fusion partner can here be any foreign protein of any size from 10 amino acids to several hundred kD. However, it is usually best to use a fusion partner of similar size to the self-protein.

Alternativt kan en immunodominant peptid sättas in i den valda IL-33 sekvensen vilket resulterar i ett fusionsprotein med själv IL-33 sekvenser på båda sidor om den främmande peptiden.Alternatively, an immunodominant peptide may be inserted into the selected IL-33 sequence resulting in a fusion protein with itself IL-33 sequences on both sides of the foreign peptide.

Som ett tredje alternativ kan det icke modifierade IL-33 produceras i en eukaryots eller prokaryot värd eller värdcell och sedan kovalent kopplas till ett bärarprotein genom kemisk koppling.As a third alternative, the unmodified IL-33 can be produced in a eukaryotic or prokaryotic host or host cell and then covalently linked to a carrier protein by chemical coupling.

Det fjärde alternativet som i vår mening är mindre fördelaktigt är att producera en utvald del av IL-33s aminosyrasekvens som en syntetisk peptid och sedan koppla denna peptid till ett främmande bärarprotein genom kemisk koppling. Detta alternativ resulterar normalt, efter injektion i patienten, i antikroppssvar som uppvisar låg bindningsaktivitet mot nativt rätt veckat protein vilket därmed ger sämre klinisk effekt.The fourth alternative, which in our opinion is less advantageous, is to produce a selected part of the amino acid sequence of IL-33 as a synthetic peptide and then couple this peptide to a foreign carrier protein by chemical coupling. This alternative normally results, after injection in the patient, in antibody responses that show low binding activity against native correctly folded protein, which thus gives a poorer clinical effect.

Efter produktion av vaccinantigenet skall det sedan testas för pyrogeninnehåll och möjligt innehåll av andra kontaminerande ämnen. För att erhålla ett tillräckligt starkt immunsvar mot självproteinet kan det blandas med en immunstimulerande substans, ett adjuvans före injektion i patienten. Efter injektion i patienten induceras vaccinet ett immunsvar mot vaccinantigenet. På grund av närvaron av självepitoper i vaccinantigenet så inducerar detta protein ett antikroppssvar mot denna målmolekyl, i detta fall IL-33, vilket leder till en sänkning av nivåerna av detta protein i patienten.After production of the vaccine antigen, it must then be tested for pyrogenic content and possible content of other contaminants. To obtain a sufficiently strong immune response to the self-protein, it can be mixed with an immunostimulatory substance, an adjuvant before injection into the patient. After injection into the patient, the vaccine induces an immune response to the vaccine antigen. Due to the presence of self-epitopes in the vaccine antigen, this protein induces an antibody response to this target molecule, in this case IL-33, which leads to a decrease in the levels of this protein in the patient.

Referencer [1] Hellman L. Profound reduction in allergen sensitivity following treatment with a novel allergy vaccine. Eur J Immunol 1994;24(2):4l5-20. [2] Hellman L. Is vaccination against IgE possible? Adv Exp Med Biol l996;409:33742. 532 250 fw' [3] 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. [5] 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(1):66-74. [6] Schmitz J, Owyang A, Oldham E, Song Y, Murphy E, McClanahan TK, et al. lL-33, an interleukin-1-like cytokine that signals via the lL-l receptor-related protein ST2 and induces T helper type 2-associated cytokines. Immunity 2005 ;23(5):479-90. [7] 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 rnaintains endotoxin tolerance. Nat Immunol 2004;5(4):373-9. [8] 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 Z-mediated lung mucosal immune responses. J Exp Med l999;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. References [1] Hellman L. Profound reduction in allergen sensitivity following treatment with a novel allergy vaccine. Eur J Immunol 1994; 24 (2): 41-20. [2] Hellman L. Is vaccination against IgE possible? Adv Exp Med Biol l996; 409: 33742. 532 250 fw '[3] 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. Phase J 2002; 16 (8): 875-7. [5] 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 (1): 66-74. [6] Schmitz J, Owyang A, Oldham E, Song Y, Murphy E, McClanahan TK, et al. lL-33, an interleukin-1-like cytokine that signals via the lL-1 receptor-related protein ST2 and induces T helper type 2-associated cytokines. Immunity 2005; 23 (5): 479-90. [7] 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. [8] 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 Z-mediated lung mucosal immune responses. J Exp Med l999; 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.

[10] 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. [10] 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.

[11] Moritz DR, Rodewald HR, Gheyselinck J, Klemenz R. The IL-1 receptor- related Tl antigen is expressed on immature and mature mast cells and on fetal blood mast cell progenitors. J Immunol 1998;16l(9):4866-74. [11] Moritz DR, Rodewald HR, Gheyselinck J, Klemenz R. The IL-1 receptor-related Tl antigen is expressed on immature and mature mast cells and on fetal blood mast cell progenitors. J Immunol 1998; 16l (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. [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.

[13] Xu D, Chan WL, Leung BP, Huang F, Wheeler R, Piedraﬁta D, et al.[13] Xu D, Chan WL, Leung BP, Huang F, Wheeler R, Piedra ﬁ ta 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.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

1 532 250 Patent claims

A vaccine containing IL-33 or a fragment thereof in its original or multimerized form linked to a non-native carrier molecule which is preferably administered together with a pharmacologically acceptable adjuvant. A vaccine according to claim 1 wherein IL-33 is from a human, dog, cat or horse. Vaccine against IL-33 according to claims 1 and 2 for the treatment of allergy, asthma or other inflammatory conditions. Use of a fusion protein containing all or part of IL-33 from the species to be treated coupled with a foreign carrier protein for the manufacture of a vaccine for the treatment of allergy, asthma or other inflammatory conditions.