HUE035187T2 - Triszaccharid-származékok és adjuvánsként történõ alkalmazásuk - Google Patents

Description

(12) EUROPEAN PATENT SPECIFICATION (45) Date of publication and mention (51) IntCI.: of the grant of the patent: C07H 13106 <2006 01> A61K 39139 <2006 01> 29.11.2017 Bulletin 2017/48 (86) International application number: (21) Application number: 11725217.1 PCT/NL2011/050393 (22) Date of filing: 03.06.2011 (87) International publication number: WO 2011/155822 (15.12.2011 Gazette 2011/50)

(54) TRISACCHARIDE DERIVATES, AND THEIR USE AS ADJUVANTS

TRISACCHARIDDERIVATE UND IHRE VERWENDUNG ALS ADJUVANTIEN DERIVES DE TRIHOLOSIDE ET LEUR UTILISATION ΕΝ TANT QU’ADJUVANTS (84) Designated Contracting States: (74) Representative: Nederlandsch Octrooibureau AL AT BE BG CH CY CZ DE DK EE ES FI FR GB P.O. Box 29720 GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO 2502 LS The Hague (NL)

PL PT RO RS SE SI SK SM TR (56) References cited: (30) Priority: 11.06.2010 EP 10165707 EP-A1- 1 104 767 EP-A1- 1 792 607 WO-A1-88/06143 US-A- 4 232 150 (43) Date of publication of application: US-A1-2009 042 816 17.04.2013 Bulletin 2013/16 • YAO, Q. ET AL.: "Chemoenzymatic synthesis of (73) Proprietor: Immunovo B.V. iGb3 and Gb3", ORGANIC LETTERS, vol. 8, no. 6, 5223 DE ’s-Hertogenbosch (NL) 2006, pages 911-914, XP002629317, • ΑΚΟΗ, C.C. AND SWANSON, B.G.: "One stage (72) Inventors: synthesis of raffinose fatty acid polyesters", • VAN BREE, Johannes, Gernardus, Mathias, Marie JOURNAL OF FOOD SCIENCE, vol. 52, no. 6, NL-5223 DE ’s-Hertogenbosch (NL) 1987, pages 1570-1576, XP9146242, • SCHENKELAARS, Everardus, Joannes, Peter, · MOGEMARK, M. ET AL.: "Influence of saccharide

Maria size on the cellular immune response to

NL-5223 DE ’s-Hertogenbosch (NL) glycopeptides", ORGANIC &amp; BIOMOLECULAR • TURKSTRA, Jouwert, Anne CHEMISTRY, vol. 1, 2003, pages 2063-2069, NL-8252 HA Dronten (NL) XP002629319, • KRIEK, Maria, Aldegonda, Jacoba · ARNDT P ET AL: "Preparation of Cellulose NL-8245 CV Leylstad (NL) Oligomers from Cellulose Triacetate (Standard

• HOF, Robert, Patrick Procedure)", CELLULOSE, KLUWER ACADEMIC NL-9745 DA Groningen (NL) PUBLISHERS, DO, vol. 12, no. 3,1 June 2005 • SCHAAPER, Wilhelmus, Martinus, Maria (2005-06-01), pages317-326,XP019234560, ISSN: NL-1313 Almere (NL) 1572-882X, DOI: DOI:10.1007/S10570-004-3387-6

Note: Within nine months of the publication of the mention of the grant of the European patent in the European Patent Bulletin, any person may give notice to the European Patent Office of opposition to that patent, in accordance with the Implementing Regulations. Notice of opposition shall not be deemed to have been filed until the opposition fee has been paid. (Art. 99(1) European Patent Convention).

Description

FIELD OF THE INVENTION

[0001] The present invention relates to novel trisaccharide derivates and their use as adjuvants, which trisaccharide derivates comprise a substituted trisaccharide core, which trisaccharide core is fully substituted with fatty acid ester groups, and optionally one or more anionic groups, the invention further relates to a method for preparing these trisaccharide derivates, to trisaccharides obtainable by this method, and to the use of the adjuvant in a vaccine.

BACKGROUND OF THE INVENTION

[0002] Antibodies are substances contained in the blood and other fluids of the body, as well as in the tissues, and which bind to antigen to make it innocuous. Antibodies constitute one of the natural defence mechanisms of the body. They are highly specific and they kill, bind or make innocuous the antigen which induced their formation.

[0003] The antigen in contact with the immune system, thus activates a complex series of cellular interactions to eliminate the antigen and/or to re-establish the preceding equilibrium.

[0004] Two of the characteristic features of antigens are their immunogenicity, that is their capacity to induce an immune response in vivo (including the formation of specific antibodies), and their antigenicity, that is their capacity to be selectively recognized by the antibodies whose origins are the antigens.

[0005] It is known how to stimulate the immune response deliberately by administering a specific antigen by means of a vaccine. The procedure allows the retention of a state of immune response in the organism which allows a more rapid and more effective response of the organism during subsequent contact with the antigen.

[0006] However, some antigens have only a weak immunogenicity and they induce an insufficient immune response to produce an effective protection for the organism. This immunogenicity can significantly be improved if an antigen is co-administered with an adjuvant.

[0007] Adjuvants are substances that enhance the immune response to antigens, but are not necessarily immunogenic themselves. Adjuvants may act by retaining the antigen locally near the site of administration to produce a depot effect facilitating a slow, sustained release of antigen to cells of the immune system. Adjuvants can also attract cells of the immune system to an antigen depot and stimulate such cells to elicit immune responses.

[0008] Adjuvants have been used for many years to improve the host immune reponse to, for example, vaccines. Intrinsic adjuvants are normally the components of the killed or attenuated bacteria used as vaccines. Extrinsic adjuvants are immunomodulators which are typically non-covalently linked to antigens and are formulated to enhance the host immune response.

[0009] Aluminium hydroxide and aluminium phosphate (collectively referred to as alum) are routinely used as adjuvants in human and veterinary vaccines. The efficacy of alum in increasing antibody responses to diphtheria and tetanus toxoids is well established and, more recently, a HBsAg vaccine has been adjuvanted with alum.

[0010] A wide range of extrinsic adjuvants can provoke immune responses to antigens. These include saponins complexed to membrane protein antigens (immune stimulating complexes), pluronic polymers with mineral oil, killed mycobacteria in mineral oil, Freund’s complete adjuvant, bacterial products, such as muramyl dipeptide (MDP).

[0011] Chemically defined adjuvants, such as monophosphoryl lipid A, phospholipid conjungates have been investigated (see Goodman-Snitkoff et al., J. Immunol. 147:410-415(1991) as has encapsulation of the protein with a proteol-iposome (see Miller et al., J. Exp. Med. 176:1739-1744 (1992)).

[0012] Synthetic polymers have also been evaluated as adjuvants. These include the homo- and copolymers of lactic and glycolic acid, which have been used to produce microspheres that encapsulate antigens (see Eldridge et al., Mol. Immunol. 28:287-294 (1993)).

[0013] Nonionic block copolymers are another synthetic adjuvant being evaluated. Adjuvant effects have also been investigated for low molecular weight copolymers in oil-based emulsions (see Hunter et al., The Theory and Partical Application of Adjuvants (Ed. Stewart-Tull, D.E.S.) John Wiley and Sons, NY. Pp. 51-94 (1995)) and for high molecular weight copolymers in aqueous formulations (Todd et al., Vaccin 15:564-570 (1997)).

[0014] Desirable characteristics of ideal adjuvants are lack of toxicity and an ability to stimulate a long lasting immune response. One of the most commonly used adjuvants in humans is alum. Other adjuvants, such as Saponin, Quil A and the water in oil adjuvant, Freund’s with killed tubercle bacilli (Freund’s complete) or without bacilli (Freud’s incomplete), have had limited use in humans due to their toxic effects; and concerns have been raised as to undesirable effects in animals.

[0015] Simply said, many adjuvant formulations have been described but most are never accepted for routine vaccines, and few have been approved for use in humans. This is mainly due to their toxicity. For example, the mineral oils used as adjuvants in certain animal vaccines are not readily degraded and persist at the site of injection thereby causing unacceptable granulomas; and, in general adjuvant formulations such as mineral compounds oil emulsions, liposomes and biodegradable polymer microspheres cause local reactions due to depot formation at the site of injection.

[0016] Examples of adjuvants presently approved in human vaccines include Alum, MF59 (an oil in water emulsion), MPL (a glycolipid), VLR, Immunopotentiating Reconstituted Influenza Virosomes (IRIV) and cholera toxin (see Reed et al. Trends in Immunology 30:23-32 (2008).

[0017] One group of adjuvants known in the art are the so called sulpholipopolysaccharides, i.e. polysaccharides containing both fatty acid esters and sulphate esters (Hilgers et al., Immunology 60., pp. 141-146, 1986). A method for preparing these compounds has been described in the international patent application WO96/20222 and WO 96/20008. These methods for preparing sulpholipopolysaccharides result in the formation of different sulpholipopolysaccharides derivates varying in the number of fatty acids esters present per polysaccharide molecule, the number of sulphate esters present per polysaccharide molecule, the number of hydroxyl groups per polysaccharide molecule and the distribution of the fatty acid esters, the sulphate esters and the hydroxyl groups over the polysaccharide molecule. This means that during preparation of these sulpholipopolysaccharides a mixture is obtained of many different sulpholipopolysaccharides. Consequently, the yield of the desired sulpholipopolysaccharide is relatively low or the adjuvant needs to be used as a difficult to characterise mixture causing regulatory issues.

[0018] In the European patent EP 1233969 an adjuvant composition is claimed which adjuvant comprises sulpholipo-disaccharides. Also a method is described for preparing these sulpholipodisaccharides. In one of the embodiments the sulpholipodisaccharides prepared are fully substituted with fatty acid ester or sulphate ester groups. However, as will be further described in the following, when these sulpholipodisaccharides are used as adjuvants in animals, undesired side effects such as occurrence of mean body temperature rise (including fever) and local irritation (tissue swelling) occur. [0019] EP1104767 A1 discloses mono- and disaccharides containing both fatty acid ester and sulphate ester groups useful inter alia as adjuvants for vaccines.

DETAILED DESCRIPTION OF THE INVENTION

[0020] In view of the foregoing it is an object of the present invention to provide compounds which are relatively easy and inexpensive to prepare, have good adjuvating properties and induce a minimum of undesired side effects when used clinically. It is a further object of the present invention to provide compounds that can be used in an adjuvant composition, for exam pie in combination with a vaccine, which compounds have an excellent safety and side effect profile. [0021] A first and second aspect of the present invention relates to trisaccharide derivates and their use as an adjuvant. The trisaccharide derivates according to the present invention comprise a substituted trisaccharide core, which trisaccharide core is fully substituted with fatty acid ester groups, and optionally one or more anionic groups.

[0022] The trisaccharide derivates according to the present invention are highly suitable for use as adjuvant for vaccines. They have a side effect profile which is surprisingly significantly better than the side effect profile of other adjuvants which are based on polysaccharide derivates, such as for example adjuvants based on disaccharides. Animals which have been vaccinated with an antigen composition and an adjuvant composition comprising the trisaccharide derivates according to the invention show less increase in mean body temperature compared with for example the disaccharide derivates of EP 1233969. Also the occurrence of local reactions (tissue swelling) around the area of injection is lower when an adjuvant is used which comprises the trisaccharide derivates according the invention.

[0023] The term antigen as used herein, refers to any component or material that induces a immunological reaction in the human or animal body, such as a virus, a bacterium, mycoplasma, a parasite or a tumor cell, a subunit of a microorganism, such as a protein, polysaccharide, peptide, glycoprotein, polysaccharide-protein conjugate, peptide-protein-conjugate.

[0024] The antigen can for example consist of or contain one or more live organisms, inactivated organisms, or so-called subunits (the latter e. g. prepared synthetically, or by recombinant DNA methods, or isolated from the organisms). The term antigen further refers to any component that can induce an immune reaction in the human or animal body. [0025] The trisaccharide core of the derivates of the present invention is preferably derived from raffinose, melezitose, maltotriose, nigerotriose, maltotriulose or kestose. It is particularly preferred that the trisaccharide core is derived from raffinose, melezitose or maltotriose, most preferably raffinose or maltotriose. These trisaccharides have in their normal, i.e. unsubstituted form eleven OH-groups which are available for reactions such as for example esterificaction with fatty acids. However, it is also possible that one or more, preferably one, of the OH-groups has reacted with an anionic group, such that for exam pie a sulphate ester or phosphate ester group is obtained, preferably a sulphate ester group is obtained. [0026] In a preferred embodiment of the present invention the trisaccharide derivates according to the invention comprises no anionic groups but only fatty acids groups, preferably identical fatty acid groups.

[0027] According to another preferred embodiment the trisaccharide derivates according to the invention comprise one or two anionic groups with ten or nine fatty acid groups, respectively, per substituted trisaccharide core. Preferably the fatty acid groups are identical.

[0028] The term anionic group as used herein refers to a negatively charged moiety (i.e. negatively charged at neutral pH or the pH of the environment in which the derivate is applied). Such an anionic group may for example be a sulphate, a sulphonate or a phosphate. Preferred anionic groups include sulphate ester groups or phosphate ester groups. Examples of such groups are-O-SO2-ONa or-O-SO2-ONH4, -O-SO2-OTEA (i.e.sulphate triethylammonium).

[0029] In a preferred embodiment of the present invention the fatty acid ester group which is covalently bound to the substituted trisaccharide core is an ester of a straight, branched, saturated or unsaturated fatty acid with a chain length of 4 to 20 carbon atoms, preferably 6 to 18, more preferably 8 to 16 carbon atoms, most preferably 10-14 and highly preferred 12 carbon atoms.

[0030] Although it is within the scope of the present invention that the substituted trisaccharide core is substituted with more than one type of fatty acid ester, it is preferred that only one type is used, i.e. that all fatty acid esters are identical. [0031] The use of fatty acids is highly preferred, however it is also envisaged by the present invention that other carboxylic acids, preferably closely related to fatty acids, may provide favourable results.

[0032] Preferably, the fatty acid ester is the ester of saturated fatty acids, monounsaturated fatty acids or polyunsaturated fatty acids, such as butyric acid, caproic acid, caprylic acid, capric acid, lauric acid, myristic acid, palmitic acid, palmitoleic acid, stearic acid, ricinoleic acid, vaccenic acid, arachidic acid, gadoleic acid, arachidonic acid, oleic acid or linoleic acid. Most preferably lauric acid.

[0033] In a preferred embodiment the substituted trisaccharide core is derived from raffinose, melezitose or maltotriose and is the trisaccharide derivate fully substituted with identical fatty acid ester groups per substituted trisaccharide, i.e. the trisaccharide core is substituted with eleven identical fatty acid ester groups.

[0034] In another preferred embodiment the substituted trisaccharide core is derived from raffinose, melezitose or maltotriose and comprises one or two anionic groups, such as a sulphate ester or a phosphate ester group, and ten or nine, respectively, identical fatty acid ester groups per substituted trisaccharide. Most preferably the fatty acid ester is the ester of lauric acid.

[0035] A third aspect of the present invention relates to a method for preparing a trisaccharide derivate comprising the steps of: i) providing a trisaccharide and dissolving it in a solvent; andii) esterifying all OH-groups of the trisaccharide with a fatty acid, or source thereof, optionally reacting at least one of the OH-groups of the trisaccharide with an anionic agent.

[0036] Due to the fact that all the OH-groups are reacted with anionic groups and/or fatty acids, few impurities are being formed. This means that it is possible to obtain without extensive purification steps a pharmaceutically acceptable pure form of an envisaged trisaccharide derivate. If only one kind of fatty acid is used, such as lauric acid, even less purification is needed to obtain the desired trisaccharide derivate in a pharmaceutically acceptable pure form. Another advantage of the method of the present invention is that the desired trisaccharide derivates are easily obtained in relatively large quantities, making the method economically attractive.

[0037] In a preferred embodiment of the present invention the trisaccharide derivates prepared comprise no anionic groups but only fatty acids groups, preferably identical fatty acid groups, i.e. all OH-groups have reacted with a fatty acid or source thereof.

[0038] According to another preferred embodiment the trisaccharide derivates prepared comprise one or two anionic groups with ten or nine fatty acid groups, respectively, per substituted trisaccharide core. Preferably the fatty acid groups are identical.

[0039] The trisaccharides used in the above mentioned method are preferably raffinose, melezitose, maltotriose, nigerotriose, maltotriulose or kestose. More preferably, raffinose, melezitose or maltotriose are used, most preferably maltotriose or raffinose.

[0040] The meaning of fatty acids as used in the above mentioned method refers to any source of fatty acids, including fatty acid salts, fatty acids halides, fatty acid esters and derivates. Preferably the fatty acids used in the claimed method are straight, branched, saturated or unsaturated fatty acids with a chain length of between 4 to 20 carbon atoms, preferably between 6 to 18, more preferably from 8 to 16 carbon atoms, most preferably 10 to 14 carbon atoms, highly preferred 12 carbon atoms.

[0041] Preferably, the fatty acids used are saturated fatty acids, monounsaturated fatty acids or polyunsaturated fatty acids, such as butyric acid, caproic acid, caprylic acid, capric acid, lauric acid, myristic acid, palmitic acid, palmitoleic acid, stearic acid, ricinoleic acid, vaccenic acid, arachidic acid, gadoleic acid, arachidonic acid, oleic acid or linoleic acid. Most preferably lauric acid.

[0042] As mentioned above, in a preferred embodiment of the present invention at least one of the OH groups is reacted with an anionic reagent. Preferably, the anionic agent used is a sulphating or phosphating agent such as gaseous SO3, HCLSO3, SO3.pyridine, SO3-2-methylpyridine, SO3-2,6-dimethylpyridine, SO3-dimethylformamide, SO3-trimethy-lamide, SO3-triethylamine, SO3-dimethylanaline, SO3-N-ethylmorpholine, SO3-diethylalanine, SO3-dioxane and combinations thereof. Most preferably the sulphonating agent is SO3.pyridine or SO3.triethylamine.

[0043] The most preferred sulphating agent is SO3. pyridine. It is further preferred that reacting at least one of the OH- groups of the trisaccharide with a sulphating agent is carried out before the esterification of the trisaccharide with a fatty acid. The advantage of first carrying out the reaction with a sulphating agent is that the sulphating agent reacts first with the so called primary OH-groups before reacting with other OH groups, thereby reducing the number of isomers formed. [0044] Inapreferredembodimenttheratiotrisaccharide : anionic agent: fatty acid equivalents is 1 :0-3 :8-11, preferably 1:0-1:10-11. Within these claimed ranges the complete substitution of the OH-groups of the trisaccharide with fatty acid esters and optionally the anionic group, such as a sulphate ester, is efficiently obtained.

[0045] Preferably, the solvent used for carrying out the reaction is a mixture of pyridine and dimethylformamide. [0046] In an additional step of the present method the trisaccharide derivates are subjected to an additional step of mixing them with a pharmaceutically acceptable excipient or diluent, such that an adjuvant composition is obtained. [0047] A fourth aspect of the present invention relates to adjuvant compositions comprising trisaccharide derivates according to the invention, or a mixture thereof. When such trisaccharide derivates are formulated into an adjuvant composition they are preferably mixed with pharmaceutically acceptable excipients or diluents. Preferably the adjuvant composition is formulated as an oil in water emulsion. Suitable oils to be used are amongst others animal oils, vegetable oils and mineral oils, such as fish oil, vitamine E, sqalane, squalene. Preferably, use is made of squalane, preferably in combination with polysorbate.

[0048] Although it is possible to only use one kind of trisaccharide derivate it is also within the scope of the invention to use in the adjuvant composition a mixture of different trisaccharide derivates according to the invention. In a preferred embodiment a mixture is used of trisaccharide derivates according to the invention with an anionic group, such as a sulphate ester group, and the same trisaccharide derivates without an anionic group, e.g. sulpho-lipo-raffinose and lipo-raffinose. Most preferably the fatty acid esters of the trisaccharides derivates used in such a mixture are the same, such as for example the ester of lauric acid.

[0049] A fifth aspect of the present invention relates to a vaccine comprising the adjuvant composition, or trisaccharide derivate as mentioned above.

[0050] Both the adjuvant composition, antigen composition or vaccine are preferably administered parenterally. Suitable means for parenteral administration include intramuscular, subcutaneous, subdermal and intradermal administration. Suitable devices for parenteral administration include needle (including microneedle) injectors, and transdermal delivery systems.

[0051] The parenteral formulation may readily be prepared by someone skilled in the art according to standard methods. Preferably the parenteral formulation is prepared as an oil in water emulsion.

[0052] The preparation of parenteral formulations under sterile conditions, for example, by filtration may readily be accomplished using standard pharmaceutical techniques well known to those skilled in the art.

[0053] The vaccine or the adjuvant composition according to the invention can be administered to humans and many different target animals, such as for example pigs, cattle, poultry, dogs, cats, horses and the like.

[0054] A sixth aspect of the present invention relates to a kit comprising the above mentioned adjuvant composition and an antigen composition.

[0055] It may be desirable to administer a combination of the adjuvant composition and antigen composition or vaccine separately. In such a case the adjuvant composition and antigen composition may conveniently be combined in the form of a kit. Such a kit could for example be a two vial system or a dual chamber syringe.

[0056] The invention will now be further described by the following, non-limiting examples

EXAMPLES

Preparation of trisaccharide derivates according to the invention

Example 1; general synthesis of sulpho-lipo-trisaccharides according to the invention [0057] A trisaccharide is dried in a vacuum oven to remove the crystal water. The trisaccharide (5 g) is subsequently dissolved, under a stream of nitrogen, in 30 mL of DMF and 14 mL of pyridine in a 100 mL three-neck round-bottom flask, equipped with a reflux-condensor. 1.05 equivalent of pyridine.SO3 is added under vigorous stirring. After 1 hr the flask is cooled in ice water and under vigorous stirring lauroyl chloride is added drop-wise to prevent heating of the reaction mixture. After 15 min the ice bath is slowly heated to 40°C. The progress of the reaction is monitored by HPLC. After completion of the reaction the mixture is concentrated in vacuo in a rotary evaporator (heated up to 60°C). The crude product is taken up in 300 mL heptane and 150 mL brine. The organic layer is separated in a 500 mL separatory funnel, dried on sodium sulfate and filtered. The heptane solution is concentrated in vacuo. The oil obtained is dissolved in 200 mL heptane and triethylamine (2.37 mL) is added drop wise. The solution obtained is filtered and concentrated in vacuo. The oil is dissolved again in 100 mL heptane, filtered and the solution is concentrated in vacuo.

Example 2; synthesis of sulpho-lipo-maltotriose (S1L1 Ο-Ma) via pyridine.SO3 route [0058] The title product was synthesized following the general method described in Example 1 with the following details: maltotriose (5.1 g, 10 mmol) was dried in a vacuum stove at <10 mbarfor 19 hrs at 40°C and 48 hrs at 70°C, yield 4.9 g. The dried maltotriose was dissolved in 30 mL of DMF and 14 mL of pyridine and sulfated with 1.6 g (1.05 eq) of pyridine.SO3, suspended in 2 mL of DMF. After 1 hr the sulfo-trisaccharide was esterified in an ice bath with 11 equivalents (25.5 mL) of lauroyl chloride. The mixture was slowly heated to 40°C and reacted for 3 hrs. Reaction steps were followed by HPLC-ELSD. The product was isolated after extraction and triethylamine exchange. Yield of the thick brown-yellow syrup: 15.5 g + 5.5 g (second crop from evaporation flask after heating to50°C). HPLC-ELSD chromatogram: see fig 1 A.

Example 3; alternative synthesis of sulpho-lipo-maltotriose (S1L10-Ma) via pyridine. S O3 route [0059] The title product was synthesized following the general method described in Example 1 with the following details: maltotriose (5.0 g, 10 mmol) was dried in a vacuum stove at <10 mbar for 20 hrs at 40°C and 90 hrs at 70°C, yield 4.9 g . The dried maltotriose was dissolved in 30 mL of DMF and 1.6 g (1.05 eq) of pyridine.SO3 was added as a suspension in 14 mL of pyridine . After 1 hr the sulpho-trisaccharide was esterified in an ice bath with 11 equivalents (25.2 mL) of lauroyl chloride and was slowly heated to 40°C. Samples for HPLC-ELSD analysis were taken at regular time points in the process. The reaction with lauroyl chloride was left overnight at ambient temperature Work up was done as described in example 1. Yield of the thick brown-yellow syrup: 24.7 g. HPLC-ELSD chromatogram: see fig 1B.

Example 4 Synthesis of sulpho-lipo-raffinose (S1L10-Ra) via pyridine.SO3 route [0060] The title product was synthesized as described in example 1 with the following details: raffinose pentahydrate (5.0 g, 10 mmol) was dried in a vacuum stove at <10 mbar for 24 hrs at 30°C and 90 hrs at 60°C, yield 4.3 g. The dried raffinose was dissolved in 30 mL of DMF and 14 mL of pyridine . Pyridine.SO3 (1.6 g, 1.05 eq) was added in one batch. After 1 hr the sulpho-trisaccharide was esterified in an ice bath with 12 equivalents (23.5 mL) of lauroyl chloride and was slowly heated to 40°C. After 4 hrs, the mixture was concentrated in vacuo, extracted and treated with TEA as described in example 1 .Yield of the thick yellow-brown syrup: 18.0 g. HPLC-ELSD chromatogram: see fig.2.

Example 5 Synthesis of lipo-maltotriose (L11-Ma) [0061] The title product was synthesized following the general method described in Example 1 with the following details: Maltotriose hydrate (0.50 g) was dried in a vacuum oven . The dried trisaccharide (0.49 g, 0.97 mmol) was dissolved in pyridine (1,4 mL) and DMF (3 mL), cooled in an ice bath and reacted with lauroyl chloride (3.36 mL, 15 eq) for 1 hrs atO °C, followed by 16 hrs at room temperature. The reaction mixture became a gel, upon the addition of 3 mL of heptanes and sonification the product dissolved. The addition of heptane was repeated twice. The organic phase (75 mL) was washed with water and a three layer system was formed. Only the organic layer was isolated, dried over sodium sulphate, filtered and concentrated to yield a thick yellow-brown syrup: 1.39 g. HPLC-ELSD chromatogram: see fig.3A.

Example 6 Synthesis of lipo-raffinose (L11-Ra) [0062] Raffinose pentahydrate (0.50 g) was dried in a vacuum oven. The dried raffinose (0.41 g, 0.86 mmol) in pyridine (1,4 mL) and DMF (3 mL) was cooled in an ice bath and reacted with lauroyl chloride (2.97 ml, 15 eq) for 1 hrs atO °C, followed by 18 hrs at room temperature. The reaction mixture was diluted with heptane (50 ml) and washed with water (25 ml). The organic phase was dried over sodium sulphate, filtered and concentrated to yield a thick yellow-brown syrup: 2.33 g. HPLC-ELSD chromatogram: see fig.3B.

Effect of adjuvants comprising the trisaccharide derivates according to the invention on anti GnRH titers, serum testosterone levels and occurrence of adverse effects.

[0063] Animal experiments have been carried out to assess the efficacy and possible adverse effects associated with the use of the trisaccharides according to the invention as an adjuvant. In this study in rats, three (sulpho-) lipo-trisac-charides based adjuvants were tested: sulpho-lipo-maltotriose (one sulphate-ester group and ten lauroyl ester groups, S1L10-Ma), prepared according to Example 3; sulpho-lipo-raffinose (one sulphate-ester group and ten lauroyl ester groups, S1L10-Ra), prepared according to Example 4; and lipo-raffinose (raffinose fully substituted with lauroyl ester groups, L11-Ra), prepared according to Example 6. The adjuvants comprising sulpho-lipo-maltotriose also comprised lipo-maltotriose (fully substituted with lauroyl ester groups, L11-Ma). The adjuvants comprising sulpho-lipo-raffinose also

comprised lipo-raffinose (fully substituted with lauroyl ester groups, L11-Ra).

[0064] Thesulpho-lipo based adjuvants were tested in different doses varying from 0.008-8 mg per dose. The adjuvants were tested in combination with a GnRH-KLH conjugate with 0.7 μg conjugated GnRH per dose. Adjuvanticity of the adjuvants was compared with the positive control adjuvant, sulpho-lipo-sucrose (disaccharide with one sulphate ester group and seven lauroyl acid ester groups, S1L7-Su) and with negative control adjuvant consisting of an squalane-in-water emulsion without saccharide compound and one group receiving PBS only.

[0065] Efficacy was determined by antibody titers and biological effects of the induced antibodies on testosterone levels. In order to determine adverse effects of immunization with the adjuvants, daily clinical observations were made and body temperature and injection site reactions was determined.

Preparation of adjuvant emulsions [0066] « Saccharides: Sulpho-lipo-maltotriose (S1L10-Ma)

Sulpho-lipo-raffinose (S1L10-Ra)

Lipo-raffinose (L11-Ra) « Squalane (A&amp;E Connock) « Polysorbate-80 (Fagron) « Sterile PBS-wit pH 7.4 (Mediabereiding ASG, Lelystad) « MilliQ water « Millex syringe driven filter unit 0.22 μίτι PES, 33 mm, 4,5 cm2 (Millipore)

« Microfluidizer M-110S equipped with interaction chamber type: F20Y « Microtrac Nanotrac Analyzer System NPA-253 [0067] The experimental adjuvants were prepared as follows.

Table 1. Composition of the emulsions for the different SLS

(continued)

[0068] The oil phase components (Saccharide, squalane, polysorbate-80 and MilliQ water, amounts as indicated in table 1) were weighed in a 50 ml Falcon tube. The components were mixed using a vortex and heated in a water bath at 50 °C until the saccharide was dissolved. The warm oil phase was added to the water phase (PBS) and the two phases were mixed by vortex and ultra-turrax at 24000 min-1 for approximately 30 sec with intervals. Subsequently, the emulsion was formed by Microflu id izer processing. The operating pressure was set to 500 kPa (5 bar) and each mixture was passed three times undercooling of the interaction chamber in an ice bath. Each emulsion was subjected to manual sterile filtration. The particle size of the emulsion was measured using a Nanotrac particle sizer.

[0069] Vaccines 1-13 were formulated by adding equal volume of adjuvant (comprising the saccharide compounds as shown in table 2) to the water phase (containing 0.7 μg conjugated GnRH). Vaccine 14 consists of PBS only. The following vaccines were prepared:

Table 2 Experimental design

Animals and immunization [0070] Male Wistar rats, 10 weeks of age, were housed with 3 rats per cage. Rats had ad libitum access to food en water. Rats were immunized at day 0, 14 and 28, according to the experimental design (Table 2) with 400 μΙ vaccine comprising 200 μΙ water phase and 200 μΙ adjuvant. Two intramuscular injections (100 μΙ each) were injected at the left and right inner thigh and 2χ100μΙ was injected subcutaneously in the neck region. Each group consists of 5 rats.

Blood samples for serum were collected from all animals prior to the immunizations and at day 41 and 56

Efficacy of the vaccines

Assays [0071] GnRH specific antibodies were measured by ELISA. Plates (96 wells) were precoated with 0.2% glutardialde-hyde in phosphate buffer (pH 5) for 3 hours at room temperature, washed with 0.1 M phosphate buffer (pH 8) and coated with 100 μΙ perwell of a solution containing 10 μg GnRH (Pepscan Presto, Lelystad, The Netherlands) per ml phosphate buffer (pH 8) and incubated for 3 hours at 37°C. Coated plates were washed with 0.05% Tween-80. Serum samples were diluted (1/10) in PEM (1% Tween-80 with 4% horse serum). This dilution was further diluted in the 96-well plate (100 μΙ perwell, 8 steps) and incubated 1 hour at 37°C. After washing with 0.05% Tween-80, 100 μΙ of goat-anti-rat antiserum conjugated with peroxidase in PEM was added to the wells. Plates were incubated for 1 h at 37°C and washed 12 times with 0.05% Tween-80. Subsequently, 150 μΙ of a substrate solution containing 2,2-azino-bis-(3-benzthiazoline-6-sulphonicacid) (ABTS) plus H2O2 was added to the wells of the plates. Plates were incubated for 45 minutes at ambient room temperature and absorbance was measured at 405 nm. Antibody titer was expressed as the 10 log of the dilution factor that gives an optical density of 4 times background (approx. 100).

[0072] Serum testosterone levels were measured using a commercially available Testosterone EIA (Beckman Coulter, Woerden, The Netherlands) according to the instructions of the manufacturer.

GnRH antibody titers [0073] GnRH antibody titers of groups treated with sulpho-lipo-maltotriose (S1L10-Ma) and sulpho-lipo-raffinose (S1L10-Ra) are depicted in Figure 4A and 4B. Both sulpho-lipo-trisaccharides induced dose dependent antibody titers against GnRH. Antibody titers were substantially higher for rats treated with 0.08-8 mg sulpho-lipo-trisaccharide than in rats treated with adjuvant without saccharide (NS, group 11), demonstrating the significant contribution of the sulpho-lipo-trisaccharide to the immune response.

[0074] Figure 4C clearly shows that GnRH antibody titers of rats that received 8 mg saccharide compounds according to the invention, i.e. S1 L10-Ma-8, S1L10-Ra-8 and L11-Ra-8 were substantially higher than in rats treated with squalane emulsion only (NS), emphasizing strong adjuvanticity of both S1L10- and L11-trisaccharides.

Serum Testosterone [0075] Immunization with GnRH-KLH conjugate emulsified with the adjuvant comprising the sulpho-lipo-trisaccharides according to the invention, i.e.S1L10-Ma and S1L10-Ra, resulted in a dramatic decline of serum testosterone levels from 0.08 mg sulpho-lipo-trisaccharide onwards (Figure 5 A-B), while the effects of the lowest sulpho-lipo-trisaccharide dose (0.008 mg) on serum testosterone were similar to the oil-in-water emulsion without saccharide compound. Immunization with the lipidated trisaccharide (L11-Ra-8) also induced decreasing testosterone levels (Figure 5C).

Adverse effects

Rise of Mean Body Temperature [0076] At least once daily clinical observations were made in all animals. Mean bodytemperature(MBT) was determined for all animals before and after each immunization by rectal temperature. Mean body temperature per group (MBT) are depicted as to preimmunization levels Figure 6.

[0077] At 3 hours after each vaccination, a slight increase in MBT was observed in rats treated with sulpho-lipo-trisaccharides only at 8 and 2 mg dose, while no effects on MBT were noted at 0.8, 0.08 and 0.008 mg. The next day 21 hours after vaccination however, MBT was dropped to almost normal values again (figure 6A and 6B). In contrast, immunization with the disaccharide compound (S1L7-Su-8) which caused a slightly higher increase in MBT than the trisaccharide compounds at 3 hours post vaccination, did not show a decrease in MBT one day post immunization, MBT was still increased at 21 hours after vaccination (figure 6C). The lipidated trisaccharide (LI 1-Ra-8) did not induce any increase in MBT. From Figure 6 it is thus clear that the vaccines comprising the compounds according to the invention (S1L10-trisaccharides) as adjuvants induced surprisingly significantly shorter temperature effects when compared to the disaccharide derivates known in the prior art, while the lipidated trisaccharide (LI 1-trisaccharide) in similarity to the oil-in-water emulsion without a saccharide compound (NS) did not induce any temperature effects at all.

Injection site reactions [0078] Before vaccination the injection sites were assessed for the presence of abnormalities or existing local reactions.

If such abnormalities or local reactions were absent, the animal was injected on that site. After each immunization, injection sites were inspected for tissue swelling. Size of subcutaneous injection sites was measured (diameter in mm). Since the intramuscular injection site reactions in the hind leg is difficult to determine, only the presence of intramuscular injections sites (tissue swelling) was determined and expressed in arbitrary values (present = 10 mm, not present = 0 mm). Scores of injection site reactions were added up per rat for the four injection sites at each inspection,means per group were calculated The results thereof are presented in Figure 7.

[0079] After immunization, minor dose dependent injection site reactions were observed in rats treated with sulpho-lipo-trisaccharides, mainly in rats treated with 8 and /or 2 mg (figure 7A and 7B). Size of the injection site reactions gradually decreased at day 3 after immunization and were almost undetectable at day 5 after the subsequent immunization. Injection site reactions caused by immunization with sulpho-lipo-disaccharide (S1 L7-Su-8) showed a completely different pattern: Injections site reactions increased in size up to day 4 and were more than 4 times bigger than sulpho-lipo-trisaccharides (see figure 7C), moreover at the final inspection 5 days after immunization still significant injection site reactions were present.

[0080] The lipidated trisaccharide (LI 1-Ra-8) did not induce any adverse effects at the site of injection. Clearly, sulpho-lipo-disaccharide comprising vaccine formulations induced more injection site reactions than vaccines comprising sulpho-lipo-trisaccharides according to the invention.

Figures [0081]

Figure 1 A-B: HPLC-ELSD chromatogram of sulpho-lipo-maltotriose, synthesized via pyridine.SO3 route Figure 2: HPLC-ELSD chromatogram of sulpho-lipo-raffinose, synthesized via pyridine.SO3 route Figure 3 A-B: HPLC-ELSD chromatogram of lipo-maltotriose and lipo-raffinose

Figure 4 A-C: GnRH antibody titer (mean) of rats immunized with various vaccine formulations Figure 5 A-C: Serum testosterone levels of rats immunized with various vaccine formulations Figure 6: Mean body temperature of rats immunized with various vaccines formulations Figure 7: Injection site reaction of rats immunized with various vaccine formulations

Claims 1. Use of a trisaccharide derivate comprising a substituted trisaccharide core, which trisaccharide core is fully substituted with fatty acid ester groups, and optionally one or more anionic groups as an adjuvant. 2. Use according to claim 1 .wherein the substituted trisaccharide core is derived from raffinose, melezitose, maltotriose, nigerotriose, maltotriulose or kestose, preferably raffinose, melezitose or maltotriose, most preferably raffinose or maltotriose. 3. Use according to claim 1 or 2, wherein the substituted trisaccharide core comprises one or two sulphate ester or phosphate ester groups as anionic groups. 4. Use according to any of the claims 1-3, wherein the anionic group is a sulphate ester. 5. Use according to any of the claims 1-4, wherein the fatty acid ester group is an esterof a straight, branched, saturated or unsaturated fatty acid with a chain length of 4 to 20 carbon atoms, preferably 6 to 18, more preferably 8 to 16 carbon atoms, most preferably 10 to 14 carbon atoms, highly preferred 12 carbon atoms. 6. Use according to any of the claims 1-5, wherein the fatty acid ester is the ester of lauric acid, myristic acid, palmitic acid, stearic acid or arachidic acid, preferably lauric acid. 7. Use according to any of the claims 1-6, wherein the fatty acid ester groups of the substituted trisaccharide core are all identical. 8. Use according to any of the claims 1-7, wherein the substituted trisaccharide core is derived from raffinose, melezitose or maltotriose and wherein the trisaccharide derivate is fully substituted with identical fatty acid ester groups per substituted trisaccharide. 9. Use according to any of the claims 1-7, wherein the substituted trisaccharide core is derived from raffinose, melezitose or maltotriose and wherein the trisaccharide core comprises one sulphate ester or phosphate ester group and ten identical fatty acid ester groups per substituted trisaccharide or two sulphate or phosphate ester groups and nine identical fatty acid ester groups per substituted trisaccharide. 10. Use according to any of the claims 1-9, wherein the fatty acid ester groups are the esters of lauric acid. 11. Adjuvant composition comprising a trisaccharide derivate as referred to in claims 1-10 or a mixture thereof and a pharmaceutical acceptable recipient and/or diluent. 12. Adjuvant composition according to claim 11 formulated as an oil in water emulsion. 13. Adjuvant composition according to claim 12 wherein the oil phase of the emulsion comprises squalane and/or polysorbate. 14. Vaccine formulation comprising an adjuvant composition according to claims 11-13. 15. A kit comprising an antigen composition and an adjuvant composition according to claims 11-13. 16. Adjuvant composition according to claim 11-13, vaccine formulation according to claim 14 or a kit according to claim 15 for use as a medicament. 17. Trisaccharide derivate comprising a substituted trisaccharide core, which trisaccharide core is fully substituted with fatty acid ester groups and one or more anionic groups, wherein the substituted trisaccharide core is derived from raffinose, melezitose or maltotriose and wherein the trisaccharide core comprises one sulphate ester or phosphate ester group and ten identical fatty acid ester groups per substituted trisaccharide or two sulphate or phosphate ester groups and nine identical fatty acid ester groups per substituted trisaccharide, wherein the fatty acid ester group is an ester of a straight, branched, saturated or unsaturated fatty acid with a chain length of 4 to 20 carbon atoms. 18. Trisaccharide derivate according to claim 17, wherein the fatty acid ester is the ester of lauric acid, myristic acid, palmitic acid, stearic acid or arachidic acid. 19. Trisaccharide derivate according to claim 18, wherein the fatty acid ester groups are the esters of lauric acid. 20. Trisaccharide derivate according to any of the claims 17-19 for use as a medicament. 21. Method for preparing a trisaccharide derivate according to any of the claims 17-19, comprising the steps of: a) providing a trisaccharide and dissolving it in a solvent, wherein the trisaccharide is raffinose, melezitose or maltotriose; and b) esterifying all OH-groups of the trisaccharide, wherein at least one of the OH-groups of the trisaccharide is reacted with an anionic agent, wherein the trisaccharide is esterified with on average one sulphate ester or phosphate estergroup and ten identical fatty acid ester groups per trisaccharide or with two sulphate or phosphate ester groups and nine identical fatty acid ester groups per trisaccharide, wherein the fatty acid is a straight, branched, saturated or unsaturated fatty acid with a chain length of between 4 to 20 carbon atoms.

Patentansprüche 1. Verwendung eines Trisaccharidderivats, das einen substituierten Trisaccharidkern umfasst, welcher Trisaccharid-kern mit Fettsäureestergruppen und gegebenenfalls einer oder mehreren anionischen Gruppen voll substituiert ist, als ein Adjuvans. 2. Verwendung nach Anspruch 1, wobei der substituierte Trisaccharidkern aus Raffinose, Melezitose, Maltotriose, Nigerotriose, Maltotriulose oder Kestose, bevorzugt Raffinose, Melezitose oder Maltotriose, am stärksten bevorzugt Raffinose oder Maltotriose abgeleitet ist. 3. Verwendung nach Anspruch 1 oder 2, wobei der substituierte Trisaccharidkern einen oder zwei Sulfatester- oder

Phosphatestergruppen als anionische Gruppen umfasst. 4. Verwendung nach einem beliebigen der Ansprüche 1-3, wobei die anionische Gruppe ein Sulfatester ist. 5. Verwendung nach einem beliebigen der Ansprüche 1-4, wobei die Fettsäureestergruppe ein Ester einer geraden, verzweigten, gesättigten oder ungesättigten Fettsäure mit einer Kettenlänge von 4 bis 20 Kohlenstoffatomen, bevorzugt 6 bis 18, stärker bevorzugt 8 bis 16 Kohlenstoffatomen, am stärksten bevorzugt 10 bis 14 Kohlenstoffatomen, hoch bevorzugt 12 Kohlenstoffatomen ist. 6. Verwendung nach einem beliebigen der Ansprüche 1-5, wobei der Fettsäureester der Ester von Laurinsäure, Myristinsäure, Palmitinsäure, Stearinsäure oder Arachinsäure, bevorzugt Laurinsäure ist. 7. Verwendung nach einem beliebigen der Ansprüche 1-6, wobei die Fettsäureestergruppen des substituierten Tri-saccharidkerns alle identisch sind. 8. Verwendung nach einem beliebigen der Ansprüche 1-7, wobei der substituierte Trisaccharidkern aus Raffinose, Melezitose oder Maltotriose abgeleitet ist, und wobei das Trisaccharidderivat mit identischen Fettsäureestergruppen pro substituiertem Trisaccharid voll substituiert ist. 9. Verwendung nach einem beliebigen der Ansprüche 1-7, wobei der substituierte Trisaccharidkern aus Raffinose, Melezitose oder Maltotriose abgeleitet ist, und wobei der Trisaccharidkern eine Sulfatester- oder Phosphatestergruppe und zehn identische Fettsäureestergruppen pro substituiertem Trisaccharid oder zwei Sulfat- oder Phosphatestergruppen und neun identische Fettsäureestergruppen pro substituiertem Trisaccharid umfasst. 10. Verwendung nach einem beliebigen der Ansprüche 1-9, wobei die Fettsäureestergruppen die Estervon Laurinsäure sind. 11. Adjuvanszusammensetzung, umfassend ein Trisaccaridderivatwie in Ansprüchen 1-10 genannt oder eine Mischung davon und einen pharmazeutisch verträglichen Rezipient und/oderein Verdünnungsmittel. 12. Adjuvanszusammensetzung nach Anspruch 11, als eine ÖI-in-Wasser-Emulsion formuliert. 13. Adjuvanszusammensetzung nach Anspruch 12, wobei die Ölphase der Emulsion Squalan und/oder Polysorbat umfasst. 14. Vakzinformulierung, die eine Adjuvanszusammensetzung gemäß Ansprüchen 11-13 umfasst. 15. Kit, das eine Antigenzusammensetzung und eine Adjuvanszusammensetzung gemäß Ansprüchen 11-13 umfasst. 16. Adjuvanszusammensetzung gemäß Anspruch 11-13, Vakzinformulierung gemäß Anspruch 14 oder ein Kit gemäß Anspruch 15 zur Verwendung als ein Medikament. 17. Trisaccharidderivat, das einen substituierten Trisaccharidkern umfasst, welcher Trisaccharidkern mit Fettsäureestergruppen und einer oder mehreren anionischen Gruppen voll substituiert ist, wobei der substituierte Trisaccharidkern aus Raffinose, Melezitose oder Maltotriose abgeleitet ist, und wobei der Trisaccharidkern eine Sulfatester- oder Phosphateestergruppe und zehn identische Fettsäureestergruppen pro substituiertem Trisaccharid oderzwei Sulfatoder Phosphateestergruppen und neun identische Fettsäureestergruppen pro substituiertem Trisaccharid umfasst, wobei die Fettsäureestergruppe ein Ester einer geraden, verzweigten, gesättigten oder ungesättigten Fettsäure mit einer Kettenlänge von 4 bis 20 Kohlenstoffatomen ist. 18. Trisaccharidderivat nach Anspruch 17, wobei der Fettsäureester der Estervon Laurinsäure, Myristinsäure, Palmitinsäure, Stearinsäure oder Arachinsäure ist. 19. Trisaccharidderivat nach Anspruch 18, wobei die Fettsäureestergruppen die Estervon Laurinsäure sind. 20. Trisaccharidderivat nach einem beliebigen der Ansprüche 17-19, zur Verwendung als ein Medikament. 21. Verfahren zum Herstellen eines Trisaccharidderivats gemäß einem beliebigen der Ansprüche 17-19, umfassend die Schritte: a) Bereitstellen eines Trisaccharids und Lösen dessen in einem Lösungsmittel, wobei das Trisaccharid Raffi-nose, Melezitose oder Maltotriose ist; und b) Verestern aller OH-Gruppen des Trisaccharids, wobei mindestens eine der OH-Gruppen des Trisaccharids mit einem anionischen Mittel umgesetzt wird, wobei das Trisaccharid mit durchschnittlich einer Sulfatester- oder Phosphatestergruppe und zehn identischen Fettsäureestergruppen pro Trisaccharid, oder mit zwei Sulfatesteroder Phosphatestergruppen und neun identischen Fettsäureestergruppen pro Trisaccharid verestert wird, wobei die Fettsäure eine gerade, verzweigte, gesättigte oder ungesättigte Fettsäure mit einer Kettenlänge zwischen 4 bis 20 Kohlenstoffatomen ist.

Revendications 1. Utilisation d’un dérivé de trisaccharide comprenant un noyau de trisaccharide substitué, lequel noyau de trisaccharide est entièrement substitué par des groupes d’ester d’acide gras, et de manière facultative un ou plusieurs groupes anioniques comme adjuvant. 2. Utilisation selon la revendication 1, dans laquelle le noyau de trisaccharide substitué est dérivé de raffinose, mélé-zitose, maltotriose, nigerotriose, maltotriulose ou kestose, de préférence de raffinose, mélézitose ou maltotriose, de manière la plus préférée de raffinose ou maltotriose. 3. Utilisation selon la revendication 1 ou 2, dans laquelle le noyau de trisaccharide substitué comprend un ou deux groupes d’ester de sulfate ou d’ester de phosphate en tant que groupes anioniques. 4. Utilisation selon l’une quelconque des revendications 1 à 3, dans laquelle le groupe anionique est un ester de sulfate. 5. Utilisation selon l’une quelconque des revendications 1 à 4, dans laquelle le groupe d’ester d’acide gras est un ester d’un acide gras linéaire, ramifié, saturé ou insaturé ayant une longueur de chaîne de 4 à 20 atomes de carbone, de préférence de 6 à 18, de manière préférée de 8 à 16 atomes de carbone, de manière la plus préférée de 10 à 14 atomes de carbone, de manière fortement préférée de 12 atomes de carbone. 6. Utilisation selon l’une quelconque des revendications 1 à 5, dans laquelle l’ester d’acide gras est l’ester de l’acide laurique, de l’acide myristique, de l’acide palmitique, de l’acide stéarique ou de l’acide arachidique, de préférence de l’acide laurique. 7. Utilisation selon l’une quelconque des revendications 1 à 6, dans laquelle les groupes d’ester d’acide gras du noyau de trisaccharide substitué sont tous identiques. 8. Utilisation selon l’une quelconque des revendications 1 à 7, dans laquelle le noyau de trisaccharide substitué est dérivé de raffinose, mélézitose ou maltotriose et dans lequel le dérivé de trisaccharide est entièrement substitué par des groupes d’ester d’acide gras identiques par trisaccharide substitué. 9. Utilisation selon l’une quelconque des revendications 1 à 7, dans laquelle le noyau de trisaccharide substitué est dérivé de raffinose, mélézitose ou maltotriose et dans lequel le noyau de trisaccharide comprend un groupe d’ester de sulfate ou d’ester de phosphate et dix groupes d’ester d’acide gras identiques par trisaccharide substitué ou deux groupes d’ester de sulfate ou de phosphate et neuf groupes d’ester d’acide gras identiques par trisaccharide substitué. 10. Utilisation selon l’une quelconque des revendications 1 à 9, dans laquelle les groupes d’ester d’acide gras sont les esters de l’acide laurique. 11. Composition adjuvante comprenant un dérivé de trisaccharide tel que mentionné dans les revendications 1 à 10 ou un mélange de celui-ci et d’un récipient et/ou diluant pharmaceutique acceptable. 12. Composition adjuvante selon la revendication 11 formulée sous forme d’une émulsion huile dans eau. 13. Composition adjuvante selon la revendication 12 dans laquelle la phase huileuse de l’émulsion comprend du squa- lane et/ou du polysorbate. 14. Formulation de vaccin comprenant une composition adjuvante selon les revendications 11 à 13. 15. Kit comprenant une composition d’antigène et une composition adjuvante selon les revendications 11 à 13. 16. Composition adjuvante selon les revendications 11 à 13, formulation de vaccin selon la revendication 14 ou kit selon la revendication 15 pour une utilisation comme médicament. 17. Dérivé de trisaccharide comprenant un noyau de trisaccharide substitué, lequel noyau de trisaccharide est entièrement substitué par des groupes d’ester d’acide gras ; et un ou plusieurs groupes anioniques, dans lequel le noyau de trisaccharide substitué est dérivé de raffinose, mélézitose ou maltotriose et dans lequel le noyau de trisaccharide comprend un groupe d’ester de sulfate ou d’ester de phosphate et dix groupes d’ester d’acide gras identiques par trisaccharide substitué ou deux groupes d’ester de sulfate ou de phosphate et neuf groupes d’ester d’acide gras identiques partrisaccharide substitué, dans lequel le groupe d’esterd’acide gras est un esterd’un acide gras linéaire, ramifié, saturé ou insaturé ayant une longueur de chaîne de 4 à 20 atomes de carbone. 18. Dérivé de trisaccharide selon la revendication 17, dans lequel l’ester d’acide gras est l’ester de l’acide laurique, de l’acide myristique, de l’acide palmitique, de l’acide stéarique ou de l’acide arachidique. 19. Dérivé de trisaccharide selon la revendication 18, dans lequel les groupes ester d’acide gras sont les esters de l’acide laurique. 20. Dérivé de trisaccharide selon l’une quelconque des revendications 17 à 19, destiné à être utilisé comme médicament. 21. Procédé de préparation d’un dérivé de trisaccharide selon l’une quelconque des revendications 17 à 19, comprenant les étapes consistant à : a) fournir un trisaccharide et le dissoudre dans un solvant, dans lequel le trisaccharide est du raffinose, mélézitose ou maltotriose ; et b) estérifier tous les groupes OH du trisaccharide, dans lequel on fait réagir au moins un des groupes OH du trisaccharide avec un agent anionique, dans lequel le trisaccharide est estérifié avec en moyenne un groupe d’ester de sulfate ou d’ester de phosphate et dix groupes d’ester d’acide gras identiques par trisaccharide ou avec deux groupes d’ester de sulfate ou de phosphate et neuf groupes d’ester d’acide gras identiques par trisaccharide, dans lequel l’acide gras est un acide gras linéaire, ramifié, saturé ou insaturé ayant une longueur de chaîne comprise entre 4 et 20 atomes de carbone.

REFERENCES CITED IN THE DESCRIPTION

This list of references cited by the applicant is for the reader’s convenience only. It does not form part of the European patent document. Even though great care has been taken in compiling the references, errors or omissions cannot be excluded and the EPO disclaims all liability in this regard.

Patent documents cited in the description • WO 9620222 A [0017] · EP 1233969 A [0018] [0022] • WO 9620008 A [0017] · EP 1104767 A1 [0019]

Non-patent literature cited in the description • GOODMAN-SNITKOFFetal. J. /mmuno/., 1991, vol. · HUNTER et al. The Theory and Partical Application 147, 410-415 [0011] of Adjuvants. John Wiley and Sons, 1995, 51-94 • MILLER et al. J. Exp. Med., 1992, vol. 176, [0013] 1739-1744 [0011] · TODD et al. Vaccin, 1997, vol. 15, 564-570 [0013] • ELDRIDGE et al. Mol. Immunol., 1993, vol. 28, · REED et al. Trends in Immunology, 2008, vol. 30, 287-294 [0012] 23-32 [0016] • HILGERS eta\. Immunology, 1986, vol. 60, 141-146 [0017]

Claims (11)

1. Triszaechatld-származekok és adjuvánském törtéő alkalmazásuk Sza^ahni it™> ROMA l'gv 'Vobs/ttmah Uu's.’.hcha U antget tot,t ma m 5' i,\/<u,,hmm aram„u ,4, uh a, msUnt történő alkalmazása, mely triszaccharid mag teljes mértékben zsirsavésztet csoportokkal és adott esetben \<u> töbh ammm\ e.s,merttal s.mhs, tanait
2. 2, Az L igénypont szerinti alkalmazás, ahol a szobsztitoált trls2aceharid mag rafiBöőzbél me~ lezitózbói, mahotrtózból, nígerottlózból, maUotthtlúzhől vagy kestözhök preferablv rafímoz-ből, meiezitőzből vagy maltotriőzből., legelőnyösebben raftinőzból vagy maltotriőzből származik, ? Az 1 vagy ~ topent ?'U',nts aih,d?n',ti· ahvt,-, x m-v/tmuk tn\, accund m a; ,ιποη,ψ csoportként egy v agy több szulfátészter vagy ibszfátészter csoportot tartalmaz,
3. 4, Az i-.'k igénypontok bármelyike szerinti alkalmazás, ahol az aníonos csoport egy szulfát-észter,
4. 5, Az. 1-4, igénypontok bármelyike szerinti alkalmazás, ahol a zslrsavészter csoport egy egyenes, elágazó, telített vagy telítetlen 4-20 szénatomos, előnyösen 6-18 szénatomos, előnyösebben 8’16 szénatomos, legelőnyösebben 10-14 szénatomos, különösen előnyösen 12 szénato-mos láncú zsírsav észtere,
5. 6, Az 1-5.. Igénypontok bármelyike szerinti alkalmazás, áltól a zsírsav észter a íaurínsav, mi- ί,ν,ηην,ο, pabu tm<„' s/uaon'.,^ xa<?x ,>!λ>λ"λ,κ ehmxosera mse
6. 7, Az 1-6, igénypontok bármelyike szerinti alkalmazás, ahol a szubsztituált tríszacchand mag zsírsavészter csoportjai trond azonosak. 8 V ; -' pontok hamumuke ,bkabmo,vz ,·, ml a v'tns’híuah t'^auehemd mag raftinőzból, meiezitőzből vagy maltotriőzből származik, és ahol a tríszaecharid származék szubszdtuált tnszaccba?időnként teljes mértékben azonos zsirsavészter csoportokkal azubszb-· mák.
7. 9. Az l·?. igénypontok bármelyike szerinti alkalmazás, ahol a szabszhtuáh triszaeebarid mag raffittózbol, melezitózból vagy maltotriózbél származik, és ahol a triszaeeharid mag egy szül· tátéazer vagy foszfátészter csoportot és tíz azonos zsirsavészte? csoportot tartalmaz. sz.ab.sztn ν,υ is.ku.'uitvtiu»' \,\vü\t «. \ aioci1 xtn mm <n >s mí -e "> t 5 ,s » ό > *' ta r \ ' uK> " d i u ο μ. i, w s \co
8. 10, Az 1-9. igénypontok bármelyike szerinti alkalmazás, ahol a zsirsavészter csoportok a lau-rmsav észteréi, 11 ví u\an\ ke^. nmem, r.ee, er> l-lrt syeort\Otok-eenn-hsA s,m"on "kv aeeb unl származékot vagy azok egy keverékét és egy gyógyászatiig elfogadható reelpienst és/vagy hígítószert tartalmaz. 12. A 1 i, igénypont szerinti adjuváns készítmény, mely olaj a vízben emulzióként formáik I' .A 12 igénypont szerinti adpoaos kcszameio, ahol ez emul/m -latos iamsa ezksak-nl és/vagy poliszorhátot tartalmaz. BWii,,'. mén η,<Λ c <- í'tw átok \ md-ike ' s. fiú mányi tartalmaz.
9. 15. Egy antigén készítményt és egy 11-13. igénypontok bármelyike szerinti adjuváns készít'· ' \ λ* ..,ϊΟιοηοΑΊ ko ló. Az 11-13. igénypontok bármelyike szerinti adiuvárts készítmény, a 14, igénypont szerinti vakcina készítmény, vagy egy 15. igénypont szerinti kit gyógyszerként történő alkalmazásra. 1 rt-.s. accbartd .··,'an.. .mk meri ep s'ex, ι.ι sa t n sa .u eb tn-l magot kir,a.’n\. méh trie-z ie^. barid mag tejes mértekben zsn-so.s. tér exportokkal e\ ad-at esetben cm több annmoa „sopvrtíal sztibsebteab. ahol .t s/iib.vníteih ní\z.aeeh,irid mag rafímezbd. mele/ito?' böl vagy mahotriőzböl származik, és ahol a triszaceharid mag agy sznltateszer vagy főszám s mo e\eyoo,m m tt' a ro < sotx'rtm kekün,,^ nkxnm ül vux r< ?a< .· deákom vags két xottat- \agy tosztaR'smn· es^sportm c< kilenc azonos zsírsavvá >mr csapnom tartalmaz szubszfituáh triszacelmridonkéttt, ahol a zsirsavészteresoport egy egyenes, elágazó, , le Έ *\'t 4 11 xerxo'v > \av « ·, itu 18, A 17. igénypont szerinti nászaccharid szárntazák, ahol á zsirsávészter a iaüriüsgv, ntlrisZ" ttnsav, palmitinsav, sztearinsav vagy arachtdonsav észtere.
10. 19. A IS. igénypont szerinti triszaceharid származék, ahol a zsirsavészter csoportok a laurin-sav észterei. 29, A 17-19. Igénypontok bármelyike szerinti triszaeebarid származék gyógyszerkém történő alkalmazásra.
11. 21. Eljárás egy 17-19, igénypontok bármelyike szerinti oi.x emberid származék előállítására, meri nek sotan: at egy un. eecnmtóot ho nyitónk e$... t ec\ «'Idoxethu, okitok, Ja t a ktxam remi mtúnv/, nek ' to, so.o m , ion;, x tn o trixncdurid összes Oll-csoportpt? e.x tv torzok, .duü a tris. aeeh trió legaldb·' οχ mri. csoportját egy anionos szerrel reagáltatjuk. ahol a trtszaeeharidot átlagoson egy ezulíátészter vagy íosziátészw csoporttá! és óz ázones zsírsávészter csoporttal észterezzük trlszaeehari-donkénk vagy két szulfátészter "agy foszfátészter csoporttal, és kilenc azonos zsirsavésztér csoporttal észterezzök triszaccharidonként, ahol a zsirsavészteresoport egy egyenes, elágazó, telített vagy telítetlen, 4-20 szénatomes zsírsav észtere.