Drug – bio-affecting and body treating compositions – Antigen – epitope – or other immunospecific immunoeffector
Reexamination Certificate
2001-06-04
2003-01-14
Scheiner, Laurie (Department: 1648)
Drug, bio-affecting and body treating compositions
Antigen, epitope, or other immunospecific immunoeffector
C424S278100, C424S283100, C514S025000
Reexamination Certificate
active
06506386
ABSTRACT:
The present invention provides an improved adjuvant formulation and a process for producing said adjuvant. The adjuvant comprises an ISCOM structure comprising a saponin, said ISCOM structure being devoid of additional detergent. Also provided is an improved method of producing an adjuvant, and vaccines comprising the adjuvant of the present invention.
For many vaccines it is generally accepted that in order to generate significant levels of antigen specific immune responses, it is necessary to help the immune system by the inclusion of an adjuvant. The term adjuvant comes from the Latin of the verb “to Help” which is adjuvare. A number of adjuvants which help the immune response to a co-administered antigen to achieve greater magnitude than that observed if the antigen was given alone are known in the art. These include metallic salts, such as aluminium hydroxide or phosphate; liposomes, the bacterially derived monophosphoryl lipid A, Cholera toxin, and numerous others. Adjuvants may be classed as immunostimulants which have a direct stimulatory effect on the cells of the immune system, or may be classed as “vehicles” which function as carriers which present antigen to the immune system more efficiently than when the antigen is given alone. Alternatively, adjuvants may function in a combination of these mechanisms.
Specific adjuvants may also be used to drive the immune response into a particular desired characteristic. In theory, any given immune response may characterised into two mutually exclusive extremes of immune effector mechanisms. One extreme being predominantly a humoral response (characterised by the generation of Th2-type cytokines and immunoglobulin production) and a second extreme of a predominantly cell-mediated immune response (characterised by the generation of Th1-type cytokines and cytotoxic T cells). Generally speaking what is actually observed in real life is a balance of these two extremes, with any given response being described as being predominantly humoral (Th2-type) or predominantly cell-mediated (Th1-type). Thus for any particular pathogen, if it is desired that a vaccine should induce a predominantly Th1-type immune response, then the vaccine should be formulated with a known Th1-type inducing adjuvant.
One such adjuvant which is known to induce a balance of humoral and cell mediated immune response, which may include strong cell-mediated and also strong humoral responses, are the Immune-stimulating complexes (so called ISCOMs).
ISCOMs are three dimensional ‘cage-like’ structures which have been shown to form upon detergent removal from mixtures of saponins, detergents and cholesterol. ISCOMs and their use in vaccines are disclosed in EP 0 109 942 “Immunogenic protein or peptide complex, method of producing said complexes and use thereof as an immune stimulant and as a vaccine” . This patent discloses ISCOMs comprising antigen with hydrophobic regions and a glycoside (saponin), characterised in that the complex has an open spherical structure consisting of circular subunits or parts of the spheric structure. ISCOMs are thus open structures of around 30 nm in diameter with a morphology which is different from liposomal structures. The ISCOMs and parts thereof also usually have a lower sedimentation constant than corresponding micelles and a higher sedimentation constant than the corresponding monomeric form of protein or peptide, and a higher sedimentation constant than the corresponding liposome. The classical “cage-like” structure of ISCOMs can be seen in the electron microscopy studies of EP 0 242 380 B1 and EP 0 180 564 B1.
During their manufacture, phospholipids or additional protein antigens may be included in the structure. These ISCOM-protein complexes have been used as very potent vaccines (EP 0 109 942 B1). Alternatively, preformed ISCOMs without any additional antigen may be mixed with extraneous antigen to form a vaccine wherein the antigen is not in a complex with the ISCOM (EP 0 436 620 B1). These vaccine formulations have also been shown to induce high levels of immune responses.
ISCOM/protein complexes have also been formed by the covalent conjugation of the protein antigen onto the surface of the ISCOM (EP 0 180 564 B1). The use of ISCOMs for mucosal vaccination has also been described Mowat et al. Immunology, 72, 317-322 (1991). The ISCOM structure has been improved for use in mucosal applications by the incorporation of membrane targeting proteins (WO 97/30728).
The saponins are plant derived glycosides, a number of which have been studied extensively for their biological properties (The Plant Glycosides, McIlroy, R. J., Edward Arnold and co., London, 1951). The saponins used most predominantly in the art for the production of ISCOMs are those derived from the plants Quillaja saponaria molina,
Aesculus hippocastanum
or
Gyophilla struthium
. Extracts of the bark of Quillaja saponaria molina which are known to have adjuvant activity are known in the art, for example Quil A (Dalsgaard, K., 1974, Saponin adjuvants III, Archiv.für dis Gesamte Virusforschung, 44, 243-254). Also pure fractions of Quil A have been described which retain adjuvant activity whilst being less toxic than Quil A, for example QS21 (EP 0 362 279 B1, and U.S. Pat. No. 5,057,540). QS21 is also described in Kensil et al. (1991. J. Immunology vol 146, 431-437).
ISCOMs comprising other purified less toxic fractions of Quil A have been used in the manufacture of vaccines. These structures have been reported to have adjuvant activity (WO 96/11711). Alternative particulate structures containing a saponin and a sterol, other than ISCOMs which are also less toxic than the saponin alone, have also been described (WO 96/33739).
Other saponins which have been described in the literature include Escin, which has been described in the Merck index (12
th
ed: entry 3737) as a mixture of saponins occuring in the seed of the horse chestnut tree, Lat:
Aesculus hippocastanum
. Its isolation is described by chromatography and purification (Fiedler, Arzneimittel-Forsch. 4, 213 (1953)), and by ion-exchange resins (Erbring et al., U.S. Pat. No. 3,238,190). Fractions of escin have been purified and shown to be biologically active (Yoshikawa M, et al. (Chem Pharm Bull (Tokyo) 1996 August;44(8):1454-1464)). Sapoalbin from
Gypsophilla struthium
(R. Vochten et al., 1968, J. Pharm.Belg., 42, 213-226) has also been described in relation to ISCOM production.
ISCOMs are conventionally formed through two steps (e.g. as described in EP 0 109 942 ). 1, solubilisation of membrane and membrane proteins with detergent; 2, removal of solubilising agent by several means whilst at the same time contacting the membrane components with the saponin whose concentration is at least equal to the critical micellular concentration of the saponin, or removing the solubilising agent and directly transferring the antigen to the solution of saponin. U.S. Pat. No. Patent No. 4,578,269 teaches particular methods of separating the antigen from the solubilising agent. These methods include, amongst others: centrifugation through a gradient of solubilisation agent into an inverse gradient of saponin; or alternatively the solubilised antigen can be mixed with saponin followed by centrifugation of the mixture and dialysis to remove excess detergent.
EP 0 242 380 teaches of an improvement in this manufacturing process. This patent tells how the addition of lipids to the process prevents the formation of antigen/glycoside micelles, and ensures that the antigen/glycoside structures are all ISCOM-like. The specification states that the lipids may be added at any stage, at a molar ratio of at least 0.1:1 of lipids to antigen, and preferably 1:1. Examples of lipids include cholesterol and phosphatidyl choline. Thus, a method for producing an immunogenic complex between an antigen and a polar triterpensaponin, associated by the attraction between the hydrophobic regions of the triterpensaponin, lipid and antigen,
said complex must be formed by:
(i) mixing antigen and lipid with a solubilising agent, thus forming complexe
Friede Martin
Garcon Nathalie
Gimmi Edward R.
Kinzig Charles M.
Scheiner Laurie
SmithKline Beecham Biologicals (S.A.)
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