RECOMBINANT ALLERGEN WITH REDUCED ENZYMATIC ACTIVITY

Abstract: The present invention provides a novel treatment for allergy comprising the provision of a recombinant allergen with reduced enzymatic activity. Vaccines comprising said mutant allergens stimulate a Th1-type immune response in allergic or naïve individuals, thereby reducing the potential for an allergic response upon contact with wild-type allergen. Preferably said allergen is DerP1.

Description: RECOMBINANT ALLERGEN WITH REDUCED ENZYMATIC ACTIVITY The present invention relates to novel therapeutic formulations, said formulations being effective in the reduction of allergic responses to specific allergens. Further, this invention relates to novel polynucleotides, polypeptides encoded by them and to the use of such polynucleotides and polypeptides, and to their production. In particular, novel vaccines are provided comprising polypeptides and to their use in the treatment of humans suffering from allergies or prevention of individuals at risk from allergies, preferably said vaccines comprising a recombinant mutant Dermatophagoides pteronyssinus allergen Der P 1. Allergic responses in humans are common, and may be triggered by a variety of allergens. Allergic individuals are sensitised to allergens, and are characterised by the presence of high levels of allergen specific IgE in the serum, and possess allergen specific T-cell populations which produce Th2-type cvtokines (IL-4, IL-5, and IL-13). Binding of IgE, in the presence of allergen, to Fc receptors present on the surface of mastocytes and basophils, leads to the rapid degranulation of the cells and the subsequent release of histamine, and other preformed and neoformed mediators of the inflammatory reaction. In addition to this, the stimulation of the T-cell recall response results in the production of IL-4 and IL-13, together cooperating to switch B-cell responses further towards allergen specific IgE production. For details of the generation of early and late phase allergic responses see Joost Van Neeven et al.. 1996, Immunology Today, 17, 526. In non-allergic individuals, the immune response to the same antigens may additionally include Thl-type cvtokines such as IFN-γ. These cvtokines may prevent the onset of allergic responses by the inhibition of high levels of Th2-type immune responses, including high levels of allergen specific IgE. Importantly in this respect, is the fact that IgE synthesis may be controlled by an inhibitory feedback mechanism mediated by the binding of IgE/allergen complexes to the CD23 receptor on B-cells (Luo et al.. J.Immunol.. 1991, 146(7), 2122-9; Yu et al., 1994, Nature, 369(6483):753-6). In systems that lack cellular bound CD23, this inhibition of IgE svnthesis does not occur. Current strategies in the treatment of such allergic responses include means to prevent the symptomatic effects of histamine release by anti-histamine treatments and/or local administration of anti-inflammatory corticosteroids. Other strategies which are under development include those which use the hosts immune system to prevent the degranulation of the mast cells, Stanworth et al., EP 0 477 231 B 1. Other forms of immunotherapy have been described (Hoyne et al, J.Exp.Med., 1993, 178, 1783-1788; Holt et al, Lancet, 1994, 344, 456-458). Some common allergens present in bee venom, house dust mite emanations and parasite proteins have been found to induce mast cell degranulation, and to stimulate interleukin-4 synthesis and secretion, even in the absence of allergen-specific IgE (Machado et al, 1996, Eur. J.Immunol. 26, 2972-2980). This non-immunological degranulation by proteolytic allergens, such as bee venom phospholipase A2 or proteases associated with house dust mite emanations is dependent on enzymatic activity. The present invention provides recombinant mutant allergens having significantly reduced proteolytic activity relative to the wild-type proteolytically active allergen, as well as nucleic acids encoding the same, and their use as a prophylactic or immunotherapeutic agent against allergy. A preferred allergen is the house dust mite allergen Der pi. The present invention relates to the provision of formulations for the treatment and prophylaxis of allergy, by providing means to down-regulate the production of IgE, as well as modifying the cell mediated response to the allergen, through a shift from a Th2 type to a Thl type of response (as measured by the reduction of ratio of IL-4 : IFN-γ producing DerPl specific T-cells, or alternatively a reduction of the IL-5:IFN-γ ratio). This is achieved by the provision and use of recombinant mutant allergens with impaired enzymatic activity. DerPl, a group 1 protease allergen of the house dust mite Dermatophagoides pteronyssinus (Topham et al.. 1994, Protein Engineering, 7, 7, 869-894; Simpson et al.. 1989, Protein Sequences and Data .Analyses. 2, 17-21) is one such allergen. It is a 30 KDa protein and has been cloned and sequenced (Chua et al, 1988, J.Exp.Med., 167, 175-182). It is known to contain 222 amino acid residues in the mature protein. The sequence of DerPl shares 31% homology to Papain, and importantly shares homology in the enzymatically active regions, most notably the Cys34-Hisl70 ion pair {Topham et al, supra). DerPl is produced in the mid-gut of the mite, where its role is probably related to the digestion of food. Up to 0.2 ng or proteolytically active DerPl is incorporated into each fecal pellet, each around 10-40 μm in diameter and, therefore, easily inspired into the human respiratory tract. Overnight storage of purified DerPl preparations at room temperature results in almost complete loss of enzymatic activity due to autoproteolytic degradation (Machado et al, 1996, Eur.J.Immunol. 26, 2972- 2980). DerPl has been found to cleave the low affinity immunoglobulin IgE Fc receptor from the surface of human B lymphocytes (CD23, Hewitt et al, 1995, J.Exp.Med., 182, 1537-1544) and CD25 (Schultz et al, J.Exp.Med, 1998, 187(2):271-5) the alpha subunit of the human T cell interleukin-2 receptor. Cleavage of the receptor from the B cell surface was associated with a parallel increase in soluble CD23 in the culture supernatant. It has been suggested that the loss of cell surface CD23 from IgE-secreting B cells may promote and enhance IgE immune responses by ablating the important inhibitory feedback mechanism that normally limits IgE synthesis (Hewitt et al, 1995, J.Exp.Med., 182, 1537-1544). Furthermore, since soluble CD23 has been shown to promote IgE production, fragments of CD23 released by DerPl may directly enhance the synthesis of IgE. In addition to the effects of CD23 cleavage, the cleavage of CD25 from the surface of T-cells induces a decrease in proliferation and INF-gamma secretion, which, consequently, may bias the immune response toward a Th2 type response. Recent papers which relate to the DerPl antigen are Machado et al. Eur. J. Immunol. (1996) 26: 2972-2980; Hewitt et al, J. Exp. Med. (1995) 182: 1537-1544; and Schulz et al. Eur. J. Immunol. (1995) 25: 3191-3194. Other mutant allergens having reduced proteolytic activity which form part of the present invention may be based upon other group I cyteine proteases, such as Der fl from Dermatophagoides farinae (80% homology to DerPl), as well as the groups III allergens (serine proteases) including DerpIII (Stewart et al. 1992. Immunology, 75, 29-35) and DerpIV (Yaseuda et al, 1993, Clin.Exp.Allergy, 23, 384-390); and the group IV allergens (amylases). The allergens of the present invention are recombinantly produced. Der pi proteolytic activity can be impaired by introducing mutations into the cDNA or genomic DNA, either at the enzymatically active site, or at the site of cleavage between the propeptide and the mature molecule. Said mutant allergen having the following advantages over the wild-type allergen: 1) increases the Thl-type aspect of the immune responses in comparison to those stimulated by the wild type allergen, thereby leading to the suppression of allergic potential of the vaccinated host, and 2) having reduced allergenicity thus being more suitable for systemic administration of high doses of the immunogen, 3) will induce DerPl specific IgG which compete with IgE for the binding of native DerPl. The allergens of the present invention are also more stable than isolated or recombinant active DerPl, as measured by the lack of autoproteolytic degradation. Thus, the present invention also provides allergens which are stable compared to the wild-type form of the allergen, said allergens having significantly reduced proteolytic activity and being substantially full length proteins, optionally said allergens further comprising the pro- form of allergen. One aspect of the present invention provides a nucleic acid encoding mutated Der pi as set out above, and a further aspect of the invention provides mutated Der pi per se. A yet further aspect of the present invention provides substantially stable recombinant DerP 1. Said stable DerP 1 being of substantially full length mature protein, or mature protein further comprising the pro-DerPl section. The term "stable" in the context of the present invention is a product which does not undergo a substantial amount of decomposition by autoproteolysis when incubated overnight at room temperature in comparison to proteolytically active wild-type DerPl, as evidenced by SDS PAGE analysis. A still further aspect of the invention provides a process for the preparation of a mutated Der pi protein, which process comprises expressing DNA encoding the said protein in a recombinant host cell and recovering the product. A DNA molecule encoding a mutated Der pi (or other mutated allergen) forms a further aspect of the invention and can be synthesized by standard DNA synthesis techniques, such as by enzymatic ligation as described by D.M. Roberts et al in Biochemistry 1985, 24, 5090-5098, by chemical synthesis, by in vitro enzymatic polymerization, or by a combination of these techniques. Enzymatic polymerisation of DNA may be carried out in vitro using a DNA polymerase such as DNA polymerase I (Klenow fragment) in an appropriate buffer containing the nucleoside triphosphates dATP, dCTP, dGTP and dTTP as required at a temperature of 10°-37°C, generally in a volume of 50ml or less. Enzymatic ligation of DNA fragments may be carried out using a DNA ligase such as T4 DNA ligase in an appropriate buffer, such as 0.05M Tris (pH 7.4), 0.01M MgCl2, 0.01M dithiothreitol, ImM spermidine, ImM ATP and O.lmg/ml bovine serum albumin, at a temperature of 4°C to ambient, generally in a volume of 50ml or less. The chemical synthesis of the DNA polymer or fragments may be carried out by conventional phosphotriester, phosphite or phosphoramidite chemistry, using solid phase techniques such as those described in 'Chemical and Enzymatic Synthesis of Gene Fragments - A Laboratory Manual' (ed. H.G. Gassen and A. Lang), Verlag Chemie, Weinheim (1982),or in other scientific publications, for example M.J. Gait, H.W.D. Matthes, M. Singh, B.S. Sproat, and R.C. Titmas, Nucleic Acids Research, 1982, 10, 6243: B.S. Sproat and W. Bannwarth, Tetrahedron Letters, 1983, 24, 5771; M.D. Matteucci and M.H Caruthers, Tetrahedron Letters, 1980, 21, 719; M.D. Matteucci and M.H. Caruthers, Journal of the American Chemical Society, 1981, 103, 3185; S.P. Adams et al., Journal of the American Chemical Society,1983, 105, 661; N.D. Sinha, J. Biernat, J. McMannus, and H. Koester, Nucleic Acids Research, 1984, 12, 4539; and H.W.D. Matthes et al., EMBO Journal, 1984, 3, 801. Alternatively, the coding sequence can be derived from DerP 1 mRNA, using known techniques (e.g. reverse transcription of mRNA to generate a complementary cDNA strand), and commercially available cDNA kits. The invention is not limited to the specifically disclosed sequence, but includes any proteolytic allergen which has been mutated to remove some or all of its proteolytic activity, whilst retaining the ability to stimulate an immune response against the wild- type allergen. The proteolytic activity of the mutant allergens may be compared to the wild type by a CD23 cleavage assay according to Shultz et al, 1995, European Journal of Immunology, 25, 3191-3194), or enzymatic degradation of substrates described in Machado et al., 1996, Eur.J.Immunol., 26, 2972-2980. The immunogenicity of the mutant allergen may be compared to that of the wild-type allergen by various immunologicals assays. The cross-reactivity of the mutant and wild-type allergens may be assayed by in vitro T-cell assays after vaccination with either mutant or wild-type allergens. Briefly, splenic T-cells isolated from vaccinated animals may be restimulated in vitro with either mutant or wild-type allergen followed by measurement of cytokine production with commercially available ELISA assays, or proliferation of allergen specific T cells may be assayed over time by incorporation of tritiated thymidine. Also the immunogenicity may be determined by ELISA assay, the details of which may be easily determined by the man skilled in the art. Briefly, two types of ELISA assay are envisaged. First, to assess the recognition of the mutant DerPl by sera of mice immunized with the wild type Der pi; and secondly by recognition of wild type DerPl allergen by the sera of animals immunised with the mutant allergen. Briefly, each wells will be coated with 100 ng of purified wild type or mutated Der pi overnight at 4°C. After incubating with a blocking solution (TBS-Tween 0.1% with 1 % BSA) successive dilutions of sera will be incubated at 37°C for 1 hour. The wells are washed 5 times, and total IgG revealed by incubating with an anti-IgG antibody conjugated with Alkaline phosphatase. The reduction of enzymaticaly active allergen or DerPl may be performed by introducing mutations into the native sequence before recombinantly producing the inactivated mutants. This may be achieved by: introducing substitutions, deletions, or additions into the active sites: by inserting, deleting, or substituting residues in regions of processing the inactive pro-enzyme into the active mature protein; or by altering the three dimensional structure of the protein such that enzymatic activity is lost, this may be achieved, amongst others, by expressing the protein in fragments, or by deleting cysteine residues involved in disulphide bridge formation, or by deleting or adding residues such that the tertiary structure of the protein is substantially altered. Alternatively, mutations may be generated with the effect of altering the interaction between the Cys and the His residues, at positions 34 and 170 of the mature protein respectively (corresponding to positions 132 and 268 of the pre-pro-protein respectively) in the resultant fully folded recombinant protein. The invention is illustrated herein, but not limited to, three specific mutations which are are given as examples of proteolytically inactive DerPl. First, the enzymatic activity of DerPl is abrogated by substituting a Cysteine residue in the active site for an alanine. This substitution occurs at Cysl32— Alal32 of the pro-DerPl protein sequence, and is set out in SEQ ID NO. 1. Second, the DerPl allergen is recombinantly expressed and retained in its inactive pro-protein form by deletion of four amino acid residues at the linker region between the pro- and mature proteins. This deletion removes amino acid residues NAET from the site 96-99 inclusive, from the Pro-DerPl protein sequence. This sequence is set out in SEQ ID NO. 2. Third, enzymatic activity of DerPl is abrogated by substituting a Histidine residue in the active site for an alanine. This substitution occurs at His268→Ala268 of the pro-DerPl protein sequence, and is set out in SEQ ID NO. 3. The active sites of each wild-type enzymatic allergen may be determined from the literature, or by reference to homologues. For example, the active sites of DerPl, being a cysteine protease, may be putatively inferred by reference to other known cysteine proteases such as Papain. DerPl shares essential structural and mechanistic features with other papain-like cysteine proteinases, including cathepsin B. The active site thiolate-imidazolium ion pair comprises the side chains of Cys34 and Hisl70 (Topham et al., 1994, Protein Engineering, 7, 7, 869-894). Mutated versions of Der p 1 may be prepared by site-directed mutagenesis of the cDNA which codes for the Der p 1 protein by conventional methods such as those described by G. Winter et al in Nature 1982. 299, 756-758 or by Zoller and Smith 1982; Nucl. Acids Res., K), 6487-6500, or deletion mutagenesis such as described by Chan and Smith in Nucl. Acids Res., 1984, 12, 2407-2419 or by G. Winter et al in Biochem. Soc. Trans., 1984, 12, 224-225. The process of the invention may be performed by conventional recombinant techniques such as described in Maniatis et. al., Molecular Cloning - A Laboratory Manual; Cold Spring Harbor, 1982-1989. In particular, the process may comprise the steps of: 1. Preparing a replicable or integrating expression vector capable, in a host cell, of expressing a DNA polymer comprising a nucleotide sequence that encodes the said mutant Der pi protein; 2. Altering the enzymatic activity of the resultant protein by one of the following techniques: replacing the cysteine or histidine residues (or other residues interacting with other residues within the active site) from the active site with an alanine residue using site directed mutagenesis; replacement of a cDNA fragment by a pair of oligonucleotides whose sequence differ from the natural one; or alternatively, deleting four residues at the junction between the propeptide and the mature enzyme using site directed mutagenesis 3. Transforming a host cell with the said vector 4. Culturing the transformed host cell under conditions permitting expression of the DNA polymer to produce the protein; and 5. Recovering the protein. The term 'transforming' is used herein to mean the introduction of foreign DNA into a host cell by transformation, transfection or infection with an appropriate plasmid or viral vector using e.g. conventional techniques as described in Genetic Engineering; Eds. S.M. Kingsman and A.J. Kingsman; Blackwell Scientific Publications; Oxford, England, 1988. The term 'transformed' or 'transformant' will hereafter apply to the resulting host cell containing and expressing the foreign gene of interest. The expression vector is novel and also forms part of the invention. The replicable expression vector may be prepared in accordance with the invention, by cleaving a vector compatible with the host cell to provide a linear DNA segment having an intact replicon, and combining said linear segment with one or more DNA molecules which, together with said linear segment encode the desired product, such as the DNA polymer encoding the Der p 1 protein under ligating conditions. Thus, the DNA polymer may be preformed or formed during the construction of the vector, as desired. The choice of vector will be determined in part by the host cell, which may be prokaryotic or eukaryotic. Suitable vectors include plasmids, bacteriophages, cosmids and recombinant viruses. The preparation of the replicable expression vector may be carried out conventionally with appropriate enzymes for restriction, polymerisation and ligation of the DNA, by procedures described in, for example, Maniatis et al cited above. The recombinant host cell is prepared, in accordance with the invention, by transforming a host cell with a replicable expression vector of the invention under transforming conditions. Suitable transforming conditions are conventional and are described in, for example, Maniatis et al cited above, or "DNA Cloning" Vol. II, D.M. Glover ed., IRL Press Ltd, 1985. The choice of transforming conditions is determined by the host cell. Thus, a bacterial host such as E. coli may be treated with a solution of CaCl2 (Cohen et al, Proc. Nat. Acad. Sci., 1973, 69. 2110) or with a solution comprising a mixture of RbCl, MnCl2, potassium acetate and glycerol, and then with 3-[N-morpholino]-propane-sulphonic acid, RbCl and glycerol. Mammalian cells in culture may be transformed by calcium co-precipitation of the vector DNA onto the cells, by lipofection, or by electroporation. The invention also extends to a host cell transformed with a replicable expression vector of the invention. Culturing the transformed host cell under conditions permitting expression of the DNA polymer is carried out conventionally, as described in, for example, Maniatis et al and "DNA Cloning" cited above. Thus, preferably the cell is supplied with nutrient and cultured at a temperature below 45°C. The product is recovered by conventional methods according to the host cell. Thus, where the host cell is bacterial, such as E. coli it may be lysed physically, chemically or enzymatically and the protein product isolated from the resulting lysate. Where the host cell is mammalian, the product may generally be isolated from the nutrient medium or from cell free extracts. Conventional protein isolation techniques include selective precipitation, absorption chromatography, and affinity chromatography including a monoclonal antibody affinity column. Alternatively, the expression may be carried out either in insect cells using a suitable vector such as a baculovirus, in transformed drosophila cells, or mammalian CHO cells. The novel protein of the invention may also be expressed in yeast cells as described for the CS protein in EP-A-0 278 941. The vaccine of the invention comprises an immunoprotective amount of the mutated version of the Der pi (or other) allergenic protein. The term "immunoprotective" refers to the amount necessary to elicit an immune response against a subsequent challenge such that allergic disease is averted or mitigated. In the vaccine of the invention, an aqueous solution of the protein can be used directly. Alternatively, the protein, with or without prior lyophilization, can be mixed, adsorbed, or covalently linked with any of the various known adjuvants. Preferably, the adjuvant may be a preferential inducer of Thl-type immune responses. An immune response is generated to an antigen through the interaction of the antigen with the cells of the immune system. The resultant immune response may be broadly distinguished into two extreme categories, being a humoral or cell mediated immune responses (traditionally characterised by antibody and cellular effector mechanisms of protection respectively). These categories of response have been termed Thl-type responses (cell-mediated response), and Th2-type immune responses (humoral response). In mice Thl-type responses are characterised by the generation of antibodies of the IgG2a subtype, whilst in the human these correspond to IgGl type antibodies. Th2-type immune responses are characterised by the generation of a broad range of immunoglobulin isotypes including in mice IgE, IgGl, IgA, and IgM. It can be considered that the driving force behind the development of these two types of immune responses are cytokines, a number of identified protein messengers which serve to help the cells of the immune system and steer the eventual immune response to either a Thl or Th2 response. Thus Thl-type cytokines induce a cell mediated immune response to the given antigen, whilst Th2-type cytokines induce a humoral immune response to the antigen. It is important to remember that the distinction of Thl and Th2-type immune responses is not absolute. In reality an individual will support an immune response which is desribed as being predominantly Thl or predominantly Th2. However, it is often convenient to consider the families of cytokines in terms of that described in murine CD4 T cell clones by Mosmann and Coffman {Mosmann, T.R. and Coffman, R.L. (1989) TH1 and TH2 cells: different patterns oflymphokine secretion lead to different functional properties. Annual Review of Immunology, 7, pl45-173). Traditionally, Thl- type responses are associated with cell mediated effector mechanisms such as cytotoxic lymphocytes (CTL) and can be characterised by the production of the INF-γ and IL-2 cytokines by T-lymphocytes. Other cytokines often directly associated with the induction of Thl-type immune responses are not produced by T-cells, such as IL-12. In contrast, Th2- type responses are associated with humoral mechanisms and the secretion of IL-4, IL-5, IL-6, IL-10 and tumour necrosis factor-β (TNF-β). It is known that certain vaccine adjuvants are particularly suited to the stimulation of either Thl or Th2 - type cytokine responses. This weighting of cytokine production translates into the generation of either a predominantly Thl-type ot Th2-type immune responses. Traditionally the best indicators of the Thl :Th2 balance of the immune response after a vaccination or infection includes direct measurement of the production of Thl or Th2 cytokines by T lymphocytes in vitro after restimulation with antigen, and measurement of the IgGl :IgG2a ratio of antigen specific antibody responses. Thus, a Thl-type adjuvant is one which stimulates isolated T-cell populations to produce high levels of Thl-type cytokines when re-stimulated with antigen in vitro, and induces antigen specific immunoglobulin responses associated with Thl-type mechanisms (IgG2a in mice, IgGl in the human). Adjuvants include, but are not limited to, aluminium hydroxide, muramyl dipeptide and saponins such as Quil A, 3D-MPL (3-O-deacylated monophosphoryl lipid A), or TDM. As a further exemplary alternative, the protein can be encapsulated within microparticles such as liposomes. Particularly preferred adjuvants which preferentially stimulate Thl-type immune responses are combinations of 3D-MPL and QS21 (EP 0 671 948 Bl), oil in water emulsions comprising 3D-MPL and QS21 (WO 95/17210), 3D-MPL formulated with other carriers (EP 0 689 454 Bl), or QS21 formulated in cholesterol containing liposomes (WO 96/33739), or immunostimulatory oligonucleotides (WO 96/02555). In yet another exemplary alternative, the protein can be conjugated to a carrier protein which is capable of providing T-cell help to the generation of the anti-allergen immune response, such as tetanus toxoid. Use of Quil A is disclosed by Dalsgaard et al., Acta Vet Scand, _18:349 (1977). Vaccine preparation is generally described in New Trends and Developments in Vaccines. Voller et al. (eds.), University Park Press, Baltimore. Maryland. 1978. Encapsulation within liposomes is described by Fullerton, US Patent 4,235,877. Conjugation of proteins to macromolecules is disclosed, for example, by Likhite, US Patent 4,372,945 and Armor et al., US Patent 4,474,757. The amount of the protein of the present invention present in each vaccine dose is selected as an amount which induces an immunoprotective response without significant, adverse side effects in typical vaccines. Such amount will vary depending upon which specific immunogen is employed and whether or not the vaccine is adjuvanted. Generally, it is expected that each dose will comprise 1-1000 μg of protein, preferably 1-200 μg. An optimal amount for a particular vaccine can be ascertained by standard studies involving observation of antibody titres and other responses in subjects. Following an initial vaccination, subjects will preferably receive a boost in about 4 weeks, followed by repeated boosts every six months for as long as a risk of allergic responses exists. The vaccines of the present invention may be administered to adults or infants, however, it is preferable to vaccinate individuals soon after birth before the establishment of substantial Th2-type memory responses. A further aspect of the invention provides a method of preventing or mitigating an allergic disease in man, which method comprises administering to a subject in need thereof an immunogenically effective amount of a mutated allergen of the invention, or of a vaccine in accordance with the invention. The examples which follow are illustrative but not limiting of the invention. Restriction enzymes and other reagents were used substantially in accordance with the vendors' instructions. Example 1 - Expression in Pichia pastoris: Construction of pNI V4811 pNIV4811 is designed to promote the expression of mature Der pi in fusion with the prepropeptide of Pichia pastoris MF . Plasmid ATCC87307 contains the sequence for mature DerPl . The full Derpl restriction map is given in figure 7. Ligate with T4 DNA Ligase: - Sphl-Xhol from pPIC9k (INVITROGEN VI 75-20) - Xhol-Pstl oligonucleotides whose sequences follow (n° 97038 and n° 97039) - Pstl-Xbal from pNIV4810 (plasmid ATCC87307) - Avrll-Sphl from pPIC9k Sequences of the oligonucleotides: n° 97038 5 'TCGAGAAAAGAGAGGCTGAAGCTACTAACGCCTGCA 3 ' n° 97039 5 'GGCGTTAGTAGCTTCAGCCTCTCTTTTC3' Results Pichia Pastoris transfected with pNIV4811 leads to the expression of a protein of 43 5 kD, comprising uncieaved proMFα-mature Der pi fusion protein, has been detected in several clones (Figure 1). Construction of pNIV4817 pNIV4817 is derived from pNIV4811. It is designed to promote the expression of the 10 mature Der p 1 in fusion with the prepeptide of Pichia pastoris MF a.. Ligate: - BstEϊl-BamHI from pNIV4811 - BamHl-Psii oligonucleotides n° 97262 and n°97263 whose sequence follows 15 - Pstl-BstΩl from pNIV4811 Sequences of the oligonucleotides n° 97262 5 'GATCCAAACGATGAGATTTCCTTCAATTTTTACTGCAGTTTTATTCGC 0 AGC ATCCTCCGCATTAGCTGCTCCAACTAACGCCTGCA 3 ' n° 97263 5 'GGCGTTAGTTGGAGCAGCTAATGCGGAGGATGCTGCGAATAAAACTGCAG TAAAAATTGAAGGAAATCTCATCGTTTG 3 ' 25 Results Several clones expressed the mature form of Der pi protein with an apparent molecular weight of 30 kDa, which was secreted into the supernatant (Figure 2). Construction of pNIV4815 Starting from pNIV4811, the following construction is designed to delete four residues [N-A-E-T (T is the first residue of the mature protein)] at the junction between the propeptide and the mature enzyme. Ligate : Blnl - BamHl fragment from pPIC9k (the vector used for expression in P. pastoris) BamHl - Eael fragment from pNIV4811 Eael - EcoRl fragment generated by RT-PCR with primers No 97142 and 97143. Residues : Ag to E74. EcoRl - Pstl oligonucleotides whose sequence follows (No 97140 and 97141). Residues : F75 to C 102 except N96AET99 Pstl - Xbal fragment from pNIV4810. Sequence of the oligonucleotides allowing the NAET deletion: No 97140 5 'AATTCAAAAACCGATTTTTGATGAGTGCAGAAGCTTTTGAACACCTA AAACTCAATTCGATTTGAACGCCTGCA 3 ' 75 bases No 97141 S'GGCGTTCAAATCGAATTGAGTTTTGAGGTGTTCAAAAGCTTCTGCAT CATCAAAAATCGGTTTTTG 3 ' 67 bases RT-PCR Primers No 97142 5'CATGAAAATTGTTTTGGCCATCGCC3' 25 bases Eαel No 97143 5 'CGGTTTTTGAATTCATCCAACGAC3' 24 bases EcoRl Construction of pNIV4819 Starting from pNIV4817, an expression plasmid designed to produce the mature form of Der pi in Pichia pastoris, the following construction, is made to replace the cysteine residue from the active site by an alanine residue (corresponding to the Cys 34 mutation in the mature protein). Ligate: - Bpul 1021-AseI fragment from pNIV4817 - Asel-Tfil synthetic fragment resulting from hybridization of oligonucleotides n° 97121 and n° 97122 whose sequence follows: correspondig to residues I \ 04 to E 142 of the proDerPl (Ig. of mature DerPl protein) - Tfll-BstEll fragment from pNIV4810 (ATCC 87307) - BstEll-Bpul 1021 fragment from pNIV4817 Sequences of the oligonucleotides n° 97121 5 'TAATGGAAATGCTCCAGCTGAAATCGATTTGCGACAAATGCGAACTGTCA CTCCCATTCGTATGCAAGGAGGCTGTGGTTCAGCTTGGGCTTTCTCTGGTGT TGCCGCAACTG 3 ' Ala 113 bases n° 97122 5 'ATTCAGTTGCGGCAACACCAGAGAAAGCCCAAAGCTGAACCACAGCCTCC TTGCATACGAATGGGAGTGACAGTTCGCATTTGTCGCAAATCGATTTCAGCT GGAGCATTTCCAT 3 ' 114 bases Construction of pNIV4815 Starting from pNIV4811, the following construction is made to delete four residues [N- A-E-T (T is the first residue of the mature protein)] at the junction between the propeptide and the mature enzyme. Ligate : Blnl - BamHl fragment from pPIC9k (the vector used for expression in PPIChia pastoris) BamHl - Eael fragment from pNIV4811 Eael - EcoRl fragment generated by RT-PCR with primers No 97142 and 97143. Residues : Aβ to E74. EcoRl - Pstl oligonucleotides whose sequence follows (No 97140 and 97141). Residues : F75 to C 102 except N96AET99 Pstl -Xbal fragment from pNIV4810. Sequence of the oligonucleotides : allowing the NAET deletion. No 97140 5'AATTCAAAAACCGATTTTTGATGAGTGCAGAAGCTTTTGAACACCTCA AAACTCAATTCGATTTGAACGCCTGCA 3 ' 75 bases No 97141 5 'GGCGTTCAAATCGAATTGAGTTTTGAGGTGTTCAAAAGCTTCTGCACT CATCAAAAATCGGTTTTTG 3 ' 67 bases RT-PCR Primers No 97142 5'CATGAAAATTGTTTTGGCCATCGCC3' 25 bases Eαel No 97143 5'CGGTTTTTGAATTCATCCAACGAC 3 ' 24 bases EcoRl Example 2 - Expression in mammalian cells Construction of pNIV4812 pNIV4812, an expression plasmid based on pEE14 (CellTech, Cockett et al, 1990 Biotechnology, vol 8,662-667) designed to produce the mature form of Der pi in CHO- Kl, codes for a pre-Der pi followed by the mature Der pi sequence (no pro-protein). Ligate: - Hindlll-Xbal from pEE14 - Hind ll-Pstl oligonucleotides n°97040 and 97041 whose sequence follows -Pstl-Xbal from pNIV4810 (plasmid ATCC 87307) Sequence of the oligonucleotides n° 97040 5 'AGCTTACCATGAAAATTGTTTTGGCCATCGCCTCATTGTTGGCATTGAGCG CTGTTTATGCTCGTACTAACGCCTGCA 3 ' n° 97041 5'GGCGTTAGTACGAGCATAAACAGCGCTCAATGCCAACAATGAGGCGATGG CCAAAACAATTTTCATGGTA 3 ' Results The expression of a protein of an apparent molecular weight of 30 kDa has been detected in several extracts (Figure 3). No protein has been detected in the culture supernatants (data not shown), which suggests that the protein was not secreted from CHO-K1 cells. Construction of pNIV4814 Starting from pNTV4812, the following construction is made to replace the cysteine residue from the active site by an alanine residue. Ligate : - Aflll - Asel fragment from pNI V4812. - Asel-Tfil oligonucleotides as in pNIV4819 construction (No 97121 and 97122) - Tfil - BstEll fragment from pNIV4810 (ATCC 87307) - BstEll - Aflll fragment from pNIV4812. Construction of pNIV4819 and pNIV4814 was made possible, thanks to the discovery that in pNIV4810 the codon encoding isoleucine 6 of the mature protein was ATT instead of ATC as published. This sequence is responsible for the presence of the Asel restriction site. Construction of pNIV4816 Starting from pNIV4812, designed to expressed in CHO-K1, pNIV4816 has the same deletion as for pNIV4815. This construct results in the production of recombinant proDerPl with the deletion of the NAET residues from the junction between the pro and mature protein. Ligate : Xbal - Aflll fragment from pEE14 Aflll - Eael fragment from pNIV4812 Eαel - EcoRl fragment generated by RT-PCR using primers No 97142 and 97143 EcoRl - Pstl oligonucleotides No 97140 and 97141 (same oligonucleotides as used in pNIV4815) Pstl -Xbal fragment from pNIV4810. Example 3 - Expression in Drosophila cells Construction of pNIV4827 pNIV4827 has ' been designed to promote the expression and secretion of mature Der pi from baculovirus infected insect cells. Ligate : pAcGP67A vector linearized with Pstl Pstl fragment from pNIV4810 (ATCC 87307) The expression of Der pi from pNIV4827 has been demonstrated by western blot. Construction of pNIV4828 pNIV4828 has been designed to promote the expression and secretion of ProDer pi from baculovirus infected insect cells. Ligate : Sapl-BamΑl from pAcGP67A (Pharmingen ref. 21220P) 5 /κHl-EcoRI 172 bp synthetic fragment EcoRl-BssSl from pNIV4820 BssSl-Sapl from pNIV4827 Sequence of the synthetic fragment : a) coding oligonucleotide N° 97520 5' GAT CCC CGG CCG TCA TCGATC AAA ACTTTT GAA GAATAC AAA AAA GCC TTC AAC AAA AGT TAT GCTACC TTC GAA GAT GAA GAA GCT GCC CGTAAA AAC TTTTTG GAA TCA GTA AAA TAT GTT CAA TCAAAT GGA GGT GCC ATC AAC CATTTG TCC GAT TTG TCG TTG GAT G 3' 172 mer b) complementary sequence N° 97521 5' AAT TCATCC AAC GAC AAA TCG GAC AAATGGTTGATG GCA CCT CCA TTT GATTGA ACATAT TTT ACT GAT TCC AAA AAG TTT TTA CGG GCA GCT TCT TCATCTTCG AAG GTA GCA TAA CTTTTG TTG AAG GCT TTTTTG TAT TCT TCAAAA GTTTTG ATC GAT GAC GGC CGG G 3' 172 mer The expression of ProDer pi from pNIV4828 has been demonstrated by western blot. Construction of pNIV4832 This plasmid codes for a Der pi propeptide followed by the mature Der pi (ProDer pi) sequence and is designed to be expressed in drosophila cells. Ligate : - Asp7\8-Bamlil fragment from expression vector pDS47/V5-His (INVITROGΕN V41 15-20) - Asp71$-Spel synthetic fragment resulting from hybridization of 98023 and 98024 oligonucleotides - Spel-BgRl fragment from pNIV4828 Sequences of the oligonucleotides n° 98023 5' GTA CCC TTA AGA TGC TA 3' n° 98024 5' CTA GTA GCA TCT TAA GG 3' NB : pNIV4828 is a plasmid designed for the isolation of recombinant baculoviruses expressing the pro-Derp 1 fused to gp67 signal peptide. Results Transitory expression of pro-DerPl in drosophila cells has been detected (data not shown). Construction of pNIV4840 pNIV4840 differs from pNIV4832 in that the expression vector used is stable and inducible (pMT/V5-His) Ligate : - Aspl 18-Not/ fragment from pΝIV4832 - Notl- Asp7\8 from pMT/V5-His (INVITROGEN V4120-20) Expression of proDerp 1 in drosophila cells has been shown (Figure 4) Construction of pNIV4842 pNIV4842 was designed to promote the expression and secretion of ProDer pi from recombinant drosophila cells. ProDer pi coding sequence was engineered to impair the cleavage of the propeptide. To achieve this goal, four nucleotide triplets coding for NAET including the cleavage site were deleted. Ligate : - Notl-EcoRI from pNIV4840 - EcoRI-Pstl synthetic fragment resulting from hybridization of oligonucleotides n°98136 and n°98137 - Pstl-BstEII from pNIV4840 - BsfEII-Notl from pNIV4840 Sequence of the synthetic oligonucleotides a) Coding sequence N° 98136 5' AAT TCA AAA ACC GAT TTT TGA TGA GTG CAG AAG CTT TTG AAC ACC TCA AAA CTC AAT TCG ATT TGA ACG CCT GCA 3' 75 mer Complementary sequence N° 98137 5' GGC GTT CAAATC GAA TTGAGT TTT GAG GTG TTC AAAAGC TTC TGC ACT CAT CAAAAA TCG GTT TTT G 3' 67 mer Results Detection of Der pi in fusion with its propeptide has been detected in the supematants after induction (Figure 5). The sequence of this recombinant mutant DerPl is given in SEQ ID NO. 4. Construction of pNIV4843 pNIV4843 has been designed to promote the expression and secretion from recombinant drosophila cells of a ProDer pi form in which the cysteine residue of the active site has been mutated to an alanine. 1") Ligate : - NotI-Asp718 from pMT/V5-His - Asp718-Pstl from pNIV4832 - PstI-TfιI from pNIV4819 - Tfil-Notl from pNIV4832 Results Detection of Der pi in fusion with its propeptide has been detected in the supematants after induction (Figure 6). The sequence of this recombinant mutant DerPl is given in SEQ ID NO. 5. Example 3, Purification procedure of recombinant ProDer pi secreted from recombinant drosophila cells Proteins from the spent culture medium (1 liter) were concentrated at 4°C by overnight ammonium sulfate precipitation to 60% saturation. After centrifugation at 17000g during 30 min., the precipitate was resuspended in 20 ml of 20 mM Tris-HCl pH8.0 and dialyzed against 5 liters of the same buffer. Insoluble proteins were discarded by centrifugation at 20000g during 30 min. The dialysate was loaded onto a Q sepharose XL column (3 x 1.6 cm, Pharmacia) equilibrated in 20 mM Tris-HCl pH8.0. After washing the column with the same buffer, bound proteins were eluted by steps of 100 mM increases of NaCl concentration. ProDer pi mainly eluted at 200mM NaCl. Enriched ProDer pi fractions were pooled and loaded onto an hydroxyapatite type 1 column (1 x 1.6 cm, Biorad) conditionned in 5 mM potassium phosphate buffer pH 7.0. Unbound material containing ProDer pi was concentrated by ultrafiltration using Omega membrane (cut-off : lOkD, Filtron). The concentrate was loaded onto a superdex 75 FPLC column (30 x 1 cm, Pharmacia) in PBS pH 7.3. Eluted ProDer pi from the gel filtration column was more than 80% pure. Example 4, Vaccine formulation Vaccines comprising the mutant DerP 1 or allergens may be formulated with many common adjuvants. One prefered adjuvant system is an oil in water emulsion described below : The oil in water emulsion adjuvant formulations used in the present invention are made comprising following oil in water emulsion component: 5% Squalene, 5% α- tocopherol, 2.0% polyoxyethylene sorbitan monooleate (TWEEN 80). The emulsions are prepared as a 2 fold concentrate. All examples used in the immunological experiments are diluted with the addition of extra components and diluents to give either a lx concentration (equating to a squalene:QS21 ratio (w/w) of 240:1) or further dilutions thereof. Briefly, TWEEN 80 is dissolved in phosphate buffered saline (PBS) to give a 2% solution in the PBS. To provide 100 ml of a two fold concentrate emulsion, 5ml of DL alpha tocopherol and 5ml of squalene are vortexed to mix thoroughly. 95ml of PBS/TWEEN solution is added to the oil and mixed thoroughly. The resulting emulsion is then passed through a syringe needle and finally microfluidised by using an MHOS Micro fluidics machine. The resulting oil droplets have a size of approximately 145-180 nm (expressed as z av. measured by PCS). The other adjuvant vaccine components (QS21, 3D-MPL and antigen) are added to the emulsion in simple admixture. The antigen containing vaccines used herein are formulated either with full dose SB62 adjuvant to give a high squalene:QS21 ratio (240: 1) or with a lower amount of SB62 to give a low ratio formulation (48:1). Other vaccines may optionally be formulated with the addition of cholesterol to the oil phase of the emulsion. These vaccines are assayed in groups of Balb/c mice. Briefly, groups of 10 mice are immunised intramuscularly 2 times at 3 weeks interval with 2 μg mutant allergen combined with oil in water emulsion adjuvant. 14 days following the second immunisation the production of cytokines (IL-4, IL5 and IFN-γ) are analysed after in vitro restimulation of spleen and lymph nodes cells with allergen. Antibody response to wild-type allergen and the isotypic profile induced are monitored by ELISA at 21 days post II and 14 days post IV.

Claims: Claims 1. A recombinant mutant allergen wherein said mutant allergen has reduced enzymatic activity compared to the wild-type allergen. 2. A recombinant mutant allergen as claimed in claim 1, wherein said allergen is based upon a type I cysteine protease allergen. 3. A recombinant mutant allergen as claimed in claim 1, wherein said allergen is based upon DerPl from Dermatophagoides pteronyssinus. 4. A recombinant mutant allergen as claimed in claim 3, wherein said mutant DerPl comprises an active site mutant. 5. A recombinant mutant allergen as claimed in claim 4, wherein said active site mutant DerPl comprises a mutation of the Cys 34 residue. 6. A recombinant mutant allergen as claimed in claim 5, wherein said mutation of the Cys 34 residue comprises an alanine substitution. 7. A recombinant mutant allergen as claimed in claim 4, wherein said active site mutant DerP 1 comprises a mutation of the His 170 residue. 8. A recombinant mutant allergen as claimed in claim 3, wherein said mutant DerPl comprises a mutation at the site of cleavage between the propeptide and the mature molecule. 9. A recombinant mutant allergen as claimed in claim 8, wherein said mutation at the site of cleavage between the propeptide and the mature molecule comprises a deletion of the residues NAET. 10. A recombinant mutant allergen as claimed in claim 3, wherein said mutation comprises the deletion or substitution of cysteine residues which are involved in disulphide bridge formation. 11. Stable recombinant DerP 1. 12. A recombinant mutant allergen having the sequence as set out in SEQ ID NO. 1 13. A recombinant mutant allergen having the sequence as set out in SEQ ID NO. 2 14. A recombinant mutant allergen having the sequence as set out in SEQ ID NO. 3 15. A recombinant mutant allergen having the sequence as set out in SEQ ID NO. 4 16. A recombinant mutant allergen having the sequence as set out in SEQ ID NO. 5 17. An isolated nucleic acid molecule encoding a mutated version of an allergen as claimed in any one of claims 1 to 16. 18. A vaccine comprising a recombinant mutant allergen as claimed in any one of claims 1 to 17, and an adjuvant. 19. A vaccine as claimed in claim 18, wherein the adjuvant is a preferential stimulator of Thl-type immune responses. 20. A vaccine as claimed in claim 18, wherein the adjuvant comprises one or both of QS21 and 3-O-deacylated monophosphoryl lipid A. 21. Use of a recombinant mutant allergen in the manufacture of a medicament for the treatment of allergy. 22. A method of treating or preventing allergic responses comprising administering to an individual suffering from or susceptible to allergy a vaccine as claimed in claim 18.

Inventors: BOLLEN ALEX, BRUCK CLAUDINE, JACOBS PAUL, MASSAER MARC, BOLLEN, ALEX, BRUCK, CLAUDINE, JACOBS, PAUL, MASSAER, MARC
Applicants: BOLLEN ALEX, BRUCK CLAUDINE, JACOBS PAUL, MASSAER MARC, SMITHKLINE BEECHAM BIOLOG, BOLLEN, ALEX, BRUCK, CLAUDINE, JACOBS, PAUL, MASSAER, MARC, SMITHKLINE BEECHAM BIOLOGICALS S.A.
Application year: 1998
Main classification: C12N15
IPC codes: A61K38, A61K39, A61P37, C07K14, C12N9, C12N15