Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/35—Allergens
  • A61K39/36—Allergens from pollen
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
  • A61P37/02—Immunomodulators
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
  • A61P37/08—Antiallergic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
  • A61K2039/577—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2 tolerising response
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
  • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/575—Hormones
  • C07K14/62—Insulins
  • C07K14/625—Extraction from natural sources
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/70—Vectors or expression systems specially adapted for E. coli
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
  • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material

Definitions

• the present invention relates to the field of the production of chimeric proteins for the prevention and treatment of allergies, in particular pollen allergies and most particularly allergies caused by allergens from the lipid transfer protein family and more specifically those found in pollen from species of Parietaria.
• Type I allergies are a significant health problem in industrialised countries. This type of allergy is caused by the formation of IgE antibodies against antigens carried by the air. These IgE antibodies interact with the mast cells and basophiles, liberating biological mediators such as histamine, and causing allergic rhinitis, conjunctivitis and bronchial asthma in about 20 % of the population [(1) Miyamoto, T. (1992). Increased prevalence of pollen allergy in Japan. In Advances in Allergology and Clinical Immunology. P. Godard, J. Bousquet, and F. B. Michel, eds . (Cornforth, UK: The Parthenon Publishing Group), pp. 343-347].
• SIT Specific immunotherapy
• Specific immunotherapy is an effective treatment for allergic reactions triggered by specific allergens and basically consists in modulating the immune response in the patient by the regular administration, in increasing concentrations, of the proteins which produce the allergy (allergenic extracts) .
• High doses of the injected allergens induce the high synthesis of IL-12 by the antigen-presenting cells, for example the dendritic cells, which preferably promote the development of T cells which are cooperative virgin cells (nT H ) toward T H 1 or T H 0.
• T H cooperative virgin cells
• This allows a deviation from the immune response of the allergic response type related to the T H 2 cells toward a T H 1/TH0 type response which leads to the production of high levels of IFN- ⁇ [(2) Akdis, CA. and Blaser, K. (2000).
• IL-IO and TGF- ⁇ [(3) Akdis, CA. , Joss, A., Akdis, M., and Blaser, K. (2001) . Mechanism of IL-10 induced cell inactivation in allergic inflammation and normal response to allergens. Int. Arch Allergy Immunol. 124; 180-182].
• the reduction in the activation and proliferation of the TH2 cells is accompanied by reduced production of IL-4, and of IgEs by the B cells.
• the reduction in the activity and infiltration of the TH2 cells into the nasal and bronchial mucus results in reduced IL-5 synthesis, allowing a reduction in eosinophile infiltration which leads to a great reduction in the liberation of inflammatory mediators such as MBP and ECP proteins .
• the new specific clones of T cells of predominant phenotype allergen THO produce a mixture of THI and TH2 type cytokines promoting the production of a large quantity of specific IgG allergen antibodies by the B cells.
• the high levels of IL-10 induce the high synthesis of specific IgG4 allergen antibodies.
• the allergen extracts isolated from natural sources are complex mixtures of proteins and other molecules. Their composition, and therefore allergenicity, depends on the material used, which varies according to the ambient conditions, the state of maturation in the case of pollens and the conditions of growth of mites, etc., in the case of fungi, etc. Some extracts can even contain an inadequate concentration of major allergens and can even be contaminated with undesirable components to which the patient is not allergic, or both.
• Current immunotherapy uses complete allergen extracts exclusively, and this leads to a number of problems such as:
• the modified allergen will be directed toward the T cells by a phagocytosis/pinocytosis-mediated antigen collection mechanism, preventing the IgE intersection and the presentation of IgE-dependent antigen.
• This induces a balance in THO or THI cytokine production by the T cells, less production of IgE and more production of IgG by the B cells; all this will lead to the induction of TH2 type T cell tolerance without a risk of anaphylaxis.
• Progress in recombinant methods of obtaining allergens and allergen derivatives has allowed a great increase in the capacity for developing new vaccines for the treatment of allergies.
• hypoallergenic derivatives in immunotherapy has previously been described using fragments of trimers of Bet v 1 [(7) Niederberger, V., Horak, F., Vrtala, S., Spitzauer, S., Krauth, M. T., Valent, P., Reisinger, J., Pelzmann, M., Hayek, B., Kronqvist, M., Gafvelin, G., Gr ⁇ nlund, H., Purohit, A., Suck, R., Fiebig, H., Cromwell, 0., Pauli, G., van Hage-Hamsten, M., and Valenta, R. (2004). Vaccination with genetically engineered allergens prevents progression of allergic disease. Proc.
• allergenic vaccines should not be made with hypoallergens since the bond to IgE could facilitate the capture and presentation of the allergen by the professional antigen-presenting cells, as dendritic cells and activated B lymphocyte cells, which express surface receptors for IgE of both high affinity and low affinity.
• This approach has a special meaning when using the sublingual route in immunotherapy.
• the intersection of high affinity receptors can also lead to a reduction in the response of the T cells toward the allergens [(9) Allam, J. P., Novak, N., Fuchs, C, Asen, S., Berge, S., Appel, T., et al.
• Parietaria is a genus of dicotyledon weed from the Urticaceae family, and the Urticales order.
• Various species of the Parietaria genus are widely and abundantly distributed along the Mediterranean coast [ (15) Colombo, P., Duro, G., Costa, M.A. , Izzo, V., Mirisola, M., Locorotondo, G., Cocchiara, R., and Geraci, D. (1998). An update on allergens. Parietaria pollen allergens. Allergy 53, 917-921].
• the most common species are P. judaica and P. officinalis, but other species such as P. lusitanica , P. mauritanica, and P.
• Parietaria A significant characteristic of Parietaria is the long pollenisation period which lasts for a plurality of months and results in the presence of almost perennial symptoms in patients who are allergic to Parietaria, ranging from slight rhinoconjunctivitis to severe asthma. It should be noted that the normal light monospecific sensitisation to Parietaria involves the sensitisation to various species of this genus, since significant crosswise reactivity has been demonstrated between differing species of Parietaria.
• Both allergens belong to the family of non-specific lipid transfer proteins (ns-LTP) and possess a signal peptide in their terminal region which, after processing, gives rise to proteins having a molecular weight of 14,726 and 11,344 Da respectively and having about 45% of identical residues.
• ns-LTP non-specific lipid transfer proteins
• the possible IgE-binding linear epitopes in both allergens, which would be situated in structurally related zones, have been described [(24) Asturias, J.A. , G ⁇ mez-Bay ⁇ n, N., Eseverri, J. L., and Martinez, A. (2003) . Par j 1 and Par j 2, the major allergens from Parietaria judaica pollen, have similar immunoglobulin E epitopes. Clinical and Experimental Allergy 33, 518-524]. These regions are the targets which will be acted upon in order to be able to obtain the optimum hypoallergenic molecules for the treatment of the allergy to P
• ns-LTP are well known for their capacity to complete in vitro the intermembrane interchange and/or the transfer of polar lipids [(25) van Ree, R. (2002). Clinical importance of non-specific lipid transfer proteins as food allergens. Biochem. Soc. Trans 30, 910-913] .
• Two main families have been characterised in plants, LTPl with a molecular mass of approximately 9 kDa and LTP2 with a molecular mass of approximately 7 kDa. Allergens belonging to the LTP family have been identified in plants other than foods, where they have been widely studied.
• Hev b 12 from the latex of Hevea brasiliensis is a basic 9.3 kDa protein which demonstrates about 65 % sequence identity with allergenic LTPs of fruits of the Rosaceae family [(26) Beezhold, D. H., Hickey, V. L., Kostyal, D.A., and et al. (2003). Lipid transfer protein from Hevea brasiliensis (Hev b 12), a cross-reactive latex protein. Ann Allergy Asthma Immunol 439-445] .
• LTPs such as Art v 3 of Artemisia vulgaris [(27) Diaz-Perales, A., Lombardero, M., Sanchez-Monge, R., and et al. (2000).
• Lipid-transfer proteins as potential plant panallergens cross-reactivity among proteins of Artemisia pollen, Castanea nut and Rosaceae fruits, with different IgE-binding capacities.
• Clin Exp Allergy 1403-1410 and Ole e 7 of Olea europaea [(28) Tejera, M. L., Villalba, M-, Batanero, E., and Rodriguez, R. (1999) .
• WO2005/085278 discloses the construction of a fusion protein comprising the two major allergens of Parietaria judaica, wherein the three-dimensional structure of the fusion protein has been disrupted by replacement of certain cysteine residues in the primary sequence of each allergen (more specifically the cysteine residues involved in the formation of disulphide bridges) so that the sequences of allergens maintain essentially the same length. According to later experiments, this should lead to a protein that is 1000 times less allergenic than natural allergens.
• the inventors of the present invention have discovered that a surprisingly large reduction in allergenicity can be obtained not only by disrupting the three-dimensional structure of the allergen but also by deletion of some IgE-binding sites (known as B epitopes) and that, most surprisingly, this does not lead to a reduction in immunogenicity.
• the present invention discloses for the first time differing chimeric proteins obtained by binding fragments of the two allergens of Parietaria judalca (Par j 1 and Par j 2) containing a smaller number of IgE-binding epitopes, as well as differing methods and intermediates for obtaining them. Not only is the three-dimensional structure of the chimeric proteins of the present invention disrupted but also certain B epitopes have been deleted.
• the chimeric proteins according to the present invention can be called hypoallergenic with an allergenicity reduced by 99.99%, as they have a lower capacity to bind IgE antibodies based on: i) in vitro ELISA, ELISA inhibition and immunodetection tests using mixtures of sera from patients allergic to P.
• judaica in vivo tests of cutaneous reactivity in patients allergic to P. judaica; and Hi) in vitro EAST inhibition test with individualised sera from patients allergic to P. judaica.
• the chimeric proteins according to the present invention maintain their immunogenic capacity, as demonstrated by lymphoproliferation tests on peripheral blood mononuclear cells (CMSP) from 13 patients allergic to P. judaica.
• CMSP peripheral blood mononuclear cells
• the allergenic extracts are complex mixtures of proteins and non-protein molecules.
• the increasing use of techniques for detecting the levels of specific IgE against the components of an extract has made it possible to demonstrate that allergic patients usually have reactivity toward various components. Cases of allergic patients who react only to a single allergen are rare. Since allergenic extracts have obvious problems in immunotherapy, one solution is to group as many therapeutic properties as possible in a single molecule.
• the present inventors have succeeded in combining two allergens in one molecule, this having not only benefits from the point of view of industrial production and therapy, but also showing significantly reduced allergenicity with no change in immunogenicity.
• the present invention relates to chimeric proteins (hereinafter called Ql, Q2, and Q3) composed of fragments of the allergens Par j 1 and Par j 2, of which the allergenic reactivity is reduced without a loss of immunogenic capacity, as they have lost some of their IgE-binding B epitopes.
• Ql, Q2, and Q3 chimeric proteins
• the resultant chimeric polypeptide has a lower molecular weight than the sum of the two individual proteins.
• the present invention also relates to the nucleotide sequence which includes the DNA that encodes the aforesaid chimeric polypeptide, the expression system comprising said sequence accompanied by the sequences required for expression and control, and the receptor cell transformed by said expression system.
• This invention also relates to the clinical use of this chimeric polypeptide, to the specific immunotherapy for the treatment of allergies, as well as to possible compositions in which this chimeric polypeptide occurs and the different methods of administration thereof.
• the hypoallergenic properties of the chimeric proteins of the present invention which allow the use thereof in immunotherapy have been widely demonstrated.
• Immunological tests carried out by the inventors show that the chimera Q2 has no recognition of IgE in sera of patients allergic to Parletaria judaica , as illustrated in Fig. 10 and 11.
• Q2 has an IgE-binding capacity which is 10,000 times less than that of the mixture of the two natural proteins, as illustrated in Fig. 12.
• the low allergenicity (IgE-binding capacity) of the chimera Q2 was corroborated by measuring the reactivity of this molecule with sera of 30 patients allergic to Parietaria juda ⁇ ca (Fig. 14). This reduction in the allergenicity was surprisingly accompanied by maintenance of the immunogenic capacity of the chimera Q2, which did not differ from that of the sum of the individual natural proteins (Fig. 16).
• the strain of the microorganism ' corresponding to the present invention was deposited in the Spanish collection of type cultures (CECT) at the University of ⁇ Valencia (Universidad de Valencia, Edificio de Investigaci ⁇ n, Campus de Burjasot, 46100 BURJASOT, Valencia) in accordance with the Budapest treaty on the international recognition of the deposit of microorganisms for the purposes of patent procedures, with the following reference :
• Fig. 1 is a constructional diagram of Ql.
• Fig. 2 shows a sequence of amino acids and nucleotides of Ql.
• the residues corresponding to the affinity tail of the vector pQE-32 are underlined.
• the residues introduced by the EcoRI (EF) section are shaded.
• the cysteine residues have double underlining.
• Fig. 3 shows a comparison of the IgE epitopes of Par j 1 and Par j 2 (boxes) and the T epitope (underlined) , indicating the Q2 deleted epitopes in thick-lined boxes.
• the identical residues (:) and similar residues (.) have been labelled.
• Fig. 4 is a constructional diagram of Q2.
• Fig. 5 shows a sequence of amino acids and nucleotides of Q2.
• the residues corresponding to the affinity tail of the vector pQE-32 are underlined.
• the residues introduced by the EcoRI (EF) Pstl (LQ) , and EcorRV (DI) section are shaded.
• the cysteine residues have double underlining.
• Fig. 6 shows a comparison of the IgE epitopes of Par j 1 and Par j 2 (boxes) and the T epitope (underlined) , indicating the Q3 deleted epitopes in double-lined boxes.
• the identical residues (:) and similar residues (.) have been labelled.
• Fig. 7 is a constructional diagram of Q3.
• Fig. 8 shows a sequence of amino acids and nucleotides of Q3.
• the residues corresponding to the affinity tail of the vector pQE-32 are underlined.
• the residues introduced by the EcoRI (EF) Pstl (LQ) , and EcorRV (DI), BgIII (RS), and Kpnl (GT) section are shaded.
• the cysteine residues have double underlining.
• Fig. 9 shows staining by Coomassie blue of a polyacrylamide gel after electrophoresis: Ql (lane 1), Q2 (lane 2) , Q3 (lane 3) .
• Fig. 10 shows the results of a Circular dichroism analysis of the pufified proteins nPar j 1-nPar j 2, Ql, Q2, and Q3.
• Fig. 11 shows immunodetection with IgE antibodies of a mixture of sera of patients allergic to P. judaica comprising: nPar j 1-nPar j 2 (lane 1) , rPar j 1
• Fig. 12 shows a binding of IgE antibodies to Ql, Q2, and Q3 using 15 sera from patients allergic to P. judaica (dilution 1/10) . The values of three experiments are shown with their deviations.
• Fig. 13 shows an ELISA inhibition test result using P. judaica extract in the solid phase and nPar j 1-Par j 2, Ql, Q2, and Q3 as inhibitors using a mixture of sera of patients allergic to P. judaica. Each value corresponds to the mean of three experiments with a standard deviation of less than 10 %.
• Fig. 14 shows the result of cutaneous tests carried out with extract of P. judaica, nPar j 1-Par j 2 and Ql (50 ⁇ g/ml) , and Q3 (250 ⁇ g/ml) . The value shown is
• Fig. 15 shows the determination of specific IgE carried out with extract of P. judaica, nPar j 1-Par j 2, and Ql (50 ⁇ g/ml), and Q2 and Q3 (250 ⁇ g/ml).
• Fig. 16 shows a determination of the optimum concentration for studying the proliferation of T lymphocytes in patients allergic to P. judaica. Stimulation index (IE) .
• Fig. 17 shows the proliferation of T lymphocytes obtained with P. judaica extract and 0,5 ⁇ g/ml of the three chimeric proteins and natural and recombinant forms of Par j 1 and Par j 2. The value shown is that of the stimulation index (%) . No significant difference (ns) .
• a chimeric protein or peptide of low allergenicity obtained by deletion of any B epitope or IgE-binding epitode or synthesis without any B epitope or IgE-binding. Deletion will preferably apply to the B epitopes in positions 28 to 53 of Par j 1 and Par j 2.
• low allergenicity and similar variants as used herein is a relative term and relates to the reduced in vivo and in vitro ability of the proteins and peptides of the invention to stimulate an allergic response when compared with the same ability of wildtype immunogens .
• the chimeric protein comprises the amino acid sequence shown in SEQ ID No.: 2, 4 or 6, most preferably the chimeric protein comprises the amino acid sequence shown in SEQ ID No.: 4.
• the chimeric protein may alternatively or additionally comprise a sequence homologous to the amino acid sequence shown in SEQ ID No. : 2, 4 or 6.
• the homologous sequence has a homology of at least 70%, more preferably at least 80%, more preferably still at least 90%, most preferably 100%, to the amino acid sequence shown in SEQ ID No.: 2, 4 or 6, most preferably to the acid sequence shown in SEQ ID No.: 4.
• Another preferred embodiment includes a chimeric protein or peptide having an amino acid sequence with at least 70%, preferably at least 80%, more preferably at least 90%, most preferably 100%, homology to the amino acid sequence shown in SEQ ID No.: 2, 4 or 6.
• the chimeric protein may also comprise a peptide sequence facilitating its purification.
• peptides are commonly known in the art, for example a polyhistidine tail.
• the fragments constituting the chimeric proteins can be synthesised by a qualified trained person following a known scheme by amplification in the polymerase chain (PCR) . Said fragments, after being digested by appropriate restriction enzymes, can integrate by ligation to an expression vector.
• This expression vector (like the commercial vector pQE32) may have the ability to fuse the chimeric protein with sequences which assist purification such as a series of histidines positioned at the terminal amino end.
• the differing DNA fragments are bound by linkers formed by recognised sequences by differing restriction enzymes, and residues which did not exist in the original sequence of the natural allergen therefore appear in the final chimeric molecule.
• a polynucleotide encoding a protein or peptide of the invention.
• polynucleotide comprises the nucleotide sequence shown in SEQ ID No.: 1, 3 or 5, most preferably the polynucleotide comprises the nucleotide sequence shown in SEQ ID No.: 3.
• the polynucleotide may alternatively or additionally comprise a sequence homologous to the nucleotide sequence shown in SEQ ID No.: 1, 3 or 5.
• the homologous sequence has a homology of at least 70%, more preferably at least 80%, more preferably still at least 90%, most preferably at least 95%, to the nucleotide sequence shown in SEQ ID No.: 1, 3 or 5.
• Another preferred embodiment includes a polynucleotide having a nucleotide sequence with at least 70%, preferably at least 80%, more preferably at least 90%, most preferably 100%, homology to the nucleotide sequence shown in SEQ ID No.: 1, 3 or 5.
• the polynucleotide may further comprise a sequence encoding a signal peptide.
• the signal peptide is an amino acid sequence which initiates transport of a protein across the membrane of the endoplasmic reticulum. Suitable signal peptides will be known to one skilled in the field of the invention.
• the invention also includes peptides encoded by a polynucleotide sequence of the invention.
• an expression system comprising a polynucleotide sequence of the invention, and a host cell transformed by said expression system capable of expressing a protein or peptide of the invention.
• the invention also includes methods of making a peptide or protein of the invention, said method including the steps of
• the invention also comprises transgenic animals capable of producing a protein or peptide of the invention, for example in their milk or in the white of their eggs.
• a protein or peptide of the invention for the treatment of an immunological disorder, particularly a hypersensitivity disorder such as allergy.
• a protein or peptide of the invention for the preparation of a medicament for the treatment of an immunological disorder, particularly a hypersensitivity disorder such as allergy.
• the medicament is a vaccine.
• the hypersensitivity disorder is an allergy to Parietaria pollen, more preferably Parietaria judaica pollen.
• treatment and variations such as x treat' or ⁇ treating' refer to any regime that can benefit a human or non-human animal .
• the treatment may be in respect of an existing condition or may be prophylactic (preventative treatment) .
• Treatment may include curative, alleviation or prophylactic effects.
• the treatment is prophylactic treatment.
• a pharmaceutical composition comprising an effective amount of a protein or peptide of the invention and a pharmaceutically acceptable excipient.
• the pharmaceutical composition is a vaccine composition.
• a method of treating an immunological disorder, particularly a hypersensitivity disorder such as allergy comprising the step of administering an effective amount of a protein or peptide of the invention to a subject in need thereof.
• a protein or peptide of the invention for use in the treatment of an immunological disorder, particularly a hypersensitivity disorder such as allergy is provided.
• the present invention covers the use of the chimeras according to the present invention, preferably the chimera Q2, or synthetic peptides derived therefrom for desensitisation treatments in mammals.
• Desensitisation methods involve the repeated administration by parenteral routes (subcutaneous, intravenous, or intramuscular) , or oral, nasal or rectal routes of the allergen in question.
• parenteral routes subcutaneous, intravenous, or intramuscular
• oral, nasal or rectal routes of the allergen in question can be administered either alone or in combination with other diluents, in accordance with the prevailing legislation and the galenical procedures for use.
• the chimeric proteins Q2 and Q3 described in the invention are hypoallergenic since, as shown in Fig. 10, 11, 12 and 14, they have lower reactivity to the serum of patients allergic to P. judaica than the complete extract or the combined natural proteins, and, moreover, this hypoallergenicity is also found in in vivo cutaneous tests (Fig. 13) .
• the chimeric molecules Ql and Q2 also have an immunogenic capacity similar to that of the combined natural proteins (Fig. 16). Both characteristics (hypoallergenicity and immunogenicity) make the chimera Q2 an excellent candidate for the preventive and curative treatment of the allergy to P. judaica pollen which can be manifested as rhinitis, conjunctivitis, asthma, urticaria, angioedema, eczema, dermatitis, or even anaphylactic shock .
• Fig. 10 shows an immunodetection test which indicates that the chimeras Q2 and Q3 (lanes 5 and 6) have a greatly reduced IgE-binding capacity in allergic patients when compared with the reactivity of the two natural proteins (lane 1) , the isolated recombinant proteins (lanes 2 and 3) or the chimera (lane 4), which shows the recombinant fusion of the two proteins which are complete but contain all their B epitopes. This should indicate that the absence of the B epitopes included between the residues 29 and 52 (chimera Q2 ) contributes to the reduction in allergenicity.
• a more direct measurement of the hypoallergenicity of the chimera Q2 was obtained by direct measurement of cutaneous reactivity in 30 patients allergic to P. judaica pollen.
• the data given in Fig. 13 shows that the chimera Q2 had a marked reduction in cutaneous reactivity.
• a comparison of the descriptives of each distribution shows that the chimera Q2 has an average size of patches 112 times smaller than that observed for the two natural proteins, and this indicates a reduction in the allergenic activity by more than 99 %.
• the low binding capacity of IgE with the chimeric protein Q2 was also demonstrated with the serum from a further 30 patients allergic to P. judaica measured by EAST (Fig. 14) . In all the patients, the IgE-binding was greatly reduced in the chimera Q2 compared to the mixture of natural proteins.
• the protein Q2 demonstrated a lymphoproliferation index which was similar to that induced by the natural extract and the mixture of the two pure natural proteins combined, as shown in Fig. 16. All this shows that the chimeric protein Q2 constructed with fragments of Par j 1 and Par j 2 contained a smaller number of IgE epitopes but maintained sufficient T epitopes to induce a protective immune response .
• the chimeric proteins were constructed by chain amplification of the polymerase (PCR) using, as a matrix, plasmids containing the sequences coding for Par j 1.0103 and Par j 2.0101 described in Gonzalez-Rioja et al., "Expression and purification of the recombinant allergens Par j 1 and Par j 2" XXIII Congreso EAACI, Abstracts Book (2004), 181-182 and specific triggers in each case.
• the triggers are composed of the hybridation zone, of various section sites for differing restrictive endonucleases
• the PCR-induced amplification reaction had the following components in a reaction volume of 50 ⁇ l : amplification buffer xlO, 5 ⁇ l; 200 ⁇ M of dNTPs; 100 pmoles of each triggering oligonucleotide: 2.5 units of polymerase Taq
• Fragment 1 FlFl, CG GGATCC TGCAAGAAACCTGCGG ⁇ BamEI) and FlRl, CG GAATTC GGCTTTTTCCGGTGCGG (EcoRI).
• Fragment 2 F1F2, CG GAATTC GAGGAGGCTTGCGGGA (EcoRI) and F1R2, CG AAGCTT CTAATAGTAACCTCTGA ⁇ HindIII ).
• Par j 1 and Par j 2 Two products of PCR corresponding to Par j 1 and Par j 2 respectively were created.
• the synthetic oligonucleotides FlFl and FlRl were used for the N and C terminal ends respectively, a size of approximately 420 pairs of bases (pb) being obtained.
• the amplified fragment was cloned in the pKS- Bluescript vector and its sequence was confirmed.
• the second fragment corresponding to Par j 2 the same procedure was employed using the oligonucleotides F1F2 and F1R2 for the N and C terminal ends respectively. On this occasion, a fragment of approximately 300 pb was obtained, which was cloned in the pKS-Bluescript vector and of which the sequence was confirmed.
• This second fragment was bound to the first by ligation through the EcoRI target, Par j 1 remaining at the N-terminal end and Par j 2 at the C-terminal end, and the resultant fragment was subsequently subcloned in the commercial expression vector pQE-32 (Qiagen) , containing an extra sequence of 13 amino acids at the N-terminal end, corresponding to the affinity tail (Fig. 2) .
• Fragment 1 FlFl, CG GGATCC TGCAAGAAACCTGCGG (BamHI) and F2R1, CG CTGCAG CCCCTTTGACGGCTCTT (Pstl) .
• Fragment 2 F2F2, CG CTGCAG ATCCAGACCGCCATGAA (Pstl) and F2R2, CG GAATTC GGCTTTTTCCGGTGCGGG (EcoRI) .
• Fragment 3 F2F3, CG GAATTC GAGGAGGCTTGCGGGAA ⁇ EcoRI) and F2R3, CG GATATC CTCCTTCGACGGCTCCTT (EcoRV) .
• Fragment 4 F2F4, CG GATATC ATAGTGCGCGCCACGAA (EcoRV) and F1R2, CG AAGCTT CTAATAGTAACCTCTGA (Hindlll) .
• the conditions of the three fragments were: 94°C, 4 min (1 cycle; 94°C, 30 s - 54°C, 30 s - 72°C, 90 s (5 cycles) ; 94°C, 30 s - 62°C, 30 s - 72°C, 90 s (35 cycles); 72°C, 10 min (1 cycle) .
• Par j 1 fragment 1
• the synthetic oligonucleotides FlFl and F2R1 were used for the N and C terminal ends respectively, a size of approximately 90 base pairs being obtained and cloned in the pKS-Bluescript vector.
• the same procedure was carried out using the oligonucleotides F2F2 and F2R2 for the N and C terminal ends respectively.
• a fragment of approximately 260 pb was obtained and was cloned in the pKS-Bluescript vector. This last fragment was bound to the first by ligation through the linker of the target carried by Pstl and its sequence was confirmed.
• fragments amplified from the sequence of Par j 2 fragments 3 and 4
• the procedure described for fragments 1 and 2 was followed.
• the oligonucleotides F2F3/F2R3 and F2F4/F1R4 for the N and C terminal ends respectively were used for the amplification thereof, the size of the products of PCR being approximately 90 and 150 pb for fragments 3 and 4.
• Fragment 4 was bound to fragment 3 by ligation through the linker of the target carried by EcoRV and its sequence was confirmed.
• the two new fragments created were bound by ligation via the linker of the target carried by EcoRI, Par j 1 remaining at the N-terminal end and Par j 2 at the C-terminal end.
• Said fragment was subsequently subcloned in the commercial expression vector pQE-32, containing an extra sequence of 13 aminoacids at the N-terminal end corresponding to the affinity tail (Fig. 5) .
• Fragment 1 FlFl, CG GGATCC TGCAAGAAACCTGCGG (BaMiI ) and F3R1, CG AGATCT GACCTCGCTGACGAG (BgIII) .
• Fragment 2 F3F2, CG AGATCT AGCAAGCTCCCGCCC (BgIII) and F3R2, CG GGTACC GGGGACCTCGGCGAC ⁇ Kpnl) .
• the conditions of the 2 fragments were: 94°C, 4 min (1 cycle); 94°C, 30 s - 54 0 C, 30 s - 72°C, 90 s (5 cycles); 94°C, 30 s - 63 0 C, 30 s - 72°C, 90 s (35 cycles); 72°C, 10 min (1 cycle) .
• Fragment 3 F3F3, CG GGTACC CTCCCGCCCATCACC (Kpnl) and F3R3, TTTAAAAAGGCCGTAATATCC.
• the construction Q2-pQE-32 was used as the matrix and approximately 150, 370 and 290 pb in size were obtained for the fragments 1, 2 and 3 respectively.
• the oligonucleotides used were F3F1 and F3R1 for the fragment 1, F3F2 and F3R2 for the fragment 2, and F3F3 and F3R3 for the fragment 3.
• the processes of sequential ligation of the fragments described in the earlier cases was repeated again.
• the new restrictive enzymes used were BgIII and Kpnl .
• a pre-inoculum of 50 ml of the same medium was produced and incubated overnight at 37 0 C with stirring (260 rpm) .
• One litre of the same medium was inoculated with said pre-inoculum, starting from an optical density (600 nm) of 0.2.
• the mixture was incubated at 37 0 C with stirring for 1 hour and 30 minutes, after which induction with a IPTG (1 mM of final concentration) was carried out for 3 h under the same incubation conditions .
• the cells were centrifuged at 10,000 rpm for 15 minutes at 4 °C and were resuspended in 50 ml of lysis buffer (phosphate 20 mM, pH 7.4; 50 mM imidazole; 0.5 M NaCl).
• the resuspended matter was treated with lysozyme (final concentration of 0.1 mg/ml) for 30 minutes at 37 0 C with stirring. Sonication (5 pulses of 20 s) was then carried out and the mixture was centrifuged at 15000 rpm for 15 minutes at 4 0 C.
• the supernatant was filtered over 0.45 ⁇ m (Millex HV, Millipore) and was applied (2.5 ml/min) to a 5 ml Hi-Trap Chelating HP column (GE-Healthcare) adapted to the AKTA Prime system (GE-Healthcare) .
• This column was chelated with nickel and had previously been balanced with 10 volumes of the lysis buffer.
• elution buffer phosphate 20 mM pH 7.4; 0.5 M imidazole
• the elute was passed through a 5 ml Hi-Trap Desalting column (GE-Healthcare) in order to remove salts and change the 20 mM pH 7 phosphate buffer sample.
• the protein was kept at -40 0 C.
• Electrophoresis was carried out in polyacrylamide gels with SDS (SDS-PAGE) .
• the method described by Laemmli was basically followed [(31) Laemmli, U.K. (1970) . Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 277,
• Hybrid protein Ql was expressed as 32 kDa His-tagged fusion protein (Fig. 9) with a final yield of 2 mg/L of bacterial culture.
• Q2 and Q3 proteins were expressed as 28 kDa His-tagged fusion proteins with a final yield of 5 and 7 mg/L culture, respectively (Fig. 9) .
• NPA spectra presented a minimum at 208 nm, a well defined shoulder at 222 nm and a maximum at 190 nm, whereas spectra of the hybrid proteins shifted towards random coil conformation reaching an almost completely unfolded state.
• Example 4 Immunological tests to demonstrate the low IgE-fixing reactivity of chimeric proteins toward a mixture of sera of patients allergic to P. judaica A) Immunodetection
• a first evaluation of the IgE-binding activity of the fusions 1, 2 and 3 was carried out by the immunotransfer method employing a mixture of sera from patients allergic to P. judaica.
• electrotransfer was carried out by the method of Towbin et al [(32) Towbin, H., Staehelin, I., and Gordon, J. (1979). Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: Procedure and some applications. Proc. Natl. Acad. Sci. USA 16, 4350-4354].
• the proteins separated by SDS-PAGE were electrically transfixed to PVDF Hybond-P membranes (GE-Healthcare) .
• the membranes had been blocked for 1 h at ambient temperature, they were incubated overnight at 4 °C with a primary antibody and, after the various washes with the same washing buffer, the membranes were incubated for 1 h at ambient temperature with a secondary peroxydase conjugate antibody. Bands were detected by the ECL chemiluminescent method (GE-Healthcare) as described by the manufacturer by exposing the membrane to a film (Hyperfilm. ECL, GE-Healthcare) .
• the reactivity of IgE to the three fusions was analysed by the ELISA method using individual sera from patients allergic to P. judaica.
• the polystyrene plates (Greiner) were incubated overnight at ambient temperature with 1 ⁇ g of P. judaica extract protein or 0.1 ⁇ g of pure protein per cup in PBS buffer (phosphate 10 mM pH 7.2; 137 mM NaCl 2.7 mM KCl). They were blocked with 200 ⁇ l/cup of PBS supplemented with 1 % BSA-0.05 % Tween 20 and kept at 37 0 C for 1 h. 100 ⁇ l/cup of serum (dilution 1/10) from allergic patients were added and incubated at 37 0 C for 90 min.
• the chimeras Q2 and Q3 reached a maximum degree of inhibition of about 60%, but this required a protein concentration 10,000 times higher than that of required for nPar j 1-Par j 2 in order to reach the same degree of inhibition, which implied that these chimeras had reduced their allergenicity by about 99.99%.
• Example 5 In vivo experiments to demonstrate the low cutaneous reactivity of the chimeric proteins Ql, Q2, and Q3
• Jn vivo skin reaction (prick) tests were carried out to evaluate the hypoallergenic nature of the chimeras 1, 2 and 3 with the aim of determining the candidate hypoallergenic molecule in order to be able to develop satisfactory immunotherapy against the allergy to P. judaica pollen.
• the cutaneous tests were carried out using P. judaica extract, nPar j 1-Par j 2, rPar j 1 and rPar j 2 expressed in E. coli, and the chimeras 1, 2 and 3.
• the purified proteins were diluted in a 0.5 % phenolated and 50 % glycerinated physiological saline solution.
• the concentrations used were 0.5, 5, and 50 ⁇ g/ml in the case of natural Par j, Par j 2, Ql; the concentrations were 5, 50 and 250 ⁇ g/ml in the case of Q2 or Q3.
• 0.9% NaCl and histamine hydrochloride (10 itig/ml) were used as negative and positive controls respectively.
• the chimeras 2 and 3 are virtually zero: Q2 (mean 0.95 mm - confidence range 95%: 0-2.89) and Q3 (mean 0.15 mm 2 confidence range 95%: 0-0.47).
• Example 6 Experiments to demonstrate the low IgE antibody-binding capacity of the chimeric proteins Ql, Q2, and Q3
• the specific IgE was determined in accordance with Ceska et al . [(33) Ceska, M. and Lundkvist, U. (1972) . A new and simple radioimmunoassay method for the determination of IgE. Immunochemistry 9, 1021-1030], by coupling the natural and recombinant proteins (50 ⁇ g/ml) as well as the P. judaica extract (2 mg/ml) to discs activated with cyanogen bromide. 50 ⁇ l of serum from the patients was subsequently added and was incubated for 1 hour at ambient temperature.
• Example 7 Induced lymphoproliferation experiments to demonstrate the immunogenic capacity of the chimeric proteins Ql, Q2 and Q3
• CMSP mononuclear peripheral blood cells
• the CMSPs of 13 patients allergic to P. judaica were isolated by centrifugation in a density gradient using a lyr ⁇ phocite separating solution (Lymphoprep,
• CMSPs were then resuspended at 1x10 viable cells/ml in culture medium (serum free medium AIM V, Gibco) and their viability was tested with 0.25% of tripan blue in PBS (Sigma Chemical Co.).
• the CMSPs prepared with viability greater than 90% were immediately used for the in vitro proliferation tests as described by the manufacturer (cell proliferation agent WST-I, Roche Diagnostics) . They were deposited in flat-bottomed microplates (Nunclon, NUNC) , 2x10 CMSP in a final volume of medium of 200 ⁇ l and measurements were taken in triplicate at 37 0 C and 5% C02 humidified atmosphere with the P.
• immunuogenic protein was scanned to determine the optimum concentration for subsequent development of the test. In all cases it was found that the protein concentration demonstrating maximum proliferation (IE %) was 0.5 ⁇ g/ml (Fig. 16) .
• the present invention relates to the use of the described hypoallergenic chimeras or synthetic peptides derived therefrom for hyposensitisation treatments in mammals.
• the method of hyposensitisation involves the repeated administration by parenteral (subcutaneous, intravenous or intramuscular) , oral, sublingual, nasal or rectal routes of the allergen in question.
• These (poly) peptides can be administered alone and in combination with other pharmaceutically acceptable diluents and excipients, in accordance with the legislation in force and the galenical procedure for use.

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