Title:  Compound and method for the prevention and/or the treatment of allergy

United States Patent:  6,602,509

Issued:  August 5, 2003

Inventors:  Saint-Remy; Jean-Marie (Grez-Doiceau, BE); Jacquemin; Marc (Sart-Bernard, BE)

Assignee:  Leuven Research & Development VZW (Leuven, BE)

Appl. No.:  362731

Filed:  July 29, 1999

Abstract

The present invention is related to a compound for the prevention and/or the treatment of allergy consisting of: at least one allergen antigenic determinant which is recognized by a B cell or an antibody secreted by a B cell of a non-atopic individual to said allergen, and at least one antigenic determinant of an antigen different from said allergen which triggers T cell activation.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a vaccination strategy by which the antibody response made by atopic individuals against allergens is deviated from the allergen major determinants that are spontaneously recognised by atopic individuals, to determinants on the same molecule that are spontaneously recognised by antibodies of non-atopic individuals, or to determinants which are not spontaneously recognised by the majority of individuals, independently of their atopic status.

The present invention is related to a compound comprising either

at least one allergen antigenic determinant which is recognised by a B cell or antibody secreted by a B cell of a non-atopic (to said allergen) individual (including cryptic determinant which is not recognised by atopics individuals, and minimally recognised by non-atopics individuals) and which is preferably not recognised by a T cell, and at least one antigenic determinant of an antigen different from said allergen, said antigenic determinant triggering T cell activation, or

a nucleotide sequence encoding said both antigenic determinants, said sequence being possibly linked to one or more regulatory sequence(s) active into a patient's cell.

The specific allergen antigenic determinants present in known main allergens are easily identified by the person skilled in the art, who may select said epitopes or antigenic determinants of said allergen which are recognised by non-atopic individuals (non-atopic individuals to said allergen) and which may differ from the other epitopes for which atopic individuals produce antibodies as above-described. Similarly, the person skilled in the art may select the specific antigenic determinant of any antigen (different from said allergen) which is known to trigger T cell activation. Preferably, said antigen is not an allergen. A preferred selection of this epitope is described in the examples presented hereafter.

The compound according to the invention will produce in atopic patients a shift of the anti-allergen immune response towards epitopes or antigenic determinants that are not spontaneously or only minimally recognised by antibodies of atopic patients.

In the compound according to the invention, the allergen antigenic determinant and the antigenic determinant of the non-allergic antigen are preferably peptidic sequences chemically bound together (in a linear tandem form or branched form), preferably by a peptidic link, which is preferably made of at least two amino-acids. The compound according to the invention is in a linear or a cyclic form, with or without additional moieties used, for instance to block peptide--peptide interactions.

Advantageously, the allergen is selected from the group consisting of Der pI and Der pII of house dust mite Dermatophagoides pteronyssinus, the major antigen of Aspergillus fumigatus, the staphylococcal B enterotoxin (SEB) and the bovine .beta.-lactoglobulin or the allergen described in the documents Clin. Exp. Allergy, No. 26, pp. 494-516 (1996); Mol. Biol. of Allergy and Immunology, ed. R. Bush, Immunology and Allergy Clinics of North American Series (August 1996).

Advantageously, in the compound according to the invention, the antigenic determinant of an antigen which triggers T cell activation is a T cell epitope (preferably a helper T cell epitope) of tetanus toxoid, diphtheria, mycobacterium, influenza or measles viruses antigens (other examples of said T cell epitopes are described in the table II of the document WO95/26365).

Preferably, the compound according to the invention is selected from the group consisting of the peptides having the following amino acid sequences:

SEQ ID NO. 1:

QYIKANSKFIGITELGGHEIKKVLVPGCHGS

SEQ ID NO. 2:

HEIKKVLVPGCHGS

SEQ ID NO. 3:

DQYIKANSKFIGITELGGQYIKANSKFIGITELSSCHGSEPCIIHRGKPFGGCHGSEPC IIHRGKPFSSCHGSEPCIIHRGKPFGGCHGSEPCIIHRGKPFSSCHGSEPCIIHRGKPF GGCHGSEPCIIHRGKPFSR

SEQ ID NO. 4:

PKYVKQNTLKLATGKKGPKYVKQNTLKLATGKKGVIIGIK

SEQ ID NO. 5:

QYIKANSKFIGITELGGCHGSEPCNIHRGKPF

or a nucleotidic sequence encoding at least one of said amino-acids sequences, preferably the nucleotide sequence

SEQ ID NO. 6: GAATTCCCACCATGGATCAGTATATAAAAGCAAATTCTAAATTT ATAGGTATAACTGAACTAGGAGGTTGCCATGGTTCAGAACCATGTATCATTCATCGTGG TAAACCATTCGGCGGTTGTCACGGAAGTGAGCCTTGCATTATACACAGAGGAAAGCCGT TCTAAGCGGCCGC.

Another aspect of the present invention is related to a pharmaceutical, cosmetical, food and/or feed composition comprising the compound according to the invention and a pharmaceutical, cosmetical, food and/or feed acceptable carrier.

Preferably, said pharmaceutical composition is a vaccine which may comprise a pharmaceutical acceptable carrier which can be any compatible non-toxic substance suitable for administering the composition (vaccine) according to the invention to a patient and obtain the desired therapeutical or prophylactic properties. The pharmaceutically acceptable carrier according to the invention suitable for oral administration are the ones well known by the person skilled in the art, such as tablets, coated or non-coated pills, capsules, solutions or syrups. Other adequate pharmaceutical carriers or vehicles may vary according to the mode of administration (cutaneous, epicutaneous, subcutaneous, intradermal, inhalation, patching, intravenous, intramuscular, parenteral, oral, etc.).

When the compound according to the invention is a nucleotidic sequence, the compound according to the invention can be administered naked or on a suitable pharmaceutical carrier such as a "vector" used for the transfection, transduction and expression of said sequence by a cell of the patient (including the expression and secretion outside the cell of the peptidic sequence encoded by said nucleotic sequence). Said "vector" is preferably selected from the group consisting of plasmids, viruses (retroviruses, adenoviruses, . . . ), lipidic vectors (such as cationic vesicles, liposomes, . . . ), molecules or devices which result in a chemical or a physical modification of the transfected cell (dextran phosphate, calcium phosphate, micro-injection device, electroporation device, etc.) or modified recombinant organisms comprising the compound according to the invention derived for instance from Salmonella or Mycobacteria strains, a nucleic acid encapsulated in the form of micro- or nanoparticles such as chirosan as described by Roy et al., Nature Medicine 5, pp. 387-391 (1999), etc.

The genetic modification of the patient's cell(s) for an ex vivo or in vivo treatment can be obtained by the person skilled in the art according to the known methods in the field of genetic therapy (such as the one described in the documents WO91/02805, WO91/18088, WO91/15501).

The pharmaceutical composition or the vaccine according to the invention may also comprise adjuvants (including helper viruses) well known by the person skilled in the art which may modulate the humoral, local, mucosal and/or cellular response of the immune system of a patient and improve the use of the compound according to the invention.

Adjuvants can be of different forms, provided they are suitable for administration to human beings. Examples of such adjuvants are oil emulsions of mineral or vegetal origin; mineral compounds such as aluminium phosphate or hydroxide, or calcium phosphate; bacterial products and derivatives, such as P40 (derived from the cell wall of Corynebacterium granulosum), monophosphoryl lipid A (MPL, derivative of LPS) and muramyl peptide derivatives and conjugates thereof (derivatives from mycobacterium components), alum, incomplete Freund's adjuvant, liposyn, saponin, squalene, etc. Recent reviews on adjuvants for human administration are described by Gupta R. K. et al. (Vaccine 11, pp. 293-306 (1993)) and by Johnson A. G. (Clin. Microbiol. Rev. 7, pp. 277-289 (1994)).

The pharmaceutical composition according to the invention is prepared by the methods generally applied by the person skilled in the art, for the preparation of various pharmaceutical compositions, especially vaccines, wherein the percentage of the active compound/pharmaceutically acceptable carrier can vary within very large ranges (generally a suitable dosage unit form contains about 0.005 .mu.g to about 1 mg of compound per kg/body weight of patient), only limited by the tolerance and the level of accointance of the patient to the compound. The limits are particularly determined by the frequency of administration and by the specific diseases or symptoms to be treated.

Preferably, the compound is present in the pharmaceutical composition in a concentration which allows at least the reduction or suppression of the signs and symptoms of allergy or of a disease of allergic origin (preferably signs and symptoms of immediate hypersensitivity allergy).

The cosmetical composition according to the invention may comprise any cosmetical acceptable carrier selected according to the specific mode of administration. For instance, for skin hygiene, the cosmetical composition could be a product in the form of a cream, an ointment or a balsam.

The food or feed composition according to the invention could be any food, feed or beverage acceptable carrier comprising the usual liquid food or feed ingredients wherein the compound according to the invention is included.

Another aspect of the present invention is related to the use of the compound according to the invention as a medicament.

The present invention is also related to the use of the compound according to the invention or the pharmaceutical composition according to the invention for the manufacture of a medicament in the prevention and/or the treatment of allergy or of a disease of allergic origin, particularly immediate hypersensitivity allergy.

Another aspect of the present invention is related to a prevention and/or treatment method of allergy or of a disease of allergic origin, particularly immediate hypersensitivity allergy, comprising the step of administering the compound or the pharmaceutical composition according to the invention to a patient preferably a human patient, especially an atopic individual to an allergen, in order to elicit or increase advantageously the production of antibodies towards antigenic determinants of the allergen that are not spontaneously or only minimally recognised by the immune system of atopic individuals.

These diseases include rhinitis and sinusitis of allergic origin, bronchial asthma, atopic dermatitis, some forms of acute and chronic urticaria, gastro-intestinal syndromes associated with the ingestion of food allergens such as .beta.-lactoglobulin, the so-called oro-pharyngeal syndrome of the same origin, anaphylactic reactions associated with drug hypersensitivity.

The present invention will be described in the following examples, in reference to the enclosed figures. These examples are presented as non-limiting illustrations of the various embodiments of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

Atopics as well as non-atopic subjects produce antibodies towards environmental allergens. These antibodies belong to all isotypes described so far, including IgE (Saint-Remy J. M. R. et al., J. Immunol. 43, pp. 338-347 (1988)). It is usually observed that atopic individuals produce 10 to 100-fold more IgE antibodies than non-atopic individuals, which can at least partly explain why atopics suffer from symptoms when encountering allergens to which they are sensitised.

It has been unexpectedly discovered that the antigenic determinants of allergens such as Der pI and Der pII--two of the main allergens of the house dust mite Dermatophagoides pteronyssinus--which are recognised by antibodies of atopics are not identical to those recognised by non-atopic individuals. This conclusion was reached by using a series of monoclonal antibodies raised in mice against purified Der pI or Der pII molecules. In a competition immunoassay, the Inventors have determined that some of the antigen determinants are recognised by anti-allergen antibodies from atopic individuals, while other determinants are recognised by anti-allergen antibodies produced by non-atopics. Further, they have shown that atopic patients whose allergic symptoms improved, either spontaneously or as a result of treatment, started producing antibodies to the very determinants recognised by non-atopic individuals, while reducing the production of initial antibodies.

The invention relates to the use of peptides derived from regions of allergen molecules that are recognised by antibodies made by non-atopics, or possibly regions which do not elicit a spontaneous antibody response. Administration of said peptides to atopic individuals results in the production of specific antibodies. Such antibodies will bind to the allergens whenever the patients are naturally exposed to them and, as a consequence, will restrict the access of antibodies made spontaneously by patients. Some atopic patients additionally produce a small proportion of antibodies to antigenic determinants recognised by non-atopics. In such cases, administration of the said peptides will increase the proportion of such antibodies so as to render them predominant in the anti-allergen immune response.

It is therefore the purpose of the present invention to provide a method by which the anti-allergen immune response is re-directed towards epitopes that are not spontaneously, or only minimally, recognised by antibodies produced by atopic patients.

The method of immunisation that is the object of the present invention provides several advantages over other methods.

Firstly, the immunisation procedure according to the invention is safe, as the peptides used do not carry determinants that can be recognised by IgE antibodies and have therefore no capacity to induce an anaphylactic reaction. This property contrasts with methods of immunisation using whole allergen molecules in their native or altered forms.

Secondly, the amount of immunising material and the number of injections required according to the invention are very much reduced as compared to alternative immunotherapeutic strategies, for the following reasons

(1) as the peptides produced by the present invention do not contain IgE binding determinants, an immunogenic dose of peptide can be given at once, which therefore significantly shorten the length of treatment. Admixture or concomitant administration of an adjuvant can increase the immunogenicity of the peptides, further reducing the number of injections (and the amount of material required) to possibly a single one;

(2) as atopic individuals can in fact produce a small amount of antibodies directed to the epitopes recognised by non-atopic individuals, injection of peptides obtained by the present invention therefore boosts a secondary immune response (a secondary immune response will result in the production of much higher antibody titres than a primary immune response);

(3) as the administration of peptides alters the immune response to allergens at an early stage, namely the allergen recognition, processing by antigen-presenting cells and presentation to T cells, a limited amount of material will be all that is required to achieve the aim of the present invention.

The above-described characteristics represent a definite advantage over conventional desensitisation which has to be administered for several months or years and which makes use of high amount of allergens. In alternative therapies, such as the use of peptides to anergise T cells, the therapy requires much higher amounts of free peptides to compensate the high rate of peptide catabolism, and repeated administration is needed to maintain the anergic state.

Thirdly, continuing exposure to the allergens present in the natural environment of patients treated by the present invention is sufficient to maintain the immune response towards the antigenic determinants corresponding to peptides used for immunisation. Experimental evidence is indeed available showing that mice immunised with a peptide derived from a antigen maintain their reactivity towards the peptide upon subsequent challenge with the whole antigen (clonal dominance phenomenon) (Benjamini E. et al. J. Immunol. 141, pp. 55-63 (1988) and Schutze M. P. et al. J. Immunol. 142, pp. 2635-2640 (1989)) and enclosed FIG. 1).

The method according to the invention also represents a clear advantage over other therapies by which tolerance to allergens rather than immunisation towards novel antigenic determinants are sought. In the former, repeated administration of tolerogens is required to maintain the state of unresponsiveness.

The precise mode of action of the present invention is not yet completely elucidated.

The number of possible antigenic determinants is high that can be recognised by antibodies on allergens. However, allergens are usually small molecules, which restricts the number of antibody molecules which can bind to allergens at the same time. Antibodies which are present at the highest concentration and/or exhibiting the highest affinity will preferentially bind to the allergen. The same holds true for specific B cells, which express at their surface membrane an immunoglobulin molecule identical to the one they secrete. An antigen will therefore be captured by B cells which have the highest affinity and/or the highest frequency. This will prevent activation of B cells recognising other epitopes on the same molecule, a phenomenon which is called the "clonal dominance phenomenon" (Schutze M. P. et al. J. Immunol. 142, pp. 2635-2640 (1989)).

If one induces a preferential immune response in atopic individuals towards epitopes that are not or only weakly recognised by spontaneously formed antibodies, the clonal dominance phenomenon indicates that the anti-allergen immune response will now be directed to these new determinants and will decrease to antigenic determinants recognised initially. Two lines of experimental evidence support this concept. First, removal of an immunodominant B cell epitope on an antigen uncovers epitopes that were not recognised on the intact antigen and towards which the antibody response is now directed (Scheerlinck J. P. Y. et al., Mol. Immunol. 30, pp. 733-739 (1993)). Second, mice immunised with an antigen use only a fraction of their potential B cell repertoire to mount a specific immune response; immunisation with a peptide activates a selected repertoire of B cells, whose reactivity will be maintained even though the animal is challenged later with the native antigen (Benjamini E. et al. J. Immunol. 141, pp. 55-63 (1988)).

These two sets of experiments illustrate what is happening as a consequence of the compound administration according to the present invention. In further support of the concept of clonal dominance and its application to the allergy, Balb/c mice were injected with a recombinant (r) allergen, Der pII. The precise specificity of antibodies produced by such mice were determined by reaction with a panel of 15-mer peptides covering the entire Der pII sequence with a 5 aminoacid overlap.

In the example shown in FIG. 1, mice are producing antibodies to rDer pII and to peptide 11-25. Further immunisation with peptide 21-35 induces an immune response to 21-35 and a significant decrease of the binding to peptide 11-25. The immune response to Der pII is therefore redirected towards determinants that were not recognised first. Further, this experiment shows that the induced "re-directed" immune response resists further immunisation with the whole rDer pII allergen.

To be fully efficient, however, the peptide carrying a B cell epitope has to be administered together with an epitope that can be recognised by T cells, which will provide the B cells with the necessary signals to allow full differentiation into mature, antibody-producing plasmocytes. The T cell epitope does not have to be derived from the same molecule as the B cell. Therefore an hetero-peptide containing a B cell epitope derived from a given allergen and a T cell epitope of another origin will maintain the required specificity at the B cell level, while ensuring that the necessary signals provided by T cells are present. Such signals include the cognate B-T cell recognition and antigen non-specific signals such as interleukine production, CD40 interaction with its ligand, B7 (CD80) interaction with CD28 (Austyn & Wood, Principles of Cellular and Molecular Immunology, Oxford University Press (1993).

The T cell epitope (or epitopes) used for the present invention is selected according to its capacity to activate T cells of a majority of patients. Preferably, it is derived from an antigen commonly used for routine immunisation, such as tetanus toxoid or diphtheria antigen. This carries two main advantages. First, a number of universal, public T cell epitopes, namely, recognised by a vast majority of patients, have been described in such molecules (Reece J. C. et al., J. Immunol. 151, pp. 6175-6184 (1993)). Second, as virtually all individuals are vaccinated against tetanus toxoid or diphtheria, priming with the T cell epitope used for the present invention is already achieved, which should increase the efficacy of the vaccination, with possible reduction in doses and number of injections.

Peptides used for immunisation in the context of the present invention are preferably produced by synthesis (see for example Grant Editions, Synthetic Peptides) by an applied biosystem peptide synthesizer model 430 A or 431 or recombinant DNA techniques for their encoding nucleic acid sequences.

The composition containing the peptides is in a form suitable for injection by the subcutaneous, intramuscular or intradermal route. However, forms for inhalation, ingestion or direct application on skin or mucosa are possible.

The peptides can be in a linear or cyclic form, with or without additional moieties used, for instance, to block peptide-peptide interactions. Peptides can also be integrated into short peptide structures which force a specific 3-D conformation such as alpha-helix.

The composition can contain other material than the peptides, such as adjuvants.

The method as described in the present invention can be used to treat human or animal diseases in which IgE antibodies are demonstrated and deemed to play a role in the triggering of symptoms.

The present invention can be also applied to patients sensitive to allergens of animal or vegetal origin, or to chemical and pharmaceutical compounds like antibiotics (penicillin).

Claim 1 of 7 Claims

What is claimed is:

1. An isolated compound consisting of one of the following amino acid sequences selected from the group consisting of SEQ ID NO. 1, SEQ ID NO. 3, SEQ ID NO. 4 and SEQ ID NO. 5.

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