WO2006075253A2 - Peptides for desensitizing subjects allergic to dog hair and dander and compositions containing said peptides. - Google Patents

Abstract

The present invention relates to peptides capable of desensitizing, in a specific manner, the great majority of subjects allergic to dog hair and dander, said peptides being defined by a sequence of 11 to 38 consecutive amino acids of a fragment of the mature Can f 1 protein which is selected from the group consisting of: the fragments from positions 3 to 30, 49 to 66 , 73 to 104, and 107 to 144, with the exclusion of the sequences from positions 15 to 26, 50 to 62 and 122 to 133, and to compositions containing said peptides.

Description

PEPTIDES FOR DESENSITIZING SUBJECTS ALLERGIC TO DOG HAIR AND DANDER AND COMPOSITIONS CONTAINING SAID PEPTIDES

The present invention relates to peptides capable of desensitizing, in a specific manner, the great majority of subjects allergic to dog hair and dander, and to compositions containing said peptides.

In addition to cats and house dust mites, dogs (Canis familiaris) are an important source of indoor allergens. Dog hair is readily transported on clothes and can be detected not only in the houses where dogs are kept but also in other places such as schools and day care centres where dogs are not present on regular basis. Chronic exposure to dog allergen may lead to allergic sensitization and contribute to the development of bronchial hyperreactivity and asthma in children. The prevalence of allergy to dogs as assessed by skin prick test is reported to occur in approximately 15 % of a young adult population.

Dog hair and dander extracts are complex mixtures containing a number of allergenic proteins but only one major allergen, Can f λ, which elicits a high frequency of positive skin test reactions among dog allergic patients and account for, on average, 70 % of IgE binding to dog dander extracts (Schou et al., Clinical and Experimental Allergy, 1991, 21, 321-328 ; De Groot et al., J. Allergy Clin. Immunol., 1991, 87, 1056-1065). Can f λ is a member of the lipocalin family of small ligand- binding proteins which includes a bovine (Bos d 2) and a murine (Mus m l) allergen. It is expressed in tongue epithelial tissue and is found in the saliva and on dog hair and dander. Can f 1 cDNA was cloned and found to encode a 18 amino acids signal sequence followed by a 156 residues mature protein (Konieczny et al., Immunol., 1997, 92, 577-586; International application WO 94/16068; SWISSPROT 018873). The antibodies involved in atopic allergy belong primarily to the IgE class of immunoglobulins. IgE appear gradually under the repeated contact with the allergen and before any symptom becomes apparent. IgE possess the property of binding, via their Fc fragment, to receptors situated on the tissue mastocytes and the blood basophils. When the allergen forms a complex with the specific IgE bound to the membrane of the basophils or mastocytes, it causes degranulation of the cells and the release of among other substances, histamine, serotonin, heparin, chemotactic factors for eosinophilic leukocytes and/or leukotrienes C4, D4 and E4 which are responsible for the allergic symptoms. Such symptoms may be systemic or local in nature, depending on the route of entry of the antigen and the pattern of deposition of IgE on mastocytes and basophils. IgE are not solely responsible for the allergy because although the IgE level is an indicator for the disease, it has no diagnostic value for the state of the patients. It is not rare for patients to have high IgE levels without showing symptoms. The appearance of IgE in allergic patients results from the production of Th2-type cytokines such as IL-4, IL-5 and IL- 13 and is inhibited by the synthesis of IFN-γ (Chretien et al., Eur. J. Immunol, 1990, 20, 243-). It is mainly the CD4⁺T lymphocytes which produce these cytokines. Specific T cells which secrete more IL-4 than IFN-γ are effectively found in allergic patients, whereas the T cells isolated from nonallergic subjects produce more IFN-γ than IL-4. The Th2-type CD4⁺ T lymphocytes specific for the allergens therefore actively participate in the appearance and the maintenance of the allergy.

Patients with dog allergy are currently treated by conventional specific immunotherapy, which consists in the injection of small, gradually increasing doses of the allergen (dog dander extracts), to desensitize the allergic subjects.

However, treatment of dog allergic patients with dog dander extracts has not proven to be as efficacious as treatment of cat allergic patients with cat dander extracts (Hedlin et al., J. Allergy, Clin. Immunol., 1991, 87, 955-964). Also, the use of isolated recombinant Can f 1 protein in replacement of dog dander extracts has not proven to be more efficacious (International application WO 94/16068). Furthermore, the use of allergen extracts or allergenic proteins for the desensitization scheme presents the drawbacks of potential anaphylaxis during treatment and the necessity of continuing therapy, often for a period of several years to build up sufficient tolerance that results in significant diminution of clinical symptoms.

In this context, there is a need for alternative molecules for specific immunotherapy of dog allergy.

The immunological mechanisms of specific immunotherapy are not totally understood. However, observations indicate that the improvement in the patient's condition, following injection of an allergen extract, results from a modification in the cytokine profile of the CD4⁺ T lymphocytes specific for the allergen; IL-4 and IL- 13 production is decreased while the production of IL-IO is increased. Since IL-4 and IL- 13 contribute to high levels of IgE, the decrease of this cytokines reduces the symptoms of allergy. Similarly, the increase of IL-IO which reduces the proliferation and the production of IFN-γ and IL-4 by T CD4⁺ lymphocytes, and stimulates the production of IgG4 by B lymphocytes instead of IgE improves the symptoms of allergy. In addition, it has been proposed that the control of allergy by IL-IO involves its association with another immunosuppressive cytokine, TGF-β (Jutel et al., Eur. J. Immunol., 2003, 33, 1205-).

Similar results were observed in patients allergic to the cat hair allergen FeI rf 1 or the bee venom phospholipase A2, which had been treated by injection of increasing doses of peptides carrying T epitopes of these allergens, without adjuvant ; IL-4 and IL- 13 production were decreased while the production of IL-IO was increased (Oldfield et al., Lancet, 2002, 360, 47-; Muller et al., J. Allergy Clin. Immunol., 1998, 101, 747-). These observations make it possible to consider the CD4⁺ T cells specific for the allergen as the target cells for immunotherapy, and the T-cell epitopes which are recognized specifically by these cells (T CD4⁺ epitopes) as the basic elements for the production of alternative molecules for specific immunotherapy. The CD4⁺ T cells possess a rearranged T receptor which allows them to selectively recognize peptide fragments derived from the degradation of the antigen by the presenting cells and presented by molecules of the Major Histocompatibility Complex class II (MHC II) or Human Leukocyte antigen class II (HLA II) in humans (Germain et al., Annu. Rev. Immunol., 1993, 11, 403-). The determinants carried by these peptide fragments and recognized by the T cells are called T-cell epitopes. T CD4⁺ epitopes are small peptides possessing a sequence of variable length which is able to bind specifically to HLA II molecules; although peptides naturally bound to HLA II molecules are 13 to 25 amino acids long, smaller peptides (11 to 20 amino acids) still bind to HLA II molecules in vitro and in vivo. T CD4⁺ epitopes are usually not antigenic for the specific IgE present in the allergic patients and thus avoid the risks of anaphylaxis encountered with the allergenic protein or the allergen extracts. In addition, their ability to modify the cytokine profile of the T CD4⁺ cells specific for the allergen should reduce the symptoms of allergy in the patients. Thus, this alternative approach is expected to be advantageous in terms of efficacy, safety and cost.

Therefore, the use of T CD4⁺ peptides specific for an allergen to desensitize allergic patients has been proposed on several occasions as an advantageous alternative to conventional specific desensitization, including for Canf 1 (International application WO 94/16068), bee venom phospholipase A2 (US Patent 6,649,166), FeI d 1 (Norman et al., Am. J. Respir. Crit. Cre Med., 1996, 154, 1623- ), ragweed (WO 93/21321; WO 96/13589), Japanese cedar pollen (WO 93/01213; WO 94/01560) and ryegrass pollen (WO 94/21675; WO 94/16068). For example, in the case of Can f 1 , three T-cell epitopes were predicted by computer analysis (C29-33, C70-76 and C122-133; Zeiler et al., J. Immunol, 1999, 162, 1415-1422) and significant areas of T cell reactivity were found in all three parts of the protein (fragments from positions 9 to 73, 64 to 116 and 98 to 156 of the mature protein) and two peptides (C 15-26 and C50-62) had T cell stimulation activity in 43 % of the patients tested (n = 12).

However, all the peptides described, except the peptides of the phospholipase A2, were selected on the basis of the stimulation of T lymphocytes in a group of allergic patients, and not on the binding to HLA II molecules.

Therefore, these peptides are specific for certain patients and are not suitable for a significant set of patients because their selection does not take into account the HLA type of the patients. Since the group of patients is not representative of the different HLA II alleles of the population, the response of the majority of patients to a given peptide may result from a bias in the sampling of the patient and not from the ability of the peptide to be recognized by the majority of patients. Accordingly, the inventors set themselves the objective of providing a set of peptides capable of desensitizing the great majority of subjects allergic to dog hair and dander. Such a set of peptides has the property of being effective in a large number of subjects, whereas the prior art peptides may be active in one allergic subject but completely ineffective for another subject because the latter does not recognize the allergen by the same determinants. However, the peptides which are effectively recognized by T CD4⁺ lymphocytes are difficult to identify due to the high degree of polymorphism of HLA II molecules.

The molecules of the Major Histocompatibility Complex (MHC class II molecules or HLA II molecules in humans) are heterodimers capable of binding a large repertoire of peptides having very different sequences, which allows them to present several peptides per antigen to the T cells. There are four different types of MHC II molecules per individual (2 HLA-DR, 1 HLA-DQ and 1 HLA-DP). The HLA-DR molecule, whose β chain is encoded by the DRBl gene (HLA-DRl) is the most highly expressed. The HLA molecules are very polymorphic and more than 200 different alleles have currently been recorded for DRB 1. The molecules encoded by the DRB3, DRB4, DRB5 and DPBl gene (HLA-DR3, HLA-DR4, HLA-DR5 and HLA-DP) are less polymorphic. The HLA isoforms (or antigens or types) encoded by these alleles possess binding properties which are different from that of another isoform and therefore bind different peptides from the same antigen. Due to the high degree of polymorphism of the HLA molecules, the set of peptides from an antigen, which are recognized by each individual depends on the HLA molecules or HLA type which characterize said individual.

Despite of a high number of HLA molecules whose repartition is not homogeneous worldwide, some alleles are predominant in a given population. For example, in France, which is representative of the Caucasian population, seven DRBl alleles only, exceed 5% (DRBl^*0101, DRB 1^*0301, DRB 1^*0401, DRBl^*0701, DRB1^*11O1, DRBl^*1301 and DRB^*1501). They represent, on their own, 64 % of the French population. These same alleles are also the most abundant in the other Caucasian populations (Table I). Their frequencies vary from 53% (in Spain) to 82% (in Denmark). For the United States and Canada, they represent 58 and 55% of the population, respectively. They therefore represent, on their own, 53 to 82% of the alleles in Caucasian populations. Table I : Frequency of the HLA-DRBl alleles in several Caucasian populations

DRBl allele FRA DAN GER ITA ROU SPA US CAN

DRBfOlOl 9.3 13.0 6.7 6.5 7.6 6.6 7.3 5.6

DRB 1^*0301 10.9 10.2 9.4 10.5 11.4 6.7 9.5 12.3

DRB 1^*0401 5.6 17.6 8.1 2.3 4.2 5.6 6.7 9.5

DRB 1^*0701 14.0 14.8 12.3 12.5 8.3 18.9 14.4 9.4

DRBf 1101 9.2 0.9 9.2 12.4 7.3 1.0 4.4 2.6

DRBfBOl 6.0 8.3 4.5 4.8 4.4 4.5 5.1 4.7

DRBl^*1501 8.0 17.6 7.8 5.6 6.2 9.4 10.3 10.4

Total 63 82 58 55 49 53 58 55

The HLA-DRB3, -DRB4 and -DRB5 molecules which are less polymorphic than HLA-DRJBl are also present at high allelic frequencies in the

Caucasian population: 9.2% for DRB3*0101, 28.4% for DRB4*0101 and 7.9% for

DRB5*0101, in the French population. They cover, on their own, 45% of the alleles in the Caucasian population. The HLA-DP4 molecules which are encoded by the

DPB 1*0401 and DPB 1*0402 alleles are the most abundant HLA II molecules in Europe and in the United States. They represent respectively 40 % and 11 % of the DP alleles in the European population, which means that they are present in 76 % of the

Europeans, in average.

Table II: HLA II alleles predominant in the Caucasian population

(allelic frequency > 5 %) as illustrated by the French population

HLA II allele FRA

DRBfOlOl 9.3

DRBl^*0301 10.9

DRB 1^*0401 5.6

DRBl^*0701 14.0

DRBf 1101 9.2

DRB1^*13O1 6.0

DRBl^*1501 8.0

DRB3^*0101 9.2

DRB4^*0101 28.4

DRB5^*0101 7.9

DPB 1*0401 40

DPBl *0402 11

The peptides from Canfλ, which bind the HLA isoforms encoded by these alleles correspond to the T CD4⁺ epitopes of the majority of the Caucasian population. The inventors have mapped four regions of Can f 1 comprising T CD4⁺ epitopes which are recognized in association with the most frequent HLA II molecules of the Caucasian population and they have shown that peptides derived from these regions are able to stimulate the proliferation of T CD4⁺ lymphocytes from the great majority of dog allergic patients; T CD4⁺ lymphocytes recognition by these peptides is indeed correlated with the ability of these peptides to bind HLA II molecules which are present on the surface of the antigen presenting cells type of the allergic patients.

These peptides which take into account the polymorphism of the MHC class II induce better desensitization which results in a significantly reduced risk of accidents (shocks) during desensitization. This represents an additional advantage for the use of these peptides for the prevention of allergy.

In addition, these peptides which recognize Canf l specific T CD4⁺ lymphocytes are useful for diagnosing sensitivity to dog hair and dander. The invention provides a peptide defined by a sequence of 11 to 38 consecutive amino acids of a fragment of the mature Canfl protein, which is selected from the group consisting of: a) the fragment from positions 3 to 30 or C3-30 (fragment I), b) the fragment from positions 49 to 66 or C49-66 (fragment II), c) the fragment from positions 73 to 104 or C73-104 (fragment III), and d) the fragment from positions 107 to 144 or C 107- 144 (fragment

IV), with the exclusion of the sequences from positions 15 to 26, 50 to 62 and 122 to

133 (C15-26, C50-62 and C122-133). In the present invention, the mature Can f 1 protein corresponds to positions 19 to 176 of the precursor, by reference to the amino acid numbering of the

Canf l sequence SWISSPROT 018873. For example, the amino acid in position 1 of the mature Canf l protein is the glutamine residue in position 19 (GIn 19 or Q 19) of the sequence SWISSPROT 018873. In the present invention, unless otherwise mentioned, a peptide is a sequence of 11 to 40 amino acids, preferably 11 to 20 amino acids. According to an advantageous embodiment of said peptide, it has a sequence of 11 to 20 amino acids.

According to another advantageous embodiment of said peptide, it has a binding affinity of less than 1000 nM for at least four different HLA II molecules whose allelic frequency exceeds 5% in the Caucasian population.

The HLA II binding activity of the peptides is assessed in a competition binding assay with a labelled peptide which serves as tracer, following the procedures described in the US Patent 6,649,166 or the International Applications WO 03/040299, WO 02/090382 and WO 2004/014936, for the HLA DR and/or DP4 molecules. The activity of each peptide is characterized by the concentration of peptide (nM) which inhibits the binding of the biotinylated peptide tracer by 50% (IC₅₀).

In the present invention, "different HLA II molecules" means HLA DP and DQ molecules which are encoded by different alleles or HLA DR molecules which are encoded by different genes or different alleles from a gene.

Preferably, said HLA II molecules are selected from the group consisting of: HLA-DRl, HLA-DR3, HLA-DR4, HLA-DR7, HLA-DRI l, HLA- DR13, HLA-DRl 5, HLA-DRB3, HLA-DRB4, HLA-DRB5 and HLA-DP4.

More preferably, said HLA II molecules are encoded by the HLA alleles: DRBl⁺OlOl, DRBl*0301, DRBl*0401, DRBl*0701, DRBl*1101, DRBl ⁺ DOl, DRBl *1501, DRB3*0101, DRB4*0101, DRB5*0101, DP4*01401 et DP4*0102, respectively ; the binding profile of a set of peptides of the invention to said twelve HLA II molecules which are encoded by the most frequent alleles in the Caucasian population, is illustrated in figure 1. Most preferably, said peptide is defined by a sequence selected from the group consisting of: a) positions 3 to 18, 9 to 24 and 15 to 30 of Canfl or C3-18, C19-24 and C15-30 (group I), b) positions 49 to 64 and 51 to 66 of Can f l or C49-64, C51-66 (group II), c) positions 73 to 88, 81 to 96, 89 to 104 of Canfl or C73-88, C81- 96 and C89-104 (group III) , d) positions 107 to 122, 123 to 138, 127 to 142, 129 to 144 of Can f 1 or C107-122, C123-138, C127-142 and C129-144 (group IV), and e) the sequences of 11 to 15 consecutive amino acids which are included in said sequences as defined in a)_, b) c) and d). According to another embodiment of said peptide it is a fragment of the Canfl protein defined by the sequence SWISSPROT O18873.

The invention encompasses the modified peptides obtained from the peptides as defined above, by introduction of any modification, providing that the modified peptide is still active for HLA II binding and T CD4⁺ recognition. The modifications which are introduced by procedures well-known in the art include with no limitation: mutation (insertion, deletion, substitution) of one or more amino acids in the peptide sequence, addition of an amino acid fusion moiety, substitution of amino acid residues by non-natural amino acids (D-amino-acids or non-amino acid analogs), modification of the peptide bond, cyclization, addition of chemical groups to the side chains (lipids, oligo-or -polysaccharides), coupling to an appropriate carrier.

Preferred modifications include those which lead to an immune regulation and/or tolerance of the Can f 1 allergen reactive CD4⁺ T cells which are present in the allergic patients; said CD4⁺ T cells secrete Th2 cytokines IL-4, IL-5, IL- 9 and IL-13 and secrete more IL-4 than IFN-γ. Immune regulation and/or tolerance of said Canf l reactive CLM⁺ T cells may be achieved by preparing variant peptides, which exhibit altered affinity for the MHC class II molecule or for the T cell receptor.

Such variant peptides are prepared according to procedures well- known in the art. For example, the mode of binding of peptides to HLA II molecules can be determined according to the protocols described in the US Patent 6,649,166. The determination of the peptide residues which are accommodated by the pockets Pl, P4, P6, P7, P9 of HLA II molecules (Pl, P4, P6, P7 and P9 residues) makes it possible to introduce mutations at these positions to modify (increase or decrease) the binding affinity of said peptide to HLA molecules. Examples of said mutations and their effect on the HLA II binding activity of the peptides are presented in the US Patent 6,649,166. Thus, the invention provides a variant of the peptide as defined above, wherein at least one of the residues Pl, P4, P6, P7 and P9 which are accommodated by the pockets (Pl, P4, P6, P7 and P9) of an HLA II molecule is mutated. In addition, at least one of the residues P-2, P-I, P2, P3, P5, P8,

PlO and PIl may be also mutated.

Preferably, the residue Pl which is mutated is selected from the group consisting of: L6, VI l, V13, Wl 7, Y18, L19, M22, V28, 152, M54, L55, 163, V65, V66, Y76, Y79, V84, V85, F86, 187, V92, Y96, 197, L98, Y99, Ll 04, 1109, Mill, L114, L115, L126, F129, F132, L138 and L143.

Immune regulation and/or tolerance of said Can f 1 reactive CD4⁺ T cells may also be achieved by preparing modified peptides, for example glycopeptides, which exhibit altered processing by the antigen presenting cells.

Other preferred modifications are those which enhance the stability and/or the solubility or facilitate the preparation of the peptides.

For example, addition of an amino acid fusion moiety (hexa-histidine tag, B cell epitope tag) and/or protease sensitive sites to the peptide backbone facilitates the purification of the peptide.

The invention provides also a polyepitopic fragment comprising the sequence of at least two different peptides as defined above.

Preferably said fragment is of about 22 to 1000 amino acids, preferably of 20 to 100 amino acids.

The polyepitopic fragment according to the present invention is a synthetic fragment which is different from any Can f 1 fragment; said synthetic polyepitopic fragment consists essentially of the concatenation of the Canf λ T CD4⁺ epitopes as defined above, and eventually of additional epitopes, for example T CD4⁺ epitopes, defined by a peptide of about 11 to 25 amino, preferably 11 to 20 amino acids.

According to an advantageous embodiment of said polyepitopic fragment, it comprises at least one T CD4⁺ epitope from another allergen, such as cat hair and dander (FeI d 1), house dust mite (Der p Y), pollen, bee venom (phospholipase A2), or mould. The polyepitopic fragment may also comprise a tag as defined above. The invention provides also a fusion protein comprising a peptide or a polyepitopic fragment as defined above; the fusion moiety may be separated from the polyepitopic fragment backbone by a protease sensitive site, to facilitate the preparation of the peptide/polyepitopic fragment. Fusion moiety which are useful for peptides preparation are well known in the art and include for example glutathione-S- transferase (GST).

The invention provides also a polynucleotide encoding a peptide, a polyepitopic fragment or a fusion protein, as defined above. The invention provides also a vector comprising said polynucleotide.

A vector refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.

Preferably said vector is an expression vector, wherein said polynucleotide encoding a peptide or a polyepitopic fragment of the invention, eventually included in a fusion protein, is placed under control of appropriate transcriptional and translational control elements to permit production or synthesis of said peptide, polyepitopic fragment or fusion protein. More particularly, the vector comprises a promoter operatively linked to said encoding polynucleotide, a ribosome site, an RNA-splicing site (when genomic DNA is used), a polyadenylation site and a transcription termination site. It also can comprise an enhancer. Selection of the promoter will depend upon the cell in which said peptide, polyepitopic fragment or fusion protein is expressed.

Large numbers of suitable vectors are known to those skilled in the art; YAC (yeast artificial chromosome), BAC (bacterial artificial), phage, phagemid, cosmid, viral vector, plasmid, RNA vector or a linear or circular DNA or RNA molecule which may consist of chromosomal, non chromosomal, semi-synthetic or synthetic DNA. The choice of the vector depends on their use and on the host cell; viral vectors and "naked"nucleic acid vectors are preferred vectors for expression in mammal cells (human and animal). The invention also concerns a prokaryotic or eukaryotic host cell which is modified by a polynucleotide or a vector as defined above, preferably an expression vector. As used herein, a cell refers to a prokaryotic cell, such as a bacterial cell, or eukaryotic cell, such as a human, animal, plant or yeast cell.

The polynucleotide sequence encoding the peptide, the polyepitopic fragment or the fusion protein of the invention may be prepared by any method known by the man skilled in the art. For example, it is amplified from a cDNA template, by polymerase chain reaction with specific primers. Preferably, the codons of said cDNA are chosen to favour the expression of said protein in the desired expression system.

The recombinant vectors comprising said polynucleotide may be obtained and introduced in a host cell by the well-known recombinant DNA and genetic engineering techniques.

The peptide or the polyepitopic fragment of the invention, eventually included in a fusion protein, may be obtained by culturing the host cell containing an expression vector comprising a polynucleotide sequence as defined above, under conditions suitable for the expression of said peptide, polyepitopic fragment or fusion protein, and recovering said peptide, polyepitopic fragment or fusion protein from the host cell culture. Advantageously, the presence of a tag allows affinity purification of the corresponding peptide, polyepitopic fragment or fusion protein and the presence of a protease sensitive allows the isolation of the peptide or polyepitopic fragment from the fusion protein. Alternatively, the peptide or the polyepitopic fragment of the invention may be prepared by chemical synthesis (fmoc chemistry).

The invention provides also a composition capable of desensitizing a subject to dog hair and dander, comprising at least one peptide, polyepitopic fragment or expression vector as defined above, and one pharmaceutically acceptable carrier. The pharmaceutical carriers are those appropriate to a planned route of administration, which are well known in the art, such as for example saline, sterile water, Ringer's solution, and isotonic sodium chloride solution.

The peptide, polyepitopic fragment or expression vector of the invention may be used to cure or to prevent allergy in individuals which have been sensitized to dog hair and dander, including humans and animals.

According to an advantageous embodiment of said composition, it comprises 2 to 6 different peptides of 11 to 38, preferably 11 to 20, consecutive amino acids of the mature Can f 1 protein, wherein said peptides in combination, have a binding affinity of less than 1000 nM for at least ten different HLA II molecules whose allelic frequency exceeds 5% in the Caucasian population.

Preferably, said composition comprises: a) a peptide from group I, a peptide from group IV and the peptide

C51-66, or b) a peptide from group I and the peptides C49-64, C 107- 122 and C123-148, or c) a peptide from group III, the peptide C9-24 or Cl 07-111, and the peptide C123-138 or C51-66.

According to another advantageous embodiment of said composition, it comprises at least one additional peptide of 11 to 40, preferably 11 to 20, consecutive amino acids of the mature Canf l protein, said peptide binding to 1 to 3 different HLA II molecules whose allelic frequency exceeds 5% in the Caucasian population.

Preferably, said additional peptide is selected from the group consisting of: C19-34, C33-48, C45-60, C61-76, C63-76, C93-108, ClOl-116, CBS- ISO, C139-154 and C141-156. More preferably, said additional peptide is selected from the group consisting of: C33-48, C45-60, ClOl-116 and C139-154.

According to another advantageous embodiment of said composition, it comprises at least one peptide comprising a T CD4 epitope from another allergen, such as cat hair and dander (FeI d 1), house dust mite (Der p 1), pollen, bee venom (phospholipase A2), or mould.

The composition according to the invention may advantageously contain a polyepitopic fragment comprising the sequence of the different peptides as defined above or an expression vector comprising a polynucleotide encoding said polyepitopic fragment. According to another advantageous embodiment of said composition, it comprises at least one drug which stimulates IL-10 secretion, such as a combination of vitamin D3 with a glucocorticoid, for example dexamethasone. The composition according to the present invention comprises an amount of peptide, polyepitopic fragment or expression vector in a form suitable for desensitizing a subject to dog hair and dander allergy, which means a form capable of reducing the allergic response (or the symptoms of allergy) to dog hair and dander. The amount of peptide, polyepitopic fragment or expression vector necessary to achieve a therapeutic effect is determined and adjusted depending on factors such as the degree of sensitivity of the subject to dog hair and dander, the age, the sex, the weight and the species of the subject.

The peptide, polyepitopic fragment or expression vector is generally administered according to known desensitization procedures used for conventional specific immunotherapy, at dosages and for periods of time effective to reduce sensitivity of the subject to dog hair and dander. The peptide, polyepitopic fragment or vector is usually administered without adjuvant. It may be administered by injection

(intradermal subcutaneous, intramuscular, intraperitoneal or intravenous), oral, sublingual or intranasal administration, inhalation or transdermal application.

The composition is in a form appropriate to a planned route of administration, including sterile injectable solutions or powders, capsule, tablet, suspension, syrup, which are prepared by procedures well known in the art.

According to another advantageous embodiment of said composition, it is in an appropriate form for sublingual or intranasal administration.

The invention relates also to the use of at least one peptide, polyepitopic fragment, expression vector as defined above, for the preparation of a medicament for the prevention and/or the treatment of dog hair and dander allergy.

The invention provides also a reagent for diagnosing sensitivity to dog hair and dander, comprising at least one peptide or polyepitopic fragment, as defined above.

According to an advantageous embodiment of the reagent of the invention, said peptide or polyepitopic fragment is labelled and/or in the form of HLA

II/peptide complex, preferably multimeric complexes such as tetrameric complexes comprising two peptides and two HLA molecules, each peptide or polyepitopic fragment being bound to one HLA molecule. Labelled peptides or polyepitopic fragments, for example with a fluorogenic molecule, are prepared according to standard procedures, known in the art.

HLA II/peptide complexes are prepared according to the procedures as described for example in Novak et al. (J. Clin. Invest., 1999, 104, 63-67; Kuroda et al., J. Virol., 2000, 74, 8751-8756).

The invention relates also to a method for diagnosing sensitivity (allergic response) to dog hair and dander in a biological sample from a patient to be tested, comprising : a) the incubation of said biological sample with a reagent as defined above, and b) the detection, by any appropriate means, of T CD4⁺ cells specific for the Canfλ protein.

According to the invention, said biological sample is preferably a blood sample. More preferably said blood sample is a suspension of peripheral blood mononuclear cells (PBMC), eventually depleted of CD8+ lymphocytes, or enriched in

CD4⁺ lymphocytes by in vitro culture in the presence of the peptides according to the invention, which is prepared according to standard procedures known in the art.

According to the invention, T CD4⁺ cells specific for the Can f 1 protein are detected by a standard proliferation assay, an ELISPOT test (International application WO 99/51630; Gahery-Segard et al., J. Virol., 2000, 74-1964-) or by flow cytometry analysis using HLA II/peptide complexes as defined above.

In addition to the preceding features, the invention further comprises other features which will emerge from the description which follows, which refers to exemplary embodiments of the peptides of the present invention, as well as to the accompanying drawings in which:

- figure 1 illustrates the binding activity of the different Can f 1 peptides (SEQ ID NO: 1 to 25) to the most frequent HLA II molecules in the Caucasian population. The IC_5O correspond to the concentration (nM) of competitor- peptide {Can f 1 peptide) that prevented binding of 50% of the labeled-peptide (tracer). Means and standard error values were deduced from at least three independent experiments. Validity of each experiment was assessed by reference peptides. Their IC₅₀ variation did not exceed a factor of three. The IC₅₀ values inferior to 1000 nM which correspond to high affinity peptides, are indicated in bold. The four different groups of peptides (group I to IV) are boxed.

- figure 2 illustrates the T CD4 cells stimulating activity of the different Canf l peptides (SEQ ID NO: 1 to 25) in HLA II-typed individuals which are allergic (RK, MS, RV, IN, JT, SR) or sensitized (HV, MT) to dog hair and dander, assessed in a standard proliferation assay. The proliferation indexes (Pr) were calculated by dividing the ³H uptake of treated cultures by ³H uptake of untreated medium controls. The affectation corresponds to the HLA II molecules which are involved in the proliferation. Stimulation indices of > 5 were regarded as positive responses.

It should be understood, however, that these examples are given by way of illustration of the subject of the invention and do not in any manner constitute a limitation thereto. Example 1: Synthesis of HLA II binding Canfl derived peptides

A set of 25 overlapping peptides of 16 amino acids, covering the entire sequence of the major dog allergen Can f 1 (SWISSPROT 018873) were synthesized by fmoc chemistry. Peptides were cleaved from the resin by 95% trifluoroacetic acid and purified by reversed-phase HPLC on a C₁₈ Vydac™ column. The size of the peptides (16 amino acids) which is compatible with that of a T CD4⁺ epitope, allows their recognition by T CD4⁺ cells. In addition, the position of the peptides in the Can f 1 amino acid sequence was chosen so that an aromatic or aliphatic residue is present in position 3 or 4, to allow their binding to the Pl pocket of the HLA II molecule. The sequence of the 25 peptides (SEQ ID NO: 1 to 25) is presented in Table III and in the sequence listing. Table IH: Sequence of the Canfl peptides (SEQ ID NO: 1 to 25)

1 C3-18* T P A L G K D T V A V S G K W Y

2 C9-24 D T V A V S G K W Y L K A M T A

3 C15-30 G K W Y L K A M T A D Q E V P E

4 C 19-34 L K A M T A D Q E V P E K P D S

5 C25-40 D Q E V P E K P D S V T P M I L

6 C33-48 D S V T P M I L K A Q K G G N L

7 C45-60 G G N L E A K I T M L T N G Q C

8 C49-64 E A K I T M L T N G Q C Q N I T

9 C51-66 K I T M L T N G Q C Q N I T V V

10 C61-76 Q N I T V V L H K T S E P G K Y

11 C63-76 I T V V L H K T S E P G K Y T A

12 C73-88 P G K Y T A Y E G Q R V V F I Q

13 C77-92 T A y E G Q R V V F I Q P S P V

14 C81-96 G Q R V V F I Q P S P V R D H Y

15 C89-104 P S P V R D H Y I L Y C E G E L

16 C93-108 R D H Y I L Y C E G E L H G R Q

17 C101-116 E G E L H G R Q I R M A K L L G

18 C107-122 R Q I R M A K L L G R D P E Q S

19 C111-126 M A K L L G R D P E Q S Q E A L

20 C123-138 Q Ξ A L E D F R E F S R A K G L

21 C 127-142 E D F R E F S R A K G L N Q E I

22 C129-144 F R E F S R A K G L N Q E I L E

23 C135-150 A K G L N Q E I L E L A Q S E T

24 C139-154 N Q E I L E L A Q S E T C S P G

25 C141-156 E I L E L A Q S E T C S P G G Q

* amino acid numbering correspond to the positions in the i nature protein.

Example 2: Analysis of peptide binding activity to the most frequent H U molecules in the Caucasian population 1) Peptide binding assay

The principles of the binding tests are described in the US Patent 6,649,166 for the DR molecules and in the International application WO 03/040299, for the DP4 molecules. Examples of these tests are presented in the US Patent 6,649,166 and in the International applications WO 02/090382, WO 03/040299 and WO 2004/014936. a) Choice of the alleles

12 HLA II molecules (10 HLA-DR molecules and 2 HLA-DP molecules) were chosen for their high frequency in the French population, which is representative of the frequencies encountered in the other Caucasian populations. - HLA-DR molecules comprising a β chain encoded by the DRBl gene

The HLA-DRl₅ -DR3, -DR4, -DR7, -DRI l, -DRl 3 and -DRl 5 molecules were chosen since they comprise a β chain which is encoded by an allele of the DRBl locus whose frequency exceeds 5 % in the French population: DRB 1*0101 (9.3 %), DRBl*0301 (10.9 %), DRBl*0401 (5.6 %), DRBl*0701 (14 %), DRB1*11O1 (9.2 %), DRB1*13O1 (6%) and DRB1*15O1 (8 %), respectively. These alleles which represent 64 % of the DR alleles in the French population are also the alleles the most frequently encountered in the other Caucasian populations. Their frequency varies from 53 % in Spain to 82 % in Denmark and represents 58 % and 55 % of the DR alleles of the population in the United States and Canada, respectively. - HLA-DR molecules comprising a β chain which is encoded by another gene The HLA-DRB3, -DRB4 and -DRB5 molecules where chosen since they comprise a β chain which is encoded by an allele whose frequency exceeds 5 % in the French population: HLA-DRB3*0101 (9.2 %), HLA-DRB4*0101 (28.4 %), and HLA-DRB5*0101 (7.9 %). These molecules, on their own, cover 45 % of the DR alleles present in the Caucasian population. - HLA-DP molecules

The HLA-DP4 molecules encoded by the DPB1*O4O1 et DPB 1*0402 alleles were chosen since they are the most abundant in Europe and in the United States. They represent respectively 40 % and 11 % of the DP alleles in the European population, which means that they are present in 76 % of the europeans, in average. b) Purification of HLA-II molecules

EBV homozygous cell lines used as sources of HLA II molecules were the following: H0M2 (DRBPOlOl, DPBl*0401), SCHU (DRBl*1501, DRB5*0101, DPBl*0402), STEILIN (DRB1*O3O1, DRB3*0101), PITOUT (DRBP0701, DBR4*0101, DPBl*0401), SWEIG (DRBl*1101), BOLETH (DRB 1*0401, DRB4*0103), HHKB (DRBl*1301, DRB3*0101, DPBl *0401). HLA- DR and HLA-DP molecules were purified by affinity chromatography using the monomorphic Mab L243 (ATCC) coupled to protein A sepharose CL 4B gel (PHARMACIA), as described in Gorga et al., J. Biol. Chem., 1987, 262, 16087- 16094. HLA-DP4 molecules were purified upon the same protocol using the monomorphic B7/21 antibody. Briefly, cells were lysed on ice at 5x10⁸ cells/ml in 150 mM NaCl, 10 mM Tris HCl pH=8.3 buffer, containing 1% Nonidet P40 (NP40), 10 mg/1 aprotinin, 5 mM EDTA and 10 μM PMSF. After centrifugation at 100 000 g for 1 h, the supernatant was applied to a sepharose 4B and protein A-sepharose 4B columns and then to the specific affinity column. HLA II molecules were eluted with 1.1 mM n-dodecyl β-D-maltoside (DM), 500 mM NaCl and 500 mM Na₂CO₃ pH=11.5. Fractions were immediately neutralized to pH=7 with 2 M Tris HCl pH=6.8 buffer and extensively dialysed against 1 mM DM, 150 mM NaCl, 10 mM phosphate pH=7 buffer. c) Synthesis of the tracer peptides

Peptides used as tracers in the binding assays which correspond to SEQ ID NO: 26 to 33 in the sequence listing, are the following: HA 306-318 (PKYVKQNTLKLAT), A3 152-166 (EAEQLRAYLDGTGVE), MT 2-16 (AKTIAYDEEARRGLE), Bl 21-36 (TERVRLVTRHIYNREE)

YKL (AAYAAAKAAALAA), LOL 191-210 (ESWGAVWRIDTPDKLTGPFT), E2/E168 (AGDLLAIETDKATI) and Oxy 271-287 (EKKYF AATQFEPLAARL). They were synthesized by fmoc chemistry and biotinylated with biotinyl-6- aminocaproic acid (Fluka Chimie) on the N-terminus. Peptides were cleaved from the resin by 95% trifluoroacetic acid and purified by reversed-phase HPLC on a C₁₈ Vydac™ column. d) HLA II peptide binding assays

HLA II molecules were diluted in 10 mM phosphate, 150 mM NaCl, 1 mM DM, 10 mM citrate, 0.003% thimerosal buffer with an appropriate biotinylated peptide (tracer) and serial dilutions of competitor peptides (peptides to be tested).

More precisely, HA 306-318 was used at pH=6 for the DRBl *0101 and DRBl*0401 alleles at 1 nM and 30 nM concentration respectively and at pH=5 for the DRB 1*1101 allele at 20 nM concentration. YKL (20 nM) was used for the DRB 1*0701 allele at pH=5. Incubation was done at pH=4.5 for the DRB1*15O1₅ DRBl*1301 and

DRB1*O3O1 alleles in the presence of A3 152-166 (10 nM), Bl 21-36 (200 nM) and

MT 2-16 (200 nM) respectively. Biotyinylated oxy peptide was used at pH=5 (10 nM) for DPBl*0401and DPBl*0402. Samples (100 μl per well) were incubated in 96- wells polypropylene plates (NUNC) at 37°C for 24 h, except for the DRB1*13O1 and DRB 1 *0301 alleles which were incubated 72 h. After neutralization with 50 μl of 450 mM Tris HCl pH=7.5_s 0.003% thimerosal, 0.3% BSA, 1 mM DM buffer, samples were applied to 96-well Maxisorp™ ELISA plates (NUNC) previously coated with 10 μg/ml L243 or B7/21 Mab and saturated with 100 niM Tris HCl pH=7.5, 0.3% BSA, 0.003% thimerosal buffer. They were allowed to bind to the antibody-coated plates for 2h at room temperature. Bound biotinylated peptide was detected by incubating streptavidin-alkaline phosphatase conjugate (AMERSHAM), and after washings, by adding 4-methylumbelliferyl phosphate substrate (SIGMA). Emitted fluorescence was measured at 450 nm upon excitation at 365 nm on a 96 well plates fluorimeter (DYNEX). Maximal binding was determined by incubating the biotinylated peptide with the HLA II molecule in the absence of competitor. Data were expressed as the competitor peptide concentration that prevented binding of 50% of the labeled peptide (IC₅₀). Means and standard error values were deduced from at least three independent experiments. Validity of each experiment was assessed by reference peptides. Their IC₅₀ variation did not exceed a factor of three. 2) Results

All the peptides except C25-40, bind with high affinity (IC₅₀ < 1000 nM) to at least one of the most frequent HLA II molecule in the Caucasian population (figure 1).

Some of the peptides which are clustered in 4 groups corresponding to positions 3 to 30 (group I)₅ 49 to 66 (group II) , 73 to 104 (group III) and 107 to 144 (group IV), bind to at least 4 different HLA II molecules (figure 1). More precisely:

- group I comprises peptides C3-18, C9-24 and C-15-30,

- group II comprises peptides C49-64 and C51-66,

- group III comprises peptides C73-88, C81-96 and C89-104,

- group IV comprises peptides Cl 07-122, C123-138, C127-142 and C129-144.

Example 3: Analysis of T cell stimulating activity of the peptides in dog-allergic and dog-sensitized patients 1) T cell proliferation assay

T CD4⁺ cells were isolated from blood samples of HLA II-typed individuals which are allergic or sensitized to dog hair and dander, according to the procedure described in Zeiler et al., J. Immunol, 1999, 162, 1415-1422). Briefly, PBMCs were separated from the heparinized peripheral blood of allergic and sensitized patients by Lymphoprep™ (NYCOMED PHARMA) density gradient centrifugation. The Ag-specific proliferation tests using PBMCs were performed as follows. The cells were seeded out and stimulated in triplicate at densities of 10⁵ cells/well (0.2 ml) of round bottomed 96-well microtiter plates (Corning Glass™, CORNING). Peptides were used at a concentration of 10 μg/ml. Culture medium was RPMI 1640 (LIFE TECHNOLOGIES) supplemented with 2 mM L-glutamine, 20 μM 2-Mercaptoethanol, sodium pyruvate (LIFE TECHNOLOGIES), nonessential amino acids (LIFE TECHNOLOGIES), 100 IU/ml penicillin, 100 μg/ml streptomycin, 10 mM HEPES (LIFE TECHNOLOGIES), and 5% inactivated human AB serum (FINNISH RED CROSS). Cultures were incubated for 5 days in a humidified 5% CO₂ incubator at 37⁰C, then pulsed for 16 h with 0.5 mCi of [³H]thymidine per well (specific activity, 2.0 Ci/mmol; AMERSHAM PHARMACIA BIOTECH). Radionuclide uptake was measured by scintillation counting, and the results expressed as a stimulation index (SI: ratio between the mean cpm in cultures stimulated with peptides and the mean cpm in untreated medium controls). Stimulation indices of ≥ 5 were regarded as positive responses. 2) Results

The results of the proliferation assay presented in figure 2 indicate that the peptides are able to stimulate the proliferation of T CD4⁺ cells from dog- allergic patients. Each group of peptide comprises a peptide which is able to stimulate the proliferation of T CD4⁺ cells in an least one of the dog-allergic patients. The T CD4⁺ cells of each dog-allergic patient are stimulated by at least a peptide from one of the groups.

Claims

1°) A peptide defined by a sequence of 11 to 38 consecutive amino acids of a fragment of the mature Can f 1 protein which is selected from the group consisting of: a) the fragment from positions 3 to 30, b) the fragment from positions 49 to 66 , c) the fragment from positions 73 to 104, and d) the fragment from positions 107 to 144, with the exclusion of the sequences from positions 15 to 26, 50 to 62 and 122 to 133. 2°) The peptide of claim I₅ which has a sequence of 11 to 20 amino acids.

3°) The peptide of claim 1 or claim 2, which has a binding affinity of less than 1000 nM for at least four different HLA II molecules whose allelic frequency exceeds 5% in the Caucasian population. 4°) The peptide of claim 3, wherein said HLA II molecules are selected from the group consisting of: HLA-DRl, HLA-DR3, HLA-DR4, HLA-DR7,

HLA-DRI l, HLA-DR13, HLA-DR15, HLA-DRB3, HLA-DRB4, HLA-DRB5 and

HLA-DP4.

5°) The peptide of claim 4, wherein said HLA II molecules are encoded by the HLA alleles: DRBPOlOl, DRBl*0301, DRBl*0401, DRBl*0701,

DRBl*1101, DRBl*1301, DRB1*15O1, DRB3*0101, DRB4*0101, DRB5*0101,

DP4*01401 et DP4*0102, respectively.

6°) The peptide of anyone of claims 1 to 5, which has a sequence selected from the group consisting of: a) positions 3 to 18, 9 to 24 and 15 to 30 of Canfl (group I), b) positions 49 to 64 and 51 to 66 of Canfl (group II), c) positions 73 to 88, 81 to 96, 89 to 104 of Canfl (group III) , d) positions 107 to 122, 123 to 138, 127 to 142, 129 to 144 of Canfl (group IV), and e) the sequences of 11 to 15 consecutive amino acids which are included in said sequences as defined in a)_, b), c) and d). 7°) The peptide of anyone of claims 1 to 6, which is a fragment of the Canfl protein defined by the sequence SWISSPROT 018873.

8°) A variant of the peptide of anyone of claims 1 to 7, wherein at least one of the residues Pl, P4, P6, P7 and P9 which are accommodated by the pockets of an HLA II molecule is mutated.

9°) The variant of claim 8, wherein the residue Pl which is mutated is selected from the group consisting of: L6, VIl₅ V13, W17, Y18, L19, M22, V28, 152, M54, L55, 163, V65, V66, Y76, Y79, V84, V85, F86, 187, V92, Y96, 197, L98, Y99, L104, 1109, Mi l 1, L114, L115, L126, F129, F132, L138 and L143. 10°) A synthetic polyepitopic fragment comprising the sequence of at least two peptides as defined in anyone of claims 1 to 9.

11°) The synthetic polyepitopic fragment according to claim 10, which comprises at least one T CD4⁺ epitope from another allergen, such as cat hair and dander (FeI d I)₅ house dust mite (Der p i), pollen, bee venom (phospholipase A2), or mould.

12°) A fusion protein, comprising a protein moiety fused to a polyepitopic fragment according to claim 10 or 11, or a peptide according to anyone of claims 1 to 9.

13°) A polynucleotide encoding a peptide according to anyone of claims 1 to 9, a polyepitopic fragment according to claim 10 or 11 , or a fusion protein according to claim 12.

14°) An expression vector comprising the polynucleotide according to claim 13.

15°) A host cell which is modified by a polynucleotide according to claim 13 or an expression vector according to claim 14.

16°) A composition capable of desensitizing a subject to dog hair and dander, comprising at least one peptide, polyepitopic fragment or expression vector according to anyone of claims 1 to 11 and 14, and one pharmaceutically acceptable carrier. 17°) The composition of claim 16, which comprises 2 to 6 different peptides of 11 to 38, preferably 11 to 20, consecutive amino acids of the mature Canf 1 protein, wherein said peptides in combination, have a binding affinity of less than 1000 nM for at least ten different HLA II molecules whose allelic frequency exceeds 5% in the Caucasian population.

18°) The composition of claim 17, which comprises: a) a peptide from group I, a peptide from group IV and the peptide C51-66, or b) a peptide from group I and the peptides C49-64, C 107- 122 and C123-148, or c) a peptide from group III, the peptide C9-24 or C 107-111, and the peptide C123-138 or C51-66.

19°) The composition of anyone of claims 16 to 18, which comprises at least one additional peptide of 11 to 40, preferably 11 to 20, consecutive amino acids of the mature Canf λ protein, said additional peptide binding to 1 to 3 different HLA II molecules whose allelic frequency exceeds 5% in the Caucasian population. 20°) The composition of claim 19, wherein said additional peptide is selected from the group consisting of: C19-34, C33-48, C45-60, C61-76, C63-76, C93-108, ClOl-116, C135-150, C139-154 and C141-156, preferably from the group consisting of: C33-48, C45-60, ClOl-1116 and C139-154.

21°) The composition of anyone of claims 16 to 20, which comprises at least one peptide comprising a T CD4⁺ epitope from another allergen, such as cat hair and dander (FeI d 1), house dust mite (Der p 1), pollen, bee venom (phospholipase A2), or mould.

22°) The composition of anyone of claims 16 to 21, which comprises at least a drug which stimulates IL-10 secretion, such as a combination of vitamin D3 with a glucocorticoid, for example dexamethasone.

23°) Use of at least one peptide, polyepitopic fragment or expression vector as defined in anyone of claims 1 to 11, 14 and 18 to 21, for the preparation of a medicament for the prevention and/or the treatment of dog hair and dander allergy.

24°) A reagent for diagnosing sensitivity to dog hair and dander, comprising at least one peptide or polyepitopic fragment as defined in anyone of claims 1 to 11 and 18 to 21. 25°) A method for diagnosing sensitivity to dog hair and dander in a biological sample from a patient to be tested, comprising: a) the incubation of said biological sample with a reagent according to claim 24, and b) the detection of T CD4+ cells specific for the Canfϊ protein.