Immunotherapy to increase cashew resistance in the target population

Abstract

The present invention relates to a method for increasing tolerance to cashew in a subject, comprising the repeated administration, by a transdermal route, of a cashew allergen together with at least one regulatory Treg peptide derived from human immunoglobulin G (IgG).

Description

Technical Field

[0001] The present invention relates to an immunotherapy for increasing tolerance to cashew in subjects with allergic constitutions.

Background Art

[0002] The cashew plant (Anacardium occidentale) is a tropical evergreen tree belonging to the Anacardiaceae family. It produces cashew seeds (nuts) that are commonly consumed by many of the world's population.

[0003] Unfortunately, cashew is also classified as one of the most potent allergenic foods, and no specific treatments are approved to address this problem. The prevalence of cashew allergy has been increasing over the past few decades in industrialized countries with the increasing consumption of this nut. Moreover, cashew allergy has been shown to cause a higher rate of severe reactions than peanut allergy, including wheezing, cardiovascular symptoms, severe dyspnea, and collapse (Clark, Allergy, 2007:62, pp. 913 - 916). Even mere exposure to cashew by smelling, touching, or tasting (but not eating) it has been reported to be sufficient to induce an allergic reaction in cashew allergy patients.

[0004] Despite its increasing prevalence, there are no available, approved, medical treatments that allow for the management of cashew allergy. This lack of treatment may be explained by the unique severity of the anaphylactic reaction induced by the consumption of cashew-containing foods in allergic individuals. As of today, the only options presented to allergic patients are strict avoidance of cashews in the diet and emergency treatment in case of allergies, such as self-injectable epinephrine or intravenous antihistamines. However, such avoidance is not easy: cashews are often packaged with other types of nuts. Moreover, cashews can be hidden in a variety of commonly consumed foods such as Asian foods, sweets, ice cream, cakes, chocolates, and sauces. [Prior art documents] [Patent Documents]

[0005] [Patent Document 1] WO2008094538 [Patent Document 2] WO2011 / 128430 [Patent Document 3] WO02 / 071950 [Patent Document 4] WO2007 / 12226 [Patent Document 5] WO2009095591 [Non-patent literature]

[0006] [Non-Patent Document 1] Clark, Allergy, 2007:62, pp. 913-916. [Non-Patent Document 2] Pelletier, 2021 (Allergy, 2021;76:1213~1222) [Non-Patent Document 3] Wang, J allergy clin Immunol, V110, pp. 160-166 (doi:10.1067 / mai.2002.125208) [Non-Patent Document 4] Wang, 2003, Int Arch Allergy Immunol, pp. 132:27-39. [Non-Patent Document 5] Robertham, J allergy clin Immunol, 2005, V115, pp. 1284-1290. [Non-Patent Document 6] Reitsma, J. Agric. Food. Chem. 2016, 64, 5, pp. 1191-1201 [Non-Patent Document 7] Cousens et al., Human Immunology, 2014, 75, pp. 1139-1146. [Non-Patent Document 8] Handbook of Pharmaceuticals Excipients, American Pharmaceutical Association (Pharmaceutical Press; revised 6th edition, 2009) [Overview of the project] [Problems that the invention aims to solve]

[0007] As of today, immunotherapy is needed to increase tolerance to cashew allergens and thus avoid severe reactions in individuals with cashew allergies. [Means for solving the problem]

[0008] The present invention relates to a method for increasing resistance to cashews in a subject, comprising the step of repeatedly administering a cashew allergen together with at least one regulatory Treg peptide derived from human immunoglobulin G (IgG) via a transdermal route. The at least one regulatory Treg peptide typically comprises or consists of a linear peptide of at most 30 amino acids in length, the linear peptide being derived from the Fc or Fab region of human IgG and having tolerogenic activity, preferably the ability to induce CD4+ / CD25+ / FoxP3+ T cell expansion in vitro. In some embodiments, the at least one regulatory Treg peptide is - A polypeptide having an sequence selected from sequence number 2 or sequence number 3, - By modifying 1, 2, 3, 4, or 5 amino acids, preferably by modifying 1, 2, or 3 amino acids, a polypeptide having a different amino acid sequence from SEQ ID NO: 2 or SEQ ID NO: 3, - A retro-inverso analog of SEQ ID NO: 2 or SEQ ID NO: 3, or a polypeptide different from the retro-inverso analog by one, two, three, four, or five amino acid modifications, preferably by one, two, or three amino acid modifications. It includes or consists of.

[0009] At least one regulatory Treg peptide may have a length of at most 50 amino acid residues, preferably at most 40 or 30 amino acids in length. At least one regulatory Treg peptide may also include chemical modifications to prevent its enzymatic degradation by proteases at its C-terminus and / or N-terminus, preferably amidation at the C-terminus and / or acetylation at the N-terminus. In some embodiments, at least one regulatory Treg peptide is selected from the peptide of SEQ ID NO: 2, the peptide of SEQ ID NO: 3, its retro-inverso, and combinations thereof. In certain embodiments, the cashew allergen is administered via a transdermal route together with a mixture of regulatory Treg peptides comprising (i) a polypeptide comprising or consisting of SEQ ID NO: 2 or its retro-inverso analog and (ii) a polypeptide comprising or consisting of SEQ ID NO: 3 or its retro-inverso analog. The mixture of regulatory Treg peptides may further comprise a polypeptide comprising or consisting of SEQ ID NO: 1 or its retro-inverso analog.

[0010] Preferably, the cashew allergen comprises at least one cashew protein selected from Ana o 1, Ana o 2, Ana o 3, its isoforms, and combinations thereof. The cashew allergen may preferably be a protein extract derived from cashew nuts, obtained from defatted cashew nut powder.

[0011] In a preferred embodiment, the cashew allergen and at least one regulatory Treg peptide are applied simultaneously to the same skin area in a subject.

[0012] Typically, the cashew allergen and at least one regulatory Treg peptide are preferably applied to intact skin by means of a skin patch.

[0013] The skin patch can be of any type. Preferably, the skin patch includes a backing having a periphery adapted to create a sealed chamber when applied to the skin of the subject. The cashew allergen and at least one regulatory Treg peptide are present in dry form on the backing of the patch facing the skin, optionally in a mixture with one or several pharmaceutically acceptable excipients. When applied to the skin, the cashew allergen and at least one regulatory Treg peptide absorbed by the backing are solubilized by transepidermal water loss, whereby they can cross the stratum corneum, penetrate into the epidermis, and reach epidermal dendritic cells. Typically, the patch preferably includes a backing made from polyethylene terephthalate (PET) and an adhesive foam crown, which is adapted to form an airtight junction between the backing of the patch and the skin of the subject, thereby obtaining a sealed chamber when the patch is applied to the skin.

[0014] In some embodiments, the cashew allergen and at least one regulatory Treg epitope are applied to the skin simultaneously, daily or once every two days, by the skin patch for at least 6 months, preferably at least 12 months.

[0015] Repeated administration of a cashew allergen in combination with at least one regulatory Treg epitope via the transdermal route can result in a decrease in the cashew-specific IgE plasma level and an increase in the cashew-specific IgG4 plasma level in the subject. Furthermore, it may be possible to more rapidly establish an increase in cashew tolerance in the subject compared to repeated administration of a cashew allergen that does not include at least one regulatory Treg epitope. The method of the present invention can protect the subject, particularly after accidental exposure to cashew by the oral route, from severe allergic reactions such as anaphylaxis and / or mast cell degranulation.

[0016] The method can be implemented in subjects diagnosed with or at risk of developing a cashew allergy. Typically, the method of the present invention is intended to provide desensitization in subjects allergic to cashews.

[0017] The present invention also relates to a cashew allergen for use in increasing resistance to cashews in a subject, which is administered via a transdermal route, preferably by a skin patch, together with at least one modulated Treg peptide.

[0018] The present invention also relates to a regulatory Treg peptide derived from human IgG for use in increasing the responsiveness of subjects to desensitization with cashew allergens, wherein the regulatory Treg peptide and cashew allergen are administered via a transdermal route, preferably by a skin patch.

[0019] A further object of the present invention is the use of a combination of a cashew allergen and a regulatory Treg peptide derived from human IgG for the production of a pharmaceutical composition for increasing cashew tolerance in a subject, wherein the pharmaceutical composition is administered via a transdermal route, preferably by a skin patch.

[0020] Finally, the present invention also relates to a skin patch comprising a lining having a periphery adapted to create a sealed chamber when applied to the skin of a subject, wherein the cashew allergen and at least one modulotropic Treg peptide are present on the lining of the patch in a dry form, optionally in a mixture with one or more pharmaceutically acceptable excipients. The patch can be used to increase tolerance to cashew in a subject. [Brief explanation of the drawing]

[0021] [Figure 1]This figure shows the evaluation of the level of protection obtained by EPIT against anaphylaxis 8 weeks after treatment. Mice were orally sensitized to cashews and treated with EPIT using VIASKIN containing either cashews with excipients, different mouse T-residopes, or a mixture of cashews or T-residopes alone. 8 weeks after EPIT, mice were orally fed cashews. (A) Evaluation of mast cell activation: Blood samples were collected 60 minutes after feeding, and plasma was isolated. mMCP-1 concentration was measured from the plasma by ELISA. (B, D, and E) Body temperature was measured every 10 minutes after 60 minutes of feeding. The area under the curve (AUC) was calculated for each mouse and shown along with the median for each group (B). The median and interquartile range of individual values ​​of the dynamics of body temperature decrease are shown (D). (C) Clinical symptoms were monitored every 10 minutes after 60 minutes of feeding. Data are SEM mean of individual values. P-values ​​were determined according to the Mann-Whitney U test. [Figure 2] This figure shows the cellular response induced by EPIT 8 weeks after treatment. Mice were orally sensitized to cashew and treated with EPIT using VIASKIN containing either cashew, a mixture of different mouse T regitopes, or a mixture of cashew alone. At the end of the experiment, the animals were sacrificed and the spleen and brachial lymph nodes were collected. Cells derived from the spleen (A) and LN (B) were isolated for Treg phenotyping by FACS (examples of staining: LiveDead, CD3, CD4, CD25, FoxP3, LAP, CD62L). Cells derived from the spleen were also used for in vitro culture with and without cashew stimulation for proliferation and cytokine production analysis (C and D). [Figure 3]This figure shows an example of a patch that can be used to carry out the present invention. Moving from the outer surface toward the surface that adheres to the skin, the layers are: a release protective paper (1) with release tabs; an adhesive dressing that ensures adhesion of the patch to the skin and covers a round lining layer; a smaller round lining layer containing the active substance (here, a modulated Treg epitope and a cashew allergen); and an adhesive foam ring (2) that surrounds the active substance and is fixed to the lining. The adhesive dressing is covered by a removable release liner (3). For application to the skin, the release liner is removed, the patch is applied to the skin, and the paper applicator is removed. The adhesive foam and lining, together with the skin surface, form a sealed condensation chamber that induces perspiration and thus allows for the solubilization of the active substance present on the surface of the lining facing the skin. [Modes for carrying out the invention]

[0022] general definition As used herein, “transdermal administration or application” refers to the application of a compound to the surface of the skin of a subject under conditions that allow contact with the surface of the skin. Transdermal administration typically includes skin application under conditions sufficient to allow the compound to penetrate or diffuse into the upper layers of the skin, preferably the epidermal layer, so that the compound can reach epidermal dendritic cells such as Langerhans cells. Transdermal administration is preferably performed on intact and / or healthy skin areas.

[0023] As used herein, “epidermis” refers to the outer layer of the skin, while the inner layers are the dermis and subcutaneous tissue. The epidermis consists of four or five layers, depending on the area of ​​skin being considered. These layers, in descending order, are the stratum corneum, stratum lucidum, stratum granulosum, stratum spinosum, and stratum basale.

[0024] The stratum corneum is mainly composed of corneocytes, which are the final stage of differentiation. Depending on the location on the skin and the age of the subject, the stratum corneum generally has a thickness ranging from 10 to 40 μm.

[0025] As used herein, “intact skin area” means a skin area that has not been pre-treated prior to the application of the compound of interest (in this specification, cashew allergen and regulatory Treg peptide).

[0026] Where used herein, “immunotherapy or immunotherapy treatment” means a treatment in which a desired immune response is induced in a patient in order to improve their condition. Immunotherapy treatment encompasses the treatment and / or prevention of disorders in a subject. Where used herein, a disorder encompasses any allergy, any disorder, symptoms or reactions caused in conjunction with or associated with an allergy.

[0027] In this specification, “transdermal immunotherapy” or “EPIT” means an immunotherapy treatment in which a compound of interest capable of inducing a desired immune response is administered via a transdermal route, for example, by a skin patch, as defined above.

[0028] As used herein, the terms “desensitization” or “desensitizing” typically refer to the sequential administration of one or more allergens to an allergic subject to induce or increase tolerance to the allergic subject to the allergens.

[0029] The term “tolerance” is defined herein as a reduction in the immunological responsiveness of a subject to a particular allergen. “Increased or improved tolerance” can be demonstrated by any means known to those skilled in the art, particularly by performing an oral challenge. When an oral challenge is used, “increased tolerance” is observed when the oral dose of the allergen required to induce an allergic reaction in the subject after treatment (EPIT, in this specification) is higher than that before treatment. Typically, such increase in dose may be at least 1.1 times, such as at least 1.5, 2.0, 3.0, 5.0, 10, or 100 times.

[0030] As used herein, “compound” refers to any molecule, such as cashew allergens and / or regulatory Treg peptides, that can induce a desired immune response in a target that requires it.

[0031] The terms “polypeptide,” “peptide,” and “protein” are used interchangeably herein to refer to polymers of amino acid residues. These terms also apply to amino acid polymers in which one or more amino acid residues may be modified or non-naturally occurring residues, such as artificial chemical mimics of corresponding naturally occurring amino acids. The term “protein” also includes fragments or variants of different allergens, such as epitope-containing fragments.

[0032] As used herein, “amino acid modification” means a change in the amino acid sequence of a polypeptide. “Amino acid modification,” which may also be called “amino acid change” herein, includes amino acid mutations such as substitutions, insertions, and / or deletions in the polypeptide sequence. “Amino acid substitution” or “substitution” herein means the replacement of an amino acid at a specific position in the parent polypeptide sequence with another amino acid. “Amino acid insertion” or “insertion” means the addition of an amino acid at a specific position in the parent polypeptide sequence. “Amino acid deletion” or “deletion” means the removal of an amino acid at a specific position in the parent polypeptide sequence. Amino acid substitutions may be conservative. A conservative substitution is the replacement of a given amino acid residue with another residue having a side chain ("R group") exhibiting similar chemical properties (e.g., charge, bulk, and / or hydrophobicity). Generally, conservative amino acid substitutions will not substantially alter the functional properties of a protein or peptide. Conservative substitutions and their corresponding rules are well documented in the most up-to-date literature.

[0033] As used herein, the term "allergen" refers to any molecule, substance, or mixture of molecules that can elicit an immune response that results in an allergic reaction in a subject. Allergens may be of various properties, forms, and origins. They may be proteins (or polypeptides or peptides), lipids, sugars, etc., in the form of extracts, recombinant forms, and / or synthetic origins. Allergens may be in their natural form, or in fragmented, modified, or otherwise.

[0034] In the context of the present invention, the term "allergen" refers to allergenic molecules derived from cashew nuts, in particular allergenic proteins and isoforms derived from cashew nuts, as well as their variants and fragments.

[0035] The allergen used to carry out the method of the present invention may include several allergenic molecules, in particular several allergenic proteins or peptides. The allergen may typically be in the form of an extract obtained from cashew nuts.

[0036] The allergen may be formulated with any excipient or carrier suitable for pharmaceutically acceptable use. The resulting allergenic preparation may be in various forms, such as liquid or solid. In one preferred embodiment, the allergen is in a dry form, i.e., lyophilized or electrosprayed form.

[0037] As used herein, “immunoglobulin” refers to the structure that constitutes the natural biological form of an antibody, including variable and constant regions. In most mammals, including humans and mice, the structure of a full-length antibody is generally a tetramer. The tetramer consists of two identical pairs of polypeptide chains, each pair having one “light” chain (typically having a molecular weight of about 25 kDa) and one “heavy” chain (typically having a molecular weight of about 50–70 kDa). In human immunoglobulins, the light chains are classified as kappa and lambda. The kappa (κ) chain is encoded by the immunoglobulin kappa locus on chromosome 2, and the lambda (λ) chain is encoded by the immunoglobulin lambda locus on chromosome 22. The two light chains in naturally occurring antibodies are identical. Each light chain consists of one constant (CL) domain and one variable (VL) domain which is important for binding to the antigen. There are several allotypes for the kappa light chain, namely Km1, Km1,2, and Km3. The heavy chain is classified as mu, delta, gamma, alpha, or epsilon, defining the antibody isotypes as IgM, IgD, IgG, IgA, and IgE, respectively. IgG has several subclasses, but is not limited to IgG1, IgG2, IgG3, and IgG4.

[0038] - The present invention Desensitization is the only available treatment that can alter the natural course of allergic diseases by reducing sensitivity to allergens. In this method, doses of the allergen are repeatedly administered to gradually induce an immune response characterized by tolerance to the allergen. This method is particularly suitable for patients exhibiting severe allergic IgE-dependent responses. Even when desensitization is performed over a long period, the exact mechanism of action remains unclear. In humans, it involves (i) an increase in IgG, particularly IgG4, which are blocking antibodies that can block IgE-mediated mechanisms by inhibiting the release of inflammatory mediators from mast cells and basophils; (ii) an increase in regulatory T cells (Tregs) that result in a better balance of the Th2 / Th1 profile; and (iii) the production of T cells that produce IL-10, which counteracts the inflammatory effects of mast cells and promotes IgG4 production. Desensitization is usually performed by sublingual (SLIT) or subcutaneous (SCIT) immunotherapy.

[0039] The applicant investigated a novel route of allergen administration to promote desensitization, namely a transdermal route. More precisely, the applicant developed an immunotherapy treatment for the management of peanut allergy based on repeated transdermal administration of peanut allergen using an in-house skin patch (called Viaskin). A marketing application for this treatment is under review under the European Medicines Agency (EMA) and the U.S. Food and Drug Administration (FDA).

[0040] The transdermal pathway allows allergens to be processed and delivered to epidermal dendritic cells, such as Langerhans cells, which migrate to lymph nodes and present epitopes to T cells. Notably, the transdermal pathway does not result in the transfer of allergens into the bloodstream. The incidence of serious systemic adverse reactions is significantly lower with percutaneous immunotherapy (EPIT) compared to traditional desensitization protocols performed via sublingual or subcutaneous pathways.

[0041] The applicant sought to evaluate transdermal immunotherapy (EPIT) in the management of cashew nut allergy. As shown in Pelletier, 2021 (Allergy, 2021;76:1213-1222), the applicant developed a robust mouse model of IgE-mediated cashew-induced anaphylaxis.

[0042] Next, the capacity of transdermal immunotherapy (EPIT) was evaluated in this model: cashew-sensitized mice were treated with repeated transdermal administration of cashew allergen via skin patches for up to 16 weeks, followed by oral loading. The applicant demonstrated that EPIT was effective in protecting sensitized mice treated with EPIT for 16 weeks from anaphylactic symptoms after oral loading. However, this protective effect was very low in mice treated for 8 or 12 weeks, while a very high level of protection against anaphylaxis was observed at 8 weeks only in a similar model of peanut-sensitized mice. Furthermore, EPIT showed a more significant increase in Treg in the inflow area lymph nodes and spleen, and a more significant decrease in allergen-specific IgE, in the peanut-sensitized mouse model than in the cashew-sensitized model. These differences in the response to EPIT in the peanut and cashew-sensitized models may stem from the higher immunogenicity of cashews. These results have raised interest in the applicability of EPIT in the management of cashew nut allergy: if a limited increase in cashew tolerance is only achieved after very long treatment periods, EPIT may not be well-received by patients.

[0043] Thus, the applicant sought strategies to enhance the therapeutic effect of EPIT in the management of cashew nut allergy.

[0044] In this context, the applicant demonstrated that the protective effect of EPIT can be enhanced by co-administration of cashew allergens via the transdermal route, together with a regulatory T epitope derived from immunoglobulin G (IgG). Co-administration of cashew extract and regulatory T-reg epitope promoted the acquisition of protective effects: the decrease in cashew-specific IgE and the increase in cashew-specific IgG2a after 8 weeks of treatment were more significant in cashew-sensitized mice co-administered with cashew extract and regulatory T epitope derived from IgG than in mice treated with the extract alone. Similarly, the decrease in body temperature and symptom scores after oral loading were significantly improved after 8 weeks of treatment in mice co-treated with cashew extract and regulatory T epitope compared to other control groups.

[0045] Notably, the increased efficacy was particularly pronounced when the allergen extract was administered co-administered with the mouse regulatory T epitopes T2 and T3, or a mixture thereof, which are autologous to the hTreg epitopes of SEQ ID NO: 2 and SEQ ID NO: 3. In this case, an increase in CD62L+ / FoxP3+ Treg induction was observed in the cutaneous inflow area lymph nodes and the spleen. This increase in Tregs was associated with decreased cashew-specific T cell proliferation and downmodulation of the Th2 cytokine (IL-4) in restimulated splenic cells.

[0046] In summary, transdermal administration of IgG-derived regulatory Treg epitopes can significantly improve the efficacy of EPIT in a mouse model of IgE-mediated cashew-induced anaphylaxis.

[0047] Accordingly, the present invention relates to a method for increasing tolerance to cashews in a subject, comprising the step of simultaneously administering a cashew allergen and at least one regulatory T peptide to the subject via a transdermal route.

[0048] More precisely, the present invention relates to an immunotherapy for increasing tolerance in subjects allergic to cashews, wherein the subject is repeatedly and simultaneously administered via a transdermal route at least one regulatory T peptide derived from a cashew allergen and preferably a G isotype human immunoglobulin.

[0049] Repeated co-administration of a cashew allergen and at least one regulatory T peptide can gradually increase tolerance to cashews in subjects. This increase in tolerance is achieved transdermally, but is higher and / or more rapidly than with repeated administration of the same cashew allergen without the administration of at least one regulatory Treg peptide.

[0050] In certain embodiments, the methods of the present invention are for providing desensitization to subjects allergic to cashews and / or for treating cashew allergies. In further embodiments, the methods of the present invention are for reducing the risk, severity, and / or frequency of the onset of allergic reactions, particularly severe reactions such as respiratory reactions, collapse, and anaphylaxis, in subjects allergic to cashews. Allergic reactions caused by cashew allergies are further described below.

[0051] In another aspect, the present invention relates to a method for providing protection against anaphylaxis in the event of accidental exposure to cashews in a subject allergic to cashews, comprising the step of repeatedly administering a cashew allergen to the subject via a transdermal route together with at least one modulated Treg epitope, wherein the repeated co-administration can gradually increase tolerance to cashews in the subject, and the increase in tolerance is obtained more or more rapidly by co-administration than by repeated administration of the same cashew allergen via a transdermal route but without the administration of a modulated Treg peptide.

[0052] In a further embodiment, the present invention relates to a method for increasing the responsiveness of a subject allergic to cashews to desensitization using a cashew allergen, wherein the cashew allergen is repeatedly administered via a transdermal route together with at least one regulatory Treg peptide.

[0053] In another aspect, the present invention relates to a method for enhancing transdermal immunotherapy (EPIT) in subjects allergic to cashews, wherein the subjects are repeatedly administered a cashew allergen via a transdermal route together with at least one regulatory Treg peptide, and the combined administration enhances the reduction of cashew-specific IgE and / or the increase of cashew-specific IgG4 and / or the induction of Treg cells, particularly CD4+ / CD25+ / FoxP3+, compared to a similar EPIT in which the cashew allergen is administered without at least one regulatory Treg peptide.

[0054] In another aspect, the present invention relates to a cashew allergen for use in immunotherapy in subjects allergic to cashew, particularly for increasing resistance to cashew in said subjects, which is administered repeatedly via a transdermal route together with a regulatory Treg peptide.

[0055] In a further embodiment, the present invention relates to a modulated Treg peptide for use in increasing the responsiveness of an allergic subject to desensitization, which is administered transdermally together with a cashew allergen.

[0056] As fully described below, cashew allergen and regulatory T peptide can be administered simultaneously, separately (i.e., diffused over a long period of time), or sequentially to the same or different skin areas in a subject. In a preferred embodiment, cashew allergen and regulatory T peptide are administered simultaneously to the same skin area, for example, by a skin patch. Regulatory T peptide and cashew allergen are typically applied to an intact area of ​​skin without any adjuvant.

[0057] In another aspect, the present invention relates to the use of a cashew allergen and a modulated Treg peptide for the preparation of a pharmaceutical composition for increasing resistance to cashew in a subject requiring it, wherein the pharmaceutical composition is repeatedly administered via a transdermal route. As fully described below, the pharmaceutical composition is preferably in a dry form and is delivered by a skin patch. The pharmaceutical composition may contain one or more pharmaceutically acceptable excipients.

[0058] Another object of the present invention also relates to a skin patch comprising both a cashew allergen and a regulatory T peptide, which is adapted to provide administration of the compounds via a transdermal route. In certain embodiments, as further described below, the skin patch device includes a lining, the periphery of which is adapted to create a sealed chamber with the skin, and the lining carries, on its skin-facing side within the chamber, a dry form of the cashew allergen and regulatory Treg mixed with, optionally, one or more pharmaceutically acceptable excipients.

[0059] - subject The subjects refer to any human being who requires immunotherapy using cashew allergens. Typically, the subjects of interest have been diagnosed with an allergy to cashews. In certain embodiments, the subjects have or are at risk of developing an allergy to cashews, particularly due to a family history of cashew allergy and / or due to the presence of previously diagnosed allergies, particularly food allergies (e.g., peanuts or pistachios) and / or disorders based on allergy patterns such as atopic dermatitis, allergic rhinitis, or asthma.

[0060] Preferably, the subject has been diagnosed with an allergy to cashews. In other words, the subject has an allergy to cashews.

[0061] Individuals allergic to cashews may experience a wide variety of reactions upon exposure to cashews, particularly upon accidental ingestion.

[0062] The aforementioned allergic reactions include cutaneous, respiratory, cardiovascular, and gastrointestinal reactions. These allergic reactions can develop into anaphylaxis, which has a high incidence in cashew allergies.

[0063] Skin manifestations are considered the most frequent and include angioedema of the eyes, angioedema of the lips, urticaria, and perioral rash.

[0064] Gastrointestinal reactions include vomiting, diarrhea, and abdominal pain.

[0065] Respiratory responses include coughing, shortness of breath, wheezing, and sneezing.

[0066] The subjects are human. Patients may be of any sex and any age. Subjects may be newborns, infants, children, teenagers, or adults. Preferably, subjects are under 18 years of age, preferably under 14 years of age. For example, subjects may be children aged 1 to 11 years, such as 4 to 11 years of age.

[0067] - Cashew allergen Cashew nuts refer to the seeds of Anacardium occidental, a tropical evergreen tree also known as the cashew tree.

[0068] As used herein, the term “cashew allergen” means any substance, molecule, or mixture of molecules derived from cashew nuts that may cause an allergic reaction in a subject upon ingestion.

[0069] The term "cashew allergen" refers not only to isolated allergenic molecules but also to combinations of several allergenic molecules, such as cashew extract or semi-purified protein preparations obtained from cashew nuts. These allergenic molecules can typically trigger IgE-mediated systemic allergic reactions.

[0070] Preferably, the cashew allergen includes, or essentially consists of, allergenic proteins, isoforms, variants, fragments, and combinations thereof derived from cashew nuts.

[0071] As described above, the target allergenic protein variant or fragment typically contains at least one IgE-binding epitope that is originally present in the allergenic protein.

[0072] The allergenic proteins or polypeptides can be isolated from cashew nuts, particularly from cashew fruit, by extraction processes. They can also be recombinantly prepared or chemically synthesized. In some embodiments, the allergenic proteins may be in their natural form. In other embodiments, the allergenic proteins can be denatured or chemically modified.

[0073] Cashew nuts are mainly composed of lipids (44% by mass) and proteins (19% by mass).

[0074] The three main allergenic proteins found in cashew nuts are: Ana o 1 is a 50 kDa 7S bicillin seed storage protein that is expected to form trimers in nature, as characterized in Wang, J allergy clin Immunol, V110, pp. 160-166 (doi:10.1067 / mai.2002.125208). Ana o 2 is an 11S globulin consisting of a 33 kDa large subunit and a 20 kDa small subunit, which are joined together by disulfide bonds to form a 53 kDa protein. Ana o 2 is also known as anacardine. For further information, see Wang, 2003, Int Arch Allergy Immunol, 132: pp. 27-39. - Ana o 3 is a 12.6 kDa 2S albumin. For further information, see Robertham, J allergy clin Immunol, 2005, V115, pp. 1284-1290.

[0075] The isoforms of these proteins are disclosed in the art and are included in the present invention. For example, the isoforms of Ana o 1 include Ana o 1.0101 and Ana o 1.0102.

[0076] The amino acid sequences of Ana o 1, Ana o 2, and Ana o 3 (including its isoforms) are known to those skilled in the art. For example, the UniprotB accession numbers for Ana o 1, Ana o 2, Ana o 3, and some of their isoforms are, for example, Q8L5L5, Q8L5L6, Q8GZP6, and Q8H2B8, respectively.

[0077] These proteins can be purified from cashew nuts (see, for example, Reitsma, J. Agric. Food. Chem. 2016, 64, 5, pp. 1191-1201), or recombinantly produced by genetically modified bacteria, such as Escherichia coli (E. coli), yeast, or any other method known to those skilled in the art.

[0078] These proteins can be used together or separately.

[0079] The term "cashew-derived protein" should be understood to also include fragments or variants of the above-mentioned allergenic proteins, such as epitope-containing fragments, recombinant proteins having one or more amino acid modifications as needed, and proteins obtained from cashew nuts that can be subjected to enzymatic, chemical, mechanical or thermal treatment as needed.

[0080] In some embodiments, the cashew allergen comprises, or essentially consists of, an allergenic protein selected from the group consisting of Ana o 1, Ana o 2, Ana o 3, isoforms, variants, fragments, and combinations thereof.

[0081] Preferred fragments of Ana o 1 include allergenic fragments containing at least one IgE-binding epitope, as shown in Table III in doi:10.1067 / mai.2002.125208.

[0082] Preferred fragments of Ana o 2 include allergenic fragments containing at least one IgE-binding epitope, as shown in Table 6 in Wang, 2003, Int Arch Allergy Immunol, 132: pp. 27-39.

[0083] Preferred fragments of Ana o 3 include allergenic fragments containing at least one IgE-binding epitope, as shown in Table II in Robertham, J allergy clin Immunol, 2005, V115, pp. 1284-1290.

[0084] In some embodiments, the cashew allergen is in the form of a composition comprising Ana o 1, Ana o 2, and Ana o 3, together with or mixed with other cashew-derived molecules, such as other cashew-derived proteins, polysaccharides, and / or lipids, as needed.

[0085] In certain embodiments, the cashew allergen is in the form of a cashew extract rich in at least one protein selected from Ana o 1, Ana o 2, and Ana o 3.

[0086] Cashew extract refers to any preparation obtained from cashew nuts or cashew fruit (including lysates, filters, homogenates, defatted powders, etc.). Cashew extract can be used directly. Alternatively, cashew protein allergens may be isolated or purified at least partially from this extract, for example, to obtain a protein extract or concentrate. Such processes may include solvent extraction, filtration, centrifugation, precipitation, chromatography, or any other technique known to those skilled in the art.

[0087] In preferred embodiments, the cashew allergen is in the form of a protein extract or concentrate derived from cashew nuts. In other words, the cashew allergen may refer to a partially isolated allergenic protein. A protein extract derived from cashew nuts typically contains at least one protein selected from Ana o 1, Ana o 2, and Ana o 3. Preferably, the cashew allergen is in the form of a protein extract derived from cashew nuts containing Ana o 1, Ana o 2, and Ana o 3.

[0088] Such a protein extract may have a total amount of protein of at least 30% by mass, preferably at least 40%, 50%, 60%, 70%, 80%, 90%, or 95% by mass, relative to the total mass of the extract.

[0089] Protein extracts can be obtained from defatted cashew powder extracted using a suitable solvent.

[0090] For example, allergenic proteins can be obtained by extracting defatted cashew nut powder in PBS for an appropriate period (e.g., under stirring and overnight at 4°C). The solution can be separated from the insoluble residue by centrifugation, and the protein extract / concentrate can be obtained from the solution, for example, by lyophilization. Alternative methods are also described in the prior art.

[0091] In addition, commercially available cashew extracts, such as those offered by Stallergenes Greer, can be used.

[0092] Cashew allergens may be in any form, such as liquid, powder, or freeze-dried solid.

[0093] - Regulatory Treg peptide As used herein, a regulatory Treg peptide means a polypeptide comprising or consisting of a regulatory Treg epitope, its variant, its retro-inverso analog, or a variant of its retro-inverso analog.

[0094] The regulatory Treg peptide exhibits tolerogenic effects: in particular, the regulatory Treg peptide according to the present invention can induce the expansion of CD4+ / CD25+ / FoxP3+ T cells in vitro.

[0095] The ability of peptides to induce the expansion of natural CD4+ / CD25+ / FoxP3+ T cells in vitro can be evaluated by methods well known to those skilled in the art. For example, those skilled in the art can proceed as follows: Human peripheral mononuclear cells (PBMCs) isolated from the blood of human volunteers are directly stimulated ex vivo for 4 days in the presence of T ligands alone, phytohemagglutinin alone (a mitotically positive control), or without stimulation. 1x10 6 Cells were stained with anti-CD4-FITC antibody (clone RPA-T 4; eBioscience) and anti-CD25-5 APC antibody (clone BC96; eBioscience) in Flow Staining Buffer (eBioscience) on ice for 30 minutes, and washed twice with buffer. After cell surface staining, cells were fixed, permeabilized, and intracellularly stained for FOXp3 (clone PCH101; eBioscience) according to the manufacturer's protocol. The frequency of FOXp3-positive CD4+ / CD25+ T cells was counted under various culture conditions. T cell activation, when accompanied by increased FOXp3 expression, is indicated by increased CD4+CD25+ expression, which indicates that activated cells are regulatory.

[0096] Alternatively, validation of regulatory Treg peptides can be performed using a adapted tetanus toxoid bystander suppression assay by measuring the potential inhibitory capacity of regulatory peptides on the recall response of human CD4 T cells to tetanus toxoid (TT). Briefly, PBMCs are labeled with CFSE cell proliferation dye (eBioscience) and rested overnight at 37°C and 5% CO2. The following day, cells are stimulated with 0.5 mg / ml tetanus toxoid (TT) (Astarte Biologics, catalog no. 1002) alone, and in combination with 8, 16, or 24 mg / ml of putative regulatory peptide or control peptide, then incubated for 6 days and analyzed by flow cytometry on day 7. CD4+ T cell proliferation, T effector activation, and the ratio of regulatory T cells to effector T cells are measured, which reflect the tolerant potential of the Treg peptides.

[0097] The regulatory Treg peptide has a length of less than 50 amino acids, preferably less than 40 amino acids, for example, 5 to 35 or 7 to 30 amino acids.

[0098] The tolerogenic activity of these peptides derives from the regulatory Treg epitopes they contain. Regulatory Treg epitopes are linear sequences (typically 15-30 amino acids long) found in common self-proteins such as IgG that can trigger a tolerogenic response. Regulatory Treg epitopes can be found in conserved regions of IgG. Regulatory Treg epitopes bind to MHC molecules, engage with circulating, naturally occurring Tregs, and / or activate them, which can lead to the expression of immunosuppressive cytokines, including, but not limited to, IL-10, TGF-β, and TNF-α.

[0099] A variant of a regulatory Treg epitope refers to a polypeptide that has a different amino acid sequence from that of the regulatory Treg epitope due to one or more amino acid modifications (e.g., 1, 2, 3, 4, and 5 modifications), while possessing tolerogenic activity, i.e., the ability to induce CD4+ / CD25hi / FoxP3+ T cells in vitro.

[0100] A retro-inverso analog of a parent peptide refers to a linear peptide in which the amino acid sequence is reversed compared to the parent peptide, and similarly, the α-center chirality of the amino acid subunits is reversed. This type of peptide helps maintain a side-chain topology similar to that of the original L-amino acid parent peptide and contains a D amino acid in the reversed sequence, making it more resistant to proteolytic degradation.

[0101] The first regulatory Treg epitopes (also called T-regitopes) were identified by Anne de Groot et coll in the Fc and Fab regions of human IgG antibodies. They demonstrated that T ligands possess the following four characteristics: (1) their sequences are highly conserved in similar self proteins; (2) almost all of them, by their in-house epitope prediction algorithm (EpiMatrix), contain a single 9-mer frame that is predicted to bind to at least four different HLA DR alleles and thus have the potential to be widely recognized in the human population; (3) in response to T ligands, T cells exhibit a T regulatory phenotype (CD4+CD25+FoxP3+); and (4) co-incubation of T cells with T ligands and immunogenic peptides inhibits the effector T cell (Teff) response to immunogenic peptides in vitro and suppresses antigen-specific secretion of effector cytokine responses (Cousens et al., Human Immunology, 2014, 75, pp. 1139-1146). Furthermore, it was shown that IgG-derived T-regitopes are conserved across non-human species such as mice, rats, cats, camels, cattle, and non-human primates.

[0102] Several T-regitopes derived from human IgG, particularly the constant region of IgG, e.g., the Fc region or, e.g., the Fab region in the kappa light chain of IgG, are described in Table 2 of WO2008094538. These T-regitopes are described as binding to MHC class II molecules, engaging with T cell receptors (TCRs) in the context of MHC class II molecules, and activating native regulatory T cells.

[0103] In preferred embodiments, the regulatory Treg peptide means that it is derived from human IgG, and that it contains a T-regitope derived from human IgG, particularly from the Fc region, or from the variable domain of Fab, such as in the VL framework of the kappa light chain of human IgG. In some embodiments, the T-regitope is selected from human IgG-derived T-regitopes, their variants, and their retro-inverso analogs shown in Table 2 on page 46 of WO2008094538. For example, the T-regitope of interest is identified as T-regitope-289, T-regitope-009, T-regitope-029, and the T-regitope of Sequence ID No. 31 in Table 2 on page 46 of WO2008094538.

[0104] Regulatory Treg peptides can be prepared by any method known in the industry, for example, recombinantly or by chemical synthesis such as solid-phase peptide synthesis.

[0105] Surprisingly, the applicant demonstrated that certain mouse T-regitopes T2 and T3, which are self-identical to the human T-regitopes of SEQ ID NO: 2 and SEQ ID NO: 3, clearly enhance the effectiveness of EPIT in providing resistance to cashew in cashew-sensitized mouse models, while other T-regitopes have little effect (e.g., the mouse T-regitope of SEQ ID NO: 4, which is a homolog of the human T-regitope of SEQ ID NO: 1).

[0106] Thus, in some embodiments, the regulatory Treg peptide is - A polypeptide having an sequence selected from sequence number 2 or sequence number 3, - By modifying 1, 2, 3, 4, 5, 6, or 7 amino acids, preferably by modifying 1, 2, 3, 4, or 5 amino acids, more preferably by modifying 1, 2, or 3 amino acids, polypeptides having an amino acid sequence different from SEQ ID NO: 2 or SEQ ID NO: 3, - A retro-inverso analog of SEQ ID NO: 2 or SEQ ID NO: 3, i.e., a polypeptide that is SEQ ID NO: 7 or SEQ ID NO: 8, and - A polypeptide having a different amino acid sequence from SEQ ID NO: 7 or SEQ ID NO: 8 by modifying 1, 2, 3, 4, 5, 6, or 7 amino acids, preferably by modifying 1, 2, 3, 4, or 5 amino acids, and more preferably by modifying 1, 2, or 3 amino acids. It includes or consists of.

[0107] Preferably, the regulatory Treg peptide has a length of at most 50 amino acid residues, preferably at most 40 or 35 amino acids, for example, at most 30 amino acids.

[0108] Regulatory Treg peptides may include chemical modifications to increase their stability and / or, in particular, to prevent their enzymatic degradation at their C-terminus or N-terminus. For example, the C-terminus of the peptide may be amidated and / or the N-terminus may be acylated.

[0109] Alternatively, a regulatory Treg peptide can be bound to the PEG moiety.

[0110] In a more preferred embodiment of the present invention, the cashew allergen is - Polypeptide of sequence number 2 or sequence number 3, - By modifying 1, 2, 3, 4, or 5 amino acids, preferably by modifying 1, 2, or 3 amino acids, polypeptides having an amino acid sequence different from SEQ ID NO: 2 or SEQ ID NO: 3, and - A polypeptide that is a retro-inverso analog of SEQ ID NO: 2 or SEQ ID NO: 3, or a polypeptide that differs in sequence from the retro-inverso analog by 1, 2, 3, 4, or 5 amino acid modifications, preferably by 1, 2, or 3 amino acid modifications. It is administered via a transdermal route along with at least one regulatory Treg peptide selected from the group consisting of the following:

[0111] In another preferred embodiment, the cashew allergen is - Polypeptide of sequence number 3, - By modifying 1, 2, 3, 4, or 5 amino acids, preferably by modifying 1, 2, or 3 amino acids, a polypeptide having a different amino acid sequence from SEQ ID NO: 3, and - A polypeptide that is a retro-inverso analog of SEQ ID NO: 3, or a polypeptide that differs in sequence from the retro-inverso analog by 1, 2, 3, 4, or 5 amino acid modifications, preferably by 1, 2, or 3 amino acid modifications. It is administered via a transdermal route along with at least one regulatory Treg peptide selected from the group consisting of the following:

[0112] In a particular embodiment of the method of the present invention, the cashew allergen is administered co-administered with at least one modulated Treg peptide selected from the peptide of SEQ ID NO: 2, the peptide of SEQ ID NO: 3, its retro-inverso analog, and combinations thereof.

[0113] In other embodiments, the cashew allergen is administered co-administered with a modulated Treg peptide selected from the peptide of SEQ ID NO: 2, the peptide of SEQ ID NO: 3, and combinations thereof.

[0114] In another specific embodiment, the cashew allergen is administered via a transdermal route along with a mixture of regulatory Treg peptides.

[0115] The mixture preferably comprises (i) a polypeptide containing or comprising SEQ ID NO: 2 or SEQ ID NO: 7 and (ii) a polypeptide containing or comprising SEQ ID NO: 3 or SEQ ID NO: 8.

[0116] The mixture may further contain, in particular, one or more additional regulatory Treg peptides derived from human IgG. For example, it may contain a sequence selected in Table 2 of WO2008094538 or a retro-inverso version thereof, or one or more polypeptides comprising the same. For example, the mixture may further contain a retro-inverso analog of SEQ ID NO: 1 or SEQ ID NO: 6, or a polypeptide comprising the same.

[0117] In some preferred embodiments, the C-terminus and N-terminus of at least one regulated Treg epitope are appropriately capped, preferably by amidation and acetylation.

[0118] As fully described below, the cashew allergen and at least one regulatory Treg peptide are administered simultaneously by a skin patch, preferably transdermally, preferably in the form of a mixture with one or more additional excipients as needed.

[0119] - Transdermal administration and means for providing such administration Transdermal application is typically carried out by applying the compounds of interest, namely cashew allergens and regulatory Treg peptides, so that the compounds come into contact with the surface of a skin area in the subject. The application is maintained for a period of time sufficient to allow the compounds to penetrate into the upper layers of the skin (i.e., the epidermis) so that they can reach epidermal dendritic cells and be processed by those cells.

[0120] The administration of cashew allergens and regulatory Treg peptides can be carried out separately, i.e., in different skin areas and / or at different times. For example, the administration of cashew allergens and regulatory Treg peptides can be carried out in the same skin area or in different parts of the skin.

[0121] In one most preferred embodiment, the cashew allergen and the regulatory Treg peptide are delivered transdermally simultaneously to the exact same skin area in the subject.

[0122] In preferred embodiments, the compound of interest is applied to intact skin. More precisely, the skin area has not undergone any preliminary treatment, such as microporation, dermabrasion, exfoliation, or chemical treatment, prior to the application of the cashew allergen and / or regulatory Treg peptide.

[0123] In some embodiments, the skin area is healthy, and the skin area to which the compound of interest is applied is free from skin disorders such as wounds, skin irritation, skin inflammation, or skin diseases such as eczema.

[0124] Transdermal administration is preferably carried out using a device suitable for maintaining contact between the compound of interest and the target skin. Such a device may include a patch, tape, dressing, sheet, gel such as a hydrogel, a hydrocolloid, or any other form known to those skilled in the art.

[0125] The device may be suitable for delivering compounds to the upper layers of the skin, primarily the epidermis, without significantly entering the bloodstream.

[0126] In some embodiments, the compound of interest is in the form of a liquid composition and can be applied using a known device such as a sealed device having a reservoir and a perforated membrane.

[0127] In some preferred embodiments, the skin device is a patch, and more preferably a sealable patch in which the compound of interest is present in a dry form. For example, the present invention can be carried out by using a skin patch device, such as those described by the applicant in WO2011 / 128430, WO02 / 071950, or WO2007 / 12226.

[0128] In certain embodiments, such devices are sealed and configured to use cashew allergens and / or modulated Treg peptides in a dry form.

[0129] In some embodiments, the patch includes a lining on which cashew allergen and / or regulatory Treg peptide is deposited, preferably in a dry form.

[0130] In some embodiments, allergens and regulatory Treg peptides can be maintained on the patch by electrostatic force without the use of adhesives.

[0131] In certain embodiments, a portion of the backing of the patch carrying the compound of interest does not come into direct contact with the skin. For the implementation of the present invention, it is particularly suitable to use a device that includes a backing adapted to create a sealed condensation chamber together with the skin. The compound of interest, i.e., the allergen and / or regulatory Treg peptide, is deposited on this backing. The compound of interest can be absorbed into the backing or adhered to it by electrostatic forces such as van der Waals forces. When the patch is applied to the skin, moisture increases in the chamber, leading to the solubilization of the allergen and regulatory Treg peptide, which then come into contact with the skin.

[0132] In other words, when applied to the skin, the patch forms a sealed, impermeable chamber in which the target compound, deposited in a dry form on the inner surface of the chamber, is solubilized by transepidermal water evaporation (sweating), and then penetrates across the stratum corneum and into the epidermis to reach epidermal dendritic cells. The term "sweating" refers to the production of fluid secreted by sweat glands in the skin of mammals. This fluid mainly contains water, but also contains various dissolved minerals and trace elements. In this embodiment, the sweat secreted by the skin evaporates and condenses within the sealed chamber.

[0133] The sealed chamber is thought to increase skin permeability, facilitating the transfer of allergens and modulated Treg epitopes to the epidermis. Notably, the applicant demonstrated that such a device does not result in significant transfer of allergens into the bloodstream.

[0134] The height of the condensation chamber, defined by the lining, the periphery of the lining, and the skin, is in the range of 0.1 mm to 1 cm, typically 0.1 mm to 5 mm, for example, 0.1 to 1 mm.

[0135] The backing of the patch (also referred to herein as the support) may be made of glass or a polymer selected from the group consisting of cellulose plastic (CA, CP), polyvinyl chloride (PVC), polypropylene, polystyrene, polyurethane, polycarbonate, polyacrylic acid, and especially poly(methyl methacrylate (PMMA), polyester, polyethylene (PE), polyethylene terephthalate (PET), fluoropolymer (e.g., PTFE), and ethylene vinyl acrylate (EVA).

[0136] In some embodiments, the occlusive skin patch may include a breathable overadhesive (also called a dressing), a lining and adhesive crown made from polyethylene terephthalate (PET), the adhesive crown and lining being adapted to create an occlusive chamber with the skin. The adhesive crown may be any suitable polymer foam, which is recovered on its underside and top surface by a suitable adhesive. The foam is typically selected to form an airtight bond between the patch lining and the skin of interest. The breathable overadhesive (or adhesive dressing) is used to ensure adhesion of the patch to the skin.

[0137] The sealing patch may further include a release liner and a paper applicator.

[0138] An example of a suitable patch for carrying out the present invention is, for example, the Viaskin patch developed by the applicant and illustrated in Figure 3.

[0139] Typically, allergens and / or regulatory Treg peptides are present in a dry form on the skin-facing side of the lining. These compounds may be deposited onto the lining by spray drying or electrospraying, as described in WO2009095591. Alternatively, the compounds of interest can be bonded to the lining with an adhesive, such as an acrylic adhesive.

[0140] When using electrospray, the backing is typically coated with a conductive layer, for example, metal particles or metal oxide particles such as titanium dioxide, aluminum, or gold, to allow for the distribution of positive or negative charges onto the backing during the electrospray process and the deposition of the desired compound.

[0141] The cashew allergen and regulatory Treg peptide may be present in pure form or as a mixture with pharmaceutically acceptable excipients, with or without additional adjuvants in the patch. Preferably, the cashew allergen and regulatory Treg peptide are not combined with any additional adjuvants in the patch.

[0142] In some embodiments, the cashew allergen and the regulatory Treg peptide are administered via the transdermal route in the form of an adjuvant-free preparation.

[0143] Pharmaceutically acceptable excipients that may be used are described in particular in the Handbook of Pharmaceuticals Excipients, American Pharmaceutical Association (Pharmaceutical Press; 6th revised edition, 2009). Suitable excipients include, but are not limited to, solvents such as water or water / ethanol mixtures, fillers, carriers, diluents, binders, penetrants, salts, buffers, stabilizers, antioxidants, preservatives, surfactants, and wetting agents.

[0144] For example, the compounds of interest, namely cashew allergens and regulatory Treg peptides, can typically be formulated in the form of a liquid formulation (e.g., as an aqueous alcohol solution) together with one or more excipients selected from buffers, diluents, surfactants, and stabilizers, and the formulation is deposited on the skin-facing lining of the patch under conditions that allow for the evaporation of the solvent and the formation of a dry deposit on the lining, for example by electrospray.

[0145] It goes without saying that the above patch is also the objective of this invention.

[0146] - Pharmacokinetics and regimens The present invention involves repeated administration of a cashew allergen combined with a modulated Treg peptide to a subject via a transdermal route, resulting in a gradual increase in the subject's tolerance to cashews.

[0147] Those skilled in the art can adapt specific doses of allergens and regulatory Treg peptides, as well as the number of applications and duration of contact, depending on the target, the properties of the allergen / regulatory Treg peptide preparation, the type of device used to provide transdermal administration, and so on.

[0148] Generally, the method involves applying the allergen and regulatory Treg peptide at least once a month for a period of several months (e.g., 6, 12, 18, 24, 36, 48 months) until a sufficient increase in tolerance is achieved.

[0149] In some embodiments, the allergen and regulatory Treg peptide are administered at least once a week, preferably once every day or every two days, for a period of at least six months, such as at least one, two, or three years.

[0150] In some preferred embodiments, the cashew allergen and the regulatory Treg peptide are preferably contained in the same skin patch as described above. In that case, the method of the present invention may involve repeated application of the patch at least once a week, for example, daily or every other day, preferably once a day.

[0151] For example, the method of the present invention may include the application of a new patch containing both cashew allergen and regulatory Treg peptide once every two days or daily. The duration of contact between the patch and the skin for each application is in the range of about 1 to 48 hours, typically about 3 to 36 hours, for example, about 6 hours, 8 hours, 12 hours, 18 hours, or 24 hours.

[0152] The amounts of cashew allergen and regulatory Treg peptide on each patch are typically measured per 1 cm of the patch surface. 2 A range of 0.1 to 1000 μg per patch, preferably 1 cm² of the patch surface. 2 A range of 20-500 μg per patch, more preferably 1 cm² of the patch surface. 2 The range is 20-200 μg per patch. The patch surface is 1 cm². 2 ~10cm 2 A range of preferably 1 cm 2 ~5cm 2 It is within the range.

[0153] The dose of cashew allergen may be 1 μg to 10 mg per patch, preferably 10 μg to 1 mg, for example, 30 μg to 800 μg or 50 μg to 500 μg.

[0154] The dosage of the regulatory Treg peptide may be 1 μg to 10 mg per patch, preferably 10 μg to 1 mg, for example, 30 μg to 800 μg or 50 μg to 500 μg.

[0155] The mass ratio of cashew allergen to the modulated Treg epitope may be 0.1 to 10, for example, 0.5 to 2 or 0.8 to 1.2.

[0156] To efficiently increase the target's tolerance to cashews, cashew allergens and regulatory Treg peptides are administered in doses sufficient to induce an immune response in the target.

[0157] In some cases, particularly at the initiation of treatment, the application of cashew allergen to the skin may involve a local inflammatory response resulting in a cascade of biochemical events, including those affecting the local vascular and immune systems. The inflammatory response may be moderate in the form of erythema (the first clinical component of the inflammatory response) or in the form of papules, also showing the presence of local edema (another component of the inflammatory response). The inflammatory response induced by the application of cashew allergen via the transdermal route may be visible to the naked eye or not.

[0158] The method of the present invention may include an initiation period in which the contact period of the patch with the skin is gradually increased, for example, from 3 hours per day to 12 hours per day. The initiation period may last for 1 to 4 weeks.

[0159] After the initial period and during routine treatment, the patch is preferably applied once daily until at least a sufficient increase in resistance is achieved.

[0160] Once sufficient tolerance has been achieved, maintenance treatment may be performed. The frequency of patch application may be reduced in maintenance treatment, for example, to once a week or once a month. Alternatively, maintenance treatment may include administration of cashew allergen only (i.e., without regulatory Treg peptide) via the dermal route. Patches containing only cashew allergen can be applied at the same frequency as usual treatment (e.g., once a day) or at a lower frequency (e.g., once a week or once a month).

[0161] For application, the patch device is applied directly to the skin, preferably to an intact area of ​​skin, more preferably to a dry, clean, healthy, and especially non-inflammatory area of ​​skin. Typically, the patch can be applied to the shoulder, shoulder blade, arm, or thigh.

[0162] Preferably, the skin receives no pretreatment before the application of the patch. Alternatively, the skin may be pretreated before the application of the cashew allergen and regulatory Treg peptide. Skin pretreatment is preferably intended when the subject is an adult. Skin pretreatment is preferably superficial. Typically, skin pretreatment can alter the stratum corneum and, if necessary, one or more epidermal cell layers while maintaining the integrity of the dermis and subcutaneous tissue of the skin. Preferably, skin pretreatment does not alter the epidermal basal layer either. Skin treatment includes, but is not limited to, skin microporation such as laser microporation, skin cleansing, mild skin exfoliation, and skin peeling. If the subject is an infant or child under 12 years of age, skin pretreatment is avoided.

[0163] The method of the present invention results in a decrease in specific IgE levels and an increase in certain specific IgG levels, particularly IgG4 levels, leading to a gradual increase in tolerance to tuberous plants. The term “specific Ig” as used herein refers to immunoglobulins that are specific to at least one allergen for which the subject is allergic. In one preferred embodiment, these immunoglobulins are specific to at least one protein derived from the tuberous plant, particularly Ana o 1, Ana o 2, Ana o 3, or their isoforms.

[0164] The method of the present invention also results in an immune bias from a dominant Th2 profile to a more balanced Th1 / Th2 profile. In other words, the method of the present invention causes an increase in the Th1 response to administered proteins. Th1 and Th2 cells are two types of CD4+ helper T cells that differ in their cytokine production patterns. Th1 cells produce IFN-γ, IL-2, and TNF-β and are involved in cell-mediated immune responses that are beneficial in host defense against intracellular pathogens and malignant cells but detrimental in mediating autoimmunity. Th2 cells secrete IL-4, IL-5, IL-9, IL-10, and IL-13, which increase antibody responses, including IgE production, and protect against parasitic invasions, but can also cause allergies and asthma. The Th1 and Th2 responses are mutually antagonistic, so that they normally exist in equilibrium and mutually regulate each other. In allergy subjects, the Th1 / Th2 balance changes, and the Th2 profile becomes dominant. An immune shift from a dominant Th2 profile to a more balanced Th1 / Th2 profile signifies a shift from an allergic state to a resistant state. This shift, mediated by an increase in Treg cells, can be assessed by any method known to those skilled in the art, such as a decrease in the IgG1 / IgG4 ratio or analysis of cytokine production.

[0165] The examples also demonstrate that the method of the present invention reduces the cashew-specific effector response after oral administration compared to the placebo group, and more precisely, inhibits the proliferation of cashew-specific T cells and mast cell degranulation.

[0166] The following examples are provided for illustrative purposes only and not for limitation.

[0167] [Table 1] [Examples]

[0168] Combination therapy using T-residope and cashews improves and enhances the therapeutic effect of transcutaneous immunotherapy in a robust mouse model of cashew allergy. Seventy mice were sensitized via the gastric route with 1 mg of cashew protein extract containing 10 μg of cholera toxin, administered once a week for six consecutive weeks. The sensitized mice were then treated with transdermal immunotherapy using a Viaskin patch. - Ten mice were treated with viaskin containing an excipient (0.1M phosphate buffer + DMSO). - Ten mice were treated with viaskin containing 50 μg of cashew protein (Stallergenes Greer, reference number XPF84D3A2.5). - Ten mice were treated with viaskin containing 50 μg of cashew protein + 50 μg of T1. - Ten mice were treated with viaskin containing 50 μg of cashew protein + 50 μg of T2. - Ten mice were treated with viaskin containing 50 μg of cashew protein + 50 μg of T3. - Ten mice were treated with viaskin containing 50 μg cashew protein + 50 μg T1 + 50 μg T2 + 50 μg T3. - Ten mice were treated with viaskin containing 50 μg of T1, 50 μg of T2, and 50 μg of T3.

[0169] mT ligands T1, T2, and T3 correspond to homologs of the human T ligands of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively. To prevent degradation by proteases, the N-terminus of the tested peptides was acetylated (CH3(C=O)-), while the C-terminus was amidated (-NH2).

[0170] - Preparation of Viaskin® excipient patches A solution of 528 μL of 0.1 M phosphate buffer and 72 μL of DMSO was prepared. A 50 μL volume was deposited onto the patch using a micropipette, and the droplet was diffused onto the surface of the backing layer of the patch. The patch was dried in a ventilated (100%) oven at 30°C. Once the deposited material was completely dry, the dried patch was stored in a petri dish with a desiccant at 5 ± 3°C.

[0171] Viaskin® was prepared by loading 50 μl of phosphate buffer + DMSO onto each patch.

[0172] Solubilization of T-resitope: All reagents were brought to room temperature.

[0173] A vial containing T-residop was centrifuged at 7500 rpm for 1 minute.

[0174] Each peptide was diluted with 100% sterile DMSO to a working concentration of 25 mg / mL or 25 μg / μl (net mass).

[0175] The peptides were aliquoted in 100% DMSO in 80 μL portions and stored at -20°C.

[0176] Viaskin® - Cashew preparation (prepared the day before application): After preparing 3 mL of 10 mg / mL cashew solution, prepare the following solutions: Cashew + T-Ligitope 1: 576 μL of 10 mg / mL cashew solution + 24 μL of T-Ligitope 1 (25 mg / mL) in DMSO Cashew + T-Ligitope 2: 576 μL of 10 mg / mL cashew solution + 24 μL of T-Ligitope 2 (25 mg / mL) in DMSO Cashew + T-Ligitope 3: 576 μL of 10 mg / mL cashew solution + 24 μL of T-Ligitope 3 (25 mg / mL) in DMSO Cashew + T-regitope mixture: 528 μL of 10 mg / mL cashew solution + 24 μL of T-regitope 1 (25 mg / mL) in DMSO + 24 μL of T-regitope 2 (25 mg / mL) in DMSO + 24 μL of T-regitope 3 (25 mg / mL) in DMSO T-residope mixture: 528 μL of phosphate buffer + 24 μL of T-residope 1 (25 mg / mL) in DMSO + 24 μL of T-residope 2 (25 mg / mL) in DMSO + 24 μL of T-residope 3 (25 mg / mL) in DMSO.

[0177] Viaskin® was prepared by loading 50 μl of the corresponding solution.

[0178] The droplets were diffused onto the surface of the backing layer of the patch and dried in a ventilated oven at 30°C. The dried patch was stored in a petri dish at 5±3°C with a desiccant as soon as the deposit was completely dry.

[0179] Mice received VIASKIN once a week for 48 hours over 8 weeks. Ten naive mice were left untreated as healthy controls. Eight weeks after treatment, mice were orally administered 45 mg of cashew protein extract. Body temperature was measured every 10 minutes for 60 minutes before and after the administration using a subcutaneous transponder. Clinical symptoms were recorded at the same frequency using the following score.

[0180] [Table 2]

[0181] Anaphylactic reactions were characterized by a decrease in body temperature and the onset of clinical symptoms. Blood samples were collected 60 minutes after loading to measure mMCP-1 and mMCP-7 levels as markers of mast cell degranulation.

[0182] For antibody response analysis, 200 μL blood samples were collected from the submandibular gland vein at the end of the sensitization period, 4 weeks after treatment, and at the end of the experiment. Cashew-specific IgE, IgG1, and IgG2a were measured by in-house ELISA.

[0183] At the end of the experiment, the animals were sacrificed, and the spleen and brachial lymph nodes were collected. Cells derived from the spleen and LN were isolated for Treg phenotyping by FACS (examples of staining: LiveDead, CD3, CD4, CD25, FoxP3, LAP, CD62L). Cells derived from the spleen were also used for in vitro culture with or without cashew stimulation for proliferation and cytokine production analysis.

[0184] result EPIT treatment with VIASKIN containing cashew protein induced a significant reduction in mast cell degranulation after oral administration, regardless of the presence of T-regitopes in VIASKIN (Figure 1A). However, patches loaded with cashew alone, or cashew and T1, did not induce protection from anaphylactic reactions at this point, i.e., at 8 weeks of treatment, whereas the addition of T2, T3, or a mixture of three T-regitopes resulted in significant protection from post-administration anaphylaxis, as evidenced by a less pronounced decrease in body temperature and lower clinical symptom scores (Figures 1B and 1C).

[0185] Cashew-specific IgE was significantly lower only in the group treated with the cashew + T3 combination compared to the control group. However, IgG2a titers measured by ELISA did not differ from control measurements regardless of the treatment, even when a tendency towards higher IgG2a was observed in mice treated with cashew + T3.

[0186] Mice treated with EPIT containing cashew along with T2, T3, or a mixture of three T-residopes showed an increased number of CD62L+ / FoxP3+ Tregs in the cutaneous aspiration lymph nodes and their spleens (Figures 2A and 2B). This increase in Tregs was associated with decreased cashew-specific T cell proliferation (Figure 2C) and downmodulation of Th2 cytokine (IL-4) in restimulated spleen cells (Figure 2D).

Claims

1. A combination of a cashew allergen and at least one regulatory Treg peptide derived from human immunoglobulin G (IgG) for increasing resistance to cashews in a subject, wherein the cashew allergen and at least one regulatory Treg peptide derived from human immunoglobulin G (IgG) are administered simultaneously or separately via a transdermal route, and at least one regulatory Treg peptide - A polypeptide having a sequence selected from sequence number 2 or sequence number 3, - By modifying 1, 2, 3, 4, or 5 amino acids, preferably by modifying 1, 2, or 3 amino acids, a polypeptide having an amino acid sequence different from SEQ ID NO: 2 or SEQ ID NO: 3, or - A retro-inverso analog of SEQ ID NO: 2 or SEQ ID NO: 3, or a polypeptide different from the retro-inverso analog by one, two, three, four, or five amino acid modifications, preferably by one, two, or three amino acid modifications. A combination consisting of the above.

2. The combination according to claim 1, wherein at least one regulatory Treg peptide includes a chemical modification to prevent its enzymatic degradation by proteases at its C-terminus and / or N-terminus, preferably amidation at the C-terminus and / or acetylation at the N-terminus.

3. The combination according to claim 1 or 2, wherein at least one regulatory Treg peptide is selected from the peptide of SEQ ID NO: 2, the peptide of SEQ ID NO: 3, its retro-inverso, and combinations thereof.

4. The combination according to any one of claims 1 to 3, wherein the cashew allergen is administered via a transdermal route together with a mixture of (i) a polypeptide comprising or comprising SEQ ID NO: 2 or its retro-inverso analog and (ii) a regulatory Treg peptide comprising or comprising SEQ ID NO: 3 or its retro-inverso analog.

5. The combination according to claim 4, wherein the mixture of regulatory Treg peptides further comprises SEQ ID NO: 1 or its retro-inverso analog, or a polypeptide comprising the same.

6. The combination according to any one of claims 1 to 5, wherein the cashew allergen comprises at least one cashew protein selected from Ana o 1, Ana o 2, Ana o 3, its isoforms and combinations thereof.

7. The combination according to any one of claims 1 to 6, wherein the cashew allergen is a protein extract derived from cashew nuts.

8. The combination according to claim 7, wherein the cashew allergen is a protein extract obtained from defatted cashew nut powder.

9. The combination according to any one of claims 1 to 8, wherein a cashew allergen and at least one regulatory Treg peptide are applied simultaneously to the same skin area in the subject.

10. The combination according to any one of claims 1 to 9, wherein the cashew allergen and at least one regulatory Treg peptide are applied to intact skin.

11. The combination according to any one of claims 1 to 10, wherein a cashew allergen and at least one regulatory Treg peptide are applied by a skin patch.

12. The combination according to claim 11, wherein the skin patch comprises a lining having a periphery adapted to create a sealed chamber when applied to the skin of interest, and the cashew allergen and at least one modulo Treg peptide are present on the lining of the patch in a dry form, optionally in a mixture with one or more pharmaceutically acceptable excipients.

13. The combination according to claim 12, wherein the skin patch comprises a lining preferably made of polyethylene terephthalate (PET) and an adhesive foam crown, the adhesive foam crown being adapted to form an airtight bond between the lining of the patch and the skin to be treated, thereby providing a sealed chamber when the patch is applied to the skin.

14. The combination according to any one of claims 1 to 13, wherein a cashew allergen and at least one moduloable Treg epitope are applied simultaneously to the skin daily or every other day for at least 6 months or at least 12 months.

15. Cashew allergen and at least one modulated Treg epitope are repeatedly administered, and the following effects are observed with said repeated administration: - Decreased cashew-specific IgE plasma levels and increased cashew-specific IgG4 plasma levels in the subjects; and / or - A more rapid increase in cashew tolerance in subjects compared to repeated administration of cashew allergens that do not contain at least one modulated Treg epitope; and / or - In particular, protection of subjects from severe allergic reactions such as anaphylaxis following accidental exposure to cashews via the oral route; and / or - In particular, protection of subjects from mast cell degranulation following accidental exposure to cashews via the oral route. A combination according to any one of claims 1 to 14, wherein at least one of the following is obtained.

16. A combination according to any one of claims 1 to 15, wherein the subject has been diagnosed with an allergy to cashews, or is at risk of developing an allergy to cashews.

17. A combination according to any one of claims 1 to 16, for the purpose of providing desensitization in a person allergic to cashews.

18. A pharmaceutical composition for increasing resistance to cashews in a subject, comprising a cashew allergen, wherein the cashew allergen is administered via a transdermal route together with at least one regulatory Treg peptide derived from human IgG, and the at least one regulatory Treg peptide is - A polypeptide having a sequence selected from sequence number 2 or sequence number 3, - By modifying 1, 2, 3, 4, or 5 amino acids, preferably by modifying 1, 2, or 3 amino acids, a polypeptide having an amino acid sequence different from SEQ ID NO: 2 or SEQ ID NO: 3, or - A retro-inverso analog of SEQ ID NO: 2 or SEQ ID NO: 3, or a polypeptide different from the retro-inverso analog by one, two, three, four, or five amino acid modifications, preferably by one, two, or three amino acid modifications. A pharmaceutical composition consisting of the following.

19. (i) the cashew allergen is as defined in claim 7, 8, or 9, and / or (ii) at least one regulatory Treg peptide is as defined in any one of claims 2 to 5, the pharmaceutical composition according to claim 18.

20. The pharmaceutical composition according to claim 18 or 19, comprising both a cashew allergen and at least one regulatory Treg peptide, and administered by a skin patch.

21. A pharmaceutical composition for increasing the response of a target to desensitization using a cashew allergen, comprising a regulatory Treg peptide derived from human IgG, wherein the regulatory Treg peptide is administered together with the cashew allergen via a transdermal route, and at least one regulatory Treg peptide is present. - A polypeptide having a sequence selected from sequence number 2 or sequence number 3, - By modifying 1, 2, 3, 4, or 5 amino acids, preferably by modifying 1, 2, or 3 amino acids, a polypeptide having an amino acid sequence different from SEQ ID NO: 2 or SEQ ID NO: 3, or - A retro-inverso analog of SEQ ID NO: 2 or SEQ ID NO: 3, or a polypeptide different from the retro-inverso analog by one, two, three, four, or five amino acid modifications, preferably by one, two, or three amino acid modifications. A pharmaceutical composition consisting of the following.

22. (i) the cashew allergen is as defined in claim 7, 8, or 9, and / or (ii) at least one regulatory Treg peptide is as defined in any one of claims 2 to 5, the pharmaceutical composition according to claim 21.

23. The pharmaceutical composition according to claim 21 or 22, comprising both a cashew allergen and at least one regulatory Treg peptide, and administered by a skin patch.

24. A skin patch comprising a lining having a periphery adapted to create a sealed chamber when applied to the target skin, wherein the cashew allergen and at least one modulated Treg peptide are present in a dry form on the skin-facing surface of the patch lining, optionally in a mixture with one or more pharmaceutically acceptable excipients, and at least one modulated Treg peptide - A polypeptide having a sequence selected from sequence number 2 or sequence number 3, - By modifying 1, 2, 3, 4, or 5 amino acids, preferably by modifying 1, 2, or 3 amino acids, a polypeptide having an amino acid sequence different from SEQ ID NO: 2 or SEQ ID NO: 3, or - A retro-inverso analog of SEQ ID NO: 2 or SEQ ID NO: 3, or a polypeptide different from the retro-inverso analog by one, two, three, four, or five amino acid modifications, preferably by one, two, or three amino acid modifications. A skin patch made of [unspecified material].

25. A skin patch according to claim 24, for increasing resistance to cashews in the subject.

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