IL-15 inhibitor useful for treating atopic dermatitis

Abstract

The present invention relates to the use of agents capable of neutralizing the activity of interleukin-15 in the treatment of atopic dermatitis. In particular, the present invention relates to IL-15 inhibitors for use in the prevention and / or treatment of atopic dermatitis.

Description

[Technical Field]

[0001] The present invention relates to the use of agents capable of neutralizing the activity of interleukin-15 in the treatment of atopic dermatitis. [Background technology]

[0002] Atopic dermatitis (AD), also known as eczema, is a chronic, primarily inflammatory skin disease characterized by dry skin, recurrent redness, and pruritus (itching).

[0003] The prevalence of AD varies widely throughout the world due to differences in region, country, age group, and data collection methods, affecting 0.2% to 36% of the pediatric population (<18 years of age) (Bylund et al., 2002, Acta Derm Venereol., 100(12):adv00160. https: / / doi.org / 10.2340 / 00015555-3510; Eichenfield et al., 2022, Pediatr Drugs 24, 293-305, https: / / doi.org / 10.1007 / s40272-022-00499-x). A recent international web survey found that the prevalence of previously diagnosed, active AD ranges from 2.1% to 4.9%, and that the prevalence of AD in adults is similar to that in adolescents and remains stable throughout adulthood (Silvenberg et al., 2020, Med. Clin. N. Am., 104, 157-176, https: / / doi.org / 10.1016 / j.mcna.2019.08.009). Globally, the prevalence of AD is often highest in high-income countries. AD is also widely observed to be overrepresented in women, particularly in adolescents and adults (Silvenberg et al., 2020, op. cit.).

[0004] The disease usually begins in childhood and may continue into adulthood, although its severity varies over the years. AD is a complex, multifactorial disease involving: i) Strong genetic component: AD can be considered a familial disease and several susceptibility genes have been identified. ii) Environmental exposures such as irritants and prurigens, pathogens, climatic factors, ultraviolet light, outdoor and indoor air pollutants, tobacco smoke exposure, water hardness, rural versus urban living, diet, breastfeeding, probiotics and prebiotics. Some of these factors may explain the increase in atopic dermatitis over the past few decades. iii) Changes in skin permeability. Approximately 30% of patients with atopic dermatitis have mutations in the gene that produces filaggrin (FLG), which increases the risk of early onset of atopic dermatitis and asthma. Filaggrin plays an important role in maintaining the skin surface at a slightly acidic level and providing antibacterial properties.

[0005] These different components interact and synergize to induce a complex immunopathology involving multiple pathways, cytokines, and cells, ultimately resulting in the signs and symptoms of atopic dermatitis. Evidence suggests that several cytokines, such as the T helper 2 cytokine interleukin-4 (IL-4) and the closely related IL-13, are important components in the pathogenesis of AD. Indeed, an antibody targeting the IL-4 receptor α subunit (IL-4Rα), shared between IL-4 and IL-13 (dupilumab), alone or in combination with corticosteroids, was the first biologic approved in Europe and the United States for the treatment of atopic dermatitis (Strowd et al., 2017, The Lancet 389, 2265-2266. https: / / doi.org / 10.1016 / S0140-6736(17)31192-3). Recently, an antibody targeting IL-13 (tralokinumab), alone or in combination with corticosteroids, has also been approved in Europe and the United States (Wollenberg et al., 2021, Br J Dermatol., 184(3):437-449, doi:10.1111 / bjd.19574; Silverberg et al., 2021, Br J Dermatol, 184(3):450-463, doi:10.1111 / bjd.19573). Nevertheless, many patients do not respond or respond inadequately, and there remains a significant unmet medical need for effective and safe treatments for atopic dermatitis. Therefore, several products targeting pathways other than IL-4 / IL-13 are under development.

[0006] Interleukin-15 (IL-15), also known as MGC9721, is a cytokine well known as a regulator of natural killer (NK) and T cell activation, survival, and proliferation (Waldmann et al., 2020, J. Exp. Med., 217, e20191062, https: / / doi.org / 10.1084 / jem.20191062). However, it has also been shown to be a modulator of other cells, such as B cells, monocytes, and eosinophils (Gill et al., 2009, Cell. Immunol., 258, 59-64, https: / / doi.org / 10.1016 / j.cellimm.2009.03.010; Mohamadzadeh et al., 2001, Journal of Experimental Medicine, 194, 1013-1020, https: / / doi.org / 10.1084 / jem.194.7.1013; Hoontrakoon et al., 2002, Am. J. Respir. Cell Mol. Biol., 26, 404-412, https: / / doi.org / 10.1165 / ajrcmb.26.4.4517). This cytokine and interleukin 2 (IL-2) share many biological activities, consistent with their shared receptor signaling components (IL-2 / 15Rβ and IL-2 / 15Rγc). However, the specificity of IL-15 relative to IL-2 is provided by a unique α-chain receptor that completes the IL-15Rαβγ heterotrimeric high-affinity receptor complex, allowing differential reactivity depending on the ligand and high-affinity receptor expressed (Fehniger and Caligiuri, 2001, Blood 97, 14-32).

[0007] The detrimental role of dysregulated IL-15 expression has been demonstrated in autoimmune diseases such as rheumatoid arthritis, psoriasis, celiac disease, eosinophilic esophagitis, alopecia areata, and vitiligo. This suggests that compounds that abolish IL-15 signaling have been shown to be effective in animal models of these diseases (Villadsen et al., 2003, J. Clin. Invest. 112, 1571-1580, https: / / doi.org / 10.1172 / JCI18986; Sestak et al., 2018, Front. Immunol. 9, 1603, https: / / doi.org / 10.3389 / fimmu.2018.01603; Laehdeaho et al., 2019, Lancet Gastroenterol Hepatol 4, 948-959; Vicari et al., 2017, MAbs 9, 927-944; Xing et al., 2014, Nature Medicine 20, 1043-1049, https: / / doi.org / 10.1038 / nm.3645; Richmond et al., 2019, Lancet Gastroenterol Hepatol 4, 948-959 ... This is based on observations that increased IL-15 expression can alleviate clinical and disease features in selected cases (Baslund et al., 2005, Arthritis Rheum. 52, 2686-2692, https: / / doi.org / 10.1002 / art.21249; Laehdeaho et al., 2019, supra) and in clinical trials. However, increased IL-15 expression may not be harmful in some cases. For example, in viral diseases, increased IL-15 expression has been associated with improved viral clearance (Verbist et al., 2011, J. Immunol. 186, 174-182, https: / / doi.org / 10.4049 / jimmunol.1002613).

[0008] Several reports describe increased expression of IL-15 or its receptor in the skin and / or blood of human patients with atopic dermatitis (Orteu et al., 2000, Clin Exp Immunol., 122, 150-156; Karlen and Simon, 2020, Int. Arch. Allergy Immunol., 181, 417-421) or dogs (Mazrier et al., 2022, Vet. Dermatol., 33, 131-e38. https: / / doi.org / 10.1111 / vde.13044).

[0009] Conversely, it has been suggested that decreased IL-15 expression may contribute to the pathogenesis of atopic dermatitis in humans (Ong et al., 2002, J. Immunol., 168, 505-510). Furthermore, a deficiency of natural killer (NK) cells in the blood has been reported in patients with atopic dermatitis, whereas activated NK cells are abundant in the skin of patients with atopic dermatitis (Mack et al., 2020, Sci. Transl. Med., 12, eaay1005. https: / / doi.org / 10.1126 / scitranslmed.aay1005). Considering that IL-15 is an important factor for the survival and activation of NK cells, the authors suggested that administration of IL-15 agonist molecules may have beneficial effects on atopic dermatitis. To further support this hypothesis, treatment of mice with the MC903 atopic dermatitis model (induced by topical application of a vitamin D3 analog) with an IL-15 agonist molecule resulted in improvements in clinical scores and other features of atopic dermatitis that were dependent on NK cells (Mack et al., 2020, supra).

[0010] In conclusion, it is unclear with existing technology whether overexpression of IL-15 in atopic dermatitis is harmful or beneficial.

[0011] Atopic dermatitis is the most common chronic inflammatory skin disease worldwide, but treatment options remain limited. Therefore, given the considerable burden that AD poses to affected subjects, there is a need to better understand the underlying causes of AD and to identify new drugs with substantial beneficial effects in the management and treatment of AD. Summary of the Invention

[0012] The present invention relates to the use of agents capable of inhibiting interleukin-15 for the treatment of atopic dermatitis, primarily in an undefined patient population worldwide, and also in patients with atopic dermatitis who do not respond to existing treatments, such as anti-IL-4R, anti-IL-4 or anti-IL-13 antibodies, or to corticoid treatment.

[0013] According to a first embodiment, there is provided an interleukin-15 inhibitor for use in the treatment of atopic dermatitis.

[0014] According to another embodiment, there is provided the use of an interleukin-15 inhibitor for the preparation of a pharmaceutical composition for the treatment of atopic dermatitis.

[0015] According to another aspect of the present invention, there is provided a method for preventing and / or treating atopic dermatitis in a subject, said method comprising administering to a subject in need thereof a therapeutically effective amount of an interleukin-15 inhibitor or a pharmaceutical composition thereof.

[0016] According to another aspect of the present invention, there is provided a pharmaceutical composition comprising an interleukin-15 inhibitor, an agent useful in the treatment of atopic dermatitis, and a pharmaceutically acceptable carrier, diluent, or excipient.

[0017] Other features and advantages of the present invention will become apparent from the following detailed description. [Brief explanation of the drawings]

[0018] [Figure 1]Figure 1 shows increased IL-15 (A) and IL-15Rα (B) gene expression in an ex vivo human atopic dermatitis model analyzed by RNAseq (relative units) of human skin explants cultured for 48 hours with control or Th2-polarizing medium as described in Example 1. Symbols represent individual explants, and group means are presented as bars ± standard deviation. [Figure 2] Figure 1 shows induced IL-15 (A) and IL-15Rα (B) gene expression in an ex vivo human atopic dermatitis model analyzed by RNAseq (relative units) of human skin explants cultured for 48 hours with control or Th2-polarizing medium, or with Th2-polarizing medium alone, or with anti-IL-4R antibody or the corticoid betamethasone, as described in Example 1. Symbols represent individual explants, and group means are presented as bars ± standard deviation. [Figure 3] Figure 1 shows increased cutaneous IL-15 and IL-15Rα expression in a humanized atopic dermatitis model analyzed in human skin xenografts 2 weeks after injection of Th2-polarized PBMCs (Th2) or LPS-activated Th2-polarized PBMCs (activated Th2), as described in Example 2. IL-15 (A) and IL-15Rα (C) expression was quantified in the epidermis as the mean intensity signal, and IL-15 expression (B) in the dermis was quantified as the number of IL-15-positive cells per 100 dermal area units. Symbols represent individual xenografted mice (grafts / PBMCs from two separate human donors), and groups are represented as violin plots showing group medians and quartiles. [Figure 4] The effect of anti-IL-15 antibodies on clinical scores was measured and analyzed in human skin xenografts at the time of injection of LPS-activated, Th2-polarized PBMCs (pretreatment) and after 2 and 4 weeks of treatment with various antibodies, as described in Example 2. As shown on the x-axis, the IgG1 group was injected with a control isotype antibody for 4 weeks, the anti-IL-15 group was injected with anti-IL-15 antibody, and the IgG1 + anti-IL-15 group was injected with a control isotype antibody for 2 weeks followed by anti-IL-15 antibody for 2 weeks. Symbols represent individual xenografted mice, and groups are represented as violin plots showing group medians and quartiles. [Figure 5] Figure 1 shows the effect of anti-IL-15 antibodies on epidermal properties in a humanized atopic dermatitis model, as described in Example 2. A: Epidermal thickness in human skin xenografts analyzed 4 weeks after injection of LPS-activated Th2-polarized PBMCs in various antibody treatment groups. As shown on the x-axis, the IgG1 group was injected with a control isotype antibody for 4 weeks, the anti-IL-15 group was injected with an anti-IL-15 antibody, and the IgG1 + anti-IL-15 group was injected with a control isotype antibody for 2 weeks followed by an anti-IL-15 antibody for 2 weeks. Symbols represent individual xenografted mice, and groups are represented as violin plots showing group medians and quartiles. B: Epidermal growth of human skin xenografts was analyzed 4 weeks after injection of LPS-activated Th2-polarized PBMCs in various antibody treatment groups by measuring Ki67 expression in the basal layer of human epidermis. As shown on the x-axis, the IgG group was injected with a control isotype antibody for 4 weeks (the IgG1 and IgG4 groups were pooled due to interanimal variability and low n per group), the anti-IL-15 group was injected with an anti-IL-15 antibody, and the IgG + anti-IL-15 group was injected with a control isotype antibody for 2 weeks followed by an anti-IL-15 antibody for 2 weeks. Symbols represent individual xenografted mice, and each group is represented as a violin plot showing group medians and quartiles. C: Epidermal filaggrin (FLG) in human skin xenografts was analyzed by immunofluorescence 4 weeks after injection of LPS-activated Th2-polarized PBMCs. As shown on the x-axis, the IgG group was injected with a control isotype antibody for 4 weeks (the IgG1 and IgG4 groups were pooled due to interanimal variability and low n per group), the anti-IL-15 group was injected with anti-IL-15 antibody, and the IgG + anti-IL-15 group was injected with a control isotype antibody for 2 weeks followed by anti-IL-15 antibody. Symbols represent individual xenografted mice, and each group is represented as a violin plot showing group medians and quartiles. [Figure 6]Anti-IL-15 antibodies do not affect the numbers of dermal T (A: CD3+), NK (B: CD56+), and NKT (C: CD3+CD56+) cells in a humanized atopic dermatitis model, as analyzed by immunofluorescence 4 weeks after injection of LPS-activated Th2-polarized PBMCs in the various antibody treatment groups, as described in Example 2. As shown on the x-axis, the IgG group was injected with a control isotype antibody for 4 weeks (the IgG1 and IgG4 groups were pooled due to interanimal variability and low n per group), the anti-IL-15 group was injected with an anti-IL-15 antibody, and the IgG + anti-IL-15 group was injected with a control isotype antibody for 2 weeks followed by an anti-IL-15 antibody for 2 weeks. Symbols represent individual xenografted mice, and each group is represented as a violin plot showing the group median and quartiles. [Figure 7] Figure 1 shows anti-IL-15-induced reduction in the expression of cytokines associated with atopic dermatitis in a humanized atopic dermatitis model, measured and analyzed in the dermis of human skin xenografts 4 weeks after injection of Th2-polarized PBMCs in various antibody treatment groups, as described in Example 2. (A: IFN-γ; B: IL-4; C: IL-17A; D: IL-22). The number of cells expressing either IL-4, IL-17A, or IL-22 was calculated for each xenograft by adding the number of cells expressing each cytokine (E). As shown on the x-axis, the IgG group was injected with a control isotype antibody for 4 weeks (the IgG1 and IgG4 groups were pooled due to interanimal variability and low n per group), the anti-IL-15 group was injected with an anti-IL-15 antibody, and the IgG + anti-IL-15 group was injected with a control isotype antibody for 2 weeks followed by an anti-IL-15 antibody for 2 weeks. Symbols represent individual xenografted mice, and each group is represented as a violin plot showing group medians and quartiles. [Figure 8]Heatmap showing the Log2FC of differentially regulated genes clustered by immune cell type (T helper cells, T cells, B cells, left) or atopic dermatitis-related genes (right) after therapeutic (IgG-CALY) or prophylactic (CALY) treatment with anti-IL-15 antibody in a humanized atopic dermatitis mouse model, compared to the control isotype alone described in Example 2. Lighter shading indicates a greater fold difference. IL32 is the only upregulated gene; all other genes are unchanged or downregulated. Asterisks indicate a significant adjusted p-value <0.05 (nonparametric Mann-Whitney test). All data are based on Nanostring transcriptome analysis (10 xenografts from three separate human donors per group). [Figure 9] Anti-IL-4R antibody and dexamethasone differentially affect IL-15 expression between the epidermis and dermis in a humanized atopic dermatitis model, as analyzed in human skin xenografts 2 weeks after injection of LPS-activated, Th2-polarized PBMCs with various treatments as described in Example 2. As shown on the x-axis, the IgG group received injections of a control isotype antibody for 4 weeks (the IgG1 and IgG4 groups were pooled due to interanimal variability and low n per group), the dexamethasone group received topical dexamethasone for the final 2 weeks, the anti-IL-4R group received anti-IL-4R antibody, and the IgG + anti-IL-4R group received injections of a control isotype antibody for 2 weeks followed by anti-IL-4R antibody for 2 weeks. In the epidermis, IL-15 expression was quantified as the mean intensity signal, and in the dermis, IL-15 expression was quantified as the number of IL-15-positive cells per 100 dermal area units. Symbols represent individual xenografted mice, and each group is represented as a violin plot showing group medians and quartiles. [Figure 10] Normalized gene expression values of IL-15 expression in skin biopsies between healthy volunteers and atopic dermatitis patient groups at baseline (W0) or after 4 weeks (W4) or 16 weeks of dupilumab treatment as described in Example 3 are shown. NL: non-lesional skin; L: lesional skin. Each symbol represents an individual, the bars represent the mean value per group, and the error bars represent the 95% confidence interval. DETAILED DESCRIPTION OF THE INVENTION

[0019] Statistical analysis was performed using Graph Pad Prism software. Figures 1, 3, and 9: The nonparametric Mann-Whitney test was used to compare the two experimental groups. (Figure 1: ****p<0.0001; Figure 4: **p<0.01, ***p<0.001; Figure 9: **p<0.01, ***p<0.001). Figures 2 and 7: One-way ANOVA with the Kruskal-Wallis test was used to compare the two groups in which anti-IL-4R antibody or betamethasone was added to the Th2-polarizing medium alone. (Figure 2: *p<0.05; Figure 7: *p<0.05, **p<0.01). Figures 4, 5, and 6: Experimental groups were compared using a one-way ANOVA test with Dunnett's multiple comparison test (Figure 4: *p<0.05; 5A: ****p<0.0001; 6B: *p<0.05; **p<0.01; 5C: *p<0.05, **p<0.01; 7: (ns: not significant). Figure 10: Experimental groups were compared using a one-way ANOVA test with Sidak's multiple comparison test ((*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001, ns: not significant).

[0020] The terms "interleukin-15," "interleukin-15," and "IL-15" refer herein to the interleukin-15 protein, also known as MGC9721, which in humans is a 14-15 kDa proinflammatory cytokine encoded by the IL-15 gene, the sequence of which is disclosed in Hugo Gene Nomenclature Committee ID 5977. The immature form of IL-15 contains 162 amino acids, with the first 29 amino acids constituting the signal peptide and amino acids 30-48 constituting the propeptide. The immature form of IL-15 is available under UniProtKB accession number P40933. The mature form of the IL-15 protein corresponds to amino acids Asn 49 to Ser 162, where the indicated positions correspond to the amino acid positions in the immature IL-15 amino acid sequence. The amino acid sequence of human mature IL-15 corresponds to the sequence disclosed in UniProtKB accession number P40933. The amino acid sequences of immature IL-15 from other species are available in the art, including, for example, mouse IL-15 (UniProtKB accession number P48346, corresponding to the mature IL-15 form), rat IL-15 (UniProtKB accession number P97604, corresponding to the mature IL-15 form), rhesus IL-15 (UniProtKB accessions NP_001038196, XP_001091166, XP_001091289 XP_001091416, corresponding to the mature IL-15 form), cynomolgus IL-15 (predicted sequence from NCBI accession number XP_005556036.1, corresponding to the mature IL-15 form), and canine IL-15 (UniProtKB accession A0A8C0NA66_CANLF, corresponding to the complete sequence of IL-15). The term "interleukin-15" also includes any variant or isoform of interleukin-15 that is naturally expressed by a cell. Of note, two alternatively spliced transcript variants of IL-15 have been reported. Although both isoforms produce the same mature protein, they differ in cellular trafficking.

[0021] The term "IL-15 inhibitor" refers to an agent capable of inhibiting interleukin-15. Inhibition of interleukin-15 can be assessed, for example, by measuring inhibition of IL-15 binding to the signaling chain of the IL-15 receptor (IL-15Rβ and / or IL-15Rγ) or the IL-15-specific IL-15Rα chain using techniques such as surface plasmon resonance or ELISA. Inhibition of interleukin-15 can also be assessed at a functional level by verifying the inhibitory effect by measuring, for example, i) inhibition of IL-15 downstream signaling events, such as STAT5 phosphorylation, which can be measured in tissue sections, whole cells, or cell extracts using Western blot, flow cytometry, or immunofluorescence techniques; ii) inhibition of IL-15-dependent cell survival or activation, cytokine secretion, or expression of specific genes in cellular assays including cell lines or primary cells from animals, healthy humans, or patients; or iii) inhibition of IL-15-dependent mechanisms in vivo, such as NK and other leukocyte homeostasis, which can be measured by flow cytometry in the blood of animals, healthy humans, or patients. Of these agents, several interleukin-15 inhibitors have been or are under active development for the treatment of various diseases, such as those described in WO 2016 / 001275, WO 2015 / 089217, WO 2005 / 044303, WO 2018 / 119246, and WO 2011 / 127324.

[0022] Interleukin-15 inhibitors may be small molecule inhibitors (e.g., small molecular weight organic compounds <900 Da), peptides, mAbs, chimeric or fusion proteins, aptamers (such as peptide aptamers, DNA aptamers, and RNA aptamers), soluble receptors, and such agents may also act by silencing or downregulating the expression of interleukin-15.

[0023] The term "antibody" as used herein refers to a polypeptide that binds to an antigen. This includes whole antibodies and antigen-binding fragments. The term "antibody" is used in its broadest sense and includes monoclonal antibodies, polyclonal antibodies, human antibodies, humanized antibodies, chimeric antibodies, bispecific antibodies, multispecific antibodies, and even engineered antibodies, so long as they retain the characteristic properties of the present invention, particularly their ability to bind to the target antigen (i.e., IL-15). Examples of antibodies are described herein. Bispecific or multispecific antibodies can be engineered as described in Sawant et al., 2020, Int. J. Mol. Sci. 2020, 21, 7496. https: / / doi.org / 10.3390 / ijms21207496. As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible minor naturally occurring mutations. Monoclonal antibodies are highly specific, directed against a single antigenic site. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method.

[0024] The term "chimeric antibody" generally refers to an antibody that contains a variable region from one source or species and at least a portion of a constant region from a different source or species, and is usually prepared by recombinant DNA technology. A typical example of a chimeric antibody is one that contains a murine variable region and a human constant region. As defined herein, the term also includes antibodies that contain at least one CDR from a first human antibody and at least a portion of the constant region from a second human antibody. It also includes antibodies that contain heavy chain CDR1, CDR2, and CDR3 from a first human antibody and light chain CDR1, CDR2, and CDR3 from a second human antibody.

[0025] The term "humanized antibody" refers to an antibody from a non-human species having one or more complementarity-determining regions (CDRs) from said non-human species and a framework region from a human immunoglobulin molecule. A humanized antibody may optionally further comprise one or more framework residues from the non-human species from which the CDRs are derived.

[0026] The term "human antibody" or "fully human antibody" refers to an antibody in which the variable and constant regions of both the heavy and light chains are all of human origin or substantially identical to sequences of human origin, although not necessarily from the same antibody.

[0027] An "isolated antibody" refers to an antibody that has been separated from a component of its natural environment. For example, an isolated antibody has been purified to greater than 95% or 99% purity as determined by methods in the art (e.g., Flatman et al., 2007, J Chromatogr B Analyt Technol Biomed Life Sci, 848:79-87), including electrophoretic methods (e.g., SDS-PAGE, isoelectric electrophoresis, capillary electrophoresis) or chromatographic methods (e.g., ion exchange or reverse-phase HPLC (high performance liquid chromatography)).

[0028] The term "variant" can apply to polynucleotides and / or polypeptides. For example, a peptide or polypeptide variant referred to herein refers to a peptide or polypeptide that is substantially homologous to a reference peptide sequence but has an amino acid sequence that differs from the reference sequence due to the deletion, insertion, and / or substitution of one or more amino acids. Substantially homologous refers to a variant amino acid sequence that is identical to the reference peptide sequence except for the deletion, insertion, and / or substitution of a few amino acids, e.g., 1, 2, 3, 4, 5, or 6 amino acids. Substantially homologous refers to a variant amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the reference amino acid sequence. A variant nucleic acid sequence can be at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a reference nucleic acid sequence. The identity of two amino acid sequences or two nucleic acid sequences can be more easily determined by visual inspection and / or mathematical calculation, or by comparing sequence information using known computer programs used for sequence comparison, such as the Clustal package version 1.83. Variants may also include sequences with at least one conservatively substituted amino acid, meaning that a given amino acid residue is replaced with a residue having similar physicochemical characteristics. Examples of conservative substitutions include the substitution of aliphatic residues, such as Ile, Val, Leu, or Ala, for one another, or the substitution of polar residues, such as Lys for Arg; Glu for Asp; or Gln for Asn. Other such conservative substitutions, for example, substitutions of entire regions with similar hydrophobicity characteristics, are well known (Kyte, et al, 1982, J. Mol. Biol., 157:105-131).For example, a "conservative amino acid substitution" can involve substituting a native amino acid residue with a non-native residue such that there is little or no effect on the polarity or charge of the amino acid residue at that position. Alternatively, the substitution of one or more amino acids present in the original polypeptide may not be conservative, resulting in a variant with altered properties compared to the reference antibody. Desirable amino acid substitutions (whether conservative or non-conservative) can be determined by one of skill in the art at the time such substitutions are desired. The term "variant" also includes peptides or polypeptides that are substantially homologous to a reference peptide sequence, but have an amino acid sequence that differs from the reference sequence because one or more amino acids have been chemically modified or substituted with an amino acid analog. The term also includes glycosylated polypeptides.

[0029] The terms "binding" or "binding" of an inhibitor to a target antigen refer to at least a transient interaction or association between the inhibitor and the target antigen (e.g., IL-15) or a fragment of the target antigen that contains the epitope recognized by the inhibitor. As used herein, an antibody that binds to IL-15 is also referred to as an anti-IL-15 antibody.

[0030] The terms "selectively bind," "specifically bind," and "specific for," when applied to an antibody, indicate that the antibody preferentially recognizes and / or binds to a target polypeptide or epitope, i.e., binds with higher affinity than other antigens or epitopes, i.e., binding to the target polypeptide can be distinguished from non-specific binding to other antigens. The binding affinity of an antibody can be readily determined by those skilled in the art, for example, by equilibrium dialysis, equilibrium binding, surface plasmon resonance, or spectroscopy (e.g., using a fluorescence assay). In particular, when using surface plasmon resonance (SPR) technology, a binding event of a biomolecule causes a change in the refractive index at the surface layer on which one of the binding partners is immobilized, which is detected as a change in the surface plasmon resonance signal, expressed as response units (RU). By measuring the real-time binding kinetics of an antibody to a target antigen, SPR technology can determine the rate of binding of the antibody to its target (k a or k on The binding strength (measured as the association constant) d or k off The affinity of an antibody for its target can be determined by the equilibrium dissociation constant, K D can be quantitatively measured by determining D is K D =k d / k a is defined as k a is the association rate (k on ) and k d is the dissociation rate (k off ) (Murphy, et al., 2006, Curr Protoc Protein Sci, Chapter 19: Unit 19.14). Comparison of the affinity and / or binding properties between two antibodies can be performed by comparing the K D Without actually determining the value of K D The affinity can be determined based on a quantitative measurement of binding proportional to the affinity (for example, by ELISA or FACS analysis), or a qualitative measurement or estimation of affinity (for example, a functional assay or an in vitro or in vivo assay).

[0031] The term "blocking" or "neutralizing" activity of an inhibitor refers to its ability to inhibit the activity of a target. The neutralizing activity of an inhibitor can be determined by in vitro or in vivo assays or functional assays. When applied to inhibitors that bind IL-15, the term generally refers to the ability of the inhibitor to neutralize IL-15 activity, which can be seen in, for example, the following: IL-15-induced proliferation and / or survival of activated T cells, natural killer cells, natural killer T cells, and B lymphocytes, or any other cells expressing heterotrimeric IL-15Rαβγ or heterodimeric IL-15Rβγ receptors (Finch, et al, 2011, Br J Pharmacol. 162:480-90), IL-15-induced immunoglobulin synthesis by B lymphocytes stimulated with anti-IgM or CD40 ligand (Litinskiy et al, 2012, Nat Immunol, 3:822-9), IL-15-induced activation of human neutrophils (Rathhe and Girard, 2004, J Leukoc Biol., 76:162-8), as well as inhibition of IL-15-induced production of inflammatory cytokines from macrophages, dendritic cells, or epithelial cells (Nanayakkara, et al, 2013, Am J Clin Nutr., 98:1123-35).

[0032] Unless otherwise constrained by the definition of the individual substituents, the term "substituted" refers to a group substituted with 1 to 5 substituents selected from the group consisting of "C1-C6 alkyl," "C3-C8-cycloalkyl," "heterocycloalkyl," "sulfonyl," "sulfonamido," "alkoxy," "alkoxycarbonyl," "halogen," "carboxy," "halomethyl," cyano, hydroxy, nitro, and the like.

[0033] The term "pharmaceutically acceptable" refers to a carrier that is composed of materials that are biologically or otherwise undesirable.

[0034] The term "carrier" refers to any ingredient, other than the active agent, present in a pharmaceutical formulation and thus includes diluents, binders, lubricants, disintegrants, fillers, coloring agents, wetting or emulsifying agents, pH buffering agents, preservatives, etc.

[0035] As used herein, "treatment" and "treating" and the like generally refer to obtaining a desired pharmacological and physiological effect. The effect may be prophylactic, in that a disease, symptom, or condition is prevented or partially prevented, and / or may be therapeutic, in that a disease, condition, symptom, or adverse effects resulting from a disease are partially or completely cured. As used herein, the term "treatment" encompasses any treatment of disease in a mammal, particularly a human, and includes: (a) preventing the onset of disease in a subject who may be predisposed to the disease, for example, based on family history, but has not yet been diagnosed with the disease; (b) inhibiting the disease, i.e., preventing its development; or (c) palliating the disease, i.e., causing regression of the disease and / or its symptoms or condition, e.g., amelioration or repair of damage. For example, treatment of celiac disease includes preventing, reducing, or eradicating symptoms of the disease or disorder, e.g., atopic dermatitis symptoms such as dry skin, itching, results from scratching or scratching (scalding, sensitive, swollen skin), partial or total alleviation of skin lesions including: i) red to brownish-gray patches (especially on the hands, feet, ankles, wrists, neck, upper chest, eyelids, inside the elbows and knees, and in infants, the face and scalp); ii) small, raised bumps that may leak fluid and form crusts when scratched, and iii) thickened, cracked, scaly skin.

[0036] As used herein, the term "subject" refers to a mammal. For example, mammals contemplated by the present invention include humans, primates, laboratory rodents, and pets such as dogs and cats.

[0037] The term "efficacy" of a treatment or method according to the invention can be measured based on the change in the course of a disease or condition in response to a use or method according to the invention. For example, the effectiveness of a treatment or method according to the invention can be measured by the effect on the signs or symptoms of the disease. A response is achieved when the patient experiences partial or total relief or alleviation of the undesired symptoms of the disease.

[0038] The term "effective amount" as used herein refers to an amount of at least one agent according to the present invention, or a pharmaceutical formulation thereof, that induces a detectable alleviation of disease symptoms in a subject to which said agent is administered.

[0039] IL-15 inhibitors IL-15 inhibitors according to the present invention include anti-IL antibodies, inhibitory peptides and small molecules.

[0040] According to one embodiment, the IL-15 inhibitor is a small molecule as described in Quemener et al., 2017, J. Med. Chem. 2017, 60, 6249-6272 DOI: 10.1021 / acs.jmedchem.7b00485, in particular the following structure: [ka] wherein R is optionally substituted phenyl and n is an integer selected from 1 to 10, especially compounds having the following structure: [ka] It is a molecule having the formula:

[0041] According to another embodiment, the IL-15 inhibitor can be a peptide such as the IL-15 antagonists described in WO 2020 / 227019 and WO 2015 / 089217, e.g., BNZ132 (also known as BNZ-1, also known as YT033, also known as EQ-101) (SEQ ID NO: 2) (IKEFLQRFIHIVQSIINTS), and the IL-15 antagonists described in U.S. Patent Application Publication No. 2019 / 0070263, e.g., PKEFLERFVHLVQMFIHQSLS (SEQ ID NO: 3), or fragments or variants of those sequences. Yet another peptidic IL-15 inhibitor is described in U.S. Patent No. 7,736,638 (SEQ ID NO: 4), or a fragment or variant of those sequences.

[0042] According to another embodiment, the IL-15 inhibitor may be an anti-IL15 antibody or an antigen-binding fragment thereof. According to a particular aspect, the anti-IL15 antibody or antigen-binding fragment thereof binds to IL-15, particularly human IL-15, or a fragment of IL-15.

[0043] Anti-IL15 antibodies according to the present invention generally exhibit high specificity for human IL-15. However, depending on the degree of sequence identity between IL-15 homologues of different species, a given antibody or antigen-binding fragment may exhibit cross-reactivity with IL-15 from at least one other species, such as primates (e.g., cynomolgus monkeys, rhesus monkeys, marmosets), mice, rats, dogs, and / or rabbits. In the case of antibodies against human IL-15, some degree of cross-reactivity with IL-15 forms from other mammals may be desirable in certain circumstances.

[0044] In certain embodiments, the anti-IL15 antibodies or fragments thereof according to the invention bind preferentially to human IL-15.

[0045] In a further embodiment, the antibody or antigen-binding fragment thereof according to the invention does not exhibit cross-reactivity with rat IL-15 and / or mouse IL-15.

[0046] In one embodiment, an inhibitor according to the invention preferentially inhibits IL-15 and optionally also exhibits some inhibition of other proteins having homology to IL-15, such as IL-2, in particular human IL-2 or IL-21 (disclosed in WO 2015 / 089217).

[0047] The ability of an antibody to block or neutralize the activity of its target protein can be assessed by its potency, as defined herein, and as such, can be measured, for example, by IC 50 Typically, the neutralizing activity of an antibody can be determined by an in vitro assay, for example, an assay to measure the level of inhibition of IL-15-induced proliferation and / or survival of a cell line, such as Kit 225 or M-07e cells, in the presence of the antibody, as described in WO 2016 / 001275.

[0048] In some embodiments, the antibodies and antigen-binding fragments thereof according to the invention have an IC of 200 nM or less, particularly less than 100 nM, particularly less than 50 nM, less than 30 nM, less than 20 nM, especially less than 10 nM, less than 8 nM, less than 7 nM, less than 5 nM, less than 4 nM, less than 3 nM, less than 2 nM, less than 1 nM, less than 0.5 nM, less than 0.3 nM, less than 0.2 nM, less than 0.1 nM, less than 0.05 nM, or less than 0.03 nM, for inhibiting IL-15 activity such as IL-15-induced proliferation and / or survival of cell lines such as Kit 225 or M-07e cells as described in WO 2016 / 001275. 50 It has.

[0049] It is understood that any variant or fragment of the antibodies described therein can bind to IL-15 and optionally neutralize IL-15 activity and can be a suitable IL-15 inhibitor according to the present invention. In certain embodiments, such variants can exhibit the same or higher binding affinity for IL-15, the same or higher potency for IL-15, the same or higher species selectivity for IL-15, the same or higher selectivity for IL-15, and / or the same or higher neutralization potency compared to the parent antibody or fragment from which the variant is derived.

[0050] In certain embodiments of the invention, the antibody to IL-15, or an antigen-binding fragment thereof that binds IL-15, is a monoclonal antibody.

[0051] In certain embodiments of the invention, the antibody against IL-15, or an antigen-binding fragment thereof that binds to IL-15, is a bispecific or multispecific antibody, particularly having dual specificity targeting IL-15 and IL-4R or IL-13 or IL-4.

[0052] In a further specific embodiment of the invention, the antibody against IL-15 or an antigen-binding fragment thereof that binds IL-15 is a humanized antibody.

[0053] In a further specific embodiment of the invention, the antibody against IL-15 or an antigen-binding fragment thereof that binds IL-15 is a recombinant antibody.

[0054] Antibodies against IL-15 or antigen-binding fragments thereof that bind to IL-15 that are suitable for use according to the present invention may be characterized by their portion that interacts with the target protein, in particular their variable region, which typically comprises a heavy chain variable region and a light chain variable region as described in WO 2016 / 001275.

[0055] According to certain embodiments, the anti-IL-15 inhibitor is: (1) A heavy chain variable region of SEQ ID NO: 5, or any variant thereof, wherein the variant has the amino acid sequence of SEQ ID NO: 5, except that one, two, three, or four amino acids are substituted with different amino acids, and the substitutions are any of the following: (i) within the heavy chain variable framework region, an arginine (R) at position H3 (VH RH3) substituted with glutamine (Q), a methionine (M) at position H5 (VH MH5) substituted with valine (V), an alanine (A) at position H6 (VH AH6) substituted with glutamic acid (E), an alanine (A) at position H49 (VH AH49) substituted with serine (S); (ii) within the heavy chain CDR2, an aspartic acid (D) at position H61 (VH DH61) substituted with a glutamic acid (E), a serine (S) at position H62 (VH SH62) substituted with a threonine (T), and (iii) within the heavy chain CDR3, a methionine (M) at position H98 (VH MH98) substituted with leucine (L), phenylalanine (F), isoleucine (I), or alanine (A); a tryptophan (W) at position H100C (VH WH100C) substituted with tyrosine (Y), phenylalanine (F), or alanine (A); a methionine (M) at position H100E (VH MH100E) substituted with leucine (L), phenylalanine (F), or isoleucine (I); a heavy chain variable region of SEQ ID NO: 5 or a variant thereof selected from the group consisting of: (2) a light chain variable region of SEQ ID NO: 9; An isolated antibody or antigen-binding fragment thereof that binds to IL-15, comprising:

[0056] According to another particular embodiment, the anti-IL-15 inhibitor is: (1) a heavy chain variable region of SEQ ID NO: 6, or any variant thereof, wherein the variant has the amino acid sequence of SEQ ID NO: 6, except that one, two, three, or four amino acids are replaced with different amino acids; and (2) a light chain variable region of SEQ ID NO: 9; An isolated antibody or antigen-binding fragment thereof that binds to IL-15, comprising:

[0057] According to another particular embodiment, the anti-IL-15 inhibitor is: (1) In the sequence of SEQ ID NO: 5: (i) VH RH3 is substituted with glutamine (Q), VH MH5 is substituted with valine (V), and VH AH6 is substituted with glutamic acid (E); (ii) VH SH62 is substituted with threonine (T); and (iii) VH WH100C is substituted with tyrosine (Y); a heavy chain variable region of the amino acid sequence of SEQ ID NO: 5 having a substitution selected from: (2) a light chain variable region of the amino acid sequence of SEQ ID NO: 9; 2. The isolated antibody or antigen-binding fragment thereof of claim 1, comprising:

[0058] According to another particular embodiment, the anti-IL-15 inhibitor is: (1) a heavy chain variable region selected from SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8; (2) a light chain variable region of SEQ ID NO: 9; An isolated antibody or antigen-binding fragment thereof that binds to IL-15, comprising:

[0059] According to another specific embodiment, the anti-IL-15 inhibitor is an isolated antibody or antigen-binding fragment thereof that binds to IL-15, comprising a heavy chain variable region of SEQ ID NO:6 and a light chain variable region of SEQ ID NO:9.

[0060] 10. An interleukin-15 inhibitor for use according to claim 1 or 6, wherein the interleukin-15 is selected from the group consisting of: - a heavy chain of SEQ ID NO: 10 and a light chain of SEQ ID NO: 11, or a variant or fragment thereof; or - a variable heavy chain region of SEQ ID NO: 12 or a variant or fragment thereof, and a variable light chain region of SEQ ID NO: 13 or a variant or fragment thereof; or - a variable heavy chain region of SEQ ID NO: 14 or a variant or fragment thereof, and a variable light chain region of SEQ ID NO: 15 or a variant or fragment thereof; or - a heavy chain of SEQ ID NO: 16 and a light chain of SEQ ID NO: 17 or a variant or fragment thereof; or - a heavy chain of SEQ ID NO: 18 and a light chain of SEQ ID NO: 17 or a variant or fragment thereof; or - a heavy chain of SEQ ID NO: 19 and a light chain of SEQ ID NO: 17 or a variant or fragment thereof 10. An interleukin-15 inhibitor selected from anti-IL-15 antibodies comprising:

[0061] According to another more specific embodiment, the anti-IL-15 inhibitor is an isolated antibody or antigen-binding fragment thereof that binds to IL-15, comprising a heavy chain of SEQ ID NO:16 and a light chain of SEQ ID NO:17.

[0062] According to another more specific embodiment, the anti-IL-15 inhibitor is an isolated antibody or antigen-binding fragment thereof that binds to IL-15, comprising a heavy chain of SEQ ID NO:18 and a light chain of SEQ ID NO:17.

[0063] According to another more specific embodiment, the anti-IL-15 inhibitor is an isolated antibody or antigen-binding fragment thereof that binds to IL-15, comprising a heavy chain of SEQ ID NO:19 and a light chain of SEQ ID NO:17.

[0064] According to another specific embodiment, the anti-IL-15 inhibitor is an isolated antibody or antigen-binding fragment thereof that binds to IL-15, comprising sequence 146B7 (also known as AMG174) as described in WO 2005 / 044303 or WO 03 / 017935, which is an IL-15 antagonist that is a monoclonal human IgG1 anti-IL-15 antibody having heavy chain and kappa light chain regions comprising the amino acid sequences of SEQ ID NO:10 and SEQ ID NO:11.

[0065] According to another particular embodiment, the anti-IL-15 inhibitor is an isolated antibody that binds to IL-15 as described in WO 2018 / 119246, comprising the variable heavy chain sequence of SEQ ID NO: 12 and the variable light chain sequence of SEQ ID NO: 13, or an antigen-binding fragment thereof.

[0066] According to another particular embodiment, the anti-IL-15 inhibitor is an isolated antibody or antigen-binding fragment thereof that binds to IL-15 as described in WO 2011 / 127324, in particular huABC2 (anti-IL-15Rβ antibody) comprising the variable heavy chain sequence of SEQ ID NO: 14 and the variable light chain sequence of SEQ ID NO: 15.

[0067] According to another specific embodiment, the anti-IL-15 inhibitor is an isolated antibody or antigen-binding fragment thereof that binds IL-15 as described in WO 2009 / 002562, or an isolated antibody, such as 146B7 or an antigen-binding fragment thereof, as described in WO 2017 / 217985 and U.S. Patent Application Publication No. 2018 / 0002417, or WO 2005 / 044303 and WO 03 / 017935.

[0068] Specific examples of antibodies according to the present invention include those shown in Table 1.

[0069] [Table 1]

[0070] Conjugates containing auxiliary molecules In another aspect of the invention, the isolated antibody or antigen-binding fragment thereof according to the invention is optionally conjugated to an auxiliary molecule and is therefore also referred to herein as a "conjugated antibody" or "conjugated antibody fragment."

[0071] The conjugated antibodies and conjugated antibody fragments according to the present invention can target the anti-IL-15 inhibitor to disease sites (e.g., sites of inflammation) in vivo, thereby allowing the anti-IL-15 inhibitor to have preferential concentration and therapeutic effect at the disease site with fewer side effects elsewhere in the body.

[0072] Accessory molecules can be conjugated to the antibody or antibody fragment directly, or via a spacer of appropriate length, for example, as described in Kellogg et al. (2011, Bioconjug Chem, 22:717-27).

[0073] In another embodiment, the accessory molecule comprises an antigen-binding fragment of an antibody which, when bound to an antibody or antibody fragment according to the invention, forms a bispecific antibody. In particular, said bispecific antibody may be directed against two different epitopes of IL-15 (thus defining a biparatopic antibody) or may have bispecific portions that target IL-15 and IL-4R or IL-13 or IL-4.

[0074] composition The IL-15 inhibitors are provided in the form of pharmaceutically acceptable compositions, which may contain one or more IL-15 inhibitors, particularly one or more antibodies or antigen-binding fragments thereof that bind to IL-15, in any of the forms described herein.

[0075] The compositions of the present invention may further comprise one or more additional pharmaceutically acceptable ingredients, such as alum, stabilizers, antimicrobial agents, buffers, colorants, flavoring agents, adjuvants, and the like.

[0076] The IL-15 inhibitor can be incorporated into the form of pharmaceutical compositions and unit dosages thereof together with conventional adjuvants, carriers, diluents, or excipients, and such forms can be used as solids, lyophilized forms such as tablets or filled capsules, or as liquids, such as solutions, suspensions, emulsions, elixirs, or filled capsules, all of which can be used orally or in the form of sterile injections for parenteral (including subcutaneous) use, particularly formulated into pre-filled syringes or auto-injectors. Such pharmaceutical compositions and unit dosage forms thereof can contain the ingredients in conventional proportions, with or without additional active compounds or principles, and such unit dosage forms can contain any suitable effective amount of the active ingredient corresponding to the intended dosage range to be used.

[0077] The compositions may be liquid preparations, including, but not limited to, aqueous or oily suspensions, emulsions, syrups, and elixirs. Liquid forms suitable for oral administration may contain a suitable aqueous or non-aqueous vehicle, including buffers, suspending and dispensing agents, colorants, flavorings, and the like. The compositions may also be formulated as a dry product for reconstitution with water or another suitable vehicle before use. Such liquid preparations may contain additives, including, but not limited to, suspending agents, emulsifying agents, non-aqueous vehicles, and preservatives. Suspending agents include, but are not limited to, sorbitol syrup, methylcellulose, glucose / sugar syrup, gelatin, hydroxyethylcellulose, carboxymethylcellulose, aluminum stearate gel, and hydrogenated edible fats and oils. Emulsifiers include, but are not limited to, lecithin, sorbitan monooleate, and acacia. Non-aqueous vehicles include, but are not limited to, edible oils, almond oil, fractionated coconut oil, oily esters, propylene glycol, and ethyl alcohol. Preservatives include, but are not limited to, methyl or propyl p-hydroxybenzoate and sorbic acid. Additional materials and processing techniques are available in Remington: The Science & Practice of Pharmacy, 23 rdEdition, 2020, Ed. Adeboye Adejare, Academic Press, which is incorporated herein by reference.

[0078] The solid composition of the present invention may be in the form of tablets or lozenges formulated in a conventional manner. For example, tablets and capsules for oral administration may contain conventional excipients, including, but not limited to, binders, fillers, lubricants, disintegrants, and wetting agents. Binders include, but are not limited to, syrup, acacia, gelatin, sorbitol, tragacanth, starch mucilage, and polyvinylpyrrolidone. Fillers include, but are not limited to, lactose, sugar, microcrystalline cellulose, corn starch, calcium phosphate, and sorbitol. Lubricants include, but are not limited to, magnesium stearate, stearic acid, talc, polyethylene glycol, and silica. Disintegrants include, but are not limited to, potato starch and sodium starch glycolate. Wetting agents include, but are not limited to, sodium lauryl sulfate. Tablets can be coated according to methods known in the art.

[0079] Injectable compositions are typically based upon injectable sterile saline or phosphate-buffered saline or other injectable carriers known in the art.

[0080] The compositions may also be formulated as transdermal formulations comprising aqueous or non-aqueous vehicles such as, but not limited to, creams, ointments, lotions, pastes, medicated plasters, patches, or membranes.

[0081] The compositions may also be formulated for parenteral administration, such as, but not limited to, injection or continuous infusion. Injectable preparations may be in the form of a suspension, solution, or emulsion in an oily or aqueous vehicle and may contain formulating agents, such as, but not limited to, suspending agents, stabilizers, and dispersing agents. The compositions may also be provided in powder form for reconstitution with a suitable vehicle, such as, but not limited to, sterile pyrogen-free water.

[0082] The compositions may also be formulated as a depot preparation, which may be administered by implantation or intramuscular injection. The compositions may be formulated with suitable polymeric or hydrophobic materials (for example, as an emulsion in an acceptable oil), ion exchange resins, or as sparingly soluble derivatives (for example, as a sparingly soluble salt).

[0083] The compounds can also be administered in sustained release dosage forms or from sustained release drug delivery systems. A description of representative sustained release materials is also found in the incorporated materials of Remington's Pharmaceutical Sciences.

[0084] Injectable formulations are particularly suitable for administering compounds or compositions according to the present invention.

[0085] combination According to the present invention, the IL-15 inhibitor according to the present invention can be administered alone or in combination with a co-agent useful in the prevention and / or treatment of atopic dermatitis, for example an anti-IL-4R such as dupilumab, an anti-IL-13 such as tralokinumab, a JAK inhibitor such as abrocitinib, upadacitinib (oral) or ruxolitinib (topical), or a corticoid such as dexamethasone.

[0086] The present invention encompasses the use of an IL-15 inhibitor, wherein the IL-15 inhibitor is administered to a subject in a therapeutically effective amount prior to, simultaneously with, or sequentially with other therapeutic regimens or co-drugs useful in the prevention and / or treatment of atopic dermatitis. IL-15 inhibitors according to the present invention administered simultaneously with said co-drugs can be administered in the same or different compositions and by the same or different routes of administration.

[0087] In a particular embodiment, an IL-15 inhibitor according to the present invention, in particular an antibody for IL-15 or an antigen-binding fragment thereof, can be administered in combination with an anti-IL-4R antibody such as dupilumab.

[0088] In a particular embodiment, an IL-15 inhibitor according to the present invention, in particular an antibody against IL-15 or an antigen-binding fragment thereof, can be administered in combination with an anti-IL-13 agent such as tralokinumab.

[0089] In a particular embodiment, an IL-15 inhibitor according to the present invention, in particular an antibody to IL-15 or an antigen-binding fragment thereof, may be administered in combination with one or more corticosteroids, such as dexamethasone.

[0090] Mode of administration The compositions of the present invention can be administered in any manner, including, but not limited to, orally, parenterally, sublingually, transdermally, transmucosally, topically, or a combination thereof.

[0091] Parenteral administration includes, but is not limited to, intravenous, subcutaneous, and intramuscular administration. The compositions of the present invention may also be administered in the form of an implant, which allows for slow release of the composition and slow, controlled IV infusion.

[0092] In certain embodiments, the IL-15 inhibitor is administered systemically or locally, for example, by topical application.

[0093] In certain embodiments, the IL-15 inhibitor, particularly an antibody or antigen-binding fragment thereof against IL-15, is administered by subcutaneous or intravenous route.

[0094] The dose administered to an individual, either as a single dose or multiple doses, will vary depending on a variety of factors, including pharmacokinetic properties, the condition and characteristics of the subject (sex, age, weight, health, size), the severity of symptoms, concurrent treatments, frequency of treatment, and the desired effect.

[0095] According to certain embodiments, the inhibitor is administered once a week to once a month.

[0096] Typically, a therapeutically effective amount of a pharmaceutically active antibody is a dosage in the range of 0.1 mg / kg to 50 mg / kg body weight.

[0097] patient In one embodiment, a subject according to the present invention suffers from atopic dermatitis.

[0098] According to certain embodiments, a subject according to the present invention exhibits high systemic or local levels of IL-15 or biomarkers of IL-15 pathway activation, as described, for example, in Karlen and Simon, 2020, Int. Arch. Allergy Immunol., 181, 417-421 and Kurowska et al., 2020, J. Clin. Med), 1555.

[0099] In another embodiment, a subject according to the present invention has atopic dermatitis and is unresponsive to or unable to use anti-IL-4R, anti-IL-13 and / or corticosteroid treatment. A subject unresponsive to anti-IL-4R, anti-IL-13 and / or corticosteroid treatment is defined by a lack of satisfactory response after treatment with anti-IL-4R, anti-IL-13 and / or corticosteroid.

[0100] Uses and methods according to the present invention In certain embodiments, methods are provided for preventing and / or treating atopic dermatitis, comprising administering a therapeutically effective amount of an IL-15 inhibitor, particularly an antibody against IL-15 or an antigen-binding fragment thereof, to a subject in need thereof.

[0101] According to a particular embodiment, the interleukin-15 inhibitor is administered in the form of a formulation according to the invention.

[0102] All references cited herein are incorporated by reference in their entirety.

[0103] The invention having been described, the following examples are offered by way of illustration and not by way of limitation. [Example]

[0104] Example 1: Expression of IL-15 and IL-15Rα in an ex vivo human model of atopic dermatitis The effect of an anti-IL-15 antibody (hu-BE29-2 comprising a heavy chain of SEQ ID NO: 16 and a light chain of SEQ ID NO: 17 described in WO 2016 / 001275) on atopic dermatitis was examined in the following experimental model.

[0105] For the experimental setup, use healthy skin punch biopsies that have been induced to exhibit an atopic dermatitis phenotype with a cocktail that activates the Th2 pathway (https: / / www.reprocell.com / drug-efficacy-safety-adme / assay-catalog / skin-culture-induced-atopic-dermatitis-model-systemic-topical), as follows:

[0106] Full-thickness skin biopsies measuring up to 36 x 3 mm were obtained from three healthy donors as surgical excisions, as described below. On day 1, the skin biopsies were prepared, randomized, and completely submerged in a minimum of 1 mL of basal medium overnight in a cell incubator. After an equilibration period, on day 0, the biopsies were transferred to individual culture wells in a 12-well plate. Next, holes were pierced in a Transwell™ filter using a pipette tip or forceps, and each biopsy was inserted into one of the filter holes. The filter containing the biopsy was placed in one well of a 12-well culture plate containing 1 mL of the appropriate medium, with the epidermis facing upward at the air-liquid interface. 1 mL of Th2-polarizing medium (Cousins et al., 2002, J. Immunol 169, 2498-2506) with or without the test substance was added to each well on day 0 and replaced on day 1. The test compounds used in the study were an anti-human IL-4R antibody with a sequence identical to dupilumab (https: / / www.genome.jp / dbget-bin / www_bget?dr:D10354) produced by transient transfection in Chinese hamster ovary cells (Evitria AG, Zurich, Switzerland), and betamethasone (Merck Life Sciences, Gillingham, United Kingdom) at a final concentration of 50 μg / mL or betamethasone at a final concentration of 10 μM.

[0107] Approximately 48 hours after application of the Th2 stimulatory cocktail and the test compounds listed above (day 2), biopsies are taken and stored in RNAlater at 2-8°C for a minimum of 24 hours. Following this, the RNAlater is removed, and the biopsies are frozen in liquid nitrogen and stored at 80°C. Tissue samples stabilized in RNAlater solution are processed for RNA extraction and isolation. RNA is quantified and quality checked using the Qubit system. Once quality checks are complete, the RNA samples are subjected to RNAseq analysis.

[0108] Skin and PBMC human donors Healthy (control) human abdominal skin was obtained from three healthy volunteers (39-52 years old) undergoing elective surgery and with no history of atopic disease. PBMCs were collected from 20 mL of venous blood from the same donors. The study was approved by the Rambam Health Care Campus Institutional Helsinki Committee (0182-14-RMB).

[0109] PBMC stimulation and characterization Autologous PBMCs were isolated as previously described (Keren et al., 2018, J. Allergy Clin. Immunol., 142, 305-308.e6. https: / / doi.org / 10.1016 / j.jaci.2018.02.015).

[0110] After isolation, they were cultured in the presence of IL-2 (10 U / mL), IL-4 (200 U / mL), and LPS (1 μg / mL; Sigma, St. Louis, MO) ("Th2-polarizing") to induce a Th2 phenotype. In a pilot experiment evaluating the induction of IL-15 expression, control PBMCs were cultured in the presence of IL-2 and IL-4, but without LPS activation.

[0111] We observed that Th2 culture conditions, which have been shown to induce genes similar to those found in atopic dermatitis lesions, can induce IL-15 gene expression, as well as IL-15Rα expression, in human skin explants in vitro, as shown in Figures 1A and 1B.

[0112] We also observed that induced IL-15 and IL-15Rα expression was not reduced by treatment with anti-IL-4R antibodies or corticoids in an ex vivo human atopic dermatitis model, as shown in Figures 2A and 2B.

[0113] Example 2: Anti-IL-15 antibodies are effective in a humanized mouse model of atopic dermatitis However, human atopic dermatitis models do not adequately represent human disease. Similarly, existing mouse experimental models are also of limited utility because they only represent human disease to a limited extent and rely on mouse skin and immune system components that differ from their human counterparts (Gilhar and Paus, 2021, Exp. Dermatol., 30, 319-336. https: / / doi.org / 10.1111 / exd.14270). Humanized mouse models offer the possibility of studying human cellular functions in vivo, and in dermatology, the humanized model of psoriasis, while complex and costly, has proven to be the most representative of human disease. Gilhar and Paus, 2021, supra, used the "humanized" model of atopic dermatitis. "Humanized" mice are defined herein as immunodeficient (SCID / beige) mice transplanted with normal human skin from a healthy donor and activated T helper (Th)2-polarized peripheral mononuclear cells (PBMCs) from the same donor to reproduce the clinical and histological features of atopic dermatitis at the level of the skin graft.

[0114] The primary objective of this study was to evaluate whether IL-15 is induced during the development of this atopic dermatitis model (pilot experiment). As can be seen from Figure 3, this model demonstrated the induction of both IL-15 and IL-15Rα expression. IL-15Rα was primarily expressed by epidermal cells, whereas IL-15 was likely expressed not only by epidermal cells but also by dermal cells (probably by cells of the monocyte and dendritic cell lineages). This analysis confirmed the presence of an IL-15 target in the humanized atopic dermatitis mouse model and, therefore, that it is suitable for testing compounds that neutralize IL-15.

[0115] A secondary objective of this study was to determine whether inhibitors of interleukin-15, such as anti-interleukin-15 antibodies, administered in various regimens, could be effective in this humanized model of atopic dermatitis.

[0116] When anti-IL-15 antibodies were administered for 4 weeks from the onset of atopic dermatitis or only for the final 2 weeks, significant effects were observed on macroscopic skin lesions, as shown in Figure 4, which is represented by a clinical score based on measurements of erythema and skin integrity, as described below. Furthermore, there was a significant effect of anti-IL-15 treatment on epidermal characteristics such as epidermal thickness (A), epidermal basal cell proliferation (B), and expression of filaggrin (C), a molecule associated with skin barrier integrity that is frequently mutated in human atopic dermatitis patients (O'Regan et al., 2008, J. Allergy Clin. Immunol., 122, 689-693. https: / / doi.org / 10.1016 / j.jaci.2008.08.002), all of which are known hallmarks of human atopic dermatitis (Figure 5).

[0117] The third objective was to analyze in treated mice the effects of anti-IL-15 antibodies on key readouts and biomarkers known to be associated with atopic dermatitis in humans.

[0118] We also observed an effect of anti-IL-15 therapy on cytokines (IFN-γ, IL-4, IL-17, and IL-22) that may be secreted by T cells and are known to be induced in human atopic dermatitis (Figure 7). In contrast, anti-IL-15 therapy did not significantly affect the numbers of T cells, NK cells, and NKT cells present in the dermis of human skin grafts (Figure 6). These cells are thought to secrete IFN-γ, IL-4, IL-17, and IL-22. This suggests that anti-IL-15 therapy is either affecting only the function of these T cells, NK cells, and NKT cells, or is affecting only the number and / or function of a small cell subset that could not be elucidated using the techniques used here. In the immune gene expression analysis described below, we observed that hu-BE29-2 (also known as CALY-002) treatment induced a decrease in the expression of many immune genes associated with T cells, T helper cells, and B cells, as well as genes known to be associated with atopic dermatitis, such as S100A9, DEFB4, and CXCL10 (Figure 8). Overall, these data suggest that blocking the IL-15 pathway may be an effective novel treatment for atopic dermatitis.

[0119] Because both anti-IL-4R therapeutic antibodies and corticoids are approved treatments for atopic dermatitis ( Strowd et al., 2017 , The Lancet, 39 (10086), 2265–2266), we investigated whether these treatments could affect IL-15 expression in the atopic dermatitis model analyzed. Interestingly, in the ex vivo human explant model described in Example 1, neither anti-IL-4R antibodies nor corticoids were able to reduce IL-15 ( Figure 9 ), but both treatments significantly reduced many other genes associated with atopic dermatitis. This profile was also observed for IL-15Rα expression; in fact, the expression of IL-15 and IL-15Rα was slightly increased by anti-IL-4R treatment. Furthermore, in the humanized atopic dermatitis mouse model described in this example, anti-IL-4R treatment only reduced IL-15 expression in the dermis, but not in the epidermis. Corticoids showed a tendency to reduce IL-15 expression restricted to the dermis, but this was not statistically significant.

[0120] These results suggest that the IL-15 pathway is not completely inhibited by treatment with anti-IL-4R antibodies or corticoids, and therefore anti-IL-15 therapy may be advantageously administered to patients who do not respond to anti-IL-4R or corticoids, or in combination with these or other treatments.

[0121] animal Sixty 2- to 3-month-old female CB-17 / IcrHsd-scid-bg (beige-SCID, Harlan Laboratories Ltd., Jerusalem, Israel) mice were used for the treatment experiments. Mice were housed under pathogen-free conditions in accordance with institutional guidelines.

[0122] Skin and PBMC donors Healthy abdominal human skin was obtained from healthy female volunteers undergoing elective surgery and with no history of atopic disease. PBMCs were collected from 20 mL of venous blood from the same donors. The study was approved by the Rambam Health Care Campus Institutional Helsinki Committee (0182-14-RMB).

[0123] Humanized mouse atopic dermatitis model Split-thickness skin xenografts were performed as described (Keren et al., 2018, supra). Briefly, human skin samples (1 cm, 0.4 mm thick) from three different donors were transplanted onto mice (one human xenograft per mouse). One month after transplantation, 1 × 10 7 Th2-polarized PBMCs were injected intradermally. Prior to injection, cell viability was measured by trypan blue staining.

[0124] In vivo treatment with anti-IL-15 antibodies and controls Two treatment regimens using the anti-IL-15 antibody hu-BE29-2 (SEQ ID NO: 16 and SEQ ID NO: 17) described in WO 2016 / 001275, or a control, were designed. Mice (10 per group) were either intravenously injected with 200 μg / mouse of the anti-IL-15 antibody (Calypso Biotech SA, Switzerland) three times a week for four weeks after PBMC injection, or first injected with a corresponding dose of an IgG1 control isotype antibody for two weeks, followed by the anti-IL-15 antibody for two weeks. Other groups of mice (10 mice per group) were similarly injected with 200 μg / mouse of an anti-IL-4 receptor (IL-4R) antibody whose sequence was identical to that of dupilumab (https: / / www.genome.jp / dbget-bin / www_bget?dr:D10354) and produced by transient transfection in Chinese hamster ovary cells (Evitria AG, Zurich, Switzerland) for 4 weeks, or with an IgG4 control isotype antibody for 2 weeks followed by an anti-IL-4R antibody for 2 weeks. Negative controls (5 mice per group) were injected with an IgG1 or IgG4 control antibody (Evitria AG, Zurich, Switzerland) for 4 weeks. As a positive control, dexamethasone was administered topically to groups of 10 mice three times a week starting 3 weeks after PBMC injection. Macroscopic photographs of the grafted skin were taken on day 0 and at weeks 1, 2, 3, and 4 after PBMC injection. At the end of the four weeks of treatment, each transplanted skin explant was harvested, cut in half, and either embedded in Tissue-Tek® OCT and frozen, or fixed in formalin, embedded in paraffin, and stored at room temperature until further evaluation.

[0125] Clinical and macroscopic evaluation To measure the severity of the atopic dermatitis-like phenotype in the xenografted skin, measurements commonly applied in clinical practice were adapted to analyze collected macroscopic photographs. Signs of erythema and skin integrity (excoriation / scaling / lichenification) were graded on an individual scale (0 = absent, 1 = moderate, 2 = severe) at day 0 and weeks 1, 2, 3, and 4 after PBMC injection. A total score (erythema score + skin integrity score) was calculated for each mouse. Scoring was performed blindly by three independent operators.

[0126] Hematoxylin and eosin (H&E) staining and epidermal thickness measurement Routine H&E staining was performed on 7-μm-thick frozen sections. For H&E-stained sections, the mean human epidermal thickness for each xenograft was determined by averaging measurements of the distance between the outermost surface of the epidermis, excluding the stratum corneum, and the dermal-epidermal junction at 10 randomly selected points along the entire length of each examined section using digital calipers and ImajeJ software. Measurements were performed blindly and averaged for each experiment.

[0127] Expression of IL-15 and IL-15Rα To examine the expression of IL-15 and IL15-Rα in the xenografts, OCT (Optimal Temperature Cutting Compound)-embedded skin xenografts were sectioned (7 μm thick) using a Leica cryostat. Tissue cryosections were fixed with acetone and preincubated with 10% goat serum in PBS. Next, sections were incubated overnight at 4°C with the corresponding primary antibodies (anti-IL-15, Ab55276, Abcam, 1:100; anti-IL-15RA, Ab91270, Abcam, 1:100). Secondary antibody incubation was performed at RT for 45 min (Goat-anti-Mouse-IgG-Alexa546, Invitrogen, 1:500). Counterstaining with DAPI (1 μg / ml) was performed to visualize nuclei.

[0128] Immunohistochemistry for atopic dermatitis markers. To measure filaggrin (FLG) expression, cryopreserved xenograft skin sections were stained with rabbit anti-filagrin polyclonal antibody (Biolegend, 1:500-1:250) overnight at 4°C, followed by visualization of nuclei with a species-specific fluorescently conjugated secondary antibody and DAPI counterstaining. Filaggrin expression (fluorescence intensity) was measured at 10x magnification using a fluorescence microscope in one to three randomly selected epidermal regions from three to six cryosections for each xenograft. Regions of interest (ROIs) containing the human epidermal stratum corneum were manually selected on each image, and the mean FLG expression value within the ROI was calculated using Image J software. The mean filaggrin expression level was calculated for each xenograft.

[0129] Infiltration of T cells, NK cells, and NKT cells was assessed by analyzing the expression of CD3 (a T cell marker) and CD56 (an NK cell marker). Cryopreserved xenograft skin sections were double-stained with mouse anti-human CD56 antibody (Dako, 1:100, overnight at 4°C) followed by tyramide signal amplification (TSA, capable of detecting low-abundance targets) and mouse anti-CD3 monoclonal antibody preconjugated with Alexa Fluor 647 (Invitrogen, 1:50, 1 hour at room temperature). The numbers of DAPI+, CD3+, CD56+, and CD3+ or CD56+ positive cells were calculated in three to five randomly selected 100 μm2 dermal areas in one cryosection for each xenograft skin. The average values were calculated for each skin sample and expressed as the percentage of DAPI+ cells / area.

[0130] Epidermal proliferation was analyzed by observing the expression of the Ki67 proliferation marker. FFPE xenograft skin sections were deparaffinized and rehydrated. After heat-induced antigen retrieval in sodium citrate buffer and blocking with normal goat serum, sections were stained overnight at 4°C with mouse anti-Ki-67 antibody (M7240, Dako, 1:10), followed by a fluorescently conjugated goat anti-mouse secondary antibody. Sections were briefly treated with DAPI to visualize nuclei. For each xenograft skin, two sections were photographed under a fluorescence microscope at 20x magnification. The innermost layer of the epidermis was selected as a region of interest (ROI), and Ki-67+ cells were counted within the ROI. To determine the total number of cells, DAPI-stained nuclei were counted using a binarized automated method coded into ImageJ image analysis software.

[0131] IL-4 expression was analyzed in FFPE xenograft skin sections that were first deparaffinized and rehydrated. After heat-induced antigen retrieval in citrate buffer and blocking with normal goat serum, sections were stained overnight at 4°C with a rabbit anti-IL-4 antibody (Ab9622, Abcam, 1:100) followed by a species-specific fluorescently conjugated goat secondary antibody. IL-4+ cells were determined with streptavidin-HRP, which targets biotin and catalyzes DAB precipitation. Sections were counterstained with hematoxylin. For each xenograft skin, two sections were photographed under a fluorescent microscope at 20x magnification. Dermal IL-4+ cells were counted, and dermal surface area was calculated using ImageJ imaging software to determine the percentage of IL-4+ cells per mm2. 2 The number of IL-4+ cells per 1000 cells was obtained.

[0132] IL-17A expression was analyzed by staining cryopreserved xenograft skin sections with rabbit anti-human IL-17A antibody (Ab79056, Abcam, 1:200), followed by species-specific fluorescently conjugated secondary antibodies and DAPI counterstaining overnight at 4°C to visualize nuclei. For each xenograft skin, two to three sections were photographed under a fluorescent microscope at 20x magnification. Dermal IL-17A+ cells were counted, and dermal surface area was calculated using ImageJ imaging software to calculate the area per mm. 2 The number of IL-17A+ cells per 1000 cells was obtained.

[0133] IL-22 expression was analyzed by staining cryopreserved xenograft skin sections with rabbit anti-human IL-22 antibody (BS-2623R, ThermoFisher, 1:200), followed by species-specific fluorescently conjugated secondary antibody and DAPI counterstain overnight at 4°C to visualize nuclei. For each xenograft skin, two to three sections were photographed under a fluorescent microscope at 20x magnification. Dermal IL-22+ cells were counted, and dermal surface area was calculated using ImageJ imaging software to calculate the dermal surface area per mm. 2 The number of IL-22+ cells per 1000 cells was obtained.

[0134] IFN-γ expression was analyzed by staining overnight at 4°C with rabbit anti-human IFN-γ antibody (Ab9657, Abcam, 1:100), followed by species-specific fluorescently conjugated secondary antibody and DAPI counterstaining to visualize nuclei. For each xenograft skin, two to three sections were photographed under a fluorescent microscope at 20x magnification. Dermal IFN-γ cells were counted, and the dermal surface area was calculated using ImageJ imaging software to calculate the area per mm. 2 The number of IFN-γ+ cells per 1000 cells was obtained.

[0135] Gene expression analysis in humanized models RNA extraction was performed from OCT-embedded xenografts. OCT-embedded xenografts from 55 mice were thawed on ice and washed twice with PBS. Total RNA was extracted using a Precellys® Evolution Homogenizer and TRI Reagent® (Sigma-Aldrich). Briefly, each xenograft was homogenized at 6800 rpm for six 10-second cycles and cooled on ice for 30 seconds. After this, 1 mL of TRI Reagent® and chloroform (250 μL) were added, the sample was mixed, and centrifuged at 12,500 × g for 15 minutes. The supernatant (approximately 500 μL) containing total RNA was mixed with an equal volume of isopropanol to precipitate the RNA. Total RNA was pelleted by centrifugation at 12,500 × g for 15 minutes, washed twice with 70% ethanol, centrifuged at 8,000 × g for 7 minutes, and suspended in 20 μL of RNase-free water. RNA was stored at −80°C until analysis.

[0136] The quality control of the extracted RNA was measured using a Tapestation Bioanalyzer and Qubit at the Nanostring Core facility in Heidelberg, Germany.

[0137] The Nanostring nCounter® analysis system is based on the digital detection of individual target molecules directly labeled with molecular barcoding. Each probe consists of a color-coded reporter and a capture probe, both of which are covalently linked to a target-specific sequence (50 nucleotides long). The color-coded reporter allows for target specificity, while the biotin-conjugated capture probe allows for target immobilization on the surface. Upon application of a voltage and creating an electric field, the target molecules align, exposing the reporter probe in its fully extended state and allowing for individual molecular recognition. Nanostring multiplexing was entrusted to the nCounter® Core Facility Heidelberg, Department of Human Molecular Genetics, Heidelberg University Hospital. Instrumentation: nCounter® SPRINT.

[0138] This study used the nCounte® Human Immunology V2 Panel, which quantifies 594 genes, including housekeeping genes. Purified RNA from each sample was hybridized overnight at 65°C using the Human Immunology V2 Profiling panel (NanoString Technologies, WA, USA). Purification of the hybridized probes and their binding to optical cartridges were performed using the nCounter® Prep Station, and the cartridges were scanned using an nCounter Digital Analyzer. The RCC files obtained from the NanoString Digital Analyzer were imported into nSolver™ 4.0.70 software (NanoString Technologies, WA, USA), and the data quality was checked using the default quality check settings. The "barcode" was used to determine mRNA levels. Background correction was then performed by subtracting the "mean + 2 standard deviations" value of the negative control from the raw counts, and the adjusted raw counts were normalized to the geometric mean of the 12 determined housekeeping genes. Bioinformatics and statistical analyses were performed using nSolver™ Analysis Software version 4.0.70 and the Human Immunology V2 Profiling Advanced Analysis module, including hierarchical clustering and volcano plots. Because statistical significance could not be detected at adjusted p values ≤ 0.05, data were analyzed using raw p values ≤ 0.05. Because the Human Immunology V2 Panel exhibits high cross-reactivity with mouse genes (≤ 85%), analyses were performed including all genes in the panel and excluding mouse genes with cross-reactivity > 85%. This was in accordance with information from the NanoString facility that cross-reactivity < 85% of all genes does not interfere with analysis. Genes associated with atopic dermatitis were selected to align the results of this study with the current state of AD gene profiling.

[0139] Ex vivo human skin explant stimulation with a Th2-polarizing cocktail Full-thickness human skin biopsies are achieved by punch biopsy of residual surgical tissue. Biopsies are taken at 3 mm 2 Biopsies are harvested at a size of 1 / 4" and transferred onto mesh transwells in a 12-well plate. The dermis is submerged in a specially enriched medium, while the epidermis is left exposed to air. Biopsies are cultured under optimal conditions for up to 24 hours, followed by an additional 24 hours in the presence of a Th2-polarizing inflammatory cocktail and the test substance.

[0140] Example 3: Analysis of the IL-15 pathway in samples from patients with atopic dermatitis Expression of IL-15 and related genes ("IL-15 pathway") will be analyzed in a diverse collection of well-characterized samples or databases from atopic dermatitis patients and comparison pairs; some samples will be collected from patients receiving dupilumab to estimate potential correlations with clinical outcomes of treatment. The purpose of these analyses is to further characterize the IL-15 pathway in AD and, potentially, identify patient subsets that may preferentially benefit from treatment with IL-15 inhibitors. Analyses using state-of-the-art techniques include: -Transcriptome analysis from public databases -qPCR analysis of 30 healthy and AD skin biopsies -Proteomic analysis of 10 healthy and AD skin biopsy samples (48 target genes) -Immunohistochemical analysis of 20 samples of healthy and AD skin biopsies -Single-cell RNA sequencing of 10 samples of healthy and AD skin biopsies -Measurement of IL-15 levels in 30 AD and healthy sera using ultrasensitive technology.

[0141] For data analysis from public databases, we used the microarray dataset GSE130588 downloaded from the Gene Expression Omnibus (GEO) public database, which contains data from human atopic dermatitis skin and healthy skin samples as described in Guttman-Yassky et al. 2019, J. Allergy Clin. Immunol., 143, 155-172.

[0142] To evaluate the efficacy and safety of dupilumab compared with placebo in patients with moderate to severe atopic dermatitis, biopsies were collected from lesional and non-lesional skin in five medical centers in the United States and Canada (ClinicalTrials.gov: NCT01979016). Patients received a 400 mg dose of dupilumab or placebo on day 1, followed by weekly subcutaneous injections of 200 mg of dupilumab or placebo for a total of 16 weeks. RNA was extracted and hybridized to Affymetrix Human U133Plus 2.0 gene arrays (Affymetrix, Santa Clara, CA). The original CEL format files were converted to expression profile format using the open-source package Affy, available from the R project website (https: / / cran.r-project.org). Data were normalized using the median method. Normalized gene expression values were calculated to compare IL-15 expression between patient groups.

[0143] Consistent with previous reports (Karlen and Simon, 2020, Int. Arch. Allergy Immunol., 181, 417-421), we observed significantly higher IL-15 expression in both non-lesional (NL) and lesional (L) skin of patients with atopic dermatitis (Figure 10). IL-15 expression was also significantly higher in lesional versus non-lesional atopic dermatitis skin. Dupilumab treatment slightly reduced IL-15 expression in lesional atopic dermatitis skin, but the effect was not significant. Indeed, even at week 16 (W16) of dupilumab treatment, IL-15 expression remained significantly higher in both lesional and non-lesional atopic dermatitis skin compared with healthy skin.

[0144] These results suggest that treatment with the anti-IL-4R antibody dupilumab does not sufficiently inhibit the IL-15 pathway in patients, as previously observed in the mouse humanized model (Figure 9), and thus further support that anti-IL-15 therapy may be advantageously administered to patients who do not respond to anti-IL-4R antibodies or in combination with such or other treatments.

[0145] Example 4: Generalization of the effect of anti-IL15 antibody hu-BE29-2 to other antibodies sharing the same binding epitope The aforementioned effects can be reasonably generalized to other antibodies sharing the same binding epitope as described in WO 2016 / 001275, in particular hu-BE29-10 and 24.

[0146] The epitope specificity of these anti-human IL15 antibodies, which is the same as that of hu-BE29-2, has been confirmed as described below.

[0147] Libraries of peptides containing IL-15 single-loop, double-loop, triple-loop, sheet-like fold, helical fold, and combinations thereof were synthesized using Pepscan's proprietary Chemically Linked Peptides on Scaffolds (CLIPS) technology (Timmerman 2007, Journal of Molecular Recognition, https: / / doi.org / 10.1002 / jmr.846). CLIPS templates were linked to the side chain thiol groups of cysteine residues to prepare peptide arrays. Binding of the antibodies hu-BE29-2, hu-BE29-10, and hu-BE29-24 to each of the synthetic peptides was examined by ELISA, and color development was quantified using a charge-coupled device (CCD) camera and image processing system. The results showed that antibodies hu-BE29-2, hu-BE29-10, and hu-BE29-24 all bind to an epitope of IL-15 with a sequence consisting of amino acids 83 to 93 of SEQ ID NO:1.

[0148] Sequence Listing Human mature IL-15 (human), GenBank: CAA71044.1 SEQ ID NO: 1 [ka] BNZ132 (also known as BNZ-1, BNZ-13, YT-033, and EQ-101), a cytokine modulator peptide from WO 2015 / 089217 SEQ ID NO: 2 IKEFLQRFIHIVQSIINTS Gamma-cytokine modulators from U.S. Patent Application Publication No. 2019 / 0070263 SEQ ID NO: 3 PKEFLERFVHLVQMFIHQSLS IL-15 inhibitor peptide U.S. Patent No. 7,736,638B2 SEQ ID NO: 4 SEQ ID NO:4 KVTAMKCFLL huVH1 from International Publication No. 2016 / 001275 SEQ ID NO:5 [ka] huVH2 from International Publication No. 2016 / 001275 SEQ ID NO:6 [ka] huVH8 from International Publication No. 2016 / 001275 SEQ ID NO:7 [ka] huVH18 from International Publication No. 2016 / 001275 SEQ ID NO:8 [ka] huVL1 from International Publication No. 2016 / 001275 SEQ ID NO:9 DVVMTQSPLSLPVTLGQPASISCRSSQSIVDITGNTYLEWYQQRPGQSPRLLIYKVFNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQDSFVPYTFGQGTKLEIK 146B7, also known as AMG 714 (WO 2005 / 044303) SEQ ID NO: 10 [ka] Light chain from 146B7, also known as AMG 714 (WO 2005 / 044303) SEQ ID NO: 11 [ka] 70a Variable heavy chain region from WO 2018 / 119246 SEQ ID NO: 12 [ka] 70a Variable light chain region from WO 2018 / 119246 SEQ ID NO: 13 [ka] huABC2 variable heavy chain region from WO 2011 / 127324 SEQ ID NO: 14 [ka] huABC2 variable light chain region from WO 2011 / 127324 SEQ ID NO: 15 [ka] Heavy chain from huBE29-2 (WO 2016 / 001275) SEQ ID NO: 16 [ka] Light chain from huBE29-2 (WO 2016 / 001275) SEQ ID NO: 17 [ka] Heavy chain from huBE29-10 (WO 2016 / 001275) SEQ ID NO: 18 [ka] Heavy chain from huBE29-24 (WO 2016 / 001275) SEQ ID NO: 19 [ka]

Claims

1. An interleukin-15 inhibitor for use in the treatment of atopic dermatitis.

2. The interleukin-15 inhibitor for use according to claim 1, wherein said interleukin-15 inhibitor is a small molecule.

3. The interleukin-15 inhibitor has the formula (I): 【Chemical 1】 wherein R is an optionally substituted phenyl and n is an integer selected from 1 to 10.

3. The interleukin-15 inhibitor for use according to claim 2, which is of the formula:

4. The interleukin-15 inhibitor for use according to claim 1, wherein said interleukin-15 inhibitor is a peptide.

5. The interleukin-15 inhibitor for use according to claim 4, wherein the interleukin-15 inhibitor is a peptide comprising a sequence selected from SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4, or a fragment or variant thereof.

6. The interleukin-15 inhibitor for use according to claim 1, wherein the interleukin-15 inhibitor is an anti-IL-15 antibody.

7. The interleukin-15 inhibitor is (1) A heavy chain variable region of SEQ ID NO: 5, or any variant thereof, wherein the variant has the amino acid sequence of SEQ ID NO: 5, except that one, two, three, or four amino acids are substituted with different amino acids, and the substitutions are any of the following: (iv) within said heavy chain variable framework region, an arginine (R) at position H3 (VH RH3) substituted with glutamine (Q), a methionine (M) at position H5 (VH MH5) substituted with valine (V), an alanine (A) at position H6 (VH AH6) substituted with glutamic acid (E), and an alanine (A) at position H49 (VH AH49) substituted with serine (S); (v) within the heavy chain CDR2, an aspartic acid (D) at position H61 (VH DH61) substituted with a glutamic acid (E), a serine (S) at position H62 (VH SH62) substituted with a threonine (T), and (vi) within the heavy chain CDR3, a methionine (M) at position H98 (VH MH98) substituted with leucine (L), phenylalanine (F), isoleucine (I), or alanine (A); a tryptophan (W) at position H100C (VH WH100C) substituted with tyrosine (Y), phenylalanine (F), or alanine (A); a methionine (M) at position H100E (VH MH100E) substituted with leucine (L), phenylalanine (F), or isoleucine (I); a heavy chain variable region of SEQ ID NO: 5 or any variant thereof selected from: (2) a light chain variable region of SEQ ID NO: 9; The interleukin-15 inhibitor for use according to any one of claims 1 to 6, which is an isolated antibody or antigen-binding fragment thereof that binds to IL-15, comprising:

8. The interleukin-15 inhibitor is an isolated antibody or antigen-binding fragment thereof that binds to IL-15, comprising: (1) a heavy chain variable region of SEQ ID NO: 6 or any variant thereof, wherein the variant has the amino acid sequence of SEQ ID NO: 6 except for one, two, three, or four amino acids being replaced with different amino acids; (2) a light chain variable region of SEQ ID NO: 9; 10. An interleukin-15 inhibitor for use according to claim 1 or 6, comprising:

9. The interleukin-15 inhibitor is (3) In the sequence of SEQ ID NO: 5, (ii) VH RH3 is substituted with glutamine (Q), VH MH5 is substituted with valine (V), and VH AH6 is substituted with glutamic acid (E); (iv) VH SH62 is substituted with threonine (T); and (v) VH WH100C is substituted with tyrosine (Y); a heavy chain variable region of the amino acid sequence of SEQ ID NO: 5 having a substitution selected from: (4) a light chain variable region of the amino acid sequence of SEQ ID NO: 9; 8. The interleukin-15 inhibitor for use according to claim 7, which is an isolated antibody or antigen-binding fragment thereof that binds to IL-15, comprising:

10. The interleukin-15 inhibitor is (1) a heavy chain variable region selected from SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8; (2) a light chain variable region of SEQ ID NO: 9; 10. The interleukin-15 inhibitor for use according to claim 1 or 6, which is an isolated antibody or antigen-binding fragment thereof that binds to IL-15, comprising:

11. The interleukin-15 inhibitor is a heavy chain of SEQ ID NO: 10 and a light chain of SEQ ID NO: 11, or a variant or fragment thereof; or - a variable heavy chain region of SEQ ID NO: 12 or a variant or fragment thereof, and a variable light chain region of SEQ ID NO: 13 or a variant or fragment thereof; or - a variable heavy chain region of SEQ ID NO: 14 or a variant or fragment thereof, and a variable light chain region of SEQ ID NO: 15 or a variant or fragment thereof; or a heavy chain of SEQ ID NO: 16 and a light chain of SEQ ID NO: 17 or a variant or fragment thereof; or a heavy chain of SEQ ID NO: 18 and a light chain of SEQ ID NO: 17 or a variant or fragment thereof; or - a heavy chain of SEQ ID NO: 19 and a light chain of SEQ ID NO: 17 or a variant or fragment thereof 10. The interleukin-15 inhibitor for use according to claim 1 or 6, selected from anti-IL-15 antibodies comprising:

12. A pharmaceutical composition comprising an interleukin-15 inhibitor, a drug useful for the prevention and / or treatment of atopic dermatitis, and a pharmaceutically acceptable carrier, diluent, or excipient thereof.

13. 13. The pharmaceutical composition of claim 12, further comprising a co-agent useful in the prevention and / or treatment of atopic dermatitis, such as at least one anti-IL-4R or at least one corticosteroid or a mixture thereof.

14. 14. The pharmaceutical composition of claim 12 or 13, further comprising an anti-IL-4R such as dupilumab, an anti-IL-13 such as tralokinumab, a JAK inhibitor such as abrocitinib, upadacitinib or ruxolitinib.

15. 14. The pharmaceutical composition of claim 12 or 13, further comprising at least one corticosteroid, such as dexamethasone.

16. A method for preventing and / or treating atopic dermatitis in a subject, said method comprising administering to a subject in need thereof a therapeutically effective amount of an interleukin-15 inhibitor or a pharmaceutical composition thereof.

17. The method according to claim 15, wherein the interleukin-15 inhibitor is at least one of those described in any one of claims 1 to 11.

18. The method according to claim 15 or 16, wherein the interleukin-15 inhibitor is administered in the form of a formulation according to any one of claims 12 to 15.

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