IL-15 inhibitors for treatment of atopic dermatitis

Abstract

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

Description

[0001] This application is a divisional application of PCT application PCT / EP2023 / 071573, filed on 03 August 2023, entitled “IL-15 inhibitors for the treatment of atopic dermatitis”, which entered the Chinese national phase on 24 January 2025, and has the application number 202380056686.3. TECHNICAL FIELD

[0002] The present invention relates to the use of an agent capable of neutralizing the activity of interleukin-15 in the treatment of atopic dermatitis. BACKGROUND

[0003] Atopic dermatitis (AD), also known as eczema, is a chronic, predominantly inflammatory skin disease. Atopic dermatitis is characterized by dry skin, recurrent flares, and pruritus (itching).

[0004] The prevalence of AD varies widely worldwide 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-based survey found a prevalence of 2.1% to 4.9% for both previously diagnosed and active AD, and that the prevalence of AD in adults was similar to that in adolescents and remained 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 generally highest in high-income countries. It is also widely observed that AD shows a female majority, particularly in adolescence and adulthood (Silvenberg et al., 2020, supra).

[0005] This condition often starts in childhood and can persist throughout adulthood, with varying severity during these age periods. AD is a complex, multifactorial disease that involves:

[0006] i) Strong genetic component: AD can be considered a familial disease, and several susceptibility genes have been identified.

[0007] ii) Environmental exposures, including irritants and pruritogens, pathogens, climatic factors, ultraviolet radiation, outdoor and indoor air pollutants, tobacco smoke exposure, water hardness, urban vs. rural living, diet, breastfeeding, probiotics and prebiotics. Some of these factors can explain the increase in atopic dermatitis over the past decades.

[0008] iii) Changes in skin permeability. Mutations in the gene for filaggrin (FLG) occur in about 30% of people with atopic dermatitis, which increases the risk of early onset of atopic dermatitis and development of asthma. Filaggrin plays an important role in keeping the skin surface slightly acidic, thus endowing it with antimicrobial effects.

[0009] These different components are interrelated and act synergistically to drive a complex immunopathology involving multiple pathways, cytokines, and cell types, ultimately leading to the signs and symptoms of atopic dermatitis. There is evidence that several cytokines, such as the T helper 2 cytokine interleukin 4 (IL-4) and the closely related IL-13, are important components of the pathogenesis of AD. In fact, an antibody targeting the IL-4 receptor alpha subunit (IL-4Ra) shared between IL-4 and IL-13 (dupilumab) was the first biologic approved in Europe and the United States to treat atopic dermatitis alone or in combination with a corticosteroid (Strowd et al., 2017, The Lancet 389, 2265-2266. https: / / doi.org / 10.1016 / S0140-6736 (17)31192-3 ), and more recently, an antibody targeting IL-13 (tralokinumab) was also approved in Europe and the United States, alone or in combination with a corticosteroid (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). However, many patients do not respond or respond inadequately, leaving a large unmet medical need for effective and safe treatments in atopic dermatitis, and several products targeting other pathways than IL-4 / IL-13 are in development.

[0010] 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). But it has also been shown to be a regulator 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 shares many biological activities with interleukin 2 (IL-2), which is consistent with the shared receptor signaling components (IL-2 / 15Rβ and IL-2 / 15Rγc) they share. However, the specificity of IL-15 from IL-2 is provided by the unique private alpha chain receptor that completes the IL-15Rαβγ heterotrimeric high affinity receptor complex, allowing different responsiveness depending on the expressed ligand and high affinity receptor (Fehniger and Caligiuri, 2001, Blood 97, 14-32).

[0011] Dysregulated IL-15 expression has been shown to have deleterious effects in autoimmune diseases such as rheumatoid arthritis, psoriasis, celiac disease, eosinophilic esophagitis, alopecia areata and vitiligo. This is based on the observation that compounds that neutralize IL-15 signaling can reduce clinical and disease characteristics 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; 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., 2018, Sci Transl Med 10, eaam7710, https: / / doi.org / 10.1126 / scitranslmed.aam7710 ), and in some cases, the above results were observed in clinical trials (Baslund et al., 2005, Arthritis Rheum., 52, 2686-2692, https: / / doi.org / 10.1002 / art.21249 ; et al., 2019, supra). On the other hand, increased IL-15 expression can not be detrimental. For example, in viral diseases, increased IL-15 expression is associated with an increased viral clearance rate (Verbist et al., 2011, J. Immunol. 186, 174-182. https: / / doi.org / 10.4049 / jimmunol.1002613).

[0012] Several reports have described 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 in the skin and / or blood of dogs with atopic dermatitis (Mazrier et al., 2022, Vet. Dermatol., 33, 131-e38. https: / / doi.org / 10.1111 / vde.13044).

[0013] In contrast, it has also been proposed that reduced human IL-15 expression can contribute to the pathogenesis of atopic dermatitis progression (Ong et al., 2002, J. Immunol. 168, 505-510). Furthermore, it has been reported that atopic dermatitis patients have a deficiency in blood natural killer (NK) cells, while atopic dermatitis patients have skin rich in activated NK cells (Mack et al., 2020, Sci. Transl. Med. 12, eaay1005. https: / / doi.org / 10.1126 / scitranslmed.aay1005 Given that IL-15 is an important factor for NK cell survival and activation, the authors proposed that providing IL-15 agonist molecules would have a beneficial effect on atopic dermatitis. To further support this hypothesis, they treated mice in a MC903 atopic dermatitis model (induced by topical application of a vitamin D3 analogue) with IL-15 agonist molecules and observed improvements in NK cell-dependent clinical scores and other atopic dermatitis characteristics (Mack et al., 2020, supra).

[0014] In summary, it cannot be determined from the prior art whether IL-15 overexpression is detrimental or beneficial in atopic dermatitis.

[0015] Atopic dermatitis is the most common chronic inflammatory skin disease worldwide, and treatment options remain limited. Therefore, given the enormous burden that AD places on affected subjects, there is a need for a better understanding of the underlying causes of AD and for new pharmaceutical agents that have a substantial beneficial effect in the management and treatment of AD. SUMMARY

[0016] The present invention relates primarily to the use of an agent capable of inhibiting interleukin-15 for the treatment of atopic dermatitis in an undefined global patient population, and in atopic dermatitis patients who are non-responsive to current treatments such as anti-IL-4R, anti-IL-4 or anti-IL-13 antibodies or class corticosteroids.

[0017] According to a first embodiment, there is provided an interleukin-15 inhibitor for use in the treatment of atopic dermatitis.

[0018] According to another embodiment, there is provided the use of an interleukin-15 inhibitor for the manufacture of a pharmaceutical composition for the treatment of atopic dermatitis.

[0019] According to another aspect of the present invention, there is provided a method of preventing and / or treating atopic dermatitis in a subject, the method comprising administering to a subject in need thereof a therapeutically effective amount of an interleukin-15 inhibitor or a pharmaceutical composition thereof.

[0020] According to another aspect of the present application, there is provided a pharmaceutical composition comprising an interleukin-15 inhibitor and an agent for treating atopic dermatitis and a pharmaceutically acceptable carrier, diluent or excipient thereof.

[0021] Other features and advantages of the present application will become apparent from the following detailed description, taken in conjunction with the accompanying drawings. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 : Increase in IL-15 (A) and IL-15Ra (B) gene expression in an ex vivo human atopic dermatitis model analyzed by RNAseq (relative units) in human skin explants cultured for 48 hours with control or Th2 polarizing medium as described in Example 1. Symbols represent individual explants and group mean values are represented as bar graphs + / - standard deviation.

[0023] Figure 2 : Induced IL-15 (A) and IL-15Ra (B) gene expression in an ex vivo human atopic dermatitis model analyzed by RNAseq (relative units) in human skin explants cultured for 48 hours with control or Th2 polarizing medium or Th2 polarizing medium alone or with anti-IL-4R antibody or the class corticosteroid betamethasone as described in Example 1. Symbols represent individual explants and group mean values are represented as bar graphs + / - standard deviation.

[0024] Figure 3 : Increase in skin IL-15 and IL-15Ra expression in a humanized atopic dermatitis model analyzed in human skin xenografts two weeks after injection of Th2 polarized PBMCs (Th2) or LPS-activated Th2 polarized PBMCs (activated Th2) as described in Example 2. In the epidermis, expression of IL-15 (A) and IL-15Ra (C) was quantified as mean intensity signal, while in the dermis, expression of IL-15 (B) was quantified as number of IL-15 positive cells per 100 dermal area units. Symbols represent individual xenografted mice (grafts / PBMCs from 2 separate human donors) and groups are represented as violin plots showing median and quartiles in groups.

[0025] Figure 4: Effect of anti-IL-15 antibody on clinical scores in human skin xenografts was measured and analyzed as described in Example 2 at the time of injection of LPS-activated Th2-polarized PBMCs (pre-treatment) and after 2 and 4 weeks of treatment with various antibodies. As indicated on the x-axis, the IgG1 group was injected with 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 2 weeks of control isotype antibody followed by 2 weeks of anti-IL-15 antibody. Symbols represent individual xenograft mice, and groups are represented as violin plots showing the median and quartiles of the groups.

[0026] Figure 5 : Effect of anti-IL-15 antibody on epidermal properties in a humanized atopic dermatitis model. A: Epidermal thickness of human skin xenografts was analyzed in various antibody treatment groups after 4 weeks of injection of LPS-activated Th2-polarized PBMCs. As indicated on the x-axis, the IgG1 group was injected with 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 2 weeks of control isotype antibody followed by 2 weeks of anti-IL-15 antibody. Symbols represent individual xenograft mice, and groups are represented as violin plots showing the median and quartiles of the groups. B: Epidermal proliferation of human skin xenografts was analyzed by measuring the expression of Ki67 in the basal layer of the human epidermis in various antibody treatment groups after 4 weeks of injection of LPS-activated Th2-polarized PBMCs. As indicated on the x-axis, the IgG group was injected with control isotype antibody for 4 weeks (IgG1 and IgG4 groups were combined due to inter-animal variability and small 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 2 weeks of control isotype antibody followed by 2 weeks of anti-IL-15 antibody. Symbols represent individual xenograft mice, and groups are represented as violin plots showing the median and quartiles of the groups; C: Epidermal filaggrin (FLG) was analyzed by immunofluorescence in human skin xenografts after 4 weeks of injection of LPS-activated Th2-polarized PBMCs. As indicated on the x-axis, the IgG group was injected with control isotype antibody for 4 weeks (IgG1 and IgG4 groups were combined due to inter-animal variability and small 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 2 weeks of control isotype antibody followed by 2 weeks of anti-IL-15 antibody. Symbols represent individual xenograft mice, and groups are represented as violin plots showing the median and quartiles of the groups.

[0027] Figure 6Anti-IL-15 antibody did not affect the number of dermal T (A: CD3+), NK (B: CD56+), and NKT (C: CD3+CD56+) cells in the humanized atopic dermatitis model when analyzed by immunofluorescence in various antibody treatment groups 4 weeks after injection of LPS-activated Th2-polarized PBMCs. As indicated on the x-axis, the IgG group was injected with control isotype antibody for 4 weeks (IgG1 and IgG4 groups were combined due to inter-animal variability and small 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 2 weeks of control isotype antibody followed by 2 weeks of anti-IL-15 antibody. Symbols represent individual xenograft mice, and groups are represented as violin plots showing the median and quartiles of the groups.

[0028] Figure 7 Anti-IL-15 induced a reduction in atopic dermatitis-associated cytokine expression in the humanized atopic dermatitis model when measured and analyzed in the dermis of human skin xenografts in various antibody treatment groups 4 weeks after injection of Th2-polarized PBMCs. A: IFN-g; B: IL-4; C: IL-17A; D: IL-22). The number of cells expressing IL-4, IL-17A, or IL-22 was calculated by adding the number of cells expressing each cytokine in each xenograft (E). As indicated on the x-axis, the IgG group was injected with control isotype antibody for 4 weeks (IgG1 and IgG4 groups were combined due to inter-animal variability and small 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 2 weeks of control isotype antibody followed by 2 weeks of anti-IL-15 antibody. Symbols represent individual xenograft mice, and groups are represented as violin plots showing the median and quartiles of the groups.

[0029] Figure 8 Heatmap showing Log2 FC of differentially regulated genes clustered by immune cell type (T helper cells, T cells, B cells, left) or atopic dermatitis-associated genes (right) in the humanized atopic dermatitis mouse model after therapeutic (IgG-CALY) or prophylactic (CALY) treatment with anti-IL-15 antibody compared to control isotype alone. The lighter the shade, the higher the fold difference. The only upregulated gene was IL32, and all other genes remained unchanged or downregulated. Asterisks indicate p-values for significant regulation (<0.05) (non-parametric Mann-Whitney test). All data are based on Nanostring transcriptomic analysis (10 xenografts from 3 individual human donors per group).

[0030] Figure 9: Anti-IL-4R antibody and dexamethasone have different effects on IL-15 expression between epidermis and dermis in a humanized atopic dermatitis model when analyzed in human skin xenografts two weeks after injection of LPS-activated Th2-polarized PBMCs in various treatments. As indicated on the x-axis, the IgG group was injected with control isotype antibody for 4 weeks (IgG1 and IgG4 groups were pooled due to inter-animal variability and small n per group), the dexamethasone group was injected with topical dexamethasone for the last 2 weeks of the study, the anti-IL-4R group was injected with anti-IL-4R antibody, and the IgG + anti-IL-4R group was injected with 2 weeks of control isotype antibody followed by 2 weeks of anti-IL-4R antibody. In the epidermis, IL-15 expression was quantified as mean intensity signal, while 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 groups are represented as violin plots showing the median and quartiles of the group.

[0031] Figure 10 : Normalized gene expression values of IL-15 expression in skin biopsy samples from healthy volunteers and atopic dermatitis patients groups at baseline (W0) or after 4 (W4) or 16 weeks of dupilumab treatment as described in Example 3. NL: non-lesional skin; L: lesional skin. Each symbol represents one individual, bars represent the mean value of each group, and error bars represent the 95% confidence interval.

[0032] Statistical analysis was performed using Graph Pad Prism software. Figure 1 , 3 , 9: Comparison between two experimental groups using the non-parametric Mann-Whitney test. Figure 1 : **** p<0.0001; 4: ** p<0.01, *** p<0.001; 9: ** p<0.01, *** p<0.001. Figure 2 , 7 : Comparison of the two groups using anti-IL-4R antibody or betamethasone with the case of Th2-polarizing medium alone using one-way ANOVA Kruskal-Wallis test Figure 2 : * p<0.05; Figure 7 : * p<0.05, ** p<0.01. Figure 4 , 5 , 6: Comparison of experimental groups using one-way ANOVA test combined 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 one-way ANOVA test combined with Sidak's multiple comparison test (*p<.05; **p<.01; ***p<.001; ****p<.0001, ns: not significant). DETAILED DESCRIPTION

[0033] The terms "interleukin 15", "interleukin-15", "IL-15" refer herein to the interleukin 15 protein, also known as MGC9721, which is a 14 to 15 kDa proinflammatory cytokine encoded in humans by the IL-15 gene, the sequence of which is disclosed in Human Gene Nomenclature Committee (Hugo Gene Nomenclature Committee) ID 5977. The immature form of IL-15 comprises 162 amino acids, of which the first 29 amino acids constitute a signal peptide, and amino acids 30 to 48 constitute a 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, with the indicated positions corresponding to the amino acid positions on 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. Amino acid sequences of immature IL-15 from other species are available in the art and include, 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 accession numbers 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 dog IL-15 (UniProtKB accession number A0A8C0NA66_CANLF, corresponding to the complete IL-15 sequence). The term "interleukin 15" also includes any variant or isoform of interleukin 15 naturally expressed by cells. Notably, two alternatively spliced transcript variants of IL-15 have been reported. Although the two isoforms produce the same mature protein, they differ in their cellular trafficking.

[0034] 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 the inhibition of the binding of IL-15 to its signaling chains (IL-15Rβ and / or IL-15Rγ) or to 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 the functional level, for example, by testing the inhibitory effect by measuring: i) the inhibition of signaling events downstream of IL-15, 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) the inhibition of IL-15-dependent cell survival or activation or cytokine secretion or expression of certain genes in cell assays including cell lines or primary cells from animals, healthy humans or patients; iii) the inhibition of IL-15-dependent mechanisms in vivo, such as NK and other leukocyte homeostasis that can be measured in the blood of animals, healthy humans or patients by flow cytometry. Among these agents, some interleukin 15 inhibitors have been developed or are being actively developed for the treatment of different diseases, such as described in WO 2016 / 001275, WO 2015 / 089217, WO 2005 / 044303, WO 2018 / 119246, WO 2011 / 127324.

[0035] The interleukin 15 inhibitors can be small molecule inhibitors (e.g., low molecular weight organic compounds of < 900 Da), peptides, monoclonal antibodies (mAbs), chimeric or fusion proteins, aptamers (including peptide aptamers, DNA and RNA aptamers), soluble receptors, and these agents can also act by silencing or downregulating the expression of interleukin 15.

[0036] The term "antibody" as referred to herein refers to a polypeptide that binds an antigen. This includes intact antibodies and any antigen binding fragment. The term "antibody" is used in the broadest sense and includes monoclonal antibodies, polyclonal antibodies, human antibodies, humanized antibodies, chimeric antibodies, bispecific antibodies, multispecific antibodies, and further engineered antibodies, as long as the characteristic properties of the present application, in particular the ability to bind the target antigen (i.e. IL-15) are retained. Examples of antibodies are described herein. Bispecific or multispecific antibodies can be designed as described in Sawant et al., 2020, Int. J. Mol. Sci. 2020, 21, 7496. https: / / doi.org / 10.3390 / ijms21207496. The term "monoclonal antibody" as used herein 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 naturally occurring mutations that can be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. The modifier "monoclonal" indicates the character of the antibody as being obtained from a population of substantially homogeneous antibodies, and is not to be construed as requiring production of the antibody by any particular method.

[0037] The term "chimeric antibody" generally refers to an antibody, typically prepared by recombinant DNA techniques, that contains a variable region from one source or species and at least a portion of a constant region derived from a different source or species. A typical example of a chimeric antibody includes an antibody that contains a mouse variable region and a human constant region. As defined herein, this term also includes an antibody that contains at least one CDR of a first human antibody and at least a portion of a constant region of a second human antibody. It also includes an antibody that contains heavy chain CDR1, CDR2, and CDR3 of a first human antibody and light chain CDR1, CDR2, and CDR3 of a second human antibody.

[0038] The term "humanized antibody" refers to an antibody from a non-human species whose complementarity determining regions (CDRs) have been replaced with human CDRs. The humanized antibody can optionally further comprise one or more framework residues derived from the non-human species from which the CDRs are derived.

[0039] The term "human antibody" or "fully human antibody" refers to an antibody in which the variable regions and the constant regions of both the heavy and light chains are of human origin, or are substantially identical to sequences of human origin, but not necessarily from the same antibody.

[0040] The term "isolated antibody" refers to an antibody that has been removed 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 including electrophoretic (e.g., SDS-PAGE, isoelectric focusing, capillary electrophoresis) or chromatographic (e.g., ion exchange or reverse phase HPLC) methods (see, e.g., Flatman et al., 2007, J Chromatogr B Analyt Technol Biomed Life Sci, 848:79-87).

[0041] The term "variant" can apply to polynucleotides and / or polypeptides. For example, as referred to herein, a variant of a peptide or polypeptide refers to a peptide or polypeptide that is substantially homologous to a reference peptide sequence, but whose amino acid sequence differs from the amino acid sequence of the reference sequence due to one or more amino acid deletions, insertions and / or substitutions. Substantially homologous refers to a variant amino acid sequence that is identical to the reference peptide sequence except for a few amino acid (e.g., 1, 2, 3, 4, 5, or 6 amino acids) deletions, insertions and / or substitutions. 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 have 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% identity to a reference nucleic acid sequence. Identity of two amino acid sequences or two nucleic acid sequences can be determined by visual inspection and / or mathematical calculation, or more easily by comparing sequence information using known computer programs for sequence comparison (e.g., the Clustal software package version 1.83). A variant can comprise a sequence with at least one conservatively substituted amino acid, which means that a given amino acid residue is replaced with a residue having a similar physicochemical property. Examples of conservative substitutions include the replacement of one aliphatic residue by another, e.g., Ile, Val, Leu, or Ala for each other, or the replacement of one polar residue by another, e.g., between Lys and Arg; Glu and Asp; or Gin and Asn. Other such conservative substitutions (e.g., replacements within a whole region having similar hydrophobic 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 non-native residue for a native amino acid residue, resulting in 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 is not conservative, which can result in a variant having modified properties compared to the reference antibody. The desired amino acid substitution(s), whether conservative or non-conservative, can be determined by one of skill in the art when such substitution is desired. The term "variant" also includes a peptide or polypeptide that is substantially homologous to a reference peptide sequence, but whose amino acid sequence differs from the amino acid sequence of the reference sequence because one or more amino acids have been chemically modified or replaced with an amino acid analog. The term also includes glycosylated polypeptides.

[0042] As used herein, the term "bind" or "binding" of an inhibitor to a target antigen refers to at least a transient interaction or association between the inhibitor and the target antigen (e.g., IL-15) or with a fragment of the target antigen containing an epitope recognized by the inhibitor. As used herein, antibodies that bind to IL-15 are also referred to as anti-IL-15 antibodies.

[0043] When applied to antibodies, the terms "selective binding," "specific binding," and "specific to" indicate that the antibody preferentially recognizes and / or binds to a target polypeptide or epitope, i.e., has a higher affinity than with any other antigen or epitope, meaning that binding to the target polypeptide can be distinguished from nonspecific 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 (SPR), or spectroscopy (e.g., using fluorescence assays). In particular, when using surface plasmon resonance (SPR) techniques, biomolecular binding events cause a change in the refractive index at a surface layer immobilized with a binding chaperone, which is detected as a change in the SPR signal expressed in response units (RU). By measuring the real-time binding kinetics of the antibody to its target antigen, SPR techniques can determine the association rate between the antibody and its target (in kJ / kb). a or k on (Association constant measurement), and its association strength (in kJ / kb). d or k off (Dissociation constant measurement). The affinity of an antibody for its target can be determined by determining its equilibrium dissociation constant K. D To measure quantitatively, K D Defined as K D =k d / k a , where k a It is the association rate (k on ), and k d It is the dissociation rate (k off (Murphy et al., 2006, Curr Protoc Protein Sci, [The Latest Guide to Protein Science Labs], Chapter 19: Unit 19.14). Comparisons of affinity and / or binding properties between two antibodies can be made without practically determining the Kelvin of each antibody. D It is established based on the value, but rather on K. D The affinity is established by quantitative measurements of proportional binding (e.g., by ELISA or FACS analysis) or qualitative measurements of affinity or inferences of affinity (e.g., in functional assays or in vitro or in vivo assays).

[0044] The term "blocking" or "neutralizing" activity of an inhibitor refers to its ability to inhibit the activity of its target. Neutralizing activity of an inhibitor can be determined by in vitro or in vivo assays or functional assays. When applied to an inhibitor that binds IL-15, the term refers to the ability of the inhibitor to substantially neutralize the activity of IL-15, which can correspond to, for example, inhibition of 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 the heterotrimeric IL-15Raβγ or heterodimeric IL-15Rβγ receptor (Finch et al., 2011, Br J Pharmacol. 162:480-90), inhibition of IL-15-induced synthesis of immunoglobulins by B lymphocytes upon anti-IgM or CD40 ligand stimulation (Litinskiy et al., 2012, Nat Immunol. 3:822-9), inhibition of IL-15-induced activation of human neutrophils (Rathhe and Girard, 2004, J Leukoc Biol. 76:162-8), and inhibition of IL-15-induced production of proinflammatory cytokines by macrophages, dendritic cells, or epithelial cells (Nanayakkara et al., 2013, Am J Clin Nutr. 98:1123-35).

[0045] Unless otherwise limited by the definition of the individual substituent, the term "substituted" means that the group is substituted with 1 to 5 substituents selected from the group consisting of "Ci-C6alkyl", "C3-C8cycloalkyl", "heterocycloalkyl", "sulfonyl", "sulfonamide", "alkoxy", "alkoxycarbonyl", "halogen", "carboxyl", "halomethyl", cyano, hydroxyl, nitro, and the like.

[0046] The term "pharmaceutically acceptable" refers to a carrier that is composed of biologically or otherwise

[0047] The term "carrier" refers to any component present in the pharmaceutical formulation other than the active agent, and thus includes diluents, binders, lubricants, disintegrants, fillers, colorants, wetting or emulsifying agents, pH buffering agents, preservatives, and the like.

[0048] As used herein, terms such as “treatment” and “treating” generally refer to achieving the desired pharmacological and physiological effects. This effect may be preventative in the prevention or partial prevention of a disease, its symptoms, or condition, and / or therapeutic in the partial or complete cure of a disease, condition, symptoms, or adverse reactions attributable to the disease. The term “treatment” as used herein encompasses any treatment of diseases in mammals, particularly humans, and includes: (a) preventing the development of a disease in a subject who may be predisposed to it based on family history but has not yet been diagnosed with it; (b) suppressing a disease, i.e., halting its development; or (c) alleviating a disease, even if the disease and / or its symptoms or condition subside, such as by improving or repairing damage. For example, treatment of celiac disease includes preventing, reducing, or even eliminating symptoms of the disease or disorder, such as partially or completely relieving symptoms of atopic dermatitis, such as dry skin, itching, scratching, and the consequences of scratching (skin stinging, sensitivity, swelling). Skin lesions include i) red to brownish-gray patches (especially on the hands, feet, ankles, wrists, neck, upper chest, eyelids, elbows and the inside of the knee creases, and on the face and scalp of infants); ii) small, raised bumps that may ooze fluid and crust when scratched; and iii) thickened, cracked, scaly skin.

[0049] As used herein, the term "subject" refers to a mammal. For example, mammals contemplated in this invention include humans, primates, laboratory rodents, and pets such as dogs and cats.

[0050] The term "efficacy" in the treatment or method according to the invention can be measured based on changes that occur during the course of a disease or symptom in response to the use or method according to the invention. For example, the efficacy of a treatment or method according to the invention can be measured by its effect on disease signs or symptoms. Relief is achieved when the patient experiences partial or complete remission, or a reduction in undesirable disease symptoms.

[0051] As used herein, the term "effective amount" refers to an amount of at least one pharmaceutical agent or pharmaceutical preparation according to the invention that causes a detectable reduction in disease symptoms in a subject to administration of said agent.

[0052] IL-15 inhibitors

[0053] The IL-15 inhibitors according to the present invention comprise anti-IL antibodies, inhibitory peptides, and small molecules.

[0054] According to one embodiment, the IL-15 inhibitor can be a small molecule, such as the small molecules described in Quemener et al., 2017, J. Med. Chem. 2017, 60, 6249-6272 DOI: 10.1021 / acs.jmedchem.7b00485, in particular the compound having the following structure:

[0055] wherein R is an optionally substituted phenyl group, and n is an integer selected from 1 to 10, in particular the molecule having the following structure:

[0056]

[0057] 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, such as BNZ132 (aka BNZ-1, aka YT033, aka EQ-101) (SEQ ID NO: 2) (IKEFLQRFIHIVQSIINTS) and the peptides described in US 2019 / 0070263, such as PKEFLERFVHLVQMFIHQSLS (SEQ ID NO: 3) or fragments or variants of these sequences. Other peptide IL-15 inhibitors are described in US 7,736,638 (SEQ ID NO: 4) or fragments or variants of these sequences.

[0058] According to another embodiment, the IL-15 inhibitor can be an anti-IL15 antibody or antigen-binding fragment thereof. According to a particular aspect, the anti-IL15 antibody or antigen-binding fragment thereof binds to IL-15, in particular human IL-15 or a fragment of IL-15.

[0059] The anti-IL15 antibodies according to the application typically 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 can exhibit cross-reactivity with IL-15 from at least one other species, such as a primate (e.g. cynomolgus monkey, rhesus monkey, marmoset), mouse, rat, dog and / or rabbit. For antibodies directed against human IL-15, a certain degree of cross-reactivity with IL-15 in other mammalian forms can be desirable in some cases.

[0060] In a particular embodiment, the anti-IL15 antibody or fragment thereof according to the application preferentially binds to human IL-15.

[0061] In yet another embodiment, the antibody or antigen-binding fragment thereof according to the application does not exhibit cross-reactivity with rat IL-15 and / or mouse IL-15.

[0062] In one embodiment, the inhibitors according to the application preferentially inhibit IL-15 and optionally additionally show some inhibitory effect on other proteins with homology to IL-15, such as IL-2, in particular human IL-2 or IL-21 (as disclosed in WO 2015 / 089217).

[0063] The ability of an antibody to block or neutralize the activity of its target protein can be assessed by its potency as defined herein, which is itself reflected, for example, by an IC 50 value. In general, the neutralizing activity of an antibody can be determined by, for example, an in vitro assay as described in WO 2016 / 001275 for measuring the level of inhibition of IL-15-induced cell line (e.g., Kit 225 or M-07e cells) proliferation and / or survival in the presence of said antibody.

[0064] In some embodiments, the antibodies and antigen-binding fragments thereof according to the application have an IC 50 equal to or lower than 200 nM, in particular lower than 100 nM, in particular lower than 50 nM, lower than 30 nM, lower than 20 nM, more particularly lower than 10 nM, lower than 8 nM, lower than 7 nM, lower than 5 nM, lower than 4 nM, lower than 3 nM, lower than 2 nM, lower than 1 nM, lower than 0.5 nM, lower than 0.3 nM, lower than 0.2 nM, lower than 0.1 nM, lower than 0.05 nM, or lower than 0.03 nM, for inhibiting IL-15 activity, such as IL-15-induced cell line (e.g., Kit 225 or M-07e cells) proliferation and / or survival, as described in WO 2016 / 001275.

[0065] It will be appreciated that any variant or fragment of an antibody described herein that is capable of binding to IL-15 and optionally neutralizing IL-15 activity will be a suitable IL-15 inhibitor according to the application. In a particular embodiment, the variant can show the same or even higher binding affinity for IL-15, and / or the same or even higher potency, and / or the same or greater species selectivity, and / or the same or greater selectivity for IL-15, and / or the same or greater neutralizing efficacy, as compared to the parent antibody or fragment from which such variant is derived.

[0066] In particular embodiments of the application, the antibody against IL-15 or an antigen-binding fragment thereof that binds to IL-15 is a monoclonal antibody.

[0067] In particular embodiments of the application, the antibody to IL-15 or antigen-binding fragment thereof that binds to IL-15 is a bispecific or multispecific antibody, in particular a bispecific antibody targeting IL-15 and IL-4R or IL-13 or IL-4.

[0068] In yet another particular embodiment of the application, the antibody to IL-15 or antigen-binding fragment thereof that binds to IL-15 is a humanized antibody.

[0069] In yet another particular embodiment of the application, the antibody to IL-15 or antigen-binding fragment thereof that binds to IL-15 is a recombinant antibody.

[0070] The antibody to IL-15 or antigen-binding fragment thereof that binds to IL-15 suitable for use according to the application can be characterized by the part of it that interacts with the target protein, in particular by its variable region, typically including the heavy chain variable region and the light chain variable region, for example the variable regions described in WO 2016 / 001275.

[0071] According to particular embodiments, the anti-IL-15 inhibitor is an isolated antibody or antigen-binding fragment thereof that binds IL-15, comprising:

[0072] (1) the heavy chain variable region of SEQ ID NO: 5 or any variant thereof having the amino acid sequence of SEQ ID NO: 5 except that 1, 2, 3 or 4 amino acids are substituted by different amino acids, wherein the substitutions are selected from:

[0073] (i) within the heavy chain variable framework region, arginine (R) at position H3 (VH RH3) is substituted by glutamine (Q), methionine (M) at position H5 (VH MH5) is substituted by valine (V), alanine (A) at position H6 (VH AH6) is substituted by glutamic acid (E), alanine (A) at position H49 (VH AH49) is substituted by serine (S),

[0074] (ii) within the heavy chain CDR2, aspartic acid (D) at position H61 (VH DH61) is substituted by glutamic acid (E), serine (S) at position H62 (VH SH62) is substituted by threonine (T), and

[0075] (iii) within the heavy chain CDR3, methionine (M) at position H98 (VH MH98) is substituted with leucine (L), phenylalanine (F), isoleucine (I), or alanine (A), tryptophan (W) at position H100C (VH WH100C) is substituted with tyrosine (Y), phenylalanine (F), or alanine (A), methionine (M) at position H100E (VH MH100E) is substituted with leucine (L), phenylalanine (F), or isoleucine (I);

[0076] and

[0077] (2) the light chain variable region of SEQ ID NO: 9.

[0078] According to another specific embodiment, the anti-IL-15 inhibitor is an isolated antibody or antigen-binding fragment thereof that binds IL-15, comprising:

[0079] (1) the heavy chain variable region of SEQ ID NO: 6, or any variant thereof that has the amino acid sequence of SEQ ID NO: 6 except that 1, 2, 3, or 4 amino acids are substituted with different amino acids, and

[0080] (2) the light chain variable region of SEQ ID NO: 9.

[0081] According to another specific embodiment, the anti-IL-15 inhibitor is an isolated antibody or antigen-binding fragment thereof as in claim 1, comprising:

[0082] (1) a heavy chain variable region of the amino acid sequence of SEQ ID NO: 5, having a substitution in the sequence of SEQ ID NO: 5 selected from:

[0083] (i) VH RH3 is substituted with glutamine (Q), VH MH5 is substituted with valine (V), VH AH6 is substituted with glutamic acid (E),

[0084] (ii) VH SH62 is substituted with threonine (T), and

[0085] (iii) VH WH100C is substituted with tyrosine (Y);

[0086] and

[0087] (2) a light chain variable region of the amino acid sequence of SEQ ID NO: 9.

[0088] According to another specific embodiment, the anti-IL-15 inhibitor is an isolated antibody or antigen-binding fragment thereof that binds IL-15, comprising:

[0089] (1) a heavy chain variable region selected from the group consisting of: SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8, and

[0090] (2) a light chain variable region of SEQ ID NO: 9.

[0091] According to another particular embodiment, the anti-IL-15 inhibitor is an isolated antibody or an antigen-binding fragment thereof that binds IL-15, comprising a heavy chain variable region of SEQ ID NO: 6 and a light chain variable region of SEQ ID NO: 9.

[0092] The interleukin-15 inhibitor for use according to claim 1 or 6, wherein said interleukin-15 inhibitor is selected from the group consisting of an anti-IL-15 antibody comprising:

[0093] - a heavy chain of SEQ ID NO: 10 and a light chain of SEQ ID NO: 11 or a variant or fragment thereof; or

[0094] - 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

[0095] - 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

[0096] - a heavy chain of SEQ ID NO: 16 and a light chain of SEQ ID NO: 17 or a variant or fragment thereof; or

[0097] - a heavy chain of SEQ ID NO: 18 and a light chain of SEQ ID NO: 17 or a variant or fragment thereof; or

[0098] - a heavy chain of SEQ ID NO: 19 and a light chain of SEQ ID NO: 17 or a variant or fragment thereof.

[0099] According to yet another particular embodiment, the anti-IL-15 inhibitor is an isolated antibody or an antigen-binding fragment thereof that binds IL-15, comprising a heavy chain of SEQ ID NO: 16 and a light chain of SEQ ID NO: 17.

[0100] According to yet another particular embodiment, the anti-IL-15 inhibitor is an isolated antibody or an antigen-binding fragment thereof that binds IL-15, comprising a heavy chain of SEQ ID NO: 18 and a light chain of SEQ ID NO: 17.

[0101] According to a further particular embodiment, the anti-IL-15 inhibitor is an isolated antibody or antigen-binding fragment thereof binding IL-15 comprising a heavy chain of SEQ ID NO: 19 and a light chain of SEQ ID NO: 17.

[0102] According to a further particular embodiment, the anti-IL-15 inhibitor is an isolated antibody or antigen-binding fragment thereof binding IL-15 comprising the sequence 146B7 (also known as AMG174) as described in WO 2005 / 044303 or in WO 03 / 017935, which as IL-15 antagonist is a monoclonal human IgGl anti-IL-15 antibody with a heavy chain and a kappa light chain region comprising the amino acid sequences of SEQ ID NO: 10 and SEQ ID NO: 11.

[0103] According to a further particular embodiment, the anti-IL-15 inhibitor is an isolated antibody or antigen-binding fragment thereof binding IL-15 as described in WO 2018 / 119246 comprising a variable heavy chain sequence of SEQ ID NO 12 and a variable light chain sequence of SEQ ID NO 13.

[0104] According to a further particular embodiment, the anti-IL-15 inhibitor is an isolated antibody or antigen-binding fragment thereof binding IL-15 as described in WO 2011 / 127324, in particular huABC2 (anti-IL-15R beta antibody) comprising a variable heavy chain sequence of SEQ ID NO 14 and a variable light chain sequence of SEQ ID NO 15.

[0105] According to a further particular embodiment, the anti-IL-15 inhibitor is an isolated antibody or antigen-binding fragment thereof binding IL-15 as described in WO 2009 / 002562, or an isolated antibody or antigen-binding fragment thereof binding IL-15 as described in WO 2017 / 217985 and US2018 / 0002417 or in WO 2005 / 044303 and WO 03 / 017935, e.g. 146B7 or an antigen-binding fragment thereof.

[0106] Particular examples of antibodies according to the present application include the antibodies listed in Table 1.

[0107] Table 1

[0108]

[0109] Conjugates comprising a helper molecule

[0110] In another aspect of the present application, the isolated antibody or antigen-binding fragment thereof according to the present application is optionally conjugated with a helper molecule and is then also referred to herein as "conjugated antibody" or "conjugated antibody fragment".

[0111] The conjugated antibodies and conjugated antibody fragments according to the present application can target the anti-IL-15 inhibitor to the site of disease (e.g. inflammation) in vivo, such that the anti-IL-15 inhibitor can have a preferential concentration and therapeutic effect at the site of disease, and fewer side effects at other sites in the body.

[0112] The accessory molecule can be conjugated to the antibody or antibody fragment directly or via a spacer of appropriate length, e.g. as described by Kellogg et al. (2011, Bioconjug Chem 22:717-27).

[0113] In another embodiment, the accessory molecule comprises an antigen binding fragment of an antibody which, when conjugated to an antibody or antibody fragment according to the present application, forms a bispecific antibody. In particular, the bispecific antibody can be directed against two different epitopes of IL-15 (thus defining a bispecific antibody), or have a bispecific portion targeting IL-15 and IL-4R or IL-13 or IL-4.

[0114] Compositions

[0115] The IL-15 inhibitor is provided in the form of a pharmaceutically acceptable composition. The pharmaceutical composition can comprise one or more IL-15 inhibitors, in particular one or more antibodies or antigen binding fragments thereof binding IL-15 in any form described herein.

[0116] The compositions of the present application can further comprise one or more pharmaceutically acceptable additional ingredients, such as alum, stabilizers, antimicrobial agents, buffers, coloring, flavoring, adjuvants, and the like.

[0117] The IL-15 inhibitor, together with a conventionally employed adjuvant, carrier, diluent or excipient can be made into a pharmaceutical composition in unit dosage form for administration orally or parenterally, including subcutaneously, and can be used in such form for full administration of the dosage. Such pharmaceutical compositions and unit dosage forms thereof can comprise 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 commensurate with the intended use. The pharmaceutical compositions can be formulated to be immediate or modified release.

[0118] The compositions can be liquid formulations, including but not limited to aqueous or non-aqueous suspensions, solutions, emulsions, syrups, and elixirs. Liquid forms suitable for oral administration can include a suitable aqueous or non-aqueous vehicle with buffers, suspending, and dispensing agents, colorants, flavorants, and the like. The compositions can also be formulated as a dry product for reconstitution with water or other suitable vehicle before use. Such liquid preparations can 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, methyl cellulose, glucose / sugar syrup, gelatin, hydroxyethylcellulose, carboxymethylcellulose, aluminum stearate gel, and hydrogenated vegetable oil. Emulsifying agents 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. Other materials and processing techniques are listed in the following document, which is incorporated herein by reference: Remington: The Science & Practice of Pharmacy, 23rdedition, 2020, Adeboye Adejare, Editor, Academic Press, which is incorporated herein by reference.

[0119] The solid compositions of the present application can be in the form of tablets or capsules prepared by conventional means. For example, tablets and capsules for oral administration can contain conventional excipients including, but not limited to, binding agents, fillers, lubricants, disintegrants, and wetting agents. Binding agents include, but are not limited to, syrup, acacia, gelatin, sorbitol, tragacanth, starch paste, and polyvinylpyrrolidone. Fillers include, but are not limited to, lactose, sugar, microcrystalline cellulose, maize 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. The tablets can be coated according to methods well known in the art.

[0120] Injectable compositions are typically based upon injectable sterile saline or phosphate buffered saline or other injectable carriers known in the art.

[0121] The compositions can also be formulated as transdermal formulations, including but not limited to creams, ointments, lotions, pastes, medicated plaster, patches, or membranes containing aqueous or non-aqueous vehicles including but not limited to those mentioned above.

[0122] The compositions can also be formulated for parenteral administration, including but not limited to by injection or continuous infusion. Formulations for injection can be in the form of suspensions, solutions, or emulsions in oily or aqueous vehicles, and can contain formulation agents including but not limited to suspending, stabilizing, and dispersing agents. Compositions can also be provided in powder form for reconstitution with a suitable vehicle, including but not limited to sterile, pyrogen-free water.

[0123] The compositions can also be formulated in a depot formulation for administration by implantation or by intramuscular injection. Compositions can be formulated with suitable polymeric or hydrophobic materials (e.g., as an emulsion in an acceptable oil), ion exchange resins, or as a sparingly soluble derivative (e.g., as a sparingly soluble salt).

[0124] The compounds can also be administered in sustained release forms or from sustained release drug delivery systems. Descriptions of representative sustained release materials can also be found in the materials contained in Remington's Pharmaceutical Sciences.

[0125] Injectable formulations are particularly suited for administration of the compounds or compositions according to the present application.

[0126] Combinations

[0127] According to the present application, the IL-15 inhibitor according to the present application can be administered alone or in combination with an adjunct useful for the prevention and / or treatment of atopic dermatitis, such as an anti-IL-4R like dupilumab, an anti-IL-13 like tralokinumab, a JAK inhibitor like abrocitinib, upadacitinib (oral) or ruxolitinib (topical) or a classi cal corticosteroid like dexamethasone.

[0128] The present application encompasses the use of an IL-15 inhibitor, wherein the IL-15 inhibitor will be administered to a subject in a therapeutically effective amount prior to, simultaneously with, or sequentially with other therapeutic regimens or adjuncts useful for the prevention and / or treatment of atopic dermatitis. The IL-15 inhibitor according to the present application administered simultaneously with said adjuncts can be in the same or different composition and administered by the same or different route of administration.

[0129] In particular embodiments, the IL-15 inhibitor, in particular an IL-15 antibody or antigen binding fragment thereof according to the present application, can be administered in combination with an anti-IL-4R antibody like dupilumab.

[0130] In particular embodiments, the IL-15 inhibitor, in particular an IL-15 antibody or antigen binding fragment thereof according to the present application, can be administered in combination with an anti-IL-13 like tralokinumab.

[0131] In particular embodiments, the IL-15 inhibitor, in particular the IL-15 antibody or antigen-binding fragment thereof according to the application, can be administered in combination with one or more corticosteroids such as dexamethasone.

[0132] Mode of administration

[0133] The compositions of the application can be administered in any way, including but not limited to orally, parenterally, sublingually, transdermally, transmucosally, topically, or a combination thereof.

[0134] Parenteral administration includes, but is not limited to, intravenous, subcutaneous and intramuscular administration. The compositions of the application can also be administered in the form of an implant, which allows slow release of the composition and slow controlled intravenous infusion.

[0135] In particular embodiments, the IL-15 inhibitor is administered systemically or locally, for example by topical application.

[0136] In particular embodiments, the IL-15 inhibitor, in particular the IL-15 antibody or antigen-binding fragment thereof, is administered by subcutaneous or intravenous route.

[0137] The dose administered to an individual will vary depending on a variety of factors including pharmacokinetic properties, subject conditions and characteristics (sex, age, weight, health, size), extent of symptoms, concurrent therapy, frequency of treatment, and desired effect.

[0138] According to particular embodiments, the frequency of administration of the inhibitor is once a week to once a month.

[0139] Generally, a therapeutically effective amount of a pharmaceutically active antibody is in the range of 0.1 mg / kg to 50 mg / kg body weight dose.

[0140] Patient

[0141] In embodiments, the subject according to the application suffers from atopic dermatitis.

[0142] According to particular embodiments, the subject according to the application exhibits high systemic or local levels of IL-15, or biomarkers of IL-15 pathway activation, for example as described in Karlen and Simon, 2020, Int. Arch. Allergy Immunol. 181, 417-421 and Kurowska et al., 2020, J. Clin. Med 1555.

[0143] In another embodiment, the subject according to the application suffers from atopic dermatitis and has no response to or cannot be treated with anti-IL-4R, anti-IL-13 and / or corticosteroid therapy. A subject having no response to anti-IL-4R, anti-IL-13 and / or corticosteroid therapy is defined as lacking satisfactory remission after treatment with anti-IL-4R, anti-IL-13 and / or corticosteroid.

[0144] Uses and methods according to the application

[0145] In a particular embodiment, a method of preventing and / or treating atopic dermatitis is provided, the method comprising administering to a subject in need thereof a therapeutically effective amount of an IL-15 inhibitor, in particular an IL-15 antibody or antigen-binding fragment thereof.

[0146] According to a particular embodiment, the interleukin-15 inhibitor is administered in the form of a formulation according to the application.

[0147] The references cited herein are all incorporated by reference in their entirety.

[0148] Having described the application, the following examples are presented by way of illustration and not limitation.

[0149] Example

[0150] Example 1 : Expression of IL-15 and IL-15Ra in an ex vivo human model of atopic dermatitis

[0151] 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, as described in WO 2016 / 001275) in atopic dermatitis was investigated in the following experimental model.

[0152] The experimental set-up used healthy skin punch biopsies which were induced to show an atopic dermatitis phenotype using a cocktail of mixtures activating the Th2 pathway as follows: https: / / www.reprocell.com / drug-efficacy-safety-adme / assay-catalog / skin-culture- induced-atopic-dermatitis-model-systemic-topical ):

[0153] Full thickness skin biopsy samples of up to 36 x 3 mm were obtained from three different healthy donors by excision through surgical operation as described hereinafter. On day -1, the skin biopsy samples were prepared, randomized and completely immersed in at least 1 mL of basal medium in a cell culture incubator overnight. After the equilibration period, the biopsy samples were transferred to individual culture wells of a 12-well plate on day 0. Then, using a pipette tip or forceps, the biopsy samples were placed in the upper compartment of a Transwell® insert (24-well format, 0.4 pm pore size, Corning®) and the lower compartment was filled with 2 mL of basal medium. The lower compartment was used to collect the supernatant of the upper compartment. TMA hole was cut in the filter and each biopsy sample was inserted into a hole of a filter. The filter containing the biopsy sample was placed into a well of a 12-well culture plate containing 1 mL of appropriate medium with the epidermis facing upwards at the air-liquid interface. 1 mL of medium containing or not Th2 polarizing medium (Cousins et al., 2002, J. Immunol 169, 2498-2506) and test article was added to each well on day 0 and changed on day 1. The test compounds used in this study were: an anti-human IL-4R antibody, whose sequence is identical to 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); and betamethasone (Merck Life Sciences, Gillingham, Unite Kingdom) at a final concentration of 50 pg / mL or betamethasone at a final concentration of 10 pM.

[0154] Approximately 48 h (day 2) after application of the Th2 stimulating cocktail and the above-mentioned test compounds, the biopsies were harvested and stored in RNAlater at 2-8 °C for at least 24 h. After this time, the RNAlater was removed and the biopsies were snap-frozen in liquid nitrogen and stored at -80 °C. The tissue samples stabilized in RNAlater solution were processed for RNA extraction and isolation. The RNA was quantified and quality checked using the Qubit system. Once the quality check was completed, the RNA samples were submitted for RNAseq analysis.

[0155] Skin and PBMC human donors

[0156] Healthy (control) abdominal human skin was obtained from three healthy volunteers (39-52 years old) who underwent elective surgery and had no history of atopy. PBMC 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).

[0157] PBMC stimulation and characterization

[0158] Autologous PBMC 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).

[0159] Following isolation, they were cultured in the presence of IL-2 (10 U / mL), IL-4 (200 U / mL), and LPS (1 pg / mL; Sigma, St. Louis, MO) (“Th2 polarization”) to induce a Th2 phenotype. In the trial experiment to assess induction of IL-15 expression, control PBMC were cultured in the presence of IL-2 and IL-4 but without LPS activation.

[0160] It was observed that Th2 culture conditions, known to induce genes similar to those found in atopic dermatitis lesions, were able to induce IL-15 gene expression and IL-15Ra expression in human skin explants in vitro, as shown in Figure 1 A and Figure 1 B.

[0161] It was also observed that treatment with anti-IL-4R antibody or a corticoid did not reduce induced IL-15 and IL-15Ra expression in an ex vivo human atopic dermatitis model, as shown in Figure 2 A and Figure 2 B.

[0162] Example 2: Anti-IL-15 antibodies are effective in a humanized mouse model of atopic dermatitis

[0163] However, human atopic dermatitis models do not represent the human disease well. Similarly, existing mouse experimental models have limited value as they represent the human disease only to a limited extent and rely on mouse skin and immune system components that are different from human skin and immune system components (Gilhar and Paus, 2021, Exp. Dermatol. 30, 319-336. https: / / doi.org / 10.1111 / exd.14270). Humanized mouse models offer the possibility to study human cell functions in vivo, and in dermatology, humanized models of psoriasis have been shown to be the most representative of the human disease, although they are complex and expensive. The "humanized" model of atopic dermatitis described in Gilhar and Paus, 2021, supra, was used. "Humanized" mice are defined here as immunodeficient (SCID / beige) mice grafted with normal human skin from a healthy donor and activated T helper (Th)2-polarized peripheral blood mononuclear cells (PBMC) from the same donor to recapitulate the clinical and histological features of atopic dermatitis at the level of the skin graft.

[0164] The first objective of this study was to assess whether IL-15 was induced during the development of this atopic dermatitis model (pilot experiment). As can be seen in Figure 3 , it was demonstrated that both IL-15 and IL-15Ra expression were induced in this model. IL-15Ra is mainly expressed by epidermal cells, while IL-15 is expressed by epidermal cells and also by dermal cells, possibly by cells of the monocyte and dendritic cell lineage. This analysis validated the presence of the IL-15 target in the humanized atopic dermatitis mouse model and thus its suitability for testing compounds that neutralize IL-15.

[0165] The secondary objective of this study was to determine whether an interleukin 15 inhibitor, as an anti-interleukin 15 antibody, administered in different regimens, is effective in this humanized model of atopic dermatitis.

[0166] As described below, a significant impact on macroscopic skin lesions was observed when the anti-IL-15 antibody was administered within 4 weeks of the onset of the atopic dermatitis lesion or only in the last 2 weeks, as represented by the clinical score based on the measurement of erythema and skin integrity Figure 4 . In addition, anti-IL-15 treatment had a significant impact on the following epidermal properties Figure 5 ), such as the expression of epidermal thickness (A), epidermal basal cell proliferation (B) and filaggrin (C) (a molecule associated with skin barrier integrity, often 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 features known to be characteristic of human atopic dermatitis.

[0167] The third objective was to analyze the impact of the anti-IL-15 antibody on key readouts and biomarkers known to be associated with human atopic dermatitis in treated mice.

[0168] Anti-IL-15 therapy also had an impact on cytokines that can be secreted by T cells and are known to be induced in human atopic dermatitis (IFN-g, IL-4, IL-17 and IL-22) Figure 7 On the other hand, anti-IL-15 therapy had no significant impact on the number of T cells, NK cells and NKT cells present in the dermis of human skin grafts Figure 6 which are believed to be responsible for the secretion of IFN-g, IL-4, IL-17 and IL-22. This suggests that anti-IL-15 only affects the function of these T cells, NK cells and NKT cells, or only affects the number and / or function of a small subset of cells that cannot be described with the techniques used herein. When looking at the immune gene expression analysis described below, it was observed that hu-BE29-2 (also known as CALY-002) treatment induced a decrease in the expression of many immune genes related to T cells, T helper cells and B cells, as well as genes known to be associated with atopic dermatitis, including S100A9, DEFB4, CXCL10 Figure 8 Overall, these data suggest that blocking the IL-15 pathway can be an effective new therapy for treating atopic dermatitis.

[0169] Since both anti-IL-4R therapeutic antibodies and corticosteroids are approved treatments for atopic dermatitis (Strowd et al., 2017, The Lancet, 39(10086), 2265-2266), it was investigated whether these treatments could impact IL-15 expression in the atopic dermatitis model analyzed. Interestingly, in the ex vivo human explant model of Example 1, both anti-IL-4R antibodies and corticosteroids failed to decrease IL-15 Figure 9 ), while both of these treatments can significantly decrease many other genes associated with atopic dermatitis. The same was true for the expression of IL-15Ra, and in fact, anti-IL-4R treatment even slightly increased the expression of IL-15 and IL-15Ra. Moreover, in the humanized atopic dermatitis mouse model of the present example, anti-IL-4R only decreased IL-15 expression in the dermis, but not in the epidermis. Corticosteroids showed a trend to decrease IL-15 expression only in the dermis, but this was not statistically significant.

[0170] These results suggest that treatment with anti-IL-4R antibodies or corticosteroids does not completely suppress the IL-15 pathway, and thus that anti-IL-15 therapy can be advantageously administered to patients who do not respond to anti-IL-4R or corticosteroids, or in combination with these or other treatments.

[0171] Animals

[0172] A total of 60 female C.B-17 / IcrHsd-scid-bg (Harlan Laboratories Ltd., Jerusalem, Israel) mice, 2-3 months of age, were used in the therapy experiments. Mice were housed under pathogen-free conditions in accordance with institutional guidelines.

[0173] Human donors of skin and PBMCs

[0174] Healthy abdominal human skin was obtained from healthy female volunteers who underwent elective surgery and had no history of atopic disease. PBMCs were collected from 20 mL of venous blood from the same donor. The study was approved by the Helsinki Committee of the Rambam Medical Campus Institutions (0182-14-RMB).

[0175] Humanized mouse atopic dermatitis model

[0176] Layered skin xenografts were performed as described (Keren et al., 2018, supra). Briefly, human skin samples (1 cm2, 0.4 mm thick) from three different donors were grafted onto mice (one human xenograft per mouse). One month after grafting, 1x10 7 Th2-polarized PBMCs. Prior to injection, cell viability was determined by Trypan Blue staining.

[0177] In vivo treatment with anti-IL-15 antibodies and controls

[0178] Two treatment regimens or controls using the anti-IL15 antibody hu-BE29-2 of SEQ ID NO: 16 and SEQ ID NO: 17 as described in WO 2016 / 001275 were considered. Mice (10 per group) were injected intravenously three times per week for 4 weeks with 200 pg / mouse of the anti-IL-15 antibody (manufactured by Calypso Biotech S.A., Switzerland) or first with a matching dose of the IgGl control isotype antibody for 2 weeks and then with the anti-IL-15 antibody for 2 weeks. Other groups of mice (10 per group) were likewise injected with 200 pg / mouse of an anti-IL-4 receptor (IL-4R) antibody for 4 weeks, the sequence of the IL-4R antibody being identical to that of dupilumab (Dupixent®) https: / / www.genome.jp / dbget-bin / www_bget?dr:D10354) and produced in Chinese hamster ovary cells by transient transfection (Evitria AG, Zurich, Switzerland), or first injected with IgG4 control isotype antibody for 2 weeks and then injected with anti-IL-4R antibody for 2 weeks. Negative controls (5 mice per group) were injected with matching doses of IgG1 or IgG4 control antibodies (Evitria AG, Zurich, Switzerland) for 4 weeks. As positive controls, a group of 10 mice was topically applied with dexamethasone three times per week starting in the third week after PBMC injection. Macroscopic images of the grafted skin were taken at day 0 and weeks 1, 2, 3, and 4 after PBMC injection. At the end of the 4-week treatment, each xenografted skin explant was collected, cut in half and embedded in O.C.T. and stored frozen, or fixed with formalin, embedded in paraffin and stored at room temperature until further evaluation. O.C.T. and stored frozen, or fixed with formalin, embedded in paraffin and stored at room temperature until further evaluation.

[0179] Clinical macroscopic evaluation

[0180] To measure the severity of the atopic dermatitis-like phenotype in the xenografted skin, a measure commonly applied in the clinic was adapted for application to the analysis of the macroscopic images collected to grade the signs of erythema and skin integrity (epidermal breaks / scale / mossy changes) on a discrete scale (0 = none, 1 = moderate, and 2 = severe) at day 0 and weeks 1, 2, 3, and 4 after PBMC injection. The total score (erythema score + skin integrity score) was calculated for each mouse. The scoring was performed independently by three operators in a blinded fashion.

[0181] Hematoxylin and eosin (H&E) staining and epidermal thickness measurement

[0182] Routine H&E staining was performed on 7 pm thick cryosections. The mean human epidermal thickness of each xenograft was determined on these H&E stained sections by averaging the measured distance between the outermost surface of the epidermis, excluding the stratum corneum, and the dermal-epidermal junction at 10 randomly selected points across the entire length of each examined section with the help of a digital caliper and the ImajeJ software. The measurements were then averaged within each experimental group in a blinded fashion.

[0183] Expression of IL-15 and IL-15Ra

[0184] To detect IL-15 and IL15-Ra expression in xenografts, OCT (Optimal Cutting Temperature Compound) embedded skin xenografts were sectioned (7 pm thick) using a Leica cryostat. Tissue cryosections were fixed in acetone and pre-incubated in PBS with 10% goat serum. Sections were then incubated with the corresponding primary antibody (anti-IL-15, Ab55276, Abeam, 1 : 100; anti-IL-15Ra, Ab91270, Abeam, 1 : 100) overnight at 4°C. Secondary antibody incubation was performed for 45 min at room temperature (Goat-anti-Mouse-IgG-Alexa 546, Invitrogen, 1 : 500). Counterstaining was performed with DAPI (1 pg / ml) to visualize the nuclei.

[0185] Immunohistochemistry of atopic dermatitis markers.

[0186] To measure filaggrin (FLG) expression, cryopreserved xenografted skin sections were stained overnight at 4°C with rabbit anti-filaggrin polyclonal antibody (Biolegend, 1 : 500-1 : 250), followed by species-specific fluorescent conjugated secondary antibody and DAPI counterstaining to visualize the nuclei. Filaggrin expression (fluorescence intensity) was measured using a fluorescence microscope at 10x magnification in 1-3 randomly selected epidermis areas of 3-6 cryosections per xenograft. Regions of interest (ROIs) including the human stratum corneum were manually selected on each image and Image J software was used to calculate the mean FLG expression value within the ROIs. For each xenograft, the mean expression level of filaggrin was calculated.

[0187] T, NK and NKT cell infiltration was assessed by analyzing CD3 (T cell marker) and CD56 (NK cell marker) expression. Cryopreserved xenografted skin sections were double-stained with mouse anti-human CD56 antibody (Dako, 1 : 100, overnight at 4°C) followed by tyramide signal amplification (TSA, enabling detection of low-abundance targets) and pre-conjugated mouse anti-CD3 monoclonal antibody Alexa Fluor 647 (Invitrogen, 1 : 50, 1 h at room temperature). In one cryosection of each xenografted skin, the number of DAPI+, CD3+, CD56+and CD3+or CD56+positive cells was calculated in 3-5 randomly selected 100 pm2dermis areas. The mean value was calculated for each skin sample and expressed as percentage of DAPI+cells per area.

[0188] Epidermal proliferation was analyzed by observing 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 staining with anti-mouse fluorescently conjugated goat secondary antibody. Sections were briefly treated with DAPI to visualize cell nuclei. For each xenograft skin, 2 sections were imaged at 20x magnification using a fluorescent microscope. The innermost layer of the epidermis was selected as the region of interest (ROI) and Ki-67+ cells were counted in the ROI. To determine the total number of cells, DAPI-stained cell nuclei were counted using an automatic method coded in ImageJ image analysis software.

[0189] IL-4 expression was analyzed on 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 rabbit anti-IL-4 antibody (Ab9622, Abcam, 1 : 100) followed by staining with species-specific biotinylated goat secondary antibody. IL-4+ cells were determined by targeting biotin with streptavidin-HRP to catalyze DAB precipitation. Sections were counterstained with hematoxylin. For each xenograft skin, 2 sections were imaged at 20x magnification using a light microscope. Dermal IL-4+ cells were counted and dermal surface area was calculated in ImageJ imaging software to derive the number of IL-4+ cells per mm 2

[0190] Expression of IL-17A was analyzed by staining cryopreserved xenograft skin sections overnight at 4°C with rabbit anti-human IL-17A antibody (Ab79056, Abeam, 1 : 200) followed by species-specific fluorescently conjugated secondary antibody and DAPI counterstaining to visualize cell nuclei. For each xenograft skin, 2-3 sections were imaged at 20x magnification using a fluorescent microscope. Dermal IL-17A+ cells were counted and dermal surface area was calculated in ImageJ imaging software to derive the number of IL-17A+ cells per mm 2

[0191] ​​IL-22 expression was analyzed by staining cryopreserved xenograft skin sections overnight at 4°C with rabbit anti-human IL-22 antibody (BS-2623R, Thermo Fisher Scientific, 1:200), followed by species-specific fluorescently conjugated secondary antibody and DAPI counterstaining to make cell nuclei visible. For each xenograft skin section, 2-3 sections were imaged using a fluorescence microscope at 20x magnification. IL-22+ cells in the dermis were counted, and the dermal surface area was calculated using ImageJ imaging software to determine the expression per mm². 2 The number of IL-22+ cells.

[0192] IFN-γ expression was analyzed by staining cryopreserved xenograft skin sections overnight at 4°C with rabbit anti-human IFN-γ antibody (Ab9657, Abogen Biosciences, 1:100), followed by species-specific fluorescently conjugated secondary antibody and DAPI counterstaining to make cell nuclei visible. For each xenograft skin, 2-3 sections were imaged using a fluorescence microscope at 20x magnification. Dermal IFN-γ+ cells were counted, and the dermal surface area was calculated using ImageJ imaging software to obtain the expression per mm². 2 The number of IFN-γ+ cells.

[0193] Gene expression analysis in humanized models

[0194] RNA was extracted from OCT-embedded xenografts. OCT-embedded xenografts from 55 mice were thawed on ice and then washed twice in PBS. Evolution Homogenizer ( Evolution Homogenisator and TRI Total RNA was extracted from Sigma-Aldrich. In short, each xenograft was homogenized at 6800 rpm for six 10-second cycles, cooled on ice for 30 seconds, and then 1 mL of TRI was added. Mix the sample with chloroform (250 μL) and centrifuge at 12,500 × g for 15 min. Mix the supernatant containing total RNA (approximately 500 μL) with an equal volume of isopropanol to precipitate the RNA. Precipitate the total RNA by centrifuging at 12,500 × g for 15 min, wash twice with 70% ethanol and centrifuge at 8,000 × g for 7 min, then resuspend in 20 μL of RNase-free water. Store the RNA at -80 °C until analysis.

[0195] Quality control of extracted RNA was measured using a tapestation bioanalyzer and Qubit at the Nanostring Core facility in Heidelberg, Germany.

[0196] Nanoskin Analysis System (Nanostring) The analysis system is based on the digital detection of individual target molecules, which are directly labeled with molecular barcodes. Each probe consists of a color-coded reporter probe and a capture probe, both covalently linked to a target-specific sequence (50 nucleotides in length). The color-coded reporter probe allows for target exclusivity, while the biotin-conjugated capture probe allows the target to be immobilized on a surface. With the application of voltage and the generation of an electric field, the target molecules align, allowing the reporter probe to fully extend and enabling the recognition of individual molecules. NanoString multiplexing is outsourced to the Department of Human Molecular Genetics, Heidelberg University Hospital. Core facilities. Instruments used. SPRINT.

[0197] In this study, the following methods were used: The Human Immunology V2 Panel quantifies 594 genes, including housekeeping genes. Purified RNA obtained from each sample was hybridized overnight at 65°C using the Human Immunology V2 Panel Analyzer (NanoString Technologies, WA, USA). The hybridization probe was further purified and bound to the optical cartridge at the Prep Station, and the optical cartridge was scanned on the nCounter digital analyzer. The RCC file obtained from the Nanoskin digital analyzer was imported into nSolver. TM Software 4.0.70 (Nanoskin Technologies, Washington, USA) was used, and data quality was checked using the default quality check settings. mRNA levels were determined using "barcodes," and background correction was performed by subtracting the negative control's "mean + 2 standard deviations" value from the raw counts. The adjusted raw counts were then normalized to the geometric mean of the 12 identified housekeeping genes. nSolver was used. TMBioinformatics and statistical analyses, including hierarchical clustering and volcano plots, were performed with the Ingenuity® software 4.0.70 version and the Ingenuity® Human Immunology V2 Analysis Advanced Analysis Module. As no statistical significance with a P-adjusted value < 0.05 could be detected, we used raw p-values < 0.05 to analyze the data. As the cross-reactivity with mouse genes was high (down to 85%) in the Ingenuity® Human Immunology V2 panel, we performed analyses including all genes in the panel and excluded mouse genes with a cross-reactivity > 85%. This was due to information conveyed to us by the Nanostring facility that any value below 85% should not interfere with the analysis. To align the findings of this study with the latest state-of-the-art of the AD gene profile, selected relevant genes in atopic dermatitis were chosen.

[0198] Ex vivo human skin explant stimulation with Th2 polarizing cocktail

[0199] Full-thickness human skin biopsies were obtained by punch biopsies of residual surgical tissue. Biopsies were obtained with a size of 3 mm 2 and transferred onto mesh transport wells in 12-well plates. The dermis of the skin was immersed in a specially reinforced medium, leaving the epidermis exposed to air. Biopsies were incubated for up to 24 hours under optimal conditions and then cultured for an additional 24 hours in the presence of a Th2 polarizing inflammatory cocktail and the test article.

[0200] Example 3: Analysis of the IL-15 pathway in samples from atopic dermatitis patients

[0201] The expression of IL-15 and related genes (“IL-15 pathway”) was analyzed in various well-characterized sample sets or databases from atopic dermatitis patients and controls; some samples were collected from patients who had received dupilumab treatment and possible correlation analyses with clinical outcomes of the treatment were performed. The aim of these analyses was to further characterize the IL-15 pathway in AD and possibly identify a subset of patients who can preferentially benefit from treatment with an IL-15 inhibitor. Analyses using state-of-the-art technologies included:

[0202] - Transcriptomic analysis from public databases

[0203] - qPCR analysis from 30 healthy and AD skin biopsies

[0204] - Proteomic analysis (48 target genes) of 10 healthy and AD skin biopsies

[0205] - Immunohistochemical analysis of 20 healthy and AD skin biopsies

[0206] - Single-cell RNA sequencing of 10 healthy and AD skin biopsies

[0207] IL-15 levels were measured in 30 cases of AD sera and healthy sera using ultrasensitive technology.

[0208] To analyze data from public databases, microarray dataset GSE130588 containing data from human atopic dermatitis skin and healthy skin samples downloaded from the Gene Expression Omnibus (GEO) public database was used as described in Guttman-Yassky et al., 2019, J. Allergy Clin. Immunol., 143, 155-172.

[0209] In a randomized, placebo-controlled, double-blind, phase 2 trial (ClinicalTrials.gov: NCT01979016) conducted at 5 medical centers in the United States and Canada, biopsy specimens were collected from lesional and non-lesional skin to assess the efficacy and safety of dupilumab compared with placebo in patients with moderate-to-severe atopic dermatitis. Patients received a 400-mg loading dose of dupilumab or placebo at day 1, followed by a once-weekly subcutaneous injection of 200 mg of dupilumab or placebo for a total of 16 weeks. RNA was extracted and then hybridized to Affymetrix Human U133Plus 2.0 gene arrays (Affymetrix, Santa Clara, CA, USA). Raw CEL format files were converted to expression profile format by 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.

[0210] It was observed that IL-15 expression was significantly increased in both non-lesional (NL) and lesional (L) skin of atopic dermatitis Figure 10 ), consistent with previous reports (Karlen and Simon, 2020, Int. Arch. Allergy Immunol., 181, 417-421). IL-15 expression was also significantly higher in lesional atopic dermatitis skin than in non-lesional atopic dermatitis skin. It was also observed that while dupilumab treatment slightly decreased IL-15 expression in lesional atopic dermatitis skin, the effect was not significant. In fact, at week 16 (W16) of dupilumab treatment, IL-15 expression in both lesional and non-lesional atopic dermatitis skin remained significantly higher compared to healthy skin.

[0211] These results indicate that, as already observed in the mouse humanized model (Figure 9 ), patient treatment with anti-IL-4R antibody dupilumab does not completely suppress the IL-15 pathway, and thus further supports that anti-IL-15 therapy can be advantageously administered to patients who are non-responsive to anti-IL-4R antibodies, or in combination with these or other therapies.

[0212] Example 4: Generalization of the effects of anti-IL15 antibody hu-BE29-2 to other antibodies sharing the same binding epitope

[0213] The above effects can reasonably be generalized to other antibodies sharing the same binding epitope, such as the antibodies described in WO 2016 / 001275, in particular hu-BE29-10 & 24.

[0214] As described below, it has been demonstrated that these anti-human IL 15 antibodies share the same epitope specificity as hu-BE29-2.

[0215] A structured peptide library of IL-15 was synthesized using the proprietary Chemical Ligation on Peptide Scaffolds (CLIPS) technology of Pepscan B.V., which peptides were designed as single loop, double loop, triple loop, sheet-like fold, helix-like fold and combinations thereof (Timmerman 2007, Journal of Molecular recognition, 20: 64-73). CLIPS templates were coupled to the side chain thiol groups of cysteine residues and peptide arrays were prepared. Antibodies hu-BE29-2, hu-BE29-10 and hu-BE29-24 were tested for binding to each of the synthetic peptides by ELISA technology, wherein color development was quantified with 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 the same epitope in IL-15 consisting of the sequence of amino acids 83 to 93 of SEQ ID NO: 1. https: / / doi.org / 10.1002 / jmr.846 ). CLIPS templates were coupled to the side chain thiol groups of cysteine residues and peptide arrays were prepared. Antibodies hu-BE29-2, hu-BE29-10 and hu-BE29-24 were tested for binding to each of the synthetic peptides by ELISA technology, wherein color development was quantified with 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 the same epitope in IL-15 consisting of the sequence of amino acids 83 to 93 of SEQ ID NO: 1.

[0216] SEQUENCE LISTING

[0217] Human mature IL-15 (Homo sapiens), GenBank: CAA71044.1

[0218] SEQ ID NO: 1

[0219] MDFQVQIFSFLLISASVIMSRANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS

[0220] Cytokine modulator peptide from WO 2015 / 089217, BNZ132 (also known as BNZ-1, BNZ-13, YT-033, EQ-101)

[0221] SEQ ID NO: 2

[0222] IKEFLQRFIHIVQSIINTS

[0223] Gamma-cytokine modulator peptide from US2019 / 0070263

[0224] SEQ ID NO: 3

[0225] PKEFLERFVHLVQMFIHQSLS

[0226] IL-15 inhibitor peptide US7,736,638B2 SEQ ID NO: 4

[0227] SEQ ID NO: 4

[0228] KVTAMKCFLL

[0229] huVH1 from WO 2016 / 001275

[0230] SEQ ID NO: 5

[0231] EVRLMASGGGLVQPGGSLRLSCAAS EFTFSNYAMS WVRQAPGKGLEWVA TISRGGDYTYYPDSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYC ARRVSMITGGWAMDY WGQGTLVTVSS

[0232] huVH2 from WO 2016 / 001275

[0233] SEQ ID NO: 6

[0234] EVQLVESGGGLVQPGGSLRLSCAAS EFTFSNYAMS WVRQAPGKGLEWVA TISRGGDYTYYPDSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYC ARRVSMITGGWAMDY WGQGTLVTVSS

[0235] huVH8 from WO 2016 / 001275

[0236] SEQ ID NO: 7

[0237] EVRLMASGGGLVQPGGSLRLSCAASEFTFSNYAMSWVRQAPGKGLEWVATISRGGDYTYYPDTVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARRVSMITGGYAMDYWGQGTLVTVSS

[0238] huVH18 from WO 2016 / 001275

[0239] SEQ ID NO: 8

[0240] EVQLVESGGGLVQPGGSLRLSCAASEFTFSNYAMSWVRQAPGKGLEWVATISRGGDYTYYPDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARRVSMITGGYAMDYWGQGTLVTVSS

[0241] huVL1 from WO 2016 / 001275

[0242] SEQ ID NO: 9

[0243] DVVMTQSPLSLPVTLGQPASISCRSSQSIVDITGNTYLEWYQQRPGQSPRLLIYKVFNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQDSFVPYTFGQGTKLEIK

[0244] Heavy chain from 146B7, also known as AMG 714 (WO 2005 / 044303)

[0245] SEQ ID NO: 10

[0246] MGWTLVFLFLLSVTAGVHSEVQLVQSGAEVKKPGESLKISCKVSGYFFTTYWIGWVRQMPGKGLEYMGIIYPGDSDTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARGGNWNCFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0247] Light chain from 146B7, also known as AMG 714 (WO 2005 / 044303)

[0248] SEQ ID NO: 11

[0249] MVSSAQFLGLLLLCFQGTRCEIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASRRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQRYGSSHTFGQGTKLEISRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Variable heavy chain region from 70a WO 2018 / 119246

[0250] SEQ ID NO: 12

[0251] QVQLQESGPGLVKPSGTLSLTCAVSGGSISSSNWWSWVRQPPGKGLEWIGEIYHYGYTNYNPSLKSRVTISVDKSKNQFSLKLSSVTAADTAVYYCAREGIGWPSFDYWGQGTLVTVSS

[0252] Variable light chain region from 70a WO 2018 / 119246

[0253] SEQ ID NO: 13

[0254] SSELTQDPAASVALGQTVRITCQGDTLRSYYASWYQQKPGQAPILVIYGKNNRPSGIPDRFSGSSSGNTASLTITGAQAEDEADYYCNSRDLSGKNLVFGGGTKLTVL

[0255] Variable heavy chain region from huABC2 WO 2011 / 127324

[0256] SEQ ID NO: 14

[0257] MKLWLNWVFLLTLLHGIQCEVQLVESGGGLVQPGGSLRLSCAASGFTFSDFYMEWVRQAPGKGLEWIAAASRNKANDYTTEYSASVKGRFIVSRDDSKNSLYLQMNSLKTEDTAVYYCARSYYRYDGMDYWGQGTTVTVSS

[0258] Variable light chain region from huABC2 WO 2011 / 127324

[0259] SEQ ID NO: 15

[0260] MDFQVQIFSFLLISASVIVSRGEIVLTQSPATLSLSPGERATLSCSAISSVSYMYWYQQKPGQAPRLLIYDTSNLVSGVPARFSGSGSGTDYTLTISSLEPEDFAYYYCQQWNTYPYTFGGGTKVEIK

[0261] Heavy chain from huBE29-2 (WO 2016 / 001275)

[0262] SEQ ID NO: 16

[0263] EVQLVESGGGLVQPGGSLRLSCAASEFTFSNYAMSWVRQAPGKGLEWVATISRGGDYTYYPDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARRVSMITGGWAMDYWGQGTLVTVSS ASTKGPSVFPLAPSSKSTSGGTAA LGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPK SCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQ YNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGF YPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0264] Heavy chain from huBE29-2 (WO 2016 / 001275)

[0265] SEQ ID NO: 17

[0266] DVVMTQSPLSLPVTLGQPASISCRSSQSIVDITGNTYLEWYQQRPGQSPRLLIYKVFNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQDSFVPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0267] Heavy chain from huBE29-10 (WO 2016 / 001275)

[0268] SEQ ID NO: 18

[0269] EVRLMASGGGLVQPGGSLRLSCAASEFTFSNYAMSWVRQAPGKGLEWVATISRGGDYTYYPDTVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARRVSMITGGYAMDYWGQGTLVTVSS ASTKGPSVFPLAPSSKSTSGGTAA LGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPK SCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQ YNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGF YPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0270] Heavy chain from huBE29-24 (WO 2016 / 001275)

[0271] SEQ ID NO: 19

[0272] EVQLVESGGGLVQPGGSLRLSCAASEFTFSNYAMSWVRQAPGKGLEWVATISRGGDYTYYPDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARRVSMITGGYAMDYWGQGTLVTVSS ASTKGPSVFPLAPSSKSTSGGTAA LGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPK SCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQ YNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGF YPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Claims

1. Use of an interleukin-15 inhibitor in the manufacture of a medicament for the treatment of atopic dermatitis, wherein the interleukin-15 inhibitor is an anti-IL-15 antibody.

2. The use of claim 1, wherein the anti-IL-15 antibody comprises: (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 1, 2, 3, or 4 amino acids are substituted with a different amino acid, wherein the substitutions are selected from: (i) within the heavy chain variable framework region, arginine (R) at position H3 (VH RH3) is substituted with glutamine (Q), methionine (M) at position H5 (VH MH5) is substituted with valine (V), alanine (A) at position H6 (VH AH6) is substituted with glutamic acid (E), alanine (A) at position H49 (VH AH49) is substituted with serine (S), (ii) within the heavy chain CDR2, aspartic acid (D) at position H61 (VH DH61) is substituted with glutamic acid (E), serine (S) at position H62 (VH SH62) is substituted with threonine (T), and (iii) within the heavy chain CDR3, methionine (M) at position H98 (VH MH98) is substituted with leucine (L), phenylalanine (F), isoleucine (I), or alanine (A), tryptophan (W) at position H100C (VH WH100C) is substituted with tyrosine (Y), phenylalanine (F), or alanine (A), methionine (M) at position H100E (VH MH100E) is substituted with leucine (L), phenylalanine (F), or isoleucine (I); and (2) a light chain variable region of SEQ ID NO:

9.

3. The use of claim 1, wherein the anti-IL-15 antibody comprises: (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 1, 2, 3, or 4 amino acids are substituted with a different amino acid, and (2) a light chain variable region of SEQ ID NO:

9.

4. The use of claim 1, wherein the anti-IL-15 antibody comprises: (1) a heavy chain variable region of the amino acid sequence of SEQ ID NO: 5 having a substitution in the sequence of SEQ ID NO: 5 selected from: (i) VH RH3 is substituted with glutamine (Q), VH MH5 is substituted with valine (V), 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); and (2) a light chain variable region of the amino acid sequence of SEQ ID NO:

9.

5. The use of any one of claims 1-4, wherein the anti-IL-15 antibody comprises: (1) a heavy chain variable region selected from the group consisting of: SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8, and (2) a light chain variable region of SEQ ID NO:

9.

6. The use of any one of claims 1-4, wherein the anti-IL-15 antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises SEQ ID NO: 6 and the light chain variable region comprises SEQ ID NO:

9.

7. The use of any one of claims 1-6, wherein the anti-IL-15 antibody comprises: - 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.

8. The use of any one of claims 1-6, wherein the anti-IL-15 antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises SEQ ID NO: 16 and the light chain comprises SEQ ID NO:

17.

9. The use of any one of claims 1-8, wherein the anti-IL-15 antibody binds to human IL-15.

10. The use of any one of claims 1-9, wherein the anti-IL-15 antibody is isolated.

11. The use of any one of claims 1-10, wherein the anti-IL-15 antibody is monoclonal.

12. The use of any one of claims 1-11, wherein the anti-IL-15 antibody is humanized.

13. The use of any one of claims 1-12, wherein the anti-IL-15 antibody is administered to a human subject having atopic dermatitis.

14. The use of any one of claims 1-13, wherein the anti-IL-15 antibody is administered orally, parenterally, sublingually, transdermally, transmucosally, or topically.

15. The use of claim 14, wherein the anti-IL-15 antibody is administered intravenously or subcutaneously.

16. The use of any one of claims 1-15, wherein the anti-IL-15 antibody is administered in combination with an agent for the prevention and / or treatment of atopic dermatitis.

17. The use of claim 16, wherein the agent is an anti-IL-4R antibody such as dupilumab, an anti-IL-13 antibody such as tralokinumab, a JAK inhibitor such as abrocitinib, upadacitinib (oral), or ruxolitinib (topical), or a corticosteroid such as dexamethasone.

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