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

Technical Field

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

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

[0003] 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 (aged <18 years) (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 that the prevalence of previously diagnosed and active AD ranged from 2.1% to 4.9%, 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. [Journal of Medical Clinics of North America], 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 predominance, especially in adolescence and adulthood (Silvenberg et al., 2020, supra).

[0004] The condition usually begins in childhood and can continue through adulthood with varying severity during these ages. AD is a complex, multifactorial disease that involves:

[0005] i) Strong genetic component: AD can be considered a familial disease, and several susceptibility genes have been identified.

[0006] ii) Environmental exposures, including irritants and pruritogens, pathogens, climatic factors, ultraviolet radiation, outdoor and indoor air pollutants, tobacco smoke exposure, water hardness, urban versus rural living, diet, breastfeeding, probiotics and prebiotics. Some of these factors may explain the increase in atopic dermatitis over the past few decades.

[0007] iii) Altered skin permeability. About 30% of people with atopic dermatitis have mutations in the gene that produces filaggrin (FLG), which increases the risk of early atopic dermatitis attacks and the development of asthma. Flaggrin plays an important role in keeping the skin surface slightly acidic, thus conferring its antimicrobial effects.

[0008] These different components are interconnected and work synergistically to drive a complex immunopathology involving multiple pathways, cytokines, and cell types, ultimately leading to the signs and symptoms of atopic dermatitis. Evidence suggests that several cytokines, such as the T helper 2 cytokine interleukin 4 (IL-4) and the closely related IL-13, are important components of AD pathogenesis. In fact, an antibody (dupirumab) targeting the IL-4 receptor α subunit (IL-4Rα) shared between IL-4 and IL-13 was the first biologic approved in Europe and the United States for the treatment of atopic dermatitis, either alone or in combination with corticosteroids (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 (troloxetine) has also been approved in Europe and the United States, alone or in combination with corticosteroids (Wollenberg et al., 2021, Br J Dermatol. [British Journal of Dermatology], 184(3):437–449, doi:10.1111 / bjd.19574; Silverberg et al., 2021, Br J Dermatol. [British Journal of Dermatology], 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 pathways other than IL-4 / IL-13 are in development.

[0009] Interleukin-15 (IL-15), also known as MGC9721, is a cytokine that is well known as a regulator of natural killer (NK) and T cell activation, survival, and proliferation (Waldmann et al., 2020, J. Exp. Med., 217, e20191062, https: / / doi.org / 10.1084 / jem.20191062). However, it has also been shown to be a 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), consistent with their shared receptor signaling components (IL-2 / 15Rβ and IL-2 / 15Rγc). However, the specificity of IL-15 versus IL-2 is provided by distinct private α-chain receptors that complete the IL-15Rαβγ heterotrimeric high-affinity receptor complex, allowing for differential responsiveness depending on the ligand and high-affinity receptor expressed (Fehniger and Caligiuri, 2001, Blood 97, 14-32).

[0010] 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 reduce clinical and disease features 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; et al., 2019, Lancet Gastroenterol Hepatol, 4, 948–959; Vicari et al., 2017, MAbs, 9, 927–944; Xing et al., 2014, Nature Medicine, 20, 1043–1049, https: / / doi.org / 10.1038 / nm.3645; Richmond et al., 2018, Sci Transl Med, 10, eaam7710, https: / / doi.org / 10.1126 / scitranslmed.aam7710 ), and in some cases, the above results have been 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 may not be harmful. For example, in viral diseases, increased IL-15 expression is associated with improved viral clearance (Verbist et al., 2011, J. Immunol. 186, 174–182. https: / / doi.org / 10.4049 / jimmunol.1002613).

[0011] Several reports have described increased expression of IL-15 or its receptors 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).

[0012] Conversely, reduced human IL-15 expression has also been proposed to contribute to the pathogenesis of atopic dermatitis progression (Ong et al., 2002, J. Immunol. [Journal of Immunology] 168, 505–510). In addition, it has been reported that blood natural killer (NK) cells are deficient in atopic dermatitis patients, while the skin of atopic dermatitis patients is enriched with activated NK cells (Mack et al., 2020, Sci. Transl. Med. [Science Translational Medicine], 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 an IL-15 agonist molecule would have a beneficial effect on atopic dermatitis. To further support this hypothesis, they treated mice with an IL-15 agonist molecule in the MC903 atopic dermatitis model (induced by topical application of a vitamin D3 analog) and observed improvements in clinical scores and other atopic dermatitis features that were dependent on NK cells (Mack et al., 2020, supra).

[0013] In conclusion, it is not possible to determine whether IL-15 overexpression is harmful or beneficial in atopic dermatitis based on the current technology.

[0014] Atopic dermatitis is the most common chronic inflammatory skin disease worldwide, and treatment options remain limited. Therefore, given the enormous burden of AD on affected subjects, there is a need to better understand the underlying causes of AD and to identify new agents with substantial beneficial effects in the management and treatment of AD. Summary of the invention

[0015] The present invention generally relates to the use of agents capable of inhibiting interleukin-15 for the treatment of atopic dermatitis in an undefined global patient population, and in atopic dermatitis patients who are unresponsive to current treatments such as anti-IL-4R, anti-IL-4 or anti-IL-13 antibodies or corticosteroids.

[0016] According to a first embodiment, an interleukin-15 inhibitor for use in treating atopic dermatitis is provided.

[0017] According to another embodiment, provided is a method for preparing a pharmaceutical composition for treating atopic dermatitis using an interleukin-15 inhibitor.

[0018] According to another aspect of the present invention, there is provided a method for preventing and / or treating atopic dermatitis in a subject, the method comprising administering a therapeutically effective amount of an interleukin-15 inhibitor or a pharmaceutical composition thereof to a subject in need thereof.

[0019] According to another aspect of the present invention, a pharmaceutical composition is provided, which comprises an interleukin-15 inhibitor and an agent for treating atopic dermatitis and a pharmaceutically acceptable carrier, diluent or excipient thereof.

[0020] Other features and advantages of the present invention will become apparent from the following detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 : Increase in IL-15 (A) and IL-15Rα (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 media as described in Example 1. Symbols represent individual explants, and group means are presented as bar graphs + / - standard deviations.

[0022] Figure 2 : IL-15 (A) and IL-15Rα (B) gene expression induced 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 corticosteroid betamethasone as described in Example 1. Symbols represent individual explants and group means are represented as bars + / - standard deviations.

[0023] Figure 3 :As described in Example 2, after injection of Th2 polarized PBMC (Th2) or LPS activated Th2 polarized PBMC (activated Th2) two weeks, skin IL-15 and IL-15Rα expression increased in the humanized atopic dermatitis model analyzed in human skin xenografts. In the epidermis, the expression of IL-15 (A) and IL-15Rα (C) was quantified as the average intensity signal, while in the dermis, the expression of IL-15 (B) was quantified as the number of IL-15 positive cells per 100 dermal area units. Symbols represent each xenograft mouse (graft / PBMC from 2 separate human donors), and groups are represented as violin plots showing group medians and quartiles.

[0024] Figure 4: As described in Example 2, the effect of anti-IL-15 antibodies on clinical scores was measured and analyzed in human skin xenografts when injected with LPS-activated Th2 polarized PBMCs (before treatment) and after 2 weeks and 4 weeks of treatment with various antibodies. As shown on the x-axis, the IgG1 group was injected with control isotype antibodies for 4 weeks, the anti-IL-15 group was injected with anti-IL-15 antibodies, and the IgG1+anti-IL-15 group was injected with control isotype antibodies for 2 weeks and then anti-IL-15 antibodies for 2 weeks. Symbols represent each xenograft mouse, and groups are represented as violin plots showing group medians and quartiles.

[0025] Figure 5 : Effects of anti-IL-15 antibodies on epidermal properties in a humanized atopic dermatitis model as described in Example 2. A: Epidermal thickness of human skin xenografts was analyzed in various antibody treatment groups 4 weeks after injection of LPS-activated Th2 polarized PBMCs. As shown on the x-axis, the IgG1 group was injected with control isotype antibodies for 4 weeks, the anti-IL-15 group was injected with anti-IL-15 antibodies, and the IgG1+anti-IL-15 group was injected with control isotype antibodies for 2 weeks and then anti-IL-15 antibodies for 2 weeks. Symbols represent each xenograft mouse, and groups are represented as violin plots showing group medians and quartiles. B: Epidermal proliferation of human skin xenografts was analyzed in various antibody treatment groups 4 weeks after injection of LPS-activated Th2 polarized PBMCs by measuring the expression of Ki67 in the basal layer of the human epidermis. As shown on the x-axis, the IgG group was injected with control isotype antibodies for 4 weeks (due to differences between animals and small n per group, the IgG1 group and the IgG4 group were combined), the anti-IL-15 group was injected with anti-IL-15 antibodies, and the IgG+anti-IL-15 group was injected with control isotype antibodies for 2 weeks and then injected with anti-IL-15 antibodies for 2 weeks. Symbols represent each xenograft mouse, and groups are represented as violin plots showing group medians and quartiles; C: Analysis of epidermal filaggrin (FLG) in human skin xenografts by immunofluorescence 4 weeks after injection of LPS-activated Th2-polarized PBMCs. As shown on the x-axis, the IgG group was injected with control isotype antibodies for 4 weeks (due to differences between animals and small n per group, the IgG1 group and the IgG4 group were combined), the anti-IL-15 group was injected with anti-IL-15 antibodies, and the IgG+anti-IL-15 group was injected with control isotype antibodies for 2 weeks and then injected with anti-IL-15 antibodies for 2 weeks. Symbols represent individual xenograft mice, and groups are represented as violin plots showing group medians and quartiles.

[0026] Figure 6: As described in Example 2, 4 weeks after injection of LPS-activated Th2 polarized PBMCs, when analyzed by immunofluorescence in various antibody treatment groups, in a humanized atopic dermatitis model, anti-IL-15 antibodies did not affect the number of dermal T (A: CD3+), NK (B: CD56+) and NKT (C: CD3+CD56+) cells. As shown on the x-axis, the IgG group was injected with control isotype antibodies within 4 weeks (due to differences between animals and small n per group, IgG1 and IgG4 groups were combined), the anti-IL-15 group was injected with anti-IL-15 antibodies, and the IgG+ anti-IL-15 group was injected with control isotype antibodies for 2 weeks and then injected with anti-IL-15 antibodies for 2 weeks. Symbols represent each xenograft mouse, and groups are represented as violin plots showing group medians and quartiles.

[0027] Figure 7 : As described in Example 2, anti-IL-15 induced a decrease in the expression of cytokines associated with atopic dermatitis in the humanized atopic dermatitis model was 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-γ; B: IL-4; C: IL-17A; D: IL-22). The number of cells expressing IL-4, IL-17A or IL-22 in each xenograft was calculated by adding the number of cells expressing each cytokine (E). As shown on the x-axis, the IgG group was injected with control isotype antibodies for 4 weeks (due to inter-animal differences and small n per group, the IgG1 group and the IgG4 group were combined), the anti-IL-15 group was injected with anti-IL-15 antibodies, and the IgG+anti-IL-15 group was injected with control isotype antibodies for 2 weeks and then injected with anti-IL-15 antibodies for 2 weeks. Symbols represent individual xenograft mice, and groups are represented as violin plots showing group medians and quartiles.

[0028] Figure 8 :As described in Example 2, heat map shows the Log2FC of differentially regulated genes clustered by immune cell type (T helper cells, T cells, B cells, left) or atopic dermatitis-related genes (right) after therapeutic treatment (IgG-CALY) or preventive treatment (CALY) with anti-IL-15 antibodies in a humanized atopic dermatitis mouse model compared to a single control isotype. The lighter the shade, the higher the fold difference. The only upregulated gene is IL32, and all other genes remain unchanged or downregulated. Asterisks indicate p values ​​(nonparametric Mann-Whitney test) of significant regulation <0.05. All data are based on Nanostring transcriptomic analysis (10 xenografts from 3 separate human donors in each group).

[0029] Fig. 9: As described in Example 2, two weeks after injection of LPS-activated Th2 polarized PBMCs in various treatments, anti-IL-4R antibodies and dexamethasone have different effects on IL-15 expression between the epidermis and dermis in a humanized atopic dermatitis model when analyzed in human skin xenografts. As shown on the x-axis, the IgG group was injected with control isotype antibodies for 4 weeks (due to inter-animal differences and small n per group, the IgG1 group and the IgG4 group were combined), the dexamethasone group was injected with topical dexamethasone in the last 2 weeks of the study, the anti-IL-4R group was injected with anti-IL-4R antibodies, and the IgG+anti-IL-4R group was injected with control isotype antibodies for 2 weeks and then anti-IL-4R antibodies for 2 weeks. In the epidermis, the expression of IL-15 was quantified as the mean intensity signal, while in the dermis, the expression of IL-15 was quantified as the number of IL-15 positive cells per 100 dermal area units. Symbols represent individual xenograft mice, and groups are represented as violin plots showing group medians and quartiles.

[0030] Fig.10 : Normalized gene expression values ​​of IL-15 expression in skin biopsy samples between healthy volunteers and atopic dermatitis patient groups at baseline (W0) or after 4 weeks (W4) or 16 weeks of dupilumab treatment as described in Example 3. NL: non-lesional skin; L: lesional skin. Each symbol represents a separate individual, the bars represent the mean of each group, and the error bars represent 95% confidence intervals.

[0031] Statistical analysis was performed using Graph Pad Prism software. Figure 1 , 3 , 9: Use the nonparametric Mann-Whitney test to compare the two experimental groups. ( Figure 1 : ****p<0.0001; 4: **p<0.01, ***p<0.001; 9: **p<0.01, ***p<0.001). Figure 2 , 7 : One-way ANOVA Kruskal-Wallis test was used to compare the two groups treated with anti-IL-4R antibody or betamethasone with the group treated with Th2 polarizing media alone ( Figure 2 : *p<0.05; Figure 7 :*p<0.05, **p<0.01). Figure 4 , 5 , 6: One-way ANOVA combined with Dunnett's multiple comparison test was used to compare the experimental groups ( 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). Fig.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

[0032] The terms "interleukin 15", "interleukin-15", "IL-15" herein refer 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, whose sequence is disclosed in 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, wherein the indicated positions correspond to 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. The amino acid sequences of immature IL-15 from other species are available in the art, and include, for example, mouse IL-15 (UniProtKB Accession No. P48346, corresponding to the mature IL-15 form), rat IL-15 (UniProtKB Accession No. P97604, corresponding to the mature IL-15 form), rhesus monkey IL-15 (UniProtKB Accession No. NP_001038196, XP_001091166, XP_001091289XP_001091416, corresponding to the mature IL-15 form), cynomolgus monkey IL-15 (predicted sequence from NCBI Accession No. XP_005556036.1, corresponding to the mature IL-15 form), and dog IL-15 (UniProtKB Accession No. 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. Of note, two alternatively spliced ​​transcript variants of IL-15 have been reported. Although these two isoforms produce the same mature protein, they differ in their cellular transport.

[0033] The term "IL-15 inhibitor" refers to an agent capable of inhibiting interleukin 15. Inhibition of interleukin 15 can be evaluated, for example, by measuring the inhibition of IL-15 binding to the signaling chain of the IL-15 receptor (Il-15Rβ and / or IL-15Rγ) or the IL-15-specific IL-15Rα chain using techniques such as surface plasmon resonance or ELISA. Inhibition of interleukin 15 can also be evaluated at the functional level, for example, by testing the inhibitory effect by measuring the following indicators: i) inhibition of signaling events downstream of IL-15, such as STAT5 phosphorylation, which can be measured using Western blot, flow cytometry or immunofluorescence techniques in tissue sections, whole cells or cell extracts; ii) 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 people or patients; iii) inhibition of IL-15-dependent mechanisms in vivo, such as NK and other leukocyte homeostasis that can be measured by flow cytometry in the blood of animals, healthy people or patients. Among these agents, some interleukin 15 inhibitors have been developed or are being actively developed for the treatment of different diseases, for example as described in WO 2016 / 001275, WO 2015 / 089217, WO 2005 / 044303, WO 2018 / 119246, WO 2011 / 127324.

[0034] Interleukin 15 inhibitors can be small molecule inhibitors (e.g., low molecular weight organic compounds <900 Da), peptides, monoclonal antibodies (mAbs), chimeric proteins 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.

[0035] The term "antibody" as used herein refers to a polypeptide that binds to an antigen. This includes complete antibodies and any antigen-binding fragments. The term "antibody" is used in its broadest sense and includes monoclonal antibodies, polyclonal antibodies, human antibodies, humanized antibodies, chimeric antibodies, bispecific antibodies, multispecific antibodies, and further engineered antibodies, as long as the characteristic properties of the present invention, especially the ability to bind to 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 the following literature: Sawant et al., 2020, Int. J. Mol. Sci. [International Journal of Molecular Sciences] 2020, 21, 7496. https: / / doi.org / 10.3390 / ijms21207496. As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous antibody population, that is, except for possible naturally occurring mutations that may be present in small amounts, the individual antibodies comprising the population are identical. Monoclonal antibodies are highly specific to a single antigenic site. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method.

[0036] The term "chimeric antibody" generally refers to an antibody, usually prepared by recombinant DNA technology, comprising a variable region from one source or species and at least a portion of a constant region derived from a different source or species. Typical examples of chimeric antibodies include antibodies comprising a mouse variable region and a human constant region. As defined herein, this term also includes antibodies comprising 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 antibodies comprising heavy chain CDR1, CDR2 and CDR3 of a first human antibody and light chain CDR1, CDR2 and CDR3 of a second human antibody.

[0037] The term "humanized antibody" refers to an antibody from a non-human species having one or more complementary determining regions (CDRs) from the non-human species and a framework region from a human immunoglobulin molecule. A humanized antibody may optionally further comprise one or more framework residues derived from the non-human species from which the CDRs were obtained.

[0038] The term "human antibody" or "fully human antibody" refers to antibodies in which the variable and 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.

[0039] The term "isolated antibody" refers to an antibody that has been separated from components of its natural environment. For example, an isolated antibody has been purified to a purity greater than 95% or 99% when measured by methods in the art including electrophoresis (e.g., SDS-PAGE, isoelectric focusing, capillary electrophoresis) or chromatography (e.g., ion exchange or reversed phase high performance liquid chromatography (HPLC)) methods (see, e.g., Flatman et al., 2007, J Chromatogr B Analyt Technol Biomed Life Sci [Chromatography Magazine B: Analytical Technology in Biomedicine and Life Sciences], 848:79-87).

[0040] The term "variant" can be applied to polynucleotides and / or polypeptides. For example, as mentioned 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 that 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 a reference peptide sequence except for the deletion, insertion and / or substitution of a few amino acids (e.g., 1, 2, 3, 4, 5, or 6 amino acids). Substantially homologous refers to a variant amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to a reference amino acid sequence. Variant nucleic acid sequences may 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 with a reference nucleic acid sequence. The identity of two amino acid sequences or two nucleic acid sequences can be determined by visual inspection and / or mathematical calculations, or more easily by using a known computer program for sequence comparison (e.g., Clustal software package version 1.83) to compare sequence information and determine. Variants may include sequences with at least one conservatively substituted amino acid, meaning that a given amino acid residue is replaced by a residue with similar physicochemical characteristics. Examples of conservative substitutions include replacing one aliphatic residue with another, such as Ile, Val, Leu or Ala replacing each other, or replacing another with a polar residue, such as Lys with Arg; Glu with Asp; or replacing between Gln and Asn. Other such conservative substitutions (e.g., substitutions of entire regions with similar hydrophobicity characteristics) are well known (Kyte et al., 1982, J. Mol. Biol. [Journal of Molecular Biology] 157: 105-131). For example, a "conservative amino acid substitution" may involve replacing a natural amino acid residue with a non-natural residue, thereby having little or no effect on the polarity or charge of the amino acid residue at that position. Alternatively, substitutions of one or more amino acids present in the original polypeptide are not conservative, which may produce a variant with modified properties compared to the reference antibody. The desired amino acid substitutions (whether conservative or non-conservative) can be determined by one skilled in the art when such substitutions are desired. The term "variant" also includes peptides or polypeptides that are substantially homologous to a reference peptide sequence, but whose amino acid sequence differs from that of the reference sequence because one or more amino acids have been chemically modified or substituted with an amino acid analog. The term also includes glycosylated polypeptides.

[0041] As used herein, the term "binding" of an inhibitor to a target antigen refers to at least a transient interaction or association of the inhibitor with the target antigen (e.g., IL-15) or with a fragment of the target antigen comprising an epitope recognized by the inhibitor. As used herein, antibodies that bind to IL-15 are also referred to as anti-IL-15 antibodies.

[0042] When applied to antibodies, the terms "selectively bind", "specifically bind" and "specific for" indicate that the antibody preferentially recognizes and / or binds to a target polypeptide or epitope, i.e., has a higher affinity than to any other antigen or epitope, i.e., binding to the target polypeptide can be distinguished from non-specific binding to other antigens. The binding affinity of an antibody can be readily determined by a person skilled in the art, for example, by equilibrium dialysis, equilibrium binding, surface plasmon resonance or spectroscopy (e.g., using fluorescence measurement). In particular, when using the surface plasmon resonance (SPR) technique, a biomolecule binding event results in a change in the refractive index at a surface layer to which a binding partner is immobilized, which is detected as a change in the surface plasmon resonance signal expressed in response units (RU). By measuring the real-time binding kinetics of an antibody to its target antigen, the SPR technique can determine the association rate (expressed in k) between the antibody and its target. a or k on association constant), its association strength (measured in k d or k off The affinity of an antibody for its target can be determined by determining its equilibrium dissociation constant, K D To quantitatively measure, K D Defined as K D =k d / k a , where k a is the association rate (k on ), and k d is the dissociation rate (k off ) (Murphy et al., 2006, Curr Protoc Protein Sci, [Latest Protein Science Experiment Guide], Chapter 19: Unit 19.14). Comparison of affinity and / or binding properties between two antibodies can be performed without actually determining the K of each antibody. D value, but is based on K D The binding affinity of a protein can be determined by quantitative measurement of the binding ratio (e.g., by ELISA or FACS analysis) or by qualitative measurement of affinity or inference of affinity (e.g., in a functional assay or in vitro or in vivo assay).

[0043] The term "blocking" or "neutralizing" activity of an inhibitor refers to its ability to inhibit the activity of its target. The neutralizing activity of an inhibitor can be determined by in vitro assays or in vivo assays or functional assays. When applied to inhibitors that bind IL-15, the term refers to the ability of the inhibitor to substantially neutralize IL-15 activity, which can correspond, for example, to 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-15Rαβγ or heterodimeric IL-15Rβγ receptor (Finch et al., 2011, Br J Pharmacol. [British Journal of Pharmacology] 162:480-90), inhibition of IL-15-induced B lymphocyte synthesis of immunoglobulins upon stimulation with anti-IgM or CD40 ligand (Litinskiy et al., 2012, Nat Immunol. [Natural Immunology], 3:822-9), inhibition of IL-15-induced activation of human neutrophils (Rathhé and Girard, 2004, J Leukoc Biol. [Journal of Leukocyte Biology], 76: 162-8), and inhibiting IL-15-induced production of proinflammatory cytokines by macrophages, dendritic cells or epithelial cells (Nanayakkara et al., 2013, Am J Clin Nutr. [American Journal of Clinical Nutrition], 98: 1123-35).

[0044] Unless otherwise limited by the definition of individual substituents, the term "substituted" means that the group is substituted by 1 to 5 substituents selected from the group consisting of "C1-C6 alkyl", "C3-C8 cycloalkyl", "heterocycloalkyl", "sulfonyl", "sulfonamide", "alkoxy", "alkoxycarbonyl", "halogen", "carboxyl", "halomethyl", cyano, hydroxyl, nitro, etc.

[0045] The term "pharmaceutically acceptable" refers to a carrier composed of a biologically or otherwise undesirable material.

[0046] The term "carrier" refers to any component present in a 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.

[0047] As used herein, "treatment" and "treating" and the like generally mean obtaining the desired pharmacological and physiological effects. The effect may be prophylactic, in terms of preventing or partially preventing a disease, its symptoms or conditions, and / or therapeutic, in terms of partially or completely curing a disease, condition, symptom or adverse reaction attributable to the disease. The term "treatment" as used herein encompasses any treatment of a disease in mammals, particularly humans, and includes: (a) preventing the onset of the disease in a subject who may be susceptible to the disease, for example based on family history, but has not yet been diagnosed with the disease; (b) inhibiting the disease, i.e., arresting its development; or (c) alleviating the disease, i.e., resolving the disease and / or its symptoms or conditions, e.g., improving or repairing damage. For example, treatment of celiac disease includes preventing, reducing, or even eliminating symptoms of the disease or disorder, such as partial or complete relief of atopic dermatitis symptoms, such as dry skin, itching, scratching, and the consequences of scratching (skin irritation, sensitivity, swelling), skin lesions including i) red to brown-gray patches (particularly on the hands, feet, ankles, wrists, neck, upper chest, eyelids, inside the bends of the elbows and knees, and on the face and scalp of infants); ii) small, raised bumps that may ooze fluid and form crusts when scratched, and iii) thickened, cracked, scaly skin.

[0048] The term "subject" as used herein refers to a mammal. For example, mammals contemplated by the present invention include humans, primates, laboratory rodents, and pets, such as dogs and cats.

[0049] The term "therapeutic effect" of a treatment or method according to the invention can be measured based on the changes that occur in the course of a disease or condition in response to the use or method according to the invention. For example, the therapeutic effect of a treatment or method according to the invention can be measured by its effect on disease signs or symptoms. Remission is achieved when a patient experiences partial or complete remission, or a reduction in undesirable disease symptoms.

[0050] As used herein, the term "effective amount" refers to an amount of at least one agent according to the present invention, or a pharmaceutical formulation thereof, which results in a detectable reduction in disease symptoms in a subject to which the agent is administered.

[0051] IL-15 inhibitors

[0052] IL-15 inhibitors according to the present invention include anti-IL antibodies, inhibitory peptides and small molecules.

[0053] According to one embodiment, the IL-15 inhibitor may be a small molecule, such as the small molecule described in Quemener et al., 2017, J. Med. Chem. [Journal of Medicinal Chemistry] 2017, 60, 6249-6272 DOI: 10.1021 / acs.jmedchem.7b00485, in particular a compound having the following structure:

[0054] wherein R is an optionally substituted phenyl group and n is an integer selected from 1 to 10, in particular molecules having the following structure:

[0055] 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 (also known as BNZ-1, also known as YT033, also known as EQ-101) (SEQ ID NO: 2) (IKEFLQRFIHIVQSIINTS) and the peptides described in US2019 / 0070263, such as PKEFLERFVHLVQMFIHQSLS (SEQ ID NO: 3) or fragments or variants of these sequences. Other peptide IL-15 inhibitors (SEQ ID NO: 4) or fragments or variants of these sequences are described in US 7,736,638.

[0056] According to another embodiment, the IL-15 inhibitor may be an anti-IL15 antibody or an antigen-binding fragment thereof. According to a specific aspect, the anti-IL15 antibody or an antigen-binding fragment thereof binds to IL-15, in particular human IL-15 or a fragment of IL-15.

[0057] The anti-IL15 antibodies according to the present invention generally exhibit high specificity for human IL-15. However, depending on the degree of sequence identity between IL-15 homologs of different species, a given antibody or antigen-binding fragment may exhibit cross-reactivity with IL-15 from at least one other species (e.g., primates (e.g., cynomolgus monkeys, rhesus monkeys, marmosets), mice, rats, dogs and / or rabbits). For antibodies against human IL-15, a certain degree of cross-reactivity with other mammalian forms of IL-15 may be desirable in some cases.

[0058] In a specific embodiment, the anti-IL15 antibodies or fragments thereof according to the present invention preferentially bind to human IL-15.

[0059] In yet another embodiment, the antibody or antigen-binding fragment thereof according to the present invention shows no cross-reactivity with rat IL-15 and / or mouse IL-15.

[0060] In one embodiment, the inhibitor according to the present invention preferentially inhibits IL-15, and optionally additionally exhibits some inhibitory effect on other proteins having homology to IL-15 (e.g. IL-2, in particular human IL-2 or IL-21) (as disclosed in WO2015 / 089217).

[0061] 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 itself can be assessed, for example, by IC 50 The neutralizing activity of the antibody can be reflected by the value. Generally, the neutralizing activity of the antibody can be determined by, for example, the following in vitro assay: an assay for measuring the level of inhibition of proliferation and / or survival of a cell line (e.g., Kit 225 or M-07e cells) induced by IL-15 in the presence of the antibody as described in WO 2016 / 001275.

[0062] In some embodiments, the IC of the antibodies and antigen-binding fragments thereof according to the present invention 50 Equal to or below 200 nM, in particular below 100 nM, in particular below 50 nM, below 30 nM, below 20 nM, more particularly below 10 nM, below 8 nM, below 7 nM, below 5 nM, below 4 nM, below 3 nM, below 2 nM, below 1 nM, below 0.5 nM, below 0.3 nM, below 0.2 nM, below 0.1 nM, below 0.05 nM, or below 0.03 nM for inhibiting IL-15 activity, such as IL-15 induced proliferation and / or survival of cell lines (e.g. Kit 225 or M-07e cells), as described in WO 2016 / 001275.

[0063] 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 present invention. In a particular embodiment, the variant may exhibit 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, compared to the parent antibody or fragment from which the variant is derived.

[0064] In a specific embodiment of the invention, the antibody against IL-15 or an antigen-binding fragment thereof that binds to IL-15 is a monoclonal antibody.

[0065] In a specific embodiment of the present invention, the antibody against IL-15 or an antigen-binding fragment thereof that binds to IL-15 is a bispecific or multispecific antibody, in particular an antibody that bispecifically targets IL-15 and IL-4R or IL-13 or IL-4.

[0066] In yet another specific embodiment of the present invention, the antibody against IL-15 or an antigen-binding fragment thereof that binds to IL-15 is a humanized antibody.

[0067] In another specific embodiment of the present invention, the antibody against IL-15 or the antigen-binding fragment thereof that binds to IL-15 is a recombinant antibody.

[0068] Antibodies against IL-15 or antigen-binding fragments thereof that bind to IL-15 suitable for use according to the present invention may be characterized by the portion thereof that interacts with the target protein, in particular its variable region, generally including a heavy chain variable region and a light chain variable region, such as the variable regions described in WO 2016 / 001275.

[0069] According to certain embodiments, the anti-IL-15 inhibitor is an isolated antibody or antigen-binding fragment thereof that binds IL-15, comprising:

[0070] (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 by different amino acids, wherein the substitution is selected from:

[0071] (i) in the heavy chain variable framework region, arginine (R) at position H3 (VH RH3) is replaced by glutamine (Q), methionine (M) at position H5 (VH MH5) is replaced by valine (V), alanine (A) at position H6 (VH AH6) is replaced by glutamic acid (E), and alanine (A) at position H49 (VH AH49) is replaced by serine (S),

[0072] (ii) in the heavy chain CDR2, aspartic acid (D) at position H61 (VH DH61) is replaced by glutamic acid (E), and serine (S) at position H62 (VH SH62) is replaced by threonine (T), and

[0073] (iii) in the heavy chain CDR3, methionine (M) at position H98 (VH MH98) is substituted by leucine (L), phenylalanine (F), isoleucine (I) or alanine (A), tryptophan (W) at position H100C (VH WH100C) is substituted by tyrosine (Y), phenylalanine (F) or alanine (A), and methionine (M) at position H100E (VH MH100E) is substituted by leucine (L), phenylalanine (F) or isoleucine (I);

[0074] as well as

[0075] (2) The light chain variable region of SEQ ID NO:9.

[0076] According to another specific embodiment, the anti-IL-15 inhibitor is an isolated antibody or antigen-binding fragment thereof that binds IL-15, comprising:

[0077] (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 by different amino acids, and

[0078] (2) The light chain variable region of SEQ ID NO:9.

[0079] According to another specific embodiment, the anti-IL-15 inhibitor is the isolated antibody or antigen-binding fragment thereof as claimed in claim 1, comprising:

[0080] (1) A heavy chain variable region of the amino acid sequence of SEQ ID NO: 5, which has a substitution in the sequence of SEQ ID NO: 5 selected from the following:

[0081] (i) VH RH3 was replaced by glutamine (Q), VH MH5 was replaced by valine (V), and VH AH6 was replaced by glutamic acid (E),

[0082] (ii) VH SH62 is substituted with threonine (T), and

[0083] (iii) VH WH100C was substituted with tyrosine (Y);

[0084] as well as

[0085] (2) A light chain variable region having the amino acid sequence of SEQ ID NO:9.

[0086] According to another specific embodiment, the anti-IL-15 inhibitor is an isolated antibody or antigen-binding fragment thereof that binds IL-15, comprising:

[0087] (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

[0088] (2) The light chain variable region of SEQ ID NO:9.

[0089] According to another specific embodiment, the anti-IL-15 inhibitor is an isolated antibody or antigen-binding fragment thereof that binds IL-15, comprising the heavy chain variable region of SEQ ID NO:6 and the light chain variable region of SEQ ID NO:9.

[0090] The interleukin-15 inhibitor for use according to claim 1 or 6, wherein the interleukin-15 inhibitor is selected from anti-IL-15 antibodies, which comprises:

[0091] - a heavy chain of SEQ ID NO: 10 and a light chain of SEQ ID NO: 11 or a variant or fragment thereof; or

[0092] - the variable heavy chain region of SEQ ID NO: 12 or a variant or fragment thereof and the variable light chain region of SEQ ID NO: 13 or a variant or fragment thereof; or

[0093] - the variable heavy chain region of SEQ ID NO: 14 or a variant or fragment thereof and the variable light chain region of SEQ ID NO: 15 or a variant or fragment thereof; or

[0094] - the heavy chain of SEQ ID NO: 16 and the light chain of SEQ ID NO: 17 or a variant or fragment thereof; or

[0095] - a heavy chain of SEQ ID NO: 18 and a light chain of SEQ ID NO: 17 or a variant or fragment thereof; or

[0096] - the heavy chain of SEQ ID NO: 19 and the light chain of SEQ ID NO: 17 or a variant or fragment thereof.

[0097] According to yet another specific embodiment, the anti-IL-15 inhibitor is an isolated antibody or antigen-binding fragment thereof that binds IL-15, comprising the heavy chain of SEQ ID NO:16 and the light chain of SEQ ID NO:17.

[0098] According to yet another specific embodiment, the anti-IL-15 inhibitor is an isolated antibody or antigen-binding fragment thereof that binds IL-15, comprising the heavy chain of SEQ ID NO:18 and the light chain of SEQ ID NO:17.

[0099] According to yet another specific embodiment, the anti-IL-15 inhibitor is an isolated antibody or antigen-binding fragment thereof that binds IL-15, comprising the heavy chain of SEQ ID NO:19 and the light chain of SEQ ID NO:17.

[0100] According to another specific embodiment, the anti-IL-15 inhibitor is an isolated antibody or an antigen-binding fragment thereof that binds to IL-15, which comprises the sequence 146B7 (also known as AMG174) as described in WO 2005 / 044303 or in WO 03 / 017935, which as an IL-15 antagonist is a monoclonal human IgG1 anti-IL-15 antibody having a heavy chain and a kappa light chain region, which comprises the amino acid sequences of SEQ ID NO: 10 and SEQ ID NO: 11.

[0101] According to another specific embodiment, the anti-IL-15 inhibitor is an isolated antibody or antigen-binding fragment thereof that binds to IL-15 as described in WO 2018 / 119246, which comprises a variable heavy chain sequence of SEQ ID NO 12 and a variable light chain sequence of SEQ ID NO 13.

[0102] According to another specific embodiment, the anti-IL-15 inhibitor is an isolated antibody or antigen-binding fragment thereof that binds to IL-15 as described in WO 2011 / 127324, in particular huABC2 (anti-IL-15Rβ antibody) comprising the variable heavy chain sequence of SEQ ID NO 14 and the variable light chain sequence of SEQ ID NO 15.

[0103] According to another specific embodiment, the anti-IL-15 inhibitor is an isolated antibody or an antigen-binding fragment thereof that binds to IL-15 as described in WO 2009 / 002562, or an isolated antibody or an antigen-binding fragment thereof that binds to 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.

[0104] Specific examples of the antibodies according to the present invention include the antibodies listed in Table 1.

[0105] Table 1

[0106]

[0107] Conjugates containing helper molecules

[0108] In another aspect of the invention, the isolated antibody or antigen-binding fragment thereof according to the invention is optionally conjugated to an auxiliary molecule, and is then also referred to herein as a "conjugated antibody" or a "conjugated antibody fragment".

[0109] The conjugated antibodies and conjugated antibody fragments according to the present invention can target anti-IL-15 inhibitors to disease sites (e.g., inflammatory sites) in vivo, so that the anti-IL-15 inhibitors can have preferential concentrations and therapeutic effects at the disease sites and have fewer side effects in other parts of the body.

[0110] The accessory molecule can be bound to the antibody or antibody fragment directly or via a spacer of appropriate length, for example as described by Kellogg et al. (2011, Bioconjug Chem, 22:717-27).

[0111] In another embodiment, the auxiliary molecule comprises an antigen-binding fragment of an antibody, which, when conjugated to an antibody or antibody fragment according to the invention, forms a bispecific antibody. In particular, the bispecific antibody may be directed against two different epitopes of IL-15 (thus defining a two-site antibody), or have a bispecific portion that targets IL-15 and IL-4R or IL-13 or IL-4.

[0112] Composition

[0113] The IL-15 inhibitor is provided in the form of a pharmaceutically acceptable composition. The pharmaceutical composition may comprise one or more IL-15 inhibitors, in particular one or more antibodies or antigen-binding fragments thereof that bind to IL-15 in any form described herein.

[0114] The composition of the present invention may further comprise one or more pharmaceutically acceptable additional ingredients, such as alum, stabilizers, antimicrobial agents, buffers, colorants, flavoring agents, adjuvants, and the like.

[0115] IL-15 inhibitors can be made into the form of pharmaceutical compositions and unit dosage forms thereof together with conventionally used adjuvants, carriers, diluents or excipients, and can be used in this form as solids (e.g., tablets or filled capsules, lyophilized forms) or liquids (e.g., solutions, suspensions, emulsions, elixirs or capsules filled with these substances) for oral use, or in the form of sterile injection solutions for parenteral (including subcutaneous) use, especially formulated in prefilled syringes or automatic injectors. Such pharmaceutical compositions and unit dosage forms thereof may contain ingredients in conventional proportions, may or may not contain additional active compounds or ingredients, and such unit dosage forms may contain any suitable effective amount of active ingredients commensurate with the dosage range intended for use.

[0116] The composition can be a liquid preparation, including but not limited to aqueous or oily suspensions, solutions, emulsions, syrups and elixirs. Liquid forms suitable for oral administration may include suitable aqueous or non-aqueous vehicles containing buffers, suspending agents and dispensing agents, colorants, flavoring agents, etc. The composition can also be formulated into a dry product so that it can be reconstituted with water or other suitable vehicles before use. Such liquid preparations may contain additives, including but not limited to suspending agents, emulsifiers, non-aqueous vehicles and preservatives. Suspending agents include but are not limited to sorbitol syrup, methylcellulose, glucose / syrup, gelatin, hydroxyethylcellulose, carboxymethylcellulose, aluminum stearate gel and hydrogenated edible fats. Emulsifiers include but are not limited to lecithin, sorbitan monooleate and gum arabic. Non-aqueous vehicles include but are not limited to edible oils, almond oil, fractionated coconut oil, oily esters, propylene glycol and ethanol. Preservatives include but are not limited to methyl or propyl parabens and sorbic acid. Other materials, processing techniques, etc. are listed in the following document incorporated by reference into this article: Remington: The Science & Practice of Pharmacy, 23rd edition, 2020, edited by Adeboye Adejare, Academic Press, which is incorporated by reference into this article.

[0117] The solid compositions of the present invention may be in the form of tablets or lozenges formulated in a conventional manner. For example, tablets and capsules for oral administration may contain conventional excipients, including but not limited to binders, fillers, lubricants, disintegrants and wetting agents. Binders include but are not limited to syrups, gum arabic, gelatin, sorbitol, tragacanth, starch mucus and polyvinyl pyrrolidone. Fillers include but are not limited to lactose, sugar, microcrystalline cellulose, corn starch, calcium phosphate and sorbitol. Lubricants include but are not limited to magnesium stearate, stearic acid, talc, polyethylene glycol and silicon dioxide. Disintegrants include but are not limited to potato starch and sodium starch glycolate. Wetting agents include but are not limited to sodium lauryl sulfate. Tablets may be coated according to methods well known in the art.

[0118] Injectable compositions are typically based on injectable sterile saline or phosphate-buffered saline or other injectable carriers known in the art.

[0119] The composition may also be formulated into a transdermal formulation comprising an aqueous or non-aqueous vehicle including, but not limited to, a cream, ointment, lotion, paste, medicated plaster, patch or membrane.

[0120] The composition can also be formulated for parenteral administration, including but not limited to by injection or continuous infusion. The formulation for injection can be in the form of a suspension, solution or emulsion in an oily or aqueous vehicle, and can contain formulation agents, including but not limited to suspending agents, stabilizers and dispersants. The composition can also be provided in powder form for reconstitution with a suitable vehicle including but not limited to sterile, pyrogen-free water.

[0121] The composition can also be formulated as a storage preparation that can be administered by implantation or by intramuscular injection. The composition can be formulated with a suitable polymer or hydrophobic material (e.g., formulated as an emulsion in an acceptable oil), an ion exchange resin, or formulated as a sparingly soluble derivative (e.g., formulated as a sparingly soluble salt).

[0122] The compounds can also be administered in sustained release form or from a sustained release drug delivery system. Descriptions of representative sustained release materials can also be found in the materials contained in Remington's Pharmaceutical Sciences.

[0123] Injectable formulations are particularly suitable for administering the compounds or compositions according to the invention.

[0124] combination

[0125] According to the present invention, the IL-15 inhibitor according to the present invention can be administered alone or in combination with an adjuvant that can be used for the prevention and / or treatment of atopic dermatitis, such as anti-IL-4R such as dupilumab, anti-IL-13 such as trorocitumab, JAK inhibitors such as abrocitinib, upadacitinib (oral) or ruxolitinib (topical) or corticosteroids such as dexamethasone.

[0126] The present invention encompasses the use of IL-15 inhibitors, wherein the IL-15 inhibitors are administered to a subject in a therapeutically effective amount prior to, simultaneously with, or sequentially with other treatment regimens or adjuvants useful for preventing and / or treating atopic dermatitis. The IL-15 inhibitor according to the present invention administered simultaneously with the adjuvant may be administered in the same or different compositions and by the same or different routes of administration.

[0127] In certain embodiments, an IL-15 inhibitor (particularly an IL-15 antibody or antigen-binding fragment thereof according to the present invention) may be administered in combination with an anti-IL-4R antibody such as dupilumab.

[0128] In a specific embodiment, an IL-15 inhibitor (particularly an IL-15 antibody or antigen-binding fragment thereof according to the present invention) may be administered in combination with an anti-IL-13 such as trorucizumab.

[0129] In certain embodiments, an IL-15 inhibitor (particularly an IL-15 antibody or antigen-binding fragment thereof according to the invention) may be administered in combination with one or more corticosteroids such as dexamethasone.

[0130] Mode of administration

[0131] The compositions of the present invention can be administered in any manner, including but not limited to, orally, parenterally, sublingually, transdermally, transmucosally, topically, or a combination thereof.

[0132] Parenteral administration includes, but is not limited to, intravenous, subcutaneous, and intramuscular administration.The compositions of the present invention may also be administered in the form of implants, which allow for slow release of the composition as well as slow controlled intravenous infusion.

[0133] In specific embodiments, the IL-15 inhibitor is administered systemically or locally, such as by topical application.

[0134] In specific embodiments, the IL-15 inhibitor (particularly an IL-15 antibody or antigen-binding fragment thereof) is administered subcutaneously or intravenously.

[0135] The dosage administered to an individual in single or multiple doses will vary depending on various factors including pharmacokinetic properties, subject condition and characteristics (sex, age, weight, health status, size), degree of symptoms, concurrent co-therapy, frequency of treatment and desired effect.

[0136] According to specific embodiments, the inhibitor is administered once a week to once a month.

[0137] Typically, the therapeutically effective amount of the pharmaceutically active antibody is in the dosage range of 0.1 mg / kg to 50 mg / kg body weight.

[0138] patient

[0139] In an embodiment, the subject according to the present invention suffers from atopic dermatitis.

[0140] According to a specific embodiment, the subject according to the present invention exhibits high systemic or local levels of IL-15, or biomarkers of IL-15 pathway activation, such as described in the following documents: Karlen and Simon, 2020, Int. Arch. Allergy Immunol. [International Archives of Allergy and Immunology], 181, 417–421 and Kurowska et al., 2020, J. Clin. Med [Clinical Medicine Journal], 1555.

[0141] In another embodiment, the subject according to the present invention has atopic dermatitis and is unresponsive to or cannot be treated with anti-IL-4R, anti-IL-13 and / or corticosteroids. A subject who is unresponsive to anti-IL-4R, anti-IL-13 and / or corticosteroids is defined as a lack of satisfactory relief after treatment with anti-IL-4R, anti-IL-13 and / or corticosteroids.

[0142] Use and method according to the invention

[0143] In a specific embodiment, a method for preventing and / or treating atopic dermatitis is provided, comprising administering a therapeutically effective amount of an IL-15 inhibitor, particularly an IL-15 antibody or an antigen-binding fragment thereof, to a subject in need thereof.

[0144] According to a specific embodiment, the interleukin-15 inhibitor is administered in the form of a formulation according to the invention.

[0145] All references cited herein are incorporated by reference in their entirety.

[0146] Having described the invention, the following examples are presented by way of illustration and not limitation.

[0147] Examples

[0148] Example 1: Expression of IL-15 and IL-15Rα in an in vitro human model of atopic dermatitis

[0149] The effect of an anti-IL-15 antibody (hu-BE29-2, comprising the heavy chain of SEQ ID NO: 16 and the light chain of SEQ ID NO: 17, as described in WO 2016 / 001275) in atopic dermatitis was investigated in the following experimental model.

[0150] Experimental setup: Healthy skin punch biopsy samples were used, and these skin punch biopsy samples were induced to show an atopic dermatitis phenotype using a cocktail that activates the Th2 pathway as follows ( https: / / www.reprocell.com / drug-efficacy-safety-adme / assay-catalog / skin-culture- induced-atopic-dermatitis-model-systemic-topical ):

[0151] Full-thickness skin biopsies up to 36x3 mm were obtained from three different healthy donors by surgical excision as described below. On day -1, skin biopsies were prepared, randomized, and completely immersed in at least 1 mL of basal medium in a cell culture incubator overnight. After an equilibration period, biopsies were transferred to individual wells of a 12-well plate on day 0. Then, using a pipette tip or forceps, the biopsies were placed in a Transwell plate. TMA hole was cut in the filter and each biopsy sample was inserted into one of the filter holes. The filter containing the biopsy sample was placed in a well of a 12-well culture plate containing 1 mL of the appropriate culture medium, with the epidermis facing up at the air-liquid interface. 1 mL of culture medium with or without Th2 polarization medium (Cousins ​​et al., 2002, J. Immunol [Journal of Immunology] 169, 2498-2506) and the test article was added to each well on day 0 and replaced on day 1. The test compounds used in this study were: anti-human IL-4R antibody, whose sequence is the same as 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 μg / mL, or betamethasone at a final concentration of 10 μM.

[0152] Approximately 48 h (day 2) after application of the Th2 stimulation mixture and the test compounds mentioned above, biopsy samples were collected and stored in RNAlater at 2°C-8°C for at least 24 h. After this, the RNAlater was removed and the biopsy samples were quickly frozen in liquid nitrogen and stored at 80°C. Tissue samples stabilized in RNAlater solution were processed for RNA extraction and isolation. RNA was quantified and quality checked using the Qubit system. Once the quality check was completed, the RNA samples were submitted for RNAseq analysis.

[0153] Skin and PBMC human donors

[0154] Healthy (control) abdominal human skin was obtained from three healthy volunteers (39-52 years old) 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 has been approved by the Rambam Health Care Campus Institutional Helsinki Committee (0182-14-RMB).

[0155] PBMC stimulation and characterization

[0156] Autologous PBMCs were isolated as previously described (Keren et al., 2018, J. Allergy Clin. Immunol., 142, 305-308.e6. https: / / doi.org / 10.1016 / j.jaci.2018.02.015).

[0157] After isolation, they were cultured in the presence of IL-2 (10 U / mL), IL-4 (200 U / mL) and LPS (1 μg / mL; Sigma, St. Louis, MO) ("Th2 polarization") to induce a Th2 phenotype. In a pilot experiment to assess the induction of IL-15 expression, control PBMCs were cultured in the presence of IL-2 and IL-4 but without LPS activation.

[0158] 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 as well as IL-15Rα expression in human skin explants in vitro, e.g. Figure 1 A and Figure 1 As shown in B.

[0159] It was also observed that treatment with anti-IL-4R antibodies or corticosteroids did not reduce the induced IL-15 and IL-15Rα expression in an ex vivo human atopic dermatitis model. Figure 2 A and Figure 2 As shown in B.

[0160] Example 2: Anti-IL-15 antibody is effective in a humanized mouse model of atopic dermatitis

[0161] However, human models of atopic dermatitis do not represent human disease very well. Similarly, existing mouse experimental models are of limited value because they represent human disease only to a limited extent and rely on mouse skin and immune system components that differ from those of humans (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 function in vivo, and in dermatology, humanized models of psoriasis have proven to be the most representative of human disease, despite their complexity and cost. A "humanized" model of atopic dermatitis as described above in Gilhar and Paus, 2021 was used. "Humanized" mice are defined here as immunodeficient (SCID / beige) mice transplanted with normal human skin from a healthy donor and activated T helper (Th)2-polarized peripheral blood mononuclear cells (PBMCs) from the same donor to recapitulate the clinical and histological features of atopic dermatitis at the skin graft level.

[0162] The first aim of this study was to evaluate whether IL-15 was induced during the development of this atopic dermatitis model (pilot experiment). Figure 3 As can be seen in the Figure 2, it has been demonstrated that the expression of both IL-15 and IL-15Rα is induced in this model. IL-15Rα is primarily expressed by epidermal cells, while IL-15 is expressed by epidermal cells as well as by dermal cells, presumably by cells of the monocyte and dendritic cell lineages. This analysis validates the presence of an IL-15 target in the humanized atopic dermatitis mouse model and is therefore suitable for testing compounds that neutralize IL-15.

[0163] A secondary objective of this study was to determine whether interleukin-15 inhibitors given at different schedules as anti-interleukin-15 antibodies would be effective in this humanized model of atopic dermatitis.

[0164] As described below, when anti-IL-15 antibodies were administered within 4 weeks of atopic dermatitis lesion onset or only within the last 2 weeks, significant effects on macroscopic skin lesions, as indicated by clinical scores based on measures of erythema and skin integrity, were observed. Figure 4 In addition, anti-IL-15 treatment had an effect on the following epidermal properties: Figure 5 ) had significant effects on epidermal thickness (A), epidermal basal cell proliferation (B), and expression of filaggrin (C), a molecule associated with skin barrier integrity that is 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.

[0165] The third aim was to analyze the effects of anti-IL-15 antibodies on key readouts and biomarkers known to be associated with human atopic dermatitis in treated mice.

[0166] Anti-IL-15 therapy also has effects on cytokines that may be secreted by T cells and are known to be induced in human atopic dermatitis (IFN-γ, IL-4, IL-17, and IL-22). Figure 7 On the other hand, anti-IL-15 therapy had no significant effect on the number of T cells, NK cells, and NKT cells present in the dermis of human skin grafts ( Figure 6 ), which are cells thought to be responsible for the secretion of IFN-γ, IL-4, IL-17, and IL-22. This suggests that anti-IL-15 affects only the function of these T cells, NK cells, and NKT cells, or only the number and / or function of a small subset of cells that cannot be characterized using the techniques used in this article. When observing 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 may be an effective new treatment for atopic dermatitis.

[0167] 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 affect the expression of IL-15 in the atopic dermatitis models analyzed. Interestingly, in the ex vivo human explant model of Example 1, neither anti-IL-4R antibodies nor corticosteroids were able to reduce IL-15 ( Fig. 9 ), while both treatments significantly reduced many other genes associated with atopic dermatitis. The same was true for IL-15Rα expression, and in fact, anti-IL-4R treatment even slightly increased the expression of IL-15 and IL-15Rα. Moreover, in the humanized atopic dermatitis mouse model of this example, anti-IL-4R only reduced IL-15 expression in the dermis, not in the epidermis. Corticosteroids showed a trend to reduce IL-15 expression only in the dermis, but it was not statistically significant.

[0168] These results suggest that treatment with anti-IL-4R antibodies or corticosteroids does not completely inhibit the IL-15 pathway and that anti-IL-15 therapy may therefore be advantageously administered to patients who do not respond to anti-IL-4R or corticosteroids, or used in combination with these or other treatments.

[0169] animal

[0170] A total of 60 female CB-17 / IcrHsd-scid-bg (beige-SCID, Harlan Laboratories Ltd., Jerusalem, Israel) mice aged 2 to 3 months were used in the therapy experiments. The mice were housed under pathogen-free conditions in accordance with institutional guidelines.

[0171] Skin and PBMC human donors

[0172] 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 Health Campus Agency (0182-14-RMB).

[0173] Humanized mouse atopic dermatitis model

[0174] Split-thickness 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 transplanted into mice (one human xenograft per mouse). One month after transplantation, 1x10 7 Th2 polarized PBMCs. Before injection, cell viability was determined by Trypan Blue staining.

[0175] In vivo treatment with anti-IL-15 antibody and controls

[0176] 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 a week with 200 μg / mouse of anti-IL-15 antibody (manufactured by Calypso Biotech SA, Switzerland) for 4 weeks after PBMC injection, or first injected with a matching dose of IgG1 control isotype antibody for 2 weeks and then injected with anti-IL-15 antibody for 2 weeks. Other groups of mice (10 per group) were also injected with 200 μg / mouse of anti-IL-4 receptor (IL-4R) antibody for 4 weeks. The sequence of IL-4R antibody is the same as that of dupilumab ( 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 a positive control, dexamethasone was applied topically three times a week to a group of 10 mice starting from the third week after PBMC injection. Macroscopic images of the transplanted skin were taken on day 0 and 1, 2, 3 and 4 weeks after PBMC injection. At the end of the 4-week treatment, each xenografted skin explant was collected, cut in half and embedded in The samples were either fixed in OCT and stored frozen or fixed in formalin, embedded in paraffin and stored at room temperature until further evaluation.

[0177] Clinical macroscopic evaluation

[0178] To measure the severity of the atopic dermatitis-like phenotype in xenograft skin, measurements commonly used in the clinic were adapted for analysis of collected macroscopic images to grade signs of erythema and skin integrity (epidermal breakdown / scaly / lichenification) on a discrete scale (0 = none, 1 = moderate, and 2 = severe) at day 0 and 1, 2, 3, and 4 weeks after PBMC injection. A total score (erythema score + skin integrity score) was calculated for each mouse. Scoring was performed independently by three operators in a blinded manner.

[0179] Hematoxylin and eosin (H&E) staining and epidermal thickness measurement

[0180] Routine H&E staining was performed on 7-μm-thick frozen sections. The mean human epidermal thickness for each xenograft was determined on these H&E-stained sections by averaging the measured distances between the outermost surface of the epidermis excluding the stratum corneum and the dermal-epidermal junction at 10 randomly selected points over the entire length of each section examined with the aid of a digital caliper and ImajeJ software. The measurements performed in a blinded manner were then averaged within each experimental group.

[0181] Expression of IL-15 and IL-15Rα

[0182] To detect the expression of IL-15 and IL15-Rα in xenografts, OCT (optimal temperature cutting compound) embedded skin xenografts were sectioned (7 μm thick) using a Leica cryostat. Tissue cryosections were fixed in acetone and pre-incubated with 10% goat serum in PBS. The sections were then incubated with the corresponding primary antibodies (anti-IL-15, Ab55276, Abcam, 1:100; anti-IL-15RA, Ab91270, Abcam, 1:100) at 4°C overnight. Secondary antibody incubation was performed at room temperature for 45 minutes (Goat-anti-Mouse-IgG-Alexa546, Invitrogen, 1:500). Counterstaining was performed with DAPI (1 μg / ml) to visualize the nuclei.

[0183] Immunohistochemistry for atopic dermatitis markers.

[0184] To measure the expression of filaggrin (FLG), cryopreserved xenograft skin sections were stained overnight at 4°C with rabbit anti-filagrin polyclonal antibody (Biolegend, 1:500-1:250), followed by species-specific fluorescent conjugated secondary antibody and DAPI counterstaining to visualize cell nuclei. Filagrin expression (fluorescence intensity) was measured in 1-3 randomly selected epidermal areas of 3-6 frozen sections of each xenograft at 10x magnification using a fluorescence microscope. A region of interest (ROI) including the stratum corneum of the human epidermis was manually selected on each image, and Image J software was used to calculate the average FLG expression value within the ROI. For each xenograft, the average expression level of filaggrin was calculated.

[0185] T, NK and NKT cell infiltration was assessed by analyzing the expression of CD3 (T cell marker) and CD56 (NK cell marker). Cryopreserved xenograft skin sections were double stained with the following antibodies: 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, 1h at room temperature). In one frozen section of each xenograft skin, the number of DAPI+, CD3+, CD56+ and CD3+ or CD56+ positive cells was calculated in 3-5 randomly selected 100μm2 dermal areas. The average value for each skin sample was calculated and expressed as the percentage of DAPI+ cells / area.

[0186] Epidermal proliferation was analyzed by observing the expression of the Ki67 proliferation marker. FFPE xenograft skin sections were dewaxed and rehydrated. After heat-induced antigen retrieval in sodium citrate buffer and blocking with normal goat serum, the sections were stained overnight at 4°C with mouse anti-Ki-67 antibody (M7240, Dako, 1:10) and then stained with anti-mouse fluorescent-conjugated goat secondary antibody. The sections were briefly treated with DAPI to visualize the cell nuclei. For each xenograft skin, 2 sections were imaged at 20× 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 nuclei were counted using a thresholding automated method encoded in ImageJ image analysis software.

[0187] IL-4 expression was analyzed on FFPE xenograft skin sections that were first dewaxed 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) and then stained with species-specific biotinylated goat secondary antibodies. IL-4+ cells were identified by targeting biotin with streptavidin-HRP to catalyze DAB precipitation. Sections were counterstained with hematoxylin. For each xenograft skin, two sections were imaged at 20× magnification using a light microscope. Dermal IL-4+ cells were counted and the dermal surface area was calculated in ImageJ imaging software to give per mm 2 The number of IL-4+ cells.

[0188] IL-17A expression was analyzed by staining cryopreserved xenograft skin sections with rabbit anti-human IL-17A antibody (Ab79056, Abcam, 1:200) at 4°C overnight, followed by species-specific fluorescently conjugated secondary antibodies 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 give the expression of IL-17A per mm 2 The number of IL-17A+ cells.

[0189] IL-22 expression was analyzed by staining cryopreserved xenograft skin sections with rabbit anti-human IL-22 antibody (BS-2623R, ThermoFisher, 1:200) overnight at 4°C, followed by species-specific fluorescent 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-22+ cells were counted and dermal surface area was calculated in ImageJ imaging software to give the expression of IL-22+ cells per mm2. 2 The number of IL-22+ cells.

[0190] IFN-γ expression was analyzed by staining cryopreserved xenograft skin sections with rabbit anti-human IFN-γ antibody (Ab9657, Abcam, 1:100) at 4°C overnight, followed by species-specific fluorescent conjugated secondary antibodies and DAPI counterstaining to visualize cell nuclei. For each xenograft skin, 2-3 sections were imaged at 20x magnification using a fluorescent microscope. Dermal IFN-γ+ cells were counted and dermal surface area was calculated in ImageJ imaging software to give the expression of IFN-γ per mm2. 2 The number of IFN-γ+ cells.

[0191] Gene expression analysis in humanized models

[0192] RNA extraction 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 using Sigma-Aldrich. Briefly, each xenograft was homogenized at 6800 rpm for 6 cycles of 10 s and cooled on ice for 30 s, and 1 mL of TRI and chloroform (250 μL), the samples were mixed and centrifuged at 12,500×g for 15 minutes. The supernatant containing total RNA (approximately 500 μL) was mixed with an equal volume of isopropanol to precipitate the RNA. The total RNA was precipitated by centrifugation at 12,500×g for 15 minutes, washed twice with 70% ethanol and centrifuged at 8,000×g for 7 minutes, and then resuspended in 20 μL of RNase-free water. The RNA was stored at -80°C until analysis.

[0193] Quality control of the extracted RNA was measured using the Tapestation Bioanalyzer and Qubit at the Nanostring Core facility in Heidelberg, Germany.

[0194] Nanoskin Analysis System (Nanostring The NanoString analysis system is based on the digital detection of single target molecules, which are directly labeled with molecular barcodes. Each probe consists of a color-coded reporter probe and a capture probe, both of which are covalently linked to a target-specific sequence (50 nucleotides in length). The color-coded reporter probes allow for singularity of the target, while the capture probes conjugated to biotin allow for the target to be fixed on the surface. With the application of voltage and the generation of an electric field, the target molecules align, allowing the reporter probes to be fully extended and allowing the identification of single molecules. NanoString multiplexing was outsourced to the Department of Human Molecular Genetics, Heidelberg University Hospital Heidelberg Core Facilities. Instruments used SPRINT.

[0195] In this study, the Human Immunology V2 Panel, which quantifies 594 genes, including housekeeping genes. Purified RNA obtained from each sample was hybridized overnight at 65°C using the Human Immunology V2 Assay Panel (NanoString Technologies, WA, USA). The hybridization probes are further purified on the Prep Station and combined with the optical cartridge, and the optical cartridge is scanned on the nCounter digital analyzer. The RCC file obtained from the Nanoskin digital analyzer is imported into nSolver TM 4.0.70 software (NanoSkin Technologies, Inc., WA, USA), and the data quality was checked using the default quality check settings. mRNA levels were determined using "barcodes", and background correction was then performed by subtracting the "mean + 2 standard deviations" value of the negative control from the raw counts, and the adjusted raw counts were then normalized to the geometric mean of 12 identified housekeeping genes. nSolver was used TMBioinformatics and statistical analyses, including hierarchical clustering and volcano plots, were performed using the Human Immunology V2 Analysis software version 4.0.70 and the Human Immunology V2 Analysis Advanced Analysis module. As statistical significance could not be detected with P-adjusted values ​​≤ 0.05, we used raw p-values ​​≤ 0.05 to analyze the data. Due to the high cross-reactivity with mouse genes in the Human Immunology V2 panel assay (down to 85%), we performed analyses including all genes in the panel assay and excluded mouse genes with cross-reactivity > 85%. This was due to the information conveyed to us by the Nanoskin facility that any value below 85% should not interfere with the analysis. Selected relevant genes in atopic dermatitis were selected in order to align the findings of this study with the state of the art in AD gene profiling).

[0196] Ex vivo stimulation of human skin explants with a Th2 polarizing cocktail

[0197] Full-thickness human skin biopsy specimens were obtained by punch biopsy of residual surgical tissue. 2 Biopsy samples were obtained from 100 μm-sized samples and transferred to mesh transfer wells in 12-well plates. The dermis of the skin was immersed in a specially fortified medium, leaving the epidermis exposed to air. The biopsy samples were incubated under optimal conditions for up to 24 hours and then incubated for an additional 24 hours in the presence of a Th2-polarized inflammatory cocktail and the test article.

[0198] Example 3: Analysis of the IL-15 pathway in samples from patients with atopic dermatitis

[0199] The expression of IL-15 and related genes (the "IL-15 pathway") is analyzed in various well-characterized sample sets or databases from patients with atopic dermatitis and controls; some samples are collected from patients who have been treated with dupilumab and will be analyzed for possible correlation with clinical outcomes of treatment. The goal of these analyses is to further characterize the IL-15 pathway in AD and potentially identify subsets of patients who may preferentially benefit from treatment with IL-15 inhibitors. Analyses using state-of-the-art technology include:

[0200] - Transcriptomic analysis from public databases

[0201] -qPCR analysis from 30 healthy and AD skin biopsy samples

[0202] - Proteomic analysis of 10 healthy and AD skin biopsy samples (48 target genes)

[0203] - Immunohistochemical analysis of 20 healthy and AD skin biopsy samples

[0204] - Single-cell RNA sequencing of 10 healthy and AD skin biopsy samples

[0205] -Using ultrasensitive technology to measure IL-15 levels in 30 AD sera and healthy sera.

[0206] To analyze data from public databases, the 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 the following literature: Guttman-Yassky et al., 2019, J. Allergy Clin. Immunol., 143, 155–172.

[0207] In a randomized, placebo-controlled, double-blind, phase 2 trial conducted at 5 medical centers in the United States and Canada (ClinicalTrials.gov: NCT01979016), biopsy specimens were collected from lesional and nonlesional skin to evaluate the efficacy and safety of dupilumab as compared with placebo in patients with moderate-to-severe atopic dermatitis. Patients received a 400-mg loading dose or placebo on day 1 followed by subcutaneous injections of 200 mg of dupilumab or placebo once weekly 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). The original CEL format files were converted to expression profile format using the open-source package Affy, which is 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.

[0208] It was observed that the expression of IL-15 was significantly increased in both non-lesional (NL) and lesional (L) skin of atopic dermatitis ( Fig.10 ), consistent with previous reports (Karlen and Simon, 2020, Int. Arch. Allergy Immunol. [International Archives of Allergy and Immunology], 181, 417–421). IL-15 expression in lesional atopic dermatitis skin was also significantly higher than that in non-lesional atopic dermatitis skin. It was also observed that although dupilumab treatment slightly reduced 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 lesional and non-lesional atopic dermatitis skin remained significantly higher compared with healthy skin.

[0209] These results suggest that, as has been observed in mouse humanized models ( Fig. 9 ), treatment of patients with the anti-IL-4R antibody dupilumab did not completely inhibit the IL-15 pathway, and thus further supports that anti-IL-15 therapy may be advantageously administered to patients who do not respond to anti-IL-4R antibodies, or used in combination with these or other treatments.

[0210] Example 4: Extending the effect of anti-IL15 antibody hu-BE29-2 to other antibodies sharing the same binding epitope

[0211] The above effects can reasonably be extended to other antibodies sharing the same binding epitope, such as the antibodies described in WO 2016 / 001275, in particular hu-BE29-10&24.

[0212] As described below, these anti-human IL15 antibodies were shown to have the same epitope specificity as hu-BE29-2.

[0213] Using Pepscan's proprietary chemically linked peptides on scaffolds (CLIPS) technology, a library of structured peptides of IL-15 was synthesized. These peptides were designed to be single-ring, double-ring, triple-ring, sheet-like, helical, and combinations thereof (Timmerman 2007, Journal of Molecular Recognition, 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. The binding of antibodies hu-BE29-2, hu-BE29-10, and hu-BE29-24 to each synthetic peptide was tested by ELISA technology, in which color development was quantified using a charge coupled device (CCD) camera and an image processing system. The results showed that antibodies hu-BE29-2, hu-BE29-10, and hu-BE29-24 all bound to an epitope in IL-15 consisting of the sequence of amino acids 83 to 93 of SEQ ID NO: 1.

[0214] Sequence Listing

[0215] Human mature IL-15 (Homo sapiens), gene pool: CAA71044.1

[0216] SEQ ID NO:1

[0217] MDFQVQIFSFLLISASVIMSRANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS

[0218] Cytokine modulator peptide from WO 2015 / 089217, BNZ132 (also known as BNZ-1, BNZ-13, YT-033, EQ-101)

[0219] SEQ ID NO:2

[0220] IKEFLQRFIHIVQSIINTS

[0221] γ-cytokine modulator peptides from US2019 / 0070263

[0222] SEQ ID NO:3

[0223] PKEFLERFVHLVQMFIHQSLS

[0224] IL-15 inhibitor peptide US7,736,638B2 SEQ ID NO:4

[0225] SEQ ID NO:4

[0226] KVTAMKCFLL

[0227] huVH1 from WO 2016 / 001275

[0228] SEQ ID NO:5

[0229] EVRLMASGGGLVQPGGSLRLSCAAS EFTFSNYAMS WVRQAPGKGLEWVA TISRGGDYTYYPDSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYC ARRVSMITGGWAMDY WGQGTLVTVSS

[0230] huVH2 from WO 2016 / 001275

[0231] SEQ ID NO:6

[0232] EVQLVESGGGLVQPGGSLRLSCAAS EFTFSNYAMS WVRQAPGKGLEWVA TISRGGDYTYYPDSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYC ARRVSMITGGWAMDY WGQGTLVTVSS

[0233] huVH8 from WO 2016 / 001275

[0234] SEQ ID NO:7

[0235] EVRLMASGGGLVQPGGSLRLSCAASEFTFSNYAMSWVRQAPGKGLEWVATISRGGDYTYYPDTVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARRVSMITGGYAMDYWGQGTLVTVSS

[0236] huVH18 from WO 2016 / 001275

[0237] SEQ ID NO:8

[0238] EVQLVESGGGLVQPGGSLRLSCAASEFTFSNYAMSWVRQAPGKGLEWVATISRGGDYTYYPDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARRVSMITGGYAMDYWGQGTLVTVSS

[0239] huVL1 from WO 2016 / 001275

[0240] SEQ ID NO:9

[0241] DVVMTQSPLSLPVTLGQPASISCRSSQSIVDITGNTYLEWYQQRPGQSPRLLIYKVFNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQDSFVPYTFGQGTKLEIK

[0242] Heavy chain from 146B7, also known as AMG 714 (WO 2005 / 044303)

[0243] SEQ ID NO:10

[0244] MGWTLVFLFLLSVTAGVHSEVQLVQSGAEVKKPGESLKISCKVSGYFFTTYWIGWVRQMPGKGLEYMGIIYPGDSDTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARGGNWNCFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0245] Light chain from 146B7, also known as AMG 714 (WO 2005 / 044303)

[0246] SEQ ID NO:11

[0247] MVSSAQFLGLLLLCFQGTRCEIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASRRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQRYGSSHTFGQGTKLEISRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLS SPVTKSFNRGEC

[0248] Variable heavy chain region from 70a WO 2018 / 119246

[0249] SEQ ID NO:12

[0250] QVQLQESGPGLVKPSGTLSLTCAVSGGSISSSNWWSWVRQPPGKGLEWIGEIYHYGYTNYNPSLKSRVTISVDKSKNQFSLKLSSVTAADTAVYYCAREGIGWPSFDYWGQGTLVTVSS

[0251] Variable light chain region from 70a WO 2018 / 119246

[0252] SEQ ID NO:13

[0253] SSELTQDPAASVALGQTVRITCQGDTLRSYYASWYQQKPGQAPILVIYGKNNRPSGIPDRFSGSSSSGNTASLTITGAQAEDEADYYCNSRDLSGKNLVFGGGTKLTVL

[0254] Variable heavy chain region from huABC2 WO 2011 / 127324

[0255] SEQ ID NO:14

[0256] MKLWLNWVFLLTLLHGIQCEVQLVESGGGLVQPGGSLRLSCAASGFTFSDFYMEWVRQAPGKGLEWIAAASRNKANDYTTEYSASVKGRFIVSRDDSKNSLYLQMNSLKTEDTAVYYCARSYYRYDGMDYWGQGTTVTVSS

[0257] Variable light chain region from huABC2 WO 2011 / 127324

[0258] SEQ ID NO:15

[0259] MDFQVQIFSFLLISASVIVSRGEIVLTQSPATLSLSPGERATLSCSAISSVSYMYWYQQKPGQAPRLLIYDTSNLVSGVPARFSGSGSGTDYTLTISSLEPEDFAYYYCQQWNTYPYTFGGGTKVEIK

[0260] Heavy chain from huBE29-2 (WO 2016 / 001275)

[0261] SEQ ID NO:16

[0262] EVQLVESGGGLVQPGGSLRLSCAASEFTFSNYAMSWVRQAPGKGLEWVATISRGGDYTYYPDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARRVSMITGGWAMDYWGQGTLVTVSS ASTKGPSVFPLAPSSKSTSGGTAA LGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPK SCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQ YNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGF YPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0263] Light chain from huBE29-2 (WO 2016 / 001275)

[0264] SEQ ID NO:17

[0265] DVVMTQSPLSLPVTLGQPASISCRSSQSIVDITGNTYLEWYQQRPGQSPRLLIYKVFNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQDSFVPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0266] Heavy chain from huBE29-10 (WO 2016 / 001275)

[0267] SEQ ID NO:18

[0268] EVRLMASGGGLVQPGGSLRLSCAASEFTFSNYAMSWVRQAPGKGLEWVATISRGGDYTYYPDTVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARRVSMITGGYAMDYWGQGTLVTVSS ASTKGPSVFPLAPSSKSTSGGTAA LGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPK SCDKTHTCPPCPAPELLGGPSVFLFPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQ YNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGF YPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0269] Heavy chain from huBE29-24 (WO 2016 / 001275)

[0270] SEQ ID NO:19

[0271] EVQLVESGGGLVQPGGSLRLSCAASEFTFSNYAMSWVRQAPGKGLEWVATISRGGDYTYYPDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARRVSMITGGYAMDYWGQGTLVTVSS ASTKGPSVFPLAPSSKSTSGGTAA LGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPK SCDKTHTCPPCPAPELLGGPSVFLFPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQ YNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGF YPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Claims

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

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

3. The interleukin-15 inhibitor for use according to claim 2, wherein the interleukin-15 inhibitor has the formula wherein R is an optionally substituted phenyl group, and n is an integer selected from 1 to 10.

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

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

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

7. The interleukin-15 inhibitor for use according to claim 1 or 6, wherein the interleukin-15 inhibitor is an isolated antibody or an antigen-binding fragment thereof that binds to IL-15, comprising: (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 by different amino acids, wherein the substitution is selected from: (iv) in the heavy chain variable framework region, arginine (R) at position H3 (VH RH3) is replaced by glutamine (Q), methionine (M) at position H5 (VH MH5) is replaced by valine (V), alanine (A) at position H6 (VH AH6) is replaced by glutamic acid (E), and alanine (A) at position H49 (VH AH49) is replaced by serine (S), (v) in the heavy chain CDR2, aspartic acid (D) at position H61 (VH DH61) is replaced by glutamic acid (E), and serine (S) at position H62 (VH SH62) is replaced by threonine (T), and (vi) in the heavy chain CDR3, methionine (M) at position H98 (VH MH98) is substituted by leucine (L), phenylalanine (F), isoleucine (I) or alanine (A), tryptophan (W) at position H100C (VH WH100C) is substituted by tyrosine (Y), phenylalanine (F) or alanine (A), and methionine (M) at position H100E (VH MH100E) is substituted by leucine (L), phenylalanine (F) or isoleucine (I); as well as (2) The light chain variable region of SEQ ID NO:

9.

8. The interleukin-15 inhibitor for use according to claim 1 or 6, wherein the interleukin-15 inhibitor is an isolated antibody or an antigen-binding fragment thereof that binds to IL-15, comprising: (1) a heavy chain variable region of SEQ ID NO: 6 or any variant thereof, wherein the variant has the amino acid sequence of SEQ ID NO: 6 except that 1, 2, 3 or 4 amino acids are substituted by different amino acids, and (2) The light chain variable region of SEQ ID NO:

9.

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

9.

10. The interleukin-15 inhibitor for use according to claim 1 or 6, wherein the interleukin-15 inhibitor is an isolated antibody or an antigen-binding fragment thereof that binds to IL-15, comprising: (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) The light chain variable region of SEQ ID NO:

9.

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

12. A pharmaceutical composition comprising an interleukin-15 inhibitor and an agent for preventing and / or treating atopic dermatitis and a pharmaceutically acceptable carrier, diluent or excipient thereof.

13. The pharmaceutical composition according to claim 12, further comprising an adjuvant for preventing and / or treating atopic dermatitis, such as at least one anti-IL-4R antibody or at least one corticosteroid or a mixture thereof.

14. The pharmaceutical composition of claim 12 or 13, further comprising at least one anti-IL-4R antibody such as dupilumab, anti-IL-13 such as trorocitinib, or a JAK inhibitor such as abrocitinib, upadacitinib, or ruxolitinib.

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

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

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

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

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