Treatment for Prurigo Nodularis

Abstract

Described herein are treatments and preventions for prurigo nodularis (PN), antibodies and pharmaceutical compositions for use in the treatment or prevention of PN, and the use of anti-IL-31RA antibodies (e.g., nemolizumab) in the manufacture of a medicament for the treatment or prevention of PN. Also described herein are biomarkers for PN and methods of altering or ameliorating these biomarkers through treatment with an antibody that binds to IL-31RA (e.g., nemolizumab).

Description

[Technical Field]

[0001] Related Applications This application claims priority under 35 U.S.C. §119(e) to (i) U.S. Provisional Application No. 63 / 403,483, filed September 2, 2022, and (ii) U.S. Provisional Application No. 63 / 534,558, filed August 24, 2023, the entire contents of both applications being incorporated herein by reference.

[0002] Field Described herein are treatment and prevention of prurigo nodularis (PN), antibodies and pharmaceutical compositions for use in treating or preventing PN, and the use of anti-IL-31RA antibodies (e.g., nemolizumab) in the manufacture of a medicament for treating or preventing PN. Also described herein are biomarkers for PN and methods of altering or ameliorating these biomarkers through treatment with antibodies that bind to IL-31RA (e.g., nemolizumab). [Background technology]

[0003] background The following discussion is provided merely to aid the reader in understanding the present disclosure and is not admitted to describe or constitute prior art to the present disclosure.

[0004] Chronic prurigo (CP) is a skin disorder caused by sensitization of nerve cells to itch and an extended itch-scratch cycle. Prurigo nodularis (PN), a subtype of CP, is a skin disorder that causes hard, itchy nodules (nodules) to form on the skin. The itching (scratching) can be intense and can lead to bleeding or painful scratching. Scratching can cause more skin lesions to appear. Itching is worsened by heat, sweating, or irritation from clothing. In some cases, people with PN have a history of other diseases, including eczema (atopic dermatitis), diabetes, lymphoma, HIV infection, severe anemia, or kidney disease.

[0005] The exact cause of PN is unknown, and diagnosis of the disease is based on observing symptoms such as extremely itchy skin with the formation of nodules. In some cases, a skin biopsy is used to confirm the diagnosis. Currently, treatments may include corticosteroid creams, oral medications, cryotherapy, or photochemotherapy.

[0006] Treatment for PN remains necessary, and patients are likely to respond to or are responding to such treatment. Summary of the Invention

[0007] overview Described herein are treatments and preventions for prurigo nodularis (PN) that achieve specific therapeutic results, such as reduced TNF signaling. Generally, the treatments and preventions involve administering an anti-IL-31RA antibody (e.g., nemolizumab) to a subject with PN. Also described herein are biomarkers for PN and methods of using the disclosed biomarkers to determine whether a subject will respond to treatment.

[0008] In a first aspect, the disclosure provides a method of treating or preventing prurigo nodularis (PN) in a subject, the method comprising administering an anti-IL-31RA antibody to a subject having PN, wherein the subject exhibits activation of tumor necrosis factor (TNF) signaling in lesional skin cells compared to a reference level of TNF activation.

[0009] In a second aspect, the present disclosure provides a method of normalizing tumor necrosis factor (TNF) gene expression in a subject with PN, comprising administering an anti-IL-31RA antibody to the subject with PN, wherein the subject exhibits tumor necrosis factor (TNF) activation in lesional skin cells compared to a reference level of TNF activation, and wherein administration of the anti-IL-31RA antibody normalizes activation of TNF signaling. In some embodiments, normalization is determined about 4 weeks, about 8 weeks, or about 12 weeks after administration of the anti-IL-31RA antibody.

[0010] In some embodiments, differential expression was determined by RT-qPCR, RT-PCR, RNA-seq, Northern blotting, serial analysis of gene expression (SAGE), or DNA or RNA microarrays, or differential expression was determined at the protein level by Western blotting, ELISA, surface plasmon resonance, or mass spectrometry.

[0011] In some embodiments, activation of TNF signaling in the lesional skin cells is increased compared to the reference level, hi some embodiments, the lesional skin cells are fibroblasts.

[0012] In some embodiments, the reference level is an activation level that is the level of activation of TNF signaling in skin cells of a human not having PN. In some embodiments, the skin cells of a human not having PN are fibroblasts.

[0013] In some embodiments, the reference level is an activation level that is the level of activation of TNF signaling in non-lesional skin cells of the subject.

[0014] In a third aspect, the present disclosure provides a method of reducing inflammation in the skin of a subject with prurigo nodularis (PN), the method comprising administering an anti-IL-31RA antibody to a subject with PN, thereby reducing inflammation associated with tumor necrosis factor (TNF) signaling in the skin.

[0015] In some embodiments, TNF signaling in the subject's skin is overexpressed compared to a reference level of activation of TNF signaling, and optionally, the TNF gene is overexpressed by fibroblasts.

[0016] In some embodiments, the reference level is an activation level that is the level of activation of TNF signaling in skin cells of a human not having PN. In some embodiments, the skin cells of a human not having PN are fibroblasts.

[0017] In some embodiments, the reference level is an activation level that is the level of activation of TNF signaling in non-lesional skin cells of the subject.

[0018] In some embodiments, the inflammation further involves IL-1 pathway signaling, IL-6 pathway signaling, TGFβ pathway signaling, or any combination thereof.

[0019] In a fourth aspect, the present disclosure provides a method of treating or preventing prurigo nodularis (PN) in a subject, the method comprising administering an anti-IL-31RA antibody to a subject with PN, wherein treatment with the anti-IL-31RA antibody results in reduced tumor necrosis factor (TNF) pathway activation. In some embodiments, the reduced TNF pathway activation occurs in lesional skin of the subject. In some embodiments, the reduced TNF pathway activation occurs in fibroblasts of the subject.

[0020] In some embodiments, a further outcome of the treatment is: (a) a decrease in leukocyte migration or cell movement of leukocytes; (b) inhibition of the STAT3 pathway; (c) inhibition of the STAT5b pathway; (d) downregulation of IL-1 or the IL-1 pathway; (e) downregulation of IL-6 or the IL-6 pathway; (f) downregulation of VEGF or the VEGF pathway; (g) a decrease in TGFB1 pathway activation, or (h) A combination thereof.

[0021] In some embodiments, (a) decreased leukocyte migration or leukocyte cell movement, (b) inhibition of the STAT3 pathway, (c) inhibition of the STAT5b pathway, (d) downregulation of IL-1 or the IL-1 pathway, (e) downregulation of IL-6 or the IL-6 pathway, (f) downregulation of VEGF or the VEGF pathway, (g) decreased TGFBl pathway activation, or (h) a combination thereof is determined relative to (i) a control sample obtained from one or more individuals without PN, or (ii) a biological sample obtained from the subject prior to administration of an anti-IL-31RA antibody.

[0022] In some embodiments, (a) a decrease in leukocyte migration or leukocyte cell movement, (b) inhibition of the STAT3 pathway, (c) inhibition of the STAT5b pathway, (d) downregulation of IL-1 or the IL-1 pathway, (e) downregulation of IL-6 or the IL-6 pathway, (f) downregulation of VEGF or the VEGF pathway, (g) a decrease in TGFB1 pathway activation, or (h) a combination thereof is assessed about 4 weeks, about 8 weeks, or about 12 weeks after administration of the anti-IL-31RA antibody.

[0023] In some embodiments, (a) a decrease in leukocyte migration or leukocyte cell movement, (b) inhibition of the STAT3 pathway, (c) inhibition of the STAT5b pathway, (d) downregulation of IL-1 or the IL-1 pathway, (e) downregulation of IL-6 or the IL-6 pathway, (f) downregulation of VEGF or the VEGF pathway, (g) a decrease in TGFB1 pathway activation, or (h) a combination thereof is determined by mass spectrometry performed on one or more biological samples obtained from the subject.

[0024] In some embodiments, the one or more biological samples are plasma samples or skin samples.

[0025] In some embodiments, the subject exhibits at least two, at least three, at least four, at least five, at least six, or all seven of the following: (a) decreased leukocyte migration or leukocyte cell movement; (b) inhibition of the STAT3 pathway; (c) inhibition of the STAT5b pathway; (d) downregulation of IL-1 or the IL-1 pathway; (e) downregulation of IL-6 or the IL-6 pathway; (f) downregulation of VEGF or the VEGF pathway; and (g) decreased TGFB1 pathway activation.

[0026] In a fifth aspect, the present disclosure provides a method of inactivating, reducing the activation of, or reducing the number of COL11A1+ fibroblasts in a subject with prurigo nodularis (PN), comprising administering an anti-IL-31RA antibody to the subject, wherein administering the anti-IL-31RA antibody results in the inactivation, reduction of the activation, or reduction in the number of COL11A1+ fibroblasts in the skin of the subject. In some embodiments, the COL11A1+ fibroblasts are found in the papillary dermis.

[0027] In a sixth aspect, the present disclosure provides a method for reducing TGFβ expression in at least one cell type in a subject with PN, comprising administering an anti-IL-31RA antibody to the subject, wherein administering the anti-IL-31RA antibody results in a reduction in TGFβ expression in at least one cell type in the skin of the subject. In some embodiments, the at least one cell type comprises fibroblasts, endothelial cells, pericytes, neurons, or any combination thereof. In some embodiments, the reduction in TGFβ expression comprises a reduction in expression of TGFB1, TGFB2, TGFB3, or any combination thereof.

[0028] In a seventh aspect, the present disclosure provides a method for reducing the expression of at least one inflammatory gene expressed by keratinocytes in a subject with PN, comprising administering an anti-IL-31RA antibody to the subject, wherein administering the anti-IL-31RA antibody results in a reduction in the expression of at least one inflammatory gene expressed by keratinocytes in the skin of the subject. In some embodiments, the at least one inflammatory gene is selected from KRT6, KRT16, KRT17, S100A8, S100A9, and any combination thereof. In some embodiments, the keratinocytes express Th2 cytokines. In some embodiments, administering the anti-IL-31RA antibody results in a reduction in reactive oxygen species and / or cellular stress to which the keratinocytes are exposed.

[0029] In an eighth aspect, the present disclosure provides a method for reducing infiltration of at least one type of immune cell in skin lesions of a subject with PN, the method comprising administering an anti-IL-31RA antibody to the subject, wherein administering the anti-IL-31RA antibody results in a reduction in infiltration of at least one type of immune cell in at least one lesion in the skin of the subject. In some embodiments, the at least one type of immune cell comprises a macrophage. In some embodiments, the macrophage is a lipid-associated macrophage characterized by expression of APOE and TREM2. In some embodiments, the at least one type of immune cell is selected from the group consisting of T cells, NK cells, CD8 + cells, Tregs, and any combination thereof. In some embodiments, the method comprises administering an anti-IL-31RA antibody, resulting in a decrease in expression of ICAM1, E-selectin (SELE), IL6, CCL2, CCL3, CCL4, CCL13, CCL18, CXCL2, CXCL12, and any combination thereof, in at least one cell type in the lesion. In some embodiments, the at least one cell type in the lesion comprises myeloid cells, pericytes, endothelial cells, and any combination thereof.

[0030] In some embodiments of any of the aforementioned aspects, the anti-IL-31RA antibody is administered subcutaneously.

[0031] In some embodiments of any of the foregoing aspects, the anti-IL-31RA antibody is administered once per week, once per two weeks, once per three weeks, once per four weeks, once per five weeks, once per six weeks, once per seven weeks, or once per eight weeks.

[0032] In some embodiments of any of the foregoing aspects, the anti-IL-31RA antibody is administered at a dose of about 0.01 mg / kg to about 0.1 mg / kg, about 0.1 mg / kg to about 0.5 mg / kg, about 0.5 mg / kg to about 1.5 mg / kg, about 1.5 mg / kg to about 2.5 mg / kg, or about 2.5 mg / kg to about 10 mg / kg. Alternatively, in some embodiments, the anti-IL-31RA antibody is administered at a dose of about 10 mg, about 15 mg, about 20 mg, about 25 mg, about 30 mg, about 35 mg, about 40 mg, about 45 mg, about 50 mg, about 55 mg, about 60 mg, about 65 mg, about 70 mg, about 75 mg, about 80 mg, about 85 mg, or about 90 mg.

[0033] In some embodiments of any of the foregoing aspects, the anti-IL-31RA antibody is administered according to a flat dosing regimen. Alternatively, in some embodiments, the anti-IL-31RA antibody is administered according to a loading dose regimen.

[0034] In some embodiments of any of the foregoing aspects, the anti-IL-31RA antibody comprises a heavy chain variable region comprising an HCDR1 comprising SEQ ID NO: 8, an HCDR2 comprising SEQ ID NO: 9, and an HCDR3 comprising SEQ ID NO: 10, and a light chain variable region comprising an LCDR1 comprising SEQ ID NO: 12, an LCDR2 comprising SEQ ID NO: 13, and an LCDR3 comprising SEQ ID NO: 14. In some embodiments, the anti-IL-31RA antibody is nemolizumab or a fragment or variant thereof. In some embodiments, the anti-IL-31RA antibody is nemolizumab.

[0035] The foregoing summary and the following detailed description are exemplary and explanatory and are intended to provide further explanation of the present disclosure as claimed. Other objects, advantages, and novel features will become readily apparent to those skilled in the art from the following brief description of the drawings and detailed description of the present disclosure. [Brief explanation of the drawings]

[0036] [Figure 1A] Figures 1A-1E show that prurigo nodularis (PN) is characterized by immune activation and abnormal keratinocyte differentiation. The number of differentially expressed genes (DEGs) in PN lesional versus non-lesional skin (n = 62, FC >= 2 or FC <= -2, FDR <= 0.1) (A). The enriched GO categories in PN lesional skin (B). A literature-based gene network derived from the top 1,000 DEGs in PN skin was generated using the Genomatix Pathway System (GePS, Genomatix.de). The picture shows the top 50 most connected genes co-cited in PubMed abstracts in the same sentence linked to function terms (most relevant genes / interactions). Orange represents up-regulated genes, and green represents down-regulated genes in PN lesional versus non-lesional skin. Significant nodes included the proliferation marker Ki67 (MKI67), IL-1 family members IL36G and IL1A, and CXCL8 and CDKN1A (C). Number of genes and intracluster correlation in modules identified from weighted co-expression network (WCGNA) analysis of non-lesional and lesional PN skin (D). Enriched functions in major co-expression modules from PN skin (modules #5, #6, and #8) (E). [Figure 1B] See legend to Figure 1A. [Figure 1C] See legend to Figure 1A. [Figure 1D] See legend to Figure 1A. [Figure 1E] See legend to Figure 1A. [Figure 2A]Figures 2A-2C show enriched transcriptomic cellular signatures and their overlap with psoriasis and AD. Cell-type inference analysis for non-lesional (NL) and lesional (L) PN skin samples using xCell. Enriched cellular signatures are shown in red, while underrepresented cellular signatures are shown in blue. The bars on the left indicate the statistical difference in enrichment between lesional and non-lesional PN skin, with colors representing different p-value thresholds (A). Comparison of PN-associated DEGs to DEGs in psoriasis (Pso) and atopic dermatitis (AD) for increased and decreased DEGs (B). Correlation analysis between effect sizes in lesional PN and effect sizes in psoriasis (Pso) and atopic dermatitis (AD). Spearman's rank correlation was included. Genes significant on the x-axis, y-axis, and both axes are colored red, blue, and purple, respectively (C). [Figure 2B] See legend to Figure 2A. [Figure 2C] See legend to Figure 2A. [Figure 3A] Figures 3A-3B show transcriptomic changes associated with the anti-IL31R inhibitor nemolizumab. Principal component analysis (PCA) of transcriptomic data from PN biopsies before and after 12 weeks of a prospective, placebo-controlled, double-blind clinical trial using the anti-IL31R inhibitor nemolizumab. Different colors represent different treatment groups, with lesional samples shown as triangles and non-lesional skin shown as circles (A). Heatmap showing two-way clustering (using genes differentially expressed between non-lesional and lesional skin at baseline) for all samples (B). [Figure 3B] See legend to Figure 3A. [Figure 4A] Figures 4A-4C show the effect of nemolizumab on PN-related transcriptomic changes. (A) Three-way Bend plot of increases and decreases in DEGs in PN skin and their overlap with DEGs in the nemolizumab and placebo cohorts (compared to baseline). (B) Correlation analysis between different groups (PN baseline vs. placebo and nemolizumab DEGs) (Spearman's rank correlation). [Figure 4B] See legend to Figure 4A. [Figure 4C] See legend to Figure 4A. [Figure 5A] Figures 5A-5D show that nemolizumab treatment resulted in normalized epidermal differentiation and reduced IL-31 / Th2 responses in PN skin. Nemolizumab treatment resulted in reduced IL-31 and IL-13 responses in PN skin compared to placebo, along with reduced expression of IL-17A-responsive genes (A). Nemolizumab treatment was accompanied by reduced transcriptomic signatures of Th1, Th17, and Th2 cells (B). Cross-comparison of the transcriptomic responses in PN skin to cellular signatures obtained from single-cell data from healthy epidermis demonstrated that nemolizumab treatment resulted in normalized epidermal gene expression associated with the epidermal differentiation layer (KRT10+), corresponding to normalized epidermal differentiation. The different nomenclature corresponds to different layers of the epidermis, with "basal" corresponding to KRT5+ basal cells, KRT10+ "differentiated" corresponding to the epidermal spinous layer, and "keratinized" corresponding to the granular layer (FLG+) (C). There was greater normalization of transcription factor binding sites (TFBS) of PN-associated DEGs between nemolizumab downregulated genes compared to placebo (D). [Figure 5B] See legend to Figure 5A. [Figure 5C] See legend to Figure 5A. [Figure 5D] See legend to Figure 5A. [Figure 6A] Figures 6A-6B show a nemolizumab-driven reduction in pruritus scores after 12 weeks of treatment, accompanied by tighter clustering of PN samples in PCA analysis. The Peak Pruritus Numerical Rating Scale (PP-NRS) was overlaid onto transcriptomic data from PN lesion skin and evaluated using PCA analysis. The nemolizumab group is shown as large dots, and the placebo group is shown as small dots. Baseline (top) and week 12 (bottom) of treatment are shown (A). Nemolizumab treatment resulted in tighter clustering of PN samples in PCA analysis compared to biopsy samples from the placebo-treated cohort (B). [Figure 6B] See legend to Figure 6A. [Figure 7] Figure 1 shows the cytokine signature in PN in placebo vs. nemolizumab-treated skin. A literature-based network generated using the Genomatix Pathway System (GePS, genomatix.de) with a function term co-citation filter shows key cytokines as key nodes in PN skin. [Figure 8] 1 shows the expression of selected TFs in PN skin before and after nemolizumab treatment. [Figure 9] PN, distances between PCA components before and after nemolizumab treatment are shown. [Figure 10] Clinical scores of patients selected for mass spectrometry are shown. Left panel: PNR score at baseline. Right panel: NRS change at week 12. [Figure 11] Enriched canonical pathways from mass spectrometry analysis are shown. Enriched canonical pathways were sorted according to z-score only (right) and z-score and p-value (p<0.05) (left). [Figure 12] Upstream regulator analysis from mass spectrometry analysis. Top: Upstream regulators sorted according to z-score only (right) and z-score and p-value (p<0.05) (left). [Figure 13] The enriched biological functions, as identified by mass spectrometry, are shown. The enriched biological function ontologies were sorted according to z-score and p-value (p<0.05). [Figure 14] Hierarchical clustering heatmaps based on scRNA seq for samples obtained from healthy skin (H), diseased PN (LPN), and non-diseased PN (NPN) skin samples are shown. The Y axis represents single cells, and the X axis represents the genes expressed by them. The right side of the heatmap shows annotations of cell source and cell type. [Figure 15] UMAP representation of fibroblast clusters based on scRNAseq. Clusters annotated by disease type, fibroblast subtype, cell differentiation trajectory, and pseudotime. [Figure 16]Figure 1 shows a graphical summary of lesional PN fibroblast DEGs using Ingenuity pathway analysis. Orange indicates pathways, regulators, or processes that are activated in lesional PN skin compared to healthy skin, and blue indicates inactivation. [Figure 17] Figure 1 shows the cell-cell communication network in PN-lesioned skin. Data are derived from scRNAseq data. The higher the number of interactions / interaction strengths, the thicker the line connecting two cell types. [Figure 18] Figure 1 shows a graphical summary of results from Ingenuity pathway analysis of bulk RNA-seq data from a Phase II study of nemolizumab. Orange indicates pathways, regulators, or processes that are activated in lesional PN skin of patients treated with nemolizumab compared to healthy skin, and blue indicates inactivation. [Figure 19] Figures 19A-19E show the cell types and their spatial locations observed in PN skin. Panel A shows a UMAP plot showing 72,782 cells color-coded by cell type. Panel B shows a UMAP plot showing cells color-coded by skin condition (H: healthy control, NPN: non-lesional sample from a patient with PN, LPN: lesioned sample from a patient with PN). Panel C shows a bar plot showing the abundance composition across skin conditions for each cell type in scRNA-seq. Panel D shows a dot plot showing representative marker genes for each cell type. The color scale represents the scaled expression of each gene. The size of the dot represents the percentage of cells expressing each gene of interest. Panel E shows a spatial plot showing the prediction score for each cell type. The coordinates of the spots correspond to their location within the tissue. [Figure 20]Figures 20A-20C show the spatial location of the major cell types detected in PN skin. Panel A shows hematoxylin and eosin (H&E) staining of the PN skin biopsy used for spatial sequencing. Panel B shows a scatter plot showing the cell type composition for each spot in the spatial-seq sample. Each spot is represented as a pie chart showing the relative proportion of the cell type. Panel C shows a spatial plot showing the extracellular matrix score in the spatial-seq sample. [Figure 21]Figures 21A-21J show the identification of fibroblast subtypes. Panel A shows a UMAP plot of 15,084 fibroblasts color-coded by subtype. Panel B shows a UMAP plot of fibroblasts color-coded by skin condition. Panel C shows a bar plot illustrating the abundance of each fibroblast subtype across skin conditions. Panel D shows a dot plot depicting the top marker genes for each fibroblast subtype. The color scale represents the scaled expression of each gene. The size of the dot represents the percentage of cells expressing the gene of interest. Panel E shows a violin plot depicting the extracellular matrix module score in fibroblast subtypes divided by skin condition. Panel F shows a dot plot depicting upstream regulators of COL11A+FB-identified DEGs by comparing LPN with healthy cells. The color scale represents the -log10 (p-value) from the enrichment analysis. The size of the dot represents the number of differentially expressed genes downstream of the upstream regulator. Panel G shows a bar plot depicting the top 10 pathways enriched using upregulated DEGs identified in COL11A+ FBs by comparing LPNs with healthy cells. Panel H shows a dot plot depicting the expression of all collagen genes across fibroblast subtypes. The color scale represents the scaled expression of each gene. The size of the dot represents the percentage of cells expressing the gene of interest. Panel I shows immunohistochemical staining for trichrome, procollagen I, and COL11A1 in PN and healthy tissues. Panel J shows a violin plot depicting the extracellular matrix module scores in fibroblast subtypes divided by healthy, PN, and AD skin conditions. [Figure 22] Figure 1 shows the identification of fibroblast subtypes. This figure provides immunohistochemical staining of SFRP4, SFRP2, and RAMP1 in PN and healthy tissue. [Figure 23]Figures 23A-H show the identification of endothelial subtypes. Panel A shows a UMAP plot depicting 3,840 endothelial cells color-coded by subcluster. Panel B shows a dot plot depicting the top marker genes for each endothelial subcluster. The color scale represents the scaled expression of each gene. The size of the dot represents the percentage of cells expressing the gene of interest. Panel C shows a UMAP plot depicting endothelial cells color-coded by skin condition. Panel D shows a bar plot depicting the enrichment composition across skin conditions for each endothelial subcluster. Panel E shows a dot plot depicting upstream regulators of cluster marker genes for endothelial subcluster 2. The color scale represents -log10(p-value) from the enrichment analysis. The size of the dot represents the number of differentially expressed genes downstream of the upstream regulator. Panel F shows a bar plot depicting the top 10 pathways enriched using cluster marker genes for endothelial subcluster 2. Panel G shows a dot plot depicting upstream regulators of cluster marker genes for endothelial subcluster 5. The color scale represents the -log10(p-value) from the enrichment analysis. The size of the dot represents the number of differentially expressed genes downstream of the upstream regulator. Panel H shows a bar plot showing the top 10 pathways enriched using cluster marker genes for endothelial subcluster 5. [Figure 24]Figures 24A-24J show the identification of pericyte subtypes. Panel A shows a UMAP plot depicting 3,052 pericytes color-coded by subcluster. Panel B shows a UMAP plot depicting pericytes color-coded by skin condition. Panel C shows a bar plot depicting the abundance composition of each pericyte subcluster across skin conditions. Panel D shows a dot plot depicting the top marker genes for each pericyte subcluster. The color scale represents the scaled expression of each gene. The size of the dot represents the percentage of cells expressing the gene of interest. Panel E shows a violin plot depicting the extracellular matrix module score in pericyte subclusters divided by skin condition. Panel F shows a dot plot depicting the expression of all collagen genes across pericyte subclusters. The color scale represents the scaled expression of each gene. The size of the dot represents the percentage of cells expressing the gene of interest. Panel G shows a dot plot depicting upstream regulators of cluster marker genes for pericyte subcluster 3. The color scale represents -loglO(p-value) from the enrichment analysis. The size of the dot represents the number of differentially expressed genes downstream of the upstream regulator. Panel H shows a dot plot showing upstream regulators of cluster marker genes for pericyte subcluster 7. The color scale represents -loglO(p-value) from the enrichment analysis. The size of the dot represents the number of differentially expressed genes downstream of the upstream regulator. Panel I shows a bar plot showing the top 10 pathways enriched using cluster marker genes for pericyte subcluster 3. Panel J shows a bar plot showing the top 10 pathways enriched using cluster marker genes for pericyte subcluster 7. [Figure 25]Figures 25A-25F show the identification of keratinocyte subtypes. Panel A shows a UMAP plot depicting 40,277 keratinocytes color-coded by subtype. Panel B shows a UMAP plot depicting keratinocytes color-coded by skin condition. Panel C shows a bar plot depicting the abundance composition of each keratinocyte subtype across skin conditions. Panel D shows a dot plot depicting the top marker genes for each keratinocyte subtype. The color scale represents the scaled expression of each gene. The size of the dot represents the percentage of cells expressing the gene of interest. Panel E shows a bar plot depicting the top 10 pathways enriched using cluster marker genes for inflammatory keratinocytes. Panel F shows a dot plot depicting upstream regulators of cluster marker genes for inflammatory keratinocytes. The color scale represents -log10(p-value) from the enrichment analysis. The size of the dot represents the number of differentially expressed genes downstream of the upstream regulator. [Figure 26]Figures 26A-J show the identification of myeloid and T cell subtypes. Panel A shows a UMAP plot of 2,130 myeloid cells color-coded by subtype. Panel B shows a UMAP plot of myeloid cells color-coded by skin condition. Panel C shows a dot plot showing the top marker genes for each myeloid subtype. The color scale represents the scaled expression of each gene. The size of the dot represents the percentage of cells expressing the gene of interest. Panel D shows a bar plot showing the abundance composition of each myeloid subtype across skin conditions. Panel E shows immunohistochemical staining of TREM2 and CD138 in PN skin tissue. Panel F shows a UMAP plot of 5,817 T cells color-coded by subtype. Panel G shows a UMAP plot of T cells color-coded by skin condition. Panel H shows a dot plot showing the top marker genes for each T cell subtype. The color scale represents the scaled expression of each gene. The size of the dot represents the percentage of cells expressing the gene of interest. Panel I shows a bar plot demonstrating the abundance composition of each T cell subtype across skin conditions. Panel J shows immunohistochemical staining for CD8 and CD4 in PN skin tissue. [Figure 27]Figures 27A-F show cell-cell interactions revealed by ligand-receptor analysis. Panel A shows a heat map depicting the number of ligand-receptor pairs in healthy samples. Rows, cell types expressing the ligand; columns, cell types expressing the receptor. Color scale, number of ligand-receptor pairs. Panel B shows a heat map depicting the number of ligand-receptor pairs in NPN samples. Rows, cell types expressing the ligand; columns, cell types expressing the receptor. Color scale, number of ligand-receptor pairs. Panel C shows a heat map depicting the number of ligand-receptor pairs in LPN samples. Rows, cell types expressing the ligand; columns, cell types expressing the receptor. Color scale, number of ligand-receptor pairs. Panel D shows dot plots depicting the expression of specific ligands (left) and receptors (right) with higher interaction scores in LPN compared to healthy samples. The color scale indicates the expression level within the cells, and the size of the dot reflects the percentage of cells expressing the gene. Panel D shows cell-cell interactions based on interaction weight / strength using CellChat. Panel F shows a heatmap of the TGFb signaling pathway network in healthy, NPN, and LPN skin, outlining source (sender) and target cells (receivers, mediators, influencers). [Figure 28-1] Figures 28A-28F show a comparison of atopic dermatitis (AD) and prurigo nodularis (PN) keratinocyte subtypes. Panel A shows a UMAP plot showing 68,451 keratinocytes color-coded by skin condition. Panel B shows a UMAP plot showing keratinocytes color-coded by clustering. Panel C shows a UMAP plot showing keratinocytes color-coded by keratinocyte subtype. Panel D shows dot plots showing the top marker genes for each keratinocyte cluster. The color scale represents the scaled expression of each gene. The size of the dot represents the percentage of cells expressing the gene of interest. Panel E shows a bar plot showing the number of keratinocytes in each cluster for each skin condition. Panel F shows the enriched GO BP processes in AD vs. PN skin for each keratinocyte subtype. [Figure 28-2] See description of Figure 28-1. [Figure 28-3] See description of Figure 28-1. [Figure 29] Figures 29A-29H show a comparison of atopic dermatitis (AD) and prurigo nodularis (PN) T cell and myeloid subtypes. Panel A shows a UMAP plot of 10,389 T cells color-coded by cluster. Panel B shows a UMAP plot color-coded by T cell subtype. Panel C shows a UMAP plot of T cells color-coded by disease state. Panel D shows a bar plot showing the abundance and number of each T cell subtype across each disease state. Panel E shows a UMAP plot of 5,752 myeloid cells color-coded by cluster. Panel F shows a UMAP plot of myeloid cells color-coded by myeloid subtype. Panel G shows a UMAP plot of bone marrow color-coded by disease state. Panel H shows a bar plot showing the abundance and number of each myeloid subtype across each disease state. [Figure 30-1] Figures 30A-B show the identification of myeloid and T cell subtypes. Panel A shows UMAP plot expression of specific immune cells in lesional AD (LAD) and lesional PN skin (LPN). Panel B shows violin plots showing expression in each T cell subset for specific immune genes. [Figure 30-2] See description of Figure 30-1. [Figure 31-1]Figures 31A-31H show the effects of nemolizumab on keratinocyte and fibroblast subtypes. Panel A shows a UMAP plot showing IL31RA expression primarily in keratinocytes and fibroblasts. Panel B shows a UMAP plot showing OSMR expression primarily in keratinocytes, fibroblasts, endothelial cells, and pericytes. Panel C shows a UMAP overlay of DEGs increased by nemolizumab treatment (nemolizumab-positive). Panel D shows a UMAP overlay of DEGs decreased by nemolizumab treatment (nemolizumab-negative). Panel E shows a violin plot showing nemolizumab-upregulated gene module scores in fibroblast subtypes divided by skin condition. Panel F shows a violin plot showing nemolizumab-downregulated gene module scores in fibroblast subtypes divided by skin condition. Panel G shows a violin plot illustrating nemolizumab up-regulated gene module scores in keratinocyte subtypes divided by skin condition. Panel H shows a violin plot illustrating nemolizumab down-regulated gene module scores in keratinocyte subtypes divided by skin condition. [Figure 31-2] See description of Figure 31-1. [Figure 32] Figures 32A-B show the effect of nemolizumab across major cell types in scRNA-seq. Panel A shows a violin plot showing nemolizumab up-regulated gene module scores across all cell types divided by skin condition. Panel B shows a violin plot showing nemolizumab down-regulated module scores across all cell types divided by skin condition. DETAILED DESCRIPTION OF THE INVENTION

[0037] Detailed Description Described herein are treatments and preventions of prurigo nodularis (PN) using anti-IL-31RA antibodies (e.g., nemolizumab) and previously unknown biomarkers and gene signatures associated with PN. The disclosed biomarkers, including differentially expressed genes (DEGs), PN-specific gene ontologies (GOs), and other inflammatory and hyperproliferative markers, can be used to identify subjects with PN, determine whether a subject is likely to respond to treatment (e.g., with an anti-IL-31RA antibody), and track the subject's responsiveness to treatment. The disclosed treatments and preventions achieve therapeutic endpoints (e.g., normalizing DEGs, normalizing epidermal hyperproliferation, normalizing epidermal differentiation, and / or reducing inflammatory responses in the skin) previously unknown or unattainable with conventional treatments for PN.

[0038] Furthermore, histological analysis of the PN nodules described herein reveals epidermal dysregulation (hyperkeratosis and hyperplasia), dermal fibrosis, and inflammatory cell infiltration. Thus, the present disclosure also provides the role of fibroblasts in the pathogenesis of PN and the associated clinical changes, outcomes, and endpoints achieved by nemolizumab.

[0039] I. Definition It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0040] Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Unless otherwise specified, materials and / or methodology known to those of ordinary skill in the art may be utilized in carrying out the methods described herein, based on the guidance provided herein.

[0041] As used herein, the singular terms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Reference to an object in the singular is not intended to mean "one and only one," unless expressly so stated, but rather "one or more."

[0042] As used herein, when used in conjunction with a numerical value, "about" means the stated numerical value, as well as plus or minus 10% of the numerical value. For example, "about 10" should be understood as both "10" and "9 to 11."

[0043] As used herein, "and / or" refers to and includes any and all possible combinations with one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative ("or").

[0044] As used herein, a phrase of the form "A / B" or of the form "A and / or B" means (A), (B), or (A and B), and a phrase of the form "at least one of A, B, and C" means (A), (B), (C), (A and B), (A and C), (B and C), or (A, B and C).

[0045] As used herein, the phrase "therapeutically effective amount" with respect to an anti-IL31R antibody (e.g., nemolizumab) refers to a dose of the antibody that provides the specific pharmacological effect for which the drug is administered to a subject in need of such treatment. A therapeutically effective amount may be effective to reduce, ameliorate, or eliminate itching, scratching, and / or lesion or nodule formation, and / or improve quality of life in a subject with PN. It is emphasized that in certain cases, a therapeutically effective amount of an anti-IL31R (e.g., nemolizumab) may not always be effective in treating PN in an individual subject, even if it is considered a therapeutically effective amount by those skilled in the art. Those skilled in the art can adjust what is considered a therapeutically effective amount according to standard practices as needed to treat a particular subject. A therapeutically effective amount may vary based on, for example, the age and weight of the subject, and / or the subject's general health, and / or the severity of the subject's PN.

[0046] The terms "treat," "treatment," or "treating" as used herein with respect to PN refer to reducing, improving, or eliminating itching, scratching, and / or lesion or nodule formation, and / or improving quality of life in a subject with PN.

[0047] The terms "prevent," "preventing," or "prevention," as used herein with respect to PN, refer to eliminating or reducing the risk of developing lesions or nodules, or preventing the development of a biomarker signature of the present disclosure associated with PN. Prevention can also refer to the prevention of PN flares or recurrences after an initial flare has been treated or cured.

[0048] As used herein, the terms "individual," "subject," and "patient" are used interchangeably and refer to any individual mammalian subject, e.g., a cow, dog, cat, horse, or human. In specific embodiments, the subject, individual, or patient is a human.

[0049] II. Prurigo Nodularis (PN) and Biomarkers Prurigo nodularis (or "PN") is a skin disorder that causes hard, itchy nodules to form on the skin. The itching (scratching) can be intense and can lead to bleeding or painful scratching. Scratching can cause more skin lesions to appear. The itching is exacerbated by heat, sweating, or irritation from clothing. In some cases, people with PN have a history of other diseases, including eczema (atopic dermatitis), diabetes, lymphoma, HIV infection, severe anemia, or kidney disease. The exact cause of PN was previously poorly understood. It was thought that nodules were likely to form if the skin was injured or irritated in some way. Therefore, scratching the skin can cause nodules to form. However, the original cause of the intense itching was unknown.

[0050] Approximately 50% of people with PN have a history of atopy. The primary symptom of PN is the formation of hard, extremely itchy lumps (nodules) on the skin. Nodules can range in size from very small to approximately half an inch in diameter. Nodules often have a rough, dry top and can range in number from a few to hundreds. Nodules most commonly form on the outer arms, shoulders, and legs. Nodules can also form on the neck and torso, rarely on the face and palms. They may be lighter or darker in color than the surrounding skin. Scarring may occur after the nodules begin to heal. PN symptoms can begin at any age but are most common in adults over the age of 50. People with PN can be very concerned about the appearance of the nodules, and the intensely itchy skin can interfere with sleep or daily activities. This can lead to stress and depression in people with PN.

[0051] Pruritus refers to the itchy and / or itchy sensation of the skin. It can be caused by other diseases or conditions, such as PN or dry skin. In some cases, itching involves generalized itching of the skin throughout the body. In some cases, itching is localized to a specific area of ​​the body, such as the arms or legs. It can be chronic or acute. Symptoms of pruritus include, but are not limited to, peeling, redness, bumps, spots, blisters, dry skin, cracked skin, and a leathery or scaly texture to the skin. In some cases, itching does not result in detectable changes to the skin. Behavioral responses to pruritus include, but are not limited to, scratching and / or massaging the skin. In some cases, scratching the skin can result in peeling ranging from mild to severe. In some cases, patients with pruritus refrain from scratching and / or massaging their skin. Conventional treatments for PN include, but are not limited to, skin moisturizers, topical emollients, antihistamines such as diphenhydramine, topical corticosteroids, topical calcineurin inhibitors, and phototherapy, narrow-band UVB, and systemic immunosuppressants such as cyclosporine or methotrexate.

[0052] The present disclosure illuminates for the first time underlying gene expression patterns that are associated with PN and can be used to diagnose PN, identify subjects likely to respond to treatment (e.g., treatment with anti-IL-31RA antibodies), and determine whether a subject is responding appropriately to treatment.

[0053] In particular, the present disclosure demonstrates that subjects with PN may differentially express at least 5,934 genes (known as differentially expressed genes, or DEGs), as shown in FIG. 1A and Table 1 below. This differential gene expression may be observed in the subject's skin, particularly in skin samples that contain or consist of nodules or lesions. In some embodiments, the differential gene expression may be at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 300, at least 350, at least 400, at least 450, at least 500, at least 550, at least 600, at least 650, at least 700, at least 750, at least 800, at least 800, at least 900, at least 1000, at least 1500, at least 2000, at least 2500, at least 3000, at least 3500, at least 4000, at least 4500, at least 5000, at least 5500, at least 6000, at least 6500, at least 7000, at least 7500, at least 8000, at least 9000, at least 10 ... 50, at least 900, at least 950, at least 1000, at least 1100, at least 1200, at least 1300, at least 1400, at least 1500, at least 1600, at least 1700, at least 1800, at least 1900, at least 2000, at least 2100, at least 2200, at least 2300, at least 2400, or at least 2500, and up to 2500, 3000, 3500, 4000, 4500, 5000, 5500, or about 6000 DEGs of the present disclosure may be differentially expressed in subjects with PN. Of these DEGs, 2,060 may be increased (i.e., overexpressed), and 3,874 may be decreased (i.e., not expressed). Genes that may be most increased include: KRT6C, which may be increased by at least 100-fold, at least 150-fold, at least 200-fold, at least 250-fold, at least 300-fold, at least 350-fold, at least 400-fold, at least 450-fold, at least 500-fold, at least 550-fold, or 588-fold compared to expression levels in a sample (e.g., a skin sample) from an individual without PN; DEFB4A, which may be increased by at least 25-fold, at least 50-fold, at least 75-fold, at least 100-fold, at least 125-fold, or at least 150-fold compared to expression levels in a sample (e.g., a skin sample) from an individual without PN, and KRT16, which may be increased by at least 10-fold, at least 20-fold, at least 30-fold, at least 40-fold, at least 50-fold, at least 60-fold, at least 70-fold, at least 80-fold, or at least 90-fold compared to expression levels in a sample (e.g., a skin sample) from an individual without PN.

[0054] Decreased genes include: LCE5A, which may be decreased by at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, or at least 11-fold compared to its expression level in a sample (e.g., a skin sample) from an individual without PN, and AQP7, which may be reduced by at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, or at least 7.9-fold compared to expression levels in a sample (e.g., a skin sample) from an individual without PN.

[0055] Genes encoding cytokines may also be overexpressed in subjects with PN. For cytokine genes, the most prominent upregulated genes are IL-36 family members and IL-20 family members. These upregulated or overexpressed genes may include: IL36A (e.g., approximately 6.8 fold, FDR = 1.8 × 10 -4 ), IL36G (e.g., approximately 8.4 fold, FDR = 3.9 × 10 -25 ), IL19 (e.g., approximately 5.1 fold, FDR = 7.4 × 10 -4 ), IL20 (e.g., approximately 3.5 times, FDR = 1.7 × 10 -3), IL22 (e.g., approximately 2.7 times, FDR = 2.9 × 10 -2 ), IL24 (e.g., approximately 5.8 times, FDR = 3.8 × 10 -10 ), and IL26 (e.g., approximately 4.9 fold, FDR = 3.3 × 10 -3 ). Each of these IL-36 and IL-20 family member cytokine genes can be overexpressed by at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 3.5-fold, at least about 4-fold, at least about 4.5-fold, at least about 5-fold, at least about 5.5-fold, at least about 6-fold, at least about 6.5-fold, at least about 7-fold, at least about 7.5-fold, at least about 8-fold, or at least about 8-5-fold compared to expression levels in a sample (e.g., a skin sample) from an individual without PN.

[0056] Other factors that may be upregulated or overexpressed include IL1A (e.g., approximately 4.7-fold, FDR=1.0×10 -12 ) and IL1B (e.g., approximately 4.1-fold, FDR = 3.7 × 10 -6 In addition, the IL4R gene has a FDR of about 2.6 times (FDR = 6.3 × 10 -19 ) can be increased. Table 1 at the end of the Examples section of this specification provides a more comprehensive list of DEGs.

[0057] In addition to the aforementioned genes, the present disclosure also demonstrates that certain plasma markers or signatures may be altered as a result of successful treatment of PN with an anti-IL-31RA antibody such as nemolizumab. Such markers or signatures may be detectable, for example, by mass spectrometry and other protein evaluation methods (e.g., ELISA, Western blot, etc.). Circulating plasma protein markers or signatures that may be modulated as a result of treatment with an anti-IL-31RA antibody such as nemolizumab may include leukocyte migration and cell movement, the IL-6 pathway, the vascular endothelial growth factor (VEGF) pathway, the STAT3 (signal transducer and activator of transcription 3) pathway, the STAT5b (signal transducer and activator of transcription 5b) pathway, the TGFB1 (transforming growth factor beta-1) pathway, and neuroontology.

[0058] The STAT3 pathway, a direct target of IL-31 signaling, is also inhibited in the nemolizumab responder signature of the present disclosure, suggesting target engagement. STAT3 activity and expression may be relatively higher in subjects with PN or in subjects prior to initiating treatment with an anti-IL-31RA antibody, such as nemolizumab, compared to individuals or populations without PN.

[0059] The amount of circulating proinflammatory cytokines may be relatively high in subjects with PN, or in subjects prior to initiating treatment with an anti-IL-31RA antibody, such as nemolizumab, compared to individuals or populations without PN. Such proinflammatory cytokine signatures include, but are not limited to, IL-6 and VEGF. In subjects with PN treated with an anti-IL-31RA antibody, such as nemolizumab, the treatment may result in a decrease in one or both of IL-6 and VEGF, or a decrease or inhibition of the signaling pathways of IL-6, VEGF, or both, compared to baseline levels. The baseline levels may be determined with respect to (i) a control sample obtained from one or more individuals (i.e., a population) without PN, or (ii) a biological sample obtained from the subject prior to administration of the anti-IL-31RA antibody.

[0060] TGFB1 activity is also inhibited in the nemolizumab responder signature of the present disclosure. TGFB1 activity and expression may be relatively higher in subjects with PN or in subjects prior to initiating treatment with an anti-IL-31RA antibody, such as nemolizumab, compared to individuals or populations without PN.

[0061] For purposes of plasma protein markers of the present disclosure, the amount of a protein marker of the present disclosure in the plasma of a subject with PN may be at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 30-fold, at least 35-fold, at least 40-fold, at least 45-fold, or at least 50-fold higher than the baseline level. The baseline level may be determined with respect to (i) a control sample obtained from one or more individuals (i.e., a population) without PN, or (ii) a biological sample obtained from the subject prior to administration of an anti-IL-31RA antibody. Similarly, after a subject with PN is treated with an anti-IL-31RA antibody, such as nemolizumab (e.g., 2, 4, 6, 8, 10, or 12 weeks after administration of the antibody), the amount of a plasma protein marker of the present disclosure in the subject may be decreased by at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 30-fold, at least 35-fold, at least 40-fold, at least 45-fold, or at least 50-fold compared to baseline levels. Baseline levels may be determined with respect to (i) a control sample obtained from one or more individuals (i.e., a population) without PN, or (ii) a biological sample obtained from the subject prior to administration of the anti-IL-31RA antibody.

[0062] Additionally, neuronal ontologies (e.g., CREB signaling in neurons, synaptogenesis signaling pathways, neuroglial cell death, and neuroglial apoptosis) may be upregulated in subjects with PN and subsequently downregulated in nemolizumab-responder subjects, thus highlighting the impact of IL-31 as a neuroinflammatory cytokine in PN.

[0063] Thus, the present disclosure provides a method for diagnosing PN, comprising detecting the expression levels of at least one, at least two, at least three, at least four, or at least five of the differentially expressed genes (DEGs) in Table 1 (e.g., KRT6C, DEFB4A, KRT16, LCE5A, AQP7, IL-36 family members, IL-20 family members, etc.) in a sample obtained from a subject suspected of having PN, and comparing the expression levels of the DEGs to a reference level based on gene expression levels in a sample (e.g., a skin sample) from an individual without PN. In some embodiments, the sample obtained from a subject suspected of having PN is a skin sample that may contain lesions or nodules. If certain genes are upregulated or overexpressed (e.g., KRT6C, DEFB4A, KRT16) and / or other genes are downregulated or underexpressed (e.g., LCE5A, AQP7), the subject may be diagnosed with PN.

[0064] The present disclosure also provides methods for determining the likelihood of a positive response to treatment (e.g., treatment with an anti-IL-31RA antibody such as nemolizumab) in a subject with PN, the method comprising detecting the expression levels of at least one, at least two, at least three, at least four, or at least five of the differentially expressed genes (DEGs) in Table 1 (e.g., KRT6C, DEFB4A, KRT16, LCE5A, AQP7, IL-36 family members, IL-20 family members, etc.) in a sample obtained from the subject with PN, and comparing the expression levels of the DEGs to a reference level based on gene expression levels in a sample (e.g., a skin sample) from an individual without PN. In some embodiments, the sample obtained from the subject with PN is a skin sample that may contain lesions or nodules. If certain genes are upregulated or overexpressed (e.g., KRT6C, DEFB4A, KRT16) and / or other genes are downregulated or underexpressed (e.g., LCE5A, AQP7), the subject is more likely to respond to treatment.

[0065] The present disclosure also provides methods for determining whether a subject with PN is responding to treatment (e.g., treatment with an anti-IL-31RA antibody such as nemolizumab), comprising detecting the expression levels of at least one, at least two, at least three, at least four, or at least five of the differentially expressed genes (DEGs) in Table 1 (e.g., KRT6C, DEFB4A, KRT16, LCE5A, AQP7, IL-36 family members, IL-20 family members, etc.) in a sample obtained from the subject with PN before treatment is initiated, and comparing the expression levels of the DEGs to a baseline level of expression, where the baseline level of gene expression is from a sample (e.g., a skin sample) from the same individual. In some embodiments, the sample may be a skin sample that may contain lesions or nodules. The subject is responding to treatment if the expression levels of certain genes (e.g., KRT6C, DEFB4A, KRT16) decrease and / or the expression levels of other genes (e.g., LCE5A, AQP7) increase.

[0066] The present disclosure also provides a method of determining whether a subject with PN is responding to treatment (e.g., treatment with an anti-IL-31RA antibody such as nemolizumab), comprising detecting one or more biomarkers selected from leukocyte migration and cell movement, IL-6 pathway, VEGF pathway, STAT3 pathway, STAT5b pathway, TGFB1 pathway, and a neuroontology of the disclosure in a post-treatment plasma sample obtained from a subject with PN administered at least one dose of an anti-IL-31RA antibody, wherein a decrease in leukocyte migration and cell movement, a decrease in IL-6 or a decrease in IL-6 pathway signaling, a decrease in VEGF or a decrease in VEGF pathway signaling, a decrease in STAT3 or a decrease in STAT3 pathway signaling, a decrease in STAT5b or a decrease in STAT5b pathway signaling, a decrease in TGFB1 or a decrease in TGFB1 pathway signaling, or an increase in a neuroontology of the disclosure indicates responsiveness to treatment relative to a baseline amount, wherein the baseline amount was determined from a plasma sample obtained from the same subject before treatment was initiated.

[0067] This disclosure also defines for the first time the biological processes enriched in the PN skin gene ontology (GO). The most prominent GO categories associated with PN are: ● "Cornified envelope" (FDR=1.5×10 -12 ), ●“Epidermal cell differentiation” (FDR=6.4×10 -10 ), - Keratinization (FDR=1.6×10 -12 ), ● "Peptidase regulatory factor activity" (FDR = 1.1 × 10 -4 ), ● "Interleukin-4 and 13 signaling" (FDR = 6.8 × 10 -7 ), "Interferon alpha / beta signaling" and "response to interferon gamma" (FDR=4.1×10 -7 and FDR = 4.1 × 10 -6 ), ● "IL23 pathway" (FDR = 2 × 10 -5 ), and ● "Metaphase and anaphase" (FDR = 3.8 × 10 -10 ).

[0068] These GO categories are defined in more detail in Figure 1B and Table 2 at the end of the Examples section of this specification. These categories reflect the hyperproliferative nature of PN, which was found to be associated with alterations in epidermal differentiation and inflammatory components. For example, an inflammatory network including the proliferation marker Ki67 (MKI67), the cell cycle gene CDKN1A, and IL-1 and IL-36 was found to be potentially relevant to the pathogenesis of PN, as shown in Figure 1C.

[0069] The present disclosure also discloses 20 co-expression modules identified in non-lesional skin and 10 clusters in lesional PN skin, as shown in Figure 1D and Table 3 at the end of the Examples section herein. "Immune response" (FDR = 1.8 x 10 -47 ), "defense response" (FDR = 1.2 × 10 -39 ), the most prominent involved immunological processes (module #8), including "cell cycle" (FDR=2.9×10 -94 ), "DNA metabolic process" (FDR = 8.7 × 10 -67 ), including cell proliferation (module #6), and "epidermal development" (FDR 3.5 × 10 -10 ), “keratinization” (FDR=1.7×10 -6 Distinct functions can be defined for these co-expressed gene modules, including epidermal processes such as cutaneous neuropathy (module #5), and cutaneous neuropathy (module #6). See also Figure 1E. Another notable finding is the high expression of "extracellular matrix" (FDR = 1.16 × 10). -59 , module #2), including genes such as MMP14, MMP16, COL1A1, COL1A2, and COL3A1, which were slightly elevated in lesional skin (FC ≥ 1.4, FDR ≤ 6 × 10), consistent with the association between PN and dermal fibrosis. -2 ).

[0070] The present disclosure also provides specific cell type signatures for non-lesional and lesional PN skin samples. As shown in Figure 2A, enrichment of transcriptomic signatures associated with epithelial cells and keratinocytes was observed (p<0.001 and p<0.0001, respectively). Subjects with PN may exhibit a Th2-related signature (p<0.0001), consistent with the enriched GO category of IL-4 / IL-13 (as described above and shown in Figure 1B). Other inflammatory signatures, such as macrophages (p<0.01), may be more diverse.

[0071] As shown in Figure 2B, a three-way comparison of the PN transcriptomes of atopic dermatitis (AD) and psoriasis showed that all three diseases shared significant overlap in both up- and down-regulated genes. However, the correlation in effect sizes in lesional skin was more pronounced between PN and psoriasis (Spearman correlation ρ = 0.64) than between PN and AD (ρ = 0.55). Commonly up-regulated genes in both psoriasis and PN include those involved in cytokine activity (e.g., CCL3, CXCL10, IFNG, IL12B, IL19, IL1B, IL20) and keratinization (e.g., KRT16, KRT17, LCE3A, LCE3E) (Table 4).

[0072] Thus, the present disclosure provides methods for identifying or diagnosing PN based on gene ontology, co-expression modules, and / or gene signatures. For example, a subject may exhibit the PN skin gene ontology (GO) shown in FIG. 1B or Table 2. Additionally or alternatively, the skin sample may also exhibit upregulation or overexpression of Ki67 (MKI67), CDKN1A, and / or the inflammatory network, including IL-1 and IL-36. Additionally or alternatively, the subject may exhibit a co-expression module as shown in FIG. 1D or Table 3. Additionally or alternatively, a subject with PN may exhibit (e.g., in a skin sample) the transcriptomics or Th2 signature disclosed in FIG. 2A.

[0073] Furthermore, this disclosure provides unprecedented insight into the pathogenesis of PN and the associated tissue- and cell-type-specific changes that occur in PN skin both during disease development and in response to treatment with anti-IL31RA antibodies. Because PN pathogenesis is characterized by an increase in profibrotic responses accompanied by an immune shift away from IL-13 and IL-22 responses, changes in PN skin can be observed across both immune and stromal cell populations, including keratinocytes, endothelial cells, and most significantly, fibroblasts and fibroblast subpopulations, during treatment.

[0074] A characteristic histopathological feature of PN is papillary dermal fibrosis with vertically arranged collagen fibers, including a marked increase in compact collagen and increased expression of procollagen I in the papillary dermis of lesional PN skin by trichrome staining. Furthermore, COL11A1+ fibroblasts may be the primary source of an activated and enriched profibrotic response, including increased mRNA expression of both collagen I and collagen III. Consistent with their profibrotic function, COL11A1+ fibroblasts may be found primarily in the papillary dermis, where the fibrotic response is most pronounced with either trichrome or procollagen I staining, indicative of active collagen I biosynthesis.

[0075] The expansion of the COL11A1+ fibroblast subpopulation is unique to PN and is not seen in atopic dermatitis (AD) skin. Furthermore, the profibrotic effects of this population are not observed in AD COL11A1+ fibroblasts. Of the two components of the heterodimeric IL-31 receptor, IL31RA expression was detected in both keratinocytes and fibroblasts, whereas OSMRB expression was found to be more widespread in different cell populations. Thus, the two major cell types responding to IL-31 in PN are likely fibroblasts and keratinocytes, consistent with the observed transcriptomic shifts driven by treatment with anti-IL-31RA antibodies (e.g., nemolizumab) that can be attributed to these two cell types.

[0076] Other cell types, including endothelial cells and pericytes, also contribute to fibrosis in prurigo nodularis (LPN) skin. Endothelial changes are known in LPN, but the nature of these shifts has not been previously detailed. This disclosure demonstrates that endothelial cells, likely under the influence of proinflammatory and profibrotic cytokines such as TGFB, contribute to extracellular matrix reorganization. TGFβ may be an upstream promoter of fibrosis in PN skin, as it was observed to be expressed in a wide range of cell types in PN skin, including endothelial cells, fibroblasts, and neurons for TGFB1, and fibroblasts and pericytes for TGFB2 and TGFB3. Notably, TGFB2 and TGFB3 are more strongly involved in fibrosis than TGFB1.

[0077] One of the most distinctive histological features of PN is the presence of compact orthohyperkeratosis accompanied by irregular epidermal proliferation. Keratinocytes exhibit striking transcriptomic changes in lesional PN skin, with the most prominent shift seen in inflammatory keratinocytes, defined by expression of KRT6, KRT16, KRT17, S100A8, and S100A9. This analysis demonstrates the critical role of Th2 cytokines (e.g., IL-13 and IL-22) in this transition. The most enriched biological categories in inflammatory keratinocyte subsets were associated with mitochondrial function and protein translation, suggesting the generation of reactive oxygen species and cellular stress that may contribute to the inflammatory response in skin.

[0078] Immune cell infiltration also occurs in lesional PN skin and is characterized by a shift in specific immune cell populations. The most prominent shift can be observed in macrophage populations, particularly lipid-associated macrophages, characterized by the expression of APOE and TREM2. Lipid metabolites from lipid-associated macrophages have been shown to induce the production of proinflammatory cytokines in atherosclerosis, thereby amplifying the inflammatory response. T cells are also prominent in PN lesions, including cycling T cells, as well as NK and CD8 +The number of Tregs and T cell proliferation was increased. Furthermore, various stromal cell populations, particularly endothelial and pericytes, increased the expression of various proinflammatory cytokines, chemokines, and adhesion molecules, indicating their likely role in immune trafficking and amplification in PN. This includes increased expression of the adhesion molecules ICAM1, E-selectin (SELE), and IL6 on endothelial cells, as well as increased expression of CCL2, CCL3, CCL4, CCL13, CCL18, CXCL2, and CXCL12 expressed by various cell types (e.g., myeloid cells, pericytes, and endothelial cells) in the PN skin. CCL2 and IL-6 have established roles in the development of fibrosis. CCL2 is the most potent profibrotic chemokine, and through CCR2, CCL2 acts directly on fibroblasts to stimulate collagen synthesis. Similarly, IL-6 trans-signaling enhances lung fibroblast proliferation and extracellular matrix protein production.

[0079] This disclosure demonstrates that shifts in epidermal cell populations are highly similar between PN and AD, with both diseases possessing a distinct "inflammatory" keratinocyte subset characterized by increased expression of proinflammatory molecules, including S100A8 and S100A9, along with increased expression of the inflammatory keratins KRT6 and KRT16. While altered expression of S100A8, S100A9, and KRT16 has been described in AD skin, their expression has not been addressed in PN skin. These epidermal changes are accompanied by subtle changes in gene expression in PN versus AD skin, with regulators of immune-related processes, such as antimicrobial responses, and T cell trafficking, observed only in LAD but not LPN keratinocytes.

[0080] Alterations in T cell phenotype were observed between LPN and LAD skin, particularly within the CD4 effector T cell population, and mRNA expression of IL-13 and IL-22 was significantly lower in LPN skin compared with LAD skin. IL-22 is known to promote epidermal proliferation and activate innate immune and antimicrobial responses in the skin. Thus, although PN is an inflammatory-driven disease, it may not be centered on the IL-13 / IL-22 response to the same extent as AD.

[0081] The present disclosure provides a mechanism of action for IL-31 receptor antagonism. In particular, transcriptomic shifts indicative of stabilization of extracellular matrix remodeling and normalization of epidermal differentiation can be observed as a result of the disclosed therapeutic methods. Normalization of the pathological transcriptomic signature observed in COL11A1+ fibroblasts and inflammatory keratinocyte subsets may be associated with clinical improvement of PN skin lesions during treatment, which may also reverse the cellular, genetic, and molecular underpinnings of PN development and progression.

[0082] Thus, the present disclosure provides a method for inactivating, reducing the activation of, or decreasing the number of COL11A1+ fibroblasts in a subject with PN, comprising administering an anti-IL-31RA antibody to the subject, wherein administering the anti-IL-31RA antibody results in the inactivation, reduction of activation, or reduction in the number of COL11A1+ fibroblasts in the skin of the subject. In some embodiments, COL11A1+ fibroblasts are found in the papillary dermis. The total number of such reduced inactivated or activated fibroblasts can be determined compared to the number and activation state of fibroblasts in the skin lesion before treatment with an anti-IL-31RA antibody (e.g., nemolizumab).

[0083] The present disclosure also provides a method for reducing TGFβ expression in at least one cell type in a subject with PN, comprising administering an anti-IL-31RA antibody to the subject, wherein administering the anti-IL-31RA antibody results in a reduction in TGFβ expression in at least one cell type in the subject's skin. In some embodiments, the at least one cell type comprises fibroblasts, endothelial cells, pericytes, neurons, or any combination thereof. In some embodiments, the reduction in TGFβ expression comprises a reduction in expression of TGFB1, TGFB2, TGFB3, or any combination thereof. Such a reduction in TGFβ expression can be determined relative to the expression level in the corresponding cell type in the PN skin lesion before treatment with an anti-IL-31RA antibody (e.g., nemolizumab).

[0084] The present disclosure provides methods for reducing the expression of at least one inflammatory gene expressed by keratinocytes in a subject with PN, comprising administering an anti-IL-31RA antibody to the subject, wherein administering the anti-IL-31RA antibody results in a reduction in at least one inflammatory gene expressed by keratinocytes in the subject's skin. In some embodiments, the at least one inflammatory gene is selected from KRT6, KRT16, KRT17, S100A8, S100A9, and any combination thereof. In some embodiments, the keratinocytes express Th2 cytokines. In some embodiments, administering the anti-IL-31RA antibody results in a reduction in reactive oxygen species and / or cellular stress to which the keratinocytes are exposed. Such a reduction in inflammatory gene expression can be determined relative to the expression level in the corresponding cell type in the PN skin lesion before treatment with an anti-IL-31RA antibody (e.g., nemolizumab).

[0085] The present disclosure provides methods for reducing infiltration of at least one type of immune cell in skin lesions of a subject with PN, the method comprising administering an anti-IL-31RA antibody to the subject, wherein administering the anti-IL-31RA antibody results in a reduction in infiltration of at least one type of immune cell in at least one lesion in the subject's skin. In some embodiments, the at least one type of immune cell comprises a macrophage. In some embodiments, the macrophage is a lipid-associated macrophage characterized by expression of APOE and TREM2. In some embodiments, the at least one type of immune cell comprises a T cell, a NK cell, a CD8 + In some embodiments, the method comprises administering an anti-IL-31RA antibody, resulting in a decrease in the expression of ICAM1, E-selectin (SELE), IL6, CCL2, CCL3, CCL4, CCL13, CCL18, CXCL2, CXCL12, and any combination thereof, in at least one cell type in the lesion. In some embodiments, the at least one cell type in the lesion comprises myeloid cells, pericytes, endothelial cells, and any combination thereof. Such a decrease in immune cell infiltration or expression of ICAM1, E-selectin (SELE), IL6, CCL2, CCL3, CCL4, CCL13, CCL18, or CXCL2, CXCL12 can be determined by comparing the expression levels of the corresponding cell types in the PN skin lesion before treatment with an anti-IL-31RA antibody (e.g., nemolizumab) and the amount of infiltration observed in the skin lesion before treatment.

[0086] The expression levels of the genes and markers disclosed herein can be determined by any suitable method known in the art, including, but not limited to, RT-qPCR, RT-PCR, RNA-seq, Northern blotting, serial analysis of gene expression (SAGE), DNA or RNA microarrays, and in situ hybridization. At the protein level, the biomarkers of the present disclosure may be detected or measured using, for example, Western blotting, ELISA (enzyme-linked immunosorbent assay), surface plasmon resonance, and mass spectrometry.

[0087] Subjects with or suspected of having PN that display any of the DEGs, gene ontology, co-expression modules, or gene signatures of the present disclosure are suitable for treatment or prevention with an anti-IL-31RA antibody, such as nemolizumab, as described in further detail herein.

[0088] The present disclosure provides a combination of: (1) a pharmaceutical composition for use in the treatment or prevention of prurigo nodularis (PN), comprising an anti-IL31RA antibody (e.g., nemolizumab or a fragment or variant thereof) as an active ingredient; and (2) a diagnostic agent for detecting the expression level of at least one gene selected from the genes disclosed in Table 1 in a subject suspected of having PN, compared to a reference expression level of the at least one gene.

[0089] III. Therapeutic Antibodies and Interleukin-31 Receptor Subunit Alpha (IL-31RA) Interleukin-31 (IL-31) is a neuroinflammatory cytokine that can activate both structural and immune cells and peripheral nerves. IL-31 is involved in numerous chronic inflammatory diseases, including atopic dermatitis. IL-31 is produced by a variety of cells, including type 2 helper (Th2) T cells. IL-31 signals through a receptor complex consisting of the IL-interleukin-31 receptor subunit alpha ("IL-31RA," also known as NR10, glm-r, and GPL) and oncostatin M receptor beta (OSMRβ), which are expressed in immune and epithelial cells, as well as a subset of neurons.

[0090] When IL-31RA functions as an IL-31 receptor, it forms a heterodimer with the oncostatin M receptor (OSMR). There are several known splice variants of human-derived IL-31RA (WO 00 / 075314): NR10.1 consists of 662 amino acids and contains a transmembrane domain. NR10.2 is a soluble receptor-like protein consisting of 252 amino acids without a transmembrane domain. Further known IL-31RA splice variants that function as transmembrane receptor proteins include NR10.3 and IL-31RAv3. Preferred IL-31RA variants include NR10.3 (also referred to as ILRAv4 (Nat Immunol 5, 752-60, 2004)) and IL-31RAv3. NR10.3 (IL31RAv4) consists of 662 amino acids (WO00 / 075314, Nat Immunol 5, 752-60, 2004), and IL31RAv3 consists of 732 amino acids (GenBank accession number: NM-139017).

[0091] The amino acid sequence of IL31RAv4 is as follows: (SEQ ID NO: 1).

[0092] The amino acid sequence of IL31RAv3 is as follows: (SEQ ID NO: 2).

[0093] Mouse-derived IL-31RA contains the following amino acid sequence: (SEQ ID NO: 3).

[0094] Cynomolgus monkey-derived IL-31RA contains the following amino acid sequence: (SEQ ID NO: 4).

[0095] For purposes of this disclosure, an anti-IL-31RA antibody (ie, a therapeutic antibody) such as nemolizumab must bind to at least human IL-31RA or a splice variant thereof.

[0096] As used herein, the term "antibody" refers collectively to immunoglobulin or immunoglobulin-like molecules, including IgA, IgD, IgE, IgG, and IgM, combinations thereof, or fragments thereof. Antibody fragments may include, for example, Fab fragments and single-chain variable fragments (scFv). Antibodies generally contain heavy (H) and light (L) chains interconnected by disulfide bonds. There are two types of light chains: lambda (λ) and kappa (κ). There are five major heavy chain classes (or isotypes) that determine the functional activity of antibody molecules: IgG, IgA, IgM, IgD, and IgE. Each heavy and light chain contains a constant region and a variable region (also known as a "domain"). Combined, the heavy and light chain variable regions, also referred to as "Fab regions," specifically bind to a given antigen. The light and heavy chain variable regions contain a "framework" region interrupted by three hypervariable regions, also referred to as "complementarity-determining regions" or "CDRs." The extent of the framework regions and CDRs are defined (see Kabat et al., Sequences of Proteins of Immunological Interest, USDapartment of Health and Human Services, 1991). The Kabat database is currently maintained online. The sequences of the framework regions of different light or heavy chains are relatively conserved within a species, and the framework regions act to form a scaffold for orienting the CDRs through non-covalent interactions between the chains.

[0097] CDRs are primarily responsible for binding to an epitope on an antigen. The CDRs of each chain are typically referred to as CDR1, CDR2, and CDR3, are numbered sequentially starting from the N-terminus, and are typically identified by the chain in which the particular CDR is located. Thus, HCDR3 is located in the variable domain of the heavy chain of the antibody in which it is found, while LCDR1 is the CDR1 from the variable domain of the light chain of the antibody in which it is found. Antibodies that bind to IL-31RA have a specific V H Area and V LEach antibody has a specific CDR sequence, and therefore a specific CDR sequence.Antibodies with different specificities generally have different CDRs.Although CDRs are different for each antibody, only a limited number of amino acid positions within CDRs are directly involved in antigen binding.These positions within CDRs are called specificity-determining residues (SDRs).

[0098] The Fc fragment region (Fc) of an antibody plays a role in regulating immune cell activity. The Fc region ensures that each antibody generates an appropriate immune response to a given antigen by binding to specific classes of proteins found on specific cells, such as B lymphocytes, follicular dendritic cells, natural killer cells, macrophages, and neutrophils. These proteins are called "Fc receptors." Because the constant domains of the heavy chains make up the Fc region of an antibody, the class of heavy chain in an antibody determines its class effect. Antibody heavy chains include alpha, gamma, delta, epsilon, and mu, which correlate with the antibody's isotypes: IgA, IgG, IgD, IgE, and IgM, respectively. Therefore, different antibody isotypes have different class effects because their different Fc regions bind to and activate different types of receptors.

[0099] IgG, the most abundant antibody isotype in human serum, has four subclasses: IgG1, IgG2, IgG3, and IgG4. The amino acid sequences of the constant regions of these peptides are known in the art; see, for example, Rutishauser, U. et al. (1968) "Amino acid sequence of the Fc region of a human gamma G-immunoglobulin" PNAS 61(4):1414-1421, Shinoda et al. (1981) "Complete amino acid sequence of the Fc region of a human delta chain" PNAS 78(2):785-789, and Robinson et al. (1980) "Complete amino acid sequence of a mouse immunoglobulin alpha chain (MOPC511)" PNAS 77(8):4909-4913.

[0100] All therapeutic antibodies for purposes of the methods and pharmaceutical uses of the present disclosure are antibodies or fragments thereof that bind to IL-31RA, but the specific anti-IL-31RA antibody is not limited. Nemolizumab is a preferred anti-IL-31RA antibody, but other anti-IL-31RA antibodies can be used as well. Therapeutic antibodies suitable for use in the methods and pharmaceutical uses of the present disclosure can be human, humanized, or chimeric, and can be IgA, IgG (i.e., IgG1, IgG2, IgG3, and IgG4), IgD, IgE, or IgM.

[0101] Nemolizumab is a humanized monoclonal antibody that binds to IL-31RA. Nemolizumab is annotated as follows: Immunoglobulin G2-kappa, anti-[Homo sapiens IL31RA (Interleukin-31 Receptor Subunit Alpha)], humanized monoclonal antibody; gamma 2 heavy chain (1-445) [humanized VH (Homo sapiens IGHV1-2*02 (83.70%)-(IGHD)-IGHJ5*01) [8.8.14] (1-121)-Homo sapiens IGHG2*01 (CH1 C10>S(135), R12>K(137), E16>G(141), S17>G(142) (122-219), hinge C4>S(223) (220-231), CH2 H30>Q(268) (232-340), CH3 R11>Q(355), Q98>E(419)(341-445))(122-445)], (224-214')-kappa light chain disulfide containing, (1'-214') [humanized V-kappa (Homo sapiens IGKV1-39*01 (82.10%)-IGKJ4*01) [6.3.9] (1'-107')-Homo sapiens IGKC*01 (108'-214')], dimer (227-227":230-230")-bisdisulfide. Nemolizumab has disulfide bridges at the following positions: Intra-H (C23-C104) 22-96 148-204 261-321 367-425 22''-96'' 148''-204'' 261''-321'' 367''-425'', Intra-L (C23-C104) 23'-88' 134'-194' 23'''-88''' 134'''-194'''', Inter-HL (h5-CL126) 224-214' 224''-214'''', Inter-HH (h8,h11) 227-227'' 230-230''. Nemolizumab has N-glycosylation sites at the following positions: H CH2 N84.4:297,297''. Nemolizumab lacks the C-terminal glycine and lysine of the heavy chain (CHS G1>del, K2>del).

[0102] Nemolizumab contains the following heavy chain amino acid sequence: QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYIMNWVRQAPGQGLEWMGLINPYNGGTDYNPQFQDRVTITADKSTSTAYMELSSLRSEDTAVYYCARDGYDDGPYTLETWGQ GTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKSCVE CPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSP (SEQ ID NO: 5).

[0103] Nemolizumab contains the following light chain amino acid sequence: DIQMTQSPSSLSASVGDRVTITCQASEDIYSFVAWYQQKPGKAPKLLIYNAQTEAQGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQHHYDSPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 6).

[0104] The heavy chain variable region of nemolizumab comprises the following amino acid sequence: QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYIMNWVRQAPGQGLEWMGLINPYNGGTDYNPQFQDRVTITADKSTSTAYMELSSLRSEDTAVYYCARDGYDDGPYTLETWGQGTLVTVSS (SEQ ID NO: 7).

[0105] HCDR1 of nemolizumab comprises the amino acid sequence GYIMN (SEQ ID NO: 8), HCDR2 comprises the amino acid sequence LINPYNGGTDYNPQFQD (SEQ ID NO: 9), and HCDR3 comprises the amino acid sequence DGYDDGPYTLET (SEQ ID NO: 10).

[0106] The light chain variable region of nemolizumab comprises the following amino acid sequence: DIQMTQSPSSLSASVGDRVTITCQASEDIYSFVAWYQQKPGKAPKLLIYNAQTEAQGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQHHYDSPLTFGGGTKVEIKR (SEQ ID NO: 11).

[0107] LCDR1 of nemolizumab comprises the amino acid sequence QASEDIYSFVA (SEQ ID NO: 12), LCDR2 comprises the amino acid sequence NAQTEAQ (SEQ ID NO: 13), and LCDR3 comprises the amino acid sequence QHHYDSPLT (SEQ ID NO: 14).

[0108] For purposes of this disclosure, a "variant antibody" or "variant" of nemolizumab includes (i) an antibody having a heavy chain that comprises at least 55%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% amino acid sequence identity to the heavy chain sequence of nemolizumab; (ii) an antibody having a light chain that comprises at least 55%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% amino acid sequence identity to the light chain sequence of nemolizumab; (ii) antibodies having variable regions with at least 55%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% amino acid sequence identity to the variable region sequences of nemolizumab, (iv) antibodies having CDRs with at least 55%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% amino acid sequence identity to the CDR sequences of nemolizumab, and (v) combinations thereof. For example, suitable variants include immunoglobulins or immunoglobulin-like molecules with heavy and light chain amino acid sequences identical or substantially similar to those of nemolizumab. Other suitable therapeutic antibodies may bind to the same isoform of IL-31RA as nemolizumab (e.g., IL31-RAv3), optionally to the same epitope of IL-31RA, block or neutralize IL-31RA, or a combination thereof. Additional exemplary therapeutic antibodies are described, for example, in WO2010 / 064697.

[0109] Nemolizumab variants and suitable therapeutic antibodies may be monoclonal or polyclonal. Such monoclonal antibodies having IL31-RA binding activity and / or neutralizing activity can be obtained, for example, by the following procedure: anti-IL31-RA monoclonal antibodies are prepared by known methods using IL31-RA or a fragment thereof derived from a mammal, such as a human or mouse, as an antigen, and antibodies having IL31-RA binding activity and / or neutralizing activity are then selected from the anti-IL31-RA monoclonal antibodies thus obtained. Specifically, a desired antigen or cells expressing the desired antigen are used as a sensitizing antigen for immunization by conventional immunization methods. Anti-IL31-RA monoclonal antibodies can be prepared by fusing the obtained immune cells with known parent cells using conventional cell fusion methods and screening for monoclonal antibody-producing cells (hybridomas) using conventional screening methods. Animals to be immunized include mammals such as mice, rats, rabbits, sheep, monkeys, goats, donkeys, cattle, horses, and pigs. Antigens can be prepared using known IL31-RA gene sequences according to known methods, for example, by baculovirus-based methods (e.g., WO98 / 46777). Variants of nemolizumab and suitable therapeutic antibodies include intrabodies, peptibodies, nanobodies, single-domain antibodies, multispecific antibodies (e.g., bispecific antibodies, diabodies, triabodies, tetrabodies, tandem di-scFvs, tandem tri-scFvs), derpins, heavy chain monomers, heavy chain dimers, or single-domain antibodies (i.e., V ... H H-fragment or "lactam-like" antibodies).

[0110] Hybridomas can be prepared, for example, according to the method of Milstein et al. (Kohler, G. and Milstein, C., Methods Enzymol. (1981) 73:3-46). When an antigen has low immunogenicity, the antigen may be conjugated to an immunogenic macromolecule, such as albumin, before immunization. The antigen used to prepare a monoclonal antibody having binding activity and / or neutralizing activity against human IL31-RA is not particularly limited, as long as it allows the preparation of an antibody having binding activity and / or neutralizing activity against human IL31-RA. For example, numerous mutants of human IL31-RA are known to exist, and any mutant can be used as an immunogen as long as it allows the preparation of an antibody having binding activity and / or neutralizing activity against human IL31-RA. Alternatively, under the same conditions, a peptide fragment of IL31-RA or a protein in which artificial mutations have been introduced into the native IL31-RA sequence may be used as an immunogen. Human IL31-RA.3 is one of the preferred immunogens for preparing antibodies having the activity of binding to and / or neutralizing IL31-RA in the present disclosure.

[0111] The IL31-RA binding activity of a therapeutic antibody can also be determined by methods known to those skilled in the art. Methods for determining the antigen-binding activity of an antibody include, for example, ELISA (enzyme-linked immunosorbent assay), EIA (enzyme immunoassay), RIA (radioimmunoassay), and immunofluorescence. For example, when using an enzyme immunoassay, an antibody-containing sample, such as purified antibody or culture supernatant of antibody-producing cells, is added to an antigen-coated plate. A secondary antibody labeled with an enzyme such as alkaline phosphatase is added, and the plate is incubated. After washing, an enzyme substrate such as p-nitrophenyl phosphate is added, and the absorbance is measured to evaluate the antigen-binding activity. The binding activity and / or neutralizing activity of a therapeutic antibody against IL31-RA can be measured, for example, by observing its effect of suppressing the proliferation of an IL-31-dependent cell line. For example, the activity of a purified mouse IL-31 antibody can be assayed by evaluating the IL-31-dependent growth of Ba / F3 cells transfected with mouse IL-31 receptor α and mouse OSMR genes.

[0112] Any of the anti-IL31RA antibodies (i.e., "therapeutic antibodies") disclosed herein, including nemolizumab and fragments or variants thereof, can be used to treat and / or prevent PN and achieve the therapeutic endpoints of the disclosure. Optimal dosages and routes of administration may vary.

[0113] IV. Pharmaceutical Compositions Provided herein are pharmaceutical compositions for use in the treatment or prevention of prurigo nodularis (PN), including skin lesions, nodules, or itching caused by PN. The pharmaceutical compositions comprise, as an active ingredient, an anti-IL31RA antibody (i.e., a "therapeutic antibody"), such as nemolizumab, or a fragment or variant thereof.

[0114] The phrase "comprising nemolizumab or a fragment or variant thereof as an active ingredient" means that nemolizumab or a fragment or variant thereof is included as at least one of the active ingredients, and does not limit the percentage of the antibody. Furthermore, the therapeutic agent for PN of the present disclosure may contain other ingredients that enhance the treatment or prevention of PN in combination with nemolizumab or a fragment or variant thereof. For example, the composition may include one or more of a topical corticosteroid cream or injection, an ointment containing menthol or phenol for cooling and soothing itchy skin, a capsaicin cream, an oral corticosteroid, a selective serotonin reuptake inhibitor (SSRI), and an oral antihistamine.

[0115] Pharmaceutical compositions of nemolizumab or a fragment or variant thereof can be prepared as formulations according to standard methods (see, for example, Remington's Pharmaceutical Sciences, Mark Publishing Company, Easton, USA). Pharmaceutical compositions generally contain carriers and / or excipients in addition to the antibody. For example, in some embodiments, the pharmaceutical composition comprises one or more surfactants (e.g., PEG and Tween), excipients, antioxidants (e.g., ascorbic acid), colorants, flavoring agents, preservatives, stabilizers, buffers (e.g., phosphoric acid, citric acid, and other organic acids), chelating agents (e.g., EDTA), suspending agents, isotonizing agents, binders, disintegrants, lubricants, flow enhancers, Corrigent, light anhydrous silicic acid, lactose, crystalline cellulose, mannitol, starch, carmellose calcium, carmellose sodium, hydroxypropyl cellulose, hydroxypropyl methylcellulose, polyvinyl acetal diethylaminoacetate, polyvinylpyrrolidone, gelatin, medium-chain triglycerides, polyoxyethylene hydrogenated castor oil 60, sucrose, carboxymethyl cellulose, corn starch, and inorganic salts. In some embodiments, the pharmaceutical composition comprises one or more other low molecular weight polypeptides, proteins such as serum albumin, gelatin, and immunoglobulins, and amino acids such as glycine, glutamine, asparagine, arginine, and lysine.

[0116] Nemolizumab or a fragment or variant thereof may be prepared as an aqueous solution for injection, and may be dissolved in an isotonic solution containing, for example, saline, dextrose, or other excipients or isotonic agents (i.e., tonicity agents). Isotonicity agents may include, for example, D-sorbitol, D-mannose, D-mannitol, and sodium chloride. In addition, suitable solubilizers, such as alcohols (e.g., ethanol), polyalcohols (e.g., propylene glycol and PEG), and nonionic detergents (polysorbate 80 and HCO-50), may be used simultaneously.

[0117] In some embodiments, nemolizumab or a fragment or variant thereof is encapsulated in microcapsules (microcapsules made of hydroxymethylcellulose, gelatin, polymethylmethacrylate, etc.), and may be made into components of colloid drug delivery systems (liposomes, albumin microparticles, microemulsions, nanoparticles, and nanocapsules) (see, e.g., "Remington's Pharmaceutical Science 16th edition" &, Oslo Ed. (1980)). Furthermore, methods for producing sustained-release drugs are known and can be applied to nemolizumab or a fragment or variant thereof (Langer et al., J. Biomed. Mater. Res. (1981) 15, 167-277; Langer, Chem. Tech. (1982) 12, 98-105; U.S. Pat. No. 3,773,919; European Patent Application (EP) No. 58,481; Sidman et al., Biopolymers (1983) 22, 547-56; EP 133,988).

[0118] The pharmaceutical compositions of the present disclosure may be administered orally or parenterally, preferably parenterally. Specifically, the pharmaceutical compositions are administered to patients by injection or transdermal administration. Injections include, for example, intravenous, intramuscular, and subcutaneous injections for systemic or local administration. The pharmaceutical composition may be administered to the site where inflammation and / or pruritus is to be suppressed, or to the area surrounding the site by local injection or intramuscular or subcutaneous injection. In some embodiments, the pharmaceutical composition is administered at the site of one or more skin abrasions, lesions, or nodules, or proximal to the site of one or more skin abrasions, lesions, or nodules.

[0119] The administration method can be appropriately selected depending on the age, weight, and condition of the patient. The single administration dose can be selected, for example, within the range of 0.0001 to 100 mg of antibody (e.g., nemolizumab or a fragment or variant thereof) per kg of body weight. Alternatively, for example, when the antibody is administered to a human patient, the dose of the antibody can be selected within the range of 0.001 to 1,000 mg / kg of body weight. In some embodiments, the composition may be administered at a concentration of, for example, about 0.01 to 50 mg / kg, about 0.01 mg / kg to about 0.1 mg / kg, about 0.05 mg / kg to 0.15 mg / kg, about 0.1 mg / kg to about 0.6 mg / kg, about 0.1 mg / kg to about 1 mg / kg, about 0.25 mg / kg to about 0.75 mg / kg, about 0.4 mg / kg to about 0.8 mg / kg, about 0.4 mg / kg to about 1.8 mg / kg, about 0.5 to about 2.5 mg / kg, about 0.8 mg / kg to about 2.2 mg / kg, about 1 mg / kg to about 2.5 mg / kg, or about 1 mg / kg to about 3.5 mg / kg. The formulation is formulated to administer a dose containing nemolizumab or a fragment or variant thereof of about 1 mg / kg to about 5 mg / kg, about 2 mg / kg to about 4 mg / kg, about 2.5 mg / kg to about 10 mg / kg, about 5 mg / kg to about 10 mg / kg, about 10 mg / kg to about 20 mg / kg, about 10 mg / kg to about 40 mg / kg, about 20 mg / kg to about 50 mg / kg, about 25 mg / kg to about 75 mg / kg, about 50 mg / kg to about 100 mg / kg, about 100 mg / kg to about 500 mg / kg, or about 100 mg / kg to about 1000 mg / kg of body weight. In preferred embodiments, the dosage ranges from about 0.01 mg / kg to about 0.1 mg / kg, from about 0.1 mg / kg to about 0.5 mg / kg, from about 0.5 mg / kg to about 1.5 mg / kg, from about 1.5 mg / kg to about 2.5 mg / kg, or from about 2.5 mg / kg to about 10 mg / kg.In some embodiments, the dosage is about 0.01 mg / kg, about 0.02 mg / kg, about 0.03 mg / kg, about 0.04 mg / kg, about 0.05 mg / kg, about 0.06 mg / kg, about 0.07 mg / kg, about 0.08 mg / kg, about 0.09 mg / kg, about 0.1 mg / kg, about 0.2 mg / kg, about 0.3 mg / kg, about 0.4 mg / kg, about 0.5 mg / kg, about 0.6 mg / kg, about 0.7 mg / kg, about 0.8 mg / kg, about 0.9 mg / kg, about 1 mg / kg, about 1.1 mg / kg, about 1.2 mg / kg, about 1.3 mg / kg, about 1.4 mg / kg, about 1.5 mg / kg, about 1.6 mg / kg, about 1.7 mg / kg, about 1.8 mg / kg, about 1.9 mg / kg, about 2.0 mg / kg, about 2.1 mg / kg, about 2.2 mg / kg, about 2.3 mg / kg, about 2.4 mg / kg, about 2.5 mg / kg, about 2.6 mg / kg, about 2.7 mg / kg, about 2.8 mg / kg, about 2.9 mg / kg, about 3.0 mg / kg, about 3.1 mg / kg, about 3.2 mg / kg, about 3.3 mg / kg, about 3.4 mg / kg, about 3.5 mg / kg, about 3.6 mg / kg, about 3.7 mg / kg, about 3.8 mg / kg, about 3.9 mg / kg, about 4.0 mg / kg, about 4.1 mg / kg, about 4.2 mg / kg, about 4.3 mg / kg, about 4.4 mg / kg, about 4.5 mg / kg, about 4.6 mg / kg, about mg / kg, about 1.8 mg / kg, about 1.9 mg / kg, about 2 mg / kg, about 2.1 mg / kg, about 2.2 mg / kg, about 2.3 mg / kg, about 2.4 mg / kg, about 2.5 mg / kg, about 2.6 mg / kg, about 2.7 mg / kg, about 2.8 mg / kg, about 2.9 mg / kg, about 3 mg / kg, about 3.5 mg / kg, about 4 mg / kg, about 4.5 mg / kg, about 5 mg / kg, about 6 mg / kg, about 7 mg / kg, about 8 mg / kg, about 9 mg / kg, about 10 mg / kg, about 15 mg / kg, about 25 mg / kg, about 50 mg / kg, about 75 mg / kg, about 100 mg / kg, about 500 mg / kg or about 1,000 mg / kg. In certain embodiments, the effective amount of nemolizumab or a fragment or variant thereof is about 0.1 mg / kg, about 0.5 mg / kg, about 1 mg / kg, about 1.5 mg / kg, about 2 mg / kg, or about 2.5 mg / kg. In a preferred embodiment, the dosage is about 0.5 mg / kg.

[0120] The present disclosure provides a pharmaceutical composition comprising an anti-IL31RA antibody (e.g., nemolizumab or a fragment or variant thereof) as an active ingredient for use in treating or preventing prurigo nodularis (PN) in a subject, wherein the subject differentially expresses at least one gene selected from the genes disclosed in Table 1 compared to a reference expression level of the at least one gene.

[0121] The present disclosure provides a pharmaceutical composition for use in treating or preventing prurigo nodularis (PN) in a subject, comprising an anti-IL31RA antibody (e.g., nemolizumab or a fragment or variant thereof) as an active ingredient, wherein the subject differentially expresses at least one gene selected from the genes disclosed in Table 1 compared to a reference expression level of the at least one gene, and the subject is diagnosed as having PN by detecting expression levels of at least one, at least two, at least three, at least four, or at least five of the differentially expressed genes (DEGs) in Table 1 in a sample obtained from a subject suspected of having PN, and comparing the expression levels of the DEGs to a reference level, wherein the reference level is the corresponding gene expression level for each DEG in a sample from an individual without PN.

[0122] Any of the pharmaceutical compositions disclosed herein comprising nemolizumab and fragments or variants thereof can be used to treat and / or prevent PN and achieve the therapeutic endpoints of the disclosure. Optimal dosages and routes of administration may vary.

[0123] V. Methods for treating / preventing PN and compositions for treating or preventing PN The present disclosure provides methods for treating or preventing pruritus in a subject with prurigo nodularis (PN), the methods comprising, consisting of, or consisting essentially of administering to the subject an anti-IL-31RA antibody (i.e., a "therapeutic antibody"), such as nemolizumab or a fragment or variant thereof. The disclosed methods can be carried out to achieve specific therapeutic endpoints, which are described in more detail below. Also disclosed herein is the use of an anti-IL-31RA antibody (i.e., a "therapeutic antibody"), such as nemolizumab or a fragment or variant thereof, in a subject to treat or prevent PN and / or achieve the therapeutic endpoints of the disclosure. Also disclosed herein is the use of an anti-IL-31RA antibody (i.e., a "therapeutic antibody"), such as nemolizumab or a fragment or variant thereof, in a subject to treat or prevent PN and / or achieve the therapeutic endpoints of the disclosure. In addition, certain subgroups of subjects with PN may be particularly suitable for treatment with the methods and uses of the present disclosure (e.g., patients exhibiting any of the DEGs disclosed in Table 1).

[0124] The present disclosure is the first to report a transcriptomic signature of PN that can not only identify and positively diagnose PN, but also identify subjects with PN who are likely to respond to treatment with an anti-IL-31RA antibody (e.g., nemolizumab) and track the response to treatment with an anti-IL-31RA antibody (e.g., nemolizumab).

[0125] This disclosure is also the first to report the plasma proteome signature of subjects with PN successfully treated with an anti-IL-31RA antibody, such as nemolizumab. This "responder signature" can be used not only as a marker for positive clinical endpoints, but also to identify subjects with PN who are likely to respond to treatment with an anti-IL-31RA antibody (e.g., nemolizumab) and track their response to treatment with an anti-IL-31RA antibody (e.g., nemolizumab).

[0126] A. Subjects to be treated A subject treated for PN according to the methods and uses of the present disclosure may demonstrate one or more of the underlying gene expression patterns disclosed herein. In particular, a subject with PN treated according to the methods and uses of the present disclosure may differentially express up to 5,943 genes (known as differentially expressed genes or DEGs) shown in Figure 1A and Table 1 below. This differential gene expression may be observed in the subject's skin, particularly in skin samples containing or consisting of nodules or lesions. Of these DEGs, 2,060 may be increased (i.e., overexpressed), and 3,874 may be decreased (i.e., not expressed). Genes that may be most increased include: KRT6C, which may be increased by at least 100-fold, at least 150-fold, at least 200-fold, at least 250-fold, at least 300-fold, at least 350-fold, at least 400-fold, at least 450-fold, at least 500-fold, at least 550-fold, or 588-fold compared to expression levels in a sample (e.g., a skin sample) from an individual without PN; DEFB4A, which may be increased by at least 25-fold, at least 50-fold, at least 75-fold, at least 100-fold, at least 125-fold, or at least 150-fold compared to expression levels in a sample (e.g., a skin sample) from an individual without PN, and KRT16, which may be increased by at least 10-fold, at least 20-fold, at least 30-fold, at least 40-fold, at least 50-fold, at least 60-fold, at least 70-fold, at least 80-fold, or at least 90-fold compared to expression levels in a sample (e.g., a skin sample) from an individual without PN.

[0127] Decreased genes include: LCE5A, which may be decreased by at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, or at least 11-fold compared to its expression level in a sample (e.g., a skin sample) from an individual without PN, and AQP7, which may be reduced by at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, or at least 7.9-fold compared to expression levels in a sample (e.g., a skin sample) from an individual without PN.

[0128] Genes encoding cytokines may also be overexpressed in subjects suffering from PN, which can be treated or prevented according to the methods and uses of the present disclosure. For cytokine genes, the most prominent upregulated genes are IL-36 family members and IL-20 family members. These upregulated or overexpressed genes may include: IL36A (e.g., approximately 6.8 fold, FDR = 1.8 × 10 -4 ), IL36G (e.g., approximately 8.4 fold, FDR = 3.9 × 10 -25 ), IL19 (e.g., approximately 5.1 fold, FDR = 7.4 × 10 -4 ), IL20 (e.g., approximately 3.5 times, FDR = 1.7 × 10 -3 ), IL22 (e.g., approximately 2.7 times, FDR = 2.9 × 10 -2 ), IL24 (e.g., approximately 5.8 times, FDR = 3.8 × 10 -10 ), and IL26 (e.g., approximately 4.9 fold, FDR = 3.3 × 10 -3 ).

[0129] Each of these IL-36 and IL-20 family member cytokine genes can be overexpressed by at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 3.5-fold, at least about 4-fold, at least about 4.5-fold, at least about 5-fold, at least about 5.5-fold, at least about 6-fold, at least about 6.5-fold, at least about 7-fold, at least about 7.5-fold, at least about 8-fold, or at least about 8-5-fold compared to expression levels in a sample (e.g., a skin sample) from an individual without PN.

[0130] Other factors that may be upregulated or overexpressed in samples (e.g., skin samples) obtained from subjects with PN being treated include IL1A (e.g., about 4.7-fold, FDR=1.0×10 -12 ), IL1B (e.g., approximately 4.1-fold, FDR = 3.7 × 10 -6 ), and IL4R (e.g., approximately 2.6-fold (FDR = 6.3 × 10 -19 ) can be mentioned. Table 1 at the end of the Examples section of this specification provides a more comprehensive list of DEGs.

[0131] Certain Gene Ontology (GO) categories may also be evident in the skin of subjects with PN treated according to the methods and uses of the present disclosure. These GO categories are as follows: ● "Cornified envelope" (FDR=1.5×10 -12 ), ●“Epidermal cell differentiation” (FDR=6.4×10 -10 ), - Keratinization (FDR=1.6×10 -12 ), ● "Peptidase regulatory factor activity" (FDR = 1.1 × 10 -4 ), ● "Interleukin-4 and 13 signaling" (FDR = 6.8 × 10 -7 ), "Interferon alpha / beta signaling" and "response to interferon gamma" (FDR=4.1×10 -7 and FDR = 4.1 × 10 -6 ), ● "IL23 pathway" (FDR = 2 × 10 -5 ), and ● "Metaphase and anaphase" (FDR = 3.8 × 10 -10 ).

[0132] These categories reflect the hyperproliferative nature of PN, which has been found to be associated with alterations in epidermal differentiation and inflammatory components. In some embodiments, subjects with PN treated according to the methods and uses of the present disclosure may overexpress the proliferation marker Ki67 (MKI67), the cell cycle gene CDKN1A, and / or an inflammatory network including IL-1 and IL-36.

[0133] A subject with PN treated according to the methods or uses of the present disclosure may additionally or alternatively present one or more of the co-expression modules or clusters shown in Figure 1D and Table 3 at the end of the Examples section herein. Different functions are defined for these co-expressed gene modules as described herein.

[0134] The present disclosure also provides specific cell type signatures for non-lesional and lesional PN skin that may be present in subjects with PN treated according to the methods and uses of the present disclosure. For example, subjects with PN may have a transcriptomic signature associated with epithelial cells and keratinocytes, as described herein, as observed in Figure 2A, and / or may have a signature associated with Th2. Other inflammatory signatures, such as macrophages, may also be prominent in PN lesions and the skin of subjects with PN.

[0135] Generally, the methods and uses of the present disclosure can treat or prevent PN in subjects suffering from mild, moderate, or severe pruritus. In some embodiments, PN may be classified as moderate to severe. In some embodiments, PN may be classified as moderate, and in other embodiments, PN may be classified as severe. In some embodiments, pruritus may be scored as none, mild, moderate, or severe. "None," "mild," "moderate," and "severe" are terms of the art used to describe the presence, degree, and / or intensity of pruritus. Those skilled in the art are aware of the boundaries and boundaries of these terms. For example, pruritus can be characterized according to one or more of the following methods known to those skilled in the art: Intensity can be quickly measured using a unidimensional scale routinely used in clinical care. See Pereira et al., Allergology International (2017) 66:3-78. Additionally or alternatively, patients can be asked to rate their itch intensity using a numeric rating scale (NRS) ranging from 0 ("no itch") to 10 ("worst imaginable itch"). Another unidimensional scale, the visual analog scale (VAS), provides patients with the opportunity to indicate itch intensity by marking a 10-cm ruler-like scale. Both endpoints are marked with numerical values ​​corresponding to intensity, with 0 representing "no itch" and 10 representing "worst imaginable itch." A score below 3.0 VAS / NRS points is generally associated with mild itch, while a score above 6.9 indicates severe itch. A score above 9.0 represents very severe itch. The verbal rating scale (VRS) is a further unidimensional scale that allows patients to describe their itch intensity using ascending adjectives (0 - no itch, 4 - worst imaginable itch). The NRS, VAS, and VRS have been validated in large studies of patients with chronic pruritus and pruritic skin diseases of various origins. These instruments are highly reproducible, and there was a high correlation between scales 6, 7, and 8. Chronic pruritus can significantly reduce patients' quality of life. For this reason, the Dermatology Life Quality Index (DLQI) has been widely used and validated.DLQI scores range from 0 to 30, with higher scores indicating poorer quality of life. Investigator Global Assessment (IGA) scores range from 0 (clear) to 5 (very severe disease) and are presented as a percentage of patients in the indicated population. In this study, IGA scores range from 0 to 4.

[0136] A subject treated for PN according to the methods and uses of the present disclosure may demonstrate one or more of the plasma protein signature alterations of the present disclosure. In particular, a subject with PN treated according to the methods and uses of the present disclosure may exhibit (a) decreased leukocyte migration and cell movement, (b) inhibition of the STAT3 and STAT5b pathways, (c) downregulation of the IL-6 and VEGF pathways, (d) decreased TGFB1 pathway, or (e) a combination thereof. The aforementioned reduction or inhibition may be observed at a specific time point after initiation of treatment, for example, 2 weeks, 4 weeks, 6 weeks, 8 weeks, 10 weeks, or 12 weeks. The reduction or inhibition may be determined with respect to (i) a control sample obtained from one or more individuals without PN, or (ii) a biological sample obtained from the subject prior to administration of an anti-IL-31RA antibody. Additionally or alternatively, the subject may exhibit upregulation of neuronal terms, including neuronal CREB signaling, synaptogenesis signaling pathways, neuroglial cell death and neuroglial apoptosis, and combinations thereof, following administration of an anti-IL-31RA antibody.

[0137] Subjects with PN suitable for treatment with the methods and uses of the present disclosure may exhibit high levels of leukocyte migration or leukocyte cell trafficking relative to individuals or populations without PN, or in subjects prior to initiating treatment with an anti-IL-31RA antibody such as nemolizumab. Additionally or alternatively, suitable subjects may exhibit relatively high STAT3 pathway activity or expression compared to individuals or populations without PN, or in subjects prior to initiating treatment with an anti-IL-31RA antibody such as nemolizumab. Additionally or alternatively, suitable subjects may exhibit relatively high TGFB1 pathway activity or expression compared to individuals or populations without PN, or in subjects prior to initiating treatment with an anti-IL-31RA antibody such as nemolizumab. Additionally or alternatively, suitable subjects may exhibit relatively high amounts of circulating cytokine signatures compared to individuals or populations without PN, or in subjects prior to initiating treatment with an anti-IL-31RA antibody such as nemolizumab. Such cytokine pathways include, but are not limited to, the IL-6 and VEGF pathways.

[0138] Prior to treatment with an anti-IL-31Ra antibody, such as nemolizumab, the amount of a protein marker of the present disclosure in the plasma of a subject with PN may be at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 30-fold, at least 35-fold, at least 40-fold, at least 45-fold, or at least 50-fold higher than the baseline level. The baseline level may be determined with respect to (i) a control sample obtained from one or more individuals (i.e., a population) without PN, or (ii) a biological sample obtained from the subject prior to administration of the anti-IL-31RA antibody.

[0139] In some embodiments, the subject has been diagnosed with PN for at least about 6 months. In certain embodiments, the subject has at least about 20 nodules in the body with a bilateral distribution. In certain embodiments, the subject has pruritus lesions on the upper extremities with or without lesions on the trunk or lower extremities. In some embodiments, the pruritus is assigned a score of at least 7 on the Numerical Rating Scale (NRS). In some embodiments, the mean worst day intensity of the NRS score is at least 7 compared to the previous three days. In some embodiments, the mean worst day intensity of the NRS score is at least 7 compared to the previous week.

[0140] In some embodiments, the subject does not have atopic dermatitis (AD). In some embodiments, the subject does not have chronic pruritus due to a condition other than PN, such as scabies, bites, lichen simplex chronicus, psoriasis, acne, folliculitis, habitual picking, lymphomatoid papulosis, chronic actinic dermatitis, dermatitis herpetiformis, sporotrichosis, or bullous disease. In some embodiments, the subject does not have neurogenic or psychogenic pruritus, such as hypoesthesia, palmar pruritus, delusional parasitosis, or pathological pruritus.

[0141] B. Therapeutic Endpoints for Treatment Based on the data provided herein, IL-31 signaling appears to be upstream of IL-17 and IL-4 signaling, because treatment with the anti-IL-31RA antibody nemolizumab was able to alter the transcriptome expression signatures of IL-17 and IL-4, at least in subjects with PN. Accordingly, the present disclosure provides a method for normalizing differentially expressed genes (DEGs) in subjects with PN, comprising administering an anti-IL-31RA antibody, such as nemolizumab or a fragment or variant thereof, to the subject. As shown in the Examples section below, treatment or prevention with nemolizumab results in the normalization of several PN-associated DEGs, both in terms of gene increases (e.g., approximately 969 genes) and decreases (e.g., approximately 1,268 genes). See Figure 4A. In some embodiments, the hydroxylase activity is about 5, about 10, about 15, about 20, about 25, about 50, about 75, about 100, about 150, about 200, about 250, about 300, about 350, about 400, about 450, about 500, about 550, about 600, about 650, about 700, about 750, about 800, about 850, about 900, about 950, about 1000, about 1100, about 120 0, about 1300, about 1400, about 1500, about 1600, about 1700, about 1800, about 1900, about 2000, about 2100, about 2200, about 2300, about 2400, or about 2500 DEGs may be normalized in subjects with PN after treatment with an anti-IL-31RA antibody, such as nemolizumab or a fragment or variant thereof.In some embodiments, at least 5, at least 10, at least 15, at least 20, at least 25, at least 50, at least 75, at least 100, at least 150, at least 200, at least 250, at least 300, at least 350, at least 400, at least 450, at least 500, at least 550, at least 600, at least 650, at least 700, at least 750, at least 800, at least 850, at least 900, at least 950, at least 1000, at least 1100, at least At least 1200, at least 1300, at least 1400, at least 1500, at least 1600, at least 1700, at least 1800, at least 1900, at least 2000, at least 2100, at least 2200, at least 2300, at least 2400, or at least 2500 DEGs, and up to 2500, 3000, 3500, 4000, 4500, 5000, 5500, or about 6000 DEGs, can be normalized in subjects with PN after treatment with an anti-IL-31RA antibody, such as nemolizumab or a fragment or variant thereof. In some embodiments, treatment or prevention can involve administration of an anti-IL-31RA antibody (e.g., nemolizumab) once per week, once every two weeks, once every three weeks, once every four weeks, once every five weeks, once every six weeks, once every seven weeks, or once every eight weeks. Administration about once every 4 weeks may be preferred. Administration may be via injection, such as subcutaneous injection. The time frame for determining / comparing normalization of DEG may be 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, or 12 weeks or more.For example, at baseline (i.e., at or before treatment initiation), a sample (e.g., a skin sample) may be obtained from a subject to detect the expression levels of some or all of the putative DEGs disclosed in Table 1 (e.g., KRT6C, DEFB4A, KRT16, LCE5A, AQP7, IL-36 family members, IL-20 family members, etc.), and another sample may be taken and evaluated 4, 5, 6, 7, 8, 9, 10, 11, 12, or more weeks after treatment initiation, at a reference expression level of the DEG associated with normal, healthy skin (e.g., expression levels obtained from a skin sample from a subject without PN) or at a more consistent (i.e., "normalized") expression level to determine whether the expression level of the DEG has changed. Such methods can be used to track treatment and assess whether a subject is responding to treatment with an anti-IL-31RA antibody (e.g., nemolizumab).

[0142] GO categories enriched among DEGs may also be decreased or altered as a result of treatment with an anti-IL-31RA antibody (e.g., nemolizumab). For example, after 12 weeks of treatment, the following GO categories were decreased: "cell cycle" (FDR = 5.6 × 10 -14 ), "keratinocyte differentiation" (FDR = 1.8 × 10 -4 ), and “Interleukin-4 and 13 signaling” (FDR = 1.5 × 10 -2) was observed. Indeed, the data provided in the Examples section demonstrate that nemolizumab treatment (and treatment with other anti-IL-31RA antibodies) normalizes epidermal hyperproliferation, normalizes differentiation, and reduces inflammatory responses, particularly those associated with Th2 responses. Accordingly, the present disclosure provides methods for normalizing epidermal hyperproliferation, normalizing epidermal differentiation, and / or reducing inflammatory responses in the skin, comprising administering an anti-IL-31RA antibody (e.g., nemolizumab) to a subject with PN. In some embodiments, the inflammatory response may be a Th2 response. In some embodiments, administration of the anti-IL-31RA antibody (e.g., nemolizumab) may be once a week, once every two weeks, once every three weeks, once every four weeks, once every five weeks, once every six weeks, once every seven weeks, or once every eight weeks. Administration approximately once every four weeks may be preferred. Administration may be via injection, such as subcutaneous injection.

[0143] In some embodiments, the disclosed methods for reducing inflammatory responses in the skin of a subject with PN may alter the cytokine response signature generated in human epidermal rafts in the subject's skin. For example, IL17A mRNA expression was not significantly different in non-lesional versus lesional skin, even after 12 weeks of treatment with nemolizumab. A consistent decrease in IL-31 responses, alone or in combination with other proinflammatory cytokines, including the Th2 cytokines IL-13 or IL-17A, can be seen in Figure 5A, thus providing evidence of IL-31 pathway blockade by nemolizumab. However, IL-17A-responsive genes are shown herein to be enriched in PN skin and downregulated by nemolizumab. This suggests that IL-17A itself is not the predominant cytokine in PN, but is downstream of IL-31 signaling. Indeed, a consistent decrease in IL-31 responses, alone or in combination with other proinflammatory cytokines (including the Th2 cytokines IL-13 or IL-17A), was observed with nemolizumab treatment (Figure 5A), and even more robust decreases were observed in Th1, Th17, and Th2 markers (Figure 5B). Thus, IL-31 signaling is likely upstream of IL-17 and IL-4 signaling.

[0144] In some embodiments, the basal keratinocyte (KRT14+) signature may be elevated in PN lesional skin, and this signature may be restored to normal (i.e., non-PN) when treated according to the methods and uses of the present disclosure. In some embodiments, the epidermal spinous layer (KRT10+) signature may be elevated in PN lesional skin, and this signature may be restored to normal (i.e., non-PN) when treated according to the methods and uses of the present disclosure (FIG. 5C).

[0145] In some embodiments, transcription factor binding sites (TFBSs) enriched among genes upregulated in baseline lesional skin are more likely to be enriched among genes downregulated by nemolizumab after treatment with an anti-IL-31RA antibody such as nemolizumab (e.g., by 12 weeks post-treatment, as shown in Figure 5D). In some embodiments, transcription factors downregulated by treatment can be EGR4 (a member of the EGF family of zinc finger transcription factors), STAT3, and / or KLF16.

[0146] The present disclosure further provides plasma protein markers identified in subjects successfully treated with nemolizumab; thus, the resulting changes in biomarkers can be considered a "responder signature" for establishing success and a means of tracking responsiveness. Accordingly, disclosed herein is a method for treating or preventing prurigo nodularis (PN) in a subject, comprising administering an anti-IL-31RA antibody to a subject with PN, wherein treatment with the anti-IL-31RA antibody results in (a) decreased leukocyte migration and cell movement, (b) decreased IL-6 or decreased IL-6 pathway signaling, (c) decreased VEGF or decreased VEGF pathway signaling, (d) decreased STAT3 or decreased STAT3 pathway signaling, (e) decreased STAT5b or decreased STAT5b pathway signaling, (f) decreased TGFB1 or decreased TGFB1 pathway signaling, or (g) a combination thereof. In some embodiments, the subject may also exhibit an increase in the neuron ontology of the present disclosure. Additionally, disclosed herein is a method of altering the immune response in a subject with PN, comprising administering an anti-IL-31RA antibody to the subject, wherein treatment with the anti-IL-31RA antibody results in (a) decreased leukocyte migration and cell movement, (b) decreased IL-6 or decreased IL-6 pathway signaling, (c) decreased VEGF or decreased VEGF pathway signaling, (d) decreased STAT3 or decreased STAT3 pathway signaling, (e) decreased STAT5b or decreased STAT5b pathway signaling, (f) decreased TGFB1 or decreased TGFB1 pathway signaling, or (g) a combination thereof. In some embodiments, the subject may also exhibit an increase in the neuron ontology of the present disclosure. The disclosed plasma protein markers are detectable, for example, by mass spectrometry and other protein evaluation methods (e.g., ELISA, Western blot, etc.).

[0147] In particular, when subjects with PN are treated with an anti-IL-31RA antibody, such as nemolizumab, immune cells (eg, leukocytes) may experience decreased migration or cell migration, or both.

[0148] STAT3 pathway activity may be decreased in subjects with PN treated with an anti-IL-31RA antibody, such as nemolizumab, compared to individuals or populations without PN, or compared to the subject before initiating treatment with an anti-IL-31RA antibody, such as nemolizumab.

[0149] The amount of cytokine activity may be relatively high in subjects with PN, or in subjects prior to initiating treatment with an anti-IL-31RA antibody, such as nemolizumab, compared to individuals or populations without PN. Such cytokine pathways include, but are not limited to, the IL-6 and VEGF pathways. In subjects with PN treated with an anti-IL-31RA antibody, such as nemolizumab, treatment may result in a decrease in the IL-6 or VEGF signature, or both, compared to baseline levels. The baseline level may be determined with respect to (i) a control sample obtained from one or more individuals (i.e., a population) without PN, or (ii) a biological sample obtained from the subject prior to administration of the anti-IL-31RA antibody.

[0150] TGFB1 pathway activity may be decreased in subjects with PN who are treated with an anti-IL-31RA antibody, such as nemolizumab, compared to individuals or populations without PN, or compared to the subject before initiating treatment with an anti-IL-31RA antibody, such as nemolizumab.

[0151] For purposes of plasma protein markers of the present disclosure, the amount of a protein marker of the present disclosure in the plasma of a subject with PN may be at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 30-fold, at least 35-fold, at least 40-fold, at least 45-fold, or at least 50-fold higher than the baseline level. The baseline level may be determined with respect to (i) a control sample obtained from one or more individuals (i.e., a population) without PN, or (ii) a biological sample obtained from the subject prior to administration of an anti-IL-31RA antibody. Similarly, after a subject with PN is treated with an anti-IL-31RA antibody, such as nemolizumab (e.g., 2, 4, 6, 8, 10, or 12 weeks after administration of the antibody), the amount of a plasma protein marker of the present disclosure in the subject may be decreased by at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 30-fold, at least 35-fold, at least 40-fold, at least 45-fold, or at least 50-fold compared to baseline levels. Baseline levels may be determined with respect to (i) a control sample obtained from one or more individuals (i.e., a population) without PN, or (ii) a biological sample obtained from the subject prior to administration of the anti-IL-31RA antibody.

[0152] In addition, neural ontologies (e.g., CREB signaling in neurons, synaptogenesis signaling pathways, neuroglial cell death, and neuroglial apoptosis) may be upregulated in subjects with PN and subsequently downregulated in nemolizumab responder subjects.

[0153] In some embodiments of the methods and uses of the present disclosure, treatment or prophylaxis with an anti-IL-31RA antibody, such as nemolizumab or a fragment or variant thereof, results in a reduction in pruritus scores. The reduction in scores may be measured, for example, by the Peak Pruritus Numerical Rating Scale (PP-NRS). See, for example, FIG. 6A. Indeed, the data provided herein show that all subjects with PN treated with nemolizumab showed an improvement in pruritus scores. In some embodiments, the measured distance between Principal Component 1 and Principal Component 2 (i.e., PC1 / PC2 components) may be shorter than that of a placebo group not treated with nemolizumab or another anti-IL-31RA antibody. More specifically, at week 12 (i.e., 12 weeks after the start of treatment), the proportion of patients achieving a weekly average 4-point reduction in the PP NRS was significantly higher in the nemolizumab group compared to the placebo group (52.9% vs. 8.3%, p<0.001). At week 12, the proportion of subjects achieving IGA success (defined as IGA 0 [clear] or 1 [almost clear]) was significantly higher in the nemolizumab group compared with placebo (20.6% vs. 2.8%, p=0.02).

[0154] C. Fibroblast Response to Treatment As described herein, this disclosure is the first to establish that PN is an inflammatory and fibrotic disease, and that antagonizing IL-31 signaling (e.g., via treatment with nemolizumab) can exert anti-fibrotic effects by inhibiting key signaling pathways. In particular, compared with healthy skin, lesional PN fibroblasts exhibit a profibrotic and pro-inflammatory state, which can result in differential expression of genes in lesional PN fibroblasts involved in the activation of inflammatory (TNF, IL1B, IL6) and profibrotic (TGFβ) signaling pathways. Indeed, PN fibroblasts play a central role in intracellular crosstalk, and treatment with an anti-IL-31RA antibody, such as nemolizumab or its fragment or variant, reverses neuroregulatory dysfunction and reduces inflammation and fibrosis.

[0155] For example, the disclosure provides a method of treating or preventing prurigo nodularis (PN) in a subject, comprising administering an anti-IL-31RA antibody to a subject with PN, wherein the subject exhibits activation of tumor necrosis factor (TNF) signaling in lesional skin cells compared to a reference level of activation of TNF signaling.

[0156] Similarly, the present disclosure provides a method of normalizing activated tumor necrosis factor (TNF) expression in a subject with PN, comprising administering an anti-IL-31RA antibody to the subject with PN, wherein the subject exhibits activated tumor necrosis factor (TNF) signaling in lesional skin cells compared to a reference level of expression of the TNF gene, and administration of the anti-IL-31RA antibody normalizes the expression level of the TNF gene. For purposes of such methods, normalization can be determined about 4 weeks, about 8 weeks, or about 12 weeks after administration of the anti-IL-31RA antibody.

[0157] For purposes of these methods, differential expression and pathway activation can be determined by RT-qPCR, RT-PCR, RNA-seq, Northern blotting, serial analysis of gene expression (SAGE), or DNA or RNA microarrays. Additionally or alternatively, differential expression and pathway activation can also be determined at the protein level by Western blotting, ELISA, surface plasmon resonance, or mass spectrometry.

[0158] TNF activation in lesional skin cells, such as fibroblasts, can be elevated before treatment compared to a reference level of expression. The reference level can be an activation level that is the level of activation of TNF signaling in skin cells (e.g., fibroblasts) of a human without PN. Additionally or alternatively, the reference level is the activation level of the TNF gene in non-lesional skin cells of the subject.

[0159] The present disclosure also provides a method for reducing inflammation in the skin of a subject with prurigo nodularis (PN), comprising administering an anti-IL-31RA antibody to the subject with PN, thereby reducing inflammation associated with tumor necrosis factor (TNF) signaling in the skin. In such a method, TNF signaling in the subject's skin is overexpressed compared to a reference level of activated TNF signaling, and optionally, TNF signaling is activated in fibroblasts. The reference level can be the activated level of TNF signaling in skin cells (e.g., fibroblasts) of a human not having PN. Alternatively, the reference level can be the activated level of TNF signaling in non-lesional skin cells of the subject.

[0160] In some embodiments of these methods, the inflammation further involves IL-1 pathway signaling, IL-6 pathway signaling, TGFβ pathway signaling, or any combination thereof.

[0161] The present disclosure also provides methods for treating or preventing prurigo nodularis (PN) in a subject, comprising administering an anti-IL-31RA antibody to a subject with PN, wherein treatment with the anti-IL-31RA antibody results in reduced tumor necrosis factor (TNF) pathway activation. In some embodiments, the reduced TNF pathway activation occurs in lesional skin of the subject. In some embodiments, the reduced TNF pathway activation occurs in fibroblasts of the subject.

[0162] As the present disclosure demonstrates, there can often be overlapping signaling pathways involved in the pathogenesis of PN. Therefore, for purposes of the methods of the present disclosure, additional outcomes of treatment include: (a) a decrease in leukocyte migration or cell movement of leukocytes; (b) inhibition of the STAT3 pathway; (c) inhibition of the STAT5b pathway; (d) downregulation of IL-1 or the IL-1 pathway; (e) downregulation of IL-6 or the IL-6 pathway; (f) downregulation of VEGF or the VEGF pathway; (g) a decrease in TGFB1 pathway activation, or (h) A combination thereof.

[0163] In some embodiments, a decrease in leukocyte migration or leukocyte cell movement, (b) inhibition of the STAT3 pathway, (c) inhibition of the STAT5b pathway, (d) downregulation of IL-1 or the IL-1 pathway, (e) downregulation of IL-6 or the IL-6 pathway, (f) downregulation of VEGF or the VEGF pathway, (g) a decrease in TGFBl pathway activation, or (h) a combination thereof is determined with respect to (i) a control sample obtained from one or more individuals without PN, or (ii) a biological sample obtained from the subject prior to administration of an anti-IL-31RA antibody.

[0164] In some embodiments, (a) a decrease in leukocyte migration or leukocyte cell movement, (b) inhibition of the STAT3 pathway, (c) inhibition of the STAT5b pathway, (d) downregulation of IL-1 or the IL-1 pathway, (e) downregulation of IL-6 or the IL-6 pathway, (f) downregulation of VEGF or the VEGF pathway, (g) a decrease in TGFB1 pathway activation, or (h) a combination thereof is assessed about 4 weeks, about 8 weeks, or about 12 weeks after administration of the anti-IL-31RA antibody.

[0165] In some embodiments, (a) decreased leukocyte migration or leukocyte cell movement, (b) inhibition of the STAT3 pathway, (c) inhibition of the STAT5b pathway, (d) downregulation of IL-1 or the IL-1 pathway, (e) downregulation of IL-6 or the IL-6 pathway, (f) downregulation of VEGF or the VEGF pathway, (g) decreased TGFBl pathway activation, or (h) a combination thereof is determined by mass spectrometry performed on one or more biological samples obtained from the subject. In some embodiments, the one or more biological samples are plasma samples or skin samples.

[0166] In some embodiments, the subject exhibits at least two, at least three, at least four, at least five, at least six, or all seven of the following: (a) decreased leukocyte migration or leukocyte cell movement; (b) inhibition of the STAT3 pathway; (c) inhibition of the STAT5b pathway; (d) downregulation of IL-1 or the IL-1 pathway; (e) downregulation of IL-6 or the IL-6 pathway; (f) downregulation of VEGF or the VEGF pathway; and (g) decreased TGFB1 pathway activation.

[0167] D. Dosages and Administration Regimens for the Disclosed Methods and Uses An effective amount of an anti-IL-31RA antibody, such as nemolizumab or a fragment or variant thereof, is an amount sufficient to produce a beneficial or desired result, such as alleviating at least one or more symptoms of PN. As used herein, an effective amount would also include an amount sufficient to delay or prevent the onset of pruritus, alter the course of PN symptoms, or reverse PN symptoms. Thus, an exact "effective amount" cannot be specified. However, an appropriate "effective amount" in any given case can be determined by one of ordinary skill in the art using only routine experimentation.

[0168] An effective amount can be administered in one or more administrations, applications, or dosages. Such delivery depends on several variables, including the duration for which individual dosage units are used, the bioavailability of the therapeutic agent, the route of administration, and the like. However, it is understood that the specific dosage level of the therapeutic agent of the present disclosure for any particular subject will depend on a variety of factors, including the activity of the specific compound used, the subject's age, weight, general health, sex, and diet, the time of administration, the rate of excretion, the drug combination, and the severity and administration form of the particular disorder being treated. Treatment and prophylactic dosages can generally be titrated to optimize safety and effectiveness. Dosages are determined by a physician and can be adjusted, if necessary, to the observed effects of treatment. Typically, dosage-effect relationships from in vitro and / or in vivo studies can initially provide useful guidance regarding appropriate dosages for patient administration. Generally, it is desired to administer an amount of the compound effective to achieve serum levels commensurate with concentrations found to be effective in vitro. Determination of these parameters is within the skill of the art. These considerations, as well as effective formulation and administration procedures, are well known in the art and are described in standard textbooks.

[0169] Dosing regimens for treating or preventing PN may involve flat dosing (i.e., administering the same dose repeatedly at predetermined intervals) or may involve a loading dose (i.e., administering an initial dose that is higher or different than subsequent successive doses). For purposes of either type of dosing regimen, an effective dose may be administered topically, parenterally, subcutaneously, intradermally, or intramuscularly. In a preferred embodiment, administration involves subcutaneous injection.

[0170] In some embodiments, the loading dose and subsequent consecutive doses may be administered via the same route (e.g., subcutaneously), while in some embodiments, the loading dose and subsequent consecutive doses may be administered via different routes (e.g., parenteral and subcutaneous, respectively). In some embodiments, the loading dose may be about 5 mg, about 10 mg, about 15 mg, about 20 mg, about 25 mg, about 30 mg, about 35 mg, about 40 mg, about 45 mg, about 50 mg, about 55 mg, about 60 mg, about 65 mg, about 70 mg, about 75 mg, about 80 mg, about 85 mg, about 90 mg, about 95 mg, about 100 mg, about 105 mg, about 110 mg, about 115 mg, about 120 mg, or more. In some embodiments, the loading dose can be 5 mg, 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, 35 mg, 40 mg, 45 mg, 50 mg, 55 mg, 60 mg, 65 mg, 70 mg, 75 mg, 80 mg, 85 mg, 90 mg, 95 mg, 100 mg, 105 mg, 110 mg, 115 mg, 120 mg or more.In some embodiments, the loading dose is about 0.01 mg / kg, about 0.02 mg / kg, about 0.03 mg / kg, about 0.04 mg / kg, about 0.05 mg / kg, about 0.06 mg / kg, about 0.07 mg / kg, about 0.08 mg / kg, about 0.09 mg / kg, about 0.1 mg / kg, about 0.2 mg / kg, about 0.3 mg / kg, about 0.4 mg / kg, about 0.5 mg / kg, about 0.6 mg / kg, about 0.7 mg / kg, about 0.8 mg / kg, about 0.9 mg / kg, about 1 mg / kg, about 1.1 mg / kg, about 1.2 mg / kg, about 1.3 mg / kg, about 1.4 mg / kg, about 1.5 mg / kg, about 1.6 mg / kg, about 1.7 mg / kg, or about 1.8 mg / kg. g / kg, about 1.8 mg / kg, about 1.9 mg / kg, about 2 mg / kg, about 2.1 mg / kg, about 2.2 mg / kg, about 2.3 mg / kg, about 2.4 mg / kg, about 2.5 mg / kg, about 2.6 mg / kg, about 2.7 mg / kg, about 2.8 mg / kg, about 2.9 mg / kg, about 3 mg / kg, about 3.5 mg / kg, about 4 mg / kg, about 4.5 mg / kg, about 5 mg / kg, about 6 mg / kg, about 7 mg / kg, about 8 mg / kg, about 9 mg / kg, about 10 mg / kg, about 15 mg / kg, about 25 mg / kg, about 50 mg / kg, about 75 mg / kg, about 100 mg / kg, about 500 mg / kg, or about 1,000 mg / kg.In some embodiments, the loading dose is 0.01 mg / kg, 0.02 mg / kg, 0.03 mg / kg, 0.04 mg / kg, 0.05 mg / kg, 0.06 mg / kg, 0.07 mg / kg, 0.08 mg / kg, 0.09 mg / kg, 0.1 mg / kg, 0.2 mg / kg, 0.3 mg / kg, 0.4 mg / kg, 0.5 mg / kg, 0.6 mg / kg, 0.7 mg / kg, 0.8 mg / kg, 0.9 mg / kg, 1 mg / kg, 1.1 mg / kg, 1.2 mg / kg, 1.3 mg / kg, 1.4 mg / kg, 1.5 mg / kg, 1.6 mg / kg, 1.7 mg / kg, 1.8 mg / kg, 1.9 mg / kg, 2.0 mg / kg, 2.1 mg / kg, 2.2 mg / kg, 2.3 mg / kg, 2.4 mg / kg, 2.5 mg / kg, 2.6 mg / kg, 2.7 mg / kg, 2.8 mg / kg, 2.9 mg / kg, 2.9 mg / kg, 3.0 mg / kg, 3.1 mg / kg, 3.2 mg / kg, 3.3 mg / kg, 3.4 mg / kg, 3.5 mg / kg, 3.6 mg / kg, 3.7 mg / kg, 3.8 mg / kg, 3.9 ... The loading dose may be 1.8 mg / kg, 1.9 mg / kg, 2 mg / kg, 2.1 mg / kg, 2.2 mg / kg, 2.3 mg / kg, 2.4 mg / kg, 2.5 mg / kg, 2.6 mg / kg, 2.7 mg / kg, 2.8 mg / kg, 2.9 mg / kg, 3 mg / kg, 3.5 mg / kg, 4 mg / kg, 4.5 mg / kg, 5 mg / kg, 6 mg / kg, 7 mg / kg, 8 mg / kg, 9 mg / kg, 10 mg / kg, 15 mg / kg, 25 mg / kg, 50 mg / kg, 75 mg / kg, 100 mg / kg, 500 mg / kg, or 1,000 mg / kg. In some embodiments, the loading dose is administered as a single injection. In some embodiments, the loading dose is administered as multiple injections, which may be administered simultaneously or spaced apart by defined intervals.

[0171] Subsequent consecutive doses in a loading dose regimen are generally lower than the loading dose. For example, in some embodiments, a dosing regimen may include a loading dose of 60 mg and consecutive doses of 30 mg, which may be administered at defined intervals, such as every four weeks. In some embodiments, the consecutive doses in the dosing regimen may be about 5 mg, about 10 mg, about 15 mg, about 20 mg, about 25 mg, about 30 mg, about 35 mg, about 40 mg, about 45 mg, about 50 mg, about 55 mg, about 60 mg, about 65 mg, about 70 mg, about 75 mg, about 80 mg, about 85 mg, about 90 mg, about 95 mg, about 100 mg, about 105 mg, about 110 mg, about 115 mg, about 120 mg, or more. In some embodiments, the continuous dose can be 5 mg, 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, 35 mg, 40 mg, 45 mg, 50 mg, 55 mg, 60 mg, 65 mg, 70 mg, 75 mg, 80 mg, 85 mg, 90 mg, 95 mg, 100 mg, 105 mg, 110 mg, 115 mg, 120 mg or more.In some embodiments, the sequential doses are about 0.01 mg / kg, about 0.02 mg / kg, about 0.03 mg / kg, about 0.04 mg / kg, about 0.05 mg / kg, about 0.06 mg / kg, about 0.07 mg / kg, about 0.08 mg / kg, about 0.09 mg / kg, about 0.1 mg / kg, about 0.2 mg / kg, about 0.3 mg / kg, about 0.4 mg / kg, about 0.5 mg / kg, about 0.6 mg / kg, about 0.7 mg / kg, about 0.8 mg / kg, about 0.9 mg / kg, about 1 mg / kg, about 1.1 mg / kg, about 1.2 mg / kg, about 1.3 mg / kg, about 1.4 mg / kg, about 1.5 mg / kg, about 1.6 mg / kg, about 1.7 mg / kg, about 1.8 mg / kg, about 1.9 mg / kg, about 2.0 mg / kg, about 2.1 mg / kg, about 2.2 mg / kg, about 2.3 mg / kg, about 2.4 mg / kg, about 2.5 mg / kg, about 2.6 mg / kg, about 2.7 mg / kg, about 2.8 mg / kg, about 2.9 mg / kg, about 2.9 mg / kg, about 3.0 mg / kg, about 3.1 mg / kg, about 3.2 mg / kg, about 3.3 mg / kg, about 3.4 mg / kg, about 3.5 mg / kg, about 3.6 mg / kg, about 3.7 mg / kg, about 3.8 mg / kg, about 3.9 mg / kg, about 4.0 mg / kg, about 4.1 mg / kg, about 4.2 mg / kg, about 4.3 mg / kg, about 4.4 mg / kg, about 4.5 mg / kg, g / kg, about 1.8 mg / kg, about 1.9 mg / kg, about 2 mg / kg, about 2.1 mg / kg, about 2.2 mg / kg, about 2.3 mg / kg, about 2.4 mg / kg, about 2.5 mg / kg, about 2.6 mg / kg, about 2.7 mg / kg, about 2.8 mg / kg, about 2.9 mg / kg, about 3 mg / kg, about 3.5 mg / kg, about 4 mg / kg, about 4.5 mg / kg, about 5 mg / kg, about 6 mg / kg, about 7 mg / kg, about 8 mg / kg, about 9 mg / kg, about 10 mg / kg, about 15 mg / kg, about 25 mg / kg, about 50 mg / kg, about 75 mg / kg, about 100 mg / kg, about 500 mg / kg, or about 1,000 mg / kg.In some embodiments, the sequential doses are 0.01 mg / kg, 0.02 mg / kg, 0.03 mg / kg, 0.04 mg / kg, 0.05 mg / kg, 0.06 mg / kg, 0.07 mg / kg, 0.08 mg / kg, 0.09 mg / kg, 0.1 mg / kg, 0.2 mg / kg, 0.3 mg / kg, 0.4 mg / kg, 0.5 mg / kg, 0.6 mg / kg, 0.7 mg / kg, 0.8 mg / kg, 0.9 mg / kg, 1 mg / kg, 1.1 mg / kg, 1.2 mg / kg, 1.3 mg / kg, 1.4 mg / kg, 1.5 mg / kg, 1.6 mg / kg, 1.7 mg / kg, 1.8 mg / kg, 1.9 mg / kg, 2.0 mg / kg, 2.1 mg / kg, 2.2 mg / kg, 2.3 mg / kg, 2.4 mg / kg, 2.5 mg / kg, 2.6 mg / kg, 2.7 mg / kg, 2.8 mg / kg, 2.9 mg / kg, 2.9 mg / kg, 3.0 mg / kg, 3.1 mg / kg, 3.2 mg / kg, 3.3 mg / kg, 3.4 mg / kg, 3.5 mg / kg, 3.6 mg / kg, 3.7 mg / kg, 3.8 mg / kg, 3.9 ... mg / kg, 1.8 mg / kg, 1.9 mg / kg, 2 mg / kg, 2.1 mg / kg, 2.2 mg / kg, 2.3 mg / kg, 2.4 mg / kg, 2.5 mg / kg, 2.6 mg / kg, 2.7 mg / kg, 2.8 mg / kg, 2.9 mg / kg, 3 mg / kg, 3.5 mg / kg, 4 mg / kg, 4.5 mg / kg, 5 mg / kg, 6 mg / kg, 7 mg / kg, 8 mg / kg, 9 mg / kg, 10 mg / kg, 15 mg / kg, 25 mg / kg, 50 mg / kg, 75 mg / kg, 100 mg / kg, 500 mg / kg, or 1,000 mg / kg.

[0172] For purposes of a loading dose regimen, the first consecutive dose may be administered 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, or 10 weeks after the initial loading dose. In some embodiments, the first consecutive dose is administered 4 weeks after the initial loading dose. In some embodiments, subsequent consecutive doses are administered every 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, or 10 weeks. In some embodiments, the consecutive doses are spaced four weeks apart (ie, nemolizumab or a fragment or variant thereof is administered once every four weeks).

[0173] In some embodiments, the dosage of nemolizumab or a fragment or variant thereof administered to a subject can be within the range of 0.001 to 1,000 mg / kg of the subject's body weight. In some embodiments, the dosage is about 0.01 to 50 mg / kg, about 0.01 mg / kg to about 0.1 mg / kg, about 0.05 mg / kg to 0.15 mg / kg, about 0.1 mg / kg to about 0.6 mg / kg, about 0.1 mg / kg to about 1 mg / kg, about 0.25 mg / kg to about 0.75 mg / kg, about 0.4 mg / kg to about 0.8 mg / kg, about 0.4 mg / kg to about 1.8 mg / kg, about 0.5 to about 2.5 mg / kg, about 0.8 mg / kg to about 2.2 mg / kg, about 1 mg / kg to about 2.5 mg / kg, or about 1 mg / kg to about 3. The range of nemolizumab or a fragment or variant thereof is about 5 mg / kg, about 1 mg / kg to about 5 mg / kg, about 2 mg / kg to about 4 mg / kg, about 2.5 mg / kg to about 10 mg / kg, about 5 mg / kg to about 10 mg / kg, about 10 mg / kg to about 20 mg / kg, about 10 mg / kg to about 40 mg / kg, about 20 mg / kg to about 50 mg / kg, about 25 mg / kg to about 75 mg / kg, about 50 mg / kg to about 100 mg / kg, about 100 mg / kg to about 500 mg / kg, or about 100 mg / kg to about 1000 mg / kg of body weight. In preferred embodiments, the dosage ranges from about 0.01 mg / kg to about 0.1 mg / kg, from about 0.1 mg / kg to about 0.5 mg / kg, from about 0.5 mg / kg to about 1.5 mg / kg, from about 1.5 mg / kg to about 2.5 mg / kg, or from about 2.5 mg / kg to about 10 mg / kg.In some embodiments, the dosage is about 0.01 mg / kg, about 0.02 mg / kg, about 0.03 mg / kg, about 0.04 mg / kg, about 0.05 mg / kg, about 0.06 mg / kg, about 0.07 mg / kg, about 0.08 mg / kg, about 0.09 mg / kg, about 0.1 mg / kg, about 0.2 mg / kg, about 0.3 mg / kg, about 0.4 mg / kg, about 0.5 mg / kg, about 0.6 mg / kg, about 0.7 mg / kg, about 0.8 mg / kg, about 0.9 mg / kg, about 1 mg / kg, about 1.1 mg / kg, about 1.2 mg / kg, about 1.3 mg / kg, about 1.4 mg / kg, about 1.5 mg / kg, about 1.6 mg / kg, about 1.7 mg / kg, or about 1.8 mg / kg. g / kg, about 1.8 mg / kg, about 1.9 mg / kg, about 2 mg / kg, about 2.1 mg / kg, about 2.2 mg / kg, about 2.3 mg / kg, about 2.4 mg / kg, about 2.5 mg / kg, about 2.6 mg / kg, about 2.7 mg / kg, about 2.8 mg / kg, about 2.9 mg / kg, about 3 mg / kg, about 3.5 mg / kg, about 4 mg / kg, about 4.5 mg / kg, about 5 mg / kg, about 6 mg / kg, about 7 mg / kg, about 8 mg / kg, about 9 mg / kg, about 10 mg / kg, about 15 mg / kg, about 25 mg / kg, about 50 mg / kg, about 75 mg / kg, about 100 mg / kg, about 500 mg / kg, or about 1,000 mg / kg. In certain embodiments, the dosage of nemolizumab or a fragment or variant thereof is about 0.1 mg / kg, about 0.5 mg / kg, about 1 mg / kg, about 1.5 mg / kg, about 2 mg / kg, or about 2.5 mg / kg. In a preferred embodiment, the dosage is about 0.5 mg / kg.

[0174] In some embodiments, the dose of nemolizumab or a fragment or variant thereof administered to a subject is in the range of 1 to 100 mg, 25 to 75 mg, 30 to 60 mg, 40 to 80 mg, 20 to 80 mg, 1 to 25 mg, 1 to 50 mg, 10 to 90 mg, or 15 to 85 mg, or any range therebetween. In some embodiments, the dose can be about 5 mg, about 10 mg, about 15 mg, about 20 mg, about 25 mg, about 30 mg, about 35 mg, about 40 mg, about 45 mg, about 50 mg, about 55 mg, about 60 mg, about 65 mg, about 70 mg, about 75 mg, about 80 mg, about 85 mg, about 90 mg, about 95 mg, about 100 mg, about 105 mg, about 110 mg, about 115 mg, about 120 mg, or more. In some embodiments, the dosage can be 5 mg, 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, 35 mg, 40 mg, 45 mg, 50 mg, 55 mg, 60 mg, 65 mg, 70 mg, 75 mg, 80 mg, 85 mg, 90 mg, 95 mg, 100 mg, 105 mg, 110 mg, 115 mg, 120 mg or more.

[0175] In some embodiments of the methods and uses of the present disclosure, a loading dose of about 60 mg of nemolizumab or a fragment or variant thereof may be administered to a subject with PN, followed by continuous doses of about 30 mg of a nemolizumab fragment or variant thereof every four weeks. In some embodiments of the methods and uses of the present disclosure, a first dose of about 60 mg of nemolizumab or a fragment or variant thereof may be administered to a subject with PN, followed by continuous doses of about 60 mg of a nemolizumab fragment or variant thereof every four weeks (i.e., the dose remains constant or is a "flat" dosing regimen). In some embodiments of the methods and uses of the present disclosure, a first dose of about 30 mg of nemolizumab or a fragment or variant thereof may be administered to a subject with PN, followed by continuous doses of about 30 mg of a nemolizumab fragment or variant thereof every four weeks.

[0176] In some embodiments of the methods and uses of the present disclosure, nemolizumab or a fragment or variant thereof is administered by a topical or parenteral route. In some embodiments, nemolizumab or a fragment or variant thereof is administered subcutaneously. In some embodiments, the dose is administered subcutaneously at or near the site of one or more nodules, lesions, or excisions.

[0177] In some embodiments of the methods and uses of the present disclosure, nemolizumab or a fragment or variant thereof is administered daily, every other day, twice a week, three times a week, four times a week, five times a week, six times a week, once a week, once every two weeks, once every three weeks, once every four weeks, once every five weeks, once every six weeks, once every seven weeks, once every eight weeks, once every nine weeks, once every ten weeks, once every eleven weeks, once every twelve weeks, twice a year, once a year, and / or as needed based on the onset of symptoms of PN. In a preferred embodiment, nemolizumab or a fragment or variant thereof is administered every four weeks or every eight weeks.

[0178] In some embodiments of the methods and uses of the present disclosure, the duration of treatment or prevention is about 1 day, about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 9 weeks, about 10 weeks, about 11 weeks, about 12 weeks, about 13 weeks, about 14 weeks, about 15 weeks, about 16 weeks, about 17 weeks, about 18 weeks, about 19 weeks, about 20 weeks, about 24 weeks, about 30 weeks, about 36 weeks, about 40 weeks, about 48 weeks, about 50 weeks, about 1 year, about 2 years, about 3 years, about 4 years, about 5 years, or as needed based on the appearance of symptoms of PN. In preferred embodiments, the duration of treatment or prevention is about 12 weeks to about 24 weeks, about 12 weeks to about 36 weeks, about 12 weeks to about 48 weeks, or about 24 weeks to about 36 weeks.

[0179] The present disclosure provides the use of nemolizumab or a fragment or variant thereof in the manufacture of a medicament for treating or preventing PN, for normalizing differentially expressed genes (DEGs) in a subject with PN, for normalizing epidermal hyperproliferation, for normalizing epidermal differentiation, and / or for reducing inflammatory responses in the skin. All of the dosages, administration regimens, administration routes, biomarkers, and treatment endpoints of the present disclosure are also applicable to these uses.

[0180] The following examples are provided to illustrate the present disclosure, it being understood that the invention should not be limited to the particular conditions or details set forth in these examples. [Example]

[0181] Example 1 Treatment of prurigo nodularis with nemolizumab method Patient cohort The current study was conducted in patients with moderate to severe PN. Briefly, 70 patients were randomized 1:1 to placebo (36 patients) or nemolizumab (34 patients), the latter at a dose of 0.5 mg / kg body weight administered at baseline, Week 4, and Week 8. Peak pruritus scores on a numerical rating scale (PP-NRS) were recorded: pruritus severity on the numerical rating scale ranged from 0 (no pruritus) to 10 (worst imaginable pruritus). Peak pruritus was estimated using the worst score every 24 hours for 7 days, with the highest score recorded as the peak score. The primary outcome of the study was the percent change from baseline in PP-NRS at Week 4.

[0182] Reconstructed human epidermis (RHE) model A three-dimensional RHE model was generated. Briefly, normal human dermal fibroblasts (NHDF) and normal human epidermal keratinocytes (NHEK) were used to generate RHE cultures. The RHE cultures used were full-thickness with dermis and epidermis and composed of autologous fibroblasts and keratinocytes. They were scaffold-free (no exogenous matrix), allowing cells to self-organize the different layers of the skin and avoiding some potential inhibitors contained in the collagen matrix for RNA extraction. RHE cultures were then placed in inserts (1.2 cm). 2 ) for 42 days to obtain at least two layers of dermis and six layers of epidermis. RHE cultures from six different donors were either unstimulated or stimulated with seven different cytokines or cytokine combinations (three replicates per donor per condition): IL-31, IL-13, IL-17A, IFNg, IL-31 + IL-13, IL-31 + IL-17A, IL-31 + IFNg, and IL-13 + IL-17A. The concentrations of each cytokine used were as follows: IL-31 (500 ng / mL), IL-13 (100 ng / mL), IL-17A (200 ng / mL), and IFNg (50 ng / mL). RHE cultures were lysed 72 hours after stimulation, and RNA extraction was performed using the MagMAX mirVana Total RNA Isolation from Tissue Kit (ThermoFisher Scientific). RNA was purified and concentrated using the RNA Clean & Concentrator-5 kit (Zymo Research) according to the protocol, with RNA concentrations ranging from 3 to 380 ng / µL. Total RNA was quantified using the QuantiFluor One RNA kit (Promega) on a GloMax-Multi+ detection system (Promega). Library preparation was performed using the Illumina® Stranded mRNA Prep Ligation Kit (Illumina) according to the manufacturer's recommendations.

[0183] Skin biopsy processing and RNA isolation Skin biopsies were collected from 16 placebo-treated and 15 nemolizumab-treated subjects. Samples included lesional and non-lesional biopsies at baseline, as well as lesional samples (placebo or nemolizumab) after 12 weeks of treatment. RNA was extracted from the skin biopsies using Tripure Isolation Reagent (Sigma-Aldrich) according to the manufacturer's instructions. DNase treatment was applied to these RNA samples using an RNase-Free DNase Kit (Cat. No. 79254, Qiagen) followed by an RNeasy® MinElute® Cleanup Kit (Cat. No. 74204, Qiagen).

[0184] Total RNA was quantified using the QuantiFluor One RNA kit (Promega). The resulting RNA concentrations ranged from 4 to 20 ng / μl. Total RNA was qualified using the Fragment Analyzer 5300 (Agilent) with the Agilent HS RNA Fragment Kit (Agilent). The resulting RNA quality numbers (RQNs) ranged from 1 to 6. Library preparation was performed using the SMARTer Stranded Total RNA-Seq Kit V2-Pico Input Mammalian Kit (TaKaRa).

[0185] RNA-Seq Libraries were quantified using the QuantiFluor One dsDNA kit (Promega), and library analysis was performed using the Agilent HS NGS Fragment kit (Agilent) on a Fragment Analyzer 5300 (Agilent). Following size selection with AMPure XP beads, shotgun libraries were sequenced using NextSeq (Illumina) on an Illumina NextSeq500 sequencer at 2 × 75 bp (High Output Kit v2, 150 cycles).

[0186] RNA-seq data processing After adapter trimming, sequence reads from 83 unique samples were aligned to the human genome (GRCh37) using STAR. Reads that uniquely mapped to a single genomic location were then used to quantify gene (GENCODE v29) expression levels with HTSeq. Two RNA-seq samples were identified as outliers, and 81 samples were used for subsequent analysis. Only genes with an average of at least one read per sample were retained. DESeq2 was used for expression normalization, and a negative binomial distribution was used to model expression levels for differential expression analysis. For non-lesional vs. lesional and baseline vs. week 12 comparisons, individual effects were included as covariates, and for placebo vs. nemolizumab comparisons, age and sex were controlled. A false discovery rate (FDR) ≤ 5% and |log2 fold change| >= 1 were used as criteria for declaring significant differentially expressed genes (DEGs).

[0187] Cytokine, cellular signature, and functional inference analysis The most significant DEGs were compared with cytokine-induced transcripts in keratinocytes (defined by FDR <= 10% and a fold change (FC) of 1.5). A fair comparison was performed using the top 1,000 most significant DEGs from the PN data. For comparison with the epidermal compartment gene signature, scRNA-seq was performed on the epidermal layers of skin biopsies to identify the top 50 marker genes for the basal, differentiated, and keratinized layers, respectively. The effect size (log2FC) was then examined for each marker gene in each differential expression comparison. For transcriptional analysis, the promoter region was defined as 5,000 base pairs upstream of the transcription start site, and enrichment statistics for transcription factor binding were calculated using the meme suite.

[0188] Weighted Gene Correlation Network Analysis (WGCNA) Genes expressed in at least 20% of the samples were used in the WGCNA dataset. The "softPower" parameter was selected as the minimum value achieving at least r2 >= 0.75. Spearman correlation was used to calculate correlations, and the minimum module size was set to 100. When modules were confluent, a high cutoff of 0.2 was used.

[0189] result Prurigo nodularis is characterized by abnormal keratinocyte differentiation and immune activation After quality control, we performed RNA sequencing data analysis of biopsy tissues from patients with prurigo nodularis (PN). There were 31 PN patients with transcriptomics data for both lesional and non-lesional skin samples. Using a false discovery rate (FDR) of ≤10% and |log2| ≥ 1 as criteria, 5,943 differentially expressed genes (DEGs) were identified when comparing uninvolved and lesional skin at baseline, of which 2,060 genes were increased and 3,874 genes were decreased (Figure 1A). Genes that showed the most robust increase included KRT6C (588-fold, FDR = 8.2 × 10). -80 ), DEFB4A (150x, FDR=1.1×10 -12 ), and KRT16 (90-fold, FDR = 1.9 × 10 -52 ). Decreased genes included LCE5A (11-fold decrease, FDR = 8.1 × 10 -18 ) and AQP7 (7.9-fold decrease, FDR = 2.6 × 10 -17 Regarding cytokines, the most significantly upregulated gene was the IL36 family member: IL36A (6.8-fold, FDR = 1.8 × 10 -4 ) and IL36G (8.4-fold, FDR = 3.9 × 10 -25 ), IL-20 family member: IL19 (5.1-fold, FDR = 7.4 × 10 -4 ), IL20 (3.5-fold, FDR = 1.7 × 10 -3 ), IL22 (2.7-fold, FDR = 2.9 × 10 -2 ), IL24 (5.8-fold, FDR = 3.8 × 10 -10), and IL26 (4.9-fold, FDR = 3.3 × 10 -3 ) were included. Other contributing factors were IL1A (4.7-fold, FDR = 1.0 × 10 -12 ), and IL1B (4.1-fold, FDR = 3.7 × 10 -6 The Th2 cytokines IL4 and IL13 did not reach significance, but IL4R increased 2.6-fold (FDR = 6.3 × 10 -19 ) (See Table 1 at the end of the Examples section herein).

[0190] Functional enrichment analysis was then performed on the DEGs to define biological processes associated with PN skin. The most prominent Gene Ontology (GO) terms included "cornified envelope" (FDR = 1.5 × 10 -12 ), “Epidermal cell differentiation” (FDR=6.4×10 -10 ), “keratinization” (FDR=1.6×10 -12 ), "peptidase regulatory factor activity" (FDR = 1.1 × 10 -4 ), "Interleukin-4 and 13 signaling" (FDR = 6.8 × 10 -7 ), "interferon alpha / beta signaling" and "response to interferon gamma" (FDR=4.1×10 -7 , and FDR = 4.1 × 10 -6 ), "IL23 pathway" (FDR = 2 × 10 -5 ), and "metaphase and anaphase" (FDR = 3.8 × 10 -10 ) (Figure 1B) (see also Table 2 at the end of the Examples section of this specification). These reflect the hyperproliferative nature of PN associated with altered epidermal differentiation and inflammatory components. Focusing on key expression modules in the lesional skin transcriptome revealed an inflammatory network including the proliferation marker Ki67 (MKI67), the cell cycle gene CDKN1A, and IL-1 and IL-36 (Figure 1C).

[0191] To better understand the disease regulatory network involved in PN skin, a weighted gene co-expression network analysis (WGCNA) was performed. 20 co-expression modules were identified in non-lesional skin and 10 clusters were identified in lesional PN skin (Figure 1D) (see Table 3 at the end of the Examples section herein). This analysis allowed us to identify the "immune response" (FDR = 1.8 × 10 -47 ), "defense response" (FDR = 1.2 × 10 -39 ), the most prominent involved immunological processes (module #8), including "cell cycle" (FDR=2.9×10 -94 ), "DNA metabolic process" (FDR = 8.7 × 10 -67 ), including cell proliferation (module #6), and "epidermal development" (FDR 3.5 × 10 -10 ), “keratinization” (FDR=1.7×10 -6 We were able to assign distinct functions to these co-expressed gene modules, including epidermal processes such as cutaneous neuropathy (module #5), particularly those co-expressed in PN-lesioned skin (Figure 1E). Another notable finding was the co-expressed modules in the "extracellular matrix" (FDR = 1.16 × 10 -59 , module #2), including genes such as MMP14, MMP16, COL1A1, COL1A2, and COL3A1, which were slightly elevated in lesional skin (FC ≥ 1.4, FDR ≤ 6 × 10), consistent with the association between PN and dermal fibrosis. -2 ).

[0192] Transcriptomic changes in PN lesions are enriched for keratinocyte and T cell signatures Using an in silico approach (xCell), we estimated specific cell-type signatures for each non-lesional and lesional PN skin sample. We observed enrichment of transcriptomic signatures associated with epithelial cells and keratinocytes (p<0.001 and p<0.0001, respectively) (Figure 2A). Consistent with the enriched IL-4 / IL-13 GO category (Figure 1B), there was also an increased prominence of Th2-related signatures (p<0.0001). Other inflammatory signatures, such as macrophages (p<0.01), were more variable (Figure 2A). To address the relationship of PN to other hyperproliferative skin diseases with strong inflammatory signatures, we also compared the PN transcriptome with those of atopic dermatitis (AD) and psoriasis. In a three-way comparison, there were numerous genes shared between all three diseases, both up- and down-regulated (Figure 2B). The correlation of effect sizes in lesional skin was more pronounced between PN and psoriasis (Spearman correlation ρ = 0.64) than between PN and AD (ρ = 0.55). Genes commonly upregulated in both psoriasis and PN include those involved in cytokine activity (e.g., CCL3, CXCL10, IFNG, IL12B, IL19, IL1B, IL20) and keratinization (e.g., KRT16, KRT17, LCE3A, LCE3E). See Table 4 at the end of the Examples section herein.

[0193] Transcriptomic changes in PN skin induced by IL-31 receptor inhibitor nemolizumab Clinical results in patients with PN showed that nemolizumab resulted in a higher percentage of improvement in pruritus and skin lesions with an overall favorable safety profile. At week 12, the proportion of patients achieving a 4-point reduction in the weekly average PP-NRS score was significantly higher in the nemolizumab group compared to placebo (52.9% vs. 8.3%, p<0.001). At week 12, the proportion of subjects achieving IGA success (defined as IGA 0 [clear] or 1 [almost clear]) was significantly higher in the nemolizumab group compared to placebo (20.6% vs. 2.8%, p=0.02).

[0194] To address the therapeutic effect of the IL-31 receptor (IL-31R) inhibitor nemolizumab, we performed RNA-seq data from PN biopsies before and after 12 weeks of treatment, using a placebo control in a double-blind study. At baseline, there were 16 and 15 individuals in the placebo and nemolizumab groups, respectively. At week 12, lesion samples were obtained from 18 patients (11 placebo and 7 nemolizumab). Principal component analysis (PCA) was used to observe the intermixing of samples from patients with PN at baseline between the two treatment groups (placebo vs. nemolizumab). After 12 weeks of treatment, there was a trend toward clustering in the nemolizumab cohort, but not the placebo cohort (Figure 3A). This involved sample clustering using genes identified as differentially expressed in non-lesional versus lesional skin, grouping six of the seven nemolizumab samples (86%) at week 12 with baseline non-lesional skin samples, compared with 57% (4 of 7) in the placebo group (Figure 3B). Notably, nemolizumab treatment resulted in normalization of more PN-associated DEGs compared with placebo for both up-regulated genes in PN lesional skin (969 genes with nemolizumab vs. 211 with placebo) and down-regulated genes in PN lesional skin (1,268 genes with nemolizumab vs. 166 with placebo) (Figure 4A). This was also reflected in the correlation between placebo and nemolizumab-treated DEGs, with much greater overlap between nemolizumab-treated and PN DEGs compared with placebo vs. PN for both increased and decreased DEGs (Figure 4B). The GO categories enriched among DEGs by week 12 were only decreased in the nemolizumab treatment group, with “cell cycle” (FDR=5.6×10 -14 ), "keratinocyte differentiation" (FDR = 1.8 × 10 -4 ), and “Interleukin-4 and 13 signaling” (FDR = 1.5 × 10 -2 ), whereas these GO categories were not found in the placebo control group (see Table 2 at the end of the Examples section herein). These data demonstrate that nemolizumab treatment normalized both epidermal hyperproliferation and differentiation in addition to reducing inflammatory responses, particularly those associated with Th2 responses.

[0195] Nemolizumab response is associated with a decrease in IL-31 / Th2 responses in PN skin To address the effect of nemolizumab treatment on inflammatory responses in PN skin, nemolizumab and placebo treatment responses were examined against cytokine response signatures generated in RHE cultures and human epidermal rafts. A consistent reduction in IL-31 responses was observed, either alone or in combination with other inflammatory cytokines, including the Th2 cytokines IL-13 or IL-17A (Figure 5A), providing clear evidence of IL-31 pathway blockade by nemolizumab. Notably, IL-17A-responsive genes were enriched in PN skin (Table 4), likely corresponding to the overlap of specific downstream immunological cascades between psoriasis and PN. IL17A mRNA expression itself was not significantly different in non-lesional versus lesional skin or with nemolizumab treatment up to week 12 (see Table 1 at the end of the Examples section herein), suggesting that IL-17A is not the dominant cytokine in the PN IL-17A signature but is downstream of IL-31 signaling. In terms of changes in cellular transcriptomics, a more robust reduction was observed in the nemolizumab group, including Th1 and Th17 (Figure 5B).

[0196] To determine the tissue compartment with the greatest contribution to the cellular response to anti-IL-31R blockade, transcriptomics data from the placebo and nemolizumab groups were compared with the gene signature of the epidermal compartment obtained from single-cell RNA-seq data. Results show that while the basal keratinocyte (KRT14+) signature was elevated in lesional skin with PN, the latter recovered to a similar extent in both the placebo and treatment groups, whereas induction of the epidermal spinous (KRT10+) signature in lesional skin with PN was only restored by treatment, but not in the placebo group (Figure 5C). Transcription factor binding site (TFBS) analysis was then performed to further understand the transcriptional regulators of transcriptomics changes after PN and placebo or nemolizumab treatment. Results demonstrated that binding sites enriched among genes upregulated in baseline lesional skin were more likely to be enriched among genes downregulated by nemolizumab by week 12 (Figure 5D). The most significantly regulated transcription factors included EGR4, a member of the EGF family of zinc finger transcription factors (p = 4.5 × 10) and EGR5, a member of the EGF family of zinc finger transcription factors (p = 4.5 × 10) enriched in the promoters of up-regulated genes and nemolizumab-down-regulated genes, respectively. -6 and p = 1.2 × 10 -8 ), STAT3 (respectively, p = 2.2 × 10 -4 and p = 2.5 × 10 -5 ), and KLF16 (p = 4.5 × 10 -5 and p = 2 × 10 -5 ) were included (see Table 5 at the end of the Examples section herein).

[0197] Nemolizumab results in a reduction in pruritus scores The Peak Pruritus Numerical Rating Scale (PP-NRS) was correlated with the transcriptome data (Figure 6A). While PP-NRS scores were similar at baseline in both groups, a consistent reduction in pruritus was observed only in the nemolizumab group, whereas a broader response was observed in the placebo group. Furthermore, all patients in the nemolizumab group showed improvement, whereas only a subset of patients in the placebo group showed significant changes. Notably, the measured distance between the principal component 1 and principal component 2 (PC1 / PC2) components in the nemolizumab-treated group was much shorter than that in the placebo group (Figure 6B), consistent with a treatment response.

[0198] Consideration The data presented here are the first to provide a comprehensive view of global transcriptomic changes in PN skin and reveal novel and important insights into the mechanism of action and efficacy of the anti-IL-31 receptor inhibitor nemolizumab. Notably, these data demonstrate at the transcriptomic level that many of the characteristic histological changes observed in PN, including epidermal changes, inflammatory responses (Figures 2A-C), fibrosis, and pruritus (Figures 6A-B), are normalized with nemolizumab treatment.

[0199] Many of the gene expression changes in PN skin are related to abnormal keratinocyte proliferation and differentiation (Figures 1A-1E). These epidermal changes account for a large portion of the overlap between PN and both AD and psoriasis (Figures 2A-2C, Table 4), both of which are also characterized by marked epidermal proliferation and altered epidermal differentiation. Notably, these changes showed significant improvement by week 12 of treatment in the nemolizumab-treated group but not in the placebo group (Table 2). Furthermore, consistent with the therapeutic effect of nemolizumab, the greatest effect of normalization was observed in the differentiated layer of the epidermis (Figure 5C), which may reflect a decrease in keratinocyte proliferation and restoration of normal epidermal differentiation.

[0200] Consistent with PN being an inflammation-driven disease process, immune responses such as Th2 (IL-4 / IL-13) responses and type I and type II IFN responses were prominent (Figures 1A-1E). Th2 responses are closely correlated with pruritus in diseases such as atopic dermatitis, a common predisposing condition to PN development. Interestingly, anti-IL-31 receptor blockade not only significantly reduced IL-31 responses in keratinocytes of PN skin (Figure 5A), but also resulted in reduced Th2 responses and reduced Th17 responses (Figure 5B), corresponding to reduced IL-13 and IL-17 responses in keratinocytes (Figure 5A). The enriched IL-17 response in PN skin, and contributions from IL-36, likely explain the greater overlap of PN skin with plaque psoriasis, as opposed to AD (Figures 2B and 2C). No significant changes in IL17A mRNA expression were observed in the PN data, suggesting that IL-17A is not the dominant cytokine in PN. In addition, the alteration of both Th2 and Th17 responses, and to a lesser extent type II IFN responses, with nemolizumab treatment suggests that these cytokines act downstream of IL-31 in PN.

[0201] Fibrosis is a hallmark of PN and is most prominent in the papillary dermis and less common in the reticular dermis. Fibrosis in PN is characterized by the deposition of vertically oriented collagen fibrils. A module of genes involved in extracellular matrix biology was found to be enriched in PN skin, including both collagen 1 and collagen 3 genes (Table 3). COL1A1, COL1A2, and COL1A3 mRNAs were increased in PN skin at baseline (1.7-fold, 1.44-fold, and 1.52-fold, respectively; see Table 1) but showed no significant changes with nemolizumab treatment at week 12.

[0202] These data also demonstrate how nemolizumab-induced changes in the transcriptome of PN correlate with improvements in pruritus. Chronic pruritus is a debilitating symptom of PN and severely impacts quality of life. While the pathogenesis of pruritus in PN remains unclear, possible contributing factors include Th2 cytokines, IL-4 and IL-13, the primary prurigens in atopic dermatitis, as well as alterations in cutaneous nerves, which have been shown to decrease intraepidermal nerve fiber density in both lesional and non-lesional skin. Data from nemolizumab treatment are consistent with both of these scenarios contributing to pruritus. Thus, nemolizumab treatment results in suppression of Th2 and IL-4 / IL-13 responses in PN skin, as well as decreased expression of factors such as KLF16, which has been shown to inhibit neurite outgrowth. Additionally, nerve growth factor (NGF), which has been shown to be increased in PN skin, is also normalized to a greater extent with nemolizumab treatment at week 12 compared to the placebo group (Table 1). No changes in CGRP or substance P (TAC1) expression were observed. These data are highly suggestive of a broad effect of nemolizumab on pruritus, which may explain the prolonged period of pruritus improvement seen beyond the last dose of nemolizumab (>2 months).

[0203] In summary, PN is a debilitating and difficult-to-treat condition, and there are currently no approved therapies for its treatment. This study provides a detailed characterization of transcriptomic changes in PN skin and demonstrates a broad mechanism of action for the anti-IL-31 receptor inhibitor nemolizumab. These data demonstrate the broad therapeutic effects of anti-IL-31 receptor inhibition with nemolizumab on multiple aspects of PN pathogenesis, including epidermal differentiation, inflammatory responses, pruritus, and extracellular remodeling, confirming the upstream role of IL-31 in PN pathogenesis.

[0204] Example 2 Plasma proteomic analysis of patients with moderate to severe prurigo nodularis treated with nemolizumab This example details a randomized, double-blind, phase 2 study of the anti-human IL31Ra blocking antibody nemolizumab at a dose of 0.5 mg / kg body weight administered subcutaneously at baseline, week 4, and week 8 compared with placebo in patients with moderate to severe prurigo nodularis. The objective of this study was to characterize the effects of nemolizumab on the entire plasma proteome using mass spectrometry.

[0205] Materials and Methods Patient dataset Nineteen placebo non-responders and 19 nemolizumab responders were selected based on the change in PP-NRS (Peak Pruritus Numeric Rating Scale) clinical score at week 12 (19 placebo patients with a PP-NRS change of -1.4 or greater and 19 nemolizumab patients with a PP-NRS change of less than -5.4). The PP-NRS scores at baseline and after 12 weeks of nemolizumab treatment are shown in Figure 10.

[0206] protocol After plasma gY14 ultra-depletion, samples were analyzed using Proteome Science's TMT Calibrator MS2 workflow. A pool of healthy skin biopsies was used: 1) to allow batch calibration between each plex, and 2) to trigger the mass spectrometer to detect skin-associated peptides in the blood.

[0207] statistics Only peptides corresponding to unique proteins were used for statistical analysis. Both baseline-corrected and baseline-uncorrected values ​​were used to calculate differentially expressed proteins. Three statistical tests were used to calculate the following list of differentially expressed proteins: 1) least-squares regression (with or without patient ID), 2) robust regression, and 3) general regression. 193 proteins were found to be differentially expressed (adjusted p-value < 0.05). No additional filters (e.g., logFC filter) were applied to this protein list. Enrichment analysis was performed using the 193 differentially expressed proteins using QIAGEN IPA (QIAGEN Inc., digitalinsights.qiagen.com / IPA).

[0208] result The list of 193 differentially expressed proteins was then analyzed using the enrichment software IPA. The p-value indicates the significance of pathways affected in the dataset, while the z-score provides insight into the directionality, i.e., how "activated" or "inhibited" a pathway in the dataset.

[0209] The canonical pathways were sorted according to z-score alone (Figure 11, right) or sorted by z-score and filtered by p-value (Figure 11, left; -log(p-value)>1.3 corresponds to p-value<0.05). IL-31 has been shown to induce STAT3 activation. Although STAT3 activation is not specific to IL-31, downregulation of this pathway in nemolizumab responders suggests target engagement by nemolizumab. Furthermore, compared with placebo non-responders, the signature of nemolizumab responders was characterized by downregulation of the IL-6 pathway. In addition, the vascular endothelial growth factor (VEGF) pathway, which has been shown to correlate with the severity of PN, was downregulated in nemolizumab responders, likely reflecting clinical improvement. Finally, two neuron ontologies ("CREB signaling in neurons" and "synaptogenesis signaling pathway") were identified as downregulated in nemolizumab responder subjects, highlighting the influence of IL-31 as a neuroinflammatory cytokine in PNs.

[0210] Upstream regulator analysis allowed the identification of transcription factors and small molecules whose function was affected by disease or treatment, depending on the analyzed dataset. Proteins identified in this analysis may not themselves be differentially expressed in the studied dataset, but their activation / inhibition status can explain expression changes in the dataset. Classical pathways were sorted according to z-score alone (Figure 12, right) or sorted by z-score and filtered by p-value (Figure 12, left, p-value <0.05). Profibrotic transforming growth factor beta 1 (TGFβ1) was found as a potential regulator of the nemolizumab responder signature and may indicate resolution of fibrotic nodules. Another potential regulator was signal transducer and activator of transcription 5b (STAT5b), which may reflect target engagement by nemolizumab. These data clearly suggest that nemolizumab strongly influences inflammatory and tissue remodeling processes in PN.

[0211] Biological function analysis of nemolizumab responders revealed downregulated "leukocyte migration" and downregulated "leukocyte cell migration." In addition, two terms related to the neuraxis were downregulated: "neuroglial cell death" and "neuroglial apoptosis," which in some cases reflected an improvement in pruritus (Figure 13).

[0212] Consideration This example aimed to perform a broad and exploratory analysis to identify plasma protein biomarkers that may also explain the mechanism of action of nemolizumab treatment.

[0213] To characterize the full range of plasma proteome changes, a preselected group of nemolizumab responders and placebo non-responders was evaluated. Due to this preselection of patients, the signature described in this example may reflect not only changes induced by nemolizumab but also changes due to a decrease in PP-NRS.

[0214] The dataset analyzed in this study reveals that the nemolizumab responder signature was characterized by improvements in various aspects of PN pathophysiology, including inflammation, neuroimmune function, and tissue remodeling.

[0215] Enrichment analysis revealed that the nemolizumab breath responder signature was characterized by reduced leukocyte migration and cell migration. The STAT3 pathway, a direct target of IL-31 signaling, was also inhibited in nemolizumab breath responder subjects, suggesting target engagement. Nemolizumab breath responders also showed downregulation of other proinflammatory cytokine pathways, including the IL-6 and VEGF pathways.

[0216] Pathway analysis also revealed effects of nemolizumab treatment on neuron-related processes, including CREB signaling in neurons and synaptogenesis signaling pathways, neuroglial cell death, and neuroglial apoptosis, consistent with the observed clinical effects of nemolizumab on pruritus.

[0217] Finally, the dataset showed a decrease in TGFB1 pathway activation suggesting an impact of nemolizumab treatment on tissue remodeling.

[0218] These observations strengthen the understanding that plasma proteomics can effectively capture, at least in part, the tissue-specific (i.e., skin) effects of nemolizumab in PN.

[0219] Example 3 Fibroblast involvement in PN pathogenesis We combined single-cell and bulk RNA sequencing (RNAseq) to delineate the molecular and cellular signatures of PN and investigate the impact of nemolizumab in treated patients. Single-cell RNAseq (scRNAseq) was performed on skin biopsies obtained from four patients with PN and four healthy controls (Figures 14 and 15). The role of fibroblasts in PN pathogenesis was further investigated by cell-to-cell communication analysis using CellChat. Bulk RNAseq was performed on skin biopsies obtained at baseline and week 12 from a phase 2 study of nemolizumab in patients with PN (n = 70).

[0220] The scRNAseq results showed that, compared with healthy skin, lesional PN fibroblasts exhibited a profibrotic and proinflammatory state, which was confirmed by trajectory analysis, reflecting altered fibroblast differentiation in PN skin (Figure 16). Consistent with this, functional analysis of differentially expressed genes in lesional PN fibroblasts indicated activation of inflammatory (TNF, IL1B, IL6) and profibrotic (TGFβ) signaling pathways. CellChat results emphasized the role of PN fibroblasts as central players in intracellular crosstalk (Figure 17). Combining bulk and single-cell RNAseq data, we observed that nemolizumab reversed neuromodulatory dysfunction and reduced inflammation and fibrosis (Figure 18).

[0221] This study demonstrated that PN is an inflammatory and fibrotic disease and that nemolizumab exerts its antifibrotic effects by inhibiting key signaling pathways.

[0222] Example 4 scRNA-seq in Prurigo Nodularis (PN) summary Background: Prurigo nodularis (PN) is a chronic neuroimmune-mediated skin disorder characterized by prurigo-keratotic nodules symmetrically distributed on the extremities and trunk. Neuroimmune dysregulation and chronic scratching are thought to induce and maintain the characteristic lesions.

[0223] Objective: This example provides a comprehensive view of the molecular pathogenesis of PN at the single-cell level to identify and outline the key pathological processes and cell types involved. The characteristics that distinguish PN skin from skin of patients with atopic dermatitis (AD) were of particular interest. A further objective was to determine the effects and specificity of the interleukin-31 (IL-31) receptor alpha antagonist, nemolizumab, at the single-cell level.

[0224] Methods: Single-cell RNA sequencing (scRNA-seq) of skin from 15 healthy donors and non-lesional and lesional skin from 6 patients with PN and AD, respectively, was combined with spatial sequencing (spatial-seq) using the 10x Visium platform and integrated with bulk RNA-seq data from patients treated with nemolizumab.

[0225] Results: The results described in this example demonstrate that PN is an inflammatory skin disease characterized by both keratinocyte proliferation and activation of a profibrotic response. This example demonstrates that the COL11A1+ fibroblast subset is a major contributor to fibrosis and is found primarily in the papillary dermis of PN skin. Activation of a fibrotic response is a key feature distinguishing PN from AD skin. This example further demonstrates the broad effect of nemolizumab on PN cell types, with a pronounced effect of driving COL11A1+ fibroblast and keratinocyte responses toward normal.

[0226] Conclusion: This example provides a high-resolution characterization of the cell types and cellular processes activated in PN skin, establishing PN as a chronic fibrotic inflammatory skin disease. It further demonstrates the broad effects of nemolizumab on the pathological processes of PN skin.

[0227] Introduction Prurigo nodularis (PN) is a chronic neuroimmune-mediated skin disease characterized by chronic and intense pruritus, significantly impacting quality of life. Clinically, it is characterized by multiple nodules that can cover large parts of the limbs and trunk. While the etiology of PN remains unclear, previous studies have implicated immune and neuroregulatory disorders as key circuits in its pathogenesis. PN has been suggested to share clinical and pathological overlap with atopic dermatitis (AD), which may share T helper 2 (Th2) polarization. However, direct comparison of these two conditions at the single-cell level has yet to be performed. Indeed, previous studies have demonstrated that bulk RNA-Seq analysis reveals that PN has a distinct molecular signature compared to AD. The best-characterized immune mediators in PN are the cytokines IL-31 receptor alpha (IL-31RA) and oncostatin M receptor beta (OSMRB). Although activated Th2 cells are thought to be the primary source of IL-31, other cell types can also produce IL-31, including eccrine sweat glands, mast cells, basophils, eosinophils, and monocytes / macrophages. Recent studies in human PN lesions have revealed that macrophages, in addition to T cells, are the primary cellular source of IL-31. The critical importance of IL-31 to PN pathogenesis has been demonstrated by nemolizumab, an investigational monoclonal antibody that inhibits IL-31RA, resulting in effective suppression of downstream inflammatory responses, including stabilization of Th2 responses and extracellular matrix (ECM) remodeling. Recent evidence indicates that fibrosis is a hallmark of PN, but the cause of this fibrosis in PN skin is unknown. The major fibroblast population in human skin has two major clusters characterized by the expression of SFRP2 and FMO1, and five minor fibroblasts, including COL11A1+ fibroblasts, three of which (SFRP2+, FMO1+, and COL11A1+) have been suggested to have roles in matrix deposition, inflammatory cell retention, and connective tissue cell differentiation based on their gene expression profiles.

[0228] This example provides an in-depth exploration of PN pathogenesis to characterize the core mechanisms involved, identify target cells for nemolizumab treatment, and contrast its pathogenesis with AD through a combination of single-cell RNA (scRNA) and spatial RNA sequencing approaches.

[0229] result Single-cell RNA-seq and spatial-seq reveal diverse cell types and their spatial locations in PN skin To understand the unbiased cellular composition and cell state of healthy (H) skin and lesional PN (LPN) skin, we generated single-cell suspensions of skin biopsies from 15 healthy donors and six patients with PN. Skin biopsies were also collected from peripheral non-lesional sites in four of the six PN patients (NPN), yielding a total of 25 scRNA-seq libraries. The resulting quality-controlled PN+healthy single-cell atlas contained a total of 72,782 cells, with an average of 2,379 genes and 10,417 transcripts detected per cell. To study the heterogeneity of these cells, variable genes were selected and used to perform uniform manifold approximation and projection (UMAP) dimensionality reduction and cell clustering using the R package Seurat. Cluster annotation was supported by overlapping cluster markers with canonical lineage-specific genes reported in previous skin disease scRNA-seq studies. Ten major cell types were recovered across all samples, including keratinocytes, melanocytes, eccrine sweat gland cells, endothelial cells, fibroblasts, pericytes, neurons, T cells, myeloid cells, and mast cells (Figure 19A). Most of these cell types comprised the majority of cells from the healthy, NPN, and LPN libraries, suggesting that each cell type is related to a common cell lineage rather than derived from a specific condition. Two minor cell populations, eccrine sweat gland cells and neurons, were primarily derived from healthy samples. Interestingly, a clear separation of keratinocytes, fibroblasts, and endothelial cells was observed between healthy, NPN, and LPN cells, suggesting major transcriptional differences (Figure 19B). Moderate shifts in cell type proportions were observed in LPN compared to NPN and healthy controls, with the most pronounced shifts observed in mast cells, endothelial cells, T cells, and myeloid cells in LPN skin (Figure 19C). Marker genes for each cell population showed a clear separation between each cell type (Figure 19D).

[0230] To localize the major cell types detected by scRNA-seq in systemic sclerosis (SSc) skin, spatial sequencing (spatial-seq) was performed on SSc skin samples using a 10x Visium platform. 395 spatially defined spots were detected, with an average of 2,613 genes and 4,432 transcripts per spot (Figure 20). Spatial spots were deconvolved by the major cell types detected in scRNA-seq using the Seurat anchor-based label transfer method. The deconvolved prediction scores for each cell type were displayed on the tissue (Figure 19E) and combined into a scatter plot representing the relative cell type composition for each spot (Figure 20B). Keratinocytes were localized in the epidermis and hair follicles. Myeloid cells and T cells were primarily located in the epidermis adjacent to the epidermis. Fibroblasts were distributed throughout the majority of spots within the dermis, and pericytes were located near blood vessels (Figure 20B). These two cell types were the major producers of extracellular matrix (ECM) components (Figure 20C). Other cell types represented minor populations and were under-detected in the Spatial-seq samples.

[0231] COL11A1+ fibroblasts are enriched for profibrotic responses in PN skin To characterize fibroblast heterogeneity, we subclustered all fibroblasts from the scRNA-seq dataset. Based on previously published marker genes, we annotated fibroblast subclusters into six subtypes, including SFRP2+ fibroblasts (FBs), APOE+ FBs, RAMP1+ FBs, COL11A1+ FBs, TNN+ FBs, and SFRP4+ FBs (Figures 21A and 20). Interestingly, COL11A1+ FBs were predominantly derived from LPN samples compared with healthy or NPN samples (Figures 21B and 21C). COL11A1+ FBs expressed high levels of COL11A1, POSTN, and PRSS23, suggesting a profibrotic role in PN skin (Figure 21D). To account for the ECM production capacity of different fibroblast subtypes, ECM module scores were calculated using a list of genes from the extracellular matrix pathway from Gene Ontology, identifying the highest ECM score in LPN COL11A1+ FBs (p = 4.4E-83) (Figure 21E). Differential expression analysis was then performed between LPNs and healthy COL11A+ FBs to infer upstream regulatory factors driving differential expression. High activation z scores for transforming growth factor beta-1 (TGFB1), IL-5, and IL-4 likely reflect a profibrotic response, while high tumor necrosis factor (TNF), interferon gamma (IFNG), and IL-6 activation z scores suggest an inflammatory response in LPN COL11A1+ FBs compared to their healthy counterparts (Figure 21F). Enrichment analysis also revealed top fibrosis-related pathways (i.e., extracellular matrix organization, collagen fibril organization) and inflammation-related pathways (i.e., neutrophil degranulation, neutrophil activation involved in immune response) (Figure 21G). COL11A1+FBs had the highest expression pattern of collagen genes, including COL1A1, COL1A2, COL3A1, COL5A1, COL5A2, COL6A1, COL6A2, COL6A3, COL11A1, COL12A1, COL14A1, and COL16A1 (Figure 21H). Collectively, the above results suggest a strong fibrotic potential of COL11A1+FBs in LPN skin.We confirmed the presence of major fibroblast subtypes (SFRP4, SFRP2, RAMP1, and COL11A1) (Figures 21I and 22) and validated the fibrotic phenotype by immunohistochemistry in PN skin. To compare the fibrotic inducing abilities of fibroblasts in PN and AD, we combined the PN scRNA-seq dataset with single-cell datasets from AD skin and compared the ECM scores between fibroblast subtypes in healthy (H), non-lesional AD (NAD), NPN, lesional AD (LAD), and LPN skin. While the LAD fibroblast subtype exhibited a higher ECM score compared with healthy or peripheral non-lesional fibroblasts, the LPN fibroblast subtype expressed a significantly higher ECM score than LAD cells (p = 1.8 × 10-15) (Figure 21J), especially COL11A1+FBs (p = 2.2 × 10-9), the FB subset that was most significantly increased in PN skin.

[0232] Endothelial cells and pericytes exhibit fibrotic and inflammatory responses in PN skin Next, we investigated the heterogeneity of endothelial cells and clustered these cells into six subclusters (Figures 23A and 23B). Disease composition analysis identified endothelial subclusters 2 and 5 as enriched in LPN compared with healthy or NPN samples (Figures 23C and 23D) (p = 0.0014 and 2.1 × 10-22, respectively). Subcluster 2 represents activated endothelial cells with high expression of ICAM1 and E-selectin (SELE), which also exhibit inflammatory hallmarks such as TNFAIP3 and IL6. Subcluster 5 expressed high levels of several collagen genes (i.e., COL4A1 and COL15A1), suggesting their potential involvement in fibrosis (Figure 3B). To study these two LPN-specific subclusters, we performed enrichment analysis using their cluster marker genes. Subcluster 5 marker genes were involved in proinflammatory cytokines (i.e., TNF, IL1B, IFN-G, IL6) and pathways (i.e., cytokine-mediated signaling pathways, cellular response to cytokine stimulation) (Figures 23E, 23F). Subcluster 2 marker genes were regulated by profibrotic upstream regulators (i.e., TGFB1, angiotensinogen (AGT), epidermal growth factor (EGF), IL-5) and were enriched in ECM-related pathways (i.e., extracellular matrix organization, extracellular structural organization) (Figures 23G, 23H). These results suggest that endothelial cells were actively involved in both fibrotic and inflammatory responses in PN skin.

[0233] Similarly, pericytes were subclustered, yielding nine subclusters (Figures 24A and 24D). Compositional analysis identified subclusters 3 and 8 as enriched in LPN samples compared with healthy or NPN samples (Figures 24B and 24C). ECM scores were calculated, and collagen genes across pericyte subtypes were plotted. Cells in subcluster 3 had the highest ECM score in LPN and exhibited the highest expression pattern of collagen genes (Figures 24E and 24F). LPN cells in subcluster 7 also revealed a significantly higher ECM score than healthy cells and expressed the second-highest pattern of collagen genes (Figures 24E and 24F). We then performed enrichment analysis using marker genes from subclusters 3 and 7 and found that both subclusters were involved in profibrotic upstream regulators (i.e., TGFB1, AGT, prolactin (PRL)) and pathways (i.e., extracellular matrix organization, collagen fibril organization) (Figures 22G-J). Subcluster 3 also showed an interferon-driven inflammatory response (Figures 22G, 22I). Collectively, these results demonstrate that endothelial cells and pericytes can actively contribute to fibrosis and inflammatory responses in lesional PN skin.

[0234] Keratinocyte response in PN skin Keratinocytes were subclustered into six keratinocyte subtypes: basal, spinous, supraspinous, granular, follicular, and inflammatory keratinocytes (Figures 25A and 25D). Inflammatory keratinocytes were primarily derived from LPN samples (Figures 25B and 25C). Enrichment analysis using inflammatory keratinocyte markers implicated in proinflammatory upstream regulators and mitochondrial respiratory pathways, suggesting high energy expenditure in PN keratinocytes during inflammation (Figure 25E). Upstream regulators of the inflammatory subtype of keratinocytes include Th2 cytokines (IL-4, IL-5, IL-33), and TGFB1 (Figure 25F), supporting the enrichment of Th2 responses in lesional PN skin.

[0235] scRNA-seq reveals immune subtype heterogeneity in PN skin Given the strong inflammatory response observed in fibroblasts, endothelial cells, and pericytes, we investigated immune cell heterogeneity. Myeloid cells were subclustered and annotated into nine subtypes, including cycling myeloid cells, Langerhans cells (LCs), plasmacytoid dendritic cells (pDCs), classical type 1 dendritic cells (cDCs), classical type 2 dendritic cell subset A (cDCs2A), classical type 2 dendritic cell subset B (cDCs2B), interstitial macrophages (IMs), perivascular macrophages, and lipid-associated macrophages (LAMs, also known as TREM2 macrophages) (Figures 26A-E). The p-values ​​for pDCs, cDCs2A, IMs, PVMs, and LAMs were 1.30 × 10-13, 3.33 × 10-8, 1.72 × 10-12, 9.41 × 10-5, and 4.5 × 10-11, respectively. Increased proportions of pDCs, cDC2A, and macrophage subpopulations (IM, PVM, and LAM / TREM2) were observed in lesional PN skin compared with non-lesional and healthy skin (Figures 26D-26E). The prominence of LAM / TREM2 macrophages in lesional PN skin was confirmed using immunohistochemistry (Figure 26). Seven subtypes of T cells and other lymphoid cells were identified: cycling T cells, innate lymphoid cells (ILCs), natural killer cells (NK), CD8+ T cells (CD8T), tissue-resident memory T cells (Trm), CD4+ T cells (CD4T), and regulatory T cells (Treg). Several NK and T cell populations showed increased proportions in lesional PN skin, including cycling, NK, CD8, and T regulatory cells. The presence of T cells in lesional PN skin was confirmed by IHC staining for CD8 and CD4 (Figure 26).

[0236] Ligand-receptor analysis reveals cell type-specific networks in PNs To address the observed shifts in cell type composition and transcriptional changes, we analyzed cell-cell communication changes in PN compared to healthy skin. To do this, we performed separate ligand-receptor analyses for healthy, NPN, and LPN cell types using CellphoneDB and CellChat. The greatest number of interactions, particularly between fibroblasts, endothelial cells, pericytes, myeloid cells, and keratinocytes, was observed in LPN (Figure 5C) compared to healthy (Figure 27A) or NPN skin (Figure 27B). To study specific ligand-receptor pairs in PN, we selected pairs with higher interaction scores in LPN compared to healthy or NPN, revealing various signaling pathways involved in PN arising from both immune (T cells, myeloid) and stromal cell populations (fibroblasts, endothelial cells, and pericytes). Several validated pro-inflammatory cytokines, such as IFNγ, IL-1, IL-6, and TNF, were also implicated in the pathogenesis of PN. Notably, this analysis revealed several other proinflammatory mediators, including CCL2, CCL3, CXCL2, CXCL12, and IL7, expressed by various cell types in PN skin (Figures 27D and 27E). The appearance of fibroblast growth factors (FGF2, FGF7), platelet-derived growth factor (PDGFB), transforming growth factor beta (TGFB1, TGFB2, TGFB3), and vascular endothelial growth factor (VEGFB), supported the involvement of fibrosis in PN pathogenesis. A robust signaling network related to the TGFB signaling pathway in lesional PN skin was observed, with TGFB sources observed in multiple cell types and fibroblasts being the primary target cells in LPN skin (Figure 27F). Collectively, these data indicate a profibrotic and proinflammatory shift within the interacting populations of PN skin.

[0237] Comparison of epithelial responses in PN skin and AD skin by single cell analysis As previously described, lesional PN skin exhibited an expansion of COL11A1 fibroblasts compared with lesional AD skin, suggesting a more profibrotic signature in PN pathophysiology (Figures 21J and 22B). To further compare keratinocyte responses in PN and AD skin, we compared gene expression changes in each compartment (Figures 28A-D). Both PN and AD skin had an expansion of KCs within cluster 3, corresponding to an "inflammatory" phenotype, with prominent expression of several inflammatory markers, including KRT6, KRT16, and S100A8 / A9, along with IFN signature genes such as IFI27 and IFITM3, and the inflammasome gene PYCARD, which encodes an apoptosis-associated speck-like protein containing CARD(ASC) (Figures 28A, 28D, and 28E). Using thresholds of FC>2 and FDR<0.05, assessing the enriched gene ontology categories in each KC compartment showed fairly consistent changes across "inflammatory," basal, spinous, supraspinous, and follicular keratinocytes compared to PN skin (Figure 28F). AD has enriched inflammatory responses such as "defense response to bacteria" and "neutrophil degranulation" in "inflammatory" KCs (p=2.8×E-07 and p=3.8×E-05, respectively), and T cell chemotaxis in basal and spinous AD KCs (p=3.5×10E-05 and p=4.1×10E-5, respectively). In contrast, terms related to altered epidermal differentiation were enriched in "inflammatory" and spinous keratinocytes from PN compared to AD skin (Figure 28F). Consistent with all KC subtypes, there was higher expression of the chemokines CCL27, S100A7, and S100A9 in AD compared to PN.

[0238] Comparison of immune cell responses in PN skin and AD skin by single cell analysis The proportions of T cell subsets were similar overall between PN and AD skin across several T cell subsets, including CD8, CD4 effector T cells, Tregs, and cycling T cells. In addition, prominent ILC and NK cell subsets were present in both NPN, LPN, NAD, and LAD skin (Figures 29A-D). The most striking difference between LAD and LPN skin was found in CD4+ effector T cells, with LAD CD4+ cells exhibiting increased expression of IL13 (2.7-fold higher, FDR = 8.5 × 10E-24) and IL22 (4.7-fold, FDR = 6.0 × 10E-21). Significantly fewer IL4-positive T cells were observed in LAD skin (Figure 30). In contrast, LPN CD4+ T cells had higher expression of CCL5 (2.4-fold, FDR = 2.8 × 10E-16). There was a trend towards increased expression of IL17A and IL17F in LPN compared to LAD skin, but this was not significant (Figure 30).

[0239] The same nine myeloid cell subsets were identified in PN, AD, and healthy skin as described above, with some subsets being more prominent in LPN skin compared to LAD (Figures 29E-29H). These include pDCs, interstitial macrophages (IM), and lipid-associated macrophages (LAM, TREM2). + ) were included. The most significant differences in gene expression were found only in PN IM macrophages, which increased expression of CCL3 and CCL4 (2.7-fold for both, FDR = 2.0 × 10E-09 and 3.9 × 10E-05, respectively). In contrast, LAD IM macrophages increased expression of MHC class II molecules, including HLA-DRB1, HLA-DQA1, and HLA-DQB1 (2.2-fold, 2.3-fold, and 2.7-fold, and FDR = 2.7 × 10E-10, 8.6 × 10E-14, and 9.8 × 10E-13, respectively).

[0240] Blockade of IL-31 receptor alpha with nemolizumab reverts the transcriptional profile of LPN fibroblasts and keratinocytes to a healthy state Following treatment with nemolizumab, an IL-31 receptor alpha antagonist, we demonstrated transcriptomic changes in PN skin using a bulk RNA-seq approach. To determine where the biological response to nemolizumab was most pronounced, we plotted the expression of two genes encoding heterodimeric IL-31 receptors, IL-31RA and OSMRB (Figures 31A and 31B). IL31RA was specifically expressed in fibroblasts and keratinocytes. In contrast, OSMRB was more widely expressed in fibroblasts, keratinocytes, pericytes, and endothelial cells. Strikingly, genes differentially upregulated with nemolizumab treatment compared to baseline lesions were found primarily in fibroblast clusters, whereas differentially downregulated genes were more widely localized in keratinocytes and immune cell subsets, consistent with attenuation of hyperkeratosis and a reduced inflammatory response with treatment (Figures 31C and 31D). To study the effects of nemolizumab on gene expression levels down to the single-cell level in PN skin, we generated a gene list containing genes significantly downregulated by nemolizumab compared to placebo from a recently published bulk RNA-seq study. Module scores were then calculated using the gene list across all major cell subtypes: PN skin (Figure 31D), KC subsets (Figures 31E, 31F, 31G, and 31H), and FB subsets (Figure 6F). Consistent with the expression of IL31RA and OSMR, the most prominent changes were observed in keratinocytes and fibroblasts, particularly inflammatory KCs and COL11A1+ fibroblasts. These results indicate that nemolizumab treatment reverts transcriptional profiles from PN to healthy skin across a wide range of both stromal and immune cell populations, particularly fibroblasts and keratinocytes (the cell types responsible for the most prominent histopathological changes in lesional PN skin).

[0241] Consideration This example provides detailed insight into the pathogenesis of PN and the associated tissue- and cell-type-specific changes that occur in PN skin. Surprisingly, an increase in profibrotic responses accompanied by an immune shift away from IL-13 and IL-22 responses is a key feature of PN from AD. Changes in PN skin are observed across both immune and stromal cell populations, including keratinocytes, endothelial cells, and most severely, fibroblasts and fibroblast subpopulations.

[0242] A characteristic histopathological feature of PN is papillary dermal fibrosis with vertically arranged collagen fibers. Findings consistent with this feature demonstrate a marked increase in dense collagen in the papillary dermis of lesional PN skin by trichrome staining, as well as increased expression of procollagen I in the papillary dermis. Furthermore, through single-cell analysis, this example identifies the COL11A1+ fibroblast subset as the primary source of an activated and enriched profibrotic response, including increased mRNA expression of both collagen I and collagen III. Consistent with their profibrotic function, COL11A1+ fibroblasts were found primarily in the papillary dermis, and the fibrotic response was most pronounced by either trichrome or procollagen I staining, indicating active collagen I biosynthesis. This expansion of the COL11A1+ fibroblast subpopulation is unique to PN and is not seen in AD skin. Furthermore, the profibrotic effects of this population were not observed in AD COL11A1+ fibroblasts. Recently, a subset of cancer-associated fibroblast (CAF)-like phenotypes was described in AD skin characterized by the expression of WNT5a, tenascin (TCN), and periostin (POSTN), among several other genes. Consistent with this publication, periostin (POSTN) and WNT5A, along with fibroblast activation protein (FAP), a characteristic marker of CAFs, were expressed in COL11A1+ fibroblasts in our dataset. Another recently published paper described CXCL14-IL24+ secreting papillary dermal fibroblasts as unique to PN skin. Here, we observed IL24+CXCL14-negative FBs, as well as a small subset with SFRP2+ FBs, and their enrichment in PN skin, but detectable levels in both lesional AD and healthy skin. These FBs expressed increased levels of MMP1, but low levels of COL1A1 and COL1A2, suggesting that they do not contribute to fibrosis in PN skin.

[0243] Of the two components of the heterodimeric IL-31 receptor, expression of IL31RA was detected in both keratinocytes and fibroblasts, whereas expression of OSMRB was found to be more widespread in different cell populations, suggesting that the two major cell types responding to IL-31 in PN are likely to be fibroblasts and keratinocytes, consistent with the observed transcriptomic shifts driven by nemolizumab that can be attributed to these two cell types.

[0244] Notably, this example provided evidence that other cell types, including endothelial cells and pericytes, also contribute to fibrosis in prurigo nodularis (LPN) skin. While endothelial changes are known in LPN, the nature of these shifts has not been previously detailed. These data suggest that endothelial cells, likely under the influence of pro-inflammatory and pro-fibrotic cytokines such as TGFB, contribute to extracellular matrix reorganization. TGFβ may be an upstream promoter of fibrosis in PN skin, as it was observed to be expressed in a wide range of cell types in PN skin, including endothelial cells, fibroblasts, and neurons for TGFB1, and fibroblasts and pericytes for TGFB2 and TGFB3. Notably, TGFB2 and TGFB3 are more strongly implicated in fibrosis than TGFB1.

[0245] One of the most distinctive histological features of PN is the presence of compact orthohyperkeratosis accompanied by irregular epidermal proliferation. Keratinocytes exhibit striking transcriptomic changes in lesional PN skin, with the most prominent shift seen in inflammatory keratinocytes defined by KRT6, KRT16, and KRT17 expression, along with S100A8 and S100A9 expression. This analysis indicates a critical role for Th2 cytokines (e.g., IL-13 and IL-22) in this transition, consistent with previous observations. The most enriched biological categories in inflammatory keratinocyte subsets were associated with mitochondrial function and protein translation, suggesting the generation of reactive oxygen species and cellular stress that may contribute to the inflammatory response in skin.

[0246] Immune cell infiltration was prominent in lesional PN skin and characterized by a shift in specific immune cell populations. The most prominent shift was observed in macrophage populations, particularly lipid-associated macrophages, characterized by expression of APOE and TREM2. Lipid metabolites from lipid-associated macrophages have been shown to induce the production of proinflammatory cytokines in atherosclerosis, amplifying the inflammatory response. These macrophages have recently been implicated in the pathogenesis of acne. T cells were also prominent in PN lesions, including cycling T cells, as well as NK and CD8 + The numbers of T cells, Tregs, and Tregs were increased. The role of these T cell populations in PN skin has not been previously characterized. Furthermore, various stromal cell populations, particularly endothelial and pericytes, increased expression of various proinflammatory cytokines, chemokines, and adhesion molecules, suggesting an active role in immune trafficking and amplification in PN. This includes increased expression of the adhesion molecules ICAM1, E-selectin (SELE), and IL6 on endothelial cells, as well as increased expression of CCL2, CCL3, CCL4, CCL13, CCL18, CXCL2, and CXCL12 expressed by various cell types (e.g., myeloid cells, pericytes, and endothelial cells) in PN skin. It is noteworthy that CCL2 and IL-6 have established roles in the development of fibrosis. CCL2 is the most potent profibrotic chemokine, and through CCR2, CCL2 acts directly on fibroblasts to stimulate collagen synthesis. Similarly, IL-6 trans-signaling enhances lung fibroblast proliferation and extracellular matrix protein production.

[0247] These data further outline the differences between PN and AD. Notably, shifts in cell populations in the epidermis are very similar between PN and AD, with both diseases possessing a prominent "inflammatory" keratinocyte subset characterized by increased expression of proinflammatory molecules, including S100A8 and S100A9, along with increased expression of the inflammatory keratins KRT6 and KRT16. While altered expression of S100A8, S100A9, and KRT16 has been described in AD skin, their expression has not been addressed in PN skin. Interestingly, epidermal changes were accompanied by subtle changes in gene expression in PN versus AD skin, with regulators of immune-related processes, such as antimicrobial responses, and T cell trafficking, observed only in LAD but not LPN keratinocytes. Alterations in T cell phenotype were also observed between LPN and LAD skin, particularly within the CD4 effector T cell population, with IL13 and IL22 mRNA expression significantly lower in LPN skin compared with LAD skin. IL-22 is known to promote epidermal proliferation and activate innate immune and antibacterial responses in the skin. IL-13 is a key effector cytokine in AD skin and is the therapeutic target of three biologics: the IL-4Ra blocker dupilumab, and the anti-IL13 mAbs lebrikizumab and tralokinumab. These data suggest that PN is an inflammatory-driven disease, but may not be centered on the IL-13 / IL-22 response to the same extent as AD.

[0248] These data also provide information about the mechanism of action of the IL-31 receptor antagonist, nemolizumab. Bulk RNA-seq analysis of LPN skin from patients treated with nemolizumab was performed, and the observed transcriptomic shifts indicated stabilization of extracellular matrix remodeling and normalization of epidermal differentiation. By cross-referencing single-cell data with nemolizumab bulk RNA-seq data, we were able to demonstrate the broad effects of nemolizumab on the aberrant transcriptomic activation of various cell types in PN skin. Therefore, the normalization of the pathological transcriptomic signature observed in COL11A1+ fibroblasts and inflammatory keratinocyte subsets likely reflects the observed clinical improvement of PN skin lesions during nemolizumab treatment. These results also validate our observations regarding the molecular and cellular impact of nemolizumab treatment on the pathophysiological pillars of PN disease, including inflammation, altered epidermal differentiation, and fibrosis.

[0249] In summary, these data provide unique insights into the pathogenesis of PN, highlighting it as a chronic neuroimmune skin disease involving complex immune-stromal cell crosstalk, likely promoting and driving aberrant keratinocyte proliferation and activation. This is accompanied by a marked shift toward a profibrotic response, primarily within the papillary dermis, involving activation of COL11A1+ fibroblasts, endothelial cells, and pericytes. Remarkably, these changes are reversible by blocking IL-31 receptor alpha. Thus, these new insights expand our understanding of PN pathogenesis and the mode of action of anti-IL-31R therapy for this debilitating disease.

[0250] Materials and Methods Human sample acquisition Six patients with prurigo nodularis, six patients with AD, and 15 healthy donors were recruited for single-cell RNA sequencing. Six-mm punch biopsies were obtained from affected and non-lesional AD and PN skin. Patients had not received active topical treatment at least 2 weeks prior to enrollment. None of the patients had received prior systemic treatment. Patients with PN did not have comorbid active AD. This study was approved by the University of Michigan Institutional Review Board (IRB), and all patients provided written consent. This study was conducted in accordance with the principles of the Declaration of Helsinki. See patient demographics in the table below. TIFF2025530773000001.tif159151

[0251] Single-cell RNA-seq library preparation, sequencing, and alignment Single-cell suspensions for scRNA-seq were generated as follows: skin biopsies were incubated overnight in 0.4% dispase (Life Technologies) in Hank's Balanced Saline (Gibco) at 4°C. The epidermis and dermis were separated. The epidermis was digested in 0.25% trypsin-EDTA (Gibco) containing 10 U / mL DNase I (Thermo Scientific) for 1 hour at 37°C, quenched with FBS (Atlanta Biologicals), and filtered through a 70 μM mesh. The dermis was minced and digested in 0.2% collagenase II (Life Technologies) and 0.2% collagenase V (Sigma) in plating medium for 1.5 hours at 37°C, and filtered through a 70 μM mesh. Epidermal and dermal cells were combined at a 1:1 ratio, and libraries were constructed on the 10x Chromium system with chemistry v3 by the University of Michigan Advanced Genomics Core. The libraries were then sequenced on an Illumina NovaSeq 6000 sequencer to generate 150-bp paired-end reads. Data processing, including quality control, read alignment (hg38), and gene quantification, was performed using 10x Cell Ranger software.

[0252] Cell clustering and cell type annotation The R package Seurat (v4.1.1) was used to cluster cells within the merged matrix. Cells with fewer than 500 transcripts, fewer than 100 genes, more than 1e5 transcripts, or mitochondrial expression greater than 10% were first filtered out as low-quality cells. The NormalizeData function was used to normalize the expression level of each cell using default parameters. The FindVariableFeatures function was used to select variable genes with default parameters. The ScaleData function was used to scale and center the counts in the dataset. Principal component analysis (PCA) was performed on the variable genes. To remove potential batch effects between samples processed in different batches, the RunHarmony function from the Harmony package was applied. Uniform manifold approximation and projection (UMAP) dimensionality reduction was performed using the RunUMAP function. Clusters were obtained using the FindNeighbors and FindClusters functions with the resolution set to 0.6. Cluster marker genes were found using the FindAllMarkers function. Cell types were annotated by overlapping cluster markers with canonical cell type signature genes. To calculate disease composition based on cell type, the number of cells for each cell type from each disease state was counted. The counts were then divided by the total number of cells for each disease state and scaled to 100% for each cell type. Differential expression analysis between any two cell groups was performed using the FindMarkers function. All differential expression analysis comparisons were displayed as mean log2 fold change and corrected for multiple testing using a false discovery rate (FDR) adjustment.

[0253] Cell type subclustering Subclustering was performed on abundant cell types. Subclusters were obtained using the same function as above. Subclusters defined exclusively by mitochondrial gene expression, which exhibited low quality, were removed from further analysis. Subtypes were annotated by overlapping the marker genes of the subcluster with canonical subtype signature genes. Module scores were calculated using the AddModuleScore function on the intended gene list. ECM scores were calculated for genes from the extracellular matrix pathway from the Gene Ontology database. Cytokine scores for fibroblast subtypes were calculated for induced genes in fibroblasts after stimulation with TGF-β or IL-4. Nemolizumab-induced or decreased genes were obtained from a previous bulk RNA-seq study by Tsoi et al. Differentially expressed genes or cluster marker genes were used for enrichment analysis to obtain potential upstream regulators using Ingenuity Pathway Analysis (QIAGEN Inc., qiagenbioinformatics.com / products / ingenuity-pathway-analysis) or the canonical pathway using Enrichr.

[0254] Ligand-receptor interaction analysis CellphoneDB (v3) and CellChat were used for ligand-receptor analysis. Each cell type was separated by its disease classification (healthy, non-lesional, and lesional), and a separate run was performed for each disease classification. Pairs with a p-value >0.05 were excluded from further analysis. The number of interactions between each cell type pair was then calculated for each condition. To compare the healthy and lesional conditions, pairs that showed higher interaction scores in the lesional condition were used to indicate lesion-specific interactions.

[0255] Immunohistochemical staining Paraffin-embedded tissue sections (lesional and healthy skin) were heated at 60°C for 30 minutes, deparaffinized, and rehydrated. Slides were placed in PH9 antigen retrieval buffer and heated at 125°C for 30 seconds in a pressure cooker water bath. After cooling, slides were treated with 3% H2O2 (5 minutes) and blocked with 10% goat serum (30 minutes). Subsequently, anti-human primary antibodies were used for overnight incubation at 4°C. The antibodies used were anti-COL11A1 (ThermoFisher Scientific, catalog no. PA5-68410), anti-SFRP2 (Lifespan Biosciences, catalog no. LS-C794043), anti-SFRP4 (Lifespan Biosciences, catalog no. LC-C408100), anti-TREM2 (ThermoFisher Scientific, catalog no. PA5-18763), anti-RAMP1 (Abcam, catalog no. AB64409), anti-CD4 (ThermoFisher Scientific, catalog no. 14-244-82), anti-CD8 (ThermoFisher Scientific, catalog no. MA5-13473), and anti-CD3 (Origene, catalog no. UM500048). Slides were then washed and treated with secondary antibodies, peroxidase (30 min), and diaminobenzidine substrate. After counterstaining with hematoxylin, the sections were dehydrated, mounted, and examined under a microscope.

[0256] Spatial sequencing library preparation Skin samples were frozen in OCT medium and stored at -80°C until sectioning. Tissue permeabilization was optimized on 20 μm sections using the Visium Spatial Tissue Optimization Reagents Kit (10x Genomics, Pleasanton, CA, USA), with the optimal permeabilization time set at 9 min. Samples were mounted on gene expression slides (10x Genomics), fixed in ice-cold methanol, stained with hematoxylin and eosin, and scanned under a microscope (Keyence, Itasca, IL, USA). Tissue permeabilization was performed to release polyA mRNA for capture by poly(dT) primers pre-coated on slides and containing Illumina TruSeq Read, spatial barcodes, and unique molecular identifiers (UMIs). The Visium Spatial Gene Expression Reagent Kit (10x Genomics) was used for reverse transcription to generate spatially barcoded full-length cDNA, which was then used for second-strand synthesis, followed by denaturation, allowing the cDNA to be transferred from the slide to a tube for amplification and library construction. A Visium spatial single-cell 3' gene expression library consisting of Illumina paired-end sequences flanking P5 / P7 was constructed after enzymatic fragmentation, size selection, end repair, A-tailing, adapter ligation, and PCR. The Dual Index Kit TT Set A (10x Genomics) was used to add unique i7 and i5 sample indexes and generate TruSeq Read 1 for sequencing spatial barcodes and UMIs, and TruSeq Read 2 for sequencing cDNA inserts, respectively. The library was then sequenced on an Illumina NovaSeq 6000 sequencer to generate 150-bp paired-end reads.

[0257] Spatial sequencing data analysis After sequencing, the reads were aligned to the human genome (hg38), and an expression matrix was extracted using the spaceranger pipeline (10x Genomics). The expression matrix was then analyzed using Seurat. Specifically, the SCTransform function was used to scale the data and find variable genes with default parameters. PCA and UMAP were applied for dimensionality reduction. The FindTransferAnchors function was used to find a set of anchors between the spatial-seq and scRNA-seq data, which were then transferred from the scRNA-seq to the spatial-seq data using the TransferData function. These two functions construct a weight matrix that defines the association between each query cell and each anchor. These weights were summed to 1 per spot and used as the percentage of cell types within the spot. The ECM score was calculated using the AddModuleScore function for genes from the extracellular matrix pathway from the Gene Ontology database.

[0258] [Table 1] TIFF2025530773000003.tif196145TIFF2025530773000004.tif196146TIFF2025530773000005.tif196146TIFF2025530773000006.tif196145TIFF2025530773000007.tif196145TIFF2025530773000008.tif196147TIFF2025530773000009.tif197147TIFF2025530773000010.tif196145TIFF2025530773000011.tif196147TIFF2025530773000012.tif196145TIFF2025530773000013.tif196146TIFF2025530773000014.tif196147TIFF2025530773000015.tif196146TIFF2025530773000016.tif196145TIFF2025530773000017.tif196145TIFF2025530773000018.tif196146TIFF2025530773000019.tif196145TIFF2025530773000020.tif196145TIFF2025530773000021.tif196145TIFF2025530773000022.tif196147TIFF2025530773000023.tif196146TIFF2025530773000024.tif196145TIFF2025530773000025.tif196145TIFF2025530773000026.tif196146TIFF2025530773000027.tif196147TIFF2025530773000028.tif196147TIFF2025530773000029.tif196146TIFF2025530773000030.tif196145TIFF2025530773000031.tif196146TIFF2025530773000032.tif196146TIFF2025530773000033.tif196146TIFF2025530773000034.tif196146TIFF2025530773000035.tif196145TIFF2025530773000036.tif196145TIFF2025530773000037.tif196146TIFF2025530773000038.tif196146TIFF2025530773000039.tif196145TIFF2025530773000040.tif196146TIFF2025530773000041.tif196147TIFF2025530773000042.tif196145TIFF2025530773000043.tif197146TIFF2025530773000044.tif196146TIFF2025530773000045.tif196146TIFF2025530773000046.tif196146TIFF2025530773000047.tif196146TIFF2025530773000048.tif196146TIFF2025530773000049.tif196146TIFF2025530773000050.tif196146TIFF2025530773000051.tif196146TIFF2025530773000052.tif196146TIFF2025530773000053.tif196146TIFF2025530773000054.tif196146TIFF2025530773000055.tif196145TIFF2025530773000056.tif196146TIFF2025530773000057.tif196147TIFF2025530773000058.tif196145TIFF2025530773000059.tif196145TIFF2025530773000060.tif196146TIFF2025530773000061.tif196146TIFF2025530773000062.tif196146TIFF2025530773000063.tif196146TIFF2025530773000064.tif196147TIFF2025530773000065.tif196146TIFF2025530773000066.tif197146TIFF2025530773000067.tif197145TIFF2025530773000068.tif196147TIFF2025530773000069.tif197145TIFF2025530773000070.tif196146TIFF2025530773000071.tif196145TIFF2025530773000072.tif196146TIFF2025530773000073.tif196147TIFF2025530773000074.tif196147TIFF2025530773000075.tif196145TIFF2025530773000076.tif196145TIFF2025530773000077.tif196146TIFF2025530773000078.tif196147TIFF2025530773000079.tif196145TIFF2025530773000080.tif196146TIFF2025530773000081.tif196145TIFF2025530773000082.tif196146TIFF2025530773000083.tif196146TIFF2025530773000084.tif196145TIFF2025530773000085.tif196147TIFF2025530773000086.tif196146TIFF2025530773000087.tif196145TIFF2025530773000088.tif196145TIFF2025530773000089.tif196147TIFF2025530773000090.tif196147TIFF2025530773000091.tif196145TIFF2025530773000092.tif196146TIFF2025530773000093.tif196145TIFF2025530773000094.tif196147TIFF2025530773000095.tif196147TIFF2025530773000096.tif196146TIFF2025530773000097.tif196146TIFF2025530773000098.tif196146TIFF2025530773000099.tif196146TIFF2025530773000100.tif196145TIFF2025530773000101.tif196145TIFF2025530773000102.tif196146TIFF2025530773000103.tif196145TIFF2025530773000104.tif196146TIFF2025530773000105.tif196145TIFF2025530773000106.tif196145TIFF2025530773000107.tif196147TIFF2025530773000108.tif196147TIFF2025530773000109.tif196146TIFF2025530773000110.tif196147TIFF2025530773000111.tif196146TIFF2025530773000112.tif196145TIFF2025530773000113.tif196146TIFF2025530773000114.tif196145TIFF2025530773000115.tif197145TIFF2025530773000116.tif196145TIFF2025530773000117.tif196146TIFF2025530773000118.tif196147TIFF2025530773000119.tif196146TIFF2025530773000120.tif196145TIFF2025530773000121.tif196147TIFF2025530773000122.tif196147TIFF2025530773000123.tif196147TIFF2025530773000124.tif196148TIFF2025530773000125.tif196146TIFF2025530773000126.tif196147TIFF2025530773000127.tif196147TIFF2025530773000128.tif196147TIFF2025530773000129.tif196146TIFF2025530773000130.tif196146TIFF2025530773000131.tif196146TIFF2025530773000132.tif196146TIFF2025530773000133.tif196146TIFF2025530773000134.tif197146TIFF2025530773000135.tif196145TIFF2025530773000136.tif196146TIFF2025530773000137.tif196146TIFF2025530773000138.tif196146TIFF2025530773000139.tif196146TIFF2025530773000140.tif196146TIFF2025530773000141.tif196145TIFF2025530773000142.tif196145TIFF2025530773000143.tif196146TIFF2025530773000144.tif196146TIFF2025530773000145.tif196146TIFF2025530773000146.tif196145TIFF2025530773000147.tif196145TIFF2025530773000148.tif196146TIFF2025530773000149.tif196145TIFF2025530773000150.tif196146TIFF2025530773000151.tif196146TIFF2025530773000152.tif196145TIFF2025530773000153.tif196146TIFF2025530773000154.tif196146TIFF2025530773000155.tif196146TIFF2025530773000156.tif196145TIFF2025530773000157.tif196148TIFF2025530773000158.tif196146TIFF2025530773000159.tif196146TIFF2025530773000160.tif196146TIFF2025530773000161.tif196147TIFF2025530773000162.tif197146TIFF2025530773000163.tif196146TIFF2025530773000164.tif196147TIFF2025530773000165.tif196146TIFF2025530773000166.tif196145TIFF2025530773000167.tif196147TIFF2025530773000168.tif196146TIFF2025530773000169.tif196145TIFF2025530773000170.tif197146TIFF2025530773000171.tif196148TIFF2025530773000172.tif196145TIFF2025530773000173.tif196148TIFF2025530773000174.tif196146TIFF2025530773000175.tif197148TIFF2025530773000176.tif196145TIFF2025530773000177.tif196146TIFF2025530773000178.tif196146TIFF2025530773000179.tif196147TIFF2025530773000180.tif196146TIFF2025530773000181.tif196146TIFF2025530773000182.tif196147TIFF2025530773000183.tif196146TIFF2025530773000184.tif196148TIFF2025530773000185.tif196145TIFF2025530773000186.tif196146TIFF2025530773000187.tif196148TIFF2025530773000188.tif196147TIFF2025530773000189.tif196146TIFF2025530773000190.tif196146TIFF2025530773000191.tif196146TIFF2025530773000192.tif196146TIFF2025530773000193.tif196146TIFF2025530773000194.tif196147TIFF2025530773000195.tif196147TIFF2025530773000196.tif196145TIFF2025530773000197.tif196145TIFF2025530773000198.tif196146TIFF2025530773000199.tif196145TIFF2025530773000200.tif196146TIFF2025530773000201.tif196146TIFF2025530773000202.tif197146TIFF2025530773000203.tif196146TIFF2025530773000204.tif196146TIFF2025530773000205.tif196146TIFF2025530773000206.tif196147TIFF2025530773000207.tif196146TIFF2025530773000208.tif196148TIFF2025530773000209.tif196147TIFF2025530773000210.tif196146TIFF2025530773000211.tif196147TIFF2025530773000212.tif196148TIFF2025530773000213.tif196147TIFF2025530773000214.tif196146TIFF2025530773000215.tif196148TIFF2025530773000216.tif196148TIFF2025530773000217.tif196148TIFF2025530773000218.tif196147TIFF2025530773000219.tif196145TIFF2025530773000220.tif196146TIFF2025530773000221.tif196145TIFF2025530773000222.tif196146TIFF2025530773000223.tif196146TIFF2025530773000224.tif196147TIFF2025530773000225.tif196146TIFF2025530773000226.tif196145TIFF2025530773000227.tif196147TIFF2025530773000228.tif196146TIFF2025530773000229.tif196146TIFF2025530773000230.tif196148TIFF2025530773000231.tif196148TIFF2025530773000232.tif196147TIFF2025530773000233.tif196147TIFF2025530773000234.tif196146TIFF2025530773000235.tif196147TIFF2025530773000236.tif196148TIFF2025530773000237.tif196146TIFF2025530773000238.tif196145TIFF2025530773000239.tif196147TIFF2025530773000240.tif196149TIFF2025530773000241.tif196146TIFF2025530773000242.tif196146TIFF2025530773000243.tif196147TIFF2025530773000244.tif196147TIFF2025530773000245.tif196146TIFF2025530773000246.tif196148TIFF2025530773000247.tif196146TIFF2025530773000248.tif196146TIFF2025530773000249.tif196145TIFF2025530773000250.tif196147TIFF2025530773000251.tif196147TIFF2025530773000252.tif196145TIFF2025530773000253.tif196147TIFF2025530773000254.tif196149TIFF2025530773000255.tif196147TIFF2025530773000256.tif196147TIFF2025530773000257.tif196146TIFF2025530773000258.tif196147TIFF2025530773000259.tif196146TIFF2025530773000260.tif196147TIFF2025530773000261.tif196147TIFF2025530773000262.tif196147TIFF2025530773000263.tif196147TIFF2025530773000264.tif196146TIFF2025530773000265.tif196147TIFF2025530773000266.tif197147TIFF2025530773000267.tif196148TIFF2025530773000268.tif196147TIFF2025530773000269.tif92145.

[0259]

Table 2

[0260]

Table 3

[0261]

Table 4

[0262]

Table 5

Claims

1. 1. A method of treating or preventing prurigo nodularis (PN) in a subject, comprising administering an anti-IL-31RA antibody to a subject having PN, wherein the subject exhibits activation of tumor necrosis factor (TNF) signaling in lesional skin cells compared to a reference level of activation of TNF signaling.

2. 1. A method of normalizing activation of tumor necrosis factor (TNF) signaling in a subject with PN, comprising administering an anti-IL-31RA antibody to the subject with PN, wherein the subject exhibits activated tumor necrosis factor (TNF) signaling in lesional skin cells compared to a reference level of activated TNF signaling, and wherein administration of the anti-IL-31RA antibody normalizes the activated TNF signaling.

3. The method of claim 2, wherein normalization is determined about 4 weeks, about 8 weeks, or about 12 weeks after administration of the anti-IL-31RA antibody.

4. 4. The method of any one of claims 1 to 3, wherein differential expression is determined by RT-qPCR, RT-PCR, RNA-seq, Northern blotting, serial analysis of gene expression (SAGE), or DNA or RNA microarray, or wherein differential expression is determined at the protein level by Western blotting, ELISA, surface plasmon resonance, or mass spectrometry.

5. The method of any one of claims 1 to 4, wherein said activation of TNF signaling in said lesional skin cells is increased compared to said reference level of activation.

6. The method of any one of claims 1 to 5, wherein the lesional skin cells are fibroblasts.

7. The method of any one of claims 1 to 6, wherein the reference level is an activation level that is the level of activation of TNF signaling in skin cells of a human who does not have PN.

8. The method of claim 7, wherein the skin cells of the human not having PN are fibroblasts.

9. The method of any one of claims 1 to 6, wherein the reference level is the level of activation of TNF signaling in non-lesional skin cells of the subject.

10. 1. A method of reducing inflammation in the skin of a subject with prurigo nodularis (PN), comprising: administering an anti-IL-31RA antibody to a subject with PN, thereby reducing inflammation associated with tumor necrosis factor (TNF) signaling in the skin. A method comprising:

11. 11. The method of claim 10, wherein TNF signaling in the subject's skin is overexpressed compared to a reference level of activation of the TNF signaling, and optionally, the TNF signaling is activated in fibroblasts.

12. 12. The method of claim 11, wherein the reference level is an activation level that is a level of activation of TNF signaling in skin cells of a human who does not have PN.

13. The method of claim 12, wherein the skin cells of the person without PN are fibroblasts.

14. 12. The method of claim 11, wherein the reference level is a level of activation of TNF signaling in non-lesional skin cells of the subject.

15. 15. The method of any one of claims 10 to 14, wherein the inflammation further involves IL-1 pathway signaling, IL-6 pathway signaling, TGFβ pathway signaling, or any combination thereof.

16. A method of treating or preventing prurigo nodularis (PN) in a subject, comprising administering an anti-IL-31RA antibody to a subject having PN, wherein administering the anti-IL-31RA antibody results in a decrease in tumor necrosis factor (TNF) pathway activation.

17. 17. The method of claim 16, wherein the reduction in TNF pathway activation occurs in lesional skin of the subject.

18. 18. The method of claim 16 or 17, wherein the reduction in TNF pathway activation occurs in fibroblasts of the subject.

19. A further outcome of said treatment is (a) a decrease in leukocyte migration or leukocyte cell movement; (b) inhibition of the STAT3 pathway; (c) inhibition of the STAT5b pathway; (d) downregulation of IL-1 or the IL-1 pathway; (e) downregulation of IL-6 or the IL-6 pathway; (f) downregulation of VEGF or the VEGF pathway; (g) a decrease in TGFB1 pathway activation, or (h) Combinations thereof The method according to any one of claims 16 to 18, wherein

20. 20. The method of claim 19, wherein (a) the decrease in leukocyte migration or leukocyte cell movement, (b) the inhibition of the STAT3 pathway, (c) the inhibition of the STAT5b pathway, (d) the downregulation of IL-1 or the IL-1 pathway, (e) the downregulation of IL-6 or the IL-6 pathway, (f) the downregulation of VEGF or the VEGF pathway, (g) the decrease in TGFB1 pathway activation, or (h) a combination thereof, is determined with respect to (i) a control sample obtained from one or more individuals without PN, or (ii) a biological sample obtained from the subject prior to administration of the anti-IL-31RA antibody.

21. 21. The method of claim 19 or 20, wherein (a) the reduction in leukocyte migration or leukocyte cell movement, (b) the inhibition of the STAT3 pathway, (c) the inhibition of the STAT5b pathway, (d) the downregulation of IL-1 or the IL-1 pathway, (e) the downregulation of IL-6 or the IL-6 pathway, (f) the downregulation of VEGF or the VEGF pathway, (g) the reduction in TGFB1 pathway activation, or (h) a combination thereof, is assessed about 4 weeks, about 8 weeks, or about 12 weeks after administration of the anti-IL-31RA antibody.

22. 22. The method of any one of claims 19-21, wherein (a) the decrease in leukocyte migration or leukocyte cell movement, (b) the inhibition of the STAT3 pathway, (c) the inhibition of the STAT5b pathway, (d) the downregulation of IL-1 or the IL-1 pathway, (e) the downregulation of IL-6 or the IL-6 pathway, (f) the downregulation of VEGF or the VEGF pathway, (g) the decrease in TGFB1 pathway activation, or (h) a combination thereof, is determined by mass spectrometry performed on one or more biological samples obtained from the subject.

23. 23. The method of claim 22, wherein the one or more biological samples are a plasma sample or a skin sample.

24. 24. The method of any one of claims 19-23, wherein the subject exhibits at least two, at least three, at least four, at least five, at least six, or all seven of: (a) the decrease in leukocyte migration or leukocyte cell movement; (b) the inhibition of the STAT3 pathway; (c) the inhibition of the STAT5b pathway; (d) the downregulation of IL-1 or the IL-1 pathway; (e) the downregulation of IL-6 or the IL-6 pathway; (f) the downregulation of VEGF or the VEGF pathway; and (g) the decrease in TGFB1 pathway activation.

25. A method for inactivating, reducing the activation of, or reducing the number of COL11A1+ fibroblasts in a subject with prurigo nodularis (PN), comprising administering an anti-IL-31RA antibody to the subject, wherein administering the anti-IL-31RA antibody results in the inactivation, reduction of the activation, or reduction in the number of COL11A1+ fibroblasts in the skin of the subject.

26. 26. The method of claim 25, wherein the COL11A1+ fibroblasts are found within the papillary dermis.

27. 1. A method of reducing TGFβ expression in at least one cell type in a subject with prurigo nodularis (PN), comprising administering to the subject an anti-IL-31RA antibody, wherein administering the anti-IL-31RA antibody results in a reduction of TGFβ expression in at least one cell type in the skin of the subject.

28. 28. The method of claim 27, wherein the at least one cell type comprises fibroblasts, endothelial cells, pericytes, neurons, or any combination thereof.

29. 29. The method of claim 27 or 28, wherein the reduction in TGFβ expression comprises a reduction in expression of TGFB1, TGFB2, TGFB3, or any combination thereof.

30. 1. A method for reducing the expression of at least one inflammatory gene expressed by keratinocytes in a subject with prurigo nodularis (PN), comprising administering an anti-IL-31RA antibody to the subject, wherein administering the anti-IL-31RA antibody results in a reduction of at least one inflammatory gene expressed by keratinocytes in the skin of the subject.

31. 31. The method of claim 30, wherein the at least one inflammatory gene is selected from KRT6, KRT16, KRT17, S100A8, S100A9, and any combination thereof.

32. 32. The method of claim 30 or 31, wherein the keratinocytes express Th2 cytokines.

33. The method of any one of claims 30 to 32, wherein administering the anti-IL-31RA antibody results in a decrease in reactive oxygen species and / or cellular stress to which the keratinocytes are exposed.

34. A method for reducing infiltration of at least one type of immune cell in skin lesions of a subject having prurigo nodularis (PN), comprising administering to the subject an anti-IL-31RA antibody, wherein administering the anti-IL-31RA antibody results in a reduction in infiltration of at least one type of immune cell in at least one lesion in the skin of the subject.

35. 35. The method of claim 34, wherein the at least one type of immune cell comprises a macrophage.

36. 36. The method of claim 35, wherein the macrophages are lipid-associated macrophages characterized by expression of APOE and TREM2.

37. The at least one type of immune cell is a T cell, a NK cell, a CD8 ＋ The method of any one of claims 34 to 36, comprising cells, Tregs, and any combination thereof.

38. 38. The method of any one of claims 34-37, comprising administering the anti-IL-31RA antibody, which results in a decrease in expression of ICAM1, E-selectin (SELE), IL6 CCL2, CCL3, CCL4, CCL13, CCL18, CXCL2, CXCL12, and any combination thereof, in at least one cell type in the lesion.

39. 39. The method of claim 38, wherein the at least one cell type in the lesion comprises myeloid cells, pericytes, endothelial cells, and any combination thereof.

40. The method of any one of claims 1 to 39, wherein the anti-IL-31RA antibody is administered subcutaneously.

41. 41. The method of any one of claims 1 to 40, wherein the anti-IL-31RA antibody is administered once per week, once per two weeks, once per three weeks, once per four weeks, once per five weeks, once per six weeks, once per seven weeks, or once per eight weeks.

42. 43. The method of any one of claims 1-42, wherein the anti-IL-31RA antibody is administered at a dose of about 0.01 mg / kg to about 0.1 mg / kg, about 0.1 mg / kg to about 0.5 mg / kg, about 0.5 mg / kg to about 1.5 mg / kg, about 1.5 mg / kg to about 2.5 mg / kg, or about 2.5 mg / kg to about 10 mg / kg.

43. 42. The method of any one of claims 1-41, wherein the anti-IL-31RA antibody is administered at a dose of about 10 mg, about 15 mg, about 20 mg, about 25 mg, about 30 mg, about 35 mg, about 40 mg, about 45 mg, about 50 mg, about 55 mg, about 60 mg, about 65 mg, about 70 mg, about 75 mg, about 80 mg, about 85 mg, or about 90 mg.

44. The method of any one of claims 1 to 43, wherein the anti-IL-31RA antibody is administered according to a flat dosing regimen.

45. The method of any one of claims 1 to 43, wherein the anti-IL-31RA antibody is administered according to a loading dose regimen.

46. The method of any one of claims 1 to 45, wherein the anti-IL-31RA antibody comprises a heavy chain variable region comprising HCDR1 comprising SEQ ID NO:8, HCDR2 comprising SEQ ID NO:9, and HCDR3 comprising SEQ ID NO:10, and a light chain variable region comprising LCDR1 comprising SEQ ID NO:12, LCDR2 comprising SEQ ID NO:13, and LCDR3 comprising SEQ ID NO:

14.

47. The method of any one of claims 1 to 46, wherein the anti-IL-31RA antibody is nemolizumab or a fragment or variant thereof.

48. The method of claim 47, wherein the anti-IL-31RA antibody is nemolizumab.

Images