Treatment of prurigo nodularis

Abstract

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

Description

[0001] Related applications

[0002] This application claims priority under 35 U.S.C. §119(e) to (i) U.S. Provisional Application No. 63 / 403,483, filed on September 2, 2022, and (ii) U.S. Provisional Application No. 63 / 534,558, filed on August 24, 2023, the entire contents of which are incorporated herein by reference. Technical Field

[0003] Described herein are methods for treating and preventing prurigo nodularis (PN), antibodies and pharmaceutical compositions for treating or preventing PN, and the use of anti-IL-31RA antibodies (e.g., nemolizumab) in the manufacture of medicaments for treating or preventing PN. Also described herein are biomarkers for PN and methods for altering or improving these biomarkers by treatment with antibodies that bind to IL-31RA (e.g., nemolizumab). Background Art

[0004] The following discussion is provided to assist the reader in understanding the present disclosure and is not admitted to describe or constitute prior art thereto.

[0005] Prurigo chronicus (CP) is a skin condition caused by the sensitivity of neurons to itching and the development of an itch-scratch cycle. Prurigo nodularis (PN), a subtype of CP, is a skin condition in which hard, itchy bumps (nodules) develop on the skin. The itching (pruritus) can be intense, causing people to scratch themselves to the point of bleeding or pain. Scratching can lead to more skin lesions. Itching can be made worse by heat, sweating, or irritation from clothing. In some cases, people with PN have a history of other medical conditions, including eczema (atopic dermatitis), diabetes, lymphoma, HIV infection, severe anemia, or kidney disease.

[0006] The exact cause of PN is unknown, and the diagnosis of the disease is based on observation of signs such as extremely itchy skin and the formation of nodules. In some cases, a skin biopsy is used to confirm the diagnosis. Current treatments may include corticosteroid creams, oral medications, cryotherapy, or photochemotherapy.

[0007] There is still a need to treat PN and determine whether the patient is likely to respond or is responding to such treatment. Summary of the Invention

[0008] Described herein are treatments and preventions for prurigo nodularis (PN) that achieve specific therapeutic outcomes, such as reduction in TNF signaling. Generally, the treatments and preventions comprise administering an anti-IL-31RA antibody (e.g., nemolizumab) to a subject suffering from PN. Also described herein are biomarkers for PN and methods of using the disclosed biomarkers to determine whether a subject is responsive to treatment.

[0009] In a first aspect, the present disclosure provides 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 diseased skin cells compared to a reference level of TNF activation.

[0010] In a second aspect, the present disclosure provides a method for normalizing tumor necrosis factor (TNF) gene expression in a subject with PN, comprising administering an anti-IL-31RA antibody to a subject with PN, wherein the subject exhibits activation of TNF in diseased 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 at about 4 weeks, about 8 weeks, or about 12 weeks after administration of the anti-IL-31RA antibody.

[0011] In some embodiments, differential expression is determined by RT-qPCR, RT-PCR, RNA-seq, Northern blot, serial analysis of gene expression (SAGE), or DNA or RNA microarray; or wherein differential expression is determined at the protein level by Western blot, ELISA, surface plasmon resonance, or mass spectrometry.

[0012] In some embodiments, activation of TNF signaling is higher in diseased skin cells compared to a reference level. In some embodiments, the diseased skin cells are fibroblasts.

[0013] In some embodiments, the reference level is an activation level of TNF signaling in skin cells of a person not suffering from PN. In some embodiments, the skin cells of a person not suffering from PN are fibroblasts.

[0014] In some embodiments, the reference level is an activation level, which is the activation level of TNF signaling in non-lesional skin cells of the subject.

[0015] In a third aspect, the present disclosure provides a method of reducing skin inflammation in a subject with prurigo nodularis (PN), comprising administering an anti-IL-31RA antibody to the subject with PN, thereby reducing inflammation in the skin involving tumor necrosis factor (TNF) signaling.

[0016] In some embodiments, TNF signaling is overexpressed in the skin of the subject relative to a reference level of TNF signaling activation, optionally wherein the TNF gene is overexpressed by fibroblasts.

[0017] In some embodiments, the reference level is an activation level of TNF signaling in skin cells of a person not suffering from PN. In some embodiments, the skin cells of a person not suffering from PN are fibroblasts.

[0018] In some embodiments, the reference level is an activation level, which is the activation level of TNF signaling in non-lesional skin cells of the subject.

[0019] In some embodiments, inflammation further involves IL-1 pathway signaling, IL-6 pathway signaling, TGFβ pathway signaling, or any combination thereof.

[0020] In a fourth aspect, the present disclosure provides a method for treating or preventing prurigo nodularis (PN) in a subject, comprising administering an anti-IL-31RA antibody to a subject suffering from PN, wherein treatment with the anti-IL-31RA antibody results in a decrease in activation of the tumor necrosis factor (TNF) pathway. In some embodiments, the decrease in TNF pathway activation occurs in the subject's lesional skin. In some embodiments, the decrease in TNF pathway activation occurs in the subject's fibroblasts.

[0021] In some embodiments, treatment further results in:

[0022] (a) Decreased leukocyte migration or cell movement of leukocytes;

[0023] (b) inhibiting the STAT3 pathway;

[0024] (c) inhibiting the STAT5b pathway;

[0025] (d) downregulation of IL-1 or IL-1 pathway;

[0026] (e) downregulation of IL-6 or IL-6 pathway;

[0027] (f) downregulation of VEGF or VEGF pathway;

[0028] (g) decreased activation of the TGFB1 pathway, or

[0029] (h) combinations thereof.

[0030] In some embodiments, (a) leukocyte migration or leukocyte cell motility is reduced; (b) the STAT3 pathway is inhibited; (c) the STAT5b pathway is inhibited; (d) IL-1 or the IL-1 pathway is downregulated; (e) IL-6 or the IL-6 pathway is downregulated; (f) VEGF or the VEGF pathway is downregulated; (g) TGFB1 pathway activation is reduced, 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.

[0031] In some embodiments, (a) decreased leukocyte migration or leukocyte cell motility; (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 activation of the TGFB1 pathway, or (h) a combination thereof is assessed after about 4 weeks, about 8 weeks, or about 12 weeks following administration of the anti-IL-31RA antibody.

[0032] In some embodiments, (a) leukocyte migration or leukocyte cell motility is reduced; (b) the STAT3 pathway is inhibited; (c) the STAT5b pathway is inhibited; (d) IL-1 or the IL-1 pathway is downregulated; (e) IL-6 or the IL-6 pathway is downregulated; (f) VEGF or the VEGF pathway is downregulated; (g) TGFB1 pathway activation is reduced, or (h) a combination thereof is determined by mass spectrometry analysis of one or more biological samples obtained from the subject.

[0033] In some embodiments, the one or more biological samples is a plasma sample or a skin sample.

[0034] 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 cell motility 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; and (g) decreased activation of the TGFB1 pathway.

[0035] In a fifth aspect, the present disclosure provides a method for inactivating, reducing activation, or decreasing the number of COL11A1+ fibroblasts in a subject with prurigo nodularis (PN), comprising administering an anti-IL-31RA antibody to the subject, wherein administration of the anti-IL-31RA antibody results in inactivation, reducing activation, or decreasing the number of COL11A1+ fibroblasts in the subject's skin. In some embodiments, the COL11A1+ fibroblasts are present in the papillary dermis.

[0036] In a sixth aspect, the present disclosure provides a method for reducing TGFβ expression in at least one cell type in a subject suffering from PN, comprising administering an anti-IL-31RA antibody to the subject, wherein administration of 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 the expression of TGFB1, TGFB2, TGFB3, or any combination thereof.

[0037] 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 suffering from PN, comprising administering an anti-IL-31RA antibody to the subject, wherein administration of the anti-IL-31RA antibody results in a decrease 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, keratinocytes express Th2 cytokines. In some embodiments, administration of the anti-IL-31RA antibody results in a decrease in reactive oxygen species and / or cellular stress to which keratinocytes are exposed.

[0038] In an eighth aspect, the present disclosure provides a method for reducing the infiltration of at least one type of immune cell in a skin lesion of a subject suffering from PN, comprising administering an anti-IL-31RA antibody to the subject, wherein administration of the anti-IL-31RA antibody results in a reduction in the infiltration of at least one type of immune cell in at least one lesion in the subject's skin. In some embodiments, 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, at least one type of immune cell comprises a T cell, a NK cell, a CD8 T ... +In some embodiments, the administration of an anti-IL-31RA antibody results in a decrease in the expression of ICAM1, E-selectin (SELE), IL6CCL2, 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.

[0039] In some embodiments of any of the aforementioned aspects, the anti-IL-31RA antibody is administered subcutaneously.

[0040] In some embodiments of any of the aforementioned aspects, the anti-IL-31RA antibody is administered 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.

[0041] 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.

[0042] In some embodiments of any of the aforementioned aspects, the anti-IL-31RA antibody is administered according to a steady-state dosing regimen. Alternatively, in some embodiments, the anti-IL-31RA antibody is administered according to a loading dose regimen.

[0043] In some embodiments of any of the foregoing aspects, the anti-IL-31RA antibody comprises a heavy chain variable region comprising: a HCDR1 comprising SEQ ID NO: 8, a HCDR2 comprising SEQ ID NO: 9, and a HCDR3 comprising SEQ ID NO: 10; and a light chain variable region comprising: a LCDR1 comprising SEQ ID NO: 12, a LCDR2 comprising SEQ ID NO: 13, and a 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.

[0044] The foregoing general description and the following detailed description are exemplary and explanatory and are intended to provide further explanation of the disclosure as claimed. Other objects, advantages and novel features will be apparent to those skilled in the art from the following brief description of the drawings and embodiments of the disclosure. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 A-1E shows that prurigo nodularis (PN) is characterized by immune activation and abnormal keratinocyte differentiation. The number of differentially expressed genes (DEGs) in PN lesional skin relative to non-lesional skin (n=62, FC>=2 or FC<=-2, FDR<=0.1) (A). GO categories enriched in PN lesional skin (B). A literature-based gene network obtained from the top 1,000 DEGs in PN skin was generated using the Genomatix Pathway System (GePS, Genomatix.de). The image shows the top 50 best-connected genes co-cited in the same sentence associated with the functional word (most relevant gene / interaction) in the PubMed abstract. Orange represents genes that are upregulated in PN lesional skin relative to non-lesional skin, while green represents downregulated genes. Key nodes include the proliferation marker Ki67 (MKI67), IL-1 family members IL36G and IL1A, and CXCL8 and CDKN1A (C). The number of genes in the modules and intracluster correlations identified by weighted co-expression gene network analysis (WCGNA) of non-lesional and lesional PN skin (D). Functions enriched in the key co-expression modules (modules 5, 6, and 8) of PN skin (E).

[0046] Figure 2 A-2C shows enriched transcriptomic cell features and overlap with psoriasis and AD. Cell type inference analysis was performed on non-lesional (NL) and lesional (L) PN skin samples using xCell. Enriched cell features are shown in red, while underrepresented cell features are shown in blue. The bar chart on the left shows 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 with DEGs in psoriasis (Pso) and atopic dermatitis (AD) regarding increased and decreased DEGs (B). Correlation analysis between effect sizes in PN lesions and effect sizes in psoriasis (Pso) and atopic dermatitis (AD). Spearman rank correlation is included. Genes significant on the x-axis, y-axis, and both axes are colored red / blue / purple, respectively (C).

[0047] Figure 3A-3B shows transcriptome changes associated with the anti-IL31R inhibitor nemolizumab. Principal component analysis (PCA) of transcriptome data from PN biopsies before and after a 12-week prospective placebo-controlled, double-blind clinical trial with the anti-IL-31R inhibitor nemolizumab. Different colors represent different treatment groups, with lesion samples shown as triangles and non-lesion skin shown as circles (A). A heat map shows 2-way clusters of all samples (using genes differentially expressed between non-lesion and lesion skin at baseline) (B).

[0048] Figure 4 A-4C shows the effect of nemolizumab on PN-related transcriptome changes. A 3-way Venn diagram of increased and decreased DEGs in PN skin and overlap with DEGs in the nemolizumab and placebo groups (compared to baseline) (A). Correlation analysis between different groups (PN baseline vs. placebo and nemolizumab DEGs) (Spearman rank correlation) (B).

[0049] Figure 5 A-5D shows that nemolizumab treatment normalizes epidermal differentiation and reduces IL-31 / Th2 responses in PN skin. Compared with placebo, nemolizumab treatment reduced IL-31 and IL-13 responses in PN skin, while reducing the expression of IL-17A responsive genes (A). Nemolizumab treatment was accompanied by a reduction in the transcriptomic signatures of Th1, Th17, and Th2 cells (B). Cross-comparison of the transcriptomic responses of PN skin with cell signatures obtained from single-cell data of healthy epidermis showed that nemolizumab treatment normalized the expression of epidermal genes associated with the epidermal differentiation layer (KRT10+), corresponding to the normalization of epidermal differentiation. The different nomenclature corresponds to different layers of the epidermis, with "basal" corresponding to KRT5 + Basal cells, KRT10 + “Differentiation” corresponds to the spinous layer, and “keratinization” corresponds to the granular layer (FLG + )(C). Among genes downregulated by nemolizumab, normalization of transcription factor binding sites (TFBS) of PN-associated DEGs was greater compared with placebo (5D).

[0050] Figure 6A-6B shows that the decrease in pruritus scores driven by nemolizumab was accompanied by tighter clustering of PN samples on PCA analysis after 12 weeks of treatment. The Peak Pruritus Numeric Rating Scale (PP-NRS) was superimposed on transcriptome data from PN lesion skin and assessed using PCA analysis. The nemolizumab group is shown as large dots, while the placebo group is shown as small dots. Baseline (top) and week 12 of treatment (bottom) are shown (A). Nemolizumab treatment resulted in tighter clustering of PN samples on PCA analysis compared to biopsy samples from the placebo-treated group (B).

[0051] Figure 7 The cytokine signature of PN in placebo- and nemolizumab-treated skin is shown. A literature-based network was generated using the Genomatix Pathway System (GePS, genomatix.de) using function word co-citation filters and showing key cytokines as major nodes in PN skin.

[0052] Figure 8 Shown are the expressions of selected TFs in PN skin before and after nemolizumab treatment.

[0053] Figure 9 Shown are the distances between PCA components in PN before and after nemolizumab treatment.

[0054] Figure 10 Shown are clinical scores of patients selected for mass spectrometry analysis. Left panel: PNR score at baseline. Right panel: Change in NRS at week 12.

[0055] Figure 11 Enriched canonical pathways from mass spectra are shown. Enriched canonical pathways are ranked according to: right) z-score only, left) z-score and p-value (p<0.05).

[0056] Figure 12 Upstream regulator analysis from mass spectrometry is shown. Above: Upstream regulators are ranked according to: right) z score only, left) z score and p value (p<0.05).

[0057] Figure 13 The enriched biological functions identified by mass spectrometry are shown. The enriched biological function ontologies are ranked according to z-score and p-value (p<0.05).

[0058] Figure 14 A heatmap showing hierarchical clustering of scRNAseq from samples obtained from healthy skin (H), lesional PN (LPN), and nonlesional PN (NPN) skin samples. The Y-axis represents single cells, while the X-axis represents the genes expressed by them. The right-hand side of the heatmap indicates the cell of origin and cell type annotation.

[0059] Figure 15 Figure 3. UMAP representation of scRNAseq-based fibroblast clusters. Clusters annotated with disease type, fibroblast subtype, cell differentiation trajectory, and pseudotime.

[0060] Figure 16 Figure 3. Graphical summary of DEGs in lesional PN fibroblasts using Ingenuity pathway analysis. Orange indicates pathways, regulators, or processes that are activated in lesional PN skin compared to healthy skin, while blue indicates inactivation.

[0061] Figure 17 Figure 3. Cell-cell communication network in PN lesion skin. Data derived from scRNA-seq data. The higher the number of interactions / interaction strength, the thicker the line connecting the two cell types.

[0062] Figure 18 A graphical summary of the results obtained from Ingenuity pathway analysis of bulk RNAseq data from a Phase II study of nemolizumab is shown. Orange indicates pathways, regulators, or processes that are activated in lesional PN skin from nemolizumab-treated patients compared to healthy skin, while blue indicates inactivation.

[0063] Figure 19 Figure 19E shows the cell types observed in PN skin and their spatial locations. Figure A shows a UMAP plot showing 72,782 cells colored by cell type. Figure B shows a UMAP plot showing cells colored by skin condition (H: healthy control; NPN: non-lesional sample from PN patient; LPN: lesional sample from PN patient). Figure C shows a bar graph showing the abundance composition of each cell type across skin conditions in scRNA-seq. Figure 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. Figure E shows a spatial plot showing the predicted score for each cell type. The coordinates of the dot correspond to the location in the tissue.

[0064] Figure 20 Figure 20C shows the spatial location of the major cell types detected in PN skin. Panel A shows hematoxylin and eosin (H&E) staining of a PN skin biopsy used for spatial sequencing. Panel B shows a scatter plot showing the cell type composition of each point in the spatial-seq sample. Each point is represented by 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.

[0065] Figure 21A-21J shows the identification of fibroblast subtypes. Panel A shows a UMAP showing 15,084 fibroblasts colored by subtype. Panel B shows a UMAP plot showing fibroblasts colored by skin condition. Panel C shows a bar graph showing the abundance composition of each fibroblast subtype across skin conditions. Panel D shows a dot plot showing 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 showing the extracellular matrix module score in fibroblast subtypes divided by skin condition. Panel F shows a dot plot showing the upstream regulators of DEGs identified in COL11A+FB 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 graph showing the top 10 pathways enriched using upregulated DEGs identified in COL11A+FB by comparing LPN to healthy cells. Panel H shows a dot plot showing the expression of all collagen genes in 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 tissue. Panel J shows a violin plot showing the extracellular matrix module score in fibroblast subtypes by healthy, PN, and AD skin conditions.

[0066] Figure 22 Identification of fibroblast subtypes is shown. This figure provides immunohistochemical staining of SFRP4, SFRP2, and RAMP1 in PN and healthy tissue.

[0067] Figure 23A-23H shows identification of endothelial subtypes. Panel A shows a UMAP plot showing 3,840 endothelial cells colored by subcluster. Panel B shows a dot plot showing the top marker genes for each endothelial subcluster. The color scale indicates the scaled expression of each gene. The size of the dot indicates the percentage of cells expressing the gene of interest. Panel C shows a UMAP plot showing endothelial cells colored by skin condition. Panel D shows a bar plot showing the abundance composition of each endothelial subcluster across skin conditions. Panel E shows a dot plot showing the upstream regulators of the cluster marker genes for endothelial subcluster 2. The color scale indicates the -log10 (p-value) from the enrichment analysis. The size of the dot indicates the number of differentially expressed genes downstream of the upstream regulator. Panel F shows a bar plot showing the top 10 pathways enriched using the cluster marker genes for endothelial subcluster 2. Panel G shows a dot plot showing the upstream regulators of the cluster marker genes for endothelial subcluster 5. The color scale represents -log10 (p-value) from the enrichment analysis. The size of the dots represents the number of differentially expressed genes downstream of the upstream regulator. Panel H shows a bar graph showing the top 10 pathways enriched using cluster marker genes for endothelial subcluster 5.

[0068] Figure 24 A-24J shows identification of pericyte subtypes. Panel A shows a UMAP plot showing 3,052 pericytes colored by subcluster. Panel B shows a UMAP plot showing pericytes colored by skin condition. Panel C shows a bar graph showing the abundance composition of each pericyte subcluster across skin conditions. Panel D shows a dot plot showing the top marker genes for each pericyte subcluster. The color scale indicates the scaled expression of each gene. The size of the dot indicates the percentage of cells expressing the gene of interest. Panel E shows a violin plot showing the extracellular matrix module score in pericyte subclusters by skin condition. Panel F shows a dot plot showing the expression of all collagen genes in pericyte subclusters. The color scale indicates the scaled expression of each gene. The size of the dot indicates the percentage of cells expressing the gene of interest. Panel G shows a dot plot showing the upstream regulators of cluster marker genes for pericyte subcluster 3. The color scale indicates the -log10 (p-value) from the enrichment analysis. The size of the dot indicates 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 -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 I shows a bar chart showing the top 10 pathways enriched using cluster marker genes for pericyte subcluster 3. Panel J shows a bar chart showing the top 10 pathways enriched using cluster marker genes for pericyte subcluster 7.

[0069] Figure 25A-25F shows identification of keratinocyte subtypes. Panel A shows a UMAP showing 40,277 keratinocytes colored by subtype. Panel B shows a UMAP plot showing keratinocytes colored by skin condition. Panel C shows a bar graph showing the abundance composition of each keratinocyte subtype across skin conditions. Panel D shows a dot plot showing 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 graph showing the top 10 pathways enriched using cluster marker genes for inflammatory keratinocytes. Panel F shows a dot plot showing 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.

[0070] Figure 26 A-26J shows identification of myeloid and T cell subtypes. Panel A shows a UMAP plot showing 2,130 myeloid cells colored by subtype. Panel B shows a UMAP plot showing myeloid cells colored by skin condition. Panel C shows a dot plot showing the top marker genes for each myeloid subtype. The color scale indicates the scaled expression of each gene. The size of the dot indicates 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 showing 5,817 T cells colored by subtype. Panel G shows a UMAP plot showing T cells colored by skin condition. Panel H shows a dot plot showing the top marker genes for each T cell subtype. The color scale indicates the scaled expression of each gene. The size of the dot indicates the percentage of cells expressing the gene of interest. Panel I shows a bar plot showing the abundance composition of each T cell subtype across skin conditions. Panel J shows immunohistochemical staining of CD8 and CD4 in PN skin tissue.

[0071] Figure 27A-27F shows cell-cell interactions revealed by ligand-receptor analysis. Panel A shows a heat map showing 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 showing 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 showing 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 a dot plot showing 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 in the cell, while the dot size 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 heat map of the TGFb signaling pathway network in healthy, NPN, and LPN skin, outlining source (sender) and target cells (receivers, mediators, influencers).

[0072] Figure 28 A-28F shows a comparison of keratinocyte subtypes in atopic dermatitis (AD) and prurigo nodularis (PN). Panel A shows a UMAP plot showing 68,451 keratinocytes colored by skin state. Panel B shows a UMAP plot showing keratinocytes colored by cluster. Panel C shows a UMAP plot showing keratinocytes colored by keratinocyte subtype. Panel D shows a dot plot 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 graph showing the number of keratinocytes in each cluster for each skin state. Panel F shows the enriched GO BP processes in AD versus PN skin for each keratinocyte subtype.

[0073] Figure 29 A-29H shows a comparison of T cell and myeloid subtypes in atopic dermatitis (AD) and prurigo nodularis (PN). Panel A shows a UMAP plot showing 10,389 T cells colored by cluster. Panel B shows a UMAP plot showing coloring by T cell subtype. Panel C shows a UMAP plot showing T cells colored by disease state. Panel D shows a bar chart showing the abundance and number of each T cell subtype across each disease state. Panel E shows a UMAP plot showing 5,752 myeloid cells colored by cluster. Panel F shows a UMAP plot showing myeloid cells colored by myeloid subtype. Panel G shows a UMAP plot showing myeloid colored by disease state. Panel H shows a bar chart showing the abundance and number of each myeloid subtype across each disease state.

[0074] Figure 30 A-30B shows the identification of myeloid and T cell subtypes. Panel A shows a UMAP graph of the expression of specific immune cells in lesional AD (LAD) and lesional PN skin (LPN). Panel B shows a violin plot showing the expression of specific immune genes in each T cell subset.

[0075] Figure 31 A-31H shows the effect of nemolizumab in keratinocytes and fibroblast subtypes. Figure A shows a UMAP diagram, which shows the expression of IL31RA mainly in keratinocytes and fibroblasts. Figure B shows a UMAP diagram, which shows the expression of OSMR mainly in keratinocytes, fibroblasts, endothelial cells and pericytes. Figure C shows a UMAP, which shows the superposition of DEGs that increase with nemolizumab treatment (nemolizumab positive). Figure D shows a UMAP, which shows the superposition of DEGs that decrease with nemolizumab treatment (nemolizumab negative). Figure E shows a violin diagram, which shows the gene module score of nemolizumab upregulation in fibroblast subtypes divided by skin condition. Figure F shows a violin diagram, which shows the gene module score of nemolizumab downregulation in fibroblast subtypes divided by skin condition. Panel G shows a violin plot showing the module scores of genes upregulated by nemolizumab in keratinocyte subtypes according to skin condition. Panel H shows a violin plot showing the module scores of genes downregulated by nemolizumab in keratinocyte subtypes according to skin condition.

[0076] Figure 32 A-32B shows the effects of nemolizumab in major cell types from scRNA-seq. Panel A shows a violin plot showing the module scores of genes upregulated by nemolizumab across all cell types by skin condition. Panel A shows a violin plot showing the module scores of genes downregulated by nemolizumab across all cell types by skin condition. DETAILED DESCRIPTION

[0077] This article describes the treatment and prevention of prurigo nodularis (PN) using anti-IL-31RA antibodies (e.g., nemolizumab), as well as previously unknown biomarkers and gene signatures associated with PN. The disclosed biomarkers include differentially expressed genes (DEGs), PN-specific gene ontologies (GOs), and other inflammatory and hyperproliferative markers that 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 that were previously unknown or unattainable with conventional PN treatments (e.g., normalizing DEGs, normalizing epidermal hyperproliferation, normalizing epidermal differentiation, and / or reducing inflammatory responses in the skin).

[0078] In addition, histological analysis of PN nodules described herein revealed epidermal dysregulation (excessive orthokeratosis and hyperplasia), dermal fibrosis and inflammatory cell infiltration. Therefore, the present disclosure also provides the role of fibroblasts in the pathogenesis of PN and the related clinical changes, results and endpoints achieved by nemolizumab.

[0079] I. Definition

[0080] 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.

[0081] Unless otherwise defined, technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art. Unless otherwise specified, materials and / or methods known to one of ordinary skill in the art can be used to perform the methods described herein based on the guidance provided herein.

[0082] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Reference to an item in the singular is not intended to mean "one and only one," but rather "one or more," unless explicitly stated otherwise.

[0083] As used herein, when used with a numerical value, "about" means the numerical value and ±10% of the numerical value. For example, "about 10" should be understood as "10" and "9-11".

[0084] Additionally, as used herein, "and / or" refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of a combination ("or") when interpreted in the alternative.

[0085] As used herein, a phrase in the form “A / B” or “A and / or B” means (A), (B), or (A and B); a phrase in 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).

[0086] As used herein, the phrase "therapeutically effective amount" with respect to an anti-IL31R antibody (e.g., nemolizumab) means an antibody dosage that provides a specific pharmacological effect by administering the drug to a subject in need of such treatment. A therapeutically effective amount can effectively reduce, improve, or eliminate pruritus, scratching, and / or lesion or nodule formation and / or improve the quality of life of a subject suffering from PN. It should be emphasized that a therapeutically effective amount of an anti-IL31R antibody (e.g., nemolizumab) may not always be effective in treating PN in each individual subject, even if such dosage is considered a therapeutically effective amount by a person skilled in the art. Those skilled in the art can adjust the amount that is considered a therapeutically effective amount according to standard procedures, based on the needs of treating a particular subject. A therapeutically effective amount can vary based on, for example, the age and weight of the subject, and / or the overall health of the subject, and / or the severity of the subject's PN.

[0087] As used herein, the terms "treat," "treatment," and "treating" with respect to PN refer to reducing, ameliorating, or eliminating itching, scratching, and / or lesion or nodule formation and / or improving the quality of life of a subject suffering from PN.

[0088] As used herein, the terms "prevent" or "preventing" with respect to PN refer to excluding or reducing the risk of developing lesions or nodules or preventing the development of a disclosed biomarker signature associated with PN. Prevention can also refer to preventing the onset or recurrence of PN once the initial episode has been treated or cured.

[0089] The terms "individual," "subject," and "patient" are used interchangeably herein and refer to any individual mammalian subject, such as bovine, canine, feline, equine, or human. In certain embodiments, the subject, individual, or patient is human.

[0090] II. Prurigo nodularis (PN) and biomarkers

[0091] Prurigo nodularis (or "PN") is a skin condition in which hard, itchy bumps (nodules) form on the skin. The itching (pruritus) can be intense, causing people to scratch themselves to the point of bleeding or pain. Scratching can cause more skin lesions to develop. Heat, sweating, or irritation from clothing can make the itching worse. In some cases, people with PN have a history of other medical conditions, including eczema (atopic dermatitis), diabetes, lymphoma, HIV infection, severe anemia, or kidney disease. The exact cause of PN was not previously well understood. It is thought that nodules are more likely to form when the skin is scratched or irritated in some way. Therefore, the act of a person scratching their skin can cause the nodules to form. However, the reason why the skin becomes so itchy in the first place is unclear.

[0092] About 50% of people with PN have a history of atopy. The main symptom of PN is the formation of hard, very itchy bumps (nodules) on the skin. The nodules can range in size from very small to about half an inch in diameter. The nodules usually have rough, dry tops and can range in number from a few to hundreds. Nodules most commonly form on the outside of the arms, shoulders, and legs. Nodules can also form on the neck and trunk, and rarely on the face and palms. They may be lighter or darker in color than the surrounding skin. Scars may appear after the nodules begin to heal. Symptoms of PN can begin at any age but are most common in adults after the age of 50. People with PN may become very concerned about the appearance of the nodules, and the intensely itchy skin may interfere with sleep or daily activities. This can lead to stress and depression in people with PN.

[0093] Pruritus refers to itchy skin and / or itch. Pruritus can be caused by PN or other diseases or conditions (such as dry skin). In some cases, pruritus involves general itching of the skin all over the body. In some cases, pruritus is confined to specific areas of the body, such as on the arms or legs. Pruritus can be chronic or acute. Symptoms of pruritus include but are not limited to skin excoriation, redness, lumps, spots, blisters, dry skin, cracked skin and leathery or scaly texture of the skin. In some cases, pruritus does not cause detectable skin changes. Behavioral responses to pruritus include but are not limited to skin scratching and / or skin massage. In some cases, skin scratching can result in excoriation ranging from mild to severe. In some cases, patients with pruritus avoid scratching and / or massaging the skin. Traditional treatments for PN include but are not limited to skin moisturizers, topical emollients, antihistamines such as diphenhydramine, topical corticosteroids, topical calcineurin inhibitors and light therapy. Use narrowband UVB therapy and systemic immunosuppressive drugs such as cyclosporine or methotrexate.

[0094] The present disclosure elucidates for the first time the underlying gene expression patterns associated with PN and can be used to diagnose PN, identify subjects who may respond to treatment (such as treatment with anti-IL-31RA antibodies), and determine whether a subject has responded appropriately to treatment.

[0095] Specifically, the present disclosure shows that subjects with PN can differentially express at least 5,934 genes (referred to as differentially expressed genes or DEGs), which are shown in Figure 1A and Table 1 below. Such differential gene expression can be observed in the skin of a subject, and particularly in a skin sample comprising or consisting of a nodule or lesion. In some embodiments, 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 850, 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 at most 2500, 3000, 3500, 4000, 4500, 5000, 5500 or about 6000 disclosed DEGs can be differentially expressed in subjects with PN. Of these DEGs, 2,060 may be increased (i.e., overexpressed) and 3,874 may be reduced (i.e., underexpressed). The genes that may be most increased include:

[0096] ●KRT6C 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 the expression level in a sample (e.g., a skin sample) from an individual without PN.

[0097] DEFB4A 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 the expression level in a sample (e.g., a skin sample) from an individual without PN; and

[0098] ●KRT16 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 the expression level in a sample (e.g., a skin sample) from an individual without PN.

[0099] The genes that were reduced included:

[0100] LCE5A 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, at least 8-fold, at least 9-fold, at least 10-fold, or at least 11-fold compared to the expression level in a sample (e.g., a skin sample) from an individual without PN; and

[0101] AQP7 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.

[0102] Genes encoding cytokines may also be overexpressed in subjects with PN. Among cytokine genes, the most prominent upregulated genes are IL-36 family members and IL-20 family members. These upregulated or overexpressed genes may include:

[0103] IL36A (e.g., approximately 6.8-fold, FDR = 1.8 × 10 -4 );

[0104] IL36G (e.g., approximately 8.4-fold, FDR = 3.9 × 10 -25 );

[0105] IL19 (e.g., approximately 5.1-fold, FDR = 7.4 × 10 -4 );

[0106] IL20 (e.g., about 3.5-fold, FDR = 1.7 × 10 -3 );

[0107] IL22 (e.g., approximately 2.7-fold, FDR = 2.9 × 10 -2 );

[0108] IL24 (e.g., approximately 5.8-fold, FDR = 3.8 × 10 -10 );and

[0109] IL26 (e.g., approximately 4.9-fold, FDR = 3.3 × 10 -3 ).

[0110] Each of these IL-36 and IL-20 family member cytokine genes may be overexpressed 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.

[0111] 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., about 4.1-fold, FDR = 3.7 × 10 -6 ). In addition, the IL4R gene may increase, for example, by about 2.6 times (FDR = 6.3 × 10 -19 ). Table 1 at the end of the Examples section of this specification provides a more comprehensive list of DEGs.

[0112] In addition to the aforementioned genes, the present disclosure also shows that certain plasma markers or characteristics may be altered by successful treatment of PN with anti-IL-31RA antibodies (such as nemolizumab). Such markers or characteristics can be detected, for example, by mass spectrometry and other protein assessment methods (e.g., ELISA, Western blot, etc.). Circulating plasma protein markers or characteristics that may be modulated by treatment with anti-IL-31RA antibodies (such as nemolizumab) may include leukocyte migration and cell motility, IL-6 pathway, vascular endothelial growth factor (VEGF) pathway, STAT3 (signal transducer and activator of transcription 3) pathway, STAT5b (signal transducer and activator of transcription 5b) pathway, TGFB1 (transforming growth factor beta-1) pathway, and neuronal ontology.

[0113] The STAT3 pathway is a direct target of IL-31 signaling and is also inhibited in the disclosed nemolizumab responder signature, suggesting target engagement. STAT3 activity and expression may be relatively high in subjects with PN or in subjects before initiation of treatment with an anti-IL-31RA antibody, such as nemolizumab, relative to individuals or populations without PN.

[0114] The amount of circulating proinflammatory cytokines may also be relatively high in subjects with PN or in subjects before starting treatment with an anti-IL-31RA antibody (such as nemolizumab) relative to individuals or populations without PN. Such proinflammatory cytokine profiles may include, but are not limited to, IL-6 and VEGF. In subjects with PN receiving treatment with an anti-IL-31RA antibody (such as nemolizumab), treatment may result in a decrease in one or both of IL-6 and VEGF, or a decrease or inhibition of IL-6, VEGF, or both signaling pathways relative to baseline levels. Baseline levels can be determined relative to (i) a control sample obtained from one or more individuals (i.e., a population) without PN or (ii) a biological sample obtained from a subject prior to administration of the anti-IL-31RA antibody.

[0115] TGFB1 activity is also suppressed in the disclosed nemolizumab responder profile. TGFB1 activity and expression may be relatively high in subjects with PN or in subjects before initiating treatment with an anti-IL-31RA antibody, such as nemolizumab, relative to individuals or populations without PN.

[0116] With respect to the disclosed plasma protein markers, the amount of the disclosed protein marker in the plasma of a subject with PN can 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 greater than the baseline level. The baseline level can be determined relative to (i) a control sample obtained from one or more individuals without PN (i.e., a population) or (ii) a biological sample obtained from a subject prior to administration of an anti-IL-31RA antibody. Similarly, after treatment of a subject with PN with an anti-IL-31RA antibody (such as nemolizumab) (e.g., 2 weeks, 4 weeks, 6 weeks, 8 weeks, 10 weeks, or 12 weeks after administration of the antibody), the amount of the disclosed plasma protein markers in the subject can 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, 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 relative to baseline levels. Baseline levels can be determined relative to (i) a control sample obtained from one or more individuals without PN (i.e., a population) or (ii) a biological sample obtained from the subject prior to administration of the anti-IL-31RA antibody.

[0117] Additionally, neuronal entities (e.g., CREB signaling in neurons, synaptogenic signaling pathways, glial cell death, and glial apoptosis) may be upregulated in subjects with PN and subsequently downregulated in nemolizumab responder subjects, thus emphasizing the impact of IL-31 as a neuroinflammatory cytokine in PN.

[0118] Thus, the present disclosure provides a method for diagnosing PN, comprising detecting the expression level of at least one, at least two, at least three, at least four, or at least five differentially expressed genes (DEGs) listed 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 level of the DEGs to a reference level, which can be based on the gene expression level in a sample (e.g., a skin sample) from an individual without PN. In some embodiments, the sample obtained from the subject suspected of having PN is a skin sample, which may contain lesions or nodules. If certain genes are upregulated or overexpressed (e.g., KRT6C, DEFB4A, KRT16) and / or if other genes are downregulated or underexpressed (e.g., LCE5A, AQP7), the subject can be diagnosed with PN.

[0119] The present disclosure also provides methods for determining the likelihood that a subject with PN will respond positively to treatment (e.g., treatment with an anti-IL-31RA antibody, such as nemolizumab), comprising detecting the expression level of at least one, at least two, at least three, at least four, or at least five differentially expressed genes (DEGs) listed 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 with PN, and comparing the expression level of the DEGs to a reference level, which can be based on the gene expression level 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, which can contain lesions or nodules. If certain genes are upregulated or overexpressed (e.g., KRT6C, DEFB4A, KRT16) and / or if other genes are downregulated or underexpressed (e.g., LCE5A, AQP7), the subject is likely to respond to treatment.

[0120] The present disclosure also provides methods for determining whether a subject with PN is responsive to treatment (e.g., treatment with an anti-IL-31RA antibody, such as nemolizumab), comprising detecting the expression level of at least one, at least two, at least three, at least four, or at least five differentially expressed genes (DEGs) listed 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 with PN, and comparing the expression level of the DEGs to a baseline expression level, wherein the baseline expression level of the genes is obtained from a sample (e.g., a skin sample) from the same individual before starting treatment. In some embodiments, the sample can be a skin sample, which can contain lesions or nodules. If the expression level of certain genes is decreased (e.g., KRT6C, DEFB4A, KRT16) and / or if the expression level of other genes is increased (e.g., LCE5A, AQP7), the subject is responsive to treatment.

[0121] The present disclosure also provides methods for determining whether a subject with PN is responsive to treatment (e.g., treatment with an anti-IL-31RA antibody, such as nemolizumab), comprising detecting one or more biomarkers selected from the group consisting of leukocyte migration and cell motility, the IL-6 pathway, the VEGF pathway, the STAT3 pathway, the STAT5b pathway, the TGFB1 pathway, and a disclosed neuronal entity in a post-treatment plasma sample obtained from a subject with PN who has been administered at least one dose of an anti-IL-31RA antibody; wherein a decrease in leukocyte migration and cell motility, 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 disclosed neuronal entity relative to a baseline amount indicates responsiveness to treatment, wherein the baseline amount is determined from a plasma sample obtained from the same subject before initiation of treatment.

[0122] 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:

[0123] "Keratinized capsule" (FDR = 1.5 × 10 -12 ),

[0124] Epidermal cell differentiation (FDR = 6.4 × 10 -10 ),

[0125] ● “Keratinization” (FDR = 1.6 × 10 -12 ),

[0126] ● “Peptidase regulator activity” (FDR = 1.1 × 10 -4 ),

[0127] ● “Interleukin-4 and 13 signaling” (FDR = 6.8 × 10 -7 ),

[0128] ● “Interferon α / β signaling” and “response to interferon γ” (FDR = 4.1 × 10 -7 and FDR = 4.1 × 10 -6 ),

[0129] ●“IL23 pathway” (FDR=2×10 -5 ),and

[0130] ●“Mitosis metaphase and anaphase” (FDR = 3.8 × 10 -10 ).

[0131] These GO categories are represented by Figure 1 B and Table 2 at the end of the Examples section of this specification are defined in more detail. These categories reflect the hyperproliferative nature of PN, which has been found to be associated with altered epidermal differentiation and inflammatory components. For example, the proliferation marker Ki67 (MKI67), the cell cycle gene CDKN1A, and the inflammatory network involving IL-1 and IL-36 have now been exposed as potentially involved in the pathogenesis of PN, such as Figure 1 As shown in C.

[0132] The present disclosure also discloses 20 co-expression modules identified in non-lesional skin and 10 clusters identified in lesional PN skin, such as Figure 1 D and Table 3 at the end of the Examples section of this specification. Different functions can be defined for these co-expressed gene modules, especially those in PN lesional skin, the most prominent of which are related to immune processes (module 8), including "immune response" (FDR = 1.8 × 10 -47 ), “defense response” (FDR = 1.2 × 10 -39 ); Cell proliferation (module 6) including “cell cycle” (FDR = 2.9 × 10 -94 ), “DNA metabolic process” (FDR = 8.7 × 10 -67 ); and epidermal processes (module 5), such as “epidermal development” (FDR 3.5×10 -10 ), “keratinization” (FDR = 1.7 × 10 -6 ). See also Figure 1 Other notable findings were changes in the extracellular matrix (FDR = 1.16 × 10 -59; module no. 2) and included genes such as MMP14, MMP16, COL1A1, COL1A2, and COL3A1, which were moderately elevated in lesional skin (FC ≥ 1.4; FDR ≤ 6 × 10 -2 ), which is consistent with the association between PN and skin fibrosis.

[0133] The present disclosure also provides specific cell type characteristics of non-lesional and lesional PN skin samples. Figure 2 As shown in A, enrichment of transcriptomic signatures associated with epithelial cells and keratinocytes was observed (p < 0.001 and p < 0.0001, respectively). Subjects with PN showed a Th2-related signature (p < 0.0001), consistent with the enriched GO categories of IL-4 / IL-13 (as discussed above and Figure 1 Other inflammatory features, such as macrophages (p < 0.01), may be more variable.

[0134] like Figure 2 As shown in Figure B, a three-way comparison of the PN transcriptome with that of atopic dermatitis (AD) and psoriasis revealed significant overlap in upregulated and downregulated genes across all three diseases. However, the correlation of effect sizes in lesional skin was more pronounced between PN and psoriasis (Spearman correlation p = 0.64) than between PN and AD (p = 0.55). Genes commonly upregulated in psoriasis and PN included those involved in cytokine activity (CCL3, CXCL10, IFNG, IL12B, IL19, IL1B, IL20, etc.) and keratinization (KRT16, KRT17, LCE3A, LCE3E, etc.) (Table 4).

[0135] Therefore, 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 have Figure 1 B or PN skin gene ontology (GO) shown in Table 2. Additionally or alternatively, the skin sample may also show upregulation or overexpression of Ki67 (MKI67), CDKN1A and / or inflammatory networks involving IL-1 and IL-36. Additionally or alternatively, the subject may have Figure 1 D or a co-expression module shown in Table 3. Additionally or alternatively, a subject with PN may have (e.g., in a skin sample) Figure 2 The transcriptome or Th2 signature disclosed in A.

[0136] In addition, the present disclosure provides unprecedented insights into the pathogenesis of PN and the associated tissue-specific and cell type-specific changes that occur in PN skin during disease progression and in response to treatment with anti-IL31RA antibodies. During treatment, changes in PN skin can be observed in immune and stromal cell populations (including keratinocytes, endothelial cells, and most profoundly, fibroblasts and fibroblast subsets), as PN pathogenesis is characterized by an increased profibrotic response accompanied by an immune shift away from IL-13 and IL-22 responses.

[0137] The characteristic histopathological feature of PN is fibrosis of the papillary dermis with vertically aligned collagen fibers, including a significant increase in dense collagen in the papillary dermis of lesional PN skin by trichrome staining and increased expression of procollagen I in the papillary dermis. In addition, COL11A1+ fibroblasts can be the main source of activated and enriched profibrotic responses, including increased mRNA expression of both collagen I and collagen III. Consistent with their profibrotic function, COL11A1+ fibroblasts are predominantly found in the papillary dermis, where the fibrotic response is most pronounced, with trichrome or procollagen I staining indicating active collagen I biosynthesis.

[0138] Expansion of the COL11A1+ fibroblast subset is specific to PN and is not seen in atopic dermatitis (AD) skin. Furthermore, a profibrotic role for this population was not observed in AD COL11A1+ fibroblasts. Of the two components of the heterodimeric IL-31 receptor, IL31RA expression was detected on both keratinocytes and fibroblasts, whereas OSMRB expression was found to be more widespread across different cell populations. Therefore, the two key cell types responsive to IL-31 in PN may be fibroblasts and keratinocytes, consistent with the observation that treatment with anti-IL-31RA antibodies (e.g., nemolizumab) drives transcriptome shifts attributable to these two cell types.

[0139] Other cell types, including endothelial cells and pericytes, also contribute to fibrosis in the skin of lesional prurigo nodularis (LPN). Endothelial changes in LPN are known, but the nature of these changes has not been previously detailed. The present disclosure shows that endothelial cells, possibly under the action of pro-inflammatory and pro-fibrotic cytokines such as TGFB, contribute to extracellular matrix reorganization. TGFβ may be an upstream promoter of fibrosis in PNs, as TGFβ 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.

[0140] One of the most characteristic histological features of PN is the presence of dense orthokeratosis accompanied by irregular epidermal proliferation. Keratinocytes exhibit significant transcriptome changes in diseased PN skin, with the most prominent changes observed in inflammatory keratinocytes, defined by the expression of KRT6, KRT16, KRT17, S100A8, and S100A9, and this analysis suggests a key role for Th2 cytokines (such as IL13 and IL22) in this transition. The most abundant biological classes in the inflammatory keratinocyte subset are related to mitochondrial function and protein translation, suggesting the production of reactive oxygen species and cellular stress, which can contribute to the inflammatory response in the skin.

[0141] Immune cell infiltration also occurs in lesional PN skin and is characterized by changes in specific immune cell populations. The most obvious changes can be observed in macrophage populations, especially lipid-associated macrophages characterized by the expression of APOE and TREM2. Lipid metabolites from lipid-associated macrophages have been shown to trigger the production of proinflammatory cytokines in atherosclerosis, which in turn amplify the inflammatory response. T cells are also prominent in PN lesions, with circulating T cells as well as NK, CD8 + and Treg numbers. In addition, various stromal cell populations, particularly endothelial cells and pericytes, have increased expression of various proinflammatory cytokines, chemokines, and adhesion molecules, suggesting a possible role in immune trafficking and immune amplification in PNs. This includes increased expression of the adhesion molecules ICAM1, E-selectin (SELE), and IL6 in endothelial cells, as well as increased expression of CCL2, CCL3, CCL4, CCL13, CCL18, CXCL2, and CXCL12 expressed by various cell types in PN skin (e.g., myeloid cells, pericytes, and endothelial cells). CCL2 and IL-6 play an established role in the development of fibrosis. CCL2 is the most potent profibrotic chemokine; CCL2 acts directly on fibroblasts via CCR2 to stimulate collagen synthesis. Similarly, IL-6 trans-signaling enhances lung fibroblast proliferation and extracellular matrix protein production.

[0142] This disclosure shows that changes in cell populations in the epidermis are highly similar between PN and AD, with both diseases having a prominent "inflammatory" keratinocyte subpopulation characterized by increased expression of pro-inflammatory molecules (including S100A8 and S100A9) and the inflammatory keratin proteins KRT6 and KRT16. Changes in the expression of S100A8, S100A9, and KRT16 have been described in AD skin, but their expression in PN skin has not been addressed. These epidermal changes are accompanied by subtle changes in gene expression between PN and AD skin, with immune-related processes (e.g., antimicrobial responses) and T cell trafficking regulators observed only in LAD but not in LPN keratinocytes.

[0143] Changes in T cell phenotype were observed between LPN and LAD skin, particularly within the CD4 effector T cell population, with significantly lower mRNA expression of IL13 and IL22 in LPN skin compared to LAD skin. IL-22 is known to promote epidermal proliferation and activate innate immune and antimicrobial responses in the skin. Therefore, while PN is an inflammation-driven disease, it may not be centered around the same degree of IL-13 / IL-22 response as AD.

[0144] 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 considered as a consequence of the disclosed therapeutic methods. The normalization of the pathological transcriptomic signature observed in COL11A1+ fibroblasts and inflammatory keratinocyte subsets may be associated with the clinical improvement of PN skin lesions during treatment, which may also reverse the cellular, genetic, and molecular basis of PN development and progression.

[0145] Thus, the present disclosure provides methods for inactivating, reducing activation, or decreasing the number of COL11A1+ fibroblasts in a subject with PN, comprising administering an anti-IL-31RA antibody to the subject, wherein administration of the anti-IL-31RA antibody causes inactivation, reduced activation, or a decrease in the number of COL11A1+ fibroblasts in the subject's skin. In some embodiments, the COL11A1+ fibroblasts are present in the papillary dermis. This decrease in activation or total number of activated fibroblasts can be determined relative to the number and activation state of fibroblasts in the skin lesion prior to treatment with an anti-IL-31RA antibody (e.g., nemolizumab).

[0146] The present disclosure also provides a method for reducing TGFβ expression in at least one cell type in a subject suffering from PN, comprising administering an anti-IL-31RA antibody to the subject, wherein administration of 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 the expression of TGFB1, TGFB2, TGFB3, or any combination thereof. This reduction in TGFβ expression can be determined relative to the expression level of the corresponding cell type in the PN skin lesion prior to treatment with an anti-IL-31RA antibody (e.g., nemolizumab).

[0147] The present disclosure provides a method for reducing the expression of at least one inflammatory gene expressed by keratinocytes in a subject suffering from PN, comprising administering an anti-IL-31RA antibody to the subject, wherein administration of 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, keratinocytes express Th2 cytokines. In some embodiments, administration of the anti-IL-31RA antibody results in a reduction in reactive oxygen species and / or cellular stress to which keratinocytes are exposed. This reduction in inflammatory gene expression can be determined relative to the expression level of the corresponding cell type in PN skin lesions prior to treatment with the anti-IL-31RA antibody (e.g., nemolizumab).

[0148] The present disclosure provides a method for reducing the infiltration of at least one type of immune cell in a skin lesion of a subject suffering from PN, comprising administering an anti-IL-31RA antibody to the subject, wherein administration of the anti-IL-31RA antibody causes a reduction in the infiltration of at least one type of immune cell in at least one lesion of the subject's skin. In some embodiments, 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, at least one type of immune cell comprises a T cell, a NK cell, a CD8 T ... + Cells, Tregs, and any combination thereof. In some embodiments, administration of an anti-IL-31RA antibody results 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, at least one cell type in the lesion comprises myeloid cells, pericytes, endothelial cells, and any combination thereof. This decrease in immune cell infiltration or expression of ICAM1, E-selectin (SELE), IL6 CCL2, CCL3, CCL4, CCL13, CCL18, or CXCL2, CXCL12 can be determined relative to the amount of infiltration observed in the skin lesion before treatment or relative to the expression level of the corresponding cell type in the PN skin lesion before treatment with an anti-IL-31RA antibody (e.g., nemolizumab).

[0149] 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 blot, serial analysis of gene expression (SAGE), DNA or RNA microarrays, and in situ hybridization. At the protein level, the disclosed biomarkers can be detected or measured using, for example, Western blot, ELISA (enzyme-linked immunosorbent assay), surface plasmon resonance, and mass spectrometry.

[0150] Subjects with or suspected of having PN and the presence of any of the disclosed DEGs, gene ontologies, co-expression modules, or gene signatures are suitable for treatment or prevention with an anti-IL-31RA antibody, such as nemolizumab, as described in further detail herein.

[0151] The present disclosure provides the following combination: (1) a pharmaceutical composition for treating or preventing 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 that detects the expression level of at least one gene selected from the genes disclosed in Table 1 in a subject suspected of having PN, and compares it with a reference expression level of the at least one gene.

[0152] III. Therapeutic Antibodies and Interleukin-31 Receptor Subunit α (IL-31RA)

[0153] Interleukin-31 (IL-31) is a neuroinflammatory cytokine that activates structures, immune cells, and peripheral nerves. It has been implicated in a variety of 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 composed 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 is expressed on a subset of immune and epithelial cells and neurons.

[0154] IL-31RA forms a heterodimer with the oncostatin M receptor (OSMR) when acting as an IL-31 receptor. Human IL-31RA has several known splice variants (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 and lacks a transmembrane domain. Additionally, known IL-31RA splice variants that act as transmembrane receptor proteins include NR10.3 and IL-31RAv3. Preferred IL-31RA variants include NR10.3 (also known as ILRAv4 (Nat Immunol 5, 752-60, 2004) and IL-31RAv3. NR10.3 (IL31RAv4) consists of 662 amino acids (WO 00 / 075314; Nat Immunol 5, 752-60, 2004), while IL31RAv3 consists of 732 amino acids (GenBank Accession No.: NM-139017).

[0155] The amino acid sequence of IL31RAv4 is:

[0156]

[0157] The amino acid sequence of IL31RAv3 is:

[0158]

[0159] Mouse-derived IL-31RA contains the amino acid sequence:

[0160]

[0161]

[0162] Cynomolgus macaque-derived IL-31RA comprises the amino acid sequence:

[0163]

[0164] For purposes of the present disclosure, an anti-IL-31RA antibody (ie, therapeutic antibody), such as nemolizumab, must bind to at least human IL-31RA or a splice variant thereof.

[0165] As used herein, the term "antibody" refers collectively to immunoglobulins 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 comprise a heavy (H) chain and a light (L) chain interconnected by disulfide bonds. There are two types of light chains, lambda (λ) and kappa (κ). There are five major classes (or isotypes) of heavy chains, which determine the functional activity of antibody molecules: IgM, IgD, IgG, IgA, and IgE. Each heavy and light chain contains a constant region and a variable region (also called a "domain"). Combined, the heavy and light chain variable regions, also called the "Fab region," specifically bind to a given antigen. The light and heavy chain variable regions contain a "framework" region interspersed with three hypervariable regions, also called "complementarity determining regions" or "CDRs." The extent of the framework regions and CDRs has been defined (see Kabat et al., Sequences of Proteins of Immunological Interest, US Department of Health and Human Services, 1991). The Kabat database is now maintained online. The sequences of the framework regions of different light or heavy chains are relatively conserved within species, and the framework regions serve to form a scaffold that positions the CDRs in the correct orientation through non-covalent interactions between the chains.

[0166] The CDRs are primarily responsible for binding to the epitope on the antigen. The CDRs of each chain are typically referred to as CDR1, CDR2, and CDR3, and are numbered sequentially starting from the N-terminus and are often also identified by the chain in which a particular CDR is located. Thus, HCDR3 is located in the variable domain of the antibody heavy chain in which it is located, while LCDR1 is CDR1 from the variable domain of the antibody light chain in which it is located. Antibodies that bind to IL-31RA will have a specific V H Area and V L Region sequence, and therefore has a specific CDR sequence. Antibodies with different specificities generally have different CDRs. Although CDRs vary from antibody to antibody, only a limited number of amino acid positions within the CDRs are directly involved in antigen binding. These positions within the CDRs are called specificity determining residues (SDRs).

[0167] The Fc fragment region (Fc) of an antibody plays a role in regulating immune cell activity. The Fc region is used to bind to proteins of a specific class found on certain cells (such as B lymphocytes, follicular dendritic cells, natural killer cells, macrophages, neutrophils, etc.), ensuring that each antibody produces an appropriate immune response to a given antigen, and is referred to as a "Fc receptor". Because the constant domain of the heavy chain constitutes the Fc region of the antibody, the class of the heavy chain in the antibody determines its class effect. The heavy chain in the antibody includes α, γ, δ, ε and μ, and is respectively associated with the isotype IgA, IgG, IgD, IgE and IgM of the antibody. Therefore, due to different Fc regions binding and activating different types of receptors, antibodies of different isotypes have different class effects.

[0168] IgG has four subclasses and is the most abundant antibody isotype found in human serum. The four subclasses, IgG1, IgG2, IgG3, and IgG4, are highly conserved. The amino acid sequences of the constant regions of these peptides are known in the art, for example, see 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 aminoacid 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 (MOPC 511)" PNAS 77(8): 4909-4913.

[0169] For the disclosed methods and medical uses, all therapeutic antibodies 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 may also be used. Therapeutic antibodies suitable for the disclosed methods and medical uses can be human, humanized, or chimeric, and can be IgA, IgG (i.e., IgG1, IgG2, IgG3, and IgG4), IgD, IgE, or IgM.

[0170] Nemolizumab is a humanized monoclonal antibody that binds to IL-31RA. Nemolizumab is annotated as follows: Immunoglobulin G2-κ, anti-[Homo sapiens IL31RA (interleukin 31 receptor subunit α)], humanized monoclonal antibody; γ2 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 (CH1C10>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), CH3R11>Q(355), Q98>E(419)(341-445))(122-445)], (224-214′)-disulfide bond with κ light chain (1′-214′) [humanized V-κ(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")-double disulfide bond. Nemolizumab has disulfide bridges at the following positions: within H (C23-C104) 22-96 148-204 261-321 367-425 22"-96" 148"-204" 261"-321" 367"-425"; within L (C23-C104) 23'-88' 134'-194' 23"'-88"' 134"'-194"'; between HL (h5-CL126) 224-214' 224"-214"'; between HH (h8, h11) 227-227" 230-230". Nemolizumab has an N-glycosylation site at the following position: H CH2N84.4: 297, 297". Nemolizumab lacks the C-terminal glycine and lysine of the H chain (CHS G1>del, K2>del).

[0171] Nemolizumab contains the following heavy chain amino acid sequence:

[0172]

[0173] Nemolizumab contains the following light chain amino acid sequence:

[0174]

[0175] The heavy chain variable region of nemolizumab comprises the amino acid sequence:

[0176]

[0177] 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).

[0178] The light chain variable region of nemolizumab comprises the amino acid sequence:

[0179]

[0180] 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).

[0181] For purposes of the present disclosure, "variant antibodies" or "variants" of nemolizumab may include, but are not limited to: (i) antibodies 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) antibodies 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

[0015] The present invention also provides antibodies that have light chains that have 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 that have CDRs that have 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 that have the same or substantially similar heavy and light chain amino acid sequences as nemolizumab. Other suitable therapeutic antibodies may bind to the same IL-31RA isoform as nemolizumab (e.g., IL31-RAv3), optionally bind to the same epitope of IL-31RA, block or neutralize IL-31RA, or a combination thereof. Other exemplary therapeutic antibodies are described, for example, in WO 2010 / 064697.

[0182] Variants of nemolizumab and suitable therapeutic antibodies can be monoclonal or polyclonal antibodies. Such monoclonal antibodies having IL31-RA binding and / or neutralizing activity can be obtained, for example, by the following procedure: using an antigen IL31-RA or a fragment thereof derived from a mammal such as a human or mouse to prepare an anti-IL31-RA monoclonal antibody by a known method, and then selecting an antibody having IL31-RA binding and / or neutralizing activity from the anti-IL31-RA monoclonal antibody thus obtained. Specifically, according to conventional immunization methods, the desired antigen or a cell expressing the desired antigen is used as a sensitizing antigen for immunization. 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) by conventional screening methods. Animals to be immunized include, for example, mammals such as mice, rats, rabbits, sheep, monkeys, goats, donkeys, cows, horses, and pigs. Antigens can be prepared according to known methods, for example, by using baculovirus methods (e.g., WO 98 / 46777) using known IL31-RA gene sequences. Variants of nemolizumab and suitable therapeutic antibodies may also include intrabodies, peptibodies, nanobodies, single domain antibodies, multispecific antibodies (e.g., bispecific antibodies, bifunctional antibodies, trifunctional antibodies, tetrafunctional antibodies, tandem two scFvs, tandem three scFvs), darpins, heavy chain monomers, heavy chain dimers, or single domain antibodies (i.e., V H H fragment or "camelid-like" antibody), either of which may be derived from the sequence and / or binding domain of nemolizumab.

[0183] Hybriomas 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 the immunogenicity of the antigen is low, immunization can be performed after linking the antigen to an immunogenic macromolecule (such as albumin). The antigen used to prepare monoclonal antibodies with binding and / or neutralizing activity against human IL31-RA is not particularly limited, as long as it can produce antibodies with binding and / or neutralizing activity against human IL31-RA. For example, human IL31-RA is known to have many variants, and any variant can be used as an immunogen as long as it can produce antibodies with binding and / or neutralizing activity against human IL31-RA. Alternatively, under the same conditions, a peptide fragment of IL31-RA or a protein with artificial mutations introduced into the native IL31-RA sequence can be used as an immunogen. Human IL31-RA.3 is one of the preferred immunogens for preparing antibodies with binding and / or neutralizing activity against IL31-RA in the present disclosure.

[0184] The IL31-RA binding activity of therapeutic antibodies can be determined by methods known to those skilled in the art. Methods for determining the antigen binding activity of antibodies include, for example, ELISA (enzyme-linked immunosorbent assay), EIA (enzyme immunoassay), RIA (radioimmunoassay), and fluorescent antibody assays. For example, when using enzyme immunoassay, a sample containing the antibody, such as purified antibody and 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 assess antigen binding activity. The binding and / or neutralizing activity of therapeutic antibodies against IL31-RA can be measured, for example, by observing the effect of inhibiting the growth of IL-31-dependent cell lines. For example, the activity of purified mouse IL-31 antibodies can be analyzed by assessing the IL-31-dependent growth of Ba / F3 cells transfected with mouse IL-31 receptor α and mouse OSMR genes.

[0185] Any anti-IL31RA antibody disclosed herein (ie, a "therapeutic antibody"), including nemolizumab and fragments or variants thereof, can be used to treat and / or prevent PN and achieve the disclosed therapeutic endpoints. The optimal dosage and route of administration may vary.

[0186] IV. Pharmaceutical Compositions

[0187] Provided herein are pharmaceutical compositions for treating or preventing prurigo nodularis (PN), including lesions or nodules or pruritus caused by PN, comprising an anti-IL31RA antibody (ie, a "therapeutic antibody"), such as nemolizumab or a fragment or variant thereof, as an active ingredient.

[0188] The phrase "comprising nemolizumab or a fragment or variant thereof as an active ingredient" means comprising nemolizumab or a fragment or variant thereof as at least one of the active ingredients, and does not limit the ratio of the antibody. In addition, the therapeutic agent for PN in the present disclosure may further comprise 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 comprise one or more topical corticosteroid creams or injections, ointments with menthol or phenol to cool and soothe itchy skin, capsaicin creams, oral corticosteroids, selective serotonin reuptake inhibitors (SSRIs), and oral antihistamines.

[0189] The pharmaceutical composition of nemolizumab or its fragment or variant disclosed herein can be prepared into formulations (see, for example, Remington's Pharmaceutical Science, Mark Publishing Company, Easton, USA) according to standard methods. In addition to the antibody, the pharmaceutical composition generally includes a carrier and / or an additive. For example, in some embodiments, the pharmaceutical composition includes one or more surfactants (such as PEG and Tween), excipients, antioxidants (such as ascorbic acid), colorants, flavorings, preservatives, stabilizers, buffers (such as phosphoric acid, citric acid and other organic acids), chelating agents (such as EDTA), suspending agents, isotonic agents, adhesives, disintegrants, lubricants, flow promoters, correctives, light anhydrous silicic acid, lactose, crystalline cellulose, mannitol, starch, carboxymethylcellulose calcium, sodium carboxymethylcellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, polyvinyl acetal diethylamino acetate, polyvinyl pyrrolidone, gelatin, medium-chain fatty acid 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).

[0190] When nemolizumab or its fragment or variant can be prepared as an aqueous solution for injection, nemolizumab or its fragment or variant can be dissolved in an isotonic solution containing, for example, normal saline, dextrose or other excipients or tonicity agents. Tonicity agents can include, for example, D-sorbitol, D-mannose, D-mannitol and sodium chloride. In addition, suitable solubilizing agents such as alcohols (e.g., ethanol), polyols (e.g., propylene glycol and PEG) and non-ionic detergents (polysorbate 80 and HCO-50) can be used simultaneously.

[0191] In some embodiments, nemolizumab or a fragment or variant thereof can be encapsulated in microcapsules (microcapsules made of hydroxymethylcellulose, gelatin, polymethyl methacrylate, etc.) and made into components of colloidal drug delivery systems (liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) (see, for example, "Remington's Pharmaceutical Science 16th edition" &, Oslo Ed. (1980)). In addition, methods for making sustained-release drugs are known, and these methods can be applied to nemolizumab or fragments or variants thereof (Langer et al., J. Biomed. Mater. Res. (1981) 15, 167-277; Langer, Chem. Tech. (1982) 12, 98-105; U.S. Patent No. 3,773,919; European Patent Application (EP) No. 58,481; Sidman et al., Biopolymers (1983) 22, 547-56; EP 133,988).

[0192] The pharmaceutical compositions of the present disclosure can be administered orally or parenterally, but parenteral administration is preferred. Specifically, the pharmaceutical compositions are administered to the patient by injection or transdermal administration. Injections include, for example, intravenous, intramuscular, and subcutaneous injections for systemic or local administration. The pharmaceutical compositions can be administered to the site to be inhibited from inflammation and / or pruritus or to the area around the site by local infusion or intramuscular or subcutaneous injection. In some embodiments, the pharmaceutical composition is administered to one or more skin exfoliations, lesions, or nodules, or is administered near one or more skin exfoliations, lesions, or nodules.

[0193] The administration method can be appropriately selected according to the patient's age, weight, and condition. The single administration dose can be selected, for example, from 0.0001 to 100 mg of the antibody (e.g., nemolizumab or a fragment or variant thereof) per kilogram of body weight. Alternatively, for example, when the antibody is administered to a human patient, the dose of the antibody can be selected from the range of 0.001 to 1,000 mg / kg body weight. In some embodiments, the composition is formulated for administration containing, for example, about 0.01 mg / kg 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 mg / kg 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, A dosage of nemolizumab, or a fragment or variant thereof, of about 1 mg / kg to about 3.5 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, or 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 dose range is 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.In some embodiments, the 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. / 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 dose is about 0.5 mg / kg.

[0194] The present disclosure provides a pharmaceutical composition for 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.

[0195] The present disclosure provides a pharmaceutical composition for 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 wherein the subject is diagnosed as having prurigo nodularis (PN) by detecting the expression level of at least one, at least two, at least three, at least four, or at least five differentially expressed genes (DEGs) in Table 1 in a sample obtained from a subject suspected of having PN, and comparing the expression level of the DEGs to a reference level, wherein the reference level is the corresponding gene expression level of each DEG in a sample from an individual without PN.

[0196] Any pharmaceutical composition disclosed herein comprising nemolizumab and its fragments or variants can be used to treat and / or prevent PN and achieve the disclosed therapeutic endpoints. The optimal dosage and route of administration may vary.

[0197] V. Treatment / Prevention Methods and Compositions for Treatment or Prevention

[0198] The present disclosure provides methods for treating or preventing pruritus in a subject with prurigo nodularis (PN), 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 performed to achieve specific therapeutic endpoints, which are discussed in more detail below. Also disclosed herein are the uses of anti-IL-31RA antibodies (i.e., "therapeutic antibodies"), such as nemolizumab or a fragment or variant thereof, in subjects for treating or preventing PN and / or achieving the disclosed therapeutic endpoints. Also disclosed herein are anti-IL-31RA antibodies (i.e., "therapeutic antibodies"), such as nemolizumab or a fragment or variant thereof, administered to a subject for treating or preventing PN and / or achieving the disclosed therapeutic endpoints. In addition, specific subgroups of subjects with PN may be particularly suitable for treatment according to the disclosed methods and uses (e.g., patients with any of the DEGs disclosed in Table 1).

[0199] The present disclosure reports for the first time a transcriptomic signature of PN that is not only capable of identifying and proactively diagnosing PN, but also of identifying subjects with PN who will likely respond to treatment with anti-IL-31RA antibodies (e.g., nemolizumab), and tracking responses to treatment with anti-IL-31RA antibodies (e.g., nemolizumab).

[0200] This disclosure also reports for the first time the plasma proteomic signature of PN subjects successfully treated with anti-IL-31RA antibodies (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 anti-IL-31RA antibodies (e.g., nemolizumab) and to track responses to treatment with anti-IL-31RA antibodies (e.g., nemolizumab).

[0201] A. Subjects Treated

[0202] A subject treated for PN according to the disclosed methods and uses may exemplify one or more of the basal gene expression patterns disclosed herein. Specifically, a subject suffering from PN and treated according to the disclosed methods and uses may differentially express up to or at least 5,943 genes (referred to as differentially expressed genes or DEGs) as shown in Figure 1 A and Table 1 below. This differential gene expression can be observed in the skin of subjects, and particularly in skin samples containing or consisting of nodules or lesions. Of these DEGs, 2,060 are likely to be increased (i.e., overexpressed) and 3,874 are likely to be decreased (i.e., underexpressed). The genes that are likely to be most increased include:

[0203] KRT6C 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 the expression level in a sample (e.g., a skin sample) from an individual without PN;

[0204] DEFB4A 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 the expression level in a sample (e.g., a skin sample) from an individual without PN; and

[0205] ●KRT16 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 the expression level in a sample (e.g., a skin sample) from an individual without PN.

[0206] The genes that were reduced included:

[0207] LCE5A 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, at least 8-fold, at least 9-fold, at least 10-fold, or at least 11-fold compared to the expression level in a sample (e.g., a skin sample) from an individual without PN; and

[0208] AQP7 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.

[0209] Genes encoding cytokines may also be overexpressed in subjects suffering from PN and can be treated or prevented according to the disclosed methods and uses. With respect to cytokine genes, the most prominent upregulated genes are IL-36 family members and IL-20 family members. These upregulated or overexpressed genes may include:

[0210] IL36A (e.g., approximately 6.8-fold, FDR = 1.8 × 10 -4 );

[0211] IL36G (e.g., approximately 8.4-fold, FDR = 3.9 × 10 -25 );

[0212] IL19 (e.g., approximately 5.1-fold, FDR = 7.4 × 10 -4 );

[0213] IL20 (e.g., about 3.5-fold, FDR = 1.7 × 10 -3 );

[0214] IL22 (e.g., approximately 2.7-fold, FDR = 2.9 × 10 -2 );

[0215] IL24 (e.g., approximately 5.8-fold, FDR = 3.8 × 10 -10 );and

[0216] IL26 (e.g., approximately 4.9-fold, FDR = 3.3 × 10 -3 ).

[0217] Each of these IL-36 and IL-20 family member cytokine genes can be overexpressed 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 the expression level in a sample (e.g., a skin sample) from an individual without PN.

[0218] Other factors that may be upregulated or overexpressed in samples (e.g., skin samples) obtained from subjects with PN to be treated may include IL1A (e.g., approximately 4.7-fold, FDR = 1.0 × 10 -12 ), IL1B (e.g., about 4.1-fold, FDR = 3.7 × 10 -6) and IL4R (e.g., about 2.6-fold (FDR = 6.3 × 10 -19 ). Table 1 at the end of the Examples section of this specification provides a more comprehensive list of DEGs.

[0219] Certain gene ontology (GO) categories may also be evident in the skin of subjects suffering from PN and treated according to the disclosed methods and uses. These GO categories are:

[0220] "Keratinized capsule" (FDR = 1.5 × 10 -12 ),

[0221] Epidermal cell differentiation (FDR = 6.4 × 10 -10 ),

[0222] ● “Keratinization” (FDR = 1.6 × 10 -12 ),

[0223] ● “Peptidase regulator activity” (FDR = 1.1 × 10 -4 ),

[0224] ● “Interleukin-4 and 13 signaling” (FDR = 6.8 × 10 -7 ),

[0225] ● “Interferon α / β signaling” and “response to interferon γ” (FDR = 4.1 × 10 -7 and FDR = 4.1 × 10 -6 ),

[0226] ●“IL23 pathway” (FDR=2×10 -5 ),and

[0227] ●“Mitosis metaphase and anaphase” (FDR = 3.8 × 10 -10 ).

[0228] These categories reflect the hyperproliferative nature of PN, which has been found to be associated with altered epidermal differentiation and an inflammatory component. In some embodiments, subjects suffering from PN and treated according to the disclosed methods and uses may overexpress the proliferation marker Ki67 (MKI67), the cell cycle gene CDKN1A, and / or an inflammatory network involving IL-1 and IL-36.

[0229] A subject suffering from PN and treated according to the disclosed methods or uses may additionally or alternatively have Figure 1 D and one or more of the co-expression modules or clusters shown in Table 3 at the end of the Examples section of this specification. As described herein, different functions are defined for these co-expression gene modules.

[0230] The present disclosure also provides a characterization of specific cell types in non-lesional and lesional PN skin that may be present in a subject suffering from PN and treated according to the disclosed methods and uses. For example, a subject suffering from PN may have Figure 2 The transcriptomic signature observed in A is associated with epithelial cells and keratinocytes, and / or may have a Th2-associated signature, as described herein. Other inflammatory signatures, such as macrophages, may also be prominent in PN lesions and the skin of subjects with PN.

[0231] In general, the disclosed methods and uses can treat or prevent PN in subjects suffering from mild, moderate or severe pruritus. In some embodiments, PN can be classified as moderate to severe. In some embodiments, PN can be classified as moderate, while in other embodiments, PN can be classified as severe. In some embodiments, pruritus can be scored as none, mild, moderate or severe. "None", "mild", "moderate" and "severe" are technical terms that describe the presence, extent and / or intensity of exfoliation. Those skilled in the art are aware of the boundaries and limits 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 the intensity of their pruritus using a numerical rating scale (NRS) from 0 ("no itch") to 10 ("worst itch imaginable"). Another unidimensional scale, the visual analog scale (VAS), provides patients with the opportunity to indicate the intensity of their itch by marking on a 10 cm long ruler. Both endpoints are marked with numbers corresponding to the intensity, where 0 represents "no itch" and 10 represents "the worst itch imaginable". Scores below 3.0 VAS / NRS points are generally associated with mild itch, while scores above 6.9 indicate severe itch. Scores above 9.0 represent extremely severe itch. The verbal rating scale (VRS) is another unidimensional scale that allows patients to describe their itch intensity using a gradually ascending adjective scale (0 - no itch, 4 - the worst itch imaginable). NRS, VAS and VRS have been validated in large-scale studies consisting of patients with chronic pruritus who suffer from pruritic skin diseases or pruritus of various causes. These tools have high reproducibility and there is a high correlation between grades 6, 7 and 8. Chronic pruritus can greatly reduce the quality of life of patients. For this reason, the Dermatology Life Quality Index (DLQI) is widely used and has been validated. The DLQI score ranges from 0 to 30, with higher scores indicating lower quality of life. The Investigator Global Assessment (IGA) score ranges from 0 (clear) to 5 (extremely severe disease) and is presented as a percentage of patients in a given population. In this study, the IGA score ranged from 0 to 4.

[0232] A subject treated for PN according to the disclosed methods and uses may exhibit one or more of the changes in plasma protein profiles disclosed herein. Specifically, a subject suffering from PN and to be treated according to the disclosed methods and uses may exhibit (a) decreased leukocyte migration or cell motility; (b) inhibition of the STAT3 and STAT5b pathways; (c) downregulation of the IL-6 and VEGF pathways; (d) reduction in the TGFB1 pathway, or (e) a combination thereof. The aforementioned reduction or inhibition may be observed at a specified time point after the start of treatment, such as 2 weeks, 4 weeks, 6 weeks, 8 weeks, 10 weeks, or 12 weeks. The reduction or inhibition may be 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. Additionally or alternatively, following administration of an anti-IL-31RA antibody, the subject may exhibit upregulation of neuronal entity terms, including CREB signaling in neurons, synaptogenesis signaling pathways, glial cell death, and glial cell apoptosis, and combinations thereof.

[0233] Subjects with PN and suitable for treatment according to the disclosed methods and uses may exhibit elevated levels of leukocyte migration or motility relative to individuals or populations without PN or prior to initiation of treatment with an anti-IL-31RA antibody (such as nemolizumab). Additionally or alternatively, suitable subjects may exhibit relatively elevated STAT3 pathway activity or expression relative to individuals or populations without PN or prior to initiation of treatment with an anti-IL-31RA antibody (such as nemolizumab). Additionally or alternatively, suitable subjects may exhibit relatively elevated TGFB1 pathway activity or expression relative to individuals or populations without PN or prior to initiation of treatment with an anti-IL-31RA antibody (such as nemolizumab). Additionally or alternatively, suitable subjects may exhibit relatively elevated levels of circulating cytokine signatures relative to individuals or populations without PN or prior to initiation of treatment with an anti-IL-31RA antibody (such as nemolizumab). Such cytokine pathways may include, but are not limited to, IL-6 and VEGF pathways.

[0234] Prior to treatment with an anti-IL-31Ra antibody (such as nemolizumab), the amount of a disclosed protein marker 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 can be determined relative to (i) a control sample obtained from one or more individuals without PN (i.e., a population) or (ii) a biological sample obtained from a subject prior to administration of the anti-IL-31RA antibody.

[0235] In certain embodiments, the subject has been diagnosed with PN for at least about 6 months. In a specific embodiment, the subject has at least about 20 nodules distributed bilaterally on his / her body. In a specific embodiment, the subject has prurigo lesions on the upper limbs, with or without lesions on the trunk or lower limbs. In certain embodiments, the scoring of pruritus assigned on a numerical rating scale (NRS) is at least 7. In certain embodiments, the mean value of the worst day intensity of the NRS scoring was at least 7 within the first 3 days. In certain embodiments, the mean value of the worst day intensity of the NRS scoring was at least 7 within the previous week.

[0236] In some embodiments, the subject does not have atopic dermatitis (AD). In some embodiments, the subject does not have chronic pruritus caused by conditions other than PN, such as scabies, insect bites, chronic lichen simplex, psoriasis, acne, folliculitis, habitual scratching, lymphomatoid papulosis, chronic actinic dermatitis, dermatitis herpetiformis, sporotrichosis, or bullous disease. In some embodiments, the subject does not have neurological or psychogenic pruritus, such as paresthesia, brachioradial pruritus, delusional parasitosis, or mimicry.

[0237] B. Therapeutic Endpoints of Treatment

[0238] Based on the data provided herein, it is believed that IL-31 signaling is upstream of IL-17 and IL-4 signaling, at least in subjects with PN, because treatment with the anti-IL-31RA antibody nemolizumab can alter the transcriptomic expression profiles of IL-17 and IL-4. Thus, the present disclosure provides methods 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 a subject with PN. As shown in the Examples section below, treatment or prevention with nemolizumab normalized multiple PN-associated DEGs for both increased genes (e.g., approximately 969 genes) and decreased genes (e.g., approximately 1,268 genes). See Figure 4A. In some embodiments, after treatment with an anti-IL-31RA antibody (such as nemolizumab) or fragment or variant thereof, 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 800, about 900, about 1000, about 15 ... In some embodiments, the present invention relates to normalizing the expression of about 0, about 700, about 750, about 800, about 850, about 900, about 950, about 1000, about 1100, about 1200, 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. In some embodiments, after treatment with an anti-IL-31RA antibody (such as nemolizumab) or fragment or variant thereof, 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, or more of the subjects with PN may have their PN reduced. In some embodiments, 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 DEGs and up to 2500, 3000, 3500, 4000, 4500, 5000, 5500, or about 6000 DEGs are normalized. In some embodiments, treatment or prevention may comprise administration of an anti-IL-31RA antibody (e.g., nemolizumab) once a week, once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 5 weeks, once every 6 weeks, once every 7 weeks, or once every 8 weeks. Administration once about every 4 weeks may be preferred. Administration may be by injection, such as subcutaneous injection. The time frame for determining / comparing DEG normalization may be 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks or 12 weeks or longer.For example, a baseline sample (e.g., a skin sample) can be obtained from the subject (i.e., at the start of treatment or before) to detect the expression level 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 4, 5, 6, 7, 8, 9, 10, 11, 12, or more weeks after the start of treatment, another sample can be taken and evaluated to determine whether the expression level of the DEG has changed and is more consistent with (i.e., "normalized") a reference expression level of a DEG associated with normal healthy skin (e.g., expression levels obtained from a skin sample of a subject without PN). Such methods can be used to track treatment and assess whether a subject responds to treatment with an anti-IL-31RA antibody (e.g., nemolizumab).

[0239] The enriched GO categories in DEGs may also be reduced or altered by treatment with anti-IL-31RA antibodies (e.g., nemolizumab). For example, after 12 weeks of treatment, the following GO categories were observed to be reduced: “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 ). In fact, the data provided in the Examples section indicate 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. Therefore, 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, the administration of an anti-IL-31RA antibody (e.g., nemolizumab) may be once a week, once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 5 weeks, once every 6 weeks, once every 7 weeks, or once every 8 weeks. Administration approximately every 4 weeks may be preferred. Administration may be by injection, such as subcutaneous injection.

[0240] In connection with the disclosed methods of reducing the inflammatory response in the skin of a subject suffering from PN, in some embodiments, the cytokine response profile produced in the human epidermal rafts of the subject's skin can be altered. For example, IL17A mRNA expression was not significantly different between non-lesional and lesional skin, nor was there a difference after 12 weeks of treatment with nemolizumab. Figure 5A sustained decrease in IL-31 response can be seen in A, either alone or in combination with other inflammatory cytokines, including the Th2 cytokines IL-13 or IL-17A, thus providing evidence that nemolizumab blocks the IL-31 pathway. However, IL-17A responsive genes are shown herein to be enriched in PN skin and downregulated by nemolizumab. This suggests that while IL-17A itself is not the primary cytokine in PN, it is downstream of IL-31 signaling. In fact, a sustained decrease in IL-31 response was observed with nemolizumab treatment, either alone or in combination with other inflammatory cytokines, including the Th2 cytokines IL-13 or IL-17A ( Figure 5 A), and a more robust reduction in Th1, Th17, and Th2 markers was observed ( Figure 5 B) Therefore, IL-31 signaling may be upstream of IL-17 and IL-4 signaling.

[0241] In some embodiments, the basal keratinocyte (KRT14+) profile in PN lesional skin may be elevated, and when treated according to the disclosed methods and uses, this profile may return to normal (i.e., non-PN). In some embodiments, the spinous layer (KRT10+) profile in PN lesional skin may be elevated, and when treated according to the disclosed methods and uses, this profile may return to normal (i.e., non-PN). Figure 5 C).

[0242] In some embodiments, transcription factor binding sites (TFBS) enriched in genes upregulated in baseline lesional skin are upregulated following treatment with an anti-IL-31RA antibody, such as nemolizumab (e.g., week 12 after treatment, such as week 12 after treatment). Figure 5 D) are more likely to be enriched in genes downregulated by nemolizumab. In some embodiments, the transcription factor downregulated by treatment can be EGR4 (which is a member of the EGF family of zinc finger transcription factors), STAT3 and / or KLF16.

[0243] The present disclosure further provides plasma protein markers that have been identified in subjects successfully treated with nemolizumab, such that changes in the resulting biomarkers can be considered a means of determining a successful "responder signature" and tracking responsiveness. Thus, disclosed herein are methods for treating or preventing prurigo nodularis (PN) in a subject, comprising administering an anti-IL-31RA antibody to a subject suffering from PN, wherein treatment with the anti-IL-31RA antibody results in: (a) decreased leukocyte migration or cell motility, (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 disclosed neuronal population. Additionally, disclosed herein are methods for altering an immune response in a subject with PN, 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 or cell motility, (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 number of the disclosed neuronal entities. The disclosed plasma protein markers can be detected, for example, by mass spectrometry and other protein assessment methods (e.g., ELISA, Western blotting, etc.).

[0244] Specifically, when subjects with PN are treated with an anti-IL-31RA antibody (such as nemolizumab), immune cells (eg, white blood cells) may experience a decrease in migration or cell motility, or both.

[0245] STAT3 pathway activity may be reduced in subjects with PN and treated with an anti-IL-31RA antibody, such as nemolizumab, relative to individuals or populations without PN or subjects before initiating treatment with an anti-IL-31RA antibody, such as nemolizumab.

[0246] The amount of cytokine activity may also be relatively high in subjects with PN or in subjects prior to initiation of treatment with an anti-IL-31RA antibody (such as nemolizumab) relative to individuals or populations without PN. Such cytokine pathways may include, but are not limited to, IL-6 and VEGF pathways. In subjects with PN receiving treatment with an anti-IL-31RA antibody (such as nemolizumab), treatment may result in a decrease in IL-6 or VEGF signatures, or both, relative to baseline levels. Baseline levels can be determined relative to (i) a control sample obtained from one or more individuals (i.e., a population) without PN or (ii) a biological sample obtained from a subject prior to administration of the anti-IL-31RA antibody.

[0247] Subjects with PN and treated with an anti-IL-31RA antibody, such as nemolizumab, may have reduced TGFB1 pathway activity relative to individuals or populations without PN or subjects before initiating treatment with an anti-IL-31RA antibody, such as nemolizumab.

[0248] With respect to the disclosed plasma protein markers, the amount of the disclosed protein marker 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 greater than the baseline level. The baseline level can be determined relative to (i) a control sample obtained from one or more individuals without PN (i.e., a population) or (ii) a biological sample obtained from a subject prior to administration of an anti-IL-31RA antibody. Similarly, after treatment of a subject with PN with an anti-IL-31RA antibody (such as nemolizumab) (e.g., 2 weeks, 4 weeks, 6 weeks, 8 weeks, 10 weeks, or 12 weeks after administration of the antibody), the amount of the disclosed plasma protein markers in the subject can 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, 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 relative to baseline levels. Baseline levels can be determined relative to (i) a control sample obtained from one or more individuals without PN (i.e., a population) or (ii) a biological sample obtained from the subject prior to administration of the anti-IL-31RA antibody.

[0249] Additionally, neuronal entities (e.g., CREB signaling in neurons, synaptogenic signaling pathways, glial cell death, and glial cell apoptosis) may be upregulated in subjects with PN and subsequently downregulated in nemolizumab responder subjects.

[0250] In some embodiments of the disclosed methods and uses, treatment or prevention with an anti-IL-31RA antibody (such as nemolizumab) or a fragment or variant thereof results in a decrease in pruritus score. The decrease in score can be measured, for example, by the Peak Pruritus Numeric Rating Scale (PP-NRS). See, e.g., Figure 6 A. In fact, the data presented herein show that all subjects with PN and 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 component) may be less than the measured distance of the placebo group not treated with nemolizumab or other anti-IL-31RA antibodies. More specifically, at week 12 (i.e., 12 weeks after the start of treatment), the proportion of patients who achieved an average weekly reduction of 4 points in the PPNRS 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 who achieved 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).

[0251] C. Fibroblast response to treatment

[0252] As described herein, the present disclosure establishes for the first time that PN is an inflammatory and fibrotic disease and that by antagonizing IL-31 signaling (e.g., by treatment with nemolizumab), an anti-fibrotic effect can be exerted by inhibiting key signaling pathways. In particular, compared to healthy skin, diseased PN fibroblasts exhibit a pro-fibrotic and pro-inflammatory state, which can lead to differential expression of genes involved in activation of inflammatory (TNF, IL1B, IL6) and pro-fibrotic (TGFβ) signaling pathways in diseased PN fibroblasts. Indeed, PN fibroblasts play a central role in this intracellular crosstalk, and treatment with an anti-IL-31RA antibody (e.g., nemolizumab or a fragment or variant thereof) restores neural dysfunction and reduces inflammation and fibrosis.

[0253] For example, the present disclosure provides 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 diseased skin cells compared to a reference level of activation of TNF signaling.

[0254] Similarly, the present disclosure provides a method for normalizing activation of tumor necrosis factor (TNF) expression in a subject with PN, comprising administering an anti-IL-31RA antibody to a subject with PN, wherein the subject exhibits activation of tumor necrosis factor (TNF) signaling in diseased skin cells compared to a reference expression level of the TNF gene, and wherein administration of the anti-IL-31RA antibody normalizes the expression level of the TNF gene. For purposes of this method, normalization can be determined at about 4 weeks, about 8 weeks, or about 12 weeks after administration of the anti-IL-31RA antibody.

[0255] For the purposes of these methods, differential expression and pathway activation can be determined by RT-qPCR, RT-PCR, RNA-seq, Northern blot, 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 blot, ELISA, surface plasmon resonance, or mass spectrometry.

[0256] Compared to the reference expression level, TNF activation in diseased skin cells (e.g., fibroblasts) may be higher before treatment. The reference level can be an activation level that is the activation level of TNF signaling in skin cells (e.g., fibroblasts) of a person not suffering from PN. Additionally or alternatively, the reference level is the activation level of the TNF gene in non-lesional skin cells of a subject.

[0257] The present disclosure also provides a method for reducing skin inflammation in a subject with prurigo nodularis (PN), comprising administering an anti-IL-31RA antibody to a subject with PN, thereby reducing inflammation in the skin involving tumor necrosis factor (TNF) signaling. In such methods, TNF signaling in the subject's skin is overexpressed relative to a reference level of activation of TNF signaling, optionally wherein TNF signaling is activated in fibroblasts. The reference level can be the activation level of TNF signaling in skin cells (e.g., fibroblasts) of a person who does not have PN. Alternatively, the reference level can be the activation level of TNF signaling in non-lesional skin cells of the subject.

[0258] 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.

[0259] The present disclosure also provides a method for treating or preventing prurigo nodularis (PN) in a subject, comprising administering an anti-IL-31RA antibody to a subject suffering from PN, wherein treatment with the anti-IL-31RA antibody results in a decrease in activation of the tumor necrosis factor (TNF) pathway. In some embodiments, the decrease in TNF pathway activation occurs in the subject's lesional skin. In some embodiments, the decrease in TNF pathway activation occurs in the subject's fibroblasts.

[0260] As indicated by the present disclosure, overlapping signaling may often be involved in the pathogenesis of PN. Therefore, for the purposes of the disclosed methods, treatment further results in:

[0261] (a) Decreased leukocyte migration or cell movement of leukocytes;

[0262] (b) inhibiting the STAT3 pathway;

[0263] (c) inhibiting the STAT5b pathway;

[0264] (d) downregulation of IL-1 or IL-1 pathway;

[0265] (e) downregulation of IL-6 or IL-6 pathway;

[0266] (f) downregulation of VEGF or VEGF pathway;

[0267] (g) decreased activation of the TGFB1 pathway, or

[0268] (h) combinations thereof.

[0269] In some embodiments, the reduction in leukocyte migration or leukocyte cell motility; (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 activation of the TGFB1 pathway, 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.

[0270] In some embodiments, (a) decreased leukocyte migration or leukocyte cell motility; (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 activation of the TGFB1 pathway, or (h) a combination thereof is assessed after about 4 weeks, about 8 weeks, or about 12 weeks following administration of the anti-IL-31RA antibody.

[0271] In some embodiments, (a) decreased leukocyte migration or leukocyte motility; (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 activation of the TGFB1 pathway, or (h) a combination thereof is determined by mass spectrometry analysis of one or more biological samples obtained from the subject. In some embodiments, the one or more biological samples are plasma samples or skin samples.

[0272] 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 cell motility 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; and (g) decreased activation of the TGFB1 pathway.

[0273] D. Dosages and Administration Regimens for the Disclosed Methods and Uses

[0274] An effective amount of an anti-IL-31RA antibody (such as nemolizumab) or a fragment or variant thereof is an amount sufficient to achieve a beneficial or desired result (such as alleviating at least one or more symptoms of PN). As used herein, an effective amount also includes an amount sufficient to delay or prevent the onset of pruritus, alter the course of PN symptoms, or reverse PN symptoms. Therefore, it is not possible to specify an exact "effective amount." However, for any given situation, one of ordinary skill in the art can determine an appropriate "effective amount" using only routine experimentation.

[0275] The effective amount can be administered in one or more doses, applications, or dosage forms. Such delivery depends on many variables, including the time period over which a single dosage unit is used, the bioavailability of the therapeutic agent, the route of administration, etc. However, it should be understood that the specific dosage level of the therapeutic agent disclosed herein for any particular subject depends on a variety of factors, including the activity of the specific compound employed, the subject's age, weight, general health, sex, and diet, time of administration, rate of excretion, drug combination, and the severity and form of administration of the specific condition being treated. Generally, therapeutic and preventive doses can be titrated to optimize safety and efficacy. The dosage can be determined by a physician and adjusted as necessary to accommodate the observed therapeutic effect. Typically, the dose-effect relationship of in vitro and / or in vivo testing can initially provide useful guidance for the appropriate dosage administered to the patient. Generally speaking, it is desirable to administer a compound that effectively achieves an amount of serum levels commensurate with the effective concentration found in vitro. The determination of these parameters is fully within the skill of the art. These considerations, as well as effective formulations and administration procedures, are well known in the art and are described in standard textbooks.

[0276] The dosage regimen for treating or preventing PN can comprise steady administration (i.e., repeated administration of the same dose at predetermined time intervals) or comprise a loading dose (i.e., administration of an initial dose that is higher than or different from subsequent continuous doses). With regard to any type of dosage regimen, the effective dose can be administered topically, parenterally, subcutaneously, subdermally, intradermally, or intramuscularly. In a preferred embodiment, administration comprises subcutaneous injection.

[0277] In some embodiments, the loading dose and subsequent continuous doses can be administered by the same route (e.g., subcutaneously), while in some embodiments, the loading dose and subsequent continuous doses can be administered by different routes (e.g., parenteral and subcutaneously, respectively). In some embodiments, the loading 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 loading dose may 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 can be 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 In some embodiments, the present invention provides an oral dosage of at least about 1 mg / kg, about 1.8 mg / kg, about 1.9 mg / kg, about 2 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 can be 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, 2.1 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. In some embodiments, the loading dose is administered as a single injection. In some embodiments, the loading dose is administered as multiple injections, which can be administered simultaneously or separately at defined time intervals.

[0278] The subsequent continuous dose of the loading dose regimen is generally lower than the loading dose. For example, in some embodiments, the dosage regimen can include a loading dose of 60 mg and a continuous dose of 30 mg, which can be administered at a limited interval of, for example, once every 4 weeks. In certain embodiments, the continuous dose of the dosage regimen 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 higher. 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 continuous dose can be 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 In some embodiments, the present invention provides an oral dosage of at least about 1 mg / kg, about 1.8 mg / kg, about 1.9 mg / kg, about 2 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 continuous dose can be 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, 2.1 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. 7mg / kg, 1.8mg / kg, 1.9mg / kg, 2mg / kg, 2.1mg / kg, 2.2mg / kg, 2.3mg / kg, 2.4mg / kg, 2.5mg / kg, 2.6mg / kg, 2.7mg / kg, 2.8mg / kg, 2.9mg / kg, 3mg / kg, 3.5m g / kg, 4mg / kg, 4.5mg / kg, 5mg / kg, 6mg / kg, 7mg / kg, 8mg / kg, 9mg / kg, 10mg / kg, 15mg / kg, 25mg / kg, 50mg / kg, 75mg / kg, 100mg / kg, 500mg / kg or 1,000mg / kg.

[0279] In terms of the loading dose regimen, the first continuous dose can 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 certain embodiments, the first continuous dose is administered 4 weeks after the initial loading dose. In certain embodiments, subsequent continuous doses are administered once 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 certain embodiments, continuous doses are administered once every 4 weeks (i.e., nemolizumab or its fragment or variant is administered once every 4 weeks).

[0280] In some embodiments, the dose of nemolizumab or a fragment or variant thereof administered to a subject can be in the range of 0.001 to 1,000 mg / kg of the subject's body weight. In some embodiments, the dose range is about 0.01 mg / kg 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 mg / kg 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, about 1

[0014] In some embodiments, the present invention provides an antibody or fragment thereof in an amount of from about 1 mg / kg to about 3.5 mg / kg, from about 1 mg / kg to about 5 mg / kg, from about 2 mg / kg to about 4 mg / kg, from about 2.5 mg / kg to about 10 mg / kg, from about 5 mg / kg to about 10 mg / kg, from about 10 mg / kg to about 20 mg / kg, from about 10 mg / kg to about 40 mg / kg, from about 20 mg / kg to about 50 mg / kg, from about 25 mg / kg to about 75 mg / kg, from about 50 mg / kg to about 100 mg / kg, or from about 100 mg / kg to about 500 mg / kg, or from about 100 mg / kg to about 1000 mg / kg of body weight of nemolizumab or a fragment or variant thereof. In preferred embodiments, the dose range is 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.In some embodiments, the 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. / 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.

[0281] In some embodiments, the dose of nemolizumab or its fragment or variant 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, 15 to 85 mg, or a 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 may 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.

[0282] In some embodiments of the disclosed methods and uses, 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 subsequent consecutive doses of about 30 mg of nemolizumab, or a fragment or variant thereof, administered once every 4 weeks. In some embodiments of the disclosed methods and uses, 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 subsequent consecutive doses of about 60 mg of nemolizumab, or a fragment or variant thereof, administered once every 4 weeks (i.e., the dose remains constant or is a "steady-state" dosing regimen). In some embodiments of the disclosed methods and uses, 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 subsequent consecutive doses of about 30 mg of nemolizumab, or a fragment or variant thereof, administered once every 4 weeks.

[0283] In some embodiments of the disclosed methods and uses, nemolizumab or a fragment or variant thereof is administered topically or parenterally. In some embodiments, nemolizumab or a fragment or variant thereof is administered subcutaneously. In some embodiments, the dose is administered subcutaneously at or near one or more nodules, lesions, or excoriations.

[0284] In some embodiments of the disclosed methods and uses, 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 10 weeks, once every 11 weeks, once every 12 weeks, twice a year, once a year, and / or as needed based on the onset of PN symptoms. In a preferred embodiment, nemolizumab, or a fragment or variant thereof, is administered once every four weeks or every eight weeks.

[0285] In some embodiments of the disclosed methods and uses, the duration of treatment or prevention is about one day, about one week, about two weeks, about three weeks, about four weeks, about five weeks, about six weeks, about seven weeks, about eight weeks, about nine 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 one year, about two years, about three years, about four years, about five years, or as needed based on the onset of PN symptoms. In preferred embodiments, the duration of treatment or prevention is about 12 weeks to about 24 weeks, about 12 to about 36 weeks, about 12 to about 48 weeks, or about 24 to about 36 weeks.

[0286] The present disclosure provides uses of nemolizumab or a fragment or variant thereof for 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 disclosed doses, dosing regimens, routes of administration, biomarkers, and therapeutic endpoints also apply to these uses.

[0287] The following examples are given to illustrate the present disclosure. It should be understood that the invention is not to be limited to the specific conditions or details described in these examples.

[0288] Examples

[0289] Example 1 - Treatment of prurigo nodularis with nemolizumab

[0290] method

[0291] Patient groups

[0292] 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 administered at a dose of 0.5 mg / kg body weight at baseline, week 4, and week 8. Peak pruritus scores on a numeric rating scale (PP-NRS) were recorded: the severity of pruritus on the numeric rating scale ranges from 0 (no itch) to 10 (worst itch imaginable), and peak pruritus was estimated using the worst score per 24 hours over a 7-day period, with the highest score recorded as the peak score. The primary outcome of the study was the percentage change from baseline in the PP-NRS at week 4.

[0293] Reconstructed Human Epidermis (RHE) Model

[0294] Generate a three-dimensional RHE model. Briefly, RHE cultures were generated using normal human dermal fibroblasts (NHDF) and normal human epidermal keratinocytes (NHEK). The RHE cultures used were full-thickness, with both dermis and epidermis, and were composed of autologous fibroblasts and keratinocytes. They were scaffold-free (no exogenous matrix), allowing the cells to self-assemble into the different skin layers and avoiding some potential inhibitors contained in the collagen matrix for RNA extraction. RHE was grown in a 1.2 cm insert. 2 ) in 12-well plates for 42 days to obtain a minimum of two dermal layers and six epidermal layers. RHE cultures from six different donors were left unstimulated or stimulated with seven different cytokines or cytokine combinations (three replicates per condition per donor): 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: IL-31 (500 ng / mL), IL-13 (100 ng / mL), IL-17A (200 ng / mL), and IFNg (50 ng / mL). 72 hours after stimulation, RHE cultures were lysed and RNA was extracted using the MagMAX mirVana Tissue Total RNA Isolation 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 OneRNA kit (Promega) on the GloMax-Multi+ detection system (Promega). Library preparation was performed using the Stranded mRNA Prep Ligation Kit (Illumina) according to the manufacturer's recommendations.

[0295] Skin biopsy processing and RNA isolation

[0296] Skin biopsies were collected from 16 placebo- and 15 nemolizumab-treated subjects. The samples included lesional and nonlesional biopsies at baseline and lesional samples after 12 weeks of treatment (placebo or nemolizumab). RNA was extracted from the skin biopsies using Tripure isolation reagent (Sigma-Aldrich) according to the manufacturer's instructions. In these RNA samples, DNase treatment was applied using the RNase-Free DNase Kit (Cat. No. 79254, Qiagen) and then Cleanup kit (Cat. No. 74204, Qiagen).

[0297] Total RNA was quantified using the QuantiFluor One RNA Kit (Promega). The resulting RNA concentration ranged from 4 to 20 ng / μl. Total RNA was identified using the Fragment Analyzer 5300 (Agilent) and the Agilent HS RNAFragment Kit (Agilent). The resulting RNA quality number (RQN) ranged from 1 to 6. Library preparation was performed using the SMARTer Stranded Total RNA-Seq Kit V2 - Pico Input Mammalian Kit (TaKaRa).

[0298] RNA-Seq

[0299] The library was quantified using the QuantiFluor One dsDNA kit (Promega) and analyzed using the FragmentAnalyzer 5300 (Agilent) with the Agilent HS NGS Fragment kit (Agilent). After size selection with AMPure XP beads, the shotgun library was sequenced using NextSeq (Illumina) on an Illumina NextSeq500 sequencer at 2 × 75 bp (High Output Kit v2, 150 cycles).

[0300] RNA-seq data processing

[0301] After adapter trimming, sequence reads from 83 unique samples were aligned to the human genome (GRCh37) using STAR. Gene (GENCODEv29) expression levels were subsequently quantified using HTSeq using reads uniquely mapped to a genomic location. 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. Expression normalization was performed using DESeq2, and expression levels were modeled using a negative binomial distribution for differential expression analysis. For comparisons of non-lesional vs. lesional and baseline vs. week 12, individual effects were included as covariates; for comparisons of placebo vs. nemolizumab, age and sex were controlled. A false discovery rate (FDR) ≤ 5% and |log2 fold change| > = 1 were used as criteria for declaring significantly differentially expressed genes (DEGs).

[0302] Cytokine, cell signature, and functional inference analysis

[0303] The most significant DEGs were compared to cytokine-induced transcripts in keratinocytes (defined by FDR <= 10% and 1.5-fold change (FC)). The top 1,000 most significant DEGs from the PN data were used for fair comparison. To compare with the epidermal compartment gene signature, scRNA-seq was performed on the epidermal layer of skin biopsies, and the top 50 marker genes for the basal layer, differentiated layer, and cornified layer were identified separately. The effect size (in log2FC) of each marker gene was then studied 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 the enrichment statistics for transcription factor binding were calculated using the MEME suite.

[0304] Weighted gene correlation network analysis (WGCNA)

[0305] Genes expressed in at least 20% of the samples were used in the WGCNA dataset. The "softPower" parameter was selected as the minimum value to achieve at least r² >= 0.75. Correlations were calculated using Spearman correlation, and the minimum module size was set to 100. When merging modules, a height cut of 0.2 was used.

[0306] result

[0307] Prurigo nodularis is characterized by abnormal keratinocyte differentiation and immune activation

[0308] After quality control, RNA sequencing data were analyzed from biopsies of patients with prurigo nodularis (PN). Transcriptome data were available for both lesional and nonlesional skin samples from 31 PN patients. Using a false discovery rate (FDR) ≤ 10% and |log2| ≥ 1 as criteria, 5,943 differentially expressed genes (DEGs) were identified when comparing non-involved skin and lesional skin at baseline, of which 2,060 genes were increased and 3,874 genes were decreased ( Figure 1 A). Genes showing the most robust increase included KRT6C (588-fold, FDR = 8.2 × 10 -80 ), DEFB4A (150-fold, FDR = 1.1 × 10 -12 ) and KRT16 (90-fold, FDR = 1.9 × 10 -52 Reduced genes included LCE5A (reduced 11-fold, FDR = 8.1 × 10 -18 ) and AQP7 (reduced 7.9-fold, FDR = 2.6 × 10 -17 For cytokines, the most prominent upregulated genes included IL36 family members: IL36A (6.8-fold, FDR = 1.8 × 10 -4 ) and IL36G (8.4-fold, FDR = 3.9 × 10 -25 ); IL-20 family members: IL19 (5.1-fold, FDR = 7.4 × 10 -4 ), IL20 (3.5 times, FDR=1.7×10 -3 ), IL22 (2.7 times, FDR=2.9×10 -2 ), IL24 (5.8 times, FDR=3.8×10 -10 ) and IL26 (4.9-fold, FDR = 3.3 × 10 -3 Other factors included IL1A (4.7-fold, FDR = 1.0 × 10 -12 ) and IL1B (4.1-fold, FDR = 3.7 × 10 -6 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 of this specification).

[0309] Functional enrichment analysis of the DEGs was then performed to define biological processes associated with PN skin. The most prominent gene ontology (GO) terms included: “keratinized 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 α / β signaling” and “response to interferon γ” (FDR = 4.1 × 10 -7 and FDR = 4.1 × 10 -6 ), “IL23 pathway” (FDR = 2 × 10 -5 ) and “mitosis metaphase and anaphase” (FDR = 3.8 × 10 -10 )( Figure 1 B) (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; by focusing on key expression modules of the lesion skin transcriptome, we revealed the proliferation marker Ki67 (MKI67), the cell cycle gene CDKN1A, and an inflammatory network involving IL-1 and IL-36 ( Figure 1 C).

[0310] To better understand the disease regulatory networks involved in PN skin, weighted gene co-expression network analysis (WGCNA) was performed. Twenty co-expression modules were identified in non-lesional skin and 10 clusters were identified in lesional PN skin ( Figure 1 D) (see Table 3 at the end of the Examples section of this specification). This analysis allowed the inventors to assign different functions to these co-expressed gene modules, especially those in PN lesional skin, the most prominent of which involved immune processes (module no. 8), including "keratin response" (FDR = 1.8 × 10 -47 ), “defense response” (FDR = 1.2 × 10 -39 ); Cell proliferation (module 6) including “cell cycle” (FDR = 2.9 × 10 -94 ), “DNA metabolic process” (FDR = 8.7 × 10 -67 ); and epidermal processes (module 5), such as “epidermal development” (FDR 3.5×10 -10 ), “keratinization” (FDR = 1.7 × 10 -6 )( Figure 1 E). Other notable findings were changes in the extracellular matrix (FDR = 1.16 × 10 -59 ; module no. 2) and included genes such as MMP14, MMP16, COL1A1, COL1A2, and COL3A1, which were moderately elevated in lesional skin (FC ≥ 1.4; FDR ≤ 6 × 10 -2 ), which is consistent with the association between PN and skin fibrosis.

[0311] Transcriptome changes in PN lesions are enriched for keratinocyte and T cell signatures

[0312] A computer simulation approach (xCell) was used to infer specific cell type signatures for each non-lesional and lesional PN skin sample. Enrichment of transcriptomic signatures associated with epithelial cells and keratinocytes was observed (p < 0.001 and p < 0.0001, respectively) ( Figure 2 A). Th2-related features were also more prominent (p<0.0001), consistent with the enriched GO categories of IL-4 / IL-13 ( Figure 1 B). Other inflammatory features, such as macrophages (p<0.01), showed greater changes ( Figure 2 A). To address the relationship between PN and other hyperproliferative skin diseases that also have strong inflammatory features, the PN transcriptome was compared with the transcriptomes of atopic dermatitis (AD) and psoriasis. In a 3-way comparison, a large number of common genes were found between all three diseases for both up-regulated and down-regulated genes ( Figure 2 B). The correlation of effect sizes in lesional skin was more pronounced between PN and psoriasis (Spearman correlation p = 0.64) than between PN and AD (p = 0.55). Genes commonly upregulated in psoriasis and PN included genes involved in cytokine activity (CCL3, CXCL10, IFNG, IL12B, IL19, IL1B, IL20, etc.) and keratinization (KRT16, KRT17, LCE3A, LCE3E, etc.). See Table 4 at the end of the Examples section of this specification.

[0313] Transcriptome changes in PN skin treated with the IL31 receptor inhibitor nemolizumab

[0314] Clinical results for PN patients showed that nemolizumab resulted in a higher percentage of improvement in pruritus and skin lesions, with an overall good safety profile. At week 12, a significantly higher proportion of patients in the nemolizumab group achieved a mean weekly reduction of 4 points on the PP-NRS compared to placebo (52.9% vs. 8.3%, p < 0.001). At week 12, a significantly higher proportion of subjects in the nemolizumab group achieved IGA success (defined as IGA 0 [clear] or 1 [almost clear]) compared to placebo (20.6% vs. 2.8%, p = 0.02).

[0315] To address the therapeutic effect of the IL-31 receptor (IL-31R) inhibitor nemolizumab, RNA-seq data were obtained from PN biopsies before and after 12 weeks of treatment with placebo control in a double-blind study. At baseline, there were 16 and 15 individuals in the placebo and nemolizumab groups, respectively, and lesion samples were obtained from 18 patients (11 placebo and 7 nemolizumab) at week 12. Using principal component analysis (PCA), it was observed that the samples of PN patients were mixed between the two treatment groups (placebo vs. nemolizumab) at baseline. After 12 weeks of treatment, there was a trend towards grouping in the nemolizumab group, but not in the placebo group ( Figure 3 A). This was accompanied by sample clustering using genes identified as differentially expressed in the non-lesional versus lesional skin comparison, where 6 of 7 nemolizumab samples (86%) at week 12 were grouped with baseline non-lesional skin samples, compared to 57% (4 of 7) in the placebo group ( Figure 3 B). Notably, compared with placebo, nemolizumab treatment normalized a greater number of PN-related DEGs, namely genes upregulated in PN lesional skin (969 with nemolizumab vs. 211 with placebo) and genes downregulated in PN lesional skin (1,268 with nemolizumab vs. 166 with placebo) ( Figure 4 A). This is also reflected in the correlation between placebo- and nemolizumab-treated DEGs, with much greater overlap in increased and decreased DEGs between the nemolizumab-treated group and PN compared to placebo and PN ( Figure 4 B). GO categories enriched in the DEGs that were reduced only in the nemolizumab-treated group by week 12 included “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 ), while none of these GO categories were found in the placebo control group (see Table 2 at the end of the Examples section of this specification). These data suggest that nemolizumab treatment, in addition to reducing inflammatory responses, particularly those associated with Th2 responses, also normalizes epidermal hyperproliferation and differentiation.

[0316] Nemolizumab response is accompanied by a reduction in IL-31 / Th2 responses in PN skin

[0317] To address the role of nemolizumab treatment on the inflammatory response in PN skin, we interrogated the cytokine responses produced in RHE cultures and human epidermal rafts in response to nemolizumab and placebo. Consistently reduced IL-31 responses were observed, either alone or in combination with other inflammatory cytokines, including the Th2 cytokines IL-13 or IL-17A ( Figure 5 A), providing clear evidence that nemolizumab blocks the IL-31 pathway. Notably, IL-17A response genes were enriched in PN skin, possibly corresponding to specific downstream immune cascades that overlap between psoriasis and PN (Table 4). IL17A mRNA expression itself was not significantly different in non-lesional and lesional skin, or in nemolizumab treatment by week 12 (see Table 1 at the end of the Examples section of this specification), indicating that although IL-17A is not the main cytokine in PN, IL-17A is characterized as downstream of IL-31 signaling. In terms of cellular transcriptome changes, a more robust reduction was observed in the nemolizumab group, including Th1 and Th17 ( Figure 5 B).

[0318] To identify the tissue compartments that contribute most to the cellular response against IL-31R blockade, transcriptomic data from the placebo and nemolizumab groups were compared with gene signatures of the epidermal compartment obtained from single-cell RNA-seq data. The results showed that the basal keratinocyte (KRT14+) signature was elevated in PN lesional skin, which was restored to a similar extent in both the placebo and treatment groups, while the induction of the spinous (KRT10+) signature in PN lesional skin was restored only by treatment but not by the placebo group ( Figure 5 C). Transcription factor binding site (TFBS) analysis was then performed to further understand the transcriptional regulators underlying transcriptome changes following PN and placebo or nemolizumab treatment. The results showed that binding sites enriched in genes upregulated in lesional skin at baseline were more likely to be enriched in genes downregulated by nemolizumab at week 12 ( Figure 5 D). The most significantly regulated transcription factors included EGR4 (p = 4.5 × 10 enriched in the promoters of up-regulated and nemolizumab-downregulated genes, respectively). -6 and p = 1.2 × 10 -8 , ), a member of the EGF family of zinc finger transcription factors; STAT3 (p = 2.2 × 10 -4 and p = 2.5 × 10 -5 ); and KLF16 (p = 4.5 × 10 -5 and p = 2 × 10 -5 ) (See Table 5 at the end of the Examples section of this specification).

[0319] Nemolizumab reduced pruritus scores

[0320] Peak Pruritus Numeric Rating Scale (PP-NRS) PP-NRS was correlated with transcriptome data ( Figure 6 A). Although the PP-NRS was similar in both groups at baseline, pruritus was consistently reduced only in the nemolizumab group, whereas a wide range of responses was observed in the placebo group. In addition, although all patients in the nemolizumab group showed improvement, only a subset of patients in the placebo group showed significant changes. Notably, the measured distance between principal components 1 and 2 (PC1 / PC2) was much smaller in the nemolizumab-treated group than in the placebo group ( Figure 6 B), consistent with treatment response.

[0321] Discussion

[0322] The data presented here provide the first comprehensive understanding of the global transcriptome 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 reflect at the transcriptomic level many of the hallmark histological changes observed in PN, including epidermal alterations, inflammatory responses ( Figure 2 A-2C), fibrosis and pruritus ( Figure 6 A-6B), and how these were normalized with nemolizumab treatment.

[0323] Many gene expression changes in PN skin are associated with abnormal keratinocyte proliferation and differentiation ( Figure 1 A-1E). These epidermal changes account for most of the overlap between PN and AD and psoriasis ( Figure 2 A-2C; Table 4), AD and psoriasis are also characterized by marked epidermal hyperplasia and altered epidermal differentiation. Notably, these changes showed significant improvement by week 12 in the nemolizumab-treated group but not in the placebo group (Table 2). Furthermore, consistent with the therapeutic effect of nemolizumab, the greatest normalization effect was observed in the differentiated layers of the epidermis ( Figure 5 C), which may reflect a decrease in keratinocyte proliferation and restoration of normal epidermal differentiation.

[0324] Consistent with PN being an inflammatory-driven disease process, immune responses such as Th2 (IL-4 / IL-13) responses and type I and II IFN responses are prominent ( Figure 1 A-1E). Th2 responses are closely associated with pruritus in diseases such as atopic dermatitis, a common predisposing disease for the development of PN. Interestingly, anti-IL-31 receptor inhibition not only significantly reduced the IL-31 response of keratinocytes in PN skin ( Figure 5 A), but also reduced Th2 response and Th17 ( Figure 5 B), corresponding to a decrease in IL-13 and IL-17 responses in keratinocytes ( Figure 5 A). The enriched IL-17 response in PN skin and the contribution of IL-36 may explain the greater overlap between PN skin and plaque psoriasis compared with AD ( Figure 2 (B and 2C). No significant changes in IL17A mRNA expression were observed in the PN data, suggesting that IL-17A is not a major cytokine in PN. In addition, changes in Th2 and Th17 responses, and to a lesser extent, type II IFN responses, with nemolizumab treatment suggest that these cytokines act downstream of IL-31 in PN.

[0325] Fibrosis is a characteristic 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 gene module involved in extracellular matrix biology was found to be enriched in PN skin (Table 3), involving collagen 1 and collagen 3 genes. COL1A1, COL1A2, and COL1A3 mRNA were increased in PN skin at baseline (1.7-fold, 1.44-fold, and 1.52-fold, respectively, see Table 1) but did not show significant changes with nemolizumab treatment at week 12.

[0326] This data also demonstrates how the transcriptome changes in PN driven by nemolizumab are associated with improvements in pruritus. Chronic pruritus is a debilitating symptom of PN and has a profound impact on quality of life. The cause of pruritus in PN remains unclear, but possible factors include Th2 cytokines, IL-4 and IL-13 (the main pruritogens in atopic dermatitis) and changes in skin innervation, where a decrease in the density of nerve fibers within the epidermis has been shown to be reduced in both lesioned and non-lesioned skin. The data from nemolizumab treatment are consistent with these two scenarios that cause pruritus. Therefore, nemolizumab treatment causes inhibition of Th2 and IL-4 / IL-13 responses in PN skin, and also causes a decrease in the expression of factors such as KLF16, which has been shown to inhibit neurite outgrowth. In addition, it has been shown that nerve growth factor (NGF), which is increased in PN skin, also reached a greater degree of normalization (Table 1) than in the placebo group at week 12 with nemolizumab treatment. There was no change in the expression of CGRP or substance P (TAC1). These data are highly suggestive of a broad effect of nemolizumab on pruritus and may explain the long duration (>2 months) of pruritus improvement seen after the last dose of nemolizumab.

[0327] In summary, PN is a debilitating and difficult-to-treat condition, and there are currently no approved therapies for its treatment. This study deeply characterized the transcriptome changes in PN skin and demonstrated the broad mechanism of action of the anti-IL-31 receptor inhibitor nemolizumab. These data demonstrate the broad therapeutic effect of anti-IL-31 receptor inhibition with nemolizumab on multiple aspects of PN pathogenesis, including epidermal differentiation, inflammatory responses, pruritus, and extracellular remodeling, and confirm the upstream role of IL-31 in PN pathogenesis.

[0328] Example 2 - Plasma proteome analysis of patients with moderate to severe prurigo nodularis treated with nemolizumab

[0329] This example details a randomized, double-blind, phase 2 trial of the anti-human IL31Ra blocking antibody nemolizumab administered subcutaneously at a dose of 0.5 mg / kg body weight versus placebo in patients with moderate to severe prurigo nodularis at baseline, week 4, and week 8. The goal of this study was to characterize the effects of nemolizumab on the entire plasma proteome by using mass spectrometry.

[0330] Materials and methods

[0331] Patient dataset

[0332] Nineteen (19) placebo non-responders and nineteen (19) nemolizumab responders were selected based on the change in PP-NRS (peak pruritus score on a numeric rating scale) clinical score at Week 12 (19 placebo patients had a PP-NRS change ≥ -1.4 and 19 nemolizumab patients had a PP-NRS change < -5.4). The PP-NRS scores at baseline and after 12 weeks of nemolizumab treatment are shown in Figure 10 middle.

[0333] plan

[0334] Following plasma gY14 over-depletion, samples were analyzed using Proteome Science's TMT calibrator MS2 workflow. A pool of healthy skin biopsies was used to 1) allow batch calibration between clusters and 2) trigger the mass spectrometer to detect skin-associated peptides in blood.

[0335] statistics

[0336] 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 list of differentially expressed proteins: 1) least squares regression (including and excluding patient ID), 2) robust regression, and 3) generalized 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 on the 193 proteins found to be differentially expressed using QIAGEN IPA (QIAGEN Inc., digitalinsights.qiagen.com / IPA).

[0337] result

[0338] The list of 193 differentially expressed proteins was then analyzed using the enrichment software IPA. While the p-value indicates the significance of the affected pathway in the dataset, the z-score provides insight into the directionality, i.e., how "activated" or "repressed" the pathway is in the dataset.

[0339] Canonical paths are sorted only by z-score ( Figure 11 right), or sorted by z-score and filtered by p-value ( Figure 11 Left; -log(p-value)>1.3, corresponding to p-value<0.05). IL-31 has been shown to induce STAT3 activation. Although STAT3 activation is not specific for IL-31, the downregulation of this pathway in nemolizumab responders suggests target engagement of nemolizumab. Furthermore, the nemolizumab responder profile was characterized by downregulation of the IL-6 pathway compared to placebo non-responders. Additionally, the vascular endothelial growth factor (VEGF) pathway, which has been shown to be associated with PN severity, was downregulated in nemolizumab responders, potentially reflecting clinical improvement. Finally, two neuronal entities (“CREB signaling in neurons” and “synaptogenesis signaling pathway”) were identified as downregulated in nemolizumab responder subjects, highlighting the impact of IL-31 as a neuroinflammatory cytokine in PN.

[0340] Upstream regulator analysis allows the identification of transcription factors and small molecules whose functions are affected by disease or treatment, depending on the dataset being analyzed. Although the proteins identified in this analysis may not themselves be differentially expressed in the dataset under investigation, their activation / inhibition status may explain the expression changes in the dataset. Canonical pathways are ranked solely based on z-scores ( Figure 12 right), or sorted by z-score and filtered by p-value ( Figure 12Left; p-value < 0.05). Profibrotic transforming growth factor β1 (TGFβ1) was found to be a potential regulator of the nemolizumab responder signature, potentially indicating regression of fibrotic nodules. Another potential regulator was signal transducer and activator of transcription 5b (STAT5b), which may reflect engagement of the nemolizumab target. These data clearly demonstrate that nemolizumab strongly influences inflammatory and tissue remodeling processes in PNs.

[0341] Biofunctional analysis of nemolizumab responders revealed down-regulation of "leukocyte migration" and "leukocyte cell motility." Additionally, down-regulation of two neuronal axis-related pathways, "glial cell death" and "glial cell apoptosis," may reflect improvements in pruritus. Figure 13 ).

[0342] Discussion

[0343] The goal of this example is to conduct a broad and exploratory analysis to identify plasma protein biomarkers that could also explain the mechanism of action of nemolizumab treatment.

[0344] To characterize the full range of plasma proteomic changes, a preselected group of nemolizumab responders and placebo non-responders were assessed. Due to this preselection of patients, the features described in this example likely reflect not only nemolizumab-induced changes but also changes due to a decrease in PP-NRS.

[0345] The dataset analyzed in this study revealed that the nemolizumab responder signature is characterized by improvements in various aspects of PN pathophysiology, including inflammation, neuroimmune function, and tissue remodeling.

[0346] Enrichment analysis revealed that the nemolizumab responder signature is characterized by reduced leukocyte migration and cell motility. The STAT3 pathway, a direct target of IL-31 signaling, was also suppressed in nemolizumab responder subjects, suggesting target engagement. Nemolizumab responders also showed downregulation of other proinflammatory cytokine pathways, including the IL-6 and VEGF pathways.

[0347] Pathway analysis also revealed the effects of nemolizumab treatment on neuronal-related processes, including "CREB signaling in neurons" and "synaptogenesis signaling pathway," "glial cell death," and "glial cell apoptosis," consistent with the observed clinical effects of nemolizumab on pruritus.

[0348] Finally, the dataset showed a reduction in TGFB1 pathway activation, suggesting an effect of nemolizumab treatment on tissue remodeling.

[0349] These observations strengthen the understanding that plasma proteomics can effectively capture, at least in part, the tissue-specific (ie, skin) effects of nemolizumab in PN.

[0350] Example 3 - Fibroblasts participate in the pathogenesis of PN

[0351] Single-cell and bulk RNA sequencing (RNAseq) were combined to characterize the molecular and cellular features of PN and to investigate the impact of nemolizumab in treated patients. Single-cell RNAseq (scRNAseq) was performed on skin biopsies obtained from four PN patients and four healthy controls. Figure 14 and 15 The role of fibroblasts in the pathogenesis of PN was further investigated by cell-cell communication analysis using CellChat. Batch RNAseq was performed on skin biopsies obtained at baseline and week 12 from PN patients (n=70) in a phase 2 study of nemolizumab.

[0352] scRNAseq results showed that compared with healthy skin, pathological PN fibroblasts showed a pro-fibrotic and pro-inflammatory state, as confirmed by trajectory analysis, reflecting the altered differentiation of fibroblasts in PN skin ( Figure 16 Consistent with this, functional analysis of differentially expressed genes in diseased PN fibroblasts indicated activation of inflammatory (TNF, IL1B, IL6) and pro-fibrotic (TGFβ) signaling pathways. CellChat results highlight the role of PN fibroblasts as central players in this intracellular crosstalk ( Figure 17 Combining bulk and single-cell RNAseq data, we observed that nemolizumab restored neural dysregulation and reduced inflammation and fibrosis ( Figure 18 ).

[0353] This study demonstrates that PN is an inflammatory and fibrotic disease and that nemolizumab exerts its anti-fibrotic effects by inhibiting key signaling pathways.

[0354] Example 4 - scRNA-seq in Prurigo nodularis (PN)

[0355] summary

[0356] Background: Prurigo nodularis (PN) is a chronic neuroimmune skin disease characterized by pruritic hyperkeratotic nodules distributed symmetrically on the limbs and trunk. Neuroimmune dysregulation and chronic scratching are believed to induce and maintain the characteristic lesions.

[0357] Objective: This study aims to gain a comprehensive understanding of the molecular pathogenesis of PN at the single-cell level to identify and outline key pathological processes and the cell types involved. Features that distinguish PN skin from skin of patients with atopic dermatitis (AD) are of particular interest. An additional objective is to determine the effects of the interleukin-31 (IL-31) receptor α antagonist nemolizumab and its specificity at the single-cell level.

[0358] Methods: Using the 10X Visium platform, single-cell RNA sequencing (scRNA-seq) was performed on skin from 15 healthy donors and nonlesional and lesional skin from 6 patients with PN and AD, respectively, combined with spatial sequencing (spatial-seq). The data were integrated with bulk RNA-seq data from patients treated with nemolizumab.

[0359] Results: The results described in this example demonstrate that PN is an inflammatory skin disease characterized by keratinocyte proliferation and activation of a profibrotic response. This example demonstrates that a subpopulation of COL11A1+ fibroblasts is a major contributor to fibrosis and is primarily present in the papillary dermis of PN skin. Activation of the fibrotic response is a major distinguishing feature between PN and AD skin. This example further demonstrates the broad impact of nemolizumab on PN cell types, with prominent effects driving responses of COL11A1+ fibroblasts and keratinocytes toward normalization.

[0360] Conclusions: This study 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 impact of nemolizumab on pathological processes in PN skin.

[0361] introduce

[0362] Prurigo nodularis (PN) is a chronic neuroimmune skin disease characterized by chronic and intense itch, which significantly impacts quality of life. Clinically, it is characterized by multiple nodules that can cover large areas of the limbs and trunk. The pathogenesis of PN remains unclear, but previous studies have implicated immune and neural dysregulation as key circuits in its pathogenesis. It has been proposed that PN shares clinical and pathological overlap with atopic dermatitis (AD), potentially sharing a common T helper 2 (Th2) polarization. However, direct comparisons of these two conditions at the single-cell level have not been performed. Indeed, in previous studies, bulk RNASeq analysis revealed a distinct molecular signature of PN compared with AD. The best-characterized immune mediators in PN are the cytokines IL-31 receptor α (IL-31RA) and oncostatin M receptor β (OSMRB). Activated Th2 cells are considered the primary source of IL-31, but other cell types can also produce IL-31, including eccrine sweat glands, mast cells, basophils, eosinophils, and monocytes / macrophages. Recent studies of human PN lesions have demonstrated that, in addition to T cells, macrophages are the major cellular source of IL-31. The critical importance of IL-31 for the pathogenesis of PN has been demonstrated using nemolizumab, an investigational monoclonal antibody that inhibits the IL-31RA, which effectively inhibits downstream inflammatory responses, including Th2 responses and stabilization of extracellular matrix (ECM) remodeling. Recently published evidence suggests that fibrosis is a characteristic of PN, but the source of this fibrosis in PN skin is unknown. Major fibroblast populations have been described in human skin, with two major clusters characterized by expression of SFRP2 and FMO1, and five minor fibroblasts defined, including COL11A1+ fibroblasts, three of which (SFRP2+, FMO1+, and COL11A1+) are thought to have roles in matrix deposition, inflammatory cell retention, and connective tissue cell differentiation based on their gene expression profiles.

[0363] This example provides an in-depth exploration of PN pathogenesis to characterize the central mechanisms involved and identify target cells for nemolizumab treatment, and compares its pathogenesis with that of AD through a combination of single-cell RNA (scRNA) and spatial RNA sequencing approaches.

[0364] result

[0365] Single-cell RNA-seq and spatial-seq reveal distinct cell types and their spatial locations in PN skin

[0366] To understand the unbiased cellular composition and cellular states of healthy (H) skin and lesional PN (LPN) skin, single-cell suspensions from skin biopsies of 15 healthy donors and 6 PN patients were generated. Skin biopsies were also collected from peripheral non-lesional sites of 4 of the 6 PN patients (NPN), generating a total of 25 scRNA-seq libraries. The resulting quality-controlled PN plus 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 investigate the heterogeneity of these cells, variable genes were selected and uniform manifold approximation and projection (UMAP) dimensionality reduction and cell clustering were performed using the R package Seurat. Cluster annotations were confirmed by overlapping cluster markers with canonical lineage-specific genes reported in previous skin disease scRNA-seq studies. Ten major cell types ( Figure 19 A), including keratinocytes, melanocytes, exocrine gland cells, endothelial cells, fibroblasts, pericytes, neurons, T cells, bone marrow cells and mast cells. The majority of these cell types contained cells from most healthy, NPN and LPN libraries, indicating that each cell type is associated with a common cell lineage rather than originating from a specific condition. Two small cell populations, exocrine gland cells and neurons, were primarily derived from healthy samples. Interestingly, a clear separation was observed between keratinocytes, fibroblasts and endothelial cells in healthy, NPN and LPN cells, indicating major transcriptional differences ( Figure 19 B). Moderate changes in cell type ratios were observed in LPNs compared to NPNs and healthy controls, with the most pronounced changes in mast cells, endothelial cells, T cells, and myeloid cells in LPN skin ( Figure 19 C). Marker genes for each cell population show clear separation between each cell type ( Figure 19 D).

[0367] 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 the 10X Visium platform. 395 spatially defined spots were detected, with an average of 2,613 genes and 4,432 transcripts per spot ( Figure 20 Using a Seurat anchor-based marker transfer approach, spatial points were deconvolved by the major cell types detected in scRNA-seq. The deconvolved prediction score for each cell type is displayed on the tissue ( Figure 19 E) and combined into a scatter plot showing the relative cell type composition of each point ( Figure 20B). Keratinocytes are located in the epidermis and hair follicles. Myeloid cells and T cells are mainly located in the superficial dermis near the epidermis. Fibroblasts are distributed in most points in the dermis, and pericytes are located near blood vessels ( Figure 20 B). These two cell types are the main producers of extracellular matrix (ECM) components ( Figure 20 C) Other cell types represent minor populations and are less frequently detected in spatial-seq samples.

[0368] COL11A1+ fibroblasts are enriched in PN skin, leading to a profibrotic response

[0369] To characterize the heterogeneity of fibroblasts, all fibroblasts from the scRNA-seq dataset were sub-clustered. Based on previously published marker genes, the fibroblast sub-clusters were annotated into six subtypes, including SFRP2+ fibroblasts (FB), APOE+FB, RAMP1+FB, COL11A1+FB, TNN+FB, and SFRP4+FB ( Figure 21 A and Figure 20 Interestingly, COL11A1+FB was mainly derived from LPN samples compared with healthy or NPN samples ( Figure 21 B, C). COL11A1+FB expressed high levels of COL11A1, POSTN, and PRSS23, indicating a profibrotic role in PN skin ( Figure 21 D). To illustrate the ability of different fibroblast subtypes to produce ECM, an ECM module score was calculated using a gene list of the extracellular matrix pathway from the Gene Ontology, and the highest ECM score was identified in LPNCOL11A1+FB (p=4.4E-83) ( Figure 21 E). We then performed differential expression analysis between LPN and healthy COL11A+FB, and inferred the upstream regulators driving the differential expression. Compared with healthy counterparts, high activation z scores of transforming growth factor beta-1 (TGFB1), IL-5, and IL-4 may reflect a profibrotic response, and high tumor necrosis factor (TNF), interferon gamma (IFNG), and IL-6 activation z scores indicate an inflammatory response in LPN COL11A1+FB ( Figure 21 F). 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+FB had the highest expression pattern of collagen genes, including COL1A1, COL1A2, COL3A1, COL5A1, COL5A2, COL6A1, COL6A2, COL6A3, COL11A1, COL12A1, COL14A1, and COL16A1 ( Figure 21 H). In summary, the above results indicate a strong fibrogenic potential of COL11A1+FB in LPN skin. The presence of major fibroblast subtypes (SFRP4, SFRP2, RAMP1, and COL11A1) was confirmed. Figure 21 I and Figure 22 ), and the fibrotic phenotype in PN skin was verified by immunohistochemistry. To compare the fibrotic induction capacity of fibroblasts in PN and AD, the PN scRNA-seq dataset was combined with a single-cell dataset from AD skin, and the ECM scores between fibroblast subtypes in healthy (H), non-lesional AD (NAD), NPN, lesional AD (LAD), and LPN skin were compared. Although the LAD fibroblast subtype exhibited a higher ECM score compared to 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 21 J), particularly in COL11A1+ FBs, which were the most significantly increased FB subset in PN skin (p=2.2×10-9).

[0370] Endothelial cells and pericytes exhibit fibrotic and inflammatory responses in PN skin

[0371] Next, we investigated the heterogeneity of endothelial cells and clustered these cells into six subclusters ( Figure 23 A, B). Disease component analysis identified that endothelial subclusters 2 and 5 were enriched in LPN compared with healthy or NPN samples ( Figure 23 C, D) (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 show inflammatory features such as TNFAIP3 and IL6. Subcluster 5 expresses high levels of several collagen genes (i.e., COL4A1 and COL15A1), suggesting possible involvement in fibrosis ( Figure 3 B). To investigate these two LPN-specific subclusters, enrichment analysis was performed using their cluster marker genes. Subcluster 5 marker genes involved proinflammatory cytokines (i.e., TNF, IL1B, IFNG, IL6) and pathways (i.e., cytokine-mediated signaling pathways, cellular responses to cytokine stimulation) ( Figure 23E, F). Subcluster 2 marker genes are regulated by profibrotic upstream regulators (i.e., TGFB1, angiotensinogen (AGT), epidermal growth factor (EGF), IL-5) and enriched in ECM-related pathways (i.e., extracellular matrix organization, extracellular structural organization) ( Figure 23 G, H). These results suggest that endothelial cells actively participate in fibrosis and inflammatory responses in PN skin.

[0372] Similarly, pericytes were sub-clustered to obtain nine subclusters ( Figure 24 A, D). Compositional analysis identified that subcluster 3 and subcluster 8 were enriched in LPN samples compared with healthy or NPN samples ( Figure 24 B, C). ECM scores were calculated and all collagen genes in pericyte subtypes were plotted. Cells in subcluster 3 had the highest ECM score in LPN and showed the highest expression pattern of collagen genes ( Figure 24 E, F). LPN cells in subcluster 7 also showed a much higher ECM score than healthy cells and expressed the second highest pattern of collagen genes ( Figure 24 E, F). Enrichment analysis was then performed using the marker genes of subclusters 3 and 7, revealing that both subclusters were involved in pro-fibrotic upstream regulators (i.e., TGFB1, AGT, prolactin (PRL)) and pathways (i.e., extracellular matrix organization, collagen fibril organization) ( Figure 22 GJ). Subcluster 3 also illustrates the inflammatory response driven by interferon ( Figure 22 G, I). Taken together, these results suggest that endothelial cells and pericytes can actively contribute to fibrosis and inflammation in diseased PN skin.

[0373] Keratinocyte response in PN skin

[0374] Keratinocytes were sub-clustered to obtain six keratinocyte subtypes: basal keratinocytes, spinous keratinocytes, supraspinous keratinocytes, granular keratinocytes, follicular keratinocytes, and inflammatory keratinocytes ( Figure 25 A, D). Inflammatory keratinocytes mainly originate from LPN samples ( Figure 25 B, C). Enrichment analysis using inflammatory keratinocyte markers involved pro-inflammatory upstream regulators and mitochondrial respiratory pathways, indicating high energy consumption in PN keratinocytes during inflammation ( Figure 25 E). The upstream regulatory factors of the inflammatory subtype of keratinocytes include Th2 cytokines (IL-4, IL-5, IL-33) and TGFB1 ( Figure 25 F), Supporting enriched Th2 responses in diseased PN skin.

[0375] scRNA-seq reveals heterogeneity of immune subtypes in PN skin

[0376] Given the strong inflammatory response observed in fibroblasts, endothelial cells, and pericytes, the heterogeneity of immune cells was investigated. Myeloid cell subsets were clustered and annotated into nine subtypes, including circulating myeloid cells, Langerhans cells (LCs), plasmacytoid dendritic cells (pDCs), classical type 1 dendritic cells (cDC1s), classical type 2 dendritic cell subset A (cDC2A), classical type 2 dendritic cell subset B (cDC2B), interstitial macrophages (IMs), perivascular macrophages, and lipid-associated macrophages (LAMs, also known as TREM2 macrophages) ( Figure 26 AE) The p values for pDC, cDC2A, IM, PVM, and LAM 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 pDC, cDC2A, and macrophage subsets (IM, PVM, and LAM / TREM2) were observed in lesional PN skin compared with non-lesional and healthy skin ( Figure 26 DE). Immunohistochemistry confirmed the prominence of LAM / TREM2 macrophages in lesional PN skin ( Figure 26 ). For T cells and other lymphocytes, seven subtypes were obtained: circulating 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 circulating, NK, CD8, and T regulatory cells. The presence of T cells in lesional PN skin was confirmed by IHC staining of CD8 and CD4 ( Figure 26 ).

[0377] Ligand-receptor analysis reveals cell-type-specific networks in PNs

[0378] Given the observed changes in cell type composition and transcriptional changes, we analyzed changes in cell-cell communication in PNs compared to healthy skin. To this end, we performed separate ligand-receptor analyses for healthy, NPN, and LPN cell types using CellphoneDB and CellChat. Figure 27 A) or NPN skin ( Figure 27 B) compared to LPN ( Figure 5The greatest number of interactions was observed in LPNs (C), particularly in fibroblasts, endothelial cells, pericytes, myeloid cells, and keratinocytes. To investigate specific ligand-receptor pairs in PN, pairs with higher interaction scores in LPNs compared to healthy or NPNs were selected, which revealed various signaling pathways involved in PNs caused by immune (T cells, myeloid) and stromal cell populations (fibroblasts, endothelial cells, and pericytes). Several validated proinflammatory cytokines, such as IFNG, IL1, IL6, and TNF, are also involved in PN pathogenesis. Notably, this analysis revealed several other proinflammatory mediators expressed by various cell types in PN skin, including CCL2, CCL3, CXCL2, CXCL12, and IL7 ( Figure 27 D, E). The presence of fibroblast growth factors (FGF2, FGF7), platelet-derived growth factor (PDGFB), transforming growth factor β (TGFB1, TGFB2, TGFB3), and vascular endothelial growth factor (VEGFB) confirmed the involvement of fibrosis in PN pathogenesis. A robust signaling network associated with the TGFB signaling pathway in lesional PN skin was observed, with TGFB sources observed in multiple cell types and the primary target cells in LPN skin being fibroblasts ( Figure 27 F). Taken together, these data illustrate a profibrotic and proinflammatory shift within the interactome in PN skin.

[0379] Comparison of epithelial responses in PN and AD skin by single-cell analysis

[0380] As previously reported, lesional PN skin shows an expansion of COL11A1 fibroblasts compared with lesional AD skin, suggesting a more profibrotic feature in PN pathophysiology ( Figure 21 J and Figure 22 B). Keratinocyte responses were further compared in PN and AD skin, and gene expression changes in each compartment were compared ( Figure 28 Both PN and AD skin had an expansion of KCs in cluster 3, which corresponds to an “inflammatory” phenotype with prominent expression of several inflammatory markers, including KRT6 and KRT16, S100A8 / A9, as well as IFN signature genes such as IFI27, IFITM3, and the inflammasome gene PYCARD, which encodes apoptosis-associated speck-like protein containing CARD (ASC). Figure 28A, D, E). Evaluation of gene ontology categories enriched in each KC compartment using a threshold of FC > 2 and FDR < 0.05 demonstrated fairly consistent changes in “inflammatory”, basal, spinous, supraspinous, and interfollicular keratinocytes, with AD having enriched inflammatory responses such as “defense response to bacteria” and “neutrophil degranulation” (p = 2.8 × E-07 and p = 3.8 × E-05, respectively) in “inflammatory” KCs, as well as T cell chemotaxis in basal and spinous AD KCs (p = 3.5 × 10E-05, p = 4.1 × 10E-5, respectively) compared to PN skin. Figure 28 F). In contrast, terms related to altered epidermal differentiation were enriched in “inflammatory” and spinous keratinocytes from PN compared to AD skin ( Figure 28 F) Consistent across all KC subtypes, expression of the chemokines CCL27, S100A7, and S100A9 was higher in AD compared with PN.

[0381] Comparison of immune cell responses in PN and AD skin by single-cell analysis

[0382] The proportions of several T cell subsets, including CD8 and CD4 effector T cells, Tregs, and circulating T cells, were generally similar between PN and AD skin. In addition, significant ILC and NK cell subsets were present in NPN, LPN, NAD, and LAD skin ( Figure 29 The most significant differences between LAD and LPN skin were found in CD4+ effector T cells, with LAD CD4+ cells having increased expression of IL13 (2.7-fold higher, FDR = 8.5×10-24) and IL22 (4.7-fold higher, FDR = 6.0×E10-21). Very few IL4-positive T cells were observed in LAD skin ( Figure 30 In contrast, LPN CD4+ T cells had higher CCL5 expression (2.4-fold, FDR = 2.8 × 10-16). There was a trend towards increased expression of IL17A and IL17F in LPN compared with LAD skin, but this was not significant ( Figure 30 ).

[0383] The same nine myeloid cell subsets were identified in PN, AD, and healthy skin as above, with several subsets being more prominent in LPN skin compared to LAD ( Figure 29 EH). This includes pDCs, interstitial macrophages (IM), and lipid-associated macrophages (LAM, TREM2 +The most significant differences in gene expression were found only in PN IM macrophages, which had increased expression of CCL3 and CCL4 (both 2.7-fold, FDR = 2.0 × 10E-09 and 3.9 × 10E-05, respectively). In contrast, LAD IM macrophages had increased expression of MHC class II molecules (including HLA-DRB1, HLA-DQA1, and HLA-DQB1) (2.2-, 2.3-, and 2.7-fold, FDR = 2.7 × 10E-10, FDR = 8.6 × 10E-14, and FDR = 9.8 × 10E-13, respectively).

[0384] Blockade of IL-31 receptor α with nemolizumab restores transcriptional profiles to healthy levels in LPN fibroblasts and keratinocytes

[0385] Using a bulk RNA-seq approach, we demonstrated transcriptome changes in PN skin following treatment with the IL-31 receptor α antagonist nemolizumab. To determine where the biological response to nemolizumab was most prominent, we mapped the expression of two genes encoding the heterodimeric IL-31 receptor, IL-31RA and OSMRB ( Figure 31 A, B). IL31RA is specifically expressed in fibroblasts and keratinocytes. In contrast, OSMRB is more broadly expressed in fibroblasts, keratinocytes, pericytes, and endothelial cells. Strikingly, genes differentially upregulated with nemolizumab treatment compared with baseline lesions were primarily found in fibroblast clusters, whereas differentially downregulated genes were more broadly localized to keratinocytes and immune cell subsets, consistent with reduced hyperkeratosis and inflammatory responses with treatment ( Figure 31 C, D). To investigate the effect of nemolizumab on gene expression levels down to the single-cell level in PN skin, a gene list was generated from a recently published bulk RNA-seq study containing genes significantly downregulated by nemolizumab compared to placebo. PN skin ( Figure 31 D), KC subset ( Figure 31 E, F, G, H) and FB subsets ( Figure 6 F) Module scores were calculated for the gene lists of all major cell subtypes. Consistent with the expression of IL31RA and OSMR, the most significant changes were observed in keratinocytes and fibroblasts, particularly inflammatory KCs and COL11A1+ fibroblasts. These results suggest that nemolizumab treatment restored the transcriptional profile from PN to healthy skin in a broad range of stromal and immune cell populations, particularly in fibroblasts and keratinocytes, cell types that contribute to the most pronounced histopathological changes in diseased PN skin.

[0386] discuss

[0387] This example provides a detailed understanding of the pathogenesis of PN and the associated tissue-specific and cell-type-specific changes that occur in PN skin. Strikingly, changes in PN skin were observed in immune and stromal cell populations, including keratinocytes, endothelial cells, and most profoundly, fibroblasts and fibroblast subsets, with an increased profibrotic response being the key distinguishing feature of PN from AD, accompanied by an immune shift away from IL-13 and IL-22 responses.

[0388] The characteristic histopathological feature of PN is fibrosis of the papillary dermis with vertically aligned collagen fibers. Consistent with this characteristic, trichrome staining revealed a significant increase in dense collagen in the papillary dermis of affected PN skin, as well as increased expression of procollagen I in the papillary dermis. Furthermore, single-cell analysis identified a subset of COL11A1+ fibroblasts 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 primarily found in the papillary dermis, where the fibrotic response was most pronounced, with trichrome or procollagen I staining indicating active collagen I biosynthesis. This expansion of the COL11A1+ fibroblast subset is unique to PN and is not seen in AD skin. Furthermore, the profibrotic effect of this population was not observed in AD COL11A1+ fibroblasts. Recently, AD skin We describe a subset of cancer-associated fibroblasts (CAFs) with a phenotype characterized by the expression of WNT5a, tenascin (TCN), and periostin. Consistent with this publication, in our dataset, periostin (POSTN) and WNT5A in COL11A1+ fibroblasts together with fibroblast activation protein (FAP), a hallmark marker of CAFs Another recently published paper described CXCL14-IL24+ secreting papillary dermal fibroblasts as the primary PN skin Similar IL24+CXCL14-negative FBs were observed here as a small subset of SFRP2+ FBs, and their These FBs expressed increased levels of MMP1. However, lower levels of COL1A1 and COL1A2 indicated that they did not contribute to fibrosis in PN skin.

[0389] Of the two components of the heterodimeric IL-31 receptor, IL31RA expression was detected on both keratinocytes and fibroblasts, whereas OSMRB expression was found to be more widespread across different cell populations. This suggests that the two key cell types that respond to IL-31 in the PN may be fibroblasts and keratinocytes, consistent with the observation that nemolizumab-driven transcriptome shifts can be attributed to these two cell types.

[0390] Notably, this example provides evidence that other cell types, including endothelial cells and pericytes, also contribute to fibrosis in the skin of lesional prurigo nodularis (LPN). Endothelial changes in LPN are known, but the nature of these changes has not been previously described in detail. These data suggest that endothelial cells may contribute to extracellular matrix reorganization under the action of pro-inflammatory and pro-fibrotic cytokines such as TGFB. TGFb may be an upstream promoter of fibrosis in PN, as TGFb 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.

[0391] One of the most characteristic histological features of PN is the presence of dense orthokeratosis accompanied by irregular epidermal proliferation. Keratinocytes exhibit significant transcriptome changes in lesional PN skin, with the most prominent changes observed in inflammatory keratinocytes, defined by expression of KRT6, KRT16, and KRT17, as well as S100A8 and S100A9, and this analysis suggests a key role for Th2 cytokines (such as IL13 and IL22) in this transition, consistent with previous observations. The most abundant biological classes in the inflammatory keratinocyte subset were associated with mitochondrial function and protein translation, suggesting the production of reactive oxygen species and cellular stress, which can contribute to the inflammatory response in the skin.

[0392] Immune cell infiltration is significantly observed in lesional PN skin and is characterized by changes in specific immune cell populations. The most significant changes were 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 trigger the production of proinflammatory cytokines in atherosclerosis, which in turn amplify the inflammatory response. These macrophages have recently been implicated in the pathogenesis of acne. T cells are also prominent in PN lesions, with circulating T cells as well as NK, CD8 +and an increase in the number of Tregs. The role of these T cell populations in PN skin has not been previously characterized. In addition, various stromal cell populations, particularly endothelial cells and pericytes, have increased expression of various proinflammatory cytokines, chemokines and adhesion molecules, indicating a positive role in immune trafficking and immune amplification in PN. This includes increased expression of adhesion molecules ICAM1, E-selectin (SELE) and IL6 in endothelial cells, as well as increased expression of CCL2, CCL3, CCL4, CCL13, CCL18, CXCL2 and CXCL12 expressed by various cell types in PN skin (e.g., myeloid cells, pericytes and endothelial cells). It is worth noting that CCL2 and IL-6 have established roles in the development of fibrosis. CCL2 is the most potent profibrotic chemokine; CCL2 acts directly on fibroblasts through CCR2, stimulating collagen synthesis. Similarly, IL-6 trans-signaling enhances lung fibroblast proliferation and extracellular matrix protein production.

[0393] This data further outlines the differences between PN and AD. Notably, changes in cell populations in the epidermis were highly similar between PN and AD, with both diseases having a prominent "inflammatory" keratinocyte subpopulation characterized by increased expression of pro-inflammatory molecules (including S100A8 and S100A9) and the inflammatory keratin proteins KRT6 and KRT16. Changes in the expression of S100A8, S100A9, and KRT16 have been described in AD skin, but their expression in PN skin has not been addressed. Interestingly, the epidermal changes were accompanied by subtle changes in gene expression between PN and AD skin, with immune-related processes (such as antimicrobial responses) and T cell trafficking regulators observed only in LAD but not in LPN keratinocytes. Changes in T cell phenotype were also observed between LPN and LAD skin, particularly within the CD4 effector T cell population, with significantly lower mRNA expression of IL13 and IL22 in LPN skin compared to LAD skin. IL-22 is known to promote epidermal proliferation and activate innate immunity and antimicrobial 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 although PN is an inflammation-driven disease, it may not be centered around an IL-13 / IL-22 response to the same extent as AD.

[0394] These data also provide information about the mechanism of action of the IL-31 receptor antagonist nemolizumab. Bulk RNA-seq analysis was performed on LPN skin from patients treated with nemolizumab, and the transcriptome shifts observed indicated stabilization of extracellular matrix remodeling and normalization of epidermal differentiation. By cross-referencing single-cell data with nemolizumab bulk RNA-seq data, it was possible to show the broad effects of nemolizumab on abnormal transcriptome activation in various cell types in PN skin. Therefore, the normalization of pathological transcriptome signatures observed in COL11A1+ fibroblasts and inflammatory keratinocyte subsets may reflect the clinical improvement of PN skin lesions observed during nemolizumab treatment. These results also validate observations on the molecular and cellular effects of nemolizumab treatment on the pathophysiological pillars of PN disease, including inflammation, altered epidermal differentiation, and fibrosis.

[0395] Together, these data provide unique insights into the pathogenesis of PN, highlighting it as a chronic neuroimmune skin disease with complex immune-stromal cell crosstalk that promotes and potentially drives aberrant keratinocyte proliferation and activation. This is accompanied by a dramatic shift toward a profibrotic response primarily within the papillary dermis involving activation of COL11A1+ fibroblasts, endothelial cells, and pericytes. Notably, these changes were reversible by blocking IL-31 receptor α. Thus, these novel insights expand our understanding of the pathogenesis of PN and the mode of action of anti-IL-31R therapy for this debilitating disease.

[0396] Materials and methods

[0397] Human sample collection

[0398] Six patients with prurigo nodularis, six patients with AD, and 15 healthy donors were recruited for single-cell RNA sequencing. 6mm punch biopsies were obtained from affected lesional and non-lesional AD and PN skin. Patients had not received active topical treatment for at least 2 weeks prior to recruitment. No patients had received prior systemic treatment. PN patients had no concomitant active AD. The study was approved by the University of Michigan Institutional Review Board (IRB), and all patients gave written consent. The study was conducted in accordance with the Declaration of Helsinki Principles. See patient demographics in the table below:

[0399] Patient demographics

[0400]

[0401] Single-cell RNA-seq library preparation, sequencing, and alignment

[0402] Single-cell suspensions for scRNA-seq were generated as follows: Skin biopsies were incubated overnight at 4°C in 0.4% dispase (Life Technologies) in Hank's balanced saline solution (Gibco). The epidermis and dermis were separated. The epidermis was digested in 0.25% trypsin-EDTA (Gibco) containing 10 U / ml DNase I (Thermo Scientific) at 37°C for 1 hour, 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 normal culture medium at 37°C for 1.5 hours and filtered through a 70 μM mesh. Epidermal and dermal cells were combined in a 1:1 ratio and libraries were constructed using chemistry v3 on a 10X Chromium system by the Advanced Genomics Center at the University of Michigan. The library was 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.

[0403] Cell clustering and cell type annotation

[0404] The cells in the merged matrix were clustered using the R package Seurat (v4.1.1). Cells with less than 500 transcripts or 100 genes or more than 1e5 transcripts or 10% mitochondrial expression were first filtered out as low-quality cells. The expression levels of each cell were normalized using the NormalizeData function with default parameters. The variable genes were selected using the FindVariableFeatures function with default parameters. The counts in the dataset were scaled and centered using the ScaleData function. Principal component analysis (PCA) was performed on the variable genes. The RunHarmony function from the Harmony package was applied to remove potential batch effects between samples processed in different batches. Uniform manifold approximation and projection (UMAP) dimensionality reduction was performed using the RunUMAP function. Clusters were obtained using the FindNeighbors and FindClusters functions with a 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 of each cell type from each disease condition was counted. The counts were then divided by the total number of cells for each disease condition and scaled to 100% for each cell type. Differential expression analysis between any two groups of cells was performed using the FindMarkers function. All differential expression analysis comparisons are shown as mean log2 fold change and the false discovery rate was used. FDR adjustment was used to correct for multiple testing.

[0405] Cell type sub-clustering

[0406] Sub-clustering was performed on enriched cell types. Sub-clusters were obtained using the same function described above. Sub-clusters defined solely by mitochondrial gene expression were removed from further analysis, indicating low quality. Subtypes were annotated by overlapping the marker genes of the sub-clusters with canonical subtype signature genes. Module scores were calculated for the expected gene list using the AddModuleScore function. ECM scores were calculated for genes in 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. Genes induced or reduced by nemolizumab were obtained from a previous batch 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 classic pathways using Enrichr.

[0407] Ligand-receptor interaction analysis

[0408] CellphoneDB (v3) and CellChat were applied to ligand-receptor analysis. Each cell type was separated by its disease classification (healthy, non-lesioned, and diseased), and a separate run was performed for each disease classification. Pairs with p-values>0.05 were filtered out from further analysis. The number of interactions between each cell type pair was then calculated for each condition. To compare healthy and diseased conditions, pairs showing higher interaction scores in the diseased condition were used to display disease-specific interactions.

[0409] Immunohistochemical staining

[0410] Paraffin-embedded tissue sections (lesional and healthy skin) were heated at 60°C for 30 minutes, deparaffinized, and rehydrated. Slides were placed in pH 9 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). Anti-human primary antibodies were then incubated overnight (4°C). The antibodies used were anti-COL11A1 (ThermoFisher Scientific, catalog number PA5-68410), anti-SFRP2 (Lifespan Biosciences, catalog number LS-C794043), anti-SFRP4 (Lifespan Biosciences, catalog number LC-C408100), anti-TREM2 (ThermoFisher Scientific, catalog number PA5-18763), anti-RAMP1 (Abcam, catalog number AB64409), anti-CD4 (ThermoFisher Scientific, catalog number 14-244-82), anti-CD8 (ThermoFisher Scientific, catalog number MA5-13473), anti-CD3 (Origene, catalog number UM500048). The slides were then washed and treated with secondary antibodies, peroxidase (30 minutes) and diaminobenzidine substrate. Counterstained with hematoxylin and dehydrated, the slides were mounted and observed under a microscope.

[0411] Spatial sequencing library preparation

[0412] Skin samples were frozen in OCT medium and stored at -80 ° C until sectioning. 20 μm sections were optimized for tissue permeabilization using the Visium Spatial Tissue Optimization Kit (10X Genomics, Pleasanton, CA, USA), which determined the optimal permeabilization time to be 9 minutes. The 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 poly-A mRNA for capture by poly (dT) primers, which were pre-coated on the slides and included Illumina TruSeq reads, spatial barcodes, and unique molecular identifiers (UMIs). The Visium Spatial Gene Expression Kit (10X Genomics) was used for reverse transcription to generate full-length cDNA with spatial barcodes and for second-strand synthesis, followed by denaturation to allow cDNA to be transferred from the slides to tubes for amplification and library construction. After enzymatic fragmentation, size selection, end repair, A-tailing, adapter ligation, and PCR, a Visium spatial single-cell 3′ gene expression library consisting of Illumina paired-end sequences flanked by P5 / P7 was constructed. Unique i7 and i5 sample indexes were added using the Dual Index Kit TT Set A (10X Genomics), and TruSeq reads 1 for sequencing spatial barcodes and UMIs and TruSeq reads 2 for sequencing cDNA inserts were generated, respectively. The library was then sequenced on an Illumina NovaSeq 6000 sequencer to generate 150bp paired-end reads.

[0413] Spatial sequencing data analysis

[0414] After sequencing, the reads were aligned to the human genome (hg38) and the expression matrix was extracted using the spaceranger pipeline (10XGenomics). The expression matrix was then analyzed using Seurat. Specifically, the SCTransform function was used to scale the data and find variable genes using default parameters. PCA and UMAP were applied for dimensionality reduction. A set of anchor points was found between spatial-seq data and scRNA-seq data using the FindTransferAnchors function, and then transferred from scRNA-seq to spatial-seq data using the TransferData function. These two functions construct a weight matrix that defines the association between each query unit and each anchor point. For each point, these weights sum to 1 and are used as the percentage of the cell type in the point. The ECM score was calculated using the AddModuleScore function for genes in the extracellular matrix pathway from the Gene Ontology database.

[0415] Table 1. Differential expression analysis in five different comparisons: (A) nonlesional skin vs. lesional skin at baseline; (B) placebo group at baseline vs. week 12; (C) nemolizumab group at baseline vs. week 12; (D) placebo group vs. nemolizumab group at baseline; (E) placebo group vs. nemolizumab group at week 12. Differential expression values are shown as log2 fold change (i.e., log2FC). P values and false discovery rates (FDRs) were calculated but not shown.

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[1347] Table 3 - Gene expression modules in non-lesional and lesional PN skin.

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[1350] Table 4 - Functional enrichment of dysregulated genes in PN and psoriatic lesional skin.

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[1475] Table 5 - Binding sites are enriched for the following transcription factors: (A) genes upregulated in lesional skin at baseline; (B) genes downregulated in lesional skin by week 12 in the placebo group; (C) genes downregulated in lesional skin by week 12 in the nemolizumab group.

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Claims

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 diseased skin cells compared to a reference level of activation of TNF signaling.

2. A method for normalizing activation of tumor necrosis factor (TNF) signaling in a subject with PN, comprising administering an anti-IL-31RA antibody to a subject with PN, wherein the subject exhibits activation of TNF signaling in diseased skin cells compared to a reference level of activation of TNF signaling, and wherein administration of the anti-IL-31RA antibody normalizes the activation of TNF signaling. 3 . The method of claim 2 , wherein normalization is determined at about 4 weeks, about 8 weeks, or about 12 weeks after administration of the anti-IL-31RA antibody.

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

5. The method according to any one of claims 1 to 4, wherein the activation of TNF signaling in the diseased skin cells is higher compared to the reference level of activation.

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

7. The method according to any one of claims 1 to 6, wherein the reference level is an activation level of TNF signaling in skin cells of a person not suffering from PN.

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

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

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

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

12. The method of claim 11, wherein the reference level is an activation level of TNF signaling in skin cells of a person not suffering from PN.

13. The method of claim 12, wherein the skin cells of the human not suffering from PN are fibroblasts.

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

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 suffering from PN, wherein administration of the anti-IL-31RA antibody results in a decrease in tumor necrosis factor (TNF) pathway activation.

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

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

19. The method of any one of claims 16 to 18, wherein the treatment further results in: (a) Decreased leukocyte migration or cell movement of leukocytes; (b) inhibiting the STAT3 pathway; (c) inhibiting the STAT5b pathway; (d) downregulation of IL-1 or IL-1 pathway; (e) downregulation of IL-6 or IL-6 pathway; (f) downregulation of VEGF or VEGF pathway; (g) decreased activation of the TGFB1 pathway, or (h) combinations thereof.

20. The method of claim 19, wherein (a) the leukocyte migration or leukocyte cell motility is reduced; (b) the STAT3 pathway is inhibited; (c) the STAT5b pathway is inhibited; (d) the IL-1 or IL-1 pathway is downregulated; (e) the IL-6 or IL-6 pathway is downregulated; (f) the VEGF or VEGF pathway is downregulated; (g) the TGFB1 pathway activation is reduced, 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 the anti-IL-31RA antibody.

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

22. The method of any one of claims 19 to 21, wherein (a) the leukocyte migration or cell motility of leukocytes is reduced; (b) the STAT3 pathway is inhibited; (c) the STAT5b pathway is inhibited; (d) the IL-1 or IL-1 pathway is downregulated; (e) the IL-6 or IL-6 pathway is downregulated; (f) the VEGF or VEGF pathway is downregulated; (g) the TGFB1 pathway activation is reduced, or (h) a combination thereof is determined by mass spectrometry analysis of one or more biological samples obtained from the subject.

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

24. The method of any one of claims 19 to 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) decreased leukocyte migration or leukocyte cell motility; (b) inhibition of the STAT3 pathway; (c) inhibition of the STAT5b pathway; (d) downregulation of the IL-1 or IL-1 pathway; (e) downregulation of the IL-6 or IL-6 pathway; (f) downregulation of the VEGF or VEGF pathway; and (g) decreased activation of the TGFB1 pathway.

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

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

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

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

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

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

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

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 administration of the anti-IL-31RA antibody results in a reduction in reactive oxygen species and / or cellular stress to which the keratinocytes are exposed.

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

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

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

37. The method according to any one of claims 34 to 36, wherein the at least one type of immune cell comprises T cells, NK cells, CD8 + cells, Tregs, and any combination thereof.

38. The method of any one of claims 34 to 37, wherein administering the anti-IL-31RA antibody results in decreased 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. 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. The method of any one of claims 1 to 40, wherein the anti-IL-31RA antibody is administered 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.

42. The method of any one of claims 1 to 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. The method of any one of claims 1 to 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, 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 steady-state dosing schedule.

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