Methods and means of treating chronic inflammatory and autoimmune diseases

Abstract

The present invention relates to pharmaceutical compositions for targeting activated pathogenic T cells and / or NK cells in patients with chronic inflammatory or autoimmune diseases. The pharmaceutical compositions comprise a first molecule that specifically recognizes CD7. The present invention further describes a kit comprising these pharmaceutical compositions. The present invention further relates to a method for targeting activated pathogenic T cells and / or NK cells in patients with chronic inflammatory or autoimmune diseases.

Description

[Technical Field]

[0001] The present invention relates to the field of immune system-related diseases, and more specifically to novel means and methods for treating these diseases. More specifically, the present invention provides a pharmaceutical composition comprising an antibody that specifically recognizes CD7 for the treatment of chronic inflammatory or autoimmune diseases.

[0002] Typically, the present invention has applications in the field of systemic autoimmune diseases, particularly systemic sclerosis or Sjögren's syndrome. [Background technology]

[0003] Autoimmune diseases refer to conditions in which the body triggers an immune response against its own components, producing T lymphocytes that fight autoantibodies and / or autoantigens, thereby causing damage and dysfunction to its own tissues and cells. In some conditions, such as myasthenia gravis, type 1 diabetes, and autoimmune thyroiditis, the disease is mediated by autoantibodies directly binding to tissue-specific antigens. These diseases are called organ-specific autoimmune diseases. In other diseases, autoantibodies bind to antigens expressed on organ-nonspecific structures. Autoimmune responses occur, for example, when antigens are abnormally expressed due to infection or tissue / cellular stress, or in specific tissues / organs where circulating immune cells or immune complexes accumulate. These are called systemic autoimmune diseases, such as systemic sclerosis (SSc), Sjögren's syndrome (SS), rheumatoid arthritis (RA), dermatomyositis (DM), and lupus erythematosus (SLE).

[0004] Conventional treatments for autoimmune diseases involve the administration of drugs that nonspecifically suppress the immune response. Examples of such drugs include methotrexate, cyclophosphamide, Imuran (azathioprine), and cyclosporine A. Steroid compounds such as prednisone and methylprednisolone are also frequently used. These drugs vary in effectiveness for both organ-specific and systemic autoimmune diseases. The use of these drugs is limited by toxic side effects and by the fact that they induce fairly broad immunosuppression in patients receiving long-term administration. For example, the normal protective immune response against pathogenic microorganisms is suppressed, thereby increasing the risk of infection by these pathogens. A further drawback is the increased risk of developing malignancies in patients receiving long-term systemic immunosuppression with certain broad immunosuppressants.

[0005] Systemic sclerosis (SSc) is an autoimmune disease of unknown cause characterized by high morbidity and mortality (PMID: 34487318). SSc is a systemic autoimmune disease characterized by vascular lesions, inflammation, and progressive fibrosis of the skin and internal organs (Non-Patent Literature 1). Autoimmunity in SSc targets nuclear autoantigens. These antigens are abnormally presented by endothelial cells and fibroblasts under hypoxic stress and function as antigenic targets (Non-Patent Literature 2). This is indicated by the development of dysregulated Raynaud's phenomenon, which appears as the first major disease symptom. T lymphocytes have been detected in SSc-affected tissues, and several studies have suggested their involvement in the observed fibrosis and vascular lesions through the production of cytokines such as interleukin (IL)-4, IL-13, and IL-17 (Non-Patent Literature 3). Surprisingly, recent studies have shown that cytotoxic T cells play a significant role in mediating SSc cutaneous pathology (Non-Patent Literature 4). Furthermore, epigenetic studies have suggested that natural killer (NK) cells and CD8+ T cells are involved in the pathogenesis of SSc (Non-Patent Literature 5).

[0006] In chronic inflammatory states, T cell activation is suppressed to prevent unnecessary inflammatory side effects. Activation of antigen-specific CD4+ T cells is regulated by professional antigen-presenting cells via a process controlled by major histocompatibility complex (MHC) class II. For cytotoxic T and NK cells, the regulatory mechanisms are less clearly defined because these cells depend on receptors other than MHC class II and are universally expressed in inflammatory tissues. In chronic infections and malignancies, the activation of cytotoxic T and NK cells has been shown to be regulated by the interaction of costimulatory and inhibitory receptors (Non-Patent Literature 6). Animal models suggest that similar mechanisms may be at work in cytotoxic autoimmunity (Non-Patent Literature 7). However, the precise role of T cells in SSc pathogenesis remains unclear. On the other hand, genetic studies have demonstrated that human leukocyte antigen (HLA) genes corresponding to MHC class II confer susceptibility to SSc (Non-Patent Literature 8). On the other hand, treatment with cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) immunoglobulin (abatacept), a drug that targets T cells, has shown only limited clinical efficacy (Non-Patent Literature 9).

[0007] The inventors addressed this controversy and hypothesized that targeting specific costimulatory receptors on cells expressing costimulatory molecules could be a selective and potentially safer therapeutic strategy in SSc treatment.

[0008] The present invention aims to alleviate the above-mentioned problems. Alternatively, or additionally, the present invention aims to provide pharmaceutical compositions and methods for the treatment of chronic inflammatory or autoimmune diseases, such as SSc and SS. [Prior art documents] [Non-patent literature]

[0009] [Non-Patent Document 1] Truchetet ME, Brembilla NC, Chizzolini C. Current Concepts on the Pathogenesis of Systemic Sclerosis. Clin Rev Allergy Immunol. 2021.

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[0010] The inventors of this invention found that T cells and NK cells with upwardly regulated co-stimulatory receptor CD7 expression are key immune cells that promote the pathogenesis of SSc, identifying potential targets for selective therapy. The inventors analyzed the co-stimulatory profiles of these cells with the aim of specifically inhibiting and / or eliminating the activation of pathogenic cytotoxic immune cells in SSc skin. Using detailed high-depth single-cell transcriptome analysis of 109 SSc skin biopsies compared with 68 healthy skin biopsies, confirmed by multiplex immunohistochemistry, it was surprising to observe the significant presence of activated cytotoxic T cells and NK cells showing disease-related upward regulation of the CD7-activated receptor. Further examination of the expression profiles of known lymphocyte co-stimulatory receptors in the skin, and comparison with those of healthy cells, revealed consistent and strong upward regulation of CD7 in increased cytotoxic NK cell and proliferating cytotoxic T cell populations. Upward regulation of CD7 was directly correlated with upward regulation of pro-inflammatory, cytotoxic, and pro-fibrotic mediators in the same cells. Using in vitro models of cytotoxic and leukocytogenic fibroblastic contraction, CD7 was shown to contribute to pro-fibrotic symptoms in a disease-related manner. Treatment with compositions containing anti-CD7 with or without immunotoxin (anti-CD7 and anti-CD7-IT) was studied, and the results showed dysfunction or effective depletion of these subsets (by anti-CD7 or anti-CD7-IT). In particular, significant removal of CD7+ cell subsets in the blood and skin of SSc patients was already observed one week after administration of the pharmaceutical compositions of the present invention.

[0011] Accordingly, according to a first aspect of the present invention, a pharmaceutical composition for targeting activated pathogenic T cells and / or NK cells in patients with chronic inflammatory or autoimmune diseases is provided. The pharmaceutical composition of the present invention comprises a first molecule that specifically recognizes CD7.

[0012] Preferably, the first molecule is an antibody or a fragment of its derivative. For example, the first molecule is a monoclonal antibody. Preferably, the first molecule is an IgG2a antibody. For example, the first molecule is an anti-(human) CD7 mouse IgG2a monoclonal antibody.

[0013] Preferably, the first molecule contains a combination of complementarity-determining region (CDR) sequences, and the CDR sequences include a CDR3 sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 17. More preferably, the CDR3 of the first molecule contains or consists of the amino acid sequence of SEQ ID NO: 17.

[0014] A preferred example of the first molecule according to the present invention is WT1.

[0015] The pharmaceutical composition of the present invention may preferably further contain a second molecule that specifically recognizes CD3.

[0016] Preferably, the second molecule is an antibody or a fragment of its derivative. For example, the second molecule is a monoclonal antibody. Preferably, the second molecule is an IgG2b antibody. For example, the first molecule is an anti-(human) CD3 mouse IgG2b monoclonal antibody.

[0017] Preferably, the second molecule contains a combination of complementarity-determining region (CDR) sequences, and the CDR sequences include a CDR3 sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 7. More preferably, the CDR3 of the second molecule contains or consists of the amino acid sequence of SEQ ID NO: 7.

[0018] A preferred example of the second molecule according to the present invention is SPV-T3a.

[0019] Optionally, activated pathogenic T cells and / or NK cells are selected from the group consisting of CD8 cytotoxic T cells, CD4 helper T cells, follicular helper T cells, proliferative T cells, and combinations thereof. For example, a pharmaceutical composition targets activated pathogenic T cells and / or NK cells by selective depletion of activated pathogenic T cells and / or NK cells, or by membrane receptor inhibition, or by intracellular kinase inhibition.

[0020] Preferably, the chronic inflammatory and autoimmune disease is systemic sclerosis or Sjögren's syndrome. Preferably, the patient may exhibit symptoms of vascular lesions, fibrosis, and / or autoimmune inflammation.

[0021] Preferably, activated pathogenic T cells and / or NK cells include activated pathogenic T cells and / or NK cells in the patient's blood and / or inflamed tissue.

[0022] Preferably, the first molecule, or the second molecule, or both, comprises at least one toxic moiety, preferably a recombinant A chain (rRTA) of lysine, for example, a recombinant A chain of lysine having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 21. More preferably, the rRTA comprises or consists of the amino acid sequence of SEQ ID NO: 21.

[0023] Preferably, the pharmaceutical composition of the present invention further comprises one or more excipients, carriers, buffers, stabilizers, isotonic modifiers, preservatives, or preservatives or antioxidants.

[0024] Preferably, the pharmaceutical composition of the present invention is administered by intravenous, intradermal, or subcutaneous injection.

[0025] A second aspect of the present invention provides a method for targeting activated pathogenic T cells and / or NK cells in a patient with a chronic inflammatory or autoimmune disease. The method comprises administering an effective amount of the pharmaceutical composition of the present invention described herein to the patient.

[0026] In a third aspect, the present invention provides a lyophilized form of the pharmaceutical composition of the present invention. The lyophilized composition may be suitable for reconstitution with water or an aqueous solution, for example, to form a composition for use in the method of the present invention.

[0027] According to the fourth aspect, the present invention is A container or housing in which the composition of the present invention is contained, A label or insert containing instructions for the use of the composition in the therapeutic method of the present invention, We provide a kit that includes this.

[0028] Depending on the circumstances, the container or housing may be kept sterile by, for example, sealing or airtight closure.

[0029] Of course, it will be understood that features described in relation to one aspect of the present invention may also be incorporated into other aspects of the present invention. For example, a method of the present invention may incorporate any of the features described in relation to the apparatus of the present invention, and vice versa. [Brief explanation of the drawing]

[0030] [Figure 1]Figure 1 shows annotations of cutaneous T cell and NK cell clusters from the single-cell RNA sequencing dataset GSE195452. A Unsupervised manifold approximation and projection-based dimensionality reduction (UMAP) clustering of 7,187 cells identified eight transcriptionally distinct cell clusters: tissue-resident memory T cells (Trm), cytotoxic T cells (CTLs), regulatory T cells (Treg), impaired tissue-resident T cells (Thprm), naive / central memory (Tncm), proliferative T cells (Tprolif), NK cells (NK), and a cluster containing MAIT cells, INKT CD8+ T cells, and γδ T cells (T mix). B UMAP showing cells belonging to a healthy individual (control group) or a patient (SSc). Heatmaps showing the top 5 genes by differential expression level in each cell cluster: C Trm (CD69, ZFP36L2, CXCR4, IL7R), CTLs (GZMK, IFNG, CCL5, CCL4, CD8A), Tregs (CD4, FOXP3, CTLA4, IL2RA), Thprm (NR4A1, CD69, CXCR4, DUSP1), Tncm (TCF7, SELL, IL7R), Tprolif (MKI67), NK (NKG7, FCGR3A, FGFBP2, KLRD1, GZMB, PRF1), and Tmix (CD8A, CCL5, TRGC2, NKG7, GZMB, PRF1, FCGR3A, FGFBP2, KLRD1). A heatmap showing the top 10 genes with the most upwardly regulated expression in each of the five different clusters of isolated CD8+ T cells: D Naive (Tn) cluster: IL7R, Granzyme K+ (GZK+): GZMK, IFNG, CCL4, Granzyme B+ (GZB+): PRF1, GNLY, NKG7, GZMB, GZMZ, GZMH, Exhausted (Tex): NR4A2, NR4A3, Proliferating (Tprolif): LASP1, TMPO, ANP32B. E UMAP showing PDCD1 (PD-1) positive (red) and negative (gray) gene expression in CD8+ T cell clusters. [Figure 2]Figure 2 shows single-cell RNA sequence analysis of the GSE138669 dataset. Unsupervised manifold approximation and projection-based dimensionality reduction (UMAP) clustering of 2,500 cells identified seven transcriptionally distinct cell clusters: quiescent tissue-resident T cells (Tqcm), cytotoxic T cells (CTLs), regulatory T cells (Treg), impaired tissue-resident T cells (Thprm), naive / central memory T cells (Tncm), proliferative T cells (Tprolif), and NK cells (NK). The UMAP showing cells belonging to healthy individuals (control group) or patients (SSc) is shown at the top of this panel. B Heatmap of the top 10 genes differentially expressed in each cell cluster: Tqcm (CD69, IL7R, TCF7, SELL, ANXA1), CTLs (CD8A, GZMK, GZMA), Tregs (CD4, CD27, CTLA4, IL2RA), Thprm (NR4A1, DUSP1), Tncm (TCF7, IL7R), Tprolif (MKI67), NK (NKG7, FCGR3A, KLRD1, PRF1). C (Left) Gene set enrichment analysis using Wiki pathways as the reference dataset. Examples of the most significant pathways characteristic of each cluster are shown. Statistical information. (Right) Comparison of enrichment scores for outlines of pro-inflammatory, pro-fibrotic, and pulmonary fibrosis pathways in cutaneous T cell and NK cell clusters of HD vs. SSc. [Figure 3]A. Frequency of T cell and NK cell clusters in the skin of healthy donors (HD) (n=56) and systemic sclerosis (SSc) patients (n=97) (GSE195452). B. Frequency of T cell and NK cell clusters in the skin of HD patients (n=9) and SSc patients (n=12) (GSE138669). Abbreviations: Tissue-resident memory T cells (Trm), cytotoxic T cells (CTLs), regulatory T cells (Treg), impaired tissue-resident T cells (Thprm), naive / central memory T cells (Tncm), proliferative T cells (Tprolif), NK cells (NK), quiescent tissue-resident T cells (Tqcm). In both panels A and B, values ​​are shown as percentage change in cell number, and statistical analysis was performed using the Wilcoxon test with multiple comparison correction. Only the corrected p-value (q) for statistically significant comparisons is shown. *q<0.05, **q<0.01. C Representative multicolor immunofluorescence composite images of T helper cells (CD3+CD8-, red), cytotoxic T cells (CD8+, blue), regulatory T cells (FOXP3+, green), and natural killer cells (CD56+CD3-, yellow) in SSc lesional skin. Abbreviations: hair follicle (HF), epidermis (E), blood vessel (BV). Scale 50 μm. D Immunofluorescence composite images of infiltrating cytotoxic CD8+ (cyan) T cells and CD56+CD3- (yellow) NK cells in non-lesional and lesional skin of a representative early diffuse SSc patient. E Percentage (%) of cytotoxic T cells (CD3+CD8+) and NK cells (CD56+CD3-) in matched non-lesional and lesional SSc skin (n=24 SSc patients). The values ​​are expressed as the percentage (%) of CD8+ or CD56+ cells relative to all cells (DAPI-positive) present in each biopsy specimen (excluding the keratinocyte-rich epidermal layer). Non-parametric Wilcoxon tests were used for statistical analysis, with ***p<0.001. F (left) Gene set enrichment analysis of cutaneous T cell and NK cell clusters using the Wiki pathway as the reference dataset. Examples of superior pathways (p<0.001) shown by NK and CTL clusters are presented. Statistical analysis was performed using the Kolmogorov-Smirnov (KS) test.(Right) Comparison of enrichment scores for the "Summary of Pro-inflammatory / Pro-fibrotic Mediators" (q=0.025) and "Pulmonary Fibrosis" (q=0.0002) pathways in cutaneous T cell and NK cell clusters in HD (green) vs. SSc (red) (see this figure for GSE195452, and Supplementary Figure 2C for GSE138669). G (Left) UMAP presenting five transcriptionally distinct CD8+ T cell clusters in 56 HD and 97 SSc (cell count 977) in the skin. Based on differentially expressed topogenetics, the clusters were annotated as naive (T naive), granzyme K positive (GZMK+), granzyme B positive (GZMB+), exhausted (Texh), and proliferating (T prolif). (Right) Cell frequency of CD8+ T cell clusters in healthy individuals and scleroderma. H: Percentage of CD8+ T cells expressing granzyme B (GZMB) in peripheral blood (HD group n=15, SSc group n=30). Values ​​are expressed as a percentage of total viable peripheral blood mononuclear cells (%). *p<0.05. [Figure 4]Figure 4 shows that upward regulation of CD7 is associated with the activation of cytotoxic T cells and NK cells in the skin and lungs of scleroderma (SSc). A. Two-dimensional dot plot comparing gene expression of selected activating and inhibitory costimulatory receptors in Tprolif, CD8+GZMB+, and NK clusters between HD and SSc (circle size indicates the percentage of cells expressing each gene, color intensity represents the average expression level, and numbers indicate the average normalized count). B. (Top) UMAPs representing positive (red) and negative (gray) gene expression of CD7 in CD8+ T cells (left) and CD56+ NK cells (right). (Bottom) CD7 normalized gene expression intensity in CD8+ T cells (left) and CD56+ NK cells (right) between HD and SSc. C. Scatter plot of gene expression values ​​highlighting genes that are specifically abundant in skin T cells and NK cells of SSc patients compared to a healthy control group. D Representative photographs of CD7 immunohistochemistry (IHC) staining in lesional and non-lesional skin biopsy specimens from systemic sclerosis patients. Quantification of CD7 IHC score is also shown (n=20). Non-parametric sign test, *p<0.05. E Immunofluorescence micrographs showing co-expression of CD7 in CD8+ T cells and CD56+ NK cells in early dSSc skin. Representative experiments are shown on a 100 μm scale. F (Left) UMAP (GSE128169) showing T cells and NK cells obtained from lung tissue of a control group (healthy individuals) (n=6) and lung tissue of SSc patients with interstitial lung disease (n=7). (Center) Concentration plots showing gene expression concentrations of CD7, NCAM1 (CD56), CD4, and CD8A. (Right) CD7 gene expression counts (normalized) in T cells and NK cells from control and SSc lungs. (Each dot represents the average CD7 expression for each donor.) [Figure 5]Figure 5 shows the staining of CD3 and CD7 cells in lesioned versus non-lesioned skin from a systemic sclerosis (SSC) patient. CD7 gene expression in immune cells and stromal cells of the skin is also shown. A Representative image of CD3 immunohistochemistry (IHC) staining in lesioned and non-lesioned skin biopsy specimens from one SSC patient. Scale is 100 μm. B Quantification of CD3+ T cells in lesioned versus non-lesioned SSc skin (n=20). Error bars indicate mean ± standard deviation. Non-parametric Wilcoxon test, p=0.19. C In SSc-affected skin, a large infiltration of CD7-positive cells is observed in the perivascular region, while in control non-affected skin, the number of CD7-positive cells in the perivascular region is small. Scale is 100 micrometers. Here, a representative image of one SSc patient showing early diffuse lesions is shown. D Two-dimensional dot plot comparing CD7 gene expression in immune cells and stromal cells of the skin. Cell cluster annotations were obtained from metadata information as described in the single-cell RNA sequencing dataset GSE195452. The size of the circle indicates the percentage of cells expressing CD7, and the intensity of the color represents the average expression level. The numerical value indicates the average of the normalized counts. [Figure 6]CD7 co-stimulation plays a crucial role in the cytotoxic and pro-fibrotic effects of T cells and NK cells. A. SECTM1 log-normalized gene expression levels (GSE195452) in a subset of immune cells and stromal cells of the skin. Annotations for the illustrated cell clusters are from Gur et al. (Reference 10). B. Subgroup analysis of CD7 normalized gene expression in healthy individuals (HD) and patients with systemic sclerosis (SSc). Patients with early-stage limited cutaneous sclerosis (lSSc) or diffuse cutaneous sclerosis (dSSc) were compared between early-onset and late-onset groups. Early-onset was defined as within 3 years of initial diagnosis. One-way ANOVA with Tukey's multiple comparison test, *p<0.05, ***p<0.001. C. (Left) Scatter plot showing the correlation between CD7 normalized gene expression and skin score. Each circle represents one SSc patient. Spearman correlation coefficient r=0.34, p=0.07. (Right) Normalized CD7 gene expression in low-skin score group vs. high-skin score group in SSc patients. The distinction between the low-skin score group and the high-skin score group is as previously explained (Reference 10). D Normalized CD7 gene expression between treatment-naive SSc patients or SSc patients who have received immunosuppressant therapy, and between SSc patients with interstitial lung disease (ILD) or SSc patients without ILD. E Percentage of CD8+CD7+ and CD8+CD7- T cells in peripheral blood (HD group n=15, SSc group n=30). Values ​​are shown as a percentage (%) of the total number of CD3+ T cells. *p<0.05. F Granzyme B (GZMB) expression level (mean fluorescence intensity) and percentage of IL-4+ / IL-13+ cells in CD8+CD7+ T cells in the HD group and SSc group. GZMB expression levels are expressed as mean fluorescence intensity (MFI), and IL-4+ / IL-13+ cell values ​​are expressed as the percentage of positive cells within the CD8+CD7+ T cell compartment. Student's t-test, *p<0.05. Cytolytic activity of T cells and NK cells in co-culture with G K562 target cells was quantified by measuring lactate dehydrogenase (LDH) release from target cells. T cells and NK cells were stimulated with anti-CD3 / CD28 and IL-2 / IL-15, respectively, and anti-CD7 was added to inhibit CD7 costimulation.Unstim refers to unstimulated control cells. Statistical comparisons between groups were performed using standard one-sided, one-way analysis of variance (ANOVA) with Tukey's multiple comparison test. *p<0.05, **p<0.01, ***p<0.001. Pairwise correlation plots of CD7 and XCL1, TGB1, OSM, and MMP9 gene expression within NK cell or cytotoxic T cell (CTL) clusters in H SSc-affected skin (GSE195452). [Figure 7] Pairwise correlations between CD7 and pro-fibrotic genes. Wiki gene pathway pulmonary fibrosis and pro-inflammatory and pro-fibrotic phenotypes were integrated, and the potential correlation between CD7 gene expression and included genes was evaluated separately for each cluster: Group A (cytotoxic T cells: CTLs) and Group B (NK cells). Statistical significance for each comparison is adjusted for multiple comparisons and presented as adjusted p-values. *p<0.05, **p<0.01. [Figure 8]Removal of activated CD7+ T cells and NK cells by targeted immunotoxins prevents fibroblast contraction and reduces myofibroblast phenotype. A Flow cytometry quantification of CD3 / CD7-IT induced cell death, showing the absolute cell number (cell count / μl) of CD2+ T cells and CD56+ NK cells in peripheral blood mononuclear cells (PBMCs) isolated from SSc patients (n=5). B Normalized cell survival percentage (n=3) of CD3+ T cells and CD56+ NK cells in PBMCs isolated from SSc patients. C Flow cytometry quantification of CD3 / CD7-IT induced cell death, showing the absolute cell number (cell count / μl) of CD8+GZMB+ T cells and CD56+GZMB+ NK cells in peripheral blood mononuclear cells (PBMCs) isolated from SSc patients (n=5). D Flow cytometry quantification of CD3 / CD7-IT induced cell death in CD8+ T cells and CD56+ NK cells in ex vivo skin explants (n=4). Contrast t-test, *p<0.05, **p<0.01. E Schematic diagram of our in vitro hydrogel collagen contraction assay in a newly developed 3D model co-cultured primary cutaneous fibroblasts and peripheral blood mononuclear cells (PBMCs). Contraction levels were quantified compared to a cell-free control and plotted as a graph on the right (n=3). Bar graphs show the mean ± standard deviation. Representative experimental images are shown at the bottom of this panel. F The proportion of pro-apoptotic cytotoxic T cells (CD8+7-AAD-Annexin V+) and NK cells (CD56+7-AAD-Annexin V+) under the illustrated conditions was measured by flow cytometry of enzyme-digested collagen plugs (n=5). Peripheral blood mononuclear cells (PBMCS) treated with G IgG or CD3.CD7-IT were co-cultured with primary dermal fibroblasts in a developed 3D hydrogel collagen co-culture model, and gene expression reflecting the myofibroblast phenotype was analyzed in the fibroblasts. The values ​​represent relative gene expression levels (-ΔCt) measured by qPCR. GAPDH and RPS27A were used as reference genes. Data are expressed as mean ± standard error (SEM). Statistical comparisons of three or more groups were performed using standard one-way analysis of variance (ANOVA) with Tukey's multiple comparison test.*p<0.05, **p<0.01, ***p<0.001. Abbreviations: phytohemoaglutinin (PHA), immunotoxin (IT), peripheral blood mononuclear cells (PBMCs). [Figure 9] CD3 / CD7-IT specifically removes only activated cytotoxic T cells and NK cells in vitro. A. IL-2 concentration (pg / ml) in the cell supernatant was measured with and without α-CD3 / CD7-IT treatment. B. Pie charts showing the proportions of effector cells (CD8+CD45RA+CD27-), memory cells (CD8+CD45RA-CD27+), and naive cells (CD8+CD45RA+CD27+) in the CD8+ T cell population under the indicated stimulation and treatment culture conditions (percentages in the pie charts are mean values ​​for n=6 SSc patients). C. The response of α-CD3 / CD7-IT treated cells to TCR-mediated (PHA) restorative stimulation was evaluated by intracellular flow cytometry. Values ​​are expressed as a ratio of change after restorative stimulation compared to the pre-stimulation value. D (Bottom) Absolute number (cell count / μl) of CD19-positive B cells after in vitro treatment with CD3 / CD7-IT compared with untreated peripheral blood mononuclear cells (n=6). (Top) Representative flow cytometry plot of a particular experiment. E Normalized cell viability of M2 macrophages and CD19+ B cells isolated from the blood of SSc patients under different culture / treatment conditions as shown. Cycloheximide was used as a positive control. F Flow cytometry histogram of a representative experiment showing increased CD3 and CD7 expression in CD8+GZMB+ cells and increased CD7 expression in CD56+GZMB+ NK cells upon phytohemoaglutinin (PHA) stimulation. This increased expression is further quantified in G. G CD3 and CD7 expression is shown as mean fluorescence intensity (MFI). Statistical analysis was performed using Student's t-test. **p<0.01,****p<0.0001 H The percentages of necrotizing cytotoxic T cells (CD8+7-AAD+Annexin V+) and NK cells (CD56+7-AAD+Annexin V+) under the illustrated conditions were measured by flow cytometry of enzyme-digested collagen plugs (n=5). [Figure 10] Anti-CD3 / CD7-IT treatment reduces cytotoxic T cells and NK cells in the blood and skin of patients with early scleroderma. A. Percentage of CD3+ T cells and CD56+ NK cells in peripheral blood of SSc patients before and after anti-CD3 / CD7-IT treatment. B. Percentage of CD4+ T cells and CD8+ T cells in peripheral blood of SSc patients before and after anti-CD3 / CD7-IT treatment. (Right) CD4+ / CD8+ T cell ratio before and after treatment. C. Percentage of CD8+ perforin+ T cells and CD56+ perforin+ NK cells in peripheral blood of SSc patients before and after anti-CD3 / CD7-IT treatment. D. Representative immunofluorescence images of lesional skin of patients before and after treatment. E. Quantification of lymphocyte subsets in lesional skin of patients before and after treatment. [Figure 11]The SECTM1-CD7 axis in the activation of cytotoxic T cells and NK cells in lesional skin of systemic sclerosis (SSc). A schematic diagram model (created with BioRender.com) illustrating the proposed SECTM1-CD7 axis's involvement in the activation of cytotoxic T cells and NK cells. In SSc-affected skin, CD7 and IFNG are predominantly expressed in cytotoxic T cells and NK cells, while SECTM1 and IFNGR are expressed in antigen-presenting cells (APCs) and stromal cells (primarily fibroblasts). This suggests the existence of a cytokine-mediated positive feedback loop between CD7-positive cytotoxic immune cells and SECTM1-producing APCs and fibroblasts, with IFN-γ as the major cytokine. B A two-dimensional dot plot comparing the expression levels of selected genes in immune cell (myeloid and lymphoid) and stromal cell populations in skin. Cell cluster annotations were obtained from metadata information as described in the single-cell RNA sequencing dataset GSE195452. The size of the circles indicates the percentage of cells expressing each gene, and the intensity of the color represents the average expression level. The numerical values ​​represent the average of the normalized counts. The pairwise correlation plot in the skin of a C SSc affected area (GSE195452) shows a positive correlation between CD7 / IFNG and SECTM1 / IFNGR1. Abbreviations: TCR: T cell receptor, NKR: NK cell receptor, MHC: major histocompatibility complex, APC: antigen-presenting cell. [Figure 12]When fibroblasts are co-cultured with CD7-positive T cells and NK cells, their contractility increases, and a myofibroblast-like phenotype is enhanced. A. Schematic diagram of the experimental design in the developed 3D in vitro collagen contractile fibroblast:immune cell co-culture model. B. Flow cytometry-based gate setting strategy used to fractionate CD7-positive versus CD7-negative T cell and NK cell populations from healthy peripheral blood (n=3). First, lymphocyte populations were gated based on cell size (FSC) and granularity (SSC). Next, dead cells were excluded based on 7-AAD-positive staining, and then CD19-CD14-negative lymphocytes with positive or negative CD7 expression were fractionated. C. Fibroblast contractility levels were quantified compared to a cell-free control and plotted on a graph (n=3). Error bars indicate mean ± standard deviation. Representative experimental images are shown on the right side of this panel. D (Left) Representative images of immunohistochemical staining of type I collagen in collagen plugs containing only fibroblasts, or in collagen plugs co-cultured with fibroblasts, CD7-positive T cells, and NK cells. (Right) Quantification of type I collagen-positive fibroblasts in the control group and in fibroblast groups co-cultured with CD7-negative or CD7-positive cells (n=3). Error bars indicate mean ± standard deviation. E The percentage of CD45-α-SMA+IL-6+ fibroblasts under the indicated conditions was measured by flow cytometry of enzyme-digested collagen plugs (n=3). F The αSMA expression level (mean fluorescence intensity - MFI) of CD45-αSMA+ fibroblasts under the indicated conditions was measured by flow cytometry of enzyme-treated collagen plugs (n=3). (Right) A flow cytometry histogram of one representative experiment showing increased α-SMA expression in fibroblasts co-cultured with CD7+ T cells compared to CD7- T cells and NK cells. G CD7+ T cells and NK cells versus CD7- T cells and NK cells (n=3) were co-cultured with primary dermal fibroblasts in a developed 3D hydrogel collagen co-culture model, and the expression of genes reflecting myofibroblast-like phenotypes, such as COL1A1 and ACTA2, was analyzed in the fibroblasts. The values ​​represent relative gene expression levels (-ΔCt) measured by qPCR.GAPDH and RPS27A were used as reference genes. Data are presented as mean ± standard error (SEM). [Modes for carrying out the invention]

[0031] (Detailed description of the invention) In this specification, a pharmaceutical composition is defined as any composition that can be administered to an individual, whether as a single dose or as multiple doses according to a dosage schedule, via any effective route, preferably intravenously, and optionally containing a standard administration medium and / or standard therapeutic components for related chronic inflammatory or autoimmune diseases. For example, the pharmaceutical compositions of the present invention have the ability to remove or reduce the number of unwanted CD7-positive cells.

[0032] Unwanted cells are those that contain CD7 molecules (and many others) on their cell surface, and these cells are involved in pathological conditions in an individual. Typically, these cells are T cells, NK cells, and other cells involved in chronic inflammatory or autoimmune diseases. Abnormal cells containing CD7 (e.g., T-cell leukemia and lymphoma) can also be removed or suppressed according to the present invention.

[0033] The pharmaceutical composition of the present invention comprises a first molecule that specifically recognizes CD7. Optionally, the pharmaceutical composition of the present invention further comprises a second molecule that specifically recognizes CD3. Additionally or alternatively, the pharmaceutical composition according to the present invention may further comprise at least one further molecule that specifically recognizes CD5, CD2, CD4, CD8, or IL-2 receptors.

[0034] CD3 / CD7 or other ligand-receptors, such as molecules that specifically recognize another cell surface antigen, are terms well known to those skilled in the art.

[0035] This refers to any molecule that has relatively high binding affinity and specificity to CD3, CD7, or other receptors, such as cell surface antigens. In the context of the present invention, CD3, CD7, and other cell surface antigens described herein are human antigens. Typically, these are ligands for receptors, or antibodies against CD3, CD7, and other receptors (defined as any molecule capable of specific interaction with a receptor). Such antibodies may be cleaved, humanized, or otherwise modified without losing their specificity (such modifications are defined herein as derivatives and / or fragments).

[0036] Preferably, one or more of the first molecule, second molecule, and further molecules according to the present invention are fragments of an antibody or a derivative thereof. As used in all aspects of the present invention, the terms “antibody” or “antibody molecule” include all immunoglobulins, whether naturally occurring or partially or entirely synthetically produced. The terms “antibody” or “antibody molecule” include monoclonal antibodies (mAbs) and polyclonal antibodies (including polyclonal antisera). Antibodies may be in their complete form or fragments derived from a complete antibody (see below). Antibodies may be human antibodies, humanized antibodies, or non-human antibodies. A “monoclonal antibody” is a homogeneous population of antibodies that reacts very specifically to a single antigenic site or “determinant” of a target molecule. A “polyclonal antibody” includes a heterogeneous population of antibodies directed to different antigenic determinants of a target molecule. “Antiserum (one or more)” refers to serum containing antibodies obtained from an immunized animal. It has been demonstrated that fragments of a whole antibody can exhibit antigen-binding function. Accordingly, references to antibodies in this specification, and to the methods, arrays, and kits of the present invention, cover not only complete antibodies but also any polypeptide or protein containing antibody-binding fragments. Examples of binding fragments include (i) Fab fragments consisting of VL, VH, CL, and CH1 domains; (ii) Fd fragments consisting of VH and CH1 domains; (iii) Fv fragments consisting of VL and VH domains of a single antibody; (iv) dAb fragments consisting of a VH domain; (v) isolated CDR regions; (vi) F(ab')2 fragments, which are bivalent fragments containing two linked Fab fragments, with the VH and VL domains linked by a peptide linker so that both domains bind to form an antigen-binding site; (vii) single-chain Fv molecules (scFv); (viii) bispecific single-chain Fv dimers (WO 93 / 11161); and (ix) "diabodies," polyvalent or polyspecific fragments constructed by gene fusion (WO94 / 13804; 58). Fv, scFv, or diabody molecules can sometimes be stabilized by incorporating a disulfide bond linking the VH and VL domains. Minibodies containing scFv with a CH3 domain can also be constructed.

[0037] The antibody molecules and immunotoxins used herein are intended to include recombinant antibodies and recombinant immunotoxins, respectively (e.g., SC monoclonal antibodies conjugated to recombinant ribosome inhibitory proteins via Fab, scFv, or cleavable peptide linkers). Additionally, or alternatively, the first and second antibody molecules may be provided as a single bispecific (anti-CD3 / anti-CD7) antibody, thereby providing a bispecific immunotoxin such as anti-CD3 / CD7-rRTA.

[0038] With respect to antibody molecules, the term "selectively binding" in this specification may be used to refer to a situation in which one component of a particular binding pair does not show significant binding to molecules other than its specific binding partner. This term also applies, for example, when an antigen-binding site is specific to a particular epitope present in multiple antigens. In this case, a specific binding molecule having an antigen-binding site can bind to various antigens that have that epitope.

[0039] Preferably, when the first molecule is an antibody, it is a monoclonal antibody, such as a mouse monoclonal antibody.

[0040] In a preferred embodiment, the first molecule comprises a combination of complementary determination regions (CDRs). The CDR sequence comprises a CDR3 sequence having at least 95, 96, 97, 98, or 99% sequence identity with the amino acid sequence of SEQ ID NO: 17 (ARWAYFYGSSPYFFDY). Most preferably, the CDR3 sequence comprises or consists of the amino acid sequence of SEQ ID NO: 17. Optionally, the CDR sequence further comprises a CDR1 sequence having at least 95, 96, 97, 98, or 99% sequence identity with the amino acid sequence of SEQ ID NO: 15 (GYTFTNYG). Preferably, the CDR1 sequence comprises or consists of the amino acid sequence of SEQ ID NO: 15. The CDR sequence may further comprise a CDR2 sequence having at least 95, 96, 97, 98, or 99% sequence identity with the amino acid sequence of SEQ ID NO: 16 (INTYTGEP). Preferably, the CDR2 sequence comprises or consists of the amino acid sequence of SEQ ID NO: 16.

[0041] Sequence identity and sequence similarity can be determined by aligning two peptide sequences or two nucleotide sequences using a whole-sequence alignment algorithm or a local-sequence alignment algorithm according to the lengths of the two sequences. Sequences of similar lengths are preferably aligned using a whole-sequence alignment method (e.g., the Needleman-Wunsch method) that optimally aligns the entire sequence. On the other hand, sequences of significantly different lengths are preferably aligned using a local-sequence alignment method (e.g., the Smith-Waterman method). Subsequently, sequences (when optimally aligned using default parameters in a program such as GAP or BESTFIT) may be described as "substantially identical" or "essentially similar" when they share at least a certain minimum sequence identity percentage (described later). GAP uses the Needleman-Wunsch whole-sequence alignment algorithm to align two sequences over their entire length (full length). This maximizes the number of matching locations and minimizes the number of gaps. Whole-sequence alignment is appropriately used to determine sequence identity when the lengths of two sequences are similar. Generally, the default parameters for GAP are used: a gap generation penalty of 50 (nucleotides) / 8 (proteins) and a gap extension penalty of 3 (nucleotides) / 2 (proteins). For nucleotides, nwsgapdna is used as the default scoring matrix, and for proteins, Blosum62 (Henikoff & Henikoff, 1992, PNAS 89, 915-919) is used as the default scoring matrix.Sequence alignment and sequence identity percentage scores may be calculated using, for example, the GCG Wisconsin Package version 10.3 available from Accelrys Inc. (address: 9685 Scranton Road, San Diego, CA 92121-3752 USA), or computer programs such as the open-source software "needle" (using the full-sequence Needleman-Wunsch algorithm) or "water" (using the local Smith-Waterman method), run with the same parameters as the GAP described above, or with default settings (both "needle" and "water," for both protein and DNA alignments, default gap opening penalty is 10.0, default gap extension penalty is 0.5, default scoring matrices are Blossum62 for protein and DNAFull for DNA). When the full-length sequences are substantially different, local alignment using the Smith-Waterman algorithm is preferred.

[0042] Alternatively, the percentage of homology or identity may be determined by searching public databases using algorithms such as FASTA or BLAST. Therefore, the nucleic acid and protein sequences of the present invention can be further used as "query sequences" to perform searches on public databases, for example, to identify other family members or related sequences. Such searches can be performed using the BLASTn and BLASTx programs (version 2.0) described in Altschul et al. (1990) J. Mol. Biol. 215:403-10. BLAST nucleotide searches can be performed using the NBLAST program (score = 100, word length = 12), which yields nucleotide sequences homologous to the oxidoreductase nucleic acid molecule of the present invention. BLAST protein searches can be performed using the BLASTx program (score = 50, word length = 3), which yields amino acid sequences homologous to the protein molecule of the present invention. To obtain a gapped alignment for comparison, Gapped BLAST is used according to the method described in Altschul et al., (1997) Nucleic Acids Res. 25(17): 3389-3402. When using the BLAST and Gapped BLAST programs, the default parameters of each program (e.g., BLASTx and BLASTn) can be used. See the National Center for Biotechnology Information website: http: / / www.ncbi.nlm.nih.gov / .

[0043] Optionally, the first numerator is: - WT1, mouse IgG2a (WO9948534A1), - TXU-7 Mouse IgG1 (CA2292426A1) - RFT2, mouse IgG2a (Kirkham et al 1988) - CHT2, SDZCHH380, mouse IgG2a (Heinrich et al 1989, Lazarovits et al 1993) - FITC-S9.1, mouse IgG2b (Fishwild et al 1992) - 3A1e, mouse IgG2b (Vallera et al 1996); and - HB2, mouse IgG1 (Flavel et al 2001) It is selected from the group consisting of the following.

[0044] Preferably, the first molecule is a monoclonal IgG2a antibody. In some embodiments, the first molecule may include a VH domain having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence of SEQ ID NO: 13 and / or a VL domain having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence of SEQ ID NO: 14. In certain embodiments, the first molecule may be a WT1 antibody having the heavy chain of SEQ ID NO: 11 and the light chain of SEQ ID NO: 12.

[0045] <WT1> WT1 (INN name grisnilimab) is a mouse IgG2a monoclonal antibody that selectively binds to human CD7 (UniProt: P09564). CD7 is a transmembrane protein belonging to the immunoglobulin superfamily and is present on thymocytes and mature T cells. WT1, as well as its manufacturing and characterization, are described in EP 0945139 A1. Tax et al., Monoclonal antibodies against human thymocytes and T lymphocytes. Protides of the Biological Fluids, 29th Colloquium, 1981, edited by Peeters H, Pergamon Press, Oxford and New York, 1982; Tax et al., Hamatol Bluttransfus, 1983, Vol. 28, pp. 139-141; and Tax et al., Clin Exp Immunol, 1984, Vol. 55, pp. 427-436 (all of these descriptions are incorporated expressly by reference herein). WT1 is commercially available. For example, the anti-CD7 antibody (clone WT1) is available for research use (e.g., immunofluorescence and immunohistochemistry) from LifeSpan BioSciences, Inc. under catalog number LS-C122885-1000 (PBS, 1000 μl in 0.1% sodium azid).

[0046] The monomeric molecular weight of WT1 is approximately 150 kDa, consisting of two heavy chains of approximately 50 kDa and two lambda light chains of approximately 25 kDa. The amino acid sequences of the WT1 light and heavy chains were determined by extracting mRNA from a hybridoma cell pellet, performing RT-PCR, and then DNA sequencing using an ABI3130x1 gene analyzer. The amino acid sequences were predicted and confirmed by mass spectrometry. Complementarity-determining regions (CDRs) were determined according to the IMGT numbering system (Lefranc, M.-P. et al., Nucleic Acids Research, 1999, Vol. 27, pp. 209-212, incorporated herein by reference).

[0047] The amino acid sequences of the WT1 heavy and light chains are shown below. WT1 heavy chain: [ka]

[0048] The VH domain is underlined. CDRH1-H3 is shown in bold and underlined with a wavy line.

[0049] WT1 Light Chain: [ka]

[0050] The VL domain is underlined. CDRL1-L3 is shown in bold and underlined with a wavy line.

[0051] WT1-VH: [ka]

[0052] WT1-VL: [ka]

[0053] WT1-CDRH1: [ka]

[0054] WT1-CDRH2: [ka]

[0055] WT1-CDRH3: [ka]

[0056] WT1-CDRL1: [ka]

[0057] WT1-CDRL2: [ka]

[0058] WT1-CDRL3: [ka]

[0059] The pharmaceutical formulations of the present invention optionally, or preferably, include a second molecule that specifically recognizes CD3. Preferably, if the second molecule is an antibody, it is a monoclonal antibody, for example, a mouse monoclonal antibody. In some embodiments of the present invention, the second molecule is an IgG2b antibody. This antibody does not immobilize human complement and does not bind to human Fc receptors, and therefore does not induce cytokine release by target T cells and / or NK cells. In some embodiments, the second molecule is a mouse antibody. For example, the second molecule is an anti-(human)CD3 mouse IgG2b monoclonal antibody.

[0060] In a preferred embodiment, the second molecule comprises a combination of complementary determination regions (CDRs). The CDR sequence includes a CDR3 sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence of SEQ ID NO: 7 (ARGSRYDYYGMDY). Most preferably, the CDR3 sequence includes or consists of the amino acid sequence of SEQ ID NO: 7. Optionally, the CDR sequence further comprises a CDR1 sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence of SEQ ID NO: 5 (GYTFTSYT). Preferably, the CDR1 sequence includes or consists of the amino acid sequence of SEQ ID NO: 15. The CDR sequence may further comprise a CDR2 sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence of SEQ ID NO: 6 (INPSSGYT). Preferably, the CDR2 sequence includes or consists of the amino acid sequence of SEQ ID NO: 6.

[0061] Optionally, the first numerator is as follows: - SPV-T3a, mouse IgG2b (Spits et al., 1983) - hOKT3γ4, humanized IgG4 (Richards et al., 1999) - Visilizumab, humanized IgG2 Fc modified compound (Carpenter et al., 2002) - Teplizumab, humanized, Fc modified (Herald et al., 2019) - Otelixizumab, humanized, Fc modified (Keymeulen et al., 2020) It is selected from the group consisting of the following.

[0062] In some embodiments, the first molecule may include a VH domain having at least 95, 96, 97, 98, or 99% sequence identity with the amino acid sequence of SEQ ID NO: 3, and / or a VL domain having at least 95, 96, 97, 98, or 99% sequence identity with the amino acid sequence of SEQ ID NO: 4. In certain embodiments, the second molecule may be an SPV-T3a antibody having the heavy chain of SEQ ID NO: 1 and the light chain of SEQ ID NO: 2.

[0063] <SPV-T3a> SPV-T3a (generic name: dafsolimab) is a mouse IgG2b monoclonal antibody that selectively binds to human CD3, a T cell surface glycoprotein composed of a CD3γ chain (UniProt: P09693), a CD3δ chain (UniProt: P04234), and two CD3ε chains (UniProt: P07766). SPV-T3a and its manufacturing and characterization are described in EP 0945139 A1 and Spits et al., Hybridoma, 1983, Vol. 2, pp. 423-437 (the entire contents of both documents are explicitly incorporated herein by reference).

[0064] SPV-T3a, with a monomer molecular weight of approximately 150 kDa, consists of two heavy chains of approximately 50 kDa and two κ-light chains of approximately 25 kDa. The amino acid sequences of the light and heavy chains of SPV-T3a were determined by extracting mRNA from a hybridoma cell pellet, performing RT-PCR, and then DNA sequencing using an ABI3130x1 gene analyzer. The amino acid sequences were predicted and confirmed by mass spectrometry. The complementarity-determining regions (CDRs) were determined according to the IMGT numbering system (Lefranc, M.-P. et al., Nucleic Acids Research, 1999, Vol. 27, pp. 209-212, incorporated herein by reference).

[0065] The amino acid sequences of the SPV-T3a heavy chain and light chain are shown below, respectively.

[0066] SPV-T3a heavy chain: [ka]

[0067] The VH domain is underlined. CDRH1-H3 is shown in bold and underlined with a wavy line.

[0068] SPV-T3a light chain: [ka]

[0069] The VL domain is underlined. CDRL1-L3 is shown in bold and underlined with a wavy line.

[0070] SPV-T3a-VH: [ka]

[0071] SPV-T3a-VL: [ka]

[0072] SPV-T3a-CDRH1: [ka]

[0073] SPV-T3a-CDRH2: [ka]

[0074] SPV-T3a-CDRH3: [ka]

[0075] SPV-T3a-CDRL1: [ka]

[0076] SPV-T3a-CDRL2: [ka]

[0077] SPV-T3a-CDRL3: [ka]

[0078] Optionally, when the pharmaceutical composition of the present invention includes a second molecule, the ratio of the first molecule to the second molecule may be within the range of 1:10 to 10:1, 1:9 to 9:1, 1:8 to 8:1, 1:7 to 7:1, 1:6 to 6:1, 1:5 to 5:1, 1:4 to 4:1, 1:3 to 3:1, 1:2 to 2:1, for example, 1:1.

[0079] Preferably, one or more of the first molecule, the second molecule, and further molecules according to the present invention comprises at least one toxic moiety. This may result in high efficacy, but it may also be used in combination with, for example, prodrug therapy to provide high specificity to cell populations.

[0080] The toxic portion essentially refers to any molecule that directly or indirectly causes toxicity to target cells, and includes, but is not limited to, lectins, lysine, abrin, PE toxin, diphtheria toxin, radioisotopes, cell proliferation inhibitors such as adriamycin, apoptosis inducers, and prodrugs such as the combination of thymidine kinase and ganciclovir, as well as prodrug conversions.

[0081] The first, second, or further molecules according to the present invention may comprise the same or different toxic moieties. Optionally, the toxic moieties may be independently selected from the group consisting of lysine, deglycosylated lysine A (dgRTA), and non-glycosylated recombinant lysine A. Deglycosylated lysine A is preferred in some embodiments of the present invention because it can inhibit the binding of lysine A to glucose receptors expressed by hepatocytes. In prodrug embodiments, the prodrug may typically comprise a converter to one of CD3 or CD7 or a molecule that specifically recognizes the receptor, with the other being the prodrug. However, CD3 does not need to bind to the toxic site to exert its effect because it inhibits the interaction between the T cell receptor and APC. This is one of the advantages of the present invention that is not present in the prior art.

[0082] In one preferred embodiment of the present invention, the first molecule, for example WT-1, comprises recombinant lysine A (rRTA). In another preferred embodiment of the present invention, the pharmaceutical composition comprises both the first molecule and the second molecule (for example, both WT-1 and SPV-T3a) (each comprising recombinant lysine A (rRTA)). For example, the pharmaceutical composition of the present invention comprises WT-1 conjugated with RTA (generic name: grisnilimab setaritox) and SPV-T3a conjugated with RTA (generic name: dafsolimab setaritox).

[0083] The binding ratio of toxin (e.g., rRTA) to molecule (e.g., antibody molecule) can range from 0.5:1 to 5:1. In particular, the binding ratio of toxin (e.g., rRTA) to molecule (e.g., antibody molecule) can range from 0.5:1 to 2:1, for example, from 0.75:1 to 2:1, or from 0.75:1 to 1.5:1.

[0084] <rRTA> RTA stands for lysine toxin A chain. In its natural form, the plant toxin lysine consists of two disulfide-bonded chains, each approximately 32 kDa in length, with an A chain (RTA) that inhibits protein synthesis and a B chain (RTB) that promotes cell binding and membrane translocation.

[0085] In clinical applications, RTB is replaced with cell-targeting ligands specifically directed towards the target cells, such as mAb SPV-T3a and / or WT1. After being introduced into the cell by the mAb, RTA dissociates from the mAb, and a portion of the released RTA is transported to the cytoplasm via the trans-Golgi network and the endoplasmic reticulum (ER). In the cytoplasm, RTA catalytically inhibits protein synthesis through N-glycosidase activity and 28S ribosomal RNA A 4324 Remove the base. This prevents the binding of elongation factors 1 (EF-1) and EF-2.

[0086] The rRTA according to the present invention may be produced from recombinant E. coli cell lines constructed for this purpose. The RTA protein has a monomeric molecular weight of 30 kDa and contains 268 amino acids.

[0087] A preferred embodiment of the rRTA of the present invention may have at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence of SEQ ID NO: 21. In one preferred embodiment of the present invention, the rRTA comprises or consists of the amino acid sequence of SEQ ID NO: 21.

[0088] Sequence ID 21 [ka]

[0089] The toxic portion may be linked to a molecule that specifically binds using any suitable conjugation or linker chemistry, such as creating a fusion protein by recombinant means that typically includes a protease cleavage site between the binding molecule and the toxic (protein) portion. However, due to ease of manufacture and freedom in selecting the toxic portion, chemical binding, optionally by an acid-degradable linker, is preferred. (The conjugate typically passes through lysosomes during internalization.) Other linkers suitable for use in the pharmaceutical compositions of the present invention may be selected from the group consisting of cleavable linkers (e.g., enzyme-cleavable peptide linkers, acid-sensitive hydrazone linkers, glutathione-sensitive disulfide linkers) and non-cleavable linkers (e.g., linkers based on maleimidocaproyl [MC] and 4-maleimidomethylcyclohexane-1-carboxylate [MCC]).

[0090] In particular, conjugation may be performed using N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP; Pharmacia) or 4-succinimidyloxycarbonyl-α-methyl-α(2-pyridyldithio)toluene (SMPT). SMPT is a so-called "second-generation crosslinking agent" characterized by having a disulfide bond inhibited by the presence of a phenyl ring. As a result, the SMPT linker is less susceptible to extracellular reduction by thiols present in tissues and blood, and consequently, the serum half-life of the immunotoxin is extended. Thorpe et al. demonstrated in vivo experiments using a mouse tumor model that the antitumor effect of dgRTA-based ITs was significantly improved by using SMPT instead of the first-generation crosslinking agent SPDP (WO9948534A1). Although SMPT contains a disulfide bond, this bond plays an important role in the intracellular dissociation of the molecule and the toxic portion.

[0091] The present invention also provides a pharmaceutical composition in which the same or different toxic moieties are conjugated to at least two molecules that specifically recognize different receptors. Using different toxic moieties offers significant advantages when the side effects of the toxic moieties may differ, because it allows for the administration of higher doses. Typically, the pharmaceutical composition of the present invention may further include at least one additional molecule that specifically recognizes CD5, CD2, CD4, CD8, or IL-2 receptors. This additional molecule can also be conjugated to the toxic moiety. This results in high efficacy and can also be used, for example, in combination with prodrug therapy to provide high specificity to cell populations.

[0092] The dosage used is described in detail herein. The dose limit of an immunotoxin in a dosage scheme like the present invention is usually dependent on the immunotoxin itself. This is due not only to the specificity and affinity of the specific binding molecule, but also because the acceptable dose differs for each drug. Expressed in terms of RTA equivalents, the limit is generally per 1 m² of body surface area. 2 No more than 10 mg of RTA equivalent per unit, preferably 1 ml 2 No more than 5 mg of RTA equivalent per unit, more preferably 1 mg 2 The amount is within 2.5 mg RTA equivalent per unit, for example, within 2.0 mg RTA equivalent. Preferably, the RTA equivalent dose of the pharmaceutical composition of the present invention is within 1 m² of body surface area. 2 Each dose contains 0.3 to 2.0 mg of RTA equivalent, more preferably with a body surface area of ​​1 m². 2 Each dose contains 0.5 to 1.5 mg of RTA (Rapid-Reactive Acid) equivalent, for example, for a body surface area of ​​1 m². 2 Each serving contains 0.6-1.2 mg of RTA equivalent.

[0093] Typically, the compositions of the present invention are used to treat patients with chronic inflammatory or autoimmune diseases, or other CD7-positive malignancies. For example, the compositions of the present invention can be used to treat autoimmune diseases selected from the group of diseases including systemic lupus, rheumatoid arthritis, systemic sclerosis, localized sclerosis, IgG4-related disease, myositis, Sjögren's syndrome, primary sclerosing cholangitis, primary biliary cirrhosis, autoimmune hepatitis, vasculitis, nephritis, encephalomyelitis, neuropathy, (sclero)itis; chronic inflammatory diseases including axial spondyloarthritis, peripheral spondyloarthritis, psoriatic arthritis, inflammatory bowel disease, psoriasis; and atopic allergic diseases including atopic dermatitis, atopic asthma, nasal polyps. Preferably, systemic autoimmune diseases are systemic sclerosis or Sjögren's syndrome. Patients eligible for treatment of the present invention may exhibit symptoms of vascular lesions, fibrosis, and / or autoimmune inflammation.

[0094] Therefore, such a dosage plan is also part of the present invention.

[0095] The compositions of the present invention target activated pathogenic T cells and / or NK cells in a patient, preferably activated pathogenic T cells and / or NK cells in the patient's blood and / or inflammatory tissue. Preferably, the present invention targets activated pathogenic T cells and / or NK cells by selective depletion, membrane receptor inhibition, or intracellular kinase inhibition. Optionally, activated pathogenic T cells are selected from the group consisting of CD8 cytotoxic T cells, CD4 helper T cells, follicular helper T cells, proliferative T cells, and combinations thereof. Preferably, activated pathogenic T cells include CD8 cytotoxic T cells.

[0096] The pharmaceutical composition of the present invention is administered to a patient, preferably a human, via various routes. All modes of administration are conceivable, such as oral, rectal, intravenous, intradermal, intramuscular, subcutaneous, intrauterine, or intraventricular injection. Preferably, the pharmaceutical composition of the present invention is administered intravenously. For example, by continuous intravenous infusion, over periods of up to 6 hours, 4 hours, 2 hours, 1 hour, or 45 minutes, 30 minutes. In one embodiment, the pharmaceutical composition of the present invention is administered by continuous intravenous infusion over a period of 4 hours.

[0097] The pharmaceutical composition of the present invention may optionally be administered, for example, by intravenous infusion.

[0098] When using complete antibodies, the dosage of the first molecule per dose can be 1-50 mg, 1-40 mg, 1-30 mg, 1-25 mg, 1-20 mg, 1-10 mg, 3-10 mg, 3-8 mg, 4-9 mg, 4-8 mg, 5-7 mg, for example, about 6 mg.

[0099] When using a complete antibody-RTA conjugate, the dosage can be 0.01-1 mg per kg of body weight, 0.05-5 mg per kg of body weight, 0.05-2 mg per kg of body weight, 0.05-1 mg per kg of body weight, 0.05-0.5 mg per kg of body weight, 0.05-0.2 mg per kg of body weight, for example, approximately 0.1 mg per kg of body weight.

[0100] The drug may be administered once a day, every two days, every three days, every four days, every five days, every six days, or every seven days. For example, the drug may be administered once a day on days 0, 2, 4, and 6 for 14 days, or once a day on days 0, 2, 6, 11, 17, and 24. In one embodiment of the present invention, the drug is administered once a day on days 0, 2, 4, and 6.

[0101] The pharmaceutical compositions of the present invention optionally further comprise one or more excipients, carriers, buffers, stabilizers, isotonic modifiers, preservatives, or preservatives or antioxidants. Such materials should be nontoxic and should not interfere with the efficacy of the active ingredient. The exact properties of carriers and other materials may depend on the route of administration (e.g., intravenous injection).

[0102] In a preferred embodiment of the present invention, the composition comprises (i) a first molecule in an amount of 0.05 to 0.5 mg / mL, optionally 0.1 to 0.5 mg / mL, for example 0.2 mg / mL, and optionally one or more of the following: (ii) a second molecule in an amount of 0.05 to 0.5 mg / mL, optionally 0.1 to 0.5 mg / mL, for example 0.2 mg / mL; (iii) a citrate buffer in an amount of 5 to 20 mM, optionally 5 (iv) 50-300 mM, optionally 75-200 mM, for example 125 mM, of L-arginine or a pharmaceutically acceptable salt thereof; (v) 0.01-0.1% (w / v), optionally 0.01-0.08% (w / v), for example 0.05% (w / v), of polysorbate, for example Tween® 20; and (vi) 120-160 mM of maltose. Optionally, the composition is an aqueous solution. Preferably, the pH of the composition is in the range of 6-7.5, optionally 6-7, for example 6.5.

[0103] In some embodiments, the composition further includes at least one agent selected from the following: Maltose at 120-160 mM; Trehalose at 100-150 mM, optionally 125 mM; 25-75 mM, optionally 50 mM glycine; and 80-120 mM, optionally 100 mM mannitol Includes.

[0104] In particular, the composition may contain 130-150 mM, optionally 140 mM, of maltose monohydrate.

[0105] In some embodiments, the composition is as follows: (1) SPV-T3a-rRTA at 0.2 mg / mL and WT1-rRTA at 0.2 mg / mL; (2) 10 mM sodium citrate / citrate buffer; (3) 125 mM arginine hydrochloride; (4) 0.05% (by weight) Tween® 20; (5) Contains 140 mM maltose monohydrate, Here, the composition is an aqueous solution for injection, and its pH is 6.5.

[0106] In some embodiments, the composition is sterile. In some embodiments, the composition is suitable for injection.

[0107] The present invention also provides a method for targeting activated pathogenic T cells in patients with chronic inflammatory or autoimmune diseases. The method of the present invention comprises administering an effective amount of the pharmaceutical composition of the present invention as described herein to a patient. The method of the present invention may be combined with other immunosuppressive therapies and / or agents that enhance efficacy and / or safety. Optionally, the method of the present invention may be used as a pre-treatment for trelogogenic therapies, such as cell therapy using trelogogenic nanoparticles.

[0108] The present invention also provides a kit of pharmaceutical compositions for treating chronic inflammatory or autoimmune diseases, comprising compositions described herein. The present invention reduces the number of unwanted cell populations, such as activated pathogenic T cells, including CD7+ cells, CD8+ T cells, CD4+ T cells, or NK cells, to at least 20% of the original amount, and typically to 5% or less. Typically, this reduction is maintained at a low level over a long period, in contrast to the results achieved by prior art therapies. A further advantage of the present invention is that the exemplified compositions target not only T cells but also NK cells.

[0109] Although the present invention has been described and illustrated with reference to specific embodiments, those skilled in the art will understand that the invention is also applicable to many different modifications not specifically illustrated herein. For illustrative purposes only, certain possible modifications are described below.

[0110] (Examples) <material and method> immunotoxin The anti-CD3 / CD7 immunotoxin combination (CD3 / CD7-IT) mentioned in this paper consists of a 1:1 mixture (W / W) of mouse monoclonal antibodies SPV-T3a (anti-CD3) and WT1 (anti-CD7), both of which are conjugated to recombinant lysine toxin A, as previously reported (36,40).

[0111] Isolation, cryopreservation, and culture of peripheral blood mononuclear cells (PBMCs) PBMCs were isolated from the peripheral blood of patients (n=30) and healthy donors (n=15) by Ficoll Pacque PLUS density centrifugation and cultured in complete RPMI1640+ GlutaMAX® medium (Gibco, catalog number 72400-021) supplemented with 100 IU / ml penicillin, 100 mg / ml streptomycin, 100 mg / L sodium pyruvate, and 10% human mixed serum. PBMCs not processed immediately were cryopreserved and stored in liquid nitrogen until further use. To generate phytohemoaglutinin (PHA)-activated T cells, PBMCs were seeded at a cell density of 100,000 cells / well in 96-well U-bottom plates (Greiner) and stimulated with 5 μg / ml PHA (Roche, catalog number 11082132001) at 37°C under 5% CO2 conditions for 24 hours. To evaluate cytokine production, PBMCs were stimulated at 37°C under 5% CO2 conditions for 4 hours in the presence of 5 μg / ml brefelzin A (Merck), with 12.5 ng / ml foerball myristate acetate (Sigma) and 500 ng / ml ionomycin (Merck).

[0112] immunohistochemistry Immunohistochemical analysis was performed on skin biopsy specimens (formalin-fixed, paraffin-embedded (FFPE)) from 20 patients with systemic sclerosis. Skin biopsy specimens were obtained by surgical excision using a 6 mm diameter punch biopsy from lesional and non-lesional areas of the forearm, based on diagnosis by a specialist. CD3 staining was used to evaluate T cell infiltration in all skin specimens, and CD7 staining was used to evaluate activated T lymphocyte and NK cell infiltration. For CD3 staining, slides were deparaffinized by washing with xylene and rehydrated with ethanol. Antigens were recovered at room temperature (RT) in 10 mM sodium citrate buffer (pH 6.0). Peroxidase activity blocking was performed by incubation in 3% hydrogen peroxide solution for 30 minutes. Next, sections were incubated overnight at room temperature (RT) with mouse anti-human CD3 monoclonal antibody as the primary antibody, diluted 1:200 in PBS containing 1% BSA (clone F7.2.38, Dako, catalog number M7254). The tissue was then incubated with secondary antibody (BrightVision Poly-HRP, Immunologic DPVO55HRP) at room temperature for 60 minutes. Antibody visualization was performed using 3,3'-diaminobenzene (Bright DAB, Immunologic). Nuclei on all slides were counterstained with hematoxylin and mounted on coverslips (Permount, Thermo-Fischer, Waltham, MA, USA). CD7 was immunohistochemically evaluated using an Omnis automated immunohistochemical system (DAKO) according to the manufacturer's standard procedure. Briefly, FFPE tissues were deparaffinized, rehydrated, and then subjected to heat-mediated antigen recovery (97°C for 30 minutes). Next, endogenous peroxidase was inhibited, and a mouse anti-human CD7 monoclonal antibody (ready-to-use, diluted in Envision Flex Antibody Diluent, clone CBC.37, DAKO; catalog number GA64361-2) was applied as the primary antibody for 20 minutes at room temperature. The tissue was then incubated with a secondary antibody (BrightVision Poly-HRP, Immunologic DPVO55HRP) at room temperature for 60 minutes. 3'3'-diaminobenzene (bright DAB, Immunologic) was used to visualize the antibodies.All slide nuclei were counterstained with hematoxylin and mounted on coverslips (Permount, Thermo-Fischer, Waltham, MA, USA). CD7 was immunohistochemically evaluated using an Omnis automated immunohistochemical system (DAKO) according to the manufacturer's standard procedure. Briefly, FFPE tissue was deparaffinized, rehydrated, and then antigen was recovered by heat treatment (97°C for 30 minutes). Subsequently, endogenous peroxidase was inhibited, and mouse anti-human CD7 monoclonal antibody (ready to use, diluted with Envision Flex antibody diluent, clone CBC.37, DAKO; catalog number GA64361-2) was added as the primary antibody at room temperature for 20 minutes. Then, the secondary antibody (Envision Flex HRP, DAKO) was applied at room temperature for 20 minutes. The antibody conjugates were stained using Envision Flex substrate working solution (DAKO), and the nuclei were counterstained with hematoxylin. Human synovial / tonsil specimens were used as positive controls, and untreated skin sections were used as negative controls. The entire surface of all mounted sections (n=4) for each donor and symptom was observed and imaged using CaseViewer (v2.3.0.99276). CD3-positive cells were counted in four blinded fields of view by two independent observers, and the total number of positive cells was plotted as mean ± standard deviation. CD7-positive staining was evaluated using an arbitrary 0-4 semi-quantitative scoring system for positively stained areas. This scoring was performed blindly by two independent observers. The expression of type I collagen (Goat Anti-Type I Collagen-UNLB, Southern Biotech, catalog number 1310-01) in fibroblasts was also evaluated. Staining was performed in the same manner as for the CD3 marker, except that a reaction using rabbit biotinylated anti-goat IgG antibody (Vector Laboratories, PK-6101) as a secondary antibody was performed at room temperature for 30 minutes. The values ​​shown in the graph represent the mean ± standard deviation (SD).

[0113] Multiplex immunohistochemical staining and imaging of skin in systemic sclerosis (SSc) patients. For multiplex immunofluorescence staining, 5 μm thick sections were included from corresponding lesional and non-lesioned skin samples taken from 24 patients with systemic sclerosis (SSc). The slides were stained using an automated platform on the BOND RX IHC & ISH Research platform (Leica Biosystems) with the Opal 7-color Automation IHC kit (NEL801001KT; PerkinElmer). This followed the previously described method (41). Incubation with primary and secondary antibodies was performed at room temperature (RT) for 1 hour and 30 minutes, respectively. The following antibodies are used to detect cutaneous lymphocyte cell populations: Opal620-labeled anti-CD56 (Cell Marque, 156R-94, clone MRQ-42), Opal690-labeled anti-CD8 (Dako, M7103, clone C8 / 144B, 1:200), Opal480-labeled anti-CD7 (Dako, GA64361-2, clone CBC.37, 1:30), Opal520-labeled anti-CD3 (Thermo Fisher, RM-9107, clone RM-9107, 1:200), Opal570-labeled anti-FOXP3 (eBioscience Affymetrix (14-4777, clone 236A / E7, 1:100) and Opal 570-labeled anti-CD20 (ThermoFisher, MS-340, clone L26, 1:600) were used. Slides were stained with DAPI for 5 minutes, washed, and mounted with Fluoromount-G (SouthernBiotech, 0100-01). Subsequently, slides were scanned at 4x magnification using an automated quantitative pathology imaging system (Vectra V.3.0.4, PerkinElmer). Multispectral images of skin tissue were annotated using Phenochart (V.1.0.9, PerkinElmer) and scanned at 20x magnification. Spectral decomposition of Opal fluorescent dyes was performed using InForm software (V.2.4.2, PerkinElmer), and multichannel images were digitally synthesized. For quantitative analysis, digital scans including the entire skin biopsy (n=3 sections per biopsy for each donor and symptom) were quantified using QuPath-0.4.4(42).

[0114] Single-cell RNA sequencing analysis Cell-by-gene matrices were obtained from two publicly available datasets: GSE195452, GSE138669, and GSE128169. Seurat (version 4.3.0) was used for data preprocessing (40). Quality control measures were taken by excluding cells with high mitochondrial gene content (>5%) and cells with gene counts per cell less than 200 or greater than 2000. Subsequently, CD3+ and / or CD7+ cells were selected, and 2126 and 5061 high-quality cells were recovered from each dataset, respectively. A CD8+ subset was then isolated from these cells and analyzed separately.

[0115] Non-negative matrix decomposition was used for principal dimensionality reduction, followed by the application of UMAP (Uniform Manifold Approximation and Projection) with Louvain clustering (43, 44), as previously described by Singh et al. (45). Next, differentially expressed genes (DEGs) within each cluster were identified using Seurat's FindAllMarkers function, and annotations were performed based on the characteristics of these DEGs. The R package pheatmap (Kolde, R. (2019). pheatmap: Pretty Heatmaps (R package version 1.0.12)) was used to visualize DEGs between cell types / groups.

[0116] To evaluate the altered expression levels (DEGs) between healthy individuals and patients with the disease, we used Seurat's FindConservedMarker function. Furthermore, to test for differences in cell frequency between healthy individuals and patients with systemic sclerosis (SSc), we applied the Wilcox test (46).

[0117] Single-sample gene set enrichment and correlation analysis To gain insights into the functional characteristics of each cell type, we performed single-sample gene set enrichment analysis (ssGSEA) using the Escape R package (47) and the WikiPathways reference gene set collection from MsigDB (48).

[0118] We used the geom_tile R function included in the ggplot2 package (Wickham, H. (2016). ggplot2: Elegant Graphics for Data Analysis (2nd ed.). Springer). The geom_tile R function from Springer was used to visualize pathways between different cell types / groups. Differences in the distribution of normalized enrichment scores (NES) between the control group and the SSc group were tested using the Kolmogorov-Smirnov (KS) test (49). To capture pathways associated with fibrosis and inflammation, we obtained a list of genes associated with these processes and further enhanced it by including CD7.

[0119] Correlation analysis at the single-cell level is noisy and prone to bias due to technical factors. Therefore, we used the WGCNA R package (50) to construct a metacell object containing CD3 / CD7+ cells (from the previously described Seurat object). This object includes a weighted gene co-expression framework for identifying modules of highly correlated genes. This metacell object was then used to obtain pairwise correlations and p-values ​​(Harrell Jr., FE, & with contributions from Charles Dupont and many others (2020)). Hmisc: Harrell Miscellaneous (R package version 4.8.0)). For visualization of correlations, the R package ComplexHeatmap (Gu, Z. (2016). ComplexHeatmap: Making Complex Heatmaps in R (R package version 2.10.0)) was used.

[0120] Collection and culture of primary fibroblasts Half of a 4mm diameter skin biopsy was placed in a 24-well plate containing 2ml of DMEM Glutamax medium (Gibco, Waltham, Massachusetts, USA) supplemented with 100 U / ml penicillin, 100 mg / ml streptomycin, 100 mg / L sodium pyruvate, and 20% fetal bovine serum. The plate was cultured for 2 weeks under normal culture conditions (5% CO2, 37°C, 95% humidity). During this time, primary dermal fibroblasts spontaneously emerged from the skin biopsy and proliferated. The medium was changed every 3-4 days. After proliferation, the primary fibroblasts were cultured in DMEM Glutamax medium (Gibco) supplemented with 100 U / ml penicillin, 100 mg / ml streptomycin, 100 mg / L sodium pyruvate, and 10% fetal bovine serum, and used in experiments after 5 passages.

[0121] Isolation of single cells from skin biopsy Six-mm diameter skin punch biopsies collected from healthy individuals were used to obtain single-cell suspensions containing cutaneously infiltrating lymphocytes for phenotypic characterization and functional assays. The protocol used was a combination of mechanical and enzymatic dispersion methods for skin tissue, as extensively described by He et al. (PMID: 27166763).

[0122] Flow cytometry analysis For each donor, 1 x 10 6Each peripheral blood mononuclear cell (PBMC) was first labeled with ViaKrome 808 fixable vital stain (1.5:1000 in PBS) at 4°C for 30 minutes to remove dead cells. Then, it was stained with fluorescently labeled extracellular antibodies (see Additional Table x) at room temperature for 20 minutes. For intracellular staining (see Additional Table x), cells were fixed and permeabilized using Cyto-Fast® Fix / Perm Buffer Set (Biolegend) according to the manufacturer's instructions. To facilitate the detection of intracellular cytokines, cells were pre-stimulated with 12.5 ng / ml Forbor 12-myristate 13-acetate (PMA), 500 ng / ml ionomycin, and 5 μg / ml brefelzin A before staining. Samples were acquired immediately after staining using a Beckman Coulter Cytoflex LX 21-color flow cytometer.

[0123] Isolation, culture, and cell viability of T cells, B cells, and NK cells derived from SSc peripheral blood. Cryopreserved peripheral blood mononuclear cells (PBMCs) from patients with systemic sclerosis were thawed and washed according to previously reported methods, and specific immune cell populations were isolated. CD3+ T cells were isolated by magnetically negative selection according to the manufacturer's instructions (MojoSort pan CD3+ T cell isolation kit; catalog number 480021). CD19+ B cells were also isolated by negative selection using the MojoSort® Human Pan B cell isolation kit (catalog number 480082). Isolation of uncontacted CD56-positive NK cells from SSc peripheral blood mononuclear cells was performed using an NK cell isolation kit (Miltenyi Biotec, catalog number 130-092-657) according to the manufacturer's protocol. After isolation, enriched CD3+ T cell, CD19+ B cell, and CD56+ NK cell fractions were evaluated by flow cytometry staining for CD3, CD19, and CD56 markers, showing purity of over 95%. The isolated immune cell populations were cultured in 96-well U-bottom plates (Greiner) at a cell density of 50,000 cells / well using XVIVO™ 15 medium (Lonza, catalog number 04-418Q). Cell viability after various stimulation (24 hours) and treatment conditions (48 hours) was evaluated using the CellTiter-Glo® 2.0 Cell Viability Assay (Promega) as directed by the manufacturer. Cells treated with 5 mM cycloheximide (Sigma, catalog number 01810-1G) were also used as a positive control. Luminescence was measured using CLARIOstar Plus (BMG LABTECH). Four technical replicates were performed for each experimental condition, and the mean value was used for subsequent analysis. Experimental values ​​were corrected for medium luminescence and normalized to the unstimulated, untreated control condition.

[0124] Monocyte isolation and differentiation into M2 macrophages CD14+ monocytes were isolated from peripheral blood mononuclear cells (PBMCs) using a positive selection kit (Miltenyi Biotec, catalog number 130-050-201) according to the manufacturer's instructions. The monocytes were seeded in 6-well plates at a cell density of 1 million cells per well using 2 ml of XVIVO™15 medium supplemented with 100 IU / ml penicillin, 100 mg / ml streptomycin, and 2% human mixed serum. Differentiation into M2-like macrophages was stimulated by adding 20 ng / ml rhM-CSF (R&D Systems, catalog number 216-MC) and 10 ng / ml rhIL-4 (Biolegend, catalog number 500815). The culture period was 7 days, with the cytokine-containing medium replaced on day 3. The cell viability of M2-like macrophages was evaluated using the previously described method (CellTiter-Glo® 2.0 Cell Viability Assay, Promega).

[0125] Multiparameter flow cytometry quantification of CD3 / CD7-IT induced cell death To evaluate the in vitro killing effect of CD3 / CD7-IT on activated T cells and NK cells, a model mimicking disease-associated T cell activation was developed using 24-hour stimulation of PBMCs with PHA (Roche). PHA stimulation resulted in increased surface expression of CD3 antigen (2-fold increase in MFI) and CD7 antigen (3-fold increase in MFI) in cytotoxic CD8+GZMB+ T cells, and increased surface expression of CD7 antigen (2-fold increase in MFI) in CD56+GZMB+ NK cells (Figures 6A, B). Inactivated or PHA-activated (5 μg / ml) PBMCs were cultured at 37°C under 5% CO2 for 24 hours, followed by treatment with CD3 / CD7 immunotoxin (IT) for 48 hours. Based on previous studies, the clinical therapeutic concentration in vitro was in the range of 1-5 nM. Based on the killing effect on primary T cells, drug concentrations (0-10 nM) were titrated, and the lowest concentration showing the maximum killing effect was selected. The drug concentration used in the in vitro experiment was 0.33 nM. After treatment, cells were collected in 15 ml conical tubes, washed with PBS, and then treated with flow cytometry staining. The staining protocols for live / dead cells, extracellular and intracellular markers followed the previously described methods. Considering the possibility of modulation of the CD3 antigen by CD3 / CD7-IT treatment, CD2 was used instead of CD3 for the identification and characterization of T cell populations. To enable the quantification of absolute cell numbers, a fixed amount of counting beads (Precision Count Beads®, Biolegend, catalog number #424902) was added to each sample before acquisition. Samples were acquired immediately after staining using a Beckman Coulter Cytoflex LX 21-color flow cytometer.

[0126] In vitro skin culture Full-thickness skin punch biopsies, 6 mm in diameter, were collected from the abdomen of four healthy patients who had undergone reconstructive surgery. All patients signed informed consent for the use of their surgically retained tissue for research purposes. Four to six punch biopsies were collected from each skin tissue sample and cut in half. Considering the possibility of heterogeneity in immune cell infiltration between skin biopsies, all skin samples were pooled and then evenly distributed under different experimental conditions. Skin samples were cultured in 24-well plates in 1 ml of RPMI 1640 medium containing 100 IU / ml rhIL-2 (Thermo Fischer, cat# 16-7027-85), 5 μg / ml PHA (Roche), growth cofactors, and antibiotics. After 24 hours, samples were treated with 0.33 nM α-CD3 / CD7-IT. After 48 hours, single-cell suspensions containing skin-infiltrating lymphocytes for functional assays were obtained using skin samples from each condition. The protocol used combines mechanical and enzymatic dispersion methods of skin tissue, as described in detail by He et al. (51).

[0127] Apoptosis assay To distinguish early apoptotic cells from non-apoptotic and late apoptotic / necrotic cells, peripheral blood mononuclear cells (PBMCs) isolated, cultured, and prepared as described were first extracellularly stained with the monoclonal antibody of interest (see table) at room temperature for 20 minutes. The cells were then washed twice with cold PBS and resuspended in 100 μl of PBS buffer containing 7-AAD (5 μl), Annexin V:FITC-labeled (5 μl), and CaCl2 (1 M) (0.15 μl). Samples were incubated in the dark at room temperature for 10 minutes and measured immediately after staining using a flow cytometer (Gallios). Cells positive for both 7-AAD and Annexin V were referred to as late apoptotic / necrotic cells, while cells negative for 7-AAD and positive for Annexin V were referred to as early apoptotic cells. Viable cells were negative for both 7-AAD and Annexin V.

[0128] Cytokine measurement Quantification of human cytokines and chemokines in the culture supernatant was measured by the Luminex method. According to the manufacturer's instructions, a Bio-Plex kit was used. Samples were analyzed with Bio-Plex Manager 4 software (Bio-Rad Laboratories, Hercules, CA, USA).

[0129] LDH cytotoxicity assay To evaluate the cytotoxic abilities of cytotoxic T cells and NK cells, lactate dehydrogenase (LDH) in the co-culture supernatant of peripheral blood mononuclear cells (PBMCs) and K562 cells was measured using an LDH-cytox kit according to the manufacturer's protocol (Biolegend #426401). PBMCs and K562 cells were seeded at a ratio of 10:1 in 96-well plates (F bottom), and 3 wells were used in duplicate. To enhance the cytotoxic abilities of CD8+ T cells and NK cells, PBMCs were stimulated overnight with α-CD3 / CD28 (1 μg / ml) or IL-2 (500 IU / ml), IL-15 (10 ng / ml), respectively. To evaluate the involvement of the CD7 receptor in the cytotoxicity of T cells and NK cells, a 330 nM anti-CD7 (WT1) blocking antibody was used. The percentage of cytotoxic ability was calculated according to the following formula: cytotoxic ability (%) = (experimental value - low control value) / (high control value - low control value) × 100. The low control value and high control value corresponded to the LDH levels of K562 cells alone without or after the addition of lysis solution, respectively.

[0130] In vitro co-culture collagen contraction assay of fibroblasts and immune cells Healthy human primary fibroblasts were detached with trypsin, and then the cell density was adjusted to 2×10 6The cells / ml were adjusted. Peripheral blood mononuclear cells (PBMCs) collected from five healthy individuals were thawed and washed as previously described, and then stimulated / treated under various experimental conditions as described in the results section. A cell suspension was prepared containing a 5:1 mixture of PBMCs and fibroblasts. To create a 3D collagen hydrogel, for each plug, 20 μl of Minimum Essential Medium (Sigma-Aldrich, St. Louis, California, USA), 10 μl of sodium bicarbonate (Gibco, Waltham, Massachusetts, USA), 150 μl of soluble collagen (PureCol, Type 1 collagen), and 90 μl of the cell suspension were sequentially mixed in the specified order in a separate tube. After carefully homogenizing the suspension, 250 μl was added to each well of a 48-well plate. Subsequently, 750 μl of complete RPMI medium was added, and the plugs were cultured under standard conditions for 24 or 48 hours. Spontaneous fibroblast contraction was macroscopically assessed by scanning plates with a standard office flatbed scanner. To quantify the contraction area, the generated images were analyzed in Fiji ImageJ. To further study the phenotype and function of lymphocytes and fibroblasts in this model, after macroscopic assessment, collagen plugs were enzymatically digested for 1 hour on a roller at 37°C with a mixture of collagenase D, dispase, and DNase in unenhanced RPMI medium supplemented with penicillin 100 U / ml and streptomycin 100 mg / ml. The reaction was stopped by adding complete RPMI medium containing 10% HPS, and the single-cell suspension was washed twice with PBS before being used for flow cytometry analysis. To co-culture fibroblasts with CD7+ or CD7- T cells and NK cells (additional Figure 7), these cells were FACS fractionated from PBMCs and seeded on 3D collagen hydrogels.

[0131] RNA isolation and quantitative real-time PCR RNA isolation was performed using 500 μl of TRIzol (Sigma-Aldrich) according to the manufacturer's guidelines. After extraction, RNA concentration was quantified using a Nanodrop spectrophotometer (Thermo Scientific, Waltham, MA, USA), and genomic DNA was removed using DNAse I. Next, up to 1 μg of RNA was reverse transcribed to cDNA by one-step reverse transcription PCR at 37°C using an oligo dT primer and 200U M-MLV reverse transcriptase (all from Life Technologies). Gene expression in this cDNA was measured using quantitative real-time polymerase chain reaction (qPCR) with 0.25 mM validated primers (Biolegio, Nijmegen, Netherlands: see Supplemental Table 3) and SYBR Green master mix (Applied Biosystems, Waltham, Massachusetts, USA). Relative gene expression levels (-ΔCt) were calculated based on the mean values ​​of the reference genes GAPDH and RPS27A.

[0132] Research design The aim of this study was to elucidate the role of costimulatory receptors that regulate pathological processes induced by cytotoxic cells in the lesional skin of systemic sclerosis (SSc) patients, and to investigate whether targeting these receptors therapeutically can halt SSc pathology. To address these questions, we performed single-cell RNA sequencing analysis including skin cells from two separate SSc cohorts (109 SSc patients and 65 healthy controls). Multiplex immunohistochemistry (24 cases) was used for spatial imaging, and multicolor flow cytometry was used to confirm protein levels. Furthermore, we analyzed the effects of costimulatory regulation in (i) stimulation / inhibition using recombinant proteins in primary lymphocytes of SSc patients, (ii) blocking antibodies in co-culture of lymphocytes and K562 target cells, and (iii) functional assays using a fibroblast / immune cell co-culture collagen contraction assay, a disease-related in vitro model that mimics the rigid, hard skin of SSc. The therapeutic efficacy of a novel combination of bispecific anti-CD3 / CD7 immunotoxins targeting immunotoxins was evaluated in (i) blood-derived lymphocytes from SSc patients, (ii) in vitro skin cultures, and (iii) severe SSc patients treated with a novel anti-CD3 / 7 immunotoxin (CD3 / CD7-IT) in exceptional use. Functional experiments were performed using multiple biological and technical replicates as described in the captions for each figure and the methods for each assay.

[0133] Patient and public involvement This study incorporated active patient participation in its design and implementation. Two patient research partners actively participated in the design of key research topics and patient recruitment methods through structured interviews and regular interactive discussions. The patient research partners were trained within the framework of STAP ("Keys to Active Participation"), an initiative of the Department of Rheumatology at Radboud University Medical Center (Nijmegen, Netherlands), which involves establishing patient panels within the hospital to support rheumatology research (38). Efforts by patients and their families to disseminate the study findings within patient organizations played a central role in promoting community engagement during and after the study.

[0134] patient This study was approved by the local research ethics committee of Radboud University Medical Center in the Netherlands (Study numbers: NL57997.091.16, NL67672.091.18). All procedures regarding patient participation were carried out in accordance with the principles of the Declaration of Helsinki and the relevant Dutch legislation regarding review by accredited research ethics committees (file number 2021-8193). All patients (18 years of age or older) provided whole blood and skin biopsies and were diagnosed with systemic sclerosis as confirmed according to the ACR 1980 preliminary classification criteria (39). SSc patients with overlapping syndromes were not included in this study. Blood samples from age- and sex-matched healthy volunteers were collected through the Sankin Blood Bank (project number: NVT 0397-02) from individuals who consented to donate blood for medical research. All patients consented to participate in this study before blood collection or skin biopsy. For the analysis examining the relationship between normalized mean CD7 gene expression and selected patient clinical characteristics, clinical data from SSc patients were obtained as part of a previous paper (10).

[0135] statistics Statistical significance comparisons between experimental groups were performed using Prism 7 software (Graphpad 9.0.0, San Diego, California, USA). The exact statistical tests performed in all experiments are clearly stated in the figure captions.

[0136] How to obtain the code The single-cell RNA sequencing analyses presented in this study were performed using standard workflows and open-source R packages and software (Methods). Some analyses involved the development of custom code. All code is available at: https: / / github.com / PrashINRA

[0137] Additional ingredients [Table 1]

[0138] [Table 2]

[0139] [Table 3]

[0140] Example 1: Expansion culture of activated cytotoxic T cells and NK cells in SSc skin Subsets of T cells and NK cells may induce autoimmune inflammatory processes in SSc by upwardly modulating costimulatory receptors. To obtain comprehensive i-profiling of cutaneous infiltrating lymphocytes, we analyzed T cell and NK cell clusters (n=5,061) obtained from single-cell RNA sequencing datasets of lesional skin and blood, comparing 97 SSc patients and 56 healthy controls as part of a recently published large dataset (GSE195452) (PMID: 35381199) (Figure 1). Since skin is the predominantly affected tissue in SSc, studying immunoprofiling of cutaneous infiltrating T lymphocytes will help understand the pathogenesis of the disease. To enhance reliability, a single-cell RNA transcriptome dataset (GSE138669) (PMID:34031030) containing 2,500 T cells obtained from 9 healthy individuals and 12 SSc skin biopsy specimens was used for a comparable analysis compared with the same dataset (Figure 2). In addition to sc-RNA sequencing data, multicolor immunofluorescence staining was performed on a third cohort consisting of 24 SSc patients. This allowed for confirmation of the transcriptome analysis results at the protein level and further spatial visualization of immune cell infiltration into the skin.

[0141] First, we analyzed T cells and NK cells in single-cell skin datasets based on differential gene expression of known lineage-specific genes (GSE195452, GSE138669). Of the transcriptionally distinct cell subtypes detected (Figure 1A, 2A), the following three—proliferating T cells, CD8+ cytotoxic T cells, and NK cells—were significantly increased in SSc compared to healthy skin in both datasets (q<0.05 in all comparisons) (Figure 3A, B). We confirmed the presence of these T cell and NK cell subsets at the protein level in the skin of SSc-affected areas in an additional cohort of 24 SSc patients (Figure 3C). Furthermore, in 71% of SSc patients, cytotoxic CD8+ T cell infiltration was increased in lesional skin biopsy specimens compared to the corresponding non-lesional skin, and in 83% of SSc patients, CD56+ NK cell infiltration was increased (n=24, p=0.06 and p<0.001, respectively) (Figure 3D, E). In SSc-affected skin, cytotoxic T cells and NK cells were mainly located in the perivascular region. In contrast, the amount of these cells infiltrating the perivascular region was small in non-lesional skin (Figure 3D).

[0142] Next, we analyzed the potential functions of these enriched cell populations in SSc skin. For this purpose, gene set enrichment analysis was performed using the Wiki pathway as the reference dataset, based on each cluster. The cutaneous cytotoxic T cell and NK cell clusters from each sc dataset were not only associated with cytolytic pathways, but were also the only clusters derived from SSc skin that were particularly rich in gene sets associated with pulmonary fibrosis, pro-inflammatory, and pro-fibrotic phenotypes compared to healthy skin (Figure 3F, Figure 2C). These pathways included pro-fibrotic genes such as TGFB1, XCL1, OSM, CCL4, IL4, IL17, FGF, and PDGF (see Figure 7 for a complete overview). This indicates that cytotoxic cells not only participate in cytotoxicity but also play a role in inducing pro-fibrotic pathophysiological processes.

[0143] Recent studies on chronic inflammatory symptoms have shown that CD8+ T cells exert their functions primarily through cytokines rather than through conventional cytotoxicity, with granzyme K playing a crucial role (12). Therefore, we focused our analysis on CD8+ T cells. In the skin, at the sc-RNAseq level, the following CD8+ subclusters were formed: naive, proliferating, resident skin cells, and exhausted cells, all positive for granzyme K (GZMK+) and granzyme B (GZMB+). Of these, only the CD8 effector GZMB+ cell subset was significantly abundant in SSc skin (Figure 3G, Figure 1D). Flow cytometry analysis of blood also showed a twofold increase in the presence of CD8+ GZMB+ cells in SSc compared to healthy donors (Figure 3H).

[0144] Example 2: Increased CD8 from the skin and lungs of systemic sclerosis patients, characterized by upward modulation of CD7 co-stimulatory molecules. + T cells and NK cells The activity of cytotoxic T cells and NK cells is tightly regulated by the interaction of activating and inhibitory receptors. In chronic infections and malignancies, the cytotoxic function of T cells and NK cells has been shown to be limited by inhibitory receptors (13-15). Therefore, we compared the expression of known T cell and NK cell activating and inhibitory receptors in immune cells from healthy skin and SSc skin. Among the inhibitory receptors, LAG3 was expressed in some CD8+GZMB+ T cells in SSc skin. The expression of TIGIT, CTLA4, and HAVCR2 (TIM-3) did not show significant differences between healthy skin and SSc skin, but PDCD1 (PD-1) was expressed only in FOXP3 and a very small number of naive / central memory type CD8+ T cells (Figure 1E). Among the activating receptors, CD69 and CD7 were upregulated in CD8+GZMB+ T cells. On the other hand, CD7, TNFRSF9 (CD137), and CD28 expression were upregulated in SSc NK cells. In proliferating SSc T cell clusters, CD40LG was downregulated, and CD28 expression was reduced in SSc compared to healthy skin (Figure 4A). Of these, CD7 was expressed in almost all cells within the cluster and showed the strongest upregulation in the patient group compared to the control group (q<0.001) (Figure 4A, B). To identify differences in cytotoxic T cell and NK cell activation between healthy individuals and SSc patients, an alternative unbiased method based on the FindConservedMarker function implemented in Seurat was used (to find conserved features between groups, i.e., between healthy donors and SSc patients). This method confirmed enrichment of cytotoxic genes and CD7 in cytotoxic T cells and NK cells of SSc patients compared to the healthy control group. In this analysis, other activating and repressing receptors were not enriched in SSc (Figure 4C). These observations suggest that CD7 costimulation may be involved in the activation of T cells and NK cells in SSc skin.

[0145] To verify these results at the protein level, immunohistochemical staining for CD7 and CD3 was performed on SSc skin tissue. The total number of CD3-positive T cells was higher in lesional SSc skin, although not statistically significant (mean number of CD3-positive T cells: lesional 15.8, non-lesional 6.1) (Figure 5A, B). Of particular note was the specific increase in CD7-positive cell infiltration in the perivascular region (Figure 7C) of lesional SSc skin (Figure 4D) compared to non-lesional areas. Furthermore, CD7 was confirmed to be co-expressed with CD8-positive and CD56-positive cells in SSc skin, but not expressed in CD3-positive CD8-negative cells (Figure 4E).

[0146] Recently, an increased presence of tissue-resident cytotoxic T cells and NK cells has also been reported in SSc lung tissue (16). Therefore, we next evaluated CD7 expression in SSc lung tissue compared to healthy tissue. Similar to our data in skin, CD7 was selectively expressed in lung cytotoxic T cells and NK cells, and its expression in CD8+ and CD56+ cells of SSc patients was significantly higher (2-fold increase) compared to healthy individuals (Figure 4F). In conclusion, CD7 is an activating receptor that is significantly upregulated in disease-associated cytotoxic immune cell populations in both lesional skin and lungs of SSc patients.

[0147] Example 3: CD7 costimulation in the cytotoxic and fibrotic processes of T cells and NK cells CD7 is selectively expressed in cutaneous T cells and NK cells (Figure 8D). Its receptor expression is upregulated after TCR ligand binding and is activated by its ligand, SECTM1 (17). SECTM1 is a transmembrane protein produced by thymic epithelial cells and fibroblasts, and is induced by IFN-γ in professional antigen-presenting cells. SECTM1-mediated CD7 activation has been shown to enhance the effector function of CD4+ and CD8+ T cells (18). To elucidate the function of CD7 in SSc patients, we analyzed SECTM1 expression in cutaneous stromal cells and immune cells. As expected, in our dataset, SECTM1 was detected primarily in cutaneous myeloid cells, including monocytes, macrophages, and dendritic cells. Furthermore, SECTM1 was also expressed in cells of a fibroblast cluster characterized by increased PTGDS expression (Figure 6A). Interestingly, it has been previously reported that this fibroblast subtype is characterized by high expression of MHC class I genes compared to other dermal fibroblast subsets, suggesting that the SECTM1-CD7 axis may be important in T cell and NK cell activation (Figure 11A). Notably, CD7 expression in T cells and NK cells showed a positive correlation with IFNG, while the expression of its receptor (IFNGR1) showed a positive correlation with SECTM1 in fibroblasts and antigen-presenting cells (Figures 11B, C). This suggests the existence of an IFN-γ-driven SECTM1-CD7 axis in SSc skin.

[0148] Clinically, SSc is a heterogeneous disease with various disease subtypes and stages. Therefore, we next analyzed CD7 gene expression in subgroup classifications of SSc patients: focal cutaneous type (lSSc) vs. diffuse cutaneous type (dSSc), and early stage (within 3 years of the first onset of non-Raynaud's symptoms) vs. late stage. We found that CD7 expression was significantly upregulated in early diffuse SSc compared to late stage disease (Figure 6B). Furthermore, CD7 expression was associated with patients showing elevated skin scores (p=0.03) (Figure 6C). Skin expression of CD7 was not associated with the presence of interstitial lung disease (ILD). Additionally, CD7 expression was similar between treatment-naive patients and patients taking immunosuppressants, suggesting that currently used therapeutic approaches do not directly target this activation axis (Figure 6D).

[0149] To further elucidate the function of CD7+ T cells, we analyzed the activation response of cells purified from blood. More CD8+CD7+ cells were detected in the blood of SSc patients compared to healthy individuals (18% of all CD3+ cells in SSc patients compared to 12% in healthy individuals) (Figure 6E). CD7+CD8+ T cells derived from SSc patients produced significantly more granzyme B after short-term (t=4 hour) stimulation with follbor myristate acetate (PMA) and ionomycin (MFI: SSc 40000 vs HD 34000). Furthermore, CD8+CD7+ T cells from SSc patients were also characterized by increased co-expression of the pro-fibrotic cytokines IL-4 and IL-13 (2.5% IL-13+ and 40% IL-4+ in CD8+ T cells). This is a higher value compared to CD8+CD7+ cells in the healthy control group (1% of CD8+ T cells were IL-13+ and 30% were IL-4+). Taken together, these data suggest that CD8+ T cells and NK cells exhibiting cytotoxic and pro-fibrotic properties in SSc are characterized by increased CD7 expression.

[0150] To investigate the involvement of CD7 in the cytotoxicity of T cells and NK cells, healthy peripheral blood mononuclear cells (PBMCs) (n=6) were co-cultured with K562 cancer cells, and the cytotoxic activity of T cells and NK cells was evaluated by measuring the amount of lactate dehydrogenase (LDH) released from damaged target cells. Interestingly, blockade of the CD7 receptor did not affect the cell viability of T cells and NK cells, but it was accompanied by a significant decrease in cytotoxicity against K562 cells (Figure 6G). This observation suggests that CD7 co-stimulation is important in an efficient cytotoxic response.

[0151] Furthermore, we observed above that cytotoxic T cells and NK cells in SSc skin were disease-specifically enriched with pathways associated with pulmonary fibrosis and pro-inflammatory / pro-fibrotic states. To explore the potential involvement of CD7 in the observed pro-fibrotic states of cytotoxic skin cells, we obtained and integrated gene lists related to these pathways and performed pairwise correlation analysis with CD7 (Figure 7). Notably, CD7 gene expression in SSc-affected skin showed a positive correlation with the expression of pro-fibrotic mediators such as XCL1 (19, 20) and CCL3 (21, 22) in cytotoxic lymphocytes (CTLs), and TGFB1 (23, 24) and OSM (25, 26) in NK cells (Figure 6H). These observations suggest that CD7 co-stimulation regulates both cytotoxicity and fibrosis by T cells and NK cells.

[0152] Example 4: In vitro removal of increased and activated subsets of CD7+ T cells and NK cells by targeted immunotoxin treatment inhibits fibroblast modification. Selective upward regulation of CD7 expression in cytotoxic T cells and NK cells in SSc skin can be a target for therapeutic modulation as well as for selective depletion of these cells. Therefore, we utilized a combination of anti-CD3 / CD7 immunotoxins (CD3 / CD7-IT) developed to target alloreactive activated T cells and NK cells in graft-versus-host disease (GvHD) (27). In cultured PBMCs isolated from patient blood, the significant killing effect of CD3 / CD7-IT (over 85% cell removal) was observed only against activated T cells and NK cells (Figure 8A). The combination of CD3 and CD7 immunotoxins showed an additive effect in killing T cells, but as expected, NK cells (CD3-CD56+CD7+) were predominantly targeted by CD7-IT (Figure 8B). Notably, treatment with CD3 / CD7-IT effectively depleted potentially pathogenic CD8+GZMB+ T cells and CD56+GZMB+ NK cells (Figure 8C). IL-2 production decreased ninefold during treatment (Figure 9A). This supports the idea that anti-CD3 / CD7-IT treatment selectively depleted activated T cells and NK cells. CD8+ T cells that survived under CD3 / CD7-IT treatment showed clear changes in memory / maturity. Specifically, CD8 effector cells decreased, while memory and naive phenotypes increased, demonstrating specific killing ability against effector cells (Figure 9B). Furthermore, upon PHA stimulation after treatment, CD8+ T cells that survived treatment showed reduced cell proliferation (decreased CD8+ Ki-67+ cells) and reduced production of cytotoxic molecules (GZMB) and pro-fibrotic molecules (IL-4) compared to the untreated control group (Figure 9C). Importantly, treatment with anti-CD3 / CD7-IT did not affect the number or viability of CD19+ B cells and CD14+ M2 monocytes / macrophages (Figure 9D,E). Next, using whole-skin in vitro cultures, we showed that anti-CD3 / CD7-IT treatment significantly reduced the number of both CD8+ T cells and CD56+ NK cells compared to the untreated state (Figure 8D).

[0153] Having successfully eliminated potentially pathogenic CD7+ T cells and NK cells, we then evaluated whether this depletion had therapeutic significance. Since fibrosis with skin tightening is a key pathological feature of SSc, we developed a novel 3D collagen fibroblast:immune cell co-culture hydrogel model to study fibroblast contractility (Figure 8E). In this model, spontaneous contraction of fibroblasts was observed in the presence of allogeneic PBMCs, with significantly greater contraction levels in the presence of PHA-activated PBMCs. PHA upregulates CD3 and CD7 expression on T cells and CD7 expression on NK cells (Figure 9F, G). Therefore, this model in vitro mimics the effector function of a potentially pathogenic immunocell subset on fibroblasts. Fibroblasts co-cultured with selected CD7+ T cells and NK cells showed increased contractility and elevated expression of IL-6, type I collagen, and alpha-smooth muscle actin (a-SMA) compared to fibroblasts co-cultured with CD7- cells (Figure 12). Next, when PHA-activated PBMCs were pre-treated with 0.33 nM a-CD3 / CD7 antibody or CD3 / CD7-IT, only immunotoxin treatment significantly reduced fibroblast contractility compared to PHA-activated PBMCs (Figure 8E). Under these conditions (24 hours of co-culture), the percentage of necrotic CD8+ or CD56+ cells was (still) not significantly affected (Figure 9H). However, a rapid increase in apoptotic CD8+ and CD56+ cells was observed (Figure 8F). Interestingly, fibroblasts co-cultured with CD3 / CD7-IT treated PBMCs showed decreased gene expression of COL1A1, FN1, and ACTA2 (Figure 8G). This suggests a decrease in the pro-fibrotic phenotype.

[0154] Example 5: Administration of bispecific CD3 / CD7-IT treatment to the first SSc patient effectively eliminates pathogenic CD7+ cells in the blood and skin. The pharmaceutical composition consists of a fixed-dose combination formulation containing equal amounts (by weight) of SPV-T3a-rRTA and WT1-rRTA at a concentration of 0.2 mg protein / mL, in 10 mM citrate buffer (pH 6.5) to which 2.63% (by weight / volume) arginine hydrochloride (125 mM), 5.04% (w / v) maltose monohydrate (140 mM), and 0.05% (w / v) Tween-20 have been added.

[0155] The pharmaceutical compositions were delivered to hospital pharmacists in packs and had to be stored at -20°C or below. Each pack contained two glass vials with rubber stoppers and aluminum flip-off caps, each containing 2.5 mg of purified SPV-T3a-rRTA and 2.5 mg of purified WT1-rRTA at a concentration of 0.2 mg protein / mL with a filling volume of 25 mL per vial.

[0156] The pharmaceutical composition is administered to patients at a body surface area (BSA) of 4 mg / m². 2 It was administered in the following dose.

[0157] For administration, the pharmaceutical composition was thawed at room temperature and transferred to an infusion syringe connected to a central catheter via an extension tube with a 0.2-micrometer inline filter. Four intravenous doses were administered over 4 hours at 2-day intervals using an automated infusion device.

[0158] A 34-year-old male patient with severe focal systemic sclerosis (dc-SSc) experienced disease progression after autologous peripheral blood stem cell transplantation (ASCT) following a failure to respond to mycophenolate mofetil, prednisone, and rituximab. The patient presented with severe physical disability, diffuse end-stage dermatofibrosis (modified Rodnan skin score 27), high inflammatory parameters including ESR 49 mm / Hour (normal range <15 mm / Hour), CRP 78 mg / L, and joint contractures. Due to his extremely poor prognosis and bedridden state, CD3 / CD7-IT therapy was administered as a last resort. The treatment depleted circulating and cutaneous resident T cells and NK cells. C-reactive protein (CRP) levels normalized from 131 mg / L to 27 mg / L after 4 weeks and further decreased to remain within the normal range after 5 months. The patient's functional status was stable and their quality of life improved, but they still had persistent functional impairment due to severe skin contractures and joint contractures. The patient died 1.5 years after CD3 / CD7-IT treatment due to disease complications.

[0159] The biological response to CD3 / CD7-IT treatment was measured by flow cytometry in patients' blood and multiplex immunofluorescence staining in the skin before and after drug administration. Consistent with the expected in vitro effects, treatment with CD3 / CD7-IT resulted in a significant reduction of circulating T cells and NK cells. Post-treatment circulating T cell and NK cell volumes are shown as percentages relative to baseline levels. Within just one week after administration, circulating T cell and NK cell volumes decreased by 86% and 77%, respectively (Figure 10A). CD3 / CD7-IT preferentially targeted CD8+ T cells compared to CD4+ T cells. Specifically, the percentage of CD8+ T cells decreased 37-fold, while CD4+ T cells decreased 8-fold. Furthermore, the CD4 / CD8 ratio increased from 0.7 at baseline to 3.6 after treatment (Figure 10B). Given our previous results suggesting proliferation and activation of classical cytotoxic CD8+ T cells and NK cells in SSc, we further investigated the killing effect of CD3 / CD7-IT on effector cytotoxic T cell and NK cell populations. Effector cytotoxic T cells were characterized as CD8+ perforin+, and NK cells as CD56+ perforin+. Interestingly, both CD8+ perforin+ and CD56+ perforin+ cell populations were completely depleted (100%) in the blood of this patient (Figure 10C). To evaluate the therapeutic effect on cutaneous commensal T cells and NK cells, skin biopsy specimens were analyzed by mIF staining before and after treatment. After treatment, immune cell infiltration was significantly reduced in skin biopsy specimens (Figure 10D). Specifically, the absolute cell counts of CD3+ T cells, CD8+ T cells, and CD56+ NK cells were all significantly reduced after treatment. Importantly, the number of CD3+FOXP3+ regulatory T cells and CD20+ B cells remained unaffected (Figure 10E). While the treatment outcomes were deemed favorable and clinically significant, even greater effects could be expected if CD3 / CD7-IT were applied in the early stages of the disease, when inflammatory components are more pronounced and fibrosis is not yet irreversible.

[0160] <Conclusion> Here, high-depth analysis of publicly available single-cell RNA sequencing datasets from two independent SSc immune cells reveals a significant increase in proliferative T cells, cytotoxic T cells, and NK cells in SSc skin. Among the increased immune cell clusters, cytotoxic T cells and NK cells exhibit gene signatures rich in pro-fibrotic and pro-inflammatory phenotypes. This suggests that SSc skin disease is driven by T cells and NK cells that not only produce cytotoxic proteins such as GZMB and perforin, but are also major producers of well-described pro-fibrotic mediators such as TGFB1, XCL1, and OSM. We recently revealed the proliferation of NK cells and IFN-γ-producing effector T cells in SSc skin. Our analysis focusing on cytotoxic T cells and NK cells in skin further reveals heterogeneity in the effector T cell response, showing that the disease-induced increased effector T cells are classical cytotoxic CD8+ T cells that produce not only IFN-γ but also GZMB and perforin. These findings contradict recent observations that non-classical CD8+ GZMK+ cells, which are not GZMB+ cytotoxic populations, form the core of the effector CD8 response in human inflammatory tissues, including RA synovial membrane and inflammatory UC colon. Furthermore, Maehara et al. quantified a subset of T cells infiltrating SSc skin using conventional immunohistochemical methods and, surprisingly based on our previous findings, found that SSc skin is predominantly infiltrated by cytotoxic GZMA-producing T cells, not type 2 helper T cells. These findings are supported by our own data, because, unlike the prominent cytotoxic features in SSc skin, the single-cell datasets we analyzed showed little to no production of Th2 cytokines such as IL-4, IL-5, and IL-13.

[0161] Furthermore, when comparing the expression of activating and inhibitory receptors in the increased cytotoxic cell population, these cells in SSc skin showed a strongly activated profile. Interestingly, unbiased transcriptome analysis with protein-level validation confirmed that CD7 was upwardly regulated in the activation of T cells and NK cells in SSc skin. This upward regulation was accompanied by increased cytotoxic and pro-fibrotic signatures. CD7 is an extracellular costimulatory molecule expressed in mature human T cells and NK cells, but its precise role in T cell and NK cell function is not well understood. To our knowledge, CD7 expression in SSc has not been previously described. Notably, we are the first to report the proliferation of CD7+ cells in SSc lesional skin. The role of CD7 in T cell biology has not been sufficiently investigated. Our findings indicate that CD7 is significantly increased in effector cells of SSc compared to healthy skin and blood. Therefore, CD7 co-stimulation appears to reflect the activation state of cytotoxic T cells, likely activated by the presentation of skin autoantigens. This contrasts with previous studies that characterized CD7 expression on CD8 T cells in the blood using limited flow cytometry techniques, demonstrating that CD7 is abundant in CD8+ naive / central memory cells but low in CD8+ effector cells. Our findings differ from those of Aandahl et al., as their study focused on peripheral blood mononuclear cells (PBMCs) from healthy donors lacking autoimmunity. The suggestion that CD7 functions as a T cell activation receptor is further supported by the fact that all highly proliferating T cells (MKI67-producing) in our data were positive for CD7 gene expression. Furthermore, blocking CD7 co-stimulation impairs the cytotoxicity of T and NK cells, again highlighting the important role of CD7 in the effector function of these cells. The finding that the CD7 antigen is specifically abundant in potentially disease-associated cytotoxic T cells and NK cells supports the development of novel therapies that specifically target cells rich in this antigen.

[0162] To explore novel therapies that may target abnormal CD7+ T cell and NK cell populations, we further investigated the killing effects of anti-CD3 / CD7-IT combination therapy. This drug was developed to deplete activated alloreactive T cells and NK cells for the treatment of graft-versus-host disease and is currently in Phase 3 clinical trials. This approach, as an attempt to target CD7-positive activated autoreactive T cells within affected tissues, is considered potentially beneficial for treating patients with systemic sclerosis. Interestingly, in vitro treatment of patients' peripheral blood mononuclear cells (PBMCs) with anti-CD3 / CD7-IT selectively depleted activated CD7+ cytotoxic T cells and NK cells. Psittacytotic T cells and NK cells, as well as other immune cell subsets (B cells and macrophages), remained unaffected. However, is this depletion of CD7+ cytotoxic T cells and NK cells related to the cessation of disease symptoms?

[0163] To model the skin tightening symptoms observed in SSc, which are partially caused by hypercontractile (myo)fibroblasts, we developed a three-dimensional co-culture system of fibroblasts and immune cells and studied immune cell-mediated fibroblast contraction. This model was first described by Dorst et al. (2022) and was modified in our setting to also include peripheral blood mononuclear cells (PBMCs). We showed that treatment of activated PBMCs with an anti-CD3 / CD7-IT antibody (depleting CD7+ cells) inhibited fibroblast contraction. This result suggests that fibroblast contraction is driven by CD7+ T cells and NK cells. However, further research is needed to rule out the possibility that other immune cell subsets, such as B cells or macrophages, also contribute to this effect.

[0164] Finally, from a clinical perspective, the results from the first SSc patients treated with CD3 / CD7-IT demonstrated that this drug effectively and selectively reduces activated cytotoxic T cells and NK cells in the blood and skin. Interestingly, this treatment does not affect resting T / NK cells or other immune cell subsets. This fact suggests that this drug may be superior to existing general-purpose immunosuppressive therapies, which are associated with many side effects and often make patients vulnerable to opportunistic infections. Since CD7 expression is enhanced in alloimmunely reactive T cells and NK cells, CD7-targeted therapies have shown clinical efficacy and safety in kidney transplant patients. In vivo depletion of cytotoxic T cells and NK cells needs to be confirmed in more SSc patients. Interestingly, our data provide evidence to support a proof-of-concept clinical trial of CD3 / CD7-IT therapy in SSc patients. Such a clinical trial should rigorously monitor disease outcomes such as mRSS and pulmonary function and evaluate the effect of a-CD3 / CD7-IT therapy on improvement of major SSc disease symptoms.

[0165] This study demonstrates that SSc-affected skin contains proliferative T cells, cytotoxic T cells, and NK cells. These cells exhibit cytotoxic, pro-inflammatory, and pro-fibrotic gene signatures. Focusing on the expression of co-stimulatory and inhibitory molecules, these cells express the co-stimulatory molecule CD7 in association with pro-inflammatory and pro-fibrotic genes, particularly in the early stages of the disease and in severe cases. Furthermore, we showed that CD7 modulates the cytolytic activity of cytolytic T cells and NK cells, and selectively removes CD7-positive cells, thereby halting the pro-fibrotic phenotype and preventing fibroblast contraction by cytotoxic cells. Finally, CD3 / CD7-targeted depletion therapy depleted CD7+ cells and stabilized disease symptoms in severe SSc patients.

[0166] The role of T cells in mediating the pathogenesis of SSc has been a subject of debate. However, recent observations showing that SSc symptoms can be achieved in long-term remission with ASCT treatment have highlighted the importance of the immune system (27). Genetic studies have shown that certain MHC class II polymorphisms confer the risk of developing SSc, leading to the consideration of CD4+ T cells as the main effector cells (5, 8, 28). However, in recent years, it has been shown that MHC class II polymorphisms confer the risk of producing disease-related autoantibodies that precede the onset of clinical symptoms, rather than the risk of developing SSc itself (29). Due to the fibrous clinical symptoms, SSc has been considered a disease involving T helper type 2 (Th2) cells (30, 31). However, epigenetic studies have revealed gene transcription in cytotoxic T cells and NK cells in SSc patients with disease risk loci (5). Furthermore, it has been revealed that cytotoxic T cells predominantly infiltrate the skin of SSc patients and are located near pre-apoptotic vascular endothelial cells (4). Another recent study has shown that increased infiltration of IFN-γ-producing effector T cells and NK cells in SSc skin is associated with fibrotic activation of a subset of fibroblasts (10). Our study supports these data, and functional analysis suggests that SSc skin lesions are driven by T cells and NK cells that produce cytotoxic proteins such as granzyme B and perforin, inducing fibroblast contractility and myofibroblast-like phenotypes, and producing well-explained pro-fibrotic mediators such as TGFB1, XCL1, CCL3, and OSM. This suggests that increased cytotoxicity in SSc skin may be associated with the induction of the fibrotic pathogenesis of the disease.

[0167] Our study addresses the question of how the cytotoxic immune response is regulated in systemic sclerosis (SSc). Cytotoxic T cells and NK cells are central effector cells in cancer and infection. Their effector responses are tightly controlled by the expression of activating and suppressing receptors (32). We found that cytotoxic cells in SSc consistently express high levels of CD7. Interestingly, interferon-γ, a major cytokine in the cytotoxic immune response, is the main inducer of SECTM1, a ligand for CD7 (18). This suggests that the SECTM1-CD7 interaction is part of an interferon-γ-driven feedback loop that enhances the cytotoxic response in SSc skin.

[0168] On the other hand, in chronic viral infections and cancer, cytotoxic cells undergo a process called "depletion," resulting in a decrease or alteration of effector function. Depletion involves increased expression of inhibitory receptors such as PD-1, LAG-3, TIM-3, and CTLA-4 (33). The degree of depletion varies from dysfunction to unresponsiveness or clonal disappearance, and is determined by factors such as antigen load and TCR affinity. The mechanism in autoimmunity is more uncertain. In autoimmune models, activation of autoreactive CD8+ cytotoxic T cells was suppressed by LAG-3 (7). T cell depletion in patients with systemic autoimmune diseases has been investigated and explained mainly in peripheral blood samples, not in tissues where autoantigen presentation occurs (34, 35). In our study, SSc skin showed that some cytotoxic T cells expressed LAG3 compared to healthy skin, suggesting a suppressed phenotype. Only a small number of cytotoxic T cells express both PD-1 and FOXP3 simultaneously, and these are likely regulatory T cells. Overall, cytotoxic lymphocytes in SSc skin exhibit characteristics of an activated state rather than a depleted state.

[0169] This study reaffirms the importance of autoimmunity in promoting SSc pathogenesis. This is clinically significant because autologous transplantation (ASCT), while curative, is a high-risk procedure applicable to only a very limited patient population (less than 10%) (27). Other broad immunosuppressive therapies currently in use do not cure the disease and have limited effect in delaying fibrosis. Selective targeting of activated lymphocytes may be a more selective and safer treatment for SSc. Therefore, we utilized a novel combination of anti-CD3 / CD7-IT developed to deplete activated allogeneic reactive T cells and NK cells for the treatment of graft-versus-host disease (36). We demonstrated that treatment with α-CD3 / CD7-IT selectively depletes activated cytotoxic T cells and NK cells in the blood and in SSc-affected skin. Due to the nature of its depletion effect, anti-CD3 / CD7-IT requires only a single dose, further supporting its favorable safety profile. In line with this concept, CD7-targeted therapies have demonstrated clinical efficacy and safety in kidney transplant patients (36, 37). Previously, we showed that anti-CD3 / CD7 immunotoxin therapy was well-tolerated and improved survival in patients with acute graft-versus-host disease (GvHD). Similar to autologous bone marrow transplantation (ASCT), it significantly increased T cell repertoire diversity with a new polyclonal T cell population, suggesting the efficacy of this therapy in readjusting the immune configuration (36).

[0170] In summary, using single-cell transcriptome data validated by multiplex immunofluorescence, we identified the strong presence of CD7-positive cytotoxic T cells and NK cells in the skin and blood of SSc patients. Furthermore, we demonstrated that targeted removal of these cells prevented myofibroblast contraction. Our results are the first to demonstrate that SSc fibrosis may be preventable with T cell-mediated immunotoxin therapy, opening the way for a new approach to inhibiting histofibrosis. CD7 activation was found to modulate cytotoxic-driven pathological processes in SSc. In summary, these findings suggest that co-stimulatory molecules are key regulators of cytotoxic-driven pathology in systemic autoimmune diseases, raising the banner for selective depletion of pathogenic cells.

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[0193] (Note) Note 1 A pharmaceutical composition for targeting activated pathogenic T cells and / or NK cells in patients with chronic inflammatory or autoimmune diseases, comprising a first molecule that specifically recognizes CD7.

[0194] Note 2 The activated pathogenic T cells and / or NK cells are selected from the group consisting of CD8 cytotoxic T cells, CD4 helper T cells, follicular helper T cells, proliferative T cells, and combinations thereof, in the pharmaceutical composition for the use described in Appendix 1.

[0195] Note 3 The aforementioned chronic inflammatory and autoimmune disease is systemic sclerosis or Sjögren's syndrome, and the pharmaceutical composition for the use described in Appendix 1 or 2.

[0196] Note 4 The patient exhibits symptoms of vascular lesions, fibrosis, and / or autoimmune inflammation; the pharmaceutical composition for the use described in any one of Appendix 1 to 3.

[0197] Note 5 The pharmaceutical composition is a pharmaceutical composition for the use described in any one of Appendix 1 to 4, which targets activated pathogenic T cells and / or NK cells by selective depletion of the activated pathogenic T cells and / or NK cells, or by membrane receptor inhibition, or by intracellular kinase inhibition.

[0198] Note 6 The activated pathogenic T cells and / or NK cells are a pharmaceutical composition for the use described in any one of Appendix 1 to 5, comprising activated pathogenic T cells and / or NK cells in the blood and / or inflamed tissue of the patient.

[0199] Appendix 7 The pharmaceutical composition further comprises a second molecule that specifically recognizes CD3, for use as described in any one of appendices 1 to 6.

[0200] Note 8 A pharmaceutical composition for the use described in any one of Appendix 1 to 7, wherein the first molecule and / or the second molecule is a fragment of an antibody or a derivative thereof.

[0201] Note 9 A pharmaceutical composition for the use described in Appendix 8, wherein the first molecule is a monoclonal antibody, preferably an IgG2a monoclonal antibody, more preferably an anti-(human) CD7 mouse IgG2a monoclonal antibody, and / or the second molecule is a monoclonal antibody, preferably an IgG2b monoclonal antibody, more preferably an anti-(human) CD3 mouse IgG2b monoclonal antibody.

[0202] Note 10 (a) The first molecule comprises a combination of complementary determination region (CDR) sequences, the CDR sequence comprising a CDR3 sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 17, and / or (b) The second molecule comprises a combination of complementary determination region (CDR) sequences, the CDR sequence comprising a CDR3 sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 7. A pharmaceutical composition for the uses described in Appendix 8 or 9.

[0203] Note 11 A pharmaceutical composition for the use described in any one of appendices 1 to 10, wherein the first molecule is WT1 and / or the second molecule is SPV-T3a.

[0204] Note 12 A pharmaceutical composition for the use described in any one of appendices 1 to 11, wherein the first molecule, or the second molecule, or both thereof, comprises at least one toxic moiety, preferably a recombinant A chain of lysine, for example, a recombinant A chain of lysine having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 21.

[0205] d Note 13 The pharmaceutical composition is a pharmaceutical composition for the use described in any one of Appendix 1 to 12, comprising one or more excipients, carriers, buffers, stabilizers, isotonic modifiers, preservatives, or preservatives or antioxidants.

[0206] Note 14 The pharmaceutical composition is administered by intravenous, intradermal, or subcutaneous injection and is a pharmaceutical composition for the use described in any one of Appendix 1 to 12.

[0207] Note 15 A method for targeting activated pathogenic T cells and / or NK cells in a patient with a chronic inflammatory or autoimmune disease, comprising administering to the patient an effective amount of a pharmaceutical composition comprising a first molecule that specifically recognizes CD7.

Claims

1. A pharmaceutical composition for targeting activated pathogenic T cells and / or NK cells in patients with chronic inflammatory or autoimmune diseases, comprising a first molecule that specifically recognizes CD7.

2. The pharmaceutical composition for the use according to claim 1, wherein the activated pathogenic T cells and / or NK cells are selected from the group consisting of CD8 cytotoxic T cells, CD4 helper T cells, follicular helper T cells, proliferative T cells, and combinations thereof.

3. The pharmaceutical composition for the use according to claim 1 or 2, wherein the chronic inflammatory and autoimmune disease is systemic sclerosis or Sjögren's syndrome.

4. A pharmaceutical composition for the use described in any one of claims 1 to 3, wherein the patient exhibits symptoms of vascular lesions, fibrosis, and / or autoimmune inflammation.

5. The pharmaceutical composition for the use according to any one of claims 1 to 4, wherein the pharmaceutical composition targets activated pathogenic T cells and / or NK cells by selective depletion of the activated pathogenic T cells and / or NK cells, or by membrane receptor inhibition, or by intracellular kinase inhibition.

6. The activated pathogenic T cells and / or NK cells comprise activated pathogenic T cells and / or NK cells in the patient's blood and / or inflamed tissue, the pharmaceutical composition for the use according to any one of claims 1 to 5.

7. The pharmaceutical composition for the use according to any one of claims 1 to 6, further comprising a second molecule that specifically recognizes CD3.

8. A pharmaceutical composition for the use according to any one of claims 1 to 7, wherein the first molecule and / or the second molecule is a fragment of an antibody or a derivative thereof.

9. The pharmaceutical composition for the use of claim 8, wherein the first molecule is a monoclonal antibody, preferably an IgG2a monoclonal antibody, more preferably an anti-(human) CD7 mouse IgG2a monoclonal antibody, and / or the second molecule is a monoclonal antibody, preferably an IgG2b monoclonal antibody, more preferably an anti-(human) CD3 mouse IgG2b monoclonal antibody.

10. (a) The first molecule comprises a combination of complementary determination region (CDR) sequences, the CDR sequence comprising a CDR3 sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 17, and / or (b) The second molecule comprises a combination of complementary determination region (CDR) sequences, the CDR sequence comprising a CDR3 sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO:

7. A pharmaceutical composition for the use described in claim 8 or 9.

11. A pharmaceutical composition for the use according to any one of claims 1 to 10, wherein the first molecule is WT1 and / or the second molecule is SPV-T3a.

12. A pharmaceutical composition for the use according to any one of claims 1 to 11, wherein the first molecule, or the second molecule, or both thereof, comprises at least one toxic moiety, preferably a recombinant A chain of lysine, for example, a recombinant A chain of lysine having at least 95% sequence identity with the amino acid sequence of SEQ ID NO:

21.

13. The pharmaceutical composition for the use described in any one of claims 1 to 12 comprises one or more excipients, carriers, buffers, stabilizers, isotonic modifiers, preservatives, or preservatives or antioxidants.

14. The pharmaceutical composition is administered by intravenous, intradermal or subcutaneous injection, and is a pharmaceutical composition for the use described in any one of claims 1 to 12.

15. A method for targeting activated pathogenic T cells and / or NK cells in a patient with a chronic inflammatory or autoimmune disease, comprising administering to the patient an effective amount of a pharmaceutical composition comprising a first molecule that specifically recognizes CD7.

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