Methods and means for treating chronic inflammatory and autoimmune diseases

Abstract

The present invention relates to a pharmaceutical composition for targeting activated pathogenic T cells and / or NK cells in a patient suffering from a chronic inflammatory or autoimmune disease. The pharmaceutical composition comprises a first molecule that specifically recognizes CD7. The present invention further describes kits comprising these pharmaceutical compositions. The invention further relates to a method of activating pathogenic T cells and / or NK cells for targeting a chronic inflammatory or autoimmune disease in a patient.

Description

TECHNICAL FIELD

[0001] The present invention relates to the field of immune system related diseases, in particular to the field of novel means and methods for the treatment of these diseases. More in particular, the present invention provides a pharmaceutical composition for the treatment of a chronic inflammatory or autoimmune disease, said pharmaceutical composition comprising an antibody specifically recognizing CD7.

[0002] Typically, the present invention finds its application in the field of systemic autoimmune diseases, in particular systemic sclerosis or Sjogren's syndrome. BACKGROUND BACKGROUND

[0003] Autoimmune diseases are characterized by an immune response of the body against its own constituents, resulting in autoantibodies and / or T lymphocytes directed against self-antigens, causing damage and dysfunction of its own tissues or cells. In some conditions, the pathology is mediated by the direct binding of autoantibodies to tissue-restricted antigens, as in myasthenia gravis, type I diabetes and autoimmune thyroiditis. These conditions are referred to as organ-specific autoimmune diseases. In other conditions, autoantibodies bind to antigens expressed in non-organ specific structures. The autoimmune response occurs in specific tissues or organs when the antigens are abnormally expressed, for example due to infection or stress of the tissues or cells, or when circulating immune cells and / or immune complexes accumulate. These are referred to as systemic autoimmune diseases, such as systemic sclerosis (SSc), Sjogren's syndrome (SS), rheumatoid arthritis (RA), (dermatomyo)sis (DM) and (systemic) lupus erythematosus (SLE).

[0004] Conventional treatment for autoimmune diseases involves the administration of drugs that non-specifically suppress the immune response. Examples of such drugs are methotrexate, cyclophosphamide, Imuran (azathioprine) and cyclosporin A. In many cases, steroid compounds are also used, such as prednisone and methylprednisilone. The efficacy of these drugs varies for organ-specific and systemic autoimmune diseases. The use of such drugs is limited due to their toxic side effects and the induction of a general immune suppression in patients receiving long-term treatment with the drugs, for example the down-regulation of the normal protective immune response against pathogenic microorganisms, thereby increasing the risk of infections caused by these pathogens. An additional disadvantage is that the risk of malignancies will increase in patients receiving long-term general immune suppression with certain universal immune suppressive drugs.

[0005] Systemic sclerosis (SSc), also known as scleroderma, is an autoimmune disease of unknown etiology characterized by high morbidity and mortality (PMID: 34487318). SSc is a systemic autoimmune disease characterized by vasculopathy, inflammation, and progressive fibrosis of the skin and internal organs (1). Autoimmunity in SSc is directed against nuclear autoantigens that can be aberrantly presented by endothelial cells and fibroblasts due to hypoxic stress and serve as antigenic targets (2). This is exemplified by the occurrence of dysregulated Raynaud’s phenomenon as the first and major disease manifestation. T lymphocytes have been detected in SSc-affected tissues and multiple studies have suggested that they can participate in the observed fibrosis and vasculopathy by producing cytokines such as interleukin (IL)-4, IL-13, and IL-17 (3). Surprisingly, a recent study showed that cytotoxic T cells play an important role in mediating SSc skin pathology (4). Furthermore, an epigenetic study suggested natural killer (NK) cells and CD8+ T cells in the pathogenesis of SSc (5).

[0006] In chronic inflammatory conditions, T cell activation is limited to prevent unwanted inflammatory side effects. Activation of antigen-specific CD4+ T cells is regulated by professional antigen-presenting cells via a major histocompatibility complex (MHC) class II-controlled process. The regulatory mechanisms of cytotoxic T cells and NK cells are not well understood, as these cells depend on non-MHC class II receptors, and these receptors are ubiquitously expressed in inflamed tissues. In chronic infections and malignancies, activation of cytotoxic T cells and NK cells has been shown to be regulated by the interplay between costimulatory and inhibitory receptors (6). Animal models suggest that similar mechanisms can also play a role in cytotoxic autoimmunity (7). However, the exact role of T cells in the pathogenesis of SSc remains to be defined. On one hand, genetic studies have demonstrated that human leukocyte antigen genes (HLA) corresponding to MHC class II confer susceptibility to SSc (8). On the other hand, treatment with a drug targeting T cells, cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) immunoglobulin (abatacept), showed limited clinical efficacy (9).

[0007] The inventors have addressed this controversy and put forward the hypothesis that targeting specific costimulatory receptors on cells expressing costimulatory molecules can represent a selective and potentially safer therapeutic strategy for treating SSc.

[0008] The present invention aims at alleviating the above-mentioned problems. Alternatively or additionally, the present invention aims at providing pharmaceutical compositions and methods for treating chronic inflammatory or autoimmune diseases, such as SSc and SS. SUMMARY

[0009] The inventors of the present application found that T cells and NK cells with upregulated expression of the costimulatory receptor CD7 are the key immune cells driving the pathogenesis of SSc and are therefore considered as potential targets for selective therapy. The inventors of the present application analyzed the costimulatory profile of pathogenic cytotoxic immune cells in SSc skin with the aim to specifically block the activation of these cells and / or to deplete said cells. Using an in-depth single cell transcriptomic analysis of 109 SSc versus 68 healthy skin biopsies confirmed by multiplex immunohistochemistry, a significant presence of activated cytotoxic T cells and NK cells was surprisingly observed that exhibit a disease-associated upregulation of the CD7 activating receptor. The expression profile of known lymphocyte costimulatory receptors in the skin was further examined and compared to the expression profile of healthy cells, showing an expanded cytotoxic NK cell population as well as a sustained and significant upregulation of CD7 in the proliferating and cytotoxic T cell population. The CD7 upregulation was directly correlated with the upregulation of proinflammatory, cytotoxic and profibrotic mediators in the same cells. The disease-associated role of CD7 on profibrotic performance was revealed using in vitro models of cytotoxicity as well as leukocyte-induced fibroblast contraction. Treatment with compositions comprising anti-CD7 with and without an immunotoxin (anti-CD7 and anti-CD7-IT) was investigated and the results show that these subpopulations are functionally impaired or effectively depleted (with anti-CD7 or anti-CD7-IT). Notably, a significant depletion of CD7 + cell subpopulations has been observed in the blood and skin of SSc patients one week after administration of the pharmaceutical composition of the present application.

[0010] Thus, according to a first aspect of the present application, a pharmaceutical composition for targeting activated pathogenic T cells and / or NK cells in a patient suffering from a chronic inflammatory or autoimmune disease is provided. The pharmaceutical composition of the present application comprises a first molecule specifically recognizing CD7.

[0011] Preferably, the first molecule is an antibody, or a fragment of a derivative thereof. 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 murine IgG2a monoclonal antibody.

[0012] Preferably, the first molecule comprises a combination of complementarity determining region (CDR) sequences, wherein the CDR sequences comprise a CDR3 sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 17. More preferably, the CDR3 of the first molecule comprises or consists of the amino acid sequence of SEQ ID NO: 17.

[0013] A preferred example of a first molecule according to the present application is WT1.

[0014] The pharmaceutical composition of the present application can preferably further comprise a second molecule specifically recognizing CD3.

[0015] Preferably, the second molecule is an antibody, or a fragment of a derivative thereof. 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 murine IgG2b monoclonal antibody.

[0016] Preferably, the second molecule comprises a combination of complementarity determining region (CDR) sequences, wherein the CDR sequences comprise a CDR3 sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 7. More preferably, the CDR3 of the second molecule comprises or consists of the amino acid sequence of SEQ ID NO: 7.

[0017] A preferred example of a second molecule according to the present application is SPV-T3a.

[0018] Optionally, 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. For example, the pharmaceutical composition targets the 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.

[0019] Preferably, the chronic inflammatory and autoimmune disease is systemic sclerosis or Sjogren’s syndrome. Preferably, the patient can exhibit symptoms of vasculopathy, fibrosis, and / or autoimmune inflammation.

[0020] Preferably, the activated pathogenic T cells and / or NK cells comprise activated pathogenic T cells and / or NK cells in the blood and / or inflamed tissue of the patient.

[0021] Preferably, the first molecule or the second molecule or both have at least one toxin moiety, preferably a recombinant A chain of ricin (rRTA), for example a recombinant A chain of ricin having at least 95% sequence identity to 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.

[0022] Preferably, the pharmaceutical composition of the present application further comprises one or more excipients, carriers, buffers, stabilizers, tonicity adjusting agents, preservatives, or antioxidants.

[0023] Preferably, the pharmaceutical composition of the present application is administered by intravenous, cutaneous, or subcutaneous injection.

[0024] According to a second aspect of the application, there is provided a method of targeting activated pathogenic T cells and / or NK cells in a patient having a chronic inflammatory or autoimmune disease. The method comprises administering to the patient an effective amount of a pharmaceutical composition of the application as described herein.

[0025] According to a third aspect, the application provides a freeze-dried lyophilized form of a pharmaceutical composition of the application. The lyophilized composition can be suitable for reconstitution, for example, with water or an aqueous solution to form a composition for use in a method of the application.

[0026] According to a fourth aspect, the application provides a kit comprising:

[0027] a container or housing having the composition of the application therein; and

[0028] a label or leaflet having instructions for use of the composition in a method of treatment of the application.

[0029] In some cases, the container or housing is kept sterile, for example, by being sealed and / or hermetically closed.

[0030] It will of course be appreciated that features described in relation to one aspect of the application can be incorporated into other aspects of the application. For example, the methods of the application can incorporate any of the features described with reference to the apparatus of the application, and vice versa. BRIEF DESCRIPTION OF DRAWINGS

[0031] Figure 1 : Annotation of skin T cell and NK cell clusters from single-cell RNA sequencing dataset GSE195452. A7,187 cells unsupervised uniform manifold approximation and projection (UMAP) clustering detected 8 transcriptionally distinct cell clusters: tissue-resident memory T cells (Trm), cytotoxic T cells (CTL), regulatory T cells (Treg), dysfunctional tissue-resident T cells (Thprm), naive / central memory (Tncm), proliferative T cells (Tprolif), and NK cells (NK), and a cluster containing MAIT, INKT CD8 +Clusters of mixtures of T and gd T cells (T mix). B UMAP shows cells belonging to healthy individuals (controls) or patients (SSc). C Heatmap displaying the top 5 differentially expressed genes in each of the distinguished cell clusters: Trm (CD69, ZFP36L2, CXCR4, IL7R), CTL (GZMK, IFNG, CCL5, CCL4, CD8A), Treg (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). D Heatmap displaying the top 10 upregulated genes in each of the 5 different clusters of cells: isolated CD8 + T cells; naive (Tn): IL7R; Granzyme K + (GZK + ): GZMK, IFNG, CCL4; Granzyme B + (GZB + ): PRF1, GNLY, NKG7, GZMB, GZMZ, GZMH; exhausted (Tex): NR4A2, NR4A3; and proliferative (Tprolif): LASP1, TMPO, ANP32B. E UMAP represents PDCD1 (PD-1) positive (red) and negative (gray) gene expression in CD8 + T cell clusters.

[0032] Figure 2Single-cell RNA sequencing analysis of dataset GSE138669. A 2,500-cell unsupervised Uniform Manifold Approximation and Projection (UMAP) clustering identified 7 transcriptionally distinct cell clusters: quiescent tissue-resident T cells (Tqcm), cytotoxic T cells (CTL), regulatory T cells (Treg), functionally-dysfunctional tissue-resident T cells (Thprm), naive / central memory (Tncm), proliferative T cells (Tprolif), and NK cells (NK). UMAP depictions of cells belonging to either healthy individuals (controls) or patients (SSc) are shown at the top of this panel. B Heatmap of the top 10 differentially expressed genes in each of the distinguished cell clusters: Tqcm (CD69, IL7R, TCF7, SELL, ANXA1), CTL (CD8A, GZMK, GZMA,), Treg (CD4, CD27, CTLA4, IL2RA), Thprm (NR4A1, DUSP1), Tncm (TCF7, IL7R), Tprolif (MKI67), NK (NKG7, FCGR3A, KLRD1, PRF1). C (left) Gene set enrichment analysis with Wiki pathways as the reference dataset. Examples of the most significant pathways unique to each cluster are shown. Statistical data (right) Comparison of the enrichment scores of the summary of pro-inflammatory and pro-fibrotic as well as lung fibrosis pathways in T and NK cell clusters of HD vs. SSc skin.

[0033] Figure 3: Frequency of T and NK cell clusters in the skin of (n=56) healthy donors (HD) compared to (n=97) patients with systemic sclerosis (SSc). B Frequency of T and NK cell clusters in the skin of (n=9) HD compared to (n=12) patients with SSc. Abbreviations: tissue-resident T cells (Trm), cytotoxic T cells (CTL), regulatory T cells (Treg), dysfunctional tissue-resident T cells (Thprm), naive / central memory (Tncm), proliferative T cells (Tprolif), NK cells (NK), quiescent tissue-resident T cells (Tqcm). For both panels A and B, values are expressed as change in cell counts in % and statistics were performed using the Wilcox-Test with correction for multiple comparisons. Adjusted p-values (q) for statistically significant comparisons are shown, * q < 0.05, ** q < 0.01. C Representative multicolor immunofluorescence composite images of T helper CD3+CD8- (red), cytotoxic CD8+ (cyan), regulatory FOXP3+ (green) T cells and CD56+CD3- (yellow) NK cells in SSc-involved skin. Abbreviations: hair follicle (HF), epidermis (E), blood vessels (BV). Scale bar 50 pm. D Immunofluorescence composite images of infiltrating cytotoxic CD8+ (cyan) T cells and CD56+CD3- (yellow) NK cells in uninvolved versus involved skin from a representative SSc patient with early diffuse disease. E Percentage (%) of cytotoxic T (CD3+CD8+) cells and NK cells (CD56+CD3-) in matched uninvolved versus involved SSc skin (n=24 SSc patients). Values are expressed as % of CD8+ or CD56+ cells present in each biopsy compared to all cells (DAPI+) (exclusion of the keratinocyte-rich epidermis layer). Statistics were performed using the non-parametric Wilcox-Test, *** p < 0.001. F (left) Gene set enrichment analysis of skin T and NK cell clusters using Wiki pathways as reference dataset. Examples of top pathways represented by NK and CTL clusters are shown (p < 0.001). Statistics were performed using the Kolmogorov-Smirnov (KS) test. (right) Comparison of enrichment scores of “Overview of proinflammatory and profibrotic mediators” (q=0.025) and “Pulmonary fibrosis” (q=0.0002) pathways in HD (green) versus SSc (red) T and NK cell clusters (here for GSE195452, supplementary Figure 2C UMAPs showing 5 transcriptionally distinct CD8+ T cell clusters in the skin of (n=56) HDs and (n=97) SScs, collectively n=977 cells. Clusters were labeled as naive (Tnaive), granzyme K+ (GZMK+), granzyme B+ (GZMB+), exhausted (Texh), and proliferative (Tprolif) according to previously differentially expressed genes. (Right) Cell frequencies of CD8+ T cell clusters between HDs and SScs. Percentage of granzyme B (GZMB)-expressing CD8+ T cells in the peripheral blood of (n=15) HDs and (n=30) SScs. Values expressed as % of total viable peripheral blood mononuclear cells, *p<0.05.

[0034] Figure 4 : CD7 upregulation is associated with activation of cytotoxic T cells and NK cells in SSc-affected skin and lungs. A2-D Dot plots comparing gene expression of selected activating and inhibitory costimulatory receptors in Tprolif, CD8+GZMB+, and NK clusters between HDs and SScs (circle size represents percentage of cells expressing each gene, and color intensity depicts mean expression, while numbers indicate mean values of normalized counts). B (top) UMAPs representing positive (red) and negative (gray) gene expression of CD7 in CD8+ T cells (left) and CD56+ NK cells (right). (bottom) Intensity of CD7 normalized gene expression between HDs and SScs in CD8+ T cells (left) and CD56+ NK cells (right). C Scatter plot with gene expression values highlighting genes specifically enriched in skin T cells and NK cells of patients with SSc compared to HDs. D Representative pictures of CD7 immunohistochemistry (IHC) staining of affected and unaffected skin biopsies from SSc patients, with quantification of CD7 IHC score (n=20). Non-parametric sign test, *p<0.05. E Immunofluorescence microscopy showing co-expression of CD7 with CD8+ T cells and CD56+ NK cells in early dSSc skin. Representative experiment depicted with a ruler of 100 pm. F (left) UMAPs showcasing T cells and NK cells from control (healthy) (n=6) and SSc (n=7) lung tissues from patients with interstitial lung disease (GSE128169). (middle) Density plots showing gene expression density of CD7, NCAM1 (CD56), CD4, and CD8A. (right) CD7 gene expression counts (normalized) between control and SSc lung T cells and NK cells (each dot represents mean CD7 expression per donor). Figure 5SSc lesions in patients with systemic sclerosis and CD3 and CD7 cell staining in non-lesional skin. CD7 gene expression in skin immune cells and stromal cells is also depicted. Representative images of CD3 immunohistochemistry (IHC) staining of involved and uninvolved skin biopsies from one SSc patient. Scale bar is 100 pm. B Quantification of CD3+ T cells in involved versus non-lesional SSc skin (n = 20). Bars are mean ± SD. Mann-Whitney test, p = 0.19. C In SSc involved skin, a massive infiltration of CD7+ cells is found in the perivascular area, while in matched uninvolved skin, the number of CD7+ cells present around the vessels is less. Scale bar is 100 pm. Here, representative images of one SSc patient with early diffuse disease are depicted. D 2-D dot plot comparing CD7 gene expression in skin immune cells and stromal cells. Cell cluster annotations were taken from the metadata information, as described in the single-cell RNA sequencing dataset GSE195452. Circle size represents the percentage of cells expressing CD7, and color intensity depicts the mean expression. Numbers indicate the mean of normalized counts.

[0035] Figure 6: CD7 co-stimulation plays an important role in T cell and NK cell cytotoxicity and pro-fibrotic manifestations. A SECTM1 gene expression (GSE195452) in skin immune cell and stromal cell subsets, normalized to log. The annotation of the depicted cell clusters was taken from Gur et al. (10). B Subgroup analysis of CD7 normalized gene expression in healthy individuals (HD) and in systemic sclerosis (SSc) patients with early versus late limited cutaneous (lSSc) or diffuse (dSSc) disease. Early disease was defined as < 3 years from first diagnosis. One-way ANOVA with Tukey’s multiple comparison test, *p < 0.05, ***p < 0.001. C (Left) Scatter plot showing the correlation of CD7 normalized gene expression with skin score. Each circle represents a single SSc patient. Spearman r = 0.34, p = 0.07. (Right) Normalized CD7 gene expression between SSc patients with low skin score versus high skin score. The distinction between low versus high skin score was taken as previously described (10). D Normalized CD7 gene expression between SSc patients treated or not with immunosuppressive drugs, and between SSc patients with or without interstitial lung disease (ILD). E Percentage of CD8+CD7+ versus CD8+CD7- T cells in the peripheral blood of (n = 15) HD and (n = 30) SSc. Values are expressed as % of total CD3+ T cells, *p < 0.05. F Expression (mean fluorescence intensity) of granzyme B (GZMB) and percentage of IL-4+ / IL-13+ cells between HD and SSc CD8+CD7+ T cells. GZMB expression level is expressed as mean fluorescence intensity (MFI) and IL-4+ / IL-13+ cells values as percentage of positive cells in the CD8+CD7+ T cell compartment. Student’s t-test, *p < 0.05. G Cytolytic activity of T cells and NK cells in co-cultures with 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 block CD7 co-stimulation. Unstim refers to unstimulated control cells. Statistical comparison between groups was performed with ordinary one-way ANOVA with Tukey’s multiple comparison test, *p < 0.05, **p < 0.01, ***p < 0.001. H Pairwise correlation plot between CD7 and XCL1, TGB1, OSM, MMP9 gene expression within NK cell or cytotoxic T cell (CTL) clusters in SSc affected skin (GSE195452).

[0036] Figure 7: Pairwise correlation of CD7 with pro-fibrotic genes. Wiki gene pathways lung fibrosis and pro-inflammatory and pro-fibrotic expression were combined and the potential correlation of CD7 gene expression with the included genes was evaluated for clusters of A cytotoxic T cells (CTLs) and B NK cells, respectively. Statistical significance of each comparison was corrected for multiple comparisons and is indicated as adjusted p-value, *p<0.05, **p<0.01.

[0037] Figure 8 : Activated CD7 + Targeted immunotoxin-mediated depletion of T cells and NK cells prevents fibroblast contraction and reduces myofibroblast phenotype. A Flow cytometric quantification of CD3 / CD7-IT-induced cell death in CD2+T cells and CD56+NK cells in PBMCs isolated from (n=5) SSc patients. B Percentage of normalized cell viability of CD3+T cells and CD56+NK cells isolated from SSc (n=3) PBMCs. C Flow cytometric quantification of CD3 / CD7-IT-induced cell death in CD8+GZMB+T cells and CD56+GZMB+NK cells in PBMCs isolated from (n=5) SSc patients. D Flow cytometric quantification of CD3 / CD7-IT-induced cell death in CD8+T cells and CD56+NK cells in ex vivo skin explants (n=4). Paired t-test, *p<0.05, **p<0.01. E Schematic representation of the in vitro hydrogel collagen contraction assay in a developed 3D model with co-cultured primary skin fibroblasts and PBMCs. The degree of contraction was quantified compared to non-cell controls and plotted in a graph at the right (n=3). Bars are mean ± SD. Images of a representative experiment are depicted at the bottom of this panel. F Percentage of pro-apoptotic cytotoxic T cells (CD8+7-AAD-Annexin V+) and NK cells (CD56+7-AAD-Annexin V+) was measured by flow cytometry of enzymatically digested collagen plugs under the depicted conditions (n=5). G PBMCs treated with IgG or CD3.CD7-IT were co-cultured with primary dermal fibroblasts in a developed 3D hydrogel collagen co-culture model and analyzed for expression of genes reflecting myofibroblast phenotype in fibroblasts. Values represent relative gene expression (-ACt) as measured with qPCR. GAPDH and RPS27A were used as reference genes. Data represent mean ± SEM. Statistical comparisons between three or more groups were performed with ordinary one-way ANOVA with Dunnett’s multiple comparison test, *p<0.05, **p<0.01, ***p<0.001. Abbreviations: phytohemagglutinin (PHA), immunotoxin (IT), peripheral blood mononuclear cells (PBMCs).

[0038] Figure 9 : CD3 / CD7-IT specifically depletes only activated cytotoxic T cells and NK cells in vitro. A Concentration of IL-2 (pg / ml) in cell supernatants of cells treated with a-CD3 / CD7-IT or not treated. B Pie charts showing the proportion of effector (CD8+CD45RA+CD27-), memory (CD8+CD45RA-CD27+) and naive (CD8+CD45RA+CD27+) cells in the CD8+ T cell population under the depicted stimulation and treatment culture conditions (percentage in pie charts are mean values of n=6 SSc patients). C Response of cells treated with a-CD3 / CD7-IT to TCR-mediated (PHA) re-stimulation assessed by intracellular flow cytometry. Values expressed as fold change of re-stimulation compared to values before stimulation. D (bottom) Comparison of absolute counts of CD19+ B cells (cells / μl) after in vitro treatment with CD3 / CD7-IT compared to untreated peripheral blood mononuclear cells (n=6). (top) Representative flow cytometry plot of one experiment. E Normalized cell viability of M2 macrophages and CD19+ B cells isolated from blood of SSc patients for the depicted different culture / treatment conditions. Cycloheximide was used as positive control. F Flow cytometry histograms of a representative experiment showing the increase of CD3 and CD7 expression in CD8+GZMB+ cells and CD7 in CD56+GZMB+ NK cells after stimulation with phytohemagglutinin (PHA), further quantified in G. CD3 and CD7 expression is expressed as mean fluorescence intensity (MFI). Statistics performed with Student t-test, **p<0.01, ****p<0.0001. H Percentage of necrotic cytotoxic T cells (CD8+7-AAD+Annexin V+) and NK cells (CD56+7-AAD+Annexin V+) measured by flow cytometry of enzymatically digested collagen plugs under the depicted conditions (n=5).

[0039] Figure 10 . Treatment with a-CD3 / CD7-IT depletes cytotoxic T cells and NK cells in blood and skin of early dSSc patients. A Percentage of CD3+ T cells and CD56+ NK cells, B CD4+ T cells and CD8+ T cells, C CD8+ perforin+ T cells and CD56+ perforin+ NK cells in peripheral blood of SSc patients before and after treatment with a-CD3 / CD7-IT. Values expressed as % of total viable peripheral blood mononuclear cells (PBMC). B (right) Ratio of CD4+ / CD8+ T cells before and after treatment. D Representative immunofluorescence images and E quantification of lymphocyte subsets in the affected skin of patients before and after treatment.

[0040] Figure 11 The SECTM1-CD7 axis of cytotoxic T cell and NK cell activation in SSc-affected skin. Schematic model of the proposed involvement of the SECTM1-CD7 axis in cytotoxic T cell and NK cell activation (created with BioRender.com). In SSc-affected skin, CD7 and IFNG are mainly expressed in cytotoxic T cells and NK cells, while SECTM1 and IFNGR are expressed in antigen-presenting cells (APCs) and stromal cells (mainly fibroblasts). This suggests the presence of a positive feedback loop mediated by cytokines in the communication between CD7+ cytotoxic immune cells and SECTM1-producing APCs and fibroblasts, with IFN-gamma as the key cytokine. B 2-D dot plot comparing the expression levels of selected genes in skin immune cells (myeloid and lymphoid) and stromal cell populations. Cell cluster annotations were taken from the metadata information, as described in the single-cell RNA sequencing dataset GSE195452. Circle size represents the percentage of cells expressing each gene, and color intensity depicts the average expression. Numbers indicate the mean of normalized counts. C Pairwise correlation plot (GSE195452) in SSc-affected skin showing 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.

[0041] Figure 12Fibroblasts co-cultured with CD7+ T cells and NK cells exhibit enhanced contractility, accompanied by elevated myofibroblast-like phenotype. A Schematic representation of the experimental design in the developed 3D in vitro collagen contraction fibroblast: immune cell co-culture model. B Flow cytometry gating strategy used to sort CD7+ versus CD7- T and NK cell populations from healthy peripheral blood (n=3). First, lymphocyte population was gated based on cell size (FSC) and granularity (SSC). Subsequently, dead cells were excluded by 7-AAD+ staining and then CD19-CD14- lymphocytes positive or negative for CD7 expression were sorted. C Level of fibroblast contraction was quantified compared to non-cell control and plotted in graph (n=3). Bars are mean ± SD. Images of a representative experiment are depicted in the right part of this panel. D (Left) Representative images of collagen type 1 immunohistochemistry of collagen plugs containing either fibroblasts alone or fibroblasts co-cultured with CD7+ T cells and NK cells. (Right) Quantification of collagen type 1 positive fibroblasts in control versus fibroblasts co-cultured with CD7- or CD7+ cells (n=3). Bars are mean ± SD. E Percentage of CD45-a-SMA+ IL-6+ fibroblasts under the depicted conditions was measured by flow cytometry on enzymatically digested collagen plugs (n=3). F Expression level (mean fluorescence intensity - MFI) of a-SMA in CD45-a-SMA+ fibroblasts under the depicted conditions was measured by flow cytometry on enzymatically digested collagen plugs (n=3). (Right) Flow cytometry histograms of a representative experiment showing increased expression of a-SMA in fibroblasts co-cultured with CD7+ cells compared to CD7- T cells and NK cells. G CD7+ versus CD7- T cells and NK cells were co-cultured with primary dermal fibroblasts in the developed 3D hydrogel collagen co-culture model (n=3) and expression of genes reflecting myofibroblast phenotype such as COL1A1 and ACTA2 were analyzed in fibroblasts. Values represent relative gene expression (-ACt) as measured with qPCR. GAPDH and RPS27A were used as reference genes. Data represent mean ± SEM. DETAILED DESCRIPTION

[0042] A pharmaceutical composition is defined herein as any composition that can be administered to an individual, as a single dose or a series of doses by any feasible route, preferably intravenously, optionally containing usual administration vehicles and / or ingredients of usual treatments of the concerned chronic inflammatory or autoimmune disease. For example, the pharmaceutical composition of the application is able to eliminate or reduce the number of unwanted CD7 positive cells.

[0043] Unwanted cells are any cells comprising CD7 (as well as of course many others) molecules associated with the cell surface which are involved in a pathological condition of the individual. Typically, these cells are T cells or NK cells or other cells which play a role in chronic inflammatory or autoimmune diseases. According to the present application, it is also possible to eliminate or suppress abnormal cells comprising CD7 (e.g. T cell leukemias or lymphomas).

[0044] The pharmaceutical composition of the present application comprises a first molecule specifically recognizing CD7. Optionally, the pharmaceutical composition of the present application further comprises a second molecule specifically recognizing CD3. Additionally or alternatively, the pharmaceutical composition according to the present application can further comprise at least one additional molecule specifically recognizing CD5, CD2, CD4, CD8 or the IL-2 receptor.

[0045] Molecule specifically recognizing CD3 / CD7 or another ligand receptor, such as another cell surface antigen, is a term widely understood in the art.

[0046] It means any molecule having a relatively high binding affinity and specificity for CD3, CD7 or another receptor (e.g. cell surface antigen). In the context of the present application, CD3, CD7 and any other cell surface antigen mentioned herein are human antigens. The molecule can typically be a ligand of the receptor, or an antibody against CD3, CD7 or another receptor (receptor defined as any molecule capable of specifically interacting), which can be truncated or humanized or altered in any other way without losing its specificity (such alterations defined herein as derivatives and / or fragments).

[0047] Preferably, one or more of the first molecule, the second molecule, and the further molecule according to the application is an antibody, or a fragment of a derivative thereof. As used herein in relation to all aspects of the application, the term "antibody" or "antibody molecule" includes any immunoglobulin, whether naturally occurring or produced in part or wholly by synthetic means. The term "antibody" or "antibody molecule" includes monoclonal antibodies (mAbs) and polyclonal antibodies (including polyclonal antisera). Antibodies can be intact, or fragments derived from whole antibodies (see below). Antibodies can be human, humanised or of non-human origin. A "monoclonal antibody" is a homogeneous, highly specific population of antibodies directed against a single antigenic site or "determinant" of a target molecule. A "polyclonal antibody" comprises a heterogeneous population of antibodies directed against different antigenic determinants of a target molecule. The term "antiserum" or "antisera" refers to antibody-containing serum obtained from an immunized animal. It has been shown that fragments of whole antibodies can perform the function of binding antigens. Thus, antibodies referred to herein, and methods, arrays and kits of the application referred to, encompass whole antibodies, and also encompass any polypeptide or protein comprising an antibody binding fragment. Examples of binding fragments are: (i) a Fab fragment, consisting of VL, VH, CL and CH1 domains; (ii) a Fd fragment consisting of VH and CH1 domains; (iii) a Fv fragment consisting of the VL and VH domains of a single antibody; (iv) a dAb fragment, which consists of a VH domain; (v) isolated CDR regions; (vi) a F(ab')2 fragment, a bivalent fragment comprising two linked Fab fragments; (vii) a single chain Fv molecule (scFv), wherein a VH domain and a VL domain are connected by a peptide linker that allows the two domains to associate to form an antigen binding site; (viii) a bispecific single chain Fv dimer (WO 93 / 11161); and (ix) a "diabody", a multivalent or multispecific fragment produced by genetic fusion of two or more Fv regions (WO 94 / 13804; 58). Fv, scFv or diabody molecules can be stabilised by the introduction of a disulphide bridge linking VH and VL domains. Minibodies comprising scFv joined to CH3 domains can also be made.

[0048] As used herein, antibody molecules and immunotoxins are intended to encompass recombinant antibodies and recombinant immunotoxins, respectively (e.g., a Fab, scFv or SC mAb linked to a recombinant ribosomal inhibiting protein by a cleavable peptide linker). Additionally or alternatively, the first antibody molecule and the second antibody molecule can be provided as a single bispecific (anti-CD3 / anti-CD7) antibody, thereby providing a bispecific immunotoxin, such as anti-CD3 / CD7-rRTA.

[0049] With respect to antibody molecules, the term "selectively binds" can be used herein to refer to situations in which one member of a specific binding pair will not exhibit any significant binding to molecules other than its specific binding partner. The term also applies to situations in which, for example, an antigen binding site is specific for a particular epitope carried by a variety of antigens, in which case the specific binding member carrying the antigen binding site will be able to bind to the various antigens carrying the epitope.

[0050] Preferably, when the first molecule is an antibody, the molecule is a monoclonal antibody, such as a murine monoclonal antibody.

[0051] In preferred embodiments, the first molecule comprises a combination of complementarity determining regions (CDRs). The CDR sequence comprises a CDR3 sequence having at least 95, 96, 97, 98, or 99% sequence identity to 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 to 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 can further comprise a CDR2 sequence having at least 95, 96, 97, 98, or 99% sequence identity to 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.

[0052] "Sequence identity" and "sequence similarity" can be determined by aligning two peptides or two nucleotide sequences using a global or local alignment algorithm, depending on the length of the two sequences. Sequences of similar length are preferably aligned using a global alignment algorithm (e.g. Needleman Wunsch) which performs an optimal alignment of sequences over their entire length, while sequences which differ greatly in length are preferably aligned using a local alignment algorithm (e.g. Smith Waterman) which performs an optimal alignment of sequences in a window of optimal alignment. Sequences can be referred to as "essentially identical" or "substantially similar" (as defined below) when they share at least some minimum percentage of sequence identity when optimally aligned (using, for example, the program GAP or BESTFIT using default parameters). GAP uses the Needleman and Wunsch global alignment algorithm to align two sequences over their entire length (full length), maximizing the number of matches and minimizing the number of gaps. Global alignment is suitable for determining sequence identity when the lengths of the two sequences are similar. Typically, using GAP default parameters, the gap creation penalty = 50 (nucleotides) / 8 (proteins), and the gap extension penalty = 3 (nucleotides) / 2 (proteins). For nucleotides, the default scoring matrix used is nwsgapdna, and for proteins, the default scoring matrix is Blosum62 (Henikoff and Henikoff, 1992, PNAS 89, 915-919). Scoring of sequence alignments and percentage sequence identity can be determined using computer programs, such as the GCG Wisconsin Package Version 10.3, available from Accelrys Inc., 9685 Scranton Road, San Diego, CA 92121-3752, USA, or using open source software, such as the programs "needle" (using the global Needleman-Wunsch algorithm) or "water" (using the local Smith-Waterman algorithm) in EmbossWIN Version 2.10.0, using the same parameters as for GAP above, or using default settings (for both "needle" and "water", and for both protein as well as DNA alignments, the default gap opening penalty is 10.0, and the default gap extension penalty is 0.5; the default scoring matrix is Blossum62 for proteins and DNAFull for DNA). Local alignment, such as alignment using the Smith-Waterman algorithm, is preferred when the overall length of the sequences differs greatly.

[0053] Alternatively, percent similarity or identity can be determined by querying public databases using algorithms such as FASTA, BLAST, and the like. Thus, the nucleic acid and protein sequences of the present application can further be used as "query sequences" to perform a search against 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) of Altschul et al. (1990) J. Mol. Biol. 215:403-10. BLAST nucleotide searches can be performed with the NBLAST program (score = 100, word- length = 12) to obtain nucleotide sequences homologous to the oxidoreductase nucleic acid molecules of the present application. BLAST protein searches can be performed with the BLASTx program (score = 50, word-length = 3) to obtain amino acid sequences homologous to the protein molecules of the present application. To obtain gapped alignments for comparison purposes, one can utilize gapped BLAST, as described in Altschul et al., (1997), Nucleic Acids Res. 25(17):3389-3402. When utilizing BLAST and gapped BLAST programs, the default parameters of the respective programs (e.g., BLASTx and BLASTn) can be used. The home page of the National Center for Biotechnology Information can be accessed at http: / / www.ncbi.nlm.nih.gov / .

[0054] Optionally, the first molecule is selected from the group consisting of:

[0055] - WT1, murine IgG2a (WO9948534A1);

[0056] - TXU-7, murine IgG1 (CA2292426A1);

[0057] - RFT2, murine IgG2a (Kirkham et al. 1988);

[0058] - CHT2, SDZ CHH380, murine IgG2a (Heinrich et al. 1989, Lazarovits et al. 1993);

[0059] - FITC-S9.1, murine IgG2b (Fishwild et al. 1992);

[0060] - 3A1e, murine IgG2b (Vallera et al. 1996); and

[0061] - HB2, murine IgGl (Flavel et al. 2001).

[0062] Preferably, the first molecule is a monoclonal IgG2a antibody. In some embodiments, the first molecule can comprise a VH domain having at least 95, 96, 97, 98, or 99% sequence identity to 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 to the amino acid sequence of SEQ ID NO: 14. In certain embodiments, the first molecule can be a WT1 antibody having a heavy chain of SEQ ID NO: 11 and a light chain of SEQ ID NO: 12.

[0063] WT1

[0064] WT1 (INN Grisnilimab) is a murine IgG2a monoclonal antibody that selectively binds to human CD7 (UniProt: P09564), is a member of the immunoglobulin superfamily and is a transmembrane protein found on thymocytes and mature T cells. WT1 and its production and characterization are described in EP 0945139 Al, Tax et al., Monoclonal antibodies against human thymocytes and T lymphocytes. Protides of the biological fluids, 29th Symposium, 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 (the entire contents of all documents are hereby incorporated by reference). WT1 is commercially available. For example, an anti-CD7 antibody (clone WT1) is sold by LifeSpan BioSciences, Inc. under the catalog number: LS-C1228851000 (1000 μΐ in PBS, 0.1% sodium azide) for research use, e.g., immunofluorescence and immunohistochemistry.

[0065] The monomeric molecular weight of WT1 is approximately 150 kDa, consisting of two heavy chains of approximately 50 kDa and two light chains of approximately 25 kDa. The amino acid sequences of the WT1 light and heavy chains were determined by extracting mRNA from hybridoma cell pellets, performing RT-PCR, and DNA sequencing on an ABI 3130x1 Genetic Analyzer. The predicted amino acid sequences were verified by mass spectrometry analysis. Complementarity determining regions (CDRs) were determined as per the IMGT numbering system (Lefranc, M.-P. et al., Nucleic Acids Research, 1999, vol. 27, pp. 209-212, incorporated herein by reference).

[0066] The amino acid sequences of the WT1 heavy and light chains are shown below, respectively.

[0067] WT1 Heavy Chain:

[0068]

[0069] VH domain is underlined; CDRH1-H3 is shown in bold and curved underlining.

[0070] WT1 Light Chain:

[0071]

[0072] VL domain is underlined; CDRL1-L3 is shown in bold and curved underlining.

[0073] WT1-VH:

[0074]

[0075] WT1-VL:

[0076]

[0077] WT1-CDRH1:

[0078]

[0079] WT1-CDRH2:

[0080]

[0081] WT1-CDRH3:

[0082]

[0083] WT1-CDRL1:

[0084]

[0085] WT1-CDRL2:

[0086]

[0087] WT1-CDRL3:

[0088]

[0089] The pharmaceutical formulation of the application can optionally or preferably comprise a second molecule specifically recognizing CD3. Preferably, when the second molecule is an antibody, said molecule is a monoclonal antibody, for example a murine monoclonal antibody. In some embodiments of the application, the second molecule is an IgG2b antibody, as this antibody does not fix human complement or bind to human Fc receptors and thus does not induce the release of cytokines by targeted T cells and / or NK cells. In some embodiments, the second molecule is a murine antibody. For example, the second molecule is an anti-(human)CD3 murine IgG 2b monoclonal antibody.

[0090] In preferred embodiments, the second molecule comprises a combination of complementarity determining 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: 7 (ARGSRYDYYGMDY). Most preferably, the CDR3 sequence comprises 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 comprises or consists of the amino acid sequence of SEQ ID NO: 15. The CDR sequence can 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 comprises or consists of the amino acid sequence of SEQ ID NO: 6.

[0091] Optionally, the first molecule is selected from the group consisting of:

[0092] - SPV-T3a, murine IgG2b (Spits et al. 1983);

[0093] - hOKT3 gamma 4, humanized IgG4 (Richard et al. 1999);

[0094] - visilizumab, humanized IgG2 Fc modification (Carpenter et al. 2002);

[0095] - teplizumab, humanized, Fc-modified (Herald et al. 2019);

[0096] - otelixizumab, humanized, Fc-modified (Keymeulen et al. 2020).

[0097] In some embodiments, the first molecule can comprise a VH domain having at least 95, 96, 97, 98, or 99% sequence identity to 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 to the amino acid sequence of SEQ ID NO: 4. In certain embodiments, the second molecule can be the SPV-T3a antibody having the heavy chain of SEQ ID NO: 1 and the light chain of SEQ ID NO: 2.

[0098] SPV-T3a

[0099] SPV-T3a (INN dafsolimab) is a murine IgG2b monoclonal antibody that selectively binds to human CD3, a T cell surface glycoprotein consisting of a CD3y chain (UniProt: P09693), a CD35 chain (UniProt: P04234), and two CD3s chains (UniProt: P07766). SPV-T3a and its production and characterization are described in EP0945139 Al and Spits et al., Hybridoma, 1983, vol. 2, pp. 423-437 (the entire contents of both of which are hereby incorporated by reference).

[0100] The monomeric molecular weight of SPV-T3a is approximately 150 kDa, consisting of two heavy chains of approximately 50 kDa and two kappa light chains of approximately 25 kDa. The amino acid sequences of the SPV-T3a light and heavy chains were determined by extracting mRNA from hybridoma cell pellets, performing RT-PCR, and DNA sequencing on an ABI 3130x1 Genetic Analyzer. The predicted amino acid sequences were verified by mass spectrometric analysis. Complementarity determining regions (CDRs) were determined as per the IMGT numbering system (Lefranc, M.-P. et al., Nucleic Acids Research, 1999, vol. 27, pp. 209-212, which is incorporated herein by reference).

[0101] The amino acid sequences of the SPV-T3a heavy and light chains are shown below, respectively.

[0102] SPV-T3a heavy chain:

[0103] SPV-T3a light chain:

[0104] The VH domain is underlined; CDRH1-H3 is shown in bold and curved underlining.

[0105] SPV-T3a light chain:

[0106]

[0107] The VL domain is underlined; CDRL1-L3 is shown in bold and curved underlining.

[0108] SPV-T3a-VH:

[0109]

[0110] SPV-T3a-VL:

[0111]

[0112] SPV-T3a-CDRH1:

[0113]

[0114] SPV-T3a-CDRH2:

[0115]

[0116] SPV-T3a-CDRH3:

[0117]

[0118] SPV-T3a-CDRL1:

[0119]

[0120] SPV-T3a-CDRL2:

[0121]

[0122] SPV-T3a-CDRL3:

[0123]

[0124] Optionally, when the pharmaceutical composition of the present application comprises a second molecule, the ratio between the first molecule and the second molecule can range from 1:10 to 10:1, 1:9 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.

[0125] Preferably, one or more of the first molecule, the second molecule and the further molecule according to the application has at least one toxin moiety. This can provide for a higher therapeutic effect, but can also be used to provide for a higher specificity of the group of cells, e.g. also in combination with a prodrug regimen.

[0126] Toxic moiety essentially refers to any and all molecules that directly or indirectly cause toxicity against target cells, thus including but not limited to lectins, ricin, abrin, PE toxin, diphtheria toxin, radioisotopes, cytostatic drugs (such as adriamycin), agents that induce apoptosis, and prodrug converting substances used in combination with prodrugs, such as thymidine kinase and gancyclovir.

[0127] The first molecule, the second molecule or the further molecule according to the application can have the same or different toxin moieties. Optionally, the toxic moiety can be independently selected from the group consisting of ricin, deglycosylated ricin A (dgRTA), and non-glycosylated recombinant ricin A. Deglycosylated ricin A can prevent the binding of ricin A to the carbohydrate receptor expressed by hepatocytes, and thus can be preferred in some embodiments of the application. In prodrug embodiments, typically one of the molecules that specifically recognizes CD3 or CD7 or a receptor can have the converting agent, and the other has the prodrug. However, CD3 does not need to be coupled to a toxic moiety to be effective, as it blocks the interaction of the T cell receptor with the APC. This is an advantage of the present application, which is not present in the prior art.

[0128] In a preferred embodiment of the application, the first molecule, e.g. WT-1, has recombinant ricin A (rRTA). In yet another preferred embodiment of the application, the pharmaceutical composition comprises both the first molecule and the second molecule (e.g. both WT-1 and SPV-T3a), each having recombinant ricin A (rRTA). For example, the pharmaceutical composition of the application comprises WT-1 conjugated to RTA (INN setaritox) and SPV-T3a conjugated to RTA (INN setaritox).

[0129] The conjugation ratio of toxin (e.g. rRTA) to molecule (e.g. antibody molecule) can range from 0.5:1 to 5:1. In particular, the conjugation ratio of toxin (e.g. rRTA) to molecule (e.g. antibody molecule) can range from 0.5:1 to 2:1, e.g. range from 0.75:1 to 2:1, or range from 0.75:1 to 1.5:1.

[0130] rRTA

[0131] RTA denotes ricin A-chain. In its native form, the plant toxin ricin consists of two disulfide-linked chains of about 32 kDa each: the A chain (RTA) which inhibits protein synthesis, and the B chain (RTB) which facilitates cell binding and membrane translocation.

[0132] For clinical use, RTB is replaced by a cell targeting ligand specific for the cells to be eliminated, such as mAb SPV-T3a and / or WT1. Upon entry of the mAb into the cell, RTA dissociates from the mAb and a fraction of the free RTA is transported via the trans-Golgi network and the endoplasmic reticulum (ER) into the cytosol. In the cytosol, RTA catalytically inhibits protein synthesis by N-glycosidase activity, removing the base A4324 in the 28S rRNA, thereby preventing the association of elongation factor 1 (EF-1) and EF-2.

[0133] The rRTA according to the present application can be produced by a recombinant E. Coli cell line constructed for this purpose. The RTA protein, which has a monomeric molecular weight of 30 kDa, contains 268 amino acids.

[0134] A preferred example of the rRTA of the present application can have at least 95, 96, 97, 98, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 21. In a preferred embodiment of the present application, the rRTA comprises or consists of the amino acid sequence of SEQ ID NO: 21.

[0135] SEQ ID NO: 21

[0136]

[0137] The toxic moiety can be coupled to the specific binding molecule using any suitable conjugation or linker chemistry, such as by recombinant means to make a fusion protein, typically including a protease cleavage site between the binding molecule and the toxic (protein) moiety, but for ease of manufacture as well as freedom of choice of toxic moiety, chemical coupling is preferred, optionally through an acid-labile linker. (Upon internalization, the conjugate will typically pass through the lysosome). Other linkers that can be suitable for use in the pharmaceutical compositions of the present application can be selected from the group consisting of cleavable linkers (such as enzymatically cleavable peptide linkers, acid-sensitive hydrazone linkers, and glutathione-sensitive disulfide linkers) and non-cleavable linkers (such as linkers based on maleimidocaproyl [MC] and 4-maleimidomethylcyclohexane-1-carboxylate [MCC]).

[0138] In particular, the conjugation can use N-3-(2-pyridylthio)propionic acid succinimidyl ester (SPDP; Pharmacia) or 4-succinimidoxycarbonyl-alpha-methyl-alpha(2- pyridylthio)toluene (SMPT). SMPT is a so-called "second generation crosslinker" which is characterized by a hindered disulfide bond (due to the presence of a benzene ring). This makes the SMPT-linker less susceptible to extracellular reduction by thiols present in the tissue and blood, and thus allows for a prolonged serum half-life of the immunotoxin. Thorpe et al. demonstrated that using SMPT instead of the first generation crosslinker SPDP significantly improved the anti-tumor effect of their dgRTA-based IT in an in vivo mouse tumor model (WO9948534A1). SMPT contains a disulfide bond, which is important for the intracellular dissociation of the molecule from the toxic moiety.

[0139] The present application also provides a pharmaceutical composition wherein at least two molecules specifically recognizing different receptors have a toxic moiety, which can be the same or different toxic moieties. The use of different toxic moieties has a major advantage when the side effects of the different moieties are different, because a higher dose can be given. Typically, the pharmaceutical composition according to the present application can further comprise at least one additional molecule specifically recognizing CD5, CD2, CD4, CD8 or the IL-2 receptor, which molecule can also be coupled to a toxic moiety. This can provide a higher therapeutic effect, but can also be used to provide a higher specificity for a group of cells, for example also in combination with a prodrug regimen.

[0140] The doses used are given in the detailed description herein. The dose limitation for the immunotoxins in a regimen such as the regimen of the present application is usually dependent on the immunotoxin, both because of the specificity and affinity of the specific binding molecule, and because the tolerable dose differs for different drugs. Expressed in equivalents of RTA, the limitation will usually be within 10 mg RTA equivalents per m 2 of body surface area, preferably within 5 mg RTA equivalents per m 2 of body surface area, more preferably within 2.5 mg RTA equivalents per m 2 of body surface area, for example within 2.0 mg RTA equivalents per m 2 of body surface area. Preferably, the RTA equivalent dose of the pharmaceutical composition of the present application is within 0.3 - 2.0 mg RTA equivalents per m 2 of body surface area, more preferably within 0.5 to 1.5 mg RTA equivalents per m 2 of body surface area, for example within 0.6 to 1.2 mg RTA equivalents per m 2 of body surface area.

[0141] Typically, the composition according to the application will be used to treat a patient for a chronic inflammatory or autoimmune disease or other CD7-positive malignancy. For example, the composition of the application can be used to treat a disease selected from the group of autoimmune diseases, such as systemic lupus erythematosus, rheumatoid arthritis, systemic sclerosis, local scleroderma, IgG4-mediated disease, myositis and Sjogren syndrome, primary sclerosing cholangitis, primary biliary cirrhosis, autoimmune hepatitis, vasculitis, nephritis, encephalomyelitis, neuropathy, (epi) scleritis; chronic inflammatory diseases, including conditions such as axial and peripheral spondyloarthritis, psoriatic arthritis, inflammatory bowel disease, psoriasis; and atopic allergic conditions, including conditions such as atopic dermatitis, atopic asthma and nasal polyps. Preferably, the systemic autoimmune disease is systemic sclerosis or Sjogren syndrome. The patient receiving treatment according to the application can have shown symptoms of vasculopathy, fibrosis and / or autoimmune inflammation.

[0142] Such a regimen is therefore also part of the present application.

[0143] The composition of the application targets activated pathogenic T cells and / or NK cells in a patient, preferably activated pathogenic T cells and / or NK cells in the blood and / or inflamed tissue of a patient. Preferably, the present application targets said 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. Optionally, said 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, said activated pathogenic T cells comprise CD8 cytotoxic T cells.

[0144] The pharmaceutical composition of the application can be administered to a patient, preferably a human, by various routes. All modes of administration can be envisaged, for example by oral, rectal or intravenous, cutaneous, intramuscular, subcutaneous, intrauterine or intracerebroventricular injection. Preferably, the pharmaceutical composition of the application is administered intravenously, for example by intravenous infusion over a period of, for example, up to 6 hours, 4 hours, 2 hours or 1 hour or 45 minutes or 30 minutes. In one embodiment of the application, the pharmaceutical composition of the application is administered by intravenous infusion over a period of 4 hours.

[0145] Optionally, the pharmaceutical composition of the application can be administered, for example, by intravenous infusion.

[0146] In the case of whole antibody use, the dose of the first molecule can be 1 to 50 mg, 1 to 40 mg, 1 to 30 mg, 1 to 25 mg, 1 to 20 mg, 1 to 10 mg, 3 to 10 mg, 3 to 8 mg, 4 to 9 mg, 4 to 8 mg, 5 to 7 mg, for example about 6 mg per administration.

[0147] In the case of whole antibody-RTA conjugate use, the dose can be 0.01 to 1 mg / kg body weight, 0.05 to 5 mg / kg body weight, 0.05 to 2 mg / kg body weight, 0.05 to 1 mg / kg body weight, 0.05 to 0.5 mg / kg body weight, 0.05 to 0.2 mg / kg body weight, for example about 0.1 mg / kg body weight.

[0148] The administration can be performed once a day, once every 2, 3, 4, 5, 6 or 7 days. For example, the administration can be performed once a day for 14 days, once a day on days 0, 2, 4 and 6, or once a day on days 0, 2, 6, 11, 17 and 24. In a preferred embodiment of the application, the administration is performed once a day on days 0, 2, 4 and 6.

[0149] Optionally, the pharmaceutical composition of the application further comprises one or more excipients, carriers, buffers, stabilizers, tonicity adjusting agents, preservatives or preservative or antioxidants. Such materials should be nontoxic and should not interfere with the efficacy of the active ingredient. The precise nature of the carrier or other material can depend on the route of administration, e.g. intravenous injection.

[0150] In a preferred embodiment of the application, the composition comprises (i) 0.05 to 0.5 mg / mL, optionally 0.1 to 0.5 mg / mL, for example 0.2 mg / mL of the first molecule; and optionally one or more of the following: (ii) 0.05 to 0.5 mg / mL, optionally 0.1 to 0.5 mg / mL, for example 0.2 mg / mL of the second molecule; (iii) 5 to 20 mM, optionally 5 to 15 mM, for example 10 mM of citrate buffer, (iv) 50 to 300 mM, optionally 75 to 200 mM, for example 125 mM of L-arginine or a pharmaceutically acceptable salt thereof, and (v) 0.01 to 0.1 % (w / v), optionally 0.01 to 0.08 % (w / v), for example 0.05 % (w / v) of polysorbate, for example Tween(RTM) 20, and (vi) 120 to 160 mM of maltose. Optionally, the composition is in water. Preferably, the pH of the composition ranges from 6 to 7.5, optionally from 6 to 7, for example 6.5.

[0151] In some embodiments, the composition further comprises at least one agent selected from the group consisting of:

[0152] 120 to 160 mM maltose;

[0153] 100 to 150 mM, optionally 125 mM trehalose;

[0154] 25 to 75 mM, optionally 50 mM glycine; and

[0155] 80 to 120 mM, optionally 100 mM mannitol.

[0156] In particular, the composition can comprise 130 to 150 mM, optionally 140 mM maltose monohydrate.

[0157] In some embodiments, the composition comprises:

[0158] (1) 0.2 mg / mL of SPV-T3a-rRTA and 0.2 mg / ml WT1-rRTA;

[0159] (2) 10 mM sodium citrate / citric acid buffer;

[0160] (3) 125 mM of L-arginine.HCl;

[0161] (4) 0.05% (w / v) Tween(RTM) 20;

[0162] (5) 140 mM maltose monohydrate,

[0163] wherein the composition is in water for injection and the pH is 6.5.

[0164] In some embodiments, the composition is sterile. In some embodiments, the composition is suitable for injection.

[0165] The present application also provides a method of targeting activated pathogenic T cells in a patient with a chronic inflammatory or autoimmune disease. The method comprises administering to the patient an effective amount of a pharmaceutical composition of the present application as described herein. The method of the present application can be combined with other immune suppressive treatments and / or efficacy and / or safety enhancers. Optionally, the method of the present application can be used as a pre-treatment for tolerance therapy, for example, cell therapy using tolerogenic nanoparticles.

[0166] The present application also provides a kit of pharmaceutical compositions for the treatment of chronic inflammatory or autoimmune diseases, said kit comprising a composition as described herein. The present application allows for a reduction of the number of unwanted cells, e.g. activated pathogenic T cells, such as CD7+, CD8+T, CD4+T or NK cell populations to at least 20%, often even to 5% or less of the original number. Typically, this number remains low over a longer period of time compared to the levels achieved with prior art solutions. The exemplified compositions not only target T cells, but also NK cells, which is another advantage of the present application.

[0167] While the application has been described and illustrated with reference to specific embodiments, it will be apparent for those skilled in the art that the application is suitable for many different variations not explicitly described herein. By way of example only, certain possible variations will now be described.

[0168] Examples

[0169] Materials and Methods

[0170] Immunotoxins

[0171] The anti-CD3 / CD7 combination (CD3 / CD7-IT) of immunotoxins as referred to in the present text contains a 1 : 1 mixture (w / w) of the murine monoclonal antibodies SPV-T3a (anti-CD3) and WT1 (anti-CD7) both conjugated to recombinant ricin A as previously described (36, 40).

[0172] Peripheral blood mononuclear cells (PBMC) isolation, cryopreservation and culture

[0173] PBMCs were isolated from peripheral blood of patients (n=30) and healthy donors (n=15) by density centrifugation using Ficoll Pacque PLUS and cultured in complete RPMI medium 1640+GlutaMAX™ (Gibco, reference 72400-021) supplemented with 100 IU / ml penicillin, 100 mg / ml streptomycin, 100 mg / L sodium pyruvate, and 10% human mixed serum. PBMCs not immediately processed were cryopreserved and stored in liquid nitrogen until further use. To generate phytohemagglutinin (PHA) to activate T cells, PBMCs were first seeded at a density of 100,000 cells per well in 96-well U-shaped plates (Greiner) and then stimulated for 24 hours at 37°C and 5% CO2 with 5 μg / ml PHA (Roche, catalog number 11082132001). To evaluate cytokine production, PBMCs were stimulated for 4 hours at 37°C and 5% CO2 in the presence of 5 μg / ml brevidin A (Merck) and 12.5 ng / ml phorbol myristate acetate (Sigma) and 500 ng / ml ionomycin A (Merck) before flow cytometry staining.

[0174] Immunohistochemistry

[0175] Immunohistochemical analysis was performed on formalin-fixed paraffin-embedded (FFPE) skin biopsies from 20 patients with systemic sclerosis. Skin biopsies were obtained from both affected and unaffected areas of the forearm, as well as by a qualified clinician using [method / technology]. Surgical resection was performed for the biopsy that diagnosed. In all skin samples, CD3 staining was used as a marker to evaluate T cell infiltration, and CD7 staining was used to assess infiltration of activated T lymphocytes and NK cells. For CD3 staining, slides were washed in xylene to deparaffinize and rehydrated with ethanol. Antigen was retrieved in 10 mM sodium citrate buffer (pH 6.0) at room temperature (RT). Peroxidase activity was blocked by incubation with 3% H202 for 30 minutes or less. Then, the sections were incubated with primary mouse CD3 anti-human monoclonal antibody (1 :200 dilution in PBS with 1% BSA; clone F7.2.38; Dako; Cat. No. M7254) overnight at room temperature. Next, the tissue was incubated with secondary antibody (BrightVision Poly-HRP, Immunologic DPVO55HRP) for 60 minutes at room temperature. 3'3'-diaminobenzidine was used to visualize the antibody (bright DAB, Immunologic). Nuclei in all slides were counterstained with hematoxylin and mounted with a coverslip (Permount, Thermo-Fischer, Waltham, MA, USA). Immunohistochemical evaluation of CD7 was performed according to the standard procedure of the manufacturer using the Omnis automated immunostainer (DAKO). Briefly, FFPE tissue was deparaffinized, rehydrated, and subjected to heat-mediated antigen retrieval (30 minutes at 97°C). Subsequently, endogenous peroxidase was blocked, and primary mouse CD7 anti-human monoclonal antibody (ready-to-use, diluted in Envision Flex antibody diluent, clone CBC.37, DAKO; Cat. No. GA64361-2) was added for 20 minutes at room temperature. Subsequently, secondary antibody (Envision Flex HRP, DAKO) was applied for 20 minutes at room temperature. The antibody complex was visualized with Envision Flex substrate working solution (DAKO), and nuclei were counterstained with hematoxylin. Human synovial / tonsil samples were used as positive controls, and skin sections without primary antibody were used as negative controls. The entire surface of all sections (n = 4) mounted for each donor and condition was examined for cellular infiltrates, and imaged with CaseViewer (v2.3.0.99276). CD3-positive cells were counted by two independent observers in four randomly selected fields, and the total number of positive cells was plotted as mean ± SD. CD7-positive staining was evaluated using an arbitrary 0-4 score semiquantitative scoring system of positively stained areas. This scoring was performed blindly by two independent observers. Expression of collagen type 1 in fibroblasts was also evaluated (goat anti-collagen type 1-UNLB, Southern Biotech, Cat. No. 1310-01).Staining was performed similarly to CD3 markers, except that secondary rabbit biotinylated anti-goat IgG antibody (Vector Laboratories, PK-6101) was incubated for 30 minutes at room temperature. Values shown in graphs represent mean ± SD.

[0176] Multiplex immunohistochemical staining and imaging of SSc skin

[0177] For multiplex immunofluorescence staining, 5 pm-thick sections from matched involved and uninvolved skin from 24 SSc patients were included. Slides were stained by using the automated platform of Opal 7-color automated IHC kit (NEL801001KT; PerkinElmer) on the BOND RX IHC & ISH research platform (Leica Biosystems) as previously described (41). Incubation with primary and secondary antibodies was performed for 1 hour and 30 minutes at room temperature, respectively. To detect skin lymphocyte populations, the following antibodies were used; anti-CD56 (CellMarque, 156R-94, clone MRQ-42) with Opal620, anti-CD8 (Dako, M7103, clone C8 / 144B, 1 :200) with Opal690, anti-CD7 (Dako, GA64361-2, clone CBC.37, 1 :30) with Opal480, anti-CD3 (Thermo, RM-9107, clone RM-9107, 1 :200) with Opal520, anti-FOXP3 (eBioscience Affymetrix, 14-4777, clone 236A / E7, 1 :100) with Opal570, and anti-CD20 (Thermo, MS-340, clone L26, 1 :600) with Opal570. Slides were stained with DAPI for 5 minutes, washed, and mounted with Fluoromount-G (SouthernBiotech, 0100-01). Slides were then scanned by using the automated quantitative pathology imaging system (Vectra V.3.0.4, PerkinElmer) with 4x magnification overview. Annotation of multispectral images of skin tissues was done with Phenochart (V.1.0.9, PerkinElmer) and scanned at 20x magnification. Spectral unmixing of Opal fluorophores was performed by InForm software (V.2.4.2, PerkinElmer) and then the multichannel images were digitally merged. For quantitative analysis, digital scans containing the whole skin biopsies (n=3 sections per biopsy for each donor and condition) were quantified by QuPath-0.4.4 (42).

[0178] Single-cell RNA sequencing analysis

[0179] Single-cell count matrix (cells x genes) was obtained from two publicly available datasets, GSE195452, GSE138669 and GSE128169. Preprocessing of data was performed using Seurat (version 4.3.0) (40). Quality control measures were implemented by filtering cells with high mitochondrial gene content (> 5%) and cells with gene counts per cell values below 200 or above 2000. Subsequently, CD3+ cells and / or CD7+ cells were sorted, recovering 2126 and 5061 high-quality cells from both datasets, respectively. After that, CD8+ subpopulation of cells was sorted from these cells for separate analysis.

[0180] For preliminary dimensionality reduction, non-negative matrix factorization was applied followed by UMAP: Uniform Manifold Approximation and Projection (43, 44) and Louvain clustering as previously described by Singh et al. (45). Then the FindAllMarkers function of Seurat was used to identify differentially expressed genes (DEG) within each cluster, which were subsequently annotated 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 in cell types / groups.

[0181] To assess DEGs between healthy and diseased individuals, the FindConservedMarker function of Seurat was used. Additionally, Wilcoxon test (46) was applied to test differences in cell frequencies between healthy and individuals with systemic sclerosis (SSc).

[0182] Single-sample gene set enrichment and correlation analysis

[0183] To gain insight into the functional features of each cell type, single-sample gene set enrichment analysis (ssGSEA) was performed using the escape R package (47) and Wiki pathways from MsigDB (48) as a collection of reference gene sets.

[0184] The R function from the ggplot2 package named geom_tile (Wickham, H. (2016). ggplot2: Elegant Graphics for Data Analysis (2nd ed.). Springer) was used to visualize the pathways in different cell types / groups. Differences in the distribution of the normalized enrichment score (NES) between the control and SSc groups were tested using the Kolmogorov-Smirnov (KS) test (49). To capture pathways related to fibrosis and inflammation, lists of genes associated with these processes were retrieved and the list of genes was further expanded by including CD7.

[0185] Correlation analysis at the single-cell level can be noisy and biased by technical factors. Therefore, a meta-cellular object containing CD3 / CD7+ cells (from the Seurat object described previously) was constructed by using the WGCNA R package (50). This object contains a weighted gene co-expression framework to identify modules of highly correlated genes. Subsequently, this meta-cellular object was used to obtain pairwise correlations and p-values were calculated using the Hmisc function from the Hmisc R package (Harrell Jr., F. E. and contributions from Charles Dupont and many others (2020). Hmisc: Harrell Miscellaneous (R package version 4.8.0)). The R software ComplexHeatmap (Gu, Z. (2016). ComplexHeatmap: Making Complex Heatmaps in R (R package version 2.10.0)) was used to visualize the correlations.

[0186] Collection of primary fibroblasts and cell culture

[0187] 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 100U / ml penicillin, 100mg / ml streptomycin, 100mg / L sodium pyruvate, and 20% fetal bovine serum. The plate was incubated for 2 weeks under standard culture conditions (5% CO2, 37°C, 95% humidity), during which primary skin fibroblasts spontaneously grew. The medium was changed every 3-4 days. After the primary fibroblasts emerged, they were cultured in DMEM Glutamax medium (Gibco) supplemented with 100U / ml penicillin, 100mg / ml streptomycin, 100mg / L sodium pyruvate, and 10% fetal bovine serum, and used for experiments after 5 passages.

[0188] Single-cell isolation from skin biopsy

[0189] A 6 mm diameter skin biopsy from a healthy individual was used to obtain a single-cell suspension containing skin-infiltrating lymphocytes for phenotypic characterization and functional assays. The protocol used combined mechanical and enzymatic dissociation of skin tissue and has been described in detail by He et al. (PMID: 27166763).

[0190] Flow cytometry analysis

[0191] For each donor, first, 1 x 10⁻⁶ molten metallurgy was prepared at 4°C. 6 Each PBMC was labeled with ViaKrome 808 immobilizable viability dye (1.5:1000 in PBS) for 30 minutes to exclude dead cells, and then stained with fluorescently labeled extracellular antibodies for 20 minutes at room temperature (Supplementary Table x). For intracellular staining (Supplementary Table x), Cyto-Fast was used according to the manufacturer's guidelines. TM Cells were fixed and permeated using a Fix / Perm buffer kit (Biolegend). To facilitate the detection of intracellular cytokines, cells were pre-stimulated with 12.5 ng / ml phorbol 12-myristate 13-acetate (PMA), 500 ng / ml iomycin, and 5 μg / ml iomycin A before staining. Samples were immediately acquired using a Beckman Coulter Cytoflex LX 21-color flow cytometer after staining.

[0192] Isolation, culture, and cell viability of T cells, B cells, and NK cells from peripheral blood of SSc

[0193] Frozen stored PBMCs from patients with systemic sclerosis were thawed and washed to isolate specific immune cell populations as previously described. CD3+ T cells were isolated using magnetic negative selection according to the manufacturer’s instructions (MojoSort Human CD3+ T Cell Isolation Kit; Cat. No. 480021). CD19+ B cells were also isolated using negative selection with the MojoSort™ Human Pan B Cell Isolation Kit (Cat. No. 480082). Unaffected CD56+ NK cells were isolated from SSc PBMCs by using the NK Isolation Kit (Miltenyi Biotec, Cat. No. 130-092-657) according to the manufacturer’s protocol. After isolation, the enriched CD3 + T cells, CD19 + B cells, and CD56 + NK cell fractions exhibited greater than 95% purity as evaluated by flow cytometry staining for CD3, CD19, CD56 markers. Isolated immune cell populations were cultured in 96-well U-bottom plates (Greiner Bio-One) at a density of 50,000 cells / well with X-VIVO™ 15 Medium (Lonza, Cat. No. 04-418Q). To evaluate cell viability of cells after different stimulations (24 hours) and treatment conditions (48 hours), CellTiter 96® AQueous One Solution Cell Proliferation Assay (Promega) was used according to the manufacturer’s instructions. Cells were also treated with 5 mM cycloheximide (Sigma, Cat. No. 01810-1G) as a positive control. Luminescence was measured using a CLARIOstar Plus (BMG LABTECH). For each experimental condition, 4 technical replicates were used and the average value was used for further analysis. Experimental values were corrected for medium luminescence and normalized to the control unstimulated and untreated condition. 2.0 cell viability assay (Promega). Cells were also treated with 5 mM cycloheximide (Sigma, Cat. No. 01810-1G) as a positive control. Luminescence was measured using a CLARIOstar Plus (BMG LABTECH). For each experimental condition, 4 technical replicates were used and the average value was used for further analysis. Experimental values were corrected for medium luminescence and normalized to the control unstimulated and untreated condition.

[0194] Monocyte isolation and differentiation into M2 macrophages

[0195] CD14 monocytes were isolated from PBMCs with a positive selection kit (Miltenyi Biotec, Cat#130-050-201) according to the manufacturer’s instructions. Monocytes were then seeded in 6-well plates at a cell density of 1 million cells per well in a volume of 2 ml in XVIVO™ 15 medium supplemented with 100 IU / ml penicillin, 100 mg / ml streptomycin, and 2% human pooled serum. Differentiation towards M2-like macrophages was stimulated by the addition of 20 ng / ml rhM-CSF (R&D Systems, Cat#216-MC) and 10 ng / ml rhIL-4 (Biolegend, Cat#500815). The duration of the culture was 7 days, and the medium was changed with cytokines at day 3. At the end of the culture, cells were collected in 15 ml conical tubes, washed with PBS and processed for flow cytometry staining. The staining protocol for extracellular and intracellular markers followed the previously described method. Since CD3 / CD7-IT treatment can modulate CD3 antigen, CD2 was used to identify and characterize T cell population instead of CD3. To be able to quantify absolute cell counts, a fixed amount of counting beads (Precision Count Beads™, Biolegend, Cat#424902) was added to each sample before acquisition. Samples were acquired immediately after staining on a Beckman Coulter Cytoflex LX 21 color flow cytometer. 2.0 Cell viability assay (Promega) to evaluate cell viability of M2-like macrophages as previously described.

[0196] Multi-parameter flow cytometry quantification of CD3 / CD7-IT induced cell death

[0197] To evaluate the killing efficacy of CD3 / CD7-IT on activated T cells and NK cells in vitro, a model was established in which 24 hours PHA (Roche) stimulation of PBMCs was used to mimic disease-associated T cell activation. PHA stimulation was accompanied by an increase in surface expression of CD3 antigen (2-fold MFI increase) and CD7 antigen (3-fold MFI increase) on cytotoxic CD8+GZMB+T cells and CD7 (2-fold MFI increase) on CD56+GZMB+NK cells (supplemented Figure 6 A, B). PBMCs, either unactivated or PHA-activated (5 pg / ml), were cultured for 24 hours at 37°C, 5% CO2 prior to treatment with CD3 / CD7- immunotoxin (IT) for 48 hours. In line with previous studies, the in vitro clinical treatment concentration was 1-5 nM. Drug concentration (0-10 nM) was titrated according to the killing efficacy of the drug on primary T cells and the lowest concentration exerting maximal killing efficacy was chosen. The concentration of the drug used for in vitro experiments was 0.33 nM. Following treatment, cells were collected in 15 ml conical tubes, washed with PBS and processed for flow cytometry staining. The staining protocol for live / dead, extracellular and intracellular markers followed the previously described method. Since CD3 / CD7-IT treatment can modulate CD3 antigen, CD2 was used to identify and characterize T cell population instead of CD3. To be able to quantify absolute cell counts, a fixed amount of counting beads (Precision Count Beads™, Biolegend, Cat#424902) was added to each sample before acquisition. Samples were acquired immediately after staining on a Beckman Coulter Cytoflex LX 21 color flow cytometer.

[0198] Ex vivo skin culture

[0199] Full-thickness skin punch biopsies of 6 mm in diameter were obtained from the abdomen of 4 healthy individuals undergoing plastic surgery. All patients signed an informed consent and their surgical residual material was used for research purposes. From each piece of skin tissue, 4-6 punch biopsies were collected and cut in half. To take into account possible heterogeneous infiltration of immune cells between skin biopsies, all skin fragments were mixed together and then equally distributed under different experimental conditions. Skin tissues were cultured in 24-well plates in 1 ml of RPMI medium 1640 with 100 IU / ml rhIL-2 (Thermo Fisher, Cat. 16-7027-85), 5 ug / ml PHA (Roche), growth supplements and antibiotics. Twenty-four hours later, samples were treated with 0.33 nM a-CD3 / CD7-IT. After 48 hours, skin fragments under each condition were used to obtain single cell suspensions containing skin infiltrating lymphocytes for functional assays. The protocol used combines mechanical and enzymatic dissociation of skin tissues and has been exhaustively described by He et al. (51).

[0200] Apoptosis assay

[0201] To discriminate between early apoptotic cells and non-apoptotic cells and cells in late apoptosis / necrosis, PBMCs isolated, cultured and prepared as previously described were first extracellularly stained with the monoclonal antibody of interest (Table) for 20 minutes at room temperature. Cells were then washed twice with cold PBS and resuspended in 100 μΐ of buffer containing 5 μΐ of 7-AAD®, 5 μΐ of Annexin V: FITC label and 0.15 μΐ of CaCl2(1 M) in PBS. Samples were incubated for 10 minutes at room temperature in the dark and data were acquired in a flow cytometer (Gallios) immediately after staining. Cells referred to as 7-AAD+ Annexin V+ were considered late apoptotic / necrotic, while cells referred to as 7-AAD- Annexin V+ were considered early apoptotic. Live cells were negative for both 7-AAD and Annexin.

[0202] Cytokine measurements

[0203] Quantification of human cytokines and chemokines in culture supernatants was measured by Luminex. BioPlex kits were used according to the manufacturer's instructions. Samples were analyzed with BioPlex Manager 4 software (Bio-Rad Laboratories, Hercules, CA, USA).

[0204] LDH cytotoxicity assay

[0205] To assess the cytotoxic capacity of cytotoxic T cells and NK cells, lactate dehydrogenase (LDH) was measured in PBMC and K562 cells co-culture supernatants using the LDH-cytox kit according to the manufacturer's protocol (BioRad® n° 426401). PBMC and K562 cells were seeded in 96-well plates (F-bottom) at a 10:1 ratio, using triplicate wells. To enhance the cytotoxic function of CD8+ T cells and NK cells, PBMC were stimulated overnight with a-CD3 / CD28 (1 pg / ml) or IL-2 (500 IU / ml), IL-15 (10 ng / ml), respectively. To evaluate the role of CD7 receptor in T cells and NK cells cytotoxicity, 330 nM of anti-CD7 (WT1) blocking antibody was used. The percentage of cytotoxic capacity was calculated according to the following formula: % cytotoxicity = (experimental value - low control value) / (high control value - low control value) x 100. The low control value and the high control value correspond to the LDH level of K562 cells without lysis solution or after addition of lysis solution, respectively.

[0206] Fibroblast and immune cell in vitro co-culture collagen contraction assay

[0207] Primary healthy human fibroblasts were isolated with trypsin and then adjusted to a cell density of 2 x 106cells / ml. PBMCs from 5 healthy individuals were thawed and washed as previously described and stimulated / treated according to the different experimental conditions mentioned in the results section. Cell suspensions containing a mixture of PBMCs and fibroblasts at a 5:1 ratio were then prepared. To create 3D collagen hydrogels, for each plug, 20 mΐ Minimal Essential Medium (Sigma-Aldrich, Saint Louis, CA, USA), 10 pL sodium bicarbonate (Gibco, Waltham, MA, USA), 150 pL soluble collagen (PureCol, type 1 collagen) and 90 pL cell suspension were mixed sequentially and in corresponding order in different tubes. After gentle homogenization of the suspension, 250 mΐ were added to each well of a 48-well plate. Subsequently, 750 mΐ complete RPMI medium was added and the plugs were incubated under standard conditions for 24 or 48 hours. Spontaneous fibroblast contraction was macroscopically evaluated by scanning the plates on a standard office plate layer scanner. To quantify the area of contraction, the generated images were analyzed using Fiji ImageJ. To further investigate the phenotype and function of lymphocytes and fibroblasts in this model, after macroscopic evaluation, collagen plugs were enzymatically digested with a cocktail of collagenase D, dispase and DNase for 1 hour at 37°C on a roller. The reaction was stopped by the addition of complete RPMI medium containing 10% HPS and the single cell suspension was then washed twice with PBS and used for flow cytometry analysis. To co-culture fibroblasts with CD7+ or CD7- T cells and NK cells (supplementary appendix Figure 7 ), these cells were FACS sorted from PBMCs and seeded in 3D collagen hydrogels.

[0208] RNA isolation and quantitative real-time PCR

[0209] RNA isolation was performed according to manufacturer’s guidelines using 500 μΐ TRIzol (Sigma Aldrich). After isolation, RNA concentration was quantified with a Nanodrop spectrophotometer (Thermo Scientific, Waltham, MA, USA) and any genomic DNA was removed using DNase I. Next, up to 1 μg of RNA was reverse transcribed into cDNA in a single-step reverse transcriptase PCR using oligo dT primers and 200 U M-MLV reverse transcriptase (all from Life Technologies) using a thermocycler at 37°C. Gene expression in this cDNA was measured in a quantitative real-time polymerase chain reaction (qPCR) using 0.25 mM validated primers (Biolegio, Nijmegen, the Netherlands; see Supplementary Table 3) and SYBR green master mix (Applied Biosystems, Waltham, MA, USA). Relative gene expression (-ACt) was calculated based on the average of the following reference genes: GAPDH and RPS27A.

[0210] Study design

[0211] The aim of this study was to elucidate the role of costimulatory receptors in the cytotoxic cell-driven pathological processes in the affected skin of patients with systemic sclerosis (SSc) and to examine whether targeting such receptors can prevent SSc pathology. To answer these questions, two independent SSc cohorts comprising skin cells from SSc and healthy individuals (total n=109 SSc and n=65 healthy individuals) were subjected to single-cell RNA sequencing analysis and spatial imaging using multiplexed immunohistochemistry (n=24) as well as multicolor flow cytometry for protein level validation. The impact of costimulatory modulation in functional assays was further analyzed using: (i) stimulation / inhibition of primary lymphocytes from SSc individuals with recombinant proteins; (ii) blocking antibodies in co-cultures of lymphocytes with K562 target cells; and (iii) in a fibroblast / immune cell co-culture collagen contraction assay as a disease-relevant in vitro model to mimic SSc tight skin. The therapeutic efficacy of a novel combination of bispecific anti-CD3 / CD7 targeted immunotoxins was evaluated in: (i) lymphocytes from SSc individuals blood, (ii) ex vivo skin cultures, and (iii) severely affected SSc patients treated with compassionate use of the novel anti-CD3 / 7 immunotoxin (CD3 / CD7-IT). Functional experiments were performed in multiple biological and technical replicates as mentioned in the figure legends of each figure and in the methods description of each assay.

[0212] Patient and public involvement

[0213] Patient partners were actively involved in the design and conduct of this study. Two patient research partners were involved in the design of the main research question and patient recruitment methods through structured interviews and regular interactive discussions. The patient research partners received STAP (“Key to Active Participation”) contextual training, a project initiated by the department of rheumatic diseases of the Radboud University Medical Centre (Nijmegen, the Netherlands) to establish patient panels in a hospital setting to support rheumatology research (38). The involvement of patients and their family members in disseminating the results of this study in patient organizations played a central role in community engagement during and after the study in terms of stimulating community engagement.

[0214] Patients

[0215] The study was approved by the local research ethics committee of Radboud University Medical Center (the Netherlands) (study numbers: NL57997.091.16, NL67672.091.18). All procedures involving patient participation were reviewed by an accredited research ethics committee in accordance with the principles of the Declaration of Helsinki, file number 2021-8193, in accordance with the relevant Dutch legislation. All patients (age > 18 years) who donated whole blood and skin biopsies were diagnosed with systemic sclerosis according to the ACR 1980 preliminary classification criteria (39). SSc patients with overlap syndromes were not included in the study. Blood samples from age- and gender-matched healthy volunteers were collected from Sanquin Blood Bank (project number: NVT 0397-02) from individuals who consented to donate blood for medical research. All patients agreed to participate in this study prior to blood sampling or skin biopsy. In the analysis, the relationship between CD7-normalized mean gene expression and selected patient clinical characteristics was examined, and clinical data of SSc patients were retrieved as part of previous publications (10).

[0216] Statistics

[0217] Statistical significant comparisons between experimental groups were performed with Prism 7 software (Graphpad 9.0.0, San Diego, CA, USA). The specific statistical test performed in each experiment is indicated in the figure legends.

[0218] Code availability

[0219] Single-cell RNA sequencing analysis presented in this study was performed with standard workflows and makes use of open-source R packages and software (Methods). Some analyses involved the development of custom code. All code is available at: https: / / github.com / PrashINRA.

[0220] Supplemental materials

[0221] Table 1: List of antibodies used for cell surface staining

[0222]

[0223] Table 2: List of antibodies used for intracellular staining

[0224]

[0225] Table 3: List of primer sequences used

[0226]

[0227] Example 1 - Expansion of activated cytotoxic T cells and NK cells in SSc skin

[0228] T cell and NK cell subsets can upregulate costimulatory receptors to direct the autoimmune inflammatory process in SSc. To obtain a comprehensive profiling of skin infiltrating lymphocytes, T cell and NK cell clusters (n=5,061) from scRNAseq datasets of affected skin and blood from 97 SSc patients were analyzed in comparison to 56 healthy individuals, as part of a recently published larger dataset (GSE195452) (PMID: 35381199) Figure 1 ). In SSc, the skin is the main affected tissue and therefore studying the immune profiling of skin infiltrating T lymphocytes contributes to a better understanding of the disease pathogenesis. To improve reliability, comparative analysis was performed on a single-cell RNA transcriptomic dataset containing 2,500 T cells from 9 healthy and 12 SSc skin biopsies (GSE138669) (PMID: 34031030) Figure 2 ). In addition to sc-RNA sequencing data, multicolor immunofluorescence staining was performed in a third cohort of 24 SSc patients to confirm transcriptomic findings at the protein level and further spatially visualize immune cell skin infiltration.

[0229] First, T cells and NK cells in the skin single-cell datasets (GSE195452, GSE138669) were analyzed based on differential gene expression of known lineage-specific genes. Among the detected transcriptionally distinct cell subtypes,Figure 1 A, 2A), In both datasets, the following three cell types were significantly expanded in SSc compared to healthy skin (q < 0.05 for all comparisons): proliferative T cells, CD8+cytotoxic T cells, and NK cells Figure 3 A, B). The presence of these T cell and NK cell subsets at the protein level in SSc-affected skin was validated in an additional cohort of 24 SSc patients Figure 3 C). In addition, the infiltration of cytotoxic CD8+T cells and CD56+NK cells in biopsies from affected skin was more pronounced compared to matched unaffected skin in 71% and 83% of SSc patients, respectively (n=24, p=0.06 and p<0.001, respectively) Figure 3 D, E). In affected SSc skin, cytotoxic T cells and NK cells were predominantly present in the perivascular area, while in unaffected skin these cells were sparsely infiltrating around blood vessels Figure 3 D).

[0230] Next, the potential functions of these enriched cell populations in SSc skin were analyzed. To this end, a gene set enrichment analysis per cluster was used with Wiki Pathways as reference dataset. The cytotoxic T cell and NK cell clusters from each sc dataset were not only associated with cytolytic pathways, but were also the only clusters from SSc skin that were uniquely enriched in gene sets associated with lung fibrosis, pro-inflammatory and pro-fibrotic behavior relative to healthy skin Figure 3 F, Figure 2 C). 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 directing pro-fibrotic pathophysiological processes.

[0231] In recent studies of chronic inflammatory conditions, CD8+T cells were found to predominantly exert cytokine-mediated functions, rather than their classical cytotoxic role, with granzyme K playing an important role (12). Therefore, CD8+T cells were analyzed specifically. In skin, the following CD8+subclusters were formed at the sc-RNAseq level: naive, proliferative, skin-resident exhausted, granzyme K (GZMK+) and granzyme B (GZMB+) positive effector cells. Only a subset of CD8 effector GZMB+cells was significantly enriched in SSc skin Figure 3 G, Figure 1 D). Flow cytometry analysis of blood also showed an increased presence of CD8+GZMB+cells in SSc compared to healthy donors (2-fold) Figure 3H).

[0232] Example 2 - Enlarged CD8 in affected skin and lungs of systemic sclerosis patients + T cells and NK cells, which are characterized in that Upregulation of CD7 costimulatory molecule

[0233] The activity of cytotoxic T cells and NK cells is tightly regulated by the reciprocal influence between activating and inhibitory cell surface receptors. In chronic infections and malignancies, T cell and NK cell cytotoxic functions have been shown to be limited by inhibitory receptors (13-15). Therefore, the expression of known T cell and NK cell activating and inhibitory receptors between immune cells of healthy and SSc skin was compared. For inhibitory receptors, LAG3 was expressed in a fraction of CD8+GZMB+T cells in SSc skin. The expression of TIGIT, CTLA4 and HAVCR2 (TIM-3) did not show any significant difference between healthy and SSc skin, while PDCD1 (PD-1) was expressed only in a small fraction of naive / central memory CD8+T cells together with FOXP3 Figure 1 E) For activating receptors, CD69 and CD7 were upregulated in CD8+GZMB+T cells, while SSc NK cells showed an increased expression of CD7, TNFRSF9 (CD137) and CD28. In clusters of proliferative SSc T cells, CD40LG was downregulated and CD28 expression was decreased in SSc skin compared to healthy skin Figure 4 A) Among these receptors, CD7 was expressed in almost all cells of these clusters and showed the strongest upregulation in patients compared to controls (q<0.001) Figure 4 A, B) In a further attempt to identify differences in cytotoxic T cell and NK cell activation between healthy and SSc individuals, an alternative unbiased approach based on the FindConservedMarker function implemented in Seurat was used (to find group, i.e. conserved features between healthy donors and SSc). This approach confirmed the enrichment of cytotoxic genes and CD7 in cytotoxic T cells and NK cells of SSc patients compared to healthy controls. No other activating or inhibitory receptor was enriched in SSc in this analysis Figure 4 C) These observations suggest that CD7 costimulation can be involved in the activation of SSc skin T cells and NK cells.

[0234] To validate these results at the protein level, CD7 and CD3 immunohistochemistry was performed in SSc skin tissues. Although not statistically significant, the total number of CD3+T cells was even higher in affected SSc skin (mean number of CD3+T cells: 15.8 in affected vs. 6.1 in unaffected) Figure 5 A, B) Notably, the number of CD3+T cells was higher in SSc skin compared to healthy skin Figure 4D) compared to, especially the perivascular areas of the affected skin Figure 7 C) increased infiltration of CD7+ cells. Moreover, in SSc skin, CD7 was found to be co-expressed with CD8 and CD56 positive cells, while no expression was observed on CD3+CD8- cells Figure 4 E).

[0235] Recently, an increased number of tissue-resident cytotoxic T cells and NK cells in SSc lungs has also been described (16). Therefore, CD7 expression in SSc lung tissue was next evaluated compared to healthy individuals. In line with the data in the skin, CD7 was selectively expressed in lung cytotoxic T cells and NK cells and its expression was significantly higher in SSc CD8+ and CD56+ cells (2-fold increase) Figure 4 F). In summary, CD7 is an activation receptor that is significantly upregulated in disease-relevant cytotoxic immune cell populations in both affected skin and lungs of patients with SSc.

[0236] Example 3 - T cell and NK cell cytotoxicity and CD7 costimulation in fibrotic processes

[0237] CD7 is selectively expressed in skin T cells and NK cells Figure 8 D). This receptor is upregulated upon TCR ligation and activated by its ligand SECTM1 (17). SECTM1 is a transmembrane protein produced by thymic epithelial cells and fibroblasts and induced in professional antigen presenting cells by IFN-gamma. CD7 activation by SECTM1 has been shown to enhance CD4+ and CD8+ T cell effector functions (18). To unravel the function of CD7 in SSc, SECTM1 expression in skin stromal cells and immune cells was analyzed. In the dataset, SECTM1 was detected, as expected, mainly in skin myeloid cells including monocytes, macrophages and dendritic cells. Moreover, SECTM1 was also expressed by cells in clusters of fibroblasts characterized by increased expression of PTGDS Figure 6 A). Interestingly, it has been previously reported that this fibroblast subtype is characterized by high expression of MHC class I genes compared to other skin fibroblast subsets, suggesting that the SECTM1-CD7 axis can be important in T cell and NK cell activation Figure 11 A). Remarkably, in fibroblasts and antigen presenting cells, T cell and NK cell CD7 expression positively correlated with IFNG, while expression of its receptor (IFNGR1) positively correlated with SECTM1 Figure 11 B, C). This suggests the presence of an IFN-gamma driven SECTM1-CD7 axis in SSc skin.

[0238] From a clinical perspective, SSc is a heterogeneous disease with multiple disease subtypes and stages. Therefore, the CD7 gene expression was next analyzed in subgroupings of SSc patients with limited (lSSc) versus diffuse (dSSc) cutaneous disease and early (≤3 years from first non-Raynaud phenomenon symptom) versus late disease. It was found that CD7 was significantly upregulated in early diffuse SSc compared to late disease Figure 6 B), and CD7 expression was further associated with patients exhibiting an increase in skin score (p=0.03) Figure 6 C). CD7 skin expression was not associated with the presence of ILD. Furthermore, CD7 expression was similar between untreated and patients receiving immunosuppressive drugs, suggesting that current treatment approaches do not seem to directly target this axis of activation Figure 6 D).

[0239] To further explore the function of CD7+ T cells, the response to activation was analyzed for cells purified from blood. In SSc blood, a larger fraction of CD8+CD7+ cells was detected compared to healthy individuals (18% of total CD3+ cells in SSc compared to 12% in HD) Figure 6 E). After short (t=4 hours) stimulation with phorbol myristate acetate (PMA) and ionomycin, CD7+CD8+ T cells from SSc patients produced significantly more granzyme B (MFI: 40,000 in SSc compared to 34,000 in HD). Furthermore, SSc CD8+CD7+ T cells were also characterized by increased co-expression of the profibrotic cytokines IL-4 and IL-13 (2.5% IL-13+ and 40% IL-4+ in CD8+ T cells) compared to healthy controls (1% IL-13+ and 30% IL-4+ in CD8+ T cells) Figure 6 F). Altogether, these data suggest that CD8+ T cells and NK cells exhibiting cytotoxic and profibrotic properties in SSc are characterized by increased CD7 expression.

[0240] To examine the role of CD7 in T cell and NK cell cytotoxicity, healthy peripheral blood mononuclear cells (PBMCs) (n=6) were co-cultured with K562 cancer cells and T cell and NK cell cytolytic activity was evaluated by measuring the release of lactate dehydrogenase (LDH) from damaged target cells. Interestingly, while blocking the CD7 receptor did not affect T cell and NK cell cell viability, it was accompanied by a significant decrease in their cytolytic capacity against K562 cells Figure 6 G). This observation suggests that CD7 costimulation is important for efficient cytotoxic responses.

[0241] Furthermore, it was observed above that SSc cutaneous cytotoxic T cells and NK cells exhibit disease-associated pathway enrichment associated with lung fibrosis and pro-inflammatory / pro-fibrotic manifestations. To explore the potential role of CD7 in the pro-fibrotic manifestations of the observed cytotoxic cutaneous cells, gene lists associated with these pathways were retrieved and integrated and pair-wise correlations with CD7 were performed Figure 7 ). Notably, CD7 gene expression in SSc-affected skin positively correlates with the expression of pro-fibrotic mediators in cytotoxic lymphocytes (CTLs), such as XCL1 (19, 20) and CCL3 (21, 22), and in NK cells, such as TGFB1 (23, 24) and OSM (25, 26) Figure 6 H). Based on these observations, it is indicated that CD7 costimulation modulates both T cell and NK cell-mediated cytotoxicity and fibrosis.

[0242] Example 4 - Prevention by targeted immunotoxin treatment in vitro of elimination of expanded and activated CD7+ T cell and NK cell subsets Fibroblast contraction

[0243] The selective upregulation of CD7 expression in cytotoxic T cells and NK cells in SSc skin can serve as a therapeutic modulatory target and also for the selective depletion of these cells. To this end, a combination of anti-CD3 / CD7 immunotoxins (CD3 / CD7-IT) aimed at targeting alloactivated T cells and NK cells in graft-versus-host disease (GvHD) was used (27). In cultured PBMCs isolated from the blood of patients, a significant killing efficacy of CD3 / CD7-IT against activated T cells and NK cells was observed (>85% of cells were depleted) Figure 8 A). The killing efficacy of the combination of CD3 and CD7 immunotoxins on T cells had an additive effect, while NK cells (CD3 CD56+CD7+) were mainly targeted by CD7-IT as expected Figure 8 B). Notably, treatment with CD3 / CD7-IT also efficiently depleted the potentially pathogenic CD8+GZMB+T cells and CD56+GZMB+NK cells Figure 8 C). IL-2 production was decreased by 9-fold after treatment Figure 9 A), supporting the selective depletion of activated T cells and NK cells by anti-CD3 / CD7-IT treatment. CD8+T cells that survived under CD3 / CD7-IT treatment exhibited a clear shift in their memory / mature status: CD8 effectors were decreased and memory and naive phenotypes were increased, thus displaying a killing specificity against effector cells Figure 9B). In addition, CD8+ T cells surviving treatment showed attenuated cell proliferation (% of CD8+ Ki-67+ cells) and decreased production of cytotoxic (GZMB) and pro-fibrotic (IL-4) molecules upon stimulation with PHA compared to their untreated counterparts Figure 9 C). Importantly, treatment with anti-CD3 / CD7-IT did not affect the number and cell viability of CD19+ B cells and CD14+ M2 monocytes / macrophages Figure 9 D, E). Next, using ex vivo whole skin cultures, and showing that both CD8+ T cells and CD56+ NK cells were significantly reduced upon treatment with anti-CD3 / CD7-IT compared to untreated conditions Figure 8 D).

[0244] As specific elimination of potentially pathogenic CD7+ T cells and NK cells was achieved, it was next evaluated whether this depletion exhibited therapeutic relevance. Fibrosis with skin tightening is a major disease feature of SSc, thus a novel 3D collagen fibroblast: immune cell co-culture hydrogel model was developed, able to study fibroblast contractility Figure 8 E). In this model, spontaneous fibroblast contraction occurred in the presence of allogeneic PBMCs, and the level of contraction was significantly greater in the presence of PHA-activated PBMCs. PHA upregulates CD3 and CD7 expression on T cells and CD7 on NK cells Figure 9 F, G), thus this model mimics the effector function of potentially pathogenic immune cell subsets on fibroblasts in vitro. Fibroblasts co-cultured with sorted CD7+ T cells and NK cells exhibited increased contractility and higher expression of IL-6, collagen type 1 and alpha smooth muscle actin (a-SMA) compared to fibroblasts co-cultured with CD7- cells Figure 12 ). Next, PHA-activated PBMCs were pre-treated with 0.33 nM a-CD3 / CD7 antibody or CD3 / CD7-IT, and showed that only upon immunotoxin treatment, fibroblast contraction was significantly reduced compared to PHA-activated PBMCs Figure 8 E). Under these conditions (24h co-culture), the percentage of necrotic CD8+ or CD56+ cells was not significantly affected Figure 9 H). However, a clear increase in apoptotic CD8+ and CD56+ cells was observed Figure 8 F). Interestingly, fibroblasts co-cultured with CD3 / CD7-IT treated PBMCs exhibited decreased gene expression of COL1A1, FN1 and ACTA2 Figure 8 G), indicating a decrease in the pro-fibrotic phenotype.

[0245] Example 5 - Administration of bispecific CD3 / CD7-IT treatment effectively clears pathogenic CD7+ cells in blood and skin in a first patient with SSc Figure 10

[0246] The pharmaceutical composition consists of a fixed dose combination comprising equal amounts (w / w) of SPV-T3a-rRTA and WT1-rRTA at a concentration of 0.2 mg protein / mL and formulated in 10 mM citrate buffer (pH 6.5) supplemented with 2.63% (w / v) arginine.HCl (125 mM), 5.04% (w / v) maltose monohydrate (140 mM) and 0.05% (w / v) Tween-20.

[0247] The pharmaceutical composition is delivered to the hospital pharmacist as a package that has to be stored at -20°C or below. Each package contains two glass vials with rubber stoppers and aluminum flip-off caps containing 2.5 mg purified SPV-T3a-rRTA and 2.5 mg purified WT1-rRTA at a concentration of 0.2 mg protein / mL in a fill volume of 25 mL / vial.

[0248] The pharmaceutical composition is administered at a dose of 4 mg / m 2 The dose is administered to the patient per body surface area (BSA).

[0249] For administration, the pharmaceutical composition is thawed at room temperature and transferred to an infusion syringe connected via an extension tube with a 0.2 pm in-line filter to a central catheter. Four intravenous doses are administered at 2-day intervals over a period of 4 hours by an automated infusion device.

[0250] A 34-year-old male patient with severe dc-SSc showing disease progression after ASCT did not respond to treatment with mycophenolate mofetil, prednisone and rituximab. The patient developed severe disabling diffuse end-stage skin fibrosis (modified Rodnan skin score of 27) with high inflammatory parameters, ESR of 49 mm / hour (< 15 mm / hour) and CRP 78 mg / L, and joint contractures. The patient was long-term bedridden with a very poor prognosis and was therefore treated with CD3 / CD7-IT as a last resort. The treatment led to depletion of circulating and skin-resident T cells and NK cells and after four weeks, the normalized level of C-reactive protein (CRP) was reduced from 131 mg / L to 27 mg / L, and then further to normal levels after 5 months. The patient’s functional status was stabilized and an improvement in quality of life was observed, but there was still a loss of functionality due to irreversible severe skin tightening and joint contractures. The patient died from disease complications 1.5 years after CD3 / CD7-IT treatment.

[0251] Biological responses to CD3 / CD7-IT treatment were measured before and after drug administration by flow cytometry in patients' blood and multiplex immunofluorescence staining in their skin. Consistent with in vitro expected results, treatment with CD3 / CD7-IT resulted in deep clearance of circulating T cells and NK cells. The levels of circulating T cells and NK cells after treatment are expressed as a percentage of the levels at the start of treatment. One week after administration, the numbers of circulating T cells and NK cells decreased by 86% and 77%, respectively. Figure 10 A). With CD4 + Compared to T cells, CD8 + T cells are more readily targeted by CD3 / CD7-IT. More specifically, the percentage of CD8+ T cells showed a 37-fold decrease, while CD4+ T cells showed a 37-fold decrease. + T cell counts showed an 8-fold decrease. Furthermore, the CD4 / CD8 ratio increased from 0.7 at baseline to 3.6 after treatment. Figure 10 B). Due to previous results showing classic cytotoxic CD8 in SSc. + The expansion and activation of T cells and NK cells were investigated, and the cytotoxic effects of CD3 / CD7-IT on effector cytotoxic T cell and NK cell populations were further explored. Effector cytotoxic T cells were characterized by CD8+. + perforin + Furthermore, NK cells are characterized as CD56. + perforin + Interestingly, this patient's blood contained CD8... + Perforin+ and CD56 + perforin + The cell population was completely exhausted (100%) Figure 10 C). Then, the effect of treatment on skin-resident T cells and NK cells was evaluated using mIF staining of skin biopsies before and after treatment. After treatment, skin biopsies showed a significant reduction in immune cell infiltration (C). Figure 10 D). More specifically, after treatment, CD3 + T cells, CD8 + T cells and CD56 + The absolute cell counts of NK cells were significantly reduced. Importantly, CD3... + FOXP3 + Regulation of T cells and CD20 + The number of B cells was not affected. Conclusion E). Although the treatment outcome is considered positive and clinically significant, greater benefits are expected if CD3 / CD7-IT is applied in the early stages of the disease, when the inflammatory component is more prominent and the fibrotic process is not yet irreversible.

[0252] ​

[0253] Here, the in-depth analysis of two independent SSc immune cell discloses single-cell RNA sequencing datasets revealed a marked expansion of proliferative T cells, cytotoxic T cells, and NK cells in SSc skin. Among the expanded immune cell clusters, cytotoxic T cells and NK cells exhibited an enrichment of gene signatures of pro-fibrotic and pro-inflammatory phenotypes. This suggests that SSc dermatosis is driven by T cells and NK cells that not only produce cytotoxic proteins such as GZMB and perforin, but are also the main producers of well-described pro-fibrotic mediators such as TGFB1, XCL1, and OSM. The expansion of NK cells and IFN-gamma-producing effector T cells in SSc skin was recently demonstrated. The focused analysis on skin cytotoxic T cells and NK cells further elucidated the heterogeneity of the effector T cell response by showing that disease-expanded effector T cells are classical cytotoxic CD8+ T cells that produce not only IFN-gamma but also GZMB and perforin. These findings contradict recent observations showing that non-classical CD8+ GZMK+ rather than GZMB+ cytotoxic populations constitute the core of the effector CD8 response in human inflamed tissues, including RA synovium and inflamed UC colon. Furthermore, Maehara et al. quantified T cell subsets infiltrating SSc skin using conventional immunohistochemistry techniques and, against prior knowledge, found that SSc skin is predominantly infiltrated by cytotoxic GZMA-producing T cells, rather than type 2 helper T cells. The data also support these findings, as little or no production of Th2 cytokines such as IL-4, IL-5, and IL-13 was observed in the analyzed single-cell datasets, in addition to the cytotoxic signature prevalent in SSc skin.

[0254] The expression of activating and inhibitory receptors in the expanded cytotoxic population was further compared and it was found that these cells in SSc skin display a strong activation profile. Interestingly, unbiased transcriptomic analysis validated by confirmation at the protein level showed a pathogenic upregulation of CD7 in T cells and NK cell activation in SSc skin. This upregulation was accompanied by an enhancement of cytotoxic and pro-fibrotic signatures. CD7 is an extracellular costimulatory molecule expressed on human mature T cells and NK cells, but its precise role in T cell and NK cell function is not known. To the best of our knowledge, CD7 expression in SSc has not been described before. Notably, CD7 was reported for the first time in SSc lesional skin +Expansion of cells. The role of CD7 in T cell biology has not been fully examined. Studies show that CD7 is significantly increased in effector cytotoxic cells in SSc compared to healthy skin and blood. Thus, CD7 costimulation appears to reflect the activation status of cytotoxic T cells, which are likely to be activated by the presentation of cutaneous autoantigens. This is in contrast to earlier studies that utilized limited flow cytometry techniques to characterize CD7 expression on blood CD8 T cells and demonstrated that CD7 was enriched on CD8+naive / central memory but expressed at lower levels on CD8+effector cells. The finding that this was different from that of Aandahl et al. can be due to the fact that their study focused on PBMC from healthy donors that lack autoimmunity. The notion of CD7 as a T cell activation receptor is further supported by data that highly proliferative T cells (producing MKI67) were all positive for CD7 gene expression. Furthermore, blocking CD7 costimulation impairs the cytotoxic capacity of T cells and NK cells and further illustrates its important role in the effector function of these cells. The concept of CD7 antigen being specifically enriched on cytotoxic T cells and NK cells, which can be relevant to the disease, supports the development of novel therapeutic approaches that specifically target cells enriched for this antigen.

[0255] To explore a new treatment that can target the abnormal CD7+T and NK cell population, the killing efficacy of a combination of anti-CD3 / CD7-IT was further tested. This drug was developed to deplete activated alloreactive T cells and NK cells to treat graft versus host disease and is currently in phase 3 clinical trials. This approach is thought to have potential benefits for treating patients with SSc because it attempts to target CD7+activated autoreactive T cells in the affected tissues. Interestingly, treating patient PBMCs in vitro with anti-CD3 / CD7-IT selectively depletes activated CD7+cytotoxic T cells and NK cells, while resting T cells and NK cells and other immune cell subsets (B cells and macrophages) are unaffected. But does this depletion of CD7+cytotoxic T cells and NK cells correlate with preventing disease manifestation?

[0256] To mimic the skin tautness manifestation observed in SSc, which is partly caused by increased (myo)fibroblast contraction, a fibroblast, immune cell 3D co-culture model was developed to study immune cell-mediated fibroblast contraction. This model was originally described by Dorst et al., 2022, and was adopted in the setting to also include PBMCs. It was shown that fibroblast contraction was prevented by activating PBMCs with anti-CD3 / CD7-IT (depleting CD7+ cells). These results suggest that fibroblast contraction seems to be driven by CD7+ T cells and NK cells. However, additional studies are needed to exclude the possibility that other immune cell subsets, such as B cells or macrophages, also contribute to this effect.

[0257] Finally, from a clinical perspective, the results from the first SSc patient treated with CD3 / CD7-IT show that this drug is effective and selectively depletes activated cytotoxic T cells and NK cells in the blood and skin in vivo. Interestingly, this treatment does not affect resting T / NK cells and other immune cell subsets, suggesting that such a drug can be superior to existing universal immune suppressive treatments for SSc, which are associated with many side effects and often leave patients vulnerable to opportunistic infections. Since CD7 expression is enhanced in allogeneic immunoresponsive T cells and NK cells, a therapeutic approach targeting CD7 has 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, the data provide evidence to support a proof-of-concept clinical trial of CD3 / CD7-IT treatment in patients with SSc. In such a clinical trial, disease outcomes, such as mRSS and lung function, should be closely monitored to evaluate the effect of a-CD3 / CD7-IT treatment on improving the main SSc disease manifestations.

[0258] Here it is shown that the SSc-affected skin contains increased numbers of proliferative T cells, cytotoxic T cells, and NK cells. These cells exhibit cytotoxic, pro-inflammatory, and profibrotic gene signatures. When focusing on their co-stimulatory and inhibitory molecule expression, these cells express the co-stimulatory molecule CD7, as well as pro-inflammatory and profibrotic genes, especially in recent-onset and severe disease. Moreover, it is shown that CD7 regulates cytolytic activity of cytotoxic T cells and NK cells, and that selective depletion of CD7+ cells prevents cytotoxic cell-induced fibroblast contraction by blocking their profibrotic phenotype. Finally, CD3 / CD7-directed depleting treatment depletes CD7+ cells and stabilizes disease manifestations in a severely affected SSc patient.

[0259] The role of T cells in mediating the pathology of SSc has been controversial. However, recent observations have highlighted the importance of the immune system, as it was shown that long-term remission of SSc disease manifestations can be achieved by treatment with ASCT (27). CD4+ T cells are thought to be the main effector cells, as genetic studies have shown that some MHC class II polymorphisms increase the risk of developing SSc (5, 8, 28). However, it was recently shown that the impact of MHC class II polymorphisms on the risk of developing SSc is less than that on the development of disease-associated autoantibodies, which precede the onset of clinical disease in a fraction of cases (29). Due to its fibrotic clinical manifestations, SSc is considered a T helper type 2 (Th2)-mediated disease (30, 31). However, epigenetic studies revealed gene transcription in cytotoxic T cells and NK cells in SSc patients with disease risk loci (5). Moreover, it was found that SSc skin is mainly infiltrated by cytotoxic T cells, close to apoptotic endothelial cells (4). A recent study linked increased infiltration of IFN-γ-producing effector T cells and NK cells in SSc skin to the fibrotic activation of a subset of fibroblasts (10). The present study confirmed these data and functional analyses showed that SSc skin disease is driven by T cells and NK cells, which produce cytotoxic proteins such as granzyme B and perforin, induce fibroblast contractility and myofibroblast phenotype, and produce well-described pro-fibrotic mediators such as TGFB1, XCL1, CCL3 and OSM. This suggests that the increase in cytotoxicity in SSc skin can be linked to the induction of the fibrotic pathology of the disease.

[0260] The present study addresses the question of how cytotoxic immune responses are regulated in SSc. Cytotoxic T cells and NK cells are central effector cells in cancer and infections. Their effector responses are tightly regulated by the expression of activating and inhibitory surface receptors (32). Here it was found that cytotoxic cells in SSc continuously express high levels of CD7. Notably, interferon-gamma, a key cytokine in the cytotoxic immune response, is the main inducer of SECTM1, a ligand of CD7 (18). This suggests that the SECTM1-CD7 interaction is part of an interferon gamma-driven feedback loop that enhances the cytotoxic response in SSc skin.

[0261] On the other hand, in chronic viral infections and cancer, the effector function of cytotoxic cells is reduced and altered due to a process called exhaustion. Exhaustion involves increased expression of inhibitory receptors such as PD-1, LAG-3, TIM-3, and CTLA-4 (33). The degree of exhaustion ranges from dysfunction to anergy or clonal deletion and depends on factors such as antigen abundance and TCR avidity. The mechanisms of autoimmunity are not fully understood. In a model of autoimmunity, activation of autoreactive CD8+ cytotoxic T cells is inhibited by LAG-3 (7). T cell exhaustion in patients with systemic autoimmune diseases has been studied and described mainly in peripheral blood samples, but not in tissues where autoantigen presentation occurs (34, 35). A subset of cytotoxic T cells in SSc skin expressed LAG3 compared to healthy skin, indicating a restricted phenotype. Only a few cytotoxic T cells expressed PD-1 as well as FOXP3, indicating that the cells were regulatory T cells. Overall, cytotoxic lymphocytes in SSc skin were characterized by an activation rather than an exhaustion profile.

[0262] This study again confirms the importance of autoimmunity in driving SSc pathology. This is clinically relevant as ASCT can cure the disease, but is a high-risk procedure and is only applicable to a very limited group of SSc patients (<10%) (27). Other broad immunosuppressive treatments currently used do not cure the disease and can only slow down fibrosis to a limited extent. Selectively targeting activated lymphocytes can represent a more selective and safer treatment for SSc. Therefore, a new combination of anti-CD3 / CD7-IT was exploited, which has been developed to deplete activated alloreactive T cells and NK cells to treat graft versus host disease (36). It was shown that treatment with a-CD3 / CD7-IT can selectively deplete activated cytotoxic T cells and NK cells in the blood and SSc-affected skin. Due to its depleting properties, anti-CD3 / CD7-IT was administered as a monotherapy and this further supports its good safety profile. In line with this view, a therapeutic approach targeting CD7 has shown clinical efficacy and safety in kidney transplant patients (36, 37). It was previously shown that anti-CD3 / CD7 immunotoxin treatment was well tolerated and improved survival in patients with acute GvHD. Similar to ASCT, a significant increase in T cell repertoire diversity involving a new polyclonal T cell population was observed, indicating the efficacy of the therapeutic approach in rebalancing the immune composition (36).

[0263] In summary, using single-cell transcriptomic data verified by multiplexed immunofluorescence, and identifying the presence of large numbers of CD7+ cytotoxic T cells and NK cells in SSc skin and blood. It was further shown that targeting depletion of these cells was accompanied by prevention of myofibroblast contraction. The results demonstrate for the first time that SSc fibrosis can be prevented by T cell-mediated immunotoxin therapy and pave the way for a novel approach to prevent tissue fibrosis. It has been found that CD7 activation regulates cytotoxicity-driven pathologies in SSc. These findings collectively indicate that co-stimulatory molecules are key regulators of cytotoxicity-driven pathologies in systemic autoimmune diseases, thereby providing a marker for selective depletion of pathogenic cells.

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Claims

1. A pharmaceutical composition for targeting activated pathogenic T cells and / or NK cells in a patient suffering from a chronic inflammatory or autoimmune disease, wherein the pharmaceutical composition comprises a first molecule specifically recognizing CD7.

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

4. The pharmaceutical composition for use according to any one of claims 1 to 3, wherein the patient exhibits symptoms of vasculopathy, fibrosis, and / or autoimmune inflammation.

5. The pharmaceutical composition for use according to any one of claims 1 to 4, wherein the pharmaceutical composition targets the 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.

6. The pharmaceutical composition for use according to any one of claims 1 to 5, wherein the activated pathogenic T cells and / or NK cells comprise activated pathogenic T cells and / or NK cells in the blood and / or inflamed tissue of the patient.

7. The pharmaceutical composition for use according to any one of claims 1 to 6, wherein the composition further comprises a second molecule specifically recognizing CD3.

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

10. The pharmaceutical composition for use according to claim 8 or 9, a. wherein the first molecule comprises a combination of complementarity determining region (CDR) sequences, wherein the CDR sequences comprise a CDR3 sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 17; and / or b. wherein the second molecule comprises a combination of complementarity determining region (CDR) sequences, wherein the CDR sequences comprise a CDR3 sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:

7.

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

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

21.

13. The pharmaceutical composition for use according to any one of claims 1 to 12, wherein the composition comprises one or more excipients, carriers, buffers, stabilizers, tonicity adjusting agents, preservatives or preservative or antioxidants.

14. The pharmaceutical composition for use according to any one of claims 1 to 12, wherein the pharmaceutical composition is administered by intravenous, cutaneous or subcutaneous injection.

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

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