Adamts12 and HMCN1 as target molecules for the treatment of chronic renal insufficiency and renal fibrosis

Abstract

The present invention relates to the interaction of the protein HMCN1 (hemicentin 1 or fibulin 6) as a substrate of the protease ADAMTS12 (A disintegrin and metalloproteinase with thrombospondin motifs 12-protein) in the pathogenesis of chronic kidney and heart diseases and in particular chronic renal insufficiency and renal fibrosis or cardiac insufficiency and cardiac fibrosis. The present invention relates to methods for identifying compounds that bind to and / or inhibit the protease ADAMTS12, and to pharmaceutical compositions for use in the treatment of kidney diseases, in particular pharmaceutical compositions comprising active ingredients that inhibit the cleavage and digestion of hemicentin 1 by the metalloprotease ADAMTS12 or the interaction of hemicentin 1 with ADAMTS12.

Description

[0001] AD AMTS 12 and HMCN1 as target molecules for the treatment of chronic renal insufficiency and renal fibrosis

[0002] Technical area of ​​the invention

[0003] The present invention relates to the interaction of the protein HMCN1 (hemicentin 1 or fibulin 6) as a substrate of the protease AD AMTS 12 (a disintegrin and metalloproteinase with thrombospondin motif 12 protein) in the pathogenesis of chronic kidney and heart diseases, and in particular chronic renal insufficiency and renal fibrosis, and heart failure and cardiac fibrosis, respectively. The present invention further relates to methods for identifying compounds that bind to and / or inhibit the protease AD AMTS 12, as well as pharmaceutical compositions for use in the treatment of kidney diseases, in particular pharmaceutical compositions comprising active substances that inhibit the cleavage and digestion of hemicentin 1 by the metalloprotease AD AMTS 12 or the interaction of hemicentin 1 with AD AMTS 12.

[0004] Background and state of the art

[0005] Fibrosis is the result of an abnormal, pathological accumulation of extracellular connective tissue (extracellular matrix, ECM), which is associated with a loss of healthy tissue and impaired organ function. Fibrosis is essentially the pathological replacement of healthy tissue with extracellular matrix. ECM is primarily synthesized by myofibroblasts, which expand following organ damage. Although the initial deposition of ECM is important for maintaining tissue integrity after organ damage, uncontrolled fibrosis leads to a loss of healthy tissue and impaired organ function. Fibrosis represents the final stage of most chronic diseases across various organ systems, and current estimates suggest that fibrosis is pathophysiologically involved in up to 45% of all deaths in industrialized countries (Henderson et al. 2020).

[0006] Worldwide, the number of patients suffering from chronic kidney disease (CKD) is increasing, and current data show that up to 10% of the population in Western countries will develop CKD during their lifetime. Due to rising average life expectancy and the increasing prevalence of hypertension and diabetes, the incidence of CKD is expected to continue to rise. With declining kidney function, morbidity and mortality increase significantly. In the end stage of CKD, dialysis and kidney transplantation are the only treatment options. Due to long waiting times for donated kidneys, most of these patients undergo dialysis. However, dialysis therapy is associated with high mortality, numerous comorbidities, and a significant reduction in quality of life.Furthermore, the high costs of dialysis represent an enormous economic burden on the healthcare system. Therefore, novel therapeutic approaches are needed.

[0007] The extent of renal fibrosis is inextricably linked to the loss of kidney function and the clinical course of CKD. Renal fibrosis is characterized by high expression, secretion, and accumulation of extracellular matrix (ECM) proteins such as collagen-1.

[0008] The extent of organ dysfunction and clinical course correlates closely with the degree of fibrosis. Across organ systems, myofibroblasts, which expand following tissue damage, are the main source of secreted extracellular matrix and are considered key cells in the pathogenesis of fibrosis (Henderson et al., 2020; Kuppe et al., 2021). Inhibition of myofibroblast expansion can prevent the progression of chronic cardiac and renal fibrosis in animal models while simultaneously preserving cardiac and renal function (Kramann, Schneider et al. 2015; Kramann, Fleig et al. 2015). Currently, however, there is no approved therapy for renal fibrosis. Due to the increasing prevalence of chronic kidney disease, the development of drugs to treat fibrosis is therefore of essential importance.

[0009] Recent studies have identified the protein AD AMTS 12 as a central protease in the development of fibrosis, mediating myofibroblast activation and expansion. However, it remains critically unclear which substrate the protease AD AMTS 12 cleaves to induce myofibroblast activation and expansion.

[0010] Thus, one of the objectives underlying the present invention is to provide methods and means for identifying active substances, compounds and compositions, as well as to provide said active substances, compounds and compositions for use in the treatment of chronic kidney and heart diseases.

[0011] Recently, the identification of a molecular target for the therapy of renal fibrosis was revealed. Based on the identification and isolation of Glil-expressing fibroblasts after induction of renal fibrosis in a mouse, and an analysis of the total RNA—i.e., the expression of all genes expressed by these activated fibroblasts—using a microarray, the researchers were able to identify the protein "A Disintegrin And Metalloproteinase with ThromboSpondin motifs 12" (AD AMTS 12) as a central interface in the initial pathogenesis of fibrosis, which induces fibroblast activation and migration following organ damage, and thus as a molecular target. AD AMTS 12 belongs to the ADAMTS metalloproteinase family (Wei et al., 2014).It has been shown that the metalloprotease ADAMTS12 is an essential mediator of fibrosis, and that the knockout (KO) of ADAMTS12 inhibits the formation of fibrosis after kidney and heart damage.

[0012] The ADAMTS (“A Disintegrin And Metalloproteinase with ThromboSpondin motifs”) proteins belong to the metzincin protease superfamily, named after a conserved methionine residue near the active site of these zinc ion-dependent metalloproteinases (Kelwick et al. 2015). At least 19 different ADAMTS proteins have been identified in mammalian genomes to date. The ADAMTS proteins are sectured, extracellular zinc matrix metalloproteinases with a uniform, ordered, modular structure. The ADAMTS proteins are initially expressed as inactive pre-proenzymes, whose structures include a signal peptide, a pro-region of variable length, a catalytic metalloproteinase domain, a disintegrin-like domain, a central thrombospondin type 1-like (TSP) sequence repeat, a cysteine-rich domain, a spacer region, and a variable number of additional C-terminal TSP repeats (Kelwick et al. 2015; Lin et al. 2009).

[0013] The ADAMTS12 gene contains a total of 24 exons that encode an extracellular protein of 1594 amino acids (Mohamedi et al. 2021). Aggrecan, COMP (cartilage oligomeric matrix protein), and alpha2M (alpha 2-macroglobulin) have been identified as substrates of ADAMTS12. A role for the ADAMTS12 protein has been described in chondrogenesis, cartilage development, and gonadal differentiation, as well as in pediatric stroke, schizophrenia, tumorigenesis, and arthritis (Lin et al. 2009; Wei et al. 2014).

[0014] However, it has remained unclear which substrate the protease ADAMTS12 cleaves to induce myofibroblast activation and expansion.

[0015] Summary of the invention

[0016] The present invention provides methods and means for identifying active substances, compounds and compositions for use in the treatment of chronic renal insufficiency, in particular for identifying highly effective active substances, compounds and compositions for use in the treatment of progressive chronic kidney disease and renal fibrosis.

[0017] The present invention also provides methods and means for identifying active substances, compounds and compositions for use in the treatment of chronic heart failure, in particular for identifying highly effective active substances, compounds and compositions for use in the treatment of progressive chronic heart disease and cardiac fibrosis.

[0018] This application discloses the identification of a novel molecular target for the therapy of fibrosis. Based on a proteomic analysis of the ECM of wild-type (WT) and ADAMTS12 knockout (KO) human renal immortalized PDGFRb+ cells, the protein HMCN1 (hemicentin 1) is identified as the most highly enriched protein in the ECM of ADAMTS12-K0 cells. Using Western blot analysis of WT and ADAMTS12-K0 mice, the inventors were able to confirm that HMCN1 peptides (cut, truncated HMCN1) accumulate in the kidneys of WT mice following kidney injury via unilateral ureteral obstruction, while the same bands do not accumulate in ADAMTS12 knockout mice. These data confirm that HMCN1 is cleaved by ADAMTS12 in vivo and in vitro. Further studies have confirmed the cleavage of HMCN1 by AD AMTS 12 in vitro.Furthermore, it was shown that inhibition of HMCN1 expression inhibits migration of immortalized renal PDGFRb+ ADAMTS12-expressing cells, while inhibition of HMCN1 in immortalized renal PDGFRb+ ADAMTS12-K0 cells leads to a slightly increased migration.

[0019] HMCN1 (hemicentin-1, also called fibulin-6, FIBL-6, FIBL6, or FBLN6) is a very large extracellular matrix protein (5,635 amino acids; > 600 kDa) and a member of the fibulin family. HMCN1 is a component of the basement membrane, plays a role in cell adhesion, and is produced primarily by mesenchymal cells, such as fibroblasts. HMCN1 is expressed in the kidney, among other organs.

[0020] The inventors were the first to demonstrate the importance of HMCN1 and its cleavage in the development of fibrosis, and to prove the cleavage of fibulins, and in particular of HMCN1, by ADAMTS12. This identified HMCN1 as a previously unknown substrate of the protease ADAMTS12, the cleavage of which induces the activation and migration of fibroblasts and is thus significantly involved in the development of renal fibrosis.

[0021] In summary, the present application identifies HMCN1 and, in particular, the interaction with and cleavage of HMCN1 by AD AMTS 12 as a novel approach for the development of therapeutics for the treatment of patients with chronic renal and / or cardiac failure, and especially for patients with renal and / or cardiac fibrosis.

[0022] In view of the prior art, it was therefore an object of the present invention to provide a method for inhibiting or reducing the cleavage of HMCN1 by a protease capable of cleaving HMCN1, wherein the method comprises at least one step in which (i) the expression of said protease or the proteolytic activity of said protease is inhibited or reduced, and / or (ii) the degradation of said protease is promoted.

[0023] A further object of the present invention was in particular to provide a method for inhibiting or reducing the cleavage of HMCN1 by the protease AD AMTS 12, wherein the method comprises at least one step selected from the group consisting of

[0024] (i) Inhibition or reduction of 7 / M / M7 S72 gene expression in a cell,

[0025] (ii) Inhibition or reduction of the proteolytic activity of protease AD AMTS 12, and / or (iii) promotion of the degradation of protease AD AMTS 12.

[0026] A further object of the present invention was to provide a method for identifying an active substance that binds to the protease AD AMTS 12, or a proteolytic fragment thereof, and / or inhibits or reduces the proteolytic activity of the protease AD AMTS 12, or a proteolytic fragment thereof, wherein the method comprises at least one step in which the detection of cleaved or uncleaved HMCN1 serves as an indicator of the proteolytic activity of the protease ADAMTS12.

[0027] A further object of the present invention was to provide active substances or antibodies, antigen-binding fragments or antigen-binding derivatives thereof, or antibody-like proteins which inhibit or reduce the cleavage of HMCN1 by the protease AD AMTS 12, for use in the treatment of chronic kidney disease, in particular progressive chronic renal insufficiency and / or renal fibrosis.

[0028] A further object of the present invention was to provide active substances or antibodies, antigen-binding fragments or antigen-binding derivatives thereof, or antibody-like proteins that inhibit or reduce the cleavage of HMCN1 by the protease AD AMTS 12, for use in the treatment of chronic heart disease, in particular progressive chronic heart failure and / or cardiac fibrosis. A further object of the present invention was to provide pharmaceutical compositions comprising such active substances or antibodies and one or more pharmaceutically acceptable excipients for use in the treatment of chronic kidney disease, based on the findings described above.

[0029] Another object of the present invention was to provide pharmaceutical compositions comprising such active substances or antibodies and one or more pharmaceutically acceptable excipients for use in the treatment of chronic heart disease, based on the findings described above.

[0030] Further tasks included the provision of methods for the qualitative and / or quantitative detection of HMCN1 and / or HMCN1 cleavage products of the protease AD AMTS 12, or a proteolytic fragment thereof, in body fluids, cells and / or the extracellular matrix (ECM), as well as the use of HMCN1 to detect the proteolytic activity of the protease AD AMTS 12, or a proteolytic fragment thereof.

[0031] The described and further technical problems are solved by the devices or methods according to the independent claims of the present invention. The dependent claims describe preferred embodiments. Value ranges limited by numerical values ​​shall always include the aforementioned limit values.

[0032] The invention and general advantageous embodiments are explained in more detail below.

[0033] Description of the drawings

[0034] Fig. 1: Differentially expressed proteins in mass spectrometry (MS) measurements of the extracellular matrix (ECM) of WT- and ADAMTS12-KO-immortalized human renal PDGFRB + Cells. (Log2FC: Log2 Fold Change) Fig. 2: GAPDH and HMCN1 Western blot of kidneys from WT and Adams12 z Mice after pseudo- or unilateral ureteral obstruction (UUO).

[0035] Fig. 3: Quantification of the 56-kDa HMCN1 band in WT and Adamis 12 Mice with normalization by GAPDH.

[0036] Fig. 4: Digestive assay of HMCN1 or control immunoprecipitation (IP) lysates with ADAMTS12 or vehicle.

[0037] Fig. 5: RT-qPCR for HMCN1 of human renal immortalized PDGFRB + Cells with ADAMTS12-K0 or overexpression of ADAMTS12 after treatment with HMCN1 siRNA or non-targeting (NT) siRNA.

[0038] Fig. 6: Quantification of the average migration velocity of AD AMTS 12 KO and active ADAMTS12-expressing PDGFRB+ cells after treatment with HMCN1 siRNA or non-targeting (NT) siRNA.

[0039] Detailed description of the invention

[0040] Before the invention is described in detail, it should be noted that this invention is not limited to specific components of the described compounds or described steps of the methods, as these methods and compounds may vary. It should also be noted that the terminology used here is only employed for the purpose of describing specific embodiments and is not intentionally limited.

[0041] It should be noted that in the description and in the appended claims, simple forms such as "a" or "the" include a singular and / or plural object, unless the context clearly specifies otherwise. If a parameter range has been specified, the limiting numerical values ​​are considered limits to the disclosed or claimed numerical range. Furthermore, it should be noted that the embodiments disclosed herein are not to be understood as separate embodiments that do not relate to one another. Features discussed in connection with one embodiment are also considered disclosed in connection with other embodiments shown herein.If, in a given case, a particular feature is disclosed not with one embodiment but with another, the person skilled in the art will understand that this does not necessarily mean that this feature is not intended to be disclosed with the other embodiment. The person skilled in the art will understand that it is in accordance with the principle of this application to disclose the feature in question for the other embodiment as well, but that this has not been done for reasons of clarity and to keep the specification manageable.

[0042] Furthermore, the content of the prior art documents mentioned herein is incorporated by reference. This applies in particular to prior art documents that disclose standard or routine procedures. In this case, the primary purpose of incorporation by reference is to ensure sufficient disclosure and to avoid lengthy repetitions.

[0043] According to a first aspect, the present invention relates to a method for inhibiting or reducing the cleavage of HMCN1 by a protease capable of cleaving HMCN1, wherein the method comprises at least one step in which (i) the expression of said protease or the proteolytic activity of said protease is inhibited or reduced, and / or (ii) the degradation of said protease is promoted.

[0044] According to a second aspect, the present invention relates to a method for inhibiting or reducing the cleavage of HMCN1 by the protease AD AMTS 12, wherein the method comprises at least one step selected from the group consisting of

[0045] (i) Inhibition or reduction of / 4 / MA77 S72 gene expression in a cell,

[0046] (ii) Inhibition or reduction of the proteolytic activity of the protease AD AMTS 12, and / or

[0047] (iii) Promotion of the degradation of the protease ADAMTS12.

[0048] In a preferred embodiment of the present invention, the inhibition or reduction of rf / MA77 S72 gene expression in a cell may, for example, comprise an ADAMTS12 gene “knock-down”, a “knock-out”, a conditional “gene knock-out”, a gene alteration or mutation, RNA interference, siRNA and / or antisense RNA.

[0049] Inhibition or reduction of the proteolytic activity of the protease ADAMTS12 may involve the use of an agent that binds to the ADAMTS12 protein and / or inhibits or reduces its proteolytic activity.

[0050] Preferably, said cell is a kidney cell or a cardiac cell, particularly preferably a renal fibroblast cell or a cardiac fibroblast cell, a renal myofibroblast cell or a cardiac myofibroblast cell, or a renal pericyte or cardiac pericyte; most preferably a renal fibroblast cell or a cardiac fibroblast cell.

[0051] The ADAMTS12 protein can be a mammalian, non-primate, primate and especially a human ADAMTS12 protein or a fragment thereof, preferably a proteolytic fragment thereof.

[0052] According to a third aspect, the present invention relates to a method for identifying an active substance that binds to the protease AD AMTS 12, or a proteolytic fragment thereof, and / or inhibits or reduces the proteolytic activity of the protease AD AMTS 12, or a proteolytic fragment thereof, wherein the method comprises at least one step in which the detection of cleaved or uncleaved HMCN1 serves as an indicator of the proteolytic activity of the protease AD AMTS 12.

[0053] In a preferred embodiment of the present invention, the above-mentioned method for identifying an active ingredient comprises at least the following steps:

[0054] (i) the provision of the protease AD AMTS 12, or a proteolytic fragment thereof,

[0055] (ii) the addition of at least one active substance to be investigated for binding to the protease ADAMTS12 or a fragment thereof, or for inhibiting or reducing the proteolytic activity of the protease ADAMTS12, or a proteolytic fragment thereof, and (iii) the detection of cleaved or uncleaved HMCN1 as an indicator of the proteolytic activity of the protease ADAMTS12.

[0056] The aforementioned procedure for identifying an active ingredient may include at least the following additional step:

[0057] (iv) Identification of at least one active substance that binds to the protease AD AMTS 12, or a proteolytic fragment thereof, and / or that inhibits or reduces the proteolytic activity of the protease ADAMTS12, or a proteolytic fragment thereof.

[0058] Preferably, the active substance to be screened, added and / or identified according to the present invention is an ADAMTS12 inhibitor or antagonist, i.e., an agent that inhibits or reduces the proteolytic activity of the protease AD AMTS 12, or of a proteolytic fragment thereof.

[0059] The active ingredient according to the present invention can be selected from the group consisting of a small molecule (“small molecule” or “smol”), an organic or inorganic compound, a natural or synthetic peptide or peptide derivative, a natural or synthetic oligopeptide or oligopeptide derivative, and a biologic or biological agent. The active ingredient can be a member of a library of compounds.

[0060] In the context of the present invention, the terms “low molecular weight compound”, “small molecule” (“small molecule” or “smol”), or “chemical drug” refer to an organic compound with a low molecular weight (<10,000 Daltons, in particular <1,000 Daltons), often with a size on the order of 1 nm. Many drugs are small molecules. Such small molecules can regulate a biological process. Small molecules may be able to inhibit a specific function of a protein. In the field of pharmacology, the term “small molecule” refers in particular to molecules that bind to specific biological macromolecules and act as effectors by altering the activity or function of a target. For example, acetylsalicylic acid (ASA) is considered a low molecular weight compound that measures 180 Daltons and consists of 21 atoms.Such low-molecular-weight compounds often have only a limited ability to trigger an immune response and remain relatively stable over time.

[0061] The low molecular weight compound according to the present invention can, in addition to other chemical backbones, substituents, groups or residues, for example comprise alkyl, alkenyl, alkynyl, alkoxy, aryl, alkylene, arylene, amino, halogen, carboxylate derivative, cycloalkyl, carbonyl derivative, heterocycloalkyl, heteroaryl, heteroarylene, sulfonate, sulfate, phosphonate, phosphate, phosphine, phosphine oxide groups.

[0062] The “biologic”, “biological drug”, “biological therapeutic”, “biopharmaceutical” or the “biological active ingredient” 4According to the present invention, preferably an antibody, or an antigen-binding fragment thereof, or an antigen-binding derivative thereof, or an antibody-like molecule or protein, or an aptamer, or a nucleic acid.

[0063] In a preferred embodiment of the method for identifying an active ingredient that binds to the protease AD AMTS 12 or a proteolytic fragment thereof, and / or inhibits or reduces the activity of the protease AD AMTS 12, or a fragment thereof, the active ingredient is a member of a “library” of compounds.

[0064] The “library” (mixture) of compounds can include, for example, low molecular weight compounds, natural or synthetic peptides or peptide derivatives, or biologics or biological agents or biological compounds.

[0065] In the context of the present invention, the term “(combinatorial) compound library” or “library of compounds” refers to collections of chemical compounds, small molecules, natural or synthetic peptides or peptide derivatives, or natural or synthetic oligopeptides or oligopeptide derivatives, or macromolecules such as polypeptides or proteins or other biologics, each containing a large number of related chemical, peptide, or biological species of molecules that can be used together in specific screening assays or identification steps. Methods for the preparation of molecular libraries of small-molecule chemical compounds (“compound libraries”) and for high-throughput screening of the compounds for interaction with the target molecule are described in the prior art (for example, Volochnyuk et al. 2019).These methods also include so-called “focus libraries,” highly annotated and pre-selected chemical molecule libraries (Wassermann et al. 2014), DNA-encoded libraries of chemical compounds (Martin et al. 2020), and chemoinformatics-based virtual molecule libraries (Saldivar-Gonzalez et al. 2020). The use of so-called “phage display” technologies for the identification of suitable small-molecule drugs was described, for example, by Takakusagi et al., 2020. Numerous other peptide and antibody display technologies, such as bacterial display, yeast surface display, mammalian surface display, and ribosome display, are described in Valldorf et al., 2022.

[0066] Methods for the production of molecular libraries, their immobilization and their high-throughput screening of biological molecules, for example peptides, peptide derivatives, proteins, antibodies, antigen-binding antibody fragments, antigen-binding antibody derivatives, or antibody-like molecules, are also described in the prior art (for peptide libraries, for example, in Bozovicar and Bratkovic 2019; Schwaar et al. 2019; for antibody libraries, in Lin and Lerner 2021).

[0067] In a preferred embodiment of the method for identifying an active substance that binds to the protease AD AMTS 12 or a proteolytic fragment thereof, and / or inhibits or reduces the activity of the protease AD AMTS 12 or a proteolytic fragment thereof, the biologic is an antibody, an antigen-binding fragment thereof, an antigen-binding derivative thereof, an antibody-like molecule or protein, an aptamer, or a nucleic acid.

[0068] In a preferred embodiment of the method for identifying an active ingredient that binds to the protease AD AMTS 12 or a proteolytic fragment thereof, and / or inhibits or reduces the activity of the protease AD AMTS 12 or a proteolytic fragment thereof, the ADAMTS12 protein is bound to a solid phase or is present in solution. According to a further aspect, the present invention relates to the use of a nucleic acid encoding the ADAMTS12 protein or a fragment thereof, or the use of the ADAMTS12 protein or a fragment thereof, in a method for identifying an active ingredient as described above that binds to the protease AD AMTS 12 or a proteolytic fragment thereof, and / or inhibits or reduces the proteolytic activity of the protease AD AMTS 12 or a proteolytic fragment thereof, wherein the method comprises at least one step in which the detection of cleaved orUncleaved HMCN1 serves as an indicator for the proteolytic activity of the protease ADAMTS12.

[0069] To express ADAMTS12 metalloproteinase or a fragment thereof, a nucleic acid encoding ADAMTS12 metalloproteinase or a fragment thereof is cloned into a suitable expression vector, e.g., a suitable expression plasmid, as described (Green and Sambrook 2012). The recombinant expression plasmid is transfected into a cell suitable for the expression of ADAMTS12 or a fragment thereof, the cell is propagated in cell culture with a suitable cell culture medium, and the expressed protein is purified from the cells and / or the cell culture medium.

[0070] As used herein, the term "transfection" refers to any process of intentionally introducing a foreign nucleic acid into a eukaryotic cell. Various types of nucleic acids can be used for transfection into eukaryotic cells, in particular deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and small, non-coding RNAs such as siRNA, shRNA, and miRNA.

[0071] Regarding transfection, a distinction is made between stable and transient transfection. In stable transfection, long-term expression of the transgene is achieved through the integration of the nucleic acid introduced into the cell into the cellular genome, whereas in transient transfection, where the expression of the transgene is only temporary, no integration of the nucleic acid into the cellular genome is required.

[0072] The selection of the optimal transfection method depends on various factors, particularly the type and origin of the target or production cell, as well as the type of nucleic acid being introduced. Physical, chemical, and viral vector-based transfection methods can be used to introduce foreign (modified homologous and / or heterologous) nucleic acid encoding the desired transgene into eukaryotic cells. Physical transfection methods include, for example, electroporation, sonoporation, magnetofection, microinjection, and biolistic techniques. Chemical transfection methods include the calcium phosphate method, the use of dendrimers, cationic polymers such as diethylaminoethyl dextran (DEAE-dextran), nanoparticles, non-liposomal nanoparticles, and liposomal transfection. Transfection using viral vectors (so-called...)“Transduction”) primarily involves the use of genetically modified retroviruses and lentiviruses, adenoviruses, and adeno-associated viruses (AAV) (Fus-Kujawa et al. 2021).

[0073] According to a fourth aspect, the present invention relates to an active ingredient obtained by the above-described methods for identifying an active ingredient that binds to the protease AD AMTS 12, or a proteolytic fragment thereof, and / or inhibits or reduces the proteolytic activity of the protease AD AMTS 12, or a proteolytic fragment thereof.

[0074] Furthermore, the present invention relates to an active substance obtained by the above-described methods for identifying an active substance that binds to the protease AD AMTS 12 or a proteolytic fragment thereof, and / or inhibits or reduces the activity of the protease AD AMTS 12, or a proteolytic fragment thereof, and / or promotes the degradation of the ADAMTS12 protein.

[0075] Furthermore, the present invention relates to an active substance obtained by the above-described methods for identifying an active substance that inhibits or reduces the expression of the ADAMTS 12 gene in a kidney cell or a cardiac cell, preferably wherein the kidney cell is a renal fibroblast cell and / or the cardiac cell is a cardiac fibroblast cell.

[0076] In a preferred embodiment, the active ingredient is a low molecular weight compound (smol), a peptide or peptide derivative, or a biologic, preferably wherein the biologic is an antibody or an antigen-binding fragment thereof, or an antigen-binding derivative thereof, or an antibody-like protein, or an aptamer, or a nucleic acid. In a preferred embodiment, the active ingredient binds specifically with high or particularly high affinity and / or avidity to the protease AD AMTS 12 or a proteolytic fragment thereof. In a preferred embodiment, when bound to AD AMTS 12, the active ingredient inhibits or reduces the ADAMTS 12 activity.

[0077] The term “specifically binding”, as used here, means that the active substance has a dissociation constant KD with respect to its binding to the ADAMTS 12 protein molecule or an epitope thereof of at most about 100 pM. In one embodiment, the KD is approximately 100 pM or lower, approximately 50 pM or lower, approximately 30 pM or lower, approximately 20 pM or lower, approximately 10 pM or lower, approximately 5 pM or lower, approximately 1 pM or lower, approximately 900 nM or lower, approximately 800 nM or lower, approximately 700 nM or lower, approximately 600 nM or lower, approximately 500 nM or lower, approximately 400 nM or lower, approximately 300 nM or lower, approximately 200 nM or lower, approximately 100 nM or lower, approximately 90 nM or lower, approximately 80 nM or lower, approximately 70 nM or lower, approximately 60 nM or lower, approximately 50 nM or lower, approximately 40 nM or lower, approximately 30 nM or lower, approximately 20 nM or lower, or about 10 nm or lower, about 1 nm or lower,approximately 900 pM or lower, approximately 800 pM or lower, approximately 700 pM or lower, approximately 600 pM or lower, approximately 500 pM or lower, approximately 400 pM or lower, approximately 300 pM or lower, approximately 200 pM or lower, approximately 100 pM or lower, approximately 90 pM or lower, approximately 80 pM or lower, approximately 70 pM or lower, approximately 60 pM or lower, approximately 50 pM or lower, approximately 40 pM or lower, approximately 30 pM or lower, approximately 20 pM or lower, approximately 10 pM or lower, or approximately 1 pM or lower.

[0078] According to a fifth aspect, the present invention relates to an antibody, or an antigen-binding fragment or antigen-binding derivative thereof, or an antibody-like protein, wherein the antibody, or the antigen-binding fragment or derivative thereof, or the antibody-like protein, inhibits or reduces the proteolytic cleavage of HMCN1 by the protease ADAMTS 12.

[0079] In a preferred embodiment, the present invention relates to an antibody, or an antigen-binding fragment or antigen-binding derivative thereof, or an antibody-like protein, which binds specifically to the protease ADAMTS 12. In a preferred embodiment, the present invention relates to said antibody, or antigen-binding fragment or antigen-binding derivative thereof, or antibody-like protein, wherein the antibody, or the antigen-binding fragment or derivative thereof, or the antibody-like protein inhibits or reduces the proteolytic activity of the protease ADAMTS 12, or of a proteolytic fragment thereof, i.e., acts as an inhibitor or antagonist of ADAMTS 12.

[0080] As used herein, the term "antibody" refers to a protein composed of one or more polypeptide chains encoded by immunoglobulin genes or fragments of immunoglobulin genes, or cDNAs derived from them. These immunoglobulin genes include the light chain genes kappa, lambda and the heavy chain genes alpha, delta, epsilon, gamma and mu of the constant region, as well as each of the many different genes of the variable region.

[0081] The basic structural unit of immunoglobulin (antibody) is typically a tetramer, consisting of two identical pairs of polypeptide chains: the light chains (L, with a molecular weight of approximately 25 kDa) and the heavy chains (H, with a molecular weight of approximately 50–70 kDa). Each heavy chain comprises a variable region (abbreviated VH or VH) and a constant region (abbreviated CH or CH). The constant region consists of three domains: CH1, CH2, and CH3. Each light chain contains a variable region (abbreviated VL or VL) and a constant region (abbreviated CL or CL).The VH and VL regions can be further subdivided into hypervariable regions, also known as complementarity-determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). Each VH and VL region consists of three CDRs and four FRs, arranged from the amino terminus to the carboxy terminus in the order FR1, CDRI, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains form a binding domain that interacts with an antigen. The CDRs are most important for the binding of the antibody or its antigen-binding portion. The FRs can be replaced by other sequences, provided the three-dimensional structure required for antigen binding is maintained.

[0082] The term "antigen-binding part" of a (monoclonal) antibody refers to one or more fragments of an antibody that retain the ability to bind specifically to the antigen in its native form. Examples of antigen-binding parts of an antibody include a Fab fragment, a monovalent fragment consisting of the VL, VH, CL, and Chl domains; an F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; an Fd fragment consisting of the VH and Chl domains; an Fv fragment consisting of the VL and VH domains of a single arm of an antibody; and a dAb fragment consisting of a VH domain and an isolated complementarity-determining region (CDR).

[0083] The antibody, antibody fragment, or antibody derivative thereof according to the present invention can be a monoclonal antibody. The antibody can be of the IgA, IgD, IgE, IgG, or IgM isotype.

[0084] As used herein, the term “monoclonal antibody (mAb)” refers to an antibody composition with a homogeneous antibody population, i.e., a homogeneous population consisting of a whole immunoglobulin or a fragment or derivative thereof. Particularly preferred is such an antibody selected from the group consisting of IgG, IgD, IgE, IgA and / or IgM, or a fragment or derivative thereof.

[0085] As used herein, the term ‘fragment’ refers to fragments of such antibody that retain their target binding capacities, e.g., a CDR (complementarity-determining region), a hypervariable region, a variable domain (Fv), a heavy IgG chain (consisting of VH, CHI, hinge, CH2 and CH3 regions), a light IgG chain (consisting of VL and CL regions) and / or a Fab and / or F(ab)2.

[0086] As used here, the term "derivative" refers to protein constructs that differ structurally from the conventional antibody concept but nevertheless exhibit some structural similarity to it, e.g., scFv, Fab, and / or F(ab)2, as well as to bi-, tri-, or higher-specific antibody constructs. All of these elements are explained below.

[0087] Other antibody derivatives known to those skilled in the art include diabodies, camelid antibodies, domain antibodies, bivalent two-chain homodimers consisting of scFvs, IgAs (two IgG structures linked by a J chain and a secretory component), shark antibodies, antibodies consisting of New World primate scaffold plus non-New World primate CDR, dimerized constructs comprising CH3+VL+VH, other scaffold protein formats comprising CDRs, and antibody conjugates.

[0088] As used here, the term "antibody-like protein" refers to a protein that has been modified (e.g., by mutagenesis of Ig loops) to bind specifically to a target molecule. Typically, such an antibody-like protein comprises at least one variable peptide loop bound at both ends to a protein scaffold. This double structural constraint increases the binding affinity of the antibody-like protein to a level comparable to that of an antibody. The variable peptide loop is typically 10 to 20 amino acids long. The scaffold protein can be any protein with good solubility properties. Preferably, the scaffold protein is a small globular protein. Antibody-like proteins include, without limitation, affibodies, anticalins, and engineered ankyrin and affilin proteins. Antibody-like proteins can be derived from large libraries of mutants, e.g.,Antibody-like binding proteins can be obtained by panning from large phage display libraries and isolated analogously to regular antibodies. They can also be obtained by combinatorial mutagenesis of surface-exposed residues in globular proteins. Antibody-like proteins have been described, for example, in Binz et al. (2005) and Hosse et al. (2006).

[0089] As used here, the term "Fab" refers to an IgG fragment comprising the antigen-binding region, the fragment being composed of a constant and a variable domain, respectively, of the heavy and light chains of the antibody. As used here, the term "F(ab)2" refers to an IgG fragment consisting of two Fab fragments linked by disulfide bonds.

[0090] The term "scFv" used here refers to a variable single-chain fragment, which is a fusion of the variable regions of the heavy and light chains of immunoglobulins linked by a short linker, usually comprising serine (S) and / or glycine (G) residues. This chimeric molecule retains the specificity of the original immunoglobulin despite the removal of the constant regions and the introduction of a linker peptide.

[0091] Modified antibody formats include, for example, bi- or tri-specific antibody constructs, antibody-based fusion proteins, immunoconjugates, and similar products.

[0092] IgG, scFv, Fab and / or F(ab)2 are antibody formats well known to those skilled in the art. Detailed explanations and techniques can be found in relevant textbooks.

[0093] According to preferred embodiments of the present invention, the antibody or the antigen-binding fragment thereof or the antigen-binding derivative thereof is a murine, chimeric, humanized or human antibody or an antigen-binding fragment or an antigen-binding derivative thereof.

[0094] Monoclonal antibodies (mAbs) derived from mice can cause undesirable immunological side effects because they contain a protein from another species that can induce an immune response. To overcome this problem, methods for humanizing and maturing antibodies have been developed to produce antibody molecules with minimal immunogenicity for use in humans, while ideally preserving the specificity and affinity of the non-human parental antibody. These methods involve, for example, replacing the scaffold regions of a mouse mAb with corresponding human scaffold regions (so-called CDR grafting). W0200907861 discloses the generation of humanized forms of mouse antibodies by linking the CDR regions of non-human antibodies to human constant regions using recombinant DNA technology. US6548640 describes CDR transplantation techniques, and US5859205 describes the production of humanized antibodies.As used herein, the term “humanized antibody” refers to an antibody, fragment or derivative thereof in which at least some of the constant regions and / or scaffold regions and optionally some of the CDR regions of the antibody are derived from or adapted to human immunoglobulin sequences.

[0095] According to a sixth aspect, the present invention relates to an active substance as described above or an antibody, an antigen-binding fragment or an antigen-binding derivative thereof, or an antibody-like protein as described above, for use in the treatment of chronic kidney disease.

[0096] Chronic kidney disease is preferably a progressive chronic renal insufficiency and / or renal fibrosis.

[0097] The present invention also relates to an active substance as described above or an antibody, an antigen-binding fragment or an antigen-binding derivative thereof, or an antibody-like protein as described above, for use in the treatment of chronic heart disease.

[0098] The heart condition is preferably heart failure, a heart attack and / or cardiac fibrosis.

[0099] According to a seventh aspect, the present invention relates to a pharmaceutical composition comprising the active ingredient as described above, or the antibody, the antigen-binding fragment or antigen-binding derivative thereof, or an antibody-like protein as described above, and one or more pharmaceutically acceptable excipients, for use in the treatment of chronic kidney disease, preferably wherein the chronic kidney disease is a progressive chronic kidney disease, renal insufficiency and / or renal fibrosis.

[0100] The same applies to a pharmaceutical composition comprising the active substance as described above, or the antibody, the antigen-binding fragment or antigen-binding derivative thereof, or an antibody-like protein as described above, and one or more pharmaceutically acceptable excipients, for use in the treatment of a chronic heart condition, preferably wherein the heart condition is heart failure and / or cardiac fibrosis.

[0101] In a preferred embodiment of the present invention, the said pharmaceutically acceptable excipient(s) is / are selected from the group consisting of pharmaceutically acceptable buffers, surfactants, diluents, carriers, excipients, fillers, binders, lubricants, lubricating agents, disinfectants, adsorbents and / or preservatives.

[0102] The pharmaceutical composition in question can be administered in the form of powder, tablets, pills, capsules, or beads. In aqueous form, the pharmaceutical formulation may be ready for administration, whereas in lyophilized form, the formulation must be converted to a liquid form before administration, e.g., by adding water for injection, which may or may not contain a preservative such as, but not limited to, benzyl alcohol, antioxidants such as vitamin A, vitamin E, vitamin C, retinyl palmitate, and selenium, the amino acids cysteine ​​and methionine, citric acid and sodium citrate, or synthetic preservatives such as the parabens methylparaben and propylparaben.

[0103] The pharmaceutical formulation may further contain one or more stabilizers, which may be, for example, an amino acid, a sugar polyol, a disaccharide, and / or a polysaccharide. The pharmaceutical formulation may also contain one or more surfactants, one or more isotonic agents, one or more metal ion chelators, and / or one or more preservatives.

[0104] The pharmaceutical formulation described herein may be suitable for oral, parenteral, intravenous, intramuscular, or subcutaneous administration. Alternatively, the active ingredient or antibody according to the present invention may be provided in a depot formulation that allows for the delayed release of the active ingredient over a specific period. Furthermore, a primary packaging, such as a pre-filled syringe or pen, a vial, or an infusion bag, is provided, comprising said pharmaceutical formulation according to this aspect of the invention.

[0105] The pre-filled syringe or pen can contain the formulation either in freeze-dried form (which then needs to be reconstituted with water for injection, for example, before administration) or in aqueous form. The syringe or pen is often a single-use, disposable item and can have a volume between 0.1 and 20 ml. However, the syringe or pen can also be a reusable syringe or a multi-dose pen.

[0106] According to an eighth aspect, the present invention relates to a method for the qualitative and / or quantitative detection of HMCN1 and / or HMCN1 cleavage products of the protease AD AMTS 12, or a proteolytic fragment thereof, in body fluids, cells and / or the extracellular matrix (ECM).

[0107] In a preferred embodiment of the present invention, the aforementioned method comprises at least one method from the group consisting of gel electrophoresis, western blot, immunoprecipitation, ELISA, and mass spectrometric detection.

[0108] According to a ninth aspect, the present invention relates to the use of HMCN1 and / or HMCN1 cleavage products for the detection of the proteolytic activity of the protease ADAMTS12, or a proteolytic fragment thereof.

[0109] In a preferred embodiment of the present invention, the aforementioned use requires that HMCN1 and the protease AD AMTS 12, or a proteolytic fragment thereof, be brought into contact with a drug or antibody candidate to be tested, and

[0110] (i) the occurrence of specific HMCN1 cleavage products through the cleavage of HMCN1 demonstrates the proteolytic activity of the protease ADAMTS12, or

[0111] (ii) the absence of specific cleavage products from the cleavage of HMCN1 demonstrates that the protease AD AMTS 12, or a fragment thereof, is not or less proteolytically active, and preferably (iii), in the case of (ii), an active substance or antibody can be identified that inhibits or reduces the proteolytic activity of the protease ADAMTS 12, or a proteolytic fragment thereof.

[0112] The present invention further relates to the use of an active ingredient obtained by the above-described methods for identifying an active ingredient that binds to the protease AD AMTS 12, or a proteolytic fragment thereof, and / or inhibits or reduces the proteolytic activity of the protease AD AMTS 12, or a proteolytic fragment thereof, for the manufacture of a medicament for the treatment of chronic kidney disease and / or heart disease, wherein the chronic kidney disease is preferably progressive chronic renal insufficiency and / or renal fibrosis, and wherein the heart disease is preferably heart failure and / or cardiac fibrosis. Preferably, when bound to AD AMTS 12, the active ingredient inhibits the ADAMTS 12 activity.

[0113] Furthermore, the present invention relates to a method for treating or preventing chronic kidney disease and / or heart disease, wherein the method comprises administering an active substance obtained by the above-described methods for identifying an active substance that binds to the protease ADAMTS 12 and / or inhibits or reduces the proteolytic activity of the protease ADAMTS 12, or a proteolytic fragment thereof, in a therapeutically effective dose or amount to a human or animal subject.

[0114] As used herein, the term “effective dose” or “effective amount” means a dose or amount of the active substance necessary, with respect to dosages and administration times, to achieve the desired therapeutic effect in a patient. Effective amounts may vary depending on factors such as the patient’s condition, age, sex and / or weight, the pharmaceutical formulation, the subtype of the disease being treated, and the like, but can nevertheless be routinely determined by a person skilled in the art.

[0115] According to a tenth aspect, the present invention relates to the use of HMCN1 and / or HMCN1 cleavage products as biomarkers of chronic kidney disease, preferably progressive chronic renal insufficiency and / or renal fibrosis.

[0116] The present invention also relates to the use of HMCN1 and / or HMCN1 cleavage products as biomarkers of chronic heart disease, preferably progressive chronic heart failure and / or cardiac fibrosis.

[0117] A preferred embodiment of the present invention relates to the use of HMCN1 and / or HMCN1 cleavage products as described above, wherein at least one HMCN1 cleavage product has a molecular weight of 56 kDa.

[0118] Preferred embodiments of the present invention also relate to the methods described above, wherein at least one HMCN1 cleavage product has a molecular weight of 56 kDa.

[0119] According to a further aspect, the present invention relates to a method for producing an active substance obtained by the above-described methods for identifying an active substance that binds to the ADAMTS12 protein or a fragment thereof and / or inhibits or reduces the activity of the protease AD AMTS 12, or a proteolytic fragment thereof, wherein the method comprises at least one step in which the detection of cleaved or uncleaved HMCN1 serves as an indicator of the proteolytic activity of the protease AD AMTS 12, and further comprising the purification of said active substance.

[0120] Furthermore, the present invention relates to a method for producing a pharmaceutical composition, comprising

[0121] (i) the method for identifying said active substance which binds to the AD AMTS 12 protein or a fragment thereof and / or inhibits or reduces the activity of the protease AD AMTS 12, or a proteolytic fragment thereof, wherein the method comprises at least one step in which the detection of cleaved or uncleaved HMCN1 serves as an indicator of the proteolytic activity of the protease AD AMTS 12, and further

[0122] (ii) mixing the identified active ingredient with a pharmaceutically acceptable carrier. According to a further aspect, the present invention relates to a composition comprising a combination of

[0123] (i) the active substance obtained by the methods described above for identifying an active substance that binds to the ADAMTS12 protein or a fragment thereof and / or inhibits or reduces the activity of the protease AD AMTS 12, or a proteolytic fragment thereof, wherein the method includes at least one step in which the detection of cleaved or uncleaved HMCN1 serves as an indicator of the proteolytic activity of the protease AD AMTS 12, or the antibody or antigen-binding fragment or antigen-binding derivative thereof or the antibody-like protein as described above, or the pharmaceutical composition comprising the active substance as described above, or the antibody, the antigen-binding fragment or antigen-binding derivative thereof, or an antibody-like protein as described above, and one or more pharmaceutically acceptable excipients, and

[0124] (ii) one or more other therapeutically active compounds.

[0125] According to another aspect, the present invention relates to a kit comprising:

[0126] (i) the protease AD AMTS 12,

[0127] (ii) HMCN1, cleavage products of HMCN1, and / or recombinant peptides of HMCN1, as a control,

[0128] (iii) optionally, instructions for use.

[0129] According to another aspect, the present invention relates to a therapeutic kit comprising:

[0130] (i) the pharmaceutical composition as described above,

[0131] (ii) a device for administering said composition, and

[0132] (iii) optionally, instructions for use.

[0133] Sequences

[0134] Table 1: Amino acid sequences

[0135] SEQ ID No. 1 :

[0136] Human ADAMTS12, amino acid sequence (UniProt ID: P58397-1)

[0137] SEQ ID No. 2: Human HMCN1, Hemicentin-1, amino acid sequence (UniProt-ID: Q96RW7-1)

[0138] ZI Examples

[0139] The present invention is explained in more detail by the examples and drawings shown and discussed below. It should be noted that the examples and drawings are for descriptive purposes only and are not intended to limit the invention in any way.

[0140] The invention is not limited to the disclosed embodiments. Other variations of the disclosed embodiments can be understood and carried out by persons skilled in the art when carrying out the claimed invention by studying the drawings, the disclosure, and the appended claims. In the claims, the word "comprising" does not exclude other elements or steps, and the indefinite article "a" or "an" does not exclude a plurality. The mere fact that certain measures are cited in different dependent claims does not mean that a combination of these measures cannot be advantageously employed. Any reference numerals in the claims are not to be understood as limiting the scope of application.

[0141] All amino acid sequences revealed here are shown from the N-terminus to the C-terminus; all nucleic acid sequences revealed here are shown from 5' to 3'.

[0142] Example 1: Materials and methods

[0143] Isolation of extracellular matrix

[0144] 4x0 were placed in 10cm trays. 5 WT- or ADAMTS12-KO human immortalized renal PDGFRB +Cells were seeded and cultured overnight. This was followed by a 24-hour serum-free culture (DMEM + 0.5% fetal calf serum) and subsequent stimulation with 10 ng / ml TGFb (100-21-10 UG, Peprotech) to induce extracellular matrix synthesis. After 72 hours, the ECM was isolated according to the ECM isolation protocol of Hellewell et al. In summary, after removal of the cell culture medium and washing the cells with PBS, the cells were removed by 5 minutes of incubation with 3 ml of 20 mM ammonium hydroxide at room temperature. Subsequently, 20 ml of deionized water were added before the diluted ammonium hydroxide / cell solution was removed. The remaining ECM layer was then washed four times with deionized water. Complete cell removal was verified under a microscope before the ECM was denatured by adding 4% SDS to 100 mM Tris-HCl pH 8.5. Eight 10-cm dishes were pooled for each biological replicate.The proteins were precipitated overnight with ice-cold acetone (80% final concentration). The following day, the precipitates were centrifuged at maximum speed for 30 minutes and washed twice in 80% acetone. The pellets were suspended in 2% SDC in 100 mM Tris-HCl pH 8.5. Protein levels were normalized using the BCA Protein Assay Kit, and the proteins were reduced with 10 mM Tris(2-carboxy(ethyl)phosphine (TCEP)) and alkylated with 40 mM 2-chloroacetamide (CAA). After overnight digestion with LysC and trypsin (1:100, enzyme / protein, w / w) at 37°C, the peptides were purified using SDB-RPS stage tips.

[0145] Chromatography and mass spectrometry

[0146] Biological replicates were loaded onto 50 cm columns with C18 1.9 pM ReproSil particles (Dr. Maisch GmbH) using an EASY-nLC 1000 chromatography system (Thermo Fisher Scientific) coupled to a mass spectrometer (Exploris 480, Thermo Fisher Scientific). A column oven maintained the column temperature at 60°C. The peptides were eluted via a 120-minute gradient, starting at 5% buffer B (80% ACN, 0.1% formic acid) and gradually increasing to 30% in 95 minutes, 60% in 5 minutes, 95% in two 5-minute intervals, and 5% in two 5-minute intervals, at a flow rate of 300 nL / min. A data-independent acquisition method was used, in which a full scan (300 to 1650 m / z, R = 120,000 at 200 m / z) was first performed with a target of 3 x 10 million ions, followed by 48 windows with a resolution of 15.000, in which precursor ions were fragmented using Higher-Energy Collisional Dissociation (fixed collision energy 27%) and analyzed with a user-defined AGC target and maximum injection time in profile mode using positive polarity.

[0147] Quantification and statistical analysis of mass spectrometry data: Mass spectrometry raw files were processed using Spectronaut software version 15 (Biognosys) (Brüderer et al. 2015) with directDIA and default settings. The human UniProt-FASTA database (42,351 entries, plus 57,749 entries in 2021) was used as the forward database. N-terminal acetylation and methionine oxidation were defined as variable modifications, and cysteine ​​carbamidomethylation was defined as a fixed modification. All bioinformatic analyses were performed using Perseus software (version 1.6.2.2) (Ewels et al. 2020). Quantified proteins were filtered for at least three valid measurements per biological replicate (WT or ADAMTS12-KO). Missing values ​​were imputed, and significantly up- or downregulated proteins were calculated using Student's t-test (two-sided) (FDR = 0.05).

[0148] Mouse experiments

[0149] ADAMTS12-KO mice were a gift from C. Lopez-Otin, from whom the mice were bred (El Hour et al. 2010). Genotyping of the mice was performed by PCR. To perform unilateral ureteral ligation (UUO), the left ureter was ligated at the level of the lower pole with two 7.0 bands (Ethicon) following a flank incision. A sham operation, consisting of only an isolated flank incision, was performed as a control. The animals were euthanized on day 10 postoperatively. All animal experiment protocols were approved by the State Agency for Nature, Environment and Consumer Protection of North Rhine-Westphalia (Germany).

[0150] Western Blot

[0151] Murine kidney tissue was lysed using RIPA lysis buffer with a complete protease inhibitor cocktail and PhosphoSTOP phosphatase inhibitor (Roche, Germany) for 30 minutes at 4°C. After centrifugation at 10,000 g for 30 minutes at 4°C, the protein concentration of the lysates was quantified. 30 pg of lysate were separated by SDS-PAGE (the acrylamide concentration was selected according to the size of the protein to be detected) and transferred to nitrocellulose. Membranes were incubated overnight at 4°C with gentle shaking using specific HMCN1 (Sigma, HPA051677, 1:2000) primary antibodies and subsequently incubated for 1 hour at room temperature with horseradish peroxidase-conjugated secondary antibodies (Vector Laboratories #P 1-2000-1 and #PI-1000-1). Protein bands were visualized using Pierce™ ECL Western blotting substrate and ChemiDoc™ Touch Imaging System and quantified using Image Lab Software.

[0152] Digestion assay

[0153] ADAMTS12-K0 cells grown to 80% confluency were lysed using lysis buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% NP-40, 2 mM TCEP, and 10% glycerol) additionally containing EDTA-free protease inhibitors (Roche #11836170001) and PhosSTOP phosphatase inhibitors (Roche #4906845001). Seven percent of the total cell lysate samples were used as a loading control. Subsequently, the cell lysates were purified by incubating them with 50 µl of ProteinG Sepharose 4 Fast Flow Suspension for one hour at 4°C. The lysate was then divided into two parts and either immunoprecipitation was performed using the HMCN1 antibody (Proteintech, #18837-1-AP) or with rabbit IgG control antibodies (Cell Signaling, #2729). For each, 4 pg of antibody and 50 pl of ProteinG suspension were used; the mixtures were incubated for 1 hour at 4°C.The IP mixtures were then divided again, washed three times with lysis buffer, and equilibrated by three washes with ADAMTS12 digestive buffer (50 mM Tris-HCl pH 7.5, 100 mM NaCl, 5 mM CaCh, 2 mM ZnCh, Brij-35 0.05%). For in vitro digestion, the pellets were dissolved in 30 µl of ADAMTS12 buffer (supplemented with 90 ng of recombinant ADAMTS12 or vehicle), and the mixtures were incubated for 12 hours at 37°C. The activity of recombinant ADAMTS12 was verified by parallel digestion of 1000 ng of recombinant COMP (R&D #3134-CPB-050). The denatured samples were separated by SDS-PAGE and transferred to a nitrocellulose membrane for Western blotting. The following primary antibodies were used: anti-ADAMTS12 (Invitrogen, #PA5-68084, 1:1000), anti-HMCN1 (Sigma, #HPA051677, 1:2000), and anti-COMP (Abeam, #ab231977, 1:1000).

[0154] Secondary antibodies used were the Light Chain-specific Anti-Rabbit IgG from Jackson Immuno Research (#211-031-171; 1:5000).

[0155] Migration Analysis: Transfection with HMCN1 siRNA (L-013514-00-0010, Horizon Discovery) or non-specific siRNA (D-001810-10-05, Horizon Discovery) was performed according to the protocol provided by Horizon Discovery (DharmaFECT Transfection Reagents - siRNA transfection protocol (https: / / horizondiscovery.com / - / media / Files / Horizon / resources / Protocols / basic-dharmafect-protocol.pdf)). Subsequently, the cells were seeded into a 96-well plate (flat-bottomed, transparent, 89626, ibidi) and incubated for 24 h in culture medium (5% fetal calf serum). After 24 h, the medium of the now 50% confluent cells was transferred to CCL-independent medium (180458054, Gibco, 5% FCS). The cells were then imaged every 10 minutes for 24 hours in a 37°C chamber using a Nikon AIR confocal microscope.Subsequently, the cell coordinates for each cell at each time point in time, as well as the average speed over time, were calculated using the Image J plugins StackReg and TrackMate.

[0156] Quantitative RT-PCR

[0157] Transfection with HMCN1 siRNA (L-013514-00-0010, Horizon Discovery) or non-specific siRNA (D-001810-10-05, Horizon Discovery) was performed according to the protocol provided by Horizon Discovery (DharmaFECT Transfection Reagents - siRNA transfection protocol https: / / horizondiscovery.com / - / media / Files / Horizon / resources / Protocols / basic-dharmafect-protocol.pdf). After transfection, the cells were incubated for 24 hours. Subsequently, the cells were washed with PBS before being lysed with RNA-Easy Lysis Buffer and RNA extraction was performed according to the manufacturer's instructions using the Qiagen RNeasy Mini Kit. Subsequently, 200 ng of RNA were reverse-transcribed into complementary DNA using the Applied Biosystems High-Capacity cDNA Reverse Transcription Kit. qRT-PCR was performed using the Bio-Rad iTaq Universal SYBR Green Supermix and the Bio-Rad CFX96 Real Time System with the CI 000 Touch Thermocycler.The cycle conditions included an initial denaturation at 95 °C for 3 minutes, followed by 40 cycles at 95 °C for 15 seconds and 60 °C for 1 minute, followed by one cycle at 95 °C for 10 seconds. GAPDH served as a housekeeping gene. The data were analyzed using the 2-AACT method. Example 2: Identification of the substrate of the protease ADAMTS12 in the pathogenesis of fibrosis.

[0158] To identify the substrate of the protease AD AMTS 12, which is essential in the pathogenesis of fibrosis, the extracellular matrix of AD AMTS 12-KO and -WT immortalized human renal PDGFRB was analyzed. + Cells were analyzed using mass spectrometry. It was assumed that the substrate of AD AMTS 12 should accumulate in the ECM of ADAMTS12-K0 cells, since it is not cleaved there.

[0159] In the mass spectrometry analysis, the inventors detected no significant differences in the expression of the few known ADAMTS12 substrates CTGF, COMP, or NCAN. Instead, surprisingly, the most highly enriched protein in the ECM of ADAMTS12-K0 cells was fibulin hemicentin 1 (HMCN1), compared to ADAMTS12-WT cells (see Fig. 1).

[0160] Example 3: Inhibition or reduction of HMCN1 cleavage in ADAMTS12 knockout mice

[0161] The next step involved investigating whether the protein HMCN1 is differentially expressed in vivo in the kidneys of WT and Adamis 12 (knockout) mice 10 days after kidney damage via unilateral ureteral obstruction (UUO) or after sham surgery. Following sham surgery, an HMCN1 western blot of WT and Adamis 12 mice showed Z—Initially, no differences were observed in (KO) mice (see Fig. 2 upper Western blot; Fig. 3). However, after UUO surgery, the inventors found a significant enrichment of HMCN1 peptides at 56 kDa in WT mice, but not in Adamis 12. Z— (KO) mice (see Fig. 2 lower Western blot; Fig. 3).

[0162] Undiluted HMCN1 is typically detected at 600 kDa (not shown here), so the detected HMCN1 at 56 kDa is considered an HMCN1 cleavage product. These results show that the genetic knockout of Adamtsl2 leads to altered, reduced HMCN1 cleavage. They also show that in vivo HMCN1 peptides (cut HMCN1) accumulate in the kidney of wild-type mice following organ damage, while the same 56 kDa fragments are not accumulated in ADAMTS12-K0 mice.

[0163] Example 4: Direct interaction of ADAMTS12 and HMCN1; cleavage of HMCN1 by ADAMTS12

[0164] The protein HMCN1 was identified in previous studies as a critical component of basement membranes, which is expressed in kidney blood vessels, among other places, and can serve as an attachment point for cells to membranes (Weicker et al. 2021; Lin et al. 2020).

[0165] Based on the results obtained within the scope of the present invention, it could be suggested that the cleavage of HMCN1 by AD AMTS 12 could enable the migration and expansion of myofibroblasts from the perivascular niche, thus mediating ADAMTS12-induced myofibroblast activation.

[0166] A direct interaction between HMCN1 and AD AMTS 12 was initially demonstrated through co-immunoprecipitation. For this purpose, recombinant AD AMTS 12, which had been labeled and expressed with an "HA tag" (hemagglutinin epitope), was incubated with HMCN1 and immunoprecipitated using an anti-HA antibody. Subsequently, the immunoprecipitate was separated by gel electrophoresis, and the presence of the HMCN1 protein was detected by Western blot using an anti-HMCN1 antibody.

[0167] To confirm the cleavage of HMCN1 by AD AMTS 12 described in Example 3, a digestion assay was performed. For this purpose, HMCN1 was isolated in vitro from cell lysates of ADAMTS12 knockout cells by immunoprecipitation (IP) with an anti-HMCN1 antibody. A control IP was performed with a non-specific rabbit IgG antibody. Subsequently, the IP lysates were digested with recombinant AD AMTS 12 or a vehicle (see Fig. 4). Subsequent Western blots for HMCN1 confirmed the cleavage of HMCN1 and revealed multiple HMCN1 cleavage products (marked with *) after digestion with AD AMTS 12 compared to the vehicle (see Fig. 4). The proteolytic digestion of HMCN1-IP lysates with recombinant ADAMTS12 resulted in a pattern of new HMCN1 bands, confirming the cleavage of HMCN1 by ADAMTS12.

[0168] Example 5: Cleavage of HMCN1 or inhibition of HMCN1 expression leads to activation and migration of fibroblasts.

[0169] Finally, it was investigated whether the cleavage of HMCN1 by AD AMTS 12 mediates the activation and migration of fibroblasts.

[0170] For this purpose, the expression of HMCN1 was inhibited in vitro using HMCN1-specific siRNA in ADAMTS12-K0 and active ADAMTS12-expressing kidney cells. The successful inhibition of HMCN1 was confirmed in a parallel experiment in which cells were harvested for RT-qPCR to confirm the reduction of HMCN1 after siRNA treatment (see Fig. 5).

[0171] This experimental approach hypothesized that either uncleaved HMCN1 anchors mesenchymal cells in perivascular niches or that cleaved HMCN1 peptides induce the migration and activation of fibroblasts. In both scenarios, cleavage of HMCN1 by AD AMTS 12 would induce activation and migration of mesenchymal cells.

[0172] Indeed, inhibition of HMCN1 expression using siRNA in ADAMTS12-overexpressing cells led to a significantly reduced migration rate (see Fig. 5). This observation confirms the hypothesis that cleaved HMCN1 peptides induce migration of mesenchymal cells.

[0173] In summary, in light of the available data, it appears that cleaved HMCN1 serves as a downstream mediator of cell migration induced by AD AMTS 12.

[0174] Example 6: Screening for drugs that bind to the metalloporotease ADAMTS12 and inhibit or reduce the cleavage of HMCN1. Screening experiments enable the identification and validation of small molecule therapeutic compounds, peptides and / or biologics that bind to the protease AD AMTS 12 and inhibit or reduce its proteolytic activity with respect to the cleavage of HMCN1.

[0175] The inventive method for identifying an active substance that binds to the protease AD AMTS 12, or a proteolytic fragment thereof, and / or inhibits or reduces the proteolytic activity of the protease AD AMTS 12, or a proteolytic fragment thereof, comprises at least one step in which the detection of cleaved or uncleaved HMCN1 serves as an indicator of the proteolytic activity of the protease ADAMTS12.

[0176] This inventive process comprises at least the following successive steps:

[0177] (i) the provision of the protease AD AMTS 12, or a proteolytic fragment thereof,

[0178] (ii) the addition of at least one active substance to be investigated for inhibiting or reducing the proteolytic activity of the protease ADAMTS12, or of a proteolytic fragment thereof, and

[0179] (iii) the detection of cleaved or uncleaved HMCN1 as an indicator of the proteolytic activity of the protease ADAMTS12.

[0180] For this purpose, the protease AD AMTS 12 can be expressed as recombinant ADAMTS12 protein, or fragments thereof which may carry a . a ' for labeling, identification or purification, e.g. an HA tag, His tag or a FLAG tag, in bacterial expression systems such as E. coli, or in insect cells or mammalian cells.

[0181] The purified recombinant ADAMTS12 protein can be used in the solid or liquid phase in the process according to the invention.

[0182] The substrate HMCN1, used as an indicator for the proteolytic activity of the protease AD AMTS 12, can be added to the process according to the invention at a later time after steps (ii) and (iii). Ideally, the process should allow for the detection of the possible cleavage products in the same reaction. DNA-encoded compound libraries are generated and screened as described (Kunig et al. 2018). Furthermore, phage display technologies (Takakusagi et al. 2020), cell surface display or ribosome display technologies (Galan et al. 2016), and / or combinatorial peptide libraries (Bozovicar and Bratkovic 2019) are used.

[0183] The purified ADAMTS12 protein is incubated with the compound library in the presence and / or absence of HMCN1 and isolated by immunoprecipitation. Compounds bound to the ADAMTS12 protein are identified, for example, by Sanger sequencing of the DNA barcodes. The identified agents and compounds are then tested for their effects on the function of ADAMTS12, its proteolytic activity against HMCN1, fibroblast migration, the expression and secretion of matrix proteins such as collagen 1 and fibronectin, and the development of renal and / or cardiac fibrosis. For this purpose, experimental mouse zww'vo models of renal and cardiac fibrosis are used.

[0184] For the identification and validation of small-molecule therapeutic compounds, peptides, and / or biologics that affect ADAMTS12 protease activity against HMCN1 or its expression, an in vitro fluorochrome reporter system based on human cells can be established. This system utilizes, for example, the expression of the eGFP-ADAMTS12 fusion protein or a luciferase-based reporter to screen compound libraries in 384- to 1,536-well assays for the identification of compounds that reduce eGFP fluorescence or luciferase levels as a readout. Expression of these human ADAMTS12 fusion reporter constructs in the aforementioned cells can be achieved, for example, by transfection and selection via resistance gene cassettes or by viral transduction. For these assays, human cell lines such as 293T cells, but also established human kidney fibroblast cell lines are used.Parallel to this screening, cytotoxicity assays are performed to exclude compounds that have an effect on reporter fluorescence or activity due to non-specific toxicity or induction of apoptosis.

[0185] Example 7: Conclusions The metalloproteinase AD AMTS 12 is a particularly attractive molecular target for the treatment of fibrosis. AD AMTS 12 is virtually not expressed under homeostasis. Following the induction of renal fibrosis, AD AMTS 12 is specifically upregulated in fibroblasts, pericytes, and myofibroblasts. The cell-specific expression of AD AMTS 12 and its low to absent expression under homeostasis suggest that inhibition of AD AMTS 12 is likely to be associated with few side effects. Furthermore, from a biochemical perspective, inhibition of the metalloproteinase AD AMTS 12 offers a clear target for drug development.

[0186] The identity of the substrate of the metalloprotease AD AMTS 12, which may be significantly involved in the development of renal fibrosis, was previously completely unknown.

[0187] In summary, it can be stated that the present application has shown that, surprisingly, none of the few known ADAMTS12 substrates (CTGF, COMP or NCAN) proved to be the most enriched protein in the extracellular matrix of ADAMTS12 knockout cells, but rather the fibulin hemicentin 1 (HMCN1).

[0188] Furthermore, in vivo mouse models demonstrated that HMCN1 peptides (proteolytic cleavage products of HMCN1) accumulate in the kidneys of wild-type mice following organ damage, while the same cleavage products (especially the 56-kDa fragment) do not accumulate in ADAMTS12-KO mice. An in vitro digestive assay also confirmed that ADAMTS12 cleaves the substrate HMCN1. Finally, it was demonstrated that the cleavage of HMCN1 by ADAMTS12 induces the migration and activation of fibroblasts, and thus plays a crucial role in the development of fibrosis.

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Claims

Patent claims:

1. A method for inhibiting or reducing the cleavage of HMCN 1 by a protease capable of cleaving HMCN1, wherein the method comprises at least one step in which (i) the expression of said protease or the proteolytic activity of said protease is inhibited or reduced, and / or (ii) the degradation of said protease is promoted.

2. A method for inhibiting or reducing the cleavage of HMCN1 by the protease AD AMTS 12, wherein the method comprises at least one step selected from the group consisting of (i) Inhibition or reduction of / 4 / MA77 S72 gene expression in a cell, (ii) Inhibition or reduction of the proteolytic activity of the protease AD AMTS 12, and / or (iii) Promotion of the degradation of the protease ADAMTS12.

3. The method of claim 2, wherein the inhibition or reduction of ADAMTS12 gene expression inhibits a 4 / MA77 S72 gene I <nock-down, Knock-out, konditionalen Gen- Knockout, eine Genveränderung, RNA-Interferenz, siRNA und / oder Antisense-RNA umfasst, und / oder wobei die Hemmung oder Reduzierung der proteolytischen Aktivität der Protease AD AMTS 12 die Verwendung eines Wirkstoffs umfasst, der an das AD AMTS 12- Protein bindet und / oder seine proteolytische Aktivität hemmt oder reduziert.

4. Method according to one of claims 2 and 3, wherein said cell is a kidney cell, preferably a renal fibroblast cell, a renal myofibroblast cell, or a renal pericyte; most preferably a renal fibroblast cell.

5. A method for identifying an active substance that binds to the protease ADAMTS12, or a proteolytic fragment thereof, and / or inhibits or reduces the proteolytic activity of the protease ADAMTS12, or a proteolytic fragment thereof, wherein the method comprises at least one step in which the Detection of cleaved or uncleaved HMCN1 serves as an indicator for the proteolytic activity of the protease AD AMTS 12.

6. The method of claim 5, comprising at least the following steps: (i) Providing the protease AD AMTS 12, or a proteolytic fragment thereof, (ii) Addition of at least one active substance to be investigated for inhibiting or reducing the proteolytic activity of the protease ADAMTS12, or of a proteolytic fragment thereof, and (iii) Detection of cleaved or uncleaved HMCN1 as an indicator of the proteolytic activity of the protease ADAMTS12.

7. A method according to one of claims 5 and 6, wherein the active ingredient is selected from the group consisting of a low molecular weight compound (smol), a natural or synthetic peptide or oligopeptide, and a biologic, and / or wherein the active ingredient is a member of a library of compounds, preferably wherein the active ingredient is an ADAMTS12 inhibitor.

8. The method of claim 7, wherein the biologic is an antibody, an antigen-binding fragment thereof, an antigen-binding derivative thereof, an antibody-like molecule, a nucleic acid or an aptamer.

9. Active ingredient obtained by the method according to any one of claims 5 to 8, wherein the active ingredient inhibits or reduces the proteolytic cleavage of HMCN1 by the protease AD AMTS 12.

10. Antibody, or antigen-binding fragment or antigen-binding derivative thereof, or antibody-like protein, wherein the antibody, or antigen-binding fragment or derivative thereof, or antibody-like protein, inhibits or reduces the proteolytic cleavage of HMCN1 by the protease AD AMTS 12.

11. Active ingredient according to claim 9 or antibody, antigen-binding fragment or antigen-binding derivative thereof, or antibody-like protein according to claim 10, for use in the treatment of chronic kidney disease.

12. Active ingredient or antibody for use according to claim 11, wherein the chronic kidney disease is progressive chronic renal insufficiency and / or renal fibrosis.

13. Pharmaceutical composition comprising the active ingredient according to claim 9 or antibody, antigen-binding fragment or antigen-binding derivative thereof, or antibody-like protein according to claim 10, and one or more pharmaceutically acceptable excipients, for use in the treatment of chronic kidney disease, preferably wherein the chronic kidney disease is progressive chronic renal insufficiency and / or renal fibrosis.

14. Methods for the qualitative and / or quantitative detection of HMCN1 and / or HMCN1 cleavage products of the protease ADAMTS12, or a proteolytic fragment thereof, in body fluids, cells and / or the extracellular matrix (ECM).

15. Method according to claim 14, comprising at least one method from the group consisting of gel electrophoresis, western blot, immunoprecipitation, ELISA, and mass spectrometric detection.

16. Use of HMCN 1 and / or HMCN 1 cleavage products to detect the proteolytic activity of the protease AD AMTS 12, or a proteolytic fragment thereof.

17. Use of HMCN1 according to claim 16, wherein HMCN1 and the protease AD AMTS 12, or a proteolytic fragment thereof, are brought into contact with a drug or antibody candidate to be tested, and (i) the occurrence of specific HMCN 1 cleavage products through the cleavage of HMCN1 demonstrates the proteolytic activity of the protease AD AMTS 12, or (ii) the absence of specific cleavage products from the cleavage of HMCN1 demonstrates that the protease AD AMTS 12, or a fragment thereof, is not or less proteolytically active, and preferably (iii), in case (ii), an active substance or antibody can be identified that inhibits or reduces the proteolytic activity of the protease AD AMTS 12, or of a proteolytic fragment thereof.

18. Use of HMCN1 and / or HMCN1 cleavage products as biomarkers of chronic kidney disease, preferably progressive chronic renal failure and / or renal fibrosis.

19. Use of HMCN 1 and / or HMCN 1 cleavage products as biomarkers of chronic heart disease, preferably progressive chronic heart failure and / or cardiac fibrosis.

20. Use of HMCN 1 and / or HMCN 1 fission products according to any one of claims 16 to 19, wherein at least one HMCN 1 fission product has a molecular weight of 56 kDa.

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