ADAMTS12 as a therapeutic target for chronic renal failure and renal fibrosis

Abstract

The present invention relates to the role of the ADAMTS12 protein (A Disintegrin And Metalloproteinase with Thrombospondin motifs 12 protein) in the development of chronic kidney disease, particularly progressive chronic kidney disease and renal fibrosis. In particular, the present invention relates to methods for identifying compounds that bind to the ADAMTS12 protein and to the use of the ADAMTS12 protein for screening and identifying ADAMTS12-interacting compounds and ADAMTS12-inhibiting compounds. The present invention further relates to pharmaceutical compositions for use in treating kidney disease, particularly pharmaceutical compositions comprising an active agent that binds to and / or inhibits the ADAMTS12 protein.

Description

[Technical Field]

[0001] The present invention relates to the role of the ADAMTS12 protein (A Disintegrin And Metalloproteinase with Thrombospondin motifs 12 protein) in the development of chronic kidney disease and heart disease, particularly progressive chronic renal failure and renal fibrosis, as well as heart failure and cardiac fibrosis. In particular, the present invention relates to methods for identifying compounds that bind to and / or inhibit the ADAMTS12 protein, and to the use of the ADAMTS12 protein for screening and identifying ADAMTS12-interacting and ADAMTS12-inhibiting compounds. The present invention further relates to pharmaceutical compositions for use in treating kidney disease, particularly pharmaceutical compositions comprising an active agent that binds to and / or inhibits the ADAMTS12 protein. [Background technology]

[0002] Fibrosis is defined as the pathological deposition of extracellular connective tissue (extracellular matrix, ECM) accompanied by the displacement of healthy tissue and loss of organ function. Initial deposition of ECM is critical for maintaining tissue integrity after organ injury, whereas unregulated deposition of ECM leads to the displacement of healthy tissue and loss of organ function. Regardless of the initial injury, fibrosis is a common end stage of almost all chronic diseases across all organs. Current estimates suggest that fibrosis accounts for up to 45% of all deaths in developed countries (Henderson et al., 2020). Due to the aging population, the prevalence of fibrosis will continue to increase over the next few decades. Summary of the Invention [Problem to be solved by the invention]

[0003] The number of patients suffering from chronic kidney failure (chronic kidney disease, CKD) is increasing worldwide, and current data indicate that up to 10% of the population in Western countries will develop CKD during their lifetime (Jha et al., 2013). Due to the increasing average age and the increasing prevalence of hypertension and diabetes, the incidence of CKD is expected to continue to rise. As kidney function declines, morbidity and mortality increase significantly. In the final stages of CKD, dialysis and kidney transplantation are the only treatments available. Due to the long wait times for donated kidneys, most of these patients undergo dialysis. However, dialysis therapy is associated with high mortality (approximately 50% 5-year survival rate after new dialysis; Naylor et al., 2019), numerous comorbidities, and a significant decrease in quality of life (three times a week, 4–6 hours of dialysis therapy). Furthermore, the high cost of dialysis poses a significant economic burden to healthcare systems (Cm and F, 2017). Therefore, new treatment approaches are needed.

[0004] The degree of renal fibrosis is closely related to the decline in renal function and the clinical course of CKD. Renal fibrosis is characterized by the high expression, secretion, and accumulation of extracellular matrix (ECM) proteins, such as collagen type 1.

[0005] Myofibroblasts, which proliferate after organ injury, are the primary producers of extracellular matrix and play a key role in the development of fibrosis (Henderson et al., 2020; Kuppe et al., 2021). The origin of these myofibroblasts was unknown for a long time, but a perivascular cell population has now been identified, characterized by expression of the transcription factor Gli1, from which 50% of myofibroblasts are derived (Kramann et al., 2015a). However, it is still unclear which signals lead to the activation, expansion, and myofibroblast differentiation of these Gli1 fibroblasts.

[0006] The histological structure of the kidney is divided into three main compartments, all of which can be affected by fibrosis, specifically glomerulosclerosis in the glomeruli, interstitial fibrosis in the tubulointerstitium, and arteriosclerosis and perivascular fibrosis in the vasculature (Djudjai and Boor 2019).

[0007] In animal models, suppression of fibrosis can prevent the progression of chronic renal failure and preserve renal function (Kramann et al., 2015a, 2015b). However, there are currently no approved treatments for renal fibrosis. With the increasing incidence of chronic renal failure, the development of drugs to treat fibrosis is essential. [Means for solving the problem]

[0008] It is therefore a fundamental object of the present invention to provide methods and means for identifying active agents, compounds and compositions, and said active agents, compounds and compositions for use in the treatment of chronic kidney disease.

[0009] This application discloses the identification of a new molecular target for the treatment of renal fibrosis. We identified and isolated Gli1-expressing fibroblasts after inducing renal fibrosis in mice. Using microarray analysis of total RNA, i.e., the expression of all genes expressed by these activated fibroblasts, we surprisingly identified the protein "ADAMTS12" (A Disintegrin and Metalloproteinase with Thrombospondin motifs 12) as a new molecular target. ADAMTS12 belongs to the ADAMTS metalloprotease family and degrades the extracellular matrix protein thrombospondin-5 (Wei et al., 2014). We demonstrated for the first time that the metalloprotease ADAMTS12 is an essential mediator of fibrosis and that knockout of ADAMTS12 suppresses the progression of fibrosis after kidney and heart injury.

[0010] ADAMTS ("A Disintegrin And Metalloproteinase with Thrombospondin motifs") proteins belong to the methionin protease superfamily and are named after the conserved methionine residue near the active center of zinc-ion-dependent metalloproteinases (Kelwick et al. 2015). To date, at least 19 ADAMTS proteins have been identified in mammalian genomes. ADAMTS proteins are secreted, extracellular zinc-containing matrix metalloproteinases with a uniform and ordered modular structure. ADAMTS proteins are initially expressed as inactive preproenzymes containing a signal peptide, a variable-length proregion, a catalytic metalloproteinase domain, a disintegrin-like domain, central thrombospondin type 1-like (TSP) sequence repeats, a cysteine-rich domain, a spacer region, and a variable number of additional C-terminal TSP repeats (Figure 5) (Kelwick et al. 2015; Lin et al. 2009).

[0011] The ADAMTS12 gene contains a total of 24 exons and encodes a 1,594-amino acid extracellular protein (Mohamedi et al. 2021). Aggrecan, COMP (cartilage oligomeric matrix protein), and α2M (α2-macroglobulin) have been identified as substrates of ADAMTS12. Roles of the ADAMTS12 protein have been described in chondrogenesis, cartilage development, and gonadal differentiation, as well as in childhood stroke, schizophrenia, tumorigenesis, and arthritis (Lin et al. 2009; Wei et al. 2014; Mohamedi et al. 2021; Witten et al. 2020).

[0012] The present application discloses a new therapeutic approach for the development of therapeutic agents for patients with chronic renal failure and renal fibrosis, using ADAMTS12 as a target molecule.

[0013] The present invention provides methods and means for identifying active agents, compounds and compositions for use in the treatment of chronic renal failure, and in particular for identifying highly effective active agents, compounds and compositions for use in the treatment of progressive chronic kidney disease and renal fibrosis.

[0014] Therefore, in view of the prior art, it was an object of the present invention to provide a method for reducing the expression and / or secretion of extracellular matrix (ECM) proteins by certain cells. A further object of the present invention was to provide a method for reducing the expression, differentiation and secretion of extracellular matrix proteins by (myo)fibroblasts.

[0015] A further object of the present invention was to provide methods for identifying active agents that bind to and / or inhibit the ADAMTS12 protein or fragments thereof.

[0016] A further object of the present invention was to provide a method for utilizing the ADAMTS12 protein or a fragment thereof, or a nucleic acid encoding the ADAMTS12 protein itself or a fragment thereof, for the identification of active agents that bind to ADAMTS12 or a fragment thereof.

[0017] A further object of the present invention, based on the above findings, was to provide an active agent for use in the treatment of chronic kidney disease, in particular for use in the treatment of progressive chronic kidney disease and / or renal fibrosis.

[0018] Based on the above findings, a further object of the present invention is to provide pharmaceutical compositions containing these agents and methods for preparing such pharmaceutical compositions.

[0019] The further technical problem described is solved by the device or method according to the present invention. Preferred embodiments are described in the dependent claims. Ranges limited by numerical values ​​should always include the limiting values.

[0020] The invention and generally advantageous embodiments are described in more detail below. [Brief explanation of the drawings]

[0021] [Figure 1] Figure 1 shows a microarray of Gli1 fibroblasts after unilateral ureteral obstruction (UUO). (A) Experimental setup. (B) Hallmark Gene Set Enrichment Analysis (GSEA) of differentially expressed genes in Gli1 fibroblasts after UUO. (C) The "top 25" genes upregulated in Gli1 fibroblasts by UUO, sorted by T value. (D) Representative images of in situ hybridization (ISH) of Pdgfrb and ADAMTS12 transcripts in mouse kidneys at different time points after ischemia-reperfusion (IRI). (E) Quantification of ADAMTS12 ISH expression. (F) Quantification of Pdgfrb ISH expression. (G) Quantification of ADAMTS12 ISH expression in Pdgfrb-positive cells. **p<0.01, ***p<0.001. [Figure 2]Figure 2 shows that genetic deficiency of ADAMTS12 prevents fibrosis. (AG) Adamts12- / - or WT mice underwent unilateral ureteral obstruction (UUO) or placebo surgery (sham). Ten days after surgery, mice were sacrificed and kidneys were removed. (A) ADAMTS12 RT-qPCR. (B) Collagen type 1 RT-qPCR (Col 1a1). (C) Fibronectin (Fn1) RT-qPCR. (D) PDGFRb immunofluorescence staining (IF). (E) IF quantification of PDGFRb expression. (F) Collagen type 1 (Col 1) immunohistochemical (IHC) staining. (G) Quantification of collagen type 1 expression by IHC. (HI) Adamts12- / - or WT mice underwent myocardial infarction (MI) or placebo surgery (sham). (H) Left ventricular ejection fraction (LV-EF) measured by echocardiography in WT and Adamts12- / - mice after myocardial infarction (MI) or sham surgery. (I) Fibrosis measured by picrosirius red staining in serial sections in WT and Adamts12- / - mice after MI or sham surgery. *p<0.05, ***p<0.01, ***p<0.001, ***p<0.001. [Figure 3]Figure 3 shows ADAMTS12 CRISPR-Cas9 KO in human kidney PDGFRb-positive fibroblasts. (A-B) RT-qPCR of ADAMTS12 or COL1A1 in control (non-targeting gRNA) and ADAMTS12 CRISPR-KO (ADAMTS12-KO) human kidney PDGFRb fibroblasts after stimulation with vehicle or TGFb. (C) Migration analysis of control (non-targeting gRNA) and ADAMTS12 CRISPR-KO (ADAMTS12-KO) human kidney PDGFRb fibroblasts after stimulation with vehicle or TGFb. (D) Western blot of HA epitope, tubulin, and eGFP in human renal PDGFRb fibroblasts using ADAMTS12 CRISPR-KO (KO) and the empty expression plasmid pMIG (no protein-encoding nucleotide sequence inserted), overexpression of HA-tagged ADAMTS12 using ADAMTS12 CRISPR-KO and the pMIG expression plasmid (WT), and overexpression of catalytically inactive HA-tagged ADAMTS12 protein (Mut) using the pMIG expression plasmid. (E) Migration analysis of human renal PDGFRb fibroblasts using ADAMTS12 CRISPR-KO (ADAMTS12-KO) and the empty expression plasmid pMIG (no protein-encoding nucleotide sequence inserted), overexpression of ADAMTS12 CRISPR-KO and the HA-tagged ADAMTS12 protein (ADAMTS12-KO with WT), and overexpression of catalytically inactive HA-tagged ADAMTS12 protein (Mut). [Figure 4]Figure 4 shows ADAMTS12 expression in human kidneys. (A) ADAMTS12 expression in CD10-negative interstitial-enriched renal single cells (after proximal tubule cell depletion) isolated from 15 human kidneys by FACS. (B) ADAMTS12 expression in PDGFRb-positive single cells isolated from 8 human kidneys by FACS. (C) Representative images of ISH for PDGFRB, COL1A1, and ADAMTS12 in 43 human kidneys. (D) Quantification of ISH. Visualization of the percentage of ADAMTS12-positive cells that are also PDGFRB-positive. (E) Correlation between ADAMTS12 and PDGFRB-ISH expression in human kidney tissues. (F) Correlation between ADAMTS12 and COL1A1-ISH expression in human kidney tissues. [Figure 5] Figure 5 shows the domain structure and organization of the ADAMTS12 protein. The N-terminus of ADAMTS12 consists of a signal peptide, prodomain, and metalloprotease domain. The C-terminus of ADAMTS12 consists of a disintegrin-like domain, the first thrombospondin type 1 repeat (TSP1), a Cys-rich domain, and seven TSP1 repeats separated by two spacer domains. The second spacer domain is a mucin-like domain (from Wei et al. 2014). DETAILED DESCRIPTION OF THE INVENTION

[0022] Before describing the present invention in detail, it should be noted that the present invention is not limited to the specific components of the described apparatus or the described steps of the method, as these methods or apparatus may vary. Also, it should be noted that the terminology used herein is used only for purposes of the specific embodiments being described and is not intended to be limiting.

[0023] It should be noted that in this specification and the appended claims, simple forms such as "a" or "the" refer to singular and / or plural subjects unless the context clearly dictates otherwise. Where ranges of parameters are specified, the limiting numerical values ​​count as the limits of the disclosed or claimed numerical range.

[0024] It should also be noted that the embodiments disclosed herein are not to be understood as separate, unrelated embodiments. Features discussed in connection with one embodiment should also be considered to be disclosed in connection with other embodiments shown herein. In some cases, even if a particular feature is not disclosed in one embodiment but is disclosed in another embodiment, those skilled in the art will understand that this does not necessarily mean that the feature should not be disclosed in another embodiment. Those skilled in the art will understand that, while it would be consistent with the principles of the present application to disclose the feature in other embodiments as well, this has not been done for the sake of clarity and to keep the specification within a manageable scope.

[0025] Furthermore, the contents of the prior art documents mentioned in this specification are incorporated by reference. This applies in particular to prior art documents that disclose standard or routine methods. In this case, the main purpose of incorporating by reference is to enable a full disclosure and to avoid lengthy repetition.

[0026] According to a first aspect, the present invention provides a method for reducing the expression and / or secretion of extracellular matrix (ECM) proteins by a given cell and / or for inhibiting the migration of fibroblasts, comprising: The method comprises: (i) inhibiting or reducing the expression of the ADAMTS12 gene in said cell; (ii) inhibiting or reducing ADAMTS12 activity; (iii) inhibiting or reducing ADAMTS12 protease activity, and / or (iv) promoting the degradation of ADAMTS12 protein; The present invention relates to a method comprising at least one step selected from the group consisting of:

[0027] Inhibition or reduction of ADAMTS12 gene expression can include, for example, knockdown, knockout, conditional gene knockout, genetic modification or mutation, RNA interference, siRNA and / or antisense RNA of the ADAMTS12 gene.

[0028] Inhibiting or reducing ADAMTS12 protein activity may involve the use of active agents that bind to ADAMTS12 protein (A Disintegrin And Metalloproteinase with ThromboSpondin motifs 12 protein) and / or inhibit or reduce its activity.

[0029] Preferably, the cells are renal or cardiac cells, preferably renal or cardiac fibroblasts, renal or cardiac myofibroblasts, or renal or cardiac pericytes, most preferably renal or cardiac fibroblasts.

[0030] The ADAMTS12 protein may be a mammalian, non-primate, primate, particularly a human ADAMTS12 protein or a fragment thereof.

[0031] According to a second aspect, the present invention relates to a method for identifying active agents that bind to said ADAMTS12 protein (A Disintegrin And Metalloproteinase with Thrombospondin motifs 12 protein) or a fragment thereof and / or inhibit or reduce the activity of said ADAMTS12 protein or a fragment thereof.

[0032] The method is: (i) providing the ADAMTS12 protein or a fragment thereof; (ii) adding at least one active agent to be analyzed for binding to the ADAMTS12 protein or fragment thereof; and (iii) identifying at least one active agent that binds to said ADAMTS12 protein or fragment thereof; The method comprises at least the steps of:

[0033] Preferably, the active agents screened and identified by the present invention are ADAMTS12 inhibitors or antagonists, agents that inhibit or reduce the activity of the ADAMTS12 protein, or fragments thereof.

[0034] The active agent according to the present invention may be selected from the group consisting of small molecule compounds, natural or synthetic peptides or peptide derivatives, and biologically or biologically active agents.

[0035] In the context of this invention, the terms "small molecule," "small molecule" ("smol"), or "chemical agent" refer to organic compounds with low molecular weights (<10,000 daltons, especially <1,000 daltons), often on the order of 1 nm in size. Many pharmaceuticals are small molecules. Such small molecules can regulate biological processes. Small molecules may be able to inhibit specific protein functions. In the field of pharmacology, the term "small molecule" specifically refers to molecules that act as effectors by binding to specific biopolymers and altering the activity or function of the target. For example, acetylsalicylic acid (ASA) is considered a small molecule compound with a molecular weight of 180 daltons and 21 atoms. Such small molecules often have little ability to provoke an immune response and remain relatively stable over long periods of time.

[0036] The small molecule compounds according to the present invention may contain, in addition to other chemical backbones, substituents, groups or molecular radicals such as alkyl groups, alkenyl groups, alkynyl groups, alkoxy groups, aryl groups, alkylene groups, arylene groups, amino groups, halogen groups, carboxylic acid derivatives, cycloalkyl groups, carbonyl derivatives, heterocycloalkyl groups, heteroaryl groups, heteroarylene groups, sulfonate groups, sulfate groups, phosphonate groups, phosphate groups, phosphine groups, or phosphine oxide groups.

[0037] A "biological agent," "biological drug," "biological therapeutic agent," "biological drug" or "biologically active agent" according to the present invention is preferably an antibody, or an antigen-binding fragment or antigen-binding derivative thereof, or an antibody-like molecule or protein, or an aptamer, or a nucleic acid.

[0038] In a preferred embodiment of the method for identifying an active agent that binds to and / or inhibits or reduces the activity of the ADAMTS12 protein or fragment thereof, the active agent is a member of a "library" of compounds.

[0039] A "library" (mixture) of compounds may include, for example, small molecule compounds, natural or synthetic peptides or peptide derivatives, or biological agents or biologically active agents or biological compounds.

[0040] In the context of the present invention, the term "(combinatorial) compound library" or "compound library" refers to a collection of macromolecules, such as chemical compounds, small molecules, natural or synthetic peptides or peptide derivatives, or proteins or other biological agents, each containing a large number of related chemical, peptide or biological molecules, which can be used together in a particular screening assay or identification process.

[0041] Methods for generating molecular libraries of small molecule compounds ("compound libraries") and performing high-throughput screening of compounds for interactions with target molecules have been described in the prior art (e.g., Volochnyuk et al., 2019). These methods include highly annotated, preselected chemical molecule libraries, so-called "focus libraries" (Wassermann et al., 2014), DNA-encoded compound libraries (Martin et al., 2020), and chemoinformatics-based virtual molecular libraries (Saldivar-Gonzalez et al., 2020). The use of phage display technology to identify suitable small molecule active agents has been 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, have been described in Valldorf et al., 2022.

[0042] Methods for generating molecular libraries of biological molecules, such as peptides, peptide derivatives, proteins, antibodies, antigen-binding antibody fragments, antigen-binding antibody derivatives or antibody-like molecules, their immobilization and their high-throughput screening have also been described in the prior art (for peptide libraries, e.g., Bozovicar and Bratkovic 2019; Schwaar et al. 2019; for antibody libraries, Lin and Lerner 2021).

[0043] In a preferred embodiment of the method for identifying an active agent that binds to an ADAMTS12 protein or a fragment thereof and / or inhibits or reduces the activity of an ADAMTS12 protein or a fragment thereof, the biological agent 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.

[0044] In a preferred embodiment of the method for identifying an active agent that binds to the ADAMTS12 protein or a fragment thereof and / or inhibits or reduces the activity of the ADAMTS12 protein or a fragment thereof, the ADAMTS12 protein is bound to a solid phase or is in solution.

[0045] According to a third aspect, the present invention relates to the use of a nucleic acid encoding an ADAMTS12 protein or a fragment thereof, or the use of an ADAMTS12 protein or a fragment thereof, in a method for identifying an active agent that binds to an ADAMTS12 protein or a fragment thereof and / or inhibits or reduces the activity of an ADAMTS12 protein or a fragment thereof.

[0046] To express the ADAMTS12 metalloproteinase or a fragment thereof, a nucleic acid encoding the ADAMTS12 metalloproteinase or a fragment thereof is cloned into a suitable expression vector, such as a suitable expression plasmid, as described (Green and Sambrook 2012). The recombinant expression plasmid is transfected into cells suitable for expressing ADAMTS12 or a fragment thereof, the cells are grown in cell culture using an appropriate cell culture medium, and the expressed protein is purified from the cells and / or cell culture medium.

[0047] As used herein, the term "transfection" refers to any method for the deliberate introduction of exogenous nucleic acid into a eukaryotic cell. Various types of nucleic acid can be used to transfect eukaryotic cells, particularly deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and small non-coding RNAs such as siRNA, shRNA, and miRNA.

[0048] Regarding transfection, a distinction is made between stable and transient transfection: in stable transfection, the nucleic acid introduced into the cell is integrated into the cellular genome, thereby achieving long-term expression of the transgene, whereas in transient transfection, the expression of the transgene is temporary, and does not require integration of the nucleic acid into the cellular genome.

[0049] The optimal transfection method depends on various factors, particularly the type and origin of the target and producer cells and the type of nucleic acid to be introduced. Physical, chemical, and viral vector-based transfection methods can be used to introduce foreign (modified homologous and / or heterologous) nucleic acids encoding the desired transgene into eukaryotic cells. Physical transfection methods include electroporation, sonoporation, magnetofection, microinjection, and biolistic methods. Chemical transfection methods include calcium phosphate, the use of dendrimers, cationic polymers such as diethylaminoethyl dextran (DEAE-dextran), nanoparticles, nonliposomal nanoparticles, and liposomal transfection. Viral vector-based transfection (also known as "transduction") involves the use of genetically modified retroviruses, lentiviruses, adenoviruses, and adeno-associated viruses (AAVs), among others (Fus-Kujawa et al. 2021).

[0050] According to a fourth aspect, the present invention relates to an active agent obtainable by the method for identifying an active agent that binds to an ADAMTS12 protein or a fragment thereof and / or inhibits or reduces the activity of an ADAMTS12 protein or a fragment thereof, or an active agent obtainable by any embodiment of the method.

[0051] Furthermore, the present invention relates to active agents that bind to the ADAMTS12 protein or a fragment thereof, and / or that inhibit or reduce the activity of the ADAMTS12 protein or a fragment thereof, and / or that promote the degradation of the ADAMTS12 protein.

[0052] Furthermore, the present invention relates to active agents that inhibit or reduce the expression of the ADAMTS12 gene in kidney cells or cardiac cells, preferably wherein the kidney cells are kidney fibroblasts and / or the cardiac cells are cardiac fibroblasts.

[0053] In a preferred embodiment, the present invention relates to an active agent, wherein the active agent is a small molecule compound (smol), a peptide or peptide derivative, or a biological agent, preferably, the biological agent 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.

[0054] In a preferred embodiment, the active agent specifically binds to the ADAMTS12 protein or a fragment thereof with high or particularly high affinity and / or avidity. In a preferred embodiment, the active agent, upon binding to ADAMTS12, reduces or inhibits ADAMTS12 activity.

[0055] As used herein, the term "specifically binds" means that the active agent has a dissociation constant KD of about 100 μM or less for binding to an ADAMTS12 protein molecule or an epitope thereof. In one embodiment, the KD is about 100 μM or less, about 50 μM or less, about 30 μM or less, about 20 μM or less, about 10 μM or less, about 5 μM or less, about 1 μM or less, about 900 nM or less, about 800 nM or less, about 700 nM or less, about 600 nM or less, about 500 nM or less, about 400 nM or less, about 300 nM or less, about 200 nM or less, about 100 nM or less, about 90 nM or less, about 80 nM or less, about 70 nM or less, about 60 nM or less, about 50 nM or less, about 40 nM or less, It is about 30 nM or less, about 20 nM or less, or about 10 nM or less, about 1 nM or less, about 900 pM or less, about 800 pM or less, about 700 pM or less, about 600 pM or less, about 500 pM or less, about 400 pM or less, about 300 pM or less, about 200 pM or less, about 100 pM or less, about 90 pM or less, about 80 pM or less, about 70 pM or less, about 60 pM or less, about 50 pM or less, about 40 pM or less, about 30 pM or less, about 20 pM or less, about 10 pM or less, or about 1 pM or less.

[0056] According to a fifth aspect, the present invention relates to an antibody, or an antigen-binding fragment or derivative thereof, or an antibody-like protein, which specifically binds to the ADAMTS12 protein.

[0057] In a preferred embodiment, the present invention relates to said antibody, or antigen-binding fragment or derivative thereof, or antibody-like protein, which inhibits ADAMTS12 activity, i.e., acts as an inhibitor or antagonist of ADAMTS12.

[0058] As used herein, the term "antibody" refers to a protein consisting of one or more polypeptide chains encoded by immunoglobulin genes, fragments of immunoglobulin genes, or cDNAs derived therefrom, including the light chain constant region genes kappa and lambda, and the heavy chain genes alpha, delta, epsilon, gamma, and mu, as well as various variable region genes.

[0059] The basic structural unit of an immunoglobulin (antibody) is usually a tetramer consisting of a pair of two identical polypeptide chains: a light chain (L, molecular weight approximately 25 kDa) and a heavy chain (H, molecular weight approximately 50-70 kDa). Each heavy chain contains a variable region (VH or VH) of the heavy chain. H and the heavy chain constant region (CH or C H The heavy chain constant region consists of three domains: CH1, CH2, and CH3. Each light chain contains a variable light region (VL or V L ) and constant light chain region (CL or C L The VH and VL regions are subdivided into regions of hypervariability, also known as complementarity-determining regions (CDRs), interspersed with highly conserved regions known as framework regions (FRs). Each VH and VL region is composed of three CDRs and four FRs, arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains form the binding domain that interacts with antigen.

[0060] The CDRs are most important for the binding of an antibody or its antigen-binding portion. The FRs can be replaced with other sequences as long as the three-dimensional structure required for binding to an antigen is maintained.

[0061] The term "antigen-binding portion" of a (monoclonal) antibody refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen in its native form. Examples of antigen-binding portions of antibodies include a Fab fragment, a monovalent fragment consisting of the VL, VH, CL, and CH1 domains, an F(ab')2 fragment, a bivalent fragment consisting of two Fab fragments linked by a disulfide bridge at the hinge region, an Fd fragment consisting of the VH and CH1 domains, an Fv fragment consisting of the VL and VH domains of a single antibody arm, and a dAb fragment consisting of a VH domain and isolated complementarity-determining regions (CDRs).

[0062] The antibody, antibody fragment or antibody derivative thereof according to the present invention may be a monoclonal antibody. The antibody may be of the IgA, IgD, IgE, IgG or IgM isotype.

[0063] As used herein, the term "monoclonal antibody (mAb)" refers to an antibody composition having a homogeneous antibody population, i.e., a homogeneous population of whole immunoglobulins or fragments or derivatives thereof. Particularly preferred are antibodies selected from the group consisting of IgG, IgD, IgE, IgA, and / or IgM, or fragments or derivatives thereof.

[0064] As used herein, the term "fragment" refers to fragments of such antibodies that retain target binding ability, e.g., CDRs (complementarity determining regions), hypervariable regions, variable domains (Fv), IgG heavy chains (consisting of VH, CH1, hinge, CH2 and CH3 regions), IgG light chains (consisting of VL and CL regions), and / or Fab and / or F(ab)2.

[0065] As used herein, the term "derivative" refers to protein constructs that are structurally distinct from the typical antibody concept, but still bear some structural relationship, such as scFv, Fab and / or F(ab)2, as well as bi-, tri- or higher order specific antibody constructs, all of which are described below.

[0066] Other antibody derivatives known to those skilled in the art are diabodies, camelid antibodies, domain antibodies, bivalent homodimers with two chains consisting of scFv, IgAs (two IgG structures linked by a J chain and secretory component), Hai antibodies, antibodies consisting of a New World primate scaffold + non-New World primate CDRs, dimerized constructs consisting of CH3 + VL + VH, other scaffold protein formats consisting of CDRs, antibody conjugates.

[0067] As used herein, the term "antibody-like protein" refers to a protein that has been modified (e.g., by mutagenesis of an Ig loop) to specifically bind to a target molecule. Typically, such antibody-like proteins consist of at least one variable peptide loop attached to a protein scaffold at both ends. This dual structural constraint enhances the binding affinity of antibody-like proteins to a level comparable to that of antibodies. The variable peptide loop typically consists of 10 to 20 amino acids in length. The scaffold protein can be any soluble protein. Preferably, the scaffold protein is a small globular protein. Antibody-like proteins include, but are not limited to, affibodies, anticalins, engineered ankyrin proteins, and affilin proteins. Antibody-like proteins can be obtained from large libraries of mutants, for example, by panning from large phage display libraries, and can be isolated by mimicking conventional antibodies. Antibody-like binding proteins can also be obtained by combinatorial mutagenesis of surface-exposed groups of globular proteins. Antibody-like proteins are described, for example, in Binz et al. (2005) and Hosse et al. (2006).

[0068] As used herein, the term "Fab" refers to an IgG fragment containing the antigen-binding region, the fragment consisting of the constant and variable domains of the antibody heavy and light chains, respectively.

[0069] As used herein, the term "F(ab)2" refers to an IgG fragment consisting of two Fab fragments linked by a disulfide bond.

[0070] As used herein, the term "scFv" refers to a single-chain variable fragment that is a fusion of the heavy and light chain variable regions of an immunoglobulin, joined by a short linker, usually consisting of serine (S) and / or glycine (G) residues. This chimeric molecule retains the specificity of the original immunoglobulin despite the removal of the constant region and the introduction of the linker peptide.

[0071] Engineered antibody formats are, for example, bi- or tri-specific antibody constructs, antibody-based fusion proteins, immunoconjugates, and the like.

[0072] IgG, scFv, Fab and / or F(ab)2 are antibody formats well known to those skilled in the art. Detailed descriptions and techniques can be found in relevant textbooks.

[0073] According to a preferred embodiment of the invention, the antibody or antigen-binding fragment or antigen-binding derivative thereof is a murine antibody, a chimeric antibody, a humanized antibody or a human antibody or an antigen-binding fragment or antigen-binding derivative thereof.

[0074] Monoclonal antibodies (mAbs) derived from mice contain proteins from other species that can elicit immune responses and can therefore cause undesirable immunological side effects. To overcome this problem, antibody humanization and maturation methods have been developed to generate antibody molecules that ideally retain the specificity and affinity of the non-human parent antibody while minimizing immunogenicity when used in humans. These methods involve, for example, replacing the scaffold region of a mouse mAb with the corresponding human scaffold region (so-called CDR grafting). WO 200907861 discloses the creation of humanized forms of mouse antibodies by linking the CDR regions of a non-human antibody to human constant regions using recombinant DNA technology. US 6,548,640 describes CDR grafting technology, and US 5,859,205 describes the production of humanized antibodies.

[0075] As used herein, the term "humanized antibody" refers to an antibody, fragment or derivative thereof in which at least a portion of the antibody constant and / or scaffold regions, and optionally some of the CDR regions, are derived from or adapted to human immunoglobulin sequences.

[0076] According to a sixth aspect, the present invention relates to an active agent as defined above, or an antibody, antigen-binding fragment or antigen-binding derivative thereof, or antibody-like protein as defined above, for use in the treatment of chronic kidney disease and / or heart disease.

[0077] The chronic kidney disease in this case is preferably progressive chronic renal failure and / or renal fibrosis. The heart disease is preferably heart failure, myocardial infarction and / or cardiac fibrosis.

[0078] Furthermore, the present invention relates to a pharmaceutical composition comprising an active agent as defined above, or an antibody, antigen-binding fragment or antigen-binding derivative thereof, or antibody-like protein as defined above, and one or more pharmaceutically acceptable excipients for use in treating chronic kidney disease and / or cardiac disease, wherein preferably said chronic kidney disease is progressive chronic kidney disease, renal failure and / or renal fibrosis, and preferably said cardiac disease is heart failure and / or cardiac fibrosis.

[0079] In a preferred embodiment of the invention, the pharmaceutically acceptable excipient is selected from the group consisting of a pharmaceutically acceptable buffer, surfactant, diluent, carrier, filler, binder, lubricant, slip additive, bactericide, adsorbent and / or preservative.

[0080] The pharmaceutical composition can be administered in the form of powder, tablet, pill, capsule or beads.In aqueous form, the pharmaceutical preparation can be administered immediately, but the lyophilized preparation needs to be converted into a liquid form before administration, and can be administered by adding water for injection with or without preservatives such as benzyl alcohol and antioxidants such as vitamin A, vitamin E, vitamin C, vitamin D, etc., antioxidants such as, but not limited to, benzyl alcohol, vitamin A, vitamin E, vitamin C, retinyl palmitate, selenium, amino acids cysteine, methionine, citric acid, sodium citrate, parabens methylparaben, propylparaben, etc.

[0081] The pharmaceutical formulation may further comprise one or more stabilizers, which may be, for example, amino acids, sugar polyols, disaccharides and / or polysaccharides. The pharmaceutical formulation may further comprise one or more surfactants, one or more isotonizing agents and / or one or more metal ion chelating agents and / or one or more preservatives.

[0082] The pharmaceutical formulations described herein are suitable for at least oral, parenteral, intravenous, intramuscular, or subcutaneous administration. Alternatively, the active agents or antibodies according to the invention can be provided in sustained release formulations that allow for sustained release of the active agent over a period of time.

[0083] There is further provided a primary packaging, such as a pre-filled syringe or pen, a vial or an infusion bag, comprising the pharmaceutical formulation according to this aspect of the invention.

[0084] Prefilled syringes or pens can contain the formulation either in lyophilized form (which must be dissolved, e.g., with water for injection, before administration) or in aqueous form. Syringes and pens are often disposable and have a capacity of 0.1 to 20 ml. However, syringes or pens can also be reusable syringes or multi-dose pens.

[0085] Furthermore, the present invention relates to the use of an active agent that binds to the ADAMTS12 protein in a method for treating chronic kidney disease and / or heart disease, wherein the chronic kidney disease is preferably progressive chronic kidney disease, renal failure and / or renal fibrosis, and / or the heart disease is preferably heart failure and / or cardiac fibrosis. Preferably, upon binding to ADAMTS12, the active agent inhibits the activity of ADAMTS12.

[0086] Furthermore, the present invention relates to the use of an active agent that binds to the ADAMTS12 protein for the manufacture of a therapeutic agent for chronic kidney disease and / or cardiac disease, wherein the chronic kidney disease is preferably progressive chronic renal failure and / or renal fibrosis, and the cardiac disease is preferably heart failure and / or cardiac fibrosis. Preferably, upon binding to ADAMTS12, the active agent inhibits the activity of ADAMTS12.

[0087] Furthermore, the present invention relates to a method for treating or preventing chronic kidney disease and / or heart disease, which method comprises administering to a human or animal subject an active agent that binds to and / or inhibits ADAMTS12 protein in a therapeutically effective dose or amount.

[0088] As used herein, the term "effective dose" or "effective amount" refers to the dosage or amount of an active agent required to achieve a desired therapeutic result in a patient, in terms of dosage and administration time. The effective amount may vary depending on factors such as the disease state, the patient's age, sex, and / or weight, pharmaceutical preparation, and the subtype of the disease being treated, but can nevertheless be routinely determined by one skilled in the art.

[0089] According to a seventh aspect, the present invention provides a method for producing an active agent by the method for identifying an active agent that binds to an ADAMTS12 protein or a fragment thereof and / or inhibits or reduces the activity of an ADAMTS12 protein or a fragment thereof, as described above, which further comprises purifying the active agent.

[0090] The present invention further comprises: (i) a method for identifying an active agent that binds to an ADAMTS12 protein or a fragment thereof and / or inhibits or reduces the activity of an ADAMTS12 protein or a fragment thereof, as described above, and further (ii) mixing the identified active agent with a pharmaceutically acceptable carrier.

[0091] According to an eighth aspect, the present invention provides a method for manufacturing a semiconductor device comprising: (i) an active agent as described above, or an antibody, or an antigen-binding fragment, or an antigen-binding derivative thereof, or an antibody-like protein as described above, which binds to an ADAMTS12 protein or a fragment thereof and / or inhibits or reduces the activity of an ADAMTS12 protein or a fragment thereof, or a pharmaceutical composition comprising the active agent as described above, or an antibody, an antigen-binding fragment, or an antigen-binding derivative thereof, or an antibody-like protein as described above, and one or more pharmaceutically acceptable excipients; and (ii) one or more other therapeutically active compounds; and The present invention relates to a composition comprising a combination of:

[0092] According to a ninth aspect, the present invention provides a method for producing a medicament for the treatment of a pulmonary arthritis (i) the pharmaceutical composition described above; (ii) a device for administering the composition; and (iii) optionally, instructions for use; and The present invention relates to a treatment kit comprising:

[0093] Sequence Listing Table 1: Human ADAMTS12, amino acid sequence (UniProt-ID: P58397-1) [Table 1] [Example]

[0094] The present invention will be described in more detail with reference to the following examples and figures, which should be noted that the examples and figures are illustrative and not limiting of the present invention.

[0095] The invention is not limited to the disclosed embodiments. Other variations of the disclosed embodiments can be understood and effected by those skilled in the art in practicing the claimed invention, from a study of the drawings, the disclosure, and the appended claims. In the claims, the word "comprising" does not exclude other elements or steps, and the indefinite articles "a" and "one" do not exclude a plurality. The mere fact that certain measures are recited in different dependent claims does not indicate that a combination of these measures cannot be used to advantage. Reference signs in the claims shall not be construed as limiting the scope of the application.

[0096] All amino acid sequences disclosed herein are shown from the N-terminus to the C-terminus, and all nucleic acid sequences disclosed herein are shown from 5' to 3'.

[0097] Example 1: Materials and Methods mouse: Gli1CreERt2 (JAX Stock #007913) and Rosa26tdTomato (JAX Stock #007909) were purchased from Jackson Laboratories (Bar Harbor, ME, USA). Offspring were genotyped by PCR according to the Jackson Laboratories protocol. ADAMTS12-KO mice were developed by C. Lopez-Otin (El Hour et al., 2010). All mice were genotyped by PCR. Mice were housed under specific pathogen-free conditions at RWTH Aachen University.

[0098] Treatment of mice: For unilateral ureteral obstruction (UUO), the left ureter was ligated at the lower pole level with two 7.0 bands (Ethicon) after a flank incision. For ischemia-reperfusion surgery (IRI), the renal artery was clamped with an aneurysm clamp for 26 minutes after the flank incision. For placebo surgery (Sham), the flank was incised. Mice were sacrificed 10 days after unilateral ureteral surgery or 28 days after the ischemia-reperfusion surgery. The animal experiment protocol was approved by the State Office for Nature, Environment and Consumer Protection of North Rhine-Westphalia (Germany). All animal experiments were performed in accordance with their guidelines. For inducible fate tracing, Gli1CreER;tdTomato mice (8 weeks old) were orally administered tamoxifen three times (10 mg p.o.). Upon tamoxifen administration, Cre recombinase translocates to the cell nucleus in Gli1-expressing cells and cleaves the loxP DNA sequence. Recombination removes the stop codon, and the underlying fluorophore tdTomato is expressed in Gli1-expressing cells. As a result, Gli1-positive cells are genetically labeled after tamoxifen administration. 21 days after tamoxifen administration, UUO or sham surgery was performed, and the mice were sacrificed 10 days after surgery. The sacrificed mice were then resuspended in 0.9 pacifi-sodium NaCl solution. The left heart was perfused with 20 ml of acetaminophen to remove residual blood from the vasculature. Myocardial infarction and sham surgery were performed as previously described (Curaj et al., 2015). Briefly, mice were anesthetized with isoflurane (2-2.5%), intubated, and ventilated with oxygen using a mouse ventilator (Harvard Apparatus, March, Germany). Subcutaneous injections of metamizol (200 μg / g bw) and bupivacaine (2.5 μg / g bw) were administered for analgesia. Local analgesia was achieved by subcutaneous and intercostal injection of methaminazole (bw). After left thoracotomy, either a sham myocardial infarction procedure (no intervention) or a myocardial infarction procedure using anterior ventricular interstitial banding (RIVA) with silk sutures (0-7) was performed. The ribs, muscle layer, and skin incision were then sutured with prolene (0-6). Postoperative analgesia was achieved by administering metamizole dissolved in drinking water (1.25 mg / ml in 1% sucrose) for 3 days.

[0099] Single-cell isolation and fluorescence-activated cell sorting (FACS): Kidneys were surgically removed, cut into small pieces, placed in 15 ml tubes (Falcon), and placed on ice-cold phosphate-buffered saline with 1% fetal calf serum (PBS). The kidney tissue was then transferred to a C-tube (Miltenyi Biotec) and processed using the Spleen 4 program with gentle-MACS (Miltenyi Biotec). The tissue was digested in a digestion solution containing 25 μg / ml Liberase TL (Roche) and 50 μg / ml DNase (Sigma) in RPMI (Gibco) at 37°C for 30 minutes with shaking at 300 RPM. After incubation, the sample was processed again with the gentle-MACS (Miltenyi Biotec) using the same program. The resulting suspension was passed through a 70 μm cell strainer (Falcon), washed with 45 ml of cold PBS, and then centrifuged at 500 g for 5 minutes at 4°C. Trypan blue staining was performed and cells were counted using a hemocytometer. By this method, the overall viability was over 80%. The isolated cells were diluted to 1x10 in PBS containing 1% FBS on ice. 7 The cells were resuspended at a final concentration of 100 cells / ml. Viable single cells were isolated by FACS sorting using a FACS Aria II instrument (Becton Dickinson, Basel, Switzerland) and gating on Gli1-tdTomato-positive, DAPI-negative cells. The average time from biopsy collection to preparation of the single-cell suspension was 5 to 6 hours.

[0100] Analysis of Affymetrix microarray data: Microarray gene expression was quantified using the R package "affy" against the mouse genome database "Mouse4302.db" and normalized using Robust-Multichip Average (RMA-). The R package Limma (v.3.44.1) was used to test for differences in gene expression between UUO and placebo surgery (Sham) using the RunLimma function. When microarray samples mapped to the same gene multiple times, duplicated genes were removed. Differentially expressed genes were ranked according to their T-values. Pathway analysis was performed using the R package "fgsea," using the Hallmark signaling pathways based on all differentially expressed genes.

[0101] RNA in situ hybridization: In situ hybridization (ISH) was performed using formalin-fixed, paraffin-embedded tissue samples and the RNAScope Multiplex Detection Kit V2 (RNAScope, #323100) according to the manufacturer's protocol with minor modifications. Antigen retrieval was performed for 30 minutes. After antigen retrieval, 3 to 5 drops of Pretreatment 1 solution were added and incubated for 10 minutes at room temperature. Three 5-minute wash steps were performed. The following probes were used for the RNAscope assay: Mm-Pdgfrb #411381-C3, Mm-Adamts12 #400531, Hs-PDGFRs #548991-C1, Hs-COL1A1 #401891-C2, and HsADAMTS12 #509701-C3.

[0102] Confocal imaging: Images were taken with a Nikon A1R confocal microscope using 40x and 60x objectives (Nikon). Raw data were processed with Nikon software or ImageJ.

[0103] Image Quantification - ISH Image Analysis: Renal cortex was systematically randomly selected, with at least seven representative tubulointerstitial regions per image. Images were split into RGB channels using ImageJ, background was removed (rolling ball radius: 10.0 pixels), and fluorescent dots (transcripts) were counted. For cell classification, three representative Z-stack images (Z-stack refers to the acquisition of multiple images of the same area using a confocal microscope, taken at a distance between the first and last focal planes) were taken from each sample. The Z-stacks were overlaid as a so-called Z-project and split into RGB channels using ImageJ. Cells were segmented and classified using an algorithm trained using the ilastik object classification workflow (Berg et al., 2019).

[0104] Quantitative RT-PCR: RNA extraction from cultured cells was performed by washing the cells with PBS and then lysing them with RNA-Easy Lysis Buffer. RNA extraction from kidney tissue was performed by transferring the tissue to an Eppendorf tube containing 400 μl of RNA-Easy Lysis Buffer and digesting it using a Mixer-Mill (2x 2 min, 20 Hz). RNA extraction was performed using the RNeasy Mini Kit (Qiagen) according to the manufacturer's instructions. 200 ng of RNA was reverse transcribed using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems). qRT-PCR was performed using iTaq Universal SYBR Green Supermix (Biorad) and a Bio-Rad CFX96 Real Time System with a C1000 Touch thermal cycler. The cycling conditions were as follows: 95°C for 3 minutes, followed by 40 cycles of 95°C for 15 seconds, 60°C for 1 minute, and one cycle of 95°C for 10 seconds. GAPDH was used as a housekeeping gene. Data were analyzed using the 2-CT method. The primers used are listed in Table 2.

[0105] Table 2: RT-PCR primer sequence list (human) [Table 2]

[0106] Immunofluorescence staining including quantification: Formalin-fixed, 2-μm paraffin-embedded kidney sections were used. Slides were blocked with 10% bovine calf serum, then incubated with primary antibodies for 1 hour, washed three times with PBS for 5 minutes, and then incubated with secondary antibodies for 30 minutes. After staining with DAPI (4',6'-diamidino-2-phenylindole) (Roche, 1:10,000), slides were mounted with ImmuMount (9990402, Epredia). Four representative images of the renal cortex per sample were taken using a 40x objective on a Nikon A1R confocal microscope. For quantification, images were split into RGB channels, and the fluorescent area was quantified using ImageJ. The following antibodies were used: anti-mouse PDGFRβ (ab32570, 1:100, Abcam), AF488 donkey anti-rabbit (711-545-152, 1:200, Jackson ImmunoResearch), and AF647 donkey anti-rat (712-605-153, 1:200, Jackson ImmunoResearch).

[0107] Immunohistochemistry including quantification: After deparaffinization, 2 μm paraffin sections were subjected to antigen retrieval by heating three times for 5 minutes in antigen unmasking solution (H-33000, Vector Laboratories). Subsequently, they were incubated with 3% hydrogen peroxide for 3 minutes, avidin / biotin (VEC-SP-2001, Vector Laboratories) for 10 minutes, and then incubated with primary antibody for 1 hour. After three washes with PBS, they were incubated with secondary antibody. Detection was performed using a DAB substrate kit (SK-4100, Vector Laboratories). Finally, sections were counterstained with hematoxylin, dehydrated, and coverslipped. Seven representative images of the renal cortex were taken from each section using a 40x objective on a bright-field microscope (BZ-9000, Keyence, IHC). The following antibodies were used: anti-mouse Col1 (1310-01, 1:100, Southern Biotech), biotinylated horse anti-goat antibody (BA-9500, 1:300, Vector Laboratories).

[0108] Mouse echocardiography Left ventricular function was measured 2 days before, 4 weeks after, and 8 weeks after myocardial infarction using a small animal ultrasound scanner (Vevo 3100 with an MX550D transducer, FUJIFILM Visualsonics, Toronto, Ontario, Canada). Measurements of short and long axes, left ventricular end-diastolic and end-systolic volumes, and heart rate were performed in B-mode (2D real-time) and M-mode using a 40 MHz transducer (MX550D). Mice were anesthetized with 1 to 2% isoflurane during the procedure. All measurements were analyzed using VevoLab software.

[0109] Picrosirius Red Coloring and Quantitation: Picrosirius red staining was performed using the Morphisto-Sirius red staining kit (13425, Morphisto). The entire slide was scanned using an Aperio Slide Scanner (Leica Biosystems), and the fibrotic areas stained red with the Picrosirius kit were quantified using the Aperio eSlide Manager program.

[0110] Human tissue processing: Human kidney tissue was harvested from normal sites as previously described (Kuppe et al., 2021). Tissue was frozen on dry ice or placed in pre-cooled University of Wisconsin solution (#BTLBUW, Bridge to Life Ltd., Columbia, US) and transported to the laboratory on ice. To isolate kidney single cells, a combination of enzymatic and mechanical disruption was used, as described above for mouse single cell isolation.

[0111] FACS of human tissue: Isolated cells were stained and isolated as previously described (Kuppe et al., 2021). Briefly, isolated cells were resuspended in 1% PBS-FBS on ice to a final concentration of 1x10. 7Cells were preincubated with Fc block (TruStainFx human, TruStainFx mouse clone 91, BioLegend) and then incubated with anti-CD10 human antibody (clone HI10a, BioLegend) diluted in 2% FBS / PBS for 30 minutes on ice in the dark. For human anti-PDGFRb staining, goat anti-mouse Dyelight405 (poly24091, BioLegend) was used as the secondary antibody. All compensation was performed at the time of imaging using single-color staining, negative staining, and fluorescence minus 1 controls. Individual cells were enriched by FACS sorting, gated on DAPI-negative cells, and further enriched for fibroblasts by PDGFRs staining. Cells were sorted using a Sony SH800 sorter (Sony Biotechnology; 100 μm nozzle sorting chip, Sony) in semi-pure mode, aiming for an efficiency of 80% or higher.

[0112] 10X Genomics 3' sc-RNA-Seq (V2 and V3) Single Cell Assay: Single-cell assays were performed as previously described. Briefly, single-cell solutions of primary human kidney cells were loaded onto the Chromium Single Cell Chip Kit, and libraries were processed using the Chromium Single Cell 3' Library Kit V2 and i7 Multiplex Kit (PN-120236, PN-120237, PN-120262, 10x Genomics) according to the manufacturer's protocol. Library quality was determined using a D1000 ScreenTape on a 2200 TapeStation system (Agilent Technologies). Sequencing was performed on an Illumina NovaSeq platform using S1 and S2 flow cells (Illumina).

[0113] Microarray from human kidney tissue: Paraffin-embedded kidney microarrays were prepared as previously described (Kuppe et al., 2021). Briefly, paraffin-embedded, formalin-fixed kidney samples from Biomaterialbank Aachen were selected based on previously performed PAS staining. Random areas were selected for each sample, and 2 mm cores were taken from each kidney sample using a TMArrayer™ (Pathology Devices, Beecher Instruments, Westminster, USA). Each core measured approximately 2.5 cm. 2 The tissue was placed in a receiver block with a 2 mm grid covering the tissue, and 5-micron-thick sections were cut and processed using standard histological techniques.

[0114] Generation of human PDGFRb+ cell lines: An immortalized renal human PDGFRb-positive cell line was used for in vitro experiments. The generation of the cell line was described in a previous study ( Kuppe et al., 2021 ).

[0115] TGFb treatment experiment: TGFb (100-21-10UG, Peprotech) was added at a concentration of 10 ng / ml in PBS to 75% confluent PDGFRb cells after 24 hours of incubation in starvation medium (medium containing 0.5% fetal serum).

[0116] sgRNA:CRISPR-Cas9 vector construction, virus production, and transduction: ADAMTS12-specific guide RNA (forwards 5'-CACCGAACATCATAGATCACTCCGG-3'; backwards 5'-AAACCCGGAGTGATCTATGATGTTC-3) The vector was cloned into the pL-CRISPR.EFS.GFP plasmid (Addgene #57818) using BsmBI restriction digestion. Lentiviral particles were generated by transient co-transfection of HEK293T cells with the lentiviral transfer plasmid, packaging plasmid psPAX2 (Addgene #12260), and VSVG packaging plasmid pMD2.G (Addgene #12259) using TransIT-LT (Mirus). Viral supernatants were collected 48 to 72 hours posttransfection, clarified by centrifugation, supplemented with 10% FCS and polybrene (Sigma-Aldrich, final concentration 8 μg / ml), and filtered through a 0.45 μm sieve (Millipore; SLHP033RS). Cell transduction was performed by incubating PDGFRs cells with the viral supernatant for 48 hours. eGFP-expressing cells were individually sorted into 96-well plates. To examine the biallelic mutation events within the cultured clones, PCR products from the ADAMTS12 clones were subcloned into the pCR™ 4Blunt-TOPO vector (Thermo Scientific K287520). At least six colonies per CRISPR clone were grown and sequenced (sequencing of colonies from clone C2:30). qPCR was performed simultaneously to confirm the loss of ADAMTS12 gene expression.

[0117] Retroviral overexpression of ADAMTS12: The ADAMTS12 vector was constructed and a stable ADAMTS12-overexpressing cell line was generated as follows. Human ADAMTS12 cDNA was synthesized by combining two gBlock gene fragments (IDTs): (1) Xho-N-terminus-EcoRI and (2) EcoRI-C-terminus-1xHA-tag-EcoRI. The resulting cDNA was fused to a contiguous CDS with a C-terminal 1xHA tag in the target vector. The C-terminal fragment was codon-optimized due to its high complexity score. Both gBlock gene fragments were first blunt-ended and then ligated into the pSC-B-amp / kan plasmid using the StrataClone Blunt PCR Cloning Kit (#240207), generating vectors (a) pSC_Adamts12_AA1-160 and (b) pSC_Adamts12_AA611-1595-HA. The N-terminal fragment was cloned from the pSC vector using the restriction enzymes XhoI and EcoRI and introduced into the pMIG backbone (Addgene plasmid #9044) to create the plasmid pMIG-Adamts12_AA1-160. The N-terminus was then introduced into the target plasmid by EcoRI restriction cloning ("in-frame" cloning) from the pSC vector. The H465Q-E466A mutation was incorporated using the Q5 Site-Directed Mutagenesis Kit (NEB; #E0554) with primers Mut-H465Q-466A-F: 5'-CACAATTGCCcaagcgCTAGGACAG-3' and Mut-H465-E466A-R: 5'-AAAGCCAGAGGGAGTCCC-3'. The integrated CDS (both WT- and MUT-ADAMTS12) was controlled by sequencing. Retroviral particles were produced by transient transfection using TransIT-LT (Mirus) in combination with the packaging plasmid pUMVC (Addgene plasmid #8449) and the pseudotyped plasmid pMD2.G (Addgene plasmid #12259; http: / / n2t.net / addgene:12259; RRID:Addgene_12259).Viral supernatants were collected 48 to 72 hours after transfection, clarified by centrifugation, and supplemented with 10% FCS and polybrene (Sigma-Aldrich, final concentration 8 μg / ml) and filtered through a 0.45 μm sieve (Millipore; SLHP033RS). Cell transduction was performed by incubating PDGF-seq cells with the viral supernatant for 48 hours. eGFP-expressing cells were purified by fluorescence-activated cell sorting.

[0118] Western Blot: To isolate proteins, cells were lysed in RIPA buffer containing a protease inhibitor cocktail (Roche). The protein concentration of the lysate was determined using the Pierce BCA Protein Assay Kit (#23225, ThermoScientific). Protein lysates adjusted to equal concentrations were then denatured in SDS sample loading buffer (BioRad) at 95°C for 5 minutes and loaded onto a 10% SDS-Page gel. After gel electrophoresis, samples were transferred to PVDF membranes, and blots were probed with primary antibody (anti-HA epitope tag (BioLegend #901533) at 1:2000) in 5% blot (Thermo Fisher) for 2 hours. After washing, the blots were incubated with secondary antibody (horseradish peroxidase-HRP-conjugated anti-mouse antibody, Vector Laboratories) for 1 hour and developed using Pierce™ ECL Western Blotting Substrates A and B. Monoclonal anti-tubulin and goat anti-GFP antibodies (Rockland #600-101-215, 1:2000) were used as loading controls, followed by HRP-conjugated secondary anti-mouse and anti-goat antibodies (Vector Laboratories), respectively.

[0119] Migration Analysis: Cells were seeded on Matrigel-coated 96-well plates (flat-bottom, clear, 89626, ibidi). After 24 hours of incubation in starvation medium (0.5% fetal calf serum), cells reaching 50% confluence were stimulated with 10 ng / ml TGFβ in CO2-independent medium (18045054, Gibco). After 24 hours of stimulation, cellular autofluorescence was recorded every 10 minutes for 18 to 24 hours using a Nikon A1R confocal microscope in a 37°C chamber. Cell segmentation was performed at each time point using the ilastik pixel classification workflow and exported as so-called "prediction maps." Prediction maps from different time points were then aligned and merged by region, and cell coordinates and average velocity were calculated using the ImageJ plugins StackReg and TrackMate. Velocity was weighted according to the length of each track. Cell migration was calculated and displayed graphically using the ggplot2 package in R. The figure shows representative results from a total of three independent experiments.

[0120] Single-cell RNA analysis: Collection and analysis of single-cell RNA data, including transcript alignment, normalization, scaling, dimensionality reduction, and cell annotation, were performed as described ( Kuppe et al., 2021 ). Gene expression analysis of ADAMTS12 was performed using the Seurat package in R.

[0121] Quantification and statistical analysis for non-single cell sequencing and microarray data: Data are presented as mean ± standard deviation unless otherwise specified. Comparisons between two groups were performed using an unpaired t-test. Comparisons between multiple groups were performed using a two-way analysis of variance (ANOVA) with Tukey's multiple comparison test. Statistical analysis was performed using GraphPad Prism 8 (GraphPad Software Inc., San Diego, CA). A p value of less than 0.05 was considered significant.

[0122] Example 2: Overexpression of ADAMTS12 gene in activated fibroblasts after renal injury To identify novel signaling pathways leading to activation of Gli1 fibroblasts, fibroblasts expressing the transcription factor Gli1 (Gli1 fibroblasts) were transfected with Gli1-CreER. (t2) R26tdTomato mice were repeatedly administered tamoxifen and genetically labeled with the fluorescent dye tdTomato. Twenty-five days after tamoxifen administration, unilateral ureteral obstruction (UUO) was performed to induce renal fibrosis, or a sham operation was performed as a control. Ten days after surgery, mice were sacrificed. Gli1 fibroblasts were isolated from UUO or control kidneys using fluorescence-activated cell sorting (FACS), and their RNA transcriptomes were measured using an Affymetrix microarray assay (Figure 1A). Principal component analysis (PCA) verified that activated Gli1 fibroblasts after UUO were distinct from non-activated Gli1 fibroblasts in control kidneys (sham). Gene set enrichment analysis (GSEA) was then performed based on Hallmark Pathways (Figure 1B). GSEA revealed significant increases in the normalized enrichment scores (NES) of pro-inflammatory pathways (inflammatory immune response, IL6-STAT3, NFKB-mediated TNFA), myofibroblast-related pathways (epithelial-mesenchymal transition, TGF-β pathway), and proliferation pathways (G2M checkpoint, mitotic spindle, E2F target). These results suggest that Gli1+ fibroblasts expand and differentiate into myofibroblasts after UUO. Gene expression analysis revealed that inflammatory and extracellular matrix genes were most highly upregulated (Figure 1C). One of the most highly upregulated genes (top 6 by T value) in activated Gli1+ fibroblasts was the ADAMTS12 (A Disintegrin And Metalloproteinase with Thrombospondin motifs 12) gene.

[0123] ADAMTS12 belongs to the ADAMTS metalloprotease family, and its function in the development of fibrosis has remained unknown.

[0124] To verify these new findings, we performed RNA in situ hybridization (ISH) of ADAMTS12 and the fibroblast marker PDGFRb after inducing renal fibrosis by ischemia-reperfusion injury (IRI) (Figure 1D). ISH staining revealed that ADAMTS12 gene expression was minimal under homeostatic conditions, and dramatically increased only in PDGFRb-positive fibroblasts after renal injury (Figure 1E-G).

[0125] Based on these findings, the present inventors were surprised to find that ADAMTS12 was one of the genes whose expression was most strongly upregulated in Gli1 fibroblasts after unilateral ureteral obstruction (UUO).

[0126] Example 3: Knockout of ADAMTS12 suppresses renal and cardiac fibrosis in vivo Based on the obtained microarray data, wild-type mice and ADAMTS12 knockout (KO) mice (Adamts12 - / - Unilateral ureteral obstruction (UUO) was performed in rats. Quantitative real-time PCR (RT-qPCR) was used to measure the gene expression of ADAMTS12 and the extracellular matrix (ECM) proteins collagen 1 (Col1a1) and fibronectin (Fn1) (Figure 2A-C). Gene knockout of ADAMTS12 abolished ADAMTS12 expression at the RNA level (Figure 2A). Furthermore, ADAMTS12 knockout (KO) mice showed significantly reduced gene expression of collagen 1 and fibronectin after UUO. To validate the RT-qPCR results, immunofluorescence staining for the fibroblast marker PDGFRb and immunohistochemical staining for the ECM protein collagen 1 were performed (Figure 2D, 2F). Quantification of PDGFRb revealed that the expansion of PDGFRb fibroblasts was strongly suppressed in ADAMTS12-KO mice after UUO (Figure 2E). Furthermore, collagen 1 deposition was significantly reduced in ADAMTS12-KO mice ( Figure 2G ).

[0127] To analyze whether these results could be applied to the pathology of chronic heart failure and cardiac fibrosis, we induced myocardial infarction (MI) in wild-type and ADAMTS12-KO mice by ligating the interventricular coronary artery. Here, we again confirmed that genetic deficiency of ADAMTS12 led to improved left ventricular ejection fraction (LV-EF) and suppressed fibrosis after MI (Fig. 2H, I).

[0128] In summary, knockout of ADAMTS12 surprisingly suppressed both renal and cardiac fibrosis after organ injury, and even reduced functional decline in the case of myocardial infarction. These results suggest that ADAMTS12 plays an important role in activating Gli1 fibroblasts.

[0129] Example 4: CRISPR-CAS9-mediated knockout of ADAMTS12 inhibits myofibroblast differentiation and migration of human fibroblasts in vitro Based on the in vitro results (Example 3, Figure 2), we next investigated the function of the metalloprotease ADAMTS12 in vitro to determine whether ADAMTS12 is essential for fibroblast expansion and myofibroblast differentiation. Using CRISPR-Cas9, we induced ADAMTS12 knockout (KO) in immortalized human kidney PDGFRb-positive fibroblasts (Figure 3A). To examine the myofibroblast differentiation potential of ADAMTS12-KO fibroblasts and WT fibroblasts, we first stimulated them with transforming growth factor β (TGFb). Here, we confirmed that ADAMTS12 KO reduced the expression of collagen 1A1 (COL1A1), a marker of myofibroblast differentiation (Figure 3A, 3B). A key step in fibroblast activation is fibroblast expansion and migration from the perivascular niche to the interstitium. In a second experiment, the migration of WT and ADAMTS12-KO fibroblasts was analyzed using confocal microscopy. These results indicate that ADAMTS12 deficiency significantly reduces fibroblast migration after TGFb stimulation (Fig. 3C).

[0130] To analyze whether the observed effects of ADAMTS12 were mediated by its metalloprotease domain, catalytically active (wild-type, WT) or inactive (mutant, Mut.) ADAMTS12 was expressed by retroviral transduction of the ADAMTS12-pMIG expression vector in immortalized human kidney PDGFRb-positive fibroblasts in which ADAMTS12 had been "knocked out" using a CRISPR-Cas9 vector as previously described (Figure 3D). Overexpression of catalytically active ADAMTS12 (WT) increased fibroblast migration after activation, whereas overexpression of catalytically inactive ADAMTS12 (Mut.) had no effect on migration (Figure 3E). These results confirm that ADAMTS12 induces fibroblast migration via its metalloprotease domain.

[0131] Thus, CRISPR-Cas9-mediated knockout of ADAMTS12 suppresses myofibroblast differentiation and migration of human fibroblasts in vitro. Catalytically active ADAMTS12 induces migration of human fibroblasts in vitro.

[0132] Example 5: ADAMTS expression by specific fibroblast and myofibroblast populations in the human kidney In the next step, we analyzed ADAMTS12 expression in human kidneys. ADAMTS12 expression was analyzed in a dataset of 15 human kidneys (Kuppe et al., 2021), and CD10-negative cells were individually sequenced (to enrich for interstitial cells). The results showed that ADAMTS12 was specifically expressed in fibroblasts and myofibroblasts, with less expression in pericytes (Figure 4A). These results were confirmed in a second dataset where single-cell sequencing of PDGFRb-positive cells from eight human kidneys was performed (Figure 4B). The single-cell data indicated that a subpopulation of fibroblasts and myofibroblasts expressed ADAMTS12. To validate these results, we further performed in situ hybridization for ADAMTS12, PDGFRb, and collagen 1 (COL1A1) in 43 human kidneys (Figure 4C). This confirmed that ADAMTS12 is primarily produced by PDGFRb-positive fibroblasts (Figure 4D), and ADAMTS12 expression clearly correlated with the expression of the fibroblast marker PDGFRb and the ECM protein collagen 1 (Figures 4E and 4F).

[0133] Example 6: Screening for active agents that bind to and / or inhibit the metalloprotease ADAMTS12 Screening experiments can identify and validate small molecule therapeutic compounds, peptides and / or biologics that bind to and / or inhibit the activity of ADAMTS12 protein.

[0134] DNA-encoded substance libraries are generated and screened as described (Kunig et al. 2018). Additionally, phage display technology (Takakusagi et al. 2020), cell surface display or ribosome display technology (Galan et al. 2016), and / or combinatorial peptide libraries (Bozovicar and Bratkovic 2019) are used. For this purpose, recombinant ADAMTS12 protein or fragments thereof, which may have a tag for labeling, identification, or purification, such as a His tag or FLAG tag, are expressed in bacterial expression systems such as E. coli, or in insect or mammalian cells.

[0135] Purified ADAMTS12 protein is incubated with a substance library and isolated by immunoprecipitation. Compounds that bind to ADAMTS12 protein are identified, for example, by Sanger sequencing of DNA barcodes. The identified drugs and compounds are then tested for their effects on ADAMTS12 function, its protease activity, fibroblast migration, the expression and secretion of matrix proteins such as collagen 1 and fibronectin, and the development of renal and cardiac fibrosis. For this purpose, in vivo experimental mouse models of renal and cardiac fibrosis are used.

[0136] To identify and validate small molecule therapeutic compounds, peptides, and / or biologics that affect ADAMTS12 protease activity or expression, we establish in vitro human cell-based fluorescent dye reporter systems, for example, using eGFP-ADAMTS12 fusion protein expression or luciferase-based reporter systems, and screen substance libraries in 384- to 1536-well assays to identify compounds that reduce eGFP fluorescence or luciferase levels as a readout. Expression of these human ADAMTS12 fusion reporter constructs in these cells can be achieved, for example, by transfection and selection via a resistance gene cassette or by viral transduction. These assays use human cell lines such as 293T cells, as well as established human kidney fibroblast cell lines. In parallel with this screening, we perform cytotoxicity assays to exclude compounds that affect reporter fluorescence or activity through nonspecific toxicity or induction of apoptosis.

[0137] In summary, based on the presented experimental data, ADAMTS12 was first identified as a potential molecular target for fibrosis treatment using a Gli1 fibroblast microarray. In vivo, knockout of ADAMTS12 was shown for the first time to strongly suppress fibroblast migration and fibrosis in UUO and MI mouse models. In vitro, knockout of ADAMTS12 using CRISPR-Cas9 reduced the migration of human renal PDGFRb-positive fibroblasts, whereas overexpression of catalytically active, but not catalytically inactive, ADAMTS12 promoted migration. This confirms that the effects of ADAMTS12 are mediated by the metalloprotease domain of ADAMTS12.

[0138] In the human kidney, ADAMTS12 was shown to be produced specifically by fibroblasts, myofibroblasts, and, to a lesser extent, pericytes. Furthermore, ADAMTS12 expression correlated with the expression of the fibroblast marker PDGFRb and the fibrotic marker collagen 1.

[0139] The metalloprotease ADAMTS12 has been little studied, and there are no reports of its involvement in the pathogenesis of renal or cardiac fibrosis. Although some studies have shown that ADAMTS12 is a negative regulator of angiogenesis (E.I. Hour et al., 2010), other research groups have reported that ADAMTS12 regulates immune responses and that knockout of ADAMTS12 leads to prolonged inflammatory immune responses (Moncada-Pazos et al., 2018; Paulissen et al., 2012).

[0140] The metalloprotease ADAMTS12 is a particularly attractive molecular target for the treatment of fibrosis. ADAMTS12 is homeostatically expressed to a low or no extent. Upon induction of renal fibrosis, ADAMTS12 expression is specifically upregulated in fibroblasts, pericytes, and myofibroblasts. The cell-specific expression of ADAMTS12 and its homeostatic low or absent expression suggest that inhibition of ADAMTS12 has few side effects. Furthermore, inhibition of the metalloprotease ADAMTS12 provides a clear starting point for drug development from a biochemical perspective.

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Claims

1. 1. A method for reducing the expression and / or secretion of extracellular matrix (ECM) proteins by a given cell and / or for inhibiting fibroblast migration, comprising: The method comprises: (i) inhibiting or reducing the expression of the ADAMTS12 gene in said cell; (ii) inhibiting or reducing ADAMTS12 activity; (iii) inhibiting or reducing ADAMTS12 protease activity, and / or (iv) promoting the degradation of ADAMTS12 protein; The method comprises at least one step selected from the group consisting of:

2. The method of claim 1, wherein the inhibition or reduction of expression of the ADAMTS12 gene comprises knockdown, knockout, conditional gene knockout, gene modification, RNA interference, siRNA and / or antisense RNA of the ADAMTS12 gene.

3. 2. The method of claim 1, wherein the inhibition or reduction of ADAMTS12 activity comprises the use of an active agent that binds to the ADAMTS12 protein (A Disintegrin And Metalloproteinase with ThromboSpondin motifs 12 protein).

4. 4. The method of any one of claims 1 to 3, wherein the cells are renal or cardiac cells, preferably renal or cardiac fibroblasts, renal or cardiac myofibroblasts, or renal or cardiac pericytes, most preferably renal or cardiac fibroblasts.

5. A method for identifying active agents that bind to and / or inhibit or reduce the activity of ADAMTS12 protein (A Disintegrin And Metalloproteinase with ThromboSpondin motifs 12 protein) or a fragment thereof.

6. (i) providing the ADAMTS12 protein or a fragment thereof; (ii) adding at least one active agent to be analyzed for binding to said ADAMTS12 protein or fragment thereof; and (iii) identifying the at least one active agent bound to the ADAMTS12 protein or fragment thereof; The method of claim 5 , comprising at least the steps of:

7. 7. The method of any one of claims 5 to 6, wherein the active agent is an ADAMTS12 inhibitor.

8. 8. The method of any one of claims 5 to 7, wherein the active agent is a member of a compound library.

9. 9. The method of any one of claims 5 to 8, wherein the active agent is selected from the group consisting of small molecule compounds, peptides, and biological agents.

10. 10. The method of claim 9, wherein the biological agent is an antibody, an antigen-binding fragment thereof, an antigen-binding derivative thereof, an antibody-like molecule, or an aptamer.

11. 11. The method of any one of claims 5 to 10, wherein the ADAMTS12 protein is bound to a solid phase or is in solution.

12. Use of a nucleic acid encoding the ADAMTS12 protein or a fragment thereof, or use of the ADAMTS12 protein or a fragment thereof, in a method for identifying an active agent that binds to the ADAMTS12 protein or a fragment thereof, according to any one of claims 5 to 11.

13. An active agent obtainable by the method of any one of claims 5 to 11.

14. An active agent that inhibits or reduces the expression of said ADAMTS12 gene in kidney cells or heart cells, preferably wherein said kidney cells are kidney fibroblasts and / or said heart cells are cardiac fibroblasts.

15. An active agent that binds to the ADAMTS12 protein (A Disintegrin And Metalloproteinase with ThromboSpondin motifs 12 protein) or a fragment thereof, and / or inhibits or reduces the activity of the ADAMTS12 protein or a fragment thereof, and / or promotes the degradation of the ADAMTS12 protein.

16. 16. The active agent of any one of claims 13 to 15, wherein the active agent is a small molecule compound (smol), a peptide or a biological agent, preferably the biological agent is an antibody or a fragment thereof, a derivative thereof, an antibody-like protein, or an aptamer.

17. An antibody, or an antigen-binding fragment or derivative thereof, or an antibody-like protein, that specifically binds to the ADAMTS12 protein.

18. 18. The antibody, or antigen-binding fragment or derivative thereof, or antibody-like protein of claim 17, wherein the antibody, or antigen-binding fragment or derivative thereof, or antibody-like protein inhibits ADAMTS12 activity.

19. An active agent according to any one of claims 13 to 16, or an antibody, antigen-binding fragment or antigen-binding derivative thereof, or antibody-like protein according to any one of claims 17 to 18, for use in the treatment of chronic kidney disease and / or heart disease.

20. 20. The active agent, or antibody, antigen-binding fragment or antigen-binding derivative thereof, or antibody-like protein for use according to claim 19, wherein the chronic kidney disease is progressive chronic renal failure and / or renal fibrosis, and / or the cardiac disease is heart failure and / or cardiac fibrosis.

21. 1. Use of an active agent that binds to ADAMTS12 protein in a method for treating chronic kidney disease and / or heart disease, wherein the chronic kidney disease is preferably progressive chronic kidney disease, renal failure and / or renal fibrosis, and the heart disease is preferably heart failure and / or cardiac fibrosis.

22. Use of an active agent that binds to ADAMTS12 protein for the manufacture of a therapeutic agent for chronic kidney disease and / or heart disease, wherein the chronic kidney disease is preferably progressive chronic renal failure and / or renal fibrosis, and the heart disease is preferably heart failure and / or cardiac fibrosis.

23. 23. The use of any one of claims 21 to 22, wherein the active agent inhibits ADAMTS12 activity upon binding to ADAMTS12.

24. A method for treating or preventing chronic kidney disease and / or heart disease, comprising administering to a human or animal subject a therapeutically effective amount of an active agent that binds to and / or inhibits ADAMTS12 protein.

25. 12. A method of producing an active agent by the method of any one of claims 5 to 11, further comprising purifying the active agent.

26. 19. A pharmaceutical composition comprising an active agent according to any one of claims 13 to 16, or an antibody, or antigen-binding fragment or derivative thereof, or antibody-like protein according to any one of claims 17 and 18, and one or more pharmaceutically acceptable excipients, for use in the treatment of chronic kidney disease and / or cardiac disease, wherein preferably said chronic kidney disease is progressive chronic kidney disease, renal failure and / or renal fibrosis, and preferably said cardiac disease is heart failure and / or cardiac fibrosis.

27. 27. The pharmaceutical composition of claim 26, wherein the excipient is selected from the group consisting of a pharmaceutically acceptable buffer, surfactant, diluent, carrier, filler, binder, lubricant, glidant additive, bactericide, adsorbent and / or preservative.

28. (i) a method according to any one of claims 5 to 11; and (ii) mixing the identified active agent with a pharmaceutically acceptable carrier; 1. A method for preparing a pharmaceutical composition comprising:

29. (i) an active agent that binds to an ADAMTS12 protein according to any one of claims 13 to 16, an antibody, or antigen-binding fragment or derivative thereof, or antibody-like protein according to any one of claims 17 to 18, or a pharmaceutical composition according to any one of claims 26 to 27; (ii) one or more other therapeutically active compounds; A composition comprising a combination of:

30. (i) a pharmaceutical composition according to any one of claims 26, 27 and 29; (ii) a device for administering the composition; and (iii) optionally, instructions for use; and A treatment kit comprising:

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