Fusion protein for preventing, treating or improving kidney diseases

Abstract

The present invention relates to a protein for use in preventing, treating or improving kidney diseases or kidney injuries, and more specifically, to a case where the protein is a follistatin fusion protein.

Description

【Technical Field】 【0001】 The present invention relates to a protein for use in preventing, treating or improving kidney diseases or kidney injuries, and more specifically, to the case where the protein is a follistatin fusion protein. 【Background Art】 【0002】 Chronic kidney disease (CKD) affects approximately 10-13% of the elderly population worldwide and is associated with significant mortality and morbidity. It is estimated that 1.5-2% of people with CKD progress to end-stage kidney disease (ESKD), and the two main causes of ESKD are type 2 diabetes and hypertension that still progress despite medical management. Over the past 20 years, treatment options for CKD have relied on blood pressure and blood sugar control, but they are extremely inadequate in long-term disease management. The treatment of ESKD depends on dialysis and kidney transplantation, both of which have relatively unfavorable outcomes for patients with progressive forms of the disease. There is an urgent need for new treatment methods, especially those incorporating novel mechanisms (Yahr et al (2022); Ammirati et al (2020)). 【0003】 A common feature of kidney dysfunction is the accumulation of fibrotic areas within the tissue, which accumulates gradually over time and can ultimately lead to a progressive impairment of the normal function of the organ. Fibrotic diseases are diseases characterized by an abnormal wound healing response in which excessive fibrotic connective tissue is formed in an organ or tissue. The deposition and accumulation of excessive extracellular matrix components such as collagen and fibronectin can lead to tissue hardening and scarring, cause pathological remodeling of the organ, and ultimately lead to organ failure (Wynn & Ramalingam (2012)). 【0004】 Follistatin is a secreted glycoprotein that has as its primary function the binding and neutralization of members of the TGF-β superfamily, particularly activin. It is known to exist in several different forms, including a 315 amino acid polypeptide (referred to as FST315), and a 288 amino acid polypeptide (referred to as FST288). Both FST315 and FST288 have high affinity for activin (activin A and activin B) as well as myostatin. In particular, follistatin can bind to and inhibit myostatin, a negative regulator of skeletal muscle mass. Follistatin is a protein of interest that can be used by itself or as a fusion moiety of a fusion protein in the treatment of several conditions, including muscle disorders such as muscular dystrophy (WO 2015 / 187977 and WO 2017 / 152090), or diseases or disorders such as inflammatory bowel disease, ulcerative colitis and Crohn's disease (WO 03 / 006057). 【0005】 However, there remains a need to identify effective therapies for use in the treatment and prevention of kidney diseases or kidney injury. 【SUMMARY OF THE INVENTION】 【0006】 In a first aspect, the present invention provides a follistatin fusion protein for use in the treatment, amelioration or prevention of a kidney disease such as chronic kidney disease (CKD), or kidney injury, the follistatin fusion protein comprising: a. a follistatin moiety; b. an antibody moiety; and optionally c. a linker between the follistatin moiety and the antibody moiety. 【0007】 In a second aspect, the present invention relates to a method of treating a patient in need of treatment for the treatment, amelioration or prevention of a kidney disease such as chronic kidney disease (CKD), or kidney injury, the method comprising administering a therapeutically effective amount of a follistatin fusion protein, the follistatin fusion protein comprising: a. a follistatin moiety; b. an antibody moiety; and optionally c. a linker between the follistatin moiety and the antibody moiety. 【0008】 In a third aspect, the present invention is directed to the use of a follistatin fusion protein in the manufacture of a medicament for treating, ameliorating or preventing a renal disease such as chronic kidney disease (CKD), or renal injury, wherein the follistatin fusion protein comprises a. a follistatin portion, b. an antibody portion, and optionally c. a linker between the follistatin portion and the antibody portion. 【0009】 In particular, 1) the renal disease is preferably a chronic kidney disease (CKD) such as primary glomerulonephritis, secondary glomerulonephritis, diabetic nephropathy, hypertensive nephrosclerosis, focal segmental glomerulosclerosis (FSGS), IgA nephropathy (IgAN), mesangial proliferative glomerulonephritis, membranous nephropathy (MN), minimal change disease (MCD), polycystic kidney disease, chronic allograft nephropathy, bone mineral metabolism disorders associated with CKD (CKD-MBD), and Goodpasture's disease, and 2) the renal injury is a) renal fibrosis or b) associated with other diseases such as diabetes, hypertension, cardiovascular disease or bone disorders. The renal disease or renal injury can be the result of a therapeutic treatment such as treatment with chemotherapy. BRIEF DESCRIPTION OF THE DRAWINGS 【0010】 【Figure 1】 Figure 1 is a schematic diagram showing FST288 and FST315 molecules aligned with bound activin molecules. 【Figure 2A】 Follistatin modulation in an adriamycin-driven chronic kidney disease model test in mice by delivery of a therapeutic protein using hydrodynamic transfection is shown. In Figure 2A, the renal hydroxyproline content was determined at the end of the test using naïve = no disease, disease and saline treatment = saline, and disease and follistatin treatment = follistatin as a surrogate for renal collagen accumulation and fibrosis. 【Figure 2B】Shows follistatin regulation in a mouse model of adriamycin-induced chronic kidney disease by delivery of therapeutic proteins using hydrodynamic transfection. In Figure 2B, serum creatinine was measured at the end of the test using disease-free = naive, disease and saline treatment = saline, and disease and follistatin treatment = follistatin as a surrogate for renal function. 【Figure 2C】 Shows follistatin regulation in a mouse model of adriamycin-induced chronic kidney disease by delivery of therapeutic proteins using hydrodynamic transfection. Figure 2C is the conditional survival rate showing the duration of the test and the condition of the animals at the end of the test, and all surviving animals that survived the disease ended in either the disease group treated with saline or follistatin. 【Figure 2D】 Shows follistatin regulation in a mouse model of adriamycin-induced chronic kidney disease by delivery of therapeutic proteins using hydrodynamic transfection. Figure 2D is picrosirius red (PSR) staining as a surrogate for the collagen content of selected kidneys, showing one kidney from the naive group, three separate kidneys from the disease group treated with saline, and three separate kidneys from the disease group treated with follistatin. 【Figure 2E】 Shows follistatin regulation in a mouse model of adriamycin-induced chronic kidney disease by delivery of therapeutic proteins using hydrodynamic transfection. In Figure 2E, all kidneys from the test were stained with PSR and analyzed to determine the relative area percentages of collagen in three different groups of mice: disease-free = naive, disease and saline treatment = saline, and disease and follistatin treatment = follistatin; statistics were performed by comparing the saline group and the follistatin-treated group using ordinary one-way ANOVA with Tukey's multiple comparison test; **** represents p < 0.0001. Group sizes were naive n = 8, saline n = 21, and follistatin n = 18. 【Figure 3A】Comparison of different follistatin moieties for the modulation of the unilateral ureteral obstruction model (UUO) of acute kidney disease in mice using hydrodynamic transfection with standard methods and commercially available plasmids for the delivery of therapeutic proteins. Figure 3A shows picrosirius red (PSR) staining as a surrogate for collagen content of selected kidneys from UUO disease groups treated with one kidney without disease (no surgery) = Non-Op, as well as control plasmid, secreted alkaline phosphatase = SEAP control, plasmid containing FST315(HBM)-Fab, and plasmid containing FST315-Fab. 【Figure 3B】 Comparison of different follistatin moieties for the modulation of the unilateral ureteral obstruction model (UUO) of acute kidney disease in mice using hydrodynamic transfection with standard methods and commercially available plasmids for the delivery of therapeutic proteins. In Figure 3B, all kidneys from the study were stained with PSR and analyzed to determine the relative area percentage of collagen for the different groups described in Figure 3A; statistics were performed using ordinary one-way ANOVA with Tukey's multiple comparison test, where NS indicates non-significance and *p < 0.05. 【Figure 3C】 Comparison of different follistatin moieties for the modulation of the unilateral ureteral obstruction model (UUO) of acute kidney disease in mice using hydrodynamic transfection with standard methods and commercially available plasmids for the delivery of therapeutic proteins. Figure 3C shows the change in body weight of mice in different groups; control plasmid (SEAP), FST315(HBM)-Fab or FST315-Fab plasmid. Group sizes, no surgery n = 4, Seap control n = 8, FST315(HBM)-Fab n = 7, FST315-Fab n = 7 【Figure 4A】Comparison of FST315-Fab and FST315(HBM)-Fab, the follistatin protein moieties, with the latter in dose response, for the regulation in a unilateral ureteral obstruction model (UUO) of acute kidney disease in mice by delivery of therapeutic proteins using subcutaneous administration directly. Figure 4A shows the morphological representation at low magnification of whole kidneys stained with PSR from a UUO mouse study showing kidneys without disease or not surgically treated on the left panel = normal, and two UUO kidneys, the central panel shows UUO diseased kidneys treated with vehicle = UUO at day 21, and the right panel shows UUO kidneys treated with 10 mg / kg / day of FST315(HBM)-Fab. Associated histological scores are also shown. 【Figure 4B】 Comparison of FST315-Fab and FST315(HBM)-Fab, the follistatin protein moieties, with the latter in dose response, for the regulation in a unilateral ureteral obstruction model (UUO) of acute kidney disease in mice by delivery of therapeutic proteins using subcutaneous administration directly. Figure 4B is a morphological representation at high magnification of kidney sections stained with PSR showing UUO diseased kidneys treated with vehicle (upper panel) and UUO diseased kidneys treated with 10 mg / kg of FST315(HBM)-Fab (lower panel). 【Figure 4C】 Comparison of FST315-Fab and FST315(HBM)-Fab, the follistatin protein moieties, with the latter in dose response, for the regulation in a unilateral ureteral obstruction model (UUO) of acute kidney disease in mice by delivery of therapeutic proteins using subcutaneous administration directly. Figure 4C is a morphological representation at medium magnification of kidney sections stained with PSR showing representative normal kidneys (Non-op) and representative UUO diseased kidneys treated with vehicle or 2 mg / kg of FST315-Fab, 10, 2, 0.2, and 0.02 mg / kg of FST315(HBM)-Fab. 【Figure 4D】Comparison of the follistatin protein moieties FST315-Fab and FST315(HBM)-Fab, with the latter as a dose response, for the modulation in a unilateral ureteral obstruction model (UUO) of acute kidney disease in mice by delivery of a therapeutic protein using subcutaneous administration directly. In Figure 4D, all kidneys from the study were stained with PSR and scanned to determine the relative area percentage of collagen in different groups of UUO-affected kidneys treated with non-operated or healthy kidneys = Non Op (n = 4), vehicle (n = 8), 2 mg / kg 315FST-Fab (n = 8), 10 mg / kg 315FST(HBM)-Fab (n = 6), 2 mg / kg 315FST(HBM)-Fab (n = 8), 0.2 mg / kg 315FST(HBM)-Fab (n = 8) and 0.02 mg / kg 315FST(HBM)-Fab (n = 8); statistics were performed using ordinary one-way ANOVA with Fisher's LSD multiple comparison test without correction, NS indicates non-significance, *p < 0.05, and ****p < 0.0001. 【Figure 4E】Comparison of the follistatin protein moieties FST315-Fab and FST315(HBM)-Fab, with the latter as a dose response, for the regulation in a unilateral ureteral obstruction model (UUO) of acute kidney disease in mice by delivery of therapeutic proteins using direct subcutaneous administration. In Figure 4E, all PSR-stained kidneys of different groups were manually scored on a histological damage scale of 1 - 10, with 1 representing a normal kidney and 10 representing the most affected kidney, taking into account especially the integrity of the epithelium, as shown in Figures 4A - C. The groups were UUO-affected kidneys treated with non-operated or healthy kidneys = Non Op (n = 4), vehicle (n = 8), 2 mg / kg 315FST-Fab (n = 8), 10 mg / kg 315FST(HBM)-Fab (n = 6), 2 mg / kg 315FST(HBM)-Fab (n = 8), 0.2 mg / kg 315FST(HBM)-Fab (n = 8) and 0.02 mg / kg 315FST(HBM)-Fab (n = 8); statistics were performed using ordinary one-way ANOVA with Fisher's LSD multiple comparison test without correction, where NS indicates non-significance, *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001. 【Figure 4F】 Comparison of the follistatin protein moieties FST315-Fab and FST315(HBM)-Fab, with the latter as a dose response, for the regulation in a unilateral ureteral obstruction model (UUO) of acute kidney disease in mice by delivery of therapeutic proteins using direct subcutaneous administration. In Figure 4F, mice of different groups described in Figures 4A - E were monitored for changes in body weight over the survival period and this was plotted over time for different groups; UUO treated with non-surgery and vehicle, 2 mg / kg of 315FST-Fab or 10, 2, 0.2 or 0.02 mg / kg of FST315(HBM)-Fab. 【Figure 5A】 Figure 5A shows the principle of the assay described in Example 4. 【Figure 5B】Figure 5B shows that adriamycin treatment induced disruption of barrier integrity as measured by increased permeability to FITC-Dx, and protection was obtained in the presence of FST315(HBM)-Fab but not in the Fab control. 【Figure 6A】 In Figure 6A, the relative expression levels of follistatin fusion proteins were determined by HPLC analysis of protein G from the recovered CHO supernatants. Values are normalized to the FST-Fc expression level. N-Fab-FST = Fab antibody portion fused to the N-terminal portion of FST; FST-Fab-C = Fab antibody portion fused to the C-terminal portion of FST; FST-Fc = Fc antibody portion fused to the C-terminal portion of FST; FST-ScFv = ScFv antibody portion fused to the C-terminal portion of FST. 【Figure 6B】 Figure 6B shows the relative expression levels of FST288, FST315, FSTWT, and FST-HBM (or named HBSM) (named 288 fusion, 315 fusion, WT fusion, and HBSM fusion, respectively, in the figure) having C-terminal fusion partners. Values are normalized to the expression of the 288 fusion. 【Figure 6C】 Figure 6C shows the total expression values of the monomers, with values normalized to the FST-Fc expression level, and n = 25. The same explanation as in Figure 2A. 【Figure 7A】 Figure 7A monitored the pharmacokinetic profiles of FST315WT, FST315-Fab, and FST315HBM-Fab administered to mice (n = 3 mice per group) at 10 mg / kg via the IV administration route and serum samples for 7 days. 【Figure 7B】 Figure 7B monitored the pharmacokinetic profiles of FST288WT, FST288-Fab, and FST288HBM-Fab administered to mice (n = 3 mice per group) at 10 mg / kg via the IV administration route and serum samples for 7 days. Pharmacokinetic parameters derived from all follistatin portions of the study are summarized in Table 1. 【Mode for Carrying Out the Invention】 【0011】 The entire document is intended to be associated as a unified disclosure, and it should be understood that all combinations of the features described herein are contemplated, even if the combinations of features are not found together in the same sentence, paragraph, or section of the document. With respect to an aspect of the invention described or claimed as "one," these terms are to be understood to mean "one or more" unless the context clearly requires a more limited meaning. The term "or" is to be understood to encompass alternative or combined items unless the context clearly requires otherwise. Where an aspect of the invention is described as "comprising" a feature, embodiments are also contemplated that "consist of" or "consist essentially of" that feature. 【0012】 As used herein, the terms " follistatin" or "FST" refer to the autocrine glycoprotein (UniProt reference number: P19883), a known inhibitor of activin A and B. Follistatin also binds with lower affinity to GDF11, GDF8 (myostatin), BMP 2, 4, 6, 7, 11 and 15. There are two main alternative splicing forms of human follistatin, a shorter form (FST288, 31.6 kDa) that binds to cells and a longer circulating form (FST315, 34.8 kDa). FST315 is defined according to SEQ ID NO: 1 and FST288 is defined according to SEQ ID NO: 2. FST315 and 288 have four domains stabilized by a network of disulfide bonds (18 in total), two N-linked glycosylation sites and one heparin binding site. FST315 has an additional 27 amino acid domain (rich in acid) at its C-terminus called the acid tail. Functional fragments and / or functional variants thereof, such as those disclosed in Sidis et al., 2005, are also included in this term. When a number follows, for example in the case of FST288, this indicates that the protein is in the 288 form of follistatin (starting from residue 1 of the mature form). When a number and a letter follow, for example in the case of FST315HBM, this indicates the heparin binding mutant (HBM) form as well as the type of mutant (here, the 315 form of follistatin starting from residue 1 of the mature form and including alanine mutations at residues K76, K81 and K82). Activin is a dimeric polypeptide growth factor and belongs to the TGF-β superfamily. Activin, depending on the cell type, stimulates hormone production in ovarian and placental cells, supports the survival of neuronal cells, and can have positive or negative effects on cell cycle progression. In some tissues, activin signaling is antagonized by its associated heterodimer, inhibin. For example, during the release of follicle-stimulating hormone (FSH) from the pituitary gland, activin promotes the secretion and synthesis of FSH, while inhibin inhibits the secretion and synthesis of FSH. Activin is also involved as a negative regulator of muscle mass and function, and activin antagonists can promote muscle growth or counteract muscle loss in vivo. 【0013】 As used herein, the term "antibody" includes, but is not limited to, monoclonal antibodies, polyclonal antibodies, and recombinant antibodies produced by recombinant techniques known in the art. The term "antibody" as used herein includes antibodies of any species, particularly mammalian species; for example, antibodies produced as dimers of this basic structure including IgG1, IgG2a, IgG2b, IgG3, IgG4, IgE, IgD, as well as IgGA1, IgGA2 or pentamers such as IgM, and modified variants thereof; human antibodies of any isotype; for example, non-human primate antibodies derived from chimpanzee, baboon, rhesus monkey or cynomolgus monkey; for example, rodent antibodies derived from mouse or rat; rabbit, goat or horse antibodies; camelid antibodies (such as those derived from camel or llama, e.g., Nanobodies™) and derivatives thereof; avian antibodies such as chicken antibodies; or fish antibodies such as shark antibodies. The term "antibody" refers to both glycosylated and non-glycosylated antibodies. Further, the term "antibody portion" as used herein is intended to refer to a full-length antibody, but more generally to an antibody fragment, more specifically to its antigen-binding fragment. An antibody fragment contains at least one heavy or light chain immunoglobulin domain known in the art and binds to one or more antigens. Examples of antibody fragments according to the present invention include Fab, modified Fab, Fab', modified Fab', F(ab')2, Fv, Fab-Fv, Fab-dsFv, Fab-Fv-Fv, scFv and Bis-scFv fragments. Said fragments can also be diabodies, tribodies, triabodies, tetrabodies, minibodies, single domain antibodies (dAb), e.g., sdAb, VL, VH, VHH or camelid antibodies (such as those derived from camel or llama, e.g., Nanobody™) and VNAR fragments. Antigen-binding fragments according to the present invention can also comprise a Fab linked to one or two scFv or dsscFv, each scFv or dsscFv binding to the same or a different target (e.g., one scFv or dsscFv binding to a therapeutic target and one scFv or dsscFv increasing the half-life by binding, e.g., to albumin). 【0014】 As used herein, the term "Fab" refers to a light chain fragment comprising a VL (variable light chain) domain and a constant domain (CL) of the light chain, as well as an antibody fragment comprising a VH (variable heavy chain) domain and a first constant domain (CH1) of the heavy chain. 【0015】 As used herein, the term "Fab'" is similar to Fab, with the Fab portion replaced by Fab'. The format may be provided as its PEGylated version. The dimer of Fab' according to the present disclosure generates F(ab')2, where, for example, dimerization may occur via a hinge. 【0016】 The term "Fv" refers to two variable domains of a full-length antibody, for example, a cognate pair or an affinity matured variable domain, i.e., a cooperating variable domain pair such as a VH and VL pair. 【0017】 As used herein, the term "single-chain variable fragment" or "scFv" refers to a single-chain variable fragment stabilized by a peptide linker between the VH variable domain and the VL variable domain. 【0018】 As used herein, the term "single-domain antibody" refers to an antibody fragment consisting of a single monomeric variable domain. Examples of single-domain antibodies include VH or VL or VHH or V-NAR. 【0019】 As used herein, the term "affinity" refers to the strength of all non-covalent interaction between a protein or fragment thereof and its receptor (where the protein of interest is a ligand) or its ligand (where the protein of interest is a receptor). Unless otherwise indicated, as used herein, the term "binding affinity" refers to the intrinsic binding affinity that reflects the 1:1 interaction between members of a binding pair (e.g., a receptor and its ligand). The affinity of a molecule for its binding partner can generally be represented by the dissociation constant (KD). Affinity can be measured by common methods known in the art, including those described herein. 【0020】 As used herein in connection with antibodies and antigen-binding fragments, the term "specific" is intended to refer to an antibody that recognizes only the antigen to which it is specific, or an antibody that has a significantly higher binding affinity, e.g., at least 5, 6, 7, 8, 9, 10-fold higher binding affinity, for the antigen to which it is specific as compared to binding to an antigen to which it is non-specific. 【0021】 The term "chimeric" refers to an antibody in which at least a first portion of the heavy chain and / or light chain antibody sequence is derived from a first species and a second portion of the heavy chain and / or light chain antibody sequence is derived from a second species. Chimeric antibodies of interest herein include "primatized" antibodies that include variable domain antigen-binding sequences derived from non-human primates (e.g., Old World monkeys such as baboons, rhesus monkeys or cynomolgus monkeys) and human constant region sequences. 【0022】 A "humanized" antibody is a chimeric antibody that contains sequences derived from non-human antibodies. In most cases, a humanized antibody is a human antibody (recipient antibody) in which residues from the hypervariable regions of the recipient have been replaced with residues from the hypervariable regions [or complementarity-determining regions (CDRs)] of a non-human species, such as a mouse, rat, rabbit, chicken, or non-human primate (donor antibody), that have the desired specificity, affinity, and activity. In most cases, residues outside the CDRs, i.e., residues of the human (recipient) antibody within the framework regions (FRs), are further replaced by the corresponding non-human residues. Additionally, a humanized antibody may contain residues not found in the recipient antibody or the donor antibody. These modifications are made to further improve antibody properties. Humanization reduces the immunogenicity of non-human antibodies in humans and thus facilitates the application of antibodies to the treatment of human diseases. Humanized antibodies and several different techniques for making them are well known in the art. Unless otherwise indicated, HVR residues (CDR residues) and other residues within the variable domain (e.g., FR residues) are numbered herein according to Kabat et al. 【0023】 The term "antibody" also refers to human antibodies that can be made as an alternative to humanization. For example, it is possible to generate transgenic animals (e.g., mice) that, upon immunization, can produce a complete repertoire of human antibodies in the absence of production of endogenous mouse antibodies. Other methods for obtaining human antibodies / antibody fragments in vitro are based on display technologies such as phage display or ribosome display technologies, and recombinant DNA libraries that are at least partially artificially made or made from the immunoglobulin variable (V) domain gene repertoire of a donor are used. Phage and ribosome display technologies for making human antibodies are well known in the art. Human antibodies can also be made from isolated human B cells that can be ex vivo immunized with an antigen of interest and then fused to generate hybridomas, which can then be screened for optimal human antibodies. 【0024】 As used herein, the term "functional variant" refers to an amino acid sequence that has been modified as compared to a reference sequence but retains at least one biological function of the reference sequence. For example, a functional variant of FST retains at least one biological activity of the reference FST protein, such as the binding and inhibition of activin A and B. 【0025】 As used herein, the term "sequence identity" or "identity" refers to the number of matches (identical nucleic acid or amino acid residues) at corresponding positions from an alignment of two polynucleotide or polypeptide sequences. Sequence identity is determined by comparing the sequences when aligned so as to maximize matches and identity while minimizing sequence gaps. In particular, sequence identity can be determined using any of several mathematical global or local alignment algorithms, depending on the lengths of the two sequences being compared. Alignment for determining nucleic acid or amino acid sequence identity percentages can be achieved in a variety of ways within the skill in the art, using publicly available computer software available on Internet websites such as, for example, http: / / blast.ncbi.nlm.nih.gov / or http: / / www.ebi.ac.uk / Tools / emboss / . One of ordinary skill in the art can determine appropriate parameters for measuring alignment, including any algorithm necessary to achieve the maximum alignment over the full length of the sequences being compared. For the purposes of this specification, nucleic acid or amino acid sequence identity % values refer to values generated using the pairwise sequence alignment program EMBOSS Needle, which creates an optimal global alignment of two sequences using the Needleman-Wunsch algorithm, and all search parameters are set to default values, i.e., score matrix = BLOSUM62, gap open = 10, gap extend = 0.5, end gap penalty = false, end gap open = 10, and end gap extend = 0.5. 【0026】 The term "isolated" throughout this specification means that an antibody, antigen-binding fragment, polypeptide or polynucleotide is present in a physical environment that may in some cases be different from what may occur in nature. The term "isolated" nucleic acid refers to a nucleic acid molecule that has been isolated from its natural environment or is synthetically produced. Isolated nucleic acids can include, for example, synthetic DNA, cDNA, genomic DNA generated by chemical treatment, or any combination thereof. 【0027】 As used herein, the term "pharmaceutically acceptable" means approved by a regulatory agency or a widely recognized pharmacopoeia such as the European Pharmacopoeia for use in animals and / or humans. The term "excipient" refers to a diluent, adjuvant, carrier, and / or vehicle administered together with a therapeutic agent. 【0028】 As used herein, terms such as "treat", "treating", etc. refer to obtaining a desired pharmacological and / or physiological effect. The effect can be prophylactic in terms of completely or partially preventing a disease or its symptoms, and / or therapeutic in terms of partially or completely curing a disease and / or the adverse effects caused by the disease. Thus, treatment encompasses any treatment of a disease in a mammal, particularly a human, and includes (a) preventing a disease in a subject who may have a predisposition to the disease but has not yet been diagnosed as having it, i.e., preventing the occurrence of the disease in a human, (b) inhibiting the disease, i.e., stopping its onset, and (c) alleviating the disease, i.e., causing regression of the disease. 【0029】 The term "therapeutically effective amount" refers to an amount sufficient to effect such treatment of a disease when administered to a subject for treating the disease. 【0030】 The term "therapeutically effective amount" refers to an amount sufficient to effect such treatment of a disease when administered to a subject for treating the disease. The therapeutically effective amount will vary depending upon the protein or its active fragment, the disease and its severity, and the age, weight, and the like of the subject being treated. 【0031】 As used herein, "chemotherapy" refers to any agent that, when used alone or in combination, alleviates, reduces, improves, prevents, or maintains remission of one or more clinical symptoms or diagnostic markers associated with a neoplastic disease, tumor, and cancer. Exemplary chemotherapeutic agents include, but are not limited to, alkylating agents, toxins, anti-cancer antibiotics, antimetabolites, antimitotic agents, and the like. Specific known compounds include, but are not limited to, Ara-C, bleomycin, camptothecin, carboplatin, cisplatin, cyclophosphamide, doxorubicin (adriamycin), gemcitabine, methotrexate, paclitaxel, and vincristine. 【0032】 Reference will now be made to specific non-limiting aspects and embodiments thereof, as well as to specific figures and examples, in order to describe the invention. 【0033】 The present invention addresses the need to identify effective therapies for use in the treatment and prevention of renal diseases or renal injury. 【0034】 The present invention is based on the surprising finding of the inventors that the addition of exogenous follistatin fusion proteins in several animal models of kidney disease is associated with a dramatic improvement in markers of kidney disease (such as markers of renal fibrosis) in said animals. Such markers are, for example, hydroxyproline, serum creatinine or collagen. Subsequently, the inventors have surprisingly demonstrated that the follistatin fusion protein can not only attenuate ECM deposition in kidney sections (as shown by a decrease in collagen), but also significantly attenuate renal fibrosis in vivo when administered to UUO mice. Surprisingly, it has also been shown that epithelial cells of the kidney (such as tubular epithelial cells) are protected in the presence of injury stimuli. 【0035】 It is demonstrated herein that follistatin fusion proteins can be therapeutic for the treatment and prevention of kidney disease or kidney injury, and these fusion proteins are advantageous over conventionally known follistatin or follistatin-based fusion proteins that contain an Fc portion. In particular, the follistatin fusion proteins that can be used according to the present invention are more easily expressed in vitro and exhibit an improved half-life. 【0036】 A main object of the present invention is a follistatin fusion protein for use in the treatment, amelioration or prevention of kidney disease or kidney injury, comprising a. a follistatin portion, b. an antibody portion, and optionally c. a linker between the follistatin portion and the antibody portion. 【0037】 The present invention also provides a method of treating a patient in need of treatment for the treatment, amelioration or prevention of kidney disease or kidney injury, comprising administering a therapeutically effective amount of a follistatin fusion protein, the follistatin fusion protein comprising a. a follistatin portion, b. an antibody portion, and optionally c. a linker between the follistatin portion and the antibody portion. 【0038】 Use of a follistatin fusion protein in the manufacture of a medicament for the treatment, amelioration or prevention of renal disease or renal injury, wherein the follistatin fusion protein comprises a. a follistatin portion, b. an antibody portion, and optionally c. a linker between the follistatin portion and the antibody portion. 【0039】 Overall in connection with the present invention, renal disease or renal injury is characterized by a variation of a specific marker in a sample of a subject as compared to the specific marker in a sample of a healthy subject, wherein the sample is a cell or tissue associated with said disease / injury (e.g. kidney cell or kidney tissue), or the sample is, for example, blood, serum or urine (however, not limited thereto). For example, the variation can be an increase in a disease marker, and the marker can be any one of, for example, an increase in serum creatinine, cystatin-C, serum albumin level, blood urea nitrogen (BUN), or a decrease in estimated glomerular filtration rate (eGFR) (however, not limited thereto). Alternatively, the variation can be an increase in albumin or protein, called albuminuria and proteinuria respectively, and / or a change in urinary disease markers such as an increase in microglobulin, neutrophil gelatinase-associated lipocalin (NGAL) or kidney injury molecule-1 (KIM-1). The variation can also be an increase in one of the follistatin ligands associated with the disease or the progression of the disease or injury, which can be activin A, activin B, GDF8 (myostatin) or GDF11 (however, not limited thereto). The said variation of the marker in the sample can be determined by any means. A decrease or increase of a marker in a sample of a subject is typically determined by comparing the level of the marker in the sample of the subject to the level of the same marker in a reference sample of the same sample type (or herein called a healthy sample or normal sample) (i.e. the basal level; for example, the basal disease activity biomarker, the basal expression level of the follistatin ligand (mRNA level and / or protein level) and / or the basal level of the fibrosis marker). 【0040】 The use, method or use of a follistatin fusion protein according to the invention as a whole, for example for the treatment, amelioration or prevention of renal diseases or renal injuries in a subject, thus comprises (a) measuring at least one marker of renal impairment in a sample from the subject (e.g. kidney cells, serum or urine), wherein the marker is, for example, follistatin ligand activity, follistatin ligand expression or circulating follistatin ligand level, (b) comparing the result of the measurement obtained in (a) with the corresponding measurement in a normal sample, and c) when an increase in the expression, activity or circulating level of the follistatin ligand is observed, administering the follistatin fusion protein to the subject, thereby treating, ameliorating or preventing a renal disease or renal injury. The follistatin ligand expression measured in step (a) can be mRNA or protein amount, and the increase can be an increase in expression as discussed above. The corresponding measurement in a normal sample need not be obtained each time a comparison is made. The corresponding measurement can be obtained at any time before the comparison is made and can be the average follistatin ligand expression or follistatin ligand activity in normal cells / tissues. 【0041】 Alternatively, the use, method or use of the follistatin fusion protein according to the invention as a whole, for example for the treatment, amelioration or prevention of kidney disease or kidney injury in a subject, may thus comprise: (a) measuring at least one marker of kidney damage in a sample from the subject (e.g., kidney cells, serum or urine), wherein the marker is, for example, proteinuria, serum creatinine or cystatin-C; (b) comparing the result of the measurement obtained in (a) with the corresponding measurement in a normal sample (such as serum or urine); and (c) when an increase in the expression of said marker is observed, administering the follistatin fusion protein to the subject, thereby treating, ameliorating or preventing kidney disease or kidney injury. The expression of the marker measured in step (a) can be at the mRNA or protein level, and the increase can be an increase in expression as discussed above. The corresponding measurement in the normal sample need not be obtained each time a comparison is made. The corresponding measurement can be obtained at any time before the comparison is made and can be the average marker expression in normal cells / tissue / serum / urine. 【0042】 In another embodiment, the use, method or use of the follistatin fusion protein according to the invention as a whole, for example for the treatment, amelioration or prevention of kidney disease or kidney injury in a subject, comprises: (a) monitoring the rate of loss of estimated glomerular filtration rate (eGFR) and (b) measuring a decrease in eGFR or the slope of eGFR loss in rapid progressors of kidney function loss (e.g., >5 ml / min / 1.73 m 2When it is recognized that the decrease in / year typically indicates an acceleration of progression), an administration to a follistatin fusion protein may be included, thereby treating, improving or preventing renal disease or renal injury. Monitoring of eGFR decline can also be considered together with an increase in proteinuria or albuminuria, and values of urine collection of 30 - 300 mg / 24 hours are typically considered a moderate increase or "microalbuminuria", and an increase in urine collection of > 300 mg / 24 hours is typically considered severe or "overt albuminuria". Inclusion of albuminuria along with loss of eGFR is justified as a way to estimate the risk of progression of renal dysfunction and the potential risk of ESRF (Ammirati, 2020). Rapid progressors of renal function loss determined by any number of measures would likely benefit from follistatin fusion protein treatment as a way to treat, improve or prevent renal disease or renal injury. 【0043】 It should be understood that markers that can be measured to determine the need for treatment can be combined, such as (in non-limiting examples) measuring an increase in follistatin ligand expression in combination with an increase in serum creatinine or proteinuria or albuminuria. 【0044】 In the context of the present invention as a whole, renal diseases are preferably chronic renal diseases such as primary glomerulonephritis, secondary glomerulonephritis, diabetic nephropathy, hypertensive nephrosclerosis, focal segmental glomerulosclerosis (FSGS), IgA nephropathy (IgAN), mesangial proliferative glomerulonephritis, membranous nephropathy (MN), minimal change disease (MCD), polycystic kidney disease, chronic allograft nephropathy, mineral and bone disorder associated with CKD (CKD-MBD), and Goodpasture's disease (however, not limited thereto). Alternatively, renal diseases are chronic renal diseases selected from primary glomerulonephritis, secondary glomerulonephritis, diabetic nephropathy, hypertensive nephrosclerosis, focal segmental glomerulosclerosis (FSGS), IgA nephropathy (IgAN), mesangial proliferative glomerulonephritis, membranous nephropathy (MN), minimal change disease (MCD), polycystic kidney disease, chronic allograft nephropathy, mineral and bone disorder associated with CKD (CKD-MBD), and Goodpasture's disease. Also, in the context of the present invention as a whole, renal injury is associated with a) renal fibrosis or b) other diseases such as true diabetes, hypertension, cardiovascular disease, or bone disorder. Both primary and secondary forms of glomerulonephritis can progress to end-stage renal failure (ESRF), similar to most forms of renal disease. Conventional treatment requires dialysis, but the outcome is generally poor, and the survival rate after more than 5 years is less than 50%. Kidney transplantation is currently considered the gold standard treatment, but the organ recipient rate is low. Renal diseases or renal injuries can be the disease or injury itself or the result of therapeutic treatment such as chemotherapy. In this regard, chemotherapeutic agents can be selected from, but not limited to, alkylating agents, toxins, antineoplastic antibiotics, antimetabolites, antimitotic agents, etc. Specific compounds known to cause renal injury include, but are not limited to, Ara-C, bleomycin, camptothecin, carboplatin, cisplatin, cyclophosphamide, doxorubicin (adriamycin), gemcitabine, methotrexate, paclitaxel, and vincristine. When a follistatin fusion protein is administered in relation to renal diseases or renal injuries that may be the result of therapeutic treatment, the fusion protein can be administered simultaneously with, separately from, or sequentially to other treatments such as chemotherapy.In certain examples, the follistatin fusion protein according to the present invention is used to treat, prevent or ameliorate kidney diseases or kidney injuries resulting from treatment with doxorubicin (adriamycin). 【0045】 Without wishing to be bound by any theory, the inventors have found that follistatin-based treatments using the fusion proteins of the present invention modulate ligand signaling, resulting in increased protection of epithelial cells (such as renal tubular epithelial cells) and decreased profibrotic signaling, reducing scar tissue in the kidney. Thus, this treatment can delay or prevent tissue damage and fibrosis associated with progressive kidney disease, resulting in a reduction in eGFR loss and proteinuria. Thereby, prevention of end-stage renal disease and / or extension of the period until renal replacement therapy / transplantation can be achieved. In other words, treatment, amelioration or prevention of the kidney disease or kidney injury of interest in the context of the present invention as a whole is obtained through protection of epithelial cells (such as renal tubular epithelial cells). 【0046】 The follistatin portion of the follistatin fusion protein used in the context of the present invention as a whole comprises, or is, a naturally occurring follistatin protein. This is preferably in its mature form, i.e., this sequence lacks an N - secretion signal sequence as it is only required for production / secretion from cells. Alternatively, this is a functional fragment thereof. The follistatin portion is, for example, the FST288 protein (SEQ ID NO: 2) or the FST315 protein (SEQ ID NO: 1). Any intermediate form thereof, for example, any follistatin portion containing residues 289 - 314 of any one of SEQ ID NOs: 1 - 4, can also be used as long as they are functional, i.e., retain at least one biological activity of FST. Preferably, but not limited to, any intermediate form of the follistatin portion starts with residue 1 of SEQ ID NO: 1. As a non - limiting example, a functional FST fragment can be FST291 (i.e., comprising residues 1 - 291 of SEQ ID NO: 1) or FST303 (i.e., comprising residues 1 - 303 of SEQ ID NO: 1). In another embodiment, the follistatin portion (i.e., naturally occurring or a functional fragment thereof) according to the present invention is a functional variant and can have one or more mutations, such as mutations in the heparin - binding site (HBS). As a non - limiting example, one or more mutation sites can be selected from K76, K81, and / or K82 numbered relative to SEQ ID NO: 1 (see, for example, SEQ IDs 22 and 25). One or more mutations can include alanine (A) instead of lysine (K) (resulting in mutations selected from K76A, K81A, and / or K82A). As a further non - limiting example, heparin - binding mutants (''HBM'') (or herein named ''(HBM)'' or ''HBSM'') can use, for example, FST288HBM (SEQ ID NO: 4), FST291HBM, FST303HBM, or FST315HBM (SEQ ID NO: 3), and the mutants contain the triple mutation K76A, K81A, and K82A.Preferably, the follistatin moiety is selected from: a. SEQ ID NO: 1, b. SEQ ID NO: 2, c. SEQ ID NO: 3, d. SEQ ID NO: 4, e. any protein comprising amino acid residues comprising residues 289 - 314 of any one of SEQ ID NOs: 1 - 4, or f. a sequence having at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to any one of SEQ ID NOs: 1 - 4. 【0047】 The antibody moiety of the follistatin fusion protein used in connection with the present invention as a whole binds to albumin, preferably to serum albumin such as human serum albumin (HSA). The antibody moiety can be a chimeric, humanized or human antibody moiety. Preferably, the antibody moiety is an antigen - binding fragment of an antibody (or referred to herein as an antigen - binding portion). Preferably, this is selected from Fab, Fab’ or F(ab’)2. In a non - limiting example, the antibody moiety comprises a light - chain variable region comprising CDR - L1 comprising SEQ ID NO: 13, CDR - L2 comprising SEQ ID NO: 14 and CDR - L3 comprising SEQ ID NO: 15; and a heavy - chain variable region comprising CDR - H1 comprising SEQ ID NO: 16, CDR - H2 comprising SEQ ID NO: 17 and / or CDR - H3 comprising SEQ ID NO: 18. In an alternative non - limiting example, the antibody moiety comprises a heavy - chain variable region comprising SEQ ID NO: 5, or a sequence having at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity thereto; and a light - chain variable region comprising SEQ ID NO: 6, or a sequence having at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity thereto. SEQ ID NOs: 5 and 6 represent the variable heavy and variable light chains of an anti - albumin antibody named "CA645" (disclosed in WO 2013 / 068571). 【0048】 In the context of the present invention as a whole, the follistatin fusion protein optionally contains a linker between the follistatin portion and the antibody portion. When a linker is present, by way of non-limiting example, the linker can be selected from the group consisting of SGGGGS (SEQ ID NO: 7), SGGGGSSGGGGS (SEQ ID NO: 19), GGGGS (SEQ ID NO: 20), and GGGGSGGGGS (SEQ ID NO: 21). 【0049】 When making the fusion protein, it will be understood that there are two options for fusing / combining any parts with each other: C-terminal fusion or N-terminal fusion. The inventors have found that when the N-terminal part of the antibody part is fused to the C-terminal part of the follistatin part, the expression of the resulting fusion protein is further improved compared to any other type of fusion. In particular, the C-terminal fusion protein of the present invention shows excellent expression and a higher yield of monomeric protein compared to known Fc-based follistatin fusion proteins. However, although the C-terminal fusion protein resulted in the highest expression level, the antibody part fused to the N-terminus of follistatin can result in an expression level about 1.5 times higher compared to, for example, an Fc-based follistatin fusion protein, and can be considered by those skilled in the art. Accordingly, in a preferred embodiment, the antibody part is connected to the C-terminal part of the follistatin part. When a linker is present, the antibody part is preferably connected (or linked) to the C-terminal part of the follistatin part via the linker (in other words, the antibody part is connected (or linked) to the C-terminal part of the follistatin part and there is a linker between the two parts). 【0050】 Similarly, when making a fusion protein between one polypeptide (here the FST part) and an antibody part (here preferably Fab, Fab’ or F(ab’)2), there are two options for fusing any parts with each other, and it will be understood that the polypeptide can be fused to either the heavy chain or the light chain of the antibody part. 【0051】 Non-limiting examples of follistatin fusion proteins that can be used in accordance with the present invention are as follows: (a) i. The FST315 polypeptide defined by SEQ ID NO: 1, or a sequence having at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity thereto; ii. The Fab heavy chain defined by SEQ ID NO: 5, or a sequence having at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity thereto, linked to the C-terminus of the FST315 polypeptide; and iii. The Fab light chain defined by SEQ ID NO: 6, or a sequence having at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity thereto; (b) i. The FST288 polypeptide defined by SEQ ID NO: 2, or a sequence having at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity thereto; ii. The Fab heavy chain defined by SEQ ID NO: 5, or a sequence having at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity thereto, linked to the C-terminus of the FST288 polypeptide; and iii. The Fab light chain defined by SEQ ID NO: 6, or a sequence having at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity thereto; (c) i. The FST315 polypeptide variant defined by SEQ ID NO: 3 (FST315HBM) or SEQ ID NO: 22, or a sequence having at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity thereto; ii. The Fab heavy chain defined by SEQ ID NO: 5, or a sequence having at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity thereto, linked to the C-terminus of the FST315 polypeptide variant; and iii. The Fab light chain defined by SEQ ID NO: 6, or a sequence having at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity thereto; (d)i. The FST288 polypeptide variant defined by SEQ ID NO: 4 (FST288HBM) or SEQ ID NO: 25, or a sequence having at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity thereto; ii. The Fab heavy chain defined by SEQ ID NO: 5, which is bound to the C-terminus of the FST288 polypeptide variant, or a sequence having at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity thereto; and iii. The Fab light chain defined by SEQ ID NO: 6, or a sequence having at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity thereto; (e)i. The FST315 polypeptide defined by SEQ ID NO: 1, or a sequence having at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity thereto; ii. A linker defined by SEQ ID NO: 7, SEQ ID NO: 19, SEQ ID NO: 20 or SEQ ID NO: 21, which is bound to the C-terminus of the FST315 polypeptide; iii. The Fab heavy chain defined by SEQ ID NO: 5, which is bound to the free end of the linker, or a sequence having at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity thereto; and iv. The Fab light chain defined by SEQ ID NO: 6, or a sequence having at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity thereto; (f)i. The FST288 polypeptide defined by SEQ ID NO: 2, or a sequence having at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity thereto; ii. A linker defined by SEQ ID NO:7, SEQ ID NO:19, SEQ ID NO:20 or SEQ ID NO:21 that is bound to the C-terminus of the FST288 polypeptide; iii. A Fab heavy chain defined by SEQ ID NO:5, or a sequence having at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity thereto, that is bound to the free end of the linker; and iv. A Fab light chain defined by SEQ ID NO:6, or a sequence having at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity thereto; (g)i. An FST315 polypeptide variant defined by SEQ ID NO:3 (FST315HBM) or SEQ ID NO:22, or a sequence having at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity thereto; ii. A linker defined by SEQ ID NO:7, SEQ ID NO:19, SEQ ID NO:20 or SEQ ID NO:21 that is bound to the C-terminus of the FST315 polypeptide variant; iii. A Fab heavy chain defined by SEQ ID NO:5, or a sequence having at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity thereto, that is bound to the free end of the linker; and iv. A Fab light chain defined by SEQ ID NO:6, or a sequence having at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity thereto; (h)i. An FST288 polypeptide variant defined by SEQ ID NO:4 (FST288HBM) or SEQ ID NO:25, or a sequence having at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity thereto; ii. A linker defined by SEQ ID NO:7, SEQ ID NO:19, SEQ ID NO:20 or SEQ ID NO:21 that is bound to the C-terminus of the FST288 polypeptide variant; iii. A Fab heavy chain defined by SEQ ID NO: 5, or a sequence having at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity thereto, attached to the free end of the linker; and iv. A Fab light chain defined by SEQ ID NO: 6, or a sequence having at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity thereto; (i) Together with a Fab light chain defined by SEQ ID NO: 6, or a sequence having at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity thereto, SEQ ID NO: 8, 9, 10, 11, 24, 25, 26, 27, 32, 33, 34 or 35, or a sequence having at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity thereto and; (j) Together with a Fab light chain defined by SEQ ID NO: 6, SEQ ID NO: 8, 9, 10, 11, 24, 25, 26, 27, 32, 33, 34 or 35; or (k) A functional variant or fragment of any one of (a) to (i). 【0052】 Any subject can be treated according to the present invention. The subject is preferably a human. However, the subject may be another mammal such as a non-human primate, horse, cow, sheep, pig, dog, cat, rabbit, rat, mouse, guinea pig or hamster. Alternatively, the term patient can be used interchangeably with subject. 【0053】 Any follistatin fusion protein used in accordance with the present invention can be incorporated into a pharmaceutical composition suitable for administration to a subject by any method, such as (but not limited to) topical, intranasal, intradermal, intravenous, subcutaneous, or intramuscular routes. Typically, the pharmaceutical compositions used in accordance with the present invention comprise a follistatin fusion protein and one or more pharmaceutically acceptable adjuvants and / or carriers. Accordingly, pharmaceutical compositions for use in the treatment, amelioration, or prevention of renal disease or renal injury in a subject are also described herein, said pharmaceutical compositions comprising a follistatin fusion protein as described herein and one or more pharmaceutically acceptable adjuvants and / or carriers. The pharmaceutical compositions according to the present invention can be part of a kit provided with instructions for use, including instructions and optionally a device for intravenous, subcutaneous, or intramuscular administration to an individual in need thereof. 【0054】 As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, etc. that are physiologically compatible and suitable for administration to a subject for the methods and uses described herein. Examples of pharmaceutically acceptable carriers include one or more of water, saline, buffered saline, dextrose, glycerol, ethanol, and the like, and combinations thereof. Depending on the route of administration or the type of formulation (such as liquid, lyophilized, or spray-dried formulations), isotonic agents, such as polyhydric alcohols like sugars, mannitol, sorbitol, or sodium chloride, can be incorporated into the composition. The pharmaceutically acceptable carrier can further include minor amounts of auxiliary substances, such as wetting or emulsifying agents, preservatives, or buffering agents, that enhance the shelf life or effectiveness of the antibody or antibody portion. 【0055】 The pharmaceutical composition according to the present invention can be in various forms. These include, for example, liquid solutions (e.g., injectable and infusible solutions), dispersions or suspensions, powders, and liposomes. The preferred form depends on the intended mode of administration and therapeutic use. A typical preferred composition is in the form of an injectable or infusible solution, such as a composition similar to those used for passive immunization of humans with other antibodies. 【0056】 An appropriate dosage of the follistatin fusion protein according to the present invention can be determined by a skilled medical practitioner. The actual dosage level of the active ingredient in the pharmaceutical composition of the present invention can be varied so as to obtain an amount of the active ingredient that is not toxic to the patient and is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration. The dosage level selected depends on various pharmacokinetic factors including the route of administration, time of administration, rate of excretion of the follistatin fusion protein, duration of treatment, other drugs, compounds, and / or materials used in combination with the particular follistatin fusion protein, age, sex, weight, condition, general health, and previous medical history of the patient being treated. 【0057】 An appropriate dosage can be, for example, in the range of about 0.01 pg / kg to about 1000 mg / kg body weight of the patient being treated, typically about 0.1 pg / kg to about 100 mg / kg body weight. The dosing regimen can be adjusted to provide the optimal desired response (e.g., therapeutic response). For example, a single dose can be administered or several divided doses can be administered over time. As used herein, the dosage unit form refers to physically discrete units suitable as unitary dosages for the subject being treated, each unit containing a predetermined quantity of the active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. Administration can be by single or multiple doses. Multiple doses can be administered at the same or different locations via the same or different routes. 【0058】 In the context of the present invention as a whole, the follistatin fusion protein can be co-administered with one or more other therapeutic agents. The combined administration of two or more agents can be achieved in several different ways. They may be administered together in a single composition, or separately as part of a combination therapy in separate compositions. For example, one can be administered before the other, separately, after the other, or continuously, or in parallel with or simultaneously with the other. In particular, when kidney disease or kidney injury may be a result of chemotherapy, the chemotherapeutic agent can be selected from, but not limited to, alkylating agents, toxins, anti-cancer antibiotics, antimetabolites, antimitotic agents, etc. Specific known compounds include, but are not limited to, Ara-C, bleomycin, camptothecin, carboplatin, cisplatin, cyclophosphamide, doxorubicin (adriamycin), gemcitabine, methotrexate, paclitaxel, and vincristine. In a specific example, the follistatin fusion protein or pharmaceutical composition according to the present invention is used to treat, prevent, or ameliorate kidney disease or kidney injury resulting from treatment with doxorubicin (adriamycin). 【0059】 It should be noted that the above embodiments are illustrative rather than limiting of the present invention, and those skilled in the art can design many alternative embodiments without departing from the scope of the present invention, particularly the scope of the claims. 【0060】 【Table 1-1】 【Table 1-2】 【Table 1-3】 【Table 1-4】 【Table 1-5】 【Table 1-6】 【Examples】 【0061】 The following examples are merely illustrative of the present invention and are not intended to limit the scope or content of the present invention. 【0062】 Materials and Methods Cloning strategy: A DNA segment corresponding to the fusion between the follistatin and the heavy or light chain sequence of an anti-albumin antibody (referred to as 645 Fab) (with or without a linker sequence in between) was generated by PCR or gene synthesis and cloned using an in-house mammalian expression vector. The heavy and light chain sequences of 645 Fab were also cloned separately using an in-house mammalian expression vector. All expression vectors were confirmed by direct sequencing using primers covering the entire open reading frame. 【0063】 Culture of CHO cells: A suspension of CHOS-XE cells (Cain, K., et al., Biotechnol Prog, 2013.29(3): p.697-706.) was pre-adapted to CD CHO medium (Invitrogen) supplemented with 2 mM Glutamax. The cells were maintained in the logarithmic growth phase with stirring at 120 RPM in a shaking incubator (Kuhner AG) and cultured at 37 °C in an atmosphere containing 8% CO2. 【0064】 Protein Expression: The follistatin-Fab protein was overexpressed by transient transfection of the CHO-XE cell line. Pairs of expression plasmids were co-transfected (e.g., heavy chain and N-fab light chain-FST-C or light chain and FST-C fab heavy chain-C). Immediately prior to transfection with DNA, the CHO cells were exchanged into Expi CHO Expression Medium (Gibco) by briefly centrifuging the cells at 1500×g and resuspending the pellet. The cells were then transfected using ExpiFectamine (Gibco) according to the manufacturer's instructions. The cultures were grown with shaking at 190 RPM in an atmosphere containing 8% CO2 at 37°C for the first 24 hours and then at 32°C for the remainder of the expression cycle. The supernatant was typically harvested 9 - 14 days after transfection by centrifugation at 4000×g followed by filtration using a 0.22 μm membrane. The final protein expression level was determined by Protein G-HPLC and SDS PAGE. 【0065】 Protein Purification: The transiently expressed protein content was captured using a Mab Select column (GE Healthcare) performed under standard conditions. Briefly, the resin was washed with 10 column volumes of phosphate buffered saline (PBS, pH 7.4), and the bound protein was eluted with 5 column volumes of 0.1 M sodium citrate pH 3.1 (except where otherwise mentioned separately in the following examples). The eluate was neutralized with TRIS-HCl pH 8.5 and filter sterilized by 0.22 μm membrane exclusion chromatography (HiLoad 26 / 60 Superdex 75 column, GE Healthcare) performed under standard conditions (where the column was pre-packed with PBS pH 7.4 as the running buffer). The quality of the samples was evaluated using absorbance at 280 nm, BEH2000 analytical UPLC, and SDS PAGE (under reducing and non-reducing conditions). 【0066】 Doxorubicin-induced renal fibrosis model of chronic kidney disease (CKD): Female Balb / c mice at least 6 weeks old and weighing more than 24 g were subjected to IV injection via the tail vein of cDNA (or a pair of cDNAs for paired testing) in an appropriate hydrodynamic transfection expression vector (Five Prime Therapeutics) containing the follistatin gene. Seven days later, the mice were administered doxorubicin (doxorubicin) at 11 mg / kg IP and monitored for 49 days. The mice were monitored daily for body weight and health; mice that had lost more than 40% of their starting body weight or showed physical signs of end-stage renal failure were terminated and data were collected. At the end of the 49-day study period, all mice were terminated and kidneys and sera were collected for analysis. Renal hydroxyproline was measured using the QuickZyme assay (QuickZyme Biosciences), and serum creatinine was measured using a 3-enzyme method and read as μg / ml serum creatinine. Renal fibrosis (hydroxyproline) in the saline + doxorubicin group labeled as "saline" on the graph was considered the maximum disease response. This was set to 100% and used to normalize between treatment groups. Naïve animals were not administered doxorubicin. 【0067】 Statistical analysis methods designated for in vivo analysis: Each assay was conducted with a naïve group, a saline group, and several cDNAs. In the primary screening phase, cDNAs were paired and 15 mice per pair were treated; in the case of retesting, cDNAs were tested individually with 22 mice per cDNA. 【0068】 For each experiment, analysis of variance (ANOVA) of the log-transformed data was performed for hydroxyproline and serum creatinine. Observed values were considered outliers if their studentized residuals were less than -3 or greater than +3. When outliers were detected, they were excluded and the analysis was repeated. Then, t-tests based on the pooled residual standard deviation from ANOVA were used to test the cDNA group means against the saline control group means. In the primary screening at n = 15 per cDNA, hits were declared if the difference from saline was significant at the 10% level (P = 0.1). In the retest performed at n = 22 per cDNA, hits were declared if the difference from saline was significant at the 2.5% level (P = 0.025). This strategy was adopted to provide an acceptable false positive and false negative rate while minimizing the number of animals used. 【0069】 PSR staining of mouse kidneys: Half of each mouse kidney was placed in 10% neutral buffered formalin, dehydrated in a tissue processor, and paraffin-embedded for histological analysis. Paraffin blocks were sectioned at 4 μm with a microtome and placed on glass slides. The slides were then de-waxed, hydrated, stained with picrosirius red (PSR), and washed with acidic water. The slides were then dehydrated, cleared, and covered with coverslips. After the slides were dried, they were scanned using a Hamamatsu slide scanner. Images of the entire slide were imported into Definiens Developer XD 64 and analyzed using the brightfield module. Briefly, the cortical region was manually annotated to remove the medulla from the analysis. Marker threshold levels (e.g., PSR staining) were determined for individual markers on a training dataset of four or more images. The analysis algorithm was then run on a dedicated server to determine the marker regions that exceeded the threshold intensity for each individual marker, and the area of each marker that exceeded the threshold intensity was determined as a percentage of the cortical area. 【0070】 Unilateral ureteral obstruction (UUO)-induced renal fibrosis model in chronic kidney disease: Male C57BL / 6 mice (Charles River) weighing at least 19 g were subjected to a unilateral ureteral obstruction (UUO)-induced renal fibrosis model in chronic kidney disease. In the test where follistatin was delivered by hydrodynamic transfection, mice were transfected with 50 μg of either pLIVE-human follistatin or pLIVE SEAP vector (Mirus Bio, catalog number MIR 5320) in TransIT-EE vehicle. The solution containing the vector was rapidly (less than 2.5 seconds) injected intravenously in a volume of 2 ml. General anesthesia with isoflurane was induced before hydrodynamic injection and maintained for at least 1 minute after injection. In the test where human follistatin protein was administered to mice, this was administered via the subcutaneous route in 100 μl of physiological saline at the dose concentration specified in the figure legend. Surgery was performed using isoflurane-induced general anesthesia under aseptic conditions appropriate for the surgical procedure. After laparotomy of the mice, the left ureter was ligated using 3.0 Mersilk suture. The muscle wall was sealed in a continuous pattern using 5-0 Vicryl, and the skin was closed by subcutaneous suture using 5-0 Vicryl. Appropriate analgesics were administered before and after surgery. As detailed in the results, blood samples were collected at appropriate times. Mice were restrained using a widely recognized mouse restraint device, and 30 μl of blood was collected from the tail vein by tail puncture using a pipette to accurately measure the blood sample. This sample was placed in a 0.2 ml PCR tube (Thermo), centrifuged (20,000 g, 5 minutes) to remove as much serum as possible, placed in a new PCR tube, and stored at -80°C. On day 19 or 21, the mice were anesthetized with isoflurane, blood was removed into a serum tube (Sarstedt) by cardiac puncture, and the mice were euthanized by cervical dislocation. The left kidney was removed, rapidly frozen in liquid nitrogen for 30 minutes, and stored at -80°C. The other half was placed in 10% neutral buffered formalin, dehydrated in a tissue processor, and paraffin-embedded for histological analysis and PSR staining as described above. 【0071】 Human Proximal Tubule Barrier Integrity Assay (see Figure 5A for the principle): Primary human proximal tubule cells (Innoprot) were seeded onto a 0.4 μM pore polycarbonate membrane in a 24-well HTS Transwell plate (Corning). The cells were cultured for 14 days, and the medium (Advanced DMEM / F12 supplemented with a renal epithelial growth kit from ATCC) was changed every other day. The epithelial barrier function was disrupted by the addition of 2 μM adriamycin (also known as doxorubicin, a widely described nephrotoxic agent). To examine the effect of follistatin, either adriamycin + 100 ng / ml Fab control protein or 100 ng / ml FST315(HBM)-Fab was added to several wells. After 72 hours of incubation, 0.5 mg / ml of FITC-dextran (150 kDa) was added to the apical chamber, and the integrity of the barrier was evaluated by monitoring the appearance of the FITC signal in the basolateral chamber over time using a fluorescence plate reader. The percent permeability was calculated by dividing the fluorescence value from the test well by the mean signal detected in a transwell without cells. The graph shows the data points for individual wells containing six replicates for each condition. Statistical significance was shown by the Mann-Whitney Student's T test. 【0072】 Example 1 Follistatin is antifibrotic in a mouse adriamycin-induced model of chronic kidney disease (CKD) Mouse follistatin was initially identified in the screening of fibrotic modifying proteins in a mouse Adriamycin CKD model as one member of a cDNA pair hydrodynamically transfected (n = 15 mice). The anti-fibrotic effect of follistatin was confirmed in repeated experiments in which follistatin cDNA was independently transfected in the same model (n = 18 - 21 mice / group). Follistatin decreased renal fibrosis by 77% compared to the saline control group (p = 0.0001) as measured by total renal hydroxyproline (Figure 2A), while the loss of renal function returned to the normal range (p = 0.0001) as measured by serum creatinine (Figure 2B). The conditional survival rate as measured by the symptoms of end-stage renal failure increased from 50% in the Adriamycin group treated with saline to 100% in the follistatin-treated group (Figure 2C). 【0073】 On day 49, all kidneys from all groups (naïve (n = 8), untreated Adriamycin animals (n = 21) and follistatin hydrodynamically transfected animals (n = 18)) were fixed, paraffin-embedded, sectioned, stained with either hematoxylin and eosin (H&E) or picrosirius red (PSR), and imaged with a Hamamatsu slide scanner. Exemplary and representative images of PSR-stained kidneys for 1 naïve, 3 untreated Adriamycin and 3 follistatin-treated Adriamycin animals are shown in Figure 2D. Kidneys from untreated mice had extensive glomerulosclerosis and tubulointerstitial fibrosis, with extensive tubular dilation and atrophy associated with marked tubular basement membrane expansion, flattening of the tubular epithelium, and loss of the brush border. In contrast, follistatin-treated mice were mostly protected from both types of fibrotic remodeling. Forty-seven mouse kidney samples stained with PSR were subjected to high-content image analysis using Definiens software, and the area of PSR staining measured in the three groups was plotted as a percentage of the PSR staining area (Figure 2E). The PSR staining area in the follistatin-treated group was normalized to a 3-fold increase in untreated kidneys (p < 0.0001). 【0074】 In conclusion, follistatin delivered by hydrodynamic transfection significantly inhibited fibrosis and multiple parameters of disease in a murine adriamycin chronic kidney disease model, including collagen accumulation in mice, biomarker levels of serum creatinine kidney injury, gross morphological kidney structure, and conditional survival, during the test period. 【0075】 Example 2 Follistatin inhibits renal fibrosis in a murine unilateral ureteral obstruction (UUO) model of acute kidney disease. The main objective of this study was to evaluate the differences in efficacy between different constructs of follistatin by hydrodynamic transfection (HDT) in delaying the development of tubulointerstitial fibrosis in the UUO model. 【0076】 The UUO kidneys of animals administered the control SEAP vector by HDT showed tubulointerstitial fibrosis in the cortex typical of this model at 21 days after surgery, with extensive expansion of the tubular basement membrane, intense collagen staining with a basement membrane strongly stained especially in the medulla, extensive loss of epithelial cell volume, tubular atrophy, loss of the proximal tubular brush border, and extensive tubulointerstitial infiltration. Less than 15% of the tubules had a nearly normal structure. Changes to the glomeruli were minimal, but the medulla was virtually destroyed and lost (Figure 3A). 【0077】 The UUO kidneys of animals that received HDT with all follistatin moieties - FST315(HBM)-Fab and FST315-Fab showed a decrease in tubular basement expansion and a decrease in collagen staining compared to UUO mice that received the control SEAP vector 1 day before the UUO surgery. All follistatin treatment groups had less occurrence of epithelial flattening and atrophy and had preservation of the tubular structure (Figure 3A). 【0078】 Picrosirius red (PSR)-stained collagen sections were quantified using a definiens image analysis algorithm that demonstrated the protective effect of follistatin. FST315-Fab showed a decrease in the percentage of collagen area, which was not statistically significant; however, 315Fst(HBM)-Fab (p<0.05) showed a significant inhibition of the percentage of collagen area compared to the SEAP control. This is consistent with the visual histological evaluation of the stained sections above (Figure 3B). 【0079】 Another indicator of the efficacy of the follistatin fusion protein is the increase in mouse body weight monitored daily during the test period. Both FST315-Fab and FST315(HBM)-Fab showed less weight loss at the onset of the disease and more weight gain from day 4 until the end of the 21-day test (Figure 3C). 【0080】 In conclusion, the follistatin fusion protein delivered by hydrodynamic transfection was protective in a mouse model of acute kidney disease, i.e., UUO, and the disease parameters were PSR staining and the percentage of collagen area in the diseased kidney and weight loss as the primary readout. 【0081】 Example 3 Comparison of FST315-Fab and FST315(HBM)-Fab, with the latter showing a dose-response effect, and both follistatin moieties were delivered as purified proteins in a mouse UUO acute kidney disease model The main objective of this study was to compare different FST315 protein formats for their efficacy in delaying the development of tubulointerstitial fibrosis in a UUO model. 【0082】 When examining the macroscopic kidney morphology from PSR-stained kidneys in non-surgically treated (non-afflicted) kidneys versus day 21 UUO kidneys, dramatic differences were shown in shape, structure, and red staining, and these macroscopic changes could be reversed in the presence of 10 mg / kg of FST315(HBM)-Fab administered daily; histological blind scoring quantifies these changes shown in Figure 4A. Higher magnification of day 21 untreated UUO highlights the typical histological changes of this model and the normalization observed in the presence of high concentrations of FST315(HBM)-Fab (Figure 4B). 【0083】 Figure 4C shows the effects of FST315-Fab versus FST315(HBM)-Fab in day 21 UUO kidneys treated with the heparin-binding variant of follistatin-Fab protein (FST315(HBM)-Fab), which had a dramatically different appearance compared to vehicle treatment 1 day prior to surgery (-1 day), and also showed greater epithelial protection and less red collagen staining compared to wild-type follistatin-fab protein (FST315-Fab). Tubular atrophy was decreased, and in particular, large sections of the cortex appeared nearly normal at the 10 mg / kg dose, with minimal basement membrane expansion, collagen staining, or loss of structure. Also, the effect of FST315(HBM)-Fab on epithelial protection and collagen PSR staining was dose-dependent, with a clear change from the high dose of 10 mg / kg, less effect at the intermediate doses of 2 mg / kg and 0.2 mg / kg, and little distinguishable from untreated UUO kidneys at the 0.02 mg / kg dose. 【0084】 When picrosirius red (PSR)-stained collagen sections were quantified using the definiens image analysis algorithm to read the percentage of collagen area, a dose-dependent significant protective effect was shown at 10 mg / kg (p<0.0001) and 2 mg / kg (p<0.001) of the 315FST(HBM)-Fab fusion protein, while non-significant decreases were shown at doses of 0.2 and 0.02 mg / kg (Figure 4D); 2 mg / kg of FST315-Fab had a smaller degree of response but a statistically significant difference with p<0.05 compared to the control untreated UUO group. Importantly, 2 mg / kg of FST315-Fab was also statistically different from 2 mg / kg of FST315(HBM)-Fab (p = 0.03), which means that the protection conferred by the FST315(HBM)-Fab form had additional effects mediated through changes in the heparin-binding variant. This could be due to the better pharmacokinetic properties of FST315-Fab, which has an average residence time of 13.1 hours in mice, compared to FST315(HBM)-Fab, which is 9.3 hours, or could be due to other functional characteristics that have not yet been characterized. Histological manual scoring of the stained kidney sections of all groups in the study was performed by experts who blinded each group, and the mean connective fibrosis score was plotted in Figure 4E. The same pattern was again observed in that the FST315(HBM)-Fab fusion protein had the most prominent inhibitory effect, showing significant differences of p<0.0001, p<0.001, and p<0.01 from the vehicle-treated UUO kidneys at 10, 2, and 0.02 mg / kg, respectively. The 2 mg / kg of FST315-Fab protein was significant with p<0.05 for epithelial protection compared to vehicle-treated UUO, but this was inferior to the FST315(HBM)-Fab fusion format at the same dose, with a significant difference at p = 0.05. This was consistent with the visual histological evaluation of the above-stained sections. 【0085】 The body weights of all animals in the UUO test were measured daily, and the data were plotted over time (Figure 4F). The weight change of the follistatin moiety was closely correlated with efficacy for both the FST315(HBM)-Fab and FST315-Fab formats compared to the vehicle-treated UUO group; different 315FST(HBM)-Fab doses also showed that weight gain was a good surrogate for renal protection that could only be measured at the end of the test. 【0086】 In conclusion, when administered as a protein, FST315(HBM)-Fab showed a statistically significant improvement compared to vehicle-treated diseased animals and also showed statistically significantly superior efficacy compared to FST315-Fab in protecting the mouse kidney from the injury initiated by the UUO surgery, and this effect was dose-dependent. The excellent protective effect of FST315(HBM)-Fab on the kidney was consistent across morphological evaluation, collagen content determined by PSR, epithelial cell health, and total body weight gain. 【0087】 Example 4 Effect of FST315(HBM)-Fab on the integrity of the renal proximal tubule epithelial cell (RPTEC) barrier RPTEC cells grown in 96-well Transwell plates form a polarized non-permeable barrier that can be measured by monitoring the resistance to the diffusion of a fluorescent dye from the upper to the lower compartment of the Transwell. In the presence of the cytotoxic adriamycin, the integrity of the barrier was compromised, which could be measured by the presence of the fluorescent dye FITC-dextran (150 kDa) that appeared in the basolateral supernatant. Treatment with FST315(HBM)-Fab protected against this loss of barrier integrity, which could be measured by a partial restoration of permeability to FITC-dextran, but the Fab molecule alone did not protect; this decrease in permeability by FST315(HBM)-Fab was statistically significantly different from the media control (p < 0.0001). The figure showing the in vitro RPTEC model and the results are shown in Figures 5A and 5B, respectively. 【0088】 In conclusion, FST315(HBM)-Fab preserves the integrity of the primary RPTEC barrier from adriamycin-induced epithelial injury in an in vitro model of renal barrier function. 【0089】 Example 5 FST-Fab and Fab-FST exhibit better protein expression, higher monomer fraction yields, and longer half-lives than FST-WT or FST-Fc fusion proteins. Comparison of the relative expression levels of the follistatin moiety fused with various fusion partners in different orientations reveals that the FST-Fab construct fused to the C-terminus of the FST moiety is optimal (Figure 6A). Fusing Fc or ScFv to the C-terminus of FST is significantly inferior to using Fab at this position, with the former both resulting in a 3.5-fold decrease in the expression product (Figure 6A). Fusing the Fab moiety at the N-terminus of the FST moiety results in a ~45% decrease in the amount of the expression product but is better than the fusion protein containing FST fused to the Fc domain. Evaluation comparing the effects on expression using FST288 or FST315 fused to Fab at the C-terminus of follistatin using the wild-type HBM sequence revealed only minor differences (Figure 6B). The FST315 version had 6% lower expression than the FST288 fusion. Comparison of the fusions containing HBSM(HBM) revealed a modest 7% decrease in expression level compared to the wild-type. 【0090】 Direct investigation of various FST fusions revealed that the Fab moiety fused to the C-terminus of the FST moiety yields the highest level of final monomer yield compared to other fusions (Figure 6C). The FST-Fc fusion yielded the lowest monomer yield in the set, being approximately 6-fold lower than the FST-Fab fusion. FST-ScFv and FST fused to the N-terminus of Fab were approximately 3-fold and 4-fold lower, respectively. 【0091】 FST315WT, FST288WT and FST288-Fab administered intravenously (IV) to mice at 10 mg / kg were removed very rapidly, with mean residence times (MRT) of 1.6 hours, 2.3 hours and 5.2 hours, respectively. In comparison, FST315-Fab, FST315(HBM)-Fab and FST288HBM-Fab administered IV to mice at 10 mg / kg showed prolonged kinetics, with MRTs of 9.3 hours, 13.1 hours and 11 hours, respectively (Figures 7A and 7B). Thus, it has been shown that thanks to the fusion between the FST part and the Fab part, it was possible to significantly extend the kinetics and half-life of the FST-containing proteins. A significant contribution to the prolonged kinetics is also contributed by the mutation of the heparin-binding site in the form of the HBM version of the follistatin part. 【0092】 References JPEG2025522462000007.jpg77115

Claims

[Claim 1] A pharmaceutical composition comprising a follistatin fusion protein for the treatment, improvement or prevention of kidney disease or kidney injury, wherein the follistatin fusion protein is a. Follistatin portion, b. Antibody portion, and optionally c. Linker between the follistatin portion and the antibody portion A pharmaceutical composition containing the above. [Claim 2] The pharmaceutical composition according to claim 1, wherein the kidney disease is a chronic kidney disease. [Claim 3] The pharmaceutical composition according to claim 2, wherein the chronic kidney disease is selected from the group consisting of primary glomerulonephritis, secondary glomerulonephritis, diabetic nephropathy, hypertensive nephrosclerosis, focal segmental glomerulosclerosis (FSGS), IgA nephropathy (IgAN), mesangial proliferative glomerulonephritis, membranous nephropathy (MN), minimal change disease (MCD), polycystic kidney disease, chronic transplant nephropathy, bone mineral metabolism disorder associated with CKD (CKD-MBD), and Goodpassia disease. [Claim 4] The pharmaceutical composition according to claim 1, wherein the kidney injury is a) renal fibrosis or b) related to another disease. [Claim 5] The pharmaceutical composition according to claim 4, wherein the other disease is selected from the group consisting of diabetes mellitus, hypertension, cardiovascular disease, and bone disorders. [Claim 6] The pharmaceutical composition according to claim 1, wherein the renal disease or renal injury is the result of a therapeutic treatment. [Claim 7] The pharmaceutical composition according to claim 6, wherein the renal disease or renal injury is a result of treatment with chemotherapy. [Claim 8] The pharmaceutical composition according to claim 7, wherein the chemotherapy is selected from Ara-C, bleomycin, camptothecin, carboplatin, cisplatin, cyclophosphamide, doxorubicin (adriamycin), gemcitabine, methotrexate, paclitaxel, and vincristine. [Claim 9] The pharmaceutical composition according to claim 7, wherein the treatment or improvement of renal disease or renal injury comprises administering the fusion protein simultaneously with, separately from, or sequentially with the chemotherapy. [Claim 10] The pharmaceutical composition according to any one of claims 1 to 9, wherein the follistatin portion includes a naturally occurring protein or a functional variant thereof, or is a naturally occurring protein or a functional variant thereof. [Claim 11] The follistatin portion, a. Sequence ID 1 b. Sequence ID 2 c. Sequence ID 3 d. Sequence ID 4 e. Any protein containing amino acid residues including residues 289 to 314 of any one of sequence numbers 1 to 4; or f. Sequences having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with any one of sequence numbers 1 to 4. A pharmaceutical composition according to any one of claims 1 to 9, selected from the above. [Claim 12] The pharmaceutical composition according to any one of claims 1 to 9, wherein the antibody portion binds to albumin and optionally to human serum albumin (HSA). [Claim 13] The pharmaceutical composition according to any one of claims 1 to 9, wherein the antibody portion is a chimeric, humanized, or human antibody portion. [Claim 14] The pharmaceutical composition according to any one of claims 1 to 9, wherein the antibody portion is an antigen-binding fragment selected from Fab, Fab', or F(ab')2. [Claim 15] The antibody portion, a. Light chain variable regions including CDR-L1 containing SEQ ID NO: 13, CDR-L2 containing SEQ ID NO: 14, and CDR-L3 containing SEQ ID NO: 15; and heavy chain variable regions including CDR-H1 containing SEQ ID NO: 16, CDR-H2 containing SEQ ID NO: 17, and CDR-H3 containing SEQ ID NO: 18; or b. Heavy chain variable region containing or consisting of Sequence ID No. 5 and light chain variable region containing or consisting of Sequence ID No. 6 A pharmaceutical composition according to any one of claims 1 to 9, comprising: [Claim 16] The pharmaceutical composition according to any one of claims 1 to 9, wherein the linker is selected from the group consisting of SGGGGS (SEQ ID NO: 7), SGGGGSSGGGGS (SEQ ID NO: 19), GGGGS (SEQ ID NO: 20), and GGGGGSGGGGGS (SEQ ID NO: 21). [Claim 17] a) The antibody portion is connected to the C-terminus or N-terminus of the follistatin portion, or b) If a linker is present, the antibody portion is connected to the C-terminus of the follistatin portion by the linker, according to any one of claims 1 to 9. [Claim 18] The aforementioned fusion protein (a) i. The FST315 polypeptide as defined by Sequence ID No. 1, or a sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity therewith; ii. A Fab heavy chain, as defined by Sequence ID No. 5, attached to the C-terminus of the FST315 polypeptide, or a sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto; and iii. A Fab light chain as defined by Sequence ID No. 6, or a sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto; (b) i. The FST288 polypeptide as defined by Sequence ID No. 2, or a sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto; ii. A Fab heavy chain, as defined by Sequence ID No. 5, attached to the C-terminus of the FST288 polypeptide, or a sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto; and iii. A Fab light chain as defined by Sequence ID No. 6, or a sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto; (c) i. FST315 polypeptide variants as defined by SEQ ID NO: 3 (FST315HBM) or SEQ ID NO: 22, or sequences having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto; ii. A Fab heavy chain, as defined by Sequence ID No. 5, attached to the C-terminus of the FST315 polypeptide variant, or a sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto; and iii. A Fab light chain as defined by Sequence ID No. 6, or a sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto; (d) i. FST288 polypeptide variants as defined by SEQ ID NO: 4 (FST288HBM) or SEQ ID NO: 25, or sequences having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto; ii. A Fab heavy chain, as defined by Sequence ID No. 5, attached to the C-terminus of the FST288 polypeptide variant, or a sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto; and iii. A Fab light chain as defined by Sequence ID No. 6, or a sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto; (e) i. The FST315 polypeptide as defined by Sequence ID No. 1, or a sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto; ii. A linker defined by SEQ ID NO: 7, SEQ ID NO: 19, SEQ ID NO: 20, or SEQ ID NO: 21, attached to the C-terminus of the FST315 polypeptide; iii. A Fab heavy chain, as defined by Sequence ID No. 5, bonded to the free end of the linker, or a sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto; and iv. The Fab light chain as defined by Sequence ID No. 6, or a sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto; (f) i. The FST288 polypeptide as defined by Sequence ID No. 2, or a sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto; ii. A linker defined by SEQ ID NO: 7, SEQ ID NO: 19, SEQ ID NO: 20, or SEQ ID NO: 21, attached to the C-terminus of the FST288 polypeptide; iii. A Fab heavy chain, as defined by Sequence ID No. 5, bonded to the free end of the linker, or a sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto; and iv. The Fab light chain as defined by Sequence ID No. 6, or a sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto; (g) i. FST315 polypeptide variants as defined by SEQ ID NO: 3 (FST315HBM) or SEQ ID NO: 22, or sequences having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto; ii. A linker defined by SEQ ID NO: 7, SEQ ID NO: 19, SEQ ID NO: 20, or SEQ ID NO: 21, attached to the C-terminus of the FST315 polypeptide variant; iii. A Fab heavy chain, as defined by Sequence ID No. 5, bonded to the free end of the linker, or a sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto; and iv. The Fab light chain as defined by Sequence ID No. 6, or a sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto; (h) i. FST288 polypeptide variants as defined by Sequence ID No. 4 (FST288HBM) or Sequence ID No. 25, or sequences having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto; ii. A linker defined by SEQ ID NO: 7, SEQ ID NO: 19, SEQ ID NO: 20, or SEQ ID NO: 21, attached to the C-terminus of the FST288 polypeptide variant; iii. A Fab heavy chain, as defined by Sequence ID No. 5, bonded to the free end of the linker, or a sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto; and iv. The Fab light chain as defined by Sequence ID No. 6, or a sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto; (i) a Fab light chain as defined by SEQ ID NO: 6, or a sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it, along with SEQ ID NOs: 8, 9, 10, 11, 24, 25, 26, 27, 32, 33, 34, or 35, or a sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it; (j) together with the Fab light chain defined by SEQ ID NO: 6, SEQ ID NOs: 8, 9, 10, 11, 24, 25, 26, 27, 32, 33, 34 or 35; or (k) A functional variant or fragment of any one of (a) through (j) A pharmaceutical composition according to any one of claims 1 to 9, comprising or consisting of the following.

Images