Fusion protein for preventing, treating or ameliorating kidney disease

Abstract

The present invention relates to a protein for use in the prevention, treatment or amelioration of kidney disease or kidney injury, more particularly wherein the protein is a follicular hormone chalastatin-fusion protein.

Description

Field of the Invention

[0001] The present invention relates to a protein for preventing, treating or improving kidney disease or kidney damage, and more specifically, the protein is a follistatin-fusion protein. Background of the Invention

[0003] Chronic kidney disease (CKD) affects about 10-13% of the aging population worldwide and is associated with significant mortality and morbidity. It is estimated that 1.5-2% of the population with CKD will progress to end-stage renal disease (ESKD) and the two main causes of ESKD are diabetes and hypertension, which are still progressive despite drug control. In the past 20 years, the treatment options for CKD have relied on the control of hypertension and blood sugar but are seriously insufficient in long-term disease management. The treatment of ESKD relies on dialysis and renal transplantation, but both do not have very good results for patients with progressive forms of the disease. There is an urgent need for new therapies and especially therapies incorporated into novel mechanisms (Yahr et al. (2022); Ammirati et al. (2020)).

[0004] A common feature of failing kidneys is the establishment of fibrotic areas within the tissue that gradually establish over time and which can ultimately lead to progressive failure of the normal function of the organ. Fibrotic diseases are diseases characterized by an abnormal wound healing response in which excessive fibrous connective tissue is formed in an organ or tissue. Deposition and accumulation of excessive extracellular matrix components (e.g., collagen and fibronectin) lead to hardening and scarring of the tissue, leading to pathological remodeling of the organ and can ultimately lead to organ failure (Wynn & Ramalingam (2012)).

[0005] Follistatin is an autocrine glycoprotein whose main function is to bind and neutralize TGF-β superfamily members, especially activin. It is known to exist in several different forms, including a 315 amino acid polypeptide (named FST315); and a 288 amino acid polypeptide (named FST288). Both FST315 and FST288 have high affinity for activin (activin A and activin B) and myostatin. In particular, follistatin can bind to and inhibit myostatin, which is a negative regulator of skeletal muscle mass. Follistatin is a protein of interest for the therapy of a variety of diseases including muscle diseases such as muscular dystrophy (WO2015 / 187977 and WO2017 / 152090) or diseases or disorders such as inflammatory bowel disease, ulcerative colitis and Crohn's disease (WO03 / 006057) directly or as a fusion part of a fusion protein.

[0006] However, there remains a need to identify effective therapies for the treatment and prevention of kidney disease or kidney damage. SUMMARY OF THE INVENTION

[0008] In the first aspect, the present invention provides a follistatin-fusion protein for treating, ameliorating or preventing kidney disease (e.g., chronic kidney disease (CKD)) or kidney damage, 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 a second aspect, the present invention relates to a method for treating a patient in need of therapy to treat, improve or prevent a kidney disease (e.g., chronic kidney disease (CKD)) or kidney damage, the method comprising administering a therapeutically effective amount of a follistatin-fusion protein, 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.

[0010] In a third aspect, the present invention describes the use of a follistatin-fusion protein in the preparation of a medicament for treating, ameliorating or preventing kidney diseases (e.g., chronic kidney disease (CKD)) or kidney damage, 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.

[0011] Specifically, 1) the renal disease is preferably 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 nephropathy (MCD), polycystic kidney disease, chronic allograft nephropathy, CKD-associated mineral and bone disease (CKD-MBD) and Goodpas tures disease, and 2) renal damage is a) renal fibrosis or b) associated with other diseases such as diabetes, hypertension, cardiovascular disease or bone disease. The renal disease or renal damage may be the result of therapeutic treatment (e.g., treatment using chemotherapy).

[0012] BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 : Schematic diagram showing FST288 and FST315 aligned with bound activin molecules.

[0014] Figure 2: Follistatin regulation in a doxorubicin-driven chronic kidney disease model in mice using hydrodynamic transfection to deliver therapeutic proteins. Figure 2 A) Hydroxyproline renal content was measured at study termination as a surrogate for renal collagen accumulation and fibrosis, no disease = naive, disease and saline treatment = saline, and disease and follistatin treatment = follistatin. ( Figure 2 B) Serum creatinine was measured at study termination as a surrogate for renal function, no disease = naive, disease and saline treatment = saline, and disease and follistatin treatment = follistatin. ( Figure 2 C) Conditional survival shows the status of the animals during the duration of the study and at the termination of the study, where all living animals surviving disease in either saline or follistatin-treated disease groups were terminated. Figure 2 D) Picros irius Red (PSR) staining as a surrogate for collagen content in selected kidneys showing 1 kidney from the naive group, 3 separate kidneys from the saline-treated disease group, and 3 separate kidneys from the follistatin-treated disease group. ( Figure 2 E) All kidneys from the study were stained with PSR and analyzed to determine the relative percent area of ​​collagen in the 3 different groups, where mice without disease = naive, mice with disease and saline treatment = saline, and mice with disease and follistatin treatment = follistatin; statistics were compared using ordinary one-way analysis of variance (ANOVA) with Tukey's multiple comparison test to compare saline and follistatin treated groups and **** Indicates p < 0.0001. Group sizes, naive n = 8, saline n = 21, follistatin n = 18.

[0015] Figure 3 : We compared different follistatin fractions for modulating the unilateral ureteral obstruction (UUO) model of acute kidney disease in mice using standard methods and commercial plasmids to deliver therapeutic proteins by hydrodynamic transfection. Figure 3 A) Picrosirius red (PSR) staining as a surrogate for collagen content in selected kidneys showing 1 kidney without disease (non-operated) = Non-Op, and 4 kidneys from the UUO disease group treated with a control plasmid secreted alkaline phosphatase = SEAP control, a plasmid containing FST315(HBM)-Fab, and a plasmid containing FST315-Fab. ( Figure 3 B) All kidneys from the study were stained with PSR and analyzed to determine Figure 3The relative percentage area of ​​collagen in the different groups described in A; statistics were performed using ordinary one-way analysis of variance (ANOVA) with Tukey's multiple comparison test, where NS represents non-significant, * p<0.05. ( Figure 3 C) Body weight changes of mice in different groups; control plasmid (SEAP), FST315(HBM)-Fab or FST315-Fab plasmid. Group size, non-surgery n=4, Seap control n=8, FST315(HBM)-Fab n=7, FST315-Fab n=7.

[0016] Figure 4 : Comparison of the follistatin protein fractions FST315-Fab and FST315(HBM)-Fab, with the latter being a dose-response comparison, in the unilateral ureteral obstruction (UUO) model of acute kidney disease in mice using direct delivery of therapeutic protein by subcutaneous administration. Figure 4 A) Morphological representation of whole kidneys at low magnification stained with PSR from a UUO mouse study showing a disease-free or non-operated kidney in the left panel = normal, and 2 UUO kidneys: the middle panel is a UUO diseased kidney treated with vehicle = 21 days UUO, and the right panel is a UUO kidney treated with 10 mg / kg / day of FST315(HBM)-Fab. The associated histological scores are also shown together. ( Figure 4 B) Morphological representation of kidney fragments at high magnification stained with PSR showing UUO diseased kidney treated with vehicle (upper panel) and UUO diseased kidney treated with 10 mg / kg FST315(HBM)-Fab (lower panel). Figure 4 C) Morphological representation of kidney sections at medium magnification stained with PSR showing representative normal kidneys (non-Op) and 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) All kidneys from the study were stained with PSR and scanned to determine the relative percent area of ​​collagen in the different groups, which were non-operated or healthy kidneys = Non-Op (n=4), UUO diseased kidneys treated with 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 analysis of variance (ANOVA) with uncorrected Fisher's LSD multiple comparison test, where NS indicates non-significance, * p<0.05 and **** p<0.0001. ( Figure 4 E) All PSR-stained kidneys in different groups were manually scored on a histological damage scale of 1-10, where 1 represents a normal kidney and 10 represents the most diseased kidney. Figure 4 AC, with special consideration of epithelial integrity. Groups were non-operated or healthy kidney = non-Op (n=4), UUO diseased kidney treated with 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 analysis of variance (ANOVA) with uncorrected Fisher's LSD multiple comparison test, where NS indicates non-significance, * p<0.05, ** p<0.01, *** p<0.001 and **** p<0.0001. Figure 4 F) Monitoring throughout the life cycle Figure 4 Body weight changes of mice in the different groups described in AE and this is plotted over time for the different groups; non-operated and UUO treated with vehicle, 2 mg / kg of 315FST-Fab or 10, 2, 0.2 or 0.02 mg / kg of FST315(HBM)-Fab.

[0017] Figure 5 :( Figure 5 A) Principle of the assay described in Example 4. Figure 5B) Doxorubicin treatment induced disruption of barrier integrity as measured by increased permeability to FITC-Dx, and provided protection in the presence of FST315(HBM)-Fab but not Fab control.

[0018] Figure 6 :( Figure 6 A) Relative expression levels of follistatin fusion proteins were determined by Protein-G HPLC analysis of collected CHO supernatants. Values ​​were normalized to FST-Fc expression levels. 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 6 B) Relative expression levels of FST288, FST315, FSTWT, and FST-HBM (or HBSM) with C-terminal fusion partners (respectively named 288 fusion, 315 fusion, WT fusion, and HBSM fusion in the figure). Values ​​are normalized to the expression of 288 fusion. ( Figure 6 C) Total expression values ​​of monomers, values ​​normalized to FST-fc expression levels, n = 25. Legend and Figure 2 (A) The same.

[0019] Figure 7 :( Figure 7 A) Pharmacokinetic profiles of FST315WT, FST315-Fab and FST315HBM-Fab administered to mice (n=3 mice per group) at 10 mg / kg via IV administration route and serum samples monitored for 7 days; ( Figure 7 B) Pharmacokinetic profile of FST288WT, FST288-Fab and FST288HBM-Fab dosed to mice (n=3 mice per group) at 10 mg / kg by IV route of administration and serum samples monitored for 7 days. Pharmacokinetic parameters derived from all follistatin fractions studied are summarized in Table 1. DETAILED DESCRIPTION OF THE INVENTION

[0021] The entire document is intended to be associated as a unified disclosure, and it should be understood that all combinations of features described herein are contemplated, even if the combination of features is not found together in the same sentence, paragraph or section of this document. In regard to aspects of the invention described or claimed with "a" or "an", it should be understood that these terms refer to "one or more / one or more" unless the context clearly requires a more restricted meaning. Unless the context clearly requires otherwise, the term "or" should be understood to cover alternative items or to cover items simultaneously. If aspects of the invention are described as "comprising" features, embodiments are also considered to be "consisting of 'features'" or "consisting essentially of 'features'".

[0022] As used herein, the term "follistatin" or "FST" refers to an autocrine glycoprotein (UniProt reference: P19883) that is a known inhibitor of activin A and B. Follistatin also binds to GDF11, GDF8 (myostatin), BMP 2, 4, 6, 7, 11 and 15 with lower affinity. There are two major alternatively spliced ​​forms of human follistatin: a shorter form (FST288, 31.6 kDa) that is cell-bound 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 a heparin binding site. FST315 has an additional 27 amino acid domain (rich in acid) at its C-terminus, called the acidic tail. The term also encompasses its functional fragments and / or functional variants, such as those disclosed in Sidis et al., 2005. If FST is followed by a number, such as FST288, this indicates that the protein is 288 forms of follistatin (starting from residue 1 of the mature form). If FST is followed by numbers and letters, such as FST315HBM, this indicates the type of heparin binding mutant (HBM) form and variant (here, 315 forms 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 can stimulate hormone production in ovarian and placental cells, support neuronal cell survival and affect cell cycle progression positively or negatively according to cell type. In several 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 FSH secretion and synthesis, while inhibin blocks FSH secretion and synthesis. Activin is also considered a negative regulator of muscle mass and function, and activin antagonists can promote muscle growth or counteract muscle loss in vivo.

[0023] As used herein, the term "antibody" includes, but is not limited to, monoclonal antibodies, polyclonal antibodies, and recombinant antibodies generated by recombinant techniques as known in the art. As used herein, the term "antibody" includes antibodies of any species, particularly antibodies of mammalian species; for example, human antibodies of any isotype, including IgG1, IgG2a, IgG2b, IgG3, IgG4, IgE, IgD, and antibodies produced as dimers of the basic structure, including IgGA1, IgGA2, or pentamers such as IgM and modified variants thereof; non-human primate antibodies, such as those from chimpanzees, baboons, rhesus monkeys, or cynomolgus monkeys; rodent antibodies, such as those from mice or rats; rabbit, goat, or horse antibodies; camelid antibodies (e.g., from camels or alpacas, such as Nanobodies TM ) and derivatives thereof; antibodies of avian species such as chicken antibodies; or antibodies of fish species such as shark antibodies. The term "antibody" refers to both glycosylated and non-glycosylated antibodies. In addition, as used herein, the term "antibody portion" may refer to a full-length antibody, but is more generally intended to refer to an antibody fragment, and more specifically to an antigen-binding fragment thereof. The fragment of an antibody comprises at least one heavy chain or light chain immunoglobulin domain as 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 double-scFv fragments. The fragment may also be a bispecific antibody, a trispecific antibody, a trispecific antibody, a tetrabodies, a mini antibody, a single domain antibody (dAb) such as sdAb, VL, VH, VHH or a camelid antibody (e.g., from a camel or alpaca such as Nanobody TM ) and VNAR fragments. The antigen binding fragment according to the invention may also comprise a Fab connected to one or two scFvs or ds scFvs, each scFv or ds scFv binding to the same or different targets (e.g., one scFv or ds scFv binding to a therapeutic target, one scFv or ds scFv increasing half-life by binding to, for example, albumin).

[0024] As used herein, the term "Fab" refers to an antibody fragment comprising a light chain fragment comprising the VL (variable light) domain and the constant domain (CL) of the light chain and the VH (variable heavy) domain and the first constant domain (CH1) of the heavy chain.

[0025] As used herein, the term "Fab'" is similar to Fab, wherein the Fab portion is replaced by Fab'. This form can be provided as a pegylated form thereof. Dimers of Fab' according to the present disclosure produce F(ab')2, wherein dimerization can be performed, for example, via a hinge.

[0026] The term "Fv" refers to the two variable domains of a full length antibody, eg, cooperating variable domains, such as a cognate pair or affinity matured variable domains, ie, a VH and VL pair.

[0027] 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 and VL variable domains.

[0028] 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.

[0029] As used herein, the term "affinity" refers to the strength of all non-covalent interactions between a protein or fragment thereof and its receptor (if the protein of interest is a ligand) or its ligand (if 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 a 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 expressed in terms of a dissociation constant (KD). Affinity can be measured by conventional methods known in the art, including those described herein.

[0030] The term "specific" as used herein in the context of antibodies and antigen-binding fragments is intended to refer to an antibody that recognizes only the antigen against which it is specifically directed, or an antibody that has a significantly higher binding affinity for the antigen against which it is specifically directed than to the antigen against which it is not specifically directed, e.g., at least 5, 6, 7, 8, 9, 10 times higher binding affinity.

[0031] The term "chimeric" refers to an antibody in which at least a first portion of the heavy and / or light chain antibody sequence is from a first species and a second portion of the heavy and / or light chain antibody sequence is from a second species. Chimeric antibodies of interest herein include "primatized" antibodies comprising variable domain antigen-binding sequences derived from non-human primates (e.g., Old World Monkeys, e.g., Baboons, Rhesus Monkeys, or Cynomolgus Monkeys) and human constant region sequences.

[0032] "Humanized" antibodies are chimeric antibodies containing sequences derived from non-human antibodies. In most cases, humanized antibodies are human antibodies (receptor antibodies), wherein the residues from the hypervariable region of the receptor are replaced by residues from the hypervariable region [or complementary determining region (CDR)] of non-human species (donor antibodies) such as mice, rats, rabbits, chickens or non-human primates, which have the required specificity, affinity and activity. In most cases, the residues of human (receptor) antibodies outside CDR, i.e., residues located in the framework region (FR), are additionally replaced by corresponding non-human residues. In addition, humanized antibodies may include residues not found in receptor antibodies or donor antibodies. These modifications have been carried out to further improve antibody properties. Humanization reduces the immunogenicity of non-human antibodies in people, and therefore helps antibodies to be applied to the treatment of human diseases. Humanized antibodies and several different techniques for generating them are well known in the art. Unless otherwise stated, HVR residues (CDR residues) and other residues in the variable domain (e.g., FR residues) are numbered herein according to Kabat et al.

[0033] The term "antibody" also refers to human antibodies, which can be produced as a humanized alternative. For example, it is possible to produce transgenic animals (e.g., mice) that can produce a complete human antibody library without producing endogenous mouse antibodies after immunization. Other methods for obtaining human antibodies / antibody fragments in vitro are based on display technologies such as phage display or ribosome display technology, in which the recombinant DNA library used is at least partially artificially generated or obtained from the immunoglobulin variable (V) domain gene library of a donor. Phage and ribosome display technology for generating human antibodies is well known in the art. Human antibodies can also be generated from separated human B cells, which are immunized in vitro with an antigen of interest and subsequently fused to generate hybridomas, which can then be screened to obtain the best human antibodies.

[0034] As used herein, the term "functional variant" refers to an amino acid sequence that has been modified relative to a reference sequence, but which retains at least one biological function of the reference sequence. For example, a functional variant of FST retains at least one biological activity of a reference FST protein, such as binding to and inhibiting activator proteins A and B.

[0035] As used herein, the term "sequence identity" or "identity" refers to the quantity of matching (identical nucleic acid or amino acid residues) in the position of the comparison of two polynucleotides or polypeptide sequences. The sequence identity is determined by comparing the sequence during the comparison so that the sequence gap is minimized while maximizing overlap and identity. Specifically, according to the length of the two sequences, any of many mathematical global or local comparison algorithms can be used to determine the sequence identity. The comparison for the purpose of determining the identity of the percentage nucleic acid or amino acid sequence can be realized in various ways within the technical scope of the art, for example, using publicly available computer software such as http: / / blast.ncbi.nlm.nih.gov / or http: / / www.ebi.ac.uk / Tools / emboss / available on the Internet website. Those skilled in the art can determine the appropriate parameters for measuring the comparison, including any algorithm required for maximum comparison on the total length of the compared sequence. For the purposes of this article, % nucleic acid or amino acid sequence identity values ​​refer to the values ​​generated using the pairwise sequence alignment program EMBOSS Needle, which uses the Needleman-Wunsch algorithm to create an optimal global alignment of two sequences, wherein all search parameters are set to the default values, i.e., scoring matrix=BLOSUM62, gap opening=10, gap extension=0.5, end gap penalty=no, end gap opening=10 and end gap extension=0.5.

[0036] The term "isolated" throughout this specification refers to an antibody, antigen binding fragment, polypeptide or polynucleotide, as the case may be, that can be present in a physical environment different from that in which it can be found in nature. The term "isolated" nucleic acid refers to a nucleic acid molecule that has been separated from its natural environment or has been synthetically produced. An isolated nucleic acid may comprise synthetic DNA, such as DNA produced by chemical processing, cDNA, genomic DNA, or any combination thereof.

[0037] As used herein, the term "pharmaceutically acceptable" means approved for use in animals and / or humans by a regulatory agency or a recognized pharmacopoeia, such as the European Pharmacopoeia. The term "excipient" refers to a diluent, adjuvant, carrier and / or vehicle with which a therapeutic agent is administered.

[0038] As used herein, the terms "treatment" or "treating" and the like refer to obtaining a desired pharmacological and / or physiological effect. The effect may be preventive 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 a side effect attributable to the disease. Treatment thus encompasses any treatment of a disease in a mammal, particularly a human, and includes: (a) preventing the disease from occurring in a subject (i.e., a human) who may be susceptible to the disease but has not yet been diagnosed as having the disease; (b) inhibiting the disease, i.e., preventing its development; and (c) alleviating the disease, i.e., causing the disease to regress.

[0039] The term "therapeutically effective amount" refers to an amount that, when administered to a subject for treating a disease, is sufficient to produce such treatment for the disease.

[0040] The term "therapeutically effective amount" refers to an amount sufficient to produce such treatment for the disease when administered to a subject to treat the disease. The therapeutically effective amount will vary depending on the protein or its active fragment, the disease and its severity, and the age, weight, etc. of the subject to be treated.

[0041] As used herein, "chemotherapy" refers to any agent that, when used alone or in combination, can alleviate, reduce, ameliorate, prevent or otherwise maintain in remission one or more clinical symptoms or diagnostic markers associated with neoplastic diseases, tumors and cancers. Exemplary chemotherapeutic agents include, but are not limited to, alkylating agents, toxins, anticancer antibiotics, antimetabolites, antimitotic agents, and the like. Known specific compounds include, but are not limited to, cytosine arabinoside Ara-C, bleomycin, camptothecin, carboplatin, cisplatin, cyclophosphamide, doxorubicin (adriamycin), gemcitabine, methotrexate, paclitaxel and vincristine.

[0042] The invention will now be described with respect to specific non-limiting aspects and embodiments thereof and with reference to certain figures and examples.

[0043] The present invention addresses the need to identify effective therapies for the treatment and prevention of kidney disease or kidney damage.

[0044] The present invention is based on the surprising discovery from the inventors, that is, in many animal models of kidney disease, the addition of exogenous follistatin-fusion protein is associated with a significant improvement in the markers of kidney disease (e.g., renal fibrosis markers) in the animals. Such markers are, for example, hydroxyproline, serum creatinine, or collagen. The inventors were then able to surprisingly prove that not only follistatin-fusion protein can weaken the ECM deposition in kidney sections (as shown by the reduction of collagen), but also follistatin-fusion protein significantly weakened renal fibrosis in vivo when applied to UUO mice. It is also surprisingly shown that epithelial cells (e.g., renal tubular epithelial cells) in the kidney are protected in the presence of damaging stimuli.

[0045] It is not only demonstrated herein that follistatin-fusion proteins can be therapeutic for treating and preventing kidney disease or kidney damage, but also that these fusion proteins are more advantageous than previously known follistatin or follistatin-based fusion proteins comprising an Fc portion. Specifically, 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.

[0046] The main object of the present invention is a follistatin-fusion protein for treating, improving or preventing kidney disease or kidney damage, wherein the follistatin-fusion protein comprises: a. a follistatin part, b. an antibody part and optionally c. a linker between the follistatin part and the antibody part.

[0047] The present invention also provides a method for treating a patient in need of therapy to treat, improve or prevent renal disease or renal damage, the method comprising administering a therapeutically effective amount of a follistatin-fusion protein, 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.

[0048] Also described is the use of a follistatin-fusion protein in the preparation of a medicament for treating, ameliorating or preventing kidney disease or kidney damage, 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.

[0049] In the context of the present invention as a whole, kidney disease or kidney injury is characterized by a change in certain markers in a sample of a subject compared to said markers in a sample of a healthy subject, and wherein the sample is a cell or tissue associated with said disease / injury (e.g., a kidney cell or kidney tissue) or wherein the sample is, for example, (but not limited to) blood, serum or urine. For example, the change may be an increase in a disease marker, wherein the marker is, for example, any one of serum creatinine, cystatin C, serum albumin levels, blood urea nitrogen (BUN), (but not limited to) or a decrease in estimated glomerular filtration rate (eGFR). Alternatively, the change may be an alteration in a urinary disease marker such as an increase in albumin or protein, referring to albuminuria and proteinuria, respectively, and / or an increase in microglobulin, neutrophil gelatinase-associated lipocalin (NGAL) or kidney injury molecule-1 (KIM-1). The change can also be an increase in one of the follistatin ligands associated with a disease or disease progression or injury, and the follistatin ligand can be (but not limited to) activin A, activin B, GDF8 (myostatin) or GDF11. The change in the marker in the sample can be determined by any method. The reduction or increase of a marker in a subject's sample is usually determined by comparing the level of the marker in the subject's sample with the level of the same marker in a reference sample of the same sample type (herein alternatively referred to as a healthy sample or a normal sample) (i.e., the basal level; for example, a basal disease activity biomarker, a basal expression level of a follistatin ligand (mRNA level and / or protein level) and / or a basal level of a fibrosis marker).

[0050] The follistatin-fusion protein, method or use of the follistatin-fusion protein for the use as a whole according to the present invention, for example, for treating, improving or preventing a subject's renal disease or renal injury, may therefore include the following steps: (a) measuring at least one marker of renal disease in a sample from the subject (e.g., renal cells, serum or urine), wherein the marker is, for example, follistatin ligand activity, follistatin ligand expression or circulating follistatin ligand level, (b) comparing the measurement results obtained from a) with the corresponding measurements in a normal sample, and c) if an increase in the expression, activity or circulating level of follistatin ligand is observed, administering the follistatin-fusion protein to the subject, thereby treating, improving or preventing renal disease or renal injury. The follistatin ligand expression measured in step a) can be the amount of mRNA or protein, and the increase can be an increase in any expression as described above. The corresponding measurement in the normal sample does not need to be obtained every time the comparison is made. The corresponding measurement may be obtained at any time prior to the comparison being made and may be the average follistatin ligand expression or follistatin ligand activity in normal cells / tissues.

[0051] Alternatively, the follistatin-fusion protein, the method or use of the follistatin-fusion protein for the use as a whole according to the present invention, for example, for treating, improving or preventing a subject's renal disease or renal injury, may therefore include the following steps: (a) measuring at least one marker of renal disease in a sample from the subject (e.g., renal cells, serum or urine), wherein the marker is, for example, proteinuria, serum creatinine or cystatin C, (b) comparing the measurement results obtained from a) with the corresponding measurements in a normal sample (e.g., serum or urine), and c) if an increase in the expression of the marker is observed, administering the follistatin-fusion protein to the subject, thereby treating, improving or preventing renal disease or renal injury. The expression of the marker measured in step a) can be the amount of mRNA or protein, and the increase can be an increase in any expression as described above. The corresponding measurement in the normal sample does not need to be obtained every time the 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 / tissues / serum / urine.

[0052] In another alternative, the follistatin-fusion protein, method or use of the follistatin-fusion protein for use according to the present invention as a whole, for example, for treating, ameliorating or preventing a renal disease or renal injury in a subject, may comprise the following steps: (a) monitoring the rate of loss of estimated GFR (eGFR) and (b) if a decrease in eGFR or measurement of the slope of eGFR loss is observed, indicating a rapid progression of renal function loss (e.g. >5 ml / min / 1.73 m 2 / year reduction is generally considered to accelerate progression), administering follistatin-fusion protein to the subject, thereby treating, improving or preventing kidney disease or kidney damage. Monitoring eGFR loss can also be considered together with an increase in proteinuria or albuminuria, where a value of 30-300 mg / 24 hours of urine collection is generally considered to be a moderate increase or "microalbuminuria" and an increase in urine collection of >300 mg / 24 hours is generally considered to be severe or "macroalbuminuria". Including albuminuria with eGFR loss is rationalized as a way to assess the risk of progression of renal dysfunction and the potential risk of ESRF (Ammirati, 2020). Rapid progression of renal function loss determined by any number of measurements can benefit from follistatin-fusion protein treatment as a method for treating, improving or preventing kidney disease or kidney damage.

[0053] It must be understood that markers that can be measured to determine the need for treatment may be combined, such as (in a non-limiting example) measuring an increase in follistatin ligand expression in combination with an increase in serum creatinine or proteinuria or albuminuria.

[0054] In the context of the present invention as a whole, the kidney disease is preferably a chronic kidney disease such as (but not limited to) 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, CKD-associated mineral and bone disease (CKD-MBD) and Goodpas tures disease. Alternatively, the kidney disease is a chronic kidney disease 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 nephropathy (MCD), polycystic kidney disease, chronic allograft nephropathy, CKD-related mineral and bone disease (CKD-MBD) and Goodpas tures disease. Also in the context of the present invention as a whole, kidney damage is a) renal fibrosis, or b) related to other diseases such as diabetes, hypertension, cardiovascular disease or bone disease. Both primary and secondary forms of glomerulonephritis can develop to end-stage renal failure (ESRF), as can most forms of kidney disease. Conventional treatment calls for dialysis, but the results are generally poor, with a survival rate of less than 50% after five or more years. Kidney transplantation is currently considered to be the gold standard treatment, but organ acceptance rates are very low. Renal disease or renal injury can be its own disease or injury or can be the result of therapeutic treatment, such as the treatment of chemotherapy.In this context, chemotherapeutic agents can be selected from but not limited to alkylating agents, toxins, anticancer antibiotics, antimetabolites, antimitotic agents, etc.The specific compound known to cause renal injury includes but is not limited to cytosine arabinoside, bleomycin, camptothecin, carboplatin, cisplatin, cyclophosphamide, doxorubicin (adriamycin), gemcitabine, methotrexate, paclitaxel and vincristine.When follistatin-fusion protein is / may be the result of therapeutic treatment in the case of renal disease or renal injury is applied, the fusion protein can be used simultaneously, separately or sequentially with other treatments such as chemotherapy.In a specific example, follistatin-fusion protein according to the present invention is used to treat, prevent or improve renal disease or renal injury caused by doxorubicin (adriamycin) treatment.

[0055] Without wishing to be bound by any theory, the inventors have found that the follistatin-based therapy using the fusion protein of the present invention can regulate ligand signaling, resulting in increased protection of epithelial cells (e.g., tubular epithelial cells) and reduced profibrotic signaling, which will reduce scar tissue in the kidney. Therefore, this treatment can slow down or prevent tissue damage and fibrosis associated with progressive renal disease, resulting in a reduction in eGFR loss and a reduction in proteinuria. Thus, the prevention of end-stage renal failure and / or the extension of the time to renal replacement therapy / transplantation can be achieved. In other words, in the context of the present invention as a whole, the treatment, improvement or prevention of a subject's renal disease or renal injury is obtained by protecting epithelial cells (e.g., tubular epithelial cells).

[0056] The follistatin part of the follistatin-fusion protein to be used in the context of the present invention as a whole comprises a naturally occurring follistatin protein or is a naturally occurring follistatin protein. It is preferably its mature form, i.e. lacks the N-secretion signal sequence, because this sequence is only necessary for production / secretion from cells. Alternatively, it is a functional fragment thereof. The follistatin part is, for example, FST288 protein (SEQ ID NO:2) or FST315 protein (SEQ ID NO:1). Any intermediate form thereof may also be used, such as any follistatin part contained between residues 289 to 314 of any one of SEQ ID NOs:1 to 4, as long as it is functional, i.e. they retain at least one biological activity of FST. Although not limiting, it is preferably any intermediate form of the follistatin part starting from residue 1 of SEQ ID NO:1. As a non-limiting example, a functional FST fragment may be FST291 (i.e. comprising residues 1 to 291 of SEQ ID NO: 1) or FST303 (i.e. comprising residues 1 to 303 of SEQ ID NO: 1). In another alternative, the follistatin portion according to the invention (i.e. its naturally occurring form or a functional fragment thereof) is a functional variant, for example it may have one or more mutations, for example a mutation in a heparin binding site (HBS). As a non-limiting example, one or more mutation sites may be selected from K76, K81 and / or K82 numbered relative to SEQ ID NO: 1 (see sequences 22 and 25 as examples). One or more mutations may comprise alanine (A) in place of lysine (K) (resulting in a mutation selected from K76A, K81A and / or K82A). As another non-limiting example, a heparin binding mutant ("HBM") (alternatively designated herein as "(HBM)" or "HBSM"), such as FST288HBM (SEQ ID NO: 4), FST291HBM, FST303HBM or FST315HBM (SEQ ID NO: 3), wherein the mutant comprises the triple mutations K76A, K81A and K82A. Preferably, the follistatin portion 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 containing residues 289 to 314 of any one of SEQ ID NOs: 1 to 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 to 4.

[0057] The antibody portion of the follistatin-fusion protein used in the context of the present invention as a whole binds to albumin, and preferably binds to serum albumin, such as human serum albumin (HSA). The antibody portion can be a chimeric, humanized or human antibody portion. Preferably, the antibody portion is an antigen-binding fragment of an antibody (alternatively referred to herein as an antigen-binding portion). Preferably, it is selected from Fab, Fab' or F(ab')2. In a non-limiting example, the antibody portion comprises a light chain variable region comprising a CDR-L1 containing SEQ ID NO: 13; a CDR-L2 containing SEQ ID NO: 14 and a CDR-L3 containing SEQ ID NO: 15; and a heavy chain variable region comprising a CDR-H1 containing SEQ ID NO: 16; a CDR-H2 containing SEQ ID NO: 17 and / or a CDR-H3 containing SEQ ID NO: 18. In an alternative non-limiting example, the antibody portion comprises a heavy chain variable region comprising or consisting of 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 or consisting of 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: 5 and SEQ ID NO: 6 represent the heavy and light variable chains of the anti-albumin antibody named "CA645" (as disclosed in WO2013 / 068571).

[0058] In the context of the present invention as a whole, the follistatin-fusion protein optionally comprises a linker between the follistatin portion and the antibody portion. When a linker is present, it may 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), as non-limiting examples.

[0059] It will be understood that when fusion proteins are produced, there are two options for fusing / combining any part to each other: C-terminal fusion or N-terminal fusion. The inventors have found that by fusing the N-terminal part of the antibody part to the C-terminal part of the follistatin part, the expression of the further improved fusion protein obtained is caused by comparing with any other type of fusion, in particular, by comparing with the known Fc-based follistatin-fusion protein, the C-terminal fusion protein of the present invention shows excellent expression and higher yield of monomeric protein. However, although the C-terminal fusion protein causes the highest expression level, those skilled in the art may consider that the antibody part is fused to the N-terminal of follistatin because they cause, for example, an expression level of about 1.5 higher than that of the Fc-based follistatin-fusion protein. Therefore, in a preferred embodiment, the antibody part is connected to the C-terminal part of the follistatin part. If there is a joint, the antibody part is preferably connected (or conjugated) to the C-terminal part of the follistatin part by a joint (in other words, the antibody part is connected (or conjugated) to the C-terminal part of the follistatin part, and there is a joint between the two parts).

[0060] Similarly, it will be appreciated that when creating a fusion protein between a polypeptide (here the FST portion) and an antibody portion (here preferably Fab, Fab' or F(ab')2), there are two options for fusing any of the portions to each other: the polypeptide to the heavy chain of the antibody portion or to the light chain of the antibody portion.

[0061] Non-limiting examples of follistatin fusion proteins that can be used according to the present invention are:

[0062] (a) i. a 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;

[0063] ii. 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 thereof, conjugated to the C-terminus of the FST315 polypeptide; and

[0064] iii. 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;

[0066] (b) i. a 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;

[0067] ii. a polypeptide consisting of SEQ ID NO: 5 or having at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence thereof conjugated to the C-terminus of the FST288 polypeptide.

[0068] A Fab heavy chain defined by a sequence having the same identity as that of

[0069] iii. 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;

[0071] (c) i. SEQ ID NO: 3 (FST315HBM) or SEQ ID NO: 22 or a

[0072] At least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity

[0073] FST315 polypeptide variants defined by a unique sequence;

[0074] ii. a polypeptide having at least 95%, at least 96%, at least 97%, at least 98% or at least 99% affinity with SEQ ID NO: 5 or ... at least 99% affinity with SEQ ID NO: 5

[0075] A Fab heavy chain defined by a sequence having sequence identity; and

[0076] iii. 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;

[0078] (d) i. SEQ ID NO: 4 (FST288HBM) or SEQ ID NO: 25 or a

[0079] At least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity

[0080] FST288 polypeptide variants defined by a unique sequence;

[0081] ii. a polypeptide having at least 95%, at least 96%, at least 97%, at least 98% or at least 99% affinity with SEQ ID NO: 5 or ... at least 99% affinity with SEQ ID NO: 5

[0082] A Fab heavy chain defined by a sequence having sequence identity; and

[0083] iii. 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;

[0085] (e) i. a 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;

[0086] ii. conjugated to the C-terminus of the FST315 polypeptide by SEQ ID NO: 7, SEQ ID

[0087] NO: 19, SEQ ID NO: 20 or SEQ ID NO: 21;

[0088] iii. a moiety consisting of SEQ ID NO: 5 or a moiety having at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity thereto, conjugated to the free end of the linker

[0089] A Fab heavy chain defined by a sequence of

[0090] 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;

[0092] (f) i. a 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;

[0093] ii. conjugated to the C-terminus of the FST288 polypeptide by SEQ ID NO: 7, SEQ ID

[0094] NO: 19, SEQ ID NO: 20 or SEQ ID NO: 21;

[0095] iii. a moiety consisting of SEQ ID NO: 5 or a moiety having at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity thereto, conjugated to the free end of the linker

[0096] A Fab heavy chain defined by a sequence of

[0097] 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;

[0099] (g) i. SEQ ID NO: 3 (FST315HBM) or SEQ ID NO: 22 or a residue thereof

[0100] At least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity

[0101] FST315 polypeptide variants defined by a unique sequence;

[0102] ii. a linker defined by SEQ ID NO:7, SEQ ID NO:19, SEQ ID NO:20 or SEQ ID NO:21 conjugated to the C-terminus of the FST315 polypeptide variant;

[0104] 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 thereof, conjugated to the free terminus of a linker; and

[0105] 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;

[0106] (h) i. A 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;

[0107] ii. a linker defined by SEQ ID NO: 7, SEQ ID NO: 19, SEQ ID NO: 20 or SEQ ID NO: 21 conjugated to the C-terminus of the FST288 polypeptide variant;

[0108] 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 thereof, conjugated to the free terminus of a linker; and

[0109] 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;

[0110] (i) 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 thereof, 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 thereof;

[0111] (j) SEQ ID NO:8, 9, 10, 11, 24, 25, 26, 27, 32, 33, 34 or 35, together with a Fab light chain defined by SEQ ID NO:6; or

[0112] (k) A functional variant or functional fragment of any one of (a) to (i).

[0113] Any subject may 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 may be used indiscriminately rather than subject.

[0114] Any follistatin-fusion protein to be used according to the present invention can be incorporated into a pharmaceutical composition suitable for administration to a subject in any manner, such as, but not limited to, topical, intranasal, intradermal, intravenous, subcutaneous or intramuscular. Typically, the pharmaceutical composition to be used according to the present invention comprises follistatin-fusion protein and one or more pharmaceutically acceptable adjuvants and / or carriers. Therefore, a pharmaceutical composition for treating, improving or preventing kidney disease or kidney damage in a subject is also described herein, wherein the pharmaceutical composition comprises follistatin-fusion protein as described herein and one or more pharmaceutically acceptable adjuvants and / or carriers. The pharmaceutical composition according to the present invention can be part of a kit with instructions for use, including instructions and optionally a device for intravenous, subcutaneous or intramuscular administration to an individual in need.

[0115] As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents and absorption delaying agents, etc., which are physiologically compatible and suitable for administration to a subject for use in the methods and uses described herein. Examples of pharmaceutically acceptable carriers include one or more of water, saline, buffered saline, dextrose, glycerol, ethanol, etc., and combinations thereof. Depending on the route of administration or the type of formulation (e.g., liquid, freeze-dried or spray-dried formulations), isotonic agents may be incorporated into the composition, such as sugars, polyols (e.g., mannitol, sorbitol) or sodium chloride. Pharmaceutically acceptable carriers may further include a small amount of auxiliary substances such as wetting agents or emulsifiers, preservatives or buffers, which enhance the shelf life or effectiveness of the antibody or antibody portion.

[0116] Pharmaceutical compositions 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 expected mode of administration and therapeutic application. Typical preferred compositions are in the form of injectable or infusible solutions, for example, similar to those compositions for passive immunization of people with other antibodies.

[0117] The appropriate dosage of follistatin-fusion protein according to the present invention can be determined by skilled medical practitioners. 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 effective to achieve the desired therapeutic response of a specific patient, composition and mode of administration without being toxic to the patient. The selected dosage level will depend on various pharmacokinetic factors including route of administration, time of administration, excretion rate of follistatin-fusion protein, duration of treatment, other drugs, compounds and / or materials used in combination with a specific follistatin-fusion protein, age, sex, weight, condition, general health and previous medical history of the patient being treated.

[0118] Suitable dosages can be, for example, in the range of about 0.01 pg / kg to about 1000 mg / kg body weight of the patient to be treated, typically about 0.1 pg / kg to about 100 mg / kg body weight. The dosage regimen can be adjusted to provide the best desired response (e.g., therapeutic response). For example, a single dose can be administered over time or multiple divided doses can be administered. The dosage unit form as used herein refers to a physically separated unit suitable as a unit dose for a subject to be treated; each unit contains a predetermined amount of active compound, which is calculated to produce the desired therapeutic effect in combination with the desired drug receptor (earner). Administration can be single or multiple doses. Multiple doses can be administered to the same or different locations by the same or different routes.

[0119] In the context of the present invention as a whole, 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 a variety of different ways. Both can be administered together in a single composition, or administered in separate compositions as part of a combination therapy. For example, one of them can be administered before or separately, after or in sequence, in parallel or simultaneously with another. In particular, if kidney disease or kidney damage is / may be the result of chemotherapy, the chemotherapeutic agent can be selected from but not limited to alkylating agents, toxins, anticancer antibiotics, antimetabolites, antimitotic drugs, etc. Known specific compounds include but are not limited to cytosine arabinoside, 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 improve kidney disease or kidney damage caused by doxorubicin (adriamycin) treatment.

[0120] It should be noted that the above-mentioned embodiments illustrate rather than limit the invention, and that those skilled in the art will be able to design many alternative embodiments without departing from the scope of the invention, especially the appended claims.

[0121] Sequence Description

[0122]

[0123]

[0124]

[0125]

[0126]

[0127]

[0128] *X in SEQ ID No 22-31 represents "any amino acid" Example

[0129] The following examples are merely illustrative of the present invention and are not intended to limit the scope or content of the present invention.

[0130] Materials and methods

[0131] Cloning strategy:DNA fragments corresponding to the fusion between follistatin and anti-albumin antibody (named as 645Fab) heavy chain or light chain sequences (with or without a linker sequence in the middle) were generated by PCR or gene synthesis and cloned using internal mammalian expression vectors. The heavy chain and light chain sequences of 645Fab were also cloned using internal mammalian expression vectors, respectively. All expression vectors were confirmed by direct sequencing using primers covering the entire open reading frame.

[0132] CHO cell culture :CHOS-XE cell suspension (Cain, K. et al., Biotechnol Prog, 2013.29(3): p.697-706.) was pre-adapted in CD CHO medium (Invitrogen) supplemented with 2mM Glutamax. The cells were kept in the logarithmic growth phase at 120RPM shaking in a shaking incubator (Kuhner AG) and cultured at 37°C in a gas environment containing 8% CO2.

[0133] Protein expression :Overexpress follistatin-Fab protein by transient transfection CHO-XE cell line. Co-transfection (e.g. N-fab light chain-FST-C and heavy chain or FST-C fab heavy chain-C and light chain) was performed to paired expression plasmids. Immediately before DNA transfection, CHO cells were exchanged into Expi-CHO expression medium (Gibco) by briefly centrifuging cells and resuspending the precipitate at 1500x g. Then, according to the manufacturer's instructions, ExpiFectamine (Gibco) was used to transfect cells. Cultures were grown at 37°C for the first 24 hours and then grown with 190RPM shaking in a gas environment containing 8% CO2 at 32°C for the remainder of the expression cycle. Supernatants were usually collected by centrifugation at 4000x g for 9-14 days after transfection and subsequently filtered using a 0.22 μm membrane. Final protein expression levels were measured by protein G-HPLC and by SDS-PAGE.

[0134] Protein purification:Use Mab Select post (GE Healthcare) run under standard conditions to capture the protein content of transient expression.In brief, with 10 column volumes of phosphate buffered saline (PBS, pH 7.4) washing resin, and with 5 column volumes of 0.1M sodium citrate pH 3.1 elution bound protein (unless mentioned otherwise in the following examples).Eluate is neutralized with TRIS HCl pH 8.5 and filtered and sterilized by 0.22 μm membrane exclusion chromatography (HiLoad26 / 60Superdex 75 posts, GE Healthcare) run under standard conditions (here, post preloaded with PBS pH 7.4 as running buffer).Use the absorbance at 280nm, BEH2000 to analyze UPLC and SDS PAGE (under reducing and non-reducing conditions) to assess sample quality.

[0135] Doxorubicin-induced renal fibrosis model of chronic kidney disease (CKD) Female Balb / c mice, at least 6 weeks old and weighing greater than 24 g, were administered cDNA (or cDNA pairs for paired studies) in appropriate hydrodynamic transfection expression vectors (one of which contained the follistatin gene) (Five Prime Therapeutics) by IV injection through the tail vein. Seven days later, mice were IP-administered 11 mg / kg doxorubicin (doxorubicin) and monitored for 49 days. The weight and health of the mice were monitored daily; those mice that lost greater than 40% of their initial body weight or showed signs of end-stage renal failure were terminated and data collected. At the end of the 49-day study period, the experiment was terminated for all mice and kidneys and serum were collected for analysis. Renal hydroxyproline was measured using the QuickZyme assay (QuickZyme Biosciences), and serum creatinine was measured using the 3-enzyme method and read as μg / ml serum creatinine. Renal fibrosis (hydroxyproline) in the saline plus doxorubicin group, labeled "saline" on the graph, was considered the greatest disease response. This was set to 100% and used to normalize each treatment group. Naive animals did not receive doxorubicin.

[0136] Statistical analysis methods specified for in vivo analysis : Each assay run consisted of a naive group, a saline group, and multiple cDNAs. In the initial screening phase, cDNAs were paired and 15 mice were treated for each pair; in the retest, each cDNA was tested individually with 22 mice.

[0137] For each run, analysis of variance (ANOVA) was performed on the log-transformed data for hydroxyproline and serum creatinine. If the observed studentized residual was less than -3 or greater than +3, it was considered an outlier. If an outlier was detected, it was excluded and the analysis was rerun. The cDNA group means were then tested against the saline control group means using a t-test based on the standard deviation of the residuals pooled from the ANOVA. In the initial screening of n=15 per cDNA pair, a hit was declared if the difference from saline was significant at the 10% level (P=0.1). In the retesting runs, n=22 per cDNA, a hit was declared if the difference from saline was significant at the 2.5% level (P=0.025). This strategy was used to produce acceptable false positive and false negative rates while minimizing the number of animals used.

[0138] PSR staining of mouse kidney :Half of the kidney from each mouse was placed in 10% neutral buffered formalin, dehydrated in a tissue processor and paraffin embedded for histological analysis. Paraffin blocks were sliced ​​at 4 μm on a microtome and mounted on slides. The slides were then dewaxed and hydrated, stained with Picros ir ius red (PSR), and then washed with acidified water. The slides were then dehydrated, transparentized, and coverslipped. The slides were air-dried and scanned using a Hamamatsu slide scanner. The complete slide images were imported into Definiens Developer XD 64 and analyzed using the bright field module. In short, the cortical area 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 data set of no less than four images. The analysis algorithm was then run on a dedicated server to determine the marker area above the threshold intensity for each individual marker, and the area of ​​each marker above the threshold intensity was determined as a proportion of the cortical area.

[0139] Unilateral ureteral obstruction (UUO)-induced renal fibrosis model of chronic kidney disease:Male C57BL / 6 mice (Charles River) weighing at least 19g were subjected to a renal fibrosis model of chronic kidney disease induced by unilateral ureteral obstruction. In those studies in which follistatin was delivered by hydrodynamic transfection, mice were transfected with 50μg pLIVE-human follistatin or pLIVE SEAP vectors (Mirus Bio, Cat#MIR 5320) in Trans IT EE vehicles. The solution containing the vector was injected intravenously quickly (less than 2.5 seconds) in a volume of 2ml. General anesthesia was induced with isoflurane before hydrodynamic injection and maintained for at least 1 minute after injection. In studies in which mice were administered human follistatin protein, it was administered by subcutaneous route in 100μl saline at the dose concentration specified in the legend of the figure. General anesthesia induced by isoflurane was used and surgery was performed under sterile conditions suitable for surgical techniques. Mice underwent laparotomy, followed by ligation of the left ureter using a 3.0Mers i lk ligature. 5-0 Vicryl was used to close the muscle wall in a continuous pattern, and 5-0 Vicryl was used to close the skin by subcutaneous suture. Appropriate analgesia was applied preoperatively and postoperatively. Blood samples were collected at the appropriate time according to the detailed description in the results. Mice were restricted using a recognized rodent fixator and 30 μl of blood was taken from the tail vein by tail puncture method and using a pipette to accurately measure blood samples. The sample was placed in a 0.2 ml PCR tube (Thermo) and rotated (20,000 g, 5 minutes) to take out serum as much as possible, put it into a fresh PCR tube and stored at -80 ° C. On the 19th or 21st day, mice were anesthetized using isoflurane, blood was taken out to serum tubes (Sars tedt) by cardiac puncture and mice were killed by cervical dislocation. The left kidney was removed and two-quarters were quickly frozen in liquid nitrogen and stored at -80 ° C. The remaining half was placed in 10% neutral buffered formalin, dehydrated in a tissue processor and paraffin embedded to perform histological analysis and PSR staining as described above.

[0140] Human renal proximal tubule barrier integrity assay (see Figure 5 A):Primary human renal proximal tubule cells (Innoprot) were seeded onto 0.4uM pore polycarbonate membranes in 24-well HTS Transwell plates (Corning). The cells were cultured for 14 days, and the culture medium (supplemented with advanced DMEM / F12 from the renal epithelial growth kit from ATCC) was replaced every other day. The epithelial barrier function was destroyed by adding 2μM doxorubicin (also known as doxorubicin, a well-described nephrotoxic agent). In order to study the effect of follistatin, some wells received doxorubicin plus 100ng / ml Fab control protein or 100ng / ml FST315 (HBM)-Fab. After 72 hours of incubation, barrier integrity was assessed by adding 0.5mg / ml FITC-dextran (150kDa) to the apical chamber and using a fluorescent microplate reader to monitor the appearance of FITC signals in the basolateral chamber over time. The permeability percentage was calculated by dividing the fluorescence value from the test well by the average signal detected in the transwell without cells. Graphs show data points from individual wells with 6 replicates per condition. Statistical significance was shown by Mann-Whitney Student's T test.

[0141] Example 1 - Follistatin is antifibrotic in a doxorubicin-induced chronic kidney disease (CKD) mouse model

[0142] Mouse follistatin was initially identified as one member of a hydrodynamically transfected cDNA pair in a screen for fibrosis-modifying proteins in the mouse doxorubicin CKD model (n=15 mice). The anti-fibrotic effects of follistatin were confirmed in a replicate study in which follistatin cDNA was independently transfected in the same model (n=18-21 mice per group). Follistatin reduced renal fibrosis as measured by total renal hydroxyproline (p=0.0001) compared to 77% of saline controls (p=0.0001). Figure 2 A), while the loss of renal function returned to normal range as measured by serum creatinine (p = 0.0001) ( Figure 2 B) Conditional survival, as measured by symptoms of end-stage renal failure, increased from 50% in the saline-treated doxorubicin group to 100% in the follistatin-treated group ( Figure 2 C).

[0143] At 49 days, all kidneys from all groups (naive (n=8), untreated doxorubicin animals (n=21), and follistatin hydrodynamically transfected animals (n=18)) were fixed, paraffin-embedded, sectioned and stained with hematoxylin and eosin (H&E) or Picrosirius red (PSR), and imaged on a Hamamatsu slide scanner. Figure 2Examples and representative images of PSR-stained kidneys from one naive, three untreated doxorubicin, and three follistatin-treated doxorubicin animals are shown in D. Untreated mouse kidneys had extensive glomerular sclerosis and tubulointerstitial fibrosis with marked tubular basement membrane dilatation, flattening of the tubular epithelium, extensive tubular dilatation with loss of brush border, and tubular atrophy. In contrast, follistatin-treated mice were largely protected from both types of fibrotic remodeling. High-content image analysis of 47 mouse kidney samples stained with PSR was performed using Def iniens software, and the PSR staining area measured in the three groups was plotted as a percentage of the PSR staining area ( Figure 2 E). The area of ​​PSR staining in follistatin-treated groups was normalized to the 3-fold increase in untreated kidneys (p<0.0001).

[0144] In conclusion, follistatin delivered by hydrodynamic transfection significantly suppressed multiple parameters of fibrosis and disease in the mouse doxorubicin chronic kidney disease model, including collagen accumulation, serum creatinine renal injury biomarker levels, gross morphological renal architecture, and conditional survival of mice over the study period.

[0145] Example 2 - Follistatin inhibits renal fibrosis in the unilateral ureteral obstruction (UUO) model of acute kidney disease in mice.

[0146] The primary objective of this study was to evaluate the efficacy of different follistatin constructs delivered by hydrodynamic transfection (HDT) in delaying the onset of tubulointerstitial fibrosis in the UUO model.

[0147] UUO kidneys in animals that received the control SEAP vector by HDT showed typical tubulointerstitial fibrosis in the cortex 21 days after surgery, with extensive dilatation of the tubular basement membrane, strong collagen staining of the basement membrane, especially in the medullary line, extensive loss of epithelial cell volume, tubular atrophy, loss of proximal tubular brush border, and extensive tubulointerstitial infiltration. Fewer than 15% of the tubules had a near-normal structure. The medulla was effectively destroyed and absent with minimal changes in the glomeruli ( Figure 3 A).

[0148] UUO kidneys in animals receiving HDT of the whole follistatin moiety-FST315(HBM)-Fab and FST315-Fab showed reduced tubular basal dilatation and collagen staining 1 day before UUO surgery compared to UUO mice receiving the control SEAP vector. All follistatin treatment groups preserved tubular architecture with less epithelial flattening and atrophy ( Figure 3 A).

[0149] Quantification of Picrosirius red (PSR) stained collagen sections using the definiens image analysis algorithm showed the protective effect of follistatin. Although FST315-Fab showed a reduced collagen area percentage relative to SEAP control, this was not statistically significant; 315Fs t(HBM)-Fab (p<0.05) showed a significant inhibition of collagen area percentage. This is consistent with the visual histological assessment of the stained sections as described above ( Figure 3 B).

[0150] Another indicator of follistatin-fusion protein efficacy was the increase in mouse body weight, which was monitored daily during the study. Both FST315-Fab and FST315(HBM)-Fab showed less weight loss at the onset of disease and gained more weight from day 4 until the study was terminated on day 21 ( Figure 3 C).

[0151] In conclusion, follistatin-fusion protein delivered by hydrodynamic transfection has a protective effect in a mouse model of acute kidney disease, UUO, with disease parameters PSR staining and percentage of collagen area in diseased kidneys as well as body weight loss as primary readouts.

[0152] Example 3 - Comparison of FST315-Fab and FST315(HBM)-Fab in the mouse UUO acute kidney disease model, where the latter has a dose-response effect and both follistatin moieties are delivered as purified proteins

[0153] The main objective of this study was to compare the efficacy of different FST315 protein forms in delaying the onset of tubulointerstitial fibrosis in the UUO model.

[0154] Examination of gross renal morphology of PSR-stained kidneys in non-operated (non-diseased) and 21-day UUO kidneys revealed significant differences in shape, structure, and red staining, and these gross changes could be reversed in the presence of FST315(HBM)-Fab administered at 10 mg / kg daily; blinded histological scoring quantified the differences in shape, structure, and red staining. Figure 4 These changes are shown in A. A high-power magnification of an untreated 21-day UUO highlights the typical histological changes of this model as well as the normalization ( Figure 4 B).

[0155] Figure 4C illustrates the effect of FST315-Fab and FST315(HBM)-Fab in day 21 UUO kidneys treated with a heparin-binding mutant form of follistatin-Fab protein (FST315(HBM)-Fab) one day before surgery (day -1), which has a significantly different appearance compared to vehicle treatment and also shows its stronger epithelial protection and less red collagen staining compared to wild-type follistatin-Fab protein (FST315-Fab). There is reduced tubular atrophy and large areas of the cortex, especially at the 10 mg / kg dose, appear almost normal with minimal basement membrane expansion, collagen staining or loss of structure. Furthermore, the effects of FST315(HBM)-Fab on epithelial protection and collagen PSR staining were dose-dependent, as there was a clear change from a high dose of 10 mg / kg to a medium dose of 2 mg / kg and 0.2 mg / kg that showed a smaller effect, while a dose of 0.02 mg / kg was almost indistinguishable from untreated UUO kidneys.

[0156] Quantification of Picros irius red (PSR) stained collagen sections using the definiens image analysis algorithm as a readout of collagen area percentage showed a dose-dependent significant protective effect of 315FST(HBM)-Fab fusion protein at 10 mg / kg (p<0.0001) and 2 mg / kg (p<0.001), while 0.2 and 0.02 mg / kg doses showed insignificant reductions ( Figure 4 D); 2 mg / kg of FST315-Fab was statistically significantly different from the control untreated UUO group, p<0.05, although the magnitude of the response was smaller. Importantly, 2 mg / kg of FST315-Fab was also statistically different from 2 mg / kg of FST315(HBM)-Fab (p=0.03), meaning that the protection provided by the FST315(HBM-Fab) form has an additional effect mediated by changes in heparin binding mutations. This may be due to the better pharmacokinetic properties of FST315(HBM)-Fab, with a mean residence time of 13.1 hours in mice compared to 9.3 hours for FST315-Fab, or it may be due to other functional properties that have not yet been characterized. Stained kidney sections from all groups in the study were manually scored histologically by an expert who was blinded to the groups, and Figure 4The average integrated fibrosis score is plotted in E. The same pattern was observed again, with the FST315 (HBM) -Fab fusion protein having the strongest inhibitory effect, with 10, 2, and 0.02 mg / kg showing significant differences, p<0.0001, p<0.001, and p<0.01, respectively, compared to vehicle-treated UUO kidneys. 2 mg / kg of FST315-Fab protein did show a significance of p<0.05 compared to vehicle-treated UUO in terms of epithelial protection, but this was not as good as the FST315 (HBM) -Fab fusion form at the same dose and also had a significant difference of p=0.05. This is consistent with the visual histological assessment of the stained sections as described above.

[0157] All animals in the UUO study were weighed daily and data were plotted over time ( Figure 4 F). Weight changes in the follistatin fraction relative to vehicle-treated UUO groups correlated well with efficacy of both FST315(HBM)-Fab and FST315-Fab forms; different 315FST(HBM)-Fab doses also showed that weight gain was a good surrogate for renal protection, which could only be measured at study termination.

[0158] In summary, FST315(HBM)-Fab when administered as a protein showed statistically significant improvement over vehicle-treated disease animals in protecting mouse kidneys from cumulative damage caused by UUO surgery and showed statistically significant superior efficacy compared to FST315-Fab, and this effect was dose-dependent. The superior protective effect of FST315(HBM)-Fab on the kidney was consistent with morphological assessments, collagen content measured by PSR, epithelial cell health, and total body weight gain.

[0159] Example 4 - Effect of FST315(HBM)-Fab on the Barrier Integrity of Renal Proximal Tubular Epithelial Cells (RPTEC)

[0160] RPTEC cells grown in 96-well transwell plates form a polarized, impermeable barrier that can be measured by monitoring the resistance to diffusion of a fluorescent dye from the upper to the lower compartment of the transwell. In the presence of the cytotoxic doxorubicin, barrier integrity is impaired and can be measured by the presence of the fluorescent dye FITC-dextran (150 kDa) appearing in the basolateral supernatant. Treatment with FST315(HBM)-Fab, but not Fab molecules alone, protected against this loss of barrier integrity as measured by partial restoration of permeability to FITC-dextran; this reduction in permeability with FST315(HBM)-Fab was statistically significantly different from medium control (p<0.0001). Figures depicting the in vitro RPTEC model and results are presented in Figure 5 A and 5B.

[0161] In conclusion, FST315(HBM)-Fab preserves primary RPTEC barrier integrity against doxorubicin-induced epithelial injury in an in vitro model of renal disease barrier function.

[0162] Example 5 - FST-Fab and Fab-FST exhibit superior protein expression, higher monomer fraction yield and longer half-life than FST-WT or FST-Fc fusion protein

[0163] Comparison of the relative expression levels of the follistatin moiety fused to various fusion partners and in different orientations showed that the FST-Fab construct fused to the C-terminus of the FST moiety was optimal ( Figure 6 A). Fusion of Fc or ScFv to the C-terminus of FST was significantly inferior to using Fab at this position, with both of the former resulting in a 3.5-fold decrease in expression product ( Figure 6 A). Fusion of the Fab portion at the N-terminus of the FST portion resulted in a decrease in the amount of expressed product by about 45%, although better than the fusion protein comprising FST fused to the Fc domain. Evaluation of the effect on expression using FST288- or FST315, using the wild-type HBM sequence fused to the Fab at the C-terminus of follistatin, showed only minor differences ( Figure 6 B) Expression of the FST315 form was 6% lower than the FST288 fusion. Comparison of the fusion containing HBSM (HBM) showed a modest 7% reduction in expression levels compared to wild type.

[0164] A head-to-head examination of the various FST-fusions revealed that fusion of the Fab moiety to the C-terminus of the FST moiety resulted in the highest level of final monomer yield compared to the other fusions ( Figure 6C) FST-Fc fusion produced the lowest monomer yield among the fusion groups, which was almost one-sixth of that of FST-Fab fusion. FST-ScFv and FST fused to the N-terminus of Fab produced almost one-third and one-quarter of the monomer yield, respectively.

[0165] FST315WT, FST288WT, and FST288-Fab administered to mice intravenously (IV) at 10 mg / kg were rapidly cleared with mean residence times (MRTs) of 1.6 hours, 2.3 hours, and 5.2 hours, respectively. In contrast, FST315-Fab, FST315(HBM)-Fab, and FST288HBM-Fab administered to mice IV at 10 mg / kg exhibited prolonged kinetics with MRTs of 9.3 hours, 13.1 hours, and 11 hours, respectively. Figure 7 A and 7B). It was thus shown that it is possible to greatly extend the kinetics and half-life of FST-containing proteins due to the fusion between the FST part and the Fab part. A significant contribution to the extended kinetics was also made by mutations of the heparin binding site in the form of the HBM of the follistatin part.

[0166] References

[0167] 1. WO2015 / 187977

[0168] 2. WO2017 / 152090

[0169] 3.WO03006057

[0170] 4.Sidis et al (2005) Endocrinology 146(1):130-136

[0171] 5.http: / / blast.ncbi.nlm.nih.gov /

[0172] 6.http: / / www.ebi.ac.uk / Tools / emboss /

[0173] 7. WO2013 / 068571

[0174] 8. Cain et al. (2013) Biotechnol Prog. 29(3):697-706.

[0175] 9.Sidis et al. (2006) Endocrinology 147(7):3586-3597

[0176] 10. Yahr et al. (2022) J. Osteopath. Med. 122(1):55-63.

[0177] 11.Ammirati AL(2020)Rev.Assoc.Med.Bras.66:S3-S9.

[0178] 12.Wynn&Ramalingam(2012)Nature Medicine 18(7):1028-1040.

Claims

1. A follistatin-fusion protein or a pharmaceutical composition comprising the follistatin-fusion protein, for treating, improving or preventing kidney disease or kidney damage, wherein the follistatin-fusion protein comprises: a. Follicle-stimulating hormone part, b. Antibody portion, and optionally c. A linker between the follistatin portion and the antibody portion.

2. A method for treating a patient in need of therapy to treat, improve or prevent renal disease or renal injury, the method comprising administering a therapeutically effective amount of a follistatin-fusion protein or a pharmaceutical composition comprising a follistatin-fusion protein, wherein the follistatin-fusion protein comprises: a. Follicle-stimulating hormone part, b. Antibody portion, and optionally c. A linker between the follistatin portion and the antibody portion.

3. Use of follistatin-fusion protein in the preparation of a medicament for treating, improving or preventing kidney disease or kidney damage, wherein the follistatin-fusion protein comprises: a. Follicle-stimulating hormone part, b. Antibody portion, and optionally c. A linker between the follistatin portion and the antibody portion.

4. The follistatin-fusion protein for use according to claim 1 or the pharmaceutical composition, the method according to claim 2 or the use according to claim 3, wherein the renal disease is a chronic kidney disease 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 nephropathy (MCD), polycystic kidney disease, chronic allograft nephropathy, CKD-associated mineral and bone disease (CKD-MBD) and Goodpastures disease.

5. The follistatin fusion protein or the pharmaceutical composition for use according to claim 1, the method according to claim 2 or the use according to claim 3, wherein the kidney damage is a) renal fibrosis or b) associated with other diseases such as diabetes, hypertension, cardiovascular disease or bone disease.

6. Follistatin fusion protein or the pharmaceutical composition for use according to claim 1, 4 or 5, the method according to claim 2, 4 or 5 or the use according to claim 3, 4 or 5, wherein the kidney disease or kidney damage is the result of a therapeutic treatment.

7. The follistatin fusion protein or pharmaceutical composition for use according to claim 6, the method or use according to claim 6, wherein the kidney disease or kidney damage is the result of treatment with chemotherapy.

8. The follistatin-fusion protein or pharmaceutical composition for use according to claim 7, the method or use according to claim 7, wherein the chemotherapy is selected from cytosine arabinoside, bleomycin, camptothecin, carboplatin, cisplatin, cyclophosphamide, doxorubicin (adriamycin), gemcitabine, methotrexate, paclitaxel and vincristine.

9. The follistatin-fusion protein or pharmaceutical composition for use according to claim 7 or claim 8, the method or use according to claim 7 or claim 8, wherein the treatment or improvement of the kidney disease or kidney damage comprises administering the fusion protein simultaneously, separately or sequentially with chemotherapy.

10. A follistatin-fusion protein or a pharmaceutical composition for use according to any of the preceding claims, a method or a use according to any of the preceding claims, wherein the follistatin part comprises or is a naturally occurring protein or a functional variant thereof.

11. A follistatin fusion protein or a pharmaceutical composition for use according to any of the preceding claims, a method or a use according to any of the preceding claims, wherein 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 between 289 and 314 of any one of SEQ ID NOs: 1 to 4; or f. A sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to any one of SEQ ID NOs: 1 to 4.

12. The follistatin-fusion protein or pharmaceutical composition for use according to any of the preceding claims, the method or the use according to any of the preceding claims, wherein the antibody portion binds to albumin, and preferably wherein the antibody portion binds to human serum albumin (HSA).

13. The follistatin-fusion protein or pharmaceutical composition for use according to any of the preceding claims, the method or the use according to any of the preceding claims, wherein the antibody portion is a chimeric, humanized or human antibody portion.

14. The follistatin-fusion protein or pharmaceutical composition for use according to any of the preceding claims, the method or the use according to any of the preceding claims, wherein the antibody portion is an antigen-binding fragment selected from Fab, Fab' or F(ab')2.

15. The follistatin-fusion protein or pharmaceutical composition for use according to any of the preceding claims, the method or the use according to any of the preceding claims, wherein the antibody portion comprises: a. a light chain variable region comprising a CDR-L1 comprising SEQ ID NO: 13; a CDR-L2 comprising SEQ ID NO: 14 and a CDR-L3 comprising SEQ ID NO: 15; and a heavy chain variable region comprising a CDR-H1 comprising SEQ ID NO: 16; a CDR-H2 comprising SEQ ID NO: 17 and a CDR-H3 comprising SEQ ID NO: 18; or b. a heavy chain variable region comprising or consisting of SEQ ID NO: 5 and a heavy chain variable region comprising or consisting of SEQ ID NO:

6.

16. The follistatin-fusion protein or pharmaceutical composition for use according to any of the preceding claims, the method or the use according to any of the preceding claims, 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 GGGGSGGGGS (SEQ ID NO: 21).

17. A follistatin-fusion protein or a pharmaceutical composition for use according to any of the preceding claims, a method or the use according to any of the preceding claims, wherein a) the antibody portion is linked to the C-terminal portion or the N-terminal portion of the follistatin portion, or b) if a linker is present, the antibody portion is linked to the C-terminal portion of the follistatin portion via the linker.

18. Follistatin-fusion protein or pharmaceutical composition for use according to any of the preceding claims, a method or the use according to any of the preceding claims, wherein the fusion protein comprises or consists of: (a) i. a 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 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 thereof, conjugated to the C-terminus of the FST315 polypeptide; as well as iii. 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; (b) i. a 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 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 thereof, conjugated to the C-terminus of the FST288 polypeptide; as well as iii. 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; (c) i. a 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 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 thereof, conjugated to the C-terminus of the FST315 polypeptide variant; as well as iii. 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; (d) i. A 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 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 thereof, conjugated to the C-terminus of the FST288 polypeptide variant; as well as iii. 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; (e) i. a 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 conjugated to the C-terminus of the FST315 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 thereof, conjugated to the free end of the linker; as well as 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; (f) i. a 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 conjugated 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 thereof, conjugated to the free end of the linker; as well as 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. a 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 conjugated 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 thereof, conjugated to the free end of the linker; as well as 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. A 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 conjugated 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 thereof, conjugated to the free end of the linker; as well as 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) 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 thereof, 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 thereof; (j) SEQ ID NO:8, 9, 10, 11, 24, 25, 26, 27, 32, 33, 34 or 35, together with a Fab light chain defined by SEQ ID NO:6; or (k) A functional variant or functional fragment of any one of (a) to (i).

Images