Folistatin fusion proteins

Abstract

The present invention relates to the field of fusion proteins, and in particular to fusion proteins comprising a follistatin moiety. The invention also relates to a method for preparing the fusion protein and a pharmaceutical preparation comprising the fusion protein.

Description

Technical Field

[0001] The present invention relates to the field of fusion proteins, and in particular to a fusion protein comprising a follistatin moiety. The present invention also relates to a method for preparing the fusion protein and a pharmaceutical preparation comprising the fusion protein. Background Art

[0002] Follistatin is an autocrine glycoprotein whose main function is to bind and neutralize members of the TGF-β superfamily, particularly activin A, activin B, GDF8 (myostatin) and GDF11. It is known to exist in several different forms, including a 315 amino acid polypeptide (designated FST315); and a 288 amino acid polypeptide (designated FST288), such as Figure 1 As shown. Both FST315 and FST288 have high affinity for activins (activin A and activin B) and for myostatin (GDF8). In particular, follistatin can bind to and inhibit myostatin, which is a negative regulator of skeletal muscle mass.

[0003] Follistatin has been shown to be a potential therapeutic protein in certain situations, including the treatment of muscle diseases such as muscular dystrophy (WO2015 / 187977 and WO2017 / 152090). However, the use of follistatin in treatment encounters many obstacles, mainly based on the difficulty of expressing follistatin in vitro and the low stability / short half-life of follistatin in vivo. Attempts have been made to overcome these obstacles, and both WO2015 / 187977 and WO2017 / 152090 discuss the use of fusion proteins comprising a follistatin polypeptide fused to the Fc portion of an immunoglobulin.

[0004] There remains a need for follistatin peptides and fusion proteins that can be more readily expressed in vitro and that have increased half-lives or other beneficial effects in vivo. Summary of the invention

[0005] In a first aspect, the present invention provides a fusion protein comprising: a follistatin portion, an antibody portion, and optionally a linker between the follistatin portion and the antibody portion.

[0006] In some embodiments, the antibody portion binds to albumin (such as serum albumin (SA)), and the follistatin portion comprises or is a naturally occurring protein, a functional fragment thereof, and / or a functional variant thereof. In some examples, 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 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.

[0007] In some embodiments, the fusion protein comprises or consists of:

[0008] (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;

[0009] 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 thereto, conjugated to the C-terminus of the FST315 polypeptide; and

[0010] 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;

[0011] (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;

[0012] 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 thereto, conjugated to the C-terminus of the FST288 polypeptide; and

[0013] 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;

[0014] (c) i. a FST315HBM polypeptide defined by SEQ ID NO: 3, or a sequence having at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity thereto;

[0015] 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 thereto, conjugated to the C-terminus of the FST315HBM polypeptide; and

[0016] 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;

[0017] (d) i. a FST288HBM polypeptide defined by SEQ ID NO: 4 or a sequence having at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity thereto;

[0018] 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 thereto, conjugated to the C-terminus of the FST288 HBM polypeptide; and

[0019] 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;

[0020] (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;

[0021] 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;

[0022] iii. a Fab heavy chain defined by SEQ ID NO: 5, or a sequence having at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity thereto, conjugated to the free end of a linker; and

[0023] 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;

[0024] (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;

[0025] 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;

[0026] iii. a Fab heavy chain defined by SEQ ID NO: 5, or a sequence having at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity thereto, conjugated to the free end of a linker;

[0027] 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;

[0028] (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;

[0029] 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;

[0030] iii. a Fab heavy chain defined by SEQ ID NO: 5, or a sequence having at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity thereto, conjugated to the free end of a linker; and

[0031] 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;

[0032] (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;

[0033] 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;

[0034] iii. a Fab heavy chain defined by SEQ ID NO: 5, or a sequence having at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity thereto, conjugated to the free end of a linker; and

[0035] 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;

[0036] (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, and 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;

[0037] (j) SEQ ID NO:8, 9, 10, 11, 24, 25, 26, 27, 32, 33, 34 or 35 and a Fab light chain defined by SEQ ID NO:6; or

[0038] (k) A functional variant or fragment of any one of (a) to (j).

[0039] In another aspect, the present invention relates to: i) one or more isolated polynucleotides encoding the fusion protein of the present invention; ii) one or more clones or expression vectors comprising the one or more polynucleotides of the present invention; and iii) a host cell comprising the one or more polynucleotides of the present invention or the one or more expression vectors of the present invention.

[0040] In another aspect, the present invention provides a method for producing the fusion protein of the present invention, the method comprising culturing the host cell of the present invention under suitable conditions to produce the fusion protein and isolating the fusion protein.

[0041] In another aspect, the present invention relates to a pharmaceutical composition comprising the fusion protein of the present invention and one or more pharmaceutically acceptable carriers, excipients or diluents.

[0042] In another aspect, the fusion protein of the present invention or the pharmaceutical composition of the present invention is used for therapeutic purposes. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 : Schematic diagram illustrating the domain organization of two mature follistatin parts (FST288 and FST315; i.e. without their N-terminal secretion signal sequence), and how an exemplary pair of FST288 molecules form a complex with a disulfide-linked homodimer of activin. Both FST288 and FST315 have four identical domains, including the N-terminal domain, FSD1, FSD2, and FSD3. FST315 can also bind activin and has an additional C-terminal domain (residues 289-315).

[0044] Figure 2 : (A) Relative expression levels of follistatin fusion proteins were determined by Protein-G HPLC analysis of harvested CHO supernatants. Values ​​were normalized to the expression level of FST-Fc. 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. (B) Relative expression levels of FST288, FST315, FSTWT and FST-HBM (also known as HBSM) with C-terminal fusion partners (referred to as 288 fusion, 315 fusion, WT fusion and HBSM fusion, respectively in the figure). Values ​​were normalized to the expression of 288 fusion. (C) Total expression values ​​of monomers, values ​​were normalized to the expression level of FST-Fc, n=25. Legend to Figure 2 (A) The same.

[0045] Figure 3 : (A) Pharmacokinetic profiles of FST315WT, FST315-Fab and FST315HBM-Fab, administered to mice (n=3 mice / group) at 10 mg / kg via IV administration, and serum samples were monitored for 7 days; (B) Pharmacokinetic profiles of FST288WT, FST288-Fab and FST288HBM-Fab, administered to mice (n=3 mice / group) at 10 mg / kg via IV administration, and serum samples were monitored for 7 days. The pharmacokinetic parameters of all follistatin parts derived from this study are summarized in Table 1.

[0046] Figure 4: (A) FST315HBM-Fab and FST315WT, (B) FST288HBM-Fab and FST288WT in a reporter cell assay stimulated with any of the 4 major FST ligands (i.e., activin A, activin B, GDF8 (myostatin), and GDF11). All follistatin fusion proteins dose-dependently inhibited the signal of all 4 ligands, and representative graphs show the different potency curves of the 4 FST moieties for different ligands, with the percentage of inhibition plotted against the FST concentration. These experiments have been performed multiple times: FST315HBM-Fab, n=7; FST315WT, n=4; FST288HBM-Fab, n=4; and FST288WT, n=4; and all data are summarized in Table 3, where the data are expressed as IC50 and nM range for the 4 ligands.

[0047] Figure 5 : SDS-PAGE analysis of protein A elution and elution pool, lane (1) is the molecular weight marker, lane (2) is the acidic elution pool, lane (3) is the acidic eluate after acidic elution, lane (4) is the alkaline elution pool, lane (5) is the acidic eluate after alkaline elution, where (M) = full-length Fst-Fab monomer, (F) = Fab.

[0048] Figure 6 : Size exclusion analysis. (A) Acidic elution pool, (B) Basic elution pool, (C) Acidic eluate after acidic elution, (D) Acidic eluate after basic elution, where (M) = full length Fst-Fab monomer and (F) = Fab.

[0049] Figure 7 : Histograms show the relative expression levels (g / L) of 4 cell clones evaluated in the fed-batch process. The clones were evaluated in two different culture media (culture media A and B) and two different feed conditions (FB1 and FB2). The total protein concentration was determined by CH1HPLC. DETAILED DESCRIPTION

[0050] Unless otherwise specified, technical terms are used in their common sense. If a specific meaning is expressed for some terms, the definition of the term will be given in the context in which the term is used.

[0051] If an indefinite or definite article is used when referring to a singular noun, for example "a", "an" or "the", this includes a plural of the noun unless specifically stated otherwise. The term "comprising" as used herein does not exclude other elements. For the purposes of the present disclosure, the term "consisting of" is considered to be a preferred embodiment of the term "comprising".

[0052] As used herein, the term "follistatin" or "FST" refers to an autocrine glycoprotein (UniProt No.: P19883) that is a known inhibitor of activin A and B. Follistatin also binds to GDF11, GDF8 (myostatin), BMP2, 4, 6, 7, 11 and 15 with lower affinity. Human follistatin has two major alternative splicing forms: a shorter form (FST288, 31.6 kDa) that binds to cells and a longer circulating form (FST315, 34.8 kDa). FST315 is defined according to SEQ ID NO: 1, and FST288 is defined according to SEQ ID NO: 2 (both SEQ ID NOs: 1 and 2 are mature forms lacking an N-terminal secretory signal peptide). FST315 and FST288 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 (highly acidic) at its C-terminus, called an acidic tail. These terms also encompass functional fragments and / or their functional variants, such as those disclosed in Sidis et al., 2005. If followed by numbers, such as FST288, it is indicated that the protein is the 288 form of follistatin (starting from residue 1 of the mature form). If followed by numbers and letters, such as FST315HBM, it represents 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 positively or negatively affect cell cycle progression depending on cell type. In some tissues, activin signaling is antagonized by its associated heterodimer inhibin (inhibin). For example, during the release of follicle stimulating hormone (FSH) from the pituitary gland, activins promote FSH secretion and synthesis, whereas inhibins prevent FSH secretion and synthesis. Activins are also considered negative regulators of muscle mass and function, and activin antagonists can promote muscle growth or counteract muscle loss in vivo.

[0053] The term "antibody" as used herein includes, but is not limited to, monoclonal antibodies, polyclonal antibodies, and recombinant antibodies produced by recombinant techniques known in the art. The term "antibody" as used herein includes antibodies of any species, particularly antibodies of mammalian species; such as 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 antibodies from chimpanzees, baboons, rhesus monkeys, or cynomolgus monkeys; rodent antibodies, such as antibodies from mice or rats; rabbit, goat, or horse antibodies; camel antibodies (e.g., antibodies from camels or llamas, such as nanobodies TM (Nanobodies TM )) and derivatives thereof; antibodies of bird species, such as chicken antibodies; or antibodies of fish species, such as shark antibodies. The term "antibody" refers to glycosylated and non-glycosylated antibodies. In addition, the term "antibody portion" used herein may refer to a full-length antibody, but more generally refers to an antibody fragment, and more particularly refers to an antigen-binding fragment thereof. The antibody fragment comprises at least one heavy chain or light chain immunoglobulin domain known in the art, and binds to one or more antigens. Examples of antibody fragments of the present invention include Fab, modified Fab, Fab?, modified Fab?, F(ab?)2, Fv, Fab-Fv, Fab-dsFv, Fab-Fv-Fv, scFv and Bis-scFv fragments. The fragment may also be a diabody, a tribody, a triabody, a tetrabody, a minibody, a single domain antibody (dAb), such as an sdAb, VL, VH, VHH or a camel antibody (e.g., from a camel or llama, such as a nanobody). TM ) and VNAR fragments. The antigen binding fragments of the present invention may further comprise a Fab linked to one or two scFvs or dsscFvs, each scFv or dsscFv binding to the same or different targets (e.g., one scFv or dsscFv binds to a therapeutic target and the other increases half-life by binding to, for example, albumin).

[0054] The term "Fab" as used herein 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.

[0055] The term "Fab?" as used herein is similar to Fab, wherein the Fab portion is replaced by Fab?. This form can be provided as a pegylated version thereof. The dimer of Fab? according to the present invention produces F(ab?)2, wherein, for example, dimerization can be achieved via a hinge.

[0056] 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.

[0057] 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.

[0058] The term "single domain antibody" as used herein 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.

[0059] 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.

[0060] The term "specificity" as used herein in the context of antibodies and antigen-binding fragments refers to an antibody that recognizes only the antigen to which the antibody is specifically directed, or that the antibody has a significantly higher binding affinity for its specific antigen than the antibody binds to a non-specific antigen, such as at least 5, 6, 7, 8, 9, 10 times higher binding affinity.

[0061] The terms "albumin", "serum albumin" or "SA" refer to a globular protein abundant in vascular and extravascular compartments. The human form of serum albumin (HSA) is known by reference number P02768, while the mouse serum equivalent is reference number P07724.

[0062] 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, such as baboons, rhesus monkeys, or cynomolgus monkeys) and human constant region sequences.

[0063] "Humanized" antibodies are chimeric antibodies containing sequences derived from non-human antibodies. In most cases, humanized antibodies are such human antibodies (receptor antibodies): wherein the residues from the hypervariable region of the receptor are replaced by the residues of the hypervariable region [or complementary determining region (CDR)] with the required specificity, affinity and activity from non-human species (donor antibodies) (such as mice, rats, rabbits, chickens or non-human primates). In most cases, the residues of the human (receptor) antibody outside the CDR, i.e., in the framework region (FR), are additionally replaced by the corresponding non-human residues. In addition, humanized antibodies may be included in residues not found in the receptor antibody or the donor antibody. These modifications are carried out to further improve the antibody properties. Humanization reduces the immunogenicity of non-human antibodies in the human body, thereby promoting the application of antibodies in the treatment of human diseases. Humanized antibodies and several different technologies for producing them are well known in the art. Unless otherwise indicated, HVR residues (CDR residues) and other residues in the variable domain (such as FR residues) are numbered according to Kabat herein.

[0064] The term "antibody" also refers to human antibodies, which can be produced as a humanized alternative. For example, transgenic animals (e.g., mice) may be produced 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 technologies, wherein recombinant DNA libraries are used, and the libraries are at least partially artificially produced or from donor immunoglobulin variable (V) domain gene libraries. Phage and ribosome display technologies for producing human antibodies are well known in the art. Human antibodies can also be produced from isolated human B cells, which are immunized in vitro with an antigen of interest, and subsequently fused to produce hybridomas, which can then be screened for producing the best human antibodies.

[0065] The term "functional variant" as used herein refers to an amino acid sequence that has been modified relative to a reference sequence but retains at least one biological function of the reference sequence. For example, a functional variant of FST retains at least one biological activity of the reference FST protein, such as binding and inhibition of activin A and B.

[0066] As used herein, the term "sequence identity" or "identity" refers to the number of matches (identical nucleic acids or amino acid residues) in the position of the comparison of two polynucleotides or polypeptide sequences. Sequence identity is determined by comparing the sequence when comparing, so as to maximize overlap and identity while minimizing sequence gaps. In particular, depending on the length of the two sequences, sequence identity can be determined using any of a number of mathematical global or local comparison algorithms. Comparisons for determining nucleic acid or amino acid sequence identity percentages can be achieved in various ways within the skill of the art, for example, using computer software available to the public on an Internet website such as http: / / blast.ncbi.nlm.nih.gov / or http: / / www.ebi.ac.uk / Tools / emboss / . Those skilled in the art can determine appropriate parameters for measuring comparisons, including any algorithm for achieving maximum comparison needs over the full length of the sequence being compared. For the purposes of this article, % nucleic acid or amino acid sequence identity values ​​refer to values ​​generated using the two-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 default values, i.e., scoring matrix=BLOSUM62, gap opening=10, gap extension=0.5, terminal gap penalty=false, terminal gap opening=10, terminal gap extension=0.5.

[0067] In this specification, the term "isolated" means that the antibody, antigen binding fragment, polypeptide or polynucleotide (as the case may be) is present in a physical environment different from that in which it may exist in nature. The term "isolated" nucleic acid refers to a nucleic acid molecule that is separated from its natural environment or produced synthetically. The isolated nucleic acid may comprise synthetic DNA (e.g., produced by chemical processing), cDNA, genomic DNA, or any combination thereof.

[0068] The terms "nucleic acid" and "polynucleotide" or "nucleotide sequence" are used interchangeably to refer to any molecule consisting of or comprising monomeric nucleotides. A nucleic acid may be an oligonucleotide or a polynucleotide. A nucleotide sequence may be DNA or RNA.

[0069] As used herein, the term "vector" refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes vectors that are self-replicating nucleic acid structures as well as vectors that are incorporated into the genome of a host cell into which they are introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as "expression vectors."

[0070] 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, vehicle and / or carrier for administration of a therapeutic agent.

[0071] 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.

[0072] As used herein, the terms "treat", "treating", and the like refer to obtaining a desired pharmacological and / or physiological effect. The effect may be prophylactic, in terms of completely or partially preventing a disease or its symptoms, and / or therapeutic, in terms of partially or completely curing a disease and / or an adverse effect attributable to the disease. Thus, treatment includes any treatment of a disease in mammals, particularly humans, and includes: (a) preventing the occurrence of the disease in a subject (i.e., a human) who may be susceptible to the disease but has not yet been diagnosed with the disease; (b) inhibiting the disease, i.e., arresting its development; and (c) ameliorating the disease, i.e., causing regression of the disease.

[0073] The invention will now be described with reference to particular non-limiting aspects and embodiments thereof and with reference to certain figures and examples.

[0074] The present invention addresses the need for improved follistatin peptides and fusion proteins by providing novel follistatin fusion proteins that incorporate an antigen-binding antibody portion (eg, an antigen-binding portion) that are more easily expressed in vitro and have increased half-life or other beneficial effects in vivo.

[0075] The present invention is based on the surprising discovery of the inventors that, compared to previously known follistatin-based fusion proteins comprising an Fc portion, follistatin-based fusion proteins incorporating an antigen binding portion exhibit superior protein expression and higher monomer fraction yields. In particular, it has been shown that the fusion proteins of the present invention have expression levels at least 1.5 times higher than that of the FST-Fc fusion protein. Compared to the FST-Fc fusion protein, the fusion proteins of the present invention not only have a higher relative expression, but they also result in a much higher yield of the monomer fraction (i.e., correctly folded, usable fusion protein) (at least 1.5 times higher than that of the FST-Fc fusion protein).

[0076] The main object / aspect of the present invention is a fusion protein comprising or consisting of: a. a follistatin portion, b. an antibody portion, and optionally c. a linker between the follistatin portion and the antibody portion.

[0077] In the context of the entire invention, the follistatin portion comprises or is a naturally occurring follistatin protein. Follistatin is preferably its mature form, i.e. lacks the N-secretion signal sequence, because this sequence is only used for production / secretion from cells. Alternatively, follistatin is its functional fragment. The follistatin portion is, for example, FST288 protein (SEQ ID NO:2) or FST315 protein (SEQ ID NO:1). Any intermediate form thereof, such as any follistatin portion comprising 289 to 314 residues of any one of SEQ ID NOs:1 to 4, may also be used, as long as they are functional, i.e. they retain at least one biological activity of FST. Although not limited, preferably any intermediate form of the follistatin portion starts at 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 of the present invention (i.e., naturally occurring or a functional fragment thereof) is a functional variant, for example, it may have one or more mutations, such as 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 include alanine (A) replacing lysine (K) (resulting in a mutation selected from K76A, K81A and / or K82A). As another non-limiting example, a heparin binding mutant ("HBM", or herein named "(HBM)" or "HBSM") may be used, such as FST288HBM (SEQ ID NO: 4), FST291HBM, FST303HBM or FST315HBM (SEQ ID NO: 3), wherein the mutant comprises a triple mutation K76A, K81A and K82A.

[0078] In particular, the folliculostatin part contained in the fusion protein described in the present invention: a) contains or is composed of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, b) contains or is composed of 289 to 314 residues of any one of SEQ ID NO:1 to 4, or c) contains or is composed of an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with any one of SEQ ID NO:1 to 4.

[0079] Without wishing to be bound by any theory, the inventors hypothesize that the fusion proteins of the present invention exhibit greater stability and / or efficacy in vivo than wild-type follistatin because the antibody portion of the fusion proteins of the present invention is able to bind, for example, free HSA in a subject, thereby extending the half-life of the fusion protein.

[0080] Thus, in the overall context of the present invention, the antibody portion preferably binds to albumin, and preferably binds to serum albumin (SA), such as mouse, rat, cynomolgus monkey or human SA. More preferably, the antibody portion binds to human HSA. The antibody portion may be a chimeric antibody portion, a humanized antibody portion or a human antibody portion. Preferably, the antibody portion of the fusion protein of the present invention is an antigen binding fragment of an antibody (or referred to herein as an antigen binding portion). Preferably, such an antigen binding portion is selected from Fab, Fab? or F(ab?)2. Alternatively, the antibody portion of the fusion protein of the present invention is selected from Fab, Fab? or F(ab?)2 and comprises a human VH3 domain capable of binding to protein A.

[0081] In one embodiment, the antibody portion of the fusion protein of the present invention comprises a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises: CDR-L1 comprising SEQ ID NO: 13, CDR-L2 comprising SEQ ID NO: 14 and CDR-L3 comprising SEQ ID NO: 15; the heavy chain variable region comprises: CDR-H1 comprising SEQ ID NO: 16, CDR-H2 comprising SEQ ID NO: 17 and / or CDR-H3 comprising SEQ ID NO: 18.

[0082] In an alternative embodiment, the antibody portion of the fusion protein of the invention comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises or consists 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 the light chain variable region comprises or consists 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.

[0083] SEQ ID NO: 5 and SEQ ID NO: 6 represent the heavy and light variable chains of the anti-albumin antibody called "CA645" (as disclosed in WO2013 / 068571). As demonstrated in the examples below, the inventors surprisingly found that FST-Fab fusion proteins comprising the heavy and light variable chains of SEQ ID NO: 5 and SEQ ID NO: 6, respectively, are easier to produce and purify.

[0084] In the overall context of the present invention, the fusion protein optionally comprises a linker between the follistatin portion and the antibody portion. When the linker appears as a non-limiting example, it can be selected from the following: SGGGGS (SEQ ID NO: 7), SGGGGSSGGGGS (SEQ ID NO: 19), GGGGS (SEQ ID NO: 20) and GGGGSGGGGS (SEQ ID NO: 21).

[0085] It should be understood that when producing a fusion protein, there are two options for fusing any part to each other: C-terminal fusion or N-terminal fusion. As shown in the examples below, the inventors were surprised to find that, compared with any other type of fusion (such as the fusion of the antibody part to the N-terminal part of the follistatin part), fusing the antibody part at the C-terminal end of the follistatin part resulted in a further increase in the expression of the resulting fusion protein. In particular, compared with known Fc-based follistatin fusion proteins, the C-terminal fusion proteins of the present invention (i.e., the antibody part is fused directly or through a linker to the C-terminal end of follistatin) show excellent expression and higher monomer protein yield. However, although the C-terminal fusion protein results in the highest expression level, the technician may consider fusing the antibody part to the N-terminal end of follistatin because they result in, for example, an expression level of about 1.5 times higher than that of the Fc-based follistatin fusion protein.

[0086] 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 (here the FST) to the heavy chain of the antibody portion or to the light chain of the antibody portion.

[0087] Therefore, in a preferred embodiment of the fusion protein of the present invention, the antibody portion is connected to the C-terminal portion of the follistatin portion. If a linker is present, the antibody portion is preferably connected (or conjugated) to the C-terminal portion of the follistatin portion via a linker (in other words, the antibody portion is connected (or conjugated) to the C-terminal portion of the follistatin portion, and there is a linker between the two portions). In one example, the fusion protein will comprise (from N-terminus to C-terminus) the follistatin portion, a linker connected to the C-terminus of the follistatin portion, and then the heavy chain of the antibody portion connected to the free end of the linker (usually the C-terminus of the linker).

[0088] In some specific (but non-limiting) examples, the fusion protein of the present invention comprises or consists of:

[0089] (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;

[0090] 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 thereto, conjugated to the C-terminus of the FST315 polypeptide; and

[0091] 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;

[0092] (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;

[0093] 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 thereto, conjugated to the C-terminus of the FST288 polypeptide; and

[0094] 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;

[0095] (c) i. a FST315HBM polypeptide defined by SEQ ID NO: 3, or a sequence having at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity thereto;

[0096] 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 thereto, conjugated to the C-terminus of the FST315HBM polypeptide; and

[0097] 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;

[0098] (d) i. a FST288HBM polypeptide defined by SEQ ID NO: 4 or a sequence having at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity thereto;

[0099] 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 thereto, conjugated to the C-terminus of the FST288 HBM polypeptide; and

[0100] 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;

[0101] (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;

[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;

[0103] iii. a Fab heavy chain defined by SEQ ID NO: 5, or a sequence having at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity thereto, conjugated to the free end of a linker; and

[0104] 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;

[0105] (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;

[0106] 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;

[0107] iii. a Fab heavy chain defined by SEQ ID NO: 5, or a sequence having at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity thereto, conjugated to the free end of a linker; and

[0108] 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;

[0109] (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;

[0110] 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;

[0111] iii. a Fab heavy chain defined by SEQ ID NO: 5, or a sequence having at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity thereto, conjugated to the free end of a linker; and

[0112] 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;

[0113] (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;

[0114] 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;

[0115] iii. a Fab heavy chain defined by SEQ ID NO: 5, or a sequence having at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity thereto, conjugated to the free end of a linker; and

[0116] 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;

[0117] (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, and 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;

[0118] (j) SEQ ID NO:8, 9, 10, 11, 24, 25, 26, 27, 32, 33, 34 or 35 and a Fab light chain defined by SEQ ID NO:6; or

[0119] (k) A functional variant or fragment of any one of (a) to (i).

[0120] Alternatively, if an N-terminal fusion is preferred, the invention provides: i. a follistatin portion defined by any one of SEQ ID NOs: 1 to 4, 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 thereto, conjugated to the N-terminus of the FST315 polypeptide; and 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, and optionally a linker between the follistatin portion and the Fab heavy chain. In some specific examples, the fusion protein can be defined by: SEQ ID NO:28, 29, 30 or 31, 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 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.

[0121] The fusion proteins of the present invention as a whole are shown to have expression levels that are at least 1.5 times higher, at least 2 times higher, at least 3 times higher, at least 4 times higher, or higher than the expression levels of wild-type FST or FST-Fc fusion proteins. They are also shown to result in a total yield of monomeric protein that is at least 1.5 times higher, at least 2 times higher, at least 3 times higher, at least 4 times higher, at least 5 times higher, or at least 6 times higher than the total yield of monomeric protein of the FST-Fc fusion.

[0122] When making such comparisons, it is preferred to compare like with like, for example, FST315-Fab fusion protein should be compared with FST315-Fc fusion protein; and / or FST288-Fab fusion protein should be compared with FST288-Fc fusion protein.

[0123] Furthermore, as highlighted in the Examples, they are present in serum for a longer period of time than wild-type FST (6 days or more for the fusion proteins according to the invention, compared to 1 day for wild-type FST).

[0124] On the other hand, the present invention provides a kind of isolated polynucleotide, the isolated polynucleotide integrally encodes the fusion protein of the present invention, or its functional variant or fragment.The isolated polynucleotide of the present invention can comprise synthetic DNA (for example, synthetic DNA produced by chemical processing), cDNA, genomic DNA or any combination thereof.

[0125] Therefore, the present invention provides an isolated polynucleotide encoding the fusion protein of the present invention as a whole, wherein the fusion protein comprises a follistatin (FST) portion, an antibody portion and optionally a linker between the follistatin portion and the antibody portion. It will be understood by those skilled in the art that the polynucleotide sequence will also comprise a nucleic acid sequence encoding an N-terminal secretion signal sequence. The sequence will be selected in particular according to the host cell expressing the fusion protein.

[0126] The DNA sequence encoding the fusion protein of the present invention can be prepared using standard techniques of molecular biology. The desired DNA sequence can be synthesized completely or partially using oligonucleotide synthesis techniques. Site-directed mutagenesis and polymerase chain reaction (PCR) techniques can be appropriately used.

[0127] It should be understood that at least two separated polynucleotides will be needed to encode the fusion protein of the present invention. In fact, at least one separated polynucleotide will encode the FST portion, the antibody portion fused to the FST portion and the optional joint therebetween, and another separated polynucleotide will encode the remaining antibody portion to make the antibody portion fused to the FST portion complete. As a non-limiting example, one polynucleotide will encode the heavy chain of the FST portion, the joint and the anti-HAS-Fab portion, and one polynucleotide will encode the light chain of the anti-HAS-Fab portion.

[0128] In some specific (but non-limiting) examples, the isolated polynucleotide encodes the fusion protein of the present invention, and the fusion protein comprises or consists of:

[0129] (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;

[0130] 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 thereto, conjugated to the C-terminus of the FST315 polypeptide; and

[0131] 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;

[0132] (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;

[0133] 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 thereto, conjugated to the C-terminus of the FST288 polypeptide; and

[0134] 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;

[0135] (c) i. a FST315HBM polypeptide defined by SEQ ID NO: 3, or a sequence having at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity thereto;

[0136] 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 thereto, conjugated to the C-terminus of the FST315HBM polypeptide; and

[0137] 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;

[0138] (d) i. a FST288HBM polypeptide defined by SEQ ID NO: 4 or a sequence having at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity thereto;

[0139] 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 thereto, conjugated to the C-terminus of the FST288 HBM polypeptide; and

[0140] 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;

[0141] (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;

[0142] 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;

[0143] iii. a Fab heavy chain defined by SEQ ID NO: 5, or a sequence having at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity thereto, conjugated to the free end of a linker; and

[0144] 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;

[0145] (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;

[0146] 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;

[0147] iii. a Fab heavy chain defined by SEQ ID NO: 5, or a sequence having at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity thereto, conjugated to the free end of a linker; and

[0148] 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;

[0149] (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;

[0150] 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;

[0151] iii. a Fab heavy chain defined by SEQ ID NO: 5, or a sequence having at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity thereto, conjugated to the free end of a linker; and

[0152] 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;

[0153] (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;

[0154] 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;

[0155] iii. a Fab heavy chain defined by SEQ ID NO: 5, or a sequence having at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity thereto, conjugated to the free end of a linker; and

[0156] 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;

[0157] (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, and 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;

[0158] (j) SEQ ID NO:8, 9, 10, 11, 24, 25, 26, 27, 32, 33, 34 or 35 and a Fab light chain defined by SEQ ID NO:6; or

[0159] (k) A functional variant or fragment of any one of (a) to (i).

[0160] Alternatively, if an N-terminal fusion is preferred, the present invention provides a polynucleotide sequence encoding a fusion protein comprising or consisting of: i. a follistatin portion defined by any one of SEQ ID NOs: 1 to 4, 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 thereto, conjugated to the N-terminus of the FST315 polypeptide; and 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, and optionally a linker between the follistatin portion and the Fab heavy chain. In some specific examples, the present invention provides a polynucleotide sequence encoding a fusion protein defined by: SEQ ID NO:28, 29, 30 or 31, 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 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.

[0161] In some specific (but non-limiting) examples, the isolated polynucleotide comprises or consists of: (i) SEQ ID NO: 36, 37, 38, 39, 57 or 58 encoding the follistatin portion, or a sequence having at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity thereto; (ii) SEQ ID NO: 48, 49 and 50 encoding the CDRs of the heavy chain of the antibody portion, or a sequence having at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity thereto; (iii) SEQ ID NO: 51, 52 and 53 encoding the CDRs of the light chain of the antibody portion, and (iv) if a linker is present, SEQ ID NO: 42, 54, 55 or 56 encoding the linker.

[0162] In further specific (but non-limiting) examples, the isolated polynucleotide comprises or consists of: (i) SEQ ID NO:36, 37, 38, 39, 57 or 58 encoding the follistatin portion, or a sequence having at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity thereto; (ii) SEQ ID NO:40 encoding the heavy chain of the antibody portion, or a sequence having at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity thereto; (iii) SEQ ID NO:41 encoding the light chain of the antibody portion, or a sequence having at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity thereto; and (iv) if a linker is present, SEQ ID NO:42, 54, 55 or 56 encoding a linker.

[0163] In other specific (but non-limiting) examples, the isolated polynucleotide comprises or consists of: (i) SEQ ID NO:43, 44, 45, 46, 59, 60, 61, 62, 63, 64, 65 or 66 encoding a Fab heavy chain portion fused to a FST portion and an optional linker therebetween, or a sequence having at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity thereto; and (i) SEQ ID NO:41 encoding a Fab light chain.

[0164] In a related aspect, the present invention provides a cloning or expression vector comprising a polynucleotide encoding the fusion protein of the present invention as a whole. It should be understood that one skilled in the art can select a double gene vector (comprising two expression cassettes, one encoding the FST portion, the antibody portion fused to the FST portion, and an optional linker therebetween, and the other encoding the remaining antibody portion) or two different vectors (one encoding the FST portion, the antibody portion fused to the FST portion, and an optional linker therebetween, and the other encoding the remaining antibody portion).

[0165] The general methods of constructing vectors, transfection methods and culture methods are well known to those skilled in the art. In this regard, reference is made, for example, to "Current Protocols in Molecular Biology", 1999, FM Ausubel (ed), Wiley Interscience, New York and the Maniatis Manual produced by Cold Spring Harbor Publishing.

[0166] In a related aspect, the present invention provides a host cell comprising: a polynucleotide sequence encoding a fusion protein of the present invention, or a cloning or expression vector comprising one or more polynucleotides encoding a fusion protein of the present invention.

[0167] Any suitable host cell / vector system can be used to express the polynucleotide sequence encoding the fusion protein of the present invention. Bacteria (e.g., E. coli) and other microbial systems can be used, and eukaryotic (e.g., mammalian) host cell expression systems can also be used. Suitable mammalian host cells include CHO, myeloma, or hybridoma cells. In one embodiment, the host cell comprises (e.g., has been transformed with the following): (1) a vector comprising two expression cassettes, one expression cassette encoding the FST portion, the antibody portion fused to the FST portion, and an optional linker therebetween, and the other expression cassette encoding the remaining antibody portion, or (2) a first vector and a second vector, wherein the first vector comprises a nucleic acid encoding an amino acid sequence comprising or consisting of the following: the FST portion, the antibody portion fused to the FST portion, and an optional linker therebetween, and the second vector comprises a nucleic acid encoding an amino acid sequence comprising or consisting of the remaining antibody portion.

[0168] Suitable host cells for cloning or expressing fusion protein encoding vectors include prokaryotes or eukaryotic cells as described herein. For example, antibodies can be produced in bacteria, particularly when glycosylation and Fc effector functions are not required. For expression of antibody fragments and polypeptides in bacteria, see Charlton, Methods in Molecular Biology, Vol. 248 (BKCLo, ed., Humana Press, Totowa, NJ, 2003, pp. 245-254, describing the expression of antibody fragments in Escherichia coli). In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeast may be suitable cloning or expression hosts for fusion protein encoding vectors, including fungi and yeast strains in which glycosylation pathways have been "humanized", resulting in the production of antibodies with partial or complete human glycosylation patterns (see Gerngross et al., 2004; Li et al., 2006). Alternatively, suitable types of mammalian cells that can be used in the present invention, such as Chinese hamster ovary (CHO cells), include CHO-S, CHO-K1 cells, dhfr-CHO cells, such as CHO-DG44 cells and CHO-DXB11 cells that can be used with the DHFR selection marker, or CHO-K1 cells or CHOK1-SV cells that can be used with the glutamine synthetase selection marker. Other cell types for expressing antibodies include lymphocyte cell lines, such as NSO myeloma cells and SP2 cells, COS cells. Host cells can be stably transformed or transfected with the isolated polynucleotide sequence or expression vector described in the present invention.

[0169] In related aspects, the present invention provides a method for producing the fusion protein of the present invention, the method comprising culturing the host cell of the present invention under suitable conditions to produce the fusion protein. The method of the present invention may also include a step (harvesting step) of reclaiming the cell culture fluid (CCF) comprising the fusion protein, in other words, a step of harvesting the fusion protein. After harvesting, the fusion protein can be purified, for example, using protein A chromatography and other chromatography / filtration steps. The method also optionally includes the step of preparing a preparation of, for example, a certain protein concentration, such as 10 mg / ml or higher, for example 50 mg / ml or higher, by mixing the purified fusion protein. Without limitation, the preparation may be a liquid preparation, a lyophilized preparation or a spray-dried preparation. For all these steps, standard methods may be used.

[0170] In another aspect, the present invention provides a method for purifying the fusion protein of the present invention, the method comprising:

[0171] i. loading the clarified cell culture fluid containing the fusion protein of the present invention onto a protein A chromatography column pre-equilibrated with a buffer so that the fusion protein binds to the column,

[0172] ii. washing the column with the same wash buffer as the equilibration buffer in step i to remove impurities,

[0173] iii. eluting the fusion protein bound to the column with an elution buffer under alkaline conditions,

[0174] iv. further eluting any remaining bound fusion protein with an acidic elution buffer,

[0175] v. neutralizing the eluate in steps iii and iv to obtain a neutralized sample,

[0176] vi. The neutralized sample is subjected to an additional purification step to obtain the purified fusion protein.

[0177] In a non-limiting example, the equilibration / wash buffer of steps i and ii is a sodium acetate buffer having a concentration of at or about 30 to 70 mM, such as at or about 30, 35, 40, 45, 50, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65 or 70 mM, and a pH of at or about 5.5 to about 6.5, such as at or about 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4 or 6.5. In another non-limiting example, the elution buffer of step iii. is a glycine-based buffer, such as a glycine / NaOH buffer, having a concentration of 30 mM to 70 mM or about 30 mM to about 70 mM, such as about 30, 35, 40, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 60, 65 or 70 mM, and a pH of about 8.0 to about 9.0, such as pH 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0. In another non-limiting example, the acidic elution buffer of step iv. is a citrate buffer having a concentration of 50 to 200 mM or about 50 to about 200 mM, such as about 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 or 200 mM, and a pH of about 1.5 to about 2.5, such as about 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4 or 2.5. In another non-limiting example, the neutralization of step v. is performed at a pH of 7.0 to 9.0 (e.g., 7.0, 7.2, 7.4, 7.6, 7.8, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9 or 9.0). Neutralization is typically performed with Tris or Tris / HCl. The inventors have found that the fusion protein of the present invention not only binds to protein A, but can also be eluted under alkaline conditions. In contrast, free Fab still strongly binds to protein A under alkaline conditions. This feature provides some potential advantages for downstream processes. First, by not using acidic elution conditions, co-elution of free Fab can be avoided because Fab still strongly binds to protein A. In addition, FST-Fab will not be exposed to harsh acidic pH for a long time.Finally, alkaline elution is compatible with subsequent chromatography steps, which means reduced sample manipulation and thus allows for potential increases in yield and recovery.

[0178] As a specific (but non-limiting) example, a method for producing a fusion protein is provided herein, wherein the fusion protein comprises a follistatin portion (e.g., FST315, FST315HBM, FST288, or FST288HBM) connected at the C-terminus to the N-terminus of the heavy chain of Fab (VH-CH1) via an optional SGGGGS linker. The FST-Fab heavy chain is co-expressed with the Fab light chain (LC), and the heavy chain and the light chain are linked by an intermolecular disulfide bond.

[0179] On the other hand, the present invention provides a pharmaceutical composition, which comprises the fusion protein of the present invention and one or more pharmaceutically acceptable carriers, excipients or diluents as a whole. The pharmaceutical composition is usually prepared by mixing the active ingredient (herein, the fusion protein of the present invention) with one or more optional pharmaceutically acceptable carriers in the form of a dry preparation or an aqueous solution.

[0180] Any suitable pharmaceutically acceptable carrier, diluent and / or excipient can be used to prepare the pharmaceutical composition (e.g., see Remington: The Science and Practice of Pharmacy, Alfonso R. Gennaro (ed.) Mack Publishing Company, April 1997). The pharmaceutical composition is generally sterile and stable under the conditions of preparation and storage. The pharmaceutical composition can be formulated as a solution (e.g., saline, dextrose solution or buffered solution, or other pharmaceutically acceptable sterile fluid), a microemulsion, a liposome, or other ordered structures suitable for accommodating high product concentrations (e.g., microparticles or nanoparticles). The carrier may include, but is not limited to: a buffer; an antioxidant; a preservative; a hydrophilic polymer; an amino acid; monosaccharides, disaccharides and other carbohydrates; a chelating agent; a salt-forming counterion; and / or a nonionic surfactant.

[0181] Preferably, the pharmaceutical composition is formulated into a solution, more preferably into an optional buffer solution. Supplementary active compounds can also be incorporated into the pharmaceutical composition of the present invention. In one embodiment, the pharmaceutical composition is a composition suitable for intravenous or subcutaneous administration. These pharmaceutical compositions are only exemplary and do not limit pharmaceutical compositions suitable for other routes of administration. The pharmaceutical compositions described herein can be packaged in single unit dose or multiple dose forms.

[0182] The fusion protein or pharmaceutical composition of the present invention can be used by one or more of the various methods known in the art through one or more routes of administration. As will be appreciated by those skilled in the art, the route of administration and / or mode will vary according to the desired results. Examples of the route of administration of the fusion protein or pharmaceutical composition of the present invention include intravenous, intramuscular, intradermal, intraocular, intraperitoneal, subcutaneous, spinal or other parenteral routes of administration, such as by injection or infusion. Alternatively, the fusion protein or pharmaceutical composition of the present invention can be administered by non-parenteral routes (such as topical, epidermal or mucosal routes of administration). When the product is used for injection or infusion, it can be in the form of a suspension, solution or emulsion in an oily or aqueous vehicle, and it may contain additional agents, such as suspending agents, preservatives, stabilizers and / or dispersants. Alternatively, the fusion protein or pharmaceutical preparation of the present invention can be provided in a dry form to be reconstituted with a suitable sterile liquid before use. It can also be prepared into a solid form suitable for dissolving or suspending in a liquid vehicle before injection.

[0183] Once formulated, the fusion protein or pharmaceutical preparation of the invention can be directly administered to a subject.

[0184] In another aspect, the present invention provides a fusion protein or pharmaceutical composition for use in therapy. Alternatively, the present invention provides a method for treating a subject in need of therapy, the method comprising administering a therapeutically effective amount of the fusion protein or pharmaceutical composition of the present invention. In another alternative, the present invention provides the use of the fusion protein or pharmaceutical composition of the present invention in the preparation of a medicament for therapy.

[0185] The therapeutically effective amount will vary depending on the protein or active fragment thereof, the disease and its severity and the age, weight, etc. of the subject to be treated.

[0186] In the overall context of the present invention, "subject" generally refers to a mammal. Mammals include, but are not limited to, domestic animals (e.g., cattle, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates, such as monkeys), rabbits, and rodents (e.g., mice and rats). More preferably, the subject is a human.

[0187] 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 scope of the appended claims.

[0188] Description of amino acid sequence

[0189]

[0190]

[0191]

[0192]

[0193]

[0194] * X in SEQ ID NO:22-31 represents "any amino acid"

[0195] Example

[0196] Materials and methods

[0197] Production of follistatin-Fab fusion protein

[0198] Cloning strategy: DNA segments corresponding to the fusion between follistatin and the heavy or light chain sequence of the anti-albumin antibody (designated 645Fab) with or without a linker sequence in between were generated by PCR or gene synthesis and cloned using in-house mammalian expression vectors. The heavy and light chain sequences of 645Fab were also cloned separately using in-house mammalian expression vectors. All expression vectors were confirmed by direct sequencing using primers covering the entire open reading frame.

[0199] CHO cell culture: A suspension of CHOS-XE cells (Cain et al., 2013) was pre-adapted in CD CHO medium (Invitrogen) supplemented with 2 mM Glutamax. Cells were maintained in logarithmic growth phase and cultured in a shaking incubator (Kuhner AG) at 37°C in an environment containing 8% CO2 with shaking at 120 RPM.

[0200] Protein expression: Overexpression of follistatin-Fab protein by transient transfection CHO-XE cell line. Cotransfect paired expression plasmids (e.g., N-fab light chain-FST-C and heavy chain or FST-C fab heavy chain-C and light chain). Immediately before DNA transfection, CHO cells were exchanged into Expi CHO expression medium (Gibco) by centrifuging cells at 1500x g and resuspending the precipitate. 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, then grown at 32°C for the remainder of the expression cycle, and cultured in an environment containing 8% CO2 with 190RPM shaking. 9-14 days after transfection, supernatants were harvested, usually by centrifugation at 4000x g and then filtered using a 0.22 μm membrane. Final protein expression levels were determined by protein G-HPLC and SDS PAGE.

[0201] Protein purification:The transiently expressed protein contents were captured using Mab Select columns (GE Healthcare) run under standard conditions. In brief, the resin was washed with 10 column volumes of phosphate buffered saline (PBS, pH 7.4), and the bound protein was eluted with 5 column volumes of 0.1 M sodium citrate pH 3.1 (unless otherwise specified in the following examples). The eluate was neutralized with TRIS-HCl pH 8.5 and sterilized by filtration (here, PBS pH 7.4 was used as the running buffer preload column) by 0.22 μm membrane exclusion chromatography (HiLoad 26 / 60 Superdex 75 columns, GE Healthcare) run under standard conditions. Sample quality was assessed using the absorbance at 280 nm, BEH2000 analysis UPLC and SDS PAGE (under reducing and non-reducing conditions).

[0202] For protein G purification, the clarified supernatant was loaded onto a protein G HP (GE Healthcare) column balanced in PBS pH 7.4 and subsequently washed with the same buffer. The bound material was eluted with 0.1 M glycine-HCl pH 3.0. The acidic eluate was neutralized with 2 M Tris / HCl pH 8.5. The purified material was quantified by the absorbance at 280 nm.

[0203] Size Exclusion Chromatography (SEC): The sample was injected into BEH200. The column was loaded onto a 1.7 μm, 4.6 mm ID x 300 mm column (Aquity) and an isocratic gradient of 0.2 M phosphate, pH 7 was run at 0.35 mL / min and detected by absorbance at 280 nm and a multichannel fluorescence (FLR) detector (Waters).

[0204] SDS-PAGE:For analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), samples were prepared by adding 4× Novex NuPAGE LDS sample buffer (Life Technologies) and 100 mM N-ethylmaleimide (Sigma-Aldrich) to the purified protein and heated to 100° C. for 3 minutes. The samples were loaded onto a 10-well Novex 4-20% Tris-glycine 1.0 mm SDS-polyacrylamide gel (Life Technologies) and separated at a constant voltage of 225 V for 40 minutes in Tris-glycine SDS running buffer (Life Technologies). Novex Mark 12 wide range protein standard (Life Technologies) was used as a standard. The gel was stained with Coomassie fast stain (Generon) and destained in distilled water.

[0205] Pharmacokinetic measurements: FST protein was administered to C57BL / 6 mice at 10 mg / kg (2 mL / kg IV, 5 mL / kg SC) by intravenous administration. Blood samples were collected every day for 7 days. Serum samples were generated and analyzed using ligand binding assays for follistatin (including FST288 and FST315). Pharmacokinetic parameters were calculated based on single data using Phoenix v8.3.

[0206] Functional active reporter gene cell assay: To measure the efficacy of follistatin in blocking stimulus (activin A / B, GDF8 / 11)-induced activation of the SMAD2 / 3 signaling pathway, HEK Blue TM TGFβ reporter cells (Invivogen). Briefly, based on concentration and predicted activity, follistatin protein and its matched controls were pre-diluted in culture medium so that the resulting inhibition curve had a complete top and bottom. A 10-point serial dilution was then made in culture medium at 1 / 3, and four replicates of each dilution were aliquoted at 20 μL / well in a 384-cell culture assay plate. 10 μl of each stimulus was added to wells representing the follistatin serial dilutions, as well as to wells without follistatin representing the highest response to the stimulus. Wells with matching volumes of culture medium represented untreated. The assay plates were incubated at 37°C for one hour, and then HEK-Blue TM TGFβ cells (10,000 cells / 20 μL) were added and the assay plate was incubated at 37°C for another 17 hours. 45 μL / well of QUANTI-Blue TMThe solution was added to a fresh 384-assay plate designated as the target plate, and 5 μL of the cell supernatant was then transferred from the assay plate to the target plate using an automated liquid handler. The target plate was briefly placed on a shaker and then incubated at 37°C for one hour. The absorbance value of each well was measured at 630 nm on a plate reader and the percentage of follistatin-mediated dose-dependent inhibition of SMAD2 / 3 activity was calculated together with the Z factor.

[0207] Biacore binding data

[0208] The binding kinetics of the fusion proteins to each target were determined by surface plasmon resonance using Biacore T200 (Cytiva) (see below). For each type of assay, kinetic parameters were determined using the Biacore T200 Evaluation Software (version 3.0) using a 1:1 binding model.

[0209] In order to evaluate the binding with activin A and activin B (R&D Systems), goat anti-human F(ab?)2 fragment specific antibody (Jackson ImmunoResearch) was first fixed on a CM5 sensor chip at a level of about 5000RU by amine coupling chemistry. Using standard multi-cycle kinetics, each analysis cycle consists of the following: FST-Fab is captured on the anti-F(ab?)2 surface, then the analyte is injected, and finally the surface is regenerated using 50mM HCl and 5mM NaOH injected for 60s. 3 times serial dilutions of 30, 10, 3.3, 1.1, 0.367 and 0.122nM in HBS-EP+ running buffer (Cytiva) were injected (at 25°C with a flow rate of 300s at 30μl / min) analyte, and then dissociation was monitored for 900s. The binding reaction of the parallel blank surface was subtracted, and the buffer blank injection was included to subtract instrument noise and offset.

[0210] To evaluate binding to GDF8 and GDF11 (R&D Systems), each of GDF8 and GDF11 was immobilized on the surface of a CM5 sensor chip by amine coupling chemistry to achieve an immobilization level of approximately 250 RU. For GDF8 and GDF11, analysis was performed using a single cycle kinetic method with continuous injections of increasing concentrations (0.8, 4, 20, 100, and 500 nM) of FST-Fab in HBS-EP+ running buffer (Cytiva) at a flow rate of 30 μl / min at 25°C for 180 s, followed by monitoring dissociation for 1800 s. Binding responses to parallel blank surfaces were subtracted, and a series of buffer blank injections were performed to subtract instrument noise and offset.

[0211] To evaluate binding to albumin: Goat anti-human F(ab?)2 fragment-specific antibodies (Jackson ImmunoResearch) were first immobilized on a CM5 sensor chip at a level of approximately 5000 RU by amine coupling chemistry. Using a standard multi-cycle kinetic method, each analysis cycle consisted of capturing the fusion protein to be tested on the anti-F(ab?)2 surface, followed by injection of albumin, and finally surface regeneration using 50 mM HCl and 5 mM NaOH injected for 60 s. Analytes were injected (300 s at a flow rate of 30 μl / min at 25°C) using 2-fold serial dilutions at concentrations of 100, 50, 25, 12.5, 6.3 and 3.1 nM in HBS-EP+ running buffer (Cytiva), and dissociation was then monitored for 900 s. The binding response of the parallel blank surface was subtracted, and a buffer blank injection was included to subtract instrument noise and offset.

[0212] Example 1 - FST-Fab results in superior expression levels and monomer yields compared to other fusion partners.

[0213] Comparison of the relative expression levels of follistatin moieties fused with various fusion partners and in different orientations showed that the FST-Fab construct fused at the C-terminus of FST was optimal ( Figure 2 A). Fusion of Fc or ScFv to the C-terminus of FST was significantly inferior to using Fab at this position, with the former two expressing 3.5-fold less product ( Figure 2 A). Fusion of the Fab part at the N-terminus of the FST part resulted in a decrease of about 45% in the amount of expressed product, although superior to the fusion protein comprising FST fused to the Fc domain. Comparative evaluation of the effect on expression using FST288- or FST315-fused to the Fab at the C-terminus of follistatin showed only marginal differences using the wild-type HBM sequence ( Figure 2 B) The FST315 version had 6% lower expression than the FST288 fusion. Comparison of fusion proteins containing HBSM (HBM) showed a modest 7% reduction in expression levels compared to wild type.

[0214] Head-to-head examination of various FST-fusions showed that the Fab portion fused to the C-terminus of the FST portion provided the highest level of final monomer yield compared to the other fusions ( Figure 2 C). Among the groups, the FST-Fc fusion provided the lowest monomer yield, nearly 6-fold worse than the FST-Fab fusion. FST-ScFv and FST fused to the N-terminus of the Fab were approximately 3- and 4-fold worse, respectively.

[0215] Example 2 - In mouse studies, FST315(HBM)-Fab exhibited prolonged pharmacokinetic activity compared to FST315WT Mechanical properties

[0216] FST315WT, FST288WT, and FST288-Fab administered intravenously (IV) to mice at 10 mg / kg were cleared very rapidly, 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 IV to mice at 10 mg / kg showed prolonged kinetics, with MRTs of 9.3 hours, 13.1 hours, and 11 hours, respectively. Figure 3 A and B). All pharmacokinetic parameters are summarized in Table 1.

[0217] Conclusion: Example 2 shows that the kinetics and half-life of FST-containing proteins can be greatly extended due to the fusion between the FST part and the Fab part. In the heparin binding site (HBM) version of follistatin, mutations in the HBM also made a significant contribution to the extended kinetics.

[0218] Example 3 - FST315-Fab, FST315HBM-Fab, FST288-Fab and FST288HBM-Fab to their ligands and Albumin shows high affinity binding

[0219] Follistatin has four high affinity ligands: activin A, activin B, GDF8 (myostatin) and GDF11. The binding of FST315-Fab, FST315HBM-Fab, FST288-Fab and FST288HBM-Fab to these ligands has been confirmed using a surface plasmon resonance (SPR) binding method, and the Kd binding affinity is confirmed to be within the expected range summarized in Table 2 (in view of the literature, see Sidis et al., 2006). The affinity Kd values ​​of all follistatin fusion proteins obtained for their specific ligands are fully consistent with the literature values ​​(23.6pM and 28.7pM, respectively (Sidis et al., 2006)) described for wild-type, unconjugated FST288WT and FST315WT binding to activin A. The human albumin binding properties of the Fab domain component of FST315 (HBM)-Fab were also confirmed by SPR, and the value of 2602pM is consistent with the expectations of active albumin binders. The Kd value for FST315(HBM)-Fab albumin binding is also in good agreement with previously published values ​​for human albumin binding of individual Fabs (cited in Adams et al., 2016, 2-5 nM; referring to different forms of 645gL4gH5 Fab).

[0220] Conclusion: Example 2 emphasizes that the expected biological activity of the FST-Fab moiety is maintained, ie, the fusion between the two moieties does not affect the binding activity of the follistatin moiety to its respective biological ligand or the Fab moiety to albumin.

[0221] Example 4 - FST315(HBM)-Fab and FST288(HBM)-Fab show activity against Inhibition of its ligand function

[0222] Next, use HEK-Blue TM -Smad2 / 3 reporter cell assays in commercially available TGFβ cell lines tested the ability of FST315(HBM)-Fab to inhibit functional signaling of the four ligands. All four ligands were used at approximately their EC50 concentrations to induce stimulation of reporter gene activation, and then FST315(HBM)-Fab and FST315WT were titrated over a high concentration range to generate Figure 4A Dose-response curves are shown. Table 3 summarizes the geometric mean IC50 data for all four ligands. It was observed that the FST315(HBM)-Fab format was consistently 3-fold more potent than the parent FST315WT when the responses were induced with the ligands activin A and activin B, and 2-fold more potent when the ligands GDF8 and GDF11 were used. Similarly, FST288(HBM)-Fab and FST288WT were titrated over a high concentration range to generate dose-response curves for all four ligands used at approximately their EC50 concentrations; representative data are shown in Figure 4B The geometric mean IC50 data for all four ligands are shown in Table 3. Unlike the data for the FST315 format, the FST288(HBM)-Fab molecule format showed very similar efficacy to the FST288WT parental molecule in all four ligands.

[0223] Conclusion: This example shows not only that FST315(HBM)-Fab is not inferior to FST315WT protein in its ability to inhibit signaling induced by ligands of the Smad2 / 3 reporter pathway, but also that FST315(HBM)-Fab has improved potency compared to FST315WT, highlighting its relevance in a therapeutic setting. This is in contrast to the potency of FST288(HBM)-Fab, which is very similar to the FST288WT molecule.

[0224] Example 5 - Fusion of FST-Fab to the human VH3 domain allows recovery by Protein A chromatography

[0225] A FST315HBM-Fab fusion (FST-Fab1) was prepared in which FST was fused to an anti-albumin F(ab?) containing a human VH3 domain (fusion protein of SEQ ID NO: 8) capable of Protein A chromatography.

[0226] Two alternative FST-F(ab?) constructs (FST-Fab2 and FST-Fab3) that do not contain a human VH3 domain were prepared. All constructs were expressed and purified according to the above method. As shown in Table 4, only FST-Fab1 containing a human VH3 domain was recovered after protein A chromatography. After protein G chromatography, all three fusion proteins were able to be recovered.

[0227] Typically, elution of proteins bound to protein A affinity capture resin is performed under acidic conditions (see the "Materials and Methods" section above). However, the inventors found that FST-Fab1 of the present invention is effectively eluted from protein A under slightly alkaline conditions, while the Fab fraction remains strongly bound.

[0228] The supernatant of FST-Fab1 clarification is loaded on the MabSelect (GE Healthcare) column balanced in 50mM sodium acetate at pH 5.8, and then washed with the same buffer. The bound material is eluted under acidic (0.1M glycine-HCl pH 2.6) or alkaline (50mM glycine-NaOH pH 8.6) conditions. Then another acid elution (0.1M citrate pH 2.0) is carried out. The acid elution and elution pool are neutralized with 2M Tris / HCl pH 8.5. These elutions and elution samples are then analyzed by SDS-PAGE and size exclusion analysis.

[0229] The results are as follows Figure 5 As shown. SDS-PAGE analysis showed that after elution under acidic conditions (lane 2), Lane 3, which contains the subsequent eluate, contains no protein because it has been removed by the acid elution. Alternatively, under slightly alkaline conditions (lane 4), no Fab band is present. Fab does not elute from the column until the acid elution (lane 5), where, as observed in the acid elution, the Fab band is not present. There is a belt.

[0230] like Figure 6 As shown, size exclusion chromatography analysis showed that only the acidic elution pool had a peak corresponding to Fab ("F") (Panel A), while Fab ("F") was absent in the alkaline elution pool (Panel B). There was little / no protein in the eluate after acidic elution (Panel C), because all the protein had been eluted by acidic elution, while the eluate after alkaline elution contained a peak corresponding to Fab (Panel D).

[0231] The effective elution of the FST-Fab fusion protein of the present invention from Protein A under slightly alkaline conditions is a unique property of the molecule. It has several potential advantages in downstream processes. First, by not using acidic elution conditions, co-elution of Fab can be avoided because Fab remains strongly bound to Protein A. In addition, Fst-Fab will not be exposed to harsh acidic pH for a long time. Finally, alkaline elution is compatible with subsequent chromatography steps, which means reducing sample handling and therefore potentially increasing yield and recovery.

[0232] Example 6 - Generation of stable cell lines

[0233] While all previous examples were performed using transiently expressed FST-fusion proteins (according to the "Materials and Methods" section above), this example focuses on obtaining stable cell lines to produce the FST-fusion proteins described herein.

[0234] Transfection of host cell lines: CHO DG44 (dhfr-) host cells were transfected with DNA double gene vector plasmids to stably express human FST315 (HBM) -645 Fab molecules (i.e., encoding SEQ ID NO: 8 and SEQ ID NO: 6) and the selection marker dihydrofolate reductase (DHFR). The vector was linearized prior to electroporation. The cells were electroporated and then allowed to recover in host cell growth medium in a static, temperature-controlled, CO2 incubator for 24 hours and then cultured in selective medium.

[0235] A total of 167 micro-pools were recovered and cultured in selective medium containing methotrexate. Based on antibody titers, 70 micro-pools were evaluated in shake flask cultures. Based on 10-day bulk mAb titers, the top 24 micro-pools were selected for evaluation in the AMBR automated micro-bioreactor.

[0236] The best 7 micro-pools (MPs) were selected for single cell cloning. Cells from each MP were centrifuged and the pellet was resuspended in PBS. Each MP cell suspension was then analyzed separately by flow cytometry. After single cell cloning, the top 54 clones based on the highest antibody titer were expanded into shake flasks for batch evaluation.

[0237] Twelve high-expressing clones were selected and evaluated in several fed-batch processes in the AMBR microbioreactor system using animal-origin-free chemically defined medium to assess clonal cell growth, antibody titer, specific productivity, and product quality.

[0238] A lead clone was selected and produced in a swing bioreactor and 5 L shake flask using the optimal fed-batch process determined previously.

[0239] Shown are the total product concentrations achieved in two different media and in the fed-batch process from four different clones (see Figure 7 ). This example shows that if one wishes to possibly increase the yield of the FST-fusion protein according to the invention, an aspect to be considered is the set of culture media (basal medium and a feed medium / feed media) used for its production. Figure 7 As shown, although clone 156 generally produced at about 1 g / L on a small scale, the use of basal medium A in combination with fed-batch process 2 (FB2) resulted in a doubling of the titer compared to fed-batch process 1 (FB1). As another example, in the presence of basal medium A (regardless of the feed medium / feed media), clone 51 had a production of about 1.2-1.3 g / L, but in basal medium B, the production dropped to below 1 g / L.

[0240] Table 1 - Pharmacokinetic parameters in Example 2

[0241]

[0242] * Undetermined

[0243] Table 2 - Kd binding affinity of Example 3 (from Biacore assay)

[0244]

[0245] * Not obtained

[0246] Table 3 - IC50 data of Example 4

[0247]

[0248]

[0249] Table 4 - Recoveries of different constructs of Example 5 after affinity chromatography.

[0250]

[0251] ** All data in this table are normalized based on the results of protein G recovery of FST-Fab1 (considered as 100%).

[0252] References

[0253] 1. WO2015 / 187977

[0254] 2. WO2017 / 152090

[0255] 3.Sidis Society, (2005)Endocrinology 146(1):130–136

[0256] 7.http: / / blast.ncbi.nlm.nih.gov /

[0257] 8.http: / / www.ebi.ac.uk / Tools / emboss /

[0258] 9.WO2013 / 068571

[0259] 10.“Current Protocols in Molecular Biology”(1999)FMAusubel(ed),Wiley Interscience,New York and the Maniatis Manual produced by Cold Spring Harbor Publishing

[0260] 11.Charlton,Methods in Molecular Biology,Vol.248(BKCLo,ed.,HumanaPress,Totowa,NJ,2003,pp.245-254,describing expression of antibody fragmentsin E.coli.);

[0261] 12.Gerngross Research,(2004),Nat.Biotech.22:1409-1414

[0262] 13.Li Person,(2006)Nat.Biotech.24:210-215.

[0263] 14.Remington:The Science and Practice of Pharmacy,Alfonso R.Gennaro(Editor)Mack Publishing Company,April 1997

[0264] 15.Cain Health,(2013)Biotechnol Prog.29(3):697-706.

[0265] 16.Adams Research,(2016)MABS 8(7):1336-1346.

[0266] Endocrinology 147(7):3586–3597.

Claims

1. A fusion protein, comprising or consisting of: a. Follistatin part, b. an antibody portion, and optionally c. A linker between the follistatin portion and the antibody portion.

2. The fusion protein according to claim 1, wherein the follistatin portion comprises or is a naturally occurring protein, a functional fragment thereof and / or a functional variant thereof.

3. A fusion protein according to any one of the preceding claims, wherein the follistatin portion comprises or consists of: 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 from 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.

4. A fusion protein according to any one of the preceding claims, wherein the antibody portion binds albumin.

5. The fusion protein of any of the preceding claims, wherein the antibody portion is a chimeric antibody portion, a humanized antibody portion, or a human antibody portion.

6. A fusion protein according to any one of the preceding claims, wherein the antibody portion is: a) an antigen binding portion selected from Fab, Fab?, or F(ab?)2, or b) an antigen binding portion selected from Fab, Fab? or F(ab?)2 and further comprising a human VH3 domain capable of binding to protein A.

7. The fusion protein of any one of the preceding claims, wherein the antibody portion comprises or consists of: a. a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises at least one CDR selected from the following: a CDR-L1 comprising SEQ ID NO: 13, a CDR-L2 comprising SEQ ID NO: 14 and / or a CDR-L3 comprising SEQ ID NO: 15; and the heavy chain variable region comprises at least one CDR selected from the following: a CDR-H1 comprising SEQ ID NO: 16, a CDR-H2 comprising SEQ ID NO: 17 and / or a CDR-H3 comprising SEQ ID NO: 18; or b. a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising or consisting of: SEQ ID NO:5 or a sequence at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical thereto, the light chain variable region comprising or consisting of SEQ ID NO:6 or a sequence at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical thereto.

8. A fusion protein according to any of the preceding claims, wherein the fusion protein comprises a linker between the follistatin portion and the antibody portion, and wherein the linker is selected from: SGGGGS (SEQ ID NO: 7), SGGGGSSGGGGS (SEQ ID NO: 19), GGGGS (SEQ ID NO: 20) and GGGGSGGGGS (SEQ ID NO: 21).

9. The fusion protein according to any one of the preceding claims, wherein: a) the antibody portion is linked to the C-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.

10. The fusion protein according to any one 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 thereto, conjugated to the C-terminus of the FST315 polypeptide; and 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 thereto, conjugated to the C-terminus of the FST288 polypeptide; and 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 FST315HBM polypeptide defined by SEQ ID NO: 3 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 thereto, conjugated to the C-terminus of the FST315HBM polypeptide; and 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 FST288HBM polypeptide defined by SEQ ID NO: 4 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 thereto, conjugated to the C-terminus of the FST288 HBM polypeptide; and 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 thereto, conjugated to the free end of a linker; and iv. a Fab light chain defined by SEQ ID NO: 6, or a sequence having at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity thereto; (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 thereto, conjugated to the free end of a linker; 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 thereto, conjugated to the free end of a linker; and iv. a Fab light chain defined by SEQ ID NO: 6, or a sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto; (h) i. 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 thereto, conjugated to the free end of a linker; and iv. a Fab light chain defined by SEQ ID NO: 6, or a sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto; (i) 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, and 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 and a Fab light chain defined by SEQ ID NO:6; or (k) A functional variant or fragment of any one of (a) to (j).

11. An isolated polynucleotide encoding the fusion protein according to any one of the preceding claims.

12. A cloning or expression vector comprising one or more polynucleotides according to claim 11.

13. A host cell, comprising: a. one or more polynucleotides according to claim 11, or b. One or more expression vectors according to claim 12.

14. A method for producing the fusion protein according to any one of claims 1 to 10, the method comprising culturing the host cell according to claim 13 under suitable conditions to produce the fusion protein and isolating the fusion protein.

15. A pharmaceutical composition comprising the fusion protein according to any one of claims 1 to 10 and one or more pharmaceutically acceptable carriers, excipients or diluents.

16. The fusion protein according to any one of claims 1 to 10 or the pharmaceutical composition according to claim 15 for use in therapy.

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