Follistatin fusion protein

Abstract

The present invention relates to the field of fusion proteins, and particularly to fusion proteins containing a follistatin moiety. The present invention also relates to a method for producing the fusion protein, together with a pharmaceutical preparation containing the fusion protein.

Description

Technical Field

[0001] The present invention relates to the field of fusion proteins, and more particularly to fusion proteins containing a follistatin moiety. The present invention also relates to a method for producing the fusion protein, together with a pharmaceutical preparation containing the fusion protein.

Background Art

[0002] Follistatin is a secreted glycoprotein that has as its primary function the binding and neutralization of members of the TGF-beta superfamily, particularly activin A, activin B, GDF8 (myostatin) and GDF11. As shown in Figure 1, it is known to exist in several different forms, including a 315 amino acid polypeptide (referred to as FST315), and a 288 amino acid polypeptide (referred to as FST288). Both FST315 and FST288 have a high affinity for activin (activin A and activin B) as well as myostatin (GDF8). In particular, follistatin can bind to and inhibit myostatin, a negative regulator of skeletal muscle mass.

[0003] Follistatin has been shown to be a potential therapeutic protein in certain conditions, including the treatment of muscle disorders such as muscular dystrophy (International Publication Nos. WO 2015 / 187977 and WO 2017 / 152090). However, the use of follistatin in therapy faces several 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 International Publication Nos. WO 2015 / 187977 and WO 2017 / 152090 contemplate the use of fusion proteins containing a follistatin polypeptide fused to the Fc portion of an immunoglobulin.

[0004] There is still a need for follistatin peptides and fusion proteins that can be more readily expressed in vitro and have improved half-life 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 including residues 289-314 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 specific examples, the fusion protein is (a) i. an 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, attached 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. An 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, attached 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. An 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, attached 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. An 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, attached to the C-terminus of the FST288HBM 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. An 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 attached 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 attached to the free end of the linker; and iv. A Fab light chain defined by SEQ ID NO: 6 or a sequence having at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity thereto; (f) i. An 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 attached 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 attached to the free end of the linker; and iv. A Fab light chain defined by SEQ ID NO: 6 or a sequence having at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity thereto; (g) i. An FST315 polypeptide variant defined by SEQ ID NO: 3 (FST315HBM) or SEQ ID NO: 22, or a sequence having at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity thereto; ii. A linker defined by SEQ ID NO: 7, SEQ ID NO: 19, SEQ ID NO: 20 or SEQ ID NO: 21 attached 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, attached to the free end of the linker; and iv. A Fab light chain defined by SEQ ID NO: 6, or a sequence having at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity thereto; (h) i. An FST288 polypeptide variant defined by SEQ ID NO: 4 (FST288HBM) or SEQ ID NO: 25, or a sequence having at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity thereto; ii. A linker defined by SEQ ID NO: 7, SEQ ID NO: 19, SEQ ID NO: 20 or SEQ ID NO: 21 attached to the C-terminus of the FST288 polypeptide variant; iii. A Fab heavy chain defined by SEQ ID NO: 5, or a sequence having at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity thereto, attached to the free end of the linker; and iv. A Fab light chain defined by SEQ ID NO: 6, or a sequence having at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity thereto; (i) An Fab light chain defined by a sequence having at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to 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 to SEQ ID NO: 6, and SEQ ID NO: 6; (j) An Fab light chain defined by SEQ ID NO: 8, 9, 10, 11, 24, 25, 26, 27, 32, 33, 34 or 35 and SEQ ID NO: 6; or (k) A functional variant or fragment of any one of (a) to (j) comprising or consisting of.

[0008] 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 cloning vectors or expression vectors comprising one or more polynucleotides of the present invention; and iii) host cells comprising one or more polynucleotides according to the present invention or one or more expression vectors according to the present invention.

[0009] In yet another aspect, the present invention provides a method for producing a fusion protein according to the present invention, comprising culturing a host cell according to the present invention under suitable conditions for producing the fusion protein and isolating the fusion protein.

[0010] In a further aspect, the present invention relates to a pharmaceutical composition comprising a fusion protein according to the present invention and one or more pharmaceutically acceptable carriers, excipients or diluents.

[0011] In a further aspect, the fusion protein or pharmaceutical composition according to the present invention is for use in therapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0012]

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Mode for Carrying Out the Invention

[0013] Technical terms are used according to their common sense, unless otherwise indicated. When a specific meaning is conveyed to a specific term, the definition of the term is given in the context in which the term is used.

[0014] When an indefinite or definite article, such as "a", "an", or "the", is used to refer to a singular noun, this includes the plural form of that noun, unless otherwise specified. As used herein, the term "comprising" 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 of".

[0015] As used herein, the terms " follistatin" or "FST" refer to the autocrine glycoprotein (UniProt accession: P19883), a known inhibitor of activin A and B. Follistatin also binds with lower affinity to GDF11, GDF8 (myostatin), BMP 2, 4, 6, 7, 11, and 15. There are two major alternative splicing forms of human follistatin, a shorter cell-binding form (FST288, 31.6 kDa) 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 and lack the 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 one heparin-binding site. FST315 has an additional 27 amino acid domain (rich in acid) at its C-terminus, called the acidic tail. Functional fragments and / or functional variants thereof, such as those disclosed by Sidis et al., 2005, are also included in this term. When numbers follow, e.g., in the case of FST288, this indicates that the protein is the 288 form of follistatin (starting from residue 1 of the mature form). When numbers and letters follow, e.g., in the case of FST315HBM, this indicates the heparin-binding mutant (HBM) form as well as the type of variant (here, the 315 form of follistatin, starting from residue 1 of the mature form and containing alanine mutations at residues K76, K81, and K82). Activin is a dimeric polypeptide growth factor and belongs to the TGF-beta superfamily. Activin can stimulate hormone production in ovarian and placental cells, support the survival of neuronal cells, and have positive or negative effects on cell cycle progression, depending on the cell type. In some tissues, activin signaling is antagonized by its related heterodimer, inhibin. For example, during the release of follicle-stimulating hormone (FSH) from the pituitary gland, activin promotes the secretion and synthesis of FSH, while inhibin inhibits the secretion and synthesis of FSH.Activin is also involved as a negative regulator of muscle mass and function, and activin antagonists can promote muscle growth or counteract muscle loss in vivo.

[0016] As used herein, the term "antibody" includes, but is not limited to, monoclonal antibodies, polyclonal antibodies, and recombinant antibodies produced by recombinant techniques known in the art. The term "antibody" as used herein refers to antibodies of any species, particularly mammalian species; for example, antibodies produced as dimers of this basic structure including IgG1, IgG2a, IgG2b, IgG3, IgG4, IgE, IgD, as well as IgGA1, IgGA2, or pentamers such as IgM, and modified variants thereof; human antibodies of any isotype; for example, non-human primate antibodies from chimpanzee, baboon, rhesus monkey or cynomolgus monkey; for example, rodent antibodies from mouse or rat; rabbit, goat or horse antibodies; camelid antibodies (e.g., from camel or llama such as Nanobodies™) and derivatives thereof; avian antibodies such as chicken antibodies; or fish antibodies such as shark antibodies. The term "antibody" refers to both glycosylated and non-glycosylated antibodies. Further, the term "antibody portion" as used herein may refer to a full-length antibody, but more generally is intended to refer to a reference antibody fragment, more specifically an antigen-binding fragment thereof. Antibody fragments contain at least one heavy or light chain immunoglobulin domain as known in the art and bind to one or more antigens. Examples of antibody fragments according to the present invention include Fab, modified Fab, Fab’, modified Fab’, F(ab’)2, Fv, Fab-Fv, Fab-dsFv, Fab-Fv-Fv, scFv and Bis-scFv fragments. The fragments may also be diabodies, tribodies, triabodies, tetrabodies, minibodies, single domain antibodies (dAbs), e.g., sdAb, VL, VH, VHH or camelid antibodies (e.g., from camel or llama such as Nanobody™) and VNAR fragments. Antigen-binding fragments according to the present invention may also comprise a Fab linked to one or two scFv or dsscFv, each scFv or dsscFv binding to the same or a different target (e.g., one scFv or dsscFv that binds to a therapeutic target, and one scFv or dsscFv that increases the half-life by binding to, e.g., albumin).

[0017] As used herein, the term "Fab" refers to a light chain fragment that includes the VL (variable light chain) domain and the constant domain (CL) of the light chain, as well as an antibody fragment that includes the VH (variable heavy chain) domain and the first constant domain (CH1) of the heavy chain.

[0018] The term "Fab" as used herein is similar to Fab in which the Fab portion is replaced by Fab'. The format may be provided as its PEGylated version. The dimer of Fab' according to the present disclosure generates F(ab')2, where, for example, dimerization may be through the hinge.

[0019] The term "Fv" refers to two variable domains of a full-length antibody, such as a cognate pair or an affinity matured variable domain, i.e., a cooperative variable domain such as a VH and VL pair.

[0020] As used herein, the term "single-chain variable fragment" or "scFv" refers to a single-chain variable fragment stabilized by a peptide linker between the VH variable domain and the VL variable domain.

[0021] As used herein, the term "single-domain antibody" refers to an antibody fragment consisting of a single monomeric variable domain. Examples of single-domain antibodies include VH or VL or VHH or V-NAR.

[0022] As used herein, the term "affinity" refers to the strength of all non-covalent interaction between a protein or a fragment thereof and its receptor (when the protein of interest is a ligand) or its ligand (when the protein of interest is a receptor). Unless otherwise indicated, when used herein, the term "binding affinity" refers to the intrinsic binding affinity that reflects the 1:1 interaction between the members of the binding pair (e.g., a receptor and its ligand). The affinity of a molecule for its binding partner can generally be represented by the dissociation constant (KD). Affinity can be measured by common methods known in the art, including those described herein.

[0023] In the context of antibodies and antigen-binding fragments, as used herein, the term "specific" is intended to refer to an antibody that recognizes only the antigen to which it is specific, or an antibody that has a significantly higher binding affinity, e.g., at least 5, 6, 7, 8, 9, 10-fold higher binding affinity, for the antigen to which it is specific compared to binding to an antigen to which it is non-specific.

[0024] The terms "albumin", "serum albumin" or "SA" refer to a globular protein that is abundant in both the vascular and extravascular compartments. Human serum albumin (HSA) is known as reference P02768, while the mouse serum equivalent is referenced as P07724.

[0025] The term "chimeric" refers to an antibody in which at least a first portion of the heavy chain and / or light chain antibody sequence is derived from a first species and a second portion of the heavy chain and / or light chain antibody sequence is derived from a second species. Chimeric antibodies of interest herein include "primatized" antibodies that contain 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.

[0026] A "humanized" antibody is a chimeric antibody that contains sequences derived from non-human antibodies. In most cases, a humanized antibody is a human antibody (recipient antibody) in which residues from the hypervariable regions of the recipient have been replaced with residues from the hypervariable regions [or complementarity-determining regions (CDRs)] of a non-human species, such as a mouse, rat, rabbit, chicken, or non-human primate (donor antibody), which have the desired specificity, affinity, and activity. In most cases, outside the CDRs, i.e., within the framework regions (FRs), residues of the human (recipient) antibody are further replaced by corresponding non-human residues. Additionally, a humanized antibody can contain residues not found in the recipient antibody or the donor antibody. These modifications are made to further improve antibody properties. Humanization reduces the immunogenicity of non-human antibodies in humans and thus facilitates the application of antibodies to the treatment of human diseases. Humanized antibodies and several different techniques for making them are well known in the art. Unless otherwise indicated, HVR residues (CDR residues) and other residues of the variable domain (e.g., FR residues) are numbered herein according to Kabat.

[0027] The term "antibody" also refers to human antibodies that can be made as an alternative to humanization. For example, it is possible to generate transgenic animals (e.g., mice) that, upon immunization, can produce a complete repertoire of human antibodies in the absence of production of endogenous mouse antibodies. Other methods for obtaining human antibodies / antibody fragments in vitro are based on display technologies such as phage display or ribosome display technologies, and recombinant DNA libraries that are at least partially artificially or made from the donor's immunoglobulin variable (V) domain gene repertoire are used. Phage and ribosome display technologies for making human antibodies are well known in the art. Human antibodies can also be made from isolated human B cells that can be ex vivo immunized with an antigen of interest and then fused to generate hybridomas, which can then be screened for optimal human antibodies.

[0028] As used herein, the term "functional variant" refers to an amino acid sequence that has been modified with respect to a reference sequence but retains at least one biological function of the reference sequence. For example, a functional variant of FST retains at least one biological activity of the reference FST protein, such as the binding and inhibition of activin A and B.

[0029] As used herein, the terms "sequence identity" or "identity" refer to the number of matches (identical nucleic acid or amino acid residues) at positions from an alignment of two polynucleotide or polypeptide sequences. Sequence identity is determined by comparing the sequences when aligned so as to maximize matches and identity while minimizing sequence gaps. In particular, sequence identity can be determined using any of several mathematical global or local alignment algorithms depending on the lengths of the two sequences being compared. The alignment for determining the percent nucleic acid sequence identity or percent amino acid sequence identity can be achieved in a variety of ways within the skill in the art using publicly available computer software available on Internet websites such as, for example, http: / / blast.ncbi.nlm.nih.gov / or http: / / www.ebi.ac.uk / Tools / emboss / . One of ordinary skill in the art can determine appropriate parameters for measuring the alignment, including any algorithm necessary to achieve the maximum alignment over the full length of the sequences being compared. For the purposes of this specification, the percent nucleic acid sequence identity value or percent amino acid sequence identity value refers to the value generated using the pairwise sequence alignment program EMBOSS Needle to create an optimal global alignment of two sequences using the Needleman - Wunsch algorithm with all search parameters set to default values, i.e., Scoring matrix = BLOSUM62, Gap open = 10, Gap extend = 0.5, End gap penalty = false, End gap open = 10, and End gap extend = 0.5.

[0030] The term "isolated" means, throughout this specification, that an antibody, antigen-binding fragment, polypeptide or polynucleotide is present in a physical environment that may, in some cases, be different from what may occur in nature. The term "isolated" nucleic acid refers to a nucleic acid molecule that has been isolated from its natural environment or produced synthetically. Isolated nucleic acids can include, for example, synthetic DNA, cDNA, genomic DNA, or any combination thereof, produced by chemical treatment.

[0031] The terms "nucleic acid" and "polynucleotide" or "nucleotide sequence" can be used interchangeably to refer to any molecule composed of or containing monomeric nucleotides. Nucleic acids can be oligonucleotides or polynucleotides. Nucleotide sequences can be DNA or RNA.

[0032] As used herein, the term "vector" refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. This term includes vectors as self-replicating nucleic acid structures, as well as vectors integrated into the genome of the host cell into which they have been introduced. Certain vectors can direct the expression of nucleic acids to which they are operably linked. Such vectors are referred to herein as "expression vectors".

[0033] As used herein, the term "pharmaceutically acceptable" means approved by a regulatory agency or recognized in a recognized pharmacopoeia such as the European Pharmacopoeia for use in animals and / or humans. The term "excipient" refers to a diluent, adjuvant, carrier and / or vehicle administered with a therapeutic agent.

[0034] The term "therapeutically effective amount" refers to an amount sufficient to effect such treatment of a disease when administered to a subject for treating the disease.

[0035] As used herein, the terms "treatment", "treating", etc. refer to obtaining a desired pharmacological and / or physiological effect. The effect can be prophylactic in terms of completely or partially preventing a disease or its symptoms, and / or therapeutic in terms of partially or completely curing a disease and / or a deleterious effect caused by the disease. Thus, treatment encompasses any treatment of a disease in a mammal, particularly a human, and includes (a) preventing a disease in a subject who may have a predisposition to the disease but who has not yet been diagnosed as having it, i.e., in a human, (b) inhibiting a disease, i.e., arresting its development, and (c) alleviating a disease, i.e., causing regression of the disease.

[0036] Here, the present invention will be described with reference to specific non-limiting aspects and their embodiments, as well as to specific figures and examples.

[0037] The present invention addresses the need for improved follistatin peptides and fusion proteins by providing novel follistatin fusion proteins incorporating antigen-binding antibody portions (such as antigen-binding portions), which fusion proteins are more readily expressed in vitro and have an improved half-life or other beneficial effects in vivo.

[0038] The present invention is based on the surprising finding of the inventors that a follistatin-based fusion protein incorporating an antigen-binding portion exhibits superior protein expression and a higher monomer fraction yield compared to known follistatin-based fusion proteins containing an Fc portion. In particular, the fusion proteins of the present invention have been shown to have an expression level at least 1.5-fold greater than that of the FST-Fc fusion protein. The fusion proteins of the present invention not only have a higher relative expression compared to the FST-Fc fusion protein, but also result in a much higher yield of the monomer fraction, i.e., the correctly folded and usable fusion protein (at least 1.5-fold greater than the FST-Fc fusion).

[0039] The main object / aspect of the present invention is a fusion protein comprising, or consisting of, a. a follistatin moiety, b. an antibody moiety, and optionally c. a linker between the follistatin moiety and the antibody moiety.

[0040] In the context of the present invention as a whole, the follistatin moiety comprises, or is, a naturally occurring follistatin protein. It is preferably in its mature form, i.e., lacking an N-secretory signal sequence as this sequence is only required for production / secretion from cells. Alternatively, it is a functional fragment thereof. The follistatin moiety is, for example, the FST288 protein (SEQ ID NO: 2) or the FST315 protein (SEQ ID NO: 1). Any intermediate form thereof, for example, any follistatin moiety comprising residues 289 - 314 of any one of SEQ ID NOs: 1 to 4, can also be used as long as it is functional, i.e., retains at least one biological activity of FST. Preferably, but not limited to, any intermediate form of the follistatin moiety starts with residue 1 of SEQ ID NO: 1. As a non-limiting example, a functional FST fragment can be FST291 (i.e., comprising residues 1 to 291 of SEQ ID NO: 1) or FST303 (i.e., comprising residues 1 to 303 of SEQ ID NO: 1). In another option, the follistatin moiety (i.e., naturally occurring or a functional fragment thereof) according to the present invention can be a functional variant and can have one or more mutations, such as mutations in the heparin binding site (HBS). As a non-limiting example, one or more mutation sites can be selected from K76, K81 and / or K82 numbered with respect to SEQ ID NO: 1 (see, for example, SEQ IDs 22 and 25). One or more mutations can include alanine (A) instead of lysine (K) (resulting in a mutation selected from K76A, K81A and / or K82A). As a further non-limiting example, heparin binding mutants (referred to herein as "HBM", or " (HBM)" or "HBSM" in this specification) can use, for example, FST288HBM (SEQ ID NO: 4), FST291HBM, FST303HBM or FST315HBM (SEQ ID NO: 3), and the mutants comprise the triple mutation K76A, K81A and K82A.

[0041] In particular, the fusion protein according to the present invention comprises the following follistatin moiety: a) comprising or consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4; b) comprising or consisting of residues 289-314 of any one of SEQ ID NOs: 1-4; or c) comprising or consisting of an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to any one of SEQ ID NOs: 1-4.

[0042] Without wishing to be bound by any theory, the inventors hypothesize that the antibody moiety in the fusion protein of the present invention can bind to, for example, free HSA of a subject, thereby extending the half-life of the fusion protein, so that the fusion protein of the present invention exhibits greater stability and / or efficacy in vivo than wild-type follistatin.

[0043] Thus, in the context of the present invention, overall, the antibody moiety preferably binds to albumin, preferably serum albumin (SA), such as mouse, rat, cynomolgus monkey or human SA. More preferably, the antibody moiety binds to human HSA. The antibody moiety can be a chimeric, humanized or human antibody moiety. Preferably, the antibody moiety of the fusion protein of the present invention is an antigen-binding fragment of an antibody (or, as referred to herein, an antigen-binding portion). Preferably, such an antigen-binding portion is selected from Fab, Fab' or F(ab')2. Alternatively, the antibody moiety 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.

[0044] In one embodiment, the antibody moiety of the fusion protein of the present invention comprises a light chain variable region comprising CDR-L1 comprising SEQ ID NO: 13, CDR-L2 comprising SEQ ID NO: 14 and CDR-L3 comprising SEQ ID NO: 15, and a heavy chain variable region comprising CDR-H1 comprising SEQ ID NO: 16, CDR-H2 comprising SEQ ID NO: 17 and / or CDR-H3 comprising SEQ ID NO: 18.

[0045] In an alternative embodiment, the antibody portion of the fusion protein of the present invention comprises a heavy chain variable region comprising or consisting of a sequence having at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO: 5, and a light chain variable region comprising or consisting of a sequence having at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO: 6.

[0046] SEQ ID NO: 5 and SEQ ID NO: 6 represent the heavy and light variable chains of an anti-albumin antibody named "CA645" (disclosed in International Publication No. WO 2013 / 068571). As confirmed in the examples below, the inventors have surprisingly found that the FST-Fab fusion protein comprising the heavy and light variable chains of SEQ ID NO: 5 and SEQ ID NO: 6, respectively, is easier to manufacture and purify.

[0047] In the context of the present invention, generally, the fusion protein optionally comprises a linker between the follistatin portion and the antibody portion. When present, non-limiting examples of the linker can be selected from the group consisting of SGGGGS (SEQ ID NO: 7), SGGGGSSGGGGS (SEQ ID NO: 19), GGGGS (SEQ ID NO: 20) and GGGGSGGGGS (SEQ ID NO: 21).

[0048] When making a fusion protein, it will be understood that there are two options for fusing any parts to each other, namely C-terminal fusion or N-terminal fusion. As shown in the following examples, the inventors have surprisingly found that by fusing an antibody part to the C-terminus of the follistatin part, compared to any other type of fusion such as an antibody part fused to the N-terminal part of the follistatin part, the expression of the resulting fusion protein is further improved. In particular, the C-terminal fusion protein of the present invention (i.e., an antibody part fused to the C-terminus of follistatin, either directly or via a linker) exhibits excellent expression and a higher yield of monomeric protein compared to known Fc-based follistatin fusion proteins. However, although the C-terminal fusion protein resulted in the highest expression level, an antibody part fused to the N-terminus of follistatin would have been conceivable by those skilled in the art because it results in an expression level that is about 1.5 times higher compared to, for example, an Fc-based follistatin fusion protein.

[0049] Similarly, when making a fusion protein between one polypeptide (here the FST part) and an antibody part (here preferably Fab, Fab’ or F(ab’)2), there are two options for fusing any parts to each other, and it will be understood that the polypeptide (here FST) is fused to either the heavy chain or the light chain of the antibody part.

[0050] Thus, in a preferred embodiment of the fusion protein of the present invention, the antibody part is attached to the C-terminal part of the follistatin part. When a linker is present, the antibody part is preferably linked (or attached) to the C-terminal part of the follistatin part via the linker (in other words, the antibody part is linked (or attached) to the C-terminal part of the follistatin part and there is a linker between the two parts). In one example, the fusion protein comprises, from the N-terminus to the C-terminus, a follistatin part, a linker linked to the C-terminus of the follistatin part, and then the heavy chain of the antibody part linked to the free end of the linker (typically the C-terminus of the linker).

[0051] In some specific examples (not limiting), the fusion protein of the present invention is (a) i. An 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, which is bound 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. An 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, which is bound 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. An 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, which is bound 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. An 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, attached to the C-terminus of the FST288HBM 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. An 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, attached 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, attached to the free end of the linker; and iv. A Fab light chain defined by SEQ ID NO: 6, or a sequence having at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity thereto; (f) i. An FST288 polypeptide defined by SEQ ID NO: 2, or a sequence having at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity thereto; ii. A linker defined by SEQ ID NO:7, SEQ ID NO:19, SEQ ID NO:20 or SEQ ID NO:21 that is attached 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, attached to the free end of the linker; and iv. A Fab light chain defined by SEQ ID NO:6 or a sequence having at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity thereto; (g)i. An FST315 polypeptide variant defined by SEQ ID NO:3 (FST315HBM) or SEQ ID NO:22, or a sequence having at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity thereto; ii. A linker defined by SEQ ID NO:7, SEQ ID NO:19, SEQ ID NO:20 or SEQ ID NO:21 that is attached 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, attached to the free end of the linker; and iv. A Fab light chain defined by SEQ ID NO:6 or a sequence having at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity thereto; (h)i. An FST288 polypeptide variant defined by SEQ ID NO:4 (FST288HBM) or SEQ ID NO:25, or a sequence having at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity thereto; ii. A linker defined by SEQ ID NO:7, SEQ ID NO:19, SEQ ID NO:20 or SEQ ID NO:21 that is attached to the C-terminus of the FST288 polypeptide variant; iii. A Fab heavy chain defined by SEQ ID NO: 5 or a sequence having at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity thereto, attached to the free end of the linker; and iv. A Fab light chain defined by SEQ ID NO: 6 or a sequence having at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity thereto; (i) A Fab light chain defined by SEQ ID NO: 8, 9, 10, 11, 24, 25, 26, 27, 32, 33, 34 or 35 or a sequence having at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity thereto and 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; (j) A Fab light chain defined by SEQ ID NO: 8, 9, 10, 11, 24, 25, 26, 27, 32, 33, 34 or 35 and SEQ ID NO: 6; or (k) A functional variant or fragment of any one of (a) to (i) comprising or consisting of.

[0052] Alternatively, if one prefers to use an N-terminal fusion, the present invention provides: i. a follistatin moiety defined by any one of SEQ ID NOs: 1-4, 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, attached to the N-terminus of the FST315 polypeptide; 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 moiety and the Fab heavy chain. In some specific examples, the fusion protein may be defined by 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, together with SEQ ID NOs: 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.

[0053] The fusion proteins according to the present invention have been shown to have an expression level that is at least 1.5-fold greater, at least 2-fold greater, at least 3-fold greater, at least 4-fold greater compared to the expression level of the wild-type FST or FST-Fc fusion protein. They have also been shown to result in a total monomer yield that is at least 1.5-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, or at least 6-fold greater than the total monomer yield of the FST-Fc fusion monomeric protein.

[0054] When making such comparisons, it is preferable to compare like with like. For example, the FST315-Fab fusion protein should be compared with the FST315-Fc fusion protein and / or the FST288-Fab fusion protein should be compared with the FST288-Fc fusion protein.

[0055] Furthermore, as highlighted in the examples, they were present in the serum for a longer period compared to wild-type FST (for the fusion proteins according to the invention, more than 6 days compared to 1 day of wild-type FST).

[0056] In a further aspect, the invention provides an isolated polynucleotide encoding the fusion protein according to the invention, or a functional variant or fragment thereof. The isolated polynucleotide according to the invention can comprise, for example, synthetic DNA, cDNA, genomic DNA generated by chemical treatment or any combination thereof.

[0057] Accordingly, there is provided herein an isolated polynucleotide encoding the fusion protein according to the invention, the fusion protein comprising a follistatin (FST) portion, an antibody portion, and optionally a linker between the follistatin portion and the antibody portion. Those skilled in the art will understand that the polynucleotide sequence further comprises a nucleic acid sequence encoding an N-terminal secretion signal sequence. The sequence is selected in particular according to the host cell in which the fusion protein is to be expressed.

[0058] Using standard techniques of molecular biology, a DNA sequence encoding the fusion protein according to the invention can be prepared. 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 used as required.

[0059] It will be understood that at least two isolated polynucleotides are required to encode the fusion protein according to the present invention. In fact, at least one isolated polynucleotide encodes an FST portion, an antibody portion fused to the FST portion, and any linker therebetween, and another isolated polynucleotide encodes the remaining antibody portion to complete the one fused to the FST portion. As a non-limiting example, one polynucleotide encodes the FST portion, linker and heavy chain of an anti-HSA-Fab portion, and one polynucleotide encodes the light chain of an anti-HSA-Fab portion.

[0060] In some specific examples (not limiting), the isolated polynucleotide is (a) i. An 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, attached 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. An 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, attached 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. An 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, attached 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. An 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, attached to the C-terminus of the FST288HBM 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. An 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 attached 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, attached to the free end of the linker; and iv. A Fab light chain defined by SEQ ID NO: 6 or a sequence having at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity thereto; (f)i. An 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, attached 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, attached to the free end of the linker; and iv. A Fab light chain defined by SEQ ID NO: 6 or a sequence having at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity thereto; (g)i. An FST315 polypeptide variant defined by SEQ ID NO: 3 (FST315HBM) or SEQ ID NO: 22, or a sequence having at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity thereto; ii. A linker defined by SEQ ID NO: 7, SEQ ID NO: 19, SEQ ID NO: 20 or SEQ ID NO: 21, attached 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, attached to the free end of the linker; and iv. A Fab light chain defined by SEQ ID NO: 6, or a sequence having at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity thereto; (h) i. An FST288 polypeptide variant defined by SEQ ID NO: 4 (FST288HBM) or SEQ ID NO: 25, or a sequence having at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity thereto; ii. A linker defined by SEQ ID NO: 7, SEQ ID NO: 19, SEQ ID NO: 20 or SEQ ID NO: 21 attached to the C-terminus of the FST288 polypeptide variant; iii. A Fab heavy chain defined by SEQ ID NO: 5, or a sequence having at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity thereto, attached to the free end of the linker; and iv. A Fab light chain defined by SEQ ID NO: 6, or a sequence having at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity thereto; (i) A Fab light chain defined by SEQ ID NO: 8, 9, 10, 11, 24, 25, 26, 27, 32, 33, 34 or 35, or a sequence having at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity thereto and 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; (j) A Fab light chain defined by SEQ ID NO: 8, 9, 10, 11, 24, 25, 26, 27, 32, 33, 34 or 35 and SEQ ID NO: 6; or (k) A functional variant or fragment of any one of (a) to (i) encoding the fusion protein of the present invention comprising or consisting of the same.

[0061] Alternatively, if one prefers to use an N-terminal fusion, the present invention provides a polynucleotide sequence encoding a fusion protein comprising, i. a follistatin moiety defined by any one of SEQ ID NOs: 1-4, 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, which is bound 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 moiety and the Fab heavy chain, or a fusion protein consisting of these. In some embodiments, 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, together with a Fab light chain defined by SEQ ID NO: 6, or a sequence having at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity thereto.

[0062] In some (non-limiting) specific examples, the isolated polynucleotide comprises or consists of (i) SEQ ID NO: 36, 37, 38, 39, 57 or 58 encoding a follistatin moiety, 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 moiety, 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 moiety, and (iv) if a linker is present, SEQ ID NO: 42, 54, 55 or 56 encoding the linker.

[0063] In further (non-limiting) specific examples, the isolated polynucleotide comprises or consists of (i) SEQ ID NO: 36, 37, 38, 39, 57 or 58 encoding a follistatin moiety, 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 moiety, 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 moiety, 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 the linker.

[0064] In yet other (non-limiting) specific 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, which encodes an FST moiety and a Fab heavy chain moiety fused to any 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 (ii) SEQ ID NO: 41, which encodes a Fab light chain.

[0065] In related aspects, the invention provides a cloning vector or an expression vector that wholly contains a polynucleotide encoding a fusion protein according to the invention. Those skilled in the art will understand this as a choice between a bicistronic vector (containing two expression cassettes, one encoding an FST moiety, an antibody moiety fused to the FST moiety, and any linker therebetween, and the other encoding the remaining antibody moiety) or two different vectors (one encoding an FST moiety, an antibody moiety fused to the FST moiety, and any linker therebetween, and the other encoding the remaining antibody moiety).

[0066] General methods for constructing vectors, transfection methods, and culture methods are well known to those skilled in the art. In this regard, reference may be made, for example, to "Current Protocols in Molecular Biology", 1999, F.M. Ausubel (ed), Wiley Interscience, New York and the Maniatis Manual produced by Cold Spring Harbor Publishing.

[0067] In related aspects, the invention provides a host cell that contains a polynucleotide sequence encoding a fusion protein of the invention, or a cloning vector or an expression vector that contains one or more polynucleotides encoding a fusion protein of the invention.

[0068] Any suitable host cell / vector system may be used for the expression of the polynucleotide sequence encoding the fusion protein of the present invention. Bacteria, such as Escherichia coli (E. coli), and other microbial systems may be used, or eukaryotes, such as mammals, host cell expression systems may be used. Suitable mammalian host cells include CHO cells, myeloma cells or hybridoma cells. In one embodiment, the host cell is (e.g., transformed below) (1) a vector containing two expression cassettes (one encoding the FST portion, the antibody portion fused to the FST portion and any linker therebetween, and the other encoding the remaining antibody portion), or (2) a first vector containing a nucleic acid encoding an amino acid sequence comprising or consisting of the FST portion, the antibody portion fused to the FST portion and any linker therebetween, and a second vector containing a nucleic acid encoding an amino acid sequence comprising or consisting of the remaining antibody portion.

[0069] Host cells suitable for cloning or expressing vectors encoding fusion proteins include the prokaryotic or eukaryotic cells described herein. For example, antibodies can be produced in bacteria, particularly when glycosylation and Fc effector functions are not required. For the expression of antibody fragments and polypeptides in bacteria, see Charlton, Methods in Molecular Biology, Vol. 248 (edited by B.K.C. Lo, Humana Press, Totowa, NJ, 2003, pp. 245-254, describing expression of antibody fragments in E. coli.). In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeast may also be suitable cloning or expression hosts for vectors encoding fusion proteins, including "humanized" fungal and yeast strains that result in the production of antibodies having a partial or complete human glycosylation pattern (see Gerngross et al., 2004; Li et al., 2006). Alternatively, suitable types of mammalian cells such as Chinese hamster ovary (CHO cells) can be used in the present invention, including CHO-S, CHO-K1 cells, dhfr-CHO cells such as CHO-DG44 cells and CHO-DXB11 cells that can be used with a DHFR selection marker, or further CHO-K1 cells or CHOK1-SV cells that can be used with a glutamine synthetase selection marker. Other cell types used for antibody expression include lymphocyte cell lines such as NSO myeloma cells and SP2 cells, and COS cells. Host cells can be stably transformed or transfected with the isolated polynucleotide sequences or expression vectors according to the present invention.

[0070] In related aspects, the present invention provides a method for producing a fusion protein, which includes culturing the host cell of the present invention under appropriate conditions for producing the fusion protein of the present invention. The method according to the present invention can further include a step of recovering a cell culture fluid (CCF) containing the fusion protein (recovery step), in other words, a step of recovering the fusion protein. Following the recovery, the fusion protein can be purified using, for example, protein A chromatography and other chromatography / filtration steps. Optionally, the method further includes formulating the purified fusion protein into a formulation with a protein concentration, such as a concentration of 10 mg / ml or more, for example 50 mg / ml or more. Without limitation, the formulation can be a liquid formulation, a lyophilized formulation, or a spray-dried formulation. Standard methods can be used for all of these steps.

[0071] In a further aspect, the present invention provides a method for purifying the fusion protein according to the present invention, i. loading a clarified cell culture fluid containing the fusion protein according to the present invention onto a protein A chromatography column pre-equilibrated with a buffer to bind the fusion protein to the column; ii. washing the chromatography column with the same washing buffer as the equilibration buffer in step i to remove impurities; iii. eluting the fusion protein bound to the column with an elution buffer under alkaline conditions; iv. further eluting the remaining bound fusion protein with an acidic elution buffer; v. neutralizing the eluates from steps iii and iv to obtain a neutralized sample; vi. subjecting the neutralized sample to additional purification steps to obtain a purified fusion protein and includes.

[0072] In a non-limiting example, the equilibration / washing buffer for steps i. and ii. is a sodium acetate buffer having a concentration of from about 30 to, or up to about 70 mM, such as about 30, 35, 40, 45, 50, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65 or 70 mM, and a pH of about 5.5 to about 6.5, such as 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 for step iii. is a glycine-based buffer, such as a glycine / NaOH buffer, having a concentration of from about 30 to, or up 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 about 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0. In yet another non-limiting example, the acidic elution buffer for step iv. is a citrate buffer having a concentration of from about 50 to, or up 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 a further non-limiting example, the neutralization in step v is carried out at a pH of 7.0 to 9.0, such as 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. The neutralization is typically carried out using 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 remained strongly bound to Protein A under alkaline conditions. This feature provides several potential advantages with respect to downstream processes. First, by not using acidic elution conditions, co-elution of free Fab can be avoided because the Fab remains strongly bound to Protein A. Furthermore, the FST-Fab is not exposed to harsh acidic pH for an extended period of time.Finally, the alkaline elution is compatible with subsequent chromatography steps, which means a reduction in sample handling and thus allows for a potential increase in yield and recovery.

[0073] (Non-limiting) As a specific example, provided herein is a method for producing a fusion protein comprising a follistatin moiety (e.g., FST315, FST315HBM, FST288 or FST288HBM) linked at the N-terminus (VH-CH1) and C-terminus of the heavy chain of the Fab via any SGGGGS linker. The FST-Fab heavy chain is co-expressed with the Fab light chain (LC), and the heavy and light chains are linked by intermolecular disulfide bonds.

[0074] In yet a further aspect, the invention provides a pharmaceutical composition comprising the entire fusion protein according to the invention and one or more pharmaceutically acceptable carriers, excipients or diluents. The pharmaceutical composition is typically prepared by mixing the active ingredient (the fusion protein according to the invention herein) having the desired purity with one or more optional pharmaceutically acceptable carriers in the form of a dry formulation or an aqueous solution.

[0075] Any suitable pharmaceutically acceptable carrier, diluent and / or excipient can be used in the preparation of the pharmaceutical composition (see, e.g., Remington: The Science and Practice of Pharmacy, Alfonso R. Gennaro (Editor) Mack Publishing Company, April 1997). The pharmaceutical composition is typically sterile and stable under the conditions of manufacture 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), microemulsion, liposome, or other ordered structure suitable for accommodating high product concentrations (e.g., microparticles or nanoparticles). The carrier can include, but is not limited to, buffers, antioxidants, preservatives, hydrophilic polymers, amino acids, monosaccharides, disaccharides, and other carbohydrates, chelating agents, salt-forming counterions, and / or nonionic surfactants.

[0076] Preferably, the pharmaceutical composition is formulated as a solution, more preferably as a buffer solution optionally. 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 merely illustrative and do not limit pharmaceutical compositions suitable for other administration routes. The pharmaceutical compositions described herein may be packaged in single unit dosage forms or multiple dosage forms.

[0077] The fusion protein or pharmaceutical composition of the present invention can be administered via one or more administration routes using one or more of various methods known in the art. As will be understood by those skilled in the art, the administration route and / or mode of administration will vary depending on the desired result. Examples of administration routes of the fusion protein or pharmaceutical composition of the present invention include, for example, intravenous, intramuscular, intradermal, intraocular, intraperitoneal, subcutaneous, spinal or other parenteral administration routes by injection or infusion. Alternatively, the fusion protein or pharmaceutical composition of the present invention can be administered via an oral route such as topical, epidermal or mucosal administration routes. If the product is for injection or infusion, it can take the form of a suspension, solution or emulsion in an oily or aqueous vehicle and can contain additional agents such as suspending agents, preservatives, stabilizers and / or dispersing agents. Alternatively, the fusion protein or pharmaceutical formulation according to the present invention may be provided in dry form for reconstitution with a suitable sterile liquid before use. A solid form suitable for dissolution or suspension in a liquid vehicle prior to injection may be prepared.

[0078] Once formulated, the fusion protein or pharmaceutical formulation of the present invention can be administered directly to a subject.

[0079] In another aspect, provided herein is a fusion protein or pharmaceutical composition according to the present invention for use in therapy. Alternatively, provided herein is a method of treating a subject in need thereof, the method comprising administering a therapeutically effective amount of a fusion protein or pharmaceutical composition of the present invention. In a further alternative, the present invention provides the use of a fusion protein or pharmaceutical composition according to the present invention in the manufacture of a medicament for use in therapy.

[0080] The therapeutically effective amount varies depending on the protein or its active fragment, the disease and its severity, and the age, weight, etc. of the subject to be treated.

[0081] In the context of the present invention, generally, a "subject" refers to a mammal. Mammals include, but are not limited to, domestic animals (e.g., cows, 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.

[0082] It should be noted that the above embodiments are illustrative rather than limiting of the present invention, and those skilled in the art can design many alternative embodiments without departing from the scope of the present invention, and particularly the scope of the claims.

[0083]

Table 1-1

Table 1-2

Table 1-3

Table 1-4

Table 1-5

Table 1-6

Example

[0084] Materials and Methods Production of follistatin-Fab fusion protein Cloning strategy: A DNA segment corresponding to the fusion between follistatin and the heavy or light chain sequence of an anti-albumin antibody (referred to as 645 Fab), with or without a linker sequence in between, was generated by PCR or gene synthesis and cloned using our mammalian expression vector. The heavy and light chain sequences of 645 Fab were also cloned separately using our mammalian expression vector. All expression vectors were confirmed by direct sequencing using primers covering the entire open reading frame.

[0085] Culture of CHO cells: A suspension of CHOS-XE cells (Cain et al., 2013) was pre-adapted to CD CHO medium (Invitrogen) supplemented with 2 mM Glutamax. The cells were maintained in logarithmic growth phase while being stirred at 120 RPM on a shaking incubator (Kuhner AG) and cultured at 37 °C in an atmosphere containing 8% CO2.

[0086] Protein expression: The follistatin-Fab protein was overexpressed by transient transfection of the CHO-XE cell line. Pairs of expression plasmids were co-transfected (e.g., N-fab light chain-FST-C with the heavy chain or FST-C fab heavy chain-C with the light chain). Immediately prior to transfection with DNA, the CHO cells were exchanged to Expi CHO Expression Medium (Gibco) by briefly centrifuging the cells at 1500×g and resuspending the pellet. The cells were then transfected using ExpiFectamine (Gibco) according to the manufacturer's instructions. The cultures were grown while shaking at 190 RPM in an atmosphere containing 8% CO2 at 37°C for the first 24 hours and then at 32°C for the remainder of the expression cycle. The supernatant was typically harvested 9 - 14 days after transfection by centrifugation at 4000×g followed by filtration using a 0.22 μm membrane. The final protein expression level was determined by protein G-HPLC and SDS PAGE.

[0087] Protein purification: The transiently expressed protein content was captured using a Mab Select column (GE Healthcare) performed under standard conditions. Briefly, the resin was washed with 10 column volumes of phosphate buffered saline (PBS, pH 7.4) and the bound protein was eluted with 5 column volumes of 0.1 M sodium citrate pH 3.1 (except where otherwise mentioned in the examples below). The eluate was neutralized with TRIS-HCl pH 8.5 and filter sterilized by 0.22 μm membrane-exclusion chromatography (HiLoad 26 / 60 Superdex 75 column, GE Healthcare) run under standard conditions (where the column was pre-packed with PBS pH 7.4 as the running buffer). The quality of the sample was evaluated using absorbance at 280 nm, BEH2000 analytical UPLC and SDS PAGE (under reducing and non-reducing conditions).

[0088] For protein G purification, the clarified supernatant was loaded onto a Protein G HP (GE Healthcare) column equilibrated with PBS pH 7.4, followed by washing 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 absorbance at 280 nm.

[0089] Size exclusion chromatography (SEC): For analysis by size exclusion chromatography (SEC), the sample was injected onto a BEH200, 200 Å, 1.7 μm, 4.6 mm ID × 300 mm column (Aquity), developed with an isocratic gradient of 0.2 M phosphate pH 7 at 0.35 mL / min, and detected by absorbance at 280 nm and a multi-channel fluorescence (FLR) detector (Waters).

[0090] SDS-PAGE: For analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the sample was prepared by adding 4× Novex NuPAGE LDS sample buffer (Life Technologies) and 100 mM N-ethylmaleimide (Sigma-Aldrich) to the purified protein and heating at 100 °C for 3 min. The sample was 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 min with Tris-glycine SDS running buffer (Life Technologies). Novex Mark12 broad range protein standard (Life Technologies) was used as the standard. The gel was stained with Coomassie Quick Stain (Generon) and destained with distilled water.

[0091] Pharmacokinetic measurement: FST protein was administered to C57BL / 6 mice via intravenous injection at 10 mg / kg (2 mL / kg intravenously, 5 mL / kg subcutaneously). Blood samples were collected daily for 7 days. Serum samples were prepared and analyzed using a ligand binding assay to detect follistatin (including FST288 and FST315). Pharmacokinetic parameters were calculated based on individual data using Phoenix v8.3.

[0092] Functional activity reporter gene cell assay: HEK-Blue™ TGFβ reporter cells (Invivogen) were used to measure the efficacy of follistatin protein in blocking the stimulation-induced activation (activin A / B, GDF8 / 11) of the SMAD2 / 3 signaling pathway. Briefly, follistatin proteins and their appropriate controls were pre-diluted in culture medium based on concentration and predicted activity such that the resulting inhibition curves had complete tops and bottoms. This was followed by a 10-point serial dilution in 1:3 with culture medium before aliquoting four replicates of each dilution at 20 μL / well in a 384-cell culture assay plate. 10 μl of each stimulus was added to wells representing the serial dilution of follistatin as well as wells without follistatin representing the highest response to the stimulus. Wells with a matching volume of medium were designated as untreated. After incubating the assay plate at 37 °C for 1 hour, HEK-Blue™ TGFβ cells (10,000 cells / 20 μL) were added per well and the assay plate was incubated at 37 °C for an additional 17 hours. 45 μL / well of QUANTI-Blue™ solution was added to a new 384 assay plate designated as the destination plate, and an automated liquid handler was used to transfer 5 μL of cell supernatant from the assay plate to the destination plate. The destination plate was held on a shaker briefly and then incubated at 37 °C for 1 hour. Absorbance values from each well were measured at 630 nm with a plate reader, and the follistatin-mediated dose-dependent inhibition rate of SMAD2 / 3 activity was calculated together with the Z-factor.

[0093] Biacore Binding Data Surface plasmon resonance using a Biacore T200 (Cytiva) was used to determine the binding kinetics of the fusion protein to various targets (see below). For each type of assay, kinetic parameters were determined using a 1:1 binding model with Biacore T200 evaluation software (version 3.0).

[0094] To evaluate binding to activin A and activin B (R&D Systems), goat anti-human F(ab’)2 fragment-specific antibody (Jackson ImmunoResearch) was first immobilized onto the CM5 sensor chip by amine coupling chemistry to a level of approximately 5000 RU. Using a standard multi-cycle kinetic approach, each analysis cycle consisted of capture of FST-Fab onto the anti-F(ab’)2 surface, followed by injection of the analyte, and finally surface regeneration using a 60-second injection of 50 mM HCl and 5 mM NaOH. Analytes were injected using a three-fold serial dilution of HBS-EP+ running buffer (Cytiva) at concentrations of 30, 10, 3.3, 1.1, 0.367, and 0.122 nM (at a flow rate of 30 μl / min for 300 seconds at 25 °C), followed by monitoring dissociation for 900 seconds. Binding responses of parallel blank surfaces were subtracted, and buffer blank injections were included to subtract instrument noise and drift.

[0095] For the evaluation of binding to GDF8 and GDF11 (R&D Systems), each of GDF8 and GDF11 was immobilized onto the surface of the CM5 sensor chip by amine coupling chemistry to achieve an immobilization level of approximately 250 RU. For both GDF8 and GDF11, analysis was performed using a single-cycle kinetic approach where FST-Fab was injected continuously for 180 seconds at increasing concentrations (0.8, 4, 20, 100, and 500 nM) in HBS-EP+ running buffer (Cytiva) at a flow rate of 30 μl / min at 25 °C, followed by monitoring dissociation for 1800 seconds. Binding responses of parallel blank surfaces were subtracted, and a series of buffer blank injections were performed to subtract instrument noise and drift.

[0096] For the evaluation of the binding to albumin, goat anti-human F(ab’)2 fragment-specific antibody (Jackson ImmunoResearch) was first immobilized on the CM5 sensor chip at a level of approximately 5000 RU via an amine coupling chemical reaction. Using a standard multi-cycle kinetic approach, each analysis cycle consisted of the capture of the fusion protein of interest onto the anti-F(ab’)2 surface, followed by the injection of albumin, and finally surface regeneration using a 60-second injection of 50 mM HCl and 5 mM NaOH. Analytes were injected using a two-fold serial dilution of HBS-EP+ running buffer (Cytiva) at concentrations of 100, 50, 25, 12.5, 6.3, and 3.1 nM (for 300 seconds at 25 °C at a flow rate of 30 μl / min), and then dissociation was monitored for 900 seconds. The binding response of a parallel blank surface was subtracted, and buffer blank injections were included to subtract the noise and drift of the instrument.

[0097] Example 1. FST-Fab- results in excellent expression levels and monomer yields compared to other fusion partners. Comparing the relative expression levels of the follistatin moiety fused to various fusion partners in different orientations reveals that the FST-Fab construct fused to the C-terminus of the FST moiety is optimal (Figure 2A). Fusing Fc or ScFv to the C-terminus of FST is significantly inferior to using Fab at this position, with both resulting in a 3.5-fold decrease in the expression product (Figure 2A). Fusing the Fab moiety at the N-terminus of the FST moiety results in a ~45% decrease in the amount of the expression product, but is better than the fusion protein containing FST fused to the Fc domain. An evaluation comparing the effects on the expression of using FST288 or FST315 fused to the Fab at the C-terminus of follistatin using the wild-type HBM sequence revealed only minor differences (Figure 2B). The FST315 version had 6% lower expression than the FST288 fusion. Comparison of the fusions containing HBSM (HBM) revealed a modest 7% decrease in the expression level compared to the wild type.

[0098] A pairwise investigation of various FST fusions revealed that the Fab portion fused to the C-terminus of the FST portion yielded the highest level of final monomer yield compared to other fusions (Figure 2C). The FST-Fc fusion yielded the lowest monomer yield in the set, approximately 6-fold lower than the FST-Fab fusion. FST-ScFv and FST fused to the N-terminus of Fab were approximately 3-fold and 4-fold lower, respectively.

[0099] Example 2 - FST315(HBM)-Fab shows extended pharmacokinetic properties compared to FST315WT in mouse studies. FST315WT, FST288WT, and FST288-Fab administered intravenously (IV) to mice at 10 mg / kg were eliminated very rapidly, with mean residence times (MRT) of 1.6 hours, 2.3 hours, and 5.2 hours, respectively. In comparison, FST315-Fab, FST315(HBM)-Fab, and FST288HBM-Fab administered IV to mice at 10 mg / kg showed extended kinetics, with MRTs of 9.3 hours, 13.1 hours, and 11 hours, respectively (Figures 3A and B). All pharmacokinetic parameters are summarized in Table 1.

[0100] Conclusion: Example 2 shows that thanks to the fusion between the FST portion and the Fab portion, it was possible to significantly extend the kinetics and half-life of FST-containing proteins. A significant contribution to the extended kinetics is also contributed by the mutation of the heparin-binding site in the form of the HBM version of the follistatin portion.

[0101] Example 3 - FST315-Fab, FST315HBM-Fab, FST288-Fab, and FST288HBM-Fab show high-affinity binding to their ligands and albumin. 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 was confirmed using surface plasmon resonance (SPR) binding assays and was found to be within the expected range as summarized in Table 2 (considering the literature, Sidis et al., 2006). The Kd binding affinity values for their specific ligands obtained for all follistatin fusion proteins are in good agreement with the literature values reported for the binding of wild-type non-binding FST288WT and FST315WT to activin A at 23.6 pM and 28.7 pM, respectively (Sidis et al., 2006). The human albumin-binding properties of the Fab domain component of FST315(HBM)-Fab were also confirmed by SPR, and the value of 2602 pM was consistent with the prediction for an active albumin binder. The Kd value for FST315(HBM)-Fab albumin binding is also in close agreement with the previously published value for Fab alone human albumin binding (this is cited as 2 - 5 nM in Adams et al., 2016, referring to different formats of 645gL4gH5 Fab).

[0102] Conclusion: Example 2 emphasizes that the expected biological activity of the FST-Fab portion was maintained, i.e., the fusion between the two portions did not affect either the binding activity of the follistatin portion to its respective biological ligands or the binding activity of the Fab portion to albumin.

[0103] Example 4 - FST 315(HBM)-Fab and FST288(HBM)-Fab show functional inhibition of their ligands in reporter gene cell assays Next, the ability of FST315(HBM)-Fab to inhibit the functional signaling of four ligands was tested using a Smad2 / 3 reporter gene cell assay performed with the HEK-Blue™-TGFβ commercially available cell line. To induce stimulation of reporter gene activation, all four ligands were used at approximately their EC50 concentrations, and then FST315(HBM)-Fab and FST315WT were titrated over a wide concentration range to generate the dose-response curves represented in Figure 4A. The geometric mean IC50 data for all four ligands are summarized in Table 3. The FST315(HBM)-Fab format was observed to be consistently 3-fold more potent than the parental FST315WT when inducing a response with the ligands activin A and activin B, and 2-fold more potent when using the ligands GDF8 and GDF11. Similarly, FST288(HBM)-Fab and FST288WT were titrated over a wide concentration range to generate dose-response curves for all four ligands used at their approximate EC50 concentrations, representative data are shown in Figure 4B, and the geometric mean IC50 data for all four ligands are summarized in Table 3. Different from the data of the FST315 format, the FST288(HBM)-Fab molecular format showed very similar efficacy to the FST288WT parental molecule across all four ligands.

[0104] Conclusion: This example not only shows that FST315(HBM)-Fab was not inferior to the FST315WT protein in its ability to inhibit ligand-induced signaling via the Smad2 / 3 reporter pathway, but also that FST315(HBM)-Fab demonstrated improved potency compared to FST315WT, highlighting its relevance in a therapeutic context. This is in contrast to the FST288(HBM)-Fab efficacy, which was very comparable to the FST288WT molecule.

[0105] Example 5 - FST-Fab fusion with human VH3 domain enables recovery by protein A chromatography The FST315 HBM-Fab fusion (FST-Fab1) was prepared, where FST is fused to an anti-albumin F(ab’) containing a human VH3 domain (fusion protein of SEQ ID NO: 8) enabling protein A chromatography.

[0106] Two alternative FST-F(ab’) constructs (FST-Fab2 and FST-Fab3) lacking the human VH3 domain were prepared. All constructs were expressed and purified according to the method described above. As shown in Table 4, only FST-Fab1 containing the human VH3 domain was recovered after protein A chromatography. All three fusion proteins could be recovered after protein G chromatography.

[0107] Typically, elution of proteins bound to protein A affinity capture resin is performed under acidic conditions (see the section on materials and methods above). However, it was the inventors' finding that the FST-Fab1 of the present invention elutes efficiently from protein A under mild alkaline conditions, while the Fab fraction remains strongly bound.

[0108] The FST-Fab 1 clarified supernatant was loaded onto a MabSelect (GE Healthcare) column equilibrated with 50 mM sodium acetate pH 5.8, followed by washing with the same buffer. The bound material was eluted under acidic (0.1 M glycine-HCl pH 2.6) or alkaline (50 mM glycine-NaOH pH 8.6) conditions. This was then followed by a further acidic strip (0.1 M citrate pH 2.0). The acidic eluate and strip pool were neutralized with 2 M Tris / HCl pH 8.5. These elution and strip samples were then analyzed by SDS-PAGE and analytical size exclusion.

[0109] The results are shown in Figure 5. Analysis by SDS-PAGE shows the presence of a band of approximately 50 kDa upon elution under acidic conditions (lane 2), indicating that the Fab produced during expression elutes under these conditions. Lane 3, which includes the next strip, is protein-free as it has been completely removed by acidic elution. Alternatively, under slightly alkaline conditions (lane 4), no Fab band is present. The Fab does not elute from the column until the acidic strip (lane 5), and the acidic strip has a band of approximately 50 kDa as seen with acidic elution.

[0110] As shown in Figure 6, analysis by size exclusion chromatography shows that only the acidic elution pool (panel A) has a peak corresponding to the Fab ("F") that is not present in the alkaline elution pool (panel B). Since all proteins have already been eluted by acidic elution, there is little / no protein in the strip following acidic elution (panel C), while the strip following alkaline elution (panel D) contains a peak corresponding to the Fab.

[0111] The efficient elution of the FST-Fab fusion protein of the present invention from Protein A under slightly alkaline conditions is an inherent property of this molecule. It presents several potential advantages with respect to downstream processes. First, by not using acidic elution conditions, co-elution of the Fab can be avoided as the Fab remains strongly bound to Protein A. Furthermore, the Fst-Fab is not exposed to harsh acidic pH for extended periods. Finally, alkaline elution is compatible with subsequent chromatography steps, which means a reduction in sample handling and thus a potential increase in yield and recovery.

[0112] Example 6 - Production of Stable Cell Lines All previous examples were carried out using transiently expressed FST fusion proteins (as per the materials and methods of the above section), and this example focuses on the acquisition of stable cell lines for the production of FST fusion proteins according to the present invention.

[0113] Transfection of host cell line: CHO DG44 (dhfr-) host cells were transfected with a DNA dual gene vector plasmid for stable expression of human FST315 (HBM)-645 Fab molecule (i.e., encoding SEQ ID NO: 8 and SEQ ID NO: 6) and dihydrofolate reductase (DHFR), a selection marker. The vector was linearized before electroporation. The cells were electroporated and then allowed to recover in a static, temperature- and CO2-controlled incubator in host cell growth medium for 24 hours before culturing in selection medium.

[0114] A total of 167 minipools were recovered and cultured in selection medium containing methotrexate. Based on antibody titers, 70 minipools were evaluated in shake flask cultures. Based on 10-day batch mAb titers, the top 24 minipools were selected for evaluation in an AMBR automated microscale bioreactor.

[0115] The best 7 minipools (MPs) were selected for single cell cloning. Cells from each MP were centrifuged and the pellet was resuspended in PBS. Then, each MP cell suspension was analyzed individually by flow cytometry. After single cell cloning, the top 54 clones based on the highest antibody titers were expanded into shake flasks for batch evaluation.

[0116] To evaluate the growth, antibody titer, specific productivity and product quality of the clone cells, 12 high-expressing clones were selected in an AMBR microscale bioreactor system using a chemically defined medium without animal-derived components and evaluated in several fed-batch processes.

[0117] The lead clone was selected and the production process in a Wave Bioreactor and 5 L shake flasks was carried out using the previously determined best fed-batch process.

[0118] Shows the total product concentrations achieved in fed-batch processes from two different media and four different clones (see Figure 7). This example shows that aspects to be considered when possibly wanting to improve the yield of manufacturing FST fusion proteins according to the present invention are the set of media (basic medium and feed medium / media) used in their manufacture. As shown in Figure 7, clone 156 was generally produced at a small scale of about 1 g / L, but the use of basic 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, clone 51 was produced at about 1.2 - 1.3 g / L in the presence of basic medium A (regardless of the feed medium / media), but the production decreased to less than 1 g / L in basic medium B.

[0119]

Table 2

[0120]

Table 3

[0121]

Table 4

[0122]

Table 5

[0123] References JPEG2025520393000011.jpg94138

Claims

1. a. Follistatin portion, b. Antibody portion, and optionally c. Linker between the follistatin portion and the antibody portion A fusion protein containing or consisting of these elements.

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

3. The follistatin portion, a. Sequence ID 1 b. Sequence ID 2 c. Sequence ID 3 d. Sequence ID 4 e. Any protein containing amino acid residues including residues 289-314 of any one of sequence numbers 1 to 4, or f. Sequences having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with any one of sequence numbers 1 to 4. A fusion protein according to claim 1 or 2, comprising or consisting of the following.

4. The fusion protein according to claim 1 or 2, wherein the antibody portion binds to albumin.

5. The fusion protein according to claim 1 or 2, wherein the antibody portion is a chimeric, humanized, or human antibody portion.

6. The antibody portion, a) an antigen-binding moiety selected from Fab, Fab', or F(ab')2, b) An antigen-binding moiety further comprising a human VH3 domain selected from Fab, Fab', or F(ab')2 that can bind to protein A. The fusion protein according to claim 1 or 2.

7. The antibody portion, a. A light chain variable region comprising at least one CDR selected from CDR-L1 containing SEQ ID NO: 13; CDR-L2 containing SEQ ID NO: 14 and / or CDR-L3 containing SEQ ID NO: 15; and a heavy chain variable region comprising at least one CDR selected from CDR-H1 containing SEQ ID NO: 16; CDR-H2 containing SEQ ID NO: 17 and / or CDR-H3 containing SEQ ID NO: 18; or b. A heavy chain variable region containing, or comprising, a sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 5, and a light chain variable region containing, or comprising, a sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO:

6. A fusion protein according to claim 1 or 2, comprising or consisting of the following.

8. The fusion protein according to claim 1 or 2, wherein the fusion protein includes a linker between the follistatin portion and the antibody portion, and the linker is selected from the group consisting of SGGGGS (SEQ ID NO: 7), SGGGGSSGGGGS (SEQ ID NO: 19), GGGGS (SEQ ID NO: 20), and GGGGGSGGGGGS (SEQ ID NO: 21).

9. a) The antibody portion is ligated 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 by the linker. The fusion protein according to claim 1 or 2.

10. The aforementioned fusion protein (a) i. FST315 polypeptide defined by sequence number 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 sequence number 5, or a sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto, attached to the C-terminus of the FST315 polypeptide; and iii. Fab light chains defined by sequence number 6, or sequences having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto; (b) i. FST288 polypeptide defined by sequence number 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 sequence number 5, or a sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto, attached to the C-terminus of the FST288 polypeptide; and iii. Fab light chains defined by sequence number 6, or sequences having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto; (c) i. FST315HBM polypeptide defined by sequence number 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 sequence number 5, or a sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto, attached to the C-terminus of the FST315HBM polypeptide; and iii. Fab light chains defined by sequence number 6, or sequences having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto; (d) i. FST288HBM polypeptide defined by sequence number 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 sequence number 5, or a sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto, attached to the C-terminus of the FST288HBM polypeptide; and iii. Fab light chains defined by sequence number 6, or sequences having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto; (e) i. FST315 polypeptide defined by sequence number 1, or a sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto; ii. A linker defined by SEQ ID NO: 7, SEQ ID NO: 19, SEQ ID NO: 20, or SEQ ID NO: 21, attached to the C-terminus of the FST315 polypeptide; iii. A Fab heavy chain defined by sequence number 5, or a sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto, bonded to the free end of the linker; and iv. Fab light chains defined by sequence number 6, or sequences having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto; (f) i. FST288 polypeptide defined by sequence number 2, or a sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto; ii. A linker defined by SEQ ID NO: 7, SEQ ID NO: 19, SEQ ID NO: 20, or SEQ ID NO: 21, attached to the C-terminus of the FST288 polypeptide; iii. A Fab heavy chain defined by sequence number 5, or a sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto, bonded to the free end of the linker; and iv. Fab light chains defined by sequence number 6, or sequences having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto; (g) i. FST315 polypeptide variants defined by sequence number 3 (FST315HBM) or sequence number 22, or sequences having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto; ii. A linker defined by SEQ ID NO: 7, SEQ ID NO: 19, SEQ ID NO: 20, or SEQ ID NO: 21, attached to the C-terminus of the FST315 polypeptide variant; iii. A Fab heavy chain defined by sequence number 5, or a sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto, bonded to the free end of the linker; and iv. Fab light chains defined by sequence number 6, or sequences having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto; (h) i. FST288 polypeptide variants defined by sequence number 4 (FST288HBM) or sequence number 25, or sequences having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto; ii. A linker defined by SEQ ID NO: 7, SEQ ID NO: 19, SEQ ID NO: 20, or SEQ ID NO: 21, attached to the C-terminus of the FST288 polypeptide variant; iii. A Fab heavy chain defined by sequence number 5, or a sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto, bonded to the free end of the linker; and iv. Fab light chains defined by sequence number 6, or sequences having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto; (i) Fab light chains defined by sequences having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with sequence number 8, 9, 10, 11, 24, 25, 26, 27, 32, 33, 34, or 35, or sequences having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with sequence number 6; (j) Fab light chain as defined by SEQ ID NOs: 8, 9, 10, 11, 24, 25, 26, 27, 32, 33, 34 or 35 and SEQ ID NO: 6; or (k) Any one of the functional variants or fragments from (a) through (j) A fusion protein according to claim 1 or 2, comprising or consisting of the following.

11. An isolated polynucleotide encoding the fusion protein according to claim 1 or 2.

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

13. a. One or more polynucleotides according to claim 11 or b. One or more expression vectors according to claim 12 Host cells, including those containing the host cell.

14. A method for producing the fusion protein described in claim 1 or 2, comprising culturing the host cell described in claim 13 under suitable conditions for producing the fusion protein, and isolating the fusion protein.

15. A pharmaceutical composition comprising a fusion protein according to claim 1 or 2 and one or more pharmaceutically acceptable carriers, excipients, or diluents.

16. A pharmaceutical composition according to claim 15 for use in treatment.

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