Recombinant FST polypeptide, fusion protein comprising same, pharmaceutical composition, and use of fusion protein and pharmaceutical composition

Abstract

Provided are a recombinant follistatin polypeptide and a fusion protein comprising same. By mutating the heparin-binding domain of the FST protein, the affinity of the protein for heparan polysaccarides is reduced, which helps prolong the half-life of the FST protein. Compared with a wild-type FST-315 protein, the FST-Fc fusion protein, by means of mutation of the FST protein and fusion expression with an Fc domain, exhibits reduced affinity for heparin and increased affinity for activin, thereby improving the stability and half-life of the fusion protein and helping reduce the frequency of administration. The FST-Fc fusion protein can have broad use prospects in aspects such as fibrosis and muscle loss.

Description

A recombinant FST polypeptide, a fusion protein comprising the same, a pharmaceutical composition and its uses Technical Field

[0001] This application relates to the fields of bioengineering and biotechnology, and in particular to a recombinant FST polypeptide, an FST-Fc fusion protein, a method for preparing the same, a polynucleotide encoding the same, a pharmaceutical composition comprising the same, and uses thereof. Background Technology

[0002] Follicle-stimulating hormone (FSH) (FST) was discovered in 1987 by two scientists, Naoto Ueno and D.M. Robertson, who extracted it almost simultaneously from the follicular fluid of pigs and cows, respectively, in an attempt to find a protein that inhibits follicle-stimulating hormone. FST is a secretory glycoprotein rich in cysteine. Due to its strong affinity for FSH, activin (ACT), and members of the TGF-β superfamily, FST exerts a high level of regulatory effect on these proteins. It can bind to activin in a nearly irreversible manner, preventing activin from binding to its receptor. FST is structurally highly conserved, with 95% homology in mammals.

[0003] Mature FST proteins have three isoforms: FST-288, FST-303, and FST-315. These three isoforms have similar affinity for activin, but their binding affinity to cell surfaces is FST-288 > FST-303 > FST-315. Besides regulating FSH secretion, FST also has multiple functions, including promoting follicle maturation and spermatogenesis, promoting skeletal muscle development and differentiation, promoting adipose tissue browning, promoting brown adipose tissue differentiation, regulating hepatic homeostasis, promoting wound healing, and inhibiting inflammatory responses.

[0004] Follicle-stimulating hormone (FSH) has a very high affinity for activin, K. D The estimated values ​​range from 50 to 900 pM. Activins play a crucial role in promoting inflammation and fibrosis development; therefore, blocking the biological function of activins holds promise as a novel strategy for preventing and halting fibrosis progression. Several papers have already elucidated the role and mechanism of FST in preventing pulmonary fibrosis, liver fibrosis, and kidney fibrosis.

[0005] The complexity of FST proteins, due to their molecular form and glycosylation, increases the difficulty of research and development, which is one reason why they have not been commercialized decades after their discovery. Several companies are conducting or have conducted related research; for example, Eli Lilly has developed a variant of FST-315, and Paranta Biosciences' FST inhaler for treating cystic fibrosis is currently in Phase I clinical trials. There remains a demand for developing clinically effective FST products. Summary of the Invention

[0006] To address the issues of the short half-life and limited clinical application of wild-type FST-315 protein, this application mutates the FST-315 protein and fuses it with the Fc domain, thereby providing an FST-Fc fusion protein that can be used to treat fibrotic diseases and / or promote muscle growth in subjects in need. This disclosure reduces the affinity of the heparin-binding domain of FST for cell surface heparin polysaccharides by mutation, thereby reducing the likelihood of internalization and degradation. Furthermore, the fusion with the Fc domain results in a fusion protein with increased affinity for activin, a longer half-life, reduced dosing frequency, and favorable pharmacokinetic properties.

[0007] In one aspect, this application relates to a recombinant FST polypeptide, wherein, compared with the amino acid sequence of the wild-type FST polypeptide shown in SEQ ID NO:1, the amino acid sequence of the recombinant FST polypeptide contains at least the following multiple substitution mutations at the following positions: K76, K81, K82 and K84.

[0008] In one aspect, this application relates to a recombinant FST polypeptide, wherein, compared with the amino acid sequence of the wild-type FST polypeptide shown in SEQ ID NO:1, the amino acid sequence of the recombinant FST polypeptide contains at least the following multi-point mutations: K76V, K81E, K82Q and K84E.

[0009] In one aspect, this application relates to an FST-Fc fusion protein comprising: a recombinant FST polypeptide and an IgG Fc domain, wherein, compared with the amino acid sequence of the wild-type FST polypeptide shown in SEQ ID NO:1, the amino acid sequence of the recombinant FST polypeptide contains at least the following multi-point mutations: K76V, K81E, K82Q, and K84E.

[0010] In one aspect, this application relates to a polynucleotide encoding the aforementioned FST-Fc fusion protein.

[0011] In one aspect, this application relates to a recombinant vector comprising the aforementioned polynucleotides.

[0012] In one aspect, this application relates to a recombinant cell containing the aforementioned polynucleotide or recombinant vector.

[0013] In one aspect, this application relates to a pharmaceutical composition comprising the above-described fusion protein and optional pharmaceutically acceptable excipients.

[0014] In one aspect, this application relates to the use of the aforementioned fusion protein, polynucleotide, recombinant vector, recombinant cell, or pharmaceutical composition in the preparation of a medicament for the prevention or treatment of FST-related diseases.

[0015] In one aspect, this application relates to a method for preparing the above-described fusion protein, comprising:

[0016] (1) The above-mentioned polynucleotides or recombinant vectors are transferred into host cells to obtain recombinant cells;

[0017] (2) After culturing the recombinant cells, the obtained cell culture is subjected to separation and purification of the fusion protein to obtain the fusion protein.

[0018] The beneficial effects of this disclosure are:

[0019] This disclosure involves mutating a specific site of basic amino acids in the HBS region of wild-type FST-315 and fusing the resulting FST-315 mutant with the IgG Fc domain (especially the hIgG4 Fc domain). This process reduces the affinity of FST-315 for heparin while retaining its good antagonistic ability against proteins such as activin, resulting in a longer half-life. The protein disclosed herein can be used to treat muscle loss (including sarcopenia, muscular dystrophy, metabolic myopathy, and muscle atrophy) and fibrotic diseases (including pulmonary fibrosis, liver fibrosis, kidney fibrosis, cardiac fibrosis, pancreatic fibrosis, skin fibrosis, peritoneal fibrosis, myelofibrosis, and nervous system fibrosis). Attached Figure Description

[0020] Figure 1 is a schematic diagram of the amino acid substitutions in the mutant FST315-m protein compared to the wild-type FST-315 protein.

[0021] Figure 2 is a graph showing the affinity of each FST-Fc fusion protein (FST315-WT-Fc, FST315-HBM-Fc and FST315-m-Fc) to activin A (ACT A) (top subgraph) and heparin (bottom subgraph) in the examples.

[0022] Figure 3 shows the in vitro cell activity assay results of each FST-Fc fusion protein (FST315-WT-Fc, FST315-HBM-Fc and FST315-m-Fc) tested in the examples.

[0023] Figure 4 shows the total number of cells in the BALF of mice in each group (***p<0.001 compared with G1 group).

[0024] Figure 5 shows the protein content in BALF of mice in each group (*p<0.05 compared with G1 group).

[0025] Figure 6 shows the HE staining scores of lung tissue in each group of mice (** p < 0.01 compared with G1 group, # p < 0.05 compared with G2 group).

[0026] Figure 7 shows the HE staining results of representative mouse lung tissue.

[0027] Figure 8 shows a bar chart of representative mouse lung tissue Masson staining scores, where “FST:1mg / kg” and “FST:10mg / kg” represent the low-dose FST315-m-Fc group and the high-dose FST315-m-Fc group, respectively.

[0028] Figure 9 shows the results of Masson staining of representative mouse lung tissue. Detailed Implementation

[0029] The present application will be further described below with reference to specific embodiments. Those skilled in the art will understand that these embodiments are merely exemplary and do not limit the scope of protection of the present application. Those skilled in the art can make various modifications, alterations, substitutions, or combinations to the solutions of the present application without departing from the spirit and scope of the present application, and the solutions obtained thereby also fall within the scope of protection of the present application.

[0030] Unless otherwise defined, the technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. See, for example, Singleton et al., Dictionary of Microbiology and Molecular Biology 2nd ed., J. Wiley & Sons (New York, NY 1994); Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Springs Harbor Press (Cold Springs Harbor, NY 1989).

[0031] In this article, unless otherwise stated, the term "vector" refers to a self-replicating DNA molecule used to transfer a target gene (e.g., a foreign gene from the same or different species) into a recipient cell.

[0032] In this article, unless otherwise stated, the terms “polynucleotide,” “nucleic acid,” “nucleic acid molecule,” “nucleotide sequence,” and “gene” are used interchangeably and are essentially a chemical substance.

[0033] In this article, unless otherwise stated, the term "treatment" means curing, reducing, alleviating, slowing down, mitigating or improving a disease or disease-related symptoms in a statistically significant manner, or preventing, delaying, stopping, suspending or halting the onset or further development of a disease or disease-related symptoms.

[0034] The term "pharmaceutical acceptable" refers to compounds, materials, compositions, and / or dosage forms that, within the bounds of reliable medical judgment, are suitable for use in contact with human and animal tissues without excessive toxicity, irritation, allergic reactions, or other problems or complications, in proportion to a reasonable benefit / risk ratio.

[0035] The term "subject" encompasses any vertebrate, such as mammals and non-mammals, including humans, non-human primates (e.g., chimpanzees, rhesus monkeys, cynomolgus monkeys, etc.), sheep, dogs, cats, horses, cattle, chickens, pigs, rats, etc., with humans being preferred.

[0036] In this document, unless otherwise stated, the terms “comprise,” “comprises,” and “comprising” or their equivalents are open-ended expressions, meaning that they may cover other unspecified elements, components, and steps in addition to those listed.

[0037] Unless the context clearly indicates otherwise, singular terms encompass the plural referents, and vice versa. Similarly, unless the context clearly indicates otherwise, the word "or" is intended to include "and".

[0038] In this paper, the percentage of identity (degree of homology) between sequences can be determined by comparing more than two sequences, for example, using freely available computer programs (such as BLASTp or BLASTn with default settings) commonly used for this purpose on the World Wide Web. Recombinant FST-315 polypeptide (FST-315 mutant protein)

[0039] The wild-type FST-315 protein (its amino acid sequence is shown in SEQ ID NO:1) comprises: an N-terminal domain (aa 1-63), FST-like domain 1 (FSD1, aa 64-123), FST-like domain 2 (FSD2, aa 124-188), FST-like domain 3 (FSD3, aa 189-260), and a hydrophobic tail region (aa 261-315). Each FSD domain of FST-315 contains multiple conserved cysteine ​​residues, forming disulfide bonds to maintain the protein's three-dimensional structure and ensure biological activity. The three FSD domains work synergistically to bind with high affinity to ligands such as Activin and Myostatin, blocking their interaction with receptors and inhibiting their signal transduction. Based on the binding site of FST315 to heparin polysaccharide, it is known that the FST315 protein has a heparin-binding domain (HBS) rich in basic amino acids, located in the K75-K84 region. That is, this heparin-binding domain is located in FSD1, which enables the protein molecule to attach to the cell surface for rapid clearance.

[0040] The inventors used AI prediction to perform site-directed basic amino acid mutations in the HBS region of FST-315, obtaining the mutant FST315-m. They observed that specific mutations of basic amino acids at specific sites reduced the affinity of FST-315 for heparin polysaccharides, thereby improving the pharmacokinetic properties of FST-315 and giving it systemic effects. Fusion expression with the human IgG4 Fc domain (hIgG4 Fc) further prolonged the protein's half-life and minimized the ADCC and CDC effects of Fc. To achieve this, the inventors designed the FST315-m mutant by performing multiple site-directed mutations at K76, K81, K82, and K84 in the wild-type FST-315 molecule, thereby increasing the protein's half-life and systemic exposure.

[0041] In some embodiments, this application relates to a recombinant FST polypeptide, wherein, compared with the amino acid sequence of the wild-type FST polypeptide shown in SEQ ID NO:1, the amino acid sequence of the recombinant FST polypeptide contains at least the following multi-point mutations: K76V, K81E, K82Q, and K84E.

[0042] In some embodiments, the amino acid sequence of the recombinant FST polypeptide has only the following multiple mutations compared to the amino acid sequence of the wild-type FST polypeptide shown in SEQ ID NO:1: K76V, K81E, K82Q and K84E.

[0043] In some embodiments, the recombinant FST polypeptide comprises the amino acid sequence shown in SEQ ID NO:2.

[0044] In some embodiments, the recombinant FST polypeptide has the amino acid sequence shown in SEQ ID NO:2. Alternatively, in some embodiments, the amino acid sequence of the recombinant FST polypeptide is shown in SEQ ID NO:2.

[0045] Fusion protein

[0046] In some embodiments, this application relates to an FST-Fc fusion protein comprising: a recombinant FST polypeptide and an IgG Fc domain, wherein, compared with the amino acid sequence of the wild-type FST polypeptide shown in SEQ ID NO:1, the amino acid sequence of the recombinant FST polypeptide contains at least the following multi-point mutations: K76V, K81E, K82Q, and K84E.

[0047] In some embodiments, the amino acid sequence of the recombinant FST polypeptide has only the following multiple mutations compared to the amino acid sequence of the wild-type FST polypeptide shown in SEQ ID NO:1: K76V, K81E, K82Q and K84E.

[0048] In some embodiments, the recombinant FST polypeptide comprises the amino acid sequence shown in SEQ ID NO:2.

[0049] In some embodiments, the recombinant FST polypeptide has the amino acid sequence shown in SEQ ID NO:2. Alternatively, in some embodiments, the amino acid sequence of the recombinant FST polypeptide is shown in SEQ ID NO:2.

[0050] In some embodiments, the FST-Fc fusion protein comprises the aforementioned recombinant FST peptide and IgG Fc domain from the N-terminus to the C-terminus.

[0051] In some embodiments, the IgG Fc domain is a human IgG Fc domain or a mutant thereof, including a human IgG1 Fc domain, a human IgG2 Fc domain, a human IgG3 Fc domain, a human IgG4 Fc domain, or a mutant thereof.

[0052] In some embodiments, the IgG Fc domain is a human IgG4 Fc domain (whose amino acid sequence is SEQ ID NO:6) or a mutant thereof. In some embodiments, the human IgG4 Fc domain mutant contains at least the S228P mutation, which can further prolong the half-life of FST-315 and reduce immune-related functions.

[0053] In some embodiments, the human IgG4 Fc domain mutant comprises any one of the mutations selected from the following (according to EU designations):

[0054] (1) S228P;

[0055] (2) S228P, F234A and L235A;

[0056] (3) S228P, K196Q, F296Y, E356K, R409K, H435R and L445P;

[0057] (4) S228P and L235E; or

[0058] (5) S228P, F234A, L235A, D265A and R409K.

[0059] In some embodiments, the IgG Fc domain is a mutant of the human IgG4 Fc domain whose amino acid sequence is shown in SEQ ID NO:7.

[0060] In some embodiments, the FST-Fc fusion protein may or may not have a linker. For example, the recombinant FST peptide and the IgG Fc domain are coupled via (G4S) n Or (G5) n The linker connects the proteins, where n is an integer from 1 to 3. In some preferred embodiments, the FST-Fc fusion protein does not have a linker to further reduce potential immunogenicity and other problems associated with linkers.

[0061] In some embodiments, the FST-Fc fusion protein comprises the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with it, or a conserved modified variant thereof. In some embodiments, amino acids differing from the amino acid sequence shown in SEQ ID NO:3, having at least 95%, 96%, 97%, 98%, or 99% sequence identity, are located in the IgG Fc domain.

[0062] The conserved modified variants of the FST-Fc fusion protein described in this article retain properties such as antagonism against proteins like activin and reduced affinity for heparin.

[0063] In this paper, conservative modification refers to amino acid modification that does not significantly affect or change the antibody binding properties. For example, conservative amino acid substitution can be the substitution with another amino acid of the same class (with similar chemical properties or functions). As an example, amino acids can be classified according to the side chain properties as follows: (1) Nonpolar amino acids: Ala (A), Val (V), Leu (L), Ile (I), Pro (P), Phe (F), Trp (W), Met (M); (2) Uncharged polar amino acids: Gly (G), Ser (S), Thr (T), Cys (C), Tyr (Y), Asn (N), Gln (Q); (3) Acidic amino acids: Asp (D), Glu (E); (4) Basic amino acids: Lys (K), Arg (R), His (H). Alternatively, amino acids can be classified based on their common side chain characteristics as follows: (1) hydrophobic amino acids: Met, Ala, Val, Leu, Ile; (2) neutral hydrophilic amino acids: Cys, Ser, Thr, Asn, Gln; (3) acidic amino acids: Asp, Glu; (4) basic amino acids: His, Lys, Arg; (5) amino acids that affect chain orientation: Gly, Pro; (6) aromatic amino acids: Trp, Tyr, Phe.

[0064] The FST-Fc fusion protein described in this application is prepared by fusing recombinant FST-315 protein with Fc to form a dimer. It exhibits good in vitro affinity for activin, reduced affinity for heparin, and good in vivo activity. By expressing the FST-Fc fusion protein with good activity, it can be used for fibrosis treatment and / or to promote muscle growth, etc.

[0065] Polynucleotides, recombinant vectors and recombinant cells

[0066] In some embodiments, this application relates to a polynucleotide encoding the aforementioned FST-Fc fusion protein.

[0067] It is known to those skilled in the art that, due to codon degeneracy, the amino acid sequences of fusion proteins and their components can be encoded by a variety of nucleic acid sequences, not limited to those disclosed herein as examples. In this document, the polynucleotides can be synthesized based on the amino acid sequence of the corresponding fusion protein using conventional whole-genome synthesis methods or enzymatic synthesis techniques. In some embodiments, the polynucleotides may be codon-optimized.

[0068] In some embodiments, this application relates to a recombinant vector comprising the aforementioned polynucleotides.

[0069] In this document, any suitable expression vector known in the art can be used to construct the recombinant vector with the polynucleotide encoding the fusion protein described above. The expression vector may be, for example, pcDNA series vectors (pcDNA3.1 vector, pcDNA3.2 vector, pcDNA3.3 vector, pcDNA3.4 vector (e.g., pcDNA3.4-TOPO TA vector)), pBK-CMV vector, pEGFP-N1 vector, pGenHT 1.0-DGV vector, etc., but is not limited thereto.

[0070] In some embodiments, this application relates to a recombinant cell containing the aforementioned polynucleotide or recombinant vector.

[0071] In some embodiments, the cells used to express the fusion protein are prokaryotic or eukaryotic cells. In some embodiments, the cells are preferably eukaryotic cells, including yeasts (e.g., Saccharomyces cerevisiae, Pichia pastoris, Candida albicans, etc.), mammalian cells (e.g., CHO cells, 293 cells, etc.), or any other eukaryotic cells suitable for preparing the fusion protein.

[0072] The aforementioned polynucleotide or recombinant vector can be transferred into host cells using any suitable known technique in the art for transferring exogenous genes into host cells (e.g., electroporation, transduction, transfection, etc.), thereby enabling the resulting recombinant cells to express the fusion protein described herein with high efficiency.

[0073] Pharmaceutical Composition

[0074] In some embodiments, this application relates to a pharmaceutical composition comprising the above-described FST-Fc fusion protein and optional pharmaceutically acceptable excipients.

[0075] In this document, the pharmaceutical composition comprises a therapeutically effective amount of the fusion protein. The term "therapeutically effective amount" can be determined by a clinician based on the subject's health status, age, sex, weight, family medical history, etc.

[0076] In some embodiments, the pharmaceutically acceptable excipients may be selected from, for example, but not limited to, solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, flow aids, flavoring agents, preservatives, suspending agents, antioxidants, penetration enhancers, pH adjusters, surfactants, diluents, etc. For other pharmaceutically acceptable excipients, see, for example, *Handbook of Pharmaceutical Excipients* (4th Edition), by RC Luo et al., translated by Zheng Junmin, Chemical Industry Press, 2005.

[0077] In this document, the pharmaceutical composition may contain a therapeutically effective amount of the fusion protein. The term "therapeutically effective amount" may be determined by a clinician based on the subject's health status, age, sex, weight, family medical history, etc.

[0078] Pharmaceutical uses and preparation methods

[0079] In some embodiments, this application relates to the use of the above-described FST-Fc fusion protein, polynucleotide, recombinant vector, recombinant cell, or pharmaceutical composition in the preparation of a medicament for the prevention or treatment of FST-related diseases.

[0080] In some embodiments, this application relates to the aforementioned FST-Fc fusion protein, polynucleotide, recombinant vector, recombinant cell, or pharmaceutical composition for the prevention or treatment of FST-related diseases. Alternatively, this application relates to a method for preventing or treating FST-related diseases in a subject in need, comprising administering (e.g., a therapeutically effective amount) of the aforementioned FST-Fc fusion protein, polynucleotide, recombinant vector, recombinant cell, or pharmaceutical composition to the subject. Alternatively, this application relates to the use of the aforementioned FST-Fc fusion protein, polynucleotide, recombinant vector, recombinant cell, or pharmaceutical composition for the prevention or treatment of FST-related diseases.

[0081] In some implementations, the FST-related disease may be selected from fibrotic diseases, muscle loss, or related symptoms.

[0082] In some embodiments, the fibrotic disease or its related symptoms are selected from pulmonary fibrosis, liver fibrosis, kidney fibrosis, cardiac fibrosis, pancreatic fibrosis, skin fibrosis, peritoneal fibrosis, bone marrow fibrosis, and nervous system fibrosis or their related symptoms.

[0083] In some embodiments, the symptoms associated with muscle loss are selected from sarcopenia, muscular dystrophy, metabolic myopathy, and muscle atrophy.

[0084] In some embodiments, the fusion protein, polynucleotide, recombinant vector, recombinant cell, pharmaceutical composition, or drug of this application can be formulated into any dosage form suitable for administration to a subject. For example, the fusion protein can be administered intravenously, intramuscularly, parenterally, orally, subcutaneously, intrathecally, intraventricularly, epidurally, intraperitoneally, intrauterinely, or intranasally. In some preferred embodiments, the fusion protein, polynucleotide, recombinant vector, recombinant cell, pharmaceutical composition, or drug is in the form of a lyophilized powder or an injectable solution.

[0085] In some embodiments, this application relates to a method for preparing the above-described FST-Fc fusion protein, comprising:

[0086] (1) The above-mentioned polynucleotides or recombinant vectors are transferred into host cells to obtain recombinant cells;

[0087] (2) After culturing the recombinant cells, the obtained cell culture is subjected to separation and purification of the fusion protein to obtain the fusion protein.

[0088] For example, after codon optimization, the fusion protein is ligated into a eukaryotic expression vector. CHO cells are used as host cells. After the expression vector is transferred into the host cells, the resulting recombinant cells are cultured in suspension. The protein is then isolated and purified from the resulting cell culture.

[0089] For purposes of description and disclosure, all patents, patent applications, and other identified publications are expressly incorporated herein by reference. These publications are provided solely because their publication predates the filing date of this application. All statements regarding the dates of these documents or representations of their contents are based on information available to the applicant and do not constitute any acknowledgment of the accuracy of the dates or contents of these documents. Furthermore, in any country, any reference to these publications herein does not constitute an endorsement that such publication is part of the general knowledge in the art.

[0090] Example

[0091] The present disclosure is described in more detail below with the aid of embodiments; however, those skilled in the art will understand that the scope of protection of the present disclosure is not limited thereto. Unless otherwise specified, the reagents, materials, or instruments involved in the following embodiments are commercially available or can be prepared by those skilled in the art based on conventional knowledge.

[0092] Example 1: Screening of FST mutants and expression of fusion protein

[0093] Referring to the three-dimensional structure and amino acid sequence of the human wild-type FST-315 protein (shown as SEQ ID NO:1), the heparin-binding domain of K75-K84 was mutated using AI-zyme software from Nanjing Genscript Biotech Co., Ltd., disrupting the binding of basic amino acids to heparin. At the same time, artificially transplanted sequences were added to construct multiple FST-315 protein mutants: FST315-m (mutation details are shown in Figure 1), FST315-m1 to FST315-m15 (their amino acid sequences are shown as SEQ ID NO:2, SEQ ID NO:8-SEQ ID NO:22, respectively).

[0094] The sequences of the above mutants were fused with the sequence of the hIgG4 Fc domain with the S228P mutation (shown as SEQ ID NO:7) to obtain fusion proteins of each mutant and Fc (i.e., FST315-m-Fc, FST315-m1-Fc to FST315-m15-Fc), and the corresponding nucleotide sequences were synthesized by Nanjing Genscript Biotech Co., Ltd.

[0095] The expression vectors for each fusion protein were constructed using conventional homologous recombination methods. The operation method is described in the instruction manual of the ClonExpress II One Step Cloning Kit (Cat.C112-01) from Nanjing Novizan Biotechnology Co., Ltd. The synthesized nucleotide sequences were then ligated with the pcDNA3.4 vector to obtain recombinant expression vectors. After linearization, the recombinant expression vectors were purified by gel extraction using a gel extraction kit.

[0096] The recombinant expression vector was transiently transfected into CHO cells using Lipofectamine transfection reagent for the expression and purification of the fusion protein: CHO-K1 cells in suspension were used as host cells, encapsulated in liposomes, and the recombinant protein was transiently expressed. The cell density was adjusted to 1-2 × 10⁶ cells / year using Opti-MEM medium. 6 Cells / mL were collected to obtain a cell suspension. 50 μg of DNA (i.e., the linearized recombinant expression vector) was mixed with 100 μL of diluted Lipofectamine™ 3000 (Lipofectamine™ 3000 was diluted at a ratio of 125 μL opti-MEM: 7.5 μL Lipofectamine™ 3000) to obtain a 150 μL transfection mixture. The transfection mixture was incubated for 15 min, and then added to 50 mL of the cell suspension and incubated at 37 °C.

[0097] The supernatant was collected by centrifugation at 500g, and rapid comparison was performed using SDS-PAGE gel electrophoresis and Western blotting to select mutant fusion proteins with a high proportion of the target band, few impurity bands, and no fragmentation. Cells corresponding to mutants FST315-m, FST315-m7, FST315-m9, and FST315-m13 were then selected for subsequent amplification expression of the fusion proteins under the aforementioned culture conditions.

[0098] The obtained cell fermentation broth was centrifuged at 500g, and the collected supernatant was filtered through a 0.22μm filter. The filtrate was then collected. The filtrate was subjected to affinity chromatography on a Protein A column under the following conditions to obtain the fusion protein: The filtrate was loaded onto a Protein A column pre-equilibrated with 20mM PB and 150mM NaCl (pH 7.2), retained for 5 min, then equilibrated for 5 column volumes, and eluted with 3 column volumes of 50mM NaAc-Hac solution (pH 3.5) to obtain the fusion protein. The yields of each fusion protein are shown in Table 1 below. The eluent of the obtained fusion protein was replaced with PBS solution and then filtered through a 0.22μm filter for sterilization before use.

[0099] Table 1 Expression yield of each fusion protein

[0100] Based on the results of SDS-PAGE gel electrophoresis and Western blotting, since FST315-m-Fc had the highest expression yield and relatively few impurity bands during electrophoresis, the fusion protein FST315-m-Fc of the FST315-m mutant (its amino acid sequence is shown in SEQ ID NO:3) was selected for subsequent experiments.

[0101] Following the method described above, the wild-type FST-315 fusion protein FST315-WT-Fc and the control fusion protein FST315-HBM-Fc were constructed using the sequence of wild-type FST-315 (FST315-WT) and the hIgG4 Fc domain with the S228P mutation, or the control protein FST315-HBM (from patent application WO2023242271A1, which has undergone partial amino acid mutation in the heparin binding domain) and the sequence of wild-type hIgG4 Fc domain (shown as SEQ ID NO:6). The amino acid sequences of these two fusion proteins are shown as SEQ ID NO:4 and SEQ ID NO:5, respectively.

[0102] Example 2: In vitro affinity activity assay of FST-Fc fusion protein (activin A)

[0103] Detection was performed using OctectRED384 (Sartorius, FB-20493) in conjunction with the AHC2 biosensor (Sartorius, 18-5142). The detection was conducted at 30°C and a rotation speed of 1000 rpm. FST315-WT-Fc, FST315-HBM-Fc, and FST315-m-Fc were first immobilized on the AHC2 biosensor. Subsequently, activator A (purchased from ACRO, catalog number: ACA-H424x) was used as the analyte (hereinafter referred to as "ACT A") for binding and dissociation steps. Detection conditions are shown in Table 2. Baseline was equilibrated at 30°C using PBS buffer containing 0.03% Tween-20 (PBS-T). Immerse the AHC2 biosensor in the PBS-T buffer for 10 minutes to pre-wet the sensor (60 seconds), equilibrate to baseline I for 60 seconds, load each fusion protein to be tested for 180 seconds, equilibrate to baseline II for 60 seconds, bind activin A for 200 seconds, and dissociate for 300 seconds.

[0104] Table 2 Detection conditions (Activator A)

[0105] As shown in the upper subplot of Figure 2, the fusion protein FST315-m-Fc exhibits a stronger affinity for activin A, the analyte, than FST315-HBM-Fc and FST315-WT-Fc, indicating that the fusion protein of this disclosure has a stronger antagonistic effect on activin A. Specific results are shown in Table 3.

[0106] Table 3. Results of affinity test for fusion protein to activin A.

[0107] Example 3: In vitro affinity activity assay of FST-Fc fusion protein (heparin)

[0108] The detection was performed using a Biacore 8K (Cytiva, 29327020) in conjunction with a protein A biosensor (Cytiva, 29127555): At 25°C, using HBS-EP+ buffer, each fusion protein to be tested (FST315-WT-Fc, FST315-HBM-Fc, FST315-m-Fc) was first captured by the chip, then passed through heparin. The affinity of each fusion protein for heparin was detected. Specific experimental conditions are shown in Table 4, and the specific steps are as follows:

[0109] Preparation of flow buffer: Dilute 10×HBS-EP+ buffer with water to prepare flow buffer (1×HBS-EP+ buffer);

[0110] Preparation of regeneration buffer (10mM glycine buffer): Weigh an appropriate amount of glycine and dissolve it in ultrapure water, adjust the pH to 1.5, and obtain the regeneration buffer;

[0111] The experiment was conducted at 25°C, using 1×HBS-EP+ buffer as the flow buffer, and each fusion protein to be tested was added to the Series SProtein A sensor chip for capture.

[0112] Heparin was serially diluted with PBS buffer and injected onto the surfaces of flow cells 1 and 2 for binding phase determination, followed by injection of flow buffer for dissociation phase determination.

[0113] All data were processed using Biacore Evaluation Software. Blank injections of flow cell 1 and flow buffer were used as a double reference in each cycle to subtract resonance units (RUs).

[0114] Table 4. Detection conditions (heparin)

[0115] As shown in the lower subplot of Figure 2, FST315-m-Fc has a significantly weaker affinity for heparin, the analyte, than FST315-HBM-Fc and FST315-WT-Fc. Specific results are shown in Table 5.

[0116] Table 5. Results of Heparin Affinity Test

[0117] Example 4: In vitro affinity activity assay of FST-Fc fusion protein (activin / heparin)

[0118] To further verify the performance advantages of the fusion protein variant (FST-m-Fc) disclosed in this invention, a head-to-head comparison test was conducted between the FST-m-Fc of this invention and the best variant FS315K(76,81,82)E-hFcLALA (hereinafter referred to as "K(76,81,82)E") disclosed in the prior art (CN110914294A), while wild-type FST-WT-Fc was introduced as a control.

[0119] The three proteins described above were constructed and expressed. Their binding kinetics with activin A and heparin were determined using different biophysical methods. The in vitro affinity activity assays were the same as in Examples 2 and 3.

[0120] The affinity of activin A was detected using an OctectRED384 (Sartorius, FB-20493) in conjunction with an AHC2 biosensor (Sartorius, 18-5142). The assay was performed at 30°C and a rotation speed of 1000 rpm. FST315-WT-Fc, K(76,81,82)E, and FST315-m-Fc were first immobilized on the AHC2 biosensor. Activin A (purchased from ACRO, catalog number: ACA-H424x) was then used as the analyte for binding and dissociation steps. Baseline equilibration was performed at 30°C using PBS buffer containing 0.03% Tween-20 (PBS-T). Immerse the AHC2 biosensor in the PBS-T buffer for 10 minutes to pre-wet the sensor (60 seconds), equilibrate to baseline I for 60 seconds, load each fusion protein to be tested for 180 seconds, equilibrate to baseline II for 60 seconds, bind activin A for 200 seconds, and dissociate for 300 seconds.

[0121] The detection was performed using a Biacore 8K (Cytiva, 29327020) in conjunction with a protein A biosensor (Cytiva, 29127555): At 25°C, using HBS-EP+ buffer, each fusion protein to be tested (FST315-WT-Fc, K(76,81,82)E, FST315-m-Fc) was first captured by the chip, and then passed through heparin. The affinity of each fusion protein for heparin was detected. Specific experimental conditions are shown in Table 4.

[0122] The results of the activin A binding affinity tests (Octet data) are shown in Table 6. The control fusion protein K(76,81,82)E showed an affinity similar to the wild-type fusion protein FST315-WT-Fc(WT). D The value is approximately 2.0 nM. However, the FST-m-Fc of this disclosure exhibits unexpectedly excellent affinity, with its K... D The value was 3.7E-10M (approximately 0.4nM), which represents an approximately 5-fold increase in affinity compared to the control fusion protein K(76,81,82)E.

[0123] Kinetic parameter analysis indicates that this increase in affinity is primarily attributed to a significantly reduced dissociation rate (k). off This means that once the fusion protein of this disclosure binds to the target activin A, the resulting complex is more stable and less prone to dissociation. This is of great significance for long-term blocking of the activin A signaling pathway in vivo.

[0124] The results of heparin binding capacity assays are shown in Table 6. Wild-type FST exhibited strong heparin binding capacity, while the control fusion protein K(76,81,82)E, as expected, eliminated heparin binding. The FST-m-Fc of this disclosure also showed extremely weak or undetectable heparin binding signals. In Biacore assays, even at high concentrations (200 nM), the response value (RU) was extremely low, making it impossible to fit an effective K-binding signal. D This indicates that the fusion protein disclosed in this paper, while significantly enhancing target affinity, perfectly retains the key property of "deheparinization," which helps improve pharmacokinetics.

[0125] Table 6. Comparison of affinity between the disclosed fusion protein and the control fusion protein.

[0126] Note: ND stands for Not Detectable.

[0127] The comparative experiments described above confirm that, in eliminating heparin binding, the FST-m-Fc of this disclosure is comparable to the representative fusion protein K(76,81,82)E in the prior art. Regarding the core pharmacodynamic indicator (activin A affinity), the FST-m-Fc of this disclosure is at least 5 times superior to the representative fusion protein K(76,81,82)E in the prior art.

[0128] Example 5: In vitro cell activity assay of FST-m-Fc fusion protein

[0129] The Human Activin RII(Luc)HEK293 Reporter Cell (ACRO; catalog number: CHEK-ATF164; abbreviated as "hActivin RII(Luc)HEK293 cells") can be used to detect the activity of FST-Fc fusion protein. Activin A protein can induce the expression of the reporter gene Luciferase in this cell line. It is expected that the FST-Fc fusion protein will exhibit a neutralizing effect on the stimulation of activin A protein. Therefore, the signal value of Luciferase is inversely proportional to the amount or activity of FST-Fc fusion protein added.

[0130] hActivin RII(Luc)HEK293 cells were counted and seeded at 30,000 cells / well in 96-well blank plates. Cells were then seeded into wells A2-H11 containing experimental medium (DMEM + 10% FBS), with 100 μL of PBS buffer added to the surrounding blank wells as a control. The plates were incubated overnight at 37°C and 5% CO2. The next day, the fusion protein samples (FST315-WT-Fc, FST315-HBM-Fc, FST315-m-Fc) were serially diluted 5-fold with experimental medium (DMEM + 10% FBS). At the same time, a 0.01 μg / mL solution of activin A protein (SinoBiological; catalog number: 10429-HNAH) was prepared using experimental medium. Discard the experimental culture medium from the previous day's 96-well white plates. Add 40 μL of 0.01 μg / mL activin A protein solution, followed by 40 μL of serial dilution buffer for each of the tested FST-Fc fusion proteins (FST315-WT-Fc, FST315-HBM-Fc, FST315-m-Fc). (The initial concentration of each fusion protein sample was 15 μg / mL, with 5-fold serial dilutions for a total of 12 gradients). Incubate at 37°C and 5% CO2 for 6 h. At room temperature, add 80 μL of fluorescent reagent (Bright Lite Luciferase Assay System; DD1204-02) to each well of the 96-well white plate. After shaking for 4 min in a microplate reader, read the Luminescence signal value. Process and analyze the experimental data.

[0131] The specific results are shown in Figure 3. It can be seen that the IC50 value of FST315-m-Fc is smaller than that of FST315-WT-Fc and FST-HBM-Fc, proving that it has better binding activity with activin A protein in vitro.

[0132] Example 6: In vivo activity assay of FST-m-Fc fusion protein (acute lung injury)

[0133] Establishment of acute lung injury model: C57BL / 6 mice were acclimatized for 7 days in an SPF-grade animal room (temperature 20-26℃, humidity 40-70%, light cycle 12 hours light 12 hours dark, 4 mice per cage, cage size 350mm×160mm×120mm). The acute lung injury model (BLM model) was established by a single administration of saline or bleomycin via tracheal intubation of the mice, according to the conditions shown in Table 7.

[0134] Table 7. Acute Lung Injury Model Construction Scheme

[0135] On the day of modeling, the BLM model animals were randomly divided into three groups (model control group, low-dose group, and high-dose group) according to the dosing regimen shown in Table 8, with eight mice in each group. The mice were administered intraperitoneally daily (the model control group was given saline) for seven days. At the same time, eight healthy mice that were given saline only during the modeling period served as normal controls and were given saline intraperitoneally daily for seven days.

[0136] Table 8 Group Dosing Regimen

[0137] On day 7 after administration, the experimental animals were euthanized, and the alveoli were lavaged with PBS buffer (pH 7.2-7.4). The alveolar lavage fluid (BALF) and lung tissue were collected for subsequent bioanalysis.

[0138] The volume of collected bronchoalveolar lavage fluid was determined. The bronchoalveolar lavage fluid was then diluted 5-fold with PBS buffer (pH 7.2-7.4) and the cells were counted using a cell counter to calculate the total cell count. Additionally, the collected bronchoalveolar lavage fluid was diluted 2-fold with PBS buffer (pH 7.2-7.4) and the protein concentration was determined using the BCA method.

[0139] Left lung tissue from mice was collected, fixed with paraformaldehyde, and then analyzed by HE staining.

[0140] All data are expressed as mean ± SEM. Pathological scores were compared between groups using the One-way ANOVA Kruskal-Wallis test in GraphPad, while other data were compared between groups using the One-way ANOVA LSD(L) test in GraphPad. A p < 0.05 was considered statistically significant, *p < 0.05, #p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

[0141] Experimental results

[0142] 1. General condition observation and weight of laboratory animals

[0143] The body weight of the experimental animals was monitored daily in this experiment. In this experiment, 7 days after BLM modeling, compared with the normal control group (G1), the body weight of the three groups (G2-G4) of mice involved in the BLM model decreased significantly. The body weight changes of the mice in each treatment group are shown in Table 9.

[0144] Table 9. Body weight of mice in each group

[0145] 2. Total cell count in BALF

[0146] Bronchoalveolar lavage fluid was collected from each group of mice to determine the volume of the fluid. After a 5-fold dilution with PBS buffer (pH 7.2-7.4), cells were counted using a cell counter. The total cell count is shown in Figure 4.

[0147] Compared with the normal control group (G1), the total cell count in the bronchoalveolar lavage fluid of the model control group (G2) was significantly increased, indicating that the model was successfully established. After administration of FST315-m-Fc (G3 and G4), the total cell count decreased, showing a certain dose-dependent effect.

[0148] 3. Protein content in BALF

[0149] Bronchoalveolar lavage fluid was collected from each group of mice, diluted 2-fold with PBS buffer (pH 7.2-7.4), and the protein concentration was determined using the BCA method. The protein content is shown in Figure 5.

[0150] Compared with the normal control group (G1), the protein content in the bronchoalveolar lavage fluid of the model control group (G2) was significantly increased, indicating that the model was successfully established. After administration of FST315-m-Fc (G3 and G4), the protein content decreased, showing a certain dose-dependent effect.

[0151] 4. HE staining of lung tissue

[0152] Left lung tissues of mice from each group were collected, and after HE staining analysis, the slides were analyzed. The scoring criteria are shown in Table 10. Subsequently, the pathological staining of mice in each group was statistically analyzed (as shown in Table 11 and Figure 6), and the HE staining results of representative mouse lung tissues are shown in Figure 7.

[0153] Table 10 Pathological Scoring Criteria for Lung Injury Note: The above scoring criteria are referenced from Doi:10.1177 / 0192623312438738

[0154] Table 11 HE staining scores of mouse lung tissue in each group

[0155] As shown in Figure 6, compared with the normal control group (G1), the HE staining score of mice in the model control group (G2) was significantly increased, indicating a significantly more severe degree of lung injury, demonstrating the successful establishment of the model. Administration of FST315-m-Fc reduced the degree of lung injury and showed a certain dose-dependent effect.

[0156] Furthermore, as shown in Figure 7, the normal control group (G1) mouse #1942 showed normal alveolar structure (blue circle); the model control group (G2) mouse #1911 showed obvious lung damage (black circle), alveolar wall thickening, inflammatory cell infiltration (green arrow), and almost no normal alveolar structure; the FST315-m-Fc treatment groups (G3, G4) mice #1935 and #1914 showed a gradual reduction in the degree of lung damage, a reduction in inflammatory cell infiltration and alveolar wall thickening, and a shortening of alveolar septa (yellow circle).

[0157] Example 7: In vivo activity assay of FST-Fc fusion protein (pulmonary fibrosis)

[0158] 1. Construction of a pulmonary fibrosis model

[0159] C57BL / 6 mice were acclimatized for 7 days in an SPF-grade animal room (temperature 20–26℃, humidity 40–70%, 12-hour light-12-hour dark light cycle, 4 mice per cage, cage size 350mm×160mm×120mm). A pulmonary fibrosis model was then established by a single administration of saline or bleomycin via tracheal intubation, according to the conditions shown in Table 12. Mice were observed for 7 days after bleomycin administration to confirm successful model establishment.

[0160] Table 12 Scheme for constructing pulmonary fibrosis models

[0161] 2. Grouping and Dosing

[0162] On day 8 after modeling (D8), the resulting pulmonary fibrosis model mice were randomly divided into four groups (model control group, low-dose FST315-m-Fc group, high-dose FST315-m-Fc group, and pirfenidone group) according to the dosing regimen shown in Table 13, with eight mice in each group. FST315-m-Fc was administered intraperitoneally every two days, and pirfenidone was administered by gavage daily. Mice in the model control group were given intraperitoneal injection of physiological saline daily, for a total of 20 days. At the same time, eight healthy mice that received only physiological saline during the modeling period served as normal controls and were given physiological saline intraperitoneally every two days for a total of 20 days.

[0163] Table 13 Dosing regimens for mice in each group

[0164] In this experiment, day 8 after bleomycin modeling was considered day 1 of drug administration. Except for the normal control group (G1), all mice in the other treatment groups began to die. On day 28 after modeling, the survival rate and median survival days of the model control group (G2) were 25% and 15.5 days, respectively. Compared with the model control group (G2), administration of FST315-m-Fc (G3 and G4) increased the survival rate and median survival days, suggesting that FST315-m-Fc has an effective protective effect on mouse lung tissue and may show the potential to inhibit the progression of pulmonary fibrosis. Administration of pirfenidone (G5) did not significantly change the survival rate. The survival rates of each group are shown in Table 14.

[0165] Table 14 Survival rate and number of surviving mice in each group

[0166] 3. Masson staining of lung tissue

[0167] According to the Ashcroft evaluation criteria (Table 15) reported in the literature (DOI:10.1136 / jcp.41.4.467), the lung tissue sections of mice in each group were semi-quantitatively evaluated. Normal histology was 0 points, and the higher the severity of the lesion, the higher the score, with the most severe lesion being 8 points.

[0168] Table 15 Ashcroft Criteria for Histopathological Evaluation of Pulmonary Fibrosis

[0169] The final scoring results are shown in the bar chart in Figure 8 and the representative pathological sections in Figure 9. In the normal control group (G1), mice #2648 showed normal alveolar structure (blue circle); in the model control group (G2), mice #2610 showed a large number of collagen fibers stained blue (yellow arrow), indicating significant pulmonary fibrosis (black circle), along with alveolar wall thickening, almost no normal alveolar structure, and significant hemorrhage (green arrow); in the FST315-m-Fc treatment groups (G3, G4), mice #2615 and #2612 showed a reduction in blue-stained collagen fibers, indicating reduced pulmonary fibrosis, less alveolar wall thickening, shortened alveolar septa, and visible normal alveolar structure.

[0170] The results showed that no related lesions were observed in the blank control group; the lesions in the model control group were the most severe, followed by the pirfenidone group; the lesions in the low-dose FST315-m-Fc treatment group were reduced, and the lesions in the high-dose FST315-m-Fc treatment group were reduced to the greatest extent.

[0171] Those skilled in the art can modify, combine, or change the embodiments of this disclosure without departing from the spirit and concept of this disclosure, and the resulting solutions still fall within the scope of this disclosure.

[0172] The sequences involved in this application are shown below:

[0173] Amino acid sequence of SEQ ID NO:1 wild-type FST-315 (FST315-WT)

[0174] The amino acid sequence of SEQ ID NO:2FST315-m

[0175] Amino acid sequence of SEQ ID NO:3FST315-m-Fc fusion protein

[0176] Amino acid sequence of SEQ ID NO:4FST315-WT-Fc fusion protein

[0177] Amino acid sequence of SEQ ID NO:5FST315-HBM-Fc fusion protein

[0178] SEQ ID NO:6 Wild-type hIgG4 Fc

[0179] SEQ ID NO:7S228P mutant hIgG4 Fc

[0180] The amino acid sequence of SEQ ID NO:8FST315-m1

[0181] The amino acid sequence of SEQ ID NO:9FST315-m2

[0182] The amino acid sequence of SEQ ID NO:10FST315-m3

[0183] The amino acid sequence of SEQ ID NO:11FST315-m4

[0184] The amino acid sequence of SEQ ID NO:12FST315-m5

[0185] The amino acid sequence of SEQ ID NO:13FST315-m6

[0186] The amino acid sequence of SEQ ID NO:14FST315-m7

[0187] The amino acid sequence of SEQ ID NO:15FST315-m8

[0188] The amino acid sequence of SEQ ID NO:16FST315-m9

[0189] The amino acid sequence of SEQ ID NO:17FST315-m10

[0190] The amino acid sequence of SEQ ID NO:18FST315-m11

[0191] The amino acid sequence of SEQ ID NO:19FST315-m12

[0192] The amino acid sequence of SEQ ID NO:20FST315-m13

[0193] The amino acid sequence of SEQ ID NO:21FST315-m14

[0194] The amino acid sequence of SEQ ID NO:22FST315-m15

Claims

1. A recombinant FST polypeptide, wherein, Compared with the amino acid sequence of the wild-type FST polypeptide shown in SEQ ID NO:1, the amino acid sequence of the recombinant FST polypeptide contains at least the following multiple point mutations: K76V, K81E, K82Q and K84E; Preferably, the amino acid sequence of the recombinant FST polypeptide has only the following multiple mutations: K76V, K81E, K82Q and K84E.

2. The recombinant FST polypeptide as described in claim 1, wherein, The recombinant FST polypeptide contains the amino acid sequence shown in SEQ ID NO:

2.

3. An FST-Fc fusion protein comprising: the recombinant FST polypeptide as described in claim 1 or 2 and an IgG Fc domain.

4. The FST-Fc fusion protein as described in claim 3, wherein, The FST-Fc fusion protein comprises the recombinant FST polypeptide and the IgG Fc domain from the N-terminus to the C-terminus, and preferably, the FST-Fc fusion protein does not have a linker.

5. The FST-Fc fusion protein as described in claim 3 or 4, wherein, The IgG Fc domain is a human IgG Fc domain or a mutant thereof; Preferably, the human IgG Fc domain or its mutant includes the human IgG1 Fc domain, the human IgG2 Fc domain, the human IgG3 Fc domain, the human IgG4 Fc domain, or its mutant. More preferably, the human IgG Fc domain is the human IgG4 Fc domain or a mutant thereof.

6. The FST-Fc fusion protein as described in claim 5, wherein, The human IgG4 Fc domain mutant contains at least the S228P mutation; Preferably, the human IgG4 Fc domain mutant comprises any combination of mutations selected from the following: (1) S228P; (2) S228P, F234A and L235A; (3) S228P, K196Q, F296Y, E356K, R409K, H435R and L445P; (4) S228P and L235E; or (5) S228P, F234A, L235A, D265A and R409K; More preferably, the IgG Fc domain is a mutant of the human IgG4 Fc domain whose amino acid sequence is shown in SEQ ID NO:

7.

7. The FST-Fc fusion protein according to any one of claims 3-6, wherein, The FST-Fc fusion protein comprises the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having at least 95% sequence identity with it, or a conserved modified variant thereof.

8. A polynucleotide encoding the FST-Fc fusion protein according to any one of claims 3-7.

9. A recombinant vector comprising the polynucleotide of claim 8; Preferably, the vector is selected from pcDNA series vectors, pBK-CMV vector, pEGFP-N1 vector or pGenHT 1.0-DGV vector.

10. A recombinant cell comprising the polynucleotide of claim 8 or the recombinant vector of claim 9; Preferably, the cells are eukaryotic cells.

11. A pharmaceutical composition comprising the FST-Fc fusion protein of any one of claims 3-7 and optionally pharmaceutically acceptable excipients; Preferably, the pharmaceutically acceptable excipients are selected from solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, flow aids, flavoring agents, preservatives, suspending agents, antioxidants, penetration enhancers, pH adjusters, surfactants, and diluents.

12. The FST-Fc fusion protein of any one of claims 3-7, the polynucleotide of claim 8, the recombinant vector of claim 9, the recombinant cell of claim 10, or the pharmaceutical composition of claim 11 for the prevention or treatment of FST-related diseases; in, The FST-related diseases are preferably selected from fibrotic diseases, muscle loss, or their related symptoms.

13. The FST-Fc fusion protein, polynucleotide, recombinant vector, recombinant cell, or pharmaceutical composition for the said use as described in claim 12, wherein, The fibrotic diseases or their related symptoms are selected from pulmonary fibrosis, liver fibrosis, kidney fibrosis, cardiac fibrosis, pancreatic fibrosis, skin fibrosis, peritoneal fibrosis, myelofibrosis, and nervous system fibrosis or their related symptoms.

14. The FST-Fc fusion protein, polynucleotide, recombinant vector, recombinant cell, or pharmaceutical composition for the said use as described in claim 12, wherein, The symptoms related to muscle loss are selected from sarcopenia, muscular dystrophy, metabolic myopathy, and muscle atrophy.

15. A method for preparing the FST-Fc fusion protein according to any one of claims 3-7, comprising: (1) Transform the polynucleotide of claim 8 or the recombinant vector of claim 9 into a host cell to obtain recombinant cells; (2) After culturing the recombinant cells, the obtained cell culture is subjected to separation and purification of the fusion protein to obtain the fusion protein.

Images