Actr ii b fusion protein, preparation method therefor, and use thereof

Abstract

An ActR II B fusion protein capable of targeting TGF-β superfamily ligands and inhibiting growth differentiation factor signaling. The ActR II B fusion protein comprises a fragment of an extracellular domain of ActR II B and an optional Fc of IgG. The ActR II B fusion protein can effectively inhibit the biological activity of growth differentiation factors, and can inhibit excessively active SMAD2 / 3 signaling pathways by means of ligand traps, so as to be used as a drug for increasing the level of red blood cells or treating or preventing anemia.

Description

An ActRIIB fusion protein, its preparation method and application Technical Field

[0001] This invention relates to the field of fusion protein drug technology, specifically to an ActRIIB fusion protein that can target TGF-β superfamily ligands and inhibit growth differentiation factor-mediated signal transduction. Background Technology

[0002] Myelodysplastic syndromes (MDS) are a group of clonal diseases characterized by abnormalities in hematopoietic stem cells, accompanied by abnormalities in the quality and quantity of blood cells. The main features are ineffective bone marrow hematopoiesis, refractory cytopenia, and a high risk of transformation to acute myeloid leukemia (AML). The global incidence of MDS is approximately (2–12) / 100,000, while the incidence in China is (0.23–1.51) / 100,000. International prognostic scoring systems classify patients into four risk groups based on the cytogenetic characteristics of MDS, the degree of cytopenia, and the percentage of bone marrow blasts. Patients in the low- and intermediate-1 risk groups are considered low-risk MDS (LR-MDS), while intermediate-2 and high-risk patients are considered high-risk MDS. Both domestically and internationally, MDS patients are treated in low-risk and high-risk groups. The average survival rate for LR-MDS patients is 6 years, while for high-risk MDS patients it is only 18 months.

[0003] Anemia is the most common clinical manifestation of LR-MDS, and its treatment currently faces numerous challenges. Erythrocyte stimulants (ESAs), such as erythropoietin (EPO) and dabexin (DAR), are commonly used as first-line treatments for anemia in MDS. However, many patients respond poorly to ESAs and become transfusion-dependent. Existing traditional treatments for MDS include immunomodulatory drugs (thalidomide and lenalidomide), demethylating agents (azacitidine and decitabine), and immunosuppressive therapies (anti-thymocyte globulin and cyclosporine A). These treatments may provide some transient efficacy in specific patient subgroups, but they are accompanied by significant adverse reactions, such as neurotoxicity, which is common in some patients treated with thalidomide. Hematopoietic stem cell transplantation is currently the only method that can cure MDS; however, allogeneic transplantation has many short-term and long-term complications, and stem cell transplantation is not an ideal cure. More effective and safer treatment methods are urgently needed.

[0004] In recent years, the development of targeted drugs has been rapid. Inhibitors targeting the TGF-β superfamily are a clinically validated and feasible treatment for LR-MDS patients. The TGF-β superfamily is a class of key cytokines involved in hematopoietic regulation. After binding to activin receptors (such as ActRIIA and ActRIIB) on the cell surface, it activates the SMAD2 / 3 signaling pathway, thereby participating in the regulation of erythropoiesis. In hematopoietic progenitor cells of MDS patients, the SMAD2 / 3 signaling pathway is overactivated, resulting in downregulation of the levels of transcription mediator 1γ and transcription factor GATA-1 in the cell nucleus. This leads to apoptosis of erythroid progenitor cells, massive proliferation of megakaryocytes but impaired differentiation, and increased ineffective hematopoiesis. TGF-β inhibitors (such as recombinant ActRIIA and ActRIIB proteins) act as ligand traps, blocking the binding of TGF-β superfamily ligands to their receptors, inhibiting the activation of the SMAD2 / 3 signaling pathway in late nucleated erythrocytes, promoting the enucleation and maturation of late erythrocytes into erythrocytes, thereby reducing ineffective hematopoiesis and alleviating anemia symptoms in patients. The marketed soluble ActRIIB fusion protein drug, Luspatercept, has shown good performance.

[0005] Therefore, other drugs targeting the TGF-β pathway are urgently needed to provide patients with more treatment options. Summary of the Invention

[0006] The purpose of this invention is to provide an ActRIIB fusion protein that targets TGF-β superfamily ligands, thereby inhibiting the overactive SMAD2 / 3 signaling pathway through ligand trapping to treat LR-MDS anemia and other applicable diseases with anemia symptoms.

[0007] To achieve the above objectives, the present invention provides the following technical solution:

[0008] The first aspect of the present invention provides an ActRIIB fusion protein, the ActRIIB fusion protein comprising:

[0009] (a) A fragment of the extracellular domain of ActRIIB, wherein the fragment begins at position 19, 20, 21, 22, 23, 24, or 25 of SEQ ID NO. 1; ends at position 128, 129, 130, or 131 of SEQ ID NO. 1; and, except that the leucine at position 79 of SEQ ID NO. 1 is replaced by an acidic amino acid, the fragment further comprises one or more amino acid substitutions at other positions; and

[0010] (b) Optionally, Fc of IgG.

[0011] In another preferred embodiment, the fragment of the extracellular domain of ActRIIB begins at position 25 of SEQ ID NO.1 and ends at position 131 of SEQ ID NO.1, and the leucine at position 79 is replaced by the acidic amino acid aspartic acid, as shown in SEQ ID NO.2.

[0012] In another preferred embodiment, the amino acid substitutions at one or more other positions are selected from R40, Y100, H115, or T131.

[0013] In another preferred embodiment, the amino acid substitutions at one or more other positions are selected from R40K, Y100F, H115Y, T131A, or T131S.

[0014] In another preferred embodiment, the fragment of the ActRIIB extracellular domain comprises amino acid substitutions selected from the group consisting of:

[0015] (1)L79D+R40K;

[0016] (2)L79D+Y100F;

[0017] (3)L79D+H115Y;

[0018] (4) L79D+T131A; or

[0019] (5)L79D+T131S.

[0020] In another preferred embodiment, the fragment of the ActRIIB extracellular domain contains an amino acid substitution of L79D+R40K.

[0021] In another preferred embodiment, the fragment of the ActRIIB extracellular domain contains an amino acid substitution of L79D+Y100F.

[0022] In another preferred embodiment, the IgG is derived from mammals, preferably from humans, cynomolgus monkeys, or mice, and most preferably from humans.

[0023] In another preferred embodiment, the IgG is derived from IgG1, IgG2, IgG3 or IgG4.

[0024] In another preferred embodiment, the Fc of the IgG is wild-type or mutant.

[0025] In another preferred embodiment, the Fc amino acid sequence of the IgG is shown in SEQ ID NO.3.

[0026] In another preferred embodiment, the Fc of the IgG contains amino acid substitutions at one or more positions.

[0027] In another preferred embodiment, the Fc of the IgG contains amino acid substitutions at one or more positions of L234A, L235A, M252Y, S254T, and T256E.

[0028] In another preferred embodiment, the Fc of the IgG contains amino acid substitutions of M252Y, S254T, and T256E.

[0029] In another preferred embodiment, the Fc amino acid sequence of the IgG is shown in SEQ ID NO.7.

[0030] In another preferred embodiment, the ActRIIB fusion protein comprises monomers or dimers formed from monomers, the dimers being homologous or heterologous.

[0031] In another preferred embodiment, the ActRIIB fusion protein further includes a linker between a fragment of the ActRIIB extracellular domain and the Fc of the IgG.

[0032] In another preferred embodiment, a fragment of the ActRIIB extracellular domain is connected to the N-terminus or C-terminus of the Fc, preferably the fragment of the ActRIIB extracellular domain is connected to the N-terminus of the Fc.

[0033] In another preferred embodiment, the joint is a rigid joint or a flexible joint.

[0034] In another preferred embodiment, the connector is three glycine residues (GGG).

[0035] In another preferred embodiment, the ActRIIB fusion protein can bind to the growth differentiation factor GDF-8.

[0036] In another preferred embodiment, the ActRIIB fusion protein can bind to the growth differentiation factor GDF-11.

[0037] In another preferred embodiment, the ActRIIB fusion protein can inhibit signal transduction induced by growth differentiation factors in cell activity assays.

[0038] In another preferred embodiment, the ActRIIB fusion protein is selected from the group consisting of:

[0039] (i) Contains an amino acid sequence selected from the group consisting of: SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.8 or SEQ ID NO.9;

[0040] (ii) A polypeptide derived from (i) formed by substitution, deletion and / or addition of one or more amino acids in the amino acid sequence of (i) and having activity of binding growth differentiation factors.

[0041] In another preferred embodiment, the ActRIIB fusion protein comprises an active fragment and / or derivative of the ActRIIB fusion protein, wherein the active fragment and / or the derivative retains 70-100% (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100%) of the activity of the ActRIIB fusion protein in binding growth differentiation factors.

[0042] In another preferred embodiment, the derivative of the ActRIIB fusion protein is a polypeptide of the ActRIIB fusion protein that has undergone amino acid mutations (substitution, deletion, and / or addition) at one or more positions and maintains at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% sequence identity.

[0043] In another preferred embodiment, the amino acid mutation is a conserved amino acid substitution.

[0044] A second aspect of the present invention provides a polynucleotide molecule that encodes the ActRIIB fusion protein described in the first aspect of the present invention.

[0045] In another preferred embodiment, the polynucleotide molecule comprises a nucleotide sequence selected from the group consisting of SEQ ID NO.10, SEQ ID NO.11, SEQ ID NO.12, or SEQ ID NO.13.

[0046] A third aspect of the present invention provides an expression vector containing the polynucleotide molecule described in the second aspect of the present invention.

[0047] In another preferred embodiment, the expression vector is a virus or plasmid, preferably a bacteriophage or bacteriophage particle.

[0048] In another preferred embodiment, the expression vector is selected from the group consisting of pcDNA3.4, pDR1, pcDNA3.1(+), pcDNA3.1 / ZEO(+), pDHFR, pTT5, pDHFF, pCGS3, pGM-CSF or pCHO 1.0, preferably pcDNA3.4 or pCGS3.

[0049] A fourth aspect of the present invention provides a host cell containing the expression vector described in the third aspect of the present invention.

[0050] In another preferred embodiment, the host cell is selected from the group consisting of COS, CHO, 293F, 293E, NSO, sf9, sf21, DH5α, BL21(DE3) or TG1, preferably E. coli TG1, BL21(DE3) cells or CHO cells.

[0051] The fifth aspect of the present invention provides a method for preparing the ActRIIB fusion protein as described in the first aspect of the present invention, comprising the following steps:

[0052] 1) Under expression conditions, host cells as described in the fourth aspect of the present invention are cultured to express the ActRIIB fusion protein;

[0053] 2) Isolate and purify the ActRIIB fusion protein described in step 1).

[0054] The present invention provides an immunoconjugate comprising, in six aspects:

[0055] (I) The ActRIIB fusion protein as described in the first aspect of the present invention; and

[0056] (II) Selected from the following groups for coupling: detectable markers, drugs, toxins, cytokines, radionuclides, enzymes or combinations thereof.

[0057] In another preferred embodiment, the conjugate is partially selected from: fluorescent or luminescent markers, radioactive markers, contrast agents for magnetic resonance imaging or computed tomography, or enzymes, radionuclides, biotoxins, cytokines, antibodies, antibody scFv fragments, gold nanoparticles / nanorobars, viral particles, liposomes, or magnetic nanoparticles.

[0058] In another preferred embodiment, the immunoconjugate includes an antibody-drug conjugate.

[0059] A seventh aspect of the present invention provides a pharmaceutical composition comprising an effective amount of the ActRIIB fusion protein as described in the first aspect of the present invention or an immunoconjugate as described in the sixth aspect of the present invention and one or more pharmaceutically acceptable carriers, diluents or excipients.

[0060] In another preferred embodiment, the pharmaceutical composition further includes one or more additional pharmaceutically active agents.

[0061] In another preferred embodiment, the dosage form of the pharmaceutical composition includes a gastrointestinal dosage form or a parenteral dosage form.

[0062] In another preferred embodiment, the parenteral drug delivery dosage form includes intravitreal injection, intravenous injection, intravenous drip, subcutaneous injection, local injection, intramuscular injection, intratumoral injection, intraperitoneal injection, intracranial injection, or intracavitary injection.

[0063] The eighth aspect of the present invention provides the use of the ActRIIB fusion protein as described in the first aspect of the present invention, the immunoconjugate as described in the sixth aspect of the present invention, or the pharmaceutical composition as described in the seventh aspect of the present invention in the preparation of a medicament for increasing red blood cell levels or for treating or preventing anemia.

[0064] In another preferred embodiment, the use also includes combination with one or more other therapeutic agents.

[0065] In another preferred embodiment, the anemia is selected from: anemia associated with myelodysplastic syndrome, anemia associated with kidney dysfunction, anemia associated with chronic kidney disease, anemia associated with acute kidney disease, anemia associated with chemotherapy (taxane and / or docetaxel), anemia caused by blood loss, anemia associated with multiple myeloma, anemia associated with thalassemia, anemia associated with tumors or cancer, anemia associated with acute kidney failure, anemia associated with chronic kidney failure, anemia associated with acute kidney disease, anemia associated with chronic kidney disease, anemia associated with end-stage renal disease, anemia associated with sickle cell disease, or a combination of multiple anemias.

[0066] A ninth aspect of the invention provides a method for treating or preventing anemia, the method comprising administering to a subject in need an ActRIIB fusion protein as described in the first aspect of the invention, an immunoconjugate as described in the sixth aspect of the invention, or a pharmaceutical composition as described in the seventh aspect of the invention.

[0067] In another preferred embodiment, the method further includes administration in combination with one or more additional therapeutic agents.

[0068] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0069] Figure 1A shows the inhibitory effect of the ActRIIB-hFc-L79D mutant on GDF-8-mediated signal transduction.

[0070] Figure 1B shows the inhibitory effect of the ActRIIB-hFc-L79D mutant on GDF-11-mediated signal transduction.

[0071] Figure 1C shows the inhibitory effect of the ActRIIB-hFc-L79D mutant on Activin A-mediated signal transduction.

[0072] Figure 2A shows the binding of the ActRIIB-hFc-L79D mutant to FcRn at pH 6.0.

[0073] Figure 2B shows the binding of the ActRIIB-hFc-L79D mutant to FcRn at pH 7.4.

[0074] Figure 3A shows the inhibitory effect of the ActRIIB-hFc-L79D-YTE mutant on GDF-8-mediated signal transduction.

[0075] Figure 3B shows the inhibitory effect of the ActRIIB-hFc-L79D-YTE mutant on GDF-11-mediated signal transduction.

[0076] Figure 3C shows the inhibitory effect of the ActRIIB-hFc-L79D-YTE mutant on Activin A-mediated signal transduction.

[0077] Figure 4A shows the alleviating effect of the ActRIIB-hFc-L79D mutant on red blood cell count (RBC) in a docetaxel-induced mouse anemia model.

[0078] Figure 4B shows the alleviating effect of the ActRIIB-hFc-L79D mutant on hemoglobin concentration (HGB) in a docetaxel-induced mouse anemia model.

[0079] Figure 5A shows the long-term alleviating effect of ActRIIB-hFc-L79D-R40K-YTE on red blood cell count (RBC) in a docetaxel-induced mouse anemia model.

[0080] Figure 5B shows the long-term alleviating effect of ActRIIB-hFc-L79D-R40K-YTE on hemoglobin concentration (HGB) in a docetaxel-induced mouse anemia model.

[0081] Figure 5C shows the long-term alleviating effect of ActRIIB-hFc-L79D-R40K-YTE on hematocrit (HCT) in a docetaxel-induced mouse anemia model.

[0082] Figure 5D shows the long-term alleviating effect of ActRIIB-hFc-L79D-R40K-YTE on reticulocyte count (RET) in a docetaxel-induced mouse anemia model.

[0083] Figure 6A shows the effect of the mutant ACTRⅡB-hFc-L79D-R40K-YTE on improving anemia in a docetaxel-induced mouse anemia model.

[0084] Figure 6B shows the effect of the mutant ACTRⅡB-hFc-L79D-R40K-YTE on improving anemia in a mouse model of β-thalassemia.

[0085] Figure 6C shows the effect of the mutant ACTRⅡB-hFc-L79D-R40K-YTE on improving anemia in a rat model of acute blood loss anemia. Detailed Implementation

[0086] Through extensive and in-depth research, the inventors obtained a novel ActRIIB fusion protein that can inhibit the activity of signaling pathways mediated by growth differentiation factors GDF-8 and GDF-11, but does not inhibit the activity of signaling pathways mediated by activin A. In a docetaxel-induced mouse anemia model, the ActRIIB fusion protein of this invention significantly increased erythrocyte levels and maintained a good anemia recovery effect for 28 days. Unexpectedly, compared with the already marketed rotezip, the novel ActRIIB fusion protein of this invention showed superior efficacy in inhibiting the activity of growth differentiation factors and in treating anemia in mouse models. Therefore, the novel ActRIIB fusion protein of this invention can be developed into a drug with superior efficacy for the prevention or treatment of anemia. This invention was completed based on this.

[0087] This invention relates to the antagonism of ActRIIB receptor ligands (also known as ActRIIB ligands) with targeted ActRIIB fusion proteins, generally for the purpose of antagonizing ActRIIB ligand signal transduction in any process associated with ActRIIB receptor activity. The ActRIIB receptor or ActRIIB ligand is involved in the regulation of many key biological processes, and the use of ActRIIB fusion proteins is envisioned for the treatment or prevention of diseases or conditions associated with abnormal activity of the ActRIIB receptor or ActRIIB ligand. Optionally, the ActRIIB fusion protein of the present invention can antagonize one or more ligands of the ActRIIB receptor. Therefore, the ActRIIB fusion protein, compositions, and methods of the present invention can be used to treat diseases associated with abnormal activity of one or more ligands of the ActRIIB receptor. Ligands of the ActRIIB receptor include certain members of the TGF-β superfamily, such as activin, GDF-8, and GDF-11. Due to their key functions in these processes, they may be ideal targets for therapeutic interventions.

[0088] the term

[0089] To facilitate a clearer understanding of this disclosure, certain terms are first defined. As used herein, unless otherwise expressly specified herein, each of the following terms shall have the meaning given below.

[0090] In this invention, the term "ActRⅡB" refers to a family of activin receptor type IIB proteins and ActRⅡB-related proteins derived from any species. ActRⅡB family members are generally all transmembrane proteins, consisting of a ligand-binding extracellular domain with a cysteine-rich region, a transmembrane domain, and a cytoplasmic domain with predicted serine / threonine kinase specificity.

[0091] Two related type II receptors—ActRIIA and ActRIIB—have been identified as type II receptors for activin. In addition to activin, ActRIIA and ActRIIB can interact with several other TGF-β superfamily proteins, including GDF-8 and GDF-11.

[0092] In this invention, the term "TGF-β" refers to transforming growth factor-β. TGF-β signaling is mediated by a heteropolymeric complex of type I and type II serine / threonine kinase receptors, which phosphorylates and activates downstream SMAD proteins upon ligand stimulation. These type I and type II receptors are all transmembrane proteins, consisting of a ligand-binding extracellular domain with a cysteine-rich region, a transmembrane domain, and a cytoplasmic domain with predicted serine / threonine specificity. Type I receptors are essential for signal transduction, while type II receptors are required for ligand binding and expression of type I receptors. Upon ligand binding, type I and type II activin receptors form a stable complex, leading to phosphorylation of type I receptors by type II receptors.

[0093] In this invention, the term "Fc" refers to the ability of papain to cleave an antibody into two identical Fab fragments and one Fc fragment. The Fc fragment is a crystallizable fragment, composed of the CH2 and CH3 domains of the antibody. The Fc fragment has no antigen-binding activity and is the site where the antibody interacts with effector molecules or cells.

[0094] As used herein, the term "linker" refers to one or more amino acid residues that provide sufficient mobility for different domains of a fusion protein to fold into a functional protein capable of exerting its activity. Suitable examples of linkers include glycine-rich (e.g., 2-10, 2-5, 2-4, 2-3 glycine residues) or glycine and proline residues and may, for example, contain a single sequence of threonine / serine and glycine or a repeating sequence of threonine / serine and / or glycine. The identification and sequence of amino acid residues in the linker may vary depending on the type of secondary structural element to be achieved in the linker. In this invention, a preferred linker is the linker GGG (three glycine amino acids).

[0095] In this invention, the terms "binding" and "specific binding" refer to a non-random binding reaction between two molecules, such as the reaction between a receptor and its targeted ligand. Typically, receptors bind at a rate of less than approximately 10... -7 M, for example, less than approximately 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 A receptor-ligand binding affinity is determined by an equilibrium dissociation constant (KD) of M or less. "KD" refers to the equilibrium dissociation constant of a specific receptor-ligand interaction, used to describe the binding affinity between the receptor and ligand. The smaller the equilibrium dissociation constant, the tighter the binding and the higher the affinity. For example, the binding affinity of the receptor and ligand can be determined using surface plasmon resonance (SPR) in a BIACORE instrument or by using ELISA to determine the relative affinity of the receptor and ligand binding.

[0096] In this invention, the term "homologous," in all its grammatical forms and spelling variations, refers to the relationship between two proteins that share a "co-evolutionary origin," including proteins from the same superfamily of organisms and homologous proteins from different species. Such proteins (and their encoded nucleic acids) exhibit sequence homology, as reflected by their sequence similarity, whether in terms of the percentage of identity or based on the presence of specific residues or motifs and conserved sites.

[0097] In this invention, the terms “sequence similarity” or “sequence identity” in all their grammatical forms refer to the degree of identity or correspondence between nucleic acid or amino acid sequences that may or may not share a common evolutionary origin.

[0098] ActRⅡB fusion protein

[0099] In this invention, the term "ActRIIB fusion protein" is used to refer to any natural polypeptide containing a member of the ActRIIB family, as well as any variant (including mutants, fragments, fusions, and peptide-like forms) of the ActRIIB family that retains useful activity. For example, ActRIIB fusion proteins include polypeptides derived from any known ActRIIB sequence that have a sequence identity of at least about 80%, preferably at least 85%, 90%, 95%, 97%, 99%, or higher, with the ActRIIB polypeptide sequence.

[0100] In this invention, the ActRIIB fusion proteins involved, optionally, fragments, functional variants, and modified forms, possess similar or identical biological activities to their corresponding wild-type ActRIIB polypeptides. ActRIIB is highly conserved across almost all vertebrates, with large segments of its extracellular domains being completely conserved. Many ligands that bind ActRIIB are also highly conserved. Therefore, comparisons of ActRIIB sequences from various vertebrate organisms provide insights into modifiable residues. Thus, active human ActRIIB variants may include one or more amino acids at corresponding positions in the sequence of another vertebrate ActRIIB, or may include residues similar to those in human or other vertebrate sequences.

[0101] The ActRIIB fusion protein of the present invention is a fragment comprising (a) an ActRIIB extracellular domain, wherein the fragment begins at position 19, 20, 21, 22, 23, 24, or 25 of SEQ ID NO. 1; ends at position 128, 129, 130, or 131 of SEQ ID NO. 1; and, except that the leucine at position 79 of SEQ ID NO. 1 is replaced by an acidic amino acid, the fragment further comprises one or more amino acid substitutions at other positions; and (b) optionally, the Fc of IgG.

[0102] The fragment of the ActRIIB extracellular domain contains amino acid substitutions selected from the following positions:

[0103] (1)L79D+R40K;

[0104] (2)L79D+Y100F;

[0105] (3)L79D+H115Y;

[0106] (4) L79D+T131A; or

[0107] (5)L79D+T131S.

[0108] The Fc of the IgG contains amino acid substitutions of M252Y, S254T, and T256E.

[0109] The ActRIIB fusion protein is selected from the following group:

[0110] (i) Contains an amino acid sequence selected from the group consisting of: SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.8 or SEQ ID NO.9;

[0111] (ii) A polypeptide derived from (i) formed by substitution, deletion and / or addition of one or more amino acids in the amino acid sequence of (i) and having activity of binding growth differentiation factors.

[0112] Nucleic acid encoding and expression vector

[0113] This invention also provides a polynucleotide molecule encoding the aforementioned ActRIIB fusion protein. The polynucleotide of this invention can be in DNA or RNA form. The DNA form includes cDNA, genomic DNA, or artificially synthesized DNA. The DNA can be single-stranded or double-stranded. The DNA can be a coding strand or a non-coding strand.

[0114] Once the relevant sequence is obtained, it can be obtained in large quantities using recombination methods. This typically involves cloning it into a vector, transferring it into cells, and then isolating the sequence from the proliferated host cells using conventional methods.

[0115] Currently, the DNA sequence encoding the fusion protein (or its active fragment, or its derivative) of this invention can be obtained entirely through chemical synthesis. This DNA sequence can then be introduced into various existing DNA molecules (such as vectors) and cells known in the art. Furthermore, mutations can be introduced into the fusion protein sequence of this invention through chemical synthesis.

[0116] The present invention also relates to vectors comprising the aforementioned suitable DNA sequences and suitable promoters or control sequences. These vectors can be used to transform suitable host cells to enable them to express proteins.

[0117] Preparation method

[0118] The host cell culture method and the isolation and purification method of the fusion protein described in this invention are conventional methods in the art. For specific operating procedures, please refer to the relevant cell culture technology manual and fusion protein isolation and purification technology manual. The preparation method of the ActRIIB fusion protein disclosed in this invention includes: culturing the aforementioned host cells under expression conditions to express the ActRIIB fusion protein; and isolating and purifying the ActRIIB fusion protein. Using the above methods, the recombinant protein can be purified into a substantially homogeneous substance.

[0119] The ActRIIB fusion protein disclosed in this invention can be separated and purified using affinity chromatography. Depending on the characteristics of the affinity column used, conventional methods such as high-salt buffer or pH adjustment can be used to elute the ActRIIB fusion protein bound to the affinity column. The obtained ActRIIB fusion protein was tested, and the results showed that the anti-ActRIIB fusion protein binds well to its relevant ligands and exhibits high affinity.

[0120] Pharmaceutical Composition

[0121] The dosage of the active ingredient in the drug combination of the present invention varies depending on the target patient, the target organ, symptoms, and method of administration. It can be determined based on the doctor's judgment, taking into account factors such as the type of dosage form, method of administration, patient's age and weight, and patient's symptoms.

[0122] The pharmaceutical compositions of the present invention contain an effective amount of the ActRIIB fusion protein as described above or an immunoconjugate as described above, and one or more pharmaceutically acceptable carriers, diluents, or excipients. Such carriers include (but are not limited to): acetate, trehalose, arginine hydrochloride, Tween, and combinations thereof.

[0123] The pharmaceutical compositions of the present invention can be administered to patients via appropriate routes of administration, including but not limited to gastrointestinal or parenteral dosage forms. The term "parenteral administration" includes intravitreal injection, intravenous injection, intravenous infusion, subcutaneous injection, local injection, intramuscular injection, intratumoral injection, intraperitoneal injection, intracranial injection, or intracavitary injection, etc.

[0124] Preferably, the pharmaceutical composition of the present invention further includes other pharmaceutically active agents, such as erythropoiesis stimulants (ESAs), such as epoetin α, epoetin β, dapoetin α, methoxy polyethylene glycol erythropoietin β, pemoxacin, EPO, etc.

[0125] application

[0126] This invention also provides treatment methods and uses. In some embodiments, a method for preventing, treating, or improving anemia is provided, the method comprising administering an effective amount of the ActRIIB fusion protein of the present invention to a patient. In some embodiments, the use of the ActRIIB fusion protein in the prevention, treatment, or improvement of anemia is provided. In some embodiments, the use of the ActRIIB fusion protein in the preparation of a medicament for the prevention, treatment, or improvement of anemia is provided. In some embodiments, the anemia is selected from: anemia associated with myelodysplastic syndromes, anemia associated with kidney dysfunction, anemia associated with chronic kidney disease, anemia associated with acute kidney disease, anemia associated with chemotherapy (taxane and / or docetaxel), anemia due to blood loss, anemia associated with multiple myeloma, anemia associated with thalassemia, anemia associated with tumors or cancer, anemia associated with acute kidney failure, anemia associated with chronic kidney failure, anemia associated with acute kidney disease, anemia associated with chronic kidney disease, anemia associated with end-stage renal disease, anemia associated with sickle cell disease, or a combination of multiple anemias.

[0127] combination therapy

[0128] In some embodiments, the ActRIIB fusion protein of the present invention can be combined with other treatment or preventative regimens, including the administration of the ActRIIB fusion protein of the present invention together with one or more other therapeutic agents or methods. For combination therapy, the ActRIIB fusion protein of the present invention can be administered simultaneously or separately from other therapeutic agents. When administered separately, the ActRIIB fusion protein of the present invention can be administered before or after the administration of another therapeutic agent.

[0129] In some embodiments, therapeutic agents used in combination with the ActRIIB fusion protein of the present invention include: docetaxel, taxane, erythropoiesis stimulants (ESAs) such as epoetin α, epoetin β, dapoetin α, methoxy polyethylene glycol erythropoietin β, pemoxatide, EPO, etc.

[0130] The main advantages of this invention include

[0131] This invention provides a novel ActRIIB fusion protein (1) which can increase red blood cell levels and treat or prevent anemia by inhibiting the activity of growth differentiation factors and suppressing the overactive SMAD2 / 3 signaling pathway; (2) in terms of cellular biological activity, the inhibitory activity of the ActRIIB fusion protein of this invention is even better than that of rotezip; (3) in the treatment of anemia in mouse models, the ActRIIB fusion protein of this invention is more effective in enhancing red blood cell levels; (4) it has high specificity and good safety; (5) it has high expression level; and (6) it has a stable structure.

[0132] The present invention will be further described below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions as described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer.

[0133] The experimental materials, sources, and reagents used in the following examples are described in detail below.

[0134] Experimental materials:

[0135] Competent cells: Brand: Shenggong, Product No.: B528412.

[0136] 293F cells: Brand GIBCO, catalog number R79007.

[0137] HepG2 cells: Brand ATCC, Catalog No. HB-8065.

[0138] 0.22μm filter: brand Millipore, part number SLGV033R.

[0139] Hitrap Mabselect Sure affinity chromatography column: Brand: Sitopan, Product No. 11003493.

[0140] Waters MassPREP TM Micro Desalting Column: Brand: Waters, Model: 186004032.

[0141] Experimental reagents:

[0142] Endotoxin-free plasmid extraction kit: Brand: Tiangen, Product No.: DP117.

[0143] DNA Purification and Recovery Kit: Brand: Tiangen, Product No.: DP214.

[0144] ClonExpress™ II One Step Cloning Kit: Brand: Novizan, Product No.: C112.

[0145] HSDNA Polymerase: Brand: Takara, Product Code: R010B.

[0146] Coating solution: 1.59 g sodium carbonate, 2.93 g sodium bicarbonate, diluted to 1 L with double distilled water.

[0147] PBST: PBS + 0.05% Tween 20.

[0148] Tween 20: Brand Aladdin, item number T104863.

[0149] ELISA blocking solution: PBST + 1% BSA.

[0150] BSA: Brand Shenggong, Item No. A60332.

[0151] Human GDF-8 protein: Brand R&D, Product No. 788-G8-100 / CF.

[0152] Human GDF-11 protein: Brand R&D, catalog number 1958-GD-010 / CF.

[0153] Human Activin A protein: Brand: Kaika Biotechnology, Product No.: ACV-HM001.

[0154] Human FcRn protein: Brand: Kaika Biotechnology, Product No.: FRN-HM101.

[0155] HRP-labeled anti-human Fc antibody: brand Sigma, catalog number A0170.

[0156] HRP-labeled anti-6X-His antibody: brand abcam, catalog number ab178563.

[0157] TMB: Brand BD Biosciences, catalog number 555214.

[0158] Termination solution: 2M sulfuric acid solution.

[0159] 0.25% Pancreatic Enzyme-EDTA: Brand GIBCO, Product No. 25200-072.

[0160] FBS: Brand GIBCO, Item No. 10099-141.

[0161] Pen Strep: Brand GIBCO, Product No. 1514022.

[0162] Sodium pyruvate: Brand GIBCO, Product No. 11360-070.

[0163] Lipofectamine TM 3000: Brand Thermo Fisher, product number L3000015.

[0164] pCAGA12-TA-Luc reporter gene plasmid: Brand: Beyotime, Catalog No.: D4026-100μg.

[0165] GMOne-Step Luciferase Reporter Gene Detection Kit: Brand: Jiman Biotechnology, Product No.: GM-040513.

[0166] Experimental apparatus:

[0167] Mastercycler Nexus PCR instrument: purchased from Eppendorf.

[0168] Hitrap Mabselect Sure column: purchased from Cytiva.

[0169] HiLoad 26 / 600 Superdex 200pg column: purchased from Cytiva.

[0170] SpectraMax i3x microplate reader: purchased from Molecular Devices.

[0171] SpectraMax M5 microplate reader: purchased from Molecular Devices.

[0172] The sequence of the present invention is shown in the table below:

[0173] Example 1. Construction of ActRIIB fusion protein and its mutants

[0174] A soluble ActRIIB fusion protein with a fusion into the human IgG Fc domain was constructed by introducing the L79D mutation into a fragment of the ActRIIB extracellular domain (amino acids from positions 25 to 131 of the ActRIIB protein as shown in SEQ ID NO.1), with the amino acid sequence shown in SEQ ID NO.2. Three glycine residues (GGG) were used as linkers to connect the ActRIIB extracellular domain to the human Fc domain (amino acid sequence shown in SEQ ID NO.3). This fusion protein was named ActRIIB-hFc-L79D (amino acid sequence shown in SEQ ID NO.4). ActRIIB-hFc-L79D has the same amino acid sequence as the marketed soluble ActRIIB fusion protein drug, rotexip, and was used as a positive control in subsequent examples.

[0175] The ActRIIB-hFc-L79D fusion protein was expressed in 293F or CHO cell lines, using the following three leader sequences:

[0176] (1) Original leader sequence: MTAPWVALALLWGSLCAGSGRGEA (SEQ ID NO.14)

[0177] (2) Gaussia luciferase leader sequence: MGVKVLFALICIAVAEA (SEQ ID NO.15)

[0178] (3) Human insulin leader sequence: MALWMRLLPLLALLALWGPDPAAA (SEQ ID NO.16) Based on the PDB structure, 6MAC is predicted to increase the affinity mutation for GDF-11. A series of mutations were introduced into the ActRIIB-hFc-L79D fusion protein, mainly mutating the extracellular domain of ActRIIB. The mutation locations include R40K, T41S, F82H, F82K, S62W, V73E, V73F, V73K, V73M, V73W, K74E, D81W, V91A, V91S, V99G, V99R, Y100F, F101G, F101S, H115Y, T131A, T131S, etc.

[0179] To improve the expression efficiency of the fusion protein in CHO cells, the nucleotide sequences of the ActRIIB-hFc-L79D fusion protein and its mutants were optimized using codons, taking into account factors such as codon bias, GC content, mRNA secondary structure, and repetitive sequences. The nucleotide sequences of the antibody were synthesized by Genewiz Biotechnology Co., Ltd.

[0180] Experiments have shown that the fusion protein expression efficiency is highest when expressed in CHO cells using the human insulin leader sequence. Therefore, ActRIIB-hFc-L79D and related mutants used in subsequent experiments of this invention were all expressed in CHO cells using the human insulin leader sequence.

[0181] Example 2. Purification of ActRIIB fusion protein and its mutants

[0182] The nucleotide sequences of the ActRIIB-hFc-L79D fusion protein and its mutants were subcloned into the vector pcDNA3.4. Recombinant plasmids were extracted and co-transfected into 293F cells and / or CHO cells. After 5-7 days of cell culture, the culture medium was centrifuged at high speed, filtered through a 0.22 μm filter, and loaded onto a Hitrap Mabselect Sure affinity chromatography column. The protein was eluted in one step with 100 mM citric acid and pH 3.5 elution buffer. The target sample was recovered and dialyzed to pH 7.4 PBS. The purity of the purified protein was determined by UPLC-SEC.

[0183] Example 3. ELISA determination of the affinity of ActRIIB fusion protein and its mutants for GDF-8 and Activin A.

[0184] To detect the binding affinity of the ActRIIB-hFc-L79D fusion protein and its mutants to GDF-8 and Activin A, recombinant human GDF-8 or recombinant human Activin A was diluted to 200 ng / mL with coating buffer, and 100 μL / well was added to the microplate and incubated overnight at 4°C. The coating buffer was removed, and 200 μL / well of blocking buffer was added, and the plate was incubated at room temperature for 1-2 hours. The blocking buffer was then removed, and the ActRIIB-hFc-L79D fusion protein and its mutants were diluted again with blocking buffer to 1 mg / mL, creating a 4-fold concentration gradient. These gradients were then added sequentially to the blocked microplate at 100 μL / well and incubated at 37°C for 1 hour. The plate was washed three times with PBST, and HRP-labeled goat anti-human Fc antibody was diluted 1:3000 with blocking buffer, and 100 μL / well was added to the microplate and incubated at 37°C for 30 minutes. Wash the plate three times with PBST, add 100 μL of TMB to each well, and incubate at room temperature in the dark for 5 minutes. Add stop solution at 50 μL / well, read the OD value at 450 nm using a microplate reader, and calculate the EC50. 50 .

[0185] Using the ActRIIB-hFc-L79D fusion protein as a positive control, the EC50 of different mutants was calculated. 50 EC with ActRIIB-hFc-L79D fusion protein 50 The ratio of the two values ​​was used to compare the relative affinity of different mutants. Specific data are shown in Table 1.

[0186] Table 1. Relative affinity of ActRIIB-hFc-L79D fusion protein and its mutants to GDF-8 and Activin A ※ No activity + < 2x increased activity — < 2x decreased activity — ≈ 2-5x decreased activity — ≈ 5-10x decreased activity — > 10x decreased activity

[0187] As shown in Table 1, when the L79D mutation was retained in the ActRIIB extracellular domain fragment, the introduction of mutations at the T41S, S62W, K74E, V91A, V91S, V99G, V99R, F101G, and F101S positions resulted in the mutants losing their binding ability with GDF-8 and Activin A. The introduction of mutations at the D81W, F82H, and F82K positions significantly reduced the affinity of the mutants for Activin A, while showing virtually no affinity for GDF-8. The introduction of mutations at the V73E, V73F, V73K, V73M, and V73W positions reduced the affinity of the mutants for GDF-8 and Activin A. The binding capacity of A was reduced by more than 2 times to varying degrees; after introducing mutations at the R40K, Y100F, H115Y, T131A, and T131S positions, the mutant activity was comparable to that of the ActRIIB-hFc-L79D fusion protein.

[0188] Example 4. ELISA determination of the binding of ActRIIB fusion protein and its mutant to GDF-11.

[0189] The binding affinity of the ActRIIB-hFc-L79D fusion protein mutants R40K, Y100F, H115Y, T131A, and T131S to GDF-11 was tested. Specifically: Dilute recombinant human GDF-11 to 200 ng / mL with coating buffer, add 100 μL / well to the microplate, and incubate overnight at 4°C. Remove the coating buffer, add 200 μL / well of blocking buffer, and incubate at room temperature for 1-2 hours. Remove the blocking buffer, then dilute the ActRIIB-hFc-L79D fusion protein and its mutants to 1 mg / mL with blocking buffer, creating 12 concentration gradients (4-fold dilutions), and add 100 μL / well to each well of the diluted protein, incubating at 37°C for 1 hour. Wash the plate three times with PBST. Dilute the HRP-labeled goat anti-human Fc antibody 1:3000 with blocking buffer and add 100 μL / well to the plate. Incubate at 37°C for 30 minutes. Wash the plate three times with PBST. Add 100 μL of TMB to each well and incubate at room temperature in the dark for 5 minutes. Add 50 μL of stop solution to each well. Read the OD value at 450 nm using a microplate reader and calculate the EC50. 50 .

[0190] Using the ActRIIB-hFc-L79D fusion protein as a positive control, the EC50 of different mutants was calculated. 50 EC with ActRIIB-hFc-L79D fusion protein 50 The ratio of the values ​​was used to compare the relative activities of different mutants. Specific data are shown in Table 2.

[0191] Table 2. Relative affinity of ActRIIB-hFc-L79D fusion protein and its mutants to GDF-11 + ≈2-5 times increase in activity ++ ≈5-10 times increase in activity

[0192] The experimental results showed that, while retaining the L79D mutation in the extracellular domain of ActRIIB, the binding affinity of mutants with mutations at the R40K, Y100F, H115Y, T131A, and T131S sites was enhanced to varying degrees, with each mutant showing an affinity increase of more than 2-fold. Considering both UPLC-SEC detection and expression levels of the mutants, the R40K mutant (named ActRIIB-hFc-L79D-R40K, amino acid sequence as shown in SEQ ID NO.5, nucleotide sequence as shown in SEQ ID NO.10) and the Y100F mutant (named ActRIIB-hFc-L79D-Y100F, amino acid sequence as shown in SEQ ID NO.6, nucleotide sequence as shown in SEQ ID NO.11), which had relatively high protein expression purity and levels, were selected for subsequent experiments.

[0193] Example 5. Determination of the affinity of ActRIIB fusion protein and its mutants for Activin A using the Biacore method.

[0194] The binding activity of ActRIIB-hFc-L79D fusion protein and its mutants to recombinant human Activin A was determined using the Protein A capture method. The specific detection method is as follows: The Protein A chip was placed in the instrument's chip chamber, and the replacement buffer was HBS-EP+Buffer. The sample to be tested was diluted to 5 μg / mL with HBS-EP+Buffer. Activin A was serially diluted 2-fold with HBS-EP+Buffer, starting from 400 nM and performing eight consecutive dilutions, with a 0 concentration added. 6M guanidine hydrochloride solution was used as the regeneration buffer. Instrument parameters were set as follows: binding time 120 s, dissociation time 600 s, flow rate 30 μL / min, and regeneration contact time 30 s. The corresponding sample and buffer were placed in the wells displayed on the plate, and the program was run. After the run, the data were analyzed using Biacore 8K Evaluation Software. The data were fitted using the 1:1 binding formula in Kinetics mode to obtain the affinity of the tested sample for Activin A. Specific data are shown in Table 3.

[0195] Table 3. Affinity of ActRIIB-hFc-L79D fusion protein and its mutants to Activin A

[0196] As shown in Table 3, the KD values ​​of the ActRIIB-hFc-L79D-R40K and ActRIIB-hFc-L79D-Y100F mutants and the positive control ActRIIB-hFc-L79D are basically equivalent to those of Activin A. The Ka values ​​of the ActRIIB-hFc-L79D-R40K mutant and the positive control ActRIIB-hFc-L79D are basically the same, while the Ka value of the ActRIIB-hFc-L79D-Y100F mutant is smaller than that of the positive control, indicating that the ActRIIB-hFc-L79D-R40K mutant maintains ligand-binding activity similar to that of the ActRIIB-hFc-L79D molecule. Meanwhile, the Kd values ​​of both ActRIIB-hFc-L79D-R40K and ActRIIB-hFc-L79D-Y100F mutants were slightly smaller than those of the positive control ActRIIB-hFc-L79D, indicating that their dissociation rate from the ligand Activin A was slower.

[0197] Example 6. Bioassay of ActRIIB fusion protein and its mutants on different ligand-mediated signal transduction

[0198] (CAGA)12 is a downstream response element of the TGF-β signaling pathway, and the luciferase reporter gene vector pCAGA12-TA-Luc can detect the activation level of CAGA12 with high sensitivity. Transient transfection of pCAGA12-TA-Luc into the HepG2 cell line (human hepatocellular carcinoma cells) with high expression of ActRII proteins (ActRIIA and ActRIIB) allowed the ligands GDF-8, GDF-11, and Activin A to activate downstream signaling, thereby evaluating the inhibitory effect of the ActRIIB-hFc-L79D fusion protein and its mutants on TGF-β superfamily-dependent (CAGA)12 transcriptional activation. The specific methods are as follows:

[0199] HepG2 cells in the logarithmic growth phase were seeded into 6-well plates and transfected with pCAGA12-TA-Luc plasmid using Lipofectamine 3000 after cell adhesion. Thirty-six hours after transfection, HepG2 cells were digested into single cells with trypsin, resuspended in analytical medium (basal medium + 0.1% BSA), and transferred at 60,000 cells / well to 3903 white-bottomed 96-well plates. The ligand and ActRIIB-hFc-L79D fusion protein and its mutant were diluted with analytical medium. The working concentration of the ligand was fixed at 10 ng / mL, and the highest working concentration of ActRIIB-hFc-L79D fusion protein and its mutant was 500 nM. Eight concentration gradients were created by 4-fold dilution. The mixture was incubated at 37°C, 5% CO2 for 45 minutes and then added to 96-well plates containing HepG2 cells. After incubating the 96-well plate at 37°C with 5% CO2 for 18 hours, GMOne-Step assay reagent was added, and luciferase activity was analyzed by reading luciferase activity using a Spectramax i3. The IC50 was then calculated. 50 Values. See Table 4 for specific data.

[0200] Table 4. Effects of ActRIIB-hFc-L79D fusion protein and its mutants on GDF-8 and GDF-11 mediated signal transduction.

[0201] The experimental results are shown in Figures 1A-1C. In the absence of any inhibitors, ligands GDF-8 (Figure 1A), GDF-11 (Figure 1B), and Activin A (Figure 1C) all activated the SMAD2 / 3 signaling pathway in HepG2 cells. Upon addition of wild-type ActRIIB-hFc recombinant protein, the activation signals of all three ligands were inhibited. ActRIIB-hFc-L79D-R40K, ActRIIB-hFc-L79D-Y100F, and the positive control ActRIIB-hFc-L79D showed similar inhibitory trends towards GDF-8, GDF-11, and Activin A; that is, they all inhibited the activation signals of growth differentiation factors GDF-8 and GDF-11, but could not inhibit the activation signal of activin A. Their corresponding IC50 values ​​were... 50 As shown in Table 4, under the activation conditions of ligands GDF-8 and GDF-11, the ActRIIB fusion proteins with R40K and Y100F mutations all exhibited higher inhibitory activity than ActRIIB-hFc-L79D.

[0202] Example 7. Affinity determination of the YTE mutant introduced into the Fc region of the ActRIIB fusion protein with human FcRn.

[0203] Mutations at positions M252Y, S254T, and T256E (YTE mutation, amino acid sequence as shown in SEQ ID NO. 7) were introduced into the hFc region of the ActRIIB fusion protein to construct ActRIIB-hFc-L79D-R40K-YTE (amino acid sequence as shown in SEQ ID NO. 8, nucleotide sequence as shown in SEQ ID NO. 12) and ActRIIB-hFc-L79D-Y100F-YTE (amino acid sequence as shown in SEQ ID NO. 9, nucleotide sequence as shown in SEQ ID NO. 13). The effect of the YTE-mutated ActRIIB fusion protein on FcRn binding was verified by ELISA under different pH conditions.

[0204] To assess the binding ability of the fusion protein to FcRn under acidic pH conditions, the fusion protein was diluted to 10 μg / mL with coating buffer, and 100 μL / well was added to an ELISA plate and incubated overnight at 4°C. The coating buffer was removed, and 200 μL / well of blocking buffer (pH 6.0) was added, and the plate was incubated at room temperature for 2 hours. The blocking buffer was then removed, and recombinant human FcRn was diluted to 300 nM with blocking buffer (pH 6.0), creating a 3-fold concentration gradient. 100 μL / well of each gradient was added sequentially to the blocked ELISA plate, and the plate was incubated at 37°C for 1.5 hours. The plate was washed three times with PBST (pH 6.0), and the HRP-labeled anti-6X-His antibody was diluted 1:2000 with blocking buffer (pH 6.0), and 100 μL / well was added to the plate. The plate was incubated at 37°C for 1 hour. Wash the plate three times with PBST (pH 6.0), add 100 μL of TMB per well, and incubate at room temperature in the dark for 30 minutes. Add 50 μL of stop solution per well, read the OD value at 450 nm using a microplate reader, and calculate the EC50. 50 To detect the binding ability of ActRIIB fusion protein to FcRn under neutral pH conditions, the pH of the blocking solution and PBST in the above method was adjusted to 7.4, the initial concentration of recombinant human FcRn was adjusted to 900 nM, and other conditions remained unchanged, and the above operation steps were repeated.

[0205] The experimental results are shown in Figures 2A-2B. After introducing the YTE mutation into the mutants ActRIIB-hFc-L79D-R40K and ActRIIB-hFc-L79D-Y100F, the binding of the YTE mutant to FcRn protein was significantly enhanced at pH 6.0 (Figure 2A); however, at pH 7.4 (Figure 2B), the YTE mutant almost did not bind to FcRn. These results indicate that introducing the YTE mutation into the hFc region can significantly enhance the binding of the ActRIIB fusion protein to FcRn without affecting its normal dissociation.

[0206] Example 8. Inhibitory activity of YTE mutation in the Fc region of ActRIIB fusion protein on cell signaling.

[0207] After introducing the YTE mutation into the mutants ActRIIB-hFc-L79D-R40K and ActRIIB-hFc-L79D-Y100F, the inhibitory effect on TGF-β superfamily-dependent (CAGA)12 transcriptional activation was evaluated using HepG2 cell lines transiently transfected with the pCAGA12-TA-Luc reporter gene. The specific experimental methods were the same as in Example 6.

[0208] The results of the cell signaling assay are shown in Figures 3A-3C. The introduction of the YTE mutation into the Fc region did not alter the inhibitory activity of the ActRIIB-hFc-L79D mutant on cell signaling; that is, the mutant could still inhibit the activation signals of growth differentiation factors GDF-8 (Figure 3A) and GDF-11 (Figure 3B), but could not inhibit the activation signal of activin A (Figure 3C). The corresponding IC50 values... 50 As shown in Table 5, under the activation conditions of ligands GDF-8 and GDF-11, ActRIIB-hFc-L79D-R40K-YTE and ActRIIB-hFc-L79D-Y100F-YTE both exhibited higher inhibitory activity than ActRIIB-hFc-L79D.

[0209] Table 5. Effects of the YTE-mutated ActRIIB-hFc-L79D mutant on GDF-8 and GDF-11 mediated signal transduction.

[0210] Example 9. Introducing a YTE mutation into the Fc region of the ActRIIB fusion protein alleviated docetaxel-induced anemia in mice.

[0211] Docetaxel (DTX) is a common chemotherapeutic drug that acts on the G2 / M cell cycle, inhibiting cell division by blocking microtubule depolymerization. A DTX-induced mouse anemia model was established in B-hFcRn mice (humanized FcRn mice, background C57BL / 6). To compare the effects of the mutants ActRIIB-hFc-L79D-R40K-YTE and ActRIIB-hFc-L79D-Y100F-YTE in this mouse anemia model, mice were randomly assigned to 5 groups of 7 mice each based on body weight. The grouping types and administration methods are shown in Table 6. Blood was collected from the mice before drug treatment, and complete blood cell counts (CBCs) were measured using a five-part differential hematology analyzer to obtain D0 data. Following the administration methods shown in Table 6, each group was first administered DTX via intraperitoneal injection (ip), followed by intravenous injection (iv) via the tail vein 2 hours later. On day 3, blood was drawn and CBC was measured again to obtain the D3 data. The red blood cell count (RBC) and hemoglobin concentration (HGB) were calculated. The data were presented as the rate of change of each indicator on the third day compared to the first day, i.e. (D3-D0) / D0*100.

[0212] Table 6. Docetaxel (DTX)-induced mouse anemia model (experimental period: 3 days)

[0213] The results are shown in Figures 4A-4B. DTX injection alone significantly decreased the number of red blood cells (Figure 4A) and hemoglobin concentration (Figure 4B) in mice. When DTX was administered simultaneously with an inhibitor, the mutants ActRIIB-hFc-L79D-R40K-YTE, ActRIIB-hFc-L79D-Y100F-YTE, and the positive control ActRIIB-hFc-L79D all inhibited the decrease in red blood cell count and hemoglobin concentration. Among them, the mutant ActRIIB-hFc-L79D-R40K-YTE showed a slightly better inhibitory effect. Therefore, the mutant ActRIIB-hFc-L79D-R40K-YTE was selected for a longer-term mouse anemia model experiment.

[0214] Example 10. ActRIIB-hFc-L79D-R40K-YTE can effectively alleviate docetaxel-induced anemia in mice.

[0215] To compare the long-term effects of the mutant ActRIIB-hFc-L79D-R40K-YTE and the positive control ActRIIB-hFc-L79D in a DTX-induced mouse anemia model, a 28-day in vivo experiment was conducted. A DTX-induced mouse anemia model was established in B-hFcRn mice (humanized FcRn mice, background C57BL / 6). Mice were randomly assigned to four groups of six mice each based on body weight. The grouping type and administration route are shown in Table 7. Blood was collected from the mice before drug treatment, and complete blood cell count (CBC) and reticulocyte count (RET) were measured using a five-part differential hematology analyzer to obtain D0 data. Following the administration route in Table 7, on day 1, each group was administered DTX via intraperitoneal injection (ip), followed by intravenous injection (iv) via tail vein two hours later. The drug was then administered again on day 15. On day 28, blood was drawn and CBC and RET were measured again to obtain the data for D28. The red blood cell count (RBC), hemoglobin concentration (HGB), and hematocrit (HCT) were calculated. The data were presented as the rate of change of each indicator on day 28 compared to day 1, i.e. (D28-D0) / D0*100.

[0216] Table 7. Docetaxel (DTX)-induced mouse anemia model (experimental period: 28 days)

[0217] The results are shown in Figures 5A-5D. In mice injected with DTX alone, the number of red blood cells (Figure 5A), hemoglobin concentration (Figure 5B), hematocrit (Figure 5C), and reticulocyte count (Figure 5D) were comparable to those in the control group. When DTX and the mutant ActRIIB-hFc-L79D-R40K-YTE were administered simultaneously, the number of red blood cells, hemoglobin concentration, hematocrit, and reticulocyte count were significantly increased compared to the DTX anemia group, with changes of 20%, 10%, 12%, and 14% compared to D0, respectively. The number of red blood cells in the group simultaneously administered DTX and the positive control ActRIIB-hFc-L79D was significantly increased compared to the DTX anemia group, but there were no significant differences in hemoglobin concentration, hematocrit, and reticulocyte count. In summary, the mutant ActRIIB-hFc-L79D-R40K-YTE can effectively counteract docetaxel-induced anemia in mice, and its anemia recovery effect is superior to that of the marketed drug rotezip.

[0218] Example 11. In vivo activity of ACTRⅡB-hFc-L79D-R40K-YTE in improving anemia in different types of models.

[0219] To verify the in vivo activity of the mutant ACTRIIB-cFc-L79D-R40K-YTE in improving anemia, the inventors established a docetaxel (DTX)-induced mouse anemia model, a spontaneous β-thalassemia mouse model, and a rat acute blood loss anemia model to investigate the in vivo activity of the mutant ACTRIIB-hFc-L79D-R40K-YTE.

[0220] 1) Pharmacodynamic study of the mutant ACTRⅡB-hFc-L79D-R40K-YTE in a docetaxel-induced mouse anemia model

[0221] Docetaxel is a cytotoxic antitumor drug that inhibits cell mitosis by promoting microtubule polymerization and inhibiting depolymerization, thereby disrupting the microtubule network structure. While inhibiting tumor growth, it also causes adverse reactions in the body, including bone marrow suppression and anemia. Therefore, the inventors established a cytotoxic drug-induced anemia model by injecting docetaxel into normal mice. The therapeutic effect of the mutant ACTRⅡB-hFc-L79D-R40K-YTE on anemia was then investigated in this model.

[0222] The experimental design included a G1 blank control group, a G2 DTX-induced anemia model group, a G3 DTX-induced anemia model group combined with 1 mg / kg of the positive control drug Luspatercept, and G4-G7 groups treated with the mutant ACTRⅡB-hFc-L79D-R40K-YTE at doses of 0.3 mg / kg, 1 mg / kg, 3 mg / kg, and 10 mg / kg, respectively. On day 0 (D0), mice were weighed and randomly assigned to each group according to their body weight. 20-30 μL of blood was collected from the retro-orbital venous plexus, anticoagulated with EDTA, and used for complete blood count, differential count, and reticulocyte count (CBC+DIFF+RET mode). The results were baseline values ​​for each animal. On day 1 (D1), animals in groups G1-G7 were administered the drug subcutaneously according to the protocol. Two hours after administration, animals in groups G2-G7 were injected with DTX via the tail vein. Subsequently, patients were weighed twice weekly. A second subcutaneous drug was administered on day 14. Blood samples were collected on days 4, 7, 14, 21, and 28 to measure CBC, DIFF, and RET. Data at each time point were expressed as raw data and / or the rate of change compared to baseline (X). t -X0) / X0*100%) means.

[0223] The experimental results, as shown in Tables 8-10 and Figure 6A, indicate that the mutant ACTRⅡB-hFc-L79D-R40K-YTE effectively improved the levels of RBC, HGB, and HCT in the peripheral blood of blood-loss rats, thus improving anemia. At the start of the experiment (D0), the mean number of peripheral blood erythrocytes in each group of mice was within the normal range of 10.6 × 10⁻⁶. 6 The number of red blood cells per μL of whole blood was approximately [number missing]. On day 4 (D4), the peripheral blood red blood cell count in group G2 (DTX+PBS) decreased to 8.83 ± 0.13 × 10 [units missing]. 6 The number of red blood cells / μL was significantly lower than that in the G1 control group, indicating signs of anemia. This demonstrates that DTX injection can lead to a decrease in peripheral blood red blood cell count and anemia in animals. In the G3 group (DTX + Luspatercept 1 mg / kg), the peripheral blood red blood cell count was 9.76 ± 0.16 × 10⁻⁶ cells / μL. 6 The number of cells / μL was lower than that in the blank control group, but significantly higher than that in the G2 model group. This indicates that rotezip has a significant ameliorative effect on DTX-induced anemia. The peripheral blood erythrocyte counts in the G4-G7 groups (mutant ACTRⅡB-hFc-L79D-R40K-YTE dose groups of 0.3 mg / kg, 1 mg / kg, 3 mg / kg, and 10 mg / kg) were 8.77 ± 0.17 × 10⁻⁶ cells / μL. 6 cells / μL, 9.41±0.14×10 6 cells / μL, 9.63±0.13×10 6 9.87 ± 0.13 × 10⁻⁶ cells / μL and 9.87 ± 0.13 × 10⁻⁶ cells / μL 6The number of cells / μL was significantly higher in all groups except for group G4 (mutant ACTRIIB-hFc-L79D-R40K-YTE 0.3 mg / kg), which was essentially the same as group G2. This showed a dose-dependent increasing pattern. This indicates that the mutant ACTRIIB-hFc-L79D-R40K-YTE can dose-dependently improve DTX-induced peripheral blood erythrocyte reduction. Peripheral blood erythrocyte levels were continuously monitored on days 7, 14, 21, and 28. In group G2 (DTX+PBS), peripheral blood erythrocyte levels gradually returned to normal. On days 14, 21, and 28, peripheral blood erythrocyte levels in groups G3-G7 were higher than in group G2. At the end of the experiment (day 28), the peripheral blood erythrocyte count in group G3 (DTX+Luspatercept 1 mg / kg) was higher than that in the blank control group and the G2 model group. In groups G4-G7 (mutant ACTRIIB-hFc-L79D-R40K-YTE dose groups of 0.3 mg / kg, 1 mg / kg, 3 mg / kg, and 10 mg / kg), the peripheral blood erythrocyte counts of animals were higher than those of the G2 model group and the blank control group, except for group G4 (mutant ACTRIIB-hFc-L79D-R40K-YTE 0.3 mg / kg), which was lower than normal. This showed a dose-dependent increasing pattern. This indicates that the mutant ACTRIIB-hFc-L79D-R40K-YTE can dose-dependently and continuously improve the decrease in peripheral blood erythrocytes induced by DTX and continuously increase the number of peripheral blood erythrocytes. The same dose of mutant ACTRIIB-hFc-L79D-R40K-YTE (1 mg / kg) was superior to rotezip (1 mg / kg) in improving anemia. Peripheral blood HGB and HCT levels in all treatment groups also improved with a similar trend.

[0224] Table 8. Changes in peripheral blood erythrocyte count (RBC) in mice of each test group

[0225] Table 9. Changes in peripheral blood hemoglobin (HGB) levels in mice of each test group

[0226] Table 10. Changes in hematocrit (HCT) in peripheral blood of mice in each test group

[0227] 2) Pharmacodynamic study of the mutant ACTRⅡB-hFc-L79D-R40K-YTE in a spontaneous β-thalassemia mouse model

[0228] Thalassemia is a hereditary disease caused by autosomal gene defects leading to abnormal hemoglobin levels. Clinically, it is classified into α-thalassemia, β-thalassemia, and γ-thalassemia based on genotyping. The Hbb-bs&Hbb-bt DKO mouse is a genetically engineered mouse model created by simultaneously knocking out the Hbb-bs and Hbb-bt genes in C57BL / 6J mice using gene editing technology. Heterozygous Hbb-bs&Hbb-bt DKO mice spontaneously exhibit the typical characteristics of severe β-thalassemia, including abnormalities in RBC, HGB, HCT, and erythrocyte morphology. Therefore, the inventors investigated and evaluated the activity of the mutant ACTRⅡB-hFc-L79D-R40K-YTE in improving anemia using this spontaneous β-thalassemia mouse model.

[0229] The experimental design included a wild-type C57BL / 6 mouse control group, an Hbb-bs&Hbb-bt DKO mouse model group, and a mutant ACTRⅡB-hFc-L79D-R40K-YTE group treated with a dose of 10 mg / kg, administered subcutaneously once a week for a total of 4 weeks. Blood samples were collected on days 0, 7, 14, 21, and 28 of the experiment, and complete blood counts were performed on mice using a five-part differential hematology analyzer in CBC+RET mode. Data at each time point were expressed as raw data and / or the rate of change compared to baseline (X). t -X0) / X0*100%) means.

[0230] The results, as shown in Tables 11-13 and Figure 6B, indicate that the mutant ACTRⅡB-hFc-L79D-R40K-YTE effectively improved peripheral blood RBC, HGB, and HCT levels, demonstrating therapeutic efficacy against thalassemia. At the start of the experiment (D0), the average peripheral blood erythrocyte count in wild-type C57BL / 6 mice was 11.08 ± 0.13 × 10⁻⁶. 6 The mean peripheral blood erythrocyte count in age-matched Hbb-bs & Hbb-bt DKO mice was 7.73 ± 0.16 × 10⁶ cells / μL in group G2. 6 Cells / μL, G3 group 7.61±0.31×10 6 The RBC count / μL was significantly lower than that of wild-type mice, with no significant difference between the G2 and G3 groups. This indicates spontaneous anemia in Hbb-bs & Hbb-bt DKO mice. Subsequently, on days 7, 14, 21, and 28, the RBC counts of Hbb-bs & Hbb-bt DKO mice in the G2 group were 8.1 ± 0.25 × 10⁻⁶. 6 cells / μL, 8.06±0.2×10 6 cells / μL, 8.6±0.31×10 6 8.54 ± 0.14 × 10⁻⁶ cells / μL and 8.54 ± 0.14 × 10⁻⁶ cells / μL 6The RBC count / μL was slightly higher than the initial D0 level, but still remained at a low level; the peripheral blood RBC count in the G3 mutant ACTRⅡB-hFc-L79D-R40K-YTE treatment group was 9.54±0.15×10⁻⁶. 6 cells / μL, 9.87±0.15×10 6 cells / μL, 10.8±0.5×10 6 11.92 ± 0.3 × 10⁻⁶ cells / μL and 11.92 ± 0.3 × 10⁻⁶ cells / μL 6 The RBC count / μL showed a gradual upward trend compared to the initial level at D0. At D7, the RBC value in the treatment group was significantly higher than that in the G2 model control group; at D21 and D28, the RBC value in the treatment group was even higher than that in G1 wild-type mice. This indicates that after administration of the mutant ACTRⅡB-hFc-L79D-R40K-YTE, the RBC value continued to increase, which could restore or even exceed the RBC level in spontaneously thalassemia mice. Peripheral blood HGB and HCT levels in the treatment group also improved with a similar trend.

[0231] Table 11. Changes in peripheral blood RBCs in mice of each test group

[0232] Table 12. Changes in peripheral blood HGB in mice of each test group

[0233] Table 13. Changes in peripheral blood hematocrit (HCT) in mice of each test group

[0234] 3) Pharmacodynamic study of the mutant ACTRⅡB-hFc-L79D-R40K-YTE in a rat model of acute blood loss anemia.

[0235] Acute blood loss anemia is a common condition in the emergency department, referring to a decrease in blood volume and hemoglobin levels caused by massive bleeding within a short period. It is commonly seen in cases of trauma, surgery, gastrointestinal bleeding, and intrathoracic or abdominal hemorrhage. Following acute bleeding, the decrease in red blood cell count and hemoglobin corresponds to the severity of the bleeding, resulting in normocytic anemia. Regeneration signs (increased reticulocyte count) may appear a few days after the bleeding. Therefore, the inventors established a rat model of acute blood loss anemia using acute exsanguination and investigated the activity of the mutant ACTRⅡB-hFc-L79D-R40K-YTE in improving anemia within this model.

[0236] The experimental design included a G1 rat blank control group, a G2 acute hemorrhagic anemia model group, and G3-G5 groups of rats receiving low, medium, and high doses of the mutant ACTRⅡB-hFc-L79D-R40K-YTE at 1 mg / kg, 3 mg / kg, and 10 mg / kg. The mutant ACTRⅡB-hFc-L79D-R40K-YTE was administered subcutaneously twice weekly for a total of two administrations. On day -1 (D-1), rats were weighed and randomly assigned to groups based on their weight. Circulating blood volume was calculated for each rat based on its weight. The calculation formula was: Total blood volume (mL) = 0.062 × body weight (g) + 0.0012. 20-30 μL of blood was collected from all rats via the retroorbital venous plexus, anticoagulated with EDTA, and the complete blood cell count and reticulocyte count (CBC+RET mode) were measured using a five-part differential hematology analyzer. The results were the baseline values ​​for each animal. Subsequently, in groups G2-G5, after isoflurane anesthesia, 1 / 5 of the circulating blood volume was extracted via the jugular vein, and the same volume of physiological saline was immediately administered via the tail vein to establish an acute blood loss model. On day 0, rats were weighed, and blood was collected from the retro-orbital venous plexus to measure CBC+RET. The mutant ACTRⅡB-hFc-L79D-R40K-YTE was administered according to the protocol. On day 2, blood was collected to measure CBC+RET. On day 3, rats were weighed and administered the drug. On day 6, blood was collected to measure CBC+RET. Data at each time point are expressed as raw data and / or the rate of change compared to baseline values ​​(X). t -X0) / X0*100%) means.

[0237] The results, as shown in Tables 14-16 and Figure 6C, indicate that the mutant ACTRⅡB-hFc-L79D-R40K-YTE effectively improved the levels of RBC, HGB, and HCT in the peripheral blood of blood-loss rats, thus improving anemia. At the start of the experiment (D-1), the mean RBC value of all rats was 8.19 × 10⁻⁶. 6 The RBC count / μL showed no significant difference between groups and was considered normal for rats. After acute blood loss was administered to the model rats at D-1, the mean RBC count measured at D0 was 8.10 ± 0.06 × 10⁻⁶ in the G1 blank control group. 6 Cells / μL, G2 model group 6.85±0.19×10 6 Cells / μL, G3 group 6.82±0.10×10 6 Cells / μL, G4 group 6.73±0.10×10 6 The concentration of cells / μL in group G5 was 6.64 ± 0.20 × 10⁻⁶. 6 RBC count / μL, relative to the baseline value of D-1, shows that acute blood loss caused a significant decrease in RBC count in rats in groups G2-G5. The degree of RBC decrease was similar among groups, with no significant difference.

[0238] On the second day after drug administration (D2), the mean RBC values ​​of the rats in each group were 8.07 ± 0.06 × 10⁻⁶ in the G1 blank control group. 6 Cells / μL, G2 model group 6.63±0.12×10 6 Cells / μL, G3 group 7.56±0.05×10 6 Cells / μL, G4 group 7.58±0.07×10 6 The concentration of cells / μL in group G5 was 7.82 ± 0.13 × 10⁻⁶. 6 The RBC levels in the G2 model group remained low, while the RBC levels in the G3, G4, and G5 treatment groups were significantly higher than those in the G2 model group, approaching those in the G1 blank control group. There were no significant differences between the treatment groups. This indicates that the RBC levels in the groups treated with the mutant ACTRⅡB-hFc-L79D-R40K-YTE rapidly recovered to near normal levels.

[0239] At the end of the experiment (D6), the mean RBC values ​​of the rats in each group were measured as follows: G1 blank control group: 8.01 ± 0.08 × 10⁻⁶. 6 Cells / μL, G2 model group 7.41±0.12×10 6 Cells / μL, G3 group 8.30±0.10×10 6 Cells / μL, G4 group 8.46±0.02×10 6 The concentration of cells / μL in group G5 was 8.57 ± 0.07 × 10⁻⁶. 6 RBC levels in the G2 model group, relative to the baseline value at D-1, showed a slow increase to near the initial level. In contrast, the RBC levels in the G3, G4, and G5 treatment groups showed a sustained increase compared to D2, significantly higher than the G2 model group and also higher than the G1 blank control group. No significant differences were observed between the treatment groups. This indicates that the RBC levels in the animals treated with the mutant ACTRIIB-hFc-L79D-R40K-YTE showed a sustained increase, even exceeding the initial normal level. In this model, the mutant ACTRIIB-hFc-L79D-R40K-YTE can rapidly exert its effect, restoring the RBC / HGB / HCT levels in anemic animals to near-normal levels as early as the day after blood loss, significantly earlier than animals that recover naturally. With repeated administration, RBC, HGB, and HCT levels can recover to or even exceed normal levels.

[0240] Table 14. Changes in peripheral blood RBC counts in rats of each test group

[0241] Table 15. Changes in peripheral blood HGB in rats of different test groups

[0242] Table 16. Changes in peripheral blood hematocrit (HCT) in rats of different test groups

[0243] Experimental results showed that in different types of anemia models, the mutant ACTRⅡB-hFc-L79D-R40K-YTE could increase peripheral blood red blood cell count (RBC), hemoglobin content (HGB), and hematocrit (HCT) to varying degrees, and improve anemia symptoms in a dose-dependent manner.

[0244] The above experiments demonstrate that a novel ActRIIB-hFc fusion protein was obtained through conserved mutations of the ActRIIB extracellular domain fragment, exhibiting similar or even superior activity and function to the already marketed drug, rotegcip. In HepG2 cell bioactivity validation based on the transient transition (CAGA)12 element, the ActRIIB fusion protein of this invention maintained its inhibition of SMAD2 / 3 signaling pathway activation mediated by growth differentiation factors GDF-8 and GDF-11, while losing its inhibition of activin A-mediated signaling pathway. In a docetaxel-induced mouse anemia model, the ActRIIB fusion protein of this invention effectively inhibited the decrease in red blood cell count and hemoglobin concentration on day 3. In a 28-day in vivo experiment, the ActRIIB fusion protein of this invention significantly increased red blood cell count, hemoglobin concentration, hematocrit, and reticulocyte count, demonstrating a superior and longer-lasting effect in increasing red blood cell levels compared to rotegcip. Therefore, the novel ActRIIB fusion protein of this invention can be developed into a drug with superior efficacy for the treatment or prevention of anemia.

Claims

1. An ActRIIB fusion protein, characterized in that, The ActRIIB fusion protein comprises: (a) A fragment of the extracellular domain of ActRIIB, wherein the fragment begins at position 19, 20, 21, 22, 23, 24, or 25 of SEQ ID NO. 1; ends at position 128, 129, 130, or 131 of SEQ ID NO. 1; and, except that the leucine at position 79 of SEQ ID NO. 1 is replaced by an acidic amino acid, the fragment further comprises one or more amino acid substitutions at other positions; and (b) Optionally, Fc of IgG; Preferably, the fragment of the extracellular domain of ActRIIB begins at position 25 of SEQ ID NO.1 and ends at position 131 of SEQ ID NO.1, and the leucine at position 79 is replaced by the acidic amino acid aspartic acid, as shown in SEQ ID NO.

2.

2. The ActRIIB fusion protein as described in claim 1, characterized in that, The amino acid substitutions at one or more other positions are selected from R40, Y100, H115, or T131.

3. The ActRIIB fusion protein as described in claim 1, characterized in that, The Fc of the IgG is wild-type or mutant; preferably, the Fc of the IgG contains amino acid substitutions at one or more positions of M252, S254, and T256.

4. The ActRIIB fusion protein as described in claim 1, characterized in that, The ActRIIB fusion protein further includes a linker between a fragment of the ActRIIB extracellular domain and the Fc of the IgG; preferably, the linker is three glycine residues.

5. The ActRIIB fusion protein as described in claim 1, characterized in that, The ActRIIB fusion protein is selected from the following group: (i) Contains an amino acid sequence selected from the group consisting of: SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.8 or SEQ ID NO.9; (ii) A polypeptide derived from (i) formed by substitution, deletion and / or addition of one or more amino acids in the amino acid sequence of (i) and having activity of binding growth differentiation factors.

6. An isolated polynucleotide molecule, characterized in that, The polynucleotide molecule encodes the ActRIIB fusion protein as described in any one of claims 1-5; preferably, the polynucleotide molecule comprises a nucleotide sequence selected from the group consisting of: SEQ ID NO.10, SEQ ID NO.11, SEQ ID NO.12 or SEQ ID NO.

13.

7. An expression carrier, characterized in that, The expression vector contains the polynucleotide molecule as described in claim 6.

8. A host cell, characterized in that, The host cell contains the expression vector as described in claim 7.

9. The method for preparing the ActRIIB fusion protein according to any one of claims 1-5, characterized in that, The method includes the following steps: 1) Under expression conditions, host cells as described in claim 8 are cultured to express the ActRIIB fusion protein as described in any one of claims 1-5; 2) Isolate and purify the ActRIIB fusion protein described in step 1).

10. An immunoconjugate, characterized in that, The immunoconjugate comprises: (I) The ActRIIB fusion protein as described in any one of claims 1-5; and (II) Selected coupling portions from the following groups: detectable markers, drugs, toxins, cytokines, radionuclides, enzymes, or combinations thereof.

11. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises an effective amount of the ActRIIB fusion protein as described in any one of claims 1-5 or the immunoconjugate as described in claim 10, and one or more pharmaceutically acceptable carriers, diluents, or excipients.

12. Use of the ActRIIB fusion protein of any one of claims 1-5, the immunoconjugate of claim 10, or the pharmaceutical composition of claim 11 in the preparation of a medicament for increasing red blood cell levels or for treating or preventing anemia; preferably, the anemia is selected from: anemia associated with myelodysplastic syndrome, anemia associated with renal disorders, anemia associated with chronic kidney disease, anemia associated with acute kidney disease, anemia associated with chemotherapy, anemia caused by blood loss, anemia associated with multiple myeloma, anemia associated with thalassemia, anemia associated with tumors or cancer, anemia associated with acute renal failure, anemia associated with chronic renal failure, anemia associated with acute kidney disease, anemia associated with chronic kidney disease, anemia associated with end-stage renal disease, anemia associated with sickle cell disease, or a combination of multiple anemias.

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