Fusion protein of human thrombopoietin receptor agonist

Abstract

The present invention relates to a human thrombopoietin receptor (TPOR) binding molecule, and particularly a fusion protein specifically binding to TPOR and a fragment thereof. Furthermore, the present invention further relates to a nucleic acid or host cell containing the fusion protein or the fragment thereof, a drug containing the fusion protein or the fragment thereof, and treatment and diagnosis methods or uses by means of using the fusion protein or the fragment thereof.

Description

A fusion protein of human thrombopoietin receptor agonist

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application is based on the Chinese patent application with application number 202310763558.4 and application date June 26, 2023, and claims the priority of the Chinese patent application. The entire content of the Chinese patent application is hereby introduced into this application as a reference. Technical Field

[0003] The present disclosure relates to human thrombopoietin receptor (TPOR) binding molecules, particularly fusion proteins and fragments thereof that specifically bind to TPOR. Furthermore, the present invention relates to nucleic acids or host cells comprising such fusion proteins or fragments thereof, medicaments comprising such fusion proteins or fragments thereof, and therapeutic and diagnostic methods or uses utilizing such fusion proteins and fragments. Background Art

[0004] Thrombopoietin (TPO) is the primary cytokine that physiologically stimulates megakaryocyte proliferation and differentiation. It is a glycoprotein with a molecular weight of approximately 35.5 kDa, primarily synthesized in the liver. TPO binds to its receptor, c-Mpl (TPOR), causing c-Mpl to form homodimers, thereby activating a series of intracellular signaling pathways, leading to megakaryocyte proliferation and differentiation. TPO is a signaling peptide synthesized and secreted by the liver and released into the bloodstream. It regulates megakaryocyte differentiation and platelet production, making it a major regulator of platelet production.

[0005] Thrombocytopenia is a bleeding disorder characterized by an abnormally low number of platelets in the peripheral blood. Examples include primary immune thrombocytopenia, thrombocytopenia caused by cancer chemotherapy, thrombocytopenia associated with liver disease, and aplastic anemia. There are numerous types of thrombocytopenia, with a large patient base, including tens of millions in China. However, existing therapies have limited clinical benefits, creating a significant unmet clinical need.

[0006] Immune thrombocytopenia (ITP), also known as idiopathic thrombocytopenic purpura, is an acquired bleeding disorder characterized by excessive platelet destruction and impaired megakaryocyte maturation mediated by humoral and cellular immunity. It accounts for approximately one-third of all bleeding disorders. The annual incidence of ITP in adults in my country is 5-10 per 100,000, with the elderly over 60 years of age being the most susceptible group. It is also the most common bleeding disorder in children. ITP patients most commonly present with mucous membrane bleeding, but in severe cases, internal bleeding and even intracranial hemorrhage may occur. Severe ITP can be fatal.

[0007] Thrombocytopenia occurs in up to 76% to 84% of patients with chronic liver disease (CLD). The hemostasis problem of CLD patients is further complicated by the presence of thrombocytopenia and platelet dysfunction. Thrombocytopenia may be caused by: splenomegaly or hypersplenism after portal hypertension, decreased production of thrombopoietin by the liver, and increased platelet destruction or consumption. Studies have shown that patients with cirrhosis and severe thrombocytopenia have an increased bleeding rate when undergoing invasive surgery. Of course, the risk of bleeding is also affected by the severity of portal hypertension, concurrent coagulation abnormalities, and the degree of invasive surgery. In different clinical studies, the platelet threshold required is 40×10 9 / L to 75×10 9 / L.

[0008] Chemotherapy-induced thrombocytopenia (CIT) refers to the inhibition of bone marrow megakaryocytes by anti-tumor chemotherapy drugs, resulting in a platelet count in the peripheral blood lower than 100×10 9 CIT is one of the most common chemotherapy-related hematologic toxicities, increasing bleeding risk, prolonging hospitalization, increasing medical costs, and, in severe cases, leading to death. CIT can lead to reduced chemotherapy dose intensity, delayed treatment, or even treatment discontinuation, thereby compromising anti-tumor efficacy and adversely affecting patients' long-term survival. The pathogenesis of CIT involves multiple factors, primarily including decreased platelet production, increased platelet destruction, and abnormal platelet distribution. Specifically, the pathogenesis of CIT includes 1) decreased platelet production: Chemotherapeutic drugs can affect various stages of platelet production, including inhibiting the proliferation of hematopoietic stem cells and megakaryocyte progenitor cells, reducing megakaryocyte production, and inhibiting megakaryocyte production and platelet release, ultimately leading to thrombocytopenia; 2) increased platelet destruction: Chemotherapeutic drugs can cause drug-induced immune thrombocytopenia. Previous reports have shown that repeated use of oxaliplatin can induce and maintain an immune response, leading to immune thrombocytopenia. Platelet antibodies can be detected in patients' blood, leading to increased platelet destruction. Clinical manifestations include sudden, isolated thrombocytopenia, usually after a cumulative oxaliplatin dose of >850 mg / m 2 3) Abnormal distribution: Chemotherapy drugs cause sinusoidal damage, resulting in damage and shedding of sinusoidal endothelial cells, sinusoidal obstruction, and, in turn, portal hypertension and hypersplenism. Platelet retention and destruction in the spleen increase, leading to a decrease in peripheral blood platelet counts. This condition often manifests as a persistent decrease in platelet counts, and patients may develop complications such as splenomegaly and portal hypertension.

[0009] [Corrected 26.08.2024 according to Rule 26] Platelets are produced by megakaryocytes in the hematopoietic tissue of the bone marrow. Thrombopoietin can promote the production of platelets in the human body by stimulating the growth and differentiation of megakaryocytes. The thrombopoietin drugs currently used in clinical practice mainly include recombinant human thrombopoietin (rhTPO) such as Thrombopoietin receptor agonist (TPO-RA) (Romiplostim) and eltrombopag. It is a recombinant human thrombopoietin (rhTPO), which is expressed by Chinese hamster ovary cells using genetic recombination technology and purified. It has a similar pharmacological effect of increasing platelets as endogenous thrombopoietin and was launched in the mainland in 2005. It is a fusion protein composed of a TPO-mimicking peptide that binds to TPOR and a human IgG1 Fc carrier region. It is a TPO receptor agonist that stimulates intracellular transcriptional pathways by binding to the TPO receptor, leading to increased platelet production. Produced in Escherichia coli using recombinant DNA technology, it mimics the effects of the body's natural thrombopoietin, thereby increasing platelet counts. When bound to the TPO receptor, it promotes the growth of bone marrow megakaryocyte colony-forming cells (CFU-Meg) and activates signal transduction pathways through the JAK2 and STAT5 kinase pathways, stimulating the differentiation and proliferation of human bone marrow progenitor cells into megakaryocytes, promoting megakaryocyte maturation, and increasing platelet production.

[0010] Despite the increasing availability of treatment options for thrombocytopenia and significant progress in efficacy, its treatment remains challenging and thorny: ① Glucocorticoids, as a first-line treatment for ITP, have poor long-term efficacy and are associated with numerous adverse reactions; ② Existing TPO-RA small molecule drugs can cause liver damage with long-term use; ③ rhTPO requires daily injections, has a low response rate, is not suitable for long-term use, and is prone to relapse upon discontinuation. Given that previous treatment strategies have failed to meet patient needs, the emergence of new drugs for the treatment of thrombocytopenia remains an urgent need.

[0011] Therefore, there is a need for a new fusion protein that specifically binds to the human thrombopoietin receptor.

[0012] Summary of the Invention

[0013] The present invention provides a novel binding molecule (eg, fusion protein) capable of specifically binding to human thrombopoietin receptor (TPOR), which can activate TPOR and is further used for treating thrombocytopenia.

[0014] In some embodiments, the fusion protein of the present invention that specifically binds to TPOR is more effective than known fusion proteins such as With improved properties.

[0015] In some embodiments, the fusion proteins of the present invention that specifically bind to TPOR, for example, compared to known fusion proteins that specifically bind to TPOR, for example Having reduced affinity for binding to an Fc receptor, such as FcγRIIb, FcγRIIa H167, FcγRIIa R167, FcγRIIIa F176, FcγRIIIa V176, and / or human FcγRI.

[0016] In some embodiments, the fusion protein of the present invention that specifically binds to TPOR does not bind to C1q.

[0017] In some embodiments, the fusion protein of the present invention that specifically binds to TPOR can promote the proliferation and differentiation of platelet-producing cells, such as megakaryocytes, compared to known fusion proteins that specifically bind to TPOR, such as It has improved properties in promoting the proliferation and differentiation of platelet-producing cells, such as megakaryocytes.

[0018] In some embodiments, the fusion proteins of the present invention that specifically bind to TPOR, for example, compared to known fusion proteins that specifically bind to TPOR, for example The invention also provides a novel polypeptide having an improved half-life and / or an increased affinity for human FcRn (e.g., at pH 6.0).

[0019] In some embodiments, the fusion protein that specifically binds to TPOR of the present invention can specifically bind to TPOR (eg, human TPOR) on the cell surface and / or competitively inhibit the binding of TPO to cells expressing TPOR.

[0020] In some embodiments, the fusion protein of the present invention that specifically binds to TPOR can activate downstream signaling pathways in cells expressing TPO.

[0021] In some embodiments, the fusion protein of the present invention that specifically binds to TPOR does not have Fc-mediated effector function, such as ADCC or CDC or ADCP.

[0022] In some embodiments, the fusion protein that specifically binds to TPOR of the present invention can promote the increase of the number of platelets in blood circulation, for example, compared to the known fusion protein that specifically binds to TPOR, for example Improves the number of platelets in blood circulation.

[0023] In some embodiments, the fusion protein of the present invention that specifically binds to TPOR can bind to human and / or cynomolgus monkey platelets.

[0024] In some embodiments, the fusion protein of the present invention that specifically binds to TPOR can induce differentiation of hematopoietic progenitor cells, for example, differentiation into megakaryocyte progenitor cells and / or megakaryocytes.

[0025] In some embodiments, the present invention relates to the following specific embodiments:

[0026] 1. A thrombopoietin receptor (TPOR) agonist fusion protein comprising a TPOR-binding domain and an Fc variant domain, wherein the TPOR-binding domain is a TPO mimetic peptide, wherein the TPO mimetic peptide comprises or consists of the amino acid sequence shown in SEQ ID NO: 9, and the Fc variant domain

[0027] (i) derived from IgG1 and comprising a combination of mutations selected from the following compared to wild-type IgG1

[0028] a) L234A, L235A, D265S; or

[0029] b) L234A, L235A, D265S, M252Y, S254T, T256E;

[0030] (ii) is derived from IgG4 and comprises, compared to wild-type IgG1, a mutation or combination of mutations selected from the group consisting of:

[0031] a)S228P;

[0032] b)S228P, F234A, L235A;

[0033] c) S228P, T250Q, M428L; or

[0034] d)S228P, F234A, L235A, T250Q, M428L.

[0035] 2. The fusion protein of embodiment 1, wherein the Fc variant domain does not contain methionine at the N-terminus.

[0036] 3. The fusion protein of embodiment 1 or 2, wherein the Fc variant domain

[0037] a) comprising or consisting of an amino acid sequence of SEQ ID NO: 11, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto, preferably said Fc variant domain further comprises L234A+L235A+D265S mutations;

[0038] b) comprising or consisting of an amino acid sequence of SEQ ID NO: 12, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, preferably said Fc variant domain further comprising L234A+L235A+D265S+M252Y+S254T+T256E mutations;

[0039] c) comprising or consisting of an amino acid sequence of SEQ ID NO: 13, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, preferably said Fc variant domain further comprises an S228P mutation;

[0040] d) comprising or consisting of an amino acid sequence of SEQ ID NO: 14, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto, preferably said Fc variant domain further comprises S228P+F234A+L235A mutations;

[0041] e) comprising or consisting of an amino acid sequence of SEQ ID NO: 15, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, preferably said Fc variant domain further comprising S228P+T250Q+M428L mutations; or

[0042] f) comprising or consisting of an amino acid sequence of SEQ ID NO: 16, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto, preferably the Fc variant domain further comprises S228P+F234A+L235A+T250Q+M428L mutations.

[0043] 4. The fusion protein according to any one of embodiments 1 to 3, comprising a peptide chain containing two tandemly linked TPO mimetic peptides and an Fc variant domain or consisting of two tandemly linked TPO mimetic peptides and an Fc variant domain.

[0044] 5. The fusion protein of embodiment 4, wherein the peptide chain comprises or consists of the following from N-terminus to C-terminus: Fc variant domain-first linker-first TPO mimetic peptide-second linker-second TPO mimetic peptide, optionally, the first linker and / or the second linker may be present or absent; or the first TPO mimetic peptide and the second TPO mimetic peptide may be the same or different.

[0045] 6. The fusion protein of embodiment 5, comprising two peptide chains, wherein the two peptide chains may be the same or different.

[0046] 7. The fusion protein of embodiment 5 or 6, wherein the first linker and / or the second linker is a glycine linker comprising 5, 6, 7 or 8 glycines.

[0047] 8. The fusion protein of embodiment 7, wherein the first linker comprises 5 glycines and the second linker comprises 8 glycines.

[0048] 9. The fusion protein according to any one of embodiments 5-8, wherein the peptide chain comprises

[0049] (i) the amino acid sequence of SEQ ID NO: 2, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto, or consisting thereof, preferably comprising the L234A+L235A+D265S mutations;

[0050] (ii) the amino acid sequence of SEQ ID NO: 3, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto, or consisting thereof, preferably comprising the L234A+L235A+D265S+M252Y+S254T+T256E mutations;

[0051] (iii) an amino acid sequence of SEQ ID NO: 4, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto, or consisting thereof, preferably comprising the S228P mutation;

[0052] (iv) an amino acid sequence of SEQ ID NO: 5, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto, or consisting thereof, preferably comprising S228P+F234A+L235A mutations;

[0053] (v) an amino acid sequence of SEQ ID NO: 6, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto, or consisting thereof, preferably comprising S228P+T250Q+M428L mutations; or

[0054] (vi). the amino acid sequence of SEQ ID NO: 7, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto, or consisting thereof, preferably comprising S228P+F234A+L235A+T250Q+M428L mutations.

[0055] 10. An isolated polynucleotide encoding one or more chains of the fusion protein of any one of embodiments 1-9.

[0056] 11. An expression vector comprising the polynucleotide of embodiment 10.

[0057] 12. A host cell comprising the polynucleotide of embodiment 10 or the vector of embodiment 11, preferably the host cell is a yeast cell or a mammalian cell, in particular a HEK293 cell or a CHO cell.

[0058] 13. A method for producing the fusion protein of any one of embodiments 1-9, comprising culturing the host cell of embodiment 12 under conditions suitable for expressing the fusion protein.

[0059] 14. A pharmaceutical composition comprising the fusion protein of any one of embodiments 1-9, optionally and a pharmaceutically acceptable excipient.

[0060] 15. Use of the fusion protein described in any one of Embodiment 19 or the pharmaceutical composition described in Embodiment 14 in the preparation of a medicament for preventing and / or treating thrombocytopenia, preferably, the thrombocytopenia is primary immune thrombocytopenia, thrombocytopenia caused by tumor chemotherapy, thrombocytopenia associated with liver disease, or aplastic anemia.

[0061] 16. A method for preventing and / or treating thrombocytopenia in a subject, the method comprising administering to the subject the fusion protein of any one of embodiments 1-9 or the pharmaceutical composition of embodiment 14, preferably, the thrombocytopenia is primary immune thrombocytopenia, thrombocytopenia caused by tumor chemotherapy, thrombocytopenia associated with liver disease, or aplastic anemia. BRIEF DESCRIPTION OF THE DRAWINGS

[0062] The preferred embodiments of the present invention described in detail below will be better understood when read in conjunction with the following drawings. For the purpose of illustrating the present invention, the drawings show presently preferred embodiments. However, it should be understood that the present invention is not limited to the precise arrangements and means of the embodiments shown in the drawings.

[0063] FIG1 shows the detection of TPO-RA fusion protein promoting the proliferation of M07e cells.

[0064] FIG2 shows the binding of TPO-RA fusion protein to cell surface TPO-R.

[0065] FIG3 shows the blocking effect of TPO-RA fusion protein on the binding of TPO-Biotin to human TPO-R on the cell surface.

[0066] FIG4 shows the activation detection of the HEK Blue TPO cell signaling pathway by TPO-RA fusion protein.

[0067] FIG5 shows the ADCP functional activity detection of TPO-RA fusion protein.

[0068] FIG6 shows the platelet count results of hFcRn mice after administration of 2203F07 and Romiplostim.

[0069] FIG7 shows the serum drug concentration of romiplostim 300 μg / kg after tail vein administration to human FcRn transgenic mice (n=3).

[0070] FIG8 shows the serum drug concentration of 2203F07 300 μg / kg after tail vein administration to human FcRn transgenic mice (n=3).

[0071] FIG9 is a bar graph showing the binding of TPO-RA fusion protein to human platelets.

[0072] FIG10 shows a bar graph showing the binding of TPO-RA fusion protein to cynomolgus monkey platelets.

[0073] FIG11 shows the changes in the ratio of CD34+CD41- cells to total cells at different concentrations of TPO-RA fusion protein.

[0074] FIG12 shows the changes in the ratio of CD34+CD41+ cells to total cells at different concentrations of TPO-RA fusion protein.

[0075] FIG13 shows the changes in the ratio of CD34-CD41+ cells to total cells at different concentrations of TPO-RA fusion protein.

[0076] Detailed Description of the Invention

[0077] Before describing the present invention in detail below, it should be understood that the present invention is not limited to the specific methodology, protocols and reagents described herein, as these may vary. It should also be understood that the terms used herein are only for the purpose of describing specific embodiments and are not intended to limit the scope of the present invention, which is limited only by the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention belongs.

[0078] To interpret this specification, the following definitions will apply, and wherever appropriate, terms used in the singular may also include the plural, and vice versa. It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0079] The term "about" when used in conjunction with a numerical value is meant to encompass the numerical value within a range having a lower limit that is 5% less than the specified numerical value and an upper limit that is 5% greater than the specified numerical value.

[0080] As used herein, the term "and / or" means any one of the alternatives or two or more of the alternatives.

[0081] As used herein, the terms "comprising" or "including" are intended to include the recited elements, integers, or steps, but do not exclude any other elements, integers, or steps. Herein, when the terms "comprising" or "including" are used, unless otherwise indicated, the context of consisting of the recited elements, integers, or steps is also encompassed. For example, when reference is made to an Fc variant domain that "comprising" or "including" a certain mutation or combination of mutations, it is intended to encompass an Fc variant domain that only has the recited mutation or combination of mutations.

[0082] In this article, "percentage of sequence identity" can be determined by comparing two optimally aligned sequences within a comparison window. Preferably, sequence identity is determined over the full length of a reference sequence (e.g., SEQ ID NO: 10 or 18). Methods for comparing sequences are well known in the art. Suitable algorithms for determining percentages of sequence identity include, for example, BLAST and BLAST 2.0 algorithms (see Altschul et al., Nuc. Acids Res. 25: 3389-402, 1977 and Altschul et al. J. Mol. Biol. 215: 403-10, 1990). Software for performing BLAST analysis is publicly available through the National Center for Biotechnology Information. For the purposes of this application, percentage identity is determined using the Basic Local Alignment Search Tool available from https: / / blast.ncbi.nlm.nih.gov / Blast.cgi using default parameters.

[0083] In this article, amino acid mutations can be amino acid substitutions, deletions, insertions and additions. The term "amino acid substitution" refers to replacing at least one amino acid residue in a predetermined parent amino acid sequence with a different "substitution" amino acid residue. The replacement residue or residues can be "naturally occurring amino acid residues" (i.e., encoded by the genetic code) and are selected from: alanine (Ala); arginine (Arg); asparagine (Asn); aspartic acid (Asp); cysteine ​​(Cys); glutamine (Gln); glutamic acid (Glu); glycine (Gly); histidine (His); isoleucine (Ile); leucine (Leu); lysine (Lys); methionine (Met); phenylalanine (Phe); proline (Pro); serine (Ser); threonine (Thr); tryptophan (Trp); tyrosine (Tyr); and valine (Val). The definition of amino acid substitution herein also encompasses replacement with one or more non-naturally occurring amino acid residues. "Non-natural amino acid residues" refer to residues that are capable of covalently binding to adjacent amino acid residues in a polypeptide chain, in addition to those naturally occurring amino acid residues listed above. Examples of non-natural amino acid residues include norleucine, ornithine, norvaline, homoserine, Aib and other amino acid residue analogs.

[0084] As used herein, the term "conservative substitution" means an amino acid substitution that does not adversely affect or change the biological function of the protein / polypeptide comprising the amino acid sequence. For example, conservative substitutions can be introduced by standard techniques known in the art such as site-directed mutagenesis and PCR-mediated mutagenesis. Typical conservative amino acid substitutions refer to substitutions of one amino acid with another amino acid having similar chemical properties (e.g., charge or hydrophobicity). Functionally similar amino acid conservative substitution tables are well known in the art. In the present invention, conservative substitution residues are from the following conservative substitution tables, especially preferred conservative amino acid substitution residues in the table.

[0085] Any combination of substitutions, deletions, insertions, and additions can be performed to obtain a final mutant protein construct having the desired properties (e.g., reduced binding affinity to Fc receptors and / or C1q). Amino acid deletions and insertions include deletions and insertions at the amino and / or carboxyl termini of the polypeptide sequence, as well as deletions and insertions within the polypeptide sequence. In some embodiments, the preferred amino acid mutation is an amino acid substitution, such as a combination of single amino acid substitutions or replacement of amino acid sequence segments.

[0086] When referring to an Fc variant domain, Fc variant, or Fc mutation herein, a single amino acid substitution is described as follows: [original amino acid residue / position / substituted amino acid residue]. For example, a substitution of serine at position 228 with proline can be represented as S228P. When multiple alternative amino acid substitutions (e.g., D, E) are possible at a given position (e.g., K35), the amino acid substitution can be represented as: K35D / E. Accordingly, single amino acid substitutions can be linked by a plus sign "+" or " / " to represent a combination of mutations at multiple given positions. For example, a combination of S228P, F234A, and L235A can be represented as: S228P+F234A+L235A, or S228P / F234A / L235A.

[0087] "Affinity" or "binding affinity" can be used to reflect the intrinsic binding ability of an interaction between members of a binding pair. The affinity of a molecule X for its binding partner Y can be expressed by the equilibrium dissociation constant (K D ) indicates that the equilibrium dissociation constant is the dissociation rate constant and the association rate constant (k dis and k on Binding affinity can be measured by common methods known in the art. A specific method for measuring affinity is SEC-HPLC, HIC-HPLC, melting temperature (Tm) value analysis, iCIEF analysis Octet RED96e or FACS herein.

[0088] The term "hinge region" refers to the portion of an antibody heavy chain polypeptide that connects the CH1 and CH2 regions in a wild-type antibody heavy chain, e.g., the IgG1 hinge region, e.g., the sequence from D221 to P230 according to EU numbering. Hinge regions of other IgG subclasses can be identified by aligning the hinge region cysteine ​​residues with those of the IgG1 subclass sequence.

[0089] The term "Fc domain" or "Fc region" is used herein to define the C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions. A native immunoglobulin "Fc domain" comprises two or three constant domains, namely a CH2 domain, a CH3 domain, and an optional CH4 domain. For example, in a native antibody, an immunoglobulin Fc domain comprises the second and third constant domains (CH2 domain and CH3 domain) of two heavy chains derived from IgG, IgA, and IgD class antibodies; or the second, third, and fourth constant domains (CH2 domain, CH3 domain, and CH4 domain) of two heavy chains derived from IgM and IgE class antibodies. Unless otherwise indicated herein, the amino acid residues in the Fc region or heavy chain constant region are numbered according to the EU numbering system (also referred to as the EU index) as described in Kabat et al., Sequences of Proteins of Immunological Interes, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD, 1991. However, the C-terminal lysine (Lys447) in the Fc region may or may not exist. Two Fc regions can achieve dimerization to form a dimeric Fc, and two different Fc heterodimerizations form heterodimeric Fc. In this article, the terms "Fc region", "Fc portion" and "dimeric Fc (e.g., heterodimeric Fc)" do not include the heavy chain variable region VH and light chain variable region VL of immunoglobulin and heavy chain constant region CH1 and light chain constant region CL, but may be included in the hinge region at the N-terminal end of the heavy chain constant region in some cases. In one embodiment, the human IgG1 heavy chain Fc region extends from Asp221 to the carboxyl terminus of the heavy chain. In one embodiment, the human IgG4 heavy chain Fc region extends from Glu219 to the carboxyl-terminus of the heavy chain.

[0090] As used herein, the term "linker" refers to any molecule that enables direct connection of the different parts of the fusion protein. Examples of covalently linked linkers between different parts of the fusion protein include peptide linkers and non-protein polymers, including but not limited to polyethylene glycol (PEG), polypropylene glycol, polyoxyalkylenes or copolymers of polyethylene glycol and polypropylene glycol. According to the present invention, the term "peptide linker" refers to an amino acid sequence, wherein the sequence connects the amino acid sequence of the first part of the fusion protein to the second part of the fusion protein. For example, a peptide linker can connect the domain that binds to the thrombopoietin receptor (TPOR) in the fusion protein to an Fc variant domain or its fragment. For example, a peptide linker can also connect a TPO mimetic peptide in the domain that binds to the thrombopoietin receptor (TPOR) to an Fc variant domain. For example, a peptide linker can also connect a TPO mimetic peptide in the domain that binds to the thrombopoietin receptor (TPOR) to another TPO mimetic peptide. Preferably, the peptide linker has a length that is sufficient to connect the two entities in a manner that allows them to maintain their conformations relative to each other so as not to interfere with the desired activity. The peptide linker may or may not consist essentially of the following amino acid residues: Gly, Ser, Ala, or Thr. Useful linkers include glycine linkers or glycine-serine polymers, including, for example (G) n 、(GG) n 、(GGG) n 、(GGGG) n 、(GGGGG) n 、(GGGGGG) n 、(GGGGGGG) n 、(GGGGGGGG) n , (GS) n 、(GSGGS) n 、(GGGGS) n 、(GGGS) n and (GGGGS) n G, wherein n is an integer of at least 1 (and preferably 2, 3, 4, 5, 6, 7, 8, 9, 10). Useful linkers also include glycine-alanine polymers, alanine-serine polymers and other flexible linkers. Preferably, the linker of the present invention is a linker composed of 5-8 glycine.

[0091] The term "fusion" as used herein refers to a fusion formed by connecting two or more initially separated proteins / genes / compounds. If the entity constituting the fusion is a protein, it is referred to as a fusion protein or a recombinant protein. Fusion proteins are encompassed within the scope of the present application's fusions. For example, one or more TPO mimetic peptides connected to an Fc dimer can constitute a fusion protein. For example, one or more TPO mimetic peptides are connected to each monomer of an Fc variant domain in the form of a homodimer. Preferably, the domains that bind to the thrombopoietin receptor (TPOR) containing two TPO mimetic peptides are connected to the C-terminus of each monomer of the Fc homodimer of human IgG4, respectively, to form a fusion protein containing four TPO mimetic peptides. The connection between the two entity molecules that can constitute the fusion protein constituting the fusion can be achieved by or without a linker.

[0092] "Effector function" refers to the interaction of an antibody Fc region with an Fc receptor or ligand or the biochemical events resulting therefrom. Exemplary "effector functions" include Clq binding, complement-dependent cytotoxicity (CDC), Fc receptor binding, FcγR-mediated effector functions (such as ADCC and antibody-dependent cell-mediated phagocytosis (ADCP)) and downregulation of cell surface receptors (e.g., B cell receptor; BCR). Such effector functions generally require the Fc region in combination with a binding domain.

[0093] An “Fc receptor” or “FcR” is a receptor that binds to the Fc region of an immunoglobulin. The FcR that binds to IgG antibodies comprises the FcγR family of receptors, including allelic variants and alternatively spliced ​​forms of these receptors. The FcγR family consists of three activating receptors (FcγRI, FcγRIII, and FcγRIV in mice; FcγRIA, FcγRIIA, and FcγRIIIA in humans) and one inhibitory receptor (FcγRIIB). Most innate effector cell types co-express one or more activating FcγRs and the inhibitory FcγRIIB, while natural killer (NK) cells selectively express one activating Fc receptor (FcγRIII in mice and FcγRIIIA in humans), but do not express the inhibitory FcγRIIB in mice and humans. Human IgG1 binds to most human Fc receptors and is considered equivalent to murine IgG2a with respect to the type of activating Fc receptor it binds.

[0094] In one embodiment, the Fc receptor is selected from the group consisting of a human Fcγ receptor, a human neonatal Fc receptor, a murine Fc receptor, and a rabbit neonatal Fc receptor.

[0095] The term "drug combination" or "combination product" refers to a non-fixed combination product or a fixed combination product, including but not limited to a kit. The term "non-fixed combination" means that the active ingredients are administered to a patient simultaneously, without specific time restrictions, or at the same or different time intervals, in sequence as separate entities, wherein such administration provides two or more active agents with effective levels of prevention or treatment in the patient's body. In some embodiments, the fusion protein of the present invention and other therapeutic agents used in the drug combination are administered at levels no greater than when they are used alone. The term "fixed combination" means that two or more active agents are administered to a patient simultaneously in the form of a single entity. The dosage and / or time interval of the two or more active agents are preferably selected so that the combined use of the parts can produce an effect greater than that achieved by using any one component alone when treating a disease or condition. Each component can be in the form of a separate formulation, which can be the same or different.

[0096] II. Fusion Proteins of the Present Invention

[0097] In some embodiments, the fusion protein of the present invention that specifically binds to TPOR comprises a TPOR-binding domain and an Fc domain.

[0098] In some embodiments, the TPOR-binding domain is a TPO mimetic peptide.

[0099] In some embodiments, the TPO mimetic peptide comprises or consists of the amino acid sequence shown in SEQ ID NO:9.

[0100] In some embodiments, the TPO mimetic peptide comprises a polypeptide composed of one or more (preferably no more than 3, such as 2 or 1) amino acids added to the N-terminus and / or C-terminus of the amino acid sequence shown in SEQ ID NO:9.

[0101] In some embodiments, the TPO mimetic peptide comprises or consists of an amino acid sequence having 1-3 amino acid differences (eg, substitutions, deletions, or additions, such as conservative substitutions) from the amino acid sequence of SEQ ID NO: 9.

[0102] In some embodiments, the Fc domain comprised by the fusion protein is a variant Fc domain, wherein the variant Fc domain comprises at least one mutation compared to the wild-type Fc;

[0103] Reducing or eliminating the effector function of the Fc region, such as reducing binding to Fcγ receptors and / or C1q;

[0104] Improving the stability of the Fc region; and / or

[0105] Enhance the half-life of fusion proteins.

[0106] The Fc region fragment applicable to antibody molecules of the present invention can be any antibody Fc region. The Fc region can include native sequence Fc region and variant Fc region. The native sequence Fc domain covers naturally occurring various immunoglobulin Fc sequences, such as various Ig subtypes and the Fc regions of their allotypes (Gestur Vidarsson et al., IgG subclasses and allotypes:from structure to effector functions, 20 October 2014, doi:10.3389 / fimmu.2014.00520.). For example, the Fc region of an antibody of the present invention can include two or three constant domains, i.e., CH2 domain, CH3 domain and optional CH4 domain. In some embodiments, the antibody Fc region can also have an IgG hinge region or a partial IgG hinge region at the N-terminus, such as an IgG1 hinge region or a partial IgG1 hinge region. Mutations can be contained in the hinge region.

[0107] Preferably, the Fc region of the antibodies of the present invention comprises, from N-terminus to C-terminus, CH2-CH3, or, from N-terminus to C-terminus, hinge region-CH2-CH3. In some embodiments, the Fc region suitable for use in the antibodies or bispecific antibodies of the present invention is a human IgG Fc, such as a human IgG1 Fc, a human IgG2 Fc, a human IgG3 Fc, or a human IgG4 Fc. Reference herein to a human IgG Fc refers to an IgG from which the Fc is derived, encompassing wild-type Fc domains or Fc regions derived from such IgG, as well as Fc variant domains or Fc region variants.

[0108] In one embodiment, the wild-type Fc region comprises or consists of the amino acid sequence of SEQ ID NO: 10 or 17, or an amino acid sequence having at least 90% identity thereto, such as 95%, 96%, 97%, 99% or more identity thereto. In one embodiment, the IgG1 Fc region comprises or consists of the amino acid sequence of SEQ ID NO: 10, or an amino acid sequence having at least 90% identity thereto, such as 95%, 96%, 97%, 99% or more identity thereto. In one embodiment, the IgG4 Fc region comprises or consists of the amino acid sequence of SEQ ID NO: 17, or an amino acid sequence having at least 90% identity thereto, such as 95%, 96%, 97%, 99% or more identity thereto.

[0109] When referring to a "variant Fc" or "Fc variant", the parent Fc region can be a wild-type Fc region or an Fc region that has a certain degree of identity with the wild-type Fc region (an amino acid sequence that has at least 90% identity thereto, such as 95%, 96%, 97%, 99% or more identity thereto). Thus, the parent Fc region can be a wild-type IgG1 Fc region or a wild-type IgG4 Fc region. In some embodiments, the IgG1 Fc region serving as the parent Fc region comprises or consists of the amino acid sequence of SEQ ID NO: 10 or an amino acid sequence that has at least 90% identity thereto, such as 95%, 96%, 97%, 99% or more identity thereto. In some embodiments, the IgG4 Fc region serving as the parent Fc region comprises or consists of the amino acid sequence of SEQ ID NO: 17 or an amino acid sequence that has at least 90% identity thereto, such as 95%, 96%, 97%, 99% or more identity thereto.

[0110] In one embodiment, the variant Fc domain suitable for use in the fusion protein of the present invention has a reduced effector function mediated by the Fc region, such as reduced or eliminated ADCC or ADCP or CDC effector function, for example, comprising mutations that achieve the above functions. In some embodiments, the Fc region suitable for use in the fusion protein of the present invention comprises a modification that changes the binding affinity to one or more Fc receptors and / or C1q. In one embodiment, the Fc receptor is an Fcγ receptor, particularly a human Fcγ receptor. In one embodiment, the Fc receptor is FcγRIIb, FcγRIIa H167, FcγRIIa R167, FcγRIIIa F176, FcγRIIIa V176 and / or human FcγRI. In some embodiments, the Fc region comprises a mutation that reduces binding to Fcγ receptors and / or C1q. Exemplary mutations that reduce or eliminate Fc region-mediated effector function are selected from the group consisting of L234A / L235A mutations (IgG1 Fc region) or F234A / F235A mutations (IgG4 Fc region) or D265S mutation (in IgG1 Fc region) or a combination thereof.

[0111] In one embodiment, the Fc domain may have mutations that result in increased serum half-life, and / or mutations that improve binding of the Fc fragment to FcRn. Exemplary mutations that increase serum half-life are selected from the group consisting of YTE mutations (M252Y, S254T, and T256E) (IgG1 Fc region) or T250Q / M428L (IgG4 Fc region).

[0112] In one embodiment, the Fc domain may have mutations that enhance the stability of the fusion protein, such as comprising an S228P mutation in the hinge region of IgG4.

[0113] In one embodiment, the Fc domain suitable for use in the fusion protein of the present invention is from IgG1, and compared to the Fc region of IgG1, the Fc variant domain comprises at least one substitution mutation selected from the following amino acid positions: 234, 235, 252, 254, 256 or 265, wherein the amino acid positions are numbered according to EU. In one embodiment, compared to the IgG1 Fc region, the Fc variant domain comprises at least one substitution mutation selected from the following amino acid positions: L234A, L235A, D265S, M252Y, S254T or T256E, wherein the amino acid positions are numbered according to EU. In one embodiment, the Fc domain comprises L234A and L235A mutations. In some embodiments, the Fc domain comprises D265S mutation. In one embodiment, the Fc domain comprises YTE mutations (M252Y, S254T and T256E). In one embodiment, the Fc domain comprises L234A, L235A and D265S mutations. In one embodiment, the Fc domain comprises L234A, L235A, and YTE mutations. In one embodiment, the Fc domain comprises D265S and YTE mutations. In one embodiment, the Fc domain comprises L234A, L235A, D265S, and YTE mutations.

[0114] In one embodiment, the Fc domain suitable for use in the fusion protein of the present invention is from IgG4, and compared to the Fc region of IgG4, the Fc variant domain comprises at least one substitution mutation selected from the following amino acid positions: 228, 234, 235, 250 or 428, wherein the amino acid positions are numbered according to EU. In one embodiment, compared to the IgG4 Fc region, the Fc variant domain comprises at least one substitution mutation selected from the following amino acid positions: S228P, F234A, L235A, T250Q or M428L, wherein the amino acid positions are numbered according to EU. In one embodiment, the Fc domain comprises an S228P mutation. In one embodiment, the Fc domain comprises an F234A and an L235A mutation. In one embodiment, the Fc domain comprises an S228P mutation, an F234A and an L235A mutation. In some embodiments, the Fc domain comprises an F234A, an L235A mutation, and T250Q and M428L. In some embodiments, the Fc domain comprises S228P, F234A, L235A, T250Q, and M428L.

[0115] In one embodiment, the N-terminus of the Fc domain of the fusion protein of the present invention may comprise an initial methionine. In one embodiment, the N-terminus of the Fc domain of the fusion protein of the present invention does not comprise a methionine.

[0116] In some embodiments, the Fc variant domain of a fusion protein of the invention comprises or consists of an amino acid sequence of SEQ ID NOs: 11-16, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0117] In some embodiments, the Fc variant domain of the fusion protein of the present invention comprises or consists of the amino acid sequence of SEQ ID NO: 11, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto. In some embodiments, the Fc variant domain of the fusion protein of the present invention comprises or consists of the amino acid sequence of SEQ ID NO: 11, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto, preferably the Fc variant domain further comprises L234A+L235A+D265S mutations (e.g., compared to IgG1 Fc region).

[0118] In some embodiments, the Fc variant domain of the fusion protein of the present invention comprises or consists of the amino acid sequence of SEQ ID NO: 12, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto. In some embodiments, the Fc variant domain of the fusion protein of the present invention comprises or consists of the amino acid sequence of SEQ ID NO: 12, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto, preferably the Fc variant domain further comprises L234A+L235A+D265S+M252Y+S254T+T256E mutations (e.g., compared to IgG1 Fc region).

[0119] In some embodiments, the Fc variant domain of the fusion protein of the present invention comprises or consists of the amino acid sequence of SEQ ID NO: 13, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto. In some embodiments, the Fc variant domain of the fusion protein of the present invention comprises or consists of the amino acid sequence of SEQ ID NO: 13, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto, preferably the Fc variant domain further comprises an S228P mutation (e.g., compared to an IgG4 Fc region).

[0120] In some embodiments, the Fc variant domain of the fusion protein of the present invention comprises or consists of the amino acid sequence of SEQ ID NO: 14, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto. In some embodiments, the Fc variant domain of the fusion protein of the present invention comprises or consists of the amino acid sequence of SEQ ID NO: 14, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto, preferably the Fc variant domain further comprises S228P+F234A+L235A mutations (e.g., compared to an IgG4 Fc region).

[0121] In some embodiments, the Fc variant domain of the fusion protein of the present invention comprises or consists of the amino acid sequence of SEQ ID NO: 15, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto. In some embodiments, the Fc variant domain of the fusion protein of the present invention comprises or consists of the amino acid sequence of SEQ ID NO: 15, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto, preferably the Fc variant domain further comprises S228P+T250Q+M428L mutations (e.g., compared to IgG4 Fc region).

[0122] In some embodiments, the Fc variant domain of the fusion protein of the present invention comprises or consists of the amino acid sequence of SEQ ID NO: 16, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto. In some embodiments, the Fc variant domain of the fusion protein of the present invention comprises or consists of the amino acid sequence of SEQ ID NO: 16, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto, preferably the Fc variant domain further comprises S228P+F234A+L235A+T250Q+M428L mutations (e.g., compared to IgG4 Fc region).

[0123] In some embodiments, the fusion protein that specifically binds to TPOR of the present invention comprises one or two or four or more TPO mimetic peptides and one or two or more Fc variant domains. In some embodiments, the one or two or more mimetic peptides are connected to the N-terminus or C-terminus of the Fc variant domain. In one embodiment, the two or more TPO mimetic peptides are connected in series.

[0124] In one embodiment, the TPO mimetic peptide is connected via a linker. In one embodiment, the TPO mimetic peptide is connected to the Fc variant domain via a linker. In some embodiments, the linker is a glycine linker, for example, the linker comprises 1-8 glycines, for example 5, 6, 7 or 8 glycines.

[0125] In some embodiments, the TPO mimetic peptides constituting the fusion protein of the present invention may be the same or different. In some embodiments, the Fc variant domains constituting the fusion protein of the present invention may be the same or different.

[0126] In some embodiments, the fusion protein that specifically binds to TPOR comprises a peptide chain comprising or consisting of two tandemly connected TPO mimetic peptides and an Fc variant domain. Preferably, the peptide chain comprises or consists of the following from the N-terminus to the C-terminus:

[0127] Fc variant domain-first linker-first TPO mimetic peptide-second linker-second TPO mimetic peptide,

[0128] Optionally, the first linker and / or the second linker may be present or absent; or the first TPO mimetic peptide and the second TPO mimetic peptide may be the same or different;

[0129] Preferably, the fusion protein comprises two peptide chains, wherein the two peptide chains may be the same or different.

[0130] In some embodiments, the peptide chain comprises or consists of an amino acid sequence of SEQ ID NO: 2-7, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto.

[0131] In some embodiments, the peptide chain comprises:

[0132] (i) an amino acid sequence of SEQ ID NO: 2, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto, or consisting thereof, preferably comprising L234A+L235A+D265S mutations (e.g. compared to an IgG1 Fc region);

[0133] (ii) the amino acid sequence of SEQ ID NO: 3, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto, or consisting thereof, preferably comprising L234A+L235A+D265S+M252Y+S254T+T256E mutations (e.g. compared to an IgG1 Fc region);

[0134] (iii) an amino acid sequence of SEQ ID NO: 4, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto, or consisting thereof, preferably comprising an S228P mutation (e.g., compared to an IgG4 Fc region);

[0135] (iv) an amino acid sequence of SEQ ID NO: 5, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto, or consisting thereof, preferably comprising S228P+F234A+L235A mutations (e.g., compared to an IgG4 Fc region);

[0136] (v) an amino acid sequence of SEQ ID NO: 6, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto, or consisting thereof, preferably comprising S228P+T250Q+M428L mutations (e.g. compared to an IgG4 Fc region); or

[0137] (vi). the amino acid sequence of SEQ ID NO: 7, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto, or consisting thereof, preferably comprising S228P+F234A+L235A+T250Q+M428L mutations (e.g. compared to an IgG4 Fc region).

[0138] III. Polynucleotides, Vectors, and Hosts

[0139] The present invention provides isolated polynucleotides encoding any chain or any monomer or domain of any of the above fusion proteins that specifically bind to TPOR. When applied to a polynucleotide sequence, the term "isolated" means that the sequence has been removed from the natural organism from which it is derived and therefore does not contain foreign or unwanted coding or regulatory sequences. The isolated sequence is suitable for use in recombinant DNA processes and genetically engineered protein synthesis systems. These isolated sequences include cDNA, mRNA, and genomic clones. The isolated sequence can be limited to protein coding sequences, or may also include 5' and 3' regulatory sequences, such as promoters and transcription terminators, or untranslated sequences (UTRs).

[0140] As used herein, a "polynucleotide" is a single-stranded or double-stranded covalently linked sequence of nucleotides, wherein the 3' and 5' ends of each nucleotide are linked by a phosphodiester bond. The polynucleotide may be composed of deoxynucleotide bases or ribonucleotide bases. Polynucleotides include DNA and RNA. The term "RNA" or "RNA molecule" or "ribonucleic acid molecule" refers to a polymer of ribonucleotides (e.g., 2, 3, 4, 5, 10, 15, 20, 25, 30 or more ribonucleotides). The term "DNA" or "DNA molecule" or "deoxyribonucleic acid molecule" refers to a polymer of deoxyribonucleotides. DNA or RNA can be synthesized naturally (e.g., by DNA replication or DNA transcription, respectively). For example, RNA can be modified after transcription. DNA or RNA can also be chemically synthesized. DNA and RNA can be single-stranded (i.e., ssRNA and ssDNA, respectively) or multi-stranded (e.g., double-stranded, i.e., dsRNA and dsDNA, respectively). RNA can be messenger RNA (mRNA). "mRNA" or "messenger RNA" is a single-stranded RNA that specifies the amino acid sequence of one or more polypeptide chains. During protein synthesis, this information is translated when ribosomes bind to the mRNA.

[0141] In some embodiments, the polynucleotide of the present invention is a DNA encoding a synthetic protein, or an mRNA. The nucleic acid may further include modified DNA or RNA, such as methylated DNA or RNA or post-translationally modified RNA.

[0142] Polynucleotide sequences encoding the muteins of the present invention can be generated by de novo solid-phase DNA synthesis or by PCR mutagenesis of existing sequences encoding wild-type Fc using methods well known in the art. Furthermore, the polynucleotides and nucleic acids of the present invention may comprise a segment encoding a secretory signal peptide, operably linked to the segment encoding the mutein of the present invention, thereby directing secretory expression of the mutein of the present invention.

[0143] The present invention also provides vectors comprising nucleic acids of the present invention. In one embodiment, the vector is an expression vector, such as a eukaryotic expression vector. Vectors include, but are not limited to, viruses, plasmids, cosmids, lambda phages, or yeast artificial chromosomes (YACs). In a preferred embodiment, the expression vector of the present invention is a pcDNA3.4 expression vector.

[0144] The present invention also provides a host cell comprising the nucleic acid or the vector. Host cells suitable for replication and support expression of fusion proteins that specifically bind to TPOR are well known in the art. Such cells can be transfected or transduced with specific expression vectors, and large quantities of vector-containing cells can be grown for inoculation of large-scale fermenters, thereby obtaining sufficient amounts of fusion proteins that specifically bind to TPOR for clinical applications. In one embodiment, the host cell is eukaryotic. In another embodiment, the host cell is selected from yeast cells, mammalian cells (e.g., CHO cells, CHO-K1 cells, or 293 cells). Examples of useful mammalian host cell lines are monkey kidney CV1 line transformed by SV40 (COS-7); human embryonic kidney line (293 or 293T cells, as described, e.g., in Graham et al., J Gen Virol 36, 59 (1977)), baby hamster kidney cells (BHK), mouse Sertoli cells (TM4 cells, as described, e.g., in Mather, Biol Reprod 23, 243-251 (1980)), monkey kidney cells (CV1), African green monkey kidney cells (VERO-76), human cervical carcinoma cells (HELA), canine kidney cells (MDCK), buffalo rat liver cells (BRL3A), human lung cells (W138), human liver cells (HepG2), mouse mammary tumor cells (MMT060562), TRI cells (as described, e.g., in Mather et al., Annals N.Y. Acad Sci 383, 44-68 (1982)), MRC5 cells, and FS4 cells. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including dhfr-CHO cells (Urlaub et al., Proc Natl Acad Sci USA 77, 4216 (1980)); and myeloma cell lines such as YO, NSO, P3X63 and Sp2 / 0. In one embodiment, the host cell is a eukaryotic cell, preferably a mammalian cell such as a Chinese hamster ovary (CHO) cell, a human embryonic kidney (HEK) cell or a lymphocyte (e.g., Y0, NSO, Sp20 cell).

[0145] IV. Preparation Method

[0146] In one aspect, the present invention provides a method for preparing a fusion protein that specifically binds to TPOR according to the present invention, wherein the method comprises culturing a host cell comprising a nucleic acid encoding the fusion protein that specifically binds to TPOR, as provided above, under conditions suitable for expression of the fusion protein that specifically binds to TPOR, and optionally recovering the fusion protein that specifically binds to TPOR from the host cell (or host cell culture medium).

[0147] In one embodiment, a vector containing a nucleic acid encoding a fusion protein that specifically binds to TPOR is introduced into cells for expression, and the cells (or cell culture supernatant) are then collected, and the fusion protein that specifically binds to TPOR is extracted and purified to obtain the fusion protein that specifically binds to TPOR. In a specific embodiment, the purification method is affinity purification. In another specific embodiment, the purification method is ion exchange purification.

[0148] V. Pharmaceutical Compositions and Pharmaceutical Formulations

[0149] The present invention also includes compositions (including pharmaceutical compositions or pharmaceutical preparations) comprising fusion proteins that specifically bind to TPOR and compositions comprising polynucleotides encoding fusion proteins that specifically bind to TPOR. These compositions may also optionally contain suitable pharmaceutical excipients, such as pharmaceutical carriers and pharmaceutical excipients known in the art, including buffers.

[0150] Pharmaceutical compositions comprising the fusion protein that specifically binds to TPOR of the present invention can be prepared by conventional mixing, dissolving, emulsifying, encapsulating, encapsulating or lyophilizing processes. Pharmaceutical compositions can be formulated in a conventional manner using one or more physiologically acceptable carriers, diluents, excipients or adjuvants that facilitate processing of the protein into pharmaceutically usable preparations. Suitable formulations depend on the chosen route of administration.

[0151] The immunoconjugates can be formulated into compositions in free acid or base, neutral, or salt form. Pharmaceutically acceptable salts are salts that substantially retain the biological activity of the free acid or base. These include acid addition salts, such as those formed with free amino groups of proteinaceous compositions, or with inorganic acids (such as hydrochloric acid or phosphoric acid) or with organic acids such as acetic acid, oxalic acid, tartaric acid, or mandelic acid. Salts formed with free carboxyl groups can also be derived from inorganic bases such as sodium, potassium, ammonium, calcium, or iron hydroxide; or organic bases such as isopropylamine, trimethylamine, histidine, or procaine. Pharmaceutically acceptable salts tend to be more soluble in aqueous and other protic solvents than the corresponding free base forms.

[0152] VI. Combination Products

[0153] In one aspect, the present invention also provides a combination product comprising a fusion protein that specifically binds to TPOR according to the present invention, and one or more other therapeutic agents (e.g., chemotherapeutic agents, other antibodies, cytotoxic agents, vaccines, anti-infective agents, or thrombopoietic drugs). The combination product of the present invention can be used in the treatment methods of the present invention.

[0154] In some embodiments, the combination product is used to prevent or treat thrombocytopenia, wherein the thrombocytopenia is primary immune thrombocytopenia, thrombocytopenia caused by tumor chemotherapy, thrombocytopenia associated with liver disease, or aplastic anemia.

[0155] VII. Methods of Treatment and Use

[0156] In one aspect, the present invention relates to a method for preventing or treating a disease in a subject, such as thrombocytopenia, comprising administering to the subject an effective amount of any fusion protein that specifically binds to TPOR as described herein, or a pharmaceutical composition or combination product comprising the same. In some embodiments, the method comprises administering to the subject an effective amount of any fusion protein that specifically binds to TPOR as described herein, or a pharmaceutical composition comprising the same, and one or more other therapeutic agents (e.g., chemotherapeutic agents, other antibodies, cytotoxic agents, vaccines, anti-infective agents, or thrombopoietic drugs, etc.).

[0157] In some embodiments, the disease benefits from activation of the TPOR signaling pathway.

[0158] In some embodiments, the thrombocytopenia is primary immune thrombocytopenia, thrombocytopenia induced by cancer chemotherapy, thrombocytopenia associated with liver disease, or aplastic anemia.

[0159] The fusion protein that specifically binds to TPOR of the present invention or the pharmaceutical composition or combination product comprising the same can be administered by any suitable method, including parenteral administration, intrapulmonary administration and intranasal administration, and, if required for local treatment, intralesional administration. Parenteral infusion includes intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration.

[0160] Depending to some extent on whether the administration is short-term or long-term, the administration can be by any suitable route, for example by injection, such as intravenous or subcutaneous injection. Various administration schedules are contemplated herein, including, but not limited to, single administration or multiple administrations at multiple time points, bolus administration, and pulse infusion.

[0161] For the prevention or treatment of disease, the appropriate dosage of the fusion protein of the invention that specifically binds to TPOR (when used alone or in combination with one or more other therapeutic agents) will depend on the type of disease to be treated, the severity and course of the disease, whether the administration is for preventive or therapeutic purposes, previous treatments, the patient's clinical history and response to the antibody, and the discretion of the attending physician. The antibody is suitably administered to the patient as a single treatment or over a series of treatments. In some embodiments, the fusion protein of the invention that specifically binds to TPOR can be administered to the patient at higher doses without causing toxicity.

[0162] In another aspect, the present invention also provides use of the fusion protein that specifically binds to TPOR of the present invention in preparing a medicament for the aforementioned method (eg, for prevention or treatment).

[0163] The present invention also provides use of the fusion protein that specifically binds to TPOR of the present invention for therapy, such as for treating or preventing the diseases described in the present invention.

[0164] The following examples are described to assist understanding of the present invention. The examples are not intended to, and should not be interpreted in any way as, limiting the scope of protection of the present invention.

[0165] These and other aspects and embodiments of the present invention are described in the accompanying drawings (a brief description of the drawings follows) and the following detailed description of the invention and are exemplified in the following examples. Any or all of the features discussed above and throughout this application may be combined in various embodiments of the present invention. The following examples further illustrate the present invention, however, it should be understood that the examples are described in an illustrative and non-limiting manner, and that various modifications may be made by those skilled in the art. Example:

[0166] Example 1 Design of Thrombopoietin Receptor Agonist Fusion Protein

[0167] This design is based on The sequence of SEQ ID NO:8 was used as a blueprint for modification. This molecule is essentially an Fc-fused homodimeric fusion protein, with its monomer structurally divided into two regions: the N-terminal and C-terminal domains. The N-terminal domain begins with a methionine amino acid as a starting amino acid (designed for expression in E. coli), followed by the natural human IgG1 Fc region. The C-terminal domain consists of two 14-amino acid TPO mimetic peptides, connected from the N-terminus to the C-terminus by five glycines and eight glycines, respectively, forming a bivalent TPO receptor binding region. The following Fc region amino acid numbers are based on the EU numbering system (human IgG1 Fc: D221-K447; human IgG4 Fc: E219-K447).

[0168] 1. The first amino acid methionine at the N-terminus of the Fc vector region was removed and the following mutations were performed: L234A, L235A, D265S. The ropiplastin mimetic peptide and linker were retained unchanged. The mimetic peptide was placed at the C-terminus of the Fc region to obtain a homodimeric tetravalent Fc fusion protein named 2203F04.

[0169] 2. The first amino acid methionine at the N-terminus of the Fc vector region was removed and the following mutations were performed: L234A, L235A, D265S, M252Y, S254T, T256E. The ropiplastin mimetic peptide and linker were retained unchanged. The mimetic peptide was located at the C-terminus of the Fc region to obtain a homodimeric tetravalent Fc fusion protein named 2203F05.

[0170] 3. The Fc carrier region was replaced with the Fc region of human IgG4, the first amino acid methionine at the N-terminus was removed, and the following mutation was performed: S228P. The ropiplastin mimetic peptide and linker were retained unchanged, with the mimetic peptide at the C-terminus of the Fc region, to obtain a homodimeric tetravalent Fc fusion protein named 2203F06.

[0171] 4. The Fc carrier region was replaced with the Fc region of human IgG4, the first amino acid methionine at the N-terminus was removed, and the following mutations were performed: S228P, F234A, L235A. The ropiplastin mimetic peptide and linker were retained unchanged, with the mimetic peptide at the C-terminus of the Fc region, to obtain a homodimeric tetravalent Fc fusion protein named 2203F07.

[0172] 5. The Fc carrier region was replaced with the Fc region of human IgG4, the first amino acid methionine at the N-terminus was removed, and the following mutations were performed: S228P, T250Q, M428L. The ropiplastin mimetic peptide and linker were retained unchanged, with the mimetic peptide at the C-terminus of the Fc region, to obtain a homodimeric tetravalent Fc fusion protein named 2203F08.

[0173] 6. The Fc carrier region was replaced with the Fc region of human IgG4, the first amino acid methionine at the N-terminus was removed, and the following mutations were performed: S228P, F234A, L235A, T250Q, M428L. The ropiplastin mimetic peptide and linker were retained unchanged, with the mimetic peptide at the C-terminus of the Fc region, to obtain a homodimeric tetravalent Fc fusion protein named 2203F09.

[0174] The above-mentioned thrombopoietin receptor agonist fusion proteins are summarized in the table below.

[0175] Example 2 In vitro expression of thrombopoietin receptor agonist fusion protein

[0176] After optimizing the design sequence, we commissioned Shanghai Bio-Tech to perform transient expression and protein production. Specifically, we performed codon optimization on the full-length fusion protein sequence, synthesized the codon-optimized DNA fragment, and cloned the synthesized gene fragment into the expression vector pcDNA3.4 (Life Technologies). Expression plasmid amplification and plasmid extraction (Qiagen, Plasmid Maxi Kit, Cat. No. 12362) and then transfected into Expi293F (Thermo Fisher Scientific, A14527) or CHO-K1 cells (ECACC catalog no. 85051005). Transient expression of the fusion protein was performed according to the supplier's Expi293F or CHO-K1 expression system method. The specific process is as follows:

[0177] (I) Cell recovery: Use a 125 ml disposable sterile shake flask to thaw the frozen CHO cells in a 37°C water bath and dilute the cells to 0.3 × 10 6 / mL, volume 30mL. 125rpm (19mm orbit), 8% CO2, 37℃ culture until 4-6×10 6 About 100g / mL.

[0178] (II) Passaging: cells grow to 4-6×10 6 cells / mL, and the subculture density was 0.2-0.3×10 6 cells / ml and cultured for 3 days.

[0179] (III) Transient transfection: cells were passaged for more than 3 generations and the density was 4-6×10 6 Cells were diluted to a density of 3-4×10 6 cells / mL, overnight culture, 7-10×10 6 , diluted to 6 × 10 6 Taking the transient transfection volume of 100mL (500mL shake flask) as an example: Solution 1: dilute 100μg plasmid with culture medium (4mL) and mix well; Solution 2: dilute 320uL transfection reagent with culture medium (3.68ml) and mix well; add solution 2 to solution 1, the total volume is 8mL, mix gently and incubate at room temperature for 1-5 minutes, no more than 5 minutes, add the mixed transfection solution drop by drop to the cell solution, shaking while adding.

[0180] (IV) Protein Expression (Maximum Titer): After 18-22 hours, add 16 mL of Feed-1 and 0.6 mL of Enhancer-2 and incubate at 32°C. On the fifth day, add 16 mL of Feed-2 and incubate at 123 rpm, 5% CO2, and 32°C. On the 12th-14th day, collect the supernatant by centrifugation.

[0181] (V) Antibody Purification: Protein A affinity chromatography: 1) Equilibrate the column: 1× PBS, flow rate 1 mL / min, 20 mL; 2) Load the sample: flow rate 1 mL / min; 3) Wash: 1× PBS, flow rate 1 mL / min, 20 mL; 4) Elute: Citrate buffer (pH 3.4), 1 mL / min, collect the aliquots, approximately 500 μL per tube. Collect 10 tubes in total and read the absorbance at 280 nm using a NanoDrop instrument; 5) Dialysis: Pipette the high-concentration protein into a dialysis bag and dialyze it in a beaker containing 1× PBS. 6) Collect the protein for later use.

[0182] The protein expression results are shown in Table 1, indicating that the fusion protein molecule can be expressed using the above method.

[0183] Table 1 Platelet receptor agonist fusion protein expression data

[0184] Example 3 Evaluation of the physicochemical properties of thrombopoietin receptor agonist fusion protein

[0185] 1.1 SEC-HPLC purity analysis

[0186] The sample obtained in Example 2 was centrifuged at 12000 rpm for 5 min, and the supernatant was transferred to a sample bottle and placed in an HPLC sample tray.

[0187] Set the chromatographic conditions as follows:

[0188] The mobile phase was 200 mM phosphate buffer, pH 6.8, and the peak area percentage of each peak was calculated by the peak area normalization method.

[0189] 1.2 HIC-HPLC analysis

[0190] Dilute the sample to 1 mg / ml, centrifuge and collect the supernatant for analysis. Set the chromatographic conditions as follows:

[0191] Gradient elution was performed using mobile phase A (50 mM phosphate buffer + 1 M ammonium sulfate, pH 7.0) and mobile phase B (50 mM phosphate buffer, pH 7.0), and the retention time of the main peak was recorded.

[0192] 1.3 Melting temperature (Tm) value analysis

[0193] Dilute the test sample to 1 mg / mL with sample buffer. Add 13 μL to a reaction tube. Add 5 μL of Protein Thermal Shift™ Buffer and 2 μL of 10× staining solution to a total volume of 20 μL. Mix thoroughly and centrifuge at 12,000 rpm for 5 minutes to remove bubbles. Place the test sample in a PCR instrument for analysis and record the sample Tm value.

[0194] 1.4 iCIEF analysis

[0195] Add 40 μL of sample solution (1 mg / mL) to a thoroughly mixed solution of the following: 70 μL of 1% methylcellulose (MC), 60 μL of 10 M urea, 20 μL of ultrapure water, 8 μL of ampholyte pH 3-10, and 1 μL each of pI markers 5.5 and 9.5. Centrifuge and remove the supernatant for analysis. Perform chromatographic integration and calculate the isoelectric point and percentage of each peak.

[0196] The results of the physicochemical property analysis of the candidate fusion proteins of the present invention are shown in Table 2.

[0197] Table 2

[0198] Example 4 Evaluation of in vitro biological activity of thrombopoietin receptor agonist fusion protein

[0199] Determination of the affinity of TPO-RA fusion protein to human, cynomolgus monkey and rat TPO receptor (TPO-R)

[0200] In order to determine the affinity of TPO-RA fusion protein with human, cynomolgus monkey and rat TPO receptor (TPO-R), Biacore (cytiva, T200) was used to determine the affinity of candidate fusion protein with human (Acro, Catalog No.: THR-H52H7), cynomolgus monkey (Kactus, Catalog No.: MPL-CM101), and rat (Sino Biological, Catalog No.: 80346-R08B) TPO-R. The candidate fusion protein and receptors of different species were all detected by HBS-EP. +Buffer (cytiva; BR100669; 32351) was used for dilution. Human, cynomolgus monkey, and rat TPO-R were diluted 2-fold starting at 200 nM to obtain five concentrations. TPO-RA fusion protein was used as the stationary phase at a concentration of 1 μg / ml. Detection method: Multi-cycle kinetics; Instrument preparation: Set the flow cell temperature to 25°C and the sample chamber temperature to 15°C. First, place the S-series sensor chip Protein A (cytiva, 18-5060) and 1× HBS-EP + buffer, prime and enter standby mode, and then perform detection on the instrument. Each cycle consisted of the following steps: 1) Capture: The fusion protein was flowed through channels 2, 3, and 4 at a flow rate of 10 μl / min for 30 seconds. 2) Analyte: Different species of analyte receptors were injected into channels 1, 2, 3, and 4 at a flow rate of 30 μl / min for 180 seconds, followed by dissociation for 300 seconds. 3) Regeneration: 10 mM glycine-HCl, pH 1.5, was injected into channels 1, 2, 3, and 4 at a flow rate of 30 μl / min for 30 seconds. Data were fitted using Biacore T200 Evaluation 3.2.1 software using a 1:1 binding model.

[0201] The results are shown in Table 3. Compared with the benchmark fusion protein 2203BM01, the candidate protein has comparable affinity to human and cynomolgus monkey TPO receptors; its affinity to rat TPO receptor is weaker.

[0202] Table 3 Affinity determination of TPO-RA fusion protein with human, cynomolgus monkey and rat TPO-R

[0203] Affinity determination of TPO-RA fusion protein and human Fc receptor

[0204] To determine whether the TPO-RA fusion protein would bind to Fc receptors and induce ADCC and ADCP effects, the Octet RED96e (Fortébio) was used to measure the affinity of the candidate fusion proteins to human FcγRI (Acro, Catalog No.: FCA-H52H2), FcγRIIb (Acro, Catalog No.: CD8-H5222), FcγRIIa H167 (Acro, Catalog No.: CD1-HS223), FcγRIIa R167 (Acro, Catalog No.: CDA-H5221), FcγRIIIa F176 (Acro, Catalog No.: CDA-H5220), and FcγRIIIa V176 (Acro, Catalog No.: CD8-H52H4). The candidate fusion protein and Fc receptor were diluted in 1× PBST (1× PBS: Sangon, Catalog No. B548117-0500; 0.02% Tween 20: Sigma, Catalog No. P1379). The Fc receptor was immobilized on a HIS1K sensor (Fortébio, Catalog No. 18-0015) at a concentration of 5 μg / ml. The candidate fusion protein was diluted in a two-fold series starting at 1250 nM for a total of seven concentrations. The prepared Fc receptor and candidate fusion protein were then added to a 96-well black plate (Greiner bio-one, Catalog No. 655209) according to the experimental layout, at 200 μl / well. Software parameters were set to 30°C and a standard kinetic signal collection frequency of 5.0 Hz. The HIS1K sensor was pre-wetted with 1× PBST for 10 minutes before testing. Each cycle consisted of the following steps: 1) immersion in buffer for 60 seconds; 2) detection of nonspecific binding of the fusion protein to the sensor; 3) regeneration with 10 mM glycine solution, pH 1.7; 4) immersion in buffer for 60 seconds; 5) immobilization of the Fc receptor on the sensor for 55 seconds; 6) immersion of the sensor in buffer for 180 seconds; 7) binding of the fusion protein to the effector receptor for 60 seconds; 8) dissociation of the fusion protein from the effector receptor for 60 seconds; 9) sensor regeneration. Finally, the binding rate (K) was determined for a 1:1 antigen-antibody binding reaction using Fortébio's Data Analysis 12.0 software. on ) and dissociation rate (K off ), and the equilibrium dissociation constant (K D ).

[0205] The results are shown in Table 4 (the responses in the table show only the highest concentration response values). The affinities of the benchmark fusion protein 2203BM01 for human FcγRIIb, FcγRIIa H167, FcγRIIa R167, FcγRIIIa F176, and FcγRIIIa V176 were similar to those reported for IgG1 antibodies and these Fc receptors, while the affinities of the candidate fusion proteins (2203F04, 2203F05, 2203F07, and 2203F09) for the above Fc receptors were lower than those of the benchmark fusion protein 2203BM01. Compared with the affinity of the benchmark fusion protein 2203BM01 to human FcγRI, the affinity of 2203F07 and 2203F09 to human FcγRI was reduced by 25.6 and 24.5 times, respectively; the binding of 2203F04 and 2203F05 to human FcγRI was even weaker.

[0206] Table 4 Affinity determination of TPO-RA fusion protein and Fc receptor

[0207] Affinity determination of TPO-RA fusion protein and human C1q

[0208] To determine whether the TPO-RA fusion protein binds to human C1q and induces a CDC effect, the affinity of the candidate fusion protein for human C1q protein (Acro, Catalog No. Ab282858) was determined using the Octet RED96e (Fortébio). The biotinylated candidate protein was immobilized on a SA sensor (Fortébio, Catalog No. 18-5020) at a concentration of 5 μg / ml. Human C1q protein was diluted in a two-fold series starting at 80 nM for a total of seven concentrations. Except for a 30 s binding / dissociation time, all other steps and data processing were the same as in Example 2.

[0209] The results are shown in Table 5. The benchmark protein 2203BM01 binds to the human C1q protein, while the candidate protein does not bind to the human C1q protein.

[0210] Table 5 Affinity determination of TPO-RA fusion protein and human C1q

[0211] Affinity determination of TPO-RA fusion protein and human FcRn

[0212] To determine whether the TPO-RA fusion protein can bind to human FcRn in a pH-dependent manner, thereby extending the protein's half-life, the affinity of candidate fusion proteins for human FcRn (Acro, Catalog No. FCN-H52W7) was determined using an Octet RED96e (Fortébio) at different pH conditions. PBST buffer was prepared at pH 6.0 and 7.4, and affinity testing was performed under the two different buffer conditions. Human FcRn was first immobilized on a SA sensor (Fortébio, Catalog No. 18-5020) using a biotinylated anti-his antibody (Genscript, Catalog No. A00613) at a concentration of 5 μg / ml. The candidate fusion protein was diluted two-fold starting at a 1000 nM concentration for a total of seven dilutions. Except for the initial SA probe, which was bound to the biotinylated anti-his antibody for 290 s, all other procedures and data processing were the same as in Example 4, "Affinity Determination of TPO-RA Fusion Proteins for Human Fc Receptors."

[0213] The results are shown in Table 6. At pH 6.0, the affinities of 2203F04, 2203F07, and 2203F09 for human FcRn were comparable to those of the benchmark protein 2203BM01, while the affinity of 2203F05 was approximately 4.7-fold higher than that of the benchmark protein 2203BM01. At pH 7.4, 2203F04, 2203F07, and the benchmark protein 2203BM01 bound very weakly to human FcRn, while the affinities of 2203F05 and 2203F09 increased.

[0214] Table 6 Affinity determination of TPO-RA fusion protein and human FcRn

[0215] Compared with 2203BM01, the binding of 2203F04, 2203F05, 2203F07 and 2203F09 to human Fc receptors and human C1q is significantly reduced, thereby avoiding potential ADCC, ADCP and CDC toxicity; the affinity of 2203F05 and 2203F09 to human FcRn at pH 6.0 is improved.

[0216] Proliferation assay of M07e cells stimulated by TPO-RA fusion protein

[0217] In order to determine the biological function of TPO-RA fusion protein, M07e cells (Nanjing Kebai, CBP60791) were selected to detect the effect of TPO-RA fusion protein on its proliferation.

[0218] M07e cells were starved and cultured overnight. The next day, the cells were collected and centrifuged at 300 g for 5 min. The cells were plated at 4 × 104 Cells were suspended at a density of 50 μl / well and evenly plated in a 96-well cell culture plate (Corning, 3590). The candidate protein and the original drug Romiplostim and IgG1 isotype (IgG1 wild type as isotype control) were diluted to 100 μg / ml as the initial concentration, and 9 concentrations were obtained by 10-fold serial dilution. A total of 10 concentrations, including the 0-well control, were added to the plated cells, with 50 μl per well. The cells were then cultured in a 37°C incubator for three days, and the cells were removed. 100 μl of CellTiter-Glo Luminescent Cell Viability Assay Reagent (Promega, Cat. No.: G7571) was added to each well. The cells were incubated on a shaker in the dark for 10 minutes and detected using an Envision microplate reader (PerkinElmer, 2105-0010).

[0219] The results are shown in Figure 1 . The ability of TPO-RA fusion protein to promote the proliferation of M07e was superior to that of the original drug Romiplostim.

[0220] Binding of TPO-RA fusion protein to human TPO-R on cell surface

[0221] To determine the binding of TPO-RA fusion proteins to human TPO-R on the cell surface, FACS was used to identify the binding of candidate proteins to human TPO-R on the surface of HEK Blue TPO cells (InvivoGen, hkb-tpo).

[0222] Based on the previous results of TPO-RA fusion protein affinity with Fc receptor and the results of stimulating the proliferation of M07e cells, the candidate protein 2203F07 and the reference proteins 2203BM01 and IgG1 were selected for biotin labeling (2203BM01-biotin, 2203F07-biotin and IgG1 isotype-biotin) for detecting binding to human TPO-R on the cell surface. The protein concentration was configured as 100 μg / ml as the initial concentration, followed by 9 10-fold dilutions. Collect HEK Blue TPO cells: centrifuge at 300 g for 5 minutes, discard the supernatant, and resuspend the cells in pre-cooled FACS Buffer (PBS + 2% FBS) at a density of 1*10 6Cells were plated evenly in a 96-well U-shaped plate (Corning, 3799) at a volume of 100 μl / well. After centrifugation at 400 g for 5 minutes, the supernatant was discarded, and the diluted candidate protein and 2203BM01 were added sequentially at 100 μl / well and incubated at 4°C for 1 hour. The cells were centrifuged at 400 g for 5 minutes, the supernatant discarded, and the cells were washed twice with FACS buffer. A 1:200 dilution of SA-PE (Invitrogen, Cat. No. 12-4317-87) was added at 100 μl / well and incubated at 4°C for 0.5 hours. After centrifugation at 400 g for 5 minutes, the supernatant discarded, and the cells were washed one to two times with FACS buffer. FACS buffer was added at 40 μl / well, the cells were resuspended, and the cells were analyzed using an Intellycite Plus analyzer. The results are shown in Figure 2. Both 2203F07 and the reference fusion protein 2203BM01 bound to human TPO-R on the surface of HEK Blue TPO cells with comparable efficacy.

[0223] TPO-RA fusion protein blocks the binding of TPO-Biotin to cell surface TPO-R

[0224] In order to determine the ability of TPO-RA fusion protein to block the binding of TPO-Biotin (ACRO, product number: THR-H52H7, biotin-labeled in the laboratory) to TPO-R on the surface of HEK Blue TPO cells, FACS detection was used. Candidate proteins 2203F07, 2203BM01 and original drug Romiplostim and IgG4 isotype (IgG4 wild type as isotype control) were diluted to 100 μg / ml as the initial concentration, and then diluted 10 times in sequence, for a total of 9 gradients. Collect HEK Blue TPO cells: centrifuge at 300g for 5 minutes, discard the supernatant, and resuspend in pre-cooled FACS Buffer (PBS + 2% FBS) to a density of 1*10 6A 100 μl / well suspension of 100 cells / ml was plated evenly in a 96-well U-shaped plate (Corning, 3799). The cells were centrifuged at 400 g for 5 minutes, and the supernatant was discarded. The diluted protein and original drug were added sequentially at 100 μl / well, and the cells were incubated at 4°C for 1 hour. The cells were centrifuged at 400 g for 5 minutes, the supernatant was discarded, and the cells were washed twice with FACS buffer. 100 μl of TPO-Biotin (1 μg / ml) was added to each well, and the cells were incubated at 4°C for 1 hour. The cells were centrifuged at 400 g for 5 minutes, the supernatant was discarded, and the cells were washed twice with FACS buffer. 100 μl / well of a 1:200 dilution of SA-PE (Invitrogen, Cat. No. 12-4317-87) was added to each well, and the cells were incubated at 4°C for 0.5 hours. The cells were centrifuged at 400 g for 5 minutes, the supernatant was discarded, and the cells were washed one to two times with FACS buffer. 40 μl / well of FACS buffer was added, and the cells were resuspended and analyzed using an Intellycite Plus analyzer.

[0225] The results are shown in Figure 3. 2203F07, the original drug Romiplostim, and the benchmark fusion protein 2203BM01 all inhibited the binding of TPO-Biotin to human TPO-R on the surface of HEK Blue TPO cells with comparable effects.

[0226] Detection of activation of HEK Blue TPO cell signaling pathway by TPO-RA fusion protein

[0227] In order to further determine the biological function of TPO-RA fusion protein, TPO reporter cell line was used to detect the activation effect of TPO-RA fusion protein on its downstream signaling pathway.

[0228] Prepare HEK Blue TPO cell suspension (InvivoGen, hkb-tpo): Incubate with preheated PBS at 37°C for 2-3 minutes or gently tap the cell culture flask to obtain a cell suspension. After centrifugation, resuspend the cells in fresh, preheated experimental medium (90% DMEM + 10% FBS) to control the cell density to approximately 2.8*10 5cells / ml. The candidate protein and the original drug Romiplostim and IgG4 isotype were diluted to 100 μg / mL as the initial concentration, and 9 concentrations were obtained by 5-fold serial dilution, plus a 0-well control for a total of 10 concentrations. Prepare a 96-well cell culture plate (Corning, 3590), add 180 μl of HEK Blue TPO cell suspension (approximately 50,000 cells) and 20 μl of the sample to be tested to each well, and incubate overnight at 37°C and 5% CO2. The next day, prepare QUANTI-Blue solution (InvivoGen, catalog number: rep-qbs): add 1 ml of QB reagent and 1 ml of QB buffer to 98 ml of sterile water, vortex and mix at room temperature for 10 minutes. Prepare a 96-well U-shaped plate (Corning, 3599), add 20 μl of HEK-Blue TPO cell supernatant and 180 μl of QUANTI-Blue solution incubated overnight, incubate at 37°C for 30 minutes, and use a microplate reader ( M3) SEAP levels were measured at 620 nm.

[0229] The results are shown in Figure 4. 2203F07, the original drug Romiplostim, and the benchmark fusion protein 2203BM01 all activated the downstream signaling pathways of HEK Blue TPO cells.

[0230] Detection of ADCP functional activity of TPO-RA fusion protein

[0231] To further determine the ADCP function of the TPO-RA fusion protein, Human CD64 (Luc) Jurkat Reporter Cells (ACRO, Catalog No.: SCJUR-STF072) were used to detect the ADCP activity of the TPO-RA fusion protein.

[0232] Prepare HEK Blue TPO cell (target cell) suspension and Human CD64 (Luc) Jurkat Reporter cell (effector cell) suspension: Use experimental culture medium (1640 + 10% FBS) to adjust the target cell HEK Blue TPO cell density to 1.67*10 6 cells / ml; the density of effector cells human CD64(Luc)Jurkat Reporter Cell was adjusted to 1.67*10 6The candidate protein and the original drug Romiplostim and IgG1 isotype were diluted to 0.185 μg / ml as the initial concentration, and 14 concentrations were obtained by 3-fold serial dilution, plus the 0-well control for a total of 15 concentrations. Prepare a 96-well cell culture plate (Corning, 3590), add 30 μl of HEK Blue TPO cell suspension (i.e. 5*10 4 cells / well), 30 μl human CD64(Luc)Jurkat Reporter cell suspension (i.e. 5*10 4 Cells were plated on a plate (100 cells / well) and 30 μl of the TPO-RA candidate protein sample and incubated at 37°C for 5 hours. 40 μl of Bio-Glo luciferase reagent (Promega, Cat. No. G7940) was added to each well and the cells were incubated at room temperature for 10 minutes before detection using Envision (PerkinElmer, 2105-0010).

[0233] The results are shown in Figure 5. ADCP activity was detected only in 2203BM01.

[0234] Example 5 Pharmacodynamic Study of 2203F07 Protein in Promoting Platelet Production in hFcRn Mice

[0235] Studies have shown that half-life data for antibody drugs obtained in humanized FcRn mice (hFcRn mice) strongly correlate with half-life data in non-human primates and humans. In Example 4, it was observed that the affinity of the 2203F07 protein molecule for human FcRn was slightly improved compared to Romiplostim, suggesting that the pharmacokinetic characteristics and pharmacodynamic effects of the 2203F07 protein molecule in humans may be superior to those of Romiplostim. To predict the difference in the pharmacodynamic effects of the 2203F07 protein molecule and Romiplostim in promoting platelet production in humans, this example used hFcRn mice as a test animal model for in vivo efficacy evaluation. Twenty-five male hFcRn mice aged 6-8 weeks were randomly divided into five groups of five animals each based on body weight. The specific groupings are shown in Table 7. According to the experimental protocol, each group of animals received a single intravenous (iv) or subcutaneous (sc) injection of a control (saline as a negative control), 100 μg / kg 2203F07, 100 μg / kg Romiplostim, 10 μg / kg 2203F07, and 10 μg / kg Romiplostim. EDTA-anticoagulated blood (approximately 30-50 μl) was collected from the retinal sinus before dosing (D0) and on days 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, and 20 after dosing for platelet count.

[0236] Table 7 Experimental plan for the pharmacodynamic study of 2203F07 in hFcRn mice

[0237] The platelet count results of mice at different times after administration are shown in Figure 6 , which show that subcutaneous administration of 2203F07 and Romiplostim can dose-dependently promote the increase in the number of circulating platelets in hFcRn mice, and the in vivo platelet-stimulating effect of 2203F07 is stronger than that of Romiplostim.

[0238] Example 6 Pharmacokinetic Study of 2203F07 Protein in hFcRn Mice

[0239] Humanized FcRn mice were used as test subjects to investigate the pharmacokinetic changes of romiplostim and 2203F07 protein after a single tail vein administration. Eighteen female hFcRn mice, aged 6-8 weeks, were randomly divided into six groups of three animals each based on body weight (see Table 8 for specific groupings). Each of the three groups received a single intravenous injection (iv) of 300 μg / kg romiplostim and 300 μg / kg 2203F07 protein. Whole blood (approximately 80 μl) was collected from the retinal sinus of the mice before and at 5, 30, 1, 2, 6, 10, 24, 48, 72, 96, and 168 hours after administration. The blood was collected into low-protein-binding EP tubes and allowed to rest at room temperature for 30 minutes. Serum was then collected by centrifugation at 3000 × rpm at 4°C for 15 minutes. All samples were stored at -80°C until analysis.

[0240] Table 8 Experimental plan for pharmacokinetic study of 2203F07 protein blood concentration in hFcRn mice

[0241] The indirect ELISA method was used to detect the drug concentrations of Romiplostim and 2203F07 protein in serum. The plate was coated with 2 μg / ml TPO-R (Acro biosystems, THR-H52H7) and incubated overnight at 2-8°C. The plate was then washed with a plate washer (1×PBS + 0.05% Tween20) and blocked with 1×PBST (1×PBS + 0.05% Tween20 + 1% BSA) solution at 37°C for 1 hour. The plate was washed and the final concentration of the standard track was set to 50 ng / ml to 0.3906 ng / ml. The standard track and serum sample were first diluted with mouse serum (Sbjbio, SBJ-SE-M008-100 mL) to the appropriate concentration, then diluted 25-fold with 1×PBST diluent containing 1% BSA and added to the blocked ELISA plate. The plate was incubated at 37°C for 1 hour. The plate was washed and HRP-labeled anti-human IgG Fc secondary antibody (Abcam, Ab99759) was added. The plate was incubated at 37°C for 1 hour. The plate was washed and TMB (sera The reaction was terminated by adding a stop solution (Solarbio, C1058) and color development was performed at 37°C for 5-10 minutes. OD at 450 nm was read on an M3 microplate reader (Molecular Devices). Softmax software was used to analyze the results, and serum drug concentrations were calculated by curve back-calculation. Pharmacokinetic parameters of drug concentrations were analyzed using WinNonlin software.

[0242] The plasma drug concentration results are shown in Table 9 and Figures 7-8. The half-lives of Romiplostim and 2203F07 were 9.97 hours and 14.68 hours, respectively; compared with Romiplostim, the half-life of the 2203F07 molecule was extended by approximately 47%.

[0243] Table 9 PK parameters of serum mean drug concentrations of 2203F07 after tail vein administration of isomeric 2203F07 to human FcRn transgenic mice (300 μg / kg, n=3)

[0244] Example 7 Detection of Binding of TPO-RA Fusion Protein to Human and Cynomolgus Monkey Platelets

[0245] In order to detect the binding of TPO-RA fusion protein to platelets, FACS was used to identify the binding of candidate proteins to human platelets (Allcells, Catalog No.: FST-PLT-5) and cynomolgus monkey platelets (Pengli Bio, Catalog No.: Cyno-PRP).

[0246] Fresh human / cynomolgus monkey platelets isolated on the day of the experiment were taken out according to the platelet concentration provided, diluted with FACS buffer, mixed and evenly plated in a 96-well U-shaped plate (Corning, 3799). Each well contained 100 μl of 2*10 6 Wash platelets once with FACS Buffer, centrifuge at 400g for 7 minutes, remove the supernatant, add 5μl Fc Block (BD, Cat. No. 564220) to each well, and incubate at room temperature for 10 minutes to completely block the Fc receptors on the platelets.

[0247] Protein Preparation: Fusion proteins 2203BM01, 2203F07, and IgG4 isotype were biotinylated to produce biotinylated proteins 2203BM01-Biotin, 2203F07-Biotin, and IgG4 isotype-Biotin. For human platelet binding experiments, a 4.8 μg / ml initial concentration of biotinylated protein was used, followed by 5-fold dilutions to create six concentration gradients. For cynomolgus monkey platelet binding experiments, a 24 μg / ml initial concentration of biotinylated protein was used, followed by 5-fold dilutions to create five concentration gradients.

[0248] Diluted biotinylated proteins 2203BM01-Biotin and 2203F07-Biotin were sequentially added to blocked platelets at 100 μl / well and incubated at 4°C for 1 hour. The platelets were centrifuged at 400 g for 7 minutes, the supernatant discarded, and the platelets washed twice with FACS buffer. A 1:200 dilution of SA-PE (Invitrogen, Cat. No. 12-4317-87) was added at 100 μl / well and incubated at 4°C for 0.5 hour. The platelets were centrifuged at 400 g for 7 minutes, the supernatant discarded, and the platelets washed twice with FACS buffer. FACS buffer was then added at 40 μl / well. The cells were resuspended and analyzed using an Intellycite Plus analyzer. The results are shown in Figures 9 and 10. The 2203F07 fusion protein bound to both human and cynomolgus monkey platelets with comparable binding to the 2203BM01 protein.

[0249] Example 8 Detection of CD34+ Cell Differentiation Induced by TPO-RA Fusion Protein

[0250] Hematopoietic progenitor cells (Marker: CD34+CD41-) can differentiate into megakaryocyte progenitor cells (Marker: CD34+CD41+) and megakaryocytes (Marker: CD34-CD41+) under the influence of certain cytokines, such as TPO. To test the effect of TPO-RA fusion protein on the differentiation of hematopoietic progenitor cells, CD34+ cells (Allcells, Catalog No.: FmPB015F-C-5M) were plated and the test protein was added and cultured for 12 days. The proportions of CD34+CD41- cells, CD34+CD41+ cells, and CD34-CD41+ cells relative to the total cells were determined by FACS.

[0251] Prepare CD34+ cell suspension: Use cell culture medium (IMDM (Gibco, Catalog No.: 2511081) (which contains 20% BIT9500 (Stemcell, Catalog No.: 09500), 100 μl β-mercaptoethanol (Sigma, Catalog No.: M3148), 7.5 μg / ml cholesterol (Sigma, Catalog No.: C3045)) to adjust the cell density to 4*104 cells / ml. Take candidate proteins 2203BM01, 2203F07 and original drug Romiplostim and IgG4 isotype to 2 μg / ml as the initial concentration, and dilute them in 10-fold gradient to obtain 8 concentrations. Prepare 24-well cell culture plates (Corning, 3526) and add 250 μl to each well. CD34+ cell suspension (i.e., 1*104 cells / well) and 250 μl of diluted test sample were incubated in a 37°C, 5% CO2 incubator for 12 days. After 12 days, the cells were removed and washed twice with 0.35% BSA-PBS, centrifuged at 400g for 5 minutes, and the supernatant was removed. The experimental group was added with 50 μl FITC-labeled anti-human CD41 antibody (thermofisher, catalog number: 11-0419-42) and 50 μl APC-labeled anti-human CD34 antibody (thermofisher, catalog number: 17-0349-42), and the control group was added with 50 μl FITC-labeled Mouse IgG1 kappa isotype control (thermofisher, catalog number: 11-4714-82) and 50 μl APC-labeled Mouse IgG1 A kappa isotype control (ThermoFisher, Cat. No. 17-4714-82) was added and incubated at 4°C for 0.5 h. The cells were centrifuged at 400 g for 5 min, the supernatant discarded, and the cells washed twice with 0.35% BSA-PBS. 40 μl / well of 0.35% BSA-PBS was added to the cells and resuspended. The cells were analyzed using an Intellycite Plus assay. The results are shown in Figures 11-13. Romiplostim, 2203BM01, and 2203F07 fusion proteins all induced CD34+CD41- cells to differentiate into CD34+CD41+ and CD34-CD41+ cells, with comparable induction abilities.

[0252] Sequence Listing

Claims

1. A thrombopoietin receptor (TPOR) agonist fusion protein, comprising a TPOR-binding domain and an Fc variant domain, wherein the TPOR-binding domain is a TPO mimetic peptide, wherein the TPO mimetic peptide comprises or consists of the amino acid sequence shown in SEQ ID NO: 9, and the Fc variant domain (i) from IgG1, and compared to wild-type IgG1, comprising a combination of mutations selected from the following c) L234A, L235A, D265S; or d) L234A, L235A, D265S, M252Y, S254T, T256E; (ii) is derived from IgG4 and comprises a mutation or combination of mutations selected from the group consisting of: e) S228P; f) S228P, F234A, L235A; g) S228P, T250Q, M428L; or h)S228P, F234A, L235A, T250Q, M428L.

2. The fusion protein of claim 1, wherein the Fc variant domain does not comprise a methionine at the N-terminus.

3. The fusion protein of claim 1 or 2, wherein the Fc variant domain g) comprising or consisting of an amino acid sequence of SEQ ID NO: 11, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto, preferably said Fc variant domain further comprises L234A+L235A+D265S mutations; h) comprising or consisting of an amino acid sequence of SEQ ID NO: 12, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto, preferably said Fc variant domain further comprising L234A+L235A+D265S+M252Y+S254T+T256E mutations; i) comprising or consisting of an amino acid sequence of SEQ ID NO: 13, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto, preferably the Fc variant domain further comprises an S228P mutation; j) comprising or consisting of an amino acid sequence of SEQ ID NO: 14, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto, preferably said Fc variant domain further comprises S228P+F234A+L235A mutations; k) comprising or consisting of an amino acid sequence of SEQ ID NO: 15, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto, preferably the Fc variant domain further comprises S228P+T250Q+M428L mutations; or l) comprising or consisting of an amino acid sequence of SEQ ID NO: 16, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto, preferably the Fc variant domain further comprises S228P+F234A+L235A+T250Q+M428L mutations.

4. The fusion protein according to any one of claims 1 to 3, comprising a peptide chain containing or consisting of two tandemly connected TPO mimetic peptides and an Fc variant domain.

5. The fusion protein of claim 4, wherein the peptide chain comprises or consists of the following from N-terminus to C-terminus: Fc variant domain-first linker-first TPO mimetic peptide-second linker-second TPO mimetic peptide, Optionally, the first linker and / or the second linker may be present or absent; or the first TPO mimetic peptide and the second TPO mimetic peptide may be the same or different. The fusion protein of claim 5 , comprising two peptide chains, wherein the two peptide chains may be the same or different.

7. The fusion protein of claim 5 or 6, wherein the first linker and / or the second linker is a glycine linker comprising 5, 6, 7 or 8 glycines.

8. The fusion protein of claim 7, wherein the first linker comprises 5 glycine residues and the second linker comprises 8 glycine residues.

9. The fusion protein of any one of claims 5 to 8, wherein the peptide chain comprises (i) an amino acid sequence of SEQ ID NO: 2, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto, or consisting thereof, preferably comprising L234A+L235A+D265S mutations; (ii). an amino acid sequence of SEQ ID NO: 3 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto, or consisting thereof, preferably comprising L234A+L235A+D265S+M252Y+S254T+T256E mutations; (iii). an amino acid sequence of SEQ ID NO: 4, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto, or consisting thereof, preferably comprising the S228P mutation; (iv). an amino acid sequence of SEQ ID NO: 5 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto, or consisting thereof, preferably comprising S228P+F234A+L235A mutations; (v). an amino acid sequence of SEQ ID NO: 6, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto, or consisting thereof, preferably comprising S228P+T250Q+M428L mutations; or (vi). An amino acid sequence of SEQ ID NO:7 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto, or consisting thereof, preferably comprising S228P+F234A+L235A+T250Q+M428L mutations.

10. An isolated polynucleotide encoding one or more chains of the fusion protein of any one of claims 1-9. An expression vector comprising the polynucleotide of claim 10 .

12. A host cell comprising the polynucleotide of claim 10 or the vector of claim 11, preferably the host cell is a yeast cell or a mammalian cell, in particular a HEK293 cell or a CHO cell.

13. A method for producing the fusion protein of any one of claims 1 to 9, comprising culturing the host cell of claim 12 under conditions suitable for expressing the fusion protein.

14. A pharmaceutical composition comprising the fusion protein according to any one of claims 1 to 9, and optionally a pharmaceutically acceptable excipient.

15. Use of the fusion protein according to any one of claims 1 to 9 or the pharmaceutical composition according to claim 14 in the preparation of a medicament for preventing and / or treating thrombocytopenia, preferably, the thrombocytopenia is primary immune thrombocytopenia, thrombocytopenia caused by tumor chemotherapy, thrombocytopenia associated with liver disease, or aplastic anemia.

16. A method for preventing and / or treating thrombocytopenia in a subject, the method comprising administering to the subject the fusion protein of any one of claims 1 to 9 or the pharmaceutical composition of claim 14, preferably, the thrombocytopenia is primary immune thrombocytopenia, thrombocytopenia caused by tumor chemotherapy, thrombocytopenia associated with liver disease or aplastic anemia.

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