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WO2025002113A1 - Protéine de fusion d'agoniste du récepteur de la thrombopoïétine humaine - Google Patents

Protéine de fusion d'agoniste du récepteur de la thrombopoïétine humaine Download PDF

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Publication number
WO2025002113A1
WO2025002113A1 PCT/CN2024/101284 CN2024101284W WO2025002113A1 WO 2025002113 A1 WO2025002113 A1 WO 2025002113A1 CN 2024101284 W CN2024101284 W CN 2024101284W WO 2025002113 A1 WO2025002113 A1 WO 2025002113A1
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Prior art keywords
amino acid
fusion protein
acid sequence
seq
identity
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Chinese (zh)
Inventor
夏凯文
邓俗俊
曹晓丹
欧阳可栋
甄滢滢
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Shanghai Jemincare Pharmaceuticals Co Ltd
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Shanghai Jemincare Pharmaceuticals Co Ltd
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/16—Extraction; Separation; Purification by chromatography
    • C07K1/18—Ion-exchange chromatography
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/16—Extraction; Separation; Purification by chromatography
    • C07K1/20—Partition-, reverse-phase or hydrophobic interaction chromatography
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes

Definitions

  • the present disclosure relates to a human thrombopoietin receptor (TPOR) binding molecule, in particular, a fusion protein and a fragment thereof that specifically binds to TPOR.
  • TPOR human thrombopoietin receptor
  • the present invention also relates to a nucleic acid or host cell comprising such a fusion protein or a fragment thereof, a drug comprising such a fusion protein or a fragment thereof, and a treatment and diagnosis method or use using these fusion proteins and fragments.
  • Thrombopoietin is the main cytokine that stimulates megakaryocyte proliferation and differentiation under physiological conditions. It is a glycoprotein with a molecular weight of about 35.5 kDa and is mainly synthesized by the liver. TPO binds to its receptor c-Mpl (TPOR) to form a homodimer of c-Mpl, thereby triggering the activation of a series of signaling pathways in the cell, leading to the proliferation and differentiation of megakaryocytes. TPO is a signal peptide synthesized and secreted by the liver and released into the blood circulation to play a role. It has the function of regulating megakaryocyte differentiation and platelet production and is the main regulatory factor for platelet production.
  • TPOR receptor c-Mpl
  • Thrombocytopenia is a type of hemorrhagic disease characterized by an abnormal decrease in platelets in peripheral blood, such as primary immune thrombocytopenia, thrombocytopenia caused by tumor chemotherapy, thrombocytopenia related to liver disease, aplastic anemia, etc.
  • primary immune thrombocytopenia thrombocytopenia caused by tumor chemotherapy
  • thrombocytopenia related to liver disease aplastic anemia, etc.
  • thrombocytopenia and a large patient base, with tens of millions of patients in China.
  • the clinical benefits of existing therapies are limited, and there is a large unmet clinical need.
  • Immune thrombocytopenia also known as idiopathic thrombocytopenic purpura, is an acquired hemorrhagic disease characterized by excessive platelet destruction and megakaryocyte maturation disorders mediated by humoral immunity and cellular immunity, accounting for about 1/3 of the total number of hemorrhagic diseases.
  • the annual incidence of ITP in adults in my country is 5-10/100,000, and the elderly over 60 years old are the high-risk group for the disease; at the same time, ITP is also the most common hemorrhagic disease in children. ITP patients often suffer from skin and mucous membrane bleeding, and severe cases may cause visceral bleeding or even intracranial hemorrhage. Severe ITP may be fatal.
  • Thrombocytopenia is present in up to 76% to 84% of patients with chronic liver disease (CLD).
  • CLD chronic liver disease
  • the hemostasis problem in patients with CLD 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.
  • the risk of bleeding is also affected by the severity of portal hypertension, concurrent coagulation abnormalities, and the degree of invasive surgery.
  • the platelet threshold is required to be between 40 ⁇ 10 9 /L and 75 ⁇ 10 9 /L.
  • CIT Chemotherapy-induced thrombocytopenia
  • CIT refers to the inhibitory effect of anti-tumor chemotherapy drugs on bone marrow megakaryocytes, resulting in a platelet count in the peripheral blood of less than 100 ⁇ 10 9 /L.
  • CIT is one of the most common chemotherapy-related hematological toxicities, which can increase the risk of bleeding, prolong hospitalization, increase medical expenses, and even lead to death in severe cases.
  • CIT can lead to reduced chemotherapy dose intensity, delayed time, or even treatment termination, thereby affecting the anti-tumor efficacy and having an adverse effect on the long-term survival of patients.
  • the pathogenesis of CIT includes multiple aspects, mainly including decreased platelet production, increased platelet destruction, and abnormal platelet distribution.
  • 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.
  • a novel binding molecule eg, fusion protein
  • TPOR thrombopoietin receptor
  • the fusion protein of the invention that specifically binds TPOR does not bind to C1q.
  • 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;
  • 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.
  • 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.
  • 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.
  • a pharmaceutical composition comprising the fusion protein of any one of embodiments 1-9, optionally and a pharmaceutical excipient.
  • thrombocytopenia is primary immune thrombocytopenia, thrombocytopenia caused by tumor chemotherapy, thrombocytopenia associated with liver disease, or aplastic anemia.
  • the peptide linker may or may not mainly include 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 , (GGGGGGG) n , (GGGGGGG) n, (GGGGGGG) 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.
  • the linker of the present invention is a linker composed of 5-8 glycine.
  • the domains of the thrombopoietin receptor (TPOR) binding 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 joint.
  • the fusion protein that specifically binds to TPOR of the present invention comprises a TPOR-binding domain and an Fc domain.
  • the TPOR-binding domain is a TPO mimetic peptide.
  • the TPO mimetic peptide comprises or consists of the amino acid sequence shown in SEQ ID NO:9.
  • the TPO mimetic peptide comprises a polypeptide composition having one or more (preferably no more than 3, for example 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.
  • the TPO mimetic peptide comprises an amino acid sequence having 1-3 amino acid differences (e.g., substitutions, deletions, or additions, such as conservative substitutions) from the amino acid sequence shown in SEQ ID NO: 9, or consists of the amino acid sequence.
  • the antibody Fc region can also carry an IgG hinge region or a portion of an IgG hinge region at the N-terminus, for example, an IgG1 hinge region or a portion of an IgG1 hinge region. Mutations may be contained in the hinge region.
  • the parent Fc region can be a wild-type Fc region or an Fc region that has a certain identity with the wild-type Fc region (an amino acid sequence that has at least 90% identity, such as 95%, 96%, 97%, 99% or higher identity thereto).
  • the parent Fc region can be a wild-type IgG1 Fc region or a wild-type IgG4 Fc region.
  • the IgG1 Fc region as the parent Fc region comprises or consists of the amino acid sequence SEQ ID NO: 10 or an amino acid sequence that has at least 90% identity, such as 95%, 96%, 97%, 99% or higher identity thereto.
  • the variant Fc domain suitable for the fusion protein of the present invention has reduced effector functions mediated by the Fc region, such as reduced or eliminated ADCC or ADCP or CDC effector functions, for example, comprising mutations that achieve the above functions.
  • the Fc region suitable for the fusion protein of the present invention comprises modifications that change the binding affinity to one or more Fc receptors and/or C1q.
  • the Fc receptor is an Fc ⁇ receptor, in particular a human Fc ⁇ receptor.
  • the Fc receptor is Fc ⁇ RIIb, Fc ⁇ RIIa H167, Fc ⁇ RIIa R167, Fc ⁇ RIIIa F176, Fc ⁇ RIIIa V176 and/or human Fc ⁇ RI.
  • 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.
  • 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 and D265S and YTE mutations.
  • the Fc domain suitable for 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 L235A mutation.
  • 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.
  • the N-terminus of the Fc domain of the fusion protein of the present invention may include an initial methionine. In one embodiment, the N-terminus of the Fc domain of the fusion protein of the present invention does not include a methionine.
  • the Fc variant domain of the fusion protein of the present invention comprises or consists of the amino acid sequence of SEQ ID NO:11-16 or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto.
  • 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.
  • 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).
  • 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.
  • 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 the IgG1 Fc region).
  • 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.
  • 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 IgG4 Fc region).
  • 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.
  • 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).
  • 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).
  • 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.
  • the one or two or more mimetic peptides are connected to the N-terminus or C-terminus of the Fc variant domain.
  • the two or more TPO mimetic peptides are connected in series.
  • 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.
  • the TPO mimetic peptides constituting the fusion protein of the present invention may be the same or different.
  • the Fc variant domains constituting the fusion protein of the present invention may be the same or different.
  • the fusion protein that specifically binds to TPOR comprises a peptide chain, wherein the peptide chain comprises or consists of two tandemly connected TPO mimetic peptides and an Fc variant domain.
  • the peptide chain comprises or consists of the following from the N-terminus to the C-terminus:
  • the fusion protein comprises two peptide chains, wherein the two peptide chains may be the same or different.
  • DNA or RNA 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 messenger RNA
  • “mRNA” or “messenger RNA” is a single-stranded RNA that specifies the amino acid sequence of one or more polypeptide chains. During protein synthesis, when ribosomes bind to the mRNA, the information is translated.
  • 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.
  • the immunoconjugates may 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, for example, 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 may also be derived from inorganic bases such as, for example, 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 solvents and other protic solvents than the corresponding free base forms.
  • 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, aplastic anemia.
  • the present invention relates to a method for preventing or treating a subject's disease, such as thrombocytopenia, comprising administering to the subject an effective amount of any fusion protein specifically binding to TPOR as described herein, or a pharmaceutical composition or combination product comprising the same.
  • the method comprises administering to the subject an effective amount of any fusion protein specifically binding 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 thrombocytopoietic drugs, etc.).
  • the thrombocytopenia is primary immune thrombocytopenia, thrombocytopenia induced by tumor chemotherapy, thrombocytopenia associated with liver disease, or aplastic anemia.
  • the fusion protein specifically binding 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.
  • the administration can be by any suitable route, for example by injection, such as intravenous or subcutaneous injection.
  • 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.
  • the appropriate dosage of the fusion protein that specifically binds to TPOR of the present invention (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 it is administered 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 in a single treatment or over a series of treatments.
  • the fusion protein that specifically binds to TPOR of the present invention can be administered to the patient at higher doses without causing toxicity.
  • the sequence of (i.e., SEQ ID NO: 8) was used as a blueprint for modification.
  • the molecule is essentially an Fc-fused homodimer fusion protein, and its monomer is structurally divided into two regions, the N-terminal and C-terminal domains.
  • the first amino acid in the N-terminal domain is methionine, which serves as the starter (designed for expression in E. coli), followed by the natural human IgG1 Fc region;
  • the C-terminal domain is two TPO mimetic peptides of 14 amino acid residues, connected by 5 glycines and 8 glycines from the N-terminus to the C-terminus, forming a divalent TPO receptor binding region.
  • the following Fc region amino acid numbers are all encoded by EU, human IgG1 Fc: D221-K447; human IgG4 Fc: E219-K447).
  • the first amino acid methionine at the N-terminus of the Fc carrier region was removed and the following mutations were performed: L234A, L235A, D265S, while the roplastin mimetic peptide and linker were kept unchanged.
  • the mimetic peptide was at the C-terminus of Fc to obtain a homodimeric tetravalent Fc fusion protein named 2203F04.
  • the first amino acid methionine at the N-terminus of the Fc carrier region was removed and the following mutations were performed: L234A, L235A, D265S, M252Y, S254T, T256E, while the roplastin mimetic peptide and linker were kept unchanged.
  • the mimetic peptide was at the C-terminus of Fc to obtain a homodimeric tetravalent Fc fusion protein named 2203F05.
  • 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 roplastin mimetic peptide and linker were kept unchanged, and the mimetic peptide was at the C-terminus of Fc, to obtain a homodimeric tetravalent Fc fusion protein named 2203F06.
  • 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, while the roplastin mimetic peptide and linker were kept unchanged, and the mimetic peptide was at the C-terminus of Fc to obtain a homodimeric tetravalent Fc fusion protein named 2203F07.
  • 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, while the roplastin mimetic peptide and linker were kept unchanged, and the mimetic peptide was at the C-terminus of Fc to obtain a homodimeric tetravalent Fc fusion protein named 2203F08.
  • 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 roplastin mimetic peptide and linker were kept unchanged, and the mimetic peptide was at the C-terminus of Fc to obtain a homodimeric tetravalent Fc fusion protein named 2203F09.
  • Example 2 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 a HPLC sample tray.
  • 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.
  • Mobile phase A 50 mM phosphate buffer + 1 M ammonium sulfate, pH 7.0
  • mobile phase B 50 mM phosphate buffer, pH 7.0
  • sample buffer Dilute the sample to 1 mg/mL with sample buffer, take 13 ⁇ L and add it to the reaction tube, add 5 ⁇ L Protein Thermal shift TM Buffer, add 2 ⁇ L 10 ⁇ staining solution, make the reaction volume 20 ⁇ L, mix well, centrifuge at 12000 rpm for 5 minutes to remove bubbles. Place the test sample in the PCR instrument, perform sample analysis, and record the sample Tm value.
  • TPO-RA TPO-RA
  • TPO-RA fusion protein 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 proteins with human (Acro, Catalog No.: THR-H52H7), cynomolgus monkey (Kactus, Catalog No.: MPL-CM101), and rat (Sino Biological, Catalog No.: 80346-R08B) TPO-R.
  • Candidate fusion proteins and receptors of different species were diluted with HBS-EP + buffer (cytiva; BR100669; 32351).
  • TPO-RA fusion protein was set as stationary phase at a concentration of 1 ⁇ g/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 the standby state, and then test on the machine.
  • Each cycle contains the following steps: 1) Capture: The fusion protein flows through channels 2, 3 and 4 at a flow rate of 10 ⁇ l/min for 30s; 2) Analyte: Inject analyte receptors of different species in channels 1, 2, 3, and 4 at a flow rate of 30 ⁇ l/min for 180s and dissociate for 300s; 3) Regeneration: Inject 10mM glycine-HCl, pH 1.5, at a flow rate of 30 ⁇ l/min for 30s in channels 1, 2, 3, and 4. The data obtained in the experiment were fitted with the 1:1 binding model using Biacore T200 Evaluation3.2.1 software.
  • Octet RED96e (Fortébio) was used to measure the affinity of the candidate fusion protein to human Fc ⁇ RI (Acro, Catalog No.: FCA-H52H2), Fc ⁇ RIIb (Acro, Catalog No.: CD8-H5222), Fc ⁇ RIIa H167 (Acro, Catalog No.: CD1-HS223), Fc ⁇ RIIa R167 (Acro, Catalog No.: CDA-H5221), Fc ⁇ RIIIa F176 (Acro, Catalog No.: CDA-H5220), and Fc ⁇ RIIIa V176 (Acro, Catalog No.: CD8-H52H4).
  • Fc ⁇ RI Acro, Catalog No.: FCA-H52H2
  • Fc ⁇ RIIb Acro, Catalog No.: CD8-H5222
  • Fc ⁇ RIIa H167 Acro, Catalog No.: CD1-HS223
  • Fc ⁇ RIIa R167 Acro, Catalog No.: CDA-H5221
  • the candidate fusion protein and Fc receptor were diluted with 1 ⁇ PBST (1 ⁇ PBS: Bio-Tech, Catalog No.: B548117-0500; 0.02% Tween 20: Sigma, Catalog No.: P1379).
  • the Fc receptor was immobilized on the HIS1K sensor (Fortébio, Catalog No.: 18-0015) at a concentration of 5 ⁇ g/ml.
  • the candidate fusion protein started at a concentration of 1250nM and was diluted 2-fold to a total of 7 concentrations.
  • the configured Fc receptor and candidate fusion protein were then added to a 96-well black board (Greiner bio-one, Catalog No.: 655209) according to the experimental layout, 200 ⁇ l/well.
  • Each cycle includes the following steps: 1) immersion in buffer for 60 seconds; 2) detection of non-specific binding of the fusion protein to the sensor; 3) regeneration in 10 mM glycine solution at pH 1.7; 4) immersion in buffer for 60 seconds; 5) immobilization of 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.
  • the association rate (K on ) and dissociation rate (K off ) of the antigen-antibody in a 1:1 binding mode were determined using Fortébio's Data Analysis 12.0 software to calculate the equilibrium dissociation constant (K D ) of the antibody.
  • the results are shown in Table 4 (the response values in the table only show the highest concentration response values).
  • the affinities of the benchmark fusion protein 2203BM01 and human Fc ⁇ RIIb, Fc ⁇ RIIa H167, Fc ⁇ RIIa R167, Fc ⁇ RIIIa F176, and Fc ⁇ RIIIa V176 are similar to the reported affinities of IgG1 antibodies and these Fc receptors, while the affinities of the candidate fusion proteins (2203F04, 2203F05, 2203F07, and 2203F09) and the above Fc receptors are lower than those of the benchmark fusion protein 2203BM01.
  • Octet RED96e was used to determine the affinity of the candidate fusion protein to the human C1q protein (Acro, Catalog No.: Ab282858).
  • the biotin-labeled candidate protein was immobilized on the SA sensor (Fortébio, Catalog No.: 18-5020) at a concentration of 5 ⁇ g/ml.
  • the human C1q protein started at a concentration of 80 nM and was diluted 2-fold to a total of 7 concentrations. Except that the binding and dissociation time was changed to 30 s, the remaining method steps and data processing were the same as in Example 2.
  • the benchmark protein 2203BM01 binds to the human C1q protein, while the candidate protein does not bind to the human C1q protein.
  • the affinity of the candidate fusion protein to human FcRn was determined using Octet RED96e (Fortébio).
  • PBST buffer reagents with pH values of 6.0 and 7.4 were prepared, and affinity tests were performed under two different pH buffer conditions.
  • human FcRn was immobilized on the SA sensor (Fortébio, Catalog No.: 18-5020) using biotin-labeled anti-his antibody (Genscript, Catalog No.: A00613) at a concentration of 5 ⁇ g/ml.
  • the candidate fusion protein started at a concentration of 1000 nM and was diluted 2-fold in a gradient, with a total of 7 dilutions. Except for the first time the SA probe was used, it was first bound to the biotin-labeled anti-his antibody for 290 s. The remaining method steps and data processing were the same as the "TPO-RA fusion protein and human Fc receptor affinity determination" in Example 4.
  • M07e cells (Nanjing Kebai, CBP60791) were selected to detect the effect of TPO-RA fusion protein on its proliferation.
  • M07e cells were starved and cultured overnight. The next day, cells were collected and centrifuged at 300g for 5min. The cells were suspended at a density of 4*10 4 cells/ml in experimental culture medium (RPMI1640+1% FBS) and evenly spread in a 96-well cell culture plate (Corning, 3590) at 50 ⁇ l/well.
  • Candidate proteins and original drug Romiplostim and IgG1 isotype (IgG1 wild type as isotype control) were diluted to 100 ⁇ g/ml as the initial concentration, and 9 concentrations were obtained by 10-fold gradient dilution. A total of 10 concentrations, plus 0-well control, were added to the spread cells, 50 ⁇ l per well.
  • TPO-RA fusion protein Binding of TPO-RA fusion protein to human TPO-R on 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).
  • the candidate protein 2203F07 and the reference 2203BM01 and IgG1 were selected for biotin labeling (2203BM01-biotin, 2203F07-biotin and IgG1 isotype-biotin) for detecting the binding with human TPO-R on the cell surface.
  • the protein concentration was configured as 100 ⁇ g/ml as the initial concentration, followed by 9 10-fold dilutions.
  • HEK Blue TPO cells were collected: centrifuged at 300g for 5 minutes, the supernatant was discarded, and the cells were resuspended in pre-cooled FACS Buffer (PBS + 2% FBS) at a density of 1*10 6 cells/ml, and evenly spread in a 96-well U-shaped plate (Corning, 3799) at 100 ⁇ l/well. Centrifuge at 400g for 5min, discard the supernatant, add the diluted candidate protein and 2203BM01 in turn, 100 ⁇ l/well, incubate at 4°C for 1h; centrifuge at 400g for 5min, discard the supernatant, and wash twice with FACS Buffer.
  • FACS Buffer PBS + 2% FBS
  • TPO-RA fusion protein Blocking of TPO-RA fusion protein on the binding of TPO-Biotin to TPO-R on the cell surface
  • TPO-RA fusion protein In order to determine the ability of TPO-RA fusion protein to block the binding of TPO-Biotin (ACRO, catalog 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 were diluted to 100 ⁇ g/ml as the initial concentration, and then diluted 10 times in sequence, with a total of 9 gradients.
  • Collect HEK Blue TPO cells centrifuge at 300g for 5min, discard the supernatant, resuspend with pre-cooled FACS Buffer (PBS + 2% FBS) to a density of 1*10 6 cells/ml suspension, evenly spread in a 96-well U-shaped plate (Corning, 3799) at 100 ⁇ l/well, centrifuge at 400g for 5min, discard the supernatant, add the diluted protein and original drug in sequence, 100 ⁇ l/well, incubate at 4°C for 1h; centrifuge at 400g for 5min, discard the supernatant, and wash twice with FACS Buffer.
  • FACS Buffer PBS + 2% FBS
  • TPO reporter cell line was used to detect the activation effect of TPO-RA fusion protein on its downstream signaling pathway.
  • HEK Blue TPO cell (InvivoGen, hkb-tpo) suspension Incubate with preheated PBS at 37°C for 2-3 minutes or gently tap the cell culture bottle to obtain a cell suspension. After centrifugation, resuspend the cells in fresh, preheated experimental culture medium (90% DMEM + 10% FBS) to control the cell density to about 2.8*10 5 cells/ml. Take the candidate protein and the original drug Romiplostim and IgG4 isotype diluted to 100 ⁇ g/mL as the initial concentration, dilute 5-fold to obtain 9 concentrations, plus 0 wells for a total of 10 concentrations.
  • HEK Blue TPO cell (target cell) suspension and Human CD64 (Luc) Jurkat Reporter cell (effector cell) suspension adjust the target cell HEK Blue TPO Cell density to 1.67*10 6 cells/ml with experimental culture medium (1640+10% FBS); adjust the effector cell human CD64 (Luc) Jurkat Reporter Cell density to 1.67*10 6 cells/ml. Dilute the candidate protein and the original drug Romiplostim and IgG1 isotype to 0.185 ⁇ g/ml as the initial concentration, and dilute to 14 concentrations in 3-fold gradient, plus 0-well control for a total of 15 concentrations. Prepare a 96-well cell culture plate (Corning, 3590), add 30 ⁇ l HEK Blue TPO cell suspension (i.e.
  • mice 25 male hFcRn mice aged 6-8 weeks were randomly divided into 5 groups, 5 animals in each group, and the specific grouping is shown in Table 7. According to the experimental plan, each group of animals was given a single tail vein injection (i.v.)/subcutaneous injection (s.c.) of Control (normal saline as negative control), 100 ⁇ g/kg 2203F07, 100 ⁇ g/kg Romiplostim, 10 ⁇ g/kg 2203F07 and 10 ⁇ g/kg Romiplostim.
  • s.c. subcutaneous injection
  • EDTA anticoagulated blood (about 30-50 ⁇ l) was collected from the retinal venous sinus before administration (D0) and on the 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 10th, 12th, 14th, 16th and 20th days after administration for platelet count detection.
  • FIG6 The results of platelet counts in mice at different times after administration are shown in FIG6 , 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 that the in vivo platelet-promoting effect of 2203F07 is stronger than that of Romiplostim.
  • Humanized FcRn mice were used as test animals to study 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 6 groups according to their body weight, with 3 animals in each group. The specific grouping is shown in Table 8; each of the 3 groups of mice was given the same test drug, with a single tail vein injection (i.v.) of 300 ⁇ g/kg Romiplostim and 300 ⁇ g/kg 2203F07 protein.
  • Whole blood (about 80 ⁇ l) was collected from the test mice through the retinal venous sinus into a low-protein adsorption EP tube before administration and 5min, 30min, 1h, 2h, 6h, 10h, 24h, 48h, 72h, 96h and 168h after administration.
  • the blood was kept at room temperature for 30min and then centrifuged at 3000 ⁇ rpm and 4°C for 15min to collect serum. All samples were stored in a -80°C refrigerator for testing.
  • the indirect ELISA method was used to detect the drug concentration of Romiplostim and 2203F07 protein in serum.
  • 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).
  • Fresh human/cynomolgus monkey platelets isolated on the day of the scheduled experiment were taken out according to the platelet concentration provided, and some platelets were diluted with FACS Buffer, mixed and evenly spread in a 96-well U-shaped plate (Corning, 3799). 100 ⁇ l per well, containing 2*10 6 platelets. Wash once with FACS Buffer, centrifuge at 400g for 7min, remove the supernatant, add 5 ⁇ l Fc Block (BD, Cat. No.: 564220) to each well, incubate at room temperature for 10 minutes, and completely block the Fc receptors on the platelets.
  • FACS Buffer BD, Cat. No.: 564220
  • Protein configuration Fusion proteins 2203BM01, 2203F07, and IgG4 isotype were labeled with biotin to obtain biotin-labeled proteins 2203BM01-Biotin, 2203F07-Biotin, and IgG4 isotype-Biotin.
  • biotin-labeled proteins 2203BM01-Biotin, 2203F07-Biotin, and IgG4 isotype-Biotin.
  • 4.8 ⁇ g/ml biotin-labeled protein was selected as the initial concentration, and then diluted 5 times to obtain 6 concentration gradients.
  • 24 ⁇ g/ml biotin-labeled protein was selected as the initial concentration, and then diluted 5 times to obtain 5 concentration gradients.
  • Hematopoietic progenitor cells can differentiate into megakaryocyte progenitor cells (Marker: CD34+CD41+) and megakaryocytes (Marker: CD34-CD41+) under the action of some cytokines such as TPO.
  • CD34+ cells Allcells, Catalog No.: FmPB015F-C-5M
  • FmPB015F-C-5M FmPB015F-C-5M
  • CD34+ cell suspension adjust the cell density to 4*104 cells/ml with cell culture medium (IMDM (Gibco, Catalog No.: 2511081) (which contains 20% BIT9500 (Stemcell, Catalog No.: 09500), 100 ⁇ l ⁇ -mercaptoethanol (Sigma, Catalog No.: M3148), 7.5 ⁇ g/ml cholesterol (Sigma, Catalog No.: C3045)).
  • IMDM Gibco, Catalog No.: 2511081
  • BIT9500 Stem, Catalog No.: 09500
  • 100 ⁇ l ⁇ -mercaptoethanol Sigma, Catalog No.: M3148
  • 7.5 ⁇ g/ml cholesterol Sigma, Catalog No.: C3045
  • candidate proteins 2203BM01, 2203F07 and original drugs Romiplostim and IgG4 iso type was diluted to 2 ⁇ g/ml as the initial concentration, and 8 concentrations were obtained by 10-fold gradient dilution.

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Abstract

La présente invention concerne une molécule de liaison au récepteur de la thrombopoïétine humaine (TPOR), et en particulier une protéine de fusion se liant spécifiquement au TPOR et un fragment de celle-ci. En outre, la présente invention concerne également un acide nucléique ou une cellule hôte contenant la protéine de fusion ou le fragment de celle-ci, un médicament contenant la protéine de fusion ou le fragment de celle-ci, et des méthodes ou des utilisations thérapeutiques et diagnostiques faisant intervenir ladite protéine de fusion ou ledit fragment de celle-ci.
PCT/CN2024/101284 2023-06-26 2024-06-25 Protéine de fusion d'agoniste du récepteur de la thrombopoïétine humaine Pending WO2025002113A1 (fr)

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CN111432845A (zh) * 2017-07-26 2020-07-17 詹森药业有限公司 保护靶向放射治疗诱导下的血管完整性的方法
CN113402614A (zh) * 2021-04-22 2021-09-17 山东泉港药业有限公司 血小板生成素拟肽融合蛋白(fc-tmp)编码基因与应用
CN113999316A (zh) * 2021-10-28 2022-02-01 中国科学院大学宁波华美医院 一种血小板生成素模拟肽类似物及其应用
WO2022265331A1 (fr) * 2021-06-14 2022-12-22 고려대학교 산학협력단 Variants fc à mécanisme immunitaire contrôlé et demi-vie dans le sang accrue

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WO2002078612A2 (fr) * 2001-04-02 2002-10-10 Euro-Celtique S.A. Anticorps synthetique contenant de la thrombopoietine (tpo) pour la stimulation de la production de plaquettes
CN101103045A (zh) * 2004-09-24 2008-01-09 安姆根有限公司 修饰的Fc分子
CN101500609A (zh) * 2006-06-14 2009-08-05 中外制药株式会社 造血干细胞增加促进剂
US20150125430A1 (en) * 2013-11-06 2015-05-07 Hadasit Medical Research Services And Development Ltd. Compositions and methods for inducing thrombopoiesis
CN111432845A (zh) * 2017-07-26 2020-07-17 詹森药业有限公司 保护靶向放射治疗诱导下的血管完整性的方法
CN113402614A (zh) * 2021-04-22 2021-09-17 山东泉港药业有限公司 血小板生成素拟肽融合蛋白(fc-tmp)编码基因与应用
WO2022265331A1 (fr) * 2021-06-14 2022-12-22 고려대학교 산학협력단 Variants fc à mécanisme immunitaire contrôlé et demi-vie dans le sang accrue
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