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WO2025209465A1 - Protéine de fusion de chimère taci/bcma et son utilisation - Google Patents

Protéine de fusion de chimère taci/bcma et son utilisation

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Publication number
WO2025209465A1
WO2025209465A1 PCT/CN2025/086591 CN2025086591W WO2025209465A1 WO 2025209465 A1 WO2025209465 A1 WO 2025209465A1 CN 2025086591 W CN2025086591 W CN 2025086591W WO 2025209465 A1 WO2025209465 A1 WO 2025209465A1
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WIPO (PCT)
Prior art keywords
amino acid
taci
acid sequence
bcma
seq
Prior art date
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Pending
Application number
PCT/CN2025/086591
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English (en)
Chinese (zh)
Inventor
熊尧
周帅祥
张志敏
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Fortvita Biologics Inc
Original Assignee
Fortvita Biologics Inc
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Filing date
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Publication of WO2025209465A1 publication Critical patent/WO2025209465A1/fr
Pending legal-status Critical Current
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/12—Drugs for disorders of the urinary system of the kidneys
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P21/00—Drugs for disorders of the muscular or neuromuscular system
    • A61P21/04—Drugs for disorders of the muscular or neuromuscular system for myasthenia gravis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/04—Antihaemorrhagics; Procoagulants; Haemostatic agents; Antifibrinolytic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/06—Antianaemics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione

Definitions

  • the present invention relates to a novel TACI/BCMA chimera and fusion proteins comprising the same.
  • the present invention also relates to nucleic acids encoding the chimera or fusion protein, vectors comprising the nucleic acids, and host cells comprising the nucleic acids or vectors.
  • the present invention also relates to therapeutic methods and uses of the fusion proteins for treating immune-related diseases.
  • TNF cytokine tumor necrosis factor
  • TNFSF TNF receptor superfamily
  • TNFRSF TNF receptor superfamily
  • TNF receptor superfamily refers to a group of cell surface cytokine receptors, all of which are type I (N-terminal extracellular) transmembrane glycoproteins containing one to six cysteine-rich domains (CRDs) in their extracellular domains. Molecules are classified as members of the superfamily based on shared structural features, including one or more cysteine-rich domains (CRDs) present in their N-terminal extracellular regions, which typically play a role in the binding of proteins to their cognate binding partners or ligands. TNFRSF proteins may have only one or several CRDs (e.g., CRD1, CRD2, etc.).
  • TNFRSF members typically contain 1 to 6 CRD pseudorepeats.
  • BAFF receptor and BCMA each contain one CRD
  • TACI contains two CRDs (CRD1 and CRD2).
  • TNFRSF members are typically trimerized or multimerized complexes that are stabilized by disulfide bonds within their cysteines. Binding of TNFRSF proteins to their ligands promotes a variety of biological activities in cells, such as induction of apoptotic cell death or cell survival and proliferation.
  • BAFF B cell activating factor
  • APRIL A proliferation-inducing ligand
  • TACI transmembrane activator and CAML-interacting protein
  • TNFRSF13B tumor necrosis factor receptor superfamily member 13B
  • APRIL calcium regulator and cyclophilin ligand
  • fusion proteins containing TACI for the treatment of immune diseases.
  • Rongchang Biopharma developed an optimized TACI-Fc fusion protein, Taitasip (CN101323643B), which utilizes the full-length extracellular domain of TACI.
  • Taitasip CN101323643B
  • the resulting drug product exhibits poor molecular stability, requiring only lyophilized powder.
  • the clinical dosing cycle is two injections per week, making it impossible to compensate for the increased dosing frequency by increasing the dosage.
  • the present invention relates to a TACI/BCMA chimera and a fusion protein comprising the chimera, such as a fusion protein with an Fc region.
  • the Fc region fusion protein of the TACI/BCMA chimera of the present invention has better stability, such as thermal stability, compared with known TACI-Fc fusion proteins; it also has a longer half-life and/or a lower clearance rate.
  • the present invention relates to a TACI/BCMA chimera, comprising a chimeric protein in which the N-terminus and/or C-terminus of a functional fragment of the TACI extracellular domain ECD is replaced with the N-terminus and/or C-terminus of BCMA.
  • TACI portion BCMAN terminal amino acid-TACI portion, wherein the TACI portion is the extracellular domain ECD of TACI lacking the N-terminus or a functional fragment thereof, or
  • the extracellular ECD functional fragment of TACI in the TACI/BCMA chimera contains CRD2 of TACI and does not contain CRD1 or any fragment of CRD1.
  • the CRD2 is the amino acid sequence of TACI corresponding to positions 71-104 shown in SEQ ID NO:1
  • CRD1 is the amino acid sequence of TACI corresponding to positions 34-66 shown in SEQ ID NO:1.
  • the extracellular ECD functional fragment of TACI further contains a partial stem region of TACI and/or any amino acid sequence between positions 68-70 of TACI corresponding to SEQ ID NO:1.
  • the extracellular ECD functional fragment of TACI is or contains the following fragment of TACI corresponding to the amino acid sequence shown in SEQ ID NO:1: amino acid residues 68-110. In one embodiment, the extracellular ECD functional fragment of TACI contains
  • the N-terminus of the extracellular ECD functional fragment of TACI refers to the N-terminal amino acid before the Y79 amino acid of TACI corresponding to SEQ ID NO:1.
  • the C-terminus of the extracellular ECD functional fragment of TACI refers to the C-terminal amino acid after the 99th amino acid, the 100th amino acid, the 101st amino acid, the 102nd amino acid, the 103rd amino acid, the 104th amino acid, the 105th amino acid, the 106th amino acid, the 107th amino acid, the 108th amino acid, the 109th amino acid or the 110th amino acid of TACI corresponding to SEQ ID NO: 1, preferably the C-terminal amino acid after the 99th or 105th amino acid.
  • the N-terminal amino acid of BCMA is selected from the amino acid sequence of BCMA corresponding to positions 1-13, 2-13, 3-13, 4-13, 5-13, 6-13, or 7-13 of SEQ ID NO: 2.
  • the N-terminal amino acid sequence of BCMA comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 40-46.
  • the C-terminal amino acid sequence of BCMA is the amino acid sequence of BCMA corresponding to positions 37-47, 37-46, 37-45, 37-44 of SEQ ID NO: 2, or the amino acid sequence corresponding to positions 43-44 or 43-46 of SEQ ID NO: 2.
  • the C-terminal amino acid of BCMA comprises a mutation, such as a substitution, that improves binding affinity, increases stability and/or improves druggability, such as a mutation at position 39 and/or position 42, such as a substitution, such as N42A or N42Q or R39D or N42A-R39D.
  • the C-terminal amino acid sequence of BCMA comprises the amino acid sequence shown in any one of SEQ ID NO:47-55, or consists of the amino acid sequence.
  • the TACI/BCMA chimera comprises the amino acid sequence shown in any one of SEQ ID NO:59-80, or comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% or 98% identical thereto, or consists of the amino acid sequence.
  • mutations that reduce binding to Fc ⁇ receptors such as L234A/L235A mutations or L234A/L235E or L234A/L235E/G237A mutations;
  • amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 85 or 87 and comprises the mutations L234A/L235E/G237A;
  • amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence set forth in SEQ ID NO: 100 or 102 and comprises the mutations L234A/L235E/G237A and M252Y/S254T/T256E;
  • the TACI/BCMA chimera is fused to Fc directly or via a linker.
  • the C-terminus of the chimera is fused to the N-terminus of Fc directly or via a linker.
  • the linker is the amino acid sequence shown in SEQ ID NO:39.
  • the fusion protein in a specific embodiment, the fusion protein
  • (i) comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of any one of SEQ ID NOs: 5-28; or
  • the present invention relates to a fusion protein dimer comprising a first monomer and a second monomer, wherein the first monomer and the second monomer respectively comprise or consist of the fusion protein chain described herein, preferably the first monomer and the second monomer are identical.
  • the present invention relates to a polynucleotide encoding the TACI/BCMA chimera or fusion protein or fusion protein dimer of the present invention.
  • the present invention relates to an expression vector comprising the polynucleotide of the present invention, for example, the expression vector is a pCDNA expression vector, such as a pCDNA3.1 expression vector.
  • the present invention relates to a host cell comprising the polynucleotide or expression vector of the present invention.
  • the present invention relates to a method for preparing a TACI/BCMA chimera or a fusion protein or fusion protein dimer thereof, wherein the method comprises culturing the host cells of the present invention under conditions suitable for the expression of the TACI/BCMA chimera or its fusion protein or fusion protein dimer, and optionally recovering the TACI/BCMA chimera or its fusion protein or fusion protein dimer from the host cells (or host cell culture medium).
  • the present invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising the TACI/BCMA chimera or fusion protein or fusion protein dimer of the present invention, and optionally a pharmaceutically acceptable excipient.
  • the present invention relates to a pharmaceutical combination or combination product comprising a TACI/BCMA chimera or fusion protein or fusion protein dimer of the present invention and one or more other therapeutic agents (e.g., cytokines, hormones, cytotoxic agents or inhibitors (e.g., cytostatic agents that affect T cell and/or B cell proliferation), antibodies or small molecule drugs or immunomodulators (e.g., immunosuppressants)).
  • cytokines e.g., cytotoxic agents or inhibitors (e.g., cytostatic agents that affect T cell and/or B cell proliferation)
  • antibodies or small molecule drugs or immunomodulators e.g., immunosuppressants
  • the present invention relates to a method for preventing or treating a subject's disease, such as a B cell or autoantibody-related disease or immune system disease (e.g., an autoimmune disease) or inflammation, the method comprising administering to the subject a TACI/BCMA chimera or fusion protein or fusion protein dimer or pharmaceutical composition or combination product of the present invention.
  • a subject's disease such as a B cell or autoantibody-related disease or immune system disease (e.g., an autoimmune disease) or inflammation
  • the method comprising administering to the subject a TACI/BCMA chimera or fusion protein or fusion protein dimer or pharmaceutical composition or combination product of the present invention.
  • the B cell or autoantibody-related disease is a B cell or autoantibody-mediated disease, such as an autoimmune disease mediated by a B cell or autoantibody.
  • the B cell or autoantibody-related disease or immune system disease or inflammation is a disease (e.g., an autoimmune disease) in which B cells abnormally proliferate or are abnormally activated in an individual compared to a sample of a healthy individual.
  • a disease e.g., an autoimmune disease
  • the disease is lupus, such as systemic lupus erythematosus, chronic kidney disease such as rheumatoid arthritis, IgA nephropathy (IgAN) or membranous nephropathy, Sjögren's syndrome, myasthenia gravis, idiopathic thrombocytopenic purpura (ITP), warm antibody autoimmune hemolytic anemia (wAIHA), multiple sclerosis (MS), coronary heart disease (CAD) or thyroid eye disease.
  • IgA nephropathy IgAN
  • membranous nephropathy Sjögren's syndrome
  • myasthenia gravis idiopathic thrombocytopenic purpura
  • ITP idiopathic thrombocytopenic purpura
  • wAIHA warm antibody autoimmune hemolytic anemia
  • MS multiple sclerosis
  • CAD coronary heart disease
  • the administration further comprises the combined administration of one or more other therapeutic agents (e.g., cytokines, hormones, cytotoxic agents or inhibitors (e.g., cytostatic agents that affect T cell and/or B cell proliferation), antibodies or small molecule drugs or immunomodulators (e.g., immunosuppressants)).
  • cytokines e.g., cytokines, hormones, cytotoxic agents or inhibitors (e.g., cytostatic agents that affect T cell and/or B cell proliferation)
  • antibodies or small molecule drugs or immunomodulators e.g., immunosuppressants
  • Figure 1 shows the structural analysis of the complexes of TACI and BCMA with APRIL.
  • Figure 2 shows the structural alignment and analysis of TACI and BCMA.
  • FIG3 shows the amino acid sequence alignment of TACI and BCMA.
  • FIG4 shows the ability of TACI/BCMA chimeric fusion protein to inhibit B cell proliferation.
  • FIG5 shows that TACI/BCMA chimeric fusion protein inhibits the proliferation of mouse spleen cells and spleen B cells induced by KLH immunization.
  • FIG6 shows that TACI/BCMA chimeric fusion protein inhibits the levels of IgA, IgM and IgG in the serum of mice immunized with KLH.
  • FIG7 shows the pharmacokinetics of TACI/BCMA chimeric fusion protein in mice.
  • Figure 8 shows the toxicokinetics of TACI/BCMA chimeric fusion protein in cynomolgus monkeys.
  • Figure 9 shows the pharmacokinetics of TACI/BCMA chimeric fusion protein and its YTE molecule in cynomolgus monkeys.
  • FIG10 shows the changes in immunoglobulin levels of TACI/BCMA chimeric fusion protein and its YTE molecule in cynomolgus monkey serum.
  • the terms “comprising” or “including” mean including the recited elements, integers, or steps, but not excluding any other elements, integers, or steps.
  • the terms “comprising” or “including” are used, unless otherwise indicated, the context of consisting of the recited elements, integers, or steps is also encompassed.
  • the context of consisting of the recited elements, integers, or steps is also encompassed.
  • the parent TACI refers to a template for introducing mutations of the present invention, which can be wild-type TACI, such as naturally occurring TACI protein, such as natural TACI derived from humans, mice, rats, non-human primates, including unprocessed (e.g., signal peptide not removed) forms and processed (e.g., N-terminal methionine removed) forms; or, for example, naturally occurring TACI allelic variants and splice variants, isoforms, homologs, and species homologs; or, for example, a TACI variant, for example, the variant can have at least 95%, 96%, 97%, 98% or 99% or higher identity with natural TACI or have no more than 1-10 or 1-5 amino acid mutations (e.g., conservative substitutions), and preferably has substantially the same BAFF binding affinity and/or APRIL binding affinity as the natural TACI protein.
  • wild-type TACI such as naturally occurring TACI protein, such as natural TACI
  • the parent TACI refers to a functional fragment of TACI, such as a fragment comprising the extracellular domain (ECD) of TACI or the CRD2 domain of TACI, such as the extracellular domain (ECD) of TACI, or the CRD2 domain of TACI, or other functional fragments of TACI.
  • a functional fragment of TACI such as a fragment comprising the extracellular domain (ECD) of TACI or the CRD2 domain of TACI, such as the extracellular domain (ECD) of TACI, or the CRD2 domain of TACI, or other functional fragments of TACI.
  • amino acid position in a TACI protein or TACI sequence segment when referring to an amino acid position in a TACI protein or TACI sequence segment, it is determined by reference to the amino acid sequence of wild-type human TACI protein (also referred to as TACI WT ) SEQ ID NO: 1.
  • the corresponding amino acid position on other TACI proteins or polypeptides can be identified by amino acid sequence alignment with SEQ ID NO: 1. Therefore, in the present invention, unless otherwise indicated, the amino acid position of a TACI protein or polypeptide is the amino acid position numbered according to SEQ ID NO: 1. For example, when referring to "Y102", it refers to the tyrosine residue Y at position 102 of SEQ ID NO: 1, or the amino acid residue at the corresponding position on another TACI polypeptide sequence after alignment.
  • the amino acid position of a BCMA protein or BCMA sequence segment when referring to an amino acid position in a BCMA protein or BCMA sequence segment, it is determined by reference to the amino acid sequence of wild-type human BCMA protein (also referred to as BCMA WT ) SEQ ID NO: 2.
  • the corresponding amino acid position on other BCMA proteins or polypeptides can be identified by amino acid sequence alignment with SEQ ID NO: 2. Therefore, in the present invention, unless otherwise indicated, the amino acid position of a BCMA protein or polypeptide is the amino acid position numbered according to SEQ ID NO: 2. For example, when referring to "Q3", it refers to the tyrosine residue Q at position 3 of SEQ ID NO: 2, or the amino acid residue at the corresponding position on other TACI polypeptide sequences after alignment.
  • single amino acid substitutions are described as follows: [original amino acid residue/position/substituted amino acid residue].
  • the substitution of tyrosine at position 102 with aspartic acid can be represented as Y102D.
  • a given position e.g., position Y102
  • the amino acid substitution can be represented as: Y102D/E.
  • the single amino acid substitutions can be connected by a plus sign "+" or "-" to represent a combination mutation at multiple given positions.
  • a combination mutation at positions K77E, F78Y, and Y102D can be represented as: K77E-F78Y-Y102D, or K77E+F78Y+Y102D.
  • N-terminal amino acid or “N-terminus” are used interchangeably and refer to one or more amino acid segments starting from the N-terminus of BCMA or TACI or a functional fragment thereof.
  • C-terminal amino acid or “C-terminus” are used interchangeably and refer to one or more amino acid segments ending at the C-terminus of BCMA or TACI or a functional fragment thereof.
  • chimera refers to a fusion protein formed by genetically engineering two or more fragments of different proteins. Such chimeras typically retain the original functions of each fragment and may exhibit new biological properties or enhanced stability.
  • drugability risk site refers to a site on a pharmaceutically acceptable protein, such as the chimera or fusion protein of the present invention, that affects its drugability (e.g., stability, etc.), including post-translational modification sites such as isomerization (D), deamidation (N), glycosylation (N*S/T), free cysteine (C), oxidation (M/W); and a large area of hydrophobic amino acids or charged amino acid residues enriched in the exposed surface of the protein (patch).
  • D isomerization
  • N deamidation
  • N*S/T glycosylation
  • C free cysteine
  • M/W oxidation
  • percent sequence identity can be determined by comparing two optimally aligned sequences over a comparison window. Preferably, sequence identity is determined over the entire length of a reference sequence (e.g., SEQ ID NO: 1). Methods of sequence alignment for comparison are well known in the art. Suitable algorithms for determining percent 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 analyses is publicly available through the National Center for Biotechnology Information. For the purposes of this application, percent identity is determined using the Basic Local Alignment Search Tool available from https://blast.ncbi.nlm.nih.gov/Blast.cgi using default parameters.
  • conservative substitution means an amino acid substitution that does not adversely affect or alter the biological function of the protein/polypeptide comprising the amino acid sequence.
  • 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).
  • Conservative substitution tables for functionally similar amino acids are well known in the art. In some embodiments, the following are exemplary conservative substitutions:
  • the parent TACI protein can have conservative amino acid substitutions relative to one of SEQ ID NO: 1, or only conservative amino acid substitutions, and in a preferred embodiment, the conservative substitutions do not exceed 10 amino acid residues, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 residues.
  • the mutant TACI protein of the present invention can have conservative amino acid substitutions relative to the TACI mutant protein sequences specifically given herein, or only conservative amino acid substitutions, and in a preferred embodiment, the conservative substitutions do not exceed 10 amino acid residues, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 residues.
  • 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 represented by the equilibrium dissociation constant ( KD ), which is the ratio of the dissociation rate constant and the association rate constant ( kdis and kon , respectively).
  • KD equilibrium dissociation constant
  • kdis and kon association rate constant
  • Binding affinity can be measured by common methods known in the art. One specific method for measuring affinity is the ForteBio affinity assay technology described herein.
  • the extracellular domain typically interacts with a specific ligand or a specific cell surface receptor, for example, by specifically binding to a binding domain of a ligand or cell surface receptor.
  • binding domains include cysteine-rich domains (CRDs).
  • CRDs cysteine-rich domains
  • the extracellular domains of members of the TNFR superfamily contain TD domains (e.g., CRD domains). Therefore, reference to the ECD herein includes the full-length sequence of the ECD of a membrane protein, as well as specific binding fragments thereof containing a CRD or a portion thereof that bind to a ligand.
  • an antibody Fc fragment refers to the C-terminal region of an immunoglobulin heavy chain containing at least a portion of the constant region, and may include native sequence Fc fragments and variant Fc fragments.
  • Native sequence Fc fragments encompass various naturally occurring immunoglobulin Fc sequences, such as the Fc regions of various Ig subclasses and their allotypes (Gestur Vidarsson et al., IgG subclasses and allotypes: from structure to effector functions, 20 October 2014, doi:10.3389/fimmu.2014.00520.)
  • the Fc region is from the Fc region of IgG, such as the Fc region of human IgG.
  • the Fc region is from the Fc region of IgG1, IgG2, IgG3, or IgG4, such as the Fc region of human IgG1, IgG2, IgG3, or IgG4. In some embodiments, the Fc region is the Fc region of human IgG1, IgG2, IgG3, or IgG4. In one embodiment, the human IgG heavy chain Fc fragment extends from Cys226 or from Pro230 of the heavy chain to the carboxyl terminus. In another embodiment, the C-terminal lysine (Lys447) of the Fc-fragment may or may not be present.
  • the Fc fragment is a variant Fc fragment comprising a mutation, for example comprising an L234A-L235A mutation.
  • the numbering of amino acid residues in the Fc fragment is according to the EU numbering system, also known as the EU index, as described in Kabat, E.A. et al., Sequences of Proteins of Immunological Interest, 5th Edition, Public Health Service, National Institutes of Health, Bethesda, MD (1991), NIH Publication 91-3242.
  • the antibody Fc fragment may have an IgG1 hinge sequence or a portion of an IgG1 hinge sequence at the N-terminus, such as the sequence from E216 to T225 or the sequence from D221 to T225 according to EU numbering.
  • the hinge sequence may contain a mutation, such as C220S.
  • linker refers to any molecule that enables direct connection of the different parts of the fusion protein.
  • linkers for establishing covalent connections 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.
  • PEG polyethylene glycol
  • 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.
  • a peptide linker can connect the TACI/BCMA portion of the fusion protein to the Fc domain or a fragment thereof.
  • a peptide linker can also connect an antibody to TACI/BCMA, such as connecting the C-terminus of the antibody heavy chain to TACI/BCMA.
  • the peptide linker has a length that is sufficient to connect the two entities in a manner that allows them to maintain their conformation relative to each other so as not to interfere with the desired activity.
  • the peptide linker may or may not primarily include the following amino acid residues: Gly, Ser, Ala or Thr.
  • Useful linkers include glycine-serine polymers, including, for example, (GSGGGGS)n(SEQ ID NO:89), (GS)n(SEQ ID NO:90), (GSGGS)n(SEQ ID NO:91), (GGGGS)n(SEQ ID NO:92), or (GGGS)n(SEQ ID NO:93), where n is an integer of at least 1 (and, for example, 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 (GSGGGGS)n(SEQ ID NO:89).
  • the linker of the present invention is SEQ ID NO:39.
  • fusion refers to a fusion formed by linking two or more initially separate proteins/genes/compounds. If the entity constituting the fusion is a protein, it is referred to as a fusion protein. Fusion proteins are encompassed within the scope of the fusions of this application.
  • TACI linked to an Fc dimer can constitute a TACI-Fc fusion protein.
  • the connection between the two entity molecules constituting the fusion can be achieved with or without a linker.
  • first and second are used with respect to an Fc domain (Fc region) or monomer, etc., to facilitate distinction when there is more than one of each type of module. Unless explicitly stated otherwise, the use of these terms is not intended to confer a particular order or orientation on the fusion proteins.
  • therapeutic agent encompasses any substance effective in preventing or treating immune diseases or inflammation, such as immune diseases or inflammation, including but not limited to cytokines, hormones, cytotoxic agents or inhibitors (e.g., cytostatic agents that affect T cell and/or B cell proliferation), antibodies or small molecule drugs or immunomodulators (e.g., immunosuppressants).
  • effective amount refers to an amount or dosage of an antibody, fragment, composition, or combination of the invention that, after administration to a patient in a single or multiple doses, produces the desired effect in a patient in need of treatment or prevention. "Effective amount” may encompass a “therapeutically effective amount” or a “prophylactically effective amount.”
  • a “therapeutically effective amount” refers to an amount effective to achieve the desired therapeutic outcome at the desired dosage and for the desired period of time.
  • a therapeutically effective amount is also an amount in which any toxic or deleterious effects of the antibody or antibody fragment or composition or combination are outweighed by the therapeutically beneficial effects.
  • a “therapeutically effective amount” preferably inhibits a measurable parameter by at least about 40%, even more preferably by at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or even 100%, relative to an untreated subject.
  • a “prophylactically effective amount” refers to an amount effective to achieve the desired prophylactic outcome at the desired dosage and for the desired period of time. Typically, because prophylactic doses are used in subjects prior to or at an earlier stage of the disease, the prophylactically effective amount will be less than the therapeutically effective amount.
  • host cell refers to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells.
  • Host cells include “transformants” and “transformed cells,” which include the primary transformed cell and progeny derived therefrom, without regard to the number of passages. Progeny may not be completely identical in nucleic acid content to the parent cell, but may contain mutations. Mutant progeny screened or selected for the same function or biological activity as the initially transformed cell are included herein.
  • label refers to a compound or composition that is directly or indirectly conjugated or fused to a reagent (such as a polynucleotide probe or antibody) and promotes the detection of the reagent to which it is conjugated or fused.
  • the label itself can be detectable (e.g., a radioisotope label or a fluorescent label) or can catalyze a chemical change in a detectable substrate compound or composition in the case of an enzymatic label.
  • the term is intended to encompass direct labeling of a probe or antibody by coupling (i.e., physically connecting) a detectable substance to the probe or antibody and indirect labeling of the probe or antibody by reacting with another reagent of the direct label.
  • biological half-life refers to the length of time it takes for a substance (e.g., an immunomodulatory protein) to lose half of its pharmacological or physiological activity or concentration. Biological half-life may be affected by elimination, excretion, degradation (e.g., enzymatic degradation/digestion) of the substance, or absorption and concentration in certain organs or tissues of the body. In some embodiments, biological half-life can be assessed by determining the time it takes for the plasma concentration of the substance to reach half of its steady-state level (“plasma half-life").
  • “Individual” or “subject” includes mammals. Mammals include, but are not limited to, domestic animals (e.g., cattle, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In some embodiments, the individual or subject is a human.
  • immune system disease or disorder refers to any disease or disorder associated with a malfunction of the immune system, including but not limited to autoimmune diseases or inflammatory diseases such as systemic lupus erythematosus and rheumatoid arthritis.
  • pharmaceutical excipient refers to a diluent, adjuvant (eg, Freund's adjuvant (complete and incomplete)), excipient, carrier, stabilizer, or the like, which is administered together with the active substance.
  • adjuvant eg, Freund's adjuvant (complete and incomplete)
  • excipient eg, carrier, stabilizer, or the like
  • composition refers to a composition that is in form permitting the biological activity of the active ingredient contained therein to be effective, and that contains no additional ingredients that are unacceptably toxic to a subject to which the composition would be administered.
  • drug combination refers to a non-fixed combination or a fixed combination, including but not limited to a kit, a pharmaceutical composition.
  • non-fixed combination means that the active ingredients (e.g., (i) the chimera or fusion protein of the present invention, and (ii) other therapeutic agents) are administered to a patient simultaneously, without specific time restrictions, or sequentially at the same or different time intervals, as separate entities, wherein such administration provides prophylactically or therapeutically effective levels of two or more active agents in the patient's body.
  • 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.
  • combination therapy refers to the administration of two or more therapeutic agents or treatment modalities (e.g., radiotherapy or surgery) to treat diseases described herein.
  • This administration includes co-administering these therapeutic agents in a substantially simultaneous manner, such as in a single capsule with a fixed ratio of active ingredients.
  • this administration includes co-administration of each active ingredient in a variety of or separate containers (e.g., tablets, capsules, powders, and liquids). Powders and/or liquids can be reconstituted or diluted to the desired dose before administration.
  • this administration also includes using each type of therapeutic agent in a sequential manner at approximately the same time or at different times. In either case, the therapeutic regimen will provide the beneficial effects of the drug combination in treating disorders or conditions described herein.
  • prevention includes the inhibition of the development or progression of a disease or condition, or symptoms of a particular disease or condition.
  • subjects with a family history of cancer are candidates for a preventative regimen.
  • prevention refers to the administration of a drug before the development of signs or symptoms of cancer, particularly in a subject at risk for cancer.
  • vector refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked.
  • the term includes vectors that are self-replicating nucleic acid structures as well as vectors that are incorporated into the genome of a host cell into which they have been introduced. Some vectors are capable of directing the expression of nucleic acids to which they are operably linked. Such vectors are referred to herein as "expression vectors.”
  • Subject/patient/individual sample refers to a collection of cells or fluids obtained from a patient or subject.
  • the source of a tissue or cell sample can be solid tissue, such as an organ or tissue sample or a biopsy sample or a puncture sample from a fresh, frozen and/or preserved organ; blood or any blood component; body fluids, such as cerebrospinal fluid, amniotic fluid (amniotic fluid), peritoneal fluid (ascites), or interstitial fluid; cells from any time during the subject's pregnancy or development.
  • Tissue samples may contain compounds that are not naturally contaminated with tissue in nature, such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, etc.
  • the present invention provides a TACI mutant protein having more uniform charge on the protein surface, better stability, and better drugability.
  • the TACI mutant protein of the present invention is a TACI mutant extracellular domain or a functional fragment thereof, wherein a portion of the domain is replaced by a corresponding domain of BCMA. Therefore, the TACI mutant protein comprising such a mutation is also referred to as a TACI/BCMA chimera in the present invention.
  • the TACI/BCMA chimera of the present invention comprises the extracellular domain of TACI or a functional fragment thereof, wherein the N-terminal amino acid and/or C-terminal amino acid of the extracellular domain or the functional fragment thereof is replaced by the N-terminal amino acid and/or C-terminal amino acid of the extracellular domain of BCMA.
  • the TACI/BCMA chimera of the present invention comprises a mutated extracellular domain of TACI or a functional fragment thereof, wherein the N-terminal amino acid of the extracellular domain or the functional fragment thereof is replaced by the N-terminal amino acid of the extracellular domain of BCMA.
  • the TACI/BCMA chimera of the present invention comprises a mutated extracellular domain of TACI or a functional fragment thereof, wherein the N-terminal amino acid and C-terminal amino acid of the extracellular domain or the functional fragment thereof are replaced by the N-terminal amino acid and C-terminal amino acid of the extracellular domain of BCMA, respectively.
  • the TACI/BCMA chimeras of the present invention further comprise substitutions at druggability risk sites.
  • the druggability risk sites are selected from positions 69, 72, 73, 74, 77, 78, 85, 102, and/or 103 of TACI.
  • the amino acid at the druggability risk site is mutated to A or D, such as D.
  • the substitution at the druggability risk site is Y102D.
  • the present invention also provides a fusion protein comprising the TACI/BCMA chimera of the present invention.
  • the TACI/BCMA chimera of the present invention is fused to another polypeptide that can confer improved pharmacokinetic properties, such as albumin, preferably an antibody Fc fragment.
  • the present invention also provides a fusion protein dimer formed by dimerization of two fusion protein chains of the present invention.
  • TACI is a member of the tumor necrosis factor receptor family, characterized by an extracellular domain (ECD) containing a cysteine-rich pseudo-repeat domain (CRD).
  • ECD extracellular domain
  • CRD cysteine-rich pseudo-repeat domain
  • TACI is a membrane-bound receptor with an extracellular domain containing two cysteine-rich pseudo-repeat sequences (CRD1 and CRD2), a transmembrane domain, and a cytoplasmic domain that interacts with CAML (calcium regulator and cyclophilin ligand), an integral membrane protein located in intracellular vesicles that is a co-inducer of NF-AT activation when overexpressed in Jurkat cells.
  • CAML calcium regulator and cyclophilin ligand
  • the TACI receptor binds to two members of the tumor necrosis factor (TNF) ligand family.
  • One ligand is named BAFF (B cell activating factor of the TNF family).
  • the other ligand has been named APRIL.
  • Both ligands are also bound by the B cell maturation receptor (BCMA). Binding of the TACI receptor to its ligands BAFF or APRIL stimulates B cell responses, including T cell-independent B cell antibody responses, isotype switching, and B cell homeostasis. Binding of BAFF or APRIL stimulates B cell responses, including T cell-independent B cell antibody responses, isotype switching, and B cell homeostasis.
  • TACI is human TACI.
  • human TACI comprises, or consists of, the amino acid sequence set forth in SEQ ID NO: 1, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.
  • the full-length human TACI sequence comprises, or consists of, the amino acid sequence set forth in SEQ ID NO: 1.
  • the human TACI protein is a type III membrane protein and lacks a signal peptide; upon expression in eukaryotic cells, the N-terminal methionine is removed.
  • the mature TACI protein does not contain the N-terminal methionine set forth in SEQ ID NO: 1.
  • Human TACI typically consists of three domains: an extracellular domain, corresponding to amino acids 1-165; a transmembrane region, corresponding to amino acids 166-186; and a cytoplasmic domain, corresponding to amino acids 187-293.
  • the extracellular domain of TACI (e.g., amino acid residues 1-165 of SEQ ID NO: 1; the ECD shown in SEQ ID NO: 29) contains two cysteine-rich domains (CRDs), each of which exhibits affinity binding to BAFF and APRIL.
  • the first cysteine-rich domain (CRD1) corresponds to or contains amino acid residues 34-66 of the sequence shown in SEQ ID NO: 1.
  • the second cysteine-rich domain (CRD2) corresponds to or contains amino acids 71-104 of the sequence shown in SEQ ID NO: 1.
  • TACI also contains a stem region of approximately 60 amino acids following the second cysteine repeat sequence in the extracellular domain, which corresponds to or contains amino acid residues 105-165 of the sequence shown in SEQ ID NO: 1.
  • the extracellular domain of TACI comprises the amino acid sequence of positions 1-165 of SEQ ID NO: 1, or comprises, or consists of, an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.
  • the extracellular domain of TACI comprises the amino acid sequence of SEQ ID NO: 29, or comprises, or consists of, an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.
  • CRD2 comprises the amino acid sequence of SEQ ID NO: 58, or comprises, or consists of, an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.
  • a functional fragment of TACI ECD comprises an amino acid sequence that binds to APRIL, BAFF, or an APRIL/BAFF heterotrimer.
  • the functional fragment of TACI ECD lacks a portion of the N-terminal residues of the ECD, which residues correspond to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66 or 67 consecutive amino acids at the N-terminus of the ECD sequence shown in SEQ ID NO:1.
  • the functional fragment of TACI ECD lacks part of the stem region of ECD, for example, lacks 55, 54, 53, 52, 51, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 consecutive amino acids at the C-terminus.
  • the functional fragment of the TACI ECD comprises CRD2, or comprises only an intact CRD2. In some embodiments, the functional fragment of the TACI ECD consists of CRD2.
  • a functional fragment of a TACI ECD comprises CRD2 and a portion of the stem region. In some embodiments, a functional fragment of a TACI ECD further comprises a sequence between CRD1 and CRD2, such as any amino acid sequence corresponding to positions 68-70 of SEQ ID NO: 1. In some embodiments, a functional fragment of a TACI ECD does not comprise CRD1 or any fragment thereof.
  • the functional fragment of TACI ECD contains a portion of the stem region, for example, the following fragment of TACI corresponding to the amino acid sequence shown in SEQ ID NO: 1: amino acid residue 105, amino acid residues 105 to 106, amino acid residues 105 to 107, amino acid residues 105 to 108, amino acid residues 105 to 109, amino acid residues 105 to 110, amino acid residues 105 to 111, amino acid residues 105 to 112, amino acid residues 105 to 113 , amino acid residues 105 to 114, amino acid residues 105 to 115, amino acid residues 105 to 116, amino acid residues 105 to 117, amino acid residues 105 to 118, amino acid residues 105 to 119, amino acid residues 105 to 120, amino acid residues 105 to 121, amino acid residues 105 to 122, amino acid residues 105 to 123, amino acid residues 105 to 124, amino acid residues 105 to 125, amino acid residues 105
  • the functional fragment of TACI ECD comprises the following fragment of TACI corresponding to the amino acid sequence set forth in SEQ ID NO: 1: amino acid residues 67 to 118, amino acid residues 67 to 117, amino acid residues 67 to 116, amino acid residues 67 to 115, amino acid residues 67 to 114, amino acid residues 67 to 113, amino acid residues 67 to 112, amino acid residues 67 to 111, amino acid residues 67 to 110, amino acid residues 67 to 109, amino acid residues 67 to 108, amino acid residues 67 to 107, amino acid residues 67 to 106, amino acid residues 67 to 105, or amino acid residues 67 to 104; amino acid residues 68 to 118, amino acid residues 68 to 117, amino acid residues 68 to 116, amino acid residues 68 to 115, amino acid residues 68 to 114, amino acid residues 68 to 113, amino acid residues 68 to
  • a functional fragment of the TACI ECD comprises amino acids 13-118 of the ECD, or comprises amino acids 68-110, or consists of said contiguous amino acid sequence.
  • the functional fragment of TACI ECD comprises the amino acid sequence shown in SEQ ID NO:30, 31 or 58, or comprises an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto, or consists of the amino acid sequence.
  • the functional fragment of TACI ECD consists of the amino acid sequence shown in SEQ ID NO:31.
  • the TACI ECD or a functional fragment thereof also encompasses ECD variants or functional fragments thereof having mutations.
  • the functional fragment of the TACI ECD comprises K77E, F78Y, and/or Y102D.
  • the functional fragment of TACI ECD comprises Y102D. In some embodiments, the functional fragment of TACI ECD comprises the amino acid sequence set forth in SEQ ID NO: 33, or comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto. In some embodiments, the functional fragment of TACI ECD comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 33 and has Y102D. In some embodiments, the functional fragment of TACI ECD consists of the amino acid sequence set forth in SEQ ID NO: 33.
  • the functional fragment of TACI ECD comprises K77E, F78Y, and Y102D.
  • the functional fragment of TACI ECD comprises the amino acid sequence set forth in SEQ ID NO: 32, or comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.
  • the functional fragment of TACI ECD comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 32 and comprises K77E, F78Y, and Y102D.
  • the functional fragment of TACI ECD consists of the amino acid sequence set forth in SEQ ID NO: 32.
  • the present invention found that the protein structure of BCMA, a protein in the TACI family (6-42, PDB ID: 1XU2), is highly homologous to the TACI structure and is basically consistent with the ligand binding site. At the same time, the N-terminus and C-terminus of both are not involved in ligand binding ( Figure 1). More importantly, the analysis results of Discovery studio software show that the surface charge distribution of BCMA is balanced, and the aggregation risk is significantly lower than that of TACI.
  • the present invention designs a series of chimeric molecules of BCMA and TACI extracellular domains.
  • the N-terminal amino acid or C-terminal amino acid of TACI is replaced with the corresponding sequence of BCMA, or both the N-terminal amino acid and the C-terminal amino acid of TACI are replaced with the corresponding sequence of BCMA, and amino acids are optionally replaced at some druggability risk sites, thereby reducing the area of charge groups and hydrophobic groups on the surface of the TACI protein and reducing the aggregation risk of TACI.
  • BCMA is a member of the tumor necrosis factor receptor family, characterized by an extracellular domain (ECD) containing a cysteine-rich pseudo-repeat domain (CRD).
  • ECD extracellular domain
  • CRD cysteine-rich pseudo-repeat domain
  • BCMA is a membrane-bound receptor with an extracellular domain containing a single CRD, a transmembrane domain, and a cytoplasmic domain containing a TRAF binding site for binding to TRAF signaling molecules.
  • BCMA binds to its cognate ligands APRIL and BAFF, but with weaker affinity for BAFF.
  • BCMA binding to BAFF has been reported to be two to three orders of magnitude weaker than the binding between BAFF and its other cognate receptors, BAFF-R and TACI.
  • BCMA is human BCMA.
  • human BCMA comprises, or consists of, the amino acid sequence set forth in SEQ ID NO: 2, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.
  • the full-length human BCMA sequence comprises the amino acid sequence set forth in SEQ ID NO: 2.
  • the BCMA protein is a type II membrane protein and lacks a signal peptide; upon expression in eukaryotic cells, the N-terminal methionine is removed.
  • the mature BCMA protein does not contain the N-terminal methionine as set forth in SEQ ID NO: 2.
  • Human BCMA typically contains three domains, namely the extracellular domain, corresponding to amino acids 1-54; the transmembrane region, corresponding to amino acids 55-77; and the cytoplasmic domain, corresponding to amino acids 78-184.
  • the extracellular domain of BCMA contains a cysteine-rich domain (CRD) that exhibits affinity for binding to APRIL and, to a lesser extent, BAFF.
  • the CRD contains amino acid residues 7-41 of the sequence shown in SEQ ID NO:2.
  • the extracellular domain of BCMA comprises amino acids 1-54 of SEQ ID NO: 2, or comprises, or consists of, an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.
  • the extracellular domain of BCMA comprises amino acids 1-54 of SEQ ID NO: 2, or comprises, or consists of, an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.
  • the N-terminus of TACI ECD or a functional fragment thereof refers to the N-terminal amino acid of TACI preceding amino acid Y79 of SEQ ID NO:1.
  • the C-terminus of TACI ECD or a functional fragment thereof refers to the C-terminal amino acid of TACI corresponding to amino acid 99, amino acid 100, amino acid 101, amino acid 102, amino acid 103, amino acid 104, amino acid 105, amino acid 106, amino acid 107, amino acid 108, amino acid 109 or amino acid 110 of SEQ ID NO: 1.
  • the C-terminus of TACI ECD or a functional fragment thereof refers to the C-terminal amino acid following amino acid position 99 of TACI corresponding to SEQ ID NO: 1. In some embodiments, the C-terminus of TACI ECD or a functional fragment thereof refers to the C-terminal amino acid following amino acid position 105 of TACI corresponding to SEQ ID NO: 1.
  • the corresponding N-terminal amino acid sequence of BCMA that can be used to replace the N-terminus of TACI ECD or its functional fragment is selected from the amino acid sequence of BCMA corresponding to positions 1-13, 2-13, 3-13, 4-13, 5-13, 6-13 or 7-13 of SEQ ID NO:2.
  • the corresponding N-terminal amino acid sequence of BCMA for replacement comprises or consists of the amino acid sequence of any one of SEQ ID NOs:40-46.
  • the corresponding C-terminal amino acid sequence of BCMA that can be used to replace the C-terminus of TACI ECD or its functional fragment is selected from the amino acid sequence of BCMA corresponding to positions 37-47, 37-46, 37-45, 37-44 of SEQ ID NO: 2, or the amino acids corresponding to positions 43-44 or 43-46 of SEQ ID NO: 2.
  • the corresponding N-terminal amino acid or C-terminal amino acid sequence of BCMA used for replacement may contain mutations, such as substitutions, for improving binding affinity, increasing stability, and/or improving drugability.
  • the C-terminal amino acid sequence of BCMA used for replacement contains mutations at position 39 and/or position 42.
  • the C-terminal amino acid sequence of the BCMA used for replacement comprises N42A. In some embodiments, the C-terminal amino acid sequence of the BCMA used for replacement comprises N42Q. In some embodiments, the C-terminal amino acid sequence of the BCMA used for replacement comprises R39D. In some embodiments, the C-terminal amino acid sequence of the BCMA used for replacement comprises N42A-R39D.
  • the corresponding C-terminal amino acid sequence of BCMA for replacement comprises or consists of the amino acid sequence shown in any one of SEQ ID NO:47-55.
  • the number of amino acids in the N-terminal or C-terminal amino acid sequence of BCMA to be replaced is equal to the number of amino acids in the N-terminal or C-terminal amino acid sequence of the replaced ECD of TACI or a functional fragment thereof, or the difference is within 1-3 amino acids.
  • the TACI/BCMA chimera of the present invention comprises the following structure:
  • BCMAN terminal amino acid-TACI portion wherein the TACI portion is an ECD or a functional fragment thereof lacking the N-terminus, or
  • BCMAN terminal amino acid-TACI portion-BCMAC terminal amino acid wherein the TACI portion is the ECD or a functional fragment thereof lacking the N-terminus and C-terminus.
  • BCMAN terminal amino acid-TACI moiety refers to a polypeptide obtained by replacing the N-terminal amino acid of TACI with the N-terminal amino acid of BCMA.
  • BCMAN terminal amino acid-TACI moiety-BCMAC terminal amino acid refers to a polypeptide obtained by replacing the N-terminal amino acid of TACI with the N-terminal amino acid of BCMA and the C-terminal amino acid of TACI with the C-terminal amino acid of BCMA.
  • the TACI portion of a TACI/BCMA chimera suitable for use in the present invention may comprise a mutation, such as a substitution.
  • the TACI portion of the TACI/BCMA chimera suitable for use in the present invention further comprises a mutation at a druggability risk site, such as an amino acid site with aggregation risk, such as replacing an amino acid at one or more druggability risk sites of the TACI portion.
  • a druggability risk site such as an amino acid site with aggregation risk
  • replacing an amino acid at one or more druggability risk sites of the TACI portion if the TACI portion comprises a druggability risk site, it is replaced with an amino acid that reduces the risk.
  • the TACI portion comprises an amino acid with aggregation risk, it is replaced with an amino acid that reduces the aggregation risk.
  • the druggability risk site is selected from amino acids 69, 72, 73, 74, 77, 78, 85, 102, or 103 corresponding to SEQ ID NO: 1.
  • the amino acid at the druggability risk site is mutated to A or D.
  • the TACI moiety comprises a mutation, eg, a substitution, at position Y102, eg, Y102D.
  • the TACI portion comprises the amino acid sequence shown in any one of SEQ ID NO:31-38, or comprises an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% or 98% identity thereto, or comprises an amino acid sequence that has 1, 2, 3, 4 or 5 amino acid changes (e.g., substitutions, deletions or additions, such as conservative substitutions; or, for example, substitutions at druggability risk sites) thereto, or consists of said amino acid sequence.
  • substitutions, deletions or additions such as conservative substitutions; or, for example, substitutions at druggability risk sites
  • the TACI portion consists of the amino acid sequence shown in any one of SEQ ID NO:31-38.
  • the BCMA portion of the TACI/BCMA chimera suitable for use in the present invention (e.g., at the BCMAN end or the BCMAC end, preferably the BCMAC end) further comprises mutations for improving binding affinity, increasing stability and/or improving drugability, such as substitutions, for example, at drugability risk sites such as mutations that result in N-glycosylation sites, for example, replacing amino acids at one or several drugability risk sites of the BCMA portion.
  • the BCMA portion comprises a drugability risk site, it is replaced with an amino acid that reduces the risk.
  • the BCMA portion comprises an amino acid with glycosylation (e.g., N-glycosylation)
  • the drugability risk site is the 39th or 42nd amino acid corresponding to SEQ ID NO: 2.
  • the amino acid at the drugability risk site is mutated to D, A, or Q.
  • the BCMAN terminus comprises the amino acid sequence shown in any one of SEQ ID NO:40-46, or comprises an amino acid sequence having at least 80% or 90% identity thereto, or comprises an amino acid sequence having 1, 2, or 3 amino acid changes (e.g., substitutions, deletions, or additions, such as conservative substitutions or substitutions at druggability risk sites) thereto, or consists of the amino acid sequence.
  • the BCMAN terminus consists of the amino acid sequence shown in any one of SEQ ID NO:40-46.
  • the BCMAC terminus comprises a mutation at position R39, such as a substitution, for example, R39D. In some embodiments, the BCMAC terminus comprises a mutation at position N42, such as a substitution, for example, N42A or N42Q. In some embodiments, the BCMAC terminus comprises a mutation at position R39 and a mutation at position N42, for example, R39D-N42A.
  • the BCMAC terminus comprises the amino acid sequence shown in any one of SEQ ID NO:47-55, or comprises an amino acid sequence that has at least 80% or 90% identity thereto, or comprises an amino acid sequence that has 1 or 2 amino acid changes (e.g., substitutions, deletions, or additions, such as conservative substitutions or, for example, substitutions at druggability risk sites) thereto, or consists of said amino acid sequence.
  • the BCMAC terminus consists of the amino acid sequence shown in any one of SEQ ID NO:47-55.
  • the TACI/BCMA chimera of the present invention comprises the amino acid sequence shown in any one of SEQ ID NO:59-80, or comprises an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% or 98% identity thereto, or consists of the amino acid sequence.
  • the present invention also provides a fusion protein comprising a TACI/BCMA chimera of the present invention.
  • the TACI/BCMA chimera of the present invention is fused to another polypeptide that can confer improved pharmacokinetic properties, such as albumin, more preferably an antibody Fc fragment.
  • FcRn-mediated recycling in the body can extend the half-life of the TACI/BCMA chimera-Fc fusion protein.
  • the present invention provides a TACI/BCMA chimera fusion protein comprising the TACI/BCMA chimera of the present invention fused to an Fc region.
  • the Fc region is fused to the C-terminus of the TACI/BCMA chimera of the present invention, for example, the N-terminus of the Fc region is fused to the C-terminus of the chimera.
  • the Fc region is a human IgG Fc, e.g., human IgG1 Fc, human IgG2 Fc, human IgG3 Fc, or human IgG4 Fc.
  • the Fc region comprises a complete hinge region at its N-terminus, i.e., starting from position E216 of the human IgG1 heavy chain constant region to the C-terminus.
  • the Fc region comprises a cysteine mutated to a serine at position 220 of the N-terminal hinge region corresponding to IgG1 (i.e., comprising a 220S mutation).
  • the Fc region may also lack part of the hinge region, for example, starting from position D221 to the C-terminus corresponding to the human IgG1 heavy chain constant region.
  • the Fc region comprises or consists of an amino acid sequence shown in SEQ ID NO: 81 or 82, or an amino acid sequence that is at least 90% identical thereto, such as 95%, 96%, 97%, 98%, 99% or more identical thereto.
  • the Fc region of the fusion protein suitable for use in the present invention lacks lysine K at the C-terminus (K447del).
  • the Fc region comprises or consists of the amino acid sequence shown in SEQ ID NO: 83 or 84, or an amino acid sequence that is at least 90% identical thereto, such as 95%, 96%, 97%, 98%, 99% or more identical thereto (optionally also lacking the C-terminal lysine).
  • the Fc region of the binding molecules of the invention can also be mutated to obtain desired properties. Mutations in the Fc region are known in the art.
  • the Fc region is modified with respect to the properties of the effector functions of the Fc region (e.g., complement activation function of the Fc region).
  • the effector functions have been reduced or eliminated relative to the wild-type Fc region.
  • the effector functions are reduced or eliminated by a method selected from the following: using an Fc isotype that naturally has reduced or eliminated effector functions, and Fc region modifications.
  • the Fc region 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.
  • modifications that alter binding affinity for one or more Fc receptors may also be included in the Fc region.
  • the Fc receptor is an Fc ⁇ receptor, particularly a human Fc ⁇ receptor.
  • the Fc region comprises mutations that reduce binding to Fc ⁇ receptors.
  • the Fc region may have mutations that result in increased serum half-life, such as mutations that improve binding of the Fc fragment to FcRn.
  • the Fc region used in the present invention has a mutation that reduces binding to Fc ⁇ receptors.
  • the Fc fragment used in the present invention has a mutation that reduces binding to Fc ⁇ receptors, such as L234A/L235A mutation, L234A/L235E mutation, G237A mutation, or L234A/L235E/G237A.
  • the Fc region used in the present invention has a mutation that improves binding of the Fc fragment to FcRn, such as a YTE mutation (M252Y/S254T/T256E) or a LS mutation (M428L/N434S) that enhances binding of the Fc fragment to FcRn.
  • exemplary mutations are described in WO2002060919A2, which is incorporated herein by reference in its entirety.
  • the Fc region comprises both the mutation that reduces binding to Fc ⁇ receptors and the mutation that improves binding of the Fc fragment to FcRn.
  • the Fc region comprises L234A/L235E/G237A and M252Y/S254T/T256E, and optionally a C-terminal deleted lysine.
  • the Fc region comprises L234A/L235E/G237A and M428L/N434S, and optionally a C-terminal deleted lysine.
  • the Fc region comprises or consists of an amino acid sequence shown in any one of SEQ ID NOs: 85-88 and 100-103, or an amino acid sequence that is at least 90% identical thereto, such as 95%, 96%, 97%, 98%, 99% or more identical thereto.
  • the Fc region comprises an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO:85 or 87 and comprises the mutations L234A/L235E/G237A.
  • the Fc region comprises an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO: 100 or 102 and comprises the mutations L234A/L235E/G237A and M252Y/S254T/T256E.
  • the Fc region comprises an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence shown in SEQ ID NO: 101 or 103 and comprises the mutations L234A/L235E/G237A, M252Y/S254T/T256E and a C-terminal deleted lysine.
  • the TACI/BCMA chimera is fused directly or via a linker to the Fc, for example, the C-terminus of the chimera is fused directly or via a linker to the N-terminus of the Fc.
  • the linker suitable for connecting the TACI/BCMA and Fc regions in the fusion proteins and dimeric molecules of the present invention can be any linker known in the art.
  • the linker can comprise an IgG1 hinge, or can comprise a linker sequence selected from the following: (GSGGGGS) n (SEQ ID NO: 94), (GS) n (SEQ ID NO: 95), (GSGGS) n (SEQ ID NO: 96), (GGGGS) n (SEQ ID NO: 97), or (GGGS) n (SEQ ID NO: 98), wherein n is an integer of at least 1, for example, 1, 2, 3, 4, or 5.
  • the present invention also provides a dimeric molecule comprising a TACI/BCMA chimera of the present invention fused to an Fc region.
  • the present invention provides a TACI/BCMA chimera-Fc dimer protein, wherein the first monomer and the second monomer, respectively, comprise or consist of the following from N-terminus to C-terminus: i) a TACI/BCMA chimera; ii) a linker (optionally present or absent); and iii) an Fc region.
  • the dimer protein is a homodimer.
  • the monomers have the same amino acid sequence.
  • the TACI/BCMA chimera-Fc protein dimer of the present invention comprises two identical monomers.
  • the TACI/BCMA chimera-Fc fusion protein of the present invention or its monomer
  • (ii) comprises or consists of an amino acid sequence of any one of SEQ ID NOs: 5-28; or
  • (iii) contains one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid changes compared to the amino acid sequence of any one of SEQ ID NOs: 5-28.
  • the TACI/BCMA chimera-Fc protein of the present invention or the protein dimer monomer of the present invention consists of the amino acid sequence of any one of SEQ ID NO:5-28.
  • the present invention provides nucleic acids encoding any of the above-described TACI/BCMA chimeras, or any monomer or domain thereof in a fusion protein or fusion protein dimer.
  • 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 TACI using methods well known in the art.
  • the polynucleotides and nucleic acids of the present invention may include a segment encoding a secretory signal peptide, which can be operably linked to a segment encoding a mutein of the present invention, thereby directing secretory expression of the mutein of the present invention.
  • the present invention also provides vectors comprising the nucleic acid of the present invention.
  • 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).
  • the expression vector of the present invention is a pCDNA expression vector, such as a pCDNA3.1 expression vector.
  • the present invention also provides a host cell comprising the nucleic acid or the vector.
  • Host cells suitable for replicating and supporting the expression of TACI/BCMA chimera or its fusion protein or fusion protein dimer 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 inoculating large-scale fermenters, thereby obtaining sufficient amounts of TACI/BCMA chimera or its fusion protein or fusion protein dimer for clinical use.
  • the host cell is eukaryotic. In another embodiment, the host cell is selected from yeast cells, mammalian cells (eg, CHO cells or 293 cells).
  • useful mammalian host cell lines include monkey kidney CV1 line (COS-7) transformed by SV40, human embryonic kidney line (293 or 293T cells or HEK293 cells), baby hamster kidney cells (BHK), mouse Sertoli cells (TM4 cells), monkey kidney cells (CV1), African green monkey kidney cells (VERO-76), human cervical cancer 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, MRC5 cells, and FS4 cells.
  • Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including dhfr-CHO cells, and myeloma cell lines such as YO, NS0, P3X63, and Sp2/0.
  • the host cell is a eukaryotic cell, preferably a mammalian cell such as a Chinese hamster ovary (CHO) cell, HEK293 cell, human embryonic kidney (HEK) cell or a lymphocyte (eg, Y0, NS0, Sp20 cell).
  • a mammalian cell such as a Chinese hamster ovary (CHO) cell, HEK293 cell, human embryonic kidney (HEK) cell or a lymphocyte (eg, Y0, NS0, Sp20 cell).
  • the present invention provides a method for preparing the TACI/BCMA chimera or its fusion protein or fusion protein dimer of the present invention, wherein the method comprises culturing a host cell comprising a nucleic acid encoding the TACI/BCMA chimera or its fusion protein or fusion protein dimer, as provided above, under conditions suitable for the expression of the TACI/BCMA chimera or its fusion protein or fusion protein dimer, and optionally recovering the TACI/BCMA chimera or its fusion protein or fusion protein dimer from the host cell (or host cell culture medium).
  • a vector containing a nucleic acid encoding a TACI/BCMA chimera or its fusion protein or fusion protein dimer is transferred into cells for expression, and then the cells (or cell culture supernatant) are collected, the TACI/BCMA chimera or its fusion protein or fusion protein dimer is extracted, and purified to obtain the TACI/BCMA chimera or its fusion protein or fusion protein dimer.
  • the purification method is affinity purification. In another specific embodiment, the purification method is ion exchange purification. In some embodiments, the purification is performed by filtration using a gel filtration chromatography column.
  • the TACI/BCMA chimera or fusion protein or fusion protein dimer provided herein can be identified, screened, or characterized for its physical/chemical properties and/or biological activities by various assays known in the art.
  • the protein surface charge of the TACI/BCMA chimera obtained in the present invention is more uniform, and therefore has better stability and better drugability.
  • the TACI/BCMA chimeras and fusion proteins obtained in the present invention exhibit improved stability (e.g., thermal stability) and pharmacokinetic data, as well as a longer half-life, compared to TACI proteins and fusion proteins known in the prior art. Under accelerated conditions, the TACI/BCMA chimera fusion proteins obtained in the present invention exhibit improved stability compared to TACI fusion proteins or variants known in the prior art, with some molecules exhibiting significantly better thermal stability than ALPN303.
  • the TACI/BCMA chimeric fusion protein obtained by the present invention has one or more properties selected from the following:
  • b) specifically binds to BAFF and APRIL, e.g., with a KD for BAFF of less than or equal to about 3 nM, 2 nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, or 0.4 nM, or greater than or equal to about 0.1 nM or 0.2 nM, or any values therebetween; and with a KD for APRIL of less than or equal to about 4 nM, 3 nM, 2 nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, or 0.4 nM, or greater than or equal to about 0.1 nM or 0.2 nM, or any values therebetween, as determined by a ForteBio assay;
  • c) effectively inhibits the binding of BAFF to IM-9 cells, for example, with an IC50 of less than or equal to about 5 nM, 4 nM, 3 nM, 2 nM, or 1.5 nM when assayed by FACS; effectively inhibits the binding of APRIL to IM-9 cells, for example, with an IC50 of less than or equal to about 5 nM, 4 nM, 3 nM, 2 nM 1.5 nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, or 0.2 nM when assayed by FACS;
  • TACI BAFF/APRIL-induced surface expression of (human) TACI, for example, with an IC50 of less than or equal to about 16 nM, 15 nM, 14 nM, 13 nM, 12 nM, 11 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM or 5.5 nM as determined by a TACI/NF- ⁇ B Reporter Jurkat signal assay;
  • e) effectively inhibits BAFF/APRIL-induced surface expression of (human) BCMA, for example, with an IC50 of less than or equal to about 16 nM, 15 nM, 14 nM, 13 nM, 12 nM, 11 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 Nm, 5.5 nM, 5 nM, 4.5 nM or 4 nM as determined by a BCMA/NF- ⁇ B Reporter Jurkat signal assay;
  • f) effectively inhibiting BAFF/APRIL-induced splenocyte proliferation e.g., with an IC50 of less than or equal to about 4.5 nM, 4 nM, 3.5 nM, 3 nM, 2.5 nM, 2 nM, 1.5 nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, or 0.6 nM;
  • m) can effectively reduce the levels of IgA, IgM and/or IgG in serum induced by BAFF;
  • o have better pharmacokinetics, for example, better than known molecules RC-18 and/or ALPN-303;
  • p has a longer half-life (e.g., plasma half-life), e.g., a half-life (e.g., plasma half-life) greater than or equal to about 45 hours, e.g., greater than or equal to about 46, 47, 48, 49, or 50 hours; and/or
  • q) has a lower clearance, such as a clearance of less than about 1.8, 1.7, 1.6, or 1.5 mL/kg/h.
  • compositions comprising a TACI/BCMA chimera or a fusion protein or fusion protein dimer thereof, or compositions comprising a polynucleotide encoding a TACI/BCMA chimera or a fusion protein or fusion protein dimer thereof.
  • compositions may also optionally contain suitable pharmaceutical excipients, such as pharmaceutical carriers and excipients known in the art, including buffers.
  • 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 acceptable formulations. The appropriate formulation depends on the chosen route of administration.
  • the TACI/BCMA chimera or its fusion protein or fusion protein dimer 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 (e.g., hydrochloric acid or phosphoric acid) or with organic acids such as acetic acid, oxalic acid, tartaric acid, or mandelic acid.
  • the present invention also provides a pharmaceutical combination or combination product comprising the TACI/BCMA chimera of the present invention or its fusion protein or fusion protein dimer, and one or more other therapeutic agents (e.g., cytokines, hormones, cytotoxic agents or inhibitors (e.g., cytostatic agents that affect T cell and/or B cell proliferation), antibodies or small molecule drugs, or immunomodulators (e.g., immunosuppressants)).
  • cytokines e.g., cytotoxic agents or inhibitors (e.g., cytostatic agents that affect T cell and/or B cell proliferation)
  • antibodies or small molecule drugs e.g., immunosuppressants
  • immunomodulators e.g., immunosuppressants
  • the pharmaceutical combination or combination product is used to prevent or treat a B cell-related disease or immune system disease or inflammation.
  • the pharmaceutical combination or combination product is used to prepare a medicament for preventing or treating a B cell-related disease or an immune system disease or inflammation.
  • the present invention relates to a method for preventing or treating a subject's disease, such as a B cell or autoantibody-related disease or an immune system disease (e.g., an autoimmune disease) or inflammation, comprising administering to the subject an effective amount of any TACI/BCMA chimera or its fusion protein or fusion protein dimer or pharmaceutical composition or drug combination described herein.
  • a subject's disease such as a B cell or autoantibody-related disease or an immune system disease (e.g., an autoimmune disease) or inflammation
  • the disease is a disease associated with abnormal expression (increased concentration) or activity of BAFF and/or APRIL or BAFF/APRIL heterotrimers, for example, compared to a sample (e.g., blood or serum) of a healthy individual.
  • the B cell or autoantibody-related disease or immune system disease is a B cell or autoantibody-mediated disease, such as a B cell or autoantibody-mediated autoimmune disease.
  • the B cell-mediated disease is a disease associated with B cell proliferation or abnormal activation, such as an autoimmune disease of B cell proliferation or abnormal activation.
  • the autoantibody-mediated disease refers to the production of unwanted autoantibodies due to abnormal proliferation or activation of B cells, or the production of abnormal autoantibodies.
  • the disease associated with B cell expansion is a disease in which B cells proliferate abnormally (e.g., an autoimmune disease), for example, compared to a sample (e.g., blood or serum) of a healthy individual.
  • the disease mediated by B cells or autoantibodies is caused by excessive production of autoantibodies against self-antigens or abnormal production of autoantibodies due to abnormal activation of B cells.
  • the present invention relates to a method of inhibiting B cell proliferation in an individual, comprising administering to the subject an effective amount of any TACI/BCMA chimera described herein, or a fusion protein or fusion protein dimer thereof.
  • the treatment of the disease will benefit from inhibition of BAFF and/or APRIL or BAFF/APRIL heterotrimer-related signaling pathways. In some embodiments, the treatment of the disease will benefit from inhibition of B cell proliferation or lymphocyte proliferation or inhibition of autoantibodies.
  • the autoimmune disease includes but is not limited to lupus, such as systemic lupus erythematosus, rheumatoid arthritis, chronic kidney disease such as IgA nephropathy (IgAN) or membranous nephropathy, Sjögren's syndrome, myasthenia gravis, idiopathic thrombocytopenic purpura (ITP), warm antibody autoimmune hemolytic anemia (wAIHA), multiple sclerosis (MS), coronary heart disease (CAD), or thyroid eye disease.
  • lupus such as systemic lupus erythematosus, rheumatoid arthritis, chronic kidney disease such as IgA nephropathy (IgAN) or membranous nephropathy, Sjögren's syndrome, myasthenia gravis, idiopathic thrombocytopenic purpura (ITP), warm antibody autoimmune hemolytic anemia (wAIHA), multiple sclerosis (MS), coronary heart
  • the TACI/BCMA chimera or its fusion protein or fusion protein dimer of the present invention 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 medication can be administered by any suitable route, such as by injection, such as intravenous or subcutaneous injection.
  • Various medication schedules are contemplated herein, including, but not limited to, single administration or multiple administration at multiple time points, bolus administration, and pulse infusion.
  • the appropriate dosage of the TACI/BCMA chimera or its fusion protein or fusion protein dimer 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 type of antibody, the severity and course of the disease, whether the administration is for preventive or therapeutic purposes, previous treatment, 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.
  • the present invention also provides the use of the TACI/BCMA chimera or its fusion protein or fusion protein dimer or pharmaceutical composition or drug combination of the present invention in the preparation of a drug for the aforementioned method (e.g., for treating the B cell or autoantibody-related diseases or immune system diseases (e.g., autoimmune diseases) or inflammation).
  • a drug for the aforementioned method e.g., for treating the B cell or autoantibody-related diseases or immune system diseases (e.g., autoimmune diseases) or inflammation.
  • the present invention also provides the TACI/BCMA chimera of the present invention or its fusion protein or fusion protein dimer or pharmaceutical composition or pharmaceutical combination, which is used for therapy, such as for the aforementioned method (e.g., for treating the B cell or autoantibody-related diseases or immune system diseases (e.g., autoimmune diseases) or inflammation) or use.
  • aforementioned method e.g., for treating the B cell or autoantibody-related diseases or immune system diseases (e.g., autoimmune diseases) or inflammation
  • the present invention also provides the use of the TACI/BCMA chimera or its fusion protein or fusion protein dimer, or pharmaceutical composition or drug combination of the present invention for the aforementioned methods (e.g., for treating diseases associated with B cells or autoantibodies, or immune system diseases (e.g., autoimmune diseases), or inflammation).
  • diseases associated with B cells or autoantibodies, or immune system diseases (e.g., autoimmune diseases), or inflammation e.g., autoimmune diseases
  • inflammation e.g., for treating diseases associated with B cells or autoantibodies, or immune system diseases (e.g., autoimmune diseases), or inflammation.
  • Full-length TACI contains 293 amino acid residues, and protease cleavage sites are present at multiple locations within the extracellular domain, making it very susceptible to fragmentation after extracellular expression.
  • the TACI extracellular domain contains two cysteine-rich domains (CRDs), CRD1 and CRD2.
  • CRD2 domain alone has a high binding activity with ligands. Therefore, in this application, we selected the CRD2 domain, consisting of amino acid residues 68-110 of TACI, to construct a fusion protein with the Fc portion of human IgG1 (SEQ ID NO: 5), thereby improving the fragmentation problem of TACI expression.
  • TACI or TACI/BCMA chimeras were linked to the Fc portion of human IgG1 via GSGGGGS (SEQ ID NO: 39) and constructed into the pCDNA3.1 vector.
  • ALPN-303 and Telitacicept (RC-18) for control were also constructed into the pCDNA3.1 vector to express the following proteins listed in Table 1:
  • the vector containing the gene encoding the fusion protein was transfected into HEK293 cells using chemical transfection.
  • Cultured HEK293 cells were transiently transfected using the chemical transfection reagent PEI according to the manufacturer's protocol.
  • the cell culture medium was centrifuged at 13,000 rpm for 20 minutes, and the supernatant was collected and purified using a prepacked Hitrap Mabselect Sure column (GE, 11-0034-95).
  • the procedure was as follows: before purification, the column was equilibrated with 5 column volumes of equilibration solution (PBS buffer (Gibco, Catalog No.: 70011-044)); the collected supernatant was passed through the column, and the column was then washed with 10 column volumes of equilibration solution to remove non-specific binding proteins; the column was rinsed with 5 column volumes of elution buffer (100 mM sodium citrate, pH 3.3), and the eluate was collected.
  • PBS buffer Gibco, Catalog No.: 70011-044
  • Tris (2M Tris) was added to each 1 ml of eluate, and the column was exchanged into PBS buffer using an ultrafiltration concentrator (MILLIPORE, Catalog No.: UFC901096), and the concentration was measured. 100 ⁇ g of purified protein was taken and the concentration was adjusted to 1 mg/mL. The protein purity was determined using a gel filtration column SW3000 (TOSOH Product No.: 18675).
  • Example 2 Determination of affinity between TACI or TACI/BCMA chimeric fusion protein and ligand
  • the equilibrium dissociation constant (KD) between the TACI or TACI/BCMA chimeric fusion protein and its ligand was determined using ForteBio interferometry.
  • ForteBio affinity measurements were performed according to existing methods (Estep, P et al., High throughput solution-based measurement of antibody-antigen affinity and epitope binding. Mabs, 2013. 5(2): p. 270-8).
  • the affinity of candidate molecules for BAFF and APRIL was measured by equilibrating the sensor offline in assay buffer for 20 minutes and then monitoring online for 120 seconds to establish a baseline.
  • the candidate molecules were loaded onto an AHC sensor (Sartorius, 18-5060) for ForteBio affinity measurement.
  • the loaded sensor was placed in a solution containing 100 nM BAFF or APRIL until plateauing, after which the sensor was transferred to assay buffer for at least 2 minutes for association and dissociation rate measurements.
  • Kinetic analysis was performed using a 1:1 binding model.
  • Example 3 In vitro functional assay of TACI/BCMA chimeric fusion protein
  • Example 3.1 TACI/BCMA chimeric fusion protein blocks the binding of BAFF to IM-9 cells.
  • Example 3.2 TACI/BCMA chimeric fusion protein blocks the binding of APRIL to IM-9 cells.
  • Samples (ALPN-303, RC-18, and TACI/BCMA chimeric fusion protein) were serially diluted threefold in FACS buffer starting at 200 nM, for a total of 12 concentrations.
  • Biotin Human APRIL (Acro, Cat# APL-H82F5-200UG) was diluted to 0.2 ⁇ g/mL.
  • the diluted samples were mixed with diluted Biotin Human APRIL at a 1:1 ratio and incubated at 37°C for 30 min.
  • IM-9 cells were counted and diluted to 2 ⁇ 10 6 cells/mL, with 100 ⁇ L/well added to a U-bottom 96-well plate. The cells were centrifuged at 500 g for 5 min, and the cell culture medium was removed.
  • the samples were then added to the U-bottom plate and the cells were resuspended at 100 ⁇ L/well and incubated at 4°C for 30 min. The supernatant was removed by centrifugation at 500 g for 5 min, and the cells were washed twice with FACS buffer. Centrifuge at 500g for 5 minutes, remove the FACS buffer, and add 50 ⁇ L of PE Streptavidin secondary antibody (BD, Cat#554061) (1:200 dilution in FACS buffer) to each well. Incubate at 4°C in the dark for 30 minutes. Centrifuge at 500g for 5 minutes, remove the supernatant, and wash the cells three times with FACS buffer. Resuspend the cells in 200 ⁇ L of FACS buffer and analyze by flow cytometry.
  • PE Streptavidin secondary antibody BD, Cat#554061
  • Example 3.3 TACI/BCMA chimeric fusion protein blocks BAFF/APRIL-induced NF- ⁇ B reporter signaling in Jurkat cells overexpressing human TACI
  • the cDNA encoding human TACI was cloned into the pLenti-IRES-puro vector (Invitrogen), and then lentiviral transfection was used to generate NF- ⁇ B reporter Jurkat cells (Jiman Bio, Cat#GM-C07855) overexpressing human TACI (i.e., TACI/NF- ⁇ B reporter Jurkat cells).
  • a serial dilution assay was then used to examine the ability of the chimeric TACI/BCMA-Fc fusion protein to block BAFF/APRIL-induced TACI/NF- ⁇ B reporter Jurkat signaling.
  • samples (ALPN-303, RC-18, and TACI/BCMA chimeric fusion protein) were serially diluted threefold, starting at 1200 nM, in RPMI-1640 medium with 10% FBS, for a total of 10 concentrations.
  • Human BAFF (Acro, Cat#BAF-H52D4) + human APRIL (Acro, Cat#APL-H52D1) was diluted to 20 nM in RPMI-1640 medium with 10% FBS.
  • the diluted samples were mixed with a 20 nM human BAFF/APRIL mixture at a 1:1 ratio and incubated at 37°C for 30 min.
  • TACI/NF- ⁇ B reporter Jurkat cells were counted and diluted to 1 ⁇ 106 cells/mL, with 50 ⁇ L/well added to a white flat-bottom 96-well plate. The incubated samples were then added to a white flat-bottom 96-well plate containing cells and resuspended at 50 ⁇ L/well. The plates were incubated at 37°C for 22 h. After incubation, the luminescent signals generated by the cells were detected using the Bio-Lite Luciferase Assay System (Vazyme, #DD1201-03).
  • Example 3.4 TACI/BCMA chimeric fusion protein blocks BAFF/APRIL-induced NF- ⁇ B reporter signaling in Jurkat cells overexpressing human BCMA
  • the cDNA encoding human BCMA was cloned into the pLenti-IRES-Neo vector (Invitrogen), and then lentiviral transfection was used to generate NF- ⁇ B reporter Jurkat cells (Jiman Bio, Cat#GM-C07855) overexpressing human BCMA (i.e., BCMA/NF- ⁇ B reporter Jurkat cells).
  • a serial dilution assay was then used to examine the ability of the chimeric TACI/BCMA-Fc fusion protein to block BAFF/APRIL-induced BCMA/NF- ⁇ B reporter Jurkat signaling.
  • samples (ALPN-303, RC-18, and TACI/BCMA chimeric fusion protein) were serially diluted threefold, starting at 2400 nM, in RPMI-1640 medium with 10% FBS, for a total of 10 concentrations.
  • Human BAFF (Acro, Cat#BAF-H52D4) + human APRIL (Acro, Cat#APL-H52D1) were diluted to 20 nM in RPMI-1640 medium with 10% FBS.
  • the diluted samples were mixed with a 20 nM human BAFF/APRIL mixture at a 1:1 ratio and incubated at 37°C for 30 min.
  • BCMA/NF- ⁇ B reporter Jurkat cells were counted and diluted to 2 ⁇ 10 6 cells/mL, with 50 ⁇ L/well added to a white flat-bottom 96-well plate. The incubated samples were then added to a white flat-bottom 96-well plate containing cells and resuspended at 50 ⁇ L/well. The plates were incubated at 37°C for 5 h. After incubation, the luminescent signals generated by the cells were detected using the Bio-Lite Luciferase Assay System (Vazyme, #DD1201-03).
  • Example 3.5 Detection of the ability of TACI/BCMA chimeric fusion protein to block spleen cell proliferation
  • Mouse spleen cells were harvested, lysed, and the cell density adjusted to 2 ⁇ 106 /mL. 50 ⁇ L of sample was added to each well, and cells were plated at 1 ⁇ 105 /well. Samples (ALPN-303, RC-18, and TACI/BCMA chimeric fusion protein) were serially diluted threefold, starting at 1200 nM, in RPMI-1640 medium with 10% FBS, for a total of nine concentrations.
  • Human BAFF (Acro, Cat#BAF-H52D4) was diluted to 50 ng/mL in RPMI-1640 medium with 10% FBS. The diluted samples were mixed with diluted human BAFF at a 1:1 ratio and incubated at 37°C for 30 min.
  • Control wells were set up: IgG group (using IgG (Equitech-Bio, Cat#SLH56) as sample + cells + BAFF antigen) and blank group (RPMI-1640 + 10% FBS) group (culture medium + cells).
  • IC50 values were calculated by curve fitting based on the readings at different concentrations. The results showed that ALPN-303, RC-18, and the TACI/BCMA chimeric fusion protein all inhibited BAFF-induced spleen cell proliferation. However, the TACI/BCMA chimeric fusion protein exhibited significantly superior inhibitory activity against BAFF-induced spleen cell proliferation than RC-18 (IC50 of 4.98 ⁇ 0.95 nM), and was comparable to the inhibitory activity of ALPN-303 (IC50 of 0.49 ⁇ 0.06 nM) (Table 4).
  • Example 3.6 Detection of the ability of TACI/BCMA chimeric fusion protein to inhibit B cell proliferation
  • PBMCs AllCell, Cat#FPB004F-C
  • B cells were isolated using the EasySepTM Human B cell Enrichment kit (STEMCELL, Cat#19054). The cells were counted and adjusted to a density of 2 ⁇ 10 6 /mL.
  • rhIL-4 R&D, Cat#204-IL-050
  • 1 ⁇ g/mL Anti-human IgM Jakson, Cat#109-006-129
  • Samples (ALPN-303, RC-18, and TACI/BCMA chimeric fusion protein) were serially diluted threefold, starting at a concentration of 2400 nM, in RPMI-1640 medium supplemented with 10% FBS, for a total of nine concentration gradients.
  • Human BAFF (Acro, Cat#BAF-H52D4) + Human APRIL (Acro, Cat#APL-H52D1) were diluted to 20 nM using RPMI-1640 + 10% FBS medium.
  • the diluted sample was mixed with the diluted 20 nM human BAFF/APRIL mixture at a ratio of 1:1 and incubated at 37°C for 30 min. Control wells were set up: IgG group (IgG (Equitech-Bio, Cat#SLH56) was used as the sample). 50 ⁇ L/well of the incubated mixture was added to the cells and incubated at 37°C for 72 h. After 72 h of incubation, cell proliferation was detected using CellCount-LiteTM Luminescent Cell Viabil (Vazyme, #DD1101-02) and a dose-effect curve was drawn.
  • Example 4 Stability determination of TACI/BCMA chimeric fusion protein
  • the thermal stability of the TACI/BCMA chimeric fusion protein was evaluated by dynamic light scattering (DLS), differential scanning calorimetry (DSC), and accelerated stability.
  • a protein solution (1 mg/mL) was centrifuged at 13,000 g/min for 5 minutes and then added to a sample plate.
  • the changes in protein particle size during a continuous temperature increase were measured using a DLS acquisition time of 5 seconds, 5 acquisitions, and an experimental temperature range of 25-85°C. After the experiment, the changes in sample particle size with temperature were analyzed.
  • the TACI/BCMA chimeric fusion protein was also transferred to a PBS solution (pH 7.4) using an ultrafiltration tube at a concentration of 10 mg/ml. The solution was then placed in a 50°C incubator for accelerated stability testing. After 4 days, samples were collected for purity analysis (SEC and CE-SDS). The results are shown in Table 6.
  • TACI/BCMA chimeric fusion proteins 099017 and 099018 exhibited the best stability, maintaining purity above 95% by SEC and above 98% by non-reducing CE-SDS. These proteins exhibited significantly better stability than the control RC-18 and the unmodified TACI CRD2 molecule 99001, as well as the control ALPN-303.
  • Example 5 In vivo efficacy of TACI/BCMA chimeric fusion protein
  • Example 5.1 Pharmacological efficacy of TACI/BCMA chimeric fusion protein in a mouse KLH immune model
  • mice purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd.
  • KLH Smallbiol, Cat#KB160
  • Alum adjuvant Thermo, Cat#77161
  • This experiment set up a blank control group (Blank, without any treatment), a model control IgG group, a 99001 group, a 99006 group and a 99017 group, with 5-7 mice in each group (3 mice in the blank control group).
  • mice were intraperitoneally injected with drugs on the 4th and 11th days (the model control IgG group was injected with an equal amount of IgG antibody (purchased from Equitech-Bio, Cat#SLH56)).
  • the spleens of the mice were collected for cell detection of B cell changes and blood was collected for detection of IgA (Invitrogen, Cat#EMIGA), IgM (Invitrogen, Cat#88-50470-88) and IgG (Invitrogen, Cat#88-50400-88) in the serum.
  • IgA Invitrogen, Cat#EMIGA
  • IgM Invitrogen, Cat#88-50470-88
  • IgG Invitrogen, Cat#88-50400-88
  • the results showed that compared with the control IgG group, the drug-treated groups were able to inhibit the proliferation of KLH-immunized splenocytes and B cells in the spleen (Table 7), as well as the levels of IgA, IgM, and IgG in the serum (Table 8).
  • the results showed that compared with the 99001 group (unmodified TACI CRD2 molecule) and the 99006 group (unmodified BCMACRD2 molecule), the 99017 group (TACI/BCMA chimeric fusion protein) had a better inhibitory effect.
  • the above data suggest that the modified TACI/BCMA chimeric fusion protein is significantly better than the TACI or BCMAWT CRD2 fragment in terms of B cell and antibody levels.
  • Example 5.2 Efficacy of TACI/BCMA chimeric fusion protein in mouse BAFF stimulation model
  • Balb/c mice purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd.
  • 10 ⁇ g human BAFF protein (Sino Biological, Cat#10056-HNCHA) on days 0, 1, 2, and 3.
  • This experiment set up a blank control group (Blank, without any treatment), a model control IgG group, an RC-18 group, an ALPN-303 group, and a TACI/BCMA chimeric fusion protein group, a total of 5 groups, with 5-7 mice in each group (3 mice in the blank control group).
  • mice were intraperitoneally injected on days 0 and 2 (the model control IgG group was injected with an equal amount of IgG antibody (purchased from Equitech-Bio, Cat#SLH56)).
  • the spleens of the mice were collected for cell detection, and ELISA kits were used to detect IgA (Invitrogen, Cat#EMIGA), IgM (Invitrogen, Cat#88-50470-88), and IgG (Invitrogen, Cat#88-50400-88) in the serum.
  • the results showed that TACI/BCMA chimeric fusion protein could effectively inhibit the proliferation of spleen cells and B cells in the spleen induced by BAFF (Table 9).
  • TACI/BCMA chimeric fusion protein could effectively reduce the levels of IgA, IgM and IgG in the serum induced by BAFF (Table 10).
  • Example 5.3 Pharmacological efficacy of TACI/BCMA chimeric fusion protein in a mouse KLH immune model
  • C57BL/6N mice purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd.
  • mice purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd.
  • KLH Smallbiol, Cat#KB160
  • Alum adjuvant Thermo, Cat#77161
  • This experiment set up 5 groups, including blank control group, model control IgG group, RC-18 group, ALPN-303 group and TACI/BCMA chimeric fusion protein group (99017 and 99018, respectively), with 5-7 mice in each group (3 mice in the blank control group).
  • mice were intraperitoneally injected on the 4th and 11th days (the model control IgG group was injected with an equal amount of IgG antibody (purchased from Equitech-Bio, Cat#SLH56)).
  • the spleens of the mice were taken for cell detection and blood was collected for detection of IgA (Invitrogen, Cat#EMIGA), IgM (Invitrogen, Cat#88-50470-88) and IgG (Invitrogen, Cat#88-50400-88) in the serum.
  • IgA Invitrogen, Cat#EMIGA
  • IgM Invitrogen, Cat#88-50470-88
  • IgG Invitrogen, Cat#88-50400-88
  • mice The pharmacokinetics of the chimeric fusion protein were evaluated by intravenous injection (I.V.) in mice.
  • I.V. intravenous injection
  • mice Nine BALB/c mice weighing approximately 20 g were injected intravenously with 10 mg/kg of the test molecule.
  • Blood samples were collected from the orbital cavity 5 minutes, 0.5 hours, 2 hours, 6 hours, and on days 2, 4, 7, 14, and 21 after a single dose. Serum was collected by centrifugation after natural coagulation.
  • the antibody drug concentration in serum was determined as follows: the antigen human APRIL-his protein (Acro Biosystems, Cat#APL-H52D1) was diluted to 1 ⁇ g/mL in coating solution (dissolve one packet of carbonate powder (Thermo, cat#23282) in 400 mL of ultrapure water, mix well, and dilute to 500 mL). 100 ⁇ L was added to each well of a 96-well ELISA plate (Thermo, Cat#442404) and incubated overnight at 4°C. The coating solution was discarded, and the plates were washed three times with 1 ⁇ PBST. 200 ⁇ L of blocking solution (PBST containing 2% BSA) was added to each well and blocked at room temperature for 1 hour.
  • coating solution dissolve one packet of carbonate powder (Thermo, cat#23282) in 400 mL of ultrapure water, mix well, and dilute to 500 mL. 100 ⁇ L was added to each well of a 96-well
  • the blocking solution was discarded, and the plates were washed three times with 1 ⁇ PBST. Then, diluted mouse serum was added and incubated at room temperature for 2 hours. The solution in the ELISA plate was discarded, and the plates were washed five times with 1 ⁇ PBST. Diluted Goatx-human IgG-Fc-HRP (BETHYL, Cat#A80-104P) was added, 100 ⁇ L per well, and incubated at room temperature for 1 hour. The solution in the ELISA plate was discarded and washed 5 times with 1 ⁇ PBST. 100 ⁇ L of TMB colorimetric solution (Solebol, Cat#PR1200) was added to each well, and the color was developed for 5-10 minutes.
  • TMB colorimetric solution Solebol, Cat#PR1200
  • Subcutaneous injection of 99017 molecules of TK was performed in cynomolgus monkeys. Each dose group consisted of two macaques (one male and one female), weighing approximately 3.5-4.5 kg. Each group received five weekly subcutaneous injections of either 100 mg/kg or 200 mg/kg of the test molecule. Blood was collected 24 hours after the first dose (C1D1) and 24 hours after the fourth dose (C4D1). Blood was collected at 30 minutes, 2 hours, 6 hours, 10 hours, 24 hours, 48 hours, 72 hours, 96 hours, and 168 hours. Serum was collected by centrifugation after natural coagulation.
  • the antibody drug concentration in serum was determined as follows: Human APRIL-his protein (Acro Biosystems, Cat# APL-H52D1) was diluted to 2 ⁇ g/mL in coating solution (dissolve one packet of carbonate powder in 400 mL of ultrapure water, mix well, and dilute to 500 mL). 100 ⁇ L was added to each well of a 96-well microtiter plate and incubated overnight at 4°C. The coating solution was discarded, and the plates were washed three times with 1 ⁇ PBST. 300 ⁇ L of blocking solution (5% SM-1 in PBST) was added to each well and blocked at room temperature for 2 hours. The blocking solution was discarded, and the plates were washed three times with 1 ⁇ PBST.
  • coating solution dissolve one packet of carbonate powder in 400 mL of ultrapure water, mix well, and dilute to 500 mL. 100 ⁇ L was added to each well of a 96-well microtiter plate and incubated overnight at 4
  • OD450nm and OD620nm values were read on a microplate reader.
  • the changes in plasma drug concentrations at different time points after administration of different doses of the test molecule to cynomolgus monkeys on C1D1 and C4D1 are shown in Figure 8.
  • the relevant kinetic parameters were calculated using a non-compartmental model using Phoenix WinNonlin 8.4.
  • the main drug exposure parameters (AUC0-t, Cmax, Tmax, T1 /2 , CI/F) are shown in Table 14.
  • the pharmacokinetics and pharmacodynamics of 99017 and 99025 were evaluated in cynomolgus monkeys using intravenous injection (I.V.). Three macaques, weighing approximately 3.5-4.5 kg, were injected intravenously with 9 mg/kg of the test molecule. Blood samples were collected 5 minutes, 2 hours, 6 hours, 12 hours, 24 hours, 72 hours, 144 hours, 216 hours, 312 hours, 432 hours, 480 hours, 624 hours, 816 hours, and 984 hours after the single dose. After natural coagulation, the blood was centrifuged and serum was collected for assay of antibody drug concentration. Blood samples were collected before a single dose and at 6, 9, 12, 18, 24, 32, and 40 days after administration.
  • Serum IgA (Cat#05219205190), IgM (Cat#05220726190), and IgG (Cat#05220718190) were measured using Roche Diagnostic immunoglobulin assay kits. The percentage decrease in serum immunoglobulin levels at different time points after administration compared to pre-dose levels was calculated. The results are shown in Figure 10. The results showed that the TACI/BCMA chimeric fusion protein effectively reduced BAFF-induced serum IgA, IgM, and IgG levels.
  • the antibody drug concentration in serum was determined as follows: Human APRIL-his protein (Acro Biosystems, Cat# APL-H52D1) was diluted to 2 ⁇ g/mL in coating solution (dissolve one packet of carbonate powder in 400 mL of ultrapure water, mix well, and dilute to 500 mL). 100 ⁇ L was added to each well of a 96-well microtiter plate and incubated overnight at 4°C. The coating solution was discarded, and the plates were washed three times with 1 ⁇ PBST. 300 ⁇ L of blocking solution (5% SM-1 in PBST) was added to each well and blocked at room temperature for 2 h. The blocking solution was discarded, and the plates were washed three times with 1 ⁇ PBST.
  • coating solution dissolve one packet of carbonate powder in 400 mL of ultrapure water, mix well, and dilute to 500 mL. 100 ⁇ L was added to each well of a 96-well microtiter plate and incubated overnight at

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Abstract

L'invention concerne une nouvelle chimère TACI/BCMA et une protéine de fusion la comprenant, et un acide nucléique codant pour la chimère ou la protéine de fusion, un vecteur comprenant l'acide nucléique, et une cellule hôte comprenant l'acide nucléique ou le vecteur. L'invention concerne en outre une méthode de traitement et l'utilisation de la protéine de fusion dans le traitement de maladies immunitaires.
PCT/CN2025/086591 2024-04-02 2025-04-01 Protéine de fusion de chimère taci/bcma et son utilisation Pending WO2025209465A1 (fr)

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CN202410396507 2024-04-02
CN202410396507.7 2024-04-02
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CN202510355955 2025-03-25
CN202510355955.7 2025-03-25

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