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WO2025209465A1 - Taci/bcma嵌合体融合蛋白及其用途 - Google Patents

Taci/bcma嵌合体融合蛋白及其用途

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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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Prior art keywords
amino acid
taci
acid sequence
bcma
seq
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PCT/CN2025/086591
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English (en)
French (fr)
Inventor
熊尧
周帅祥
张志敏
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Fortvita Biologics Inc
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Fortvita Biologics Inc
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Publication of WO2025209465A1 publication Critical patent/WO2025209465A1/zh
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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

提供一种新的TACI/BCMA嵌合体，以及包含其的融合蛋白以及编码嵌合体或融合蛋白的核酸、包含核酸的载体，以及包含核酸或载体的宿主细胞；还提供融合蛋白用于治疗免疫相关疾病的治疗方法和用途。

Description

TACI/BCMA嵌合体融合蛋白及其用途
相关申请的交叉引用
本申请基于申请号为202410396507.7，申请日为2024年4月2日的中国专利申请，以及申请号为202411500688.X，申请日为2024年10月25日的中国专利申请，以及申请号为202510355955.7，申请日为2025年3月25的中国专利申请提出，并要求所述三个中国专利申请的优先权，所述三个中国专利申请的全部内容在此引入本申请作为参考。
本发明涉及一种新的TACI/BCMA嵌合体，以及包含其的融合蛋白。本发明还涉及编码所述嵌合体或融合蛋白的核酸、包含所述核酸的载体，以及包含所述核酸或载体的宿主细胞。本发明还涉及所述融合蛋白用于治疗免疫相关疾病的治疗方法和用途。
背景技术
细胞因子肿瘤坏死因子(TNF)于1968年被报道为一种细胞毒性因子，可诱导肿瘤坏死。该蛋白质以233个氨基酸长的II型跨膜蛋白形式产生，通过金属蛋白酶TNF-α转换酶(TACE/ADAM17)的蛋白水解裂解，以可溶性同源三聚体的形式在细胞外空间中释放出来。TNF-α(TNFSF1B)和TNF-β(TNFSF1A)与两种特定受体(TNFR1和TNFR2)结合。
TNF超家族(TNFSF)现在包括19个分子，而TNF受体超家族(TNFRSF)包括29个不同的蛋白质成员。除了特定配体和受体之间的一对一选择性之外，还报道了交叉反应性，这表明存在差异信号传导和多种假定的细胞和分子作用。
TNF受体超家族(TNFRSF)指细胞表面细胞因子受体的组，所述细胞因子受体都是I型(N末端细胞外)跨膜糖蛋白，在其细胞外结构域中含有一至六个富半胱氨酸结构域(CRD)。基于共有的结构特征将分子分类为该超家族的成员，所述结构特征包括存在于其N末端细胞外区域中的一个或多个富半胱氨酸结构域(CRD)，该富半胱氨酸结构域通常在蛋白质与其同源结合配偶体或配体的结合中发挥作用。TNFRSF蛋白可以仅具有一个或几个CRD(例如CRD1、CRD2等)。通常，TNFRSF成员的ECD或胞外域含有1至6个的CRD假重复序列。例如，BAFF受体和BCMA各自含有一个CRD，而TACI含有两个CRD(CRD1和CRD2)。TNFRSF成员通常为三聚或多聚复合物，它们通过其半胱氨酸内二硫键来稳定。TNFRSF蛋白与其配体的结合促进细胞中的多种生物活性，如凋亡性细胞死亡或细胞存活和增殖的诱导。
BAFF(B cell activating factor)和APRIL(Aproliferation-inducing ligand)是属于TNF家族的B细胞激活和调节因子，能够促进B细胞的发育和增殖，促进机体各种免疫球蛋白的分泌，提高其血清中的表达量，对机体的免疫反应具有重要的调节作用。它们与细胞膜受体TACI(Transmembrane activator and CAML-interactor)和BCMA(B cell maturation antigen)结合。此外，BAFF也能与另一个受体BAFFR(B cell-activating factorreceptor)结合。BAFF和APRIL经这几个受体的信号传递，调节淋巴细胞的活化、发育和增殖。BAFF和APRIL的过量表达是多种自身免疫疾病的病因之一。研究表明，系统性红斑狼疮、类风湿性关节炎等自身免疫疾病人的血清中BAFF、APRIL浓度明显升高。因此靶向BAFF/APRIL成为治疗自身免疫疾病的一种有效途径。
跨膜激活剂和CAML相互作用蛋白(TACI)，也称为肿瘤坏死因子受体超家族成员13B(TNFRSF13B)，最初被发现是因为它能够与钙调节剂和亲环蛋白配体(CAML)相互作用。后来发现TACI通过与TNF家族的两个成员BAFF和APRIL相互作用，在体液免疫中发挥至关重要的作用。
现有技术已经发现了包含TACI的融合蛋白，用于免疫疾病的治疗。例如荣昌生物制备了一种优化的TACI-Fc融合蛋白，泰它西普(CN101323643B)，它采用全长的TACI胞外段进行改造，但是最终成药分子稳定性较差，最终制剂只能以冻干粉形式，且临床给药周期为1周2针，无法通过加大给药量的策略弥补给药频率。
因此，本领域仍然存在开发成药性好、分子稳定的TACI融合蛋白的需要。
发明内容
本发明涉及一种TACI/BCMA嵌合体，以及包含其的融合蛋白，例如与Fc区的融合蛋白。
本发明的TACI/BCMA嵌合体的Fc区融合蛋白，与已知的TACI-Fc融合蛋白相比，具有更好的稳定性，例如热稳定性；同时还具有更长的半衰期和/或更低的清除率。
在一些实施方案中，本发明涉及一种TACI/BCMA嵌合体，其包含应用BCMA的N末端和/或C末端替换TACI胞外区ECD的功能性片段的N末端和/或C末端的嵌合蛋白。
在一个具体的实施方案中，所述TACI/BCMA嵌合体包含如下结构：
BCMAN末端氨基酸-TACI部分，其中TACI部分为缺失了N末端的TACI的胞外区ECD或其功能性片段，或者
BCMAN末端氨基酸-TACI部分-BCMAC末端氨基酸，其中TACI部分为缺失了N末端和C末端的TACI的胞外区ECD或其功能性片段。
在一个具体的实施方案中，所述TACI/BCMA嵌合体中TACI的胞外区ECD功能性片段包含TACI的CRD2且不包含CRD1或CRD1的任意片段。在一个实施方案中，所述CRD2为TACI对应于SEQ ID NO:1所示的第71-104位的氨基酸序列，且CRD1为TACI对应于SEQ ID NO:1所示的第34-66位的氨基酸序列。在一个实施方案中，所述TACI的胞外区ECD功能性片段还包含TACI的部分茎区和/或TACI对应于SEQ ID NO:1的第68-70位之间的任意氨基酸序列。在一个实施方案中，所述TACI的胞外区ECD功能性片段为或包含TACI对应于SEQ ID NO:1所示的氨基酸序列的如下片段：氨基酸残基68-110。在一个实施方案中，所述TACI的胞外区ECD功能性片段包含
(i)与SEQ ID NO:31、32或33所示的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％的同一性的氨基酸序列；
(ii)与SEQ ID NO:33所示的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％的同一性且具有Y102D的氨基酸序列；
(iii)与SEQ ID NO:32所示的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％的同一性且具有K77E、F78Y和Y102D的氨基酸序列；
(iv)SEQ ID NO:31、32或33所示的氨基酸序列；或
(v)由(i)-(iv)中任一项所述的序列组成。
在一个具体的实施方案中，所述TACI的胞外区ECD功能性片段的N末端是指TACI对应于SEQ ID NO:1的Y79位氨基酸之前的N末端氨基酸。
在一个具体的实施方案中，所述TACI的胞外区ECD功能性片段的C末端是指TACI对应于SEQ ID NO:1的第99位氨基酸、第100位氨基酸、第101位氨基酸、第102位氨基酸、第103位氨基酸、第104位氨基酸、第105位氨基酸、第106位氨基酸、第107位氨基酸、第108位氨基酸、第109位氨基酸或第110位氨基酸之后的C末端氨基酸，优选地第99或105位氨基酸之后的C末端氨基酸。
在一个具体的实施方案中，所述BCMA的N末端氨基酸选自BCMA对应于SEQ ID NO:2的第1-13位、第2-13位、第3-13位、第4-13位、第5-13位、第6-13位或第7-13位的氨基酸序列。在一个具体的实施方案中，所述BCMA的N末端氨基酸序列包含SEQ ID NO:40-46中所示的任一项的氨基酸序列，或由所述的氨基酸序列组成。
在一个具体的实施方案中，所述BCMA的C末端氨基酸序列是BCMA对应于SEQ ID NO:2的第37-47位、第37-46位、第37-45位、第37-44位的氨基酸序列，或对应于SEQ ID NO:2的第43-44位或43-46位的氨基酸序列。
在一个具体的实施方案中，所述BCMA的C末端氨基酸包含改善结合亲和力，增加稳定性和/或改善成药性的突变，例如取代，例如所述突变为第39位和/或第42位的突变，例如取代，例如N42A或N42Q或R39D或N42A-R39D。
在一个具体的实施方案中，所述BCMA的C末端氨基酸序列包含SEQ ID NO:47-55中任一项所示的氨基酸序列，或由所述的氨基酸序列组成。
在一个具体的实施方案中，所述TACI ECD功能性片段包含降低聚集风险的氨基酸位点突变，例如在成药性风险位点的突变如取代，例如，所述成药性风险位点选自对应于SEQ ID NO:1的第69、72、73、74、77、78、85、102或103位氨基酸，优选地，所述成药性风险位点的氨基酸被突变为A或D，例如所述突变是Y102D。
在一个具体的实施方案中，所述TACI部分包含SEQ ID NO:31-38中任一项所示的氨基酸序列，或包含与其具有至少90％、91％、92％、93％、94％、95％、96％、97％或98％的同一性的氨基酸序列，或由所述氨基酸序列组成。
在一个具体的实施方案中，所述TACI/BCMA嵌合体包含SEQ ID NO:59-80中任一项所示的氨基酸序列，或包含与其具有至少90％、91％、92％、93％、94％、95％、96％、97％或98％的同一性的氨基酸序列，或由所述氨基酸序列组成。
在一个方面，本发明涉及一种融合蛋白，其包含本文所述的TACI/BCMA嵌合体，以及Fc区。
在一个具体的实施方案中，所述Fc区为人的IgG Fc，例如，人IgG1 Fc,人IgG2 Fc,人IgG3 Fc或人IgG4 Fc。
在一个具体的实施方案中，所述Fc区包含如下一个或多个突变：
(i)在C末端缺失赖氨酸K(K447del)；
(ii)减少由Fc区介导的效应子功能的突变；
(iii)降低与Fcγ受体结合的突变，例如L234A/L235A突变或L234A/L235E或L234A/L235E/G237A突变；
(iv)增强Fc片段与FcRn结合的突变，例如M252Y/S254T/T256E和/或M482L/N434S。
在一个具体的实施方案中，所述Fc区包含
(i)SEQ ID NO:81或82所示的氨基酸序列或与其具有至少90％同一性，例如95％，96％，97％，99％或更高的同一性的氨基酸序列；
(ii)SEQ ID NO:83或84所示的氨基酸序列或与其具有至少90％同一性，例如95％，96％，97％，99％或更高的同一性的氨基酸序列，任选地缺失C末端赖氨酸；
(iii)SEQ ID NO:85-88和100-103中任一项所示的氨基酸序列或与其具有至少90％同一性，例如95％，96％，97％，98％,99％或更高的同一性的氨基酸序列；
(iv)与SEQ ID NO:85或87所示的氨基酸序列所示的氨基酸序列具有至少85％、90％、95％、96％、97％、98％、99％的同一性且包含突变L234A/L235E/G237A的氨基酸序列；
(v)与SEQ ID NO:86或88所示的氨基酸序列所示的氨基酸序列具有至少85％、90％、95％、96％、97％、98％、99％的同一性且包含突变L234A/L235E/G237A和C末端缺失赖氨酸的氨基酸序列；
(vi)与SEQ ID NO:100或102所示的氨基酸序列所示的氨基酸序列具有至少85％、90％、95％、96％、97％、98％或99％的同一性且包含突变L234A/L235E/G237A和M252Y/S254T/T256E的氨基酸序列；
(vii)与SEQ ID NO:101或103所示的氨基酸序列所示的氨基酸序列具有至少85％、90％、95％、96％、97％、98％、99％的同一性且包含突变L234A/L235E/G237A、M252Y/S254T/T256E和C末端缺失赖氨酸的氨基酸序列；或
(viii)由以上(i)-(vii)中任一项所述的氨基酸序列组成。
在一个具体的实施方案中，所述TACI/BCMA嵌合体直接或通过接头与Fc融合，优选地，所述嵌合体的C末端直接或通过接头与Fc的N末端融合。在一个具体的实施方案中，所述接头选自(GSGGGGS)n、(GS)n、(GSGGS)n、(GGGGS)n或(GGGS)n，其中n是至少1的整数，例如1、2、3、4或5；例如是(GSGGGGS)n，其中n＝1-3，例如所述接头是SEQ ID NO:39所示的氨基酸序列。
在一个具体的实施方案中，所述融合蛋白
(i)包含与SEQ ID NO:5-28中任一项的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由所述氨基酸序列组成；或者
(ii)包含SEQ ID NO:5-28中任一项的氨基酸序列或由所述氨基酸序列组成
在一个方面，本发明涉及一种融合蛋白二聚体，其包含第一单体和第二单体，所述第一单体和第二单体分别包含本文所述的融合蛋白链或由所述融合蛋白链组成，优选地第一单体和第二单体相同。
在一个方面，本发明涉及一种多核苷酸，其编码本发明的TACI/BCMA嵌合体或融合蛋白或融合蛋白二聚体。
在一个方面，本发明涉及一种表达载体，其包含本发明的多核苷酸，例如所述表达载体是pCDNA表达载体，例如pCDNA3.1表达载体。
在一个方面，本发明涉及一种宿主细胞，其包含本发明的多核苷酸或表达载体。
在一个方面，本发明涉及一种制备TACI/BCMA嵌合体或其融合蛋白或融合蛋白二聚体的方法，其中所述方法包括，在适合TACI/BCMA嵌合体或其融合蛋白或融合蛋白二聚体表达的条件下，培养本发明的宿主细胞，和任选地从所述宿主细胞(或宿主细胞培养基)回收所述TACI/BCMA嵌合体或其融合蛋白或融合蛋白二聚体。
在一个方面，本发明涉及一种药物组合物，其包含本发明的TACI/BCMA嵌合体或融合蛋白或融合蛋白二聚体，以及任选地药用辅料。
在一个方面，本发明涉及一种药物组合或组合产品，其包含本发明的TACI/BCMA嵌合体或融合蛋白或融合蛋白二聚体，以及一种或多种其它治疗剂(例如细胞因子、激素、细胞毒性剂或抑制剂(例如影响T细胞和/或B细胞增殖的细胞抑制剂)、抗体或小分子药物或免疫调节剂(例如免疫抑制剂))。
在一个方面，本发明涉及一种预防或治疗受试者疾病，例如B细胞或自身抗体相关的疾病或免疫系统疾病(例如自身免疫性疾病)或炎症的方法，所述方法包括向所述受试者施用本发明的TACI/BCMA嵌合体或融合蛋白或融合蛋白二聚体或药物组合物或组合产品。在一个具体的实施方案中，所述B细胞或自身抗体相关的疾病为B细胞或自身抗体介导的疾病，例如B细胞或自身抗体介导的自身免疫性疾病。在一个具体的实施方案中，所述B细胞或自身抗体相关的疾病或免疫系统疾病或炎症为个体中，相比健康个体的样品，B细胞异常增殖或异常活化的疾病(例如自身免疫性疾病)。在一个具体的实施方案中，所述疾病为狼疮，例如系统性红斑狼疮，类风湿性关节炎、IgA肾病(IgAN)或膜性肾病等慢性肾病、干燥综合征、重症肌无力、特发性血小板减少性紫癜(ITP)、温抗体型自身免疫性溶血性贫血(wAIHA)、多发性硬化症(MS)、冠状动脉性心脏病(CAD)或甲状腺眼病。在一个具体的实施方案中，所述施用还包括联合施用一种或多种其它治疗剂(例如细胞因子、激素、细胞毒性剂或抑制剂(例如影响T细胞和/或B细胞增殖的细胞抑制剂)、抗体或小分子药物或免疫调节剂(例如免疫抑制剂))。
附图说明
图1显示了TACI和BCMA分别与APRIL形成复合物的结构分析。
图2显示了TACI与BCMA的结构比对与分析。
图3显示了TACI与BCMA的氨基酸序列比对。
图4显示了TACI/BCMA嵌合体融合蛋白抑制B细胞增殖的能力。
图5显示了TACI/BCMA嵌合体融合蛋白抑制KLH免疫引起的小鼠脾脏细胞和脾脏B细胞的增殖。
图6显示了TACI/BCMA嵌合体融合蛋白抑制KLH免疫引起的小鼠血清中IgA、IgM和IgG的水平。
图7显示了TACI/BCMA嵌合体融合蛋白在小鼠中的药代动力学。
图8显示了TACI/BCMA嵌合体融合蛋白在食蟹猴中毒物动力学。
图9显示了TACI/BCMA嵌合体融合蛋白及其YTE分子在食蟹猴中的药代动力学。
图10显示了TACI/BCMA嵌合体融合蛋白及其YTE分子在食蟹猴血清中的免疫球蛋白水平变化。
发明详述
在下文详细描述本发明前，应理解本发明不限于本文中描述的特定方法学、方案和试剂，因为这些可以变化。还应理解本文中使用的术语仅为了描述具体实施方案，而并不意图限制本发明的范围，其仅会由所附权利要求书限制。
I.定义
除非另外定义，本文中使用的所有技术和科学术语与本发明所属领域中普通技术人员通常的理解具有相同的含义。
为了解释本说明书，将使用以下定义，并且只要适当，以单数形式使用的术语也可以包括复数，并且反之亦然。要理解，本文所用的术语仅是为了描述具体的实施方案，并且不是限制性的。
术语“约”在与数字数值联合使用时意为涵盖具有比指定数字数值小5％、4％、3％、2％或1％的下限和比指定数字数值大5％、4％、3％、2％或1％的上限的范围内的数字数值。
如本文所用，术语“和/或”意指可选项中的任一项或可选项的两项或多项。
如本文中所用，术语“包含”或“包括”意指包括所述的要素、整数或步骤，但是不排除任意其他要素、整数或步骤。在本文中，当使用术语“包含”或“包括”时，除非另有指明，否则也涵盖由所述及的要素、整数或步骤组成的情形。例如，当提及“包含”或“包括”某个嵌合体时，也旨在涵盖仅具有所述嵌合体。
在本文中，亲本TACI是指作为引入本发明的突变的模板，其可以是野生型TACI，例如天然存在的TACI蛋白，例如来源于人、小鼠、大鼠、非人灵长类动物的天然TACI，包括未经加工的(例如未去除信号肽)的形式和经加工的(例如去除了N末端甲硫氨酸)的形式；或者例如是天然存在的TACI等位基因变体和剪接变体、同种型、同源物、和物种同源物；或者例如是TACI变体，例如，所述变体可以与天然TACI具有至少95％、96％、97％、98％或99％或更高同一性或具有不超过1-10个或1-5个氨基酸突变(例如，保守取代)，并优选与天然TACI蛋白具有基本相同的BAFF结合亲和力和/或APRIL结合亲合力。在一个实施方案中，亲本TACI是指TACI的功能性片段，例如包含TACI的胞外结构域(ECD)或TACI的CRD2结构域的片段，例如TACI的胞外结构域(ECD)，或TACI的CRD2结构域，或者TACI的其他功能性片段。
在本发明中，当提及TACI蛋白或TACI序列区段中的氨基酸位置时，通过参考野生型人TACI蛋白(也称作TACIWT)的氨基酸序列SEQ ID NO:1，予以确定。可以通过与SEQ ID NO:1进行氨基酸序列比对，鉴定在其它TACI蛋白或多肽(包括全长序列或截短片段)上的对应氨基酸位置。因此，在本发明中，除非另有说明，否则TACI蛋白或多肽的氨基酸位置为根据SEQ ID NO:1编号的氨基酸位置。例如，当提及“Y102”时，是指SEQ ID NO:1的第102位酪氨酸残基Y，或经比对在其它TACI多肽序列上的对应位置的氨基酸残基。
在本发明中，当提及BCMA蛋白或BCMA序列区段中的氨基酸位置时，通过参考野生型人BCMA蛋白(也称作BCMAWT)的氨基酸序列SEQ ID NO:2，予以确定。可以通过与SEQ ID NO:2进行氨基酸序列比对，鉴定在其它BCMA蛋白或多肽(包括全长序列或截短片段)上的对应氨基酸位置。因此，在本发明中，除非另有说明，否则BCMA蛋白或多肽的氨基酸位置为根据SEQ ID NO:2编号的氨基酸位置。例如，当提及“Q3”时，是指SEQ ID NO:2的第3位酪氨酸残基Q，或经比对在其它TACI多肽序列上的对应位置的氨基酸残基。
在本文中，在提及突变蛋白时，以如下方式来描述单氨基酸取代：[原始氨基酸残基/位置/取代的氨基酸残基]。例如，位置102的酪氨酸取代为天冬氨酸，可以表示为Y102D。当在一个给定位置(例如Y102位)可以有多种可选氨基酸取代方式(例如D,E)时，该氨基酸取代可以表示为：Y102D/E。相应地，可以通过加号“+”或“-”将各单氨基酸取代连接起来，以表示在多个给定位置的组合突变。例如K77E、F78Y、Y102D位置的组合突变，可以表示为：K77E-F78Y-Y102D，或K77E+F78Y+Y102D。
在本文中，“N末端氨基酸”或“N末端”可以互换使用，是指BCMA或TACI或其功能性片段的最N末端开始的1个或1个以上的氨基酸区段。
在本文中，“C末端氨基酸”或“C末端”可以互换使用，是指BCMA或TACI或其功能性片段的以最C末端结束的1个氨基酸或1个以上的氨基酸区段。
在本文中，术语“嵌合体”是指由两个或多个不同蛋白质的片段通过基因工程手段拼接而成的一种融合蛋白。这种嵌合体通常保留了各个片段的原有功能，并且可能展现出新的生物学特性或增强的稳定性。
在本文中，术语“成药性风险位点”是指在可药用蛋白，例如本发明的嵌合体或融合蛋白上对其成药性(例如稳定性等)有影响的位点，包括翻译后修饰位点例如isomeriztion异构化(D)，Deamidation脱酰胺(N)，GlycosylationN糖基化(N*S/T)，游离半胱氨酸(C)，氧化(M/W)；以及处于蛋白表面暴露的面积较大的疏水氨基酸或者带电荷氨基酸残基的富集区域(patch)等。
在本文中，可以通过在比较窗内比较两条最佳比对的序列来确定“序列同一性百分比”。优选地，在参考序列(例如SEQ ID NO:1)的全长上确定序列同一性。用于比较的序列比对方法是本领域内公知的。适用于确定序列同一性百分比的算法包括例如BLAST和BLAST 2.0算法(参见Altschul等，Nuc.Acids Res.25:3389-402,1977和Altschul等J.Mol.Biol.215:403-10,1990)。可通过美国国家生物技术信息中心(National Center for Biotechnology Information)公众获取用于进行BLAST分析的软件。出于本申请的目的，同一性百分比采用可从https://blast.ncbi.nlm.nih.gov/Blast.cgi获得的Basic LocalAlignment Search Tool，利用默认参数来确定。
如本文中使用的，术语“保守取代”意指不会不利地影响或改变包含氨基酸序列的蛋白/多肽的生物学功能的氨基酸取代。例如，可通过本领域内已知的标准技术例如定点诱变和PCR介导的诱变引入保守取代。典型的保守型氨基酸取代是指将一种氨基酸取代为具有相似的化学性质(例如电荷或疏水性)的另一种氨基酸。功能上相似氨基酸的保守性置换表是本领域熟知的。在一些实施方案中，以下为示例性的保守取代：
例如，亲本TACI蛋白可以相对于SEQ ID NO:1之一具有保守氨基酸取代，或仅具有保守氨基酸取代，且在一个优选实施方案中，保守取代不超过10个氨基酸残基，如1,2,3,4,5,6,7,8,9,10个残基。再例如，本发明的突变TACI蛋白可以相对于本文中具体给出的TACI突变蛋白序列具有保守氨基酸取代，或仅具有保守氨基酸取代且在一个优选实施方案中，保守取代不超过10个氨基酸残基，如1、2、3、4、5、6、7、8、9或10个残基。
“亲和力”或“结合亲和力”可以用于反映结合对子的成员之间相互作用的内在结合能力。分子X对其结合配偶体Y的亲和力可以由平衡解离常数(KD)表示，平衡解离常数是解离速率常数和结合速率常数(分别是kdis和kon)的比值。结合亲和力可以由本领域已知的常见方法测量。用于测量亲和力的一个具体方法是本文中的ForteBio亲和力测定技术。
在本文中可互换使用的术语“胞外域”、“细胞外结构域”或“ECD”是指在从细胞表达全长形式的膜蛋白(如跨膜蛋白)时，所述膜蛋白的位于液泡膜外部(例如，细胞外部的空间)的区域。出于本文的目的，应理解，提及ECD时是指构成该区域的序列和结构域，且不要求含有ECD的蛋白质是膜蛋白或所述结构域存在于细胞外部。例如，可溶免疫调节蛋白可以含有与另一部分(如多聚化结构域，例如Fc区)融合的膜蛋白的ECD序列。胞外域通常与特异性配体或特异性细胞表面受体相互作用，例如通过特异性地结合至配体或细胞表面受体的结合结构域来相互作用。结合结构域的例子包括富半胱氨酸结构域(CRD)。TNFR超家族成员的胞外域含有TD结构域(例如CRD结构域)。因此，本文中提及ECD时包括膜蛋白的ECD的全长序列，以及其与配体结合的含有CRD或其部分的特异性结合片段。
在本文中，抗体Fc片段是指含有至少一部分的恒定区的免疫球蛋白重链的C-端区域，并且可以包括天然序列Fc片段和变体Fc片段。天然序列Fc片段涵盖天然存在的各种免疫球蛋白Fc序列，例如各种Ig亚型以及其同种异型的Fc区(Gestur Vidarsson等,IgG subclasses and allotypes:from structure to effector functions，20October 2014,doi:10.3389/fimmu.2014.00520.)在一些实施方案中，所述Fc区来自IgG的Fc区，例如人IgG的Fc区。在一些实施方案中，所述Fc区来自IgG1、IgG2、IgG3或IgG4的Fc区，例如人IgG1、IgG2、IgG3或IgG4的Fc区。在一些实施方案中，所述Fc区是人IgG1、IgG2、IgG3或IgG4的Fc区。在一个实施方案中，人IgG重链Fc片段从重链的Cys226或从Pro230延伸至羧基端。在另一实施方案中，Fc-片段的C-端赖氨酸(Lys447)可以存在或可以不存在。在另一些实施方案中，Fc片段是包含突变的变体Fc片段，例如包含L234A-L235A突变。除非本文中另外指出，Fc片段中的氨基酸残基的编号根据EU编号系统，也称为EU索引，如Kabat，E.A.等，Sequences of Proteins of Immunological Interest，第5版，Public Health Service,National Institutes of Health，Bethesda，MD(1991)，NIH Publication 91-3242中所述。在一些实施方案中，抗体Fc片段可以在N端带有IgG1铰链序列或部分IgG1铰链序列，例如根据EU编号，E216到T225的序列或D221到T225的序列。在所述铰链序列中可以含有突变，例如C220S。
如本文所用的术语“接头”是指使得能够直接连接融合蛋白的不同部分的任何分子。在融合蛋白不同部分之间建立共价连接的接头的实例包括肽接头和非蛋白质聚合物，包括但不限于聚乙二醇(PEG)、聚丙二醇、聚氧化烯或聚乙二醇、聚丙二醇的共聚物。根据本发明的术语“肽接头”是指氨基酸的序列，其中所述序列将融合蛋白的第一部分的氨基酸序列连接至融合蛋白的第二部分。例如，肽接头可以将融合蛋白的TACI/BCMA部分与Fc结构域或其片段连接。例如，肽接头也可以将抗体与TACI/BCMA连接，诸如将抗体重链的C末端与TACI/BCMA连接。优选地，所述肽接头具有这样的长度，其足以连接两个实体，其方式使得它们维持它们相对于彼此的构象，使得不妨碍期望的活性。肽接头可以主要包括或可以不主要包括以下氨基酸残基：Gly、Ser、Ala或Thr。有用的接头包括甘氨酸-丝氨酸聚合物，包括例如(GSGGGGS)n(SEQ ID NO:89)、(GS)n(SEQ ID NO:90)、(GSGGS)n(SEQ ID NO:91)、(GGGGS)n(SEQ ID NO:92)或(GGGS)n(SEQ ID NO:93)，其中n是至少1(且例如2、3、4、5、6、7、8、9、10)的整数。有用的接头还包括甘氨酸-丙氨酸聚合物、丙氨酸-丝氨酸聚合物和其他柔性接头。在一些实施方案中，本发明的接头是(GSGGGGS)n(SEQ ID NO:89)。优选地，本发明的接头是SEQ ID NO:39。
本文使用的术语“融合物”是指通过连接两个或多个最初分开的蛋白/基因/化合物形成的融合物。如果构成融合物的实体是蛋白质，则被称为融合蛋白。融合蛋白涵盖在本申请的融合物的范围内。例如，TACI与Fc二聚体连接可以构成TACI-Fc融合蛋白。构成融合物的两个实体分子之间的连接可以通过或不通过接头实现。
如本文中使用的,术语“第一”和“第二”就Fc结构域(Fc区)或单体等而言为了在有超过一个每类模块时便于区分而使用。除非明确如此陈述,这些术语的使用不意图赋予融合蛋白的特定次序或取向。
本文所述的术语“治疗剂”涵盖在预防或治疗免疫疾病或炎症，例如免疫疾病或炎症中有效的任何物质，包括但不限于细胞因子、激素、细胞毒性剂或抑制剂(例如影响T细胞和/或B细胞增殖的细胞抑制剂)、抗体或小分子药物或免疫调节剂(例如免疫抑制剂)。
术语“有效量”指本发明的抗体或片段或组合物或组合的这样的量或剂量，其以单一或多次剂量施用患者后，在需要治疗或预防的患者中产生预期效果。“有效量”可以涵盖“治疗有效量”或“预防有效量”。
“治疗有效量”指以需要的剂量并持续需要的时间段，有效实现所需治疗结果的量。治疗有效量也是这样的一个量，其中抗体或抗体片段或组合物或组合的任何有毒或有害作用不及治疗有益作用。相对于未治疗的对象，“治疗有效量”优选地抑制可度量参数至少约40％、甚至更优选地至少约50％、55％、60％、65％、70％、75％、80％、85％、90％甚至100％。“预防有效量”指以需要的剂量并持续需要的时间段，有效实现所需预防结果的量。通常，由于预防性剂量在对象中在疾病较早阶段之前或在疾病较早阶段使用，故预防有效量将小于治疗有效量。
术语“宿主细胞”、“宿主细胞系”和“宿主细胞培养物”可交换地使用且是指其中引入外源核酸的细胞，包括这种细胞的后代。宿主细胞包括“转化体”和“转化的细胞”，其包括初级转化的细胞和来源于其的后代，而不考虑传代的数目。后代在核酸内容上可能与亲本细胞不完全相同，而是可以包含突变。本文中包括在最初转化的细胞中筛选或选择的具有相同功能或生物学活性的突变体后代。
本文所使用的术语“标记”是指被直接或间接缀合或融合至试剂(诸如多核苷酸探针或抗体)并且促进其所缀合或融合的试剂的检测的化合物或组合物。标记本身可以是可检测的(例如，放射性同位素标记或荧光标记)或在酶促标记的情况下可以催化可检测的底物化合物或组合物的化学改变。术语旨在涵盖通过将可检测物质偶联(即，物理连接)至探针或抗体来直接标记探针或抗体以及通过与直接标记的另一种试剂反应来间接标记探针或抗体。
术语“生物半衰期”是指物质(如免疫调节蛋白)丧失其药理学或生理学活性或浓度的一半所花费的时间长度。生物半衰期可能受以下影响：物质的消除、排泄、降解(例如，酶促降解/消化)，或者在身体某些器官或组织中的吸收和浓缩。在一些实施方案中，生物半衰期可以通过确定物质的血浆浓度达到其稳态水平的一半所花费的时间(“血浆半衰期”)来评估。
“个体”或“受试者”包括哺乳动物。哺乳动物包括但不限于，家养动物(例如，牛，羊，猫，狗和马)，灵长类动物(例如，人和非人灵长类动物如猴)，兔，以及啮齿类动物(例如，小鼠和大鼠)。在一些实施方案中，个体或受试者是人。
术语“免疫系统疾病或病症”是指与免疫系统异常有关的任何疾病或病症，包括但不限于如系统红斑性狼疮、类风湿性关节炎等自身免疫性疾病或炎性疾病。
术语“药用辅料”指与活性物质一起施用的稀释剂、佐剂(例如弗氏佐剂(完全和不完全的))、赋形剂、载体或稳定剂等。
术语“药物组合物”指这样的组合物，其以允许包含在其中的活性成分的生物学活性有效的形式存在，并且不包含对施用所述组合物的受试者具有不可接受的毒性的另外的成分。
术语“药物组合”或“组合产品”是指非固定组合或固定组合，包括但不限于药盒、药物组合物。术语“非固定组合”意指活性成分(例如，(i)本发明的嵌合体或融合蛋白、以及(ii)其他治疗剂)以分开的实体被同时、无特定时间限制或以相同或不同的时间间隔、依次地施用于患者，其中这类施用在患者体内提供预防或治疗有效水平的两种或更多种活性剂。术语“固定组合”意指两种或更多种活性剂以单个实体的形式被同时施用于患者。优选对两种或更多种活性剂的剂量和/或时间间隔进行选择，从而使各部分的联合使用能够在治疗疾病或病症时产生大于单独使用任何一种成分所能达到的效果。各成分可以各自呈单独的制剂形式，其制剂形式可以相同也可以不同。
术语“组合疗法”是指施用两种或更多种治疗剂或治疗方式(例如放射疗法或手术)以治疗本文所述疾病。这种施用包括以基本上同时的方式共同施用这些治疗剂，例如以具有固定比例的活性成分的单一胶囊。或者，这种施用包括对于各个活性成分在多种或在分开的容器(例如片剂、胶囊、粉末和液体)中的共同施用。粉末和/或液体可以在施用前重构或稀释至所需剂量。此外，这种施用还包括以大致相同的时间或在不同的时间以顺序的方式使用每种类型的治疗剂。在任一情况下，治疗方案将提供药物组合在治疗本文所述的病症或病状中的有益作用。
用于本文时，“治疗”指减缓、中断、阻滞、缓解、停止、降低、或逆转已存在的症状、病症、病况或疾病的进展或严重性。
用于本文时，“预防”包括对疾病或病症或特定疾病或病症的症状的发生或发展的抑制。在一些实施方式中，具有癌症家族病史的受试者是预防性方案的候选。通常，在癌症的背景中，术语“预防”是指在癌症的病征或症状发生前，特别是在具有癌症风险的受试者中发生前的药物施用。
术语“载体”当在本文中使用时是指能够增殖与其相连的另一个核酸的核酸分子。该术语包括作为自我复制核酸结构的载体以及结合到已经引入其的宿主细胞的基因组中的载体。一些载体能够指导与其可操作连接的核酸的表达。这样的载体在本文中被称为“表达载体”。
“受试者/患者/个体样品”指从患者或受试者得到的细胞或流体的集合。组织或细胞样品的来源可以是实体组织，像来自新鲜的、冷冻的和/或保存的器官或组织样品或活检样品或穿刺样品；血液或任何血液组分；体液，诸如脑脊液、羊膜液(羊水)、腹膜液(腹水)、或间隙液；来自受试者的妊娠或发育任何时间的细胞。组织样品可能包含在自然界中天然不与组织混杂的化合物，诸如防腐剂、抗凝剂、缓冲剂、固定剂、营养物、抗生素、等等。
II.TACI/BCMA嵌合体以及其融合蛋白
在一些实施方案中，本发明提供了一种TACI突变蛋白，其在蛋白表面的电荷更加均一，具有更好的稳定性，且具有较好的成药性。
在一些实施方案中，本发明的TACI突变蛋白经由突变后，减少了表面电荷基团和疏水基团的面积，进而降低了TACI的聚集风险。
在一些实施方案中，本发明的TACI突变蛋白是TACI突变的胞外结构域或其功能性片段，其中的部分区段被BCMA的相应区段替换。因此，包含这种突变的TACI突变蛋白还在本发明中被称为TACI/BCMA嵌合体。
在一些实施方案中，本发明的TACI/BCMA嵌合体包含TACI的胞外结构域或其功能性片段，其中所述胞外结构域或其功能性片段的N末端氨基酸和/或C末端氨基酸被BCMA的胞外结构域的N末端氨基酸和/或C末端氨基酸替换。
在一些实施方案中，本发明的TACI/BCMA嵌合体包含TACI突变的胞外结构域或其功能性片段，其中所述胞外结构域或其功能性片段的N末端氨基酸被BCMA的胞外结构域的N末端氨基酸替换。
在一些实施方案中，本发明的TACI/BCMA嵌合体包含TACI突变的胞外结构域或其功能性片段，其中所述胞外结构域或其功能性片段的N末端氨基酸和C末端氨基酸分别被BCMA的胞外结构域的N末端氨基酸和C末端氨基酸替换。
在一些实施方案中，本发明的TACI/BCMA嵌合体还包含对成药性风险位点的替换。在一些实施方案中，所述成药性风险位点选自TACI的第69、72、73、74、77、78、85、102和/或103位。在一些实施方案中，所述成药性风险位点的氨基酸被突变为A或D,例如D。在一些实施方案中，对成药性风险位点的替换为Y102D。
在一些实施方案中，本发明还提供包含本发明TACI/BCMA嵌合体的融合蛋白。在一个优选的实施方案中，本发明TACI/BCMA嵌合体与可以赋予改善的药代动力学性质的另一多肽融合，所述另一多肽例如白蛋白，优选抗体Fc片段。
在一些实施方案中，本发明还提供了由本发明的两条融合蛋白链二聚化形成的融合蛋白二聚体。
以下就本发明TACI/BCMA嵌合体或其融合蛋白的组件进行详述描述。本领域技术人员可以理解，除非上下文有明确相反指示，这些组件的任何技术特征的任何组合均在本发明考虑范畴之中。并且，本领域技术人员可以理解，除非上下文有明确相反指示，本发明的TACI/BCMA嵌合体或其融合蛋白可以包含任何这样的组合特征。
II-1TACI胞外结构域或其功能性片段
TACI是肿瘤坏死因子受体家族成员，其特征为具有含有富半胱氨酸假重复序列结构域(CRD)的细胞外结构域(ECD)。TACI是膜结合受体，其具有含有两个富半胱氨酸假重复序列(CRD1和CRD2)的细胞外结构域、跨膜结构域和与CAML(钙调节物和亲环蛋白配体)相互作用的胞质结构域，CAML是位于细胞内囊泡处的整合膜蛋白，其在Jurkat细胞中过表达时是NF-AT激活的共诱导物。TACI与B细胞和T细胞的子集相关。TACI受体结合肿瘤坏死因子(TNF)配体家族的两个成员。一种配体命名为BAFF(TNF家族的B细胞激活因子)。另一种配体已经被命名为APRIL。两种配体还被B细胞成熟受体(BCMA)结合。TACI受体与其配体BAFF或APRIL的结合刺激B细胞应答，包括非T细胞依赖性B细胞抗体应答、同种型转换和B细胞稳态。BAFF或APRIL的结合刺激B细胞应答，包括非T细胞依赖性B细胞抗体应答、同种型转换和B细胞稳态。
在一些实施方案中，TACI是人TACI。在一些实施方案中，人TACI包含SEQ ID NO:1所示的氨基酸序列，或包含与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％的同一性的氨基酸序列，或由所述氨基酸序列组成。在一些实施方案中，所述人TACI全长序列包含SEQ ID NO:1所示的氨基酸序列或由其组成。在一些实施方案中，所述的人TACI蛋白质是III型膜蛋白并且缺少信号肽；在真核细胞中表达后，N末端甲硫氨酸被去除。在一些实施方案中，成熟TACI蛋白不含如SEQ ID NO:1中所示的N末端甲硫氨酸。
野生型人TACI的结构和表征可以参见Uitprot o14836(https://www.uniprot.org/uniprotkb/O14836/entry)。人TACI通常包含3个结构域，即胞外结构域，对应于1-165位氨基酸；跨膜区，对应于166-186位氨基酸；以及胞质结构域，对应于第187-293位氨基酸。
TACI的细胞外结构域(例如SEQ ID NO:1的氨基酸残基1-165；SEQ ID NO:29中所示的ECD)含有两个富半胱氨酸结构域(CRD)，它们各自展现与BAFF和APRIL结合的亲和力。第一富半胱氨酸结构域(CRD1)对应于或含有SEQ ID NO:1中所示的序列的氨基酸残基34-66。第二富半胱氨酸结构域(CRD2)对应于或含有SEQ ID NO:1中所示的序列的氨基酸71-104。TACI还在细胞外结构域含有在第二半胱氨酸重复序列后的约60个氨基酸的茎区，对应于或含有SEQ ID NO:1中所示的序列的氨基酸残基105-165。
在一些实施方案中，TACI的胞外结构域包含SEQ ID NO:1的第1-165位氨基酸序列，或包含与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％的同一性的氨基酸序列，或由所述氨基酸序列组成。在一些实施方案中，TACI的胞外结构域包含SEQ ID NO:29所示的氨基酸序列，或包含与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％的同一性的氨基酸序列，或由所述氨基酸序列组成。在一些实施方案中，CRD2包含SEQ ID NO:58所示的氨基酸序列，或包含与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％的同一性的氨基酸序列，或由所述氨基酸序列组成。
在一些实施方案中，TACI ECD的功能性片段包含与APRIL、BAFF或APRIL/BAFF异三聚体结合的氨基酸序列。
在一些实施方案中，TACI ECD的功能性片段缺失ECD的部分N末端残基，所述残基对应于SEQ ID NO:1中所示的ECD序列N末端的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或67个连续氨基酸缺失。
在一些实施方案中，TACI ECD的功能性片段缺失ECD的部分茎区，例如缺失C末端的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或1个连续氨基酸。
在一些实施方案中，TACI ECD的功能性片段包含CRD2，或仅包含完整CRD2。在一些实施方案中，TACI ECD的功能性片段由CRD2组成。
在一些实施方案中，TACI ECD的功能性片段包含CRD2和部分茎区。在一些实施方案中，TACI ECD的功能性片段还包含CRD1和CRD2之间的序列，例如对应于SEQ ID NO:1的第68-70位之间的任意氨基酸序列。在一些实施方案中，TACI ECD的功能性片段不包含CRD1或其任意片段。
在一些实施方案中，TACI ECD的功能性片段含有部分茎区，例如包含TACI对应于SEQ ID NO:1所示的氨基酸序列的如下片段：氨基酸残基105、氨基酸残基105至106、氨基酸残基105至107、氨基酸残基105至108、氨基酸残基105至109、氨基酸残基105至110、氨基酸残基105至111、氨基酸残基105至112、氨基酸残基105至113、氨基酸残基105至114、氨基酸残基105至115、氨基酸残基105至116、氨基酸残基105至117、氨基酸残基105至118、氨基酸残基105至119、氨基酸残基105至120、氨基酸残基105至121、氨基酸残基105至122、氨基酸残基105至123、氨基酸残基105至124、氨基酸残基105至125、氨基酸残基105至126、氨基酸残基105至127、氨基酸残基105至128、氨基酸残基105至129、氨基酸残基105至130、氨基酸残基105至131、氨基酸残基105至132、氨基酸残基105至133、氨基酸残基105至134、氨基酸残基105至135、氨基酸残基105至136、氨基酸残基105至137、氨基酸残基105至138、氨基酸残基105至139、氨基酸残基105至140、氨基酸残基105至141、氨基酸残基105至142、氨基酸残基105至143、氨基酸残基105至144、氨基酸残基105至145、氨基酸残基105至146、氨基酸残基105至147、氨基酸残基105至148、氨基酸残基105至149、氨基酸残基105至150、氨基酸残基105至151、氨基酸残基105至152、氨基酸残基105至153以及氨基酸残基105至154。
在一些实施方案中，TACI ECD的功能性片段包含TACI对应于SEQ ID NO:1所示的氨基酸序列的如下片段：氨基酸残基67至118、氨基酸残基67至117、氨基酸残基67至116、氨基酸残基67至115、氨基酸残基67至114、氨基酸残基67至113、氨基酸残基67至112、氨基酸残基67至111、氨基酸残基67至110、氨基酸残基67至109、氨基酸残基67至108、氨基酸残基67至107、氨基酸残基67至106、氨基酸残基67至105或者氨基酸残基67至104；氨基酸残基68至118、氨基酸残基68至117、氨基酸残基68至116、氨基酸残基68至115、氨基酸残基68至114、氨基酸残基68至113、氨基酸残基68至112、氨基酸残基68至111、氨基酸残基68至110、氨基酸残基68至109、氨基酸残基68至108、氨基酸残基68至107、氨基酸残基68至106、氨基酸残基68至105或者氨基酸残基68至104；氨基酸残基69至118、氨基酸残基69至117、氨基酸残基69至116、氨基酸残基69至115、氨基酸残基69至114、氨基酸残基69至113、氨基酸残基69至112、氨基酸残基69至111、氨基酸残基69至110、氨基酸残基69至109、氨基酸残基69至108、氨基酸残基69至107、氨基酸残基69至106、氨基酸残基69至105或者氨基酸残基69至104；氨基酸残基70至118、氨基酸残基70至117、氨基酸残基70至116、氨基酸残基70至115、氨基酸残基70至114、氨基酸残基70至113、氨基酸残基70至112、氨基酸残基70至111、氨基酸残基70至110、氨基酸残基70至109、氨基酸残基70至108、氨基酸残基70至107、氨基酸残基70至106、氨基酸残基70至105或者氨基酸残基70至104；氨基酸残基71至118、氨基酸残基71至117、氨基酸残基71至116、氨基酸残基71至115、氨基酸残基71至114、氨基酸残基71至113、氨基酸残基71至112、氨基酸残基71至111、氨基酸残基71至110、氨基酸残基71至109、氨基酸残基71至108、氨基酸残基71至107、氨基酸残基71至106、氨基酸残基71至105或者氨基酸残基71至104。在一些实施方案中，TACI ECD的功能性片段包含ECD第13-118位氨基酸，或包含68-110位氨基酸，或由所述连续氨基酸序列组成。
在一些实施方案中，TACI ECD的功能性片段包含SEQ ID NO:30、31或58所示的氨基酸序列，或包含与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％的同一性的氨基酸序列，或由所述氨基酸序列组成。
在一些实施方案中，TACI ECD的功能性片段由SEQ ID NO:31所示的氨基酸序列组成。
在本发明的一些实施方案中TACI ECD或其功能性片段还涵盖具有突变的ECD变体或其功能性片段。在一些实施方案中，TACI ECD的功能性片段包含K77E、F78Y和/或Y102D。
在一些实施方案中，TACI ECD的功能性片段包含Y102D。在一些实施方案中，TACI ECD的功能性片段包含SEQ ID NO:33所示的氨基酸序列，或包含与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％的同一性的氨基酸序列，或由所述氨基酸序列组成。在一些实施方案中，TACI ECD的功能性片段包含与SEQ ID NO:33所示的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％的同一性且具有Y102D的氨基酸序列。在一些实施方案中，TACI ECD的功能性片段由SEQ ID NO:33所示的氨基酸序列组成。
在一些实施方案中，TACI ECD的功能性片段包含K77E、F78Y和Y102D。在一些实施方案中，TACI ECD的功能性片段包含SEQ ID NO:32所示的氨基酸序列，或包含与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％的同一性的氨基酸序列，或由所述氨基酸序列组成。在一些实施方案中，TACI ECD的功能性片段包含与SEQ ID NO:32所示的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％的同一性且具有K77E、F78Y和Y102D的氨基酸序列。在一些实施方案中，TACI ECD的功能性片段由SEQ ID NO:32所示的氨基酸序列组成。
II-2TACI/BCMA嵌合体
本发明发现，TACI同家族蛋白BCMA的蛋白质结构(6-42，PDB ID:1XU2)与TACI结构同源性很高，与配体的结合位点也基本一致，同时两者的N端与C端部分未参与配体的结合(图1)，更为重要的是Discovery studio软件的分析结果显示BCMA表面电荷分布均衡，聚集风险明显低于TACI。
因此本发明设计了一系列BCMA与TACI胞外结构域的嵌合体分子，在保留TACI与配体结合位点的基础上，将TACI的N末端氨基酸或C末端氨基酸替换为BCMA的相应序列，或者同时将TACI的N末端氨基酸与C末端氨基酸都替换为BCMA的相应序列，以及任选地对部分成药性风险位点进行了氨基酸替换，从而减少TACI蛋白表面电荷基团和疏水基团的面积，降低TACI的聚集风险。
BCMA是肿瘤坏死因子受体家族成员，其特征为具有含有富半胱氨酸假重复序列结构域(CRD)的细胞外结构域(ECD)。BCMA是一种膜结合受体，其具有含有单一CRD的细胞外结构域、跨膜结构域以及含有用于与TRAF信号传导分子结合的TRAF结合位点的胞质结构域。BCMA与同源配体APRIL和BAFF结合，但是与BAFF结合的亲和力较弱。据报道，BCMA与BAFF的结合比BAFF与其他同源受体BAFF-R和TACI之间的结合弱二至三个数量级。
在一些实施方案中，BCMA是人BCMA。在一些实施方案中，人BCMA包含SEQ ID NO:2所示的氨基酸序列，或包含与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％的同一性的氨基酸序列，或由所述氨基酸序列组成。在一些实施方案中，所述人BCMA全长序列包含SEQ ID NO:2所示的氨基酸序列。在一些实施方案中，所述BCMA蛋白质是II型膜蛋白并且缺少信号肽；在真核细胞中表达后，N末端甲硫氨酸被去除。在一些实施方案中，成熟BCMA蛋白不含如SEQ ID NO:2中所示的N末端甲硫氨酸。
野生型人BCMA的结构和表征可以参见Uitprot Q02223(https://www.uniprot.org/uniprotkb/Q02223/entry)。人BCMA通常包含3个结构域，即胞外结构域，对应于1-54位氨基酸；跨膜区，对应于55-77位氨基酸；以及胞质结构域，对应于第78-184位氨基酸。BCMA的细胞外结构域含有一个富半胱氨酸结构域(CRD)，其展现对于与APRIL结合以及以较低程度与BAFF结合的亲和力。在一些实施方案中，所述CRD含有SEQ ID NO:2中所示序列的氨基酸残基7-41。
在一些实施方案中，BCMA的胞外结构域包含SEQ ID NO:2的第1-54位氨基酸序列，或包含与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％的同一性的氨基酸序列，或由所述氨基酸序列组成。在一些实施方案中，BCMA的胞外结构域包含SEQ ID NO:57所示的氨基酸序列，或包含与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％的同一性的氨基酸序列，或由所述氨基酸序列组成。
在一些实施方案中，TACI ECD或其功能性片段(例如只含CRD2的ECD，例如TACI(68-110))的N末端和/或C末端被替换为BCMA的相应序列。
在一些实施方案中，TACI ECD或其功能性片段的N末端是指TACI对应于SEQ ID NO:1的Y79位氨基酸之前的N末端氨基酸。
在一些实施方案中，TACI ECD或其功能性片段的C末端是指TACI对应于SEQ ID NO:1的第99位氨基酸、第100位氨基酸、第101位氨基酸、第102位氨基酸、第103位氨基酸、第104位氨基酸、第105位氨基酸、第106位氨基酸、第107位氨基酸、第108位氨基酸、第109位氨基酸或第110位氨基酸之后的C末端氨基酸。
在一些实施方案中，TACI ECD或其功能性片段的C末端是指TACI对应于SEQ ID NO:1的第99位氨基酸之后的C末端氨基酸。在一些实施方案中，TACI ECD或其功能性片段的C末端是指对应于SEQ ID NO:1的第105位氨基酸之后的C末端氨基酸。
在一些实施方案中，可以用于替换TACI ECD或其功能性片段的N末端的BCMA的相应N末端氨基酸序列选自BCMA对应于SEQ ID NO:2的第1-13位、第2-13位、第3-13位、第4-13位、第5-13位、第6-13位或第7-13位的氨基酸序列。
在一些实施方案中，用于替换的BCMA的相应N末端氨基酸序列包含SEQ ID NO:40-46中所示的任一项的氨基酸序列，或由所述的氨基酸序列组成。
在一些实施方案中，可以用于替换TACI ECD或其功能性片段的C末端的BCMA的相应C末端氨基酸序列选自BCMA对应于SEQ ID NO:2的第37-47位、第37-46位、第37-45位、第37-44位的氨基酸序列，或对应于SEQ ID NO:2的第43-44位或43-46位的氨基酸。
在一些实施方案中，用于替换的BCMA的相应的N末端氨基酸或C末端氨基酸序列可以包含用于改善结合亲和力，增加稳定性和/或改善成药性的突变，例如取代。在一些实施方案中，用于替换的BCMA的C末端氨基酸序列包含在第39位和/或第42位的突变。
在一些实施方案中，用于替换的BCMA的C末端氨基酸序列包含N42A。在一些实施方案中，用于替换的BCMA的C末端氨基酸序列包含N42Q。在一些实施方案中，用于替换的BCMA的C末端氨基酸序列包含R39D。在一些实施方案中，用于替换的BCMA的C末端氨基酸序列包含N42A-R39D。
在一些实施方案中，用于替换的BCMA的相应C末端氨基酸序列包含SEQ ID NO:47-55中任一项中所示的氨基酸序列，或由所述的氨基酸序列组成。
在一些实施方案中，用于替换的BCMA的N末端或C末端氨基酸序列的氨基酸个数与被替换的TACI的ECD或其功能性片段的N末端或C末端氨基酸序列的氨基酸个数相等，或差距在1-3个氨基酸之内。
因此，在一些实施方案中，本发明的TACI/BCMA嵌合体包含如下结构：
BCMAN末端氨基酸-TACI部分，其中TACI部分为缺失了N末端的ECD或其功能性片段，或者
BCMAN末端氨基酸-TACI部分-BCMAC末端氨基酸，其中TACI部分为缺失了N末端和C末端的ECD或其功能性片段。
因此，在本文中，“BCMAN末端氨基酸-TACI部分”是指TACI的N末端氨基酸替换为BCMA的N末端氨基酸后获得的多肽。“BCMAN末端氨基酸-TACI部分-BCMAC末端氨基酸”是指TACI的N末端氨基酸替换为BCMA的N末端氨基酸且TACI的C末端氨基酸替换为BCMA的C末端氨基酸后获得的多肽。
在一些实施方案中，适用于本发明的TACI/BCMA嵌合体的TACI部分可以包含突变，例如取代。
在一些实施方案中，适用于本发明的TACI/BCMA嵌合体的TACI部分还包含在成药性风险位点，例如具有聚集风险的氨基酸位点上的突变，例如替换TACI部分的一个或几个成药性风险位点的氨基酸。在一些实施方案中，如果TACI部分包含成药性风险位点，则将其替换为降低该风险的氨基酸。在一些实施方案中，如果TACI部分包含具有聚集风险的氨基酸，则将其替换为降低该聚集风险的氨基酸。在一些实施方案中，成药性风险位点选自对应于SEQ ID NO:1的第69、72、73、74、77、78、85、102或103位氨基酸。例如，所述成药性风险位点的氨基酸被突变为A或D。
在一些实施方案中，TACI部分包含Y102位突变，例如取代，例如Y102D。
在一些实施方案中，TACI部分包含SEQ ID NO:31-38中任一项所示的氨基酸序列，或包含与其具有至少90％、91％、92％、93％、94％、95％、96％、97％或98％的同一性的氨基酸序列，或包含与其具有1、2、3、4或5个氨基酸改变(例如取代、缺失或添加，例如保守性取代；或例如在成药性风险位点的取代)的氨基酸序列，或由所述氨基酸序列组成。
在一些实施方案中，TACI部分由SEQ ID NO:31-38中任一项所示的氨基酸序列组成。
在一些实施方案中，适用于本发明的TACI/BCMA嵌合体的BCMA部分(例如在BCMAN末端或BCMAC末端，优选BCMAC末端)还包含用于改善结合亲和力，增加稳定性增加和/或改善成药性的突变，例如取代，例如在成药性风险位点如导致N糖基化位点上的突变，例如替换BCMA部分的一个或几个成药性风险位点的氨基酸。在一些实施方案中，如果BCMA部分包含成药性风险位点，则将其替换为降低该风险的氨基酸。在一些实施方案中，如果BCMA部分包含具有糖基化(例如N糖基化)的氨基酸，则将其替换为其他氨基酸。在一些实施方案中，成药性风险位点为对应于SEQ ID NO:2的第39位或第42位氨基酸。例如，所述成药性风险位点的氨基酸被突变为D、A或Q。
在一些实施方案中，BCMAN末端包含SEQ ID NO:40-46中任一项所示的氨基酸序列，或包含与其具有至少80％或90％的同一性的氨基酸序列，或包含与其具有1、2、或3个氨基酸改变(例如取代、缺失或添加，例如保守性取代或例如在成药性风险位点的取代)的氨基酸序列，或由所述氨基酸序列组成。
在一些实施方案中，BCMAN末端由SEQ ID NO:40-46中任一项所示的氨基酸序列组成。
在一些实施方案中，BCMAC末端包含R39位的突变，例如取代，例如R39D。在一些实施方案中，BCMAC末端包含N42位的突变，例如取代，例如N42A或N42Q。在一些实施方案中，BCMAC末端包含R39位的突变和N42位的突变，例如R39D-N42A。
在一些实施方案中，BCMAC末端包含SEQ ID NO:47-55中任一项所示的氨基酸序列，或包含与其具有至少80％或90％的同一性的氨基酸序列，或包含与其具有1或2个氨基酸改变(例如取代、缺失或添加，例如保守性取代或例如在成药性风险位点的取代)的氨基酸序列，或由所述氨基酸序列组成。
在一些实施方案中，BCMAC末端由SEQ ID NO:47-55中任一项所示的氨基酸序列组成。
本发明的TACI/BCMA嵌合体包含SEQ ID NO:59-80中任一项所示的氨基酸序列，或包含与其具有至少90％、91％、92％、93％、94％、95％、96％、97％或98％的同一性的氨基酸序列，或由所述氨基酸序列组成。
II-3.TACI/BCMA嵌合体融合蛋白
在一个方面，本发明还提供包含本发明TACI/BCMA嵌合体的融合蛋白。在一个优选的实施方案中，本发明TACI/BCMA嵌合体与可以赋予改善的药代动力学性质的另一多肽融合，例如白蛋白，更优选抗体Fc片段。在一些实施方案中，通过FcRn介导的体内循环回收,能延长TACI/BCMA嵌合体-Fc融合蛋白的半衰期。
在一个实施方案中，本发明提供TACI/BCMA嵌合体融合蛋白，其包含与Fc区融合的本发明TACI/BCMA嵌合体，优选地，所述Fc区融合在本发明TACI/BCMA嵌合体的C末端，例如Fc区的N末端与嵌合体的C末端融合。
在一些实施方案中，Fc区为人的IgG Fc，例如，人IgG1 Fc,人IgG2 Fc,人IgG3 Fc或人IgG4 Fc。在一些实施方案中，所述Fc区在其N末端包含完整铰链区，即其对应于人IgG1重链恒定区的E216位起始到C末端。在一些实施方案中，所述Fc区在其N末端铰链区对应于IgG1的220位的半胱氨酸被突变为丝氨酸，即(包含220S突变)。
所述Fc区还可以缺乏部分铰链区，例如对应于人IgG1重链恒定区的从D221位起始到C末端。
在一个实施方案中，Fc区包含SEQ ID NO:81或82所示的氨基酸序列或与其具有至少90％同一性，例如95％、96％、97％、98％、99％或更高的同一性的氨基酸序列或由其组成。
在一个实施方案中，适用于本发明的融合蛋白的Fc区在C末端缺失赖氨酸K(K447del)。
在一个实施方案中，Fc区包含SEQ ID NO:83或84所示的氨基酸序列或与其具有至少90％同一性，例如95％、96％、97％、98％、99％或更高的同一性的氨基酸序列(任选地还缺失C末端赖氨酸)或由其组成。
还可以将本发明的结合分子例如抗体中的Fc区进行突变以获得所需的特性。本领域已知对Fc区的突变。
在一个实施方案中，在Fc区的效应子功能(例如Fc区的补体激活功能)的特性上修饰Fc区。在一个实施方案中，所述效应子功能相对于野生同种型Fc区已经被降低或消除。在一个实施方案中，通过选自以下的方法来降低或消除效应子功能：使用天然具有降低或消除的效应子功能的Fc同种型、和Fc区修饰。在一个优选的实施方案中，Fc区具有减少的由Fc区介导的效应子功能，例如减少的或消除的ADCC或ADCP或CDC效应子功能，例如包含实现上述功能的突变。
在一个实施方案中，也可以在Fc区中包含改变对一种或多种Fc受体的结合亲和力的修饰。在一个实施方案中，所述Fc受体是Fcγ受体，特别地是人Fcγ受体。在一些实施方案中，所述Fc区包含降低与Fcγ受体结合的突变。在再一优选的实施方案中，Fc区可以具有导致增加的血清半衰期的突变，例如改善Fc片段与FcRn结合的突变。
例如，在一些实施方案中，用于本发明的Fc区具有降低与Fcγ受体结合的用于本发明的Fc片段具有降低与Fcγ受体结合的L234A/L235A突变，L234A/L235E突变、G237A突变或L234A/L235E/G237A。例如，在一些实施方案中，用于本发明的Fc区具有改善Fc片段与FcRn结合的突变，例如增强Fc片段与FcRn结合的YTE突变(M252Y/S254T/T256E)或者LS突变(M428L/N434S)。示例性的突变参见例如WO2002060919A2，其在本文全文引入作为参考。
在一些实施方案中，所述Fc区同时包含所述具有降低与Fcγ受体结合的突变和所述改善Fc片段与FcRn结合的突变。
在一些实施方案中，所述Fc区包含L234A/L235E/G237A和M252Y/S254T/T256E，以及任选地C末端缺失赖氨酸。
在一些实施方案中，所述Fc区包含L234A/L235E/G237A和M428L/N434S，以及任选地C末端缺失赖氨酸。
在一个实施方案中，Fc区包含SEQ ID NO:85-88和100-103中任一项所示的氨基酸序列或与其具有至少90％同一性，例如95％，96％，97％，98％,99％或更高的同一性的氨基酸序列或由其组成。
在一个实施方案中，Fc区包含与SEQ ID NO:85或87所示的氨基酸序列所示的氨基酸序列具有至少85％、90％、95％、96％、97％、98％或99％的同一性且包含突变L234A/L235E/G237A的氨基酸序列。
在一个实施方案中，Fc区包含与SEQ ID NO:86或88所示的氨基酸序列所示的氨基酸序列具有至少85％、90％、95％、96％、97％、98％、99％的同一性且包含突变L234A/L235E/G237A和C末端缺失赖氨酸的氨基酸序列。
在一个实施方案中，Fc区包含与SEQ ID NO:100或102所示的氨基酸序列所示的氨基酸序列具有至少85％、90％、95％、96％、97％、98％或99％的同一性且包含突变L234A/L235E/G237A和M252Y/S254T/T256E的氨基酸序列。
在一个实施方案中，Fc区包含与SEQ ID NO:101或103所示的氨基酸序列所示的氨基酸序列具有至少85％、90％、95％、96％、97％、98％、99％的同一性且包含突变L234A/L235E/G237A、M252Y/S254T/T256E和C末端缺失赖氨酸的氨基酸序列。
在一些实施方案中，TACI/BCMA嵌合体直接或通过接头与Fc融合，例如所述嵌合体的C末端直接或通过接头与Fc的N末端融合。如本领域技术人员明了的，适用于本发明融合蛋白和二聚体分子中连接TACI/BCMA和Fc区的接头可以是本领域已知的任何接头。在一些实施方案中，接头可以包含IgG1铰链，或可以包含选自以下的接头序列：(GSGGGGS)n(SEQ ID NO:94)、(GS)n(SEQ ID NO:95)、(GSGGS)n(SEQ ID NO:96)、(GGGGS)n(SEQ ID NO:97)或(GGGS)n(SEQ ID NO:98)，其中n是至少1的整数，例如1、2、3、4或5。
在一个实施方案中，接头是(GSGGGGS)n(SEQ ID NO:99)，其中n＝1-3。在一个实施方案中，接头包含或者是SEQ ID NO:39所示的氨基酸序列。
在再一方面，本发明也提供包含与Fc区融合的本发明TACI/BCMA嵌合体的二聚体分子。
在一些实施方案中，本发明提供TACI/BCMA嵌合体-Fc二聚体蛋白，其中第一单体和第二单体分别自N端到C端包含或由下述组成：i)TACI/BCMA嵌合体；ii)接头(任选地存在或不存在)；和iii)Fc区。在一些实施方案中，所述二聚体蛋白是同二聚体。在一些实施方案中，所述单体具有相同的氨基酸序列。在一些实施方案中本发明的TACI/BCMA嵌合体-Fc蛋白二聚体包含两条相同的单体。
在一些实施方案中，本发明的TACI/BCMA嵌合体-Fc融合蛋白或其单体
(i)包含与SEQ ID NO:5-28中任一项的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由所述氨基酸序列组成；或者
(ii)包含SEQ ID NO:5-28中任一项的氨基酸序列或由所述氨基酸序列组成；或者
(iii)包含与SEQ ID NO:5-28中任一项的氨基酸序列相比具有1个或多个(优选不超过10个，更优选不超过5、4、3、2、1个)的氨基酸改变。
在一些实施方案中，本发明的TACI/BCMA嵌合体-Fc蛋白或本发明的蛋白二聚体单体由SEQ ID NO:5-28中任一项的氨基酸序列组成。
III.多核苷酸、载体和宿主
本发明提供编码以上任何TACI/BCMA嵌合体或其融合蛋白或融合蛋白二聚体中的任一条链或任何单体或结构域的核酸。可以采用本领域熟知的方法，通过从头固相DNA合成或通过PCR诱变编码野生型TACI的现有序列，产生编码本发明突变蛋白的多核苷酸序列。此外，本发明的多核苷酸和核酸可以包含编码分泌信号肽的区段，并与编码本发明突变蛋白的区段可操作连接，从而可以指导本发明突变蛋白的分泌性表达。
本发明也提供包含本发明核酸的载体。在一个实施方案中，载体是表达载体，例如真核表达载体。载体包括但不限于病毒、质粒、粘粒、λ噬菌体或酵母人工染色体(YAC)。在优选的实施方案中，本发明的表达载体是pCDNA表达载体，例如pCDNA3.1表达载体。
本发明也提供包含所述核酸或所述载体的宿主细胞。适用于复制并支持TACI/BCMA嵌合体或其融合蛋白或融合蛋白二聚体表达的宿主细胞是本领域中公知的。
可以用特定的表达载体转染或转导这类细胞，并且可以生长大量的含载体细胞以用于接种大规模发酵罐，从而获得充足量的TACI/BCMA嵌合体或其融合蛋白或融合蛋白二聚体用于临床应用。
在一个实施方案中，宿主细胞是真核的。在另一个实施方案中，宿主细胞选自酵母细胞、哺乳动物细胞(例如CHO细胞或293细胞)。
可用的哺乳动物宿主细胞系的例子是由SV40转化的猴肾CV1系(COS-7)；人胚胎肾系(293或293T细胞或HEK293细胞)、幼仑鼠肾细胞(BHK)、小鼠塞托利(sertoli)细胞(TM4细胞)、猴肾细胞(CV1)、非洲绿猴肾细胞(VERO-76)、人宫颈癌细胞(HELA)、犬肾细胞(MDCK)，buffalo大鼠肝细胞(BRL3A)、人肺细胞(W138)、人肝细胞(HepG2)、小鼠乳房肿瘤细胞(MMT060562)、TRI细胞、MRC5细胞和FS4细胞。其它可用的哺乳动物宿主细胞系包括中国仓鼠卵巢(CHO)细胞，包括dhfr-CHO细胞；和骨髓瘤细胞系如YO、NS0、P3X63和Sp2/0。
在一个实施方案中，宿主细胞是真核生物细胞，优选为哺乳动物细胞如中国仓鼠卵巢(CHO)细胞、HEK293细胞、人胚胎肾(HEK)细胞或淋巴细胞(例如Y0、NS0、Sp20细胞)。
IV.制备方法
再一方面，本发明提供制备本发明TACI/BCMA嵌合体或其融合蛋白或融合蛋白二聚体的方法，其中所述方法包括，在适合TACI/BCMA嵌合体或其融合蛋白或融合蛋白二聚体表达的条件下，培养包含编码所述TACI/BCMA嵌合体或其融合蛋白或融合蛋白二聚体的核酸的宿主细胞，如上文所提供的，和任选地从所述宿主细胞(或宿主细胞培养基)回收所述TACI/BCMA嵌合体或其融合蛋白或融合蛋白二聚体。
在一个实施方案中，将包含编码TACI/BCMA嵌合体或其融合蛋白或融合蛋白二聚体的核酸的载体转入细胞中使其表达，随后收集细胞(或细胞培养上清)，提取所述TACI/BCMA嵌合体或其融合蛋白或融合蛋白二聚体，并进行纯化，以获得所述TACI/BCMA嵌合体或其融合蛋白或融合蛋白二聚体。
在一个具体的实施方案中，所述纯化方法是亲和纯化方法。在另一个具体的实施方案中，所述纯化方法是离子交换纯化。在一些实施方案中，使用凝胶过滤色谱柱过滤纯化。
V.本发明的TACI/BCMA的嵌合体或其融合蛋白或融合蛋白二聚体的性质
可以通过本领域中已知的多种测定法对本文中提供TACI/BCMA的嵌合体或其融合蛋白或融合蛋白二聚体进行鉴定，筛选，或表征其物理/化学特性和/或生物学活性。
本发明获得的TACI/BCMA的嵌合体的蛋白表面电荷更加均一，因此具有更好的稳定性，以及更好的成药性。
本发明获得的TACI/BCMA的嵌合体以及其融合蛋白，相比现有技术已知的TACI蛋白和其融合蛋白，具有更好的稳定性(例如热稳定性)和药物动力学数据，同时具有更长的半衰期。本发明获得的TACI/BCMA的嵌合体融合蛋白在加速条件下，相比现有技术已知的TACI融合蛋白或其变体，均呈现了更好的稳定性，其中部分分子的热稳定性明显优于ALPN303。
在一些实施方案中，本发明获得的TACI/BCMA的嵌合体融合蛋白具有选自以下的一种或多种性质：
a)在细胞中表达量高，且易于纯化；
b)特异性结合BAFF和APRIL，例如通过生物光干涉测量(ForteBio)测定法测定的，与BAFF的KD小于等于大约3nM、2nM、1nM、0.9nM、0.8nM、0.7nM、0.6nM、0.5nM或0.4nM，或者大于等于大约0.1nM或0.2nM，或在任意所述数值之间；且与APRIL的KD小于等于大约4nM、3nM、2nM、1nM、0.9nM、0.8nM、0.7nM、0.6nM、0.5nM或0.4nM，或者大于等于大约0.1nM或0.2nM，或在任意所述数值之间；
c)有效抑制BAFF与IM-9细胞的结合，例如在FACS测定时，IC50低于或等于大约5nM、4nM、3nM、2nM或1.5nM；有效抑制APRIL与IM-9细胞的结合，例如在FACS测定时，IC50低于或等于大约5nM、4nM、3nM、2nM 1.5nM、1nM、0.9nM、0.8nM、0.7nM、0.6nM、0.5nM、0.4nM、0.3nM或0.2nM；
d)有效抑制BAFF/APRIL诱导的(人)TACI的表面表达，例如通过TACI/NF-κB ReporterJurkat信号检测测定的IC50低于或等于大约16nM、15nM、14nM、13nM、12nM、11nM、10nM、9nM、8nM、7nM、6nM或5.5nM；
e)有效抑制BAFF/APRIL诱导的(人)BCMA的表面表达，例如通过BCMA/NF-κBReporter Jurkat信号检测测定的IC50低于或等于大约16nM、15nM、14nM、13nM、12nM、11nM、10nM、9nM、8nM、7nM、6Nm、5.5nM、5nM、4.5nM或4nM；
f)有效抑制BAFF/APRIL诱导的脾脏细胞增殖，例如IC50低于或等于大约4.5nM、4nM、3.5nM、3nM、2.5nM、2nM、1.5nM、1nM、0.9nM、0.8nM、0.7nM或0.6nM；
g)有效抑制BAFF/APRIL诱导的B细胞增殖；
h)具有更好的热稳定性；
i)具有更好的成药性；
j)具有更好的加速稳定性，例如优于已知分子RC-18和/或ALPN-303；
k)有效抑制BAFF诱导的脾脏细胞和/或脾脏中B细胞和/或不同B细胞亚群的增殖；
l)有效抑制脾脏细胞和/或脾脏中B细胞和/或不同B细胞亚群的增殖，例如在KLH免疫模型中，例如优于已知分子RC-18；
m)能有效降低BAFF诱导的血清中IgA、IgM和/或IgG水平；
n)更有效地抑制MZ细胞、GC B细胞、浆细胞的增殖，例如优于已知分子ALPN-303；
o)具有更好的药代动力学，例如优于已知分子RC-18和/或ALPN-303；
p)具有更长的半衰期(例如血浆半衰期)，例如半衰期(例如血浆半衰期)大于或等于大约45h，例如大于或等于大约46、47、48、49或50小时；和/或
q)具有更低的清除率，例如清除率低于大约1.8、1.7、1.6或1.5mL/kg/h。
VI.药物组合物和药物制剂
本发明还包括包含TACI/BCMA嵌合体或其融合蛋白或融合蛋白二聚体的组合物(包括药物组合物或药物制剂)或包含编码TACI/BCMA嵌合体或其融合蛋白或融合蛋白二聚体的多核苷酸的组合物。这些组合物还可以任选地包含合适的药用辅料，如本领域中已知的药用载体、药用赋形剂，包括缓冲剂。
可以通过常规的混合、溶解、乳化、包囊、包载或冻干过程来制备包含本发明的TACI/BCMA嵌合体或其融合蛋白或融合蛋白二聚体的药学组合物。可以以常规方式配制药学组合物,其使用一种或多种有助于将蛋白质加工成可药学使用的制剂的生理学可接受载体,稀释剂,赋形剂或辅助剂。合适的配制剂依赖于选择的施用路径。
可以将TACI/BCMA嵌合体或其融合蛋白或融合蛋白二聚体以游离酸或碱,中性或盐形式配制成组合物。药学可接受盐是基本保留游离酸或碱的生物学活性的盐。这些包括酸加成盐(acid addition salt),例如与蛋白质性组合物的游离氨基基团形成的那些,或与无机酸(如例如氢氯酸或磷酸)或与有机酸如乙酸,草酸,酒石酸或扁桃酸形成的。
VII.组合
在一方面，本发明还提供了药物组合或组合产品，其包含本发明的TACI/BCMA嵌合体或其融合蛋白或融合蛋白二聚体，以及一种或多种其它治疗剂(例如细胞因子、激素、细胞毒性剂或抑制剂(例如影响T细胞和/或B细胞增殖的细胞抑制剂)、抗体或小分子药物或免疫调节剂(例如免疫抑制剂))。本发明的药物组合或组合产品可用于本发明的治疗方法中。
在一些实施方案中，所述药物组合或组合产品用于预防或治疗B细胞相关的疾病或免疫系统疾病或炎症。
在一些实施方案中，所述药物组合或组合产品用于制备预防或治疗B细胞相关的疾病或免疫系统疾病或炎症的药物的用途。
VIII.治疗方法和用途
在一方面中，本发明涉及预防或治疗受试者疾病，例如B细胞或自身抗体相关的疾病或免疫系统疾病(例如自身免疫性疾病)或炎症的方法，所述方法包括向所述受试者施用有效量的本文所述的任何TACI/BCMA嵌合体或其融合蛋白或融合蛋白二聚体或药物组合物或药物组合。在一些实施方案中，所述疾病是与BAFF和/或APRIL或BAFF/APRIL异三聚体异常表达(浓度增高)或活性相关疾病，例如相比健康个体的样品(例如血液或血清)。
在一些实施方案中，所述B细胞或自身抗体相关的疾病或免疫系统疾病是B细胞或自身抗体介导的疾病，例如B细胞或自身抗体介导的自身免疫性疾病。在一些实施方案中，所述B细胞介导的疾病是B细胞扩增或异常活化相关的疾病，例如B细胞扩增或异常活化的自身免疫性疾病。在一些实施方案中，所述自身抗体介导的疾病是指由于B细胞异常增殖或活化而导致不需要的自身抗体产生，或导致异常的自身抗体产生。
在一些实施方案中，所述B细胞扩增相关的疾病是B细胞异常增殖的疾病(例如自身免疫性疾病)，例如相比健康个体的样品(例如血液或血清)。在一些实施方案中，所述B细胞或自身抗体介导的疾病中，由于B细胞异常活化而导致过度产生针对自身抗原的自身抗体或导致异常的自身抗体产生。
在一方面中，本发明涉及抑制个体中B细胞增殖的方法，所述方法包括向所述受试者施用有效量的本文所述的任何TACI/BCMA嵌合体或其融合蛋白或融合蛋白二聚体。
在一些实施方案中，所述疾病的治疗将受益于抑制BAFF和/或APRIL或BAFF/APRIL异三聚体相关的信号通路。在一些实施方案中，所述疾病的治疗将受益于抑制B细胞增殖或淋巴细胞增殖或抑制自身抗体。
在一些实施方案中，所述自身免疫疾病包括但不限于狼疮例如系统性红斑狼疮、类风湿性关节炎、IgA肾病(IgAN)或膜性肾病等慢性肾病、干燥综合征、重症肌无力、特发性血小板减少性紫癜(ITP)、温抗体型自身免疫性溶血性贫血(wAIHA)、多发性硬化症(MS)、冠状动脉性心脏病(CAD)或甲状腺眼病等疾病。
本发明的TACI/BCMA嵌合体或其融合蛋白或融合蛋白二聚体(以及包含其的药物组合物，其任选地另外的治疗剂)可以通过任何合适的方法给药，包括肠胃外给药，肺内给药和鼻内给药，并且，如果局部治疗需要，病灶内给药。肠胃外输注包括肌内、静脉内、动脉内、腹膜内或皮下给药。在一定程度上根据用药是短期或长期性而定，可通过任何适合途径，例如通过注射，例如静脉内或皮下注射用药。本文中涵盖各种用药时程，包括，但不限于，单次给药或在多个时间点多次给药、推注给药及脉冲输注。
为了预防或治疗疾病，本发明的TACI/BCMA嵌合体或其融合蛋白或融合蛋白二聚体的合适剂量(当单独或与一种或多种其他的治疗剂组合使用时)将取决于待治疗疾病的类型、抗体的类型、疾病的严重性和进程、以预防目的施用还是以治疗目的施用、以前的治疗、患者的临床病史和对所述抗体的应答，和主治医师的判断力。所述抗体以一次治疗或经过一系列治疗合适地施用于患者。
再一方面，本发明也提供本发明TACI/BCMA嵌合体或其融合蛋白或融合蛋白二聚体或药物组合物或药物组合在制备用于前述方法(例如用于治疗所述B细胞或自身抗体相关的疾病或免疫系统疾病(例如自身免疫性疾病)或炎症)的药物中的用途。
再一方面，本发明也提供本发明TACI/BCMA嵌合体或其融合蛋白或融合蛋白二聚体或药物组合物或药物组合，其用于疗法，例如用于前述方法(例如用于治疗所述B细胞或自身抗体相关的疾病或免疫系统疾病(例如自身免疫性疾病)或炎症)或用途。
再一方面，本发明也提供本发明TACI/BCMA嵌合体或其融合蛋白或融合蛋白二聚体或药物组合物或药物组合用于前述方法(例如用于治疗所述B细胞或自身抗体相关的疾病或免疫系统疾病(例如自身免疫性疾病)或炎症)的用途。描述以下实施例以辅助对本发明的理解。不意在且不应当以任何方式将实施例解释成限制本发明的保护范围。
本发明的这些以及其它方面和实施方案在附图(附图简述紧随其后)和以下的发明详述中得到描述并且示例于以下实施例中。上文以及整个本申请中所论述的任何或所有特征可以在本发明的各种实施方案中组合。以下实施例进一步说明本发明，然而，应理解实施例以说明而非限定的方式来描述，并且本领域技术人员可以进行多种修改。
实施例
实施例1：TACI分析与改造
全长的TACI包含293个氨基酸残基，胞外结构域多个位置存在蛋白酶切位点，导致胞外全长的TACI表达后非常容易发生断裂，而根据报道，TACI胞外结构域中存在两个富含半胱氨酸结构域(Cysteine-rich domains,CRD)，分别为CRD1与CRD2，其中单独的CRD2结构域就与配体的具有较高的结合活性。因此我们在本申请中选择TACI第68-110位氨基酸残基组成的CRD2结构域与人源抗体IgG1的Fc部分构建融合蛋白(SEQ ID NO:5)，从而改善TACI的表达断裂问题。
由于TACI蛋白在中性溶液中容易发生聚集，为了进一步提高TACI的稳定性，使用Discovery studio软件，基于TACI CRD2的晶体结构(PDB ID:1XU1)，进行了表面疏水基团、电荷基团以及成药性风险位点(例如引起聚集风险的位点)进行了分析，结果显示TACI分子第69、72、73、74、77、78、85、102和/或103位氨基酸存在潜在聚集风险。同时我们发现TACI同家族蛋白BCMA的蛋白质结构(6-42，PDB ID:1XU2)与TACI结构同源性很高，与配体的结合位点也基本一致，同时两者的N端与C端部分未参与配体的结合(图1)，更为重要的是Discovery studio软件的分析结果显示BCMA表面电荷分布均衡，聚集风险明显低于TACI。因此我们设计了一系列BCMA与TACI胞外结构域的嵌合体分子，在保留TACI与配体结合位点的基础上，将TACI的N端替换为BCMA的相应序列，以及同时将TACI的N端与C端都替换为BCMA的相应序列和或部分成药性风险位点进行了氨基酸替换，减少TACI蛋白表面电荷基团和疏水基团的面积，降低TACI的聚集风险。同时构建ALPN-303(US11274140B2)与Telitacicept(RC-18,CN101323643B)作为对照。野生型TACI(uniprot:O14836,aa1-293)的序列在本申请中显示于SEQ ID NO:1中。野生型BCMA(uniprot:Q02223,aa1-184)的序列在本申请中显示于SEQ ID NO:2中。
表达质粒构建
将不同的TACI或TACI/BCMA嵌合体通过GSGGGGS(SEQ ID NO:39)与human IgG1的Fc部分连接，并构建到pCDNA3.1载体上。此外，用于对照的ALPN-303和Telitacicept(RC-18)同样构建到pCDNA3.1载体上，用于表达表1中的如下蛋白：
表1.TACI或TACI/BCMA嵌合体融合蛋白

上述蛋白分子的具体序列信息见序列表。
融合蛋白的表达纯化
使用化学转染的方法将含有编码融合蛋白基因的载体转入HEK293细胞中。使用化学转染试剂PEI，按厂商提供的方案瞬时转染培养的HEK293细胞。
首先在超净工作台中准备表达质粒DNA和转染试剂，取5mL Opti-MEM培养基(Gibco货号：31985-070)加入50mL离心管中，加入50μg对应质粒的DNA，利用0.22μm的滤头过滤含有质粒的Opti-MEM培养基，随后加入150μg PEI(1g/L)，静置20min。将DNA/PEI混合物轻柔倒入45mLHEK293细胞并混匀，在37℃，8％CO2的条件下培养20h后，补加VPA使终浓度至2mM及2％(v/v)Feed，继续培养6天。
在细胞培养后，细胞培养液以13000rpm离心20min，收集上清，用预装柱Hitrap Mabselect Sure(GE,11-0034-95)纯化上清液。操作如下：纯化前用5倍柱体积的平衡液(PBS缓冲液(Gibco，货号：70011-044))平衡填料柱；将收集的上清通过柱子，再用10倍柱体积的平衡液清洗填料柱，去除非特异性结合蛋白；用5倍柱体积的洗脱缓冲液(100mM sodium citrate，pH 3.3)冲洗填料，收集洗脱液。每1ml洗脱液加入60μLTris(2M Tris)，使用超滤浓缩管(MILLIPORE，货号：UFC901096)交换到PBS缓冲液中，并测定浓度。取100μg纯化后蛋白，调整浓度至1mg/mL，使用凝胶过滤色谱柱SW3000(TOSOH货号：18675)测定蛋白纯度。
TACI或TACI/BCMA嵌合体融合蛋白及对照样品的纯化结果见下表2。TACI或TACI/BCMA嵌合体融合蛋白在HEK293细胞中的表达量均大于100mg/mL，大部分嵌合体融合蛋白的表达量与对照样品相当。TACI或TACI/BCMA嵌合体融合蛋白的样品纯度均大于95％，与ALPN-303相当，明显优于RC-18。
表2.TACI或TACI/BCMA嵌合体融合蛋白及对照样品的表达量和纯度

实施例2：TACI或TACI/BCMA嵌合体融合蛋白与配体的亲合力测定
采用生物光干涉测量(ForteBio)测定法测定本发明上述TACI或TACI/BCMA嵌合体融合蛋白与其配体的平衡解离常数(KD)。
ForteBio亲和力测定按照现有的方法(Estep,P等人，High throughput solution Based measurement of antibody-antigen affinity and epitope binning.Mabs,2013.5(2):p.270-8)进行。测量候选分子分别与BAFF和APRIL的亲合力：传感器在分析缓冲液中线下平衡20分钟，然后线上检测120秒建立基线；加载候选分子至AHC传感器(Sartorius,18-5060)上进行ForteBio亲和测量；将已加载候选分子的传感器置于含100nM BAFF或APRIL的溶液中直至平台期，之后将传感器转移至分析缓冲液解离至少2分钟用于结合和解离速率测量。使用1:1结合模型进行动力学的分析。
实验结果见表3。相较于对照样品，所有TACI或TACI/BCMA嵌合体融合蛋白与BAFF及APRIL的亲合力均明显优于RC-18，与ALPN-303相当。
表3.TACI或TACI/BCMA嵌合体融合蛋白与BAFF和APRIL的亲合力

实施例3：TACI/BCMA嵌合体融合蛋白体外功能检测
实施例3.1 TACI/BCMA嵌合体融合蛋白阻断BAFF与IM-9细胞的结合情况。
将样品(ALPN-303，RC-18和TACI/BCMA嵌合体融合蛋白)进行二倍系列梯度稀释，起始浓度为200nM，三倍稀释于FACS buffer中，总共建立12个浓度梯度。将Biotin Human BAFF(Acro，Cat#BAF-H82Q2-200ug)稀释至0.2μg/mL；将稀释的样品与稀释的Biotin human BAFF按照1:1混合；37度孵育30min。IM-9细胞(南京科佰，Cat#CB60276)计数，并稀释至2×106个/mL，向U型底96孔板中加入100μL/孔。500g离心5min，去除细胞培养基。然后将孵育结束的样品加入U型板并重悬细胞，100μL/孔，4℃孵育30min。500g离心5min去除上清，FACS buffer洗细胞2遍。500g离心5min，去除FACS buffer，每孔加入50μLPE Streptavidin二抗(BD，Cat#554061)(1:200稀释于FACS buffer中)，4℃避光孵育30min。500g离心5min去除上清，FACS buffer洗细胞3遍。用200μLFACS buffer重悬细胞，流式细胞仪检测。
结果显示，ALPN-303、RC-18和TACI/BCMA嵌合体融合蛋白均能抑制BAFF与IM-9细胞的结合。但是，TACI/BCMA嵌合体融合蛋白显著优于RC-18抑制BAFF与IM-9细胞结合的活性(IC50为12.47±3.73nM)，且与ALPN-303抑制活性相当(IC50为1.72±0.48nM)(表4)。
实施例3.2 TACI/BCMA嵌合体融合蛋白阻断APRIL与IM-9细胞的结合情况。
将样品(ALPN-303，RC-18和TACI/BCMA嵌合体融合蛋白)进行三倍系列梯度稀释，起始浓度为200nM，三倍稀释于FACS buffer中，总共建立12个浓度梯度。将Biotin Human APRIL(Acro，Cat#APL-H82F5-200UG)稀释至0.2μg/mL；将稀释的样品与稀释的Biotin human APRIL按照1：1混合；37度孵育30min。IM-9细胞计数，并稀释至2×106个/mL，向U型底96孔板中加入100μL/孔。500g离心5min，去除细胞培养基。然后将孵育结束的样品加入U型板并重悬细胞，100μL/孔，4℃孵育30min。500g离心5min去除上清，FACS buffer洗细胞2遍。500g离心5min，去除FACS buffer，每孔加入50μLPE Streptavidin二抗(BD，Cat#554061)(1:200稀释于FACS buffer中)，4℃避光孵育30min。500g离心5min去除上清，FACS buffer洗细胞3遍。用200μLFACS buffer重悬细胞，流式细胞仪检测。
结果显示，ALPN-303、RC-18和TACI/BCMA嵌合体融合蛋白融合均能抑制APRIL与IM-9细胞的结合。但是，TACI/BCMA嵌合体融合蛋白显著优于RC-18抑制APRIL与IM-9细胞结合的活性(IC50为5.89±2.92nM)，且与ALPN-303抑制活性相当(IC50为0.35±0.29nM)(表4)。
实施例3.3 TACI/BCMA嵌合体融合蛋白阻断BAFF/APRIL诱导表面过表达人TACI的NF-κB Reporter Jurkat信号情况
将编码人TACI的cDNA克隆到pLenti-IRES-puro载体(Invitrogen)上，然后通过慢病毒转染NF-κB Jurkat report细胞(吉满生物，Cat#GM-C07855)，产生过表达人TACI的NF-κBReporter Jurkat细胞(即TACI/NF-κB Reporter Jurkat细胞)。随后利用梯度稀释法检测嵌合体TACI/BCMA-Fc融合蛋白阻断BAFF/APRIL诱导TACI/NF-κB Reporter Jurkat信号情况。
简言之，将样品(ALPN-303，RC-18和TACI/BCMA嵌合体融合蛋白)进行三倍系列梯度稀释，起始浓度为1200nM，三倍稀释于RPMI-1640+10％FBS培养基中，总共建立10个浓度梯度。将Human BAFF(Acro，Cat#BAF-H52D4)+HumanAPRIL(Acro，Cat#APL-H52D1)应用RPMI-1640+10％FBS培养基稀释至20nm；将稀释的样品与稀释的20nm human BAFF/APRILmixture按照1：1混合；37度孵育30min。TACI/NF-κB Reporter Jurkat细胞计数，并稀释至1×106个/mL，向白色平底96孔板中加入50μL/孔。然后将孵育结束的样品加入已含有细胞的白色平底96孔板并重悬，50μL/孔，37℃孵育22h，培养结束后，采用Bio-Lite LuciferaseAssay System(Vazyme,#DD1201-03)检测细胞产生的发光信号情况。
结果显示，ALPN-303、RC-18和TACI/BCMA嵌合体融合蛋白均能抑制BAFF/APRIL结合TACI诱导的NF-κB Reporter Jurkat信号。但是，TACI/BCMA嵌合体融合蛋白抑制BAFF/APRIL结合TACI诱导的NF-κB Reporter Jurkat信号的抑制活性显著优于RC-18的抑制活性(IC50为23.55±8.47nM)，且与ALPN-303抑制活性相当(IC50为6.40±2.02nM)(表4)。
实施例3.4TACI/BCMA嵌合体融合蛋白阻断BAFF/APRIL诱导表面过表达人BCMA的NF-κB Reporter Jurkat信号情况
将编码人BCMA的cDNA克隆到pLenti-IRES-Neo载体(Invitrogen)上，然后通过慢病毒转染NF-κB Jurkat report细胞(吉满生物，Cat#GM-C07855)，产生过表达人BCMA的NF-κB Reporter Jurkat细胞(即BCMA/NF-κB Reporter Jurkat细胞)。随后利用梯度稀释法检测嵌合体TACI/BCMA-Fc融合蛋白阻断BAFF/APRIL诱导BCMA/NF-κB Reporter Jurkat信号情况。
简言之，将样品(ALPN-303，RC-18和TACI/BCMA嵌合体融合蛋白)进行三倍系列梯度稀释，起始浓度为2400nM，三倍稀释于RPMI-1640+10％FBS培养基中，总共建立10个浓度梯度。将Human BAFF(Acro，Cat#BAF-H52D4)+HumanAPRIL(Acro，Cat#APL-H52D1)应用RPMI-1640+10％FBS培养基稀释至20nm；将稀释的样品与稀释的20nmhuman BAFF/APRILmixture按照1：1混合；37度孵育30min。BCMA/NF-κB Reporter Jurkat细胞计数，并稀释至2×106个/mL，向白色平底96孔板中加入50μL/孔。然后将孵育结束的样品加入已含有细胞的白色平底96孔板并重悬，50μL/孔，37℃孵育5h，培养结束后，采用Bio-Lite LuciferaseAssay System(Vazyme,#DD1201-03)检测细胞产生的发光信号情况。
结果显示，ALPN-303、RC-18和TACI/BCMA嵌合体融合蛋白均能抑制BAFF/APRIL结合BCMA诱导的NF-κB Reporter Jurkat信号。但是，TACI/BCMA嵌合体融合蛋白抑制BAFF/APRIL结合BCMA诱导的NF-κB Reporter Jurkat信号的抑制活性显著优于RC-18(IC50为53.25±8.81nM)，且与ALPN-303抑制活性相当(IC50为4.06±0.53nM)(表4)。
实施例3.5.TACI/BCMA嵌合体融合蛋白阻断脾脏细胞增殖能力的检测
取小鼠脾脏细胞，裂红，调整细胞密度至2×106/mL，每孔加样50μL，将细胞按照1x105/孔铺板。将样品(ALPN-303，RC-18和TACI/BCMA嵌合体融合蛋白)进行三倍系列梯度稀释，起始浓度为1200nM，三倍稀释于RPMI-1640+10％FBS培养基中，总共建立9个浓度梯度。将Human BAFF(Acro，Cat#BAF-H52D4)应用RPMI-1640+10％FBS培养基稀释至50ng/mL；将稀释的样品与稀释的human BAFF按照1：1混合；37度孵育30min。
设置对照孔：IgG组(应用IgG(Equitech-Bio，Cat#SLH56)作为样品+细胞+BAFF抗原)和空白组(RPMI-1640+10％FBS)组(培养基+细胞)。
取50μL/孔孵育的混合物加入到细胞中，37度孵育72h；孵育72h后，采用CellCount-LiteTM Luminescent Cell Viabil(Vazyme,#DD1101-02)检测细胞增殖情况。
基于不同浓度下的读数值进行曲线拟合计算IC50。结果显示，ALPN-303、RC-18和TACI/BCMA嵌合体融合蛋白均能抑制BAFF诱导脾脏细胞增殖。但是，TACI/BCMA嵌合体融合蛋白显著优于RC-18抑制BAFF诱导脾脏细胞增殖的活性(IC50为4.98±0.95nM)，与ALPN-303抑制活性相当(IC50为0.49±0.06nM)(表4)。
表4.TACI/BCMA嵌合体融合蛋白的体外功能活性

实施例3.6.TACI/BCMA嵌合体融合蛋白抑制B细胞增殖能力的检测
复苏PBMC(AllCell,Cat#FPB004F-C)，利用EasySepTM Human B cell Enrichment kit(STEMCELL,Cat#19054)分离B细胞，计数并调整细胞密度为2×106/mL，并加入终浓度为20ng/mL rhIL-4(R&D,Cat#204-IL-050)和1μg/mL Anti-human IgM(Jakson，Cat#109-006-129)。将样品(ALPN-303，RC-18和TACI/BCMA嵌合体融合蛋白)进行三倍系列梯度稀释，起始浓度为2400nM，三倍稀释于RPMI-1640+10％FBS培养基中，总共建立9个浓度梯度。
将Human BAFF(Acro，Cat#BAF-H52D4)+Human APRIL(Acro，Cat#APL-H52D1)应用RPMI-1640+10％FBS培养基稀释至20nm；将稀释的样品与稀释的20nm human BAFF/APRIL mixture按照1：1混合；37度孵育30min。设置对照孔：IgG组(应用IgG(Equitech-Bio，Cat#SLH56)作为样品)，取50μL/孔孵育的混合物加入到细胞中，37度孵育72h；孵育72h后，采用CellCount-LiteTM Luminescent Cell Viabil(Vazyme,#DD1101-02)检测细胞增殖情况及绘制剂量效应曲线。
结果显示，ALPN-303和TACI/BCMA嵌合体融合蛋白均能有效抑制BAFF/APRIL诱导的B细胞增殖(图4)。其中，图中抑制率为实验组/对照IgG组×100％计算而得。
实施例4：TACI/BCMA嵌合体融合蛋白的稳定性测定
通过动态光散射法(DLS)、差示扫描量热法(DSC)以及加速稳定性评估TACI/BCMA嵌合体融合蛋白的热稳定性。
动态光散射法(DLS)评估热稳定性
将蛋白(1mg/mL)溶液以13000g/min离心5min后加入至样品板中，检测连续温度上升过程中蛋白分子粒径大小的变化，检测设置为DLS采集时间5s，采集次数5次，实验温度25-85℃。实验结束后分析样品粒径随温度的变化。
差示扫描量热法(DSC)评估热稳定性
将抗体突变体样品用PBS溶液稀释至0.5-1mg/mL，对稀释后样品和PBS缓冲液进行脱气，然后将样品加入左侧样品板，并在右侧参比板中对应位置加入PBS缓冲液。起始温度30℃平衡时间10min；以1℃/min速率加热，终止温度90℃。
DLS和DSC实验结果见表5。实验结果显示，TACI/BCMA嵌合体融合蛋白99017和99018的起始聚集温度与蛋白质变性温度Tm1与Tm2均显著优于对照RC18以及未改造的TACI CRD2分子99001，热稳定性与ALPN-303的相当。
表5.TACI/BCMA嵌合体融合蛋白的成药性
TACI/BCMA嵌合体融合蛋白的加速稳定性评估
另外将TACI/BCMA嵌合体融合蛋白用超滤管置换到pH7.4的PBS溶液中，浓度为10mg/ml，置于50℃的恒温培养箱中进行加速稳定性测试，4天后取样进行纯度(SEC和CE-SDS法)分析。实验结果见表6。
实验结果表明在50℃放置4天的条件下，TACI/BCMA嵌合体融合蛋白099017和099018稳定性表现最好，蛋白SEC纯度能够保持在95％以上，非还原CE-SDS纯度能够保持98％以上。稳定性显著优于对照RC-18和未改造的TACI CRD2分子99001，也优于对照ALPN-303。
表6.CE-SDS评估TACI/BCMA嵌合体融合蛋白的强制稳定性

实施例5：TACI/BCMA嵌合体融合蛋白体内药效
实施例5.1.TACI/BCMA嵌合体融合蛋白在小鼠KLH免疫模型中的药效实验
选用C57BL/6N小鼠(购自北京维通利华实验动物技术有限公司)，在第0、12天，通过腹腔注射含有Alum佐剂(Thermo,Cat#77161)的0.2mgKLH(Solarbiol,Cat#KB160)。本实验设空白对照组(Blank，无任何处理)、模型对照IgG组、99001组、99006组和99017组，共5组，每组5-7只小鼠(空白对照组3只小鼠)，在第4和11天腹腔注射给药(模型对照IgG组注射等量IgG抗体(购自Equitech-Bio，Cat#SLH56))，第20天取小鼠脾脏进行细胞检测B细胞改变以及采血进行血清中IgA(Invitrogen,Cat#EMIGA)、IgM(Invitrogen,Cat#88-50470-88)和IgG(Invitrogen,Cat#88-50400-88)检测。
结果显示，和对照IgG组相比，给药组均能抑制KLH免疫产生的脾脏细胞、脾脏中B细胞的增殖(表7)以及血清中IgA、IgM和IgG水平(表8)。通过计算给药组相对IgG组的抑制率，结果表明，和99001组(未改造的TACI CRD2分子)、99006组(未改造的BCMACRD2分子)相比，99017组(TACI/BCMA嵌合体融合蛋白)的抑制效果更优。以上数据提示，经改造后的TACI/BCMA嵌合体融合蛋白对B细胞及抗体水平显著优于TACI或BCMAWT CRD2片段。
表7脾脏细胞和脾脏中B细胞的数目(n＝5-7只/组)
*代表99017与99001相比有显著性差异(**p<0.01,t test)。
#代表99017与99006相比有显著性差异(##p<0.01,ttest)。
表8血清中IgA、IgM和IgG的水平(n＝5-7只/组)

*代表99017与99001相比有显著性差异(**p<0.01,t test)。
#代表99017与99006相比有显著性差异(###p<0.001,ttest)。
实施例5.2.TACI/BCMA嵌合体融合蛋白在小鼠BAFF刺激模型中的药效
选用Balb/c小鼠(购自北京维通利华实验动物技术有限公司)，在第0、1、2、3天，通过腹腔注射10μg human BAFF蛋白(Sino Biological,Cat#10056-HNCHA)。本实验设空白对照组(Blank，无任何处理)、模型对照IgG组、RC-18组、ALPN-303组和TACI/BCMA嵌合体融合蛋白组，共5组，每组5-7只小鼠(空白对照组3只小鼠)，在第0和2天腹腔注射给药(模型对照IgG组注射等量IgG抗体(购自Equitech-Bio，Cat#SLH56))，第4天取小鼠脾脏进行细胞检测以及采用ELISA试剂盒对血清中IgA(Invitrogen,Cat#EMIGA)、IgM(Invitrogen,Cat#88-50470-88)和IgG(Invitrogen,Cat#88-50400-88)检测。结果显示，TACI/BCMA嵌合体融合蛋白能有效抑制BAFF诱导的脾脏细胞和脾脏中B细胞的增殖(表9)，同时，TACI/BCMA嵌合体融合蛋白能有效降低BAFF诱导的血清中IgA、IgM和IgG水平(表10)。
表9脾脏细胞和脾脏中B细胞的数目(n＝5-7只/组)
表10血清中IgA、IgM和IgG的水平(n＝5-7只/组)

实施例5.3.TACI/BCMA嵌合体融合蛋白在小鼠KLH免疫模型中的药效实验
选用C57BL/6N小鼠(购自北京维通利华实验动物技术有限公司)，在第0、12天，通过腹腔注射含有Alum佐剂(Thermo,Cat#77161)的0.2mgKLH(Solarbiol,Cat#KB160)。本实验设空白对照组、模型对照IgG组、RC-18组、ALPN-303组和TACI/BCMA嵌合体融合蛋白组(分别是99017和99018)，共5组，每组5-7只小鼠(空白对照组3只小鼠)，在第4和11天腹腔注射给药(模型对照IgG组注射等量IgG抗体(购自Equitech-Bio，Cat#SLH56))，第20天取小鼠脾脏进行细胞检测以及采血进行血清中IgA(Invitrogen,Cat#EMIGA)、IgM(Invitrogen,Cat#88-50470-88)和IgG(Invitrogen,Cat#88-50400-88)检测。
结果显示，和对照IgG组相比，给药组均能抑制KLH免疫产生的脾脏细胞、脾脏中B细胞(图5)以及不同B细胞亚群的增殖(表11)。但是，和RC-18组相比，TACI/BCMA嵌合体融合蛋白组的抑制效果更优；和ALPN-303相比，TACI/BCMA嵌合体融合蛋白能更有效地抑制MZ细胞、GC B细胞、浆细胞的增殖。同时，TACI/BCMA嵌合体融合蛋白能有效降低KLH免疫产生的血清中IgA、IgM和IgG水平(图6，表12)。
表11不同B细胞亚群占总脾脏细胞的比例(n＝5-7只/组)
*代表99017与ALPN-303相比有显著性差异(*p<0.05,**p<0.01,ttest)。
表12血清中IgA、IgM和IgG的水平(n＝5-7只/组)
实施例5.4.TACI/BCMA嵌合体融合蛋白在小鼠上的药代动力学研究
采用小鼠进行静脉注射(Intravenous Injection,I.V.)检测嵌合体融合蛋白的药代动力学。每组9只BALB/c小鼠，体重20g左右，每只小鼠通过静脉注射10mg/kg的待检测分子，分别在单次给药后5min、0.5h、2h、6h、第2天、第4天、第7天、第14天、第21天进行眼眶采集血样，血液自然凝固后离心取血清。
血清中抗体药物浓度的测定方法如下：包被液(将一包碳酸盐粉末(Thermo,cat#23282)溶解于400mL超纯水，混匀，定容至500mL)稀释抗原humanAPRIL-his蛋白(Acro Biosystems,Cat#APL-H52D1)至1μg/mL，按照每孔100μL加入到96孔酶标板(Thermo,Cat#442404)，4℃孵育过夜。弃去包被液，1×PBST洗涤3次。每孔加入200μL封闭液(含有2％BSA的PBST溶液)，室温封闭1h。弃去封闭液，1×PBST洗涤3次后，加入经稀释的小鼠血清；室温孵育2小时。弃去酶标板中的溶液，1×PBST洗涤5次。加入稀释的Goatx-human IgG-Fc-HRP(BETHYL,Cat#A80-104P)，每孔100μL，室温孵育1小时。弃去酶标板中溶液，1×PBST洗涤5次。每孔加入100μLTMB显色液(索莱宝,Cat#PR1200)，显色5-10min，每孔加入50μL终止液(索莱宝,Cat#C1058)终止。酶标仪读取OD450nm和OD620nm值。给予待测分子的小鼠(按照10mg/kg向小鼠给药)在不同时间点血药浓度浓度变化如图7所示。采用Excel PK solver计算非房室模型计算药代动力学参数，其主要药物暴露量参数(T1/2、Cmax、AUC0-t、AUC0-∞、CL和Vss)如表13所示。通过分析上述不同分子的小鼠药代动力学参数，TACI/BCMA嵌合体融合蛋白的药代动力学明显优于RC-18，且略微优于ALPN-303。
表13 T ACI/BCMA嵌合体融合蛋白的小鼠药代动力学参数
实施例6.TACI/BCMA嵌合体融合蛋白在食蟹猴上的毒物动力学研究
采用食蟹猴进行皮下注射(Subcutaneous Injection,s.c.)检测99017分子的TK。每个剂量组2只食蟹猴(1雌1雄)，体重3.5-4.5kg左右，每组食蟹猴通过每周皮下注射100mg/kg或者200mg/kg的待检测分子，给药五次，分别在第一次给药24h后(C1D1)和第四次给药后24h(C4D1)开始采血，采血时间点为30min、2h、6h、10h、24h、48h、72h、96h和168h，血液自然凝固后离心取血清。
血清中抗体药物浓度的测定方法如下：包被液(将一包碳酸盐粉末溶解于400mL超纯水，混匀，定容至500mL)稀释抗原human APRIL-his蛋白(Acro Biosystems,Cat#APL-H52D1)至2μg/mL，按照每孔100μL加入到96孔酶标板，4℃孵育过夜。弃去包被液，1×PBST洗涤3次。每孔加入300μL封闭液(含有5％SM-1的PBST溶液)，室温封闭2h。弃去封闭液，1×PBST洗涤3次后，每孔加入100μL经稀释食蟹猴的血清；室温孵育2小时。弃去酶标板中的溶液，1×PBST洗涤3次。加入稀释的Human IgG Heavy and Light chain Monkey-Adsorbed Antibody(BETHYL,Cat#A80-319P)，每孔100μL，室温孵育1小时。弃去酶标板中溶液，1×PBST洗涤5次。每孔加入100μLTMB显色液，显色5-10min，每孔加入100μL终止液终止。酶标仪读取OD450nm和OD620nm值。给予不同剂量待测分子的食蟹猴在C1D1和C4D1后不同时间点血药浓度浓度变化如图8所示。采用Phoenix WinNonlin 8.4非房室模型计算相关动力学参数，其主要药物暴露量参数(AUC0-t、Cmax、Tmax、T1/2、CI/F)如表14所示。
结果如图8所示，200mg/kg 99017其在C1D1和C4D1的动力学高于100mg/kg，具有剂量依赖性。分析计算的不同剂量在不同给药频次后的动力学参数，100mg/kg的T1/2约为5-5.9天，ALPN-303披露的150mg/kg的T1/2为2.9天(Dillon S,Evans L,Lewis K,et al.ALPN-303,an Enhanced,Potent Dual BAFF/APRIL Antagonist Engineered by Directed Evolution for the Treatment of Systemic Lupus Erythematosus(SLE)and Other B Cell-Related Diseases.Arthritis Rheumatol.2021；73(suppl 9)；https://www.alpineimmunesciences.com/wp-content/uploads/2021/12/ALPN-303_ACR-2021.pdf)。因此，99017的半衰期显著优于ALPN-303。
表14.食蟹猴毒物动力学参数

实施例7.TACI/BCMA嵌合体融合蛋白及YTE分子在食蟹猴上的药代动力学及药效学研究
实验采用食蟹猴进行静脉注射(Intravenous Injection,I.V.)检测99017和99025分子的药代动力学及药效学。每组3只食蟹猴，体重3.5-4.5kg左右，每只食蟹猴通过静脉注射9mg/kg的待检测分子，分别在单次给药后5min、2h、6h、12h、24h、72h、144h、216h、312h、432h、480h、624h、816h和984h进行采集血样，血液自然凝固后离心取血清用于检测血清中抗体药物浓度。分别在单次给药前和给药后6d、9d、12d、18d、24d、32d和40d进行采集血样，血液自然凝固后离心取血清，利用Roche Diagnostic的免疫球蛋白检测试剂盒检测血清中IgA(Cat#05219205190)、IgM(Cat#05220726190)和IgG(Cat#05220718190)，计算给药后不同时间点和给药前相比血清中免疫球蛋白下降百分比，结果见图10。结果显示，TACI/BCMA嵌合体融合蛋白能有效降低BAFF诱导的血清中IgA、IgM和IgG水平。
血清中抗体药物浓度的测定方法如下：包被液(将一包碳酸盐粉末溶解于400mL超纯水，混匀，定容至500mL)稀释抗原humanAPRIL-his蛋白(Acro Biosystems,Cat#APL-H52D1)至2μg/mL，按照每孔100μL加入到96孔酶标板，4℃孵育过夜。弃去包被液，1×PBST洗涤3次。每孔加入300μL封闭液(含有5％SM-1的PBST溶液)，室温封闭2h。弃去封闭液，1×PBST洗涤3次后，每孔加入100μL经稀释食蟹猴的血清；室温孵育2小时。弃去酶标板中的溶液，1×PBST洗涤3次。加入稀释的Human IgG Heavy and Light chain Monkey-AdsorbedAntibody(BETHYL,Cat#A80-319P)，每孔100μL，室温孵育1小时。弃去酶标板中溶液，1×PBST洗涤5次。每孔加入100μLTMB显色液，显色5-10min，每孔加入100μL终止液终止。酶标仪读取OD450nm和OD620nm值。给予待测分子的食蟹猴(按照9mg/kg向食蟹猴给药)在不同时间点血药浓度浓度变化如图9所示。
结果如图9和图10所示，和99017相比，99025的药代动力学更长，药效学更优。
同时与ALPN-303披露的数据相比(Dillon S,Evans L,Lewis K,et al.ALPN-303,an Enhanced,Potent Dual BAFF/APRIL Antagonist Engineered by Directed Evolution for the Treatment of Systemic Lupus Erythematosus(SLE)and Other B Cell-Related Diseases.Arthritis Rheumatol.2021；73(suppl 9)；Lawrence S.Evans,Katherine E.Lewis,Daniel DeMonte,et al.Povetacicept,an Enhanced Dual APRIL/BAFF Antagonist That Modulates B Lymphocytes and Pathogenic Autoantibodies for the Treatment of Lupus and Other B Cell–Related Autoimmune Diseases.https://doi.org/10.1002/art.42462)，99017和99025的Cmax和AUC更优(表15)，药效学维持时间更久。
表15.食蟹猴药代动力学Cmax和AUC比较
序列表：





Claims (18)

  1. TACI/BCMA嵌合体，其包含应用BCMA的N末端和/或C末端替换TACI的胞外区ECD的功能性片段的N末端和/或C末端的嵌合蛋白。
  2. 权利要求1的TACI/BCMA嵌合体，其包含如下结构：
    BCMAN末端氨基酸-TACI部分，其中TACI部分为缺失了N末端的TACI的胞外区ECD或其功能性片段，或者
    BCMAN末端氨基酸-TACI部分-BCMAC末端氨基酸，其中TACI部分为缺失了N末端和C末端的TACI的胞外区ECD或其功能性片段。
  3. 权利要求1或2的TACI/BCMA嵌合体，其中TACI的胞外区ECD的功能性片段包含TACI的CRD2且不包含CRD1或CRD1的任意片段；
    优选地，所述CRD2为TACI对应于SEQ ID NO:1所示的第71-104位的氨基酸序列，且CRD1为TACI对应于SEQ ID NO:1所示的第34-66位的氨基酸序列；
    任选地，所述TACI的胞外区ECD功能性片段还包含TACI的部分茎区和/或对应于SEQ ID NO:1的第68-70位之间的任意氨基酸序列；
    优选地，所述TACI的胞外区ECD功能性片段为或包含TACI对应于SEQ ID NO:1所示的氨基酸序列的如下片段：氨基酸残基68-110；例如，
    所述TACI的胞外区ECD功能性片段包含
    (i)与SEQ ID NO:31、32或33所示的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％的同一性的氨基酸序列；
    (ii)与SEQ ID NO:33所示的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％的同一性且具有Y102D的氨基酸序列；
    (iii)与SEQ ID NO:32所示的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％的同一性且具有K77E、F78Y和Y102D的氨基酸序列；
    (iv)SEQ ID NO:31、32或33所示的氨基酸序列；或
    (v)由(i)-(iv)中任一项所述的序列组成。
  4. 权利要求1-3中任一项所述的TACI/BCMA嵌合体，其中所述TACI的胞外区ECD功能性片段的N末端是指TACI对应于SEQ ID NO:1的Y79位氨基酸之前的N末端氨基酸；和/或
    所述TACI的胞外区ECD功能性片段的C末端是指TACI对应于SEQ ID NO:1的第99位氨基酸、第100位氨基酸、第101位氨基酸、第102位氨基酸、第103位氨基酸、第104位氨基酸、第105位氨基酸、第106位氨基酸、第107位氨基酸、第108位氨基酸、第109位氨基酸或第110位氨基酸之后的C末端氨基酸，优选地第99或105位氨基酸之后的C末端氨基酸。
  5. 权利要求1-4中任一项所述的TACI/BCMA嵌合体，其中BCMA的N末端氨基酸选自BCMA对应于SEQ ID NO:2的第1-13位、第2-13位、第3-13位、第4-13位、第5-13位、第6-13位或第7-13位的氨基酸序列；优选地，所述BCMA的N末端氨基酸序列包含SEQ ID NO:40-46中所示的任一项的氨基酸序列，或由所述的氨基酸序列组成；和/或
    所述BCMA的C末端氨基酸序列为BCMA对应于SEQ ID NO:2的第37-47位、第37-46位、第37-45位、第37-44位的氨基酸序列，或对应于SEQ ID NO:2的第43-44位或43-46位的氨基酸序列；
    任选地，所述BCMA的C末端氨基酸包含改善结合亲和力，增加稳定性和/或改善成药性的突变，例如取代，例如所述突变为第39位和/或第42位的突变，例如取代，例如N42A或N42Q或R39D或N42A-R39D；
    优选地，所述BCMA的C末端氨基酸序列包含SEQ ID NO:47-55中任一项所示的氨基酸序列，或由所述的氨基酸序列组成。
  6. 权利要求1-5中任一项所述的TACI/BCMA嵌合体，其中TACI ECD功能性片段包含降低聚集风险的氨基酸位点突变，例如在成药性风险位点的突变如取代，例如，所述成药性风险位点选自对应于SEQ ID NO:1的第69、72、73、74、77、85、102或103位氨基酸，优选地，所述成药性风险位点的氨基酸被突变为A或D，例如所述突变是Y102D；
    优选地，所述TACI部分包含SEQ ID NO:35、31-34或36-38中任一项所示的氨基酸序列，或包含与其具有至少90％、91％、92％、93％、94％、95％、96％、97％或98％的同一性的氨基酸序列，或由所述氨基酸序列组成。
  7. 权利要求1-6中任一项所述的TACI/BCMA嵌合体，其中所述TACI/BCMA嵌合体包含SEQ ID NO:73、59-72或74-80中任一项所示的氨基酸序列，或包含与其具有至少90％、91％、92％、93％、94％、95％、96％、97％或98％的同一性的氨基酸序列，或由所述氨基酸序列组成。
  8. 融合蛋白，其包含权利要求1-7中任一项所述的TACI/BCMA嵌合体，以及Fc区；
    优选地，所述Fc区为人的IgG Fc，例如，人IgG1 Fc,人IgG2 Fc,人IgG3 Fc或人IgG4 Fc；
    任选地，所述Fc区包含如下一个或多个突变：
    (i)在C末端缺失赖氨酸K(K447del)；
    (ii)减少由Fc区介导的效应子功能的突变；
    (iii)降低与Fcγ受体结合的突变，例如L234A/L235A突变或L234A/L235E或L234A/L235E/G237A突变；
    (iv)增强Fc片段与FcRn结合的突变，例如M252Y/S254T/T256E和/或M428L/N434S；
    例如，所述Fc区包含
    (i)SEQ ID NO:81或82所示的氨基酸序列或与其具有至少90％同一性，例如95％，96％，97％，99％或更高的同一性的氨基酸序列；
    (ii)SEQ ID NO:83或84所示的氨基酸序列或与其具有至少90％同一性，例如95％，96％，97％，99％或更高的同一性的氨基酸序列，任选地缺失C末端赖氨酸；
    (iii)SEQ ID NO:85-88和100-103中任一项所示的氨基酸序列或与其具有至少90％同一性，例如95％，96％，97％，98％,99％或更高的同一性的氨基酸序列；
    (iv)与SEQ ID NO:85或87所示的氨基酸序列所示的氨基酸序列具有至少85％、90％、95％、96％、97％、98％、99％的同一性且包含突变L234A/L235E/G237A的氨基酸序列；
    (v)与SEQ ID NO:86或88所示的氨基酸序列所示的氨基酸序列具有至少85％、90％、95％、96％、97％、98％、99％的同一性且包含突变L234A/L235E/G237A和C末端缺失赖氨酸的氨基酸序列；
    (vi)与SEQ ID NO:100或102所示的氨基酸序列所示的氨基酸序列具有至少85％、90％、95％、96％、97％、98％或99％的同一性且包含突变L234A/L235E/G237A和M252Y/S254T/T256E的氨基酸序列；
    (vii)与SEQ ID NO:101或103所示的氨基酸序列所示的氨基酸序列具有至少85％、90％、95％、96％、97％、98％、99％的同一性且包含突变L234A/L235E/G237A、M252Y/S254T/T256E和C末端缺失赖氨酸的氨基酸序列；或
    (viii)由以上(i)-(vii)中任一项所述的氨基酸序列组成。
  9. 权利要求8的融合蛋白，其中TACI/BCMA嵌合体直接或通过接头与Fc融合，优选地，所述嵌合体的C末端直接或通过接头与Fc的N末端融合；
    任选地，所述接头选自(GSGGGGS)n、(GS)n、(GSGGS)n、(GGGGS)n或(GGGS)n，其中n是至少1的整数，例如1、2、3、4或5；例如是(GSGGGGS)n，其中n＝1-3，例如所述接头是SEQ ID NO:39所示的氨基酸序列。
  10. 权利要求8或9的融合蛋白，其中所述融合蛋白
    (i)包含与SEQ ID NO:10、5-9和11-28中任一项的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由所述氨基酸序列组成；或者
    (ii)包含SEQ ID NO:10、5-9和11-28中任一项的氨基酸序列或由所述氨基酸序列组成
  11. 融合蛋白二聚体，其包含第一单体和第二单体，所述第一和第二单体分别包含权利要求8-10中任一项所述的融合蛋白链，优选地第一单体和第二单体相同。
  12. 多核苷酸，其编码权利要求1-7中任一项的TACI/BCMA嵌合体或权利要求8-10中任一项所述的融合蛋白或权利要求11所述的融合蛋白二聚体。
  13. 表达载体，其包含权利要求12所述的多核苷酸，例如所述表达载体是pCDNA表达载体，例如pCDNA3.1表达载体。
  14. 宿主细胞，其包含权利要求12的多核苷酸或权利要求13的表达载体。
  15. 制备TACI/BCMA嵌合体或其融合蛋白或融合蛋白二聚体的方法，其中所述方法包括，在适合TACI/BCMA嵌合体或其融合蛋白或融合蛋白二聚体表达的条件下，培养权利要求14的宿主细胞，和任选地从所述宿主细胞(或宿主细胞培养基)回收所述TACI/BCMA嵌合体或其融合蛋白或融合蛋白二聚体。
  16. 药物组合物，其包含权利要求1-7中任一项的TACI/BCMA嵌合体或权利要求8-10中任一项所述的融合蛋白或权利要求11所述的融合蛋白二聚体，以及任选地药用辅料。
  17. 药物组合，其包含权利要求1-7中任一项的TACI/BCMA嵌合体或权利要求8-10中任一项所述的融合蛋白或权利要求11所述的融合蛋白二聚体，以及一种或多种其它治疗剂(例如细胞因子、激素、细胞毒性剂或抑制剂(例如影响T细胞和/或B细胞增殖的细胞抑制剂)、抗体或小分子药物或免疫调节剂(例如免疫抑制剂))。
  18. 预防或治疗受试者疾病，例如B细胞或自身抗体相关的疾病或免疫系统疾病(例如自身免疫性疾病)或炎症的方法，所述方法包括向所述受试者施用权利要求1-7中任一项的TACI/BCMA嵌合体或权利要求8-10中任一项所述的融合蛋白或权利要求11所述的融合蛋白二聚体或权利要求16的药物组合物或权利要求17的药物组合，
    例如，所述B细胞或自身抗体相关的疾病或免疫系统疾病为B细胞或自身抗体介导的自身免疫性疾病；
    任选地，所述B细胞或自身抗体相关的疾病或免疫系统疾病或炎症为个体中，相比健康个体的样品，B细胞异常增殖或异常活化的疾病，如自身免疫性疾病；
    优选地，所述疾病为狼疮例如系统性红斑狼疮、类风湿性关节炎、IgA肾病(IgAN)或膜性肾病等慢性肾病、干燥综合征、重症肌无力、特发性血小板减少性紫癜(ITP)、温抗体型自身免疫性溶血性贫血(wAIHA)、多发性硬化症(MS)、冠状动脉性心脏病(CAD)或甲状腺眼病；
    任选地，所述施用还包括联合施用一种或多种其它治疗剂(例如细胞因子、激素、细胞毒性剂或抑制剂(例如影响T细胞和/或B细胞增殖的细胞抑制剂)、抗体或小分子药物或免疫调节剂(例如免疫抑制剂))。
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