WO2024046301A1 - 包含taci多肽的融合蛋白及其用途 - Google Patents

包含taci多肽的融合蛋白及其用途 Download PDF

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Publication number
WO2024046301A1
WO2024046301A1 PCT/CN2023/115448 CN2023115448W WO2024046301A1 WO 2024046301 A1 WO2024046301 A1 WO 2024046301A1 CN 2023115448 W CN2023115448 W CN 2023115448W WO 2024046301 A1 WO2024046301 A1 WO 2024046301A1
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Prior art keywords
polypeptide
taci
linker
bcma
seq
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PCT/CN2023/115448
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English (en)
French (fr)
Inventor
高焓
王欢
章崇祺
林�源
林侃
廖成
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Jiangsu Hengrui Pharmaceutical Co Ltd
Shanghai Shengdi Pharmaceutical Co Ltd
Original Assignee
Jiangsu Hengrui Pharmaceutical Co Ltd
Shanghai Shengdi Pharmaceutical Co Ltd
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Application filed by Jiangsu Hengrui Pharmaceutical Co Ltd, Shanghai Shengdi Pharmaceutical Co Ltd filed Critical Jiangsu Hengrui Pharmaceutical Co Ltd
Priority to AU2023335543A priority Critical patent/AU2023335543A1/en
Priority to US19/107,926 priority patent/US20260125451A1/en
Priority to KR1020257009244A priority patent/KR20250057823A/ko
Priority to EP23859331.3A priority patent/EP4582455A4/en
Priority to CN202380055707.XA priority patent/CN119585314A/zh
Priority to JP2025508757A priority patent/JP2025529796A/ja
Publication of WO2024046301A1 publication Critical patent/WO2024046301A1/zh
Priority to MX2025002123A priority patent/MX2025002123A/es
Anticipated expiration legal-status Critical
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    • 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
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70578—NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2866—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for cytokines, lymphokines, interferons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C07K2317/522—CH1 domain
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/71—Decreased effector function due to an Fc-modification
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/32—Fusion polypeptide fusions with soluble part of a cell surface receptor, "decoy receptors"
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction

Definitions

  • the present disclosure relates to the field of biomedicine. Specifically, the present disclosure relates to a fusion protein containing a TACI polypeptide and its use in treating diseases, especially autoimmune diseases.
  • SLE Systemic Lupus Erythematosus
  • ANA antinuclear antibody
  • Activation of the type I interferon pathway plays a key role in the pathogenesis of SLE and can promote the activation of various types of immune cells that are closely related to the pathogenesis of SLE, such as plasmacytoid dendritic cells (pDC) and B cells, ranging from 60% to 80%.
  • SLE patients are characterized by high expression of type I interferon, and type I interferon levels are positively correlated with SLE patient disease activity index (SLEDAI) scores.
  • SLEDAI SLE patient disease activity index
  • Anifrolumab developed by AstraZeneca is a monoclonal antibody targeting type I interferon (IFN) receptor subunit 1 (IFNAR1) and antagonizes all type I interferons (IFN- ⁇ , IFN- ⁇ , IFN- ⁇ , etc.) ) related activity, thereby controlling SLE disease progression.
  • IFN interferon
  • IFNAR1 monoclonal antibody targeting type I interferon receptor subunit 1
  • BICLA Comprehensive Index of Lupus Assessment
  • B lymphocyte stimulator also known as BAFF
  • APRIL proliferation-inducing ligand
  • B lymphocyte stimulator also known as BAFF
  • APRIL proliferation-inducing ligand
  • TNF tumor necrosis factor
  • TNF proliferation-inducing ligand
  • BAFF Membrane-bound BAFF is cleaved by Furin convertase to release the trimer form. BAFF and biologically active. BAFF is expressed on various cell types, including monocytes, dendritic cells, and bone marrow stromal cells. APRIL exists in a form different from members of the TNF ligand family. It is processed by furin convertase in the Golgi and directly secreted out of the cell in the form of a soluble trimer.
  • BAFF receptor BAFF receptor, BAFF- R, also called Br3
  • B cell maturation antigen BCMA
  • TACI transmembrane activator and CAMEL interactor
  • BAFF receptor BAFF receptor, BAFF- R, also called Br3
  • BCMA B cell maturation antigen
  • TACI transmembrane activator and CAMEL interactor
  • BAFF receptor BAFF receptor, BAFF- R, also called Br3
  • the extracellular region of the TNF receptor contains multiple cysteine-rich domains (CRD), each CRD has 6 cysteine
  • CRD region plays the role of binding to the ligand.
  • BAFF and APRIL have different binding abilities to these three receptors.
  • BAFF binds TACI stronger than BCMA
  • APRIL binds BCMA stronger than TACI
  • BAFF-R is BAFF's unique high-affinity receptor. Combined with APRIL.
  • TACI is a type III transmembrane protein.
  • Human TACI is a 293 amino acid polypeptide, including the N-terminal region of the protein (amino acid residues 1-165), the transmembrane region (amino acid residues 166-186) and the intracellular region (amino acid residues 166-186). Base 187-293).
  • the extracellular region contains two CRDs (CRD1, CRD2).
  • CRD1 has weaker affinity for BAFF and APRIL than CRD2.
  • exon 2 is skipped to produce a short form of TACI that lacks the CRD1 domain. This short TACI combines BAFF and APRIL as effectively as the long form.
  • Atacicept is a BAFF/APRIL Trap developed by ZymoGenetics.
  • the molecular form is the natural TACI extracellular region fused IgG1-Fc fragment (Fc ⁇ R binding activity is missing), that is, TACI-Fc.
  • Telitacicept (RCT-18) is TACI-Fc developed by Rongchang Biotechnology.
  • Both tatacept and aselcept can simultaneously target the two cytokines BAFF and APRIL.
  • TACI-Fc which inhibits BAFF/APRIL at the same time, can more effectively reduce the body's immune response and achieve therapeutic results.
  • Purpose of SLE The phase II clinical study showed that the 48-week lupus response index (SRI-4) of the high-dose treatment group of tatasercept was significantly higher than that of the placebo control group (79.2% vs 32.0%), reaching the primary endpoint, suggesting that the inhibition of APRIL may be beneficial for Treating lupus is very important.
  • BCMA is a type III transmembrane protein.
  • the human BCMA protein is a 184 amino acid polypeptide, including the N-terminal region of the protein (amino acid residues 1-50), the transmembrane region (amino acid residues 51-93) and the intracellular region (amino acid residues 51-93). residues 94-184).
  • the extracellular domain of BCMA contains a CRD, which binds APRIL stronger than TACI. BCMA can promote the survival of plasma cells and have a negative impact on autoimmune diseases. BCMA is also involved in the development of humoral immunity (such as the production of antibodies) and immune-related diseases. No BCMA extracellular domain or its fusion protein has entered clinical practice yet.
  • BAFF/APRIL Since the overexpression of BAFF/APRIL is closely related to autoimmune diseases, using it as a target molecule to block its activity can reduce the occurrence of the disease and alleviate the symptoms, thereby achieving the purpose of alleviating and treating autoimmune diseases.
  • Relevant receptors that block BAFF/APRIL signaling can specifically act on target cells and are expected to become new biological agents for the treatment of autoimmune diseases. Therefore, there is an urgent need in this field to develop new compounds that can effectively inhibit or block the BAFF/APRIL pathway.
  • the present disclosure provides fusion proteins containing TACI polypeptides, which have the potential to become clinically safe and effective treatments for autoimmune diseases (such as SLE). drug.
  • the present disclosure provides fusion proteins containing novel structures of TACI polypeptides, nucleic acids encoding them, vectors, host cells, pharmaceutical compositions, methods thereof for treating or ameliorating diseases (eg, B cell disorders or autoimmune diseases), and Its pharmaceutical uses.
  • diseases eg, B cell disorders or autoimmune diseases
  • the present disclosure provides a fusion protein selected from the following structures:
  • TACI polypeptides and antibodies e.g., anti-IFNAR1 antibodies
  • BCMA polypeptides and antibodies e.g., anti-IFNAR1 antibodies
  • the TACI polypeptide comprises CRD1 and/or CRD2 of the extracellular domain of TACI, e.g., CRD2 comprising the extracellular domain of TACI.
  • the TACI polypeptide comprises a polypeptide selected from any one of the following (1)-(6):
  • the variant has one or more amino acid mutations selected from positions 69, 72, 73, 77, 85, 102, and 103.
  • the variant has one or more amino acid substitutions selected from 69T or 69R, 72S, 73E or 73Q, 77E, 85T or 85A, 102A or 102R, 103Y.
  • the variant has an amino acid substitution or combination of substitutions selected from any one of the following: 69T; 72S; 73E; 73Q; 77E; 69R/85T; 69R/85A; 102A; 69R/85T/102R; 73E /77E; 72S/73E/77E; 69T/102A; 69T/103Y; 69T/102A, 103Y; 69T/73E/77E/102A.
  • the present disclosure uses "mutation 1/mutation 2" to indicate that mutation 1 and mutation 2 coexist in the mutated variant.
  • “69T/102A” indicates that the mutant contains the amino acid mutations of 69T and 102A
  • “69T/73E/77E/102A” indicates that the mutant contains the amino acid mutations of 69T, 73E, 77E, and 102A.
  • the variant has an amino acid substitution combination of 69T/73E/77E/102A, for example, has an amino acid substitution combination of L69T/K73E/K77E/Y102A.
  • the site of the amino acid mutation is the amino acid residue site numbered relative to the natural sequence of the TACI extracellular region (SEQ ID NO: 1).
  • the variant has an amino acid mutation selected from position 69
  • the amino acid sequence of the TACI polypeptide comprises an amino acid sequence selected from any one of SEQ ID NO: 1, 2, 6-29.
  • At least 90% (sequence) identity encompasses at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% , at least 99% or 100% (sequence) identity;
  • at least 80% (sequence) identity encompasses at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86 %, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, At least 99% or 100% (sequence) identity.
  • the BCMA polypeptide comprises CRD1 of the extracellular domain of BCMA.
  • the BCMA polypeptide comprises amino acid residues 7-41 of SEQ ID NO: 30.
  • the BCMA polypeptide comprises an amino acid sequence set forth in any one of SEQ ID NO: 30, 67, 68 or having at least 90% sequence identity thereto.
  • the antibody in the fusion protein is an anti-IFNAR1 antibody or an antigen-binding fragment thereof.
  • the anti-IFNAR1 antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain variable region comprises an amino acid sequence such as SEQ ID HCDR1, HCDR2 and HCDR3 shown in NO: 45-47, the light chain variable region includes LCDR1, LCDR2 and LCDR3 whose amino acid sequences are shown in SEQ ID NO: 48-50 respectively.
  • VH heavy chain variable region
  • VL light chain variable region
  • the heavy chain variable region comprises an amino acid sequence such as SEQ ID HCDR1, HCDR2 and HCDR3 shown in NO: 45-47
  • the light chain variable region includes LCDR1, LCDR2 and LCDR3 whose amino acid sequences are shown in SEQ ID NO: 48-50 respectively.
  • amino acid sequence of the heavy chain variable region of the anti-IFNAR1 antibody is as shown in SEQ ID NO: 43 or has at least 90% sequence identity thereto
  • amino acid sequence of the light chain variable region is as shown in SEQ ID NO. shown in NO: 44 or having at least 90% sequence identity therewith.
  • the amino acid sequence of the heavy chain of the anti-IFNAR1 antibody is as shown in SEQ ID NO: 55 or has at least 80% sequence identity thereto, and the amino acid sequence of the light chain is as shown in SEQ ID NO: 54 or has at least 80% sequence identity with it. have at least 80% sequence identity.
  • the amino acid sequence of the heavy chain of the anti-IFNAR1 antibody is as shown in SEQ ID NO: 61 or has at least 80% sequence identity thereto, and the amino acid sequence of the light chain is as shown in SEQ ID NO: 54 or has at least 80% sequence identity with it. have at least 80% sequence identity.
  • this disclosure incorporates Anifrolumab, sifalimumab, and in its entirety WO20062002177A, WO2009100309A (e.g., 3F11, 4G5, 11E2, 9D4), WO2020156474A (e.g., 7G4, 10C5, etc.), WO2020057541A (e.g., 8G11H and 485G10H) ), CN201610634601.7 (For example, H19B7+L16C11, H19B7+L8C3, H15D10+L16C11, H15D10+L8C3 or anti-IFNAR1-C1), CN201510685200.
  • WO20062002177A e.g., 3F11, 4G5, 11E2, 9D4
  • WO2020156474A e.g., 7G4, 10C5, etc.
  • WO2020057541A e.g., 8G11H and 485G10H
  • the present disclosure provides fusion proteins comprising TACI polypeptides and BCMA polypeptides, fusion proteins comprising TACI polypeptides and anti-IFNAR1 antibodies (or antigen-binding fragments thereof), fusion proteins comprising TACI polypeptides, BCMA polypeptides, anti-IFNAR1 antibodies (or antigens thereof) binding fragment) fusion protein.
  • the fusion protein further contains the heavy chain constant region (Fc region) of an immunoglobulin.
  • the Fc region contains two subunits capable of associating. In some embodiments, the two subunits are the same or different first subunit and second subunit.
  • the Fc region is an Fc region of IgG, IgA, IgM, or IgD; in some embodiments, the Fc region is an Fc region of human IgG1, IgG2, IgG3, or IgG4, such as an Fc region of human IgG1, and For example, it is the Fc region shown in SEQ ID NO:3.
  • the Fc region in the fusion protein contains one or more amino acid substitutions compared to the wild-type Fc region, and the amino acid substitutions can reduce its binding to the Fc receptor (FcR); some embodiments In, the amino acid substitution can reduce its binding to Fc ⁇ receptor (Fc ⁇ R); in some embodiments, the Fc region has YTE mutations (M252Y, S254T and T256E), S228P, L234F, L235E, L234F/L235E, L234A/L235A or L234F/L235E/P331S mutation, the mutation site numbering basis is the EU index.
  • the Fc region in the fusion protein includes a first subunit and a second subunit capable of associating with each other, the first subunit and the second subunit having one or more reduced homologs. Dimerizing amino acid substitutions.
  • the first subunit has a convex structure according to the pestle and mortar technology
  • the second subunit has a pore structure according to the pestle and mortar technology
  • the first subunit has a pore structure according to the pestle and mortar technology
  • the second subunit has a convex structure according to the pestle and mortar technique.
  • the amino acid residue substitutions of the first subunit include one or more amino acid substitutions selected from positions 354, 356, 358, and 366
  • the amino acid residues of the second subunit include selected One or more amino acid substitutions from positions 349, 356, 358, 366, 368 and 407.
  • the first subunit includes selected from 354C, One or more amino acid substitutions in 356E, 358M and 366W
  • the second subunit includes one or more amino acid substitutions selected from 349C, 356E, 358M, 366S, 368A and 407V.
  • the first subunit includes amino acid substitutions 354C, 356E, 358M, and 366W
  • the second subunit includes amino acid substitutions 349C, 356E, 358M, 366S, 368A, and 407V.
  • the TACI polypeptide is any TACI polypeptide provided by the present disclosure
  • the BCMA polypeptide is any BCMA polypeptide provided by the present disclosure.
  • the fusion protein includes one or more (e.g., 2, 3, 4, 5, 6) of the TACI polypeptides, and one or more (e.g., 2, 3, 4, 5, 6 ) of the BCMA polypeptide. In some embodiments, the fusion protein contains 1 of the TACI polypeptide and 1 of the BCMA polypeptide, or 2 of the TACI polypeptide and 2 of the BCMA polypeptide, or 3 of the TACI polypeptide and 3 of the BCMA polypeptide.
  • the TACI polypeptide and a subunit of the Fc region are connected in any order.
  • the TACI polypeptide and the first subunit or the second subunit of the Fc region are connected in any order.
  • Via connector or direct connection the BCMA polypeptide and a subunit of the Fc region are connected in any order.
  • the BCMA polypeptide is connected to the first subunit or the second subunit of the Fc region. Subunits are connected via linkers or directly.
  • the C-terminus of the TACI polypeptide is connected to the N-terminus of the first subunit or the second subunit of the Fc region through a linker or directly; or the N-terminus of the TACI polypeptide is connected to the first subunit of the Fc region.
  • the C-terminus of the base or the second subunit is connected through a linker or directly.
  • the C-terminus of the BCMA polypeptide is connected to the N-terminus of the first subunit or the second subunit of the Fc region through a linker or directly; or the N-terminus of the BCMA polypeptide is connected to the first subunit of the Fc region.
  • the C-terminus of the base or the second subunit is connected through a linker or directly.
  • the fusion protein containing a TACI polypeptide and a BCMA polypeptide includes a polypeptide chain represented by any one of the following (I)-(VIII):
  • linker 1 represents a peptide bond
  • linker 2 is a polypeptide that can realize the connection function.
  • Linker 1, linker 2, linker 3 and linker 4 can be the same or different;
  • TACI polypeptide 1 and TACI polypeptide 2 are selected from the disclosure.
  • the TACI polypeptides provided may be the same or different;
  • BCMA polypeptide 1 and BCMA polypeptide 2 are selected from the BCMA polypeptides provided by the present disclosure and may be the same or different;
  • a, b, c, d, and e are each independently 0 or 1.
  • a and b are both 1, or a is 1, b is 0, or a is 0, b is 1, and e is 0 or 1; for example, scheme In (III) or (IV), both c and d are 1, or c is 1 and d is 0, or c is 0 and d is 1, and e is 0 or 1; for example, scheme (V), ( In VI), (VII) or (VIII), a, b, c, d are all 1, or a, b, c are all 1 and d is 0, or a, b are 1 and c, d are 0, Either a is 1 and b, c, d are all 0, or a is 0 and b, c, d are all 1, or a, b are 0 and c, d are 1, or a, b, c are 0 and d is 1, or a and c are 1 and b and d are 0, or a and b and d are 0, or a and
  • the linker is (G m S n ) h or (GGNGT) h (SEQ ID NO: 75) or (YGNGT) h (SEQ ID NO: 76) or (EPKSS) h (SEQ ID NO: 77), wherein m and n are each independently selected from an integer of 1-8 (for example, 1, 2, 3, 4, 5, 6, 7 or 8), and h is independently selected from 1 - an integer of 20 (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20).
  • the linker is a (GxS) y linker, wherein x is selected from an integer of 1-5 and y is selected from an integer of 1-6, for example, any one of SEQ ID NO: 39-41 Connector shown.
  • the linker has an amino acid sequence as shown in (G 4 S) y (SEQ ID NO: 78), y is independently selected from an integer of 1 to 6 (for example, y is 1, 2, 3, 4, 5, 6).
  • a fusion protein comprising a TACI polypeptide and a BCMA polypeptide is provided, which contains a polypeptide shown in any one of SEQ ID NOs: 31-38, 69, 70 or having at least 90% sequence identity therewith.
  • the fusion protein is a multimer, such as a dimer (eg, a homodimer or heterodimer).
  • a fusion protein containing a TACI polypeptide and a BCMA polypeptide is provided.
  • the TACI polypeptide may be a variant, and the variant number is 1 to 10 (1, 2, 3, 4, 5, 6, 7, 8 , 9, 10) amino acid mutations; in some embodiments, a fusion protein containing a TACI polypeptide and a BCMA polypeptide is provided, and the BCMA polypeptide can be a variant, and the variant has 1 to 10 (1, 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid mutations; the amino acid mutations may be conservative substitutions, substitutions or modifications, and/or deletions or additions that do not affect function.
  • proteins or molecules are provided that compete with the fusion protein containing a TACI polypeptide and a BCMA polypeptide, the TACI polypeptide, or the BCMA polypeptide for binding to BAFF and /or APRIL, or blocks the fusion protein containing a TACI polypeptide and a BCMA polypeptide, the TACI polypeptide, or the BCMA polypeptide from binding to BAFF and/or APRIL.
  • the TACI polypeptide is any TACI polypeptide provided by the present disclosure
  • the anti-IFNAR1 antibody is any anti-IFNAR1 antibody provided by the present disclosure.
  • the fusion protein comprising a TACI polypeptide and an anti-IFNAR1 antibody includes one or more (eg, 2, 3, 4, 5, 6) TACI polypeptides. In some embodiments, the fusion protein comprising a TACI polypeptide and an anti-IFNAR1 antibody includes one or more (eg, 2, 3, 4) anti-IFNAR1 antibodies.
  • the fusion protein containing a TACI polypeptide and an anti-IFNAR1 antibody includes a polypeptide chain represented by any one of the following (I)-(VIII):
  • the first polypeptide chain which is sequentially from the N-terminus to the C-terminus: [TACI polypeptide]-[linker 1]a-[antibody heavy chain], and the second polypeptide chain, which is the antibody light chain;
  • the first polypeptide chain which from the N-terminus to the C-terminus is: [antibody heavy chain]-[linker 2]b-[TACI polypeptide], and the second polypeptide chain, which is the antibody light chain;
  • the first polypeptide chain, which is an antibody heavy chain, and the second polypeptide chain, which from the N-terminus to the C-terminus are: [TACI polypeptide]-[linker 3]c-[antibody light chain];
  • a first polypeptide chain which is an antibody heavy chain
  • a second polypeptide chain which from the N-terminus to the C-terminus are: [antibody light chain]-[linker 4]d-[TACI polypeptide];
  • the first polypeptide chain, which from the N-terminus to the C-terminus is: [TACI polypeptide 1]-[linker 1]a-[antibody heavy chain], and the second polypeptide chain, which from the N-terminus to The C-terminal order is: [antibody light chain]-[linker 4]d-[TACI polypeptide 2];
  • the first polypeptide chain, which from the N-terminus to the C-terminus is: [antibody heavy chain]-[linker 2]b-[TACI polypeptide 1]
  • the second polypeptide chain, which from the N-terminus to The C-terminal is: [TACI polypeptide 2]-[linker 3]c-[antibody light chain];
  • the first polypeptide chain which from N-terminus to C-terminus is: [TACI polypeptide 1]-[linker 1]a-[antibody heavy chain]-[linker 2]b-[TACI polypeptide 2] , and a second polypeptide chain, which is an antibody light chain;
  • the first polypeptide chain, which is an antibody heavy chain, and the second polypeptide chain, which from the N-terminus to the C-terminus are: [TACI polypeptide 1]-[linker 3]c-[antibody light chain] -[linker 4]d-[TACI polypeptide 2];
  • - represents a peptide bond
  • the linker is a polypeptide that can realize the connection function.
  • Linker 1, linker 2, linker 3, and linker 4 can be the same or different;
  • TACI polypeptide 1 and TACI polypeptide 2 are selected from the disclosure. Any of the aforementioned TACI polypeptides, TACI polypeptide 1 and TACI polypeptide 2 may be the same or different, and a, b, c, and d are each independently 0 or 1.
  • a is 1 or 0.
  • b is 1 or 0.
  • c is 1 or 0.
  • d is 1 or 0.
  • a and d can be 0 or 1 at the same time, or a is 0 and d is 1, or a is 1 and d is 0; in scheme (VI), b and c can be 0 or 0 at the same time. 1, or b is 0 and c is 1, or b is 1 and c is 0; in scheme (VII), a and b can be 0 at the same time or 1, or a is 0, b is 1, or a is 1, b is 0; in scheme (VIII), c and d can be 0 or 1 at the same time, or c is 0, d is 1, or c is 1 , d is 0.
  • the linker is an amino acid sequence shown as (G m S n ) h or (GGNGT) h or (YGNGT) h or (EPKSS) h , wherein m and n are each independently selected from 1- an integer of 8 (e.g., 1, 2, 3, 4, 5, 6, 7, or 8), h is independently selected from an integer of 1-20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20).
  • the linker is a (GxS) y linker, wherein x is selected from an integer of 1-5 and y is selected from an integer of 1-6, for example, any one of SEQ ID NO: 39-41 Connector shown.
  • the linker has an amino acid sequence represented by (G 4 S) y , where y is independently selected from an integer of 1 to 6 (eg, y is 1, 2, 3, 4, 5, 6).
  • a fusion protein comprising a TACI polypeptide and an anti-IFNAR1 antibody
  • a fusion protein comprising a TACI polypeptide and an anti-IFNAR1 antibody
  • a first polypeptide chain as set forth in SEQ ID NO: 55 or having at least 80%, at least 90% sequence identity thereto, and as set forth in or having at least 80% sequence identity to any of SEQ ID NO: 56-60 , a second polypeptide chain with at least 90% sequence identity; or
  • a first polypeptide chain as set forth in SEQ ID NO: 61 or having at least 80%, at least 90% sequence identity thereto, and as set forth in any of SEQ ID NO: 56-60 or having at least 80% sequence identity thereto %, a second polypeptide chain with at least 90% sequence identity.
  • the aforementioned fusion protein includes two identical first polypeptide chains, and two identical second polypeptide chains.
  • the present disclosure provides a fusion protein containing a TACI polypeptide and an anti-IFNAR1 antibody.
  • the TACI polypeptide may be a variant, and the variant number is 1 to 10 (1, 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid mutations; the amino acid mutations may be conservative substitutions, substitutions or modifications, and/or deletions or additions that do not affect function.
  • a protein or molecule that competes with the aforementioned fusion protein containing a TACI polypeptide and an anti-IFNAR1 antibody for binding to BAFF and/or APRIL, or blocks its binding to BAFF and/or APRIL.
  • the TACI polypeptide is any TACI polypeptide provided by the present disclosure
  • the BCMA polypeptide is any BCMA polypeptide provided by the present disclosure
  • the anti-IFNAR1 antibody is any anti-IFNAR1 provided by the present disclosure. Antibody.
  • the antibody fusion protein comprising a TACI polypeptide, a BCMA polypeptide and an anti-IFNAR1 antibody includes one or more (eg, 2, 3, 4, 5, 6) TACI polypeptides. In some embodiments, the antibody fusion protein comprising a TACI polypeptide, a BCMA polypeptide and an anti-IFNAR1 antibody includes one or more (eg, 2, 3, 4, 5, 6) BCMA polypeptides. In some embodiments, the composition includes The antibody fusion protein of a TACI polypeptide, a BCMA polypeptide and an anti-IFNAR1 antibody includes one or more (eg, 2, 3, or 4) anti-IFNAR1 antibodies.
  • the aforementioned antibody fusion protein comprising a TACI polypeptide, a BCMA polypeptide and an anti-IFNAR1 antibody includes 1 aforementioned TACI polypeptide and 1 aforementioned BCMA polypeptide, or 2 aforementioned TACI polypeptides and 2 aforementioned BCMA polypeptides, or 3.
  • a fusion protein containing a TACI polypeptide, a BCMA polypeptide and an anti-IFNAR1 antibody, as described in any one of the preceding items contains a polypeptide chain represented by any one of the following (I)-(VIII):
  • a first polypeptide chain which is in sequence from N-terminus to C-terminus: [TACI polypeptide and BCMA polypeptide domain]-[linker 1]a-[antibody heavy chain], and a second polypeptide chain, which is the antibody light chain;
  • the first polypeptide chain which from N-terminus to C-terminus is: [antibody heavy chain]-[linker 2]b-[TACI polypeptide and BCMA polypeptide domain], and the second polypeptide chain, which is the antibody light chain;
  • the first polypeptide chain, which is an antibody heavy chain, and the second polypeptide chain, which from N-terminus to C-terminus are: [TACI polypeptide and BCMA polypeptide domain]-[linker 3]c-[ Antibody light chain];
  • a first polypeptide chain which is an antibody heavy chain
  • a second polypeptide chain which from the N-terminus to the C-terminus are: [antibody light chain]-[linker 4]d-[TACI polypeptide and BCMA polypeptide domain];
  • V a first polypeptide chain, which from the N-terminus to the C-terminus is: [TACI polypeptide and BCMA polypeptide domain 1]-[linker 1]a-[antibody heavy chain], and the second polypeptide chain, From N-terminus to C-terminus, they are: [antibody light chain]-[linker 4]d-[TACI polypeptide and BCMA polypeptide domain 2];
  • the first polypeptide chain which from the N-terminus to the C-terminus is: [antibody heavy chain]-[linker 2]b-[TACI polypeptide and BCMA polypeptide domain 1]
  • the second polypeptide chain From N-terminus to C-terminus, they are: [TACI polypeptide and BCMA polypeptide domain 2]-[linker 3]c-[antibody light chain];
  • the first polypeptide chain which from N terminus to C terminus is: [TACI polypeptide and BCMA polypeptide domain 1]-[linker 1]a-[antibody heavy chain]-[linker 2]b- [TACI polypeptide and BCMA polypeptide domain 2], and a second polypeptide chain, which is an antibody light chain;
  • the first polypeptide chain, which is an antibody heavy chain, and the second polypeptide chain, which from the N-terminus to the C-terminus are: [TACI polypeptide and BCMA polypeptide domain 1]-[linker 3]c- [Antibody light chain]-[linker 4]d-[TACI polypeptide and BCMA polypeptide domain 2];
  • [TACI polypeptide and BCMA polypeptide domain] "TACI polypeptide and BCMA polypeptide domain 1]", [TACI polypeptide and BCMA polypeptide domain 2]" from N-terminal to C-terminal are: [TACI polypeptide] - [Linker 5]e-[BCMA polypeptide], or: [BCMA polypeptide]-[Linker 5]e-[TACI polypeptide], "TACI polypeptide]
  • the TACI in "peptide and BCMA polypeptide domain 1" and "TACI polypeptide and BCMA polypeptide domain 2" can be the same or different, and the BCMA can be the same or different;
  • - represents a peptide bond
  • the linker is a polypeptide that can realize the connection function.
  • Linker 1, linker 2, linker 3, linker 4, and linker 5 can be the same or different; a, b, c, d , e are independently 0 or 1 respectively. a, b, c, d are independently 0 or 1.
  • a is 1 or 0.
  • b is 1 or 0.
  • c is 1 or 0.
  • d is 1 or 0.
  • a and d can be 0 or 1 at the same time, or a is 0 and d is 1, or a is 1 and d is 0; in scheme (VI), b and c can be 0 or 0 at the same time. 1, or b is 0 and c is 1, or b is 1 and c is 0; in scheme (VII), a and b can be 0 or 1 at the same time, or a is 0, b is 1, or a is 1 , b is 0; in scheme (VIII), c and d can be 0 or 1 at the same time, or c is 0, d is 1, or c is 1, d is 0; in the above scheme, e can be 0 independently or 1.
  • the linker is an amino acid sequence shown as (G m S n ) h or (GGNGT) h or (YGNGT) h or (EPKSS) h , wherein m and n are each independently selected from 1 to 8. an integer (e.g., 1, 2, 3, 4, 5, 6, 7, or 8), h is independently selected from an integer of 1-20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20).
  • the linker is a (GxS) y linker, wherein x is selected from an integer of 1-5 and y is selected from an integer of 1-6, for example, any one of SEQ ID NO: 39-41 Connector shown.
  • the linker has an amino acid sequence represented by (G 4 S) y , where y is independently selected from an integer of 1 to 6 (eg, y is 1, 2, 3, 4, 5, 6).
  • a fusion protein comprising a TACI polypeptide, a BCMA polypeptide and an anti-IFNAR1 antibody
  • a fusion protein comprising a TACI polypeptide, a BCMA polypeptide and an anti-IFNAR1 antibody
  • which comprises a first polypeptide chain as set forth in SEQ ID NO: 55 or having at least 80%, at least 90% sequence identity thereto.
  • a second polypeptide chain as shown in SEQ ID NO: 63-66, 71, 72 or having at least 80%, at least 90% sequence identity therewith;
  • a first polypeptide chain as set forth in SEQ ID NO: 61 or having at least 80%, at least 90% sequence identity thereto, and as set forth in or with any of SEQ ID NO: 63-66, 71, 72
  • a second polypeptide chain having at least 80%, at least 90% sequence identity; or
  • a first polypeptide chain as set forth in SEQ ID NO: 73 or 74 or having at least 80%, at least 90% sequence identity thereto, and as set forth in SEQ ID NO: 54 or having at least 80%, A second polypeptide chain with at least 90% sequence identity.
  • the aforementioned fusion protein includes two identical first polypeptide chains, and two identical second polypeptide chains.
  • the present disclosure provides a fusion protein containing a TACI polypeptide, a BCMA polypeptide and an anti-IFNAR1 antibody.
  • the TACI polypeptide can be a variant, and the variant number is 1 to 10 (1, 2, 3, 4, 5 , 6, 7, 8, 9, 10) amino acid mutations; the amino acid mutations may be conservative substitutions, substitutions or modifications, and/or deletions or additions that do not affect function.
  • proteins or molecules are provided that compete with, or block the binding of, BAFF and/or APRIL to the aforementioned fusion protein containing a TACI polypeptide, a BCMA polypeptide, and an anti-IFNAR1 antibody. Combine BAFF and/or APRIL.
  • the fusion protein containing a TACI polypeptide and a BCMA polypeptide, a TACI polypeptide and an IFNAR1 antibody, or a fusion protein containing a TACI polypeptide, a BCMA polypeptide and an IFNAR1 antibody has one or more of the following properties or functions:
  • the TACI polypeptide-containing protein (e.g., TACI-Fc) provided by the present disclosure has one or more of the following functional activities:
  • fragments of the TACI polypeptide that are less prone to fragmentation in some embodiments, the fragmentation can be detected by mass spectrometry analysis, such as the TACI fragmentation analysis method in Example 2;
  • the IC 50 value of TACI-Fc blocking the binding of BAFF to BAFF-R is less than 23nM, less than 10nM, less than 6nM, less than 5nM, less than 4nM, less than 3nM, Less than 2nM, less than 1nM or less; the IC 50 value is detected by ELISA method; in some embodiments, the IC 50 value testing method is shown in Example 1;
  • the TACI-Fc has an EC 50 value for binding BAFF of less than 5 nM, 1 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, 0.1 nM or less, said The EC 50 value is detected by ELISA method, such as the ELISA binding experiment in Example 2 (coated antigen protein);
  • the IC 50 value of TACI-Fc for inhibiting BAFF-induced B cell proliferation is less than 0.3nM, less than 0.2nM, less than 0.1nM, 0.06nM, less than 0.05nM, less than 0.01nM or smaller.
  • the IC 50 value is detected by an ELISA method, such as the ELISA experiment in Example 17;
  • the IC 50 value of TACI-Fc for inhibiting APRIL-induced B cell proliferation is less than 3nM, less than 2nM, less than 1nM, less than 0.5nM, less than 0.4nM, less than 0.3nM, 0.2 nM, less than 0.1 nM, less than 0.05 or less; the IC 50 value is detected by ELISA method;
  • the affinity is detected by the Biacore method; in some embodiments, the KD value of the TACI-Fc fusion protein binding to human BAFF is less than 1E -10M, less than 9E-11M, 8E-11M or less; in some embodiments, the KD value of the TACI-Fc fusion protein binding to human APRIL is less than 2.3E-11M, less than 2.0E-11M, 1.9E- 11M, 1.3E-11M or smaller;
  • the in vivo half-life of TACI-Fc in rats is greater than 4 days;
  • the TACI-Fc is stored at a constant temperature of 40°C for four weeks, the purity of the TACI-Fc fusion protein can still be maintained above 94% (SEC%); in some embodiments , the pI value of the TACI-Fc is less than 9, less than 8, less than 7, less than 6 or less, and the pI value is determined by the "18cProt/TrEMBL" system analysis.
  • the aforementioned fusion protein containing a TACI polypeptide and a BCMA polypeptide provided by the present disclosure has one or more of the following functional activities:
  • fragments of the TACI polypeptide that are less prone to fragmentation in some embodiments, the fragmentation can be detected by mass spectrometry analysis, such as the TACI fragmentation analysis method in Example 2;
  • the EC 50 value of the fusion protein binding to BAFF is less than 1 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, 0.1 nM or less, and the EC 50 The value is detected by ELISA method, such as the ELISA experiment of Example 5;
  • the EC 50 value of the fusion protein binding to APRIL is less than 1 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, 0.1 nM or less, the EC 50 value Detected by ELISA method, such as the ELISA experiment of Example 5;
  • the antibody fusion protein comprising a TACI polypeptide, a BCMA polypeptide and an anti-IFNAR1 antibody provided by the disclosure has one or more of the following functional activities:
  • fragments of the TACI polypeptide that are less prone to fragmentation in some embodiments, the fragmentation can be detected by mass spectrometry analysis, such as the TACI fragmentation analysis method in Example 2;
  • the fusion protein has an inhibition rate on the activity of IFN that is consistent or similar to that of an anti-IFNAR1 antibody (such as Anifrolumab), and the inhibition rate is determined by Methods for detecting the IFN- ⁇ / ⁇ reporter gene, such as the experiment in Example 10; the inhibition rate in vivo is by detecting the mRNA expression of the IFN- ⁇ downstream gene ISG in PBMC, such as the experiment in Example 21;
  • the EC 50 value of the fusion protein binding BAFF is less than 1.5nM, 1nM, 0.5nM, 0.4nM, 0.3nM, 0.2nM, 0.1nM or less, said The EC 50 value is detected by ELISA method, such as the ELISA experiment in Example 11;
  • the EC 50 value of the fusion protein binding to APRIL is less than 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, 0.1 nM or less, and the EC 50 value is determined by ELISA Method detection, such as the ELISA experiment of Example 11;
  • the fusion protein has an effect on IFN- ⁇ cytokine secretion by plasmacytoid dendritic cells (pDC) that is consistent or similar to that of an anti-IFNAR1 antibody (eg, Anifrolumab).
  • an anti-IFNAR1 antibody eg, Anifrolumab
  • the inhibitory ability is detected using, for example, the detection method in Example 15; in some embodiments, the fusion protein has an inhibitory effect that is consistent or similar to that of an anti-IFNAR1 antibody (such as Anifrolumab).
  • the ability to inhibit IFN- ⁇ -induced B cell differentiation into plasma cells in vitro, and the inhibitory ability is detected using, for example, the detection method in Example 16;
  • the fusion protein inhibits BAFF-induced B cell proliferation with an inhibition rate of at least 50%, at least 40%, at least 30%, at least 20 %, at least 10%, the inhibition rate is the ratio of the number of B cells after adding the antibody fusion protein to the number of B cells after adding the same concentration of IgG1, and is detected by FACS, such as the method in Example 17;
  • the fusion protein inhibits APRIL-induced B cell proliferation by at least 50%, at least 40%, at least 30%, at least 20%, at least 10%
  • the inhibition rate is the ratio of the number of B cells after adding the antibody fusion protein to the number of B cells after adding the same concentration of IgG1.
  • the inhibition rate is detected by FACS, such as the method in Example 18;
  • the fusion protein inhibits the production of plasma cells induced by BAFF and APRIL, with an inhibition rate of at least 90%, at least 80%, at least 70%, at least 60%, At least 50%, at least 40%, at least 30%, at least 20%, the inhibition rate is the ratio of the number of B cells after adding the antibody fusion protein to the number of B cells after adding the same concentration of IgG1, the inhibition is detected by FACS , such as the method in Example 19;
  • the fusion protein inhibits the production of IgA in vivo by at least 80%, at least 70%, at least 60%, at least 50%, at least 40%, at least 30%, The inhibition rate is compared with PBS of the same concentration, and the inhibition is detected by ELISA, such as the method in Example 22;
  • a product or combination which includes any of the aforementioned fusion proteins of the present disclosure containing a TACI polypeptide and a BCMA polypeptide, and also includes an antibody.
  • a product or combination that includes any of the aforementioned TACI-containing polypeptides of the present disclosure, and further includes an antibody.
  • the antibody is an anti-IFNAR1 antibody, such as Anifrolumab.
  • a complex which contains any of the foregoing TACI polypeptides of the present disclosure and any of the foregoing BCMA polypeptides of the present disclosure.
  • the TACI polypeptide and BCMA polypeptide may be operably connected directly or through any of the aforementioned linkers of the present disclosure, or may not be connected.
  • a complex which contains any of the aforementioned TACI polypeptides of the present disclosure and any of the aforementioned BCMA polypeptides of the present disclosure; or contains any of the aforementioned TACI polypeptides of the present disclosure and any of the aforementioned anti-IFNAR1 Antibody; or containing any BCMA polypeptide of the aforementioned disclosure and any anti-IFNAR1 antibody of the aforementioned disclosure; or containing any TACI polypeptide of the aforementioned disclosure and any one of the anti-IFNAR1 antibody of the aforementioned disclosure and any one of the aforementioned anti-IFNAR1 antibody BCMA polypeptide.
  • the TACI polypeptide, BCMA polypeptide, and anti-IFNAR1 antibody may be operably connected directly or through any of the aforementioned linkers of the present disclosure, or may not be connected.
  • the present disclosure provides (isolated) polynucleotides encoding a TACI polypeptide, a BCMA polypeptide, a fusion protein comprising a TACI polypeptide and a BCMA polypeptide, a fusion comprising a TACI polypeptide and an antibody (e.g., an anti-IFNAR1 antibody) of any of the aforementioned disclosures Proteins, fusion proteins, products, combinations or complexes comprising a TACI polypeptide and a BCMA polypeptide and an antibody (eg, an anti-IFNAR1 antibody).
  • the polynucleotide may be DNA or RNA (eg, mRNA).
  • the nucleic acids of the present disclosure may also be in the form of a vector, may be present in a vector, and/or may be part of a vector, such as a plasmid, cohesive plasmid, YAC, or viral vector.
  • the vector may in particular be an expression vector, ie a vector providing for expression of the CD40 binding molecule in vitro and/or in vivo (ie in a suitable host cell, host organism and/or expression system).
  • the expression vector typically contains at least one nucleic acid of the present disclosure operably linked to one or more suitable expression control elements (eg, promoter, enhancer, terminator, etc.). Selection of the elements and their sequences for expression in a particular host is common knowledge to those skilled in the art.
  • TACI polypeptides BCMA polypeptides, fusion proteins comprising TACI polypeptides and BCMA polypeptides, fusion proteins comprising TACI polypeptides and antibodies (e.g., anti-IFNAR1 antibodies), TACI polypeptides and BCMA polypeptides and antibodies (e.g., anti-IFNAR1 antibodies) of the present disclosure
  • Regulatory elements and other elements useful or necessary for the expression of fusion proteins, products, combinations or complexes are, for example, promoters, enhancers, terminators, integrators, selection markers, leader sequences, and reporter genes.
  • Nucleic acids of the present disclosure may be prepared or obtained by known means (eg, by automated DNA synthesis and/or recombinant DNA technology) based on information on the amino acid sequences of the polypeptides of the present disclosure, and/or may be isolated from suitable natural sources.
  • the disclosure provides for expressing or being capable of expressing one or more TACI polypeptides, BCMA polypeptides, fusion proteins comprising a TACI polypeptide and a BCMA polypeptide, fusion proteins comprising a TACI polypeptide and an antibody (e.g., an anti-IFNAR1 antibody), comprising a TACI polypeptide of the present disclosure. and fusion proteins, products, combinations or complexes of BCMA polypeptides and antibodies (eg, anti-IFNAR1 antibodies), and/or recombinant host cells containing nucleic acids or vectors of the present disclosure.
  • an antibody e.g., an anti-IFNAR1 antibody
  • the host cell is a bacterial cell, a fungal cell, or a mammalian cell.
  • Bacterial cells include, for example, Gram-negative bacterial strains (such as Escherichia coli strains, Proteus strains, and Pseudomonas strains) and Gram-positive bacterial strains (such as Bacillus spp. (Bacillus) strains, Streptomyces strains, Staphylococcus strains and Lactococcus strains) cells.
  • Gram-negative bacterial strains such as Escherichia coli strains, Proteus strains, and Pseudomonas strains
  • Gram-positive bacterial strains such as Bacillus spp. (Bacillus) strains, Streptomyces strains, Staphylococcus strains and Lactococcus strains
  • Fungal cells include, for example, cells of species of the genus Trichoderma, Neurospora, and Aspergillus; or include cells of the genus Saccharomyces (such as Saccharomyces cerevisiae), fission yeast Schizosaccharomyces (such as Schizosaccharomyces pombe), Pichia (such as Pichia pastoris and Pichia methanolica), and Hansen of species of the genus Hansenula cell.
  • Saccharomyces such as Saccharomyces cerevisiae
  • fission yeast Schizosaccharomyces such as Schizosaccharomyces pombe
  • Pichia such as Pichia pastoris and Pichia methanolica
  • Hansen of species of the genus Hansenula cell Hansen of species of the genus Hansenula cell.
  • Mammalian cells include, for example, HEK293 cells, CHO cells, BHK cells, HeLa cells, COS cells, and the like.
  • the present disclosure may also use amphibian cells, insect cells, plant cells, and any other cells in the art for expressing heterologous proteins.
  • the cells of the present disclosure cannot develop into completed plant or animal individuals.
  • the present disclosure provides a method for preparing a TACI polypeptide, a BCMA polypeptide, a fusion protein comprising a TACI polypeptide and a BCMA polypeptide, a fusion protein comprising a TACI polypeptide and an antibody (e.g., an anti-IFNAR1 antibody), a fusion protein comprising a TACI polypeptide and a BCMA polypeptide and an antibody (e.g., an anti-IFNAR1 antibody) , anti-IFNAR1 antibody) fusion protein, product, combination or complex method, including: expressing the aforementioned polypeptide, fusion protein, product, combination or complex in a host cell as described above, and isolating it from the host cell The polypeptide, fusion protein, product, combination or complex.
  • an antibody e.g., an anti-IFNAR1 antibody
  • purification steps may also be included.
  • filtration and concentration steps may also be included. Soluble mixtures and polymers can also be removed by conventional methods, such as molecular sieves and ion exchange. The obtained product needs to be frozen immediately, such as -70°C, or freeze-dried.
  • the polypeptides, fusion proteins, products, combinations or complexes may be prepared and purified by conventional methods.
  • the cDNA sequence encoding the fusion protein can be cloned and recombined into an expression vector.
  • the recombinant expression vector can stably transfect CHO cells. Mammalian expression systems result in glycosylation of the protein (e.g., at the highly conserved N-terminus of the Fc region).
  • the positive clones were expanded and cultured in the bioreactor medium to produce proteins.
  • the culture medium secreted with proteins can be purified, collected, filtered, and concentrated using conventional techniques. Soluble mixtures and polymers can also be removed by conventional methods, such as molecular sieves and ion exchange.
  • the polypeptides, fusion proteins, products, combinations or complexes of the present disclosure may also be obtained by other methods of producing proteins known in the art, such as chemical synthesis, including solid phase or liquid phase synthesis.
  • compositions such as pharmaceutical compositions, containing a therapeutically effective amount of a TACI polypeptide, a BCMA polypeptide, a fusion protein comprising a TACI polypeptide and a BCMA polypeptide, a fusion protein comprising a TACI polypeptide and an antibody (e.g., an anti-IFNAR1 antibody), comprising Fusion proteins, products, combinations, complexes or polynucleotides of TACI polypeptides and BCMA polypeptides and antibodies (eg, anti-IFNAR1 antibodies), and one or more pharmaceutically acceptable excipients, diluents or carriers.
  • an antibody e.g., an anti-IFNAR1 antibody
  • the unit dose of the pharmaceutical composition may contain 0.01 to 99% by weight of any of the aforementioned polypeptides, fusion proteins, products, combinations, complexes or polynucleotides. In some specific embodiments, the unit dosage of the pharmaceutical composition contains any of the aforementioned polypeptides, fusion proteins, products, combinations, complexes or polynucleotides in an amount of 0.1-2000 mg, in some specific embodiments 1 -1000mg.
  • kits comprising a TACI polypeptide of the disclosure, a BCMA polypeptide, a fusion protein comprising a TACI polypeptide and a BCMA polypeptide, a fusion comprising a TACI polypeptide and an antibody (e.g., an anti-IFNAR1 antibody)
  • an antibody e.g., an anti-IFNAR1 antibody
  • a protein, fusion protein, product, combination, complex or polynucleotide comprising a TACI polypeptide and a BCMA polypeptide and an antibody eg, an anti-IFNAR1 antibody.
  • diagnostic reagents comprising the above polynucleotides are also provided, as well as related diagnostic uses.
  • the present disclosure provides a method of treating or ameliorating a disease or disorder, comprising administering to a subject in need thereof a therapeutically effective amount of a TACI polypeptide, a BCMA polypeptide, a polypeptide comprising a TACI polypeptide and a BCMA polypeptide of any one of the foregoing disclosures.
  • fusion proteins fusion proteins comprising TACI polypeptides and antibodies (e.g., anti-IFNAR1 antibodies), fusion proteins comprising TACI polypeptides and BCMA polypeptides and antibodies (e.g., anti-IFNAR1 antibodies), products, combinations, complexes, polynucleotides, or pharmaceutical compositions.
  • the disease or disorder is one associated with TACI, BCMA and/or IFN expression.
  • methods or pharmaceutical uses of any of the aforementioned TACI polypeptides, BCMA polypeptides and anti-IFNAR1 antibodies of the present disclosure are provided for the combined treatment of diseases or conditions.
  • a method or pharmaceutical use of any of the aforementioned TACI polypeptides of the present disclosure and an anti-IFNAR1 antibody in combination to treat a disease or disorder further include combinations with BCMA polypeptides.
  • a method or pharmaceutical use of a fusion protein containing a BCMA polypeptide and an anti-IFNAR1 antibody according to any of the aforementioned disclosures is provided for treating diseases or conditions.
  • Some specific embodiments further include combinations with TACI polypeptides.
  • the anti-IFNAR1 antibody is, for example, Anifrolumab.
  • a method of treating or ameliorating B cell disorders or autoimmune diseases comprising administering to a subject in need thereof a therapeutically effective amount of a TACI polypeptide, a BCMA polypeptide, a polypeptide comprising a TACI polypeptide of the present disclosure.
  • a TACI polypeptide, a BCMA polypeptide, a fusion protein comprising a TACI polypeptide and a BCMA polypeptide, a fusion protein comprising a TACI polypeptide and an antibody (e.g., an anti-IFNAR1 antibody), a TACI polypeptide and a BCMA polypeptide and an antibody (e.g., an anti-IFNAR1 antibody) of the present disclosure e.g., an anti-IFNAR1 antibody
  • an anti-IFNAR1 antibody e.g., an anti-IFNAR1 antibody
  • the disease or disorder is a B cell disorder or an autoimmune disease.
  • the disease or disorder is associated with TACI expression, or with BCMA expression, or with IFN expression, or with expression of any two or three of TACI, BCMA, IFN.
  • the expression is, for example, excessive or abnormal expression.
  • the autoimmune disease is selected from: systemic lupus erythematosus, myasthenia gravis, multiple sclerosis, insulin-dependent diabetes, Crohn's disease, rheumatoid arthritis, polyarticular juvenile rheumatoid arthritis inflammation and psoriatic arthritis.
  • the B cell disorder is selected from the group consisting of: neoplasm, chronic leukemia, multiple myeloma, non-Hodgkin lymphoma, post-transplant lymphoid hyperplasia illness as well as light chain gammopathy.
  • the autoimmune disease is systemic lupus erythematosus.
  • cytokine is a general term for proteins released by one cell population that act as intercellular mediators on other cells.
  • cytokines include lymphokines, monokines, chemokines, and traditional peptide hormones.
  • exemplary cytokines include: IL-2, IFN- ⁇ , IL-6, TNF ⁇ , IL-17, and IL-5.
  • the TACI transmembrane activator and calcium modulator and cyclophilinligand-interactor
  • Human TACI belongs to the TNFR superfamily and is a 293 amino acid polypeptide, including the N-terminal region of the protein (amino acid residues 1- 165, see SEQ ID NO: 1) of the present disclosure, the transmembrane region (amino acid residues 166-186) and the intracellular region (amino acid residues 187-293).
  • the extracellular region contains two cysteine-rich pseudo-repeats (CRD1, CRD2).
  • CRD1 of TACI in the present disclosure encompasses wild-type CRD1 of human TACI (e.g., amino acids 33-67 of SEQ ID NO: 1) and variants thereof.
  • CRD2 of TACI in the present disclosure encompasses wild-type CRD2 of human TACI (for example, amino acids 70-104 of SEQ ID NO: 1, or amino acids 69-104) and its variants (including but not limited to the present disclosure).
  • the CRD1, CRD2 and variants of the TACI are all capable of binding to APRIL and/or BAFF, for example, binding to APRIL and BAFF at the same time.
  • the detection method of the binding is well known in the art, for example, see the embodiments of the present disclosure. Provided.
  • BCMA B cell maturation antigen
  • the human BCMA protein is a polypeptide of 184 amino acids, including the N-terminal region of the protein (amino acid residues 1-50, see SEQ ID NO. : Amino acids 1-50 of 30), transmembrane region (amino acid residues 51-93) and intracellular region (amino acid residues 94-184).
  • the extracellular domain of BCMA contains a CRD (or CRD1).
  • CRD1 of BCMA in the present disclosure encompasses wild-type CRD1 of human BCMA (eg, amino acids 7-41 of SEQ ID NO: 30 of the present disclosure) and variants thereof.
  • the BCMA CRD1 or its variants are able to interact with APRIL and /or BAFF binds, for example, binds to APRIL and BAFF simultaneously.
  • the detection method of the binding is well known in the art, for example, see what is provided in the embodiments of the present disclosure.
  • TACI extracellular domain and “TACI extracellular domain” are interchangeable, and “BCMA extracellular domain” and “BCMA extracellular domain” are interchangeable.
  • interferon alpha is used interchangeably and shall refer to the IFN alpha protein encoded by the functional gene of the interferon alpha locus, which is identical to IFN alpha 1 (GenBank accession No. NP_076918 or GenBank accession no. NM_024013) has a sequence identity of 75% or more.
  • IFN ⁇ subtypes include IFN ⁇ 1, ⁇ 2a, ⁇ 2b, ⁇ 4, ⁇ 4b, ⁇ 5, ⁇ 6, ⁇ 7, ⁇ 8, ⁇ 10, ⁇ 13, ⁇ 14, ⁇ 16, ⁇ 17 and ⁇ 21.
  • Interferon alpha shall include recombinant forms of the various IFN alpha subtypes, as well as naturally occurring preparations containing IFN alpha proteins, such as leukocyte IFN and lymphoblastoid IFN.
  • interferon alpha receptor-1 interferon alpha receptor-1
  • IFNAR1 immunoglobulin-1
  • IFNAR-1 antigen include variants, isoforms, germline homologs of human IFNAR-1 , and analogs that share at least one epitope in common with IFNAR-1.
  • the human antibodies of the invention may, in some embodiments, cross-react with IFNAR-1 from species other than humans, or with other proteins structurally related to human IFNAR-1 (eg, human IFNAR-1 homologs). In other embodiments, the antibody may be completely specific for human IFNAR-1, showing no species or other type of cross-reactivity.
  • the complete cDNA sequence of human IFNAR-1 has GenBank accession number NM_000629.
  • antibody is used in the broadest sense and covers a variety of antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies; monospecific antibodies, multispecific antibodies (e.g., bispecific antibodies), full-length antibodies, and antibody fragments (or antigen-binding fragment, or antigen-binding portion) as long as they exhibit the desired antigen-binding activity.
  • Antibodies can refer to immunoglobulins, which are tetrapeptide chain structures composed of two heavy chains and two light chains connected by inter-chain disulfide bonds. The amino acid composition and sequence of the immunoglobulin heavy chain constant region are different, so their antigenicity is also different.
  • immunoglobulins can be divided into five categories, or isotypes of immunoglobulins, namely IgM, IgD, IgG, IgA and IgE, and their corresponding heavy chains are ⁇ chain, ⁇ chain and ⁇ chain respectively. , ⁇ chain and ⁇ chain.
  • IgM immunoglobulins
  • IgD immunoglobulins
  • IgG immunoglobulins
  • IgA immunoglobulins
  • IgE immunoglobulins
  • heavy chains are ⁇ chain, ⁇ chain and ⁇ chain respectively.
  • ⁇ chain and ⁇ chain The same type of Ig can be divided into different subclasses based on differences in the amino acid composition of its hinge region and the number and position of heavy chain disulfide bonds.
  • IgG can be divided into IgG1, IgG2, IgG3, and IgG4.
  • Light chains are divided into kappa or lambda chains through differences in constant regions.
  • Each of the five types of Ig can have a kappa chain or a lambda chain.
  • the sequence of about 110 amino acids near the N-terminus of antibody heavy and light chains varies greatly and is the variable region (V region); the remaining amino acid sequences near the C-terminus are relatively stable and is the constant region (C region).
  • the variable region includes 3 hypervariable regions (CDR) and 4 framework regions (FR) with relatively conserved sequences. Three hypervariable regions determine the specificity of the antibody, also known as complementarity determining regions (CDRs).
  • Each light chain variable region (VL) and heavy chain variable region (VH) consists of 3 CDR regions and 4 FR regions.
  • the order from the amino terminus to the carboxyl terminus is: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the three CDR regions of the light chain refer to LCDR1, LCDR2, and LCDR3; the three CDR regions of the heavy chain refer to HCDR1, HCDR2, and HCDR3.
  • Antibody in this disclosure encompasses "antigen-binding fragments" including: single-chain antibodies (i.e., full-length heavy and light chains); Fab, modified Fab, Fab', modified Fab' , F(ab')2, Fv, Fab-Fv, Fab-dsFv, single domain antibody (such as VH or VL or VHH), scFv, bivalent or trivalent or quadrivalent antibody, Bis-scFv, diabody, tribody , triabody, tetrabody and epitope-binding fragments of any of the above (see, for example, Holliger and Hudson, 2005, Nature Biotech. 23(9): 1126-1136; Adair and Lawson, 2005, Drug Design Reviews-Online 2(3) ,209-217).
  • the definitive delineation of the CDRs and the identification of the residues comprising the binding site of the antibody can be accomplished by resolving the structure of the antibody and/or resolving the structure of the antibody-ligand complex. This can be accomplished by any of a variety of techniques known to those skilled in the art, such as X-ray crystallography.
  • a variety of analytical methods can be used to identify CDRs, including but not limited to Kabat numbering system, Chothia numbering system, AbM numbering system, IMGT numbering system, contact definition, and conformation definition.
  • the Kabat numbering system is the standard for numbering residues in antibodies and is commonly used to identify CDR regions (see, e.g., Johnson & Wu, 2000, Nucleic Acids Res., 28:214-8).
  • the Chothia numbering system is similar to the Kabat numbering system, but the Chothia numbering system takes into account the position of certain structural ring regions. (See, for example, Chothia et al., 1986, J. Mol. Biol., 196:901-17; Chothia et al., 1989, Nature, 342:877-83).
  • the AbM numbering system uses an integrated suite of computer programs for modeling antibody structures produced by the Oxford Molecular Group (see, e.g., Martin et al., 1989, ProcNatl Acad Sci (USA), 86:9268-9272; "AbMTM, A Computer Program for ModelingVariable Regions of Antibodies," Oxford, UK; Oxford Molecular, Ltd).
  • the AbM numbering system uses a combination of knowledge databases and ab initio approaches to model the tertiary structure of antibodies from basic sequences (see Samudrala et al., 1999, "Ab” in PROTEINS, Structure, Function and Genetics Suppl., 3:194-198 Those described in "Initio Protein Structure Prediction Using a Combined HierarchicalApproach”).
  • CDR positions can be identified as residues that contribute to antigen binding (see, e.g., Makabe et al., 2008, Journal of Biological Chemistry, 283:1156-1166).
  • CDR boundary definitions may not strictly follow one of the above methods, but still overlap with at least part of the Kabat CDR, although they may be shortened or lengthened based on predictions or experimental results that a particular residue or group of residues does not significantly affect antigen binding.
  • a CDR may refer to a CDR defined by any method known in the art, including combinations of methods. The correspondence between various numbering systems is well known to those skilled in the art, and is exemplarily shown in Table 1 below.
  • Fc region or "fragment crystallizable region” is used to define the C-terminal region of an antibody heavy chain, including native Fc regions and engineered Fc regions.
  • the Fc region contains two subunits that are the same or different.
  • the Fc region of a human IgG heavy chain is defined as extending from the amino acid residue at position Cys226 or from Pro230 to its carboxy terminus.
  • Suitable Fc regions for use in the antibodies described herein include those of human IgGl, IgG2 (IgG2A, IgG2B), IgG3 and IgG4.
  • the boundaries of the Fc region may also be varied, such as deletion of the C-terminal lysine of the Fc region (residue 447 according to the EU numbering system) or deletion of the C-terminal glycine and lysine of the Fc region (according to the EU numbering system of residues 446 and 447).
  • the numbering rule for the Fc region is the EU numbering system, also known as the EU index.
  • homology refers to the sequence similarity between two polynucleotide sequences or between two polypeptides.
  • a position in two compared sequences is occupied by the same nucleotide or amino acid monomer subunit, for example, if each position of two DNA molecules is occupied by the same nucleotide, then the molecules at that position are Homogenous.
  • the percent homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared ⁇ 100%. For example, if 6 out of 10 positions in two sequences match or are homologous when the sequences are optimally aligned, then the two sequences are 60% homologous. In general, comparisons are made when aligning two sequences yields the greatest percent homology.
  • amino acid mutation includes amino acid substitutions (also called amino acid substitutions), deletions, insertions and modifications. Any combination of substitutions, deletions, insertions and modifications can be made to achieve the final construct, as long as the final construct possesses the desired properties, such as reduction or binding to Fc receptors.
  • Amino acid sequence deletions and insertions include deletions and insertions at the amino terminus and/or carboxyl terminus of the polypeptide chain. Specific amino acid mutations may be amino acid substitutions.
  • the amino acid mutation is a non-conservative amino acid substitution, ie, one amino acid is replaced by another amino acid with different structural and/or chemical properties.
  • Amino acid substitutions include substitutions by non-naturally occurring amino acids or by derivatives of the 20 natural amino acids (e.g., 4-hydroxyproline, 3-methylhistidine, ornithine, homoserine, 5-hydroxylysine) .
  • Amino acid mutations can be generated using genetic or chemical methods well known in the art. Genetic methods can include site-directed mutagenesis, PCR, gene synthesis, etc. It is anticipated that methods other than genetic engineering to alter amino acid side chain groups, such as chemical modification, may also be available. Various names may be used herein to refer to the same amino acid mutation.
  • the amino acid residue at a specific position can be represented by position + amino acid residue, for example, 366W, indicating that the amino acid residue at position 366 is W. T366W means that the amino acid residue at position 366 has been mutated from the original T to W.
  • amino acid sequence is defined in the form of position + residue in the claim, the amino acid before mutation at the site does not limit the technical solution.
  • conservative substitution refers to a substitution with another amino acid residue that has similar properties to the original amino acid residue.
  • lysine, arginine, and histidine have similar properties in that they have basic side chains
  • aspartic acid and glutamic acid have similar properties in that they have acidic side chains.
  • Gan Acid, asparagine, glutamine, serine, threonine, tyrosine, cysteine, and tryptophan have similar properties in that they have uncharged polar side chains
  • alanine, Valine, leucine, threonine, isoleucine, proline, phenylalanine, and methionine have similar properties in that they have nonpolar side chains.
  • tyrosine, phenylalanine, tryptophan, and histidine have similar properties in that they have aromatic side chains. Therefore, it will be apparent to a person skilled in the art that even when an amino acid residue in a group showing similar properties as described above is substituted, it will not show a specific change in the properties.
  • polypeptide protein
  • protein protein
  • proteins are used interchangeably herein to refer to a polymer of amino acid residues.
  • the term applies to amino acid polymers in which one or more amino acid residues are artificial chemical mimetics of the corresponding naturally occurring amino acids, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers. Unless stated otherwise, a particular polypeptide sequence also implicitly encompasses conservatively modified variants thereof.
  • nucleic acid is used interchangeably herein with the term “polynucleotide” and refers to deoxyribonucleotides or ribonucleotides and polymers thereof in single- or double-stranded form.
  • the term encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or linkages that are synthetic, naturally occurring and non-naturally occurring, have similar binding properties to the reference nucleic acid, and are Metabolized in a manner similar to the reference nucleotide.
  • nucleic acids examples include, but are not limited to, phosphorothioates, phosphoramidates, methylphosphonates, chiral-methylphosphonates, 2-O-methylribonucleotides, peptide-nucleic acids (PNA ).
  • An "isolated" nucleic acid refers to a polynucleotide that has been separated from components of its natural environment. Isolated nucleic acids include polynucleotides contained in cells that normally contain the polynucleotide but which are present extrachromosomally or at a chromosomal location that is different from its native chromosomal location.
  • An isolated nucleic acid encoding a polypeptide or fusion protein refers to one or more polynucleotides encoding a polypeptide or fusion protein, including such one or more polynucleotides in a single vector or separate vectors, and present in a host One or more such polynucleotides at one or more locations in a cell.
  • a particular nucleic acid sequence also implicitly encompasses conservatively modified variants (eg, degenerate codon substitutions) and complementary sequences thereof as well as sequences explicitly indicated.
  • degenerate codon substitutions can be obtained by generating sequences in which the third position of one or more selected (or all) codons is mixed bases and/or deoxygenated. Inosine residue substitution.
  • effector function refers to those biological activities that are attributable to the Fc region of an antibody (either a native sequence Fc region or a mutated amino acid sequence Fc region) and that vary with the antibody isotype.
  • antibody effector functions include, but are not limited to: C1q binding and complement-dependent cytotoxicity, Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, cell surface receptors (e.g., B cell receptors body) downregulation; and B cell activation.
  • binding affinity or "affinity” is used in this disclosure as a measure of the strength of a non-covalent interaction between two molecules (eg, an antibody or portion thereof and an antigen, a ligand and a receptor).
  • the binding affinity between two molecules can be quantified by determining the dissociation constant (KD).
  • K D can be determined by measuring the kinetics of complex formation and dissociation using, for example, surface plasmon resonance (SPR) methods (Biacore).
  • SPR surface plasmon resonance
  • the rate constants corresponding to the association and dissociation of monovalent complexes are called the association rate constant ka (or kon) and the dissociation rate constant kd (or koff), respectively.
  • the value of the dissociation constant can be determined directly by well-known methods and can be determined by methods such as those described in Caceci et al. (1984, Byte 9:340-362). to perform calculations for complex mixtures.
  • KD can be determined using a double filtered nitrocellulose filter binding assay such as that disclosed in Wong & Lohman (1993, Proc. Natl. Acad. Sci. USA 90:5428-5432).
  • Other standard assays to assess the binding ability of an antibody to a target antigen are known in the art and include, for example, ELISA, Western blot, RIA and flow cytometric analysis, as well as other assays exemplified elsewhere in this disclosure.
  • Binding kinetics and binding affinity of antibodies can also be evaluated by standard assays known in the art, such as surface plasmon resonance (SPR), for example by using the Biacore TM system or KinExA.
  • SPR surface plasmon resonance
  • the binding affinities associated with different molecular interactions can be compared by comparing the K values of individual antibody/antigen complexes, e.g., the binding affinities of different antibodies for a given antigen.
  • the specificity of an interaction can be determined by determining and comparing the KD value of the interaction of interest (e.g., a specific interaction between an antibody and an antigen) with that of an interaction that is not of interest (e.g., a control antibody known not to bind the antigen of interest). Evaluate the K D value.
  • linker refers to the connecting unit that connects two polypeptide fragments, which usually has a certain degree of flexibility. The use of the linker will not cause the original function of the protein domain to be lost.
  • linkers appearing in the same structure may be the same or different.
  • the linker can be a peptide linker, which contains one or more amino acids, typically about 1-30, 2-24 or 3-15 amino acids. Linkers used in the present disclosure may be the same or different.
  • fused or "linked” means that the components (eg, TACI polypeptides and BCMA polypeptides) are joined by a covalent bond, either directly or via one or more linkers.
  • linker is a peptide linker
  • the covalent bond is a peptide bond.
  • administering when applied to an animal, human, experimental subject, cell, tissue, organ or biological fluid, means the administration of an exogenous drug, therapeutic, diagnostic or composition to the animal , contact with persons, subjects, cells, tissues, organs or biological fluids.
  • administering may refer to, for example, therapeutic, pharmacokinetic, diagnostic, research and experimental methods.
  • Treatment of cells includes contact of reagents with the cells, and contact of the reagents with a fluid, wherein the fluid is in contact with the cells.
  • administer also mean treating eg a cell in vitro and ex vivo by a reagent, a diagnostic, a binding composition or by another cell.
  • Treatment when applied to human, veterinary medicine or research subjects means therapeutic treatment, prophylactic or prophylactic measures, research and diagnostic applications.
  • Treatment means administering an internal or external therapeutic agent, such as a composition comprising any antibody or antigen-binding fragment thereof or fusion protein thereof of the present disclosure, to a subject who has, is suspected of having, , is prone to suffer from one or more diseases or symptoms thereof, and the therapeutic agent is known to have a therapeutic effect on these symptoms.
  • a therapeutic agent is administered in an amount effective to alleviate one or more disease symptoms in a treated subject or population, either by inducing regression of such symptoms or inhibiting the progression of such symptoms to any clinically measurable extent.
  • the amount of a therapeutic agent effective in alleviating the symptoms of any particular disease can vary depending on a variety of factors, such as the subject's disease state, age and weight, and the extent to which the drug produces the desired therapeutic effect in the subject. ability. Whether disease symptoms have been alleviated may be assessed by any of the clinical tests commonly used by physicians or other health care professionals to assess the severity or progression of symptoms.
  • embodiments of the present disclosure may be ineffective in alleviating symptoms of a target disease in a certain subject, according to any statistical test method known in the art, such as Student t test, chi-square test, Mann and Whitney The U test, Kruskal-Wallis test (H test), Jonckheere-Terpstra test and Wilcoxon test determined that it should alleviate the target disease symptoms in a statistically significant number of subjects.
  • any statistical test method known in the art such as Student t test, chi-square test, Mann and Whitney The U test, Kruskal-Wallis test (H test), Jonckheere-Terpstra test and Wilcoxon test determined that it should alleviate the target disease symptoms in a statistically significant number of subjects.
  • an "effective amount” includes an amount sufficient to ameliorate or prevent symptoms or symptoms of a medical condition.
  • An effective amount also means an amount sufficient to allow or facilitate diagnosis.
  • the effective amount for a particular subject or veterinary subject may vary depending on factors such as the condition to be treated, the general health of the subject, the method, route and dosage of administration, and the severity of the side effects.
  • An effective amount may be the maximum dosage or dosage regimen that avoids significant side effects or toxic effects.
  • 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, regardless of the number of passages.
  • the progeny may not be identical in nucleic acid content to the parent cells, but may contain mutations. Mutant progeny having the same function or biological activity as screened or selected in the original transformed cell are included herein.
  • Host cells include prokaryotic and eukaryotic host cells, where eukaryotic host cells include, but are not limited to, mammalian cells, insect cell line plant cells, and fungal cells.
  • Mammalian host cells include human, mouse, rat, canine, monkey, porcine, goat, bovine, equine, and hamster cells, including but not limited to Chinese hamster ovary (CHO) cells, NSO, SP2 cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), A549 cells, 3T3 cells, and HEK-293 cells.
  • CHO Chinese hamster ovary
  • NSO Chinese hamster ovary
  • SP2 cells HeLa cells
  • BHK baby hamster kidney
  • COS monkey kidney cells
  • human hepatocellular carcinoma cells e.g., Hep G2
  • A549 cells 3T3 cells
  • HEK-293 cells HEK-293 cells.
  • fusion protein comprising a TACI polypeptide and a BCMA polypeptide encompasses all molecules containing a fusion protein of a TACI polypeptide and a BCMA polypeptide of the present disclosure.
  • a fusion protein comprising a TACI polypeptide and an anti-IFNAR1 antibody encompasses all molecules containing a fusion protein of a TACI polypeptide and an anti-IFNAR1 antibody (or antigen-binding fragment thereof) of the present disclosure.
  • a fusion protein comprising a TACI polypeptide and a BCMA polypeptide and an anti-IFNAR1 antibody encompasses all molecules containing a fusion protein of a TACI polypeptide and a BCMA polypeptide and an anti-IFNAR1 antibody (or antigen-binding fragment thereof) of the present disclosure.
  • the fusion protein may comprise one or more effector molecules, for example in a conjugated manner.
  • effector molecule alone may have therapeutic activity (eg, anti-tumor activity or immune activating or inhibitory activity) or detection function, and may be in any form, such as biologically active proteins (eg, enzymes), other antibodies or antibody fragments, Synthetic or naturally occurring polymers, nucleic acids and fragments thereof such as DNA, RNA and fragments thereof, radionuclides (in particular radioiodides), radioisotopes, chelated metals, nanoparticles and reporter groups (e.g. fluorescent compounds), or compounds detectable by NMR or ESR spectroscopic analysis.
  • the conjugation of effector molecules to the fusion proteins of the present disclosure can be achieved by conventional methods.
  • Figure 1 Schematic diagram of the fusion protein structure of TACI and Fc.
  • FIG. 1 Schematic diagram of the interaction between ligands BAFF and APRIL and receptors BAFF-R, TACI and BCMA.
  • BAFF is a membrane-expressed protein that functions in the form of a soluble trimer after protease cleavage. BAFF can combine with BAFF-R, TACI and BCMA.
  • APRIL is a soluble trimeric protein that can bind to TACI and BCMA. Thicker black arrows represent stronger interactions, and thinner black arrows represent weaker interactions.
  • Figure 3 Schematic diagram of different TACI and BCMA protein domains, including TACI-ECD, TACI-d2, TACI-T2, TACI-T4, and BCMA-ECD.
  • Figure 4 Schematic structural diagram of TACI-BCMA fusion protein, including B575701, B575702, B575703, and B575704.
  • Figure 7 Schematic diagram of TACI-CRD2 and TACI-19-16 domains, including TACI-CRD2 and TACI-19-16.
  • FIG 8. Anifrolumab and TACI-T4 fusion proteins (B385801, B385802, B385803, B385804, B498301, B498302), Anifrolumab and TACI-19-16 fusion proteins (B606401, B606401-LALA, B805201, B805201-LALA, the LALA Structural diagram of the L234A/L235A mutation mutated to Fc, the same below).
  • Figure 9 The results of the IFN- ⁇ activity inhibition test by the fusion protein of Anifrolumab and TACI-T4. Among them, A in Figure 9 is the inhibition result of B385801, B385802, B385803, and B385804 on IFN- ⁇ activity; B in Figure 9 is B385804 and B498301. , B498302’s inhibition results on IFN- ⁇ activity; Figure 9 C shows the results of B606401’s inhibition of IFN- ⁇ activity, using Anifrolumab and IgG1 isotypes as controls.
  • FIG. 10 ELISA measurement results of the binding ability of the fusion protein of Anifrolumab and TACI-T4 to BAFF.
  • a in Figure 10 shows the results of the binding ability of B385801, B385802, B385803, and B385804 to BAFF, using Atacicept, Telitacicept, and IgG1 isotypes as controls.
  • B in Figure 10 is the result of the binding ability of B385804, B498301, and B498302 to BAFF;
  • C in Figure 10 is the result of the binding ability of B606401 to BAFF, using Telitacicept and IgG1 isotypes as controls.
  • FIG. 11 Schematic diagram of the structure of Anifrolumab and TACI-BCMA fusion protein, including B613301, B613302, B613303, and B613304.
  • Figure 14 Results of ELISA determination of the binding ability of Anifrolumab to TACI-BCMA fusion proteins (B613301, B613302, B613303) and APRIL, using Telitacicept and IgG1 isotypes as controls.
  • FIG. 15 Anifrolumab fusion protein with TACI-19-16 and BCMA-ECD-1 (B637301), Anifrolumab fusion protein with TACI-19-16 and BCMA-ECD-2 fusion protein (B637302, B637302-LALA, B746201, B746201 -LALA) structural diagram.
  • FIG. 19 Experimental results of pDC function in PBMC.
  • Human PBMC cells cultured in vitro were stimulated with CpG-A, treated with gradient concentrations of B637302 and B606401, and Anifrolumab and IgG1 isotypes were used as controls.
  • the level of secreted IFN- ⁇ in the supernatant was measured 24 hours later.
  • Figure 20 Results of plasma cell differentiation experiments in vitro. Including using B cells sorted from human PBMC for in vitro culture, adding CpG-B and IFN- ⁇ , adding gradient concentrations of B637302, B746201, B606401, and B805201, using Anifrolumab and IgG1 isotype as controls, 4 The differentiation ratio of plasma cells (CD27+CD38+) was measured by flow cytometry after 1 day.
  • FIG. 21 Experimental results of BAFF-induced B cell proliferation in vitro. It involves using B cells sorted from human PBMCs for CTV labeling, then culturing them in vitro, adding IL-4, anti-IgM, CD40L, and IL-17 as basic stimulation signals. On this basic condition, adding BAFF induction B cell proliferation was treated with gradient concentrations of B637302, B606401, and B805201. Telitacicept and IgG1 isotypes were used as controls. B cell proliferation signals were detected by flow cytometry on the 5th day. The drug concentrations were 100nM, 300nM, and 1000nM.
  • FIG 22 APRIL-induced B cell proliferation experiment in vitro. It involves using B cells sorted from human PBMCs for CTV labeling, then culturing them in vitro, adding IL-4, anti-IgM, CD40L, and IL-17 as basic stimulating signals. On this basic condition, adding APRIL to induce B cell proliferation was treated with gradient concentrations of B637302 and B606401. Telitacicept and IgG1 isotype were used as controls. B cell proliferation signals were detected by flow cytometry on the 5th day. The drug concentrations were 100nM, 300nM, and 1000nM.
  • FIG. 23 Experiment of BAFF and APRIL jointly inducing the generation of plasma cells in vitro. Including B cells sorted from human PBMC, cultured in vitro, stimulated by adding CpG-B on days 1 to 4, removing CpG-B on days 4 to 10, and adding IL-6+IL-10+IL-21 Continue to cultivate. Based on these basic conditions, 500ng/mL BAFF and 50ng/mL APRIL were added on days 4 to 7, and 50ng/mL BAFF and 500ng/mL APRIL were added on days 7 to 10 to induce the generation of plasma cells in vitro. From days 4 to 10, different concentrations of drugs B637302, B606401, B606401, and B805201 were added at 10, 100, and 1000 nM. Telitacicept and IgG1 isotype were used as controls. On day 10, the number of plasma cells was detected by flow cytometry.
  • FIG 24 Experiment on the in vitro generation of plasma cells induced by IFN- ⁇ , BAFF and APRIL.
  • a in Figure 24 is a schematic diagram of the experimental process. B cells sorted from human PBMC are used for in vitro culture. CpG-B and IFN- ⁇ are added for stimulation on days 1 to 4, and CpG-B is removed on days 4 to 10. , add IL-6+IL-10+IL-21 and continue culturing. Based on these basic conditions, 500ng/mL BAFF and 50ng/mL APRIL were added on days 4 to 7, and 50ng/mL BAFF and 500ng/mL APRIL were added on days 7 to 10 to induce the generation of plasma cells in vitro.
  • the drugs B637302 and B606401 were added at different concentrations of 10, 100, and 1000 nM. Telitacicept and IgG1 isotype were used as controls. The number of plasma cells was detected by flow cytometry on the 10th day. B, C, and D in Figure 24 are 10 nM of the drug respectively. , 100nM, 1000nM detection results.
  • Figure 25 PEG-IFN- ⁇ induced PD experimental results in PBMC humanized mice.
  • Figure 25 A is a schematic diagram of the experimental process. B-NDG immunodeficient mice were used, PBMC were injected into the tail vein for humanization, and PEG-IFN- ⁇ was injected intraperitoneally for induction. In this model, different concentrations of drugs B637302 and B606401 were injected intraperitoneally.
  • mice were treated, using Anifrolumab and PBS as controls;
  • B in Figure 25 is a schematic diagram of the downstream signaling pathway induced by IFN- ⁇ ;
  • C in Figure 25 is the result of taking PBMC to detect pSTAT1 levels at the 2nd hour;
  • D, E, and F in Figure 25 are respectively PBMC were collected at time points of 1, 3, and 7 days, and the mRNA expression levels of IFN- ⁇ downstream genes were detected by QPCR.
  • Figure 26 Experimental results of BAFF and APRIL jointly inducing PD in mice.
  • Figure 26 A is a schematic diagram of the experimental process. C57/B6 mice were used to induce BAFF+APRIL by intraperitoneal injection. In this model, mice were treated with different concentrations of drugs B637302 and B606401 by intraperitoneal injection. Telitacicept and PBS were used as controls. Chapter 4 , peripheral blood was taken on the 7th day, and the IgA level in the plasma was detected by ELISA; Figure 26 B and C are the results of blood testing on the 4th and 7th days respectively.
  • TACI sequence fragments of different lengths were designed, and the C-terminus of TACI-ECD or its fragment was directly fused with the N-terminus of human IgG1 Fc (SEQ ID NO: 3) to construct a TACI-Fc fusion protein (including full-length TACI- ECD-Fc, TACI-9-Fc), transfected and expressed in 293E (EBNA1 gene-modified human embryonic kidney cells), and purified to obtain a TACI-Fc fusion protein containing two identical polypeptide chains (see Figure 1 for the structure).
  • RCT-18 Telitacicept
  • TACI-ECD amino acid residues 1 to 165 of wild-type human TACI
  • the ununderlined part is the TACI sequence (digits 13 to 118 of the natural count of the extracellular region of human TACI), and the underlined part is the Fc sequence.
  • the ununderlined part is the TACI sequence (positions 30 to 110 of the extracellular domain of human TACI in natural counting) and the linker EPKSS (shown in italics), and the underlined part is the Fc sequence.
  • the method is as follows: dilute the receptor protein to 2 ⁇ g/mL with PBS (Yuanbai Biotech, B320) buffer at pH 7.4, and add 100 ⁇ L/ The well volume was added to a 96-well microplate (Corning, 3590) and incubated at 4°C overnight. After discarding the liquid, add 200 ⁇ L of 1% Casein blocking solution (Thermo, 37528) to each well for blocking, and incubate at 37°C for 2 hours.
  • PBS Luanbai Biotech, B320
  • Casein blocking solution Thermo, 37528
  • PBST buffer pH 7.4 PBS containing 0.1% tween-20
  • PBST buffer pH 7.4 PBS containing 0.1% tween-20
  • the instrument reads the absorption value at 450nm, uses software to fit the curve of the binding of the inhibitory ligand and receptor, and calculates the IC 50 value.
  • the source information of the ligands and receptor proteins used is as follows: BAFF (Sino biological, 10056-HNCH), BAFF-R (Sino biological, 16079-H02H).
  • TACI-9-Fc and RCT-18 blocking the binding of BAFF and BAFF-R were 5.02nM and 27.48nM respectively.
  • the functional activity of TACI-9-Fc was significantly stronger than that of control RCT-18. Therefore, TACI-9 is considered to be a potential candidate molecule.
  • sequence length of TACI was further truncated, and the functional activity of the truncated TACI fragment was tested.
  • the sequence of the further truncated TACI fragment is shown below:
  • TACI-10 the C-terminus of TACI-9 is truncated by 1 amino acid "L" (positions 68 to 107 of human TACI-ECD according to natural counting)
  • TACI-11 the C terminus of TACI-9 is truncated by 2 amino acids "KL" (positions 68 to 106 of human TACI-ECD according to natural counting)
  • TACI-12 TACI-9's C-terminal truncated by 3 amino acids "NKL" (human TACI-ECD at positions 68 to 105 in natural counting)
  • the C-terminus of the above truncated TACI fragment was fused to the N-terminus of human IgG Fc (SEQ ID NO: 3) to construct a TACI-Fc fusion protein, transfected and expressed in 293E, and purified to obtain TACI containing two identical polypeptides.
  • -Fc fusion protein see Figure 1 for structure).
  • the TACI-Fc fusion protein constructed above was subjected to mass spectrometry analysis.
  • the experimental results are shown in Table 2.
  • the experimental results showed that no TACI polypeptide fragmentation occurred in the TACI-Fc fusion protein constructed with the TACI-9 truncated fragment.
  • the method for detecting the activity of TACI-Fc fusion protein binding to BAFF is as follows: dilute the BAFF (Sino biological, 10056-HNCH) protein to 1 ⁇ g/mL with PBS (Sino biological, B320) buffer at pH 7.4. A volume of 100 ⁇ L/well was added to a 96-well microplate (Corning, 3590) and incubated at 4°C overnight. After discarding the liquid, 300 ⁇ L of 5% skim milk (BD, 232100) diluted in PBS was added to each well for blocking, and incubated at 37°C for 2 hours. After blocking, discard the blocking solution and wash the plate three times with PBST buffer (pH 7.4PBS containing 0.1% tween-20).
  • PBST buffer pH 7.4PBS containing 0.1% tween-20
  • Table 2 show that the binding activity of TACI-10-Fc, TACI-11-Fc, and TACI-12-Fc fusion proteins constructed from TACI-9 truncated fragments TACI-10, TACI-11, and TACI-12 to BAFF and The functional activity of blocking the binding of BAFF to BAFF-R is at the same level as that of the TACI-9-Fc fusion protein.
  • TACI proteins tend to aggregate in neutral solutions.
  • MOE Molecular Operating Environment
  • group amino acid replacement of multiple amino acid residues of TACI-9, reducing the area of exposed hydrophobic groups and ionic groups of the TACI protein, reducing the aggregation of TACI, and improving the stability of TACI, but still basically retaining the properties of TACI Functional activity.
  • the amino acid sequence of the modified TACI fragment is as follows:
  • TACI-9 includes L69T replacement
  • TACI-9 contains L69R, D85T, Y102R mutations
  • TACI-9-11 contains R72S, K73E, K77E mutations
  • TACI-9-12 contains L69T, Y102A mutation
  • TACI-9-14 (TACI-9 contains L69T, Y102A, F103Y mutations)
  • TACI-9 contains L69T, K73E, K77E, Y102A mutations
  • the C-terminus of the above-mentioned TACI fragment modified by TACI-9 was fused to the N-terminus of human IgG Fc (SEQ ID NO: 3) to construct a TACI-Fc fusion protein (see Figure 1 for the structure), which was transfected and expressed in 293E. Then through Protein A affinity purification, a TACI-Fc fusion protein containing two identical polypeptide chains was obtained.
  • the mutation site in the table is the amino acid residue position (natural sequence number) relative to TACI-ECD (SEQ ID NO: 1).
  • L69T means position 69 of the sequence SEQ ID NO: 1 (natural sequence number). Number
  • the amino acid residue is mutated from L to T.
  • the partially purified TACI-Fc fusion protein was replaced into the PBS solution using an ultrafiltration tube, and the appearance of the solution was used to observe whether there was precipitation in the TACI-Fc fusion protein solution.
  • Experimental results showed that there was no precipitation in the solution of the TACI-Fc fusion protein constructed with the modified TACI fragment, but there was precipitation in the PBS solution of RCT-18 (results not shown).
  • TACI-9-15a, TACI-9-15b, TACI-9-15c amino acid sequences are as follows:
  • Atacicept and Telitacicept are both BAFF/APRIL antagonists in the form of TACI-Fc molecules.
  • both TACI and BCMA can bind BAFF and APRIL.
  • TACI has a strong binding ability with BAFF but a weak binding ability with APRIL.
  • BCMA has a strong binding ability with APRIL.
  • BCMA and TACI as a fusion protein in order to improve the neutralizing activity against APRIL.
  • TACI-ECD is the extracellular region of TACI containing amino acid residues 1-165
  • TACI-d2 is the CRD2 ligand-binding domain containing amino acid residues 69-111
  • TACI-T2 is the CRD2 ligand-binding domain containing amino acid residues 69-111.
  • TACI-T4 is the CRD1 and CRD2 ligand-binding domain containing amino acid residues 30-110
  • BCMA-ECD is the extracellular domain of BCMA containing amino acid residues 1-54.
  • fusion proteins B575701 and B575702 are connected with TACI-d2 and BCMA-ECD sequences and fused with human wild-type IgG1-Fc; Fusion proteins B575703 and B575704 combine TACI-T2 and BCMA-ECD The sequences were linked and fused to human wild-type IgG1-Fc.
  • sequence of TACI-ECD is shown in SEQ ID NO: 1.
  • the remaining sequences are as follows, where human IgG1 Fc is underlined. Among them, the G in the last digit of SEQ ID NO: 27 does not need to exist.
  • B575701, B575702, B575703, and B575704 are the Fc( SEQ ID NO: 3). In the following sequence, the underline is Fc.
  • the coding gene sequence of the above protein was synthesized and subcloned into pcDNA3.4. Use the culture medium to dilute 50 ⁇ g of expression plasmid and mix well. Use the culture medium to dilute 200 ⁇ L of transfection reagent and mix well. Combine the two and mix well and incubate at 37°C for 15 minutes. Add the mixture drop by drop to the HEK293 cell solution. The cell solution is 37°C. After one week of culture on the shaker, the supernatant was collected by centrifugation at 8000 rpm for 5 minutes.
  • Example 5 In vitro binding experiment of TACI-BCMA fusion protein to BAFF and APRIL
  • ELISA was used to detect the binding ability of TACI-BCMA fusion proteins (B575701, B575702, B575703, and B575704) to BAFF and APRIL.
  • the method is as follows: combine BAFF (PeproTech, Cat: 310-13) or ARPIL (Acro Biosystems, Cat: APL-H52D1) was diluted to 1 ⁇ g/mL with PBS, and a 96-well plate (Costar, Cat: 3590) was coated overnight at 4°C. Wash the plate with PBST and block with Blocking Buffer (PBST containing 1% BSA) at 37°C for 1 hour. Clap the plate, dilute the antibody to be tested with Blocking Buffer, and incubate at 37°C for 1.5 hours.
  • the four fusion proteins can obviously bind APRIL, and the binding abilities are basically the same; the binding abilities of the four fusion proteins to APRIL are all stronger than Telitacicept, and the EC 50 is reduced to 1/4 to 1/3 of it. s level.
  • Example 6 Construction, expression and purification of fusion protein of Anifrolumab and TACI
  • Anifrolumab is an antagonist targeting IFNAR1, blocking the activation of cells by type I interferons through IFNAR1, and is a drug used to treat systemic lupus erythematosus.
  • TACI-CRD2 is a CRD2 ligand-binding domain containing amino acid residues 68-105
  • TACI-19-16 is a polypeptide of TACI-CRD2 with several amino acid mutations (L69T, K73E, K77E, Y102A). .
  • TACI-T4 or TACI-19-16 was connected to the N-terminal or C-terminal of the Anifrolumab heavy chain or light chain through linkers of different lengths.
  • B385801 connects TACI-T4 to the N-terminus of Anifrolumab heavy chain through linker 1 of (G 4 S) 2 ;
  • B385802 connects TACI-T4 to the C-terminal of Anifrolumab heavy chain through linker 1;
  • B385803 connects TACI-T4 through linker 1.
  • Sub-1 is connected to the N-terminal of the Anifrolumab light chain;
  • B385804 connects TACI-T4 to the C-terminal of the Anifrolumab light chain through linker 1;
  • B498301 connects TACI-T4 to the C-terminal of the Anifrolumab light chain through linker 2 of (G 4 S) 3 ;
  • B498302 connects TACI-T4 to the C-terminus of the Anifrolumab light chain through linker 3 of (G 4 S) 4 .
  • B606401 connects TACI-19-16 to the C-terminal of the Anifrolumab light chain through linker 1; B805201 connects TACI-19-16 to the C-terminal of the Anifrolumab heavy chain through linker 2.
  • B606401-LALA passes TACI-19-16 Linker 1 is connected to the C-terminus of the Anifrolumab light chain, and the Fc part is human IgG1 Fc with L234A and L235A mutations.
  • B805201-LALA connects TACI-19-16 to the C-terminus of the Anifrolumab heavy chain through linker 2, and the Fc part is human IgG1 Fc with L234A and L235A mutations.
  • TACI-19-16 is shown as SEQ ID NO: 26, other sequences are as follows:
  • the underline of the heavy chain is the Fc region of IgG, the underline of the light chain is CH1, and the linker is in italics.
  • Example 2 Using the protein preparation method provided in Example 1, through transient transfection and protein expression, the protein is eluted through a Protein A affinity chromatography column. After detection, the target protein was obtained.
  • Example 7 Experiment on the activity inhibition of type I interferon by the fusion protein of Anifrolumab and TACI
  • HEK-Blue IFN- ⁇ / ⁇ cells (InvivoGen, Cat: hkb-ifnab) kit was used to detect the IFN- ⁇ / ⁇ effects of the fusion proteins B385801, B385802, B385803, B385804, B498301, B498302, and B606401 of Anifrolumab and TACI. Activity inhibitory effect.
  • the method is: digest HEK-Blue TM IFN- ⁇ / ⁇ cells, resuspend to 2.8E5/mL in DMEM medium containing 10% FBS, 1% P/S (Gibco, Cat: 11995065) and inoculate into a 96-well plate. . Add the protein to be tested and incubate at 37°C for 30 minutes. IFN- ⁇ (Sino biological, Cat: 10704-HNAS) with a final concentration of 0.01ng/mL was added and cultured at 37°C for 24 hours.
  • Example 8 In vitro binding experiment of the fusion protein of Anifrolumab and TACI to BAFF
  • ELISA was used to detect the binding ability of Anifrolumab and TACI fusion proteins B385801, B385802, B385803, B385804, B498301, B498302, and B606401 to BAFF.
  • the method is: dilute BAFF (PeproTech, Cat: 310-13) with PBS to 1 ⁇ g/mL, and coat a 96-well plate at 4°C overnight. Wash the plate with PBST and block with Blocking Buffer (PBST containing 1% BSA) at 37°C for 1 hour. Clap the plate, dilute the antibody to be tested with Blocking Buffer, and incubate at 37°C for 1.5 hours. Wash the plate with PBST and use Blocking HRP-mouse anti-human Fc antibody (GenScript, Cat: A01854-200) was diluted in Buffer and incubated at 37°C for 40 minutes.
  • PBST Blocking Buffer
  • Blocking HRP-mouse anti-human Fc antibody GeneScript, Cat: A01854-200
  • TMB chromogenic solution Biopanda, Cat: TMB-S-003
  • ELISA stop solution Solarbio, Cat: C1058
  • Example 9 Construction, expression and purification of fusion protein of Anifrolumab and TACI-BCMA
  • the TACI-BCMA fusion protein described in Example 4 was connected to the C-terminus of the Anifrolumab antibody light chain using linker 1.
  • B613301 and B613302 are fusion proteins of TACI-d2 and BCMA-ECD connected to the C-terminus of the light chain of Anifrolumab antibody through linker 1;
  • B613303 and B613304 are fusion proteins of TACI-T2 and BCMA-ECD connected to Anifrolumab through linker 1.
  • the heavy chains of B613301, B613302, B613303, and B613304 are all the full length of the heavy chain of Anifrolumab (SEQ ID NO: 55).
  • the specific sequence of the light chain is as follows, and the underline is the light chain constant region:
  • Example 4 Using the protein preparation method provided in Example 4, through transient transfection and protein expression, the protein is eluted through a Protein A affinity chromatography column. After detection, the target protein was obtained.
  • Example 10 Experiment on the activity inhibition of type I interferon by the fusion protein of Anifrolumab and TACI-BCMA
  • the method is: digest HEK-Blue TM IFN- ⁇ / ⁇ cells, resuspend to 2.8E5/mL in DMEM medium containing 10% FBS, 1% P/S (Gibco, Cat: 11995065) and inoculate into a 96-well plate. (Costar, Cat:3599). Add the antibody to be tested and incubate at 37°C for 30 minutes. IFN- ⁇ (Sino biological, Cat: 10704-HNAS) with a final concentration of 0.01ng/mL was added and cultured at 37°C for 24 hours.
  • Example 11 In vitro binding experiment of BAFF and APRIL by the fusion protein of Anifrolumab and TACI-BCMA
  • ELISA was used to detect the binding ability of Anifrolumab and TACI-BCMA fusion proteins B613301, B613302, and B613303 to BAFF and APRIL.
  • the method is: dilute BAFF (PeproTech, Cat: 310-13) or ARPIL (Acro Biosystems, Cat: APL-H52D1) with PBS to 1 ⁇ g/mL, and coat a 96-well plate (Costar, Cat: 3590) at 4°C overnight. Wash the plate with PBST and block with Blocking Buffer (PBST containing 1% BSA) at 37°C for 1 hour. Make a decision, use Blocking Dilute the antibody to be tested in Buffer and incubate at 37°C for 1.5 hours.
  • Example 12 Construction, expression and purification of fusion protein of Anifrolumab and TACI-19-16-BCMA
  • BCMA-ECD-1 (amino acid residues 1-43) and BCMA-ECD-2 (amino acid residues 1-41) remove possible glycosylation sites (amino acid residues 42-44) in the non-CRD domain of BCMA , NAS) and deamidation sites (amino acid residues 47-48, NS) and other risk points. Removing the risk points can make the fusion protein more uniform.
  • B637301 fuses TACI-19-16 to the N-terminal of BCMA-ECD-1, and then connects TACI-19-16-BCMA-ECD-1 to the C-terminal of the Anifrolumab antibody light chain through linker 1;
  • B637302 TACI-19-16 is fused to the C-terminal of BCMA-ECD-2, and then BCMA-ECD-2-TACI-19-16 is connected to the C-terminal of the Anifrolumab antibody light chain through linker 1;
  • B746201 TACI-19- 16 is fused to the C-terminal of BCMA-ECD-2, and then BCMA-ECD-2-TACI-19-16 is connected to the C-terminal of the Anifrolumab antibody heavy chain through linker 2.
  • B637302-LALA fuses TACI-19-16 to the C-terminus of BCMA-ECD-2, and then connects BCMA-ECD-2-TACI-19-16 to the C-terminus of the antibody Anifrolumab light chain through linker 1, and the Anifrolumab antibody
  • the Fc portion is human IgG1 Fc with L234A and L235A mutations.
  • B746201-LALA fuses TACI-19-16 to the C-terminus of BCMA-ECD-2, and then connects BCMA-ECD-2-TACI-19-16 to the C-terminus of the antibody Anifrolumab heavy chain through linker 2, and the Anifrolumab antibody
  • the Fc part is human IgG1 Fc with L234A and L235A mutations, see Figure 15.
  • the heavy chains of B637301 and B637302 are both Anifrolumab heavy chain SEQ ID NO: 55, and the light chain sequences are as follows:
  • B637302-LALA heavy chain is SEQ ID NO: 61.
  • the light chain of B746201 and B746201-LALA is the light chain of Anifrolumab SEQ ID NO: 54.
  • B637301, B637302 and B746201 fusion proteins were expressed and purified, and SDS-PAGE detection showed that the antibody heavy chain and light chain connected to TACI-BCMA were single bands (results not shown). The results show that the removal of glycosylation risk points in the BCMA protein sequence can make the fusion protein more uniform.
  • Example 13 Activity inhibition experiment of type I interferon by the fusion protein of Anifrolumab and TACI-19-16-BCMA
  • HEK-Blue IFN- ⁇ / ⁇ cells InvivoGen, Cat: hkb-ifnab kit to detect the IFN- ⁇ / ⁇ effects of the fusion protein of Anifrolumab and TACI-19-16-BCMA (B606401, B637302, B746201 and B805201). Activity inhibitory effect.
  • the method is: digest HEK-Blue TM IFN- ⁇ / ⁇ cells, resuspend to 2.8E5/mL in DMEM medium containing 10% FBS, 1% P/S (Gibco, Cat: 11995065) and inoculate into a 96-well plate. (Costar, Cat:3599). Add the antibody to be tested and incubate at 37°C for 30 minutes. IFN- ⁇ (Sino biological, Cat: 10704-HNAS) with a final concentration of 0.01ng/mL was added and cultured at 37°C for 24 hours.
  • Example 14 In vitro binding experiment of BAFF and APRIL by the fusion protein of Anifrolumab and TACI-19-16-BCMA
  • ELISA was used to detect the binding ability of B606401, B637302, B746201 and B805201 fusion proteins to BAFF and APRIL.
  • the method is as follows: dilute BAFF (PeproTech, Cat: 310-13) or ARPIL (Acro Biosystems, Cat: APL-H52D1) with PBS to 1 ⁇ g/mL, and coat a 96-well plate at 4°C overnight. Wash the plate with PBST and block with Blocking Buffer (PBST containing 1% BSA) at 37°C for 1 hour. Clap the plate and dilute the antibody to be tested with Blocking Buffer. body and incubate at 37°C for 1.5 hours. Wash the plate with PBST, dilute HRP-mouse anti-human Fc antibody (GenScript, Cat: A01854-200) with Blocking Buffer, and incubate at 37°C for 40 minutes.
  • TMB chromogenic solution Biopanda, Cat: TMB-S-003
  • ELISA stop solution Solarbio, Cat: C1058
  • CpG-A ODN 2216 (InvivoGen cat: tlrl-2216) was stimulated, and treated with gradient concentrations of Anifrolumab, B637302, B606401, and IgG1 isotype. After 24 hours, the human IFN- ⁇ kit (Cisbio cat: 62HIFNAPEG) was used to detect the levels in the cell culture supernatant. IFN- ⁇ cytokine secretion levels.
  • the density was inoculated into a 96-well plate, stimulated with 2 ⁇ g/mL CpG-B 2006 (InvivoGen cat: tlrl-2006) and 250 U/mL IFN- ⁇ (Biolegend cat: 592704), plus gradient concentrations of Anifrolumab, B637302, B746201, After B606401, B805201, and IgG1 isotype control treatments, the differentiation ratio of plasma cells (CD27 + CD38 + ) was measured by flow cytometry 4 days later.
  • the results show that B637302, B746201, B606401, B805201 and the control antibody Anifrolumab can effectively inhibit the in vitro differentiation of B cells into plasma cells induced by IFN- ⁇ in a dose-dependent manner, with IC 50 of 10.75nM and 1.895nM respectively. , 17.45nM, 6.208nM, 12.66nM, indicating that the inhibitory activity of the fusion protein is equivalent to that of the control antibody Anifrolumab.
  • B cells were seeded in a 96-well plate at a density of 1 ⁇ 10 5 /well, and 20 ⁇ g/mL anti-human IgM antibody (Jacksonimmuno cat:109 -006-129), 10ng/mL recombinant human IL-4 (PeproTech cat:AF-200-04-20), 100ng/mL CD40L (R&D cat:2706-CL-025/CF), 1.5 ⁇ g/mL anti-His Antibody (R&D cat:MAB050-500), 1ng/mL recombinant human IL-17 (SinoBiological cat:12047-HNAS), plus 200ng/mL recombinant human BAFF (R&D cat:7537-BF-025/CF) stimulation, here Conditionally, gradient concentrations of Telitacicept, B637302, B606401, B80521, and IgG1 isotype were added, and B cell proliferation signals were detected by flow cytometry after 5 days.
  • Example 20 Functional experiment of plasma cell production induced by IFN- ⁇ +BAFF+APRIL
  • IFN- ⁇ +BAFF+APRIL induces the generation of plasma cells in vitro experimental method.
  • the specific process is as follows: Use the B cell sorting kit (Stemcell cat: 17954) to isolate human B cells from fresh healthy human PBMC, and culture them in vitro in the basal medium of 1640GlutaMAX (Gibco cat: 72400-47).
  • the B cells are cultured at 1x10
  • the cells were seeded in a 96-well plate at a density of 5 /well and stimulated with 2 ⁇ g/mL CpG-B 2006 (InvivoGen cat: tlrl-2006) and 250 U/mL IFN- ⁇ (Biolegend cat: 592704).
  • mice were used B-NDG immunodeficient mice, injected 2 ⁇ 10 7 PBMC cells into the tail vein for humanization, and injected 0.6 ⁇ g PEG intraperitoneally.
  • -IFN- ⁇ ( Roche) mice were treated with intraperitoneal injection of different concentrations of drugs Anifrolumab, B637302, B606401 and PBS in this model.
  • PBMC were collected at 2h, and pSTAT1 (#9167, Cell Signaling Technology) levels were detected by flow cytometry.
  • Peripheral blood was collected at time points on days 1 and 3 to isolate PBMC, and IFN- ⁇ downstream genes (such as ISG-15, IFI13, MX) were detected by QPCR. -1, HERC5, etc.) mRNA expression levels, as shown in Figure 25A and Figure 25B.
  • mice were treated with different concentrations of Telitacicept, B637302, B606401 and PBS intraperitoneally. Peripheral blood was taken on days 4 and 7, and the IgA level in the plasma was detected by ELISA. (A in Figure 26).

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Abstract

本公开提供包含TACI多肽的融合蛋白及其用途，特别是用于预防或治疗自身免疫疾病的用途。

Description

包含TACI多肽的融合蛋白及其用途
本公开要求如下专利申请的优先权：于2022年08月29日提交，申请号为CN202211041405.0，发明名称为“包含TACI多肽的融合蛋白及其用途”的中国专利申请；上述专利申请的全部内容通过引用结合在本公开中。
技术领域
本公开涉及生物医药领域，具体而言，本公开涉及一种包含TACI多肽的融合蛋白及治疗疾病，特别是自身免疫疾病的用途。
背景技术
系统性红斑狼疮(Systemic Lupus Erythematosus，SLE)是一种危及多种人体组织和器官(如面部皮肤、肾脏等)的自身免疫性疾病，其明显的特征是患者会产生针对自身组织抗原的抗体(如抗核抗体antinuclear antibody(ANA)等)。SLE致病因素复杂，涉及基因遗传、环境、激素及自身抗体等诸多因素，病程长，临床症状因人而异，异质性高。此外，SLE发病率高，仅在中国SLE患着总数就超过100万，全球患病率约为(30至50)/10万人，其中女性患病率是男性10倍。
I型干扰素通路的激活在SLE发病机理中起关键作用，能够促进浆细胞样树突状细胞(pDC)、B细胞等与SLE发病紧密相关的多种类型免疫细胞活化，60％至80％的SLE患者存在I型干扰素高表达的特征，而且I型干扰素水平与SLE患者疾病活动指数(SLEDAI)评分呈正相关。阿斯利康开发的Anifrolumab是一种靶向I型干扰素(IFN)受体亚基1(IFNAR1)的单克隆抗体，拮抗所有I型干扰素(IFN-α、IFN-β和IFN-ω等)的相关活性，从而控制SLE疾病进展。在TULIP-2的III期临床试验中，接受Anifrolumab患者的狼疮综合评价指数(BICLA)出现改善的比例分别为47.8％，而安慰剂治疗患者这一评价指标为31.5％，提示患者所有累及器官的疾病活动程度都有所改善，且减少患者对糖皮质激素的用量。但在另一个以狼疮应答指数SRI-4作为考察终点的III期(TULIP-1)临床实验中，并没有看到疾病的评分的显著改善(36％vs 40％)。该结果体现了靶向I型干扰素通路治疗SLE的潜力，但仅靶向IFNAR1的疗效可能还不够。
B淋巴细胞刺激因子(B lymphocyte stimulator，BLyS，又叫BAFF)和增殖诱导配体(A proliferation-inducing ligand，APRIL)均是B淋巴细胞分化成熟和促进浆细胞生存的关键因子，属于肿瘤坏死因子(tumor necrosis factor，TNF)配体家族，这两个因子的过度表达是SLE等多种B淋巴细胞相关自身免疫疾病的重要驱动因素。TNF配体家族成员通常被合成为跨膜蛋白，并且经常观察到膜锚定蛋白经蛋白酶水解后从细胞表面释放。BAFF为II型跨膜蛋白，存在膜结合和可溶两种形式，膜结合BAFF经弗林蛋白酶转化酶(Furin convertase)酶切后，释放出三聚体形式的 BAFF并具有生物学活性。BAFF在各种细胞类型上均有表达，包括单核细胞，树突状细胞和骨髓基质细胞。APRIL存在形式不同于TNF配体家族成员，其在高尔基体内被弗林蛋白酶转化酶加工后直接以可溶的三聚体形式分泌到细胞外。
现已发现BAFF和APRIL存在三个受体，即B细胞成熟抗原(B cell maturation antigen，BCMA)、穿膜蛋白活化物(transmembrane activitor and CAMEL interactor，TACI)和BAFF受体(BAFF receptor，BAFF-R，又叫Br3)，他们均属于TNF受体家族成员，TNF受体的胞外区含有多个半胱氨酸丰富区(cysteine-rich domain，CRD)，每个CRD有6个半胱氨酸形成3对二硫键，CRD区域发挥和配体结合的功能。BAFF和APRIL对这三个受体具有不同的结合能力，BAFF对TACI的结合强于对BCMA，APRIL对BCMA的结合强于对TACI，而BAFF-R是BAFF的独有高亲和力受体，不与APRIL结合。
TACI为III型跨膜蛋白，人TACI是293个氨基酸的多肽，包含位于蛋白N端区域(氨基酸残基1-165)，跨膜区域(氨基酸残基166-186)和细胞内区域(氨基酸残基187-293)。胞外区含有两个CRD(CRD1、CRD2)，CRD1对BAFF和APRIL的亲和力比CRD2弱。研究发现仅在人TACI中，会跳过外显子2产生缺少CRD1结构域的短形式的TACI。这种短的TACI与长形式的一样可以有效结合BAFF和APRIL。由于TACI与BAFF和APRIL具有较高的亲和力，使用胞外区TACI可溶部分，融合IgG的Fc片段，可以阻断BAFF和APRIL与细胞膜表面的TACI、BCMA和BAFFR受体结合，抑制BAFF和APRIL的生物学活性，从而治疗自身免疫疾病。阿塞西普(Atacicept)是ZymoGenetics公司研发的一款BAFF/APRIL Trap，分子形式为天然的TACI胞外区融合IgG1-Fc片段(FcγR结合活性缺失)，即TACI-Fc。泰它西普(Telitacicept，RCT-18)则是荣昌生物开发的TACI-Fc。泰它西普和阿塞西普均能同时靶向BAFF和APRIL两个细胞因子，相对于只抑制BAFF的Belimumab，同时抑制BAFF/APRIL的TACI-Fc能更有效地降低机体免疫反应，达到治疗SLE的目的。II期临床研究表明泰它西普高剂量组治疗组48周狼疮应答指数(SRI-4)显著高于安慰剂对照组(79.2％vs 32.0％)，到达主要终点，提示对于APRIL的抑制可能对于治疗红斑狼疮十分重要。
BCMA为III型跨膜蛋白，人BCMA蛋白是184个氨基酸的多肽，包含位于蛋白N端区域(氨基酸残基1-50)，跨膜区域(氨基酸残基51-93)和细胞内区域(氨基酸残基94-184)。BCMA的胞外区含有一个CRD，其对APRIL的结合强于TACI。BCMA能够促进浆细胞的存活，对自身免疫性疾病产生负面影响。BCMA还参与体液免疫的发展(如产生抗体)以及免疫相关疾病。尚未有BCMA胞外区或其融合蛋白进入临床。
在SLE的常规治疗中，主要包括抗疟药、糖皮质激素和免疫抑制剂。几大类激素治疗是基础用药，临床医师据SLE患者的疾病活动度来调整激素用量，在此基础上加免疫抑制剂、生物药或其他治疗。与类风湿性关节炎、银屑病关节炎和 强直性脊柱炎相比，用于SLE的新疗法，尤其是生物药物的批准非常少。传统激素作为广泛使用的基础治疗用药，选择性差，长期服用存在副作用，贝利尤单抗(Belimumab)是近50年在我国唯一已经获批的靶向治疗新药。因此急需开发新的安全有效的靶向药物来满足SLE巨大的临床需求。
由于BAFF/APRIL的过量表达与自身免疫性疾病密切相关，以其为靶分子设法阻断其活性，可以减少疾病的发生，减轻发病的症状，从而达到缓解和治疗自身免疫疾病的目的。封闭BAFF/APRIL信号的相关受体可以特异地作用于靶细胞，有望成为治疗自身免疫疾病的新的生物制剂。因此，本领域迫切需要开发新的、可有效抑制或封闭BAFF/APRIL途径的化合物，本公开提供了含有TACI多肽的融合蛋白，有潜力成为临床上安全、有效治疗自身免疫疾病(例如SLE)的药物。
发明内容
本公开提供了含有TACI多肽的新结构的融合蛋白、其编码核酸、载体、宿主细胞、药物组合物、其用于治疗或改善疾病(例如，B细胞障碍或自身免疫性疾病)的方法，及其制药用途。
融合蛋白
本公开提供一种融合蛋白，所述融合蛋白选自如下结构：
(1)包含TACI多肽；
(2)包含BCMA多肽；
(3)包含TACI多肽和BCMA多肽；
(4)包含TACI多肽和抗体(例如，抗IFNAR1抗体)；
(5)包含BCMA多肽和抗体(例如，抗IFNAR1抗体)；
(6)包含TACI多肽、BCMA多肽和抗体(例如，抗IFNAR1抗体)。
关于融合蛋白中的TACI多肽：
一些实施方案中，所述TACI多肽包含TACI胞外区的CRD1和/或CRD2，例如，包含TACI胞外区的CRD2。
一些实施方案中，所述TACI多肽包含选自如下(1)-(6)任一项的多肽：
(1)SEQ ID NO：1的第68-105位氨基酸残基或其变体
(2)SEQ ID NO：1或与之具有至少90％序列同一性的多肽；
(3)SEQ ID NO：1的第33-67位氨基酸残基；
(4)SEQ ID NO：1的第70-104位氨基酸残基或其变体；
(5)SEQ ID NO：1的第30-110、69-111、69-112、13-118、或33-104位氨基酸残基或其变体；
(6)SEQ ID NO：1的第68-106、68-107或68-108位氨基酸残基或其变体。
一些具体实施方案中，所述变体具有选自第69、72、73、77、85、102和103位中的一个或多个氨基酸突变。
一些具体实施方案中，所述变体具有选自69T或69R、72S、73E或73Q、77E、85T或85A、102A或102R、103Y中的一个或多个氨基酸替换。
一些具体实施方案中，所述变体具有选自如下任一的氨基酸替换或替换组合：69T；72S；73E；73Q；77E；69R/85T；69R/85A；102A；69R/85T/102R；73E/77E；72S/73E/77E；69T/102A；69T/103Y；69T/102A、103Y；69T/73E/77E/102A。
一些具体实施方案中，本公开以“突变1/突变2”表示突变1和突变2共同存在于突变后的变体中。例如“69T/102A”表示突变体中包含69T和102A的氨基酸突变，“69T/73E/77E/102A”表示突变体中包含69T、73E、77E和102A的氨基酸突变。
一些具体实施方案中，所述变体具有69T/73E/77E/102A的氨基酸替换组合，例如，具有L69T/K73E/K77E/Y102A的氨基酸替换组合。
所述氨基酸突变的位点是相对于TACI胞外区(SEQ ID NO：1)的自然顺序编号的氨基酸残基位点，例如，“所述变体具有选自第69位的氨基酸突变”是指在对应于SEQ ID NO：1的第69位的氨基酸残基具有突变。
一些实施方案中，TACI多肽的氨基酸序列包含选自SEQ ID NO：1、2、6-29任一所示的氨基酸序列。
本公开中，“至少90％(序列)同一性”涵盖至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％或100％(序列)同一性；“至少80％(序列)同一性”涵盖至少80％、至少81％、至少82％、至少83％、至少84％、至少85％、至少86％、至少87％、至少88％、至少89％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％或100％(序列)同一性。
关于融合蛋白中的BCMA多肽：
一些实施方案中，所述BCMA多肽包含BCMA胞外区的CRD1。
一些实施方案中，所述BCMA多肽包含SEQ ID NO：30的第7-41位氨基酸残基。
一些实施方案中，所述BCMA多肽包含SEQ ID NO：30、67、68任一所示或与之具有至少90％序列同一性的氨基酸序列。
关于融合蛋白中的抗体：
一些实施方案中，所述融合蛋白中的抗体为抗IFNAR1抗体或其抗原结合片段。
一些实施方案中，所述抗IFNAR1抗体或其抗原结合片段包含重链可变区(VH)和轻链可变区(VL)，其中，所述重链可变区包含氨基酸序列分别如SEQ ID NO：45-47所示的HCDR1、HCDR2和HCDR3，所述轻链可变区包含氨基酸序列分别如SEQ ID NO：48-50所示的LCDR1、LCDR2和LCDR3。
一些实施方案中，所述抗IFNAR1抗体的重链可变区的氨基酸序列如SEQ ID NO：43所示或与之具有至少90％序列同一性，轻链可变区的氨基酸序列如SEQ ID  NO：44所示或与之具有至少90％序列同一性。
一些实施方案中，所述抗IFNAR1抗体的重链的氨基酸序列如SEQ ID NO：55所示或与之具有至少80％序列同一性，轻链的氨基酸序列如SEQ ID NO：54所示或与之具有至少80％序列同一性。
一些实施方案中，所述抗IFNAR1抗体的重链的氨基酸序列如SEQ ID NO：61所示或与之具有至少80％序列同一性，轻链的氨基酸序列如SEQ ID NO：54所示或与之具有至少80％序列同一性。
一些实施方案中，本公开引入Anifrolumab、sifalimumab，以及全文引入WO20062002177A、WO2009100309A(例如3F11、4G5、11E2、9D4)、WO2020156474A(例如，7G4、10C5等)、WO2020057541A(例如8G11H和485G10H)、CN201610634601.7(例如H19B7+L16C11、H19B7+L8C3、H15D10+L16C11、H15D10+L8C3或抗IFNAR1-C1)、CN201510685200.X、WO2012162367A中抗IFNAR1抗体或其抗原结合片段作为本公开的抗IFNAR1抗体。
关于融合蛋白：
如前所述，本公开提供包含TACI多肽和BCMA多肽的融合蛋白、包含TACI多肽和抗IFNAR1抗体(或其抗原结合片段)的融合蛋白、包含TACI多肽、BCMA多肽、抗IFNAR1抗体(或其抗原结合片段)的融合蛋白。
一些实施方案中，所述融合蛋白还含有免疫球蛋白的重链恒定区(Fc区)。一些具体实施方案中，所述Fc区包含能够缔合的两个亚基。一些实施方案中，所述两个亚基是相同或不同的第一亚基和第二亚基。一些实施方案中，所述Fc区为IgG、IgA、IgM、IgD的Fc区；一些实施方案中，所述Fc区为人IgG1、IgG2、IgG3、IgG4的Fc区，例如人IgG1的Fc区，又例如为SEQ ID NO：3所示的Fc区。
一些实施方案中，所述融合蛋白中的所述Fc区相比野生型Fc区包含一个或多个氨基酸取代，所述的氨基酸取代能够减少其与Fc受体(FcR)的结合；一些实施方案中，所述的氨基酸取代能够减少其与Fcγ受体(FcγR)的结合；一些实施方案中，所述Fc区具有YTE突变(M252Y、S254T和T256E)、S228P、L234F、L235E、L234F/L235E、L234A/L235A或L234F/L235E/P331S突变，所述突变位点编号依据为EU索引。
一些实施方案中，所述融合蛋白中的所述Fc区包含能够彼此缔合的第一亚基与第二亚基，所述第一亚基和第二亚基具有一个或多个减少同源二聚化的氨基酸取代。一些实施方案中，所述第一亚基具有根据杵臼技术的凸起结构，所述第二亚基具有根据杵臼技术的孔结构，或者所述第一亚基具有根据杵臼技术的孔结构，所述第二亚基具有根据杵臼技术的凸起结构。一些实施方案中，所述第一亚基的氨基酸残基取代包括选自354、356、358和366的位点中的一个或多个氨基酸取代，所述第二亚基的氨基酸残基包括选自349、356、358、366、368和407的位点中的一个或多个氨基酸取代。一些实施方案中，所述第一亚基包括选自354C、 356E、358M和366W中的一个或多个氨基酸取代，所述第二亚基包括选自349C、356E、358M、366S、368A和407V中的一个或多个氨基酸取代。一些实施方案中，所述第一亚基包括354C、356E、358M和366W的氨基酸取代，所述第二亚基包括349C、356E、358M、366S、368A和407V的氨基酸取代。
第一方面，关于包含TACI多肽和BCMA多肽的融合蛋白：
一些实施方案中，所述TACI多肽为前述本公开提供的任一TACI多肽，所述BCMA多肽为前述本公开提供的任一BCMA多肽。
一些实施方案中，所述融合蛋白中的包含一个或多个(例如2、3、4、5、6个)所述TACI多肽，和一个或多个(例如2、3、4、5、6个)所述BCMA多肽。一些实施方案中，所述融合蛋白中包含1个所述TACI多肽和1个所述BCMA多肽，或2个所述TACI多肽和2个所述BCMA多肽,或3个所述TACI多肽和3个所述BCMA多肽，或4个所述TACI多肽和4个所述BCMA多肽，或2个所述TACI多肽和1个所述BCMA多肽，或1个所述TACI多肽和2个所述BCMA多肽，或3个所述TACI多肽和1个所述BCMA多肽，或1个所述TACI多肽和3个所述BCMA多肽；当包含2个及以上所述TACI多肽时，所述TACI多肽可以是所述本公开提供的序列相同的或不同的TACI多肽；当包含2个及以上所述BCMA多肽时，所述BCMA多肽可以是所述本公开提供的序列相同的或不同的BCMA多肽。
一些实施方案中，所述融合蛋白中，所述TACI多肽和Fc区的一个亚基是以任意顺序连接的，可选地，所述TACI多肽与Fc区的第一亚基或第二亚基通过连接子或直接连接。一些实施方案中，如前任一的融合蛋白，所述BCMA多肽和Fc区的一个亚基是以任意的顺序连接的，可选地，所述BCMA多肽与Fc区的第一亚基或第二亚基通过连接子或直接连接。一些实施方案中，所述TACI多肽的C端与Fc区的第一亚基或第二亚基的N端通过连接子或直接连接；或者所述TACI多肽的N端与Fc区的第一亚基或第二亚基的C端通过连接子或直接连接。一些实施方案中，所述BCMA多肽的C端与Fc区的第一亚基或第二亚基的N端通过连接子或直接连接；或者所述BCMA多肽的N端与Fc区的第一亚基或第二亚基的C端通过连接子或直接连接。
一些实施方案中，所述含有TACI多肽和BCMA多肽的融合蛋白，其包含如下(I)-(VIII)任一所示的多肽链：
(I)[TACI多肽]-[连接子1]a-[BCMA多肽]-[连接子2]b-[Fc区]e；
(II)[BCMA多肽]-[连接子1]a-[TACI多肽]-[连接子2]b-[Fc区]e；
(III)[Fc区]e-[连接子3]c-[TACI多肽]-[连接子4]d-[BCMA多肽]；
(IV)[Fc区]e-[连接子3]c-[BCMA多肽]-[连接子4]d-[TACI多肽]；
(V)[TACI多肽1]-[连接子1]a-[BCMA多肽1]-[连接子2]b-[Fc区]e-[连接子3]c-[TACI多肽2]-[连接子4]d-[BCMA多肽2]；
(VI)[BCMA多肽1]-[连接子1]a-[TACI多肽1]-[连接子2]b-[Fc区]e-[连接子 3]c-[BCMA多肽2]-[连接子4]d-[TACI多肽2]；
(VII)[TACI多肽1]-[连接子1]a-[BCMA多肽1]-[连接子2]b-[Fc区]e-[连接子3]c-[BCMA多肽2]-[连接子4]d-[TACI多肽2]；
(VIII)[BCMA多肽1]-[连接子1]a-[TACI多肽1]-[连接子2]b-[Fc区]e-[连接子3]c-[TACI多肽2]-[连接子4]d-[BCMA多肽2]；
其中，-表示肽键，连接子为能够实现连接功能的多肽，连接子1、连接子2、连接子3和连接子4可以相同，也可以不同；TACI多肽1和TACI多肽2选自本公开提供的TACI多肽，可以相同，也可以不同；BCMA多肽1和BCMA多肽2选自本公开提供的BCMA多肽，可以相同，也可以不同；a、b、c、d、e分别独立地为0或1，例如，方案(I)或(II)中，a和b均为1，或a为1、b为0，或a为0、b为1，以及，e为0或1；例如，方案(III)或(IV)中，c和d均为1，或c为1、d为0，或c为0、d为1，以及，e为0或1；例如，方案(V)、(VI)、(VII)或(VIII)中，a、b、c、d均为1，或a、b、c均为1且d为0，或a、b为1且c、d为0，或a为1且b、c、d均为0，或a为0且b、c、d均为1，或a、b为0且c、d为1，或a、b、c为0且d为1，或a、c为1且b、d为0，或a、c为0且b、d为1，以及，e为0或1。
一些实施方案中，所述连接子为如(GmSn)h或(GGNGT)h(SEQ ID NO：75)或(YGNGT)h(SEQ ID NO：76)或(EPKSS)h(SEQ ID NO：77)所示的氨基酸序列，其中m、n各自独立地选自1-8的整数(例如，1、2、3、4、5、6、7或8)，h独立地选自1-20的整数(例如，1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19或20)。在一些具体实施方案中，所述连接子为(GxS)y连接子，其中，x选自1-5的整数，y选自1-6的整数，例如为SEQ ID NO：39-41任一所示的连接子。例如，所述连接子具有如(G4S)y(SEQ ID NO：78)所示的氨基酸序列，y独立地选自1-6的整数(例如，y为1、2、3、4、5、6)。
一些实施方案中，提供包含TACI多肽和BCMA多肽的融合蛋白，其含有SEQ ID NO：31-38、69、70任一所示或与之具有至少90％序列同一性的多肽。
一些实施方案中，所述融合蛋白为多聚体，例如为二聚体(例如为同二聚体或异二聚体)。
一些实施方案中，提供含有TACI多肽和BCMA多肽的融合蛋白，所述TACI多肽可以是变体，所述变体有1至10个(1、2、3、4、5、6、7、8、9、10个)氨基酸突变；一些实施方案中，提供含有TACI多肽和BCMA多肽的融合蛋白，所述BCMA多肽可以是变体，所述变体有1至10个(1、2、3、4、5、6、7、8、9、10个)氨基酸突变；所述氨基酸突变可以是保守的替换、取代或修饰，和/或不影响功能的缺失、添加。
一些实施方案中，提供蛋白或分子，所述蛋白或分子与所述含有TACI多肽和BCMA多肽的融合蛋白、所述TACI多肽、或所述BCMA多肽竞争结合BAFF和 /或APRIL，或阻断所述含有TACI多肽和BCMA多肽的融合蛋白、所述TACI多肽、或所述BCMA多肽结合BAFF和/或APRIL。
第二方面，关于包含TACI多肽和抗IFNAR1抗体的融合蛋白：
一些实施方案中，所述TACI多肽为前述本公开提供的任一TACI多肽，所述抗IFNAR1抗体为前述本公开提供的任一抗IFNAR1抗体。
一些实施方案中，所述包含TACI多肽和抗IFNAR1抗体的融合蛋白中包含一个或多个(例如2、3、4、5、6个)TACI多肽。一些实施方案中，所述包含TACI多肽和抗IFNAR1抗体的融合蛋白中的包含一个或多个(例如2、3、4个)抗IFNAR1抗体。
一些实施方案中，所述含有TACI多肽和抗IFNAR1抗体的融合蛋白，其包含如下(I)-(VIII)任一所示的多肽链：
(I)第一多肽链，其从N端到C端依次为：[TACI多肽]-[连接子1]a-[抗体重链]，和第二多肽链，其为抗体轻链；
(II)第一多肽链，其从N端到C端依次为：[抗体重链]-[连接子2]b-[TACI多肽],和第二多肽链，其为抗体轻链；
(III)第一多肽链，其为抗体重链，和第二多肽链，其从N端到C端依次为：[TACI多肽]-[连接子3]c-[抗体轻链]；
(IV)第一多肽链，其为抗体重链，和第二多肽链，其从N端到C端依次为：[抗体轻链]-[连接子4]d-[TACI多肽]；
(V)第一多肽链，其从N端到C端依次为：[TACI多肽1]-[连接子1]a-[抗体重链]，和第二多肽链，其从N端到C端依次为：[抗体轻链]-[连接子4]d-[TACI多肽2]；
(VI)第一多肽链，其从N端到C端依次为：[抗体重链]-[连接子2]b-[TACI多肽1]，和第二多肽链，其从N端到C端依次为：[TACI多肽2]-[连接子3]c-[抗体轻链]；
(VII)第一多肽链，其从N端到C端依次为：[TACI多肽1]-[连接子1]a-[抗体重链]-[连接子2]b-[TACI多肽2]，和第二多肽链，其为抗体轻链；
(VIII)第一多肽链，其为抗体重链，和第二多肽链，其从N端到C端依次为：[TACI多肽1]-[连接子3]c-[抗体轻链]-[连接子4]d-[TACI多肽2]；
其中，-表示肽键，连接子为能够实现连接功能的多肽，连接子1、连接子2、连接子3、连接子4可以相同，也可以不同；TACI多肽1和TACI多肽2选自本公开前述任一TACI多肽，TACI多肽1和TACI多肽2可以相同也可以不同，a、b、c、d分别独立地为0或1。方案(I)中，a为1或0。方案(II)中，b为1或0。方案(III)中，c为1或0。方案(IV)中，d为1或0。方案(V)中，a、d可以同时为0或为1，或a为0、d为1，或a为1、d为0；方案(VI)中，b、c可以同时为0或为1，或b为0、c为1，或b为1、c为0；方案(VII)中，a、b可以同时为0或为 1，或a为0、b为1，或a为1、b为0；方案(VIII)中，c、d可以同时为0或为1，或c为0、d为1，或c为1、d为0。
一些实施方案中，所述连接子为如(GmSn)h或(GGNGT)h或(YGNGT)h或(EPKSS)h所示的氨基酸序列，其中m、n各自独立地选自1-8的整数(例如，1、2、3、4、5、6、7或8)，h独立地选自1-20的整数(例如，1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19或20)。在一些具体实施方案中，所述连接子为(GxS)y连接子，其中，x选自1-5的整数，y选自1-6的整数，例如为SEQ ID NO：39-41任一所示的连接子。例如，所述连接子具有如(G4S)y所示的氨基酸序列，y独立地选自1-6的整数(例如，y为1、2、3、4、5、6)。
一些实施方案中，提供包含TACI多肽和抗IFNAR1抗体的融合蛋白，其包含如SEQ ID NO：51-53、62任一所示或与之具有至少80％、至少90％序列同一性的第一多肽链，和如SEQ ID NO：54所示或与之具有至少80％、至少90％序列同一性的第二多肽链；
包含如SEQ ID NO：55所示或与之具有至少80％、至少90％序列同一性的第一多肽链，和如SEQ ID NO：56-60任一所示或与之具有至少80％、至少90％序列同一性的第二多肽链；或
包含如SEQ ID NO：61所示的或与之具有至少80％、至少90％序列同一性的第一多肽链，和如SEQ ID NO：56-60任一所示或与之具有至少80％、至少90％序列同一性的第二多肽链。
一些实施方案中，前述融合蛋白包含两条相同的第一多肽链，和两条相同的第二多肽链。
一些实施方案中，本公开提供含有TACI多肽和抗IFNAR1抗体的融合蛋白，所述TACI多肽可以是变体，所述变体有1至10个(1、2、3、4、5、6、7、8、9、10个)氨基酸突变；所述氨基酸突变可以是保守的替换、取代或修饰，和/或不影响功能的缺失、添加。
一些实施方案中，提供蛋白或分子，所述蛋白或分子与前述含有TACI多肽和抗IFNAR1抗体的融合蛋白竞争结合BAFF和/或APRIL，或阻断其结合BAFF和/或APRIL。
第三方面，关于包含TACI多肽、BCMA多肽和抗IFNAR1抗体的融合蛋白：
一些实施方案中，所述TACI多肽为前述本公开提供的任一TACI多肽，所述BCMA多肽为前述本公开提供的任一BCMA多肽，所述抗IFNAR1抗体为前述本公开提供的任一抗IFNAR1抗体。
一些实施方案中，所述包含TACI多肽、BCMA多肽和抗IFNAR1抗体的抗体融合蛋白中的包含一个或多个(例如2、3、4、5、6个)TACI多肽。一些实施方案中，所述包含TACI多肽、BCMA多肽和抗IFNAR1抗体的抗体融合蛋白中的包含一个或多个(例如2、3、4、5、6个)BCMA多肽。一些实施方案中，所述包含 TACI多肽、BCMA多肽和抗IFNAR1抗体的抗体融合蛋白中的包含一个或多个(例如2、3、4个)抗IFNAR1抗体。一些实施方案中，前述包含TACI多肽、BCMA多肽和抗IFNAR1抗体的抗体融合蛋白中包含1个前述TACI多肽和1个前述BCMA多肽，或2个前述TACI多肽和2个前述BCMA多肽,或3个前述TACI多肽和3个前述BCMA多肽，或4个前述TACI多肽和4个前述BCMA多肽，或2个前述TACI多肽和1个前述BCMA多肽，或1个前述TACI多肽和2个前述BCMA多肽，或3个前述TACI多肽和1个前述BCMA多肽，或1个前述TACI多肽和3个前述BCMA多肽，和一个抗IFNAR1抗体；当包含2个及以上前述TACI多肽时，所述TACI多肽可以是本公开提供的序列相同或不同的TACI多肽；当包含2个及以上前述BCMA多肽时，所述BCMA多肽可以是本公开提供的序列相同或不同的BCMA多肽。
一些实施方案中，如前任一项含有TACI多肽、BCMA多肽和抗IFNAR1抗体的融合蛋白，其包含如下(I)-(VIII)任一所示的多肽链：
(I)第一多肽链，其从N端到C端依次为：[TACI多肽和BCMA多肽结构域]-[连接子1]a-[抗体重链]，和第二多肽链，其为抗体轻链；
(II)第一多肽链，其从N端到C端依次为：[抗体重链]-[连接子2]b-[TACI多肽和BCMA多肽结构域],和第二多肽链，其为抗体轻链；
(III)第一多肽链，其为抗体重链，和第二多肽链，其从N端到C端依次为：[TACI多肽和BCMA多肽结构域]-[连接子3]c-[抗体轻链]；
(IV)第一多肽链，其为抗体重链，和第二多肽链，其从N端到C端依次为：[抗体轻链]-[连接子4]d-[TACI多肽和BCMA多肽结构域]；
(V)第一多肽链，其从N端到C端依次为：[TACI多肽和BCMA多肽结构域1]-[连接子1]a-[抗体重链]，和第二多肽链，其从N端到C端依次为：[抗体轻链]-[连接子4]d-[TACI多肽和BCMA多肽结构域2]；
(VI)第一多肽链，其从N端到C端依次为：[抗体重链]-[连接子2]b-[TACI多肽和BCMA多肽结构域1]，和第二多肽链，其从N端到C端依次为：[TACI多肽和BCMA多肽结构域2]-[连接子3]c-[抗体轻链]；
(VII)第一多肽链，其从N端到C端依次为：[TACI多肽和BCMA多肽结构域1]-[连接子1]a-[抗体重链]-[连接子2]b-[TACI多肽和BCMA多肽结构域2]，和第二多肽链，其为抗体轻链；
(VIII)第一多肽链，其为抗体重链，和第二多肽链，其从N端到C端依次为：[TACI多肽和BCMA多肽结构域1]-[连接子3]c-[抗体轻链]-[连接子4]d-[TACI多肽和BCMA多肽结构域2]；
其中，[TACI多肽和BCMA多肽结构域]”、[TACI多肽和BCMA多肽结构域1]”、[TACI多肽和BCMA多肽结构域2]”从N端到C端依次为：[TACI多肽]-[连接子5]e-[BCMA多肽]，或为：[BCMA多肽]-[连接子5]e-[TACI多肽]，“TACI多 肽和BCMA多肽结构域1”和“TACI多肽和BCMA多肽结构域2”中的TACI可以相同也可以不同，BCMA可以相同也可以不同；
其中，-表示肽键，连接子为能够实现连接功能的多肽，连接子1、连接子2、连接子3、连接子4、连接子5可以相同，也可以不同；a、b、c、d、e分别独立地为0或1。a、b、c、d分别独立地为0或1。方案(I)中，a为1或0。方案(II)中，b为1或0。方案(III)中，c为1或0。方案(IV)中，d为1或0。方案(V)中，a、d可以同时为0或为1，或a为0、d为1，或a为1、d为0；方案(VI)中，b、c可以同时为0或为1，或b为0、c为1，或b为1、c为0；方案(VII)中，a、b可以同时为0或为1，或a为0、b为1，或a为1、b为0；方案(VIII)中，c、d可以同时为0或为1，或c为0、d为1，或c为1、d为0；以上方案中，e可以独立地为0或1。
一些实施方案中，连接子为如(GmSn)h或(GGNGT)h或(YGNGT)h或(EPKSS)h所示的氨基酸序列，其中m、n各自独立地选自1-8的整数(例如，1、2、3、4、5、6、7或8)，h独立地选自1-20的整数(例如，1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19或20)。在一些具体实施方案中，所述连接子为(GxS)y连接子，其中，x选自1-5的整数，y选自1-6的整数，例如为SEQ ID NO：39-41任一所示的连接子。例如，所述连接子具有如(G4S)y所示的氨基酸序列，y独立地选自1-6的整数(例如，y为1、2、3、4、5、6)。
一些实施方案中，提供包含TACI多肽、BCMA多肽和抗IFNAR1抗体的融合蛋白，其包含如SEQ ID NO：55所示或与之具有至少80％、至少90％序列同一性的第一多肽链，和如SEQ ID NO：63-66、71、72所示或与之具有至少80％、至少90％序列同一性的第二多肽链；
包含如SEQ ID NO：61所示或与之具有至少80％、至少90％序列同一性的第一多肽链，和如SEQ ID NO：63-66、71、72任一所示或与之具有至少80％、至少90％序列同一性的第二多肽链；或
包含如SEQ ID NO：73或74所示的或与之具有至少80％、至少90％序列同一性的第一多肽链，和如SEQ ID NO：54所示或与之具有至少80％、至少90％序列同一性的第二多肽链。
一些实施方案中，前述融合蛋白包含两条相同的第一多肽链，和两条相同的第二多肽链。
一些实施方案中，本公开提供含有TACI多肽、BCMA多肽和抗IFNAR1抗体的融合蛋白，所述TACI多肽可以是变体，所述变体有1至10个(1、2、3、4、5、6、7、8、9、10个)氨基酸突变；所述氨基酸突变可以是保守的替换、取代或修饰，和/或不影响功能的缺失、添加。
一些实施方案中，提供蛋白或分子，所述蛋白或分子与前述含有TACI多肽、BCMA多肽和抗IFNAR1抗体的融合蛋白竞争结合BAFF和/或APRIL，或阻断其 结合BAFF和/或APRIL。
关于前述第一方面至第三方面的融合蛋白：
一些实施方案中，所述含有TACI多肽和BCMA多肽，含有TACI多肽和IFNAR1抗体的融合蛋白，含有TACI多肽、BCMA多肽和IFNAR1抗体的融合蛋白具有以下一种或多种性质或功能：
(a)结合于BAFF的活性；
(b)结合于APRIL的活性；
(c)降低血清免疫球蛋白(例如IgE或IgM)的浓度；
(d)降低脾脏重量；
(e)抑制或封闭BAFF/APRIL途径；
(f)减少B细胞数量。
一些实施方案中，所述本公开提供的包含TACI多肽的蛋白(例如TACI-Fc)具有以下一个或多个功能活性：
a.不容易发生TACI多肽的断裂片段；在一些实施方案中，所述断裂可通过质谱分析检测，例如实施例2中的TACI断裂分析方法；
b.阻断BAFF与BAFF-R结合；在一些实施方案中，TACI-Fc阻断BAFF与BAFF-R结合的IC50值小于23nM、小于10nM、小于6nM、小于5nM、小于4nM、小于3nM、小于2nM、小于1nM或更小；所述IC50值通过ELISA方法检测；在一些实施方案中，所述IC50值测试方法见实施例1；
c.与BAFF结合的能力；在一些实施方案中，所述TACI-Fc结合BAFF的EC50值小于5nM、1nM、0.5nM、0.4nM、0.3nM、0.2nM、0.1nM或更小，所述EC50值通过ELISA方法检测，例如实施例2的ELISA结合实验(包被抗原蛋白)；
d.抑制BAFF诱导B细胞增殖；在一些实施方案中，TACI-Fc抑制BAFF诱导B细胞增殖的IC50值小于0.3nM、小于0.2nM、小于0.1nM、0.06nM、小于0.05nM、小于0.01nM或更小。所述IC50值通过ELISA方法检测，例如实施例17中的ELISA实验；
e.抑制APRIL诱导B细胞增殖；在一些实施方案中，TACI-Fc抑制APRIL诱导B细胞增殖的IC50值小于3nM、小于2nM、小于1nM、小于0.5nM、小于0.4nM、小于0.3nM、0.2nM、小于0.1nM、小于0.05或更小；所述IC50值通过ELISA方法检测；
f.高亲和力与人BAFF和/或人APRIL结合；在一些实施方案中，所述亲和力通过Biacore方法检测；在一些实施方案中，所述TACI-Fc融合蛋白与人BAFF结合的KD值小于1E-10M、小于9E-11M、8E-11M或更小；在一些实施方案中，所述TACI-Fc融合蛋白与人APRIL结合的KD值小于2.3E-11M、小于2.0E-11M、1.9E-11M、1.3E-11M或更小；
g.良好的体内药代动力；在一些实施方案中，TACI-Fc大鼠体内半衰期大于4 天；和/或
h.良好的稳定性；在一些实施方案中，所述TACI-Fc在40℃恒温条件下存放四周，TACI-Fc融合蛋白纯度依然能保持在94％以上(SEC％)；在一些实施方案中，所述TACI-Fc的pI值小于9、小于8、小于7、小于6或更小，所述pI值通过“18cProt/TrEMBL”系统分析测定。
一些实施方案中，前述本公开提供的含有TACI多肽和BCMA多肽的融合蛋白具有以下一个或多个功能活性：
a.不容易发生TACI多肽的断裂片段；在一些实施方案中，所述断裂可通过质谱分析检测，例如实施例2中的TACI断裂分析方法；
b.与BAFF结合的能力；在一些实施方案中，所述述融合蛋白结合BAFF的EC50值小于1nM、0.5nM、0.4nM、0.3nM、0.2nM、0.1nM或更小，所述EC50值通过ELISA方法检测，例如实施例5的ELISA实验；
c.与APRIL结合的能力；在一些实施方案中，所述融合蛋白结合APRIL的EC50值小于1nM、0.5nM、0.4nM、0.3nM、0.2nM、0.1nM或更小，所述EC50值通过ELISA方法检测，例如实施例5的ELISA实验；
d.良好的体内药代动力；
e.良好的稳定性。
一些实施方案中，前述本公开提供的包含TACI多肽、BCMA多肽和抗IFNAR1抗体的抗体融合蛋白具有以下一个或多个功能活性：
a.不容易发生TACI多肽的断裂片段；在一些实施方案中，所述断裂可通过质谱分析检测，例如实施例2中的TACI断裂分析方法；
b.对I型干扰素的活性抑制；在一些实施方案中，所述融合蛋白具有与抗IFNAR1抗体(例如Anifrolumab)一致或相近似的对IFN的活性的抑制率，所述抑制率在体外通过对IFN-α/β报告基因的方法检测，例如实施例10中的实验；所述抑制率在体内通过检测PBMC中IFN-α下游基因ISG的mRNA表达，例如实施例21中的实验；
c.与BAFF结合的能力；在一些实施方案中，所述融合蛋白结合BAFF的EC50值小于1.5nM、1nM、0.5nM、0.4nM、0.3nM、0.2nM、0.1nM或更小，所述EC50值通过ELISA方法检测，例如实施例11的ELISA实验；
d.与APRIL结合的能力；在一些实施方案中，所述融合蛋白结合APRIL的EC50值小于0.5nM、0.4nM、0.3nM、0.2nM、0.1nM或更小，所述EC50值通过ELISA方法检测，例如实施例11的ELISA实验；
e.抑制IFN-α分泌；一些实施方案中，所述融合蛋白具有与抗IFNAR1抗体(例如Anifrolumab)一致或相近似的对浆细胞样树突状细胞(pDC)的IFN-α细胞因子分泌的抑制能力，所述抑制能力检测使用例如实施例15中的检测方法；一些实施方案中，所述融合蛋白具有与抗IFNAR1抗体(例如Anifrolumab)一致或相近似的抑 制IFN-α诱导的B细胞向浆细胞的体外分化的能力，所述抑制能力检测使用例如实施例16中的检测方法；
f.抑制浆细胞的分化，抑制BAFF诱导的B细胞增殖；一些实施方案中，所述融合蛋白抑制BAFF诱导的B细胞增殖的抑制率为至少50％、至少40％、至少30％、至少20％、至少10％，所述抑制率是加入抗体融合蛋白后的B细胞数与加入同等浓度的IgG1的B细胞数的比值，是通过FACS检测，例如实施例17中的方法；
g.抑制APRIL诱导的B细胞增殖；一些实施方案中，所述融合蛋白抑制APRIL诱导的B细胞增殖的抑制率为至少50％、至少40％、至少30％、至少20％、至少10％，所述抑制率是加入抗体融合蛋白后的B细胞数与加入同等浓度的IgG1的B细胞数的比值，所述抑制率是通过FACS检测，例如实施例18中的方法；
h.抑制BAFF和APRIL诱导的浆细胞生成；一些实施方案中，所述融合蛋白抑制BAFF和APRIL诱导的浆细胞生成，抑制率为至少90％、至少80％、至少70％、至少60％、至少50％、至少40％、至少30％、至少20％，所述抑制率是加入抗体融合蛋白后的B细胞数与加入同等浓度的IgG1的B细胞数的比值，所述抑制是通过FACS检测，例如实施例19中的方法；
i.抑制体内IgA的生成；一些实施方案中，所述融合蛋白抑制体内IgA的生成的抑制率为至少80％、至少70％、至少60％、至少50％、至少40％、至少30％，所述抑制率是与同等浓度的PBS相比，所述抑制是通过ELISA检测，例如实施例22中的方法；
j.良好的体内药代动力；
k.良好的稳定性。
此外，一些实施方案中，提供一种产品或组合，其包含前述本公开任一的含有TACI多肽和BCMA多肽的融合蛋白，还包含抗体。一些实施方案中，提供一种产品或组合，其包含前述本公开任一的含有TACI多肽，还包含抗体。一些实施方案中，所述抗体为抗IFNAR1抗体，例如为Anifrolumab。
一些实施方案中，提供一种复合物，其含有前述本公开任一的TACI多肽和前述本公开任一的BCMA多肽。可选地，所述TACI多肽和BCMA多肽可以是直接或通过前述本公开任一的连接子可操作地相连接的，也可以是不相连接的。
一些实施方案中，提供一种复合物，其含有前述本公开任一的TACI多肽和前述本公开任一的BCMA多肽；或含有前述本公开任一的TACI多肽和前述本公开任一的抗IFNAR1抗体；或含有前述本公开任一的BCMA多肽和前述本公开任一的抗IFNAR1抗体；或含有前述本公开任一的TACI多肽和前述本公开任一的抗IFNAR1抗体和前述本公开任一的BCMA多肽。可选地，所述TACI多肽、BCMA多肽、抗IFNAR1抗体可以是直接或通过前述本公开任一的连接子可操作地相连接的，也可以是不相连接的。
多核苷酸和载体
本公开提供(经分离的)多核苷酸，其编码前述本公开任一的TACI多肽、BCMA多肽、包含TACI多肽和BCMA多肽的融合蛋白、包含TACI多肽和抗体(例如，抗IFNAR1抗体)的融合蛋白、包含TACI多肽和BCMA多肽和抗体(例如，抗IFNAR1抗体)的融合蛋白、产品、组合或复合物。所述多核苷酸可以是DNA或RNA(例如mRNA)。
本公开的核酸也可呈载体形式，可存在于载体中和/或可为载体的一部分，该载体例如质粒、粘端质粒、YAC或病毒载体。载体可尤其为表达载体，即可提供CD40结合分子体外和/或体内(即在适合宿主细胞、宿主有机体和/或表达系统中)表达的载体。该表达载体通常包含至少一种本公开的核酸，其可操作地连接至一个或多个适合的表达调控元件(例如启动子、增强子、终止子等)。针对在特定宿主中的表达对所述元件及其序列进行选择为本领域技术人员的常识。对本公开的TACI多肽、BCMA多肽、包含TACI多肽和BCMA多肽的融合蛋白、包含TACI多肽和抗体(例如，抗IFNAR1抗体)的融合蛋白、包含TACI多肽和BCMA多肽和抗体(例如，抗IFNAR1抗体)的融合蛋白、产品、组合或复合物的表达有用或必需的调控元件及其它元件例如为启动子、增强子、终止子、整合因子、选择标记物、前导序列、报告基因。
本公开的核酸可基于本公开的多肽的氨基酸序列的信息通过已知的方式(例如通过自动DNA合成和/或重组DNA技术)制备或获得，和/或可从适合的天然来源加以分离。
宿主细胞
本公开提供表达或能够表达一种或多种本公开的TACI多肽、BCMA多肽、包含TACI多肽和BCMA多肽的融合蛋白、包含TACI多肽和抗体(例如，抗IFNAR1抗体)的融合蛋白、包含TACI多肽和BCMA多肽和抗体(例如，抗IFNAR1抗体)的融合蛋白、产品、组合或复合物，和/或含有本公开的核酸或载体的重组宿主细胞。
一些实施方案中，宿主细胞为细菌细胞、真菌细胞或哺乳动物细胞。
细菌细胞例如包括革兰氏阴性细菌菌株(例如大肠杆菌(Escherichia coli)菌株、变形杆菌属(Proteus)菌株及假单胞菌属(Pseudomonas)菌株)及革兰氏阳性细菌菌株(例如芽孢杆菌属(Bacillus)菌株、链霉菌属(Streptomyces)菌株、葡萄球菌属(Staphylococcus)菌株及乳球菌属(Lactococcus)菌株)的细胞。
真菌细胞例如包括木霉属(Trichoderma)、脉孢菌属(Neurospora)及曲菌属(Aspergillus)的物种的细胞；或者包括酵母属(Saccharomyces)(例如酿酒酵母(Saccharomyces cerevisiae))、裂殖酵母属(Schizosaccharomyces)(例如粟酒裂殖酵母(Schizosaccharomyces pombe))、毕赤酵母属(Pichia)(例如巴斯德毕赤酵母(Pichia pastoris)及嗜甲醇毕赤酵母(Pichia methanolica))及汉森酵母属(Hansenula)的物种的 细胞。
哺乳动物细胞例如包括例如HEK293细胞、CHO细胞、BHK细胞、HeLa细胞、COS细胞等。
然而，本公开也可使用两栖类细胞、昆虫细胞、植物细胞及本领域中用于表达异源蛋白的任何其它细胞。
本公开的细胞不能发育成完成的植株或动物个体。
制备方法
本公开提供一种用于制备TACI多肽、BCMA多肽、包含TACI多肽和BCMA多肽的融合蛋白、包含TACI多肽和抗体(例如，抗IFNAR1抗体)的融合蛋白、包含TACI多肽和BCMA多肽和抗体(例如，抗IFNAR1抗体)的融合蛋白、产品、组合或复合物的方法，包括：在如前所述的宿主细胞中表达前述多肽、融合蛋白、产品、组合或复合物，并自该宿主细胞中分离所述多肽、融合蛋白、产品、组合或复合物。可选地，还可以包含纯化步骤。可选地，还可以包含过滤、浓缩步骤。可溶的混合物和多聚体，也可以用常规方法去除，比如分子筛、离子交换。得到的产物需立即冷冻，如-70℃，或者冻干。
所述多肽、融合蛋白、产品、组合或复合物可用常规方法制备和纯化。比如，编码含有所述融合蛋白的cDNA序列，可以克隆并重组至表达载体。重组的表达载体可以稳定地转染CHO细胞。哺乳动物类表达系统会导致蛋白的糖基化(例如，在Fc区的高度保守N端)。获得阳性的克隆在生物反应器的培养基中扩大培养以生产蛋白。分泌了蛋白的培养液可以用常规技术纯化、收集、过滤、浓缩。可溶的混合物和多聚体，也可以用常规方法去除，比如分子筛，离子交换。然而，本公开的多肽、融合蛋白、产品、组合或复合物也可以通过本领域已知的其它产生蛋白质的方法获得，例如化学合成，包括固相或液相合成。
组合物
本公开提供组合物，例如药物组合物，其含有治疗有效量的TACI多肽、BCMA多肽、包含TACI多肽和BCMA多肽的融合蛋白、包含TACI多肽和抗体(例如，抗IFNAR1抗体)的融合蛋白、包含TACI多肽和BCMA多肽和抗体(例如，抗IFNAR1抗体)的融合蛋白、产品、组合、复合物或多核苷酸，和一种或多种可药用的赋形剂、稀释或载体。
在一些具体实施方案中，所述药物组合物单位剂量中可含有0.01至99重量％的前述任一的多肽、融合蛋白、产品、组合、复合物或多核苷酸。在一些具体实施方案中，所述药物组合物单位剂量中含前述任一的多肽、融合蛋白、产品、组合、复合物或多核苷酸的量为0.1-2000mg，在一些具体实施方案中为1-1000mg。
试剂盒
本公开提供试剂盒，包含本公开的TACI多肽、BCMA多肽、包含TACI多肽和BCMA多肽的融合蛋白、包含TACI多肽和抗体(例如，抗IFNAR1抗体)的融合 蛋白、包含TACI多肽和BCMA多肽和抗体(例如，抗IFNAR1抗体)的融合蛋白、产品、组合、复合物或多核苷酸。一些实施方案中，还提供包含上述多核苷酸的诊断试剂，以及提供相关诊断用途。
治疗疾病的方法和制药用途
一些实施方案中，本公开提供一种治疗或改善疾病或病症的方法，包括向有需要的受试者施用治疗有效量的前述本公开任一的TACI多肽、BCMA多肽、包含TACI多肽和BCMA多肽的融合蛋白、包含TACI多肽和抗体(例如，抗IFNAR1抗体)的融合蛋白、包含TACI多肽和BCMA多肽和抗体(例如，抗IFNAR1抗体)的融合蛋白、产品、组合、复合物、多核苷酸，或药物组合物。一些实施方案中，所述疾病或病症是与TACI、BCMA和/或IFN表达相关的疾病或病症。
一些实施方案中，提供前述本公开任一的TACI多肽、BCMA多肽和抗IFNAR1抗体联合治疗疾病或病症的方法或制药用途。一些实施方案中，提供前述本公开任一的TACI多肽和抗IFNAR1抗体联合治疗疾病或病症的方法或制药用途。一些具体实施方案中，进一步包括与BCMA多肽的联用。一些实施方案中，提供前述本公开任一的含有BCMA多肽和抗IFNAR1抗体的融合蛋白治疗疾病或病症的方法或制药用途。一些具体实施方案中，进一步包括与TACI多肽的联用。所述抗IFNAR1抗体例如为Anifrolumab。
一些实施方案中，提供一种治疗或改善B细胞障碍或自身免疫性疾病的方法，所述方法包括向有需要的受试者施用治疗有效量的本公开的TACI多肽、BCMA多肽、包含TACI多肽和BCMA多肽的融合蛋白、包含TACI多肽和抗体(例如，抗IFNAR1抗体)的融合蛋白、包含TACI多肽和BCMA多肽和抗体(例如，抗IFNAR1抗体)的融合蛋白、产品、组合、复合物、多核苷酸，或药物组合物的步骤。
一些实施方案中，提供本公开的TACI多肽、BCMA多肽、包含TACI多肽和BCMA多肽的融合蛋白、包含TACI多肽和抗体(例如，抗IFNAR1抗体)的融合蛋白、包含TACI多肽和BCMA多肽和抗体(例如，抗IFNAR1抗体)的融合蛋白、产品、组合、复合物、多核苷酸，或药物组合物在制备治疗或预防疾病的药物中的用途。
一些实施方案中，所述疾病或病症为B细胞障碍或自身免疫性疾病。
一些实施方案中，所述疾病或病症是与TACI表达相关的，或与BCMA表达相关的，或与IFN表达相关的，或与TACI、BCMA、IFN中的任意两者或三者表达相关的。所述表达例如是超量或异常表达。
一些实施方案中，所述自身免疫性疾病选自：系统性红斑狼疮、重症肌无力、多发性硬化、胰岛素依赖性糖尿病、克罗恩氏病、类风湿关节炎、多关节型青少年类风湿关节炎和银屑病性关节炎。一些实施方案中，所述B细胞障碍选自：肿瘤、慢性白细胞性白血病、多发性骨髓瘤、非霍奇金淋巴瘤、移植后淋巴组织增 生病以及轻链丙球蛋白病。一些实施方案中，所述自身免疫性疾病为系统性红斑狼疮。
术语定义
为了更容易理解本公开，以下具体定义了某些技术和科学术语。除非在本文中另有明确定义，本文使用的所有其它技术和科学术语都具有本公开所属领域的一般技术人员通常理解的含义。
本公开所用氨基酸三字母代码和单字母代码如J.biol.chem，243，p3558(1968)中所述。
本公开所用的单数形式“一个”、“一种”和“所述”包括复数指代，除非上下文清楚表明并非如此。
除非上下文另外清楚要求，否则在专利说明书和权利要求书中，应将词语“包含”、“具有”、“包括”等理解为“包括但不仅限于”的意义，而不是排他性或穷举性意义。
术语“细胞因子”是由一个细胞群体释放的、作为细胞间介质作用于其它细胞的蛋白质的一般术语。这样的细胞因子的例子包括淋巴因子、单核因子、趋化因子和传统的多肽激素。示例性的细胞因子包括：IL-2、IFN-γ、IL-6、TNFα、IL-17和IL-5。
本公开所述的TACI(transmembrane activator and calcium modulator andcyclophilinligand-interactor)是一种膜结合受体，人TACI属于TNFR超家族，是293个氨基酸的多肽，包含位于蛋白N端区域(氨基酸残基1-165，参见本公开的SEQ ID NO：1)，跨膜区域(氨基酸残基166-186)和细胞内区域(氨基酸残基187-293)。胞外区含有两个富含半胱氨酸的重复区CRD(cysteine-rich pseudo-repeats)(CRD1、CRD2)。本公开中TACI的“CRD1”涵盖野生型的人TACI的CRD1(例如，SEQ ID NO：1的第33-67位氨基酸)及其变体。本公开中TACI的“CRD2”涵盖野生型的人TACI的CRD2(例如，SEQ ID NO：1的第70-104位、或第69-104位氨基酸)及其变体(包括但不限于本公开的SEQ ID NO：1的第69、72、73、77、85、102和103位中的一个或多个氨基酸的变体，例如SEQ ID NO：6-29所示变体)。所述TACI的CRD1、CRD2及其变体均能够与APRIL和/或BAFF结合，例如，同时与APRIL和BAFF结合，所述结合的检测方法是本领域公知的，例如可参见本公开实施例中所提供的。
本公开所述的BCMA(B cell maturation antigen)是一种膜结合受体，人BCMA蛋白是184个氨基酸的多肽，包含位于蛋白N端区域(氨基酸残基1-50，参见本公开SEQ ID NO：30的第1-50位氨基酸)，跨膜区域(氨基酸残基51-93)和细胞内区域(氨基酸残基94-184)。BCMA的胞外区含有一个CRD(或称CRD1)。本公开中BCMA的“CRD1”涵盖野生型的人BCMA的CRD1(例如，本公开SEQ ID NO：30的第7-41位氨基酸)及其变体。所述BCMA的CRD1或其变体均能够与APRIL和 /或BAFF结合，例如，同时与APRIL和BAFF结合，所述结合的检测方法是本领域公知的，例如可参见本公开实施例中所提供的。
本公开中的“TACI胞外区”与“TACI胞外结构域”可相互替换，“BCMA胞外区”与“BCMA胞外结构域”可相互替换。
术语“干扰素α”、“IFNα”、“IFNa”、“IFNA”和“IFN alpha”可互换使用，应指干扰素α基因座的功能性基因编码的IFNα蛋白质，其与IFNα1(GenBank登录号NP_076918或GenBank登录号NM_024013编码的蛋白)具有75％或以上的序列相同性。IFNα亚型的例子包括IFNα1，α2a，α2b，α4，α4bα5，α6，α7，α8，α10，α13，α14，α16，α17和α21。“干扰素α”、“IFNα”应包括各种IFNα亚型的重组形式，以及天然存在的制备物，其包含IFNα蛋白，例如白细胞IFN和淋巴母细胞IFN。
术语“干扰素α受体-1”、“IFNAR1”、“IFNAR-1”和″IFNAR-1抗原”可互换使用，包括人IFNAR-1的变体、同工型、种系同源物，以及与IFNAR-1具有至少一个共同表位的类似物。因此，本发明的人抗体在一些实施方案中可与来自除人以外的其它物种的IFNAR-1，或与人IFNAR-1结构相关的其它蛋白质(例如人IFNAR-1同源物)交叉反应。在其它实施方案中，抗体可以对人IFNAR-1完全特异，不显示物种或其它类型的交叉反应性。人IFNAR-1的完整cDNA序列具有GenBank登录号NM_000629。
术语“和/或”，意指包含“和”与“或”两种含义。例如短语“A、B和/或C”旨在涵盖以下方面中的每一个：A、B和C；A、B或C；A或C；A或B；B或C；A和C；A和B；B和C；A(单独)；B(单独)；和C(单独)。
术语“抗体”以最广义使用，涵盖各种抗体结构，包括但不限于单克隆抗体，多克隆抗体；单特异性抗体，多特异性抗体(例如双特异性抗体)，全长抗体和抗体片段(或抗原结合片段，或抗原结合部分)，只要它们展现出期望的抗原结合活性。抗体可以指免疫球蛋白，是由两重链和两条轻链通过链间二硫键连接而成的四肽链结构。免疫球蛋白重链恒定区的氨基酸组成和排列顺序不同，故其抗原性也不同。据此，可将免疫球蛋白分为五类，或称为免疫球蛋白的同种型，即IgM、IgD、IgG、IgA和IgE，其相应的重链分别为μ链、δ链、γ链、α链和ε链。同一类Ig根据其铰链区氨基酸组成和重链二硫键的数目和位置的差别，又可分为不同的亚类，如IgG可分为IgG1、IgG2、IgG3、IgG4。轻链通过恒定区的不同分为κ链或λ链。五类Ig中第每类Ig都可以有κ链或λ链。抗体重链和轻链靠近N端的约110个氨基酸的序列变化很大，为可变区(V区)；靠近C端的其余氨基酸序列相对稳定，为恒定区(C区)。可变区包括3个高变区(CDR)和4个序列相对保守的骨架区(FR)。3个高变区决定抗体的特异性，又称为互补性决定区(CDR)。每条轻链可变区(VL)和重链可变区(VH)由3个CDR区4个FR区组成，从氨基端到羧基端依次排列的顺序为：FR1，CDR1，FR2，CDR2，FR3，CDR3，FR4。轻链的3个CDR区指LCDR1，LCDR2，和LCDR3；重链的3个CDR区指HCDR1，HCDR2和HCDR3。 本公开中的“抗体”涵盖“抗原结合片段”，所述“抗原结合片段”包括：单链抗体(即全长重链和轻链)；Fab、修饰的Fab、Fab’、修饰的Fab’、F(ab’)2、Fv、Fab-Fv、Fab-dsFv、单结构域抗体(例如VH或VL或VHH)、scFv、二价或三价或四价抗体、Bis-scFv、diabody、tribody、triabody、tetrabody和上述任意一种的表位结合片段(参见例如Holliger and Hudson,2005,Nature Biotech.23(9):1126-1136；Adair and Lawson,2005,Drug Design Reviews-Online 2(3),209-217)。
对于CDR的确定或定义，能够通过分辨抗体的结构和/或分辨抗体-配体复合物的结构来完成CDR的确定性描绘和包含抗体的结合位点的残基的鉴定。这可通过本领域技术人员已知的各种技术中的任一种，例如X射线晶体学来实现。多种分析方法可用于鉴定CDR，包括但不限于Kabat编号系统、Chothia编号系统、AbM编号系统、IMGT编号系统、接触定义、构象定义。
Kabat编号系统是用于编号抗体中残基的标准并且通常用于鉴定CDR区域(参见例如Johnson&Wu，2000，Nucleic Acids Res.，28：214-8)。Chothia编号系统与Kabat编号系统类似，但Chothia编号系统考虑了某些结构环区域的位置。(参见例如Chothia等，1986，J.Mol.Biol.，196：901-17；Chothia等人，1989，Nature，342：877-83)。AbM编号系统使用建模抗体结构的由Oxford Molecular Group生产的计算机程序集成套件(参见例如Martin等，1989，ProcNatl Acad Sci(USA)，86：9268-9272；“AbMTM，A Computer Program for ModelingVariable Regions of Antibodies，”Oxford，UK；Oxford Molecular，Ltd)。AbM编号系统使用知识数据库和从头开始方法的组合，从基本序列建模抗体的三级结构(参见Samudrala等，1999，在PROTEINS，Structure，Function and Genetics Suppl.，3：194-198中的“Ab Initio Protein Structure Prediction Using a Combined HierarchicalApproach”描述的那些)。接触定义基于可用复杂晶体结构的分析(参见例如MacCallum等，1996，J.Mol.Biol.，5：732-45)。构象定义中，CDR的位置可鉴定为对抗原结合做出贡献的残基(参见例如Makabe等，2008，Journal ofBiological Chemistry，283：1156-1166)。另外其它的CDR边界定义可能不严格遵循上述方法之一，但仍然与Kabat CDR的至少一部分重叠，尽管根据特定残基或残基组不显著影响抗原结合的预测或实验结果，它们可缩短或延长。如本公开使用的，CDR可指通过本领域已知的任何方法(包括方法的组合)定义的CDR。各种编号系统之间的对应关系是本领域技术人员熟知的，示例性的，如下表1中所示。
表1.CDR编号系统之间的关系

除非另有说明，本公开中的可变区和CDR序列均适用Kabat编号系统。
术语“Fc区”或“片段可结晶区”用于定义抗体重链的C末端区域，包括天然Fc区和改造的Fc区。一些实施方案中，Fc区包含了相同或不同的两个亚基。一些实施方案中，人IgG重链的Fc区定义为从Cys226位置处的氨基酸残基或从Pro230延伸至其羧基末端。用于本文所述抗体的合适Fc区包括人IgG1、IgG2(IgG2A、IgG2B)、IgG3和IgG4的Fc区。一些实施方案中，Fc区的边界还可以变化，例如缺失Fc区的C末端赖氨酸(根据EU编号系统的残基447)或缺失Fc区的C末端甘氨酸和赖氨酸(根据EU编号系统的残基446和447)。除非另有说明，Fc区的编号规则为EU编号系统，又称作EU索引。
术语“同源性”或“同一性”是指两个多核苷酸序列之间或两个多肽之间的序列相似性。当两个比较序列中的位置均被相同核苷酸或氨基酸单体亚基占据时，例如如果两个DNA分子的每一个位置都被相同核苷酸占据时，那么所述分子在该位置是同源的。两个序列之间的同源性百分率是两个序列共有的匹配或同源位置数除以比较的位置数×100％的函数。例如，在序列最佳比对时，如果两个序列中的10个位置有6个匹配或同源，那么两个序列为60％同源。一般而言，当比对两个序列而得到最大的同源性百分率时进行比较。
术语“氨基酸突变”包括氨基酸取代(也称氨基酸替换)、缺失、插入和修饰。可以进行取代、缺失、插入和修饰的任意组合来实现最终构建体，只要最终构建体拥有期望的特性，例如降低或对Fc受体的结合。氨基酸序列缺失和插入包括在多肽链的氨基端和/或羧基端的缺失和插入。具体的氨基酸突变可以是氨基酸取代。在一个实施方案中，氨基酸突变是非保守性的氨基酸取代，即将一个氨基酸用具有不同结构和/或化学特性的另一种氨基酸替换。氨基酸取代包括由非天然存在的氨基酸或由20种天然氨基酸的衍生物(例如4-羟脯氨酸、3-甲基组氨酸、鸟氨酸、高丝氨酸、5-羟赖氨酸)替换。可以使用本领域中公知的遗传或化学方法生成氨基酸突变。遗传方法可以包括定点诱变、PCR，基因合成等。预计基因工程以外的改变氨基酸侧链基团的方法，如化学修饰也可能是可用的。本文中可使用各种名称来指示同一氨基酸突变。本文中，可采用位置+氨基酸残基的方式表示特定位点的氨基酸残基，例如366W，表示在366位点上的氨基酸残基为W。T366W则表示第366位点上的氨基酸残基由原来的T突变为了W。
应当理解，当权利要求中以位置+残基的方式限定氨基酸序列时，该位点突变前的氨基酸不对技术方案构成限制。
术语“保守性置换”指置换为具有与原始氨基酸残基相似的特性的另一个氨基酸残基。例如，赖氨酸、精氨酸和组氨酸具有相似的特性，在于它们具有碱性侧链，并且天冬氨酸和谷氨酸具有相似的特性，在于它们具有酸性侧链。此外，甘 氨酸、天冬酰胺、谷氨酰胺、丝氨酸、苏氨酸、酪氨酸、半胱氨酸和色氨酸具有相似的特性，在于它们具有不带电荷极性侧链，并且丙氨酸、缬氨酸、亮氨酸、苏氨酸、异亮氨酸、脯氨酸、苯丙氨酸和甲硫氨酸具有相似的特性，在于它们具有非极性侧链。另外，酪氨酸、苯丙氨酸、色氨酸和组氨酸具有相似的特性，在于它们具有芳族侧链。因此，本领域技术人员将显而易见，甚至当置换如上文所述的显示相似特性的组中的氨基酸残基时，它将不显示特性的特定变化。
术语“多肽”、“蛋白”或“蛋白质”在本文中可互换使用，指氨基酸残基的聚合物。该术语适用于氨基酸聚合物，其中一个或多个氨基酸残基是相应天然存在的氨基酸的人工化学模拟物，以及适用于天然存在的氨基酸聚合物和非天然存在的氨基酸聚合物。除非另外说明，否则特定的多肽序列还隐含地涵盖其保守修饰的变体。
术语“核酸”在本文中可与术语“多核苷酸”互换使用，并且是指呈单链或双链形式的脱氧核糖核苷酸或核糖核苷酸及其聚合物。所述术语涵盖含有已知核苷酸类似物或修饰的骨架残基或连接的核酸，所述核酸是合成的、天然存在的和非天然存在的，具有与参考核酸相似的结合特性，并且以类似于参考核苷酸的方式代谢。此类类似物的实例包括但不限于硫代磷酸酯、氨基磷酸酯、甲基膦酸酯、手性-甲基膦酸酯、2-O-甲基核糖核苷酸、肽-核酸(PNA)。“分离的”核酸指已经与其天然环境的组分分开的多核苷酸。分离的核酸包括在下述细胞中含有的多核苷酸，所述细胞通常含有该多核苷酸，但该多核苷酸存在于染色体外或存在于不同于其天然染色体位置的染色体位置处。编码多肽或融合蛋白的分离的核酸指编码多肽或融合蛋白的一个或更多个多核苷酸，包括在单一载体或分开的载体中的这样的一个或更多个多核苷酸，和存在于宿主细胞中一个或更多个位置的这样的一个或更多个多核苷酸。除非另有说明，否则特定的核酸序列还隐含地涵盖其保守修饰的变体(例如，简并密码子取代)和互补序列以及明确指明的序列。具体地，如下详述，简并密码子取代可以通过产生如下序列而获得，在这些序列中，一个或多个所选的(或全部)密码子的第三位被混合碱基和/或脱氧肌苷残基取代。
术语“效应子功能”指那些可归于抗体Fc区(天然序列Fc区或氨基酸序列突变的Fc区)且随抗体同种型而变化的生物学活性。抗体效应子功能的例子包括但不限于：C1q结合和补体依赖性细胞毒性、Fc受体结合、抗体依赖性细胞介导的细胞毒性(ADCC)、吞噬作用、细胞表面受体(例如B细胞受体)下调；和B细胞活化。
术语“结合亲和力”或“亲和力”在本公开中用作两个分子(例如抗体或其部分与抗原、配体与受体)之间的非共价相互作用的强度量度。两个分子之间的结合亲和力可通过确定解离常数(KD)来量化。可通过使用例如表面等离子共振(SPR)方法(Biacore)测量复合物形成和解离的动力学来确定KD。对应于单价复合物的结合和解离的速率常数分别被称为结合速率常数ka(或kon)和解离速率常数kd(或koff)。KD通过方程KD＝kd/ka与ka和kd有关。解离常数的值可通过众所周知的方法直接确定，并且可通过方法例如Caceci等人(1984，Byte 9：340-362)中所述的那些甚 至对于复杂混合物进行计算。例如，可使用双重过滤硝化纤维素滤器结合测定如Wong&Lohman(1993，Proc.Natl.Acad.Sci.USA 90：5428-5432)中公开的那种来确定KD。评估抗体针对靶抗原的结合能力的其它标准测定是本领域已知的，包括例如ELISA、蛋白质印迹、RIA和流式细胞术分析、以及本公开其它地方例举的其它测定。抗体的结合动力学和结合亲和力也可通过本领域已知的标准测定，例如表面等离子共振(SPR)，例如通过使用BiacoreTM系统或KinExA来评价。可通过比较各个抗体/抗原复合物的KD值来比较与不同分子相互作用相关的结合亲和力，例如，不同抗体对于给定抗原的结合亲和力的比较。类似地，相互作用的特异性可通过确定和比较目的相互作用(例如抗体和抗原之间的特异性相互作用)的KD值与非目的相互作用(例如已知不结合目的抗原的对照抗体)的KD值进行评价。
术语“连接子”、“Linker”或“接头”指连接两个多肽片段的连接单元，通常具有一定的柔性，接头的使用不会使蛋白质结构域原有的功能丧失。在本文中，同一结构中出现的连接子可以是相同或不同的。连接子可以是肽连接子，其包含一个或多个氨基酸，典型的约1-30个、2-24个或3-15个氨基酸。应用于本公开的连接子可以是相同或不同的。
术语“融合”或“连接”是指部件(例如TACI多肽和BCMA多肽)直接地或经由一个或多个连接子而通过共价键连接。当连接子是肽连接子时，所述共价键是肽键。
“给予”、“施用”和“处理”当应用于动物、人、实验受试者、细胞、组织、器官或生物流体时，是指外源性药物、治疗剂、诊断剂或组合物与动物、人、受试者、细胞、组织、器官或生物流体的接触。“给予”、“施用”和“处理”可以指例如治疗、药物代谢动力学、诊断、研究和实验方法。细胞的处理包括试剂与细胞的接触，以及试剂与流体的接触，其中所述流体与细胞接触。“给予”、“施用”和“处理”还意指通过试剂、诊断、结合组合物或通过另一种细胞体外和离体处理例如细胞。“处理”当应用于人、兽医学或研究受试者时，是指治疗处理、预防或预防性措施，研究和诊断应用。
“治疗”意指给予受试者内用或外用治疗剂，诸如包含本公开的任一种抗体或其抗原结合片段或其融合蛋白的组合物，所述受试者已经患有、疑似患有、倾向于患有一种或多种疾病或其症状，而已知所述治疗剂对这些症状具有治疗作用。通常，在受治疗受试者或群体中以有效缓解一种或多种疾病症状的量给予治疗剂，无论是通过诱导这类症状退化还是抑制这类症状发展到任何临床右测量的程度。有效缓解任何具体疾病症状的治疗剂的量(也称作“治疗有效量”)可根据多种因素变化，例如受试者的疾病状态、年龄和体重，以及药物在受试者产生需要疗效的能力。通过医生或其它专业卫生保健人士通常用于评价该症状的严重性或进展状况的任何临床检测方法，可评价疾病症状是否已被减轻。尽本公开的实施方案(例如治疗方法或制品)在缓解某个受试者中目标疾病症状方面可能无效，但是根据本领域已知的任何统计学检验方法如Student t检验、卡方检验、依据Mann和Whitney 的U检验、Kruskal-Wallis检验(H检验)、Jonckheere-Terpstra检验和Wilcoxon检验确定，其在统计学显著数目的受试者中应当减轻目标疾病症状。
“有效量”包含足以改善或预防医学病症的症状或病症的量。有效量还意指足以允许或促进诊断的量。用于特定受试者或兽医学受试者的有效量可依据以下因素而变化：如待治疗的病症、受试者的总体健康情况、给药的方法途径和剂量以及副作用严重性。有效量可以是避免显著副作用或毒性作用的最大剂量或给药方案。
术语“宿主细胞”、“宿主细胞系”和“宿主细胞培养物”可互换使用，并且指已经导入外源核酸的细胞，包括此类细胞的后代。宿主细胞包括“转化体”和“经转化的细胞”，其包括原代的经转化的细胞及自其衍生的后代，而不考虑传代的次数。后代在核酸内容物上可以与亲本细胞不完全相同，而是可以含有突变。本文中包括具有与在初始转化细胞中筛选或选择的相同功能或生物学活性的突变体后代。宿主细胞包括原核和真核宿主细胞，其中真核宿主细胞包括但不限于哺乳动物细胞、昆虫细胞系植物细胞和真菌细胞。哺乳动物宿主细胞包括人、小鼠、大鼠、犬、猴、猪、山羊、牛、马和仓鼠细胞，包括但不限于中国仓鼠卵巢(CHO)细胞、NSO、SP2细胞、HeLa细胞、幼仓鼠肾(BHK)细胞、猴肾细胞(COS)、人肝细胞癌细胞(例如，Hep G2)、A549细胞、3T3细胞和HEK-293细胞。
“任选”或“任选地”意味着随后所描述地事件或环境可以但不必发生，该说明包括该事件或环境发生或不发生地场合。例如，“任选包含1-3个抗体重链可变区”意味着特定序列的抗体重链可变区可以但不必须存在。
本公开的“包含TACI多肽和BCMA多肽的融合蛋白”涵盖所有含本公开TACI多肽和BCMA多肽的融合蛋白的分子。
“包含TACI多肽和抗IFNAR1抗体的融合蛋白”涵盖所有含本公开TACI多肽和抗IFNAR1抗体(或其抗原结合片段)的融合蛋白的分子。
“包含TACI多肽和BCMA多肽和抗IFNAR1抗体的融合蛋白”涵盖所有含本公开TACI多肽和BCMA多肽和抗IFNAR1抗体(或其抗原结合片段)的融合蛋白的分子。例如，所述融合蛋白可以包含一个或多个效应分子，以例如缀和的方式。
所述“效应分子”单独可以具有治疗活性(例如具有抗肿瘤活性或免疫激活或抑制活性)或具有检测功能，其可以是任何形式，例如生物活性蛋白(例如酶)、其它抗体或抗体片段、合成或天然存在的聚合物、核酸及其片段例如DNA、RNA及其片段、放射性核素(特别地放射性碘化物)、放射性同位素、螯合金属、纳米颗粒和报道基团(例如荧光化合物)、或可通过NMR或ESR光谱分析检测的化合物。效应分子与本公开的融合蛋白的缀和方式可以通过常规方法实现。
附图说明
图1.TACI与Fc的融合蛋白结构示意图。
图2.配体BAFF和APRIL与受体BAFF-R、TACI和BCMA相互作用示意图。 BAFF为膜表达蛋白，经蛋白酶切后以可溶三聚体形式发挥功能，BAFF可以与BAFF-R、TACI和BCMA结合。APRIL为可溶三聚体蛋白，可以与TACI和BCMA结合。黑色箭头越粗代表相互作用越强，黑色箭头越细代表相互作用越弱。
图3.不同TACI和BCMA蛋白结构域示意图，包括TACI-ECD、TACI-d2、TACI-T2、TACI-T4、BCMA-ECD。
图4.TACI-BCMA融合蛋白结构示意图，包括B575701、B575702、B575703、B575704。
图5.TACI-BCMA融合蛋白(B575701、B575702、B575703、B575704)与BAFF结合能力ELISA测定结果，使用Telitacicept和IgG1同种型作为对照。
图6.TACI-BCMA融合蛋白(B575701、B575702、B575703、B575704)与APRIL结合能力ELISA测定结果，使用Telitacicept和IgG1同种型作为对照。
图7.TACI-CRD2和TACI-19-16结构域示意图，包括TACI-CRD2、TACI-19-16。
图8.Anifrolumab与TACI-T4的融合蛋白(B385801、B385802、B385803、B385804、B498301、B498302)、Anifrolumab与TACI-19-16融合蛋白(B606401、B606401-LALA、B805201、B805201-LALA，所述LALA突变为Fc的L234A/L235A突变，下同)结构示意图。
图9.Anifrolumab与TACI-T4的融合蛋白对IFN-β活性抑制检测结果，其中，图9的A为B385801、B385802、B385803、B385804对IFN-β活性抑制结果；图9的B为B385804、B498301、B498302对IFN-β活性抑制结果；图9的C为B606401对IFN-β活性抑制结果，使用Anifrolumab和IgG1同种型作为对照。
图10.Anifrolumab与TACI-T4的融合蛋白与BAFF结合能力ELISA测定结果，其中图10的A为B385801、B385802、B385803、B385804与BAFF结合能力结果，使用Atacicept、Telitacicept和IgG1同种型作为对照。图10的B为B385804、B498301、B498302与BAFF结合能力结果；图10的C为B606401与BAFF结合能力结果，使用Telitacicept和IgG1同种型作为对照。
图11.Anifrolumab与TACI-BCMA融合蛋白结构示意图，包括B613301、B613302、B613303、B613304。
图12.Anifrolumab与TACI-BCMA融合蛋白(B613301、B613302、B613303)对IFN-β活性抑制检测结果，使用Anifrolumab和IgG1同种型作为对照。
图13.Anifrolumab与TACI-BCMA融合蛋白(B613301、B613302、B613303)与BAFF结合能力ELISA测定结果，使用Telitacicept和IgG1同种型作为对照。
图14.Anifrolumab与TACI-BCMA融合蛋白(B613301、B613302、B613303)与APRIL结合能力ELISA测定结果，使用Telitacicept和IgG1同种型作为对照。
图15.Anifrolumab与TACI-19-16和BCMA-ECD-1的融合蛋白(B637301)，Anifrolumab与TACI-19-16融合蛋白和BCMA-ECD-2融合蛋白(B637302、B637302-LALA、B746201、B746201-LALA)结构示意图。
图16.Anifrolumab与TACI-19-16和BCMA的融合蛋白(B637302、B746201)、Anifrolumab与TACI-19-16的融合蛋白(B606401、B805201)对IFN-β活性抑制检测结果，使用Anifrolumab和IgG1同种型作为对照。
图17.B637302、B746201、B606401、B805201与BAFF结合能力ELISA测定结果，使用Telitacicept和IgG1同种型作为对照。
图18.B637302、B746201、B606401、B805201与APRIL结合能力ELISA测定结果，使用Telitacicept和IgG1同种型作为对照。
图19.PBMC中pDC功能实验结果。利用CpG-A刺激体外培养的人PBMC细胞，加梯度浓度的B637302、B606401处理，使用Anifrolumab、IgG1同种型作为对照，24小时后检测上清中分泌的IFN-α水平。
图20.浆细胞体外分化实验结果。包括利用从人PBMC中分选出的B细胞，进行体外培养，加CpG-B和IFN-α，加梯度浓度的B637302、B746201、B606401、B805201处理，使用Anifrolumab、IgG1同种型作为对照，4天后流式检测浆细胞(CD27+CD38+)分化比例。
图21.BAFF诱导B细胞体外增殖实验结果。包括利用从人PBMC中分选出的B细胞，进行CTV标记，然后进行体外培养，加IL-4、anti-IgM、CD40L、IL-17作为基础刺激信号，在此基础条件上，加BAFF诱导B细胞增殖，加梯度浓度的B637302、B606401、B805201处理，使用Telitacicept、IgG1同种型作为对照，第5天流式检测B细胞增殖信号，药物浓度为100nM、300nM、1000nM。
图22.APRIL诱导B细胞体外增殖实验。包括利用从人PBMC中分选出的B细胞，进行CTV标记，然后进行体外培养，加IL-4、anti-IgM、CD40L、IL-17作为基础刺激信号，在此基础条件上，加APRIL诱导B细胞增殖，加梯度浓度的B637302、B606401处理，使用Telitacicept、IgG1同种型作为对照，第5天流式检测B细胞增殖信号，药物浓度为100nM、300nM、1000nM。
图23.BAFF与APRIL共同诱导浆细胞体外生成实验。包括从人PBMC中分选出的B细胞，进行体外培养，第1至4天加CpG-B刺激，第4至10天移除CpG-B，加IL-6+IL-10+IL-21继续培养。在此基础条件上，第4至7天加500ng/mL BAFF和50ng/mL APRIL，第7至10天加50ng/mL BAFF和500ng/mL APRIL诱导浆细胞体外生成。在第4至10天加10、100、1000nM不同浓度的药物B637302、B606401、B606401、B805201处理，使用Telitacicept、IgG1同种型作为对照，第10天流式检测浆细胞数目。
图24.IFN-α和BAFF、APRIL三因子共同诱导浆细胞体外生成实验。图24的A为实验过程示意图，利用从人PBMC中分选出的B细胞，进行体外培养，第1至4天加CpG-B和IFN-α刺激，第4至10天移除CpG-B，加IL-6+IL-10+IL-21继续培养。在此基础条件上，第4至7天加500ng/mL BAFF和50ng/mL APRIL，第7至10天加50ng/mL BAFF和500ng/mL APRIL诱导浆细胞体外生成。在第0 至10天加10、100、1000nM不同浓度的药物B637302、B606401处理，使用Telitacicept、IgG1同种型作为对照，第10天流式检测浆细胞数目；图24的B、C、D分别为药物10nM、100nM、1000nM检测结果图。
图25.PEG-IFN-α诱导PBMC人源化小鼠PD实验结果。图25的A为实验过程示意图，利用B-NDG免疫缺陷小鼠，尾静脉注射PBMC进行人源化，腹腔注射PEG-IFN-α进行诱导，在该模型中腹腔注射不同浓度的药物B637302、B606401处理小鼠，使用Anifrolumab、PBS作为对照；图25的B为IFN-α诱导下游信号通路示意图；图25的C为第2小时取PBMC检测pSTAT1水平结果；图25的D、E、F分别为第1、3、7天时间点取PBMC，通过QPCR检测IFN-α下游基因mRNA表达水平。
图26.BAFF和APRIL共同诱导小鼠PD实验结果。图26的A为实验过程示意图，利用C57/B6小鼠，腹腔注射BAFF+APRIL进行诱导，在该模型中腹腔注射不同浓度的药物B637302、B606401处理小鼠，使用Telitacicept、PBS作为对照，第4、7天取外周血，通过ELISA检测血浆中IgA水平；图26的B、C分别为第4、第7天取血检测血浆IgA结果图。
其中，图21-图26中，统计学方法Student’t-test，*p<0.05，**p<0.01，***p<0.001，****p<0.0001。
具体实施方式
以下结合实施例用于进一步描述本公开，但这些实施例并非限制本公开的范围。
本公开实施例或测试例中未注明具体条件的实验方法，通常按照常规条件，或按照原料或商品制造厂商所建议的条件。参见Sambrook等，分子克隆，实验室手册，冷泉港实验室；当代分子生物学方法，Ausubel等著，Greene出版协会，Wiley Interscience，NY。未注明具体来源的试剂，为市场购买的常规试剂。
实施例1.TACI序列分析和改造
由于TACI的序列上存在多个蛋白酶切位点，导致全长的TACI表达后容易发生断裂。本实验设计不同长度的TACI序列片段，将TACI-ECD或其片段的C端与人IgG1的Fc(SEQ ID NO：3)的N端直接融合，构建TACI-Fc融合蛋白(包括全长TACI-ECD-Fc、TACI-9-Fc)，在293E(EBNA1基因修饰的人胚肾细胞)中转染表达，纯化获得含两条相同多肽链的TACI-Fc融合蛋白(结构参见图1)。采用RCT-18(Telitacicept)作为阳性对照。
序列如下所示：
>TACI-ECD(野生型人TACI的第1至165位氨基酸残基)

>TACI-9(人TACI-ECD按自然计数的第68至108位)
>人IgG1的Fc序列
>Telitacicept(RCT-18)
>Atacicept
备注：Telitacicept(RCT-18)序列中，无下划线部分为TACI序列(人TACI胞外区按自然计数的第13至118位)，下划线部分为Fc序列。Atacicept序列中，无下划线部分为TACI序列(人TACI胞外区按自然计数的第30至110位)和接头EPKSS(斜体示出)，下划线部分为Fc序列。
对获得的TACI-Fc融合蛋白进行质谱分析，实验结果表明，TACI-9-Fc无断裂片段，但阳性对照RCT-18以及全长TACI-ECD-Fc均检测到TACI断裂片段(结果未示出)。
进一步检测上述TACI-Fc融合蛋白阻断BAFF与BAFF-R结合功能，方法如下：用pH7.4的PBS(源培生物，B320)缓冲液将受体蛋白稀释至2μg/mL，以100μL/ 孔的体积加入96孔酶标板(Corning，3590)中，4℃过夜孵育。弃去液体后，每孔加入200μL 1％Casein封闭液(Thermo，37528)进行封闭，37℃孵育2小时。封闭结束后，弃去封闭液，并用PBST缓冲液(pH7.4PBS含0.1％tween-20)洗板3次后备用。将固定浓度的生物素(Biotin)标记的配体蛋白与梯度稀释的抗体或融合蛋白混合后37℃预孵育30分钟后加入封闭好的酶标板中，37℃孵育1.5小时。孵育结束后用PBST洗板3次，每孔加入100μL streptavidin-HRP(Invitrogen，434323，1:4000稀释)，37℃孵育1小时。去上清，用PBST洗板3次后每孔加入100μL TMB显色底物(KPL，5120-0077)，室温孵育10～15分钟，每孔加入50μl 1M H2SO4终止反应，用酶标仪读取在450nm处的吸收值，用软件拟合出抑制配体和受体结合的曲线，计算出IC50值。所用配体和受体蛋白的来源信息如下：BAFF(Sino biological，10056-HNCH)，BAFF-R(Sino biological，16079-H02H)。
结果显示，TACI-9-Fc和RCT-18阻断BAFF与BAFF-R结合的IC50值分别为5.02nM和27.48nM。TACI-9-Fc的功能活性显著强于对照RCT-18。因此，认为TACI-9为有潜力的候选分子。
实施例2.TACI截短片段的分析
在TACI-9的基础上，进一步截短TACI的序列长度，检测截短的TACI片段的功能活性。进一步截短的TACI片段的序列如下所示：
>TACI-10(TACI-9的C末端截短1个氨基酸“L”)(人TACI-ECD按自然计数的第68至107位)
>TACI-11(TACI-9的C末端截短2个氨基酸“KL”)(人TACI-ECD按自然计数的第68至106位)
>TACI-12(TACI-9的C末端截短3个氨基酸“NKL”)(人TACI-ECD按自然计数的第68至105位)
将上述截短的TACI片段的C端与人IgG的Fc(SEQ ID NO：3)的N端融合，构建TACI-Fc融合蛋白，在293E中转染表达，纯化获得含两条相同多肽的TACI-Fc融合蛋白(结构参见图1)。
对上述构建的TACI-Fc融合蛋白进行质谱分析，实验结果见表2，实验结果表明，TACI-9截短片段构建的TACI-Fc融合蛋白均没有发生TACI多肽断裂。
另外，检测构建的TACI-Fc融合蛋白结合BAFF的活性，以及，检测TACI-Fc融合蛋白阻断BAFF与BAFF-R结合功能活性(实验方法见实施例1)。
检测TACI-Fc融合蛋白结合BAFF的活性方法如下：用pH 7.4的PBS(源培生物，B320)缓冲液将BAFF(Sino biological，10056-HNCH)蛋白稀释至1μg/mL，以 100μL/孔的体积加入96孔酶标板(Corning，3590)中，4℃过夜孵育。弃去液体后，每孔加入300μL用PBS稀释的5％脱脂牛奶(BD，232100)进行封闭，37℃孵育2小时。封闭结束后，弃去封闭液，并用PBST缓冲液(pH 7.4PBS含0.1％tween-20)洗板3次后，每孔加入100μL梯度稀释的待测抗体或融合蛋白溶液，于37℃孵育1小时。孵育结束后用PBST洗板3次，每孔加入100μL小鼠抗人IgG(H+L)(Jackson ImmunoResearch，209-035-088，1:8000稀释)，37℃孵育1小时。用PBST洗板3次后，每孔加入100μL TMB显色底物(KPL，5120-0077)，室温孵育10～15分钟，每孔加入50μL 1M H2SO4终止反应，用酶标仪读取在450nm处的吸收值，用软件拟合出抗体与抗原的结合曲线，计算出EC50值。
表2结果表明，TACI-9的截短片段TACI-10、TACI-11、TACI-12构建的TACI-10-Fc、TACI-11-Fc、TACI-12-Fc融合蛋白对BAFF的结合活性以及阻断BAFF与BAFF-R结合的功能活性与TACI-9-Fc融合蛋白在相同的水平。
表2.TACI-Fc融合蛋白功能活性及质谱断裂情况检测结果
实施例3.TACI截短片段的序列改造
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MOE(Molecular Operating Environment)软件，基于TACI的晶体结构(PDB ID:1XU1)，分析TACI片段中的疏水基团和离子基团，对TACI-9的多个氨基酸残基进行氨基酸替换，减少TACI蛋白暴露的疏水基团和离子基团的面积，降低TACI的聚集性，提高TACI的稳定性，但仍基本保留TACI的功能活性。改造的TACI片段的氨基酸序列如下：
>TACI-9-1(TACI-9含L69T替换)
>TACI-9-2(TACI-9含R72S突变)
>TACI-9-3(TACI-9含K73E突变)
>TACI-9-4(TACI-9含K73Q突变)
>TACI-9-5(TACI-9含K77E突变)
>TACI-9-6(TACI-9含L69R，D85T突变)
>TACI-9-7(TACI-9含L69R，D85A突变)
>TACI-9-8(TACI-9含Y102A突变)
>TACI-9-9(TACI-9含L69R，D85T，Y102R突变)
>TACI-9-10(TACI-9含K73E，K77E突变)
>TACI-9-11(TACI-9含R72S，K73E，K77E突变)
>TACI-9-12(TACI-9含L69T，Y102A突变)
>TACI-9-13(TACI-9含L69T，F103Y突变)
>TACI-9-14(TACI-9含L69T，Y102A，F103Y突变)
>TACI-9-15(TACI-9含L69T，K73E，K77E，Y102A突变)
备注：上述序列中，下划线部分为突变后氨基酸残基。
将上述由TACI-9改造的TACI片段的C端与人IgG的Fc(SEQ ID NO：3)的N端融合，构建TACI-Fc融合蛋白(结构见图1)，在293E中转染表达，然后通过Protein A亲和纯化，获得含两条相同多肽链的TACI-Fc融合蛋白。
检测构建的TACI-Fc融合蛋白结合BAFF的活性(实验方法参见实施例2)，实验结果见表3：
表3.TACI-Fc融合蛋白与BAFF结合活性

备注：表中突变位点为相对于TACI-ECD(SEQ ID NO：1)的氨基酸残基位点(自然顺序编号)，例如“L69T”表示序列SEQ ID NO：1的第69位(自然顺序编号)氨基酸残基由L突变为T。
另外，将部分纯化获得的TACI-Fc融合蛋白用超滤管置换到PBS溶液中，通过溶液外观观察TACI-Fc融合蛋白溶液中是否存在沉淀。实验结果表明，改造的TACI片段构建的TACI-Fc融合蛋白的溶液中没有沉淀，而RCT-18的PBS溶液中有沉淀产生(结果未示出)。
进一步提供TACI-9-15的截短形式(TACI-9-15a，TACI-9-15b，TACI-9-15c)，氨基酸序列如下：
>TACI-9-15a
>TACI-9-15b
>TACI-9-15c(即TACI-19-16)
实施例4.TACI-BCMA融合蛋白的构建、表达及纯化
如前，阿塞西普(Atacicept)和泰它西普(Telitacicept)均为TACI-Fc分子形式的BAFF/APRIL拮抗剂。如图2所示，TACI与BCMA均能结合BAFF和APRIL，TACI与BAFF的结合能力较强而与APRIL的结合能力较弱，BCMA对APRIL的结合能力较强。我们将BCMA与TACI设计成融合蛋白，以期提高对APRIL的中和活性。
如图3所示，TACI-ECD为含有氨基酸残基1-165的TACI胞外区，TACI-d2为含有氨基酸残基69-111的CRD2配体结合结构域，TACI-T2为含有氨基酸残基13-118的CRD1和CRD2配体结合结构域，TACI-T4为含有氨基酸残基30-110的CRD1和CRD2配体结合结构域，BCMA-ECD为含有氨基酸残基1-54的BCMA胞外区。
如图4所示，设计4种形式的含有TACI多肽和BCMA多肽的融合蛋白，其中，融合蛋白B575701和B575702是将TACI-d2和BCMA-ECD序列连接，并与人野生型IgG1-Fc融合；融合蛋白B575703和B575704是将TACI-T2和BCMA-ECD 序列连接，并与人野生型IgG1-Fc融合。
TACI-ECD的序列如SEQ ID NO：1所示。其余序列如下，其中，下划线为人IgG1Fc。其中，SEQ ID NO：27最后一位的G也可以不存在。
>TACI-d2(SEQ ID NO：27)
>TACI-T2(SEQ ID NO：28)
>TACI-T4(SEQ ID NO：29)
>BCMA-ECD(SEQ ID NO：30)
>TACI-d2-BCMA-ECD(SEQ ID NO：31)
>BCMA-ECD-TACI-d2(SEQ ID NO：32)
>TACI-T2-BCMA-ECD(SEQ ID NO：33)
>BCMA-ECD-TACI-T2(SEQ ID NO：34)
B575701、B575702、B575703、B575704分别是在TACI-d2-BCMA-ECD、BCMA-ECD-TACI-d2、TACI-T2-BCMA-ECD、BCMA-ECD-TACI-T2的C末端连接人IgG1的Fc(SEQ ID NO：3)。以下序列中，下划线为Fc。
>B575701(SEQ ID NO：35)
>B575702(SEQ ID NO：36)
>B575703(SEQ ID NO：37)
>B575704(SEQ ID NO：38)
合成上述蛋白的编码基因序列，亚克隆至pcDNA3.4。使用培养液稀释50μg表达质粒并混匀，使用培养液稀释200μL转染试剂并混匀，二者合并混匀后37℃孵育15分钟，将混合液逐滴加入HEK293细胞液中，细胞液37℃摇床培养一周后，8000rpm离心5分钟收集上清。使用20mL 1×PBS，以1mL/min流速平衡Protein A亲和层析柱；蛋白上清液以1mL/min流速上样；再次使用20mL 1×PBS，以1mL/min流速冲洗非特异结合蛋白；最后使用pH 3.4的柠檬酸缓冲液，以1mL/min流速洗脱，并将洗脱蛋白转移至透析袋于1×PBS中透析，置换为PBS存储缓冲液。经检测，获得目的蛋白。
实施例5.TACI-BCMA融合蛋白对BAFF和APRIL的体外结合实验
使用ELISA分别检测TACI-BCMA融合蛋白(B575701、B575702、B575703、B575704)对BAFF和APRIL的结合能力。
方法如下：将BAFF(PeproTech，Cat:310-13)或ARPIL(Acro Biosystems，Cat: APL-H52D1)用PBS稀释到1μg/mL，96孔板(Costar，Cat:3590)4℃包被过夜。PBST洗板，用Blocking Buffer(含1％BSA的PBST)37℃封闭1小时。拍板，用Blocking Buffer稀释待测抗体，37℃孵育1.5小时。PBST洗板，用Blocking Buffer稀释HRP-小鼠抗人Fc抗体(GenScript，Cat:A01854-200)，37℃孵育40分钟。PBST洗板，加入100μL TMB显色液(Biopanda，Cat:TMB-S-003)37℃显色3分钟，加入100μL ELISA终止液(Solarbio，Cat:C1058)，检测OD450。
结果如表4和图5所示，4种融合蛋白均可以明显结合BAFF，且结合能力均强于Telitacicept，其中B575702和B575704结合BAFF能力一致，相比Telitacicept，EC50降低至其1/4的水平；B575701和B575703结合BAFF的能力一致，相比Telitacicept，EC50降低至其1/2的水平。
表4.TACI-BCMA融合蛋白与BAFF的结合
如表5和图6所示，4种融合蛋白可以明显结合APRIL，且结合能力基本一致；4种融合蛋白对APRIL的结合能力均强于Telitacicept，EC50降低至其1/4至1/3的水平。
表5.TACI-BCMA融合蛋白与APRIL的结合
实施例6.Anifrolumab与TACI的融合蛋白的构建、表达及纯化
Anifrolumab是靶向IFNAR1的拮抗剂，阻断I型干扰素通过IFNAR1对细胞进行活化，用于治疗系统性红斑狼疮的药物。如图7所示，TACI-CRD2为含有氨基酸残基68-105的CRD2配体结合结构域，TACI-19-16为TACI-CRD2带有若干氨基酸突变(L69T，K73E，K77E，Y102A)的多肽。
如图8所示，将TACI-T4或TACI-19-16通过不同长度的连接子分别连接在Anifrolumab重链或轻链的N端或C端。其中，B385801将TACI-T4通过(G4S)2的连接子1连接在Anifrolumab重链N端；B385802将TACI-T4通过连接子1连接在Anifrolumab重链C端；B385803将TACI-T4通过连接子1连接在Anifrolumab轻链N端；B385804将TACI-T4通过连接子1连接在Anifrolumab轻链C端；B498301将TACI-T4通过(G4S)3的连接子2连接在Anifrolumab轻链C端；B498302将TACI-T4通过(G4S)4的连接子3连接在Anifrolumab轻链C端。B606401将TACI-19-16通过连接子1连接在Anifrolumab轻链C端；B805201将TACI-19-16通过连接子2连接在Anifrolumab重链C端。B606401-LALA将TACI-19-16通过 连接子1连接在Anifrolumab轻链C端，且Fc部分为带有L234A和L235A突变的人IgG1Fc。B805201-LALA将TACI-19-16通过连接子2连接在Anifrolumab重链C端，且Fc部分为带有L234A和L235A突变的人IgG1Fc。
TACI-19-16如SEQ ID NO：26所示，其他序列如下：
>连接子1(SEQ ID NO：39)
>连接子2(SEQ ID NO：40)
>连接子3(SEQ ID NO：41)
>TACI-CRD2(SEQ ID NO：42，同SEQ ID NO：8)
>Anifrolumab的VH(SEQ ID NO：43)
>Anifrolumab的VL(SEQ ID NO：44)
>Anifrolumab的HCDR1(SEQ ID NO：45)
>Anifrolumab的HCDR2(SEQ ID NO：46)
>Anifrolumab的HCDR3(SEQ ID NO：47)
>Anifrolumab的LCDR1(SEQ ID NO：48)
>Anifrolumab的LCDR2(SEQ ID NO：49)
>Anifrolumab的LCDR3(SEQ ID NO：50)
>B385801的重链(SEQ ID NO：51)

>B385802的重链(SEQ ID NO：52)
>B805201的重链(SEQ ID NO：53)
>B385801、B385802、B805201、B805201-LALA的轻链(SEQ ID NO：54)
>B385803、B385804、B498301、B498302、B606401的重链(SEQ ID NO：55)
>B385803的轻链(SEQ ID NO：56)
>B385804的轻链(SEQ ID NO：57)
>B498301的轻链(SEQ ID NO：58)
>B498302的轻链(SEQ ID NO：59)
>B606401、B606401-LALA的轻链(SEQ ID NO：60)
>B606401-LALA的重链(SEQ ID NO：61)

>B805201-LALA的重链(SEQ ID NO：62)
重链的下划线为IgG的Fc区，轻链的下划线为CH1，斜体为连接子。
使用实施例1中提供的蛋白制备方法，通过瞬时转染、蛋白表达，将蛋白经Protein A亲和层析柱、洗脱。经检测，获得目的蛋白。
实施例7.Anifrolumab与TACI的融合蛋白对I型干扰素的活性抑制实验
使用HEK-Blue IFN-α/β细胞(InvivoGen，Cat：hkb-ifnab)试剂盒检测所述Anifrolumab与TACI的融合蛋白B385801、B385802、B385803、B385804、B498301、B498302、B606401对IFN-α/β的活性抑制效果。
方法为：消化HEK-BlueTM IFN-α/β细胞，用含10％FBS、1％P/S的DMEM培养基(Gibco，Cat:11995065)重悬至2.8E5/mL并接种到96孔板。加入待测蛋白，37℃培养30分钟。加入终浓度为0.01ng/mL的IFN-β(Sino biological，Cat:10704-HNAS)，37℃培养24小时。取20μL培养上清，在新的96孔板中与180μL Quanti-Blue(InvivoGen，Cat:rep-qbs)混合，37℃孵育1小时，检测OD655。
结果显示，上述7种融合蛋白均对IFN-α/β的活性具有抑制效果，其中，B385801和B385803的活性较弱，B385802活性次之，B385804、B498301、B498302和B606401活性和Anifrolumab基本相当(图9的A、图9的B、图9的C)。
实施例8.Anifrolumab与TACI的融合蛋白对BAFF的体外结合实验
使用ELISA检测Anifrolumab与TACI的融合蛋白B385801、B385802、B385803、B385804、B498301、B498302、B606401对BAFF的结合能力。
方法为：将BAFF(PeproTech，Cat:310-13)用PBS稀释到1μg/mL，96孔板4℃包被过夜。PBST洗板，用Blocking Buffer(含1％BSA的PBST)37℃封闭1小时。拍板，用Blocking Buffer稀释待测抗体，37℃孵育1.5小时。PBST洗板，用Blocking  Buffer稀释HRP-小鼠抗人Fc抗体(GenScript，Cat:A01854-200)，37℃孵育40分钟。PBST洗板，加入100μLTMB显色液(Biopanda，Cat:TMB-S-003)37℃显色3分钟，加入100μL ELISA终止液(Solarbio，Cat:C1058)，检测OD450。
结果参见图10的A至图10的C和表6，显示7种融合蛋白可以明显结合BAFF。B385801、B385803和B606401结合BAFF能力最强，且强于Telitacicept和Atacicept；B385802结合BAFF能力较弱，B385804、B498301和B498302结合BAFF能力一致，稍弱于Telitacicept。连接子的长度不会影响TACI与BAFF的结合能力。
表6.Anifrolumab与TACI的融合蛋白与BAFF的结合
实施例9.Anifrolumab与TACI-BCMA的融合蛋白构建、表达及纯化
参见图11，将实施例4中所述TACI-BCMA融合蛋白分别以连接子1连接在Anifrolumab抗体轻链C端。其中，B613301和B613302为TACI-d2和BCMA-ECD的融合蛋白以连接子1连接在Anifrolumab抗体轻链C端；B613303和B613304为TACI-T2和BCMA-ECD的融合蛋白以连接子1连接在Anifrolumab抗体轻链C端。
B613301、B613302、B613303、B613304的重链均为Anifrolumab的重链全长(SEQ ID NO：55)，轻链的具体序列如下，下划线为轻链恒定区：
>B613301的轻链(SEQ ID NO：63)
>B613302的轻链(SEQ ID NO：64)
>B613303的轻链(SEQ ID NO：65)

>B613304的轻链(SEQ ID NO：66)
使用实施例4中提供的蛋白制备方法，通过瞬时转染、蛋白表达，将蛋白经Protein A亲和层析柱、洗脱。经检测，获得目的蛋白。
实施例10.Anifrolumab与TACI-BCMA的融合蛋白对I型干扰素的活性抑制实验
使用HEK-Blue IFN-α/β细胞(InvivoGen，Cat：hkb-ifnab)试剂盒检测实施例9中的Anifrolumab与TACI-BCMA的融合蛋白B613301、B613302、B613303对IFN-α/β的活性抑制效果。
方法为：消化HEK-BlueTM IFN-α/β细胞，用含10％FBS、1％P/S的DMEM培养基(Gibco，Cat:11995065)重悬至2.8E5/mL并接种到96孔板(Costar，Cat:3599)。加入待测抗体，37℃培养30分钟。加入终浓度为0.01ng/mL的IFN-β(Sino biological，Cat:10704-HNAS)，37℃培养24小时。取20μL培养上清，在新的96孔板中与180μLQuanti-Blue(InvivoGen，Cat:rep-qbs)混合，37℃孵育1小时，检测OD655。
结果参见图12，显示3种融合蛋白均能够抑制IFN-α/β的活性，3种融合蛋白活性相当且和Anifrolumab一致。说明TACI-BCMA融合在Anifrolumab，特别是轻链C端基本不影响Anifrolumab的活性。
实施例11.Anifrolumab与TACI-BCMA的融合蛋白对BAFF和APRIL的体外结合实验
使用ELISA检测Anifrolumab与TACI-BCMA的融合蛋白B613301、B613302、B613303对BAFF和APRIL的结合能力。
方法为：将BAFF(PeproTech，Cat:310-13)或ARPIL(Acro Biosystems，Cat:APL-H52D1)用PBS稀释到1μg/mL，96孔板(Costar，Cat:3590)4℃包被过夜。PBST洗板，用Blocking Buffer(含1％BSA的PBST)37℃封闭1小时。拍板，用Blocking  Buffer稀释待测抗体，37℃孵育1.5小时。PBST洗板，用Blocking Buffer稀释HRP-小鼠抗人Fc抗体(GenScript，Cat:A01854-200)，37℃孵育40分钟。PBST洗板，加入100μL TMB显色液(Biopanda，Cat:TMB-S-003)37℃显色3分钟，加入100μL ELISA终止液(Solarbio，Cat:C1058)，检测OD450。
参见图13和表7，结果显示，3种融合蛋白均明显结合BAFF。B613302结合BAFF能力显著强于Telitacicept，EC50降低至其1/8至1/7的水平。说明TACI置于BCMA的C端(比如B613302的情况)可能更有利于保持对BAFF的结合能力。
表7.Anifrolumab与TACI-BCMA的融合蛋白与BAFF的结合
参见图14和表8，结果显示，3种融合蛋白均明显结合APRIL。B613301、B613302和B613303结合APRIL基本一致，相比Telitacicept，EC50降低至其1/10至1/6的水平。结果说明，融合了BCMA的融合蛋白的确增强了对APRIL的结合。
表8.Anifrolumab与TACI-BCMA的融合蛋白与APRIL的结合
实施例12.Anifrolumab与TACI-19-16-BCMA的融合蛋白构建、表达及纯化
BCMA-ECD-1(氨基酸残基1-43)和BCMA-ECD-2(氨基酸残基1-41)去除了BCMA非CRD结构域中可能存在的糖基化位点(氨基酸残基42-44，NAS)和脱酰胺位点(氨基酸残基47-48，NS)等风险点，去除风险点可以使得融合蛋白更均一。
如图15，B637301将TACI-19-16融合在BCMA-ECD-1的N端，然后再将TACI-19-16-BCMA-ECD-1通过连接子1连接在Anifrolumab抗体轻链C端；B637302将TACI-19-16融合在BCMA-ECD-2的C端，然后再将BCMA-ECD-2-TACI-19-16通过连接子1连接在Anifrolumab抗体轻链C端；B746201将TACI-19-16融合在BCMA-ECD-2的C端，然后再将BCMA-ECD-2-TACI-19-16通过连接子2连接在Anifrolumab抗体重链C端。B637302-LALA将TACI-19-16融合在BCMA-ECD-2的C端，然后再将BCMA-ECD-2-TACI-19-16通过连接子1连接在抗体Anifrolumab轻链C端，且Anifrolumab抗体Fc部分为带有L234A和L235A突变的人IgG1Fc。B746201-LALA将TACI-19-16融合在BCMA-ECD-2的C端，然后再将BCMA-ECD-2-TACI-19-16通过连接子2连接在抗体Anifrolumab重链C端，且Anifrolumab抗体Fc部分为带有L234A和L235A突变的人IgG1Fc，见图15。
>BCMA-ECD-1(SEQ ID NO：67)
>BCMA-ECD-2(SEQ ID NO：68)
>TACI-19-16-BCMA-ECD-1(SEQ ID NO：69)
>BCMA-ECD-2-TACI-19-16(SEQ ID NO：70)
B637301、B637302的重链均为Anifrolumab的重链SEQ ID NO：55，轻链的序列如下：
>B637301的轻链(SEQ ID NO：71)
>B637302、B637302-LALA的轻链(SEQ ID NO：72)
B637302-LALA重链为SEQ ID NO：61。
>B746201的重链(SEQ ID NO：73)
B746201、B746201-LALA的轻链为Anifrolumab的轻链SEQ ID NO：54。
>B746201-LALA的重链(SEQ ID NO：74)

B637301、B637302和B746201融合蛋白经表达纯化，SDS-PAGE检测发现连接有TACI-BCMA的抗体重链、轻链均为单一条带(结果未示出)。结果说明对BCMA蛋白序列糖基化风险点的去除可以使得融合蛋白更均一。
实施例13.Anifrolumab与TACI-19-16-BCMA的融合蛋白对I型干扰素的活性抑制实验
使用HEK-Blue IFN-α/β细胞(InvivoGen，Cat：hkb-ifnab)试剂盒检测Anifrolumab与TACI-19-16-BCMA的融合蛋白(B606401、B637302、B746201和B805201)对IFN-α/β的活性抑制效果。
方法为：消化HEK-BlueTM IFN-α/β细胞，用含10％FBS、1％P/S的DMEM培养基(Gibco，Cat:11995065)重悬至2.8E5/mL并接种到96孔板(Costar，Cat:3599)。加入待测抗体，37℃培养30分钟。加入终浓度为0.01ng/mL的IFN-β(Sino biological，Cat:10704-HNAS)，37℃培养24小时。取20μL培养上清，在新的96孔板中与180μLQuanti-Blue(InvivoGen，Cat:rep-qbs)混合，37℃孵育1小时，检测OD655。
参见图16和表9，结果显示，这4种融合蛋白均能够抑制IFN-α/β的活性，4种融合蛋白活性相当且和Anifrolumab一致。
表9.Anifrolumab与TACI-19-16-BCMA的融合蛋白对I型干扰素的活性抑制结果
实施例14.Anifrolumab与TACI-19-16-BCMA的融合蛋白对BAFF和APRIL的体外结合实验
使用ELISA检测B606401、B637302、B746201和B805201融合蛋白对BAFF和APRIL的结合能力。
方法为：将BAFF(PeproTech，Cat:310-13)或ARPIL(Acro Biosystems，Cat:APL-H52D1)用PBS稀释到1μg/mL，96孔板4℃包被过夜。PBST洗板，用Blocking Buffer(含1％BSA的PBST)37℃封闭1小时。拍板，用Blocking Buffer稀释待测抗 体，37℃孵育1.5小时。PBST洗板，用Blocking Buffer稀释HRP-小鼠抗人Fc抗体(GenScript，Cat:A01854-200)，37℃孵育40分钟。PBST洗板，加入100μL TMB显色液(Biopanda，Cat:TMB-S-003)37℃显色3分钟，加入100μL ELISA终止液(Solarbio，Cat:C1058)，检测OD450。
参见图17和表10，4种融合蛋白均明显结合BAFF，4种融合蛋白结合BAFF的能力均强于Telitacicept。B606401和B637302结合BAFF能力相当，相比Telitacicept，EC50均降低至其1/4至1/3的水平；B746201和B805201结合BAFF能力相当，相比Telitacicept，EC50均降低至其1/2的水平。
表10.Anifrolumab与TACI-19-16-BCMA的融合蛋白与BAFF的结合
参见图18和表11，4种融合蛋白均明显结合APRIL，B637302结合APRIL的能力相比Telitacicept的EC50降低至其1/7至1/6的水平，B746201和B805201结合APRIL能力相当，相比Telitacicept的EC50降低至其1/3的水平，B606401结合APRIL的能力和Telitacicept相当。
表11.Anifrolumab与TACI-19-16-BCMA的融合蛋白与APRIL的结合
实施例15.浆细胞样树突状细胞(pDC)功能实验
为评估Anifrolumab与TACI-19-16-BCMA融合蛋白的体外药效，我们建立了体外测试IFNAR1拮抗剂生物活性分子的方法。检测pDC的功能实验如下：利用新鲜分离的健康人PBMC，在1640GlutaMAX(糖浓度＝11mM)的基础培养基中进行体外培养，PBMC以3x 106/mL密度接种在96孔板中，加0.5μM CpG-A ODN 2216(InvivoGen cat:tlrl-2216)刺激，加梯度浓度的Anifrolumab、B637302、B606401、IgG1isotype处理，24h后通过人IFN-α试剂盒(Cisbio cat:62HIFNAPEG)检测细胞培养上清中的IFN-α细胞因子分泌水平。
结果显示，B637302、B606401与对照抗体Anifrolumab均能够以剂量依赖的方式抑制IFN-α的产生，而且三者的抑制活性相当(图19)。
实施例16.IFN-α诱导的浆细胞体外分化功能实验
为评估Anifrolumab与TACI-19-16-BCMA融合蛋白对IFNAR1的拮抗性生物活性，我们建立了浆细胞体外分化的功能实验方法，具体如下：利用B细胞分选试剂盒(Stemcell cat:17954)从新鲜健康人PBMC分离人B细胞，在1640GlutaMAX(Gibco cat:72400-47)的基础培养基中进行体外培养，B细胞以1×105/孔 的密度接种在96孔板中，加2μg/mL CpG-B 2006(InvivoGen cat:tlrl-2006)以及250U/mL IFN-α(Biolegend cat:592704)刺激，外加梯度浓度的Anifrolumab、B637302、B746201、B606401、B805201、IgG1isotype对照处理，4天后流式检测浆细胞(CD27+CD38+)分化比例。
参见图20，结果显示，B637302、B746201、B606401、B805201与对照抗体Anifrolumab均能够以剂量依赖的方式有效抑制IFN-α诱导的B细胞向浆细胞的体外分化，IC50分别为10.75nM、1.895nM、17.45nM、6.208nM、12.66nM,提示融合蛋白与对照抗体Anifrolumab的抑制活性相当。
实施例17.BAFF诱导B细胞增殖功能实验
为评估Anifrolumab与TACI-19-16-BCMA融合蛋白的体外药效，我们建立了体外测试TACI-BCMA生物活性分子的方法。BAFF诱导B细胞体外增殖实验步骤如下：利用B细胞分选试剂盒(Stemcell cat:17954)从新鲜健康人PBMC分离人B细胞，使用CTV(Invitrogen cat:C34557)标记B细胞。在1640GlutaMAX(Gibco cat:72400-47)的基础培养基中进行体外培养，B细胞以1×105/孔的密度接种在96孔板中，加20μg/mL抗人IgM抗体(Jacksonimmuno cat:109-006-129)、10ng/mL重组人IL-4(PeproTech cat:AF-200-04-20)、100ng/mL CD40L(R&D cat:2706-CL-025/CF)、1.5μg/mL抗His抗体(R&D cat:MAB050-500)、1ng/mL重组人IL-17(SinoBiological cat:12047-HNAS)，外加200ng/mL重组人BAFF(R&D cat:7537-BF-025/CF)刺激，在此条件上，加梯度浓度的Telitacicept、B637302、B606401、B80521、IgG1isotype处理，5天后流式检测B细胞增殖信号。
结果显示，参见表12，B637302、B606401、B805201与对照Telitacicept均能够以剂量依赖的方式有效抑制BAFF诱导的B细胞体外增殖。参见图21，B637302、B805201的抑制效果明显强于Telitacicept；在10nM低浓度药物处理条件下，Telitacicept没有体现出抑制活性，而B637302、B606401、B805201均体现出明显的抑制浆细胞分化的活性。
表12.BAFF诱导B细胞增殖结果
实施例18.APRIL诱导B细胞增殖功能实验
为评估Anifrolumab与TACI-19-16-BCMA融合蛋白的体外药效，我们建立了体外测试TACI-BCMA生物活性分子的方法。APRIL诱导B细胞体外增殖实验步 骤如下：利用B细胞分选试剂盒(Stemcell cat:17954)从新鲜健康人PBMC分离人B细胞，使用CTV(Invitrogen cat:C34557)标记B细胞，在1640GlutaMAX(Gibco cat:72400-47)的基础培养基中进行体外培养，B细胞以1×105/孔的密度接种在96孔板中，加20μg/mL抗人IgM抗体(Jacksonimmuno cat:109-006-129)、10ng/mL重组人IL-4(PeproTech cat:AF-200-04-20)、100ng/mL CD40L(R&D cat:2706-CL-025/CF)、1.5μg/mL抗His抗体(R&D cat:MAB050-500)、1ng/mL重组人IL-17(SinoBiological cat:12047-HNAS)，外加20ng/mL重组人ARPIL(R&D cat:5860-AP-010/CF)刺激，在此条件上，加梯度浓度的Telitacicept、B637302、B606401、IgG1 isotype处理，5天后流式检测B细胞增殖信号。
结果显示，参见表13和图22，B637302、B606401与对照Telitacicept均能够以剂量依赖的方式有效抑制APRIL诱导的B细胞体外增殖，而且B637302药效明显优于Telitacicept；在1000nM药物处理条件下，B606401药效也明显优于对照Telitacicept。
表13.APRIL诱导B细胞增殖结果
实施例19.BAFF+APRIL诱导浆细胞生成功能实验
为评估Anifrolumab与TACI-19-16-BCMA的融合蛋白中TACI-BCMA生物活性，我们建立BAFF+APRIL诱导浆细胞体外生成实验方法，具体过程如下：利用B细胞分选试剂盒(Stemcell cat:17954)从新鲜健康人PBMC分离人B细胞，在1640GlutaMAX(Gibco cat:72400-47)的基础培养基中进行体外培养，B细胞以1×105/孔的密度接种在96孔板中，加2μg/mL CpG-B 2006(InvivoGen cat:tlrl-2006)刺激。第4天替换培养基，加入10ng/mL重组人IL-6、50ng/mL重组人IL-10(Peprotech cat:200-10)、50ng/mL重组人IL-21(Peprotech cat:AF-200-21-10)、500ng/mL重组人BAFF(R&D cat:7537-BF-025/CF)以及50ng/mL重组人ARPIL(R&D cat:5860-AP-010/CF)刺激。7天后替换一半培养基，BAFF和ARPIL浓度调整为50ng/mL和500ng/mL，其余刺激信号的浓度不变。在此过程中，第4至10天加梯度浓度的Telitacicept、B637302、B746201、B606401、B805201、IgG1isotype处理，第10天流式检测浆细胞(CD27+CD38+)数目。
参见表14，第10天流式检测浆细胞数目的实验结果显示，BAFF和ARPIL能 够有效诱导浆细胞的体外生成。参见图23，在10nM药物处理条件下，Telitacicept不能抑制浆细胞生成，而B637302、B746201、B606401、B805201能够明显抑制浆细胞生成，说明B637302、B746201、B606401、B805201抑制BAFF和ARPIL的活性优于Telitacicept；在100nM药物处理条件下,4种测试药物和Telitacicept都能明显抑制浆细胞生成；在1000nM药物处理条件下，B637302抑制浆细胞生成的活性最强，优于Telitacicept。
表14.BAFF+APRIL诱导浆细胞生成结果
实施例20.IFN-α+BAFF+APRIL诱导浆细胞生成功能实验
为评估Anifrolumab和TACI-19-16-BCMA的融合蛋白中Anifrolumab和TACI-BCMA在B细胞向浆细胞分化和生成过程中的协同生物活性，我们建立IFN-α+BAFF+APRIL诱导浆细胞体外生成实验方法。具体过程如下：利用B细胞分选试剂盒(Stemcell cat:17954)从新鲜健康人PBMC分离人B细胞，在1640GlutaMAX(Gibco cat:72400-47)的基础培养基中进行体外培养，B细胞以1x105/well密度接种在96孔板中，加2μg/mL CpG-B 2006(InvivoGen cat:tlrl-2006)和250U/mL IFN-α(Biolegend cat:592704)刺激。第4天替换培养基，加入10ng/mL重组人IL-6、50ng/mL重组人IL-10(Peprotech cat:200-10)、15ng/mL重组人IL-21(Peprotech cat:AF-200-21-10)、500ng/mL重组人BAFF(R&D cat:7537-BF-025/CF)以及50ng/mL重组人ARPIL(R&D cat:5860-AP-010/CF)刺激。7天后替换一半培养基，BAFF和ARPIL浓度调整为50ng/mL和500ng/mL，其余刺激信号的浓度不变。在此过程中，第0至10天加梯度浓度的Anifrolumab、Telitacicept、B637302、B606401、IgG1isotype处理，第10天流式检测浆细胞(CD27+CD38+)数目(图24的A)。
在浆细胞体外生成协同实验中，经过实验条件的优化，我们发现在IL-21 15ng/mL条件下，IFN-α与BAFF+APRIL能协同促进浆细胞分化与生成，而且两者的促进作用相当。因此，我们选择在该条件下比较不同浓度的各蛋白药物的抑制活性，结果发现：B637302、B606401、Telitacicept与Anifrolumab均能够以剂量依赖的方式抑制浆细胞的生成，参见表15、图24的B至D。在10nM低浓度药物处理的条件下，Telitacicept或Anifrolumab单药对浆细胞的生成都没有明显的抑制活性，而B637302、B606401有明显的抑制活性，参见图24的B。
在100nM浓度药物处理的条件下，Anifrolumab单药无明显的抑制活性， Telitacicept单药有一定的活性，而B637302的活性也明显优于Telitacicept，参见图24的C。在1000nM浓度药物处理的条件下，Anifrolumab单药有一定的活性，而B637302、B606401融合蛋白的活性也明显优于Anifrolumab，参见图24的D。
表15.IFN-α+BAFF+APRIL诱导浆细胞生成结果
实施例21.PEG-IFN-α诱导PBMC人源化小鼠PD实验
为检测Anifrolumab与TACI-19-16-BCMA融合蛋白中Anifrolumab端的体内药效，我们利用B-NDG免疫缺陷小鼠，尾静脉注射2×107个PBMC细胞进行人源化，腹腔注射0.6μg PEG-IFN-α(Roche)进行诱导，在该模型中腹腔注射不同浓度的药物Anifrolumab、B637302、B606401以及PBS处理小鼠。第2h取PBMC，通过流式检测pSTAT1(#9167,Cell Signaling Technology)水平，第1、3天时间点收集外周血分离PBMC，通过QPCR检测IFN-α下游基因(如ISG-15、IFI13、MX-1、HERC5等)mRNA表达水平，如图25的A、图25的B。
参见图25的C，诱导后第2h收集PBMC流式检测pSTAT1结果发现，B637302、B606401与Anifrolumab抑制hIFN-α的活性相当。参见图25的D和E，第1、3、7天QPCR检测ISG的mRNA表达水平也显示类似的结果，与前面体外的报告分子测试以及生物学活性实验结果一致。
实施例22.BAFF+APRIL诱导小鼠PD实验
为检测Anifrolumab与TACI-19-16-BCMA融合蛋白中TACI-19-16-BCMA端的体内药效，我们利用C57/B6小鼠，腹腔注射3μg BAFF(BAF-H52D4-1mg，AcroBio)和0.5μg APRIL(APL-H52D1-1mg,AcroBio)进行诱导，在该模型中腹腔注射不同浓度的药物Telitacicept、B637302、B606401以及PBS处理小鼠，第4、7天取外周血，通过ELISA检测血浆中IgA水平(图26的A)。
结果显示，第0、2天PBS与给药组IgA差异尚不明显(结果未示出)，而第4、7天时B637302、B606401和Telitacicept与PBS对照组相比能够明显抑制IgA的产生，参见图26的B和图26的C。并且，第7天的结果还显示在等摩尔给药的条件下，药效B637302>B606401>Telitacicept，参见图26的C，这与之前体外BAFF和APRIL的ELISA结合实验、体外生物学活性实验结果一致。

Claims (29)

  1. 融合蛋白，其包含：
    (a)TACI多肽和抗IFNAR1抗体或其抗原结合片段，或，
    (b)TACI多肽、BCMA多肽和抗IFNAR1抗体或其抗原结合片段；
    其中，所述TACI多肽包含TACI胞外区的CRD1和/或CRD2；
    优选地，所述BCMA多肽包含BCMA胞外区的CRD1。
  2. 根据权利要求1所述的融合蛋白，其中，
    所述TACI多肽包含选自如下(1)-(6)任一项的多肽：
    (1)SEQ ID NO：1的第68-105位氨基酸残基或其变体；
    (2)SEQ ID NO：1或与其具有至少95％序列同一性的多肽；
    (3)SEQ ID NO：1的第33-67位氨基酸残基；
    (4)SEQ ID NO：1的第70-104位氨基酸残基或其变体；
    (5)SEQ ID NO：1的第30-110、69-111、69-112、13-118、或33-104位氨基酸残基或其变体；
    (6)SEQ ID NO：1的第68-106、68-107或68-108位氨基酸残基或其变体；
    优选地，所述变体具有选自第69、72、73、77、85、102和103位中的一个或多个氨基酸突变；
    更优选地，所述变体具有选自69T或69R、72S、73E或73Q、77E、85T或85A、102A或102R、103Y中的一个或多个氨基酸替换；
    最优选地，所述变体具有选自如下任一的氨基酸替换或氨基酸替换的组合：
    69T；72S；73E；73Q；77E；69R和85T；69R和85A；102A；69R，85T和102R；73E和77E；72S，73E和77E；69T和102A；69T和103Y；69T，102A和103Y；69T，73E，77E和102A；
    氨基酸残基的编号是按照SEQ ID NO：1所示序列的自然顺序编号。
  3. 根据权利要求1-2任一项所述的融合蛋白，其中所述TACI多肽包含选自SEQ ID NO：1、2、6-29任一所示的氨基酸序列。
  4. 根据权利要求1-3任一项所述的融合蛋白，其中所述抗IFNAR1抗体或其抗原结合片段包含重链可变区和轻链可变区，其中，
    所述重链可变区包含氨基酸序列分别如SEQ ID NO：45-47所示的HCDR1、HCDR2和HCDR3，
    所述轻链可变区包含氨基酸序列分别如SEQ ID NO：48-50所示的LCDR1、LCDR2和LCDR3。
  5. 根据权利要求4所述的融合蛋白，其中：
    所述重链可变区包含如SEQ ID NO：43所示或与之具有至少90％序列同一性的氨基酸序列，
    所述轻链可变区包含如SEQ ID NO：44所示或与之具有至少90％序列同一性的氨基酸序列。
  6. 根据前述权利要求任一项所述的融合蛋白，其含有免疫球蛋白的Fc区，优选为IgG1的Fc区，更优选为SEQ ID NO：3所示的Fc区。
  7. 根据权利要求6所述的融合蛋白，所述Fc区包含一个或多个氨基酸突变，所述的氨基酸突变能够减少Fc与FcR的结合，优选能够减少Fc与FcγR的结合的突变，更优选为如下任一的氨基酸突变或氨基酸突变的组合：234A；235A；234F；235E；234F和235E；234A和235A；234F，235E和331S；
    突变位点是根据EU编号系统定义。
  8. 根据权利要求1-7任一项所述的融合蛋白，其中所述抗IFNAR1抗体包含重链和轻链，所述重链包含如SEQ ID NO：55或61所示或与之具有至少90％序列同一性的氨基酸序列，所述轻链包含如SEQ ID NO：54所示或与之具有至少90％序列同一性的氨基酸序列。
  9. 根据前述权利要求任一项所述的融合蛋白，其包含如下(I)-(VIII)任一所示的多肽链：
    (I)第一多肽链，其从N端到C端依次为：[TACI多肽]-[连接子1]a-[抗体重链]，和第二多肽链，其为抗体轻链；
    (II)第一多肽链，其从N端到C端依次为：[抗体重链]-[连接子2]b-[TACI多肽]，和第二多肽链，其为抗体轻链；
    (III)第一多肽链，其为抗体重链，和第二多肽链，其从N端到C端依次为：[TACI多肽]-[连接子3]c-[抗体轻链]；
    (IV)第一多肽链，其为抗体重链，和第二多肽链，其从N端到C端依次为：[抗体轻链]-[连接子4]d-[TACI多肽]；
    (V)第一多肽链，其从N端到C端依次为：[TACI多肽1]-[连接子1]a-[抗体重链]，和第二多肽链，其从N端到C端依次为：[抗体轻链]-[连接子4]d-[TACI多肽2]；
    (VI)第一多肽链，其从N端到C端依次为：[抗体重链]-[连接子2]b-[TACI多肽1]，和第二多肽链，其从N端到C端依次为：[TACI多肽2]-[连接子3]c-[抗体轻链]；
    (VII)第一多肽链，其从N端到C端依次为：[TACI多肽1]-[连接子1]a-[抗体 重链]-[连接子2]b-[TACI多肽2]，和第二多肽链，其为抗体轻链；
    (VIII)第一多肽链，其为抗体重链，和第二多肽链，其从N端到C端依次为：[TACI多肽1]-[连接子3]c-[抗体轻链]-[连接子4]d-[TACI多肽2]；
    其中，
    -表示肽键，
    连接子1、连接子2、连接子3、连接子4可以相同，也可以不同；
    所述TACI多肽、所述TACI多肽1和所述TACI多肽2各自独立地选自权利要求1-3中任一项所限定的TACI多肽，TACI多肽1和TACI多肽2可以相同也可以不同；
    a、b、c、d分别独立地为0或1；
    所述抗体是指权利要求4-5中任一项所限定的抗IFNAR1抗体；
    优选地，所述连接子1、连接子2、连接子3、连接子4各自独立地为(GxS)y，其中，x是1-5的整数，y是1-6的整数；
    更优选地，所述连接子1、连接子2、连接子3、连接子4的氨基酸序列各自独立地如SEQ ID NO：39-41任一所示。
  10. 根据前述权利要求任一项所述的融合蛋白，其包含：
    两条相同的如SEQ ID NO：51-53、62任一所示的第一多肽链，和两条相同的如SEQ ID NO：54所示的第二多肽链；或
    两条相同的如SEQ ID NO：55所示的第一多肽链，和两条相同的如SEQ ID NO：56-60任一所示的第二多肽链；或
    两条相同的如SEQ ID NO：61所示的第一多肽链，和两条相同的如SEQ ID NO：56-60任一所示的第二多肽链。
  11. 根据权利要求1所述的融合蛋白，其中所述BCMA多肽包含SEQ ID NO：30的第7-41位所示的氨基酸残基；
    优选地，所述BCMA多肽的氨基酸序列包含选自SEQ ID NO：30、67、68任一所示的序列。
  12. 根据权利要求1-11任一项所述的融合蛋白，其包含如下(i)-(viii)任一所示的多肽链：
    (i)第一多肽链，其从N端到C端依次为：[TACI多肽和BCMA多肽结构域]-[连接子1]a-[抗体重链]，和第二多肽链，其为抗体轻链；
    (ii)第一多肽链，其从N端到C端依次为：[抗体重链]-[连接子2]b-[TACI多肽和BCMA多肽结构域]，和第二多肽链，其为抗体轻链；
    (iii)第一多肽链，其为抗体重链，和第二多肽链，其从N端到C端依次为：[TACI 多肽和BCMA多肽结构域]-[连接子3]c-[抗体轻链]；
    (iv)第一多肽链，其为抗体重链，和第二多肽链，其从N端到C端依次为：[抗体轻链]-[连接子4]d-[TACI多肽和BCMA多肽结构域]；
    (v)第一多肽链，其从N端到C端依次为：[TACI多肽和BCMA多肽结构域1]-[连接子1]a-[抗体重链]，和第二多肽链，其从N端到C端依次为：[抗体轻链]-[连接子4]d-[TACI多肽和BCMA多肽结构域2]；
    (vi)第一多肽链，其从N端到C端依次为：[抗体重链]-[连接子2]b-[TACI多肽和BCMA多肽结构域1]，和第二多肽链，其从N端到C端依次为：[TACI多肽和BCMA多肽结构域2]-[连接子3]c-[抗体轻链]；
    (vii)第一多肽链，其从N端到C端依次为：[TACI多肽和BCMA多肽结构域1]-[连接子1]a-[抗体重链]-[连接子2]b-[TACI多肽和BCMA多肽结构域2]，和第二多肽链，其为抗体轻链；
    (viii)第一多肽链，其为抗体重链，和第二多肽链，其从N端到C端依次为：[TACI多肽和BCMA多肽结构域1]-[连接子3]c-[抗体轻链]-[连接子4]d-[TACI多肽和BCMA多肽结构域2]；
    其中，
    所述[TACI多肽和BCMA多肽结构域]、所述[TACI多肽和BCMA多肽结构域1]和所述[TACI多肽和BCMA多肽结构域2]从N端到C端依次为：[TACI多肽]-[连接子5]e-[BCMA多肽]，或为：[BCMA多肽]-[连接子5]e-[TACI多肽]，
    所述[TACI多肽和BCMA多肽结构域1]和所述[TACI多肽和BCMA多肽结构域2]中的TACI多肽可以相同也可以不同，所述BCMA多肽结构域1和所述BCMA多肽结构域2可以相同也可以不同；
    所述TACI多肽选自权利要求1-3中任一项所限定的TACI多肽，所述BCMA多肽选自权利要求1或11中所限定的BCMA多肽，所述抗体选自权利要求4-8中任一项所限定的抗IFNAR1抗体；
    其中：
    -表示肽键，
    所述连接子1、连接子2、连接子3、连接子4、连接子5可以相同，也可以不同；a、b、c、d、e分别独立地为0或1；
    优选地，所述连接子1、连接子2、连接子3、连接子4、连接子5各自独立地为(GxS)y，其中，x是1-5的整数，y是1-6的整数；
    更优选地，所述连接子1、连接子2、连接子3、连接子4、连接子5的氨基酸序列各自独立地如SEQ ID NO：39-41任一所示。
  13. 根据权利要求1-12任一项所述的融合蛋白，其包含两条相同的如SEQ ID NO：55所示的第一多肽链，和两条相同的如SEQ ID NO：63-66、71、72任一所 示的第二多肽链；
    其包含两条相同的如SEQ ID NO：61所示的第一多肽链，和两条相同的如SEQ ID NO：63-66、71、72任一所示的第二多肽链；或
    其包含两条相同的如SEQ ID NO：73或74所示的第一多肽链，和两条相同的如SEQ ID NO：54任一所示的第二多肽链。
  14. 融合蛋白，其包含TACI多肽和BCMA多肽，其中：
    所述TACI多肽包含TACI胞外结构域的CRD1和/或CRD2，
    所述BCMA多肽包含BCMA胞外结构域的CRD1；
    优选地，所述TACI多肽和所述BCMA多肽直接地或通过连接子相连接。
  15. 根据权利要求14所述的融合蛋白，其中，所述TACI多肽包含选自如下(1)-(6)任一项的多肽：
    (1)SEQ ID NO：1的第68-105位氨基酸残基或其变体；
    (2)SEQ ID NO：1或与其具有至少95％序列同一性的多肽；
    (3)SEQ ID NO：1的第33-67位氨基酸残基；
    (4)SEQ ID NO：1的第70-104位氨基酸残基或其变体；
    (5)SEQ ID NO：1的第30-110、69-111、69-112、13-118或33-104位氨基酸残基或其变体；
    (6)SEQ ID NO：1的第68-106、68-107或68-108位氨基酸残基或其变体；
    优选地，所述变体具有选自第69、72、73、77、85、102和103位中的一个或多个氨基酸突变；
    更优选地，所述变体具有选自69T或69R、72S、73E或73Q、77E、85T或85A、102A或102R、103Y中的一个或多个氨基酸替换；
    最优选地，所述变体具有选自如下任一的氨基酸替换或氨基酸替换的组合：69T；72S；73E；73Q；77E；69R和85T；69R和85A；102A；69R，85T和102R；73E和77E；72S，73E和77E；69T和102A；69T和103Y；69T，102A和103Y；69T，73E，77E和102A；
    氨基酸残基的编号是按照SEQ ID NO：1所示序列的自然顺序编号。
  16. 根据权利要求14或15所述的融合蛋白，其中所述TACI多肽包含选自SEQ ID NO：1、2、6-29任一所示的氨基酸序列。
  17. 根据权利要求14-16任一项所述的融合蛋白，其中所述BCMA多肽包含SEQ ID NO：30的第7-41位所示的氨基酸残基。
  18. 根据权利要求17所述的融合蛋白，其中，所述BCMA多肽的氨基酸序列 包含选自SEQ ID NO：30、67、68任一所示的序列。
  19. 根据权利要求14-18任一项所述的融合蛋白，其还含有免疫球蛋白的Fc区，优选为IgG1的Fc区，更优选为SEQ ID NO：3所示的Fc区。
  20. 根据权利要求19所述的融合蛋白，所述Fc区包含一个或多个氨基酸突变，所述的氨基酸突变能够减少Fc与FcR的结合，优选能够减少Fc与FcγR的结合的突变，更优选为如下任一的氨基酸突变或氨基酸突变的组合：234A；235A；234F；235E；234F和235E；234A和235A；234F，235E和331S；
    突变位点是根据EU编号系统定义。
  21. 根据权利要求14-20任一所述的融合蛋白，其包含如下(I)-(VIII)任一所示的多肽链：
    (I)[TACI多肽]-[连接子1]a-[BCMA多肽]-[连接子2]b-[Fc区]e；
    (II)[BCMA多肽]-[连接子1]a-[TACI多肽]-[连接子2]b-[Fc区]e；
    (III)[Fc区]e-[连接子3]c-[TACI多肽]-[连接子4]d-[BCMA多肽]；
    (IV)[Fc区]e-[连接子3]c-[BCMA多肽]-[连接子4]d-[TACI多肽]；
    (V)[TACI多肽1]-[连接子1]a-[BCMA多肽1]-[连接子2]b-[Fc区]e-[连接子3]c-[TACI多肽2]-[连接子4]d-[BCMA多肽2]；
    (VI)[BCMA多肽1]-[连接子1]a-[TACI多肽1]-[连接子2]b-[Fc区]e-[连接子3]c-[BCMA多肽2]-[连接子4]d-[TACI多肽2]；
    (VII)[TACI多肽1]-[连接子1]a-[BCMA多肽1]-[连接子2]b-[Fc区]e-[连接子3]c-[BCMA多肽2]-[连接子4]d-[TACI多肽2]；
    (VIII)[BCMA多肽1]-[连接子1]a-[TACI多肽1]-[连接子2]b-[Fc区]e-[连接子3]c-[TACI多肽2]-[连接子4]d-[BCMA多肽2]；
    其中，
    -表示肽键，所述连接子1、连接子2、连接子3和连接子4可以相同，也可以不同；
    所述TACI多肽1和所述TACI多肽2选自权利要求14-16中任一项所限定的TACI多肽，所述TACI多肽1和所述TACI多肽2可以相同也可以不同；
    所述BCMA多肽1和所述BCMA多肽2选自权利要求14-18中任一项所限定的BCMA多肽，所述BCMA多肽1和所述BCMA多肽2可以相同也可以不同；
    a、b、c、d、e分别独立地为0或1；
    优选地，所述连接子1、连接子2、连接子3、连接子4各自独立地为(GxS)y，其中，x是1-5的整数，y是1-6的整数；
    更优选地，所述连接子1、连接子2、连接子3、连接子4、连接子5的氨基 酸序列各自独立地如SEQ ID NO：39-41任一所示。
  22. 根据权利要求14-21任一项所述的融合蛋白，其含有SEQ ID NO：31-38、69、70任一所示的氨基酸序列。
  23. 根据权利要求14-22任一项所述的融合蛋白，其为二聚体。
  24. 复合物，其含有：
    权利要求1-3任一项中所限定的TACI多肽、权利要求4-8任一项中所限定的抗IFNAR1抗体，和权利要求1或11中所限定的BCMA多肽；或者，
    其含有权利要求14-23中任一项所限定的TACI多肽，和权利要求14-23中任一项所限定的BCMA多肽。
  25. 药物组合物，其含有：
    权利要求1-23中任一项所述的融合蛋白，或权利要求24所述的复合物，以及
    一种或多种药学上可接受的载体、稀释剂或赋形剂。
  26. 多核苷酸，其编码：
    权利要求1-23中任一项所述的融合蛋白、或
    权利要求24所述的复合物。
  27. 宿主细胞，其包含或表达权利要求26所述的多核苷酸。
  28. 治疗或改善疾病或病症的方法，所述方法包括向有需要的受试者施用治疗有效量的权利要求1-23中任一项所述的融合蛋白、权利要求24所述的复合物、权利要求25所述的药物组合物或权利要求26所述的多核苷酸的步骤；
    优选地，所述疾病或病症为B细胞障碍或自身免疫性疾病；
    更优选地，所述B细胞障碍或自身免疫性疾病是与TACI和/或BCMA表达相关的疾病或病症；
    最优选地，所述自身免疫性疾病为系统性红斑狼疮。
  29. 制备权利要求1-23中任一项所述的融合蛋白、或权利要求24所述的复合物的方法，包括培养权利要求27所述的宿主细胞，表达所述融合蛋白或复合物；可选地，包括分离或纯化所述融合蛋白或复合物。
PCT/CN2023/115448 2022-08-29 2023-08-29 包含taci多肽的融合蛋白及其用途 Ceased WO2024046301A1 (zh)

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US20260125451A1 (en) 2026-05-07
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