WO2023274342A1 - Molécule de liaison à l'antigène se liant spécifiquement à baff et il-12/23 et son utilisation - Google Patents

Molécule de liaison à l'antigène se liant spécifiquement à baff et il-12/23 et son utilisation Download PDF

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WO2023274342A1
WO2023274342A1 PCT/CN2022/102621 CN2022102621W WO2023274342A1 WO 2023274342 A1 WO2023274342 A1 WO 2023274342A1 CN 2022102621 W CN2022102621 W CN 2022102621W WO 2023274342 A1 WO2023274342 A1 WO 2023274342A1
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seq
amino acid
acid sequence
hcdr1
lcdr1
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PCT/CN2022/102621
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Chinese (zh)
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毛浪勇
应华
赖炜明
陶维康
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江苏恒瑞医药股份有限公司
上海恒瑞医药有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof

Definitions

  • the present disclosure belongs to the field of biotechnology, and more specifically, the present disclosure relates to an antigen-binding molecule specifically binding to BAFF and IL-12/23 and its application.
  • BAFF B cell activating factor
  • BAFF is a cell activating factor belonging to the TNF family. BAFF is mainly expressed on the surface of the bone marrow cell membrane and exists in the form of trimers. The BAFF on the surface of the cell membrane will be hydrolyzed by protease to form soluble BAFF and enter the blood circulation system. The soluble BAFF has the characteristics of multimerization and can form up to 60-mer . In addition, BAFF can also interact with another protein of the same family, APRIL, to form a heterologous trimer. It is currently known that there are three BAFF receptors on the surface of B cells, namely BAFF-R, BCMA and TACI.
  • BAFF interacts with these three receptors and participates in the differentiation, maturation, survival and regulation of B cells.
  • APRIL and BAFF have two common receptors, namely BCMA and TACI, and APRIL interacts with these two receptors to participate in the survival and regulation of B cells (Samy, E., et al., Int Rev Immunol, 2017.36: p. 3-19; Kamal, A. and M. Khamashta, Autoimmun Rev, 2014.13: p.1094-1101).
  • BAFF is important for maintaining B cell homeostasis, and overactivation of BAFF signaling leads to the survival of self-reactive B cells and the production of autoantibodies that promote autoimmune responses (Cancro, M.P., D.P.D'Cruz, and M.A. Khamashta, J Clin Invest, 2009.119: p.1066-73).
  • IL-12 and IL-23 are two cytokines belonging to the same family. Both IL-12 and IL-23 are heterodimeric proteins composed of two subunits (Moschen, A.R., H. Tilg, and T. Raine, Nat Rev Gastroenterol Hepatol, 2019.16: p.185-196; Frieder , J., et al., Clin Pharmacol Ther, 2018.103: p.88-101). IL-12 is composed of two subunits, p35 and p40, while IL-23 is composed of two subunits, p19 and p40.
  • p40 is a subunit shared by IL-12 and IL-23 (IL-12/23 P40), so antibodies targeting p40 can simultaneously inhibit both IL-12 and IL-23 signaling pathways.
  • the interaction between IL-12 and receptors mainly activates the differentiation of Th1 cells, and at the same time participates in stimulating the secretion of interferon- ⁇ and TNF by various immune cells.
  • IL-23 and receptors mainly activates the differentiation of Th17 cells, and stimulates a variety of cells to secrete cytokines such as IL-17, IL-22 and TNF (Lee, E.B., et al., Cutis, 2018.101: p.5-9 ; Floss, D.M., et al., Cytokine Growth Factor Rev, 2015.26: p.569-578).
  • cytokines produced by the activation of IL-12/23 signaling pathway further participate in the development of diseases such as systemic lupus erythematosus through their own pathways.
  • the present disclosure constructs an antigen-binding molecule that specifically binds BAFF, and constructs an antigen-binding molecule that specifically binds BAFF and IL-12 and/or IL-23.
  • the present disclosure provides an antigen-binding molecule comprising an antigen-binding module 1 that specifically binds BAFF and an antigen-binding module 2 that specifically binds IL-12 and/or IL-23, wherein the antigen-binding module 1 Comprising heavy chain variable region B-VH and light chain variable region B-VL, said B-VH comprises B-HCDR1, B-HCDR2 and B-HCDR3, said B-VL comprises B-LCDR1, B-LCDR2 and B-LCDR3, wherein: said B-HCDR1, B-HCDR2 and B-HCDR3 respectively comprise the amino acid sequences of Bv-HCDR1, Bv-HCDR2 and Bv-HCDR3 in SEQ ID NO: 63, and said B-LCDR1 , B-LCDR2 and B-LCDR3 respectively comprise the amino acid sequences of Bv-LCDR1, Bv-LCDR2 and Bv-LCDR3 in SEQ ID NO: 64, wherein, SEQ ID NO: 63 is
  • SEQ ID NO: 64 is:
  • X1 is selected from S, A , V or G
  • X2 is selected from A , I, M or S
  • X3 is selected from T or G
  • X4 is selected from Q
  • X5 is selected from N
  • X 6 is selected from L or P
  • X 7 is selected from H
  • X 8 is selected from H or D
  • X 9 is selected from A or G
  • X 10 is selected from S or L
  • X 11 selected from P or S
  • X 12 selected from Q or H
  • X 13 selected from D
  • a or N X 14 selected from S or I
  • X 15 selected from K, R or T
  • X 16 selected from S, W, T or D
  • X 17 is selected from H, Y or S
  • X 18 is selected from Y or R
  • X 19 is selected from G or S
  • X 20 is selected from A or D
  • X 21 is selected from E or S
  • B-HCDR1, B-HCDR2, B-HCDR3, B-LCDR1, B-LCDR2 and B-LCDR3 do not contain the following CDR combination: B-HCDR1, B-HCDR2 and B-HCDR3 respectively contain SEQ ID NO: The amino acid sequences of Bv-HCDR1, Bv-HCDR2 and Bv-HCDR3 in 1, and B-LCDR1, B-LCDR2 and B-LCDR3 respectively comprise Bv-LCDR1, Bv-LCDR2 and Bv-LCDR3 in SEQ ID NO: 2 amino acid sequence.
  • the B-HCDR1, B-HCDR2, B-HCDR3, B-LCDR1, B-LCDR2, B-LCDR3, Bv-HCDR1, Bv-HCDR2, Bv-HCDR3, Bv-LCDR1, Bv- LCDR2 and Bv-LCDR3 are defined according to the same numbering convention selected from Kabat, IMGT, Chothia, AbM and Contact.
  • the B-HCDR1, B-HCDR2, B-HCDR3, B-LCDR1, B-LCDR2, B-LCDR3, Bv-HCDR1, Bv-HCDR2, Bv-HCDR3, Bv-LCDR1, Bv- LCDR2 and Bv-LCDR3 are defined according to the Kabat numbering convention.
  • the B-HCDR1, B-HCDR2, B-HCDR3, B-LCDR1, B-LCDR2, B-LCDR3, Bv-HCDR1, Bv-HCDR2, Bv-HCDR3, Bv-LCDR1, Bv- LCDR2 and Bv-LCDR3 are defined according to the IMGT numbering rules.
  • the B-HCDR1, B-HCDR2, B-HCDR3, B-LCDR1, B-LCDR2, B-LCDR3, Bv-HCDR1, Bv-HCDR2, Bv-HCDR3, Bv-LCDR1, Bv- LCDR2 and Bv-LCDR3 are defined according to the Chothia numbering convention.
  • the B-HCDR1, B-HCDR2, B-HCDR3, B-LCDR1, B-LCDR2, B-LCDR3, Bv-HCDR1, Bv-HCDR2, Bv-HCDR3, Bv-LCDR1, Bv- LCDR2 and Bv-LCDR3 are defined according to the AbM numbering convention.
  • the B-HCDR1, B-HCDR2, B-HCDR3, B-LCDR1, B-LCDR2, B-LCDR3, Bv-HCDR1, Bv-HCDR2, Bv-HCDR3, Bv-LCDR1, Bv- LCDR2 and Bv-LCDR3 are defined according to the Contact Numbering Rules.
  • the antigen-binding molecules according to any one of the above, said B-HCDR1, B-HCDR2, B-HCDR3, B-LCDR1, B-LCDR2, B-LCDR3 are defined according to the Kabat numbering rules, wherein, B-HCDR1 comprises the amino acid sequence NNAIN (SEQ ID NO: 18), B-HCDR2 comprises the amino acid sequence X 1 IX 2 PMFGX 3 AKYSX 4 X 5 FQG (SEQ ID NO: 65), and B-HCDR3 comprises the amino acid sequence SRDX 6 LLFPX 7 X 8 X 9 LX 10 X 11 (SEQ ID NO: 66), B-LCDR1 comprises the amino acid sequence X 12 GX 13 X 14 LX 15 X 16 X 17 X 18 AS (SEQ ID NO: 67), B-LCDR2 Comprising the amino acid sequence GKNNRPS (SEQ ID NO: 32) and B-LCDR3 comprising the amino acid sequence X 19 SRX 20 X 21
  • the antigen binding molecule of any of the above wherein:
  • said B-HCDR1, B-HCDR2 and B-HCDR3 respectively comprise the amino acid sequences of Bv-HCDR1, Bv-HCDR2 and Bv-HCDR3 in SEQ ID NO: 47
  • said B-LCDR1, B-LCDR2 and B-LCDR3 respectively comprising the amino acid sequences of Bv-LCDR1, Bv-LCDR2 and Bv-LCDR3 in SEQ ID NO: 48, or
  • said B-HCDR1, B-HCDR2 and B-HCDR3 respectively comprise the amino acid sequences of Bv-HCDR1, Bv-HCDR2 and Bv-HCDR3 in SEQ ID NO: 5
  • said B-LCDR1, B-LCDR2 and B-LCDR3 respectively comprising the amino acid sequences of Bv-LCDR1, Bv-LCDR2 and Bv-LCDR3 in SEQ ID NO: 12, or
  • said B-HCDR1, B-HCDR2 and B-HCDR3 respectively comprise the amino acid sequences of Bv-HCDR1, Bv-HCDR2 and Bv-HCDR3 in SEQ ID NO: 6, and said B-LCDR1, B-LCDR2 and B-LCDR3 respectively comprising the amino acid sequences of Bv-LCDR1, Bv-LCDR2 and Bv-LCDR3 in SEQ ID NO: 13, or
  • said B-HCDR1, B-HCDR2 and B-HCDR3 respectively comprise the amino acid sequences of Bv-HCDR1, Bv-HCDR2 and Bv-HCDR3 in SEQ ID NO: 7
  • said B-LCDR1, B-LCDR2 and B-LCDR3 respectively comprising the amino acid sequences of Bv-LCDR1, Bv-LCDR2 and Bv-LCDR3 in SEQ ID NO: 14, or
  • said B-HCDR1, B-HCDR2 and B-HCDR3 respectively comprise the amino acid sequences of Bv-HCDR1, Bv-HCDR2 and Bv-HCDR3 in SEQ ID NO: 8
  • said B-LCDR1, B-LCDR2 and B-LCDR3 respectively comprising the amino acid sequences of Bv-LCDR1, Bv-LCDR2 and Bv-LCDR3 in SEQ ID NO: 15, or
  • said B-HCDR1, B-HCDR2 and B-HCDR3 respectively comprise the amino acid sequences of Bv-HCDR1, Bv-HCDR2 and Bv-HCDR3 in SEQ ID NO: 9
  • said B-LCDR1, B-LCDR2 and B-LCDR3 respectively comprising the amino acid sequences of Bv-LCDR1, Bv-LCDR2 and Bv-LCDR3 in SEQ ID NO: 16, or
  • said B-HCDR1, B-HCDR2 and B-HCDR3 respectively comprise the amino acid sequence of Bv-HCDR1, Bv-HCDR2 and Bv-HCDR3 in SEQ ID NO: 10
  • said B-LCDR1, B-LCDR2 and B-LCDR3 respectively comprising the amino acid sequences of Bv-LCDR1, Bv-LCDR2 and Bv-LCDR3 in SEQ ID NO: 17, or
  • said B-HCDR1, B-HCDR2 and B-HCDR3 respectively comprise the amino acid sequence of Bv-HCDR1, Bv-HCDR2 and Bv-HCDR3 in SEQ ID NO: 11
  • said B-LCDR1, B-LCDR2 and B-LCDR3 respectively comprise the amino acid sequences of Bv-LCDR1, Bv-LCDR2 and Bv-LCDR3 in SEQ ID NO:2.
  • the antigen-binding molecule of any one of the above, said B-HCDR1, B-HCDR2, B-HCDR3, B-LCDR1, B-LCDR2, B-LCDR3, Bv-HCDR1, Bv-HCDR2 , Bv-HCDR3, Bv-LCDR1, Bv-LCDR2 and Bv-LCDR3 are defined according to the Kabat numbering rules, where,
  • said B-HCDR1 comprises the amino acid sequence of SEQ ID NO: 18
  • said B-HCDR2 comprises the amino acid sequence of SEQ ID NO: 28
  • said B-HCDR3 comprises the amino acid sequence of SEQ ID NO: 29
  • said Said B-LCDR1 comprises the amino acid sequence of SEQ ID NO: 43
  • said B-LCDR2 comprises the amino acid sequence of SEQ ID NO: 32
  • said B-LCDR3 comprises the amino acid sequence of SEQ ID NO: 44;
  • the B-HCDR1 comprises the amino acid sequence of SEQ ID NO: 18
  • the B-HCDR2 comprises the amino acid sequence of SEQ ID NO: 21
  • the B-HCDR3 comprises the amino acid sequence of SEQ ID NO: 20
  • the Said B-LCDR1 comprises the amino acid sequence of SEQ ID NO: 34
  • said B-LCDR2 comprises the amino acid sequence of SEQ ID NO: 32
  • said B-LCDR3 comprises the amino acid sequence of SEQ ID NO: 35;
  • the B-HCDR1 comprises the amino acid sequence of SEQ ID NO: 18
  • the B-HCDR2 comprises the amino acid sequence of SEQ ID NO: 22
  • the B-HCDR3 comprises the amino acid sequence of SEQ ID NO: 23
  • the Said B-LCDR1 comprises the amino acid sequence of SEQ ID NO: 31
  • said B-LCDR2 comprises the amino acid sequence of SEQ ID NO: 32
  • said B-LCDR3 comprises the amino acid sequence of SEQ ID NO: 36;
  • said B-HCDR1 comprises the amino acid sequence of SEQ ID NO: 18
  • said B-HCDR2 comprises the amino acid sequence of SEQ ID NO: 24
  • said B-HCDR3 comprises the amino acid sequence of SEQ ID NO: 20
  • said Said B-LCDR1 comprises the amino acid sequence of SEQ ID NO: 37
  • said B-LCDR2 comprises the amino acid sequence of SEQ ID NO: 32
  • said B-LCDR3 comprises the amino acid sequence of SEQ ID NO: 38;
  • said B-HCDR1 comprises the amino acid sequence of SEQ ID NO: 18
  • said B-HCDR2 comprises the amino acid sequence of SEQ ID NO: 25
  • said B-HCDR3 comprises the amino acid sequence of SEQ ID NO: 20
  • said Said B-LCDR1 comprises the amino acid sequence of SEQ ID NO: 39
  • said B-LCDR2 comprises the amino acid sequence of SEQ ID NO: 32
  • said B-LCDR3 comprises the amino acid sequence of SEQ ID NO: 40;
  • said B-HCDR1 comprises the amino acid sequence of SEQ ID NO: 18
  • said B-HCDR2 comprises the amino acid sequence of SEQ ID NO: 26
  • said B-HCDR3 comprises the amino acid sequence of SEQ ID NO: 27
  • said Said B-LCDR1 comprises the amino acid sequence of SEQ ID NO: 41
  • said B-LCDR2 comprises the amino acid sequence of SEQ ID NO: 32
  • said B-LCDR3 comprises the amino acid sequence of SEQ ID NO: 42;
  • said B-HCDR1 comprises the amino acid sequence of SEQ ID NO: 18
  • said B-HCDR2 comprises the amino acid sequence of SEQ ID NO: 30
  • said B-HCDR3 comprises the amino acid sequence of SEQ ID NO: 20
  • said The B-LCDR1 comprises the amino acid sequence of SEQ ID NO: 31
  • the B-LCDR2 comprises the amino acid sequence of SEQ ID NO: 32
  • the B-LCDR3 comprises the amino acid sequence of SEQ ID NO: 33.
  • SEQ ID NO: 63 is:
  • SEQ ID NO: 64 is:
  • the antigen binding molecule of any one of the above, wherein,
  • said B-VH comprises an amino acid sequence having at least 90% (e.g., at least 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to SEQ ID NO: 47
  • said B-VL comprises an amino acid sequence having at least 90% (e.g., at least 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to SEQ ID NO: 48, or
  • said B-VH comprises an amino acid sequence having at least 90% (e.g., at least 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to SEQ ID NO: 5
  • said B-VL comprises an amino acid sequence having at least 90% (e.g., at least 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to SEQ ID NO: 12, or
  • said B-VH comprises an amino acid sequence having at least 90% (e.g., at least 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to SEQ ID NO: 6
  • said B-VL comprises an amino acid sequence having at least 90% (e.g., at least 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to SEQ ID NO: 13, or
  • said B-VH comprises an amino acid sequence having at least 90% (e.g., at least 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to SEQ ID NO: 7
  • said B-VL comprises an amino acid sequence having at least 90% (e.g., at least 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to SEQ ID NO: 14, or
  • said B-VH comprises an amino acid sequence having at least 90% (e.g., at least 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to SEQ ID NO: 8
  • said B-VL comprises an amino acid sequence having at least 90% (e.g., at least 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to SEQ ID NO: 15, or
  • said B-VH comprises an amino acid sequence having at least 90% (e.g., at least 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to SEQ ID NO: 9
  • said B-VL comprises an amino acid sequence having at least 90% (e.g., at least 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to SEQ ID NO: 16, or
  • said B-VH comprises an amino acid sequence having at least 90% (e.g., at least 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to SEQ ID NO: 10
  • said B-VL comprises an amino acid sequence having at least 90% (e.g., at least 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to SEQ ID NO: 17, or
  • said B-VH comprises an amino acid sequence having at least 90% (e.g., at least 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to SEQ ID NO: 11
  • said B-VL comprises an amino acid sequence having at least 90% (e.g., at least 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to SEQ ID NO: 2;
  • the B-VH comprises the amino acid sequence of SEQ ID NO: 5, and the B-VL comprises the amino acid sequence of SEQ ID NO: 12; or the B-VH comprises the amino acid sequence of SEQ ID NO: 6 Amino acid sequence, and the B-VL comprises the amino acid sequence of SEQ ID NO: 13; or the B-VH comprises the amino acid sequence of SEQ ID NO: 7, and the B-VL comprises the amino acid sequence of SEQ ID NO: 14 or the B-VH comprises the amino acid sequence of SEQ ID NO: 8, and the B-VL comprises the amino acid sequence of SEQ ID NO: 15; or the B-VH comprises the amino acid sequence of SEQ ID NO: 9, and The B-VL comprises the amino acid sequence of SEQ ID NO: 16; or the
  • the antigen-binding molecule according to any one of the above, wherein the antigen-binding moiety 1 that specifically binds to BAFF is scFv.
  • the scFv molecule has the following structure from N-terminus to C-terminus: VL-linker-VH or VH-linker-VL.
  • the linker is a peptide linker having the structure "L 1 -(GGGGS)nL 2 ", wherein L 1 is a bond, A, GS, GGS or GGGS, and n is 0, 1, 2 , 3, 4, 5, 6, 7, 8, 9 or 10, L2 is a bond, G, GG, GGG or GGGG, and the peptide linker is not a bond.
  • the peptide linker is 3-15 amino acid residues in length.
  • the linker structure is: (G x S) y , wherein x is selected from an integer of 1-5 (eg, 1, 2, 3, 4 or 5), and y is selected from an integer of 1-6 (eg, 1, 2, 3, 4, 5, or 6).
  • the linker is GGGGSGGGGSGGGGS (SEQ ID NO: 70).
  • the P-HCDR1, P-HCDR2, P-HCDR3, P-LCDR1, P-LCDR2, P-LCDR3, Pv-HCDR1, Pv-HCDR2, Pv-HCDR3, Pv-LCDR1, Pv- LCDR2 and Pv-LCDR3 are defined according to the Kabat numbering convention.
  • the P-HCDR1, P-HCDR2, P-HCDR3, P-LCDR1, P-LCDR2, P-LCDR3, Pv-HCDR1, Pv-HCDR2, Pv-HCDR3, Pv-LCDR1, Pv- LCDR2 and PV-LCDR3 are defined according to the IMGT numbering rules.
  • the P-HCDR1, P-HCDR2, P-HCDR3, P-LCDR1, P-LCDR2, P-LCDR3, Pv-HCDR1, Pv-HCDR2, Pv-HCDR3, Pv-LCDR1, Pv- LCDR2 and Pv-LCDR3 are defined according to the Chothia numbering convention.
  • the P-HCDR1, P-HCDR2, P-HCDR3, P-LCDR1, P-LCDR2, P-LCDR3, Pv-HCDR1, Pv-HCDR2, Pv-HCDR3, Pv-LCDR1, Pv- LCDR2 and Pv-LCDR3 are defined according to the AbM numbering convention.
  • the P-HCDR1, P-HCDR2, P-HCDR3, P-LCDR1, P-LCDR2, P-LCDR3, Pv-HCDR1, Pv-HCDR2, Pv-HCDR3, Pv-LCDR1, Pv- LCDR2 and PV-LCDR3 are defined according to the Contact Numbering Rules.
  • the antigen-binding molecule according to any one of the above, said P-HCDR1, P-HCDR2, P-HCDR3, P-LCDR1, P-LCDR2, P-LCDR3 are defined according to the Kabat numbering rules,
  • the P-HCDR1 comprises the amino acid sequence of SEQ ID NO: 53
  • the P-HCDR2 comprises the amino acid sequence of SEQ ID NO: 54
  • the P-HCDR3 comprises the amino acid sequence of SEQ ID NO: 55
  • the P-LCDR1 Comprising the amino acid sequence of SEQ ID NO: 56
  • the P-LCDR2 comprising the amino acid sequence of SEQ ID NO: 57
  • the P-LCDR3 comprising the amino acid sequence of SEQ ID NO: 58.
  • said heavy chain variable region P-VH comprises at least 90% (e.g., at least 90%, 95%, 96%, 97%, 98% or 99%) sequence identity of the amino acid sequence of the light chain variable region P-VL comprising at least 90% (such as at least 90%, 95%, 96%, 97%) with SEQ ID NO: 52 %, 98%, or 99%) sequence identity of amino acid sequences.
  • the antigen-binding molecule according to any one of the above, the heavy chain variable region P-VH comprises the amino acid sequence of SEQ ID NO: 51, and the light chain variable region P-VL comprises SEQ ID NO: Amino acid sequence of 52.
  • the antigen-binding molecule of any one of the above which comprises a heavy chain constant region CH and a light chain constant region CL.
  • the heavy chain constant region CH is a human IgG heavy chain constant region
  • the light chain constant region CL is a human lambda or kappa light chain constant region.
  • the heavy chain constant region CH is a human IgG1 heavy chain constant region
  • the light chain constant region CL is a human lambda light chain constant region.
  • the heavy chain constant region CH is a human IgG1 heavy chain constant region
  • the light chain constant region CL is a human lambda light chain constant region.
  • the heavy chain constant region CH is a human IgG1 heavy chain constant region
  • the light chain constant region CL is a human kappa light chain constant region.
  • the heavy chain constant region CH comprises the amino acid sequence of SEQ ID NO:59
  • the light chain constant region CL comprises the amino acid sequence of SEQ ID NO:60.
  • the Fc region comprises a first subunit Fc1 and a second subunit Fc2 capable of associating with each other.
  • the Fc region is an IgG Fc region.
  • the Fc region is an IgG 1 Fc region.
  • the Fc region has one or more amino acid substitutions that reduce homodimerization; and/or the Fc region has one or more amino acids that reduce binding of the Fc region to an Fc receptor replace.
  • the Fc region has YTE mutations (M252Y, S254T, and T256E), L234A, L235A mutations, and/or S228P mutations, and the numbering of the mutations is according to the EU index.
  • the Fc region comprises a first subunit and a second subunit capable of associating with each other, the first subunit and/or the second subunit having one or more amino acids that reduce homodimerization replace.
  • the first subunit has a raised structure according to the pestle and socket technique
  • the second subunit has a pore structure according to the pestle and socket technique
  • the first subunit has a pore structure according to the pestle and socket technique
  • the second subunit has a raised structure according to the pestle and socket technique.
  • the first subunit has one or more amino acid substitutions at positions selected from 354, 356, 358, and 366
  • the second subunit has positions selected from 349, 356, 358, 366, One or more amino acid substitutions at positions 368 and 407.
  • the second subunit has one or more amino acid substitutions at positions selected from 354, 356, 358, and 366
  • the first subunit has positions selected from 349, 356, 358, 366, One or more amino acid substitutions at positions 368 and 407.
  • the first subunit has one or more amino acid substitutions selected from 354C, 356E, 358M, and 366W
  • the second subunit has one or more amino acid substitutions selected from 349C, 356E, 358M, 366S, 368A, and 407V One or more amino acid substitutions.
  • the second subunit has one or more amino acid substitutions selected from 354C, 356E, 358M, and 366W
  • the first subunit has one or more amino acid substitutions selected from 349C, 356E, 358M, 366S, 368A, and 407V
  • the first subunit includes amino acid substitutions of 354C, 356E, 358M, and 366W
  • the second subunit includes amino acid substitutions of 349C, 356E, 358M, 366S, 368A, and 407V.
  • the second subunit includes amino acid substitutions of 354C, 356E, 358M, and 366W and the first subunit includes amino acid substitutions of 349C, 356E, 358M, 366S, 368A, and 407V.
  • the antigen-binding molecule is a scFv
  • the antigen-binding module 2 is a scFv comprising a heavy chain variable region P-VH, a light chain variable region P- Full-length antibody of VL, heavy chain constant region CH and light chain constant region CL
  • the antigen-binding module 1 is fused directly or through a linker to the N-terminal of the variable region of the antigen-binding module 2 or the constant region of the antigen-binding module 2
  • the N-terminus of the scFv is directly or through a linker fused to the C-terminus of the heavy chain constant region CH of the antigen binding module 2; in some embodiments, the C-terminus of the scFv is directly Or fused to the N-terminus of the heavy chain variable region P-VH of the antigen-binding module 2 through a linker.
  • the antigen-binding molecule according to any one of the above, said antigen-binding molecule comprising a first chain having a structure represented by formula (a) and a second chain having a structure represented by formula (b); or Comprising a first chain having a structure shown in formula (c) and a second chain having a structure shown in formula (b), wherein,
  • linker 1 and linker 2 are the same or different peptide linkers.
  • the peptide linker independently has the structure L 1 -(GGGGS)nL 2 , wherein L 1 is a bond, A, GS, GGS or GGGS, and n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, L2 is a bond, G, GG, GGG or GGGG, and the peptide linker is not a bond.
  • the peptide linker is 3-15 amino acid residues in length.
  • the linker 1 and the linker 2 are each independently selected from a linker of formula (d), wherein formula (d): (G x S) y , wherein x is selected from an integer of 1-5 ( For example 1, 2, 3, 4 or 5), y is selected from an integer of 1-6 (for example, 1, 2, 3, 4, 5 or 6).
  • the linker 1 is selected from: GGGS (SEQ ID NO: 69)
  • the linker 2 is selected from: GGGGSGGGGSGGGGS (SEQ ID NO: 70).
  • the antigen-binding molecule wherein the first chain comprises at least 90% (eg, at least 90%, 95%, 96%, 97%, 98%, or 99%) of SEQ ID NO: 61 %) sequence identity, and said second strand comprises at least 90% (e.g., at least 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to SEQ ID NO: 50 or said first strand comprises an amino acid sequence having at least 90% (e.g., at least 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to SEQ ID NO: 62, and The second strand comprises an amino acid sequence having at least 90% (eg, at least 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO:50.
  • the antigen binding molecule has a first strand comprising the amino acid sequence of SEQ ID NO:61 and a second strand comprising the amino acid sequence of SEQ ID NO:50, or comprising the amino acids of SEQ ID NO:62 A first strand of the sequence and a second strand comprising the amino acid sequence of SEQ ID NO:50.
  • the antigen binding molecule has:
  • the present disclosure provides an antigen-binding molecule that specifically binds to a BAFF antigen, comprising a heavy chain variable region B-VH and a light chain variable region B-VL, the B-VH comprising B-HCDR1, B-HCDR2 and B-HCDR3, the B-VL comprises B-LCDR1, B-LCDR2 and B-LCDR3, wherein: the B-HCDR1, B-HCDR2 and B-HCDR3 comprise SEQ ID NO:63 respectively The amino acid sequence of Bv-HCDR1, Bv-HCDR2 and Bv-HCDR3, and the B-LCDR1, B-LCDR2 and B-LCDR3 respectively comprise the Bv-LCDR1, Bv-LCDR2 and Bv-LCDR3 in SEQ ID NO:64 Amino acid sequence, wherein, SEQ ID NO: 63 is:
  • SEQ ID NO: 64 is:
  • X1 is selected from S, A , V or G
  • X2 is selected from A , I, M or S
  • X3 is selected from T or G
  • X4 is selected from Q
  • X5 is selected from N
  • X 6 is selected from L or P
  • X 7 is selected from H
  • X 8 is selected from H or D
  • X 9 is selected from A or G
  • X 10 is selected from S or L
  • X 11 selected from P or S
  • X 12 selected from Q or H
  • X 13 selected from D
  • a or N X 14 selected from S or I
  • X 15 selected from K, R or T
  • X 16 selected from S, W, T or D
  • X 17 is selected from H, Y or S
  • X 18 is selected from Y or R
  • X 19 is selected from G or S
  • X 20 is selected from A or D
  • X 21 is selected from E or S
  • the B-HCDR1, B-HCDR2, B-HCDR3, B-LCDR1, B-LCDR2, B-LCDR3, Bv-HCDR1, Bv-HCDR2, Bv-HCDR3, Bv-LCDR1, Bv- LCDR2 and Bv-LCDR3 are defined according to the same numbering convention selected from Kabat, IMGT, Chothia, AbM and Contact.
  • the B-HCDR1, B-HCDR2, B-HCDR3, B-LCDR1, B-LCDR2, B-LCDR3, Bv-HCDR1, Bv-HCDR2, Bv-HCDR3, Bv-LCDR1, Bv- LCDR2 and Bv-LCDR3 are defined according to the Kabat numbering convention.
  • the B-HCDR1, B-HCDR2, B-HCDR3, B-LCDR1, B-LCDR2, B-LCDR3, Bv-HCDR1, Bv-HCDR2, Bv-HCDR3, Bv-LCDR1, Bv- LCDR2 and Bv-LCDR3 are defined according to the IMGT numbering rules.
  • the B-HCDR1, B-HCDR2, B-HCDR3, B-LCDR1, B-LCDR2, B-LCDR3, Bv-HCDR1, Bv-HCDR2, Bv-HCDR3, Bv-LCDR1, Bv- LCDR2 and Bv-LCDR3 are defined according to the Chothia numbering convention.
  • the B-HCDR1, B-HCDR2, B-HCDR3, B-LCDR1, B-LCDR2, B-LCDR3, Bv-HCDR1, Bv-HCDR2, Bv-HCDR3, Bv-LCDR1, Bv- LCDR2 and Bv-LCDR3 are defined according to the AbM numbering convention.
  • the B-HCDR1, B-HCDR2, B-HCDR3, B-LCDR1, B-LCDR2, B-LCDR3, Bv-HCDR1, Bv-HCDR2, Bv-HCDR3, Bv-LCDR1, Bv- LCDR2 and Bv-LCDR3 are defined according to the Contact Numbering Rules.
  • the B-HCDR1, B-HCDR2, B-HCDR3, B-LCDR1, B-LCDR2, B-LCDR3 are defined according to the Kabat numbering rules, wherein the B-HCDR1 comprises the amino acid sequence NNAIN( SEQ ID NO: 18), B-HCDR2 comprises the amino acid sequence X 1 IX 2 PMFGX 3 AKYSX 4 X 5 FQG (SEQ ID NO: 65), and B-HCDR3 comprises the amino acid sequence SRDX 6 LLFPX 7 X 8 X 9 LX 10 X 11 (SEQ ID NO: 66), B-LCDR1 comprises the amino acid sequence X 12 GX 13 X 14 LX 15 X 16 X 17 X 18 AS (SEQ ID NO: 67), B-LCDR2 comprises the amino acid sequence GKNNRPS (SEQ ID NO: 32 ) and B-LCDR3 comprising the amino acid sequence X 19 SRX 20 X 21 X 22 GX 23 X 24 WX 25 (SEQ ID
  • the antigen-binding molecule that specifically binds to a BAFF antigen as described in any one of the above, wherein: (i) said B-HCDR1, B-HCDR2 and B-HCDR3 respectively comprise Bv in SEQ ID NO:47 - the amino acid sequence of HCDR1, Bv-HCDR2 and Bv-HCDR3, and said B-LCDR1, B-LCDR2 and B-LCDR3 respectively comprise the amino acids of Bv-LCDR1, Bv-LCDR2 and Bv-LCDR3 in SEQ ID NO: 48 sequence, or (ii) said B-HCDR1, B-HCDR2 and B-HCDR3 respectively comprise the amino acid sequences of Bv-HCDR1, Bv-HCDR2 and Bv-HCDR3 in SEQ ID NO: 5, and said B-LCDR1, B-LCDR2 and B-LCDR3 respectively comprise the amino acid sequences of Bv-LCDR1, Bv-LCDR2 and Bv-LCDR3 in SEQ ID NO: 12, or (i) said B-
  • the B-HCDR1, B-HCDR2, B-HCDR3, B-LCDR1, B-LCDR2, B-LCDR3, Bv-HCDR1, Bv-HCDR2, Bv-HCDR3, Bv-LCDR1, Bv- LCDR2 and Bv-LCDR3 are defined according to the Kabat numbering convention.
  • the B-HCDR1, B-HCDR2, B-HCDR3, B-LCDR1, B-LCDR2, B-LCDR3, Bv-HCDR1, Bv-HCDR2, Bv-HCDR3, Bv-LCDR1, Bv- LCDR2 and Bv-LCDR3 are defined according to the IMGT numbering rules.
  • the B-HCDR1, B-HCDR2, B-HCDR3, B-LCDR1, B-LCDR2, B-LCDR3, Bv-HCDR1, Bv-HCDR2, Bv-HCDR3, Bv-LCDR1, Bv- LCDR2 and Bv-LCDR3 are defined according to the Chothia numbering convention.
  • the B-HCDR1, B-HCDR2, B-HCDR3, B-LCDR1, B-LCDR2, B-LCDR3, Bv-HCDR1, Bv-HCDR2, Bv-HCDR3, Bv-LCDR1, Bv- LCDR2 and Bv-LCDR3 are defined according to the AbM numbering convention.
  • the B-HCDR1, B-HCDR2, B-HCDR3, B-LCDR1, B-LCDR2, B-LCDR3, Bv-HCDR1, Bv-HCDR2, Bv-HCDR3, Bv-LCDR1, Bv- LCDR2 and Bv-LCDR3 are defined according to the Contact Numbering Rules.
  • the B-HCDR1, B-HCDR2, B-HCDR3, B-LCDR1, B-LCDR2, B-LCDR3 are defined according to the Kabat numbering rules, wherein (i) the B-HCDR1 comprising the amino acid sequence of SEQ ID NO: 18, the B-HCDR2 comprising the amino acid sequence of SEQ ID NO: 28, the B-HCDR3 comprising the amino acid sequence of SEQ ID NO: 29, and the B-LCDR1 comprising the amino acid sequence of SEQ ID NO : the amino acid sequence of 43, the B-LCDR2 comprises the amino acid sequence of SEQ ID NO: 32, and the B-LCDR3 comprises the amino acid sequence of SEQ ID NO: 44; or (ii) the B-HCDR1 comprises the amino acid sequence of SEQ ID NO : the amino acid sequence of 18, the B-HCDR2 comprises the amino acid sequence of SEQ ID NO: 21, the B-HCDR3 comprises the amino acid sequence of SEQ ID NO: 20, and the B-LCDR1 comprises the amino acid sequence
  • the antigen-binding molecule that specifically binds to a BAFF antigen according to any one of the above, wherein the B-VH comprises an antigen-binding molecule having at least 90% (e.g., at least 90%, 95%, 96%) of SEQ ID NO: 63 %, 97%, 98%, or 99%) sequence identity
  • said B-VL comprises an amino acid sequence having at least 90% (e.g., at least 90%, 95%, 96%, 97%, 98%) sequence identity to SEQ ID NO: 64 % or 99%) sequence identity of the amino acid sequence, wherein SEQ ID NO: 63 is:
  • SEQ ID NO: 64 is:
  • the antigen-binding molecule that specifically binds to a BAFF antigen as described in any one of the above,
  • said B-VH comprises an amino acid sequence having at least 90% (e.g., at least 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to SEQ ID NO: 47
  • said B-VL comprises an amino acid sequence having at least 90% (e.g., at least 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to SEQ ID NO: 48, or
  • said B-VH comprises an amino acid sequence having at least 90% (e.g., at least 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to SEQ ID NO: 5
  • said B-VL comprises an amino acid sequence having at least 90% (e.g., at least 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to SEQ ID NO: 12, or
  • said B-VH comprises an amino acid sequence having at least 90% (e.g., at least 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to SEQ ID NO: 6
  • said B-VL comprises an amino acid sequence having at least 90% (e.g., at least 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to SEQ ID NO: 13, or
  • said B-VH comprises an amino acid sequence having at least 90% (e.g., at least 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to SEQ ID NO: 7
  • said B-VL comprises an amino acid sequence having at least 90% (e.g., at least 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to SEQ ID NO: 14, or
  • said B-VH comprises an amino acid sequence having at least 90% (e.g., at least 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to SEQ ID NO: 8
  • said B-VL comprises an amino acid sequence having at least 90% (e.g., at least 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to SEQ ID NO: 15, or
  • said B-VH comprises an amino acid sequence having at least 90% (e.g., at least 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to SEQ ID NO: 9
  • said B-VL comprises an amino acid sequence having at least 90% (e.g., at least 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to SEQ ID NO: 16, or
  • said B-VH comprises an amino acid sequence having at least 90% (e.g., at least 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to SEQ ID NO: 10
  • said B-VL comprises an amino acid sequence having at least 90% (e.g., at least 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to SEQ ID NO: 17, or
  • said B-VH comprises an amino acid sequence having at least 90% (e.g., at least 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to SEQ ID NO: 11
  • said B-VL comprises an amino acid sequence having at least 90% (e.g., at least 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to SEQ ID NO: 2;
  • the antigen-binding molecule that specifically binds to the BAFF antigen as described in any one of the above comprises the amino acid sequence of SEQ ID NO: 47 and the B-VL comprises the amino acid of SEQ ID NO: 48 sequence, or the B-VH comprises the amino acid sequence of SEQ ID NO: 5, and the B-VL comprises the amino acid sequence of SEQ ID NO: 12; or the B-VH comprises the amino acid sequence of SEQ ID NO: 6, And the B-VL comprises the amino acid sequence of SEQ ID NO: 13; or the B-VH comprises the amino acid sequence of SEQ ID NO: 7, and the B-VL comprises the amino acid sequence of SEQ ID NO: 14; or the The B-VH comprises the amino acid sequence of SEQ ID NO:8, and the B-VL comprises the amino acid sequence of SEQ ID NO:15; or the B-VH comprises the amino acid sequence of SEQ ID NO:9, and the B-VL comprises the amino acid sequence of SEQ ID NO:9
  • the antigen-binding molecule that specifically binds to a BAFF antigen as described in any one of the above includes a heavy chain constant region CH and a light chain constant region CL.
  • the heavy chain constant region CH is a human IgG heavy chain constant region
  • the light chain constant region CL is a human lambda or kappa light chain constant region.
  • the heavy chain constant region CH is a human IgG1 heavy chain constant region
  • the light chain constant region CL is a human lambda light chain constant region.
  • the heavy chain constant region CH is a human IgG1 heavy chain constant region
  • the light chain constant region CL is a human kappa light chain constant region.
  • the heavy chain constant region CH comprises the amino acid sequence of SEQ ID NO:45
  • the light chain constant region CL comprises the amino acid sequence of SEQ ID NO:46.
  • the antigen-binding molecule that specifically binds a BAFF antigen is a full-length antibody.
  • the heavy chain constant region of the antigen-binding molecule that specifically binds a BAFF antigen has one or more amino acid substitutions that reduce the binding of its Fc region to an Fc receptor.
  • the Fc region has YTE mutations (M252Y, S254T, and T256E), L234A, L235A mutations, and/or S228P mutations, and the numbering of the mutations is according to the EU index.
  • an antigen-binding molecule as described in any one of the above for example, an antigen-binding molecule comprising an antigen-binding moiety 1 that specifically binds BAFF and an antigen-binding moiety 2 that specifically binds IL-12 and/or IL-23, Or an antigen-binding molecule that specifically binds to a BAFF antigen
  • the antigen-binding molecule has at least one of the following characteristics (for example, comprising any 1, 2, 3, 4, 5, 6 or 7 characteristics of the following A-G):
  • the antigen-binding molecule can block the binding of human BAFF to human BAFF-R; preferably, the IC50 value for blocking the binding of human BAFF to human BAFF-R is less than 11 nM (for example, less than 11 nM, less than or equal to 10 nM, less than or equal to Equal to 9nM, less than or equal to 8nM, less than or equal to 7nM, less than or equal to 6nM, less than or equal to 5nM or less), the IC50 value is detected by Elisa method; in some embodiments, the IC50 value is tested according to the present disclosure Example 2 method detection;
  • the antigen-binding molecule can inhibit BAFF-induced B cell proliferation; preferably, the antigen-binding molecule can inhibit BAFF-induced B cell proliferation; , less than or equal to 0.10 nM, less than or equal to 0.09 nM, less than or equal to 0.08 nM, less than or equal to 0.07 nM, less than or equal to 0.06 nM or less) inhibits BAFF-induced B cell proliferation; in some embodiments , the IC50 value is detected according to the method of Test Example 3 of the present disclosure;
  • the antigen binding molecule can be less than 9.99E-10M (such as less than 9.99E-10M, less than or equal to 9.00E-10M, less than or equal to 8.20E-10M, less than or equal to 7.40E-10M, less than or equal to 6.60E -10M, less than or equal to 5.20E-10M, less than or equal to 4.00E-10M, less than or equal to 3.00E-10M or less) combined with human BAFF, the KD value is detected by the Biacore method; in some implementations In the method, the KD value is detected according to the method of Test Example 5 of the present disclosure;
  • the antigen-binding molecule can be combined with cynomolgus BAFF with a KD value of less than 5.00E-10M (for example, less than or equal to 4.00E-10M, less than or equal to 3.00E-10M or less), and the KD value is passed Biacore method detection; In some embodiments, the KD value is detected according to the method of Test Example 5 of the present disclosure;
  • the antigen-binding molecule can bind to mouse BAFF with a KD value of less than 2.20E-10M (for example, less than or equal to 2.00E-10M, less than or equal to 1.00E-10M or less), and the KD value is determined by the Biacore method Detection; In some embodiments, the KD value is detected according to the method of Test Example 5 of the present disclosure;
  • the antigen-binding molecule can block the binding of human BAFF to human BCMA; preferably, the IC50 value of blocking the binding of human BAFF to human BCMA is less than 0.9nM (for example, less than or equal to 0.4nM, less than or equal to 0.35nM, less than or equal to or equal to 0.31nM or less), the IC50 value is detected by Elisa method; in some embodiments, the IC50 value is detected according to the method of Test Example 2 of the present disclosure; or
  • the antigen-binding molecule can block the binding of human BAFF to human TACI; preferably, the IC50 value of blocking the binding of human BAFF to human TACI is less than 0.6nM (for example, less than or equal to 0.55nM, less than or equal to 0.4nM, less than or equal to or equal to 0.33nM, less than or equal to 0.23nM or less), the IC50 value is detected by the Elisa method; in some embodiments, the IC50 value is detected according to the method of Test Example 2 of the present disclosure.
  • the antigen-binding molecule according to any one of the above, said antigen-binding molecule comprising an antigen-binding moiety 1 that specifically binds BAFF and an antigen-binding moiety 2 that specifically binds IL-12 and/or IL-23 , which has at least one of the following characteristics (for example, including any 1, 2, 3, 4 or 5 characteristics of the following H-L):
  • the antigen-binding molecule can block the binding of human IL-12/23 p40 to human IL-12R ⁇ 1; preferably, the IC50 value for blocking the binding of human IL-12/23 p40 to human IL-12R ⁇ 1 is less than 3.6nM ( For example, less than or equal to 3.0nM, less than or equal to 2.0nM, less than or equal to 1.0nM, less than or equal to 0.5nM, less than or equal to 0.23nM or less), the IC50 value is detected by the Elisa method; in some embodiments, The IC50 value is detected according to the method of Test Example 2 of the present disclosure;
  • the antigen-binding molecule can inhibit IL-12-induced IFN ⁇ secretion; preferably, the IC50 value of the antigen-binding molecule inhibiting IL-12-induced IFN ⁇ secretion is less than 3.0nM (for example, less than or equal to 2.6nM, less than or equal to 1.6nM or less) ; In some embodiments, the IC50 value is detected according to the method of Test Example 4 of the present disclosure;
  • J. Antigen-binding molecules can inhibit IL-23-induced IL-17 secretion; preferably, the antigen-binding molecules inhibit IL-23-induced IL-17 secretion with an IC50 value of less than 0.034nM (for example, less than or equal to 0.031nM, less than or equal to 0.030nM or less); In some embodiments, the IC50 value is detected according to the method of Test Example 4 of the present disclosure;
  • the antigen-binding molecule can bind to cynomolgus monkey IL-12/23 p40 with a KD value less than 2.7E-10M (for example, less than or equal to 2.0E-10M, less than or equal to 1.0E-10M or less), said The KD value is detected by the Biacore method; in some embodiments, the KD value is detected according to the method of Test Example 5 of the present disclosure; or
  • the antigen-binding molecule can bind to human IL-23 with a KD value of less than 6.4E-11M (e.g., less than or equal to 6.3E-11M, less than or equal to 6.2E-11M or less), the KD value is determined by the Biacore method Detection; In some embodiments, the KD value is detected according to the method of Test Example 5 of the present disclosure.
  • the present disclosure provides a pharmaceutical composition
  • a pharmaceutical composition comprising: a therapeutically effective amount of any one of the above antigen-binding molecules, and one or more pharmaceutically acceptable carriers, diluents, buffers or excipient.
  • the present disclosure provides an isolated nucleic acid encoding an antigen binding molecule according to any one of the above.
  • the present disclosure provides a host cell comprising the nucleic acid of any one of the above.
  • the present disclosure provides a method for treating a disease, the method comprising the step of administering the antigen-binding molecule or the pharmaceutical composition according to any one of the above to a subject.
  • the disease is a B cell disorder or an autoimmune disease.
  • the B cell disorder or autoimmune disease is a disease or condition associated with BAFF expression.
  • the autoimmune disease is selected from the group consisting of: systemic lupus erythematosus, myasthenia gravis, multiple sclerosis, insulin-dependent diabetes mellitus, Crohn's disease, rheumatoid arthritis, polyarticular juvenile rheumatoid Arthritis and psoriatic arthritis; said B-cell disorder is selected from the group consisting of neoplasms, chronic leukaemia, multiple myeloma, non-Hodgkin's lymphoma, post-transplantation lymphoproliferative disease, and light chain gammopathies .
  • the autoimmune disease is systemic lupus erythematosus.
  • the present disclosure also provides the use of the antigen-binding molecule or the pharmaceutical composition according to any one of the above in the preparation of a medicament for treating the aforementioned diseases.
  • the present disclosure also provides an antigen-binding molecule or a pharmaceutical composition as described above for use as a medicament.
  • Figure 1 Schematic diagram of the structure of BU-1.
  • Figure 2 Schematic diagram of the structure of BU-2.
  • Figure 3 The result of the experiment of inhibiting TNF ⁇ secretion by the bispecific antibody specifically binding to BAFF and IL-12/23.
  • Figure 4 The results of the IgA secretion inhibition experiment by the bispecific antibody specifically binding to BAFF and IL-12/23.
  • Figure 5 The result of the experiment of inhibiting IL-22 secretion by the bispecific antibody specifically binding to BAFF and IL-12/23.
  • Figure 6 The result of the experiment of inhibiting the secretion of IFN ⁇ by the bispecific antibody specifically binding to BAFF and IL-12/23.
  • cytokine is a general term for proteins/polypeptides released by a population of cells that act as intercellular mediators on other cells.
  • cytokines include lymphokines, monokines, chemokines and traditional polypeptide hormones.
  • exemplary cytokines include: IL-2, IFN- ⁇ , IL-6, TNF ⁇ , IL-17, and IL-5.
  • IL-12 and/or IL-23 or “IL-12/23” all mean to cover: “IL-12”, “IL-23” and “IL-12 and IL-23” -23" each.
  • amino acid refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to naturally occurring amino acids.
  • Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, eg, hydroxyproline, gamma-carboxyglutamic acid, and O-phosphoserine.
  • Amino acid analogs are compounds that have the same basic chemical structure (i.e., the alpha carbon bonded to a hydrogen, carboxyl, amino group, and R group) as a naturally occurring amino acid, such as homoserine, norleucine, methionine sulfoxide , Methylsulfonium methionine.
  • Such analogs have modified R groups (eg, norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid.
  • An amino acid mimetic refers to a chemical compound that has a structure that differs from the general chemical structure of an amino acid, but functions in a manner similar to a naturally occurring amino acid.
  • 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 so long as the final construct possesses the desired properties, such as reduced or binding to Fc receptors.
  • Amino acid sequence deletions and insertions include deletions and insertions at the amino and/or carboxyl termini of the polypeptide chain.
  • Specific amino acid mutations may be amino acid substitutions.
  • the amino acid mutation is a non-conservative amino acid substitution, that is, replacing one amino acid with another amino acid having different structural and/or chemical properties.
  • Amino acid substitutions include substitutions with non-naturally occurring amino acids or with 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, and the like. Methods of altering amino acid side chain groups other than genetic engineering, 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 expressed in the form of position + amino acid residue, for example, 366W means that the amino acid residue at position 366 is W. T366W means that the amino acid residue at the 366th position is mutated from the original T to W.
  • antigen-binding molecule is used in the broadest sense and covers various molecules that specifically bind to an antigen, including but not limited to antibodies, other polypeptides with antigen-binding activity, and antibody fusion proteins fused thereto.
  • the antigen-binding molecules herein are bispecific antigen-binding molecules (eg, bispecific antibodies).
  • bispecific antigen binding molecule refers to an antigen binding molecule capable of specifically binding to two different antigens or at least two different epitopes of the same antigen.
  • antibody is used in the broadest sense and encompasses various 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 fragments, or antigen-binding portions) as long as they exhibit the desired antigen-binding activity.
  • Native antibody refers to a naturally occurring immunoglobulin molecule. For example, native IgG antibodies are heterotetrameric proteins of approximately 150,000 Daltons, composed of two light chains and two heavy chains joined by disulfide bonds.
  • each heavy chain has a variable region (VH), also known as variable heavy domain, heavy chain variable region, followed by a heavy chain constant region (CH), the native IgG heavy chain constant region is usually Contains three constant domains (CH1, CH2 and CH3).
  • VH variable heavy domain
  • CH heavy chain constant region
  • each light chain has a variable region (VL, also known as variable light domain, or light chain variable domain), followed by a constant light domain (light chain constant region, CL ).
  • VH variable heavy domain
  • CH2 and CH3 heavy chain constant region
  • CL constant light domain
  • full-length antibody “intact antibody” and “whole antibody” are used interchangeably herein to refer to an antibody having a structure substantially similar to that of a native antibody or having a heavy chain with an Fc region as defined herein.
  • Natural complete antibody light chain includes light chain variable region VL and constant region CL, VL is at the amino terminal of light chain, light chain constant region includes ⁇ chain and ⁇ chain; heavy chain includes variable region VH and constant region (CH1, CH2 and CH3), VH is at the amino-terminus (also called N-terminus) of the heavy chain, and the constant region is at the carboxy-terminus (also called C-terminus), where CH3 is closest to the carboxy-terminus of the polypeptide, and the heavy chain can belong to any isotype, including IgG (including IgG1, IgG2, IgG3 and IgG4 subtypes), IgA (including IgA1 and IgA2 subtypes), IgM and IgE.
  • IgG including IgG1, IgG2, IgG3 and IgG4 subtypes
  • IgA including IgA1 and IgA2 subtypes
  • IgM and IgE IgE.
  • bispecific antibody refers to an antibody (including an antibody or an antigen-binding fragment thereof, such as a single-chain antibody) capable of specifically binding to two different antigens or two different epitopes of the same antigen.
  • Bispecific antibodies of various structures have been disclosed in the prior art. According to the integrity of the IgG molecule, it can be divided into IgG-like bispecific antibodies and antibody fragment bispecific antibodies. According to the number of antigen-binding regions, bispecific antibodies can be divided into bivalent, trivalent, tetravalent or more valent. According to whether the structure is symmetrical, it can be divided into symmetrical structure bispecific antibody and asymmetric structure bispecific antibody.
  • fragment-type bispecific antibodies such as Fab fragments lacking Fc fragments, which form bispecific antibodies by combining two or more Fab fragments in one molecule, have low immunogenicity and small molecular weight , with high tumor tissue permeability, typical antibody structures of this type such as F(ab) 2 , scFv-Fab, (scFv) 2 -Fab, etc.
  • IgG-like bispecific antibody for example, with Fc fragment
  • this type of antibody has a relatively large molecular weight, and the Fc fragment helps to purify the antibody and improve its solubility and stability.
  • the Fc part may also bind to the receptor FcRn, Increase antibody serum half-life.
  • bispecific antibodies such as KiH, CrossMAb, Triomab quadroma, Fc ⁇ Adp, ART-Ig, BiMAb, Biclonics, BEAT, DuoBody, Azymetric, XmAb, 2:1 TCBs, 1Fab-IgG TDB, FynomAb, two-in- one/DAF, scFv-Fab-IgG, DART-Fc, LP-DART, CODV-Fab-TL, HLE-BiTE, F(ab)2-CrossMAb, IgG-(scFv)2, Bs4Ab, DVD-Ig, Tetravalent - Bispecific antibodies such as DART-Fc, (scFv)4-Fc, CODV-Ig, mAb2, F(ab)4-CrossMAb (see Aran F. Labrijn et al., Nature Reviews Drug Discovery volume 18, pages 585–608 (2019 ); Chen S1 et al., J
  • variable region refers to the antigen-binding domain of an antigen-binding molecule (eg, an antibody).
  • the heavy chain variable region VH and the light chain variable region VL each comprise four conserved framework regions (FRs) and three complementarity determining regions (CDRs).
  • FRs conserved framework regions
  • CDRs complementarity determining regions
  • the heavy chain variable region in the antigen-binding module that specifically binds BAFF is marked as B-VH
  • the light chain variable region is marked as B-VL
  • the antigen-binding module that specifically binds IL-12/23 p40 The heavy chain variable region is denoted as P-VH and the light chain variable region as P-VL.
  • CDR complementarity determining region
  • frame or “FR” refers to the variable domain residues other than the CDR residues.
  • VH contains 3 CDR regions: HCDR1, HCDR2 and HCDR3;
  • VL contains 3 CDR regions: LCDR1, LCDR2 and LCDR3.
  • Each VH and VL consists of three CDRs and four FRs arranged in the following order from the amino terminus (also known as the N terminus) to the carboxyl terminus (also known as the C terminus): FR1, CDR1, FR2, CDR2, FR3, CDR3 , FR4.
  • Each VH and VL consists of three CDRs and four FRs arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • a single VH or VL may be sufficient to confer antigen binding specificity.
  • the three CDR regions in B-VH are respectively marked as B-HCDR1, B-HCDR2 and B-HCDR3, or Bv-HCDR1, Bv-HCDR2 and Bv-HCDR3; the three CDR regions in B-VL Labeled as B-LCDR1, B-LCDR2, and B-LCDR3, or Bv-LCDR1, Bv-LCDR2, and Bv-LCDR3, respectively.
  • the three CDR regions in P-VH are respectively marked as P-HCDR1, P-HCDR2 and P-HCDR3, or Pv-HCDR1, Pv-HCDR2 and Pv-HCDR3; the three CDR regions in P-VL are respectively marked as P-LCDR1, P-LCDR2, and P-LCDR3, or Pv-LCDR1, Pv-LCDR2, and Pv-LCDR3.
  • amino acid sequence boundaries of CDRs can be determined by various known schemes, for example: “Kabat” numbering convention (see Kabat et al. (1991), “Sequences of Proteins of Immunological Interest", 5th Edition, Public Health Service, National Institutes of Health , Bethesda, MD), “Chothia” numbering sequence, “ABM” numbering sequence, "contact” numbering sequence (see Martin, ACR. Protein Sequence and Structure Analysis of Antibody Variable Domains [J].
  • the Kabat numbering convention is used for variable region and CDR sequences in this disclosure. Although in specific embodiments, the Kabat numbering rules are used to define amino acid residues, the corresponding technical solutions in other numbering systems will be regarded as equivalent technical solutions.
  • antibody fragment refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that retains the antigen-binding ability of the intact antibody.
  • antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab') 2 , single domain antibody, single chain Fab (scFab), diabody, linear antibody, single chain antibody (scFv) , and multispecific antibodies formed from antibody fragments.
  • Fc region or “fragment crystallizable region” is used to define the C-terminal region of an antibody heavy chain, including native and engineered Fc regions.
  • the Fc region comprises the same or different two subunits.
  • 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 carboxyl terminus.
  • Suitable Fc regions for use in the antibodies described herein include the Fc regions of human IgGl, IgG2 (IgG2A, IgG2B), IgG3 and IgG4.
  • the boundaries of the Fc region can 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 (residue 447 according to the EU numbering system). system residues 446 and 447).
  • the numbering convention for the Fc region is the EU numbering system, also known as the EU index.
  • chimeric antibody refers to an antibody in which a portion of the heavy and/or light chains is from a particular source or species and the remaining portion of the heavy and/or light chains is from another, different source or species.
  • humanized antibody is an antibody that retains the reactivity of a non-human antibody while being less immunogenic in humans. Humanization can be achieved, for example, by retaining the non-human CDR regions and replacing the remainder of the antibody with their human counterparts (ie, the constant regions and the framework portion of the variable regions).
  • human antibody “human antibody”, “fully human antibody”, and “fully human antibody” are used interchangeably to refer to antibodies whose variable and constant regions are human sequences.
  • the term encompasses antibodies that are derived from human genes but have, for example, altered sequences that reduce potential immunogenicity, increase affinity, eliminate cysteines or glycosylation sites that might cause undesired folding.
  • the term encompasses such antibodies produced recombinantly in non-human cells which may confer glycosylation not characteristic of human cells.
  • the term also encompasses antibodies that have been raised in transgenic mice containing some or all of the immunoglobulin heavy and light chain loci.
  • the meaning of human antibody expressly excludes humanized antibodies comprising non-human antigen-binding residues.
  • affinity refers to the overall strength of the non-covalent interaction between a single binding site of a molecule (eg, an antigen-binding molecule of the disclosure) and its binding partner (eg, an antigen).
  • binding affinity refers to internal binding affinity, which reflects a 1:1 interaction between members of a binding pair (eg, antibody and antigen).
  • KD dissociation constant
  • the term “kassoc” or “ka” refers to the on-rate of a particular antibody-antigen interaction and the term “kdis” or “kd” refers to the dissociation rate of a particular antibody-antigen interaction.
  • KD refers to the dissociation constant, which is obtained from the ratio of kd to ka (ie, kd/ka) and is expressed as molarity (M).
  • M molarity
  • the KD value of an antibody can be determined using methods well known in the art. For example, surface plasmon resonance is measured using biosensing systems such as the system, or affinity in solution is measured by solution equilibrium titration (SET). In some embodiments, the KD value is detected by Biacore.
  • effector function refers to those biological activities attributable to an antibody Fc region (either native sequence Fc region or amino acid sequence mutated Fc region) and which 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.
  • the term "monoclonal antibody” refers to a population of antibodies or members thereof that are substantially homogeneous, ie, the amino acid sequences of the antibody molecules comprised in the population are identical except for natural mutations that may be present in minor amounts.
  • polyclonal antibody preparations typically comprise multiple different antibodies with different amino acid sequences in their variable domains, often specific for different epitopes.
  • “Monoclonal” denotes the characteristics of an antibody obtained from a substantially homogeneous population of antibodies and should not be construed as requiring that the antibody be produced by any particular method.
  • the antibodies provided by the present disclosure are monoclonal antibodies.
  • antigen refers to a molecule or portion of a molecule capable of being selectively bound by an antigen-binding molecule (eg, an antibody).
  • An antigen may have one or more epitopes capable of interacting with different antigen-binding molecules (eg, antibodies).
  • epitope refers to an area (area or region) on an antigen capable of specifically binding to an antibody or antigen-binding fragment thereof.
  • An epitope may be formed from contiguous amino acids (linear epitope) or comprise non-contiguous amino acids (conformational epitope), such that non-contiguous amino acids are brought into spatial proximity by folding of the antigen, ie by tertiary folding of the antigen.
  • the difference between a conformational epitope and a linear epitope is that antibody binding to a conformational epitope is lost in the presence of denaturing solvents.
  • An epitope comprises at least 3, at least 4, at least 5, at least 6, at least 7, or 8-10 amino acids in a unique spatial conformation.
  • Antibodies that bind a particular epitope can be screened using methods routine in the art, such as, but not limited to, alanine scanning, peptide blotting, peptide cleavage analysis, epitope excision, epitope extraction, antigen Chemical modification of (see Prot. Sci.9 (2000) 487-496), and cross-blocking.
  • an antigen-binding molecule eg, an antibody
  • an antibody binds an antigen or an epitope thereof with an equilibrium dissociation constant (KD) of about 1 ⁇ 10 ⁇ 7 M or less (eg, about 1 ⁇ 10 ⁇ 8 M or less).
  • KD equilibrium dissociation constant
  • the antibody binds an antigen with a KD that is 10% or less (eg, 1%) of the antibody's KD for binding to a non-specific antigen (eg, BSA, casein).
  • KD can be measured using known methods, for example by measured by surface plasmon resonance.
  • an antibody that specifically binds to an antigen or an epitope thereof does not exclude cross-reactivity to other related antigens, e.g.
  • the corresponding antigens of cynomolgus, cyno), chimpanzee (Pan troglodytes) (chimpanzee, chimp)) or marmoset (Callithrix jacchus) (commonmarmoset, marmoset) are cross-reactive.
  • antigen binding moiety refers to a polypeptide molecule that specifically binds an antigen of interest.
  • Antigen binding moieties include antibodies and fragments thereof as otherwise defined herein.
  • the antigen binding moiety comprises an antigen binding domain of an antibody comprising an antibody heavy chain variable region and an antibody light chain variable region.
  • antigen-binding moiety that specifically binds BAFF refers to an antigen-binding moiety that is capable of binding BAFF with sufficient affinity.
  • the antigen binding moiety that specifically binds to human BAFF has an equilibrium dissociation constant (KD) of less than 10.00E-10M (e.g., less than 9.99E-10M, less than or equal to 9.00E-10M, less than or equal to 8.20E-10M, less than or equal to 7.40E-10M, less than or equal to 6.60E-10M, less than or equal to 5.20E-10M, less than or equal to 4.00E-10M, less than or equal to 3.00E-10M or less), which is measured by the Biacore method.
  • KD equilibrium dissociation constant
  • an antigen binding moiety that specifically binds BAFF binds a conserved epitope in BAFF from a different species.
  • antigen-binding moiety that specifically binds IL-12/23 refers to an antigen-binding moiety capable of binding IL-12/23 with sufficient affinity.
  • the antigen binding moiety that specifically binds IL-12/23 has an equilibrium dissociation constant (KD) of less than 6.4E-11M (e.g., less than or equal to 6.3E-11M, less than or equal to 6.2E -11M or less) for binding to human IL-23, which was detected by the Biacore method.
  • the antigen binding moiety that specifically binds IL-12/23 binds a conserved epitope in IL-12/23 from a different species.
  • Antigen binding moieties include antibody fragments as defined herein, eg Fab or scFv.
  • linker refers to a linking unit that joins two polypeptide fragments.
  • linkers appearing in the same structure may be the same or different.
  • the linker may be a peptide linker comprising one or more amino acids, typically about 1-30, 2-24 or 3-15 amino acids.
  • the linkers used herein may be the same or different.
  • “-" appears in the structural formula it means that the units on both sides are directly connected by covalent bonds.
  • bond appears in a structural unit, it means that the unit has no amino acids, and the units on either side of the unit are directly connected.
  • antibody-dependent cellular cytotoxicity is mechanisms for inducing cell death that rely on the interaction of antibody-coated target cells with lytically active effector cells (such as natural killer cells (NK), monocytes, macrophages, and neutrophils) via Fc ⁇ receptors (Fc ⁇ Rs) expressed on effector cells.
  • lytically active effector cells such as natural killer cells (NK), monocytes, macrophages, and neutrophils
  • Fc ⁇ receptors Fc ⁇ receptors
  • NK cells express FcyRIIIa
  • monocytes express FcyRI, FcyRII, and FcyRIIIa.
  • the ADCC activity of the antibodies provided herein can be assessed using an in vitro assay using antigen-expressing cells as target cells and NK cells as effector cells. Cell lysis is detected based on labels released from lysed cells, such as radioactive substrates, fluorescent dyes, or native intracellular proteins.
  • ADCP antibody-dependent cellular phagocytosis
  • complement-dependent cytotoxicity refers to a mechanism that induces cell death in which the Fc effector domains of bound antibodies on target cells bind and activate complement component C1q, which in turn activates the complement cascade, resulting in target cell die. Activation of complement can also result in the deposition of complement components on the surface of target cells that promote CDC by binding to complement receptors (eg, CR3) on leukocytes.
  • complement receptors eg, CR3
  • nucleic acid is used herein interchangeably 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, synthetic, naturally occurring and non-naturally occurring, having similar binding properties to the reference nucleic acid, and defined in Metabolized in a manner similar to the reference nucleotide.
  • Examples of such analogs include, but are not limited to, phosphorothioate, phosphoramidate, methylphosphonate, chiral-methylphosphonate, 2-O-methyl ribonucleotides, peptide-nucleic acid (PNA ).
  • An "isolated" nucleic acid refers to a nucleic acid molecule that has been separated from components of its natural environment.
  • An isolated nucleic acid includes a nucleic acid molecule contained in a cell that normally contains the nucleic acid molecule, but which is present extrachromosomally or at a chromosomal location other than its natural chromosomal location.
  • An isolated nucleic acid encoding an antigen-binding molecule refers to one or more nucleic acid molecules encoding an antigen-binding molecule, including such one or more nucleic acid molecules in a single vector or in separate vectors, and one or more nucleic acid molecules present in a host cell. Such one or more nucleic acid molecules at more positions.
  • nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (eg, degenerate codon substitutions) and complementary sequences as well as the explicitly indicated sequence.
  • degenerate codon substitutions can be obtained by generating sequences in which the third position of one or more selected (or all) codons is replaced by a degenerate base and/or Deoxyinosine residue substitution.
  • polypeptide and "protein” 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 the corresponding artificial chemical mimetic of a naturally occurring amino acid, and to both naturally occurring amino acid polymers and non-naturally occurring amino acid polymers. Unless otherwise stated, a particular polypeptide sequence also implicitly encompasses conservatively modified variants thereof.
  • sequence identity refers to the degree (percentage) to which the amino acids/nucleic acids of two sequences are identical at equivalent positions when the two sequences are optimally aligned; Gaps are used to obtain the maximum percent sequence identity, and any conservative substitutions are not considered part of the sequence identity.
  • alignment can be achieved by techniques known in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software. Those skilled in the art can determine suitable parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.
  • fused or “linked” refer to the joining of components (eg, B-VH and B-VL) by a covalent bond, either directly or via one or more linkers.
  • vector means a polynucleotide molecule capable of transporting another polynucleotide to which it has been linked.
  • plasmid refers to a circular double-stranded DNA loop into which additional DNA segments can be ligated.
  • viral vector such as an adeno-associated viral vector (AAV or AAV2), in which additional DNA segments can be ligated into the viral genome.
  • AAV adeno-associated viral vector
  • Certain vectors are capable of autonomous replication in the host cells into which they are introduced (eg, bacterial vectors and episomal mammalian vectors with a bacterial origin of replication).
  • vectors can integrate into the genome of the host cell after introduction into the host cell, thereby replicating along with the host genome.
  • expression vector or "expression construct” refers to a vector that can transform a host cell and contains a vector that directs and/or controls (along with the host cell) the expression of one or more heterologous coding regions operably linked thereto.
  • Expression constructs may include, but are not limited to, sequences that affect or control transcription, translation, and, when an intron is present, RNA splicing of the coding region to which it is operably linked.
  • host cell refers to a cell 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.
  • Progeny may not be identical to the parental cell in nucleic acid content, but may contain mutations.
  • mutant progeny including cells having the same function or biological activity as those screened or selected in the primary transformed cells.
  • Host cells include prokaryotic and eukaryotic host cells, where eukaryotic host cells include, but are not limited to, mammalian cells, insect cell lines, plant cells, and fungal cells.
  • Mammalian host cells include human, mouse, rat, dog, monkey, pig, goat, cow, horse, and hamster cells, including but not limited to Chinese hamster ovary (CHO) cells, NSO, SP2 cells, HeLa cells, baby hamster cells Kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (eg, Hep G2), A549 cells, 3T3 cells, and HEK-293 cells.
  • Fungal cells include yeast and filamentous fungal cells including, for example, Pichia pastoris, Pichia finlandica, Pichia trehalophila, Pichia koclamae, Pichia membranaefaciens, Pichia minuta (Ogataea minuta, Pichia lindneri), Pichia puntiae, Pichia thermotolerans, Pichia willow salictaria), Pichia guercuum, Pichia pijperi, Pichia stiptis, Pichia methanolica, Pichia, Saccharomycescerevisiae, Saccharomyces cerevisiae , Hansenula polymorpha, Kluyveromyces, Kluyveromyces lactis, Candida albicans, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Trichoderma reesei, Chrysosporium lucknowense, Fus
  • Pichia any Saccharomyces, Hansenula polymorpha, any Kluyveromyces, Candida albicans, any Aspergillus, Trichoderma reesei, Luke Mold (Chrysosporium lucknowense), any Fusarium species, Yarrowia lipolytica, and Neurospora crassa.
  • the host cell cannot develop into a plant or individual animal.
  • composition means a mixture comprising one or more antigen binding molecules described herein together with other chemical components such as physiologically/pharmaceutically acceptable carriers and excipients.
  • pharmaceutically acceptable carrier refers to an ingredient in a pharmaceutical formulation (formulation) that is different from the active ingredient and is non-toxic to the subject.
  • subject or “individual” includes humans and non-human animals.
  • Non-human animals include all vertebrates (eg, mammals and non-mammals) such as non-human primates (eg, cynomolgus monkeys), sheep, dogs, cows, chickens, amphibians, and reptiles.
  • patient or “subject” are used interchangeably herein unless otherwise indicated.
  • cyno or “cynomolgus” refers to Macaca fascicularis.
  • the individual or subject is a human.
  • administering when applied to an animal, human, experimental subject, cell, tissue, organ or biological fluid, refers to the interaction of an exogenous drug, therapeutic agent, diagnostic agent or composition with an animal, human , subjects, cells, tissues, organs or biological fluids.
  • sample refers to a collection (such as a fluid, cell, or tissue) isolated from a subject, as well as a fluid, cell, or tissue present in a subject.
  • samples are biological fluids such as blood, serum and serosal fluids, plasma, lymph, urine, saliva, cyst fluid, tears, faeces, sputum, mucous membrane secretions of secretory tissues or organs, vaginal secretions, Ascites, pleura, pericardium, peritoneum, fluids of the peritoneal cavity and other body cavities, fluid collected from bronchial lavage, synovial fluid, liquid solutions in contact with the subject or biological source, such as culture medium (including conditioned medium), perfusion Washes, etc., tissue biopsy samples, fine needle aspirations, surgically removed tissue, organ cultures, or cell cultures.
  • biological fluids such as blood, serum and serosal fluids, plasma, lymph, urine, saliva, cyst fluid, tears, faeces, sputum, mucous membrane secretions of secret
  • Treatment refers to clinical intervention applied to an individual being treated, and may be performed for prophylactic purposes, or during the course of clinical pathology. Desired effects of treatment include, but are not limited to, prevention of occurrence or recurrence of disease, alleviation of symptoms, alleviation/reduction of any direct or indirect pathological consequences of disease, prevention of metastasis, reduction of rate of disease progression, amelioration or palliation of disease state, and regression or improved prognosis .
  • the molecules of the present disclosure are used to delay the development of a disease or slow the progression of a disease.
  • an "effective amount” is generally sufficient to reduce the severity and/or frequency of symptoms, eliminate those symptoms and/or their underlying causes, prevent the occurrence of symptoms and/or their underlying causes, and/or ameliorate or ameliorate the impairment caused by or associated with the disease state amount.
  • the effective amount is a therapeutically or prophylactically effective amount.
  • a “therapeutically effective amount” is sufficient to treat a disease state or symptom, especially a state or symptom associated with the disease state, or otherwise retard, delay or reverse the progression of the disease state or any other undesirable symptom associated with the disease quantity.
  • a “prophylactically effective amount” is an amount that, when administered to a subject, will have a predetermined prophylactic effect, such as preventing or delaying the onset (or recurrence) of the disease state, or reducing the likelihood of the onset (or recurrence) of the disease state or associated symptoms .
  • Complete therapeutic or prophylactic effect does not necessarily occur after administration of one dose, but may occur after administration of a series of doses.
  • a therapeutically or prophylactically effective amount may be administered in one or more administrations.
  • “Therapeutically effective amount” and “prophylactically effective amount” can vary depending on factors such as the disease state, age, sex and weight of the individual, and the ability of the therapeutic agent or combination of therapeutic agents to elicit a desired response in the individual.
  • Exemplary indicators of an effective therapeutic agent or combination of therapeutic agents include, for example, improved health status of a patient.
  • BAFF should be broadly understood and is intended to cover various forms of molecules of BAFF in various stages of mammalian body, such as but not limited to the process of amplification, replication, transcription, splicing, processing, translation and modification of BAFF gene Produced molecules (eg, precursor BCMA, mature BAFF, membrane-expressed BAFF, BAFF splice variants, modified BAFF, or fragments thereof); the term also encompasses artificially produced or in vitro expressed BAFF.
  • IL-12 should be broadly understood and is intended to cover various forms of IL-12 molecules in various stages of mammalian body, such as but not limited to IL-12 gene in amplification, replication, transcription, splicing, processing Molecules produced during , translation, modification (e.g. precursor IL-12, mature IL-12, membrane expressed IL-12, IL-12 splice variants, modified IL-12, or fragments, subunits thereof) ; the term also encompasses artificially produced or in vitro expressed IL-12.
  • modification e.g. precursor IL-12, mature IL-12, membrane expressed IL-12, IL-12 splice variants, modified IL-12, or fragments, subunits thereof
  • IL-23 should be broadly understood and is intended to cover various forms of IL-23 molecules in various stages of mammalian body, such as but not limited to IL-23 gene in amplification, replication, transcription, splicing, processing Molecules produced during , translation, modification (e.g. precursor IL-23, mature IL-23, membrane expressed IL-23, IL-23 splice variants, modified IL-23, or fragments, subunits thereof) ; the term also encompasses artificially prepared or in vitro expressed IL-23.
  • modification e.g. precursor IL-23, mature IL-23, membrane expressed IL-23, IL-23 splice variants, modified IL-23, or fragments, subunits thereof
  • Antigen binding molecules of the present disclosure are provided.
  • the present disclosure obtains an antigen-binding molecule that specifically binds BAFF by modifying Belimumab.
  • antigen-binding molecules that specifically bind BAFF and IL-12/23 were also constructed.
  • the antigen-binding molecules of the present disclosure have many favorable properties, such as binding to antigen with high affinity, blocking the binding of BAFF to its receptors (such as BAFF-R, BCMA and/or TACI), inhibiting the induction of BAFF B cell proliferation, blocking IL-12/23 binding to its receptor, inhibiting IL-12-induced IFN ⁇ secretion, inhibiting IL-23-induced IL-17 secretion, pharmacokinetic properties and/or druggability, etc.
  • the present disclosure provides an antigen binding molecule that specifically binds BAFF, which is an anti-BAFF antibody.
  • the antibody is a full-length antibody or an antigen-binding fragment thereof (e.g., Fv, Fab, Fab', Fab'-SH, F(ab')2, single domain antibody, single chain Fab (scFab), Diabodies, linear antibodies, single chain antibodies (scFv)) having one or more of the following functional activities:
  • the antigen-binding molecule can block the binding of human BAFF to human BAFF-R; preferably, the IC50 value for blocking the binding of human BAFF to human BAFF-R is less than 11 nM (for example, less than 11 nM, less than or equal to 10 nM, less than or equal to Equal to 9nM, less than or equal to 8nM, less than or equal to 7nM, less than or equal to 6nM, less than or equal to 5nM or less), the IC50 value is detected by Elisa method; in some embodiments, the IC50 value is tested according to the present disclosure Example 2 method detection;
  • the antigen-binding molecule can inhibit BAFF-induced B cell proliferation; preferably, the antigen-binding molecule can inhibit BAFF-induced B cell proliferation; , less than or equal to 0.10 nM, less than or equal to 0.09 nM, less than or equal to 0.08 nM, less than or equal to 0.07 nM, less than or equal to 0.06 nM or less) inhibits BAFF-induced B cell proliferation; in some embodiments , the IC50 value is detected according to the method of Test Example 3 of the present disclosure;
  • the antigen binding molecule can be less than 9.99E-10M (such as less than 9.99E-10M, less than or equal to 9.00E-10M, less than or equal to 8.20E-10M, less than or equal to 7.40E-10M, less than or equal to 6.60E -10M, less than or equal to 5.20E-10M, less than or equal to 4.00E-10M, less than or equal to 3.00E-10M or less) combined with human BAFF, the KD value is detected by the Biacore method; in some implementations In the method, the KD value is detected according to the method of Test Example 5 of the present disclosure;
  • the antigen-binding molecule can be combined with cynomolgus BAFF with a KD value of less than 5.00E-10M (for example, less than or equal to 4.00E-10M, less than or equal to 3.00E-10M or less), and the KD value is passed Biacore method detection; In some embodiments, the KD value is detected according to the method of Test Example 5 of the present disclosure;
  • the antigen-binding molecule can bind to mouse BAFF with a KD value of less than 2.20E-10M (for example, less than or equal to 2.00E-10M, less than or equal to 1.00E-10M or less), and the KD value is determined by the Biacore method Detection; In some embodiments, the KD value is detected according to the method of Test Example 5 of the present disclosure;
  • the antigen-binding molecule can block the binding of human BAFF to human BCMA; preferably, the IC50 value of blocking the binding of human BAFF to human BCMA is less than 0.9nM (for example, less than or equal to 0.4nM, less than or equal to 0.35nM, less than or equal to or equal to 0.31nM or less), the IC50 value is detected by Elisa method; in some embodiments, the IC50 value is detected according to the method of Test Example 2 of the present disclosure; or
  • the antigen-binding molecule can block the binding of human BAFF to human TACI; preferably, the IC50 value of blocking the binding of human BAFF to human TACI is less than 0.6nM (for example, less than or equal to 0.55nM, less than or equal to 0.4nM, less than or equal to or equal to 0.33nM or less), the IC50 value is detected by the Elisa method; in some embodiments, the IC50 value is detected according to the method of Test Example 2 of the present disclosure.
  • the present disclosure provides an antigen binding molecule that specifically binds BAFF and IL-12/23.
  • the antigen binding molecule is a bispecific antibody that specifically binds BAFF and IL-12/23 P40 subunits.
  • the antigen binding molecule has one or more of the following functional activities:
  • the antigen-binding molecule can block the binding of human BAFF to human BAFF-R; preferably, the IC50 value for blocking the binding of human BAFF to human BAFF-R is less than 11 nM (for example, less than 11 nM, less than or equal to 10 nM, less than or equal to Equal to 9nM, less than or equal to 8nM, less than or equal to 7nM, less than or equal to 6nM, less than or equal to 5nM or less), the IC50 value is detected by Elisa method; in some embodiments, the IC50 value is tested according to the present disclosure Example 2 method detection;
  • the antigen-binding molecule can inhibit BAFF-induced B cell proliferation; preferably, the antigen-binding molecule can inhibit BAFF-induced B cell proliferation; , less than or equal to 0.10 nM, less than or equal to 0.09 nM, less than or equal to 0.08 nM, less than or equal to 0.07 nM, less than or equal to 0.06 nM or less) inhibits BAFF-induced B cell proliferation; in some embodiments , the IC50 value is detected according to the method of Test Example 3 of the present disclosure;
  • the antigen binding molecule can be less than 9.99E-10M (such as less than 9.99E-10M, less than or equal to 9.00E-10M, less than or equal to 8.20E-10M, less than or equal to 7.40E-10M, less than or equal to 6.60E -10M, less than or equal to 5.20E-10M, less than or equal to 4.00E-10M, less than or equal to 3.00E-10M or less) combined with human BAFF, the KD value is detected by the Biacore method; in some implementations In the method, the KD value is detected according to the method of Test Example 5 of the present disclosure;
  • the antigen-binding molecule can be combined with cynomolgus BAFF with a KD value of less than 5.00E-10M (for example, less than or equal to 4.00E-10M, less than or equal to 3.00E-10M or less), and the KD value is passed Biacore method detection; In some embodiments, the KD value is detected according to the method of Test Example 5 of the present disclosure;
  • the antigen-binding molecule can bind to mouse BAFF with a KD value of less than 2.20E-10M (for example, less than or equal to 2.00E-10M, less than or equal to 1.00E-10M or less), and the KD value is determined by the Biacore method Detection; In some embodiments, the KD value is detected according to the method of Test Example 5 of the present disclosure;
  • the antigen-binding molecule can block the binding of human BAFF to human BCMA; preferably, the IC50 value of blocking the binding of human BAFF to human BCMA is less than 0.9nM (for example, less than or equal to 0.4nM, less than or equal to 0.35nM, less than or equal to or equal to 0.31nM or less), the IC50 value is detected by Elisa method; in some embodiments, the IC50 value is detected according to the method of Test Example 2 of the present disclosure;
  • the antigen-binding molecule can block the binding of human BAFF to human TACI; preferably, the IC50 value of blocking the binding of human BAFF to human TACI is less than 0.6nM (for example, less than or equal to 0.55nM, less than or equal to 0.4nM, less than or equal to or equal to 0.33nM or less), the IC50 value is detected by Elisa method; in some embodiments, the IC50 value is detected according to the method of Test Example 2 of the present disclosure;
  • the antigen-binding molecule can block the binding of human IL-12/23 p40 to human IL-12R ⁇ 1; preferably, the IC50 value for blocking the binding of human IL-12/23 p40 to human IL-12R ⁇ 1 is less than 3.6nM ( For example, less than or equal to 3.0nM, less than or equal to 2.0nM, less than or equal to 1.0nM, less than or equal to 0.5nM, less than or equal to 0.23nM or less), the IC50 value is detected by the Elisa method; in some embodiments, The IC50 value is detected according to the method of Test Example 2 of the present disclosure;
  • Antigen-binding molecules can inhibit IL-12-induced IFN ⁇ secretion; preferably, the IC50 value of antigen-binding molecules inhibiting IL-12-induced IFN ⁇ secretion is less than 3.0nM (for example, less than or equal to 2.6nM, less than or equal to 1.6nM or less) ; In some embodiments, the IC50 value is detected according to the method of Test Example 4 of the present disclosure;
  • J. Antigen-binding molecules can inhibit IL-23-induced IL-17 secretion; preferably, the antigen-binding molecules inhibit IL-23-induced IL-17 secretion with an IC50 value of less than 0.034nM (for example, less than or equal to 0.031nM, less than or equal to 0.030nM or less); In some embodiments, the IC50 value is detected according to the method of Test Example 4 of the present disclosure;
  • the antigen-binding molecule can bind to cynomolgus monkey IL-12/23 p40 with a KD value less than 2.7E-10M (for example, less than or equal to 2.0E-10M, less than or equal to 1.0E-10M or less), said The KD value is detected by the Biacore method; in some embodiments, the KD value is detected according to the method of Test Example 5 of the present disclosure; or
  • the antigen-binding molecule can bind to human IL-23 with a KD value of less than 6.4E-11M (e.g., less than or equal to 6.3E-11M, less than or equal to 6.2E-11M or less), the KD value is determined by the Biacore method Detection; In some embodiments, the KD value is detected according to the method of Test Example 5 of the present disclosure.
  • the antigen-binding molecule according to any one of the above, which comprises a heavy chain variable region B-VH and a light chain variable region B-VL, said B-VH comprising B-HCDR1, B-HCDR2 and B-HCDR3, the B-VL comprises B-LCDR1, B-LCDR2 and B-LCDR3, wherein the amino acid sequences of the B-HCDR1, B-HCDR2 and B-HCDR3 are respectively the same as those of SEQ ID NO: 63
  • the amino acid sequences of Bv-HCDR1, Bv-HCDR2 and Bv-HCDR3 are identical, and the amino acid sequences of B-LCDR1, B-LCDR2 and B-LCDR3 are respectively the same as those of Bv-LCDR1, Bv-LCDR2 and Bv-LCDR3 in SEQ ID NO: 64.
  • Bv-LCDR3 The amino acid sequences of Bv-LCDR3 are identical.
  • the B-HCDR1, B-HCDR2, B-HCDR3, B-LCDR1, B-LCDR2, B-LCDR3, Bv-HCDR1, Bv-HCDR2, Bv-HCDR3, Bv-LCDR1, Bv-LCDR2 and Bv-LCDR3 are defined according to the same numbering convention selected from Kabat, IMGT, Chothia, AbM and Contact.
  • the antigen-binding molecules according to any one of the above, said B-HCDR1, B-HCDR2, B-HCDR3, B-LCDR1, B-LCDR2, B-LCDR3 are defined according to the Kabat numbering rules, wherein, the amino acid sequence of B-HCDR1 is NNAIN (SEQ ID NO: 18), the amino acid sequence of B-HCDR2 is X 1 IX 2 PMFGX 3 AKYSX 4 X 5 FQG (SEQ ID NO: 65), the amino acid sequence of B-HCDR3 SRDX 6 LLFPX 7 X 8 X 9 LX 10 X 11 (SEQ ID NO: 66), the amino acid sequence of B-LCDR1 is X 12 GX 13 X 14 LX 15 X 16 X 17 X 18 AS (SEQ ID NO: 67) , the amino acid sequence of B-LCDR2 is GKNNRPS (SEQ ID NO: 32) and the amino acid sequence of B-LCDR3 is X 19 SRX 20
  • X 1 to X 25 are not selected from the following combinations: X 1 is G, X 2 is I, X 3 is T, X 4 is Q, X 5 is N, X 6 is L, X 7 is H, X 8 is H, X 9 is A, X 10 is S, X 11 is P, X 12 is Q, X 13 is D, X 14 is S, X 15 is R, X 16 is S, X 17 is Y, X 18 is Y, X 19 is S, X 20 is D, X 21 is S, X 22 is S, X 23 is N, X 24 is H, and X 25 is V.
  • said B-HCDR1 is shown in SEQ ID NO: 18, said B-HCDR2 is shown in SEQ ID NO: 28, and said B-HCDR3 is shown in SEQ ID NO: 29 , and said B-LCDR1 is shown in SEQ ID NO: 43, said B-LCDR2 is shown in SEQ ID NO: 32, and said B-LCDR3 is shown in SEQ ID NO: 44; or (ii) The B-HCDR1 is shown in SEQ ID NO: 18, the B-HCDR2 is shown in SEQ ID NO: 21, the B-HCDR3 is shown in SEQ ID NO: 20, and the B-LCDR1 is shown in SEQ ID NO: 34, said B-LCDR2 as shown in SEQ ID NO: 32, and said B-LCDR3 as shown in SEQ ID NO: 35; or (iii) said B-HCDR1 as shown in SEQ ID NO: 18 As shown, the B-HCDR2 is shown in SEQ ID NO: 22, the B-HCDR3 is shown in SEQ ID NO: 29 ,
  • the antigen-binding molecule as described in any one of the above it comprises B-VH and B-VL, described B-VH is as shown in SEQ ID NO: 47, and described B-VL is as shown in SEQ ID NO: 48 shown.
  • the B-VH is as shown in SEQ ID NO: 5, and the B-VL is as shown in SEQ ID NO: 12; or the B-VH is as shown in SEQ ID NO: 6, and said B-VL as shown in SEQ ID NO: 13; or said B-VH as shown in SEQ ID NO: 7, and said B-VL as shown in SEQ ID NO: 14; or said B- VH as shown in SEQ ID NO: 8, and said B-VL as shown in SEQ ID NO: 15; or said B-VH as shown in SEQ ID NO: 9, and said B-VL as shown in SEQ ID NO : shown in 16; or the B-VH as shown in SEQ ID NO: 10, and the B-VL as shown in SEQ ID NO: 17; or the B-VH as shown in SEQ ID NO: 11, and said B-VL as shown in SEQ ID NO:2.
  • the antigen-binding molecule that specifically binds BAFF and IL-12/23 comprises an antigen-binding moiety 1 that specifically binds BAFF and an antigen-binding moiety 2 that specifically binds IL-12 and/or IL-23 ; wherein said antigen binding module 2 comprises heavy chain variable region P-VH and light chain variable region P-VL, said P-VH comprising P-HCDR1, P-HCDR2 and P-HCDR3, said P-VL Comprising P-LCDR1, P-LCDR2 and P-LCDR3; the amino acid sequences of P-HCDR1, P-HCDR2 and P-HCDR3 are respectively the same as those of Pv-HCDR1, Pv-HCDR2 and Pv-HCDR3 in SEQ ID NO: 51 The amino acid sequence is the same, and the amino acid sequence of the P-LCDR1, P-LCDR2 and P-LCDR3 is respectively identical to the amino acid sequence of Pv-LCDR1, Pv-LCDR2 and Pv
  • the antigen-binding molecule according to any one of the above, said P-HCDR1, P-HCDR2, P-HCDR3, P-LCDR1, P-LCDR2, P-LCDR3 are defined according to the Kabat numbering rules, wherein, the P-HCDR1 is shown in SEQ ID NO: 53, the P-HCDR2 is shown in SEQ ID NO: 54, the P-HCDR3 is shown in SEQ ID NO: 55, and the P-LCDR1 As shown in SEQ ID NO: 56, the P-LCDR2 is shown in SEQ ID NO: 57, and the P-LCDR3 is shown in SEQ ID NO: 58.
  • the P-VH of the heavy chain variable region is shown in SEQ ID NO:51
  • the P-VL of the light chain variable region is shown in SEQ ID NO:52.
  • amino acid sequence variants of the antigen binding molecules provided herein are contemplated.
  • Amino acid sequence variants of antibodies can be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody, or by peptide synthesis. Such modifications include, for example, deletions, and/or insertions, and/or substitutions of residues within the amino acid sequence of the antigen-binding molecule. Any combination of deletions, insertions, and substitutions can be made to arrive at the final construct, so long as the final construct possesses the desired characteristics, such as antigen-binding properties.
  • antigen binding molecule variants having one or more amino acid substitutions are provided.
  • Sites of interest for substitution mutagenesis include CDRs and FRs.
  • Conservative substitutions are shown in Table 2 under the heading "Preferred Substitutions”. More substantial changes are provided in Table 2 under the heading "Exemplary Substitutions" and are described further below with reference to amino acid side chain classes.
  • Amino acid substitutions can be introduced into an antibody of interest, and the products screened for desired activity, such as retained/improved antigen binding, reduced immunogenicity, or improved ADCC or CDC.
  • amino acids can be grouped as follows:
  • Non-conservative substitutions refer to the substitution of a member of one class for a member of another class.
  • substitutional variant involves substituting one or more CDR residues of a parent antibody (eg, a humanized or human antibody).
  • a parent antibody eg. a humanized or human antibody
  • the resulting variant selected for further study will have an altered (e.g. improved) certain biological property (e.g. increased affinity, reduced immunogenicity) relative to the parent antibody, and/or will be substantially Some of the biological properties of the parental antibody are retained.
  • An exemplary substitution variant is an affinity matured antibody, which can be conveniently produced, for example, using phage display-based affinity maturation techniques such as those described herein. Briefly, one or more CDR residues are mutated, and the variant antibodies are displayed on phage and screened for specific biological activity (eg, binding affinity).
  • Alterations can be made to the CDRs, e.g., to improve antibody affinity. Such changes can be made to CDR "hotspots" (i.e., residues encoded by codons that undergo mutation at high frequency during the somatic maturation process, and/or residues that contact antigen) while making changes to the resulting variant VH or VL test for binding affinity.
  • affinity maturation diversity is introduced into the variable genes selected for maturation by any of a variety of methods, such as error-prone PCR, strand shuffling, or oligonucleotide-directed mutagenesis middle. Then, create secondary libraries. The library is then screened to identify any antibody variants with the desired affinity.
  • CDR residues involved in antigen binding can be specifically identified, for example, using alanine scanning mutagenesis or modeling.
  • HCDR3 and LCDR3 are frequently targeted.
  • substitutions, insertions or deletions may occur within one or more CDRs, so long as such changes do not substantially reduce the ability of the antibody to bind antigen.
  • conservative changes eg, conservative substitutions, as provided herein
  • Such changes do not occur at antigen contact residues.
  • alanine scanning mutagenesis One method that can be used to identify residues or regions of an antibody that can be targeted for mutagenesis is called "alanine scanning mutagenesis".
  • residues e.g. charged residues such as Arg, Asp, His, Lys and Glu
  • neutral or negatively charged amino acids e.g. Ala or Polypropylene
  • Amino acid amino acids
  • substitutions may be introduced at amino acid positions showing functional sensitivity to the initial substitution.
  • contact points between antibody and antigen can be identified by studying the crystal structure of the antigen-antibody complex. These contact residues and neighboring residues can be targeted or eliminated as candidates for substitution. Variants can be screened to determine whether they contain desired properties.
  • Amino acid sequence insertions include: amino- and/or carboxy-terminal fusions of one residue or polypeptides with a length of 100 or more residues, and intrasequence insertions of single or multiple amino acid residues.
  • terminal insertions include antibodies with an N-terminal methionyl residue.
  • Other insertional variants of antibody molecules include fusions with enzymes (or polypeptides that extend the serum half-life of antibodies) at the N- or C-terminus of the antibody.
  • the Fc region of an antigen binding molecule of the disclosure comprises one or more amino acid substitutions that reduce its binding to an Fc receptor, e.g., its binding to an Fc ⁇ receptor, and reduce or Eliminate effector functions.
  • a native IgG Fc region specifically an IgG 1 Fc region or an IgG 4 Fc region, may result in the targeting of an antigen binding molecule of the present disclosure to cells expressing Fc receptors, rather than cells expressing antigen.
  • the engineered Fc regions of the present disclosure exhibit reduced binding affinity to Fc receptors and/or reduced effector functions.
  • the engineered Fc region has a binding affinity for Fc receptors that is reduced by more than 50%, 80%, 90%, or 95% compared to a native Fc region.
  • the Fc receptor is an Fc gamma receptor.
  • the Fc receptor is a human Fc ⁇ receptor, eg, Fc ⁇ RI, Fc ⁇ RIIa, Fc ⁇ RIIB, Fc ⁇ RIIIa.
  • the engineered Fc region has reduced binding affinity for complement (eg, C1q) compared to a native Fc region.
  • the engineered Fc region has no reduced binding affinity for neonatal Fc receptor (FcRn) compared to a native Fc region.
  • the engineered Fc region has reduced effector function, which may include, but is not limited to, one or more of the following: reduced complement-dependent cytotoxicity (CDC), reduced Antibody-dependent cell-mediated cytotoxicity (ADCC), decreased antibody-dependent cellular phagocytosis (ADCP), decreased cytokine secretion, decreased immune complex-mediated antigen uptake by antigen-presenting cells, decreased interaction with NK cells decreased binding to macrophages, decreased binding to monocytes, decreased binding to polymorphonuclear cells, decreased direct signaling-induced apoptosis, decreased dendritic cell maturation, or decreased T cells primed.
  • CDC complement-dependent cytotoxicity
  • ADCC Antibody-dependent cell-mediated cytotoxicity
  • ADCP antibody-dependent cellular phagocytosis
  • cytokine secretion decreased immune complex-mediated antigen uptake by antigen-presenting cells
  • decreased interaction with NK cells decreased binding to macrophages
  • monocytes decreased binding to monocytes
  • polymorphonuclear cells
  • amino acid residue substitutions at positions 238, 265, 269, 270, 297, 327, and 329 may reduce effector function.
  • the Fc region is a human IgG 1 Fc region, and the amino acid residues at positions 234 and 235 are A, and the numbering is based on the EU index.
  • amino acid residue substitutions at positions such as 228 may reduce effector function.
  • Antigen binding molecules may also comprise disulfide bond engineering, eg, 354C of the first subunit and 349C of the second subunit.
  • the Fc region of the present disclosure comprises modifications according to the knob-into-hole (KIH) technique, which involves the introduction of a knob at the interface of one subunit and A hole structure (hole) is introduced at the interface. This enables the protrusion structure to be positioned in the hole structure, promotes the formation of heterodimers and inhibits the generation of homodimers.
  • KH knob-into-hole
  • the bulge structure is constructed by replacing small amino acid side chains from the interface of one subunit with larger side chains (such as tyrosine or tryptophan). Instead, the pore structure is created in the interface of another subunit by replacing large amino acid side chains with smaller ones, such as alanine or threonine.
  • the protrusion structure and hole structure are prepared by changing the nucleic acid encoding the polypeptide, and the optional amino acid substitutions are shown in Table 3 below:
  • knob-and-hole technique In addition to the knob-and-hole technique, other techniques for modifying the CH3 domain of a heavy chain to achieve heterodimerization are known in the art, for example WO96/27011, WO98/050431, EP1870459, WO2007/110205, WO 007/ 147901, WO2009/089004, WO2010/129304, WO2011/90754, WO2011/143545, WO2012/058768, WO2013/157954 and WO013/096291.
  • the C-terminus of the Fc region may be a complete C-terminus ending with the amino acid residue PGK; it may also be a truncated C-terminus, for example, one or two C-terminal amino acid residues have been removed from the truncated C-terminus.
  • the C-terminus of the Fc region is a shortened C-terminus ending with PG.
  • a composition of intact antibodies can include a population of antibodies from which all K447 residues and/or G446+K447 residues have been removed.
  • a composition of intact antibodies can include a population of antibodies in which the K447 residue and/or the G446+K447 residues have not been removed.
  • the composition of whole antibodies has a population of antibodies with and without a K447 residue and/or a mixture of antibodies with G446+K447 residues.
  • Antigen binding molecules eg, antibodies
  • Antigen binding molecules can be produced using recombinant methods. For these methods, one or more isolated nucleic acids encoding the antigen binding molecule are provided.
  • the present disclosure provides an isolated nucleic acid encoding an antigen binding molecule as previously described. Such nucleic acids may independently encode any of the aforementioned polypeptide chains.
  • the present disclosure provides one or more vectors (eg, expression vectors) comprising such nucleic acids.
  • the disclosure provides host cells comprising such nucleic acids.
  • a method of making an antigen binding molecule comprisin said method comprises, under conditions suitable for expression, culturing a host cell comprising a nucleic acid encoding said antigen binding molecule, as provided above, and optionally The antigen-binding molecule is efficiently recovered from the host cell (or host cell culture medium).
  • nucleic acid encoding the protein is isolated and inserted into one or more vectors for further cloning and/or expression in host cells.
  • nucleic acids can be readily isolated and sequenced using conventional procedures, or produced by recombinant methods or obtained by chemical synthesis.
  • Suitable host cells for cloning or expressing vectors encoding antigen-binding molecules include prokaryotic or eukaryotic cells as described herein. For example, it can be produced in bacteria, especially when glycosylation and Fc effector functions are not required. After expression, it can be isolated from the bacterial cell paste in a soluble fraction and can be further purified.
  • eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for vectors encoding antigen-binding molecules, including fungal and yeast strains.
  • Suitable host cells suitable for expression of antigen binding molecules may also be derived from multicellular organisms (invertebrates and vertebrates); examples of invertebrate cells include plant and insect cells.
  • a number of baculovirus strains have been identified for use in combination with insect cells, particularly for the transfection of Spodoptera frugiperda cells; plant cell cultures can also be used as hosts, e.g.
  • vertebrate cells can also be used as hosts, such as mammalian cell lines adapted for growth in suspension.
  • suitable mammalian host cell lines are the SV40-transformed monkey kidney CV1 line (COS-7); the human embryonic kidney line (293 or 293T cells); baby hamster kidney cells (BHK); Sertoli) cells (TM4 cells); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical cancer cells (HELA); canine kidney cells (MDCK); buffalo rat liver cells ( BRL3A); human lung cells (W138); human hepatocytes (Hep G2); mouse mammary tumor (MMT 060562); TRI cells; MRC 5 cells; and FS4 cells.
  • Suitable mammalian host cell lines include Chinese Hamster Ovary (CHO) cells, including DHFR-CHO cells; and myeloma cell lines, such as YO, NSO and Sp2/0.
  • CHO Chinese Hamster Ovary
  • myeloma cell lines such as YO, NSO and Sp2/0.
  • Antigen binding molecules provided herein can be identified, screened or characterized for their physical/chemical characteristics and/or biological activity by a variety of assays known in the art. In one aspect, antigen binding molecules of the present disclosure are tested for activity, eg, by known methods such as ELISA, Western blot, and the like.
  • the antigen binding molecules provided by the present disclosure can be used to detect the presence or level of BAFF or IL-12/23 in a biological sample.
  • the term “detection” encompasses quantitative or qualitative detection.
  • the biological sample comprises cells or tissue, such as tumor tissue.
  • an antigen binding molecule for use in a diagnostic or detection method is provided.
  • methods of detecting the presence of BAFF or IL-12/23 in a biological sample are provided.
  • the method comprises contacting a biological sample with an antigen-binding molecule under suitable conditions, and detecting whether a complex is formed between the detection reagent and the antigen.
  • antigen binding molecules are used to select subjects suitable for treatment, for example BAFF or IL-12/23 are biomarkers for patient selection.
  • Exemplary disorders that can be diagnosed using the antigen binding molecules of the disclosure such as autoimmune diseases, for example: systemic lupus erythematosus, myasthenia gravis, multiple sclerosis, insulin-dependent diabetes, Crohn's disease, rheumatoid arthritis arthritis, polyarticular juvenile rheumatoid arthritis, or psoriatic arthritis; or B-cell disorders such as neoplasms, chronic leukocytic leukemia, multiple myeloma, non-Hodgkin's lymphoma, post-transplant lymphoproliferative disease or light chain gammopathies.
  • autoimmune diseases for example: systemic lupus erythematosus, myasthenia gravis, multiple sclerosis, insulin-dependent diabetes, Crohn's disease, rheumatoid arthritis arthritis, polyarticular juvenile rheumatoid arthritis, or psoriatic arthritis
  • B-cell disorders such as neoplasms
  • Labeled antigen binding molecules include, but are not limited to, labels or moieties for direct detection (such as fluorescent, chromogenic, electron-dense, chemiluminescent, and radioactive labels), and moieties for indirect detection (e.g., indirect detection via enzymatic reactions or molecular interactions).
  • modules such as enzymes or ligands).
  • the present disclosure provides the use of an antigen binding molecule in the manufacture or preparation of a medicament.
  • the B cell disorder or autoimmune disease is a disease or condition associated with BAFF or IL-12/23.
  • the autoimmune disease is selected from the group consisting of: systemic lupus erythematosus, myasthenia gravis, multiple sclerosis, insulin-dependent diabetes mellitus, Crohn's disease, rheumatoid arthritis, polyarticular juvenile rheumatoid Arthritis and psoriatic arthritis; said B-cell disorder is selected from the group consisting of neoplasms, chronic leukaemia, multiple myeloma, non-Hodgkin's lymphoma, post-transplantation lymphoproliferative disease, and light chain gammopathies .
  • the autoimmune disease is systemic lupus erythematosus.
  • the use further comprises administering to the subject an effective amount of at least one additional therapeutic agent (e.g., one, two, three, four, five, or six additional therapeutic agents agent).
  • a “subject” according to any of the above embodiments may be a human.
  • a subject When used for therapeutic purposes, a subject is an individual who has, or is suspected of having, the disease of interest. When used for prophylactic purposes, the subject is an individual susceptible to the disease of interest.
  • a pharmaceutical composition comprising said antigen binding molecule, eg, for any of the above pharmaceutical uses or methods of treatment.
  • a pharmaceutical composition comprises any of the antigen binding molecules provided herein and a pharmaceutically acceptable carrier.
  • the pharmaceutical composition further comprises at least one additional therapeutic agent.
  • the antigen binding molecules of the present disclosure can be used alone or in combination with other agents for therapy.
  • an antibody of the present disclosure can be administered in combination (simultaneously, or sequentially) with at least one additional therapeutic agent.
  • the antigen binding molecules of the present disclosure can be administered by any suitable means, including parenteral, intrapulmonary, intranasal, and, if local treatment is desired, intralesional.
  • Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration. Administration may be by any suitable route, eg, by injection, such as intravenous or subcutaneous injection, depending in part on whether the administration is short-term or chronic.
  • a variety of dosing schedules are contemplated herein, including, but not limited to, single or multiple administrations at multiple time points, bolus administration, and pulse infusion.
  • Antigen binding molecules of the present disclosure will be formulated, dosed and administered in a manner consistent with good medical practice (eg, GOOD MEDICAL PRACTICE Guideline, GMP). Factors considered in this context include the particular condition being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the condition, the site of delivery of the agent, the method of administration, the timing of administration, and others known to the medical practitioner. factor. Antigen binding molecules may or may not be formulated with one or more agents currently used to prevent or treat the disorder. The effective amount of such other agents depends on the amount present in the pharmaceutical composition, the type of disorder or treatment, and other factors. These are generally used at the same dosages and routes of administration as described herein, or at about 1 to 99% of the dosages described herein, or at other dosages, and any route empirically/clinically determined to be appropriate.
  • GMP Good MEDICAL PRACTICE Guideline
  • appropriate dosages of the antigen-binding molecules of the present disclosure will depend on the type of disease to be treated, the amount of the therapeutic molecule Type, severity and course of disease, whether administered for prophylactic or therapeutic purposes, previous therapy, subject's clinical history and response to the therapeutic molecule, and the judgment of the attending physician.
  • the therapeutic molecule is suitably administered to a subject at one time or over a series of treatments.
  • a daily dosage might range from about 1 ⁇ g/kg to 100 mg/kg, depending on the factors mentioned above.
  • an article of manufacture comprising materials useful for the treatment, prevention and/or diagnosis of the disorders described above.
  • the article comprises a container and a label or package insert on or associated with the container.
  • Suitable containers include, for example, bottles, vials, syringes, IV solution bags, and the like.
  • Containers can be formed from various materials such as glass or plastic.
  • the container contains a composition effective, alone or in combination with another composition, for the treatment, prophylaxis and/or diagnosis of a condition, and may have a sterile access opening (e.g., the container may have a stopper pierceable by a hypodermic needle). IV solution bag or vial).
  • At least one active agent in the composition is an antigen binding molecule of the present disclosure.
  • the label or package insert indicates that the composition is used to treat the condition of choice.
  • the article of manufacture may comprise: (a) a first container having a composition therein, wherein the composition comprises an antigen binding molecule of the present disclosure; and (b) a second container having a composition therein, wherein the combination
  • the drug contains an additional cytotoxic or other therapeutic agent.
  • the article of manufacture of this embodiment of the present disclosure may further comprise a package insert indicating that the composition may be used to treat a particular condition.
  • the article of manufacture may further comprise a second (or third) container comprising a pharmaceutically acceptable buffer. It may further comprise other materials as desired from a commercial and user standpoint, including other buffers, diluents, filters, needles and syringes.
  • the article of manufacture is prepared in the form of a kit.
  • Example 1 Belimumab mutant antibody and its activity detection
  • Belimumab was modified by phage display technology. By designing mutation primers, the CDRs of the light and heavy chains of Belimumab were mutated separately to construct a mutation library. Use biotinylated BAFF protein (Sino biological, 10056-HNCH) to enrich and screen the mutant library to obtain candidate clones, determine the amino acid sequence of the cloned light and heavy chain variable regions by sequencing, and separate the light and heavy chain variable regions of the candidate clones Fused with antibody light/heavy chain constant regions respectively to construct full-length antibodies.
  • biotinylated BAFF protein Seo biological, 10056-HNCH
  • the sequence information of the Belimumab antibody is as follows:
  • the sequence is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4
  • the underlined part is the CDR region (determined according to the Kabat numbering system)
  • the italic part is the mutated amino acid residue
  • the underlined part is not for the frame area.
  • the CDRs in the table are the CDRs determined according to the Kabat numbering system.
  • the CDR in the table is the CDR determined according to the Kabat numbering system
  • the above heavy chain variable region and light chain variable region are fused with the antibody heavy chain constant region and light chain constant region, respectively, to construct a full-length antibody.
  • the above-mentioned heavy chain variable region is fused with the heavy chain constant region of Belimumab (sequence shown in SEQ ID NO: 45), and the light chain variable region of the antibody is fused with the light chain constant region of Belimumab (sequence shown in SEQ ID NO : shown in 46) fusion to construct anti-BAFF antibodies: B1 to B7, the variable region sequences of the obtained antibodies are shown in Table 6.
  • the heavy chain variable region is B-VH6 (SEQ ID NO: 10)
  • the light chain variable region is B-VL6 (SEQ ID NO: 17)
  • the heavy chain constant region is SEQ ID NO: 45
  • the light chain constant region is SEQ ID NO: 46.
  • the framework regions of the above-mentioned anti-BAFF antibodies were modified.
  • the amino acid residue at position 44 (according to the Kabat numbering system) of the heavy chain variable region of B6 is mutated to C (ie, 44C), and at the same time, the amino acid residue at position 100 of the light chain variable region of B6 (according to The position determined by the Kabat numbering system) amino acid residue was mutated to C (i.e. 100C) in order to increase the interchain disulfide bond between VH and VL, and the variable region sequence of antibody B61 after the B6 framework region transformation was as follows:
  • Example 2 Construction of bispecific antibodies specifically binding to BAFF and IL-12/23
  • the anti-BAFF antibody obtained in Example 1 and the known anti-IL-12/23 antibody were used to construct a bispecific antibody specifically binding to BAFF and IL-12/23.
  • the IL-12/23 antibody can be derived from any suitable antibody, for example, anti-IL-12/23 p40 subunit antibody Ustekinumab (abbreviated as Umab), the sequence of Ustekinumab is as follows:
  • the double underlined part is the variable region sequence
  • the single underlined part is the CDR region sequence
  • the variable region and CDR are confirmed according to the Kabat numbering system.
  • the human IgG1 heavy chain constant region (sequence shown in SEQ ID NO: 59), human kappa light chain constant region (sequence shown in SEQ ID NO: 60), B61 antibody heavy chain variable region and Light chain variable region (sequence shown in SEQ ID NO: 47, 48) and Ustekinumab antibody heavy chain variable region and light chain variable region (sequence shown in SEQ ID NO: 51, 52), construct specific binding Bispecific antibody to BAFF and IL-12/23.
  • the C-terminal of the variable region of the light chain of the Ustekinumab antibody is fused with the N-terminal of the constant region of the human ⁇ light chain to form the second chain;
  • the C-terminal of the variable region of the heavy chain of the Ustekinumab antibody is fused with the N-terminal of the constant region of the human IgG1 heavy chain,
  • the C-terminus of the human IgG1 heavy chain constant region is fused to the N-terminus of the scFv (constructed from the heavy and light chain variable regions of the B61 antibody) to form the first chain.
  • bispecific antibodies specifically binding to BAFF and IL-12/23 were constructed: BU-1 and BU-2.
  • Both BU-1 and BU-2 are composed of 4 chains (including two identical first chains and two identical second chains), the structural diagram of BU-1 is shown in Figure 1, and the structural diagram of BU-2 is shown in Figure 1 2.
  • the amino acid sequence of the second chain of BU-1 is the same as that of the light chain of Ustekinumab, and the sequence is shown in SEQ ID NO:50.
  • the amino acid sequence of the second chain of BU-2 is the same as that of the light chain of Ustekinumab, and the sequence is shown in SEQ ID NO:50.
  • the wavy part is the heavy chain variable region of the Ustekinumab antibody
  • the underlined part is the human IgG1 heavy chain constant region
  • the double underlined part is the heavy chain of the B61 antibody.
  • the chain variable region part, the single underline part is the light chain variable region part of B61 antibody
  • the italic part is the linker sequence part.
  • the binding activity of the antibody to BAFF and IL-12/23 p40 was detected by the Elisa method.
  • the specific method is as follows:
  • the sample to be tested was diluted to 2 ⁇ g/mL with pH 7.4 PBS (B320) buffer, added to a 96-well microplate plate (Corning, 3590) at a volume of 100 ⁇ L/well, and incubated overnight at 4°C. After the liquid was discarded, 300 ⁇ L of 5% skimmed milk (BD, 232100) diluted with PBS was added to each well for blocking, and incubated at 37° C. for 2 hours.
  • PBST buffer pH7.4PBS containing 0.1% tween-20
  • BAFF ACROBiosystems, BAF-H5248
  • IL-12/ 23 p40 Sino Biological, 10052-H08H
  • the blocking activity of the antibody against the following receptors and ligands was detected by Elisa method: human BAFF and human BAFF-R, human BAFF and human BCMA, human BAFF and human TACI, and human IL-12/23 p40 and human IL-12R ⁇ 1 .
  • the specific method is as follows:
  • the receptor protein was diluted to 2 ⁇ g/mL with pH 7.4 PBS (B320) buffer, added to a 96-well microtiter plate (Corning, 3590) at a volume of 100 ⁇ L/well, and incubated overnight at 4°C. After the liquid was discarded, 200 ⁇ L of 1% Casein blocking solution (Thermo, 37528) was added to each well for blocking, and incubated at 37° C. for 2 hours. After the blocking, the blocking solution was discarded, and the plate was washed 3 times with PBST buffer (pH7.4 PBS containing 0.1% tween-20) before use.
  • PBST buffer pH7.4 PBS containing 0.1% tween-20
  • a fixed concentration of biotin-labeled ligand protein was mixed with a gradiently diluted antibody, pre-incubated at 37°C for 30 minutes, then added to the blocked microtiter plate, and incubated at 37°C for 1.5 hours. After the incubation, the plate was washed 3 times with PBST, 100 ⁇ L streptavidin-HRP (Invitrogen, 434323, diluted 1:4000) was added to each well, and incubated at 37°C for 1 hour.
  • the sources of receptors and ligand proteins used in this test example are as follows: human BAFF (Sino biological, 10056-HNCH), human IL-12/23 p40 (Sino biological, 10052-H08H), human BAFF-R (Sino biological , 16079-H02H), human BCMA (Sino biological, 10620-H02H), human TACI (ACROBiosystems, TAI-H5256), human IL-12R ⁇ 1 (ACROBiosystems, ILB-H5255).
  • the experimental results are shown in Table 10, Table 11, and Table 12.
  • the experimental results show that the antibody specifically binding to BAFF constructed in this disclosure can effectively block the binding of human BAFF and human BAFF-R, and the IC50 value is lower than that of Belimumab.
  • the bispecific antibody specifically binding to BAFF and IL-12/23 constructed in this disclosure can effectively block human BAFF and human BAFF-R, human BAFF and human BCMA, human BAFF and human TACI, and the IC50 value is less than that of Belimumab ; It can also effectively block the binding of human IL-12/23 p40 to human IL-12R ⁇ 1.
  • the B cell proliferation assay was used to detect whether the antibody could inhibit BAFF-induced B cell proliferation.
  • the experimental method is as follows:
  • the mouse spleen was taken for grinding, centrifuged at 4°C for 5 minutes to collect the lower layer of cells, washed once with washing solution (PBS+2% FBS+2mM EDTA) and centrifuged, and added RBC lysis buffer (Invitrogen, 00-4333- 57), stand at room temperature for 5 minutes until the red blood cells are completely lysed. Centrifuge again and resuspend cells for counting.
  • the cell suspension was sorted with the B cell isolation kit (Miltenyi Biotec, 130-090-862), and the isolated B cells were sorted with RPMI 1640 medium (Gibco, 11875119)+10%FBS (Gibco, 10099-141)+ 50 ⁇ M 2-mercaptoethanol (Sigma-Aldrich, M6250) was resuspended and counted, and the cells were plated in 96-well cell plates (Costar, 3903) for later use.
  • the experimental results are shown in Table 13 and Table 14 below.
  • the experimental results show that the disclosed antibody that specifically binds to BAFF and the bispecific antibody that specifically binds to BAFF and IL-12/23 can effectively inhibit the proliferation of B cells induced by BAFF, and Belimumab inhibits
  • the IC50 value of BAFF-induced B cell proliferation is more than 2 times that of the disclosed antibody specifically binding to BAFF, while ustekinumab has no function of inhibiting BAFF-induced B cell proliferation.
  • the experimental method is as follows:
  • the mouse spleen was taken for grinding, centrifuged at 4°C for 5 minutes to collect the cells in the lower layer, washed once with washing solution (PBS+2% FBS+2mM EDTA) and centrifuged, after the supernatant was removed, RBC lysis buffer (Invitrogen, 00-4333- 57), stand at room temperature for 5 minutes until the red blood cells are completely lysed. Centrifuge again and resuspend cells for counting.
  • washing solution PBS+2% FBS+2mM EDTA
  • RBC lysis buffer Invitrogen, 00-4333- 57
  • the cell suspension was sorted with the mouse CD4 cell kit (Invitrogen, 11415D), and the isolated CD4+T cells were resuspended in the medium RPMI 1640 medium (Gibco, 11875119)+10% FBS (Gibco, 10099-141) And count to spare.
  • IL-12-induced T cell differentiation 20 ⁇ g/mL anti-mouse IL-4 (BioLegend, 504122) was added to T cells, and the cell suspension was spread in a coated 96-well plate. A fixed concentration of chimeric IL-12 (human p40 fused with mouse p35) protein was mixed with serially diluted antibodies, pre-incubated at 37°C for 1 hour, added to a 96-well plate, and incubated in a 37°C cell culture incubator for 48 hours.
  • chimeric IL-12 human p40 fused with mouse p35
  • the 96-well plate was taken out, centrifuged at 1000rpm for 3 minutes, the supernatant was collected, and the content of IFN ⁇ in the supernatant was detected with mouse IFN-gamma DuoSet ELISA Kit (R&D Systems, DY485).
  • the cell suspension was spread in a well-coated 96-well plate, and a fixed concentration of IL-23 (R&D Systems, 1290-IL-010) was mixed with a serially diluted antibody for pretreatment. After incubation for 1 hour, it was added to a 96-well plate and incubated in a 37°C cell culture incubator for 48 hours. The 96-well plate was taken out, centrifuged at 1000rpm for 3 minutes, the supernatant was collected, and the IL-17 content in the supernatant was detected with the mouse IL-17 DuoSet ELISA Kit (R&D Systems, DY421).
  • the experimental results are shown in Table 15 and Table 16 below.
  • the experimental results show that the disclosed bispecific antibody that specifically binds to BAFF and IL-12/23 can effectively inhibit the secretion of IL-17 induced by IL-23, and can effectively inhibit IL-12 Induced IFN ⁇ secretion, while Belimumab could neither inhibit IL-23-induced IL-17 secretion nor IL-12-induced IFN ⁇ secretion.
  • Biosensing chip Protein A (GE, 29127556) to affinity capture a certain amount of the sample to be tested, and then flow through a series of concentration gradient antigens on the surface of the chip, and use Biacore (GE, 8K) to detect the reaction signal in real time to obtain the binding and dissolving off the curve.
  • Biacore GE, 8K
  • the biochip was cleaned and regenerated with 10 mM glycine-hydrochloric acid solution pH 1.5 (GE, BR-1003-54).
  • the experimental data was fitted with BIA evaluation version 4.1 and GE software with a 1:1 model to obtain the affinity value.
  • the relevant antigen proteins used in this test are as follows: human IL-23 (CT048-H08H, Sino biological), human BAFF (10056-HNCH, Sino biological), cynomolgus monkey IL-12/23 P40 (10215-CL, R&D Systems), cynomolgus monkey BAFF (BAF-CM412B, Kactus), mouse BAFF (BAF-M521y, Acro Biosystems).
  • the experimental results are shown in Table 17 and Table 18 below.
  • the experimental results show that, compared with Belimumab, the anti-BAFF antibody constructed in this disclosure can bind to human BAFF with a smaller KD value; in addition, the construction of this disclosure specifically binds to BAFF and IL-12
  • the /23 bispecific antibody can bind to human IL-23, human BAFF, cynomolgus monkey IL-12/23 P40, cynomolgus monkey BAFF, and mouse BAFF with high affinity.
  • mice Simultaneously stimulate mice with chimeric IL-12 (human p40 fused with mouse p35), human IL-23 and human BAFF to induce the production of cytokines such as IFN ⁇ , TNF ⁇ , IL-22 and IgA in mice, and pass the detection
  • cytokines such as IFN ⁇ , TNF ⁇ , IL-22 and IgA
  • mice SPF female C57BL/6 mice (Beijing Weitong Lihua Experimental Animal Technology Co., Ltd., 8 weeks old) were randomly divided into groups of 5 mice, and chimeric IL-12 (2 ⁇ g/hour) was mixed intraperitoneally. Rat), human IL-23 (2 ⁇ g/mouse) and human BAFF (1 mg/kg) were injected once a day for four days. The sample to be tested (Belimumab 8mpk, BU-1 10.7mpk, or BU-1 5.35mpk) was injected intraperitoneally one hour before the protein injection on the first day and the third day respectively.
  • mice in each group were collected, and the levels of IFN ⁇ , TNF ⁇ , IL-22 and IgA were detected respectively.
  • the source information of the detection kit used in this test case is as follows: Mouse IFN-gamma Quantikine ELISA Kit (R&D Systems, MIF00), Mouse TNF-alpha Quantikine ELISA Kit (R&D Systems, MTA00B), Mouse/Rat IL-22 Quantikine ELISA Kit (R&D Systems, M2200), Mouse IgA ELISA Kit (Abcam, ab157717).
  • Belimumab was used as a positive control
  • PBS was used as a negative control.
  • BU-1 10.7mpk has the same molar drug concentration as Belimumab 8mpk.
  • the experimental results are shown in Figures 3 to 6.
  • the experimental results show that the disclosed bispecific antibody specifically binding to BAFF and IL-12/23 can significantly inhibit the secretion of TNF ⁇ , IFN ⁇ , and IL-22, while Belimumab cannot inhibit the secretion of TNF ⁇ and IFN ⁇ secretion.
  • BU-1 can significantly inhibit the secretion of IgA at two doses of 10.7mpk and 5.35mpk, and the inhibitory activity is stronger than that of Belimumab.
  • the experimental results are shown in Table 19 below.
  • the experimental results show that the half-life of the disclosed bispecific antibody specifically binding to BAFF and IL-12/23 in rats is very good, and the half-life of anti-IL-12/23 p40 reaches 14 days. The half-life is longer than that of Ustekinumab.

Abstract

La présente invention concerne une molécule de liaison à l'antigène se liant spécifiquement à BAFF et IL-12/23 et son utilisation.
PCT/CN2022/102621 2021-06-30 2022-06-30 Molécule de liaison à l'antigène se liant spécifiquement à baff et il-12/23 et son utilisation WO2023274342A1 (fr)

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