WO2023165514A1 - Molécule de liaison à l'antigène se liant spécifiquement à flt3 et cd3 et son utilisation pharmaceutique - Google Patents

Molécule de liaison à l'antigène se liant spécifiquement à flt3 et cd3 et son utilisation pharmaceutique Download PDF

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WO2023165514A1
WO2023165514A1 PCT/CN2023/079002 CN2023079002W WO2023165514A1 WO 2023165514 A1 WO2023165514 A1 WO 2023165514A1 CN 2023079002 W CN2023079002 W CN 2023079002W WO 2023165514 A1 WO2023165514 A1 WO 2023165514A1
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flt3
seq
amino acid
acid sequence
antigen
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PCT/CN2023/079002
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English (en)
Chinese (zh)
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张玲
朱亚丽
金薪盛
王姝
杨莉
应华
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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Publication of WO2023165514A1 publication Critical patent/WO2023165514A1/fr

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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
    • A61P35/02—Antineoplastic agents specific for leukemia
    • 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
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants

Definitions

  • the present disclosure belongs to the field of biotechnology, and more specifically, the present disclosure relates to antigen-binding molecules and applications thereof.
  • FMS-like tyrosine kinase 3 (FLT3, also known as CD135, FLK2, STK1) is a receptor tyrosine kinase. Compared with healthy cells, it is overexpressed on most of the bulk cells of patients with acute myeloid leukemia (AML), and is highly expressed on the leukemic hematopoietic stem cells of most patients. Additionally, FLT3 is the most frequently mutated gene in AML patients, and mutations that result in constitutive activation of the receptor are associated with poor prognosis.
  • AML acute myeloid leukemia
  • FLT3 is the most frequently mutated gene in AML patients, and mutations that result in constitutive activation of the receptor are associated with poor prognosis.
  • CD3 is an isotype or heterodimer antigen expressed on T cells. Functional CD3 is formed by dimer association of two of four different chains: ⁇ , ⁇ , ⁇ , and ⁇ . CD3 dimer arrangements include ⁇ / ⁇ , ⁇ / ⁇ , and ⁇ / ⁇ . CD3 binds to the T cell receptor complex (TCR) and is required for T cell activation. Therefore, anti-CD3 antibodies that activate T cells have been proposed for therapeutic purposes. However, administration of anti-CD3 antibodies may trigger T cell activation and associated cytokine release. Excessive cytokine release leads to severe cytokine release syndrome (CRS), which is an important challenge in the clinical application of anti-CD3 antibodies.
  • TCRS severe cytokine release syndrome
  • the present disclosure provides an antigen-binding molecule that specifically binds FLT3 and CD3 and an antibody that specifically binds FLT3.
  • the present disclosure provides an antigen-binding molecule comprising at least one antigen-binding moiety that specifically binds FLT3 and at least one antigen-binding moiety that specifically binds CD3, the antigen-binding moiety that specifically binds FLT3 comprising a heavy Chain variable region (FLT3-VH) and light chain variable region (FLT3-VL), the antigen-binding module that specifically binds CD3 comprises heavy chain variable region (CD3-VH) and light chain variable region (CD3 -VL).
  • the antigen-binding molecule comprises one or two antigen-binding moieties that specifically bind FLT3 and one antigen-binding moiety that specifically binds CD3, and the antigen-binding moiety that specifically binds FLT3 comprises a heavy chain Variable region (FLT3-VH) and light chain variable region (FLT3-VL), the antigen-binding module that specifically binds CD3 comprises heavy chain variable region (CD3-VH) and light chain variable region (CD3-VL ).
  • the antigen-binding molecule comprises an antigen-binding moiety that specifically binds FLT3 and an antigen-binding moiety that specifically binds CD3, and the antigen-binding moiety that specifically binds FLT3 comprises a heavy chain variable region ( FLT3-VH) and a light chain variable region (FLT3-VL), the antigen-binding module specifically binding to CD3 comprises a heavy chain variable region (CD3-VH) and a light chain variable region (CD3-VL).
  • the antigen binding molecule as described above, wherein
  • the FLT3-VH has: FLT3-HCDR1 comprising the amino acid sequence of SEQ ID NO: 19, FLT3-HCDR2 comprising the amino acid sequence of SEQ ID NO: 20, and FLT3-HCDR2 comprising the amino acid sequence of SEQ ID NO: 21 HCDR3, and the FLT3-VL has: FLT3-LCDR1 comprising the amino acid sequence of SEQ ID NO: 83 or 22, FLT3-LCDR2 comprising the amino acid sequence of SEQ ID NO: 84 or 23 and comprising the amino acid of SEQ ID NO: 24 sequence FLT3-LCDR3, or
  • the FLT3-VH has: FLT3-HCDR1 comprising the amino acid sequence of SEQ ID NO: 13, FLT3-HCDR2 comprising the amino acid sequence of SEQ ID NO: 14, and FLT3-HCDR2 comprising the amino acid sequence of SEQ ID NO: 15 HCDR3, and the FLT3-VL has: FLT3-LCDR1 comprising the amino acid sequence of SEQ ID NO: 16, FLT3-LCDR2 comprising the amino acid sequence of SEQ ID NO: 17, and FLT3-LCDR2 comprising the amino acid sequence of SEQ ID NO: 18 LCDR3, or .
  • the FLT3-VH has: FLT3-HCDR1 comprising the amino acid sequence of SEQ ID NO: 7, FLT3-HCDR2 comprising the amino acid sequence of SEQ ID NO: 8, 76 or 77 and comprising the amino acid of SEQ ID NO: 9 sequence of FLT3-HCDR3, and the FLT3-VL has: FLT3-LCDR1 comprising the amino acid sequence of SEQ ID NO: 10, 78, 79 or 80, FLT3- comprising the amino acid sequence of SEQ ID NO: 11, 81 or 82 LCDR2 and FLT3-LCDR3 comprising the amino acid sequence of SEQ ID NO: 12.
  • the antigen-binding molecule as described above wherein the FLT3-VH has: FLT3-HCDR1 comprising the amino acid sequence of SEQ ID NO: 19, FLT3-HCDR2 comprising the amino acid sequence of SEQ ID NO: 20 and FLT3-HCDR3 comprising the amino acid sequence of SEQ ID NO: 21, and the FLT3-VL has: FLT3-LCDR1 comprising the amino acid sequence of SEQ ID NO: 83, FLT3-LCDR2 comprising the amino acid sequence of SEQ ID NO: 84 and FLT3-LCDR3 comprising the amino acid sequence of SEQ ID NO:24.
  • the FLT3-HCDR1, FLT3-HCDR2, FLT3-HCDR3, FLT3-LCDR1, FLT3-LCDR2 and FLT3-LCDR3 are defined according to the Kabat numbering convention.
  • the antigen-binding molecule described herein that specifically binds FLT3 and CD3 comprises at least one antigen-binding moiety that specifically binds FLT3 and at least one antigen-binding moiety that specifically binds CD3, and that specifically binds FLT3
  • the antigen binding module of the heavy chain variable region comprises the heavy chain variable region FLT3-VH and the light chain variable region FLT3-VL
  • the antigen binding module specifically binding to CD3 comprises the heavy chain variable region CD3-VH and the light chain variable region CD3-VH VL;
  • the FLT3-VH comprises FLT3-HCDR1 shown in SEQ ID NO: 19, FLT3-HCDR2 shown in SEQ ID NO: 20 and FLT3-HCDR3 shown in SEQ ID NO: 21, and the FLT3-
  • the VL comprises FLT3-LCDR1 set forth in SEQ ID NO: 83 or 22, FLT3-LCDR2 set forth in SEQ ID NO: 84 or 23, and FLT3-LCDR3 set forth in SEQ ID NO: 24, or
  • the FLT3-VH comprises FLT3-HCDR1 shown in SEQ ID NO: 13, FLT3-HCDR2 shown in SEQ ID NO: 14 and FLT3-HCDR3 shown in SEQ ID NO: 15, and the FLT3- VL comprising FLT3-LCDR1 set forth in SEQ ID NO: 16, FLT3-LCDR2 set forth in SEQ ID NO: 17, and FLT3-LCDR3 set forth in SEQ ID NO: 18, or
  • said FLT3-VH comprises FLT3-HCDR1 shown in SEQ ID NO: 7, FLT3-HCDR2 shown in SEQ ID NO: 8, 76 or 77, and FLT3-HCDR3 shown in SEQ ID NO: 9, and
  • the FLT3-VL comprises FLT3-LCDR1 shown in SEQ ID NO: 10, 78, 79 or 80, FLT3-LCDR2 shown in SEQ ID NO: 11, 81 or 82, and FLT3-LCDR2 shown in SEQ ID NO: 12 LCDR3.
  • the antigen-binding molecule according to any one of the preceding which binds to human FLT3 with a KD of less than 1 ⁇ 10 -8 M, 5 ⁇ 10 -9 M at 25°C, the KD is obtained through the surface measured by plasmon resonance.
  • the antigen binding molecule of any one of the preceding wherein:
  • said FLT3-VH comprises the amino acid sequence of SEQ ID NO: 53, 5, 52 or 54
  • said FLT3-VL comprises the amino acid sequence of SEQ ID NO: 56, 6 or 55, or
  • said FLT3-VH comprises the amino acid sequence of SEQ ID NO: 45, 3, 46, 47 or 48
  • said FLT3-VL comprises the amino acid sequence of SEQ ID NO: 50, 4, 49 or 51, or
  • said FLT3-VH comprises the amino acid sequence of SEQ ID NO: 1, 29, 30, 31, 32, 33, 34 or 35
  • said FLT3-VL comprises SEQ ID NO: 2, 36, 37, 38 , 39, 40, 41, 42, 43 or 44 amino acid sequence.
  • the antigen binding molecule of any one of the preceding wherein
  • said FLT3-VH comprises the amino acid sequence of SEQ ID NO: 53, 52 or 54
  • said FLT3-VL comprises the amino acid sequence of SEQ ID NO: 56 or 55, or
  • said FLT3-VH comprises the amino acid sequence of SEQ ID NO: 45, 46, 47 or 48
  • said FLT3-VL comprises the amino acid sequence of SEQ ID NO: 50, 49 or 51, or
  • said FLT3-VH comprises the amino acid sequence of SEQ ID NO: 29, 30, 31, 32, 33, 34 or 35
  • said FLT3-VL comprises SEQ ID NO: 36, 37, 38, 39, 40 , 41, 42, 43 or 44 amino acid sequence.
  • the antigen binding molecule of any one of the preceding wherein
  • said FLT3-VH comprises the amino acid sequence of SEQ ID NO: 53
  • said FLT3-VL comprises the amino acid sequence of SEQ ID NO: 56
  • the FLT3-VH comprises the amino acid sequence of SEQ ID NO: 52
  • the FLT3-VL comprises the amino acid sequence of SEQ ID NO: 55, or
  • the FLT3-VH comprises the amino acid sequence of SEQ ID NO: 52
  • the FLT3-VL comprises the amino acid sequence of SEQ ID NO: 56, or
  • the FLT3-VH comprises the amino acid sequence of SEQ ID NO: 53
  • the FLT3-VL comprises the amino acid sequence of SEQ ID NO: 55, or
  • the FLT3-VH comprises the amino acid sequence of SEQ ID NO: 54
  • the FLT3-VL comprises the amino acid sequence of SEQ ID NO: 55, or
  • the FLT3-VH comprises the amino acid sequence of SEQ ID NO: 54
  • the FLT3-VL comprises the amino acid sequence of SEQ ID NO: 56, or
  • the FLT3-VH comprises the amino acid sequence of SEQ ID NO: 5
  • the FLT3-VL comprises the amino acid sequence of SEQ ID NO: 6;
  • said FLT3-VH comprises the amino acid sequence of SEQ ID NO: 45
  • said FLT3-VL comprises the amino acid sequence of SEQ ID NO: 50
  • the FLT3-VH comprises the amino acid sequence of SEQ ID NO: 45
  • the FLT3-VL comprises the amino acid sequence of SEQ ID NO: 49, or
  • the FLT3-VH comprises the amino acid sequence of SEQ ID NO: 46
  • the FLT3-VL comprises the amino acid sequence of SEQ ID NO: 49, or
  • the FLT3-VH comprises the amino acid sequence of SEQ ID NO: 47
  • the FLT3-VL comprises the amino acid sequence of SEQ ID NO: 49, or
  • the FLT3-VH comprises the amino acid sequence of SEQ ID NO: 48
  • the FLT3-VL comprises the amino acid sequence of SEQ ID NO: 49, or
  • the FLT3-VH comprises the amino acid sequence of SEQ ID NO: 46
  • the FLT3-VL comprises the amino acid sequence of SEQ ID NO: 50, or
  • the FLT3-VH comprises the amino acid sequence of SEQ ID NO: 47
  • the FLT3-VL comprises the amino acid sequence of SEQ ID NO: 50, or
  • the FLT3-VH comprises the amino acid sequence of SEQ ID NO: 48
  • the FLT3-VL comprises the amino acid sequence of SEQ ID NO: 50, or
  • the FLT3-VH comprises the amino acid sequence of SEQ ID NO: 45
  • the FLT3-VL comprises the amino acid sequence of SEQ ID NO: 51, or
  • the FLT3-VH comprises the amino acid sequence of SEQ ID NO: 46
  • the FLT3-VL comprises the amino acid sequence of SEQ ID NO: 51, or
  • the FLT3-VH comprises the amino acid sequence of SEQ ID NO: 47
  • the FLT3-VL comprises the amino acid sequence of SEQ ID NO: 51, or
  • the FLT3-VH comprises the amino acid sequence of SEQ ID NO: 48
  • the FLT3-VL comprises the amino acid sequence of SEQ ID NO: 51, or
  • the FLT3-VH comprises the amino acid sequence of SEQ ID NO: 3, and the FLT3-VL comprises the amino acid sequence of SEQ ID NO: 4; or
  • said FLT3-VH comprises the amino acid sequence of SEQ ID NO: 29
  • said FLT3-VL comprises the amino acid sequence of SEQ ID NO: 36
  • the FLT3-VH comprises the amino acid sequence of SEQ ID NO: 30, and the FLT3-VL comprises the amino acid sequence of SEQ ID NO: 36, or
  • the FLT3-VH comprises the amino acid sequence of SEQ ID NO: 31, and the FLT3-VL comprises the amino acid sequence of SEQ ID NO: 36, or
  • the FLT3-VH comprises the amino acid sequence of SEQ ID NO: 32
  • the FLT3-VL comprises the amino acid sequence of SEQ ID NO: 36, or
  • the FLT3-VH comprises the amino acid sequence of SEQ ID NO: 33
  • the FLT3-VL comprises the amino acid sequence of SEQ ID NO: 36, or
  • the FLT3-VH comprises the amino acid sequence of SEQ ID NO: 29
  • the FLT3-VL comprises the amino acid sequence of SEQ ID NO: 37, or
  • the FLT3-VH comprises the amino acid sequence of SEQ ID NO: 30, and the FLT3-VL comprises the amino acid sequence of SEQ ID NO: 37, or
  • the FLT3-VH comprises the amino acid sequence of SEQ ID NO: 31, and the FLT3-VL comprises the amino acid sequence of SEQ ID NO: 37, or
  • the FLT3-VH comprises the amino acid sequence of SEQ ID NO: 32
  • the FLT3-VL comprises the amino acid sequence of SEQ ID NO: 37, or
  • the FLT3-VH comprises the amino acid sequence of SEQ ID NO: 33
  • the FLT3-VL comprises the amino acid sequence of SEQ ID NO: 37, or
  • the FLT3-VH comprises the amino acid sequence of SEQ ID NO: 29
  • the FLT3-VL comprises the amino acid sequence of SEQ ID NO: 38, or
  • the FLT3-VH comprises the amino acid sequence of SEQ ID NO: 30, and the FLT3-VL comprises the amino acid sequence of SEQ ID NO: 38, or
  • the FLT3-VH comprises the amino acid sequence of SEQ ID NO: 31, and the FLT3-VL comprises the amino acid sequence of SEQ ID NO: 38, or
  • the FLT3-VH comprises the amino acid sequence of SEQ ID NO: 32
  • the FLT3-VL comprises the amino acid sequence of SEQ ID NO: 38, or
  • the FLT3-VH comprises the amino acid sequence of SEQ ID NO: 33
  • the FLT3-VL comprises the amino acid sequence of SEQ ID NO: 38, or
  • the FLT3-VH comprises the amino acid sequence of SEQ ID NO: 34
  • the FLT3-VL comprises the amino acid sequence of SEQ ID NO: 39, or
  • the FLT3-VH comprises the amino acid sequence of SEQ ID NO: 34
  • the FLT3-VL comprises the amino acid sequence of SEQ ID NO: 40, or
  • the FLT3-VH comprises the amino acid sequence of SEQ ID NO: 34
  • the FLT3-VL comprises the amino acid sequence of SEQ ID NO: 41, or
  • the FLT3-VH comprises the amino acid sequence of SEQ ID NO: 34
  • the FLT3-VL comprises the amino acid sequence of SEQ ID NO: 42, or
  • the FLT3-VH comprises the amino acid sequence of SEQ ID NO: 34
  • the FLT3-VL comprises the amino acid sequence of SEQ ID NO: 43, or
  • the FLT3-VH comprises the amino acid sequence of SEQ ID NO: 34
  • the FLT3-VL comprises the amino acid sequence of SEQ ID NO: 44, or
  • the FLT3-VH comprises the amino acid sequence of SEQ ID NO: 35
  • the FLT3-VL comprises the amino acid sequence of SEQ ID NO: 39, or
  • the FLT3-VH comprises the amino acid sequence of SEQ ID NO: 35
  • the FLT3-VL comprises the amino acid sequence of SEQ ID NO: 40, or
  • the FLT3-VH comprises the amino acid sequence of SEQ ID NO: 35
  • the FLT3-VL comprises the amino acid sequence of SEQ ID NO: 41, or
  • the FLT3-VH comprises the amino acid sequence of SEQ ID NO: 35
  • the FLT3-VL comprises the amino acid sequence of SEQ ID NO: 42, or
  • the FLT3-VH comprises the amino acid sequence of SEQ ID NO: 35
  • the FLT3-VL comprises the amino acid sequence of SEQ ID NO: 43, or
  • the FLT3-VH comprises the amino acid sequence of SEQ ID NO: 35
  • the FLT3-VL comprises the amino acid sequence of SEQ ID NO: 44, or
  • the FLT3-VH comprises the amino acid sequence of SEQ ID NO: 1
  • the FLT3-VL comprises the amino acid sequence of SEQ ID NO: 2.
  • the antigen binding molecule of any one of the preceding wherein
  • said FLT3-VH comprises the amino acid sequence of SEQ ID NO: 53
  • said FLT3-VL comprises the amino acid sequence of SEQ ID NO: 56
  • said FLT3-VH comprises the amino acid sequence of SEQ ID NO: 45
  • said FLT3-VL comprises the amino acid sequence of SEQ ID NO: 50.
  • an antigen binding molecule described herein comprises at least one antigen binding moiety that specifically binds FLT3 and at least one antigen binding moiety that specifically binds CD3, wherein:
  • the antigen-binding moiety specifically binding to FLT3 comprises FLT3-VH shown in SEQ ID NO: 53, 5, 52 or 54, and FLT3-VL shown in SEQ ID NO: 56, 6 or 55, or
  • the antigen-binding module specifically binding to FLT3 comprises FLT3-VH shown in SEQ ID NO: 45, 3, 46, 47 or 48, and FLT3 shown in SEQ ID NO: 50, 4, 49 or 51 -VL, or
  • the antigen-binding module specifically binding to FLT3 comprises the FLT3-VH shown in SEQ ID NO: 1, 29, 30, 31, 32, 33, 34 or 35, and SEQ ID NO: 2, 36, 37 , 38, 39, 40, 41, 42, 43 or 44 FLT3-VL;
  • the antigen-binding moiety that specifically binds FLT3 comprises FLT3-VH shown in SEQ ID NO: 53, 52 or 54, and FLT3-VL shown in SEQ ID NO: 56 or 55, or
  • the antigen-binding moiety specifically binding to FLT3 comprises FLT3-VH shown in SEQ ID NO: 45, 46, 47 or 48, and FLT3-VL shown in SEQ ID NO: 50, 49 or 51, or
  • the antigen-binding module that specifically binds FLT3 comprises FLT3-VH shown in SEQ ID NO: 29, 30, 31, 32, 33, 34 or 35, and SEQ ID NO: 36, 37, 38, 39 , 40, 41, 42, 43 or 44 FLT3-VL;
  • the antigen-binding module specifically binding to FLT3 comprises FLT3-VH shown in SEQ ID NO: 53, and FLT3-VL shown in SEQ ID NO: 56, or
  • the antigen-binding module specifically binding to FLT3 comprises the FLT3-VH shown in SEQ ID NO: 45, and the FLT3-VL shown in SEQ ID NO: 50.
  • the antigen binding molecule of any one of the preceding wherein
  • the antigen-binding module specifically binding to CD3 comprises a heavy chain variable region CD3-VH and a light chain variable region CD3-VL
  • the CD3-VH has: CD3-HCDR1 comprising the amino acid sequence of SEQ ID NO: 57, CD3-HCDR2 comprising the amino acid sequence of SEQ ID NO: 58, and CD3-HCDR3 comprising the amino acid sequence of SEQ ID NO: 59
  • the CD3-VL has: CD3-LCDR1 comprising the amino acid sequence of SEQ ID NO: 60
  • CD3-LCDR2 comprising the amino acid sequence of SEQ ID NO:61
  • CD3-LCDR3 comprising the amino acid sequence of SEQ ID NO:62.
  • the CD3-VH comprises the amino acid sequence of SEQ ID NO:63
  • the CD3-VL comprises the amino acid sequence of SEQ ID NO:64.
  • an antigen binding molecule described herein comprises at least one antigen binding moiety that specifically binds FLT3 and at least one antigen binding moiety that specifically binds CD3, wherein:
  • the antigen-binding module that specifically binds to CD3 comprises a heavy chain variable region CD3-VH and a light chain variable region CD3-VL
  • the CD3-VH comprises CD3-HCDR1 shown in SEQ ID NO: 57, SEQ ID NO : CD3-HCDR2 shown in 58, and CD3-HCDR3 shown in SEQ ID NO: 59
  • said CD3-VL comprises CD3-LCDR1 shown in SEQ ID NO: 60, CD3 shown in SEQ ID NO: 61 -LCDR2, and CD3-LCDR3 shown in SEQ ID NO: 62;
  • the antigen-binding module specifically binding to CD3 comprises CD3-VH shown in SEQ ID NO: 63, and CD3-VL shown in SEQ ID NO: 64.
  • the antigen binding molecule according to any one of the preceding, wherein the antigen binding molecule comprises an Fc region (including an IgG Fc region or an IgG 1 Fc region).
  • the Fc region comprises one or more amino acid substitutions that reduce binding to Fc receptors, particularly Fc ⁇ receptors, compared to wild-type Fc regions.
  • 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.
  • the antigen-binding molecule according to any one of the preceding, wherein the antigen-binding molecule comprises an Fc region comprising a first subunit (Fc1) and a second subunit (Fc1) capable of associating with each other ( Fc2), each of Fc1 and Fc2 independently has one or more amino acid substitutions that reduce homodimerization of the Fc region.
  • the Fc1 and Fc2 each independently have one or more amino acid substitutions according to the pestle-and-hole technique.
  • the Fc1 has a convex structure according to the knob-and-hole technique
  • the Fc2 has a pore structure according to the knob-and-hole technique.
  • the Fc1 has a pore structure according to the knob-and-hole technique
  • the Fc2 has a convex structure according to the knob-and-hole technique.
  • the amino acid residue at position 366 of the Fc1 is W; and the amino acid residue at position 366 of the Fc2 is S, the amino acid residue at position 368 is A, and the amino acid residue at position 407 is V, numbered according to for the EU index.
  • the amino acid residue at position 354 of the Fc1 is C; and the amino acid residue at position 349 of the Fc2 is C, numbered according to the EU index.
  • the Fcl has the amino acid sequence of SEQ ID NO:27
  • the Fc2 has the amino acid sequence of SEQ ID NO:28.
  • the antigen binding molecule of any one of the preceding wherein the antigen binding moiety that specifically binds FLT3 is a Fab, and the antigen binding moiety that specifically binds CD3 is a substituted Fab; or The antigen-binding moiety specifically binding to FLT3 is a substituted Fab, and the antigen-binding moiety specifically binding to CD3 is a Fab; the substituted Fab comprises a Titin chain and an Obscurin chain capable of forming a dimer.
  • the Titin chain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 85 to SEQ ID NO: 103
  • the Obscurin chain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 104 to SEQ ID NO: 144 The amino acid sequence of the group.
  • the Titin chain comprises the amino acid sequence of SEQ ID NO: 103
  • the Obscurin chain comprises the amino acid sequence of SEQ ID NO: 138.
  • the antigen-binding molecule according to any one of the preceding, wherein the antigen-binding molecule comprises an antigen-binding moiety that specifically binds to FLT3 and an antigen-binding moiety that specifically binds to CD3, and the antigen-binding moiety that specifically binds to FLT3
  • the antigen-binding moiety of is a Fab; the antigen-binding moiety that specifically binds CD3 is a substituted Fab.
  • the antigen-binding molecule according to any one of the preceding items comprises a first chain having a structure represented by formula (a), a second chain having a structure represented by formula (b), and a chain having a structure represented by formula (c) the third strand of the structure shown and a fourth strand with the structure shown in formula (d),
  • the structures represented by formulas (a), (b), (c) and (d) are arranged from N-terminal to C-terminal, and the linker 1 and the linker 2 are the same or different peptide linkers.
  • said Linker 1 and said Linker 2 have the same number of amino acid residues.
  • said Linker 1 and said Linker 2 are the same peptide linker.
  • said linker 1 and linker 2 comprise the amino acid sequence shown in SEQ ID NO: 66.
  • the antigen-binding molecule of any one of the preceding items has: a first chain comprising the amino acid sequence of SEQ ID NO: 72, a second chain comprising the amino acid sequence of SEQ ID NO: 73, A third strand comprising the amino acid sequence of SEQ ID NO:74 and a fourth strand comprising the amino acid sequence of SEQ ID NO:75.
  • the antigen-binding molecule of any one of the preceding wherein the antigen-binding molecule The subcontains two antigen-binding moieties specifically binding to FLT3 and one antigen-binding moiety specifically binding to CD3, the antigen-binding moiety specifically binding to FLT3 is a Fab; the antigen-binding moiety specifically binding to CD3 is replaced Fab.
  • the antigen-binding molecule according to any one of the preceding items comprises a first chain having a structure represented by formula (e), two second chains having a structure represented by formula (b), and a chain having a structure represented by formula (b).
  • the 3rd chain of structure shown in formula (f) and a 4th chain with structure shown in formula (g) comprises a first chain having a structure represented by formula (e), two second chains having a structure represented by formula (b), and a chain having a structure represented by formula (b).
  • linker 3 comprises the amino acid sequence shown in SEQ ID NO: 146
  • the linker 4 comprises the amino acid sequence shown in SEQ ID NO: 145
  • the linker 5 Comprising the amino acid sequence shown in SEQ ID NO:66.
  • the antigen-binding molecule according to any one of the preceding items has: a first chain comprising the amino acid sequence of SEQ ID NO: 68, two second chains comprising the amino acid sequence of SEQ ID NO: 69 , a third strand comprising the amino acid sequence of SEQ ID NO:70 and a fourth strand comprising the amino acid sequence of SEQ ID NO:71.
  • the present disclosure also provides an isolated antibody capable of specifically binding to FLT3, said antibody comprising a heavy chain variable region FLT3-VH and a light chain variable region FLT3-VL, wherein
  • the FLT3-VH has: FLT3-HCDR1 comprising the amino acid sequence of SEQ ID NO: 19, FLT3-HCDR2 comprising the amino acid sequence of SEQ ID NO: 20, and FLT3-HCDR2 comprising the amino acid sequence of SEQ ID NO: 21 HCDR3, and the FLT3-VL has: FLT3-LCDR1 comprising the amino acid sequence of SEQ ID NO: 83 or 22, FLT3-LCDR2 comprising the amino acid sequence of SEQ ID NO: 84 or 23 and comprising the amino acid of SEQ ID NO: 24 sequence FLT3-LCDR3, or
  • the FLT3-VH has: FLT3-HCDR1 comprising the amino acid sequence of SEQ ID NO: 13, FLT3-HCDR2 comprising the amino acid sequence of SEQ ID NO: 14, and FLT3-HCDR2 comprising the amino acid sequence of SEQ ID NO: 15 HCDR3, and the FLT3-VL has: FLT3-LCDR1 comprising the amino acid sequence of SEQ ID NO: 16, FLT3-LCDR2 comprising the amino acid sequence of SEQ ID NO: 17, and FLT3-LCDR2 comprising the amino acid sequence of SEQ ID NO: 18 LCDR3, or
  • an antibody provided herein comprises a heavy chain variable region FLT3-VH and a light chain variable region FLT3-VL, wherein
  • the FLT3-VH comprises FLT3-HCDR1 shown in SEQ ID NO: 19, FLT3-HCDR2 shown in SEQ ID NO: 20 and FLT3-HCDR3 shown in SEQ ID NO: 21, and the FLT3-
  • the VL comprises FLT3-LCDR1 set forth in SEQ ID NO: 83 or 22, FLT3-LCDR2 set forth in SEQ ID NO: 84 or 23, and FLT3-LCDR3 set forth in SEQ ID NO: 24, or
  • the FLT3-VH comprises FLT3-HCDR1 shown in SEQ ID NO: 13, FLT3-HCDR2 shown in SEQ ID NO: 14 and FLT3-HCDR3 shown in SEQ ID NO: 15, and the FLT3- The VL comprises FLT3-LCDR1 set forth in SEQ ID NO: 16, FLT3-LCDR2 set forth in SEQ ID NO: 17, and FLT3-LCDR3 set forth in SEQ ID NO: 18, or
  • said FLT3-VH comprises FLT3-HCDR1 shown in SEQ ID NO: 7, FLT3-HCDR2 shown in SEQ ID NO: 8, 76 or 77, and FLT3-HCDR3 shown in SEQ ID NO: 9, and
  • the FLT3-VL comprises FLT3-LCDR1 shown in SEQ ID NO: 10, 78, 79 or 80, FLT3-LCDR2 shown in SEQ ID NO: 11, 81 or 82, and FLT3-LCDR2 shown in SEQ ID NO: 12 LCDR3.
  • the aforementioned antibody wherein the FLT3-VH has: FLT3-HCDR1 comprising the amino acid sequence of SEQ ID NO: 19, FLT3-HCDR2 comprising the amino acid sequence of SEQ ID NO: 20 and comprising The FLT3-HCDR3 of the amino acid sequence of SEQ ID NO: 21, and the FLT3-VL has: FLT3-LCDR1 comprising the amino acid sequence of SEQ ID NO: 83, FLT3-LCDR2 comprising the amino acid sequence of SEQ ID NO: 84 and comprising FLT3-LCDR3 of the amino acid sequence of SEQ ID NO:24.
  • the FLT3-HCDR1, FLT3-HCDR2, FLT3-HCDR3, FLT3-LCDR1, FLT3-LCDR2, and FLT3-LCDR3 are defined according to the Kabat numbering convention.
  • the antibody according to any one of the preceding which binds to human FLT3 with a KD of less than 1 ⁇ 10 -8 M, 5 ⁇ 10 -9 M at 25°C, said KD is obtained by surface plasmon measured by the resonance method.
  • said FLT3-VH comprises the amino acid sequence of SEQ ID NO: 53, 5, 52 or 54
  • said FLT3-VL comprises the amino acid sequence of SEQ ID NO: 56, 6 or 55, or
  • said FLT3-VH comprises the amino acid sequence of SEQ ID NO: 45, 3, 46, 47 or 48
  • said FLT3-VL comprises the amino acid sequence of SEQ ID NO: 50, 4, 49 or 51, or
  • said FLT3-VH comprises the amino acid sequence of SEQ ID NO: 1, 29, 30, 31, 32, 33, 34 or 35
  • said FLT3-VL comprises SEQ ID NO: 2, 36, 37, 38 , 39, 40, 41, 42, 43 or 44 amino acid sequence.
  • said FLT3-VH comprises the amino acid sequence of SEQ ID NO: 53, 52 or 54
  • said FLT3-VL comprises the amino acid sequence of SEQ ID NO: 56 or 55, or
  • said FLT3-VH comprises the amino acid sequence of SEQ ID NO: 45, 46, 47 or 48
  • said FLT3-VL comprises the amino acid sequence of SEQ ID NO: 50, 49 or 51, or
  • said FLT3-VH comprises the amino acid sequence of SEQ ID NO: 29, 30, 31, 32, 33, 34 or 35
  • said FLT3-VL comprises SEQ ID NO: 36, 37, 38, 39, 40 , 41, 42, 43 or 44 amino acid sequence.
  • said FLT3-VH comprises the amino acid sequence of SEQ ID NO: 53
  • said FLT3-VL comprises the amino acid sequence of SEQ ID NO: 56
  • the FLT3-VH comprises the amino acid sequence of SEQ ID NO: 52
  • the FLT3-VL comprises the amino acid sequence of SEQ ID NO: 55, or
  • the FLT3-VH comprises the amino acid sequence of SEQ ID NO: 52
  • the FLT3-VL comprises the amino acid sequence of SEQ ID NO: 56, or
  • the FLT3-VH comprises the amino acid sequence of SEQ ID NO: 53
  • the FLT3-VL comprises the amino acid sequence of SEQ ID NO: 55, or
  • the FLT3-VH comprises the amino acid sequence of SEQ ID NO: 54
  • the FLT3-VL comprises the amino acid sequence of SEQ ID NO: 55, or
  • the FLT3-VH comprises the amino acid sequence of SEQ ID NO: 54
  • the FLT3-VL comprises the amino acid sequence of SEQ ID NO: 56, or
  • the FLT3-VH comprises the amino acid sequence of SEQ ID NO: 5
  • the FLT3-VL comprises the amino acid sequence of SEQ ID NO: 6;
  • said FLT3-VH comprises the amino acid sequence of SEQ ID NO: 45
  • said FLT3-VL comprises the amino acid sequence of SEQ ID NO: 50
  • the FLT3-VH comprises the amino acid sequence of SEQ ID NO: 45
  • the FLT3-VL comprises the amino acid sequence of SEQ ID NO: 49, or
  • the FLT3-VH comprises the amino acid sequence of SEQ ID NO: 46
  • the FLT3-VL comprises the amino acid sequence of SEQ ID NO: 49, or
  • the FLT3-VH comprises the amino acid sequence of SEQ ID NO: 47
  • the FLT3-VL comprises the amino acid sequence of SEQ ID NO: 49, or
  • the FLT3-VH comprises the amino acid sequence of SEQ ID NO: 48
  • the FLT3-VL comprises the amino acid sequence of SEQ ID NO: 49, or
  • the FLT3-VH comprises the amino acid sequence of SEQ ID NO: 46
  • the FLT3-VL comprises the amino acid sequence of SEQ ID NO: 50, or
  • the FLT3-VH comprises the amino acid sequence of SEQ ID NO: 47
  • the FLT3-VL comprises the amino acid sequence of SEQ ID NO: 50, or
  • the FLT3-VH comprises the amino acid sequence of SEQ ID NO: 48
  • the FLT3-VL comprises the amino acid sequence of SEQ ID NO: 50, or
  • the FLT3-VH comprises the amino acid sequence of SEQ ID NO: 45
  • the FLT3-VL comprises the amino acid sequence of SEQ ID NO: 51, or
  • the FLT3-VH comprises the amino acid sequence of SEQ ID NO: 46
  • the FLT3-VL comprises the amino acid sequence of SEQ ID NO: 51, or
  • the FLT3-VH comprises the amino acid sequence of SEQ ID NO: 47
  • the FLT3-VL comprises the amino acid sequence of SEQ ID NO: 51, or
  • the FLT3-VH comprises the amino acid sequence of SEQ ID NO: 48
  • the FLT3-VL comprises the amino acid sequence of SEQ ID NO: 51, or
  • the FLT3-VH comprises the amino acid sequence of SEQ ID NO: 3, and the FLT3-VL comprises the amino acid sequence of SEQ ID NO: 4; or
  • said FLT3-VH comprises the amino acid sequence of SEQ ID NO: 29
  • said FLT3-VL comprises the amino acid sequence of SEQ ID NO: 36
  • the FLT3-VH comprises the amino acid sequence of SEQ ID NO: 30, and the FLT3-VL comprises the amino acid sequence of SEQ ID NO: 36, or
  • the FLT3-VH comprises the amino acid sequence of SEQ ID NO: 31, and the FLT3-VL comprises the amino acid sequence of SEQ ID NO: 36, or
  • the FLT3-VH comprises the amino acid sequence of SEQ ID NO: 32
  • the FLT3-VL comprises the amino acid sequence of SEQ ID NO: 36, or
  • the FLT3-VH comprises the amino acid sequence of SEQ ID NO: 33
  • the FLT3-VL comprises the amino acid sequence of SEQ ID NO: 36, or
  • the FLT3-VH comprises the amino acid sequence of SEQ ID NO: 29
  • the FLT3-VL comprises the amino acid sequence of SEQ ID NO: 37, or
  • the FLT3-VH comprises the amino acid sequence of SEQ ID NO: 30, and the FLT3-VL comprises the amino acid sequence of SEQ ID NO: 37, or
  • the FLT3-VH comprises the amino acid sequence of SEQ ID NO: 31, and the FLT3-VL comprises the amino acid sequence of SEQ ID NO: 37, or
  • the FLT3-VH comprises the amino acid sequence of SEQ ID NO: 32
  • the FLT3-VL comprises the amino acid sequence of SEQ ID NO: 37, or
  • the FLT3-VH comprises the amino acid sequence of SEQ ID NO: 33
  • the FLT3-VL comprises the amino acid sequence of SEQ ID NO: 37, or
  • the FLT3-VH comprises the amino acid sequence of SEQ ID NO: 29
  • the FLT3-VL comprises the amino acid sequence of SEQ ID NO: 38, or
  • the FLT3-VH comprises the amino acid sequence of SEQ ID NO: 30, and the FLT3-VL comprises the amino acid sequence of SEQ ID NO: 38, or
  • the FLT3-VH comprises the amino acid sequence of SEQ ID NO: 31, and the FLT3-VL comprises the amino acid sequence of SEQ ID NO: 38, or
  • the FLT3-VH comprises the amino acid sequence of SEQ ID NO: 32
  • the FLT3-VL comprises the amino acid sequence of SEQ ID NO: 38, or
  • the FLT3-VH comprises the amino acid sequence of SEQ ID NO: 33
  • the FLT3-VL comprises the amino acid sequence of SEQ ID NO: 38, or
  • the FLT3-VH comprises the amino acid sequence of SEQ ID NO: 34
  • the FLT3-VL comprises the amino acid sequence of SEQ ID NO: 39, or
  • the FLT3-VH comprises the amino acid sequence of SEQ ID NO: 34
  • the FLT3-VL comprises the amino acid sequence of SEQ ID NO: 40, or
  • the FLT3-VH comprises the amino acid sequence of SEQ ID NO: 34
  • the FLT3-VL comprises the amino acid sequence of SEQ ID NO: 41, or
  • the FLT3-VH comprises the amino acid sequence of SEQ ID NO: 34
  • the FLT3-VL comprises the amino acid sequence of SEQ ID NO: 42, or
  • the FLT3-VH comprises the amino acid sequence of SEQ ID NO: 34
  • the FLT3-VL comprises the amino acid sequence of SEQ ID NO: 43, or
  • the FLT3-VH comprises the amino acid sequence of SEQ ID NO: 34
  • the FLT3-VL comprises the amino acid sequence of SEQ ID NO: 44, or
  • the FLT3-VH comprises the amino acid sequence of SEQ ID NO: 35
  • the FLT3-VL comprises the amino acid sequence of SEQ ID NO: 39, or
  • the FLT3-VH comprises the amino acid sequence of SEQ ID NO: 35
  • the FLT3-VL comprises the amino acid sequence of SEQ ID NO: 40, or
  • the FLT3-VH comprises the amino acid sequence of SEQ ID NO: 35
  • the FLT3-VL comprises the amino acid sequence of SEQ ID NO: 41, or
  • the FLT3-VH comprises the amino acid sequence of SEQ ID NO: 35
  • the FLT3-VL comprises the amino acid sequence of SEQ ID NO: 42, or
  • the FLT3-VH comprises the amino acid sequence of SEQ ID NO: 35
  • the FLT3-VL comprises the amino acid sequence of SEQ ID NO: 43, or
  • the FLT3-VH comprises the amino acid sequence of SEQ ID NO: 35
  • the FLT3-VL comprises the amino acid sequence of SEQ ID NO: 44, or
  • the FLT3-VH comprises the amino acid sequence of SEQ ID NO: 1
  • the FLT3-VL comprises the amino acid sequence of SEQ ID NO: 2.
  • said FLT3-VH comprises the amino acid sequence of SEQ ID NO: 53
  • said FLT3-VL comprises the amino acid sequence of SEQ ID NO: 56
  • said FLT3-VH comprises the amino acid sequence of SEQ ID NO: 45
  • said FLT3-VL comprises the amino acid sequence of SEQ ID NO: 50.
  • provided herein is an isolated antibody wherein
  • said antibody comprises the FLT3-VH shown in SEQ ID NO: 53, 5, 52 or 54, and the FLT3-VL shown in SEQ ID NO: 56, 6 or 55, or
  • the antibody comprises the FLT3-VH shown in SEQ ID NO: 45, 3, 46, 47 or 48, and the FLT3-VL shown in SEQ ID NO: 50, 4, 49 or 51, or
  • the antibody comprises FLT3-VH shown in SEQ ID NO: 1, 29, 30, 31, 32, 33, 34 or 35, and SEQ ID NO: 2, 36, 37, 38, 39, 40, FLT3-VL as shown in 41, 42, 43 or 44;
  • the antibody comprises FLT3-VH shown in SEQ ID NO: 53, 52 or 54, and FLT3-VL shown in SEQ ID NO: 56 or 55, or
  • the antibody comprises the FLT3-VH shown in SEQ ID NO: 45, 46, 47 or 48, and the FLT3-VL shown in SEQ ID NO: 50, 49 or 51, or
  • the antibody comprises FLT3-VH shown in SEQ ID NO: 29, 30, 31, 32, 33, 34 or 35, and SEQ ID NO: 36, 37, 38, 39, 40, 41, 42, FLT3-VL as shown in 43 or 44;
  • the antibody comprises FLT3-VH shown in SEQ ID NO: 53, and FLT3-VL shown in SEQ ID NO: 56, or
  • the antibody comprises FLT3-VH shown in SEQ ID NO: 45, and FLT3-VL shown in SEQ ID NO: 50.
  • the antibody of any one of the preceding items, wherein said antibody is a bispecific antibody.
  • the bispecific antibody specifically binds FLT3 and CD3.
  • the present disclosure provides a pharmaceutical composition
  • a pharmaceutical composition comprising: a therapeutically effective amount of the antigen-binding molecule of any of the foregoing or the antibody of any of the foregoing, and one or more pharmaceutical acceptable carrier, diluent, buffer or excipient.
  • the pharmaceutical composition further comprises at least one second therapeutic agent.
  • the present disclosure also provides an isolated nucleic acid encoding the antigen-binding molecule of any of the foregoing or the antibody of any of the foregoing.
  • the present disclosure also provides a host cell comprising the aforementioned isolated nucleic acid.
  • the present disclosure also provides a method for treating or preventing a disease, the method comprising administering to a subject in need a therapeutically effective amount of any of the aforementioned antigen-binding molecules or any of the aforementioned antigen-binding molecules. Said antibody or composition thereof.
  • the present disclosure also provides the use of the antigen-binding molecule described in any one of the foregoing or the antibody or composition thereof in the preparation of a drug for treating or preventing diseases.
  • the present disclosure also provides the antigen-binding molecule of any one of the foregoing or the antibody of any one of the foregoing or a composition thereof for use as a medicament.
  • the medicament is used to treat or prevent a disease.
  • the disease of any one of the preceding is a proliferative disease, a tumor, or an immune disease.
  • the disease of any of the preceding is a tumor or cancer.
  • the disease of any one of the preceding is multiple myeloma, malignant plasmacytoma, Hodgkin's lymphoma, nodular lymphocyte-predominant Hodgkin's lymphoma, Kahler's myeloma, acute monocytic leukemia, plasma cell leukemia, plasmacytoma, B prolymphocytic leukemia, hairy cell leukemia, B cell non-Hodgkin's lymphoma (NHL), acute myeloid leukemia (AML) , chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), chronic myeloid leukemia (CML), follicular lymphoma, Burkitt's lymphoma, marginal zone lymphoma, mantle cell lymphoma, Large cell lymphoma, precursor B-lymphoblastic lymphoma, myeloid leukemia, Waldenstrom macroglobulinemia, diffuse large B-cell lymph
  • the aforementioned disease is a disease associated with FLT3; in some embodiments, the aforementioned disease is a disease expressing FLT3.
  • the antigen-binding molecule provided by the present disclosure has the characteristics of good therapeutic activity, safety, pharmacokinetic properties and druggability (such as stability).
  • Figure 1A Schematic diagram of structure-1
  • Figure 1B Schematic diagram of structure-2.
  • 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, 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. It is contemplated that methods other than genetic engineering to alter amino acid side chain groups, such as chemical modification, are also available. Various names may be used herein to refer to the same amino acid mutation.
  • 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 position 366 is mutated from the original T to W.
  • antigen-binding molecule is used in the broadest sense and encompasses 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, as long as they exhibit the desired antigen-binding activity.
  • the antigen binding molecules herein comprise a variable region (VH) and a variable region (VL), which together constitute an antigen binding domain.
  • VH variable region
  • VL variable region
  • the antigen-binding molecules herein are bispecific antigen-binding molecules (eg, bispecific antibodies).
  • antibody is used in the broadest sense and encompasses various antibody structures including, but not limited to, monoclonal antibodies antibodies, polyclonal antibodies; monospecific antibodies, multispecific antibodies (such as bispecific antibodies), full-length antibodies, and antibody fragments (or antigen-binding fragments, or antigen-binding portions), so long as they exhibit the desired antigen-binding activity .
  • native IgG antibodies are heterotetrameric glycoproteins of approximately 150,000 Daltons composed of two identical light chains and two identical heavy chains joined by disulfide bonds. From N to C-terminus, each heavy chain has a variable region (VH), also called variable heavy domain, heavy chain variable region, followed by three constant domains (CH1, CH2 and CH3). Similarly, from N to C-terminus, each light chain has a variable region (VL), also called variable light domain, or light chain variable domain, followed by a constant light domain (light chain constant region, CL ).
  • 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 at least two different epitopes of the same antigen.
  • Bispecific antibodies of various structures have been disclosed in the prior art, which can be divided into IgG-like bispecific antibodies and antibody fragment bispecific antibodies according to the integrity of the IgG molecule, and can be divided into bivalent bispecific antibodies according to the number of antigen-binding regions.
  • trivalent, tetravalent or more valent bispecific antibodies according to whether the structure is symmetrical, can be divided into symmetrical structure bispecific antibodies and asymmetric structure bispecific antibodies.
  • bispecific antibodies based on antibody fragments such as Fab fragments lacking Fc fragments, which form bispecific antibodies by combining two or more Fab fragments in one molecule, which have lower immunogenicity, and Small molecular weight, high tumor tissue permeability, typical antibody structures of this type such as F(ab)2, scFv-Fab, (scFv)2-Fab; IgG-like bispecific antibodies (for example, with Fc fragments),
  • This type of antibody has a relatively large molecular weight.
  • the Fc fragment is helpful for the purification of the antibody and improves its solubility and stability.
  • the Fc part may also bind to the receptor FcRn to increase the serum half-life of the antibody.
  • a typical bispecific antibody structure model Such as KiH, CrossMAb, Triomab quadroma, Fc ⁇ Adp, ART-Ig, BiMAb, Biclonics, BEAT, DuoBody, Azymetric, XmAb, 2:1TCBs, 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-DART-Fc, (scFv)4 -Fc, CODV-Ig, mAb2, F(ab)4-CrossMAb, etc. (see Aran F.Labrijn et al., Nature Reviews Drug Discovery volume 18, pages585–608(2019); Chen S1 et al., J Immunol
  • variable region refers to the antigen-binding domain of an antigen-binding molecule.
  • the heavy chain variable region in the antigen-binding module that specifically binds to FLT3 is designated as FLT3-VH
  • the light chain variable region is designated as FLT3-VL
  • the heavy-chain variable region in the antigen-binding module that specifically binds to CD3 Denoted as CD3-VH and light chain variable region as CD3-VL.
  • VH and VL each contain four conserved framework regions (FRs) and three complementarity determining regions (CDRs).
  • CDR complementarity determining region
  • framework 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.
  • the three CDR regions in FLT3-VH are marked as FLT3-HCDR1, FLT3-HCDR2 and FLT3-HCDR3; the three CDR regions in FLT3-VL are marked as FLT3-LCDR1, FLT3-LCDR2 and FLT3-LCDR3 ;
  • the three CDR regions in CD3-VH are marked as CD3-HCDR1, CD3-HCDR2 and CD3-HCDR3 respectively;
  • the three CDR regions in CD3-VL Labeled as CD3-LCDR1, CD3-LCDR2 and CD3-LCDR3, respectively.
  • Each VH and VL is sequenced from N-terminus to C-terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • a single VH or VL may be sufficient to confer antigen binding specificity.
  • 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].
  • Kabat numbering convention is applicable to the variable regions and CDR sequences in the examples of the present disclosure.
  • 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 molecule (e.g. scFv); and multispecific antibodies formed from antibody fragments.
  • Fab fragment of a monovalent antibody consisting of VL, VH, CL and CH1 domains; herein, an antibody, such as an anti-FLT3 antibody
  • the Fab monovalent antibody fragment can be linked to the Fc1 subunit through the C-terminal of its CH1, and the formed Fab-Fc1 can be further linked to another antibody, such as an anti-CD3 antibody, through a disulfide bond at the N-terminal of the Fc1 subunit.
  • Fab-Fc2 forms bispecific antibodies.
  • 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 native sequence Fc regions for the antibodies described herein include human IgGl, IgG2 (IgG2A, IgG2B), IgG3 and IgG4. Unless otherwise stated, the numbering convention for the Fc region is the EU index.
  • Titin chain refers to a section of Titin protein that is 78-118 amino acids in length and contains Titin Ig- Like 152 domain peptides or functional variants thereof, the Titin chain can combine with Obscurin Ig-like 1 or Obscurin-like Ig-like 1 domain to form a dimerization complex.
  • Obscurin chain refers to a peptide segment of 87-117 amino acids on the Obscurin protein that contains the Obscurin Ig-like 1 domain or a functional variant thereof, or a segment of the Obscurin-like 1 protein that is 78-118 amino acids in length
  • An amino acid peptide segment comprising an Obscurin-like Ig-like 1 domain or a functional variant thereof, the Obscurin chain can combine with a Titin Ig-like 152 domain to form a dimerization complex.
  • the Titin chain and Obscurin chain disclosed herein can be used to replace CH1 and CL in Fab to form a substituted Fab (Fab-S), and the replacement does not affect the binding of the antigen-binding molecule to the antigen.
  • chimeric antibody refers to an antibody in which a portion of the heavy and/or light chains is derived from a particular source or species, while the remaining portion of the heavy and/or light chains is derived from a different source or species.
  • humanized antibody is an antibody that retains the reactivity of a non-human antibody while being less immunogenic in humans. This 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).
  • affinity refers to the overall strength of the non-covalent interaction between a single binding site of a molecule (eg, an antibody) 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 equilibrium dissociation constant
  • KD refers to the equilibrium dissociation constant, which is obtained from the ratio of kd to ka (ie, kd/ka) and is expressed as molarity (M). KD values for antibodies can be determined using methods known in the art, such as surface plasmon resonance, ELISA, or solution equilibrium titration (SET).
  • the term “monoclonal antibody” refers to a population of substantially homogeneous antibodies, ie, the antibody molecules comprised in the population are identical in amino acid sequence, 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 bound by a selective binding agent of an antigen binding protein (eg, an antibody).
  • An antigen may have one or more epitopes capable of interacting with different antigen binding proteins (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 spatially separated by folding of the antigen (i.e. by tertiary folding of the antigen in proteinaceous nature). near.
  • the difference between conformational epitopes and linear epitopes is: In the presence of solvent, antibody binding to conformational epitopes is lost.
  • 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.
  • Screening for antibodies that bind a particular epitope can be performed using methods routine in the art, such as, but not limited to, alanine scanning, peptide blotting (see Meth. Mol. Biol. 248 (2004) 443 -463), peptide cleavage analysis, epitope excision, epitope extraction, chemical modification of antigen (see Prot.Sci.9 (2000) 487-496), and cross-blocking (see “Antibodies”, Harlow and Lane (Cold Spring Harbor Press, Cold Spring Harb., NY)).
  • an antibody is capable of binding to a certain antigen or epitope with a higher affinity than to other antigens or epitopes.
  • an antibody binds an antigen or epitope with an equilibrium dissociation constant (KD) of about 1 x 10 -8 M or less (eg, about 5 x 10 -9 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, such as by FACS or surface plasmon resonance assays.
  • antibodies that specifically bind to an antigen or an epitope within an antigen may have cross-reactivity to other related antigens, e.g. (cynomolgus, cyno), chimpanzee (Pan troglodytes) (chimpanzee, chimp)) or marmoset (Callithrix jacchus) (commonmarmoset, marmoset) are cross-reactive.
  • related antigens e.g. (cynomolgus, cyno), chimpanzee (Pan troglodytes) (chimpanzee, chimp)) or marmoset (Callithrix jacchus) (commonmarmoset, marmoset) are cross-reactive.
  • non-binding means that the antibody cannot bind to a certain antigen or an epitope within the antigen in the above-mentioned specific binding manner.
  • KD equilibrium dissociation constant
  • antigen binding moiety refers to a polypeptide molecule that specifically binds an antigen of interest.
  • Particular antigen binding moieties include the antigen binding domain of an antibody, eg, comprising a heavy chain variable region and a light chain variable region.
  • antigen binding moiety that specifically binds FLT3 refers to a moiety that is capable of binding FLT3 with sufficient affinity such that molecules containing this moiety are useful as diagnostic and/or therapeutic agents targeting FLT3.
  • an antigen binding moiety that specifically binds FLT3 has an equilibrium dissociation constant (KD) of ⁇ about 10 nM, as measured by a surface plasmon resonance assay.
  • Antigen binding moieties include antibody fragments as defined herein, eg Fab, substituted Fab or scFv.
  • linker refers to a linking unit that joins two polypeptide fragments.
  • linkers appearing in the same formula 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.
  • Tm is the melting denaturation temperature (intrinsic fluorescence). When the protein is denatured (heating or denaturant action), the tertiary structure is opened, and the microenvironment of the aromatic amino acid changes, resulting in a change in the emission fluorescence spectrum.
  • Tm1 refers to the temperature at which the fluorescence changes to half of the maximum value.
  • Tonset is the denaturation initiation temperature. It means the temperature at which the protein begins to denature, that is, the temperature at which the fluorescence value begins to change.
  • Tagg is the aggregation onset temperature. By static light scattering, at two wavelengths of 266nm and 473nm Under Detect Aggregation, monitor the temperature at which the sample begins to aggregate. Tagg 266 refers to the aggregation initiation temperature monitored at 266nm.
  • 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.
  • 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 the antigen-binding molecule refers to one or more nucleic acid molecules encoding the antibody heavy and light chains (or fragments thereof), including such one or more nucleic acids in a single vector or in separate vectors molecule, and such one or more nucleic acid molecules present at one or more locations in the host cell.
  • a particular 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.
  • 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. During the alignment process, gaps may be introduced as necessary to obtain the maximum percent sequence identity, but any conservative substitutions are not considered to form part of the sequence identity. To determine percent 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” refers to the covalent linking of components, such as an antigen binding module and an Fc domain, directly or via a linker.
  • 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 suitable for transforming a host cell and containing the expression of one or more heterologous coding regions operatively linked thereto and/or controlling (along with the host cell).
  • 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 that have the same function or biological activity as the cells screened or selected for among 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 cells of this patent do not include objects that are not authorized under the patent law.
  • the expressions "cell”, “cell line” and “cell culture” are used interchangeably and all such designations include progeny.
  • the words “transformants” and “transformed “Cell” includes primary subject cells and cultures derived therefrom, regardless of the number of passages. It is also understood that not all progeny will have the exact same DNA content due to deliberate or unintentional mutations .comprising mutant progeny that have the same function or biological activity as the original transformed cell from which they were screened.
  • composition means a mixture comprising one or more of the antigen binding molecules or antibodies described herein and other chemical components such as physiological/pharmaceutical acceptable carriers and excipients.
  • pharmaceutically acceptable carrier refers to an ingredient in a pharmaceutical preparation that is different from the active ingredient and is non-toxic to the subject.
  • Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers or preservatives.
  • 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 expressly stated otherwise.
  • 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 of similar fluids, cells, or tissues isolated from a subject, as well as fluids, cells, or tissues present in a subject.
  • exemplary samples are biological fluids such as blood, serum and serosal fluids, plasma, lymph, urine, saliva, cystic fluid, tears, faeces, sputum, mucous membrane secretions of secretory tissues and organs, vaginal secretions, ascites , pleura, pericardium, peritoneum, peritoneal and other body cavity fluids, fluid collected from bronchial lavage, synovial fluid, liquid solutions in contact with subjects or biological sources, such as cell and organ culture media (including cell or organ condition culture medium), lavage fluid, etc., tissue biopsy samples, fine needle aspirations, surgically resected tissues, organ cultures, or cell cultures.
  • biological fluids such as blood, serum and serosal fluids, plasma, lymph, urine, saliva, cystic fluid, tears, faeces, sputum, mucous membrane
  • Treatment refers to clinical intervention intended to be applied to the 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 (e.g. lung disease).
  • 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 prevent, hinder, delay or reverse the disease state or in any way related to the disease state The amount of progression of any other undesirable symptoms associated with the disease.
  • 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 .
  • a complete therapeutic or prophylactic effect does not necessarily occur after one dose, but may occur after 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.
  • Antigen binding molecules of the present disclosure are provided.
  • the present disclosure provides antigen-binding molecules that have many favorable properties, such as high affinity for FLT3 and FLT3-expressing cells, in vitro killing activity, therapeutic activity, safety, pharmacokinetic properties, and druggability (such as yield, purity and stability, etc.).
  • Antigen-binding molecules of the present disclosure include bispecific antigen-binding molecules (eg, bispecific antibodies) and anti-FLT3 antibodies that specifically bind FLT3 and CD3.
  • the antigen-binding molecules of the present disclosure have any of the following properties:
  • the antigen-binding molecule binds to human FLT3 with a KD of less than 1 ⁇ 10 -7 M, 1 ⁇ 10 -8 M, and 5 ⁇ 10 -9 M at 25°C, and the KD is measured by surface plasmon resonance .
  • the antibodies bind cell surface FLT3 with an EC50 of less than 10 pM , 1 pM or 0.5 pM, as measured by ELISA.
  • the cells in question are Monomac-6.
  • the present disclosure provides an antigen-binding molecule comprising at least one antigen-binding moiety specifically binding to FLT3 and at least one antigen-binding moiety specifically binding to CD3, the antigen-binding moiety specifically binding to FLT3 comprising FLT3-VH and FLT3 - VL, said antigen binding moiety specifically binding to CD3 comprising CD3-VH and CD3-VL.
  • the present disclosure also provides an isolated antibody capable of specifically binding to FLT3, said antibody comprising FLT3-VH and FLT3-VL.
  • the Examples herein disclose antibody series 18, 99 and 125.
  • the antigen-binding molecule containing antibody 125 will be described below as an example.
  • the FLT3-VH of the antigen-binding molecule or anti-FLT3 antibody that specifically binds to FLT3 and CD3 has: FLT3-HCDR1 having an amino acid sequence as shown in SEQ ID NO: 19, FLT3-HCDR1 having an amino acid sequence as shown in SEQ ID NO: 20 HCDR2 and the amino acid sequence of FLT3-HCDR3 as shown in SEQ ID NO: 21, and the FLT3-VL has: the amino acid sequence of FLT3-LCDR1 as shown in SEQ ID NO: 83 or 22, the amino acid sequence as shown in SEQ ID NO: 84 Or FLT3-LCDR2 shown in 23 and amino acid sequence such as SEQ ID NO: FLT3-LCDR3 shown in 24.
  • the FLT3-VH has: the amino acid sequence of FLT3-HCDR1 shown in SEQ ID NO: 19, the amino acid sequence of FLT3-HCDR2 shown in SEQ ID NO: 20 and the amino acid sequence of SEQ ID NO:
  • the FLT3-HCDR3 shown in 21, and the FLT3-VL have: the amino acid sequence of FLT3-LCDR1 shown in SEQ ID NO: 83, the amino acid sequence of FLT3-LCDR2 shown in SEQ ID NO: 84, and the amino acid sequence of SEQ ID NO: 84 ID NO: FLT3-LCDR3 shown in 24.
  • the antigen-binding molecule or antibody as described above, the FLT3-VH and/or the FLT3-VL is murine or humanized. In some embodiments, the FLT3-VH and/or the FLT3-VL are humanized.
  • FR1, FR2 and FR3 of the humanized FLT3-VH have at least 60%, 70% or 80% sequence identity to FR1, FR2 and FR3 of SEQ ID NO: 5, said FR4 of humanized FLT3-VH has at least 80% or 90% sequence identity to FR4 of SEQ ID NO: 5, and FR1, FR2 and FR3 of said humanized FLT3-VL are identical to SEQ ID NO: 6 FR1, FR2 and FR3 have at least 60%, 70% or 80% sequence identity and/or FR4 of said humanized FLT3-VL has at least 80% or 90% sequence identity with FR4 of SEQ ID NO:6 sequence identity.
  • the FLT3-VH has FR1, FR2, FR3 derived from IGHV1-46*01 and FR4 derived from IGHJ1*01, and it is unsubstituted or has a compound selected from 1E, 37L, 48I , 67A, 69W, 71V and 78A in the group consisting of one or more amino acid substitutions; and/or the FLT3-VL has FR1, FR2, FR3 derived from IGKV6-21*01 and FR4 derived from IGKJ4*01 , and it is unsubstituted or has one or more amino acid substitutions selected from the group consisting of 2N, 47W, 49Y and 71Y.
  • the variable regions and CDRs described above are defined according to the Kabat numbering convention.
  • the antigen binding molecule or antibody of any one of the preceding wherein the amino acid sequence of FLT3-VH has at least 85%, 90%, 91% of SEQ ID NO: 53, 5, 52 or 54 , 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity, and the amino acid sequence of said FLT3-VL has at least SEQ ID NO: 56, 6 or 55 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity.
  • the amino acid sequence of the FLT3-VH is shown in SEQ ID NO: 53, 5, 52 or 54, and the amino acid sequence of the FLT3-VL is shown in SEQ ID NO: 56, 6 or 55 .
  • the amino acid sequence of the FLT3-VH is shown in SEQ ID NO: 53, 52 or 54, and the amino acid sequence of the FLT3-VL is shown in SEQ ID NO: 56 or 55.
  • the antigen binding molecule or antibody of any preceding one wherein
  • amino acid sequence of the FLT3-VH is shown in SEQ ID NO: 53
  • amino acid sequence of the FLT3-VL is shown in SEQ ID NO: 56
  • amino acid sequence of the FLT3-VH is shown in SEQ ID NO: 52
  • amino acid sequence of the FLT3-VL is shown in SEQ ID NO: 55, or
  • amino acid sequence of the FLT3-VH is shown in SEQ ID NO: 53
  • amino acid sequence of the FLT3-VL is shown in SEQ ID NO: 55, or
  • amino acid sequence of the FLT3-VH is shown in SEQ ID NO: 54
  • amino acid sequence of the FLT3-VL is shown in SEQ ID NO: 55, or
  • amino acid sequence of the FLT3-VH is shown in SEQ ID NO: 54
  • amino acid sequence of the FLT3-VL is shown in SEQ ID NO: 56
  • amino acid sequence of the FLT3-VH is shown in SEQ ID NO: 52
  • amino acid sequence of the FLT3-VL is shown in SEQ ID NO: 56, or
  • the amino acid sequence of the FLT3-VH is shown in SEQ ID NO: 5
  • the amino acid sequence of the FLT3-VL is shown in SEQ ID NO: 6.
  • the antigen-binding molecule or antibody according to any one of the preceding items wherein the amino acid sequence of the FLT3-VH is as shown in SEQ ID NO: 53, and the amino acid sequence of the FLT3-VL is as in SEQ ID NO :56.
  • the antigen-binding molecule as described above has: the amino acid sequence of CD3-HCDR1 shown in SEQ ID NO: 57, the amino acid sequence of CD3-HCDR1 shown in SEQ ID NO: 58 HCDR2, and CD3-HCDR3 with an amino acid sequence as shown in SEQ ID NO: 59; and the CD3-VL has: CD3-LCDR1 with an amino acid sequence as shown in SEQ ID NO: 60, and an amino acid sequence as shown in SEQ ID NO: 61
  • the antigen binding molecule according to any one of the preceding, said CD3-VH and/or said CD3-VL is murine or humanized. In some embodiments, the CD3-VH and/or the CD3-VL are humanized. In some embodiments, the amino acid sequence of the CD3-VH is shown in SEQ ID NO: 63, and the amino acid sequence of the CD3-VL is shown in SEQ ID NO: 64. In some embodiments, the variable regions and CDRs described above are defined according to the Kabat numbering convention.
  • the bispecific antigen-binding molecule of the present disclosure is not limited to a specific molecular structure as long as it has the desired antigen-binding function.
  • the bispecific antigen binding molecules herein can be bivalent (1+1) or trivalent (2+1).
  • the antigen-binding moiety in the antigen-binding molecule can be any antibody fragment with antigen-binding activity, which is fused via a peptide linker.
  • the peptide linker of the present disclosure may be any suitable peptide chain, as long as the antigen-binding molecule can exhibit the desired antigen-binding activity.
  • a peptide linker can be a flexible peptide of 1-50, or 3-20 amino acid residues.
  • each of the peptide linkers independently has a structure of L 1 -(GGGGS)nL 2 , wherein L 1 is a bond, A, GS, GGS, GGGS, SGGGGS, GGGTKLTVLGGG, 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.
  • each of the peptide linkers independently has the structure (GGGGS)n, where n is 1, 2, or 3.
  • the peptide linker is ASTKG (SEQ ID NO: 66), GGGGS (SEQ ID NO: 145), GGGGSGGGGS (SEQ ID NO: 146) or GGGGSGGGGSGGGGS (SEQ ID NO: 147).
  • the antigen-binding molecule of the present disclosure comprises a first chain having a structure represented by formula (a), a second chain having a structure represented by formula (b), and a chain having a structure represented by formula (c-1).
  • Exemplary bivalent antigen binding molecules have:
  • the antigen-binding molecule has: a first chain with an amino acid sequence as shown in SEQ ID NO:72, a second chain with an amino acid sequence as shown in SEQ ID NO:73, and an amino acid sequence as shown in SEQ ID NO:74 The third strand and a fourth strand with an amino acid sequence as shown in SEQ ID NO:75.
  • 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.
  • Substitutions can be made in 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:
  • a non-conservative substitution would 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, eg, to improve antibody affinity. Such changes can be made to CDR "hot spots", 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.
  • substitutions, insertions or deletions may occur within one or more CDRs as long as Such changes do not substantially reduce the ability of the antibody to bind antigen.
  • conservative changes eg, conservative substitutions, as provided herein
  • each CDR is unchanged, or contains no more than 1, 2 or 3 amino acid substitutions.
  • 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 polyalanine
  • Further 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 of 100 or more residues in length; 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.
  • one of the antigen-binding moiety that specifically binds FLT3 and the antigen-binding moiety that specifically binds CD3 is a substituted Fab comprising Heavy chain variable region, light chain variable region, Titin chain and Obscurin chain.
  • the replaced Fab the original CH1 and CL of the Fab are replaced by Titin chain and Obscurin chain.
  • the sequences of Titin chain and Obscurin chain are shown in Table 3-1 and Table 3-2.
  • 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 function.
  • 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 also has reduced binding affinity for complement, such as 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.
  • disulfide bond engineering eg, 354C of the first subunit and 349C of the second subunit.
  • mutations at 252Y, 254T and 256E can be introduced.
  • 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 the first subunit and the introduction of a knob at the interface of the second subunit.
  • KH knob-into-hole
  • a hole structure is introduced at the interface of the base. This enables the protrusion structure to be positioned in the hole structure, promotes the formation of heterodimers and inhibits the generation of homodimers.
  • the bulge structure is constructed by replacing small amino acid side chains from the interface of the first subunit with larger side chains such as tyrosine or tryptophan. Instead, the pore structure is created in the interface of the second subunit by replacing large amino acid side chains with smaller ones, such as alanine or threonine.
  • Protrusion structures and hole structures are prepared by changing the nucleic acid encoding the polypeptide, and the optional amino acid substitutions are shown in the table below:
  • knob-and-hole technique other techniques for modifying the CH3 domain of a heavy chain to achieve heterodimerization are also known in the art, for example WO96/27011, WO98/050431, EP1870459, WO2007/110205, WO2007/147901 , WO2009/089004, WO2010/129304, WO2011/90754, WO2011/143545, WO2012/058768, WO2013/157954 and WO 013/096291.
  • 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 are removed from the truncated C-terminus.
  • the C-terminus of the heavy chain is a shortened C-terminus ending in 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 intact antibodies has a population of antibodies with and without a mixture of antibodies with the K447 residue and/or G446+K447 residues.
  • 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.
  • nucleic acids In the case of native antibodies, native antibody fragments or bispecific antibodies with homodimeric heavy chains, two nucleic acids are required, one for the light chain or fragment thereof and one for the heavy chain or fragment thereof.
  • nucleic acids encode an amino acid sequence comprising the VL of the antibody and/or an amino acid sequence comprising the VH of the antibody (eg, the light and/or heavy chains of the antibody). These nucleic acids can be on the same expression vector or on different expression vectors.
  • nucleic acids are required, one for the first light chain, one for the first heavy chain comprising the first heteromonomeric Fc region polypeptide, one One for the second light chain, and one for the second heavy chain comprising a second heteromonomeric Fc region polypeptide.
  • These four nucleic acids may be contained in one or more nucleic acid molecules or expression vectors, usually these nucleic acids are located on two or three expression vectors, ie one vector may contain more than one of these nucleic acids.
  • the present disclosure provides an isolated nucleic acid encoding an antibody 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 of the antibody, culturing a host cell comprising a nucleic acid encoding said antibody, as provided above, and optionally The antibody is 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 (eg, by using oligonucleotide probes that are capable of binding specifically to genes encoding the antibody heavy and light chains), or produced recombinantly or obtained by chemical synthesis.
  • Suitable host cells for cloning or expressing antibody-encoding vectors include prokaryotic or eukaryotic cells as described herein.
  • antibodies can be produced in bacteria, especially when the antibody does not require glycosylation and Fc effector functions. After expression, antibodies can be isolated from bacterial cell paste in a soluble fraction and can be further purified.
  • eukaryotic microorganisms such as filamentous fungi or yeast are suitable cloning or expression hosts for antibody-encoding vectors, including fungal and yeast strains whose glycosylation pathways have been "humanized", This results in the production of antibodies with partially or fully human glycosylation patterns.
  • Suitable host cells for expressing (glycosylated) antibodies 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, especially 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, eg 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 (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.
  • COS-7 monkey kidney CV1 line
  • BHK baby hamster kidney cells
  • sertoli) cells TM4 cells
  • CV1 African green monkey kidney cells
  • HELA human cervical cancer cells
  • BRL3A canine kidney cells
  • MDCK buffalo rat
  • 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.
  • the present disclosure also provides immunoconjugates comprising an antigen binding molecule conjugated to one or more cytotoxic agents such as chemotherapeutics or drugs, growth inhibitors, toxins (such as protein toxins, enzymatically active toxins, or fragments thereof) of bacterial, fungal, plant or animal origin, or radioactive isotopes.
  • cytotoxic agents such as chemotherapeutics or drugs, growth inhibitors, toxins (such as protein toxins, enzymatically active toxins, or fragments thereof) of bacterial, fungal, plant or animal origin, or radioactive isotopes.
  • the antigen binding molecules provided by the present disclosure can be used to detect the presence of FLT3 and/or CD3 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 FLT3 and/or CD3 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, eg, FLT3 and/or CD3 are biomarkers used to select patients.
  • Exemplary disorders that can be diagnosed using the antigen binding molecules of the disclosure such as tumors or cancers.
  • 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).
  • compositions comprising the antigen binding molecules are provided, eg, for use in any of the following methods of treatment.
  • a pharmaceutical composition comprises any of the antigen binding molecules provided herein and a pharmaceutically acceptable carrier.
  • a pharmaceutical composition comprises any of the antigen binding molecules provided herein and at least one additional therapeutic agent.
  • compositions of antigen-binding molecules described in the present disclosure are prepared by mixing such antigen-binding molecules having the desired purity with one or more optional pharmaceutically acceptable carriers, the pharmaceutical composition In the form of a lyophilized composition or an aqueous solution.
  • Formulations for in vivo administration are generally sterile. Sterility is readily achieved, for example, by filtration through sterile filters.
  • antigen binding molecules Any of the antigen binding molecules provided herein can be used in methods of treatment.
  • the present disclosure provides the use of an antigen binding molecule in the manufacture or preparation of a medicament.
  • the medicament is for the treatment of tumors or cancer.
  • the drug is in the form of an effective amount for the above diseases.
  • the effective amount is a unit daily dose or a unit weekly dose.
  • 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 pharmaceutical composition comprising said antigen binding molecule, eg, for any of the above pharmaceutical uses or methods of treatment.
  • the pharmaceutical composition further comprises at least one additional therapeutic agent.
  • antigen binding molecules of the present disclosure can be used alone or in combination with other agents for therapy.
  • an antigen binding molecule of the disclosure can be co-administered 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. 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 need not, but are optionally, 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 of antigen-binding molecule present in the pharmaceutical composition, the type of disorder or treatment, and other factors discussed above. 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 any dosage, and any route empirically/clinically determined to be suitable.
  • the antigen-binding molecules of the present disclosure when used alone or in combination with one or more when used in combination with two other additional therapeutic agents, will depend on the type of disease to be treated, the type of therapeutic molecule, the severity and course of the disease, whether it is administered for prophylactic or therapeutic purposes, previous therapy, the patient's Clinical history and response to therapeutic molecules, and the judgment of the attending physician.
  • the therapeutic molecule is suitably administered to the patient at one time or over a series of treatments.
  • about 1 ⁇ g/kg to 15 mg/kg of the antigen binding molecule may be an initial candidate dose for administration to the patient, whether for example by one or more divided administrations or by continuous infusion .
  • a typical daily dosage might range from about 1 ⁇ g/kg to 100 mg/kg or more, depending on the factors mentioned above.
  • the exemplary unit daily dose is 50 ⁇ g-5g.
  • 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.
  • Titin chain/Obscurin chain of the present disclosure can be derived from any suitable polypeptide, including polypeptides derived from WO2021139758A1 (incorporated herein by reference) and CN202110527339.7 and patents (incorporated herein by reference) as priority documents .
  • DI bispecific antibodies against hNGF and hRANKL DI-2 to DI-20, which comprise the first heavy chain, the second heavy chain, the first light chain and the second Light chain:
  • the first heavy chain from N-terminal to C-terminal: [VH1-I]-[Linker 1]-[Obscurin chain]-[Fc2],
  • the first light chain from N-terminal to C-terminal: [VL1-I]-[Linker 2]-[Titin chain],
  • Second heavy chain from N-terminus to C-terminus: [VH2-D]-[CH1]-[Fc1], and
  • the second light chain from N-terminal to C-terminal: [VL2-D]-[CL];
  • VH1-I and VL1-I are respectively the heavy chain variable region and light chain variable region of I0 in WO2021139758A1
  • VH2-D and VL2-D are respectively the heavy chain variable region and light chain variable region of D0 in WO2021139758A1. district.
  • the structures of Obscurin chain, Titin chain, linker 1 and linker 2 in the DI bispecific antibody in this example are shown in the table below.
  • Test Example 4 of WO2021139758A1 was used to detect the binding activity of DI-2 to DI-20 bispecific antibodies and their antigens. Thermostability studies were performed on antibodies. Research method: The concentration of the antibody was diluted to 5 mg/mL with PBS, and its thermal stability was measured using a high-throughput differential scanning fluorometer (UNCHAINED, specification model: Unit). The experimental results showed that the antigen-binding activity of the engineered bispecific antibody did not change significantly; and, compared with DI-2, the Tm1 of DI-4 to DI-8, DI-10 to DI-16, and DI-20 (°C) and Tonset (°C) have been significantly improved, and the thermal stability of the bispecific antibody is better.
  • PL-1 to PL-19 comprising a first heavy chain, a second heavy chain, a first light chain and a second light chain as follows:
  • the first heavy chain from N-terminal to C-terminal: [VH1-P]-[Linker 1]-[Obscurin chain]-[Fc1],
  • the first light chain from N-terminal to C-terminal: [VL1-P]-[Linker 2]-[Titin chain],
  • Second heavy chain from N-terminus to C-terminus: [VH2-L]-[CH1]-[Fc2], and
  • the second light chain from N-terminal to C-terminal: [VL2-L]-[CL];
  • VH1-P and VL1-P are the heavy chain variable region and light chain variable region of the h1831K antibody in WO2020177733A1 respectively, and the amino acid sequences of VH2-L and VL2-L are as follows.
  • Obscurin chain, Titin chain, linker 1 and linker 2 in the PL bispecific antibody in this example is shown in the table below.
  • the binding activity of the PL bispecific antibody was detected by referring to the ELISA method in Test Example 4 in WO2021139758A1, wherein the hPDL1 and hCTLA4 antigens were purchased from: Sino biology. Thermostability studies were performed on antibodies. Methods: The concentration of the antibody was diluted to 1.4-3 mg/mL with PBS, and its thermal stability was measured with a high-throughput differential scanning fluorometer (UNCHAINED, specification model: Unit).
  • the experimental results show that the PL bispecific antibody still has good binding activity to the antigen; and, compared with PL-1, the Tm1(°C), Tagg 266(°C), Tonset(°C) of PL-2 to PL-19 There is a significant improvement, and the thermal stability of the bispecific antibody is better.
  • HJ-3 to HJ11 comprising a first heavy chain, a second heavy chain, a first light chain and a second light chain as follows:
  • the first heavy chain from N-terminal to C-terminal: [VH1-H]-[Linker 1]-[Titin chain]-[Fc1],
  • the first light chain from N-terminal to C-terminal: [VL1-H]-[Linker 2]-[Obscurin chain],
  • Second heavy chain from N-terminal to C-terminal: [VH2-J]-[CH1]-[Fc2], and
  • the second light chain from N-terminal to C-terminal: [VL2-J]-[CL];
  • VH1-H and VL1-H are the heavy chain variable region and light chain variable region of H0 in WO2021139758A1, respectively
  • VH2-J and VL2-J are the heavy chain variable region and light chain variable region of J1 in WO2021139758A1, respectively. district.
  • the structure of Obscurin chain, Titin chain, linker 1 and linker 2 in the HJ bispecific antibody in this example is shown in the table below.
  • the antigen-binding activity of the HJ bispecific antibody was detected with reference to the method in Test Example 4 in WO2021139758A1.
  • the method prepare the HJ bispecific antibody dilution solution with a buffer solution of 10mM acetic acid pH5.5 and 9% sucrose, and then concentrate the bispecific antibody by ultrafiltration to obtain different concentrations The HJ bispecific antibody solution (see Table 13-2 for the concentration of the HJ bispecific antibody), and then place the concentrated solution in a 40°C incubator for incubation.
  • Embodiment 2 Preparation of mouse anti-human FLT3 (ECD) monoclonal antibody
  • mice were immunized with human FLT3-ECD (Sino Biological, 10445-H08H), FLT3-E6-mFc, FLT3-E6-Fc and CHOK1-hFLT3-E56, CHOK1-hFLT3-E6 stable transfection cells.
  • blood was taken to measure the titer of the antibody in the serum, and the mice with high antibody titer in the serum and the titer tended to plateau were selected for splenocyte fusion, and the fused hybridoma cells were plated in a 96-well cell culture plate , placed in a 37°C, 5% CO 2 incubator for cultivation.
  • the cell culture supernatant was taken for detection by laser scanning microplate cell analyzer mirrorball and flow cytometer FACS.
  • the screened positive clones were expanded, cryopreserved and subcloned two to three times until single-cell clones were obtained.
  • the selected hybridoma clones were further prepared and purified by serum-free cell culture method.
  • the obtained hybridoma antibody was detected by FACS for binding to human FLT3 protein and competition with FLT3 ligand, and hybridoma cell lines with strong binding activity and competitiveness were selected.
  • Hybridoma cells in logarithmic growth phase were collected, RNA was extracted with Trizol (Invitrogen, Cat#15596-018), and reverse transcribed into cDNA.
  • the cDNA was used as a template for PCR amplification and then sent to a sequencing company for sequencing to obtain three antibodies, mAb18, mAb99 and mAb125.
  • variable region sequences of the above mAb18, mAb99 and mAb125 candidate molecules were respectively amplified by PCR to amplify the VH/VK sequences, and then carried out the same with the expression vector pHr (with signal peptide and hIgG1/hkappa constant region gene (CH1-Fc/CL) fragment).
  • Source recombination the construction of recombinant chimeric antibody full-length expression plasmid VH-CH1-Fc-pHr/VL-CL-pHr, and then obtain its chimeric antibodies Ch18, Ch99 and Ch125.
  • VH/VL homologous sequence of the murine antibody from the human germline database, graft the CDR region of the murine antibody onto the human template, and mutate some residues of VL and VH, and transfer the murine antibody
  • the constant regions were replaced with human constant regions to obtain the final humanized molecule.
  • the humanized antibody of mouse antibody mAb18 selects FR1, FR2, FR3 of IGHV1-69*02, and FR4 of IGHJ6*01 as the template of the heavy chain framework region; selects FR1, FR2, FR3 and IGKJ4* of IGKV2-40*01 FR4 of 01 served as the template for the framework region of the light chain.
  • amino acid residues at positions 1, 24, 27, 28, 30, 38, 40, 54, 55, 69 and/or 93 of the heavy chain variable region of the humanized antibody are substituted (according to Kabat numbering system numbering, the same below) to replace the amino acid residues; and/or to replace the amino acid residues at positions 2, 28, 29, 45, 55 and/or 56 on the light chain variable region of the humanized antibody .
  • the sequence of the antibody variable region is as follows:
  • the humanized antibody of mouse antibody mAb99 selects FR1, FR2, FR3 of IGHV1-46*01, and FR4 of IGHJ6*01 as the template of the heavy chain framework region; selects FR1, FR2, FR3 and IGKJ4* of IGKV1-33*01 FR4 of 01 served as the template for the framework region of the light chain.
  • the amino acid residues at positions 1, 12, 40, 69, 71, 76 and/or 78 of the heavy chain variable region of the humanized antibody are substituted; and/or the light Amino acid residues at positions 41, 42, 43, 44, 49 and/or 71 of the chain variable region are substituted.
  • the sequence of the antibody variable region is as follows:
  • the humanized antibody of mouse antibody mAb125 selects FR1, FR2, FR3 of IGHV1-46*01, and FR4 of IGHJ1*01 as the template of the heavy chain framework region; selects FR1, FR2, FR3 and IGKJ4* of IGKV6-21*01 FR4 of 01 served as the template for the framework region of the light chain.
  • the amino acid residues at positions 1, 37, 48, 67, 69, 71 and/or 78 of the heavy chain variable region of the humanized antibody are substituted; and/or the light Amino acid residues at positions 2, 29, 47, 49, 56 and/or 71 of the chain variable region are substituted.
  • the sequence of the antibody variable region is as follows:
  • the heavy chain variable region and the light chain variable region of each of the above groups are combined with the constant regions shown in SEQ ID NO: 25 and SEQ ID NO: 26 to obtain a complete humanized antibody.
  • the CD3 binding molecules of the present disclosure may be derived from any suitable antibody. Specifically, the embodiment of the present disclosure adopts S107E.
  • the S107E variable region sequence is as follows:
  • the present disclosure employs two antibody structures. in,
  • Structure-1 is an asymmetric structure molecule, and its schematic diagram is as shown in Figure 1A (Ob represents Obscurin) which contains
  • Chain 1 VH(anti-FLT3) -IgG1 (CH1) -IgG1Fc (Knob);
  • Chain 2 VL(anti-FLT3)-CL;
  • Strand 4 VL(S107E)-linker a-Obscurin.
  • Structure-2 is an asymmetric structure molecule, and its schematic diagram is shown in Figure 1B, which contains
  • Chain 1 VH(anti-FLT3) -IgG1 (CH1) -IgG1Fc (Hole);
  • Chain 2 (two): VL (anti-FLT3)-CL;
  • Strand 4 VH(S107E)-linker a-Obscurin.
  • the bispecific antibody BsAb-Hu99-5 (ie, BsAb-99) includes five chains.
  • the bispecific antibody BsAb-Hu125-4 (ie BsAb-125) comprises four chains. > BsAb-125 chain 1 (SEQ ID NO: 72)
  • the positive control molecule used in this disclosure is AMG427, the amino acid sequence of which is shown in SEQ ID NO: 863 in WO2017021362A1.
  • Test Example 1 Binding of an antigen-binding molecule to FLT3 on the cell surface
  • the centrifuged Monomac-6 cells were resuspended with 2% FBS (ThermoFisher Scientific, 10099-141), and the cell content in the cell suspension was 1-2 ⁇ 10 6 cells/mL.
  • the 96-well cell plate was centrifuged at room temperature at 300 g for 5 min, and the plate was washed 3 times with PBS.
  • the above ratio values set the EC50 of m18 to 1.
  • the experimental results showed that the humanized antibody maintained the ability to bind to the natural cell line Monomac-6 expressing the FLT3 antigen.
  • Biacore T200 Cytiva instrument was used to measure the affinity of AMG427 and BsAb-125 bispecific antibody to human FLT3 antigen, human and monkey CD3.
  • Biosensor chip (Cat.#29127556, Cytiva) to affinity capture a certain amount of antibody to be tested, and then flow through a series of concentration gradients of human FLT3 antigen (Sino Biological, 10445-H08H), human The source CD3 antigen (Sino Biological, CT038-H2508H) and the monkey-derived FLT3 antigen (ACRO, CDD-C52W4) were used to detect the reaction signal in real time using a Biacore instrument (Biacore T200, Cytiva) to obtain the binding and dissociation curves. After each cycle of dissociation, the biosensor chip was washed and regenerated with 10mM Glycine-HCl (pH1.5).
  • the buffer solution used in the experiment is HBS-EP+10 ⁇ buffer solution (pH 7.4 (Cat.#BR-1006-69, Cytiva) diluted to 1 ⁇ (pH 7.4) with D.I.Water.
  • the data obtained in the experiment are used BIAevaluation version , Cytiva software fitted the (1:1) Langmuir model to obtain the affinity value.
  • Test Example 3 In vitro PBMC-natural cell line killing experiment
  • Bispecific antibodies can bind to tumor cells expressing FLT3 antigen through FLT3-terminal antibody molecules, and on the other hand, can recruit and activate T cells through CD3-terminal antibody molecules, and substances such as perforin and granzyme secreted by T cells can kill cancer cells.
  • the killing ability of the bispecific antibody molecule is evaluated by detecting the change in the fluorescence signal intensity of the tumor cell after the bispecific antibody molecule, T cells, tumor cells and PBMC are co-incubated.
  • the plasmid of PCDH/lucG was transfected into Monomac-6 (Nanjing Kebai, CBP60521), MoLM-13 (DSMZ, ACC 554), MV-4-11 (ATCC, CRL-9591 TM ), K562 (ATCC, CCL- 243) cells, construct a cell line stably expressing luciferase and GFP.
  • ONE-GloTM Luciferase Promega, E6120
  • Graphpad Prism8.0 software was used to analyze data and calculate antibody-mediated killing EC 50 .
  • the wells with only target cells and PBMC without antibody were set as 0% inhibition, and the maximum inhibition rate of the antibody was calculated.
  • BsAb-125 has good consistent killing activity on FLT3-positive natural cell lines, and BsAb-125 has no killing activity on cell lines that do not express FLT3 antigen. It is safer than AMG427 and has a significantly wider therapeutic window.
  • HSPCs Resuspend HSPCs (Lonza, 2M-101C) with IMDM+15% FBS (solution B, FBS inactivated), add 25 ⁇ L to each well (density 3 ⁇ 10 5 cells/mL), E:T Ratio is 10:1. After diluting the antibody with solution B, add 25 ⁇ L to each well (the initial concentration is 2 nM, 5 ⁇ dilution, 6 points). The treated cells were cultured in an incubator at 37°C with 5% CO 2 for 48 hours.
  • AMG427 has obvious killing activity on hematopoietic stem cells HSPCs, while BsAb-125 has no killing activity on HSPCs. Therefore, BsAb-125 can protect the normal hematopoietic stem cells of the patient's body from being killed, and has high safety.
  • cytokine release of the antibody molecule in vitro.
  • the specific method is to dilute the supernatant collected in the cell killing experiment (test example 3) 10 times with 1640+10% FBS medium for use. Dilute the IL-6 and IFN ⁇ standards with 1640+10% FBS medium to the required concentration. Equilibrate the kit of the cytokine to be detected (Human IL6kit: CISBIO, 62HIL06PEG; Human IFN ⁇ kit: CISBIO, 62HIFNGPEG) to room temperature, and use the detection buffer to dilute the two detection antibodies in the kit (20-fold dilution).
  • Test Example 6 Effect of FLT3-CD3 Antibody on Tumor Growth of NOG Mice Bearing Human Acute Monocytic Leukemia Cell Monomac-6 Tumor
  • NOG mice were subcutaneously inoculated with human acute monocytic leukemia Monomac-6 cells, and intraperitoneally injected with hPBMCs (Miaoshun Biotechnology, ID#: P121081003C) one day before the inoculation, to detect the effect of bispecific antibody BsAb-125 on subcutaneously transplanted tumors. growth effects.
  • 5 ⁇ 10 6 hPBMCs were intraperitoneally injected into female NOG female mice (4-8 weeks old), and 1 ⁇ 10 6 cells/mouse/100 ⁇ L (containing 50 ⁇ L Matrigel) Monomac-6 cells were inoculated into the right side of the mouse one day later. Under the skin of the ribs.
  • Test Example 7 Effect of FLT3-CD3 antibody on tumor growth of NDG mice bearing human acute monocytic leukemia cell MV-4-11-LucG orthotopically transplanted tumor
  • NDG mice Biocytogen
  • hPBMCs were injected intraperitoneally to evaluate the effect of FLT3-CD3 bispecific antibody BsAb-125 on tumor growth, and compared with Molecule AMG427 for comparison.
  • MV-4-11-LucG cells were inoculated into female NDG mice (6-8 weeks old) at 1 ⁇ 10 7 cells/mouse/200 ⁇ L tail vein, and each mouse was intraperitoneally injected with bioluminescent substrate about 10 days after inoculation. (15mg/mL, 10mL/kg), after 10 minutes, it was imaged by a small animal imaging system.
  • the hPBMCs (Sai Li Commercial, ID#: SC12220A) were collected at 3.5 ⁇ 10 6 cells/mouse were injected into the peritoneal cavity of mice.
  • the day of the grouping was defined as Day 0 of the experiment, and each antibody (Biw) was administered intraperitoneally, ip, for a total of 4 times, and the fluorescence intensity and animal weight were monitored twice a week and the data were recorded. All data were drawn and statistically analyzed using Excel and GraphPad Prism 5 software.
  • the bioluminescence signal value is Total Flux (unit, p/s), the average value is calculated in avg; the SD value is calculated in STDEV; the SEM value is calculated in STDEV/SQRT (number of animals in each group).
  • the experimental results showed that AMG427 high-dose and low-dose (0.7, 0.2mpk) groups showed strong anti-tumor effects, which were significantly different from the solvent group (p ⁇ 0.01), and the tested antibody BsAb-125 had the strongest anti-tumor effect , and the anti-tumor effects were stronger than the equimolar amount of AMG427.
  • the body weight of the tumor-bearing mice remained basically stable, and the antibodies showed no obvious toxicity and were well tolerated.
  • Test Example 8 In vivo safety evaluation of FLT3/CD3 bispecific antibody

Abstract

L'invention concerne une molécule de liaison à l'antigène se liant spécifiquement à FLT3 et CD3 et son utilisation pharmaceutique. La molécule de liaison à l'antigène peut être utilisée pour traiter des maladies associées à une tumeur.
PCT/CN2023/079002 2022-03-01 2023-03-01 Molécule de liaison à l'antigène se liant spécifiquement à flt3 et cd3 et son utilisation pharmaceutique WO2023165514A1 (fr)

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PCT/CN2023/079002 WO2023165514A1 (fr) 2022-03-01 2023-03-01 Molécule de liaison à l'antigène se liant spécifiquement à flt3 et cd3 et son utilisation pharmaceutique

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US20180230193A1 (en) * 2015-08-07 2018-08-16 Andreas Loew Treatment of cancer using chimeric cd3 receptor proteins
CN110831970A (zh) * 2017-06-02 2020-02-21 辉瑞公司 靶向flt3的嵌合抗原受体
EP3623383A1 (fr) * 2018-09-11 2020-03-18 Deutsches Krebsforschungszentrum, Stiftung des öffentlichen Rechts Protéines de liaison à l'antigène flt3xcd3 bispécifiques améliorées
CN111247167A (zh) * 2017-06-02 2020-06-05 辉瑞公司 Flt3的特异性抗体及其用途
CN111601824A (zh) * 2017-11-21 2020-08-28 诺华股份有限公司 针对肿瘤相关抗原的三特异性结合分子及其用途
CN112996817A (zh) * 2018-11-01 2021-06-18 安源医药科技（上海）有限公司 同源二聚体型双特异性抗体及其制备方法和用途
US20220056141A1 (en) * 2018-09-11 2022-02-24 Deutsches Krebsforschungszentrum Stiftung Des Offentlichen Rechts Improved Anti-FLT3 Antigen Binding Proteins

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20180230193A1 (en) * 2015-08-07 2018-08-16 Andreas Loew Treatment of cancer using chimeric cd3 receptor proteins
CN110831970A (zh) * 2017-06-02 2020-02-21 辉瑞公司 靶向flt3的嵌合抗原受体
CN111247167A (zh) * 2017-06-02 2020-06-05 辉瑞公司 Flt3的特异性抗体及其用途
CN111601824A (zh) * 2017-11-21 2020-08-28 诺华股份有限公司 针对肿瘤相关抗原的三特异性结合分子及其用途
EP3623383A1 (fr) * 2018-09-11 2020-03-18 Deutsches Krebsforschungszentrum, Stiftung des öffentlichen Rechts Protéines de liaison à l'antigène flt3xcd3 bispécifiques améliorées
US20220056141A1 (en) * 2018-09-11 2022-02-24 Deutsches Krebsforschungszentrum Stiftung Des Offentlichen Rechts Improved Anti-FLT3 Antigen Binding Proteins
CN112996817A (zh) * 2018-11-01 2021-06-18 安源医药科技（上海）有限公司 同源二聚体型双特异性抗体及其制备方法和用途

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