WO2022057888A1 - 特异性结合vegf和ang-2的双特异性抗原结合分子 - Google Patents

特异性结合vegf和ang-2的双特异性抗原结合分子 Download PDF

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WO2022057888A1
WO2022057888A1 PCT/CN2021/119017 CN2021119017W WO2022057888A1 WO 2022057888 A1 WO2022057888 A1 WO 2022057888A1 CN 2021119017 W CN2021119017 W CN 2021119017W WO 2022057888 A1 WO2022057888 A1 WO 2022057888A1
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seq
variable region
chain variable
amino acid
heavy chain
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PCT/CN2021/119017
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French (fr)
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石金平
应华
朱曼曼
陶维康
胡齐悦
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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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Priority to CN202180054890.2A priority Critical patent/CN116234574A/zh
Publication of WO2022057888A1 publication Critical patent/WO2022057888A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/22—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against growth factors ; against growth regulators

Definitions

  • the present disclosure belongs to the field of biopharmaceuticals, and in particular, the present disclosure relates to the preparation and use of anti-ANG-2 antibodies and bispecific antigen-binding molecules of anti-ANG-2 and anti-VEGF.
  • vascular endothelial growth factor VEGF is a very critical and important factor promoting angiogenesis.
  • VEGF can promote the angiogenesis of tumor cells by combining with VEGF receptors to promote cell proliferation, migration, and increase vascular permeability. Therefore, blocking VEGF can inhibit tumor angiogenesis, thereby achieving the purpose of inhibiting tumor growth and metastasis.
  • VEGF vascular endothelial growth factor
  • Avastin a monoclonal antibody against VEGF
  • soluble VEGF receptors that neutralize VEGF
  • monoclonal antibodies against VEGF receptors all of which have shown better efficacy. active.
  • tumor angiogenesis is a complex process involving many molecules and multiple signaling pathways. By blocking one pathway, the goal of completely suppressing tumors cannot be achieved, and other angiogenesis-related factors need to be blocked at the same time.
  • Tie2 is the second vascular endothelial cell-specific tyrosine kinase receptor identified, and its binding to the ligands angiopoietin-1 (ANG1) and angiopoietin-2 (ANG2) also plays a role in angiogenesis important role.
  • Both ANG1 and ANG2 bind Tie2, where ANG1 supports endothelial cell (EC) survival and promotes the integrity and stability of blood vessels, while ANG2 has the opposite effect, shedding peripheral cells from endothelial cells, resulting in increased endothelial cell permeability, Make VEGF play a role in promoting the formation of new blood vessels.
  • ANG2 and VEGF complement each other and work together in tumor angiogenesis. Therefore, blocking VEGF and ANG2 at the same time can more effectively inhibit angiogenesis, promote the normalization of blood vessels, and achieve the purpose of inhibiting tumor growth and metastasis.
  • patent applications WO1998045332, WO2007095338A2, WO201004058, CN102250247A, WO2011117329, etc. disclose anti-ANG-2 and VEGF bispecific antibodies or VEGF antibodies, but there is still a need to develop new and efficient anti-ANG-2 and VEGF bispecific antibodies sexual antibodies.
  • the present disclosure provides a bispecific antibody targeting ANG-2 and VEGF, which has better blocking activity of blocking the binding of ANG-2 to its receptor Tie2 in vitro, and can significantly inhibit VEGF-induced HUVEC The increase of intracellular phosphorylated VEGFR level inhibited the proliferation of HUVEC cells induced by VEGF.
  • the bispecific antibodies in the present disclosure have more excellent tumor growth inhibition effect in mice, and can be used to treat ocular diseases such as AMD.
  • the present disclosure provides a bispecific antigen-binding molecule comprising a first antigen-binding domain that specifically binds ANG-2 and a second antigen-binding domain that specifically binds VEGF, wherein the specificity
  • the first antigen-binding domain that binds ANG-2 comprises:
  • a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 as set forth in SEQ ID NO:7, SEQ ID NO:38 and SEQ ID NO:39, respectively, and
  • a light chain variable region comprising LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12, respectively; wherein:
  • sequence of SEQ ID NO: 38 is: TINX 1 X 2 SSYTYYPDNVKG;
  • sequence of SEQ ID NO: 39 is: X 3 X 4 ATGX 5 FDY
  • X 1 is D or E
  • X 2 is D or N
  • X 3 is D or N
  • X 4 is E or Q
  • X 5 is C, S or V;
  • a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 as set forth in SEQ ID NO: 7, SEQ ID NO: 13 and SEQ ID NO: 14, respectively, and
  • a light chain variable region comprising LCDR1, LCDR2 and LCDR3 as set forth in SEQ ID NO: 15, SEQ ID NO: 11 and SEQ ID NO: 16, respectively.
  • the aforementioned bispecific antigen-binding molecule, wherein the first antigen-binding domain that specifically binds ANG-2 comprises:
  • a heavy chain variable region comprising HCDR1 as set forth in SEQ ID NO:7, HCDR2 as set forth in SEQ ID NO:8, 22 or 24, and HCDR2 as set forth in SEQ ID NO:9, 23, 25, 26 or 27 HCDR3 shown;
  • a light chain variable region comprising LCDR1, LCDR2 and LCDR3 as set forth in SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12, respectively.
  • the aforementioned bispecific antigen-binding molecule, wherein the first antigen-binding domain that specifically binds ANG-2 comprises:
  • a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 as set forth in SEQ ID NO:7, SEQ ID NO:22 and SEQ ID NO:23, respectively;
  • a light chain variable region comprising LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12, respectively;
  • a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 as set forth in SEQ ID NO:7, SEQ ID NO:24 and SEQ ID NO:23, respectively;
  • a light chain variable region comprising LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12, respectively;
  • a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 as set forth in SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 25, respectively;
  • a light chain variable region comprising LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12, respectively;
  • a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 as set forth in SEQ ID NO:7, SEQ ID NO:8 and SEQ ID NO:26, respectively;
  • a light chain variable region comprising LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12, respectively;
  • a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 as set forth in SEQ ID NO:7, SEQ ID NO:22 and SEQ ID NO:25, respectively;
  • a light chain variable region comprising LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12, respectively;
  • a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 as set forth in SEQ ID NO:7, SEQ ID NO:22 and SEQ ID NO:26, respectively;
  • a light chain variable region comprising LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12, respectively;
  • a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 as set forth in SEQ ID NO:7, SEQ ID NO:24 and SEQ ID NO:25, respectively;
  • a light chain variable region comprising LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12, respectively;
  • h a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 as set forth in SEQ ID NO:7, SEQ ID NO:24 and SEQ ID NO:26, respectively;
  • a light chain variable region comprising LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12, respectively;
  • a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 as set forth in SEQ ID NO:7, SEQ ID NO:8 and SEQ ID NO:23, respectively;
  • a light chain variable region comprising LCDR1, LCDR2 and LCDR3 as set forth in SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12, respectively; or
  • k a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 as set forth in SEQ ID NO:7, SEQ ID NO:22 and SEQ ID NO:27, respectively;
  • a light chain variable region comprising LCDR1, LCDR2 and LCDR3 as set forth in SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12, respectively; or
  • l a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 as set forth in SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9, respectively;
  • a light chain variable region comprising LCDR1, LCDR2 and LCDR3 as set forth in SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12, respectively.
  • the aforementioned bispecific antigen binding molecules bind to human ANG-2 with a dissociation equilibrium constant equal to or less than 10-7 M, and in some embodiments, with a dissociation equilibrium constant equal to or less than 10-8 M or 10-9 M dissociation equilibrium constant for human ANG-2 binding.
  • the aforementioned bispecific antigen binding molecules bind to human VEGF with a dissociation equilibrium constant equal to or less than 10-7 M, in some embodiments, with a dissociation equilibrium constant equal to or less than 10-8 M, 10-9 M, The 10-10 M, 10-11 M, 10-12 M or 10-13 M dissociation equilibrium constants bind to human VEGF.
  • the aforementioned bispecific antigen binding molecules are cross-binding to monkey ANG-2 and VEGF.
  • the aforementioned bispecific antigen binding molecules block ANG-2 and ANG with an IC50 of less than 24.82 nM, less than 20 nM, less than 15 nM, less than 10 nM, less than 5 nM, less than 3 nM, less than 1 nM, or less than 0.5 nM -2 receptor Tie2 binding, wherein the blocking activity was detected by the ELISA assay described in Test Example 2.
  • the aforementioned bispecific antigen binding molecules inhibit ANG-2-induced Tie2 phosphorylation in Tie2-transfected CHO cells with IC50s of less than 21.27 nM, less than 15 nM, less than 10 nM, less than 8 nM, or less than 5 nM.
  • the aforementioned bispecific antigen binding molecules can significantly inhibit VEGF-induced elevation of intracellular phosphorylated VEGFR levels in HUVECs.
  • the aforementioned bispecific antigen binding molecules inhibit VEGF-induced HUVEC cell proliferation with an IC50 of less than 18 nM, less than 10 nM, or less than 5 nM.
  • the aforementioned bispecific antigen-binding molecule, wherein the first antigen-binding domain that specifically binds ANG-2 comprises:
  • a heavy chain variable region comprising the amino acid sequence of any one of SEQ ID NO: 3, 20, 21, 28-37, 72 or 73, or comprising the amino acid sequence of SEQ ID NO: 3, 20, 21, 28-37, 72 or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity, respectively, in any of the 73 sequences; and/or
  • a light chain variable region comprising the amino acid sequence of SEQ ID NO: 4, 17, 18, 19 or 74, or comprising at least 95%, 96%, 97 and SEQ ID NO: 4, 17, 18, 19 or 74, respectively %, 98% or 99% sequence identity of amino acid sequences; or
  • a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 5, 44, 45, 46 or 75, or comprising at least 95%, 96% with SEQ ID NO: 5, 44, 45, 46 or 75, respectively , amino acid sequences of 97%, 98% or 99% sequence identity;
  • a light chain variable region comprising the amino acid sequence of SEQ ID NO: 6, 40, 41, 42, 43 or 76, or comprising at least 95% with SEQ ID NO: 6, 40, 41, 42, 43 or 76, respectively, Amino acid sequences with 96%, 97%, 98% or 99% sequence identity.
  • the aforementioned bispecific antigen-binding molecule, wherein the first antigen-binding domain that specifically binds ANG-2 comprises:
  • a heavy chain variable region the amino acid sequence of which is shown in any one of SEQ ID NO: 3, 20, 21, 28-37, 72 or 73, or the same as SEQ ID NO: 3, 20, 21, 28-37 , 72 or 73 have at least 95%, 96%, 97%, 98% or 99% sequence identity, respectively;
  • a light chain variable region the amino acid sequence of which is shown in SEQ ID NO: 4, 17, 18, 19 or 74, or at least 95%, 96% with SEQ ID NO: 4, 17, 18, 19 or 74, respectively, 97%, 98% or 99% sequence identity; or
  • a heavy chain variable region the amino acid sequence of which is shown in SEQ ID NO: 5, 44, 45, 46 or 75, or at least 95% with SEQ ID NO: 5, 44, 45, 46 or 75, respectively, 96 %, 97%, 98% or 99% sequence identity;
  • a light chain variable region the amino acid sequence of which is set forth in SEQ ID NO: 6, 40, 41, 42, 43 or 76, or at least 95% with SEQ ID NO: 6, 40, 41, 42, 43 or 76, respectively , 96%, 97%, 98% or 99% sequence identity.
  • the aforementioned bispecific antigen-binding molecule, wherein the first antigen-binding domain that specifically binds ANG-2 comprises:
  • the aforementioned bispecific antigen-binding molecule wherein the first antigen-binding domain that specifically binds ANG-2 comprises:
  • the aforementioned bispecific antigen-binding molecule, wherein the first antigen-binding domain that specifically binds ANG-2 comprises:
  • the aforementioned bispecific antigen-binding molecule, wherein the first antigen-binding domain that specifically binds ANG-2 comprises:
  • a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:33, and a light chain variable region comprising the amino acid sequence of SEQ ID NO:17; or
  • the heavy chain variable region comprising the amino acid sequence of SEQ ID NO:45, and the light chain variable region, comprising the amino acid sequence of SEQ ID NO:40.
  • the aforementioned bispecific antigen-binding molecule, wherein the first antigen-binding domain that specifically binds ANG-2 comprises:
  • the heavy chain variable region is shown in SEQ ID NO:45, and the light chain variable region is shown in SEQ ID NO:40.
  • the aforementioned bispecific antigen binding molecule, wherein the second antigen binding domain that specifically binds VEGF comprises:
  • a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 as set forth in SEQ ID NO:61, SEQ ID NO:62 and SEQ ID NO:63, respectively;
  • a light chain variable region comprising LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO:64, SEQ ID NO:65 and SEQ ID NO:66, respectively.
  • the aforementioned bispecific antigen binding molecule, wherein the second antigen binding domain that specifically binds VEGF comprises:
  • the heavy chain variable region comprising the amino acid sequence of SEQ ID NO:58, and the light chain variable region, comprising the amino acid sequence of SEQ ID NO:56.
  • the aforementioned bispecific antigen binding molecule, wherein the second antigen binding domain that specifically binds VEGF comprises:
  • the heavy chain variable region is shown in SEQ ID NO:58, and the light chain variable region is shown in SEQ ID NO:56.
  • the aforementioned bispecific antigen binding molecule, wherein the second antigen binding domain that specifically binds human VEGF further comprises a heavy chain constant region and a light chain constant region.
  • the aforementioned bispecific antigen binding molecule, wherein the heavy chain constant region of the second antigen binding domain that specifically binds human VEGF comprises L234A, L235A, I253A, H310A and H435A mutations.
  • the aforementioned bispecific antigen binding molecule, wherein the heavy chain constant region of the second antigen binding domain that specifically binds human VEGF is an IgGl constant region and comprises L234A, L235A, I253A, H310A and H435A Mutation (EU numbering).
  • the aforementioned bispecific antigen binding molecule, wherein the second antigen binding domain that specifically binds VEGF comprises:
  • a heavy chain comprising the amino acid sequence of SEQ ID NO: 59 or 60, or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 59 or 60;
  • a light chain comprising the amino acid sequence of SEQ ID NO:57, or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:57.
  • the aforementioned bispecific antigen binding molecule, wherein the second antigen binding domain that specifically binds VEGF comprises:
  • a heavy chain whose amino acid sequence is set forth in SEQ ID NO: 59 or 60, or has at least 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 59 or 60;
  • the aforementioned bispecific antigen binding molecule, wherein the second antigen binding domain that specifically binds VEGF comprises:
  • An antigen-binding molecule, wherein the second antigen-binding domain that specifically binds VEGF comprises:
  • the aforementioned bispecific antigen binding molecule, wherein the second antigen binding domain that specifically binds VEGF comprises:
  • the aforementioned bispecific antigen-binding molecule may have the molecular structure of any bispecific antibody known in the art.
  • the aforementioned bispecific antigen-binding molecule may have the molecular structure of an Fc fragment-containing bispecific antibody, or the molecular structure of an Fc fragment-free bispecific antibody.
  • the molecular structure of the Fc fragment-containing bispecific antibody includes, but is not limited to, TrioMab, Crossmab/KIH, DVD-Ig, IgG-scFv, FIT-Ig, mAb-Trap.
  • Molecular structures of bispecific antibodies without Fc fragments include, but are not limited to, BiTE, DART, TandAb, ImmTAC, TriKE.
  • the aforementioned bispecific antigen binding molecule is in the form of an IgG-scFv.
  • the first antigen binding domain that specifically binds ANG-2 is an anti-ANG-2 single chain antibody (scFv) that specifically binds VEGF
  • the second antigen binding domain is a full-length IgG antibody, wherein the anti-ANG-2 single-chain antibody is linked directly or through a linker to the C-terminus of a full-length IgG antibody heavy or light chain that specifically binds VEGF.
  • the aforementioned bispecific antigen-binding molecule, wherein the first antigen-binding domain that specifically binds to ANG-2 is an anti-ANG-2 single-chain antibody.
  • the aforementioned bispecific antigen-binding molecule wherein the first antigen-binding domain that specifically binds to ANG-2 is an anti-ANG-2 single-chain antibody, comprising:
  • the aforementioned bispecific antigen binding molecule wherein said anti-ANG-2 single chain antibody comprises the amino acid sequence of SEQ ID NO: 67, 68, 69, 70, 71, 77, 78 or 79.
  • the aforementioned bispecific antigen-binding molecule wherein said first antigen-binding domain that specifically binds ANG-2 is directly, or linked via a linker, to said second antigen that specifically binds VEGF binding domain.
  • the aforementioned bispecific antigen-binding molecule wherein said first antigen-binding domain that specifically binds ANG-2 is directly, or linked via a linker, to said second antigen that specifically binds VEGF The heavy or light chain of the binding domain.
  • the aforementioned bispecific antigen-binding molecule wherein the N-terminus of the anti-ANG-2 single chain antibody is linked to the heavyweight of the second antigen-binding domain that specifically binds VEGF through a linker chain C-terminal.
  • the aforementioned bispecific antigen binding molecule wherein the linker is (GG) n , wherein n is an integer from 1-20.
  • the aforementioned bispecific antigen binding molecule comprising:
  • the first strand comprising the amino acid sequence of SEQ ID NO: 80, 81, 82 or 83, or having at least 95%, 96%, 97%, 98% with SEQ ID NO: 80, 81, 82 or 83, respectively or an amino acid sequence of 99% sequence identity;
  • a second chain comprising the amino acid sequence of SEQ ID NO:57, or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:57.
  • the aforementioned bispecific antigen binding molecule is a 4-peptide structure in the form of an IgG-scFv comprising two identical first chains and two identical second chains, wherein:
  • the first strand comprising the amino acid sequence of SEQ ID NO: 80, 81, 82 or 83, or having at least 95%, 96%, 97%, 98% with SEQ ID NO: 80, 81, 82 or 83, respectively or an amino acid sequence of 99% sequence identity;
  • a second chain comprising the amino acid sequence of SEQ ID NO:57, or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:57.
  • the aforementioned bispecific antigen binding molecule comprises:
  • the first chain the amino acid sequence of which is as set forth in SEQ ID NO: 80, 81, 82 or 83, or at least 95%, 96%, 97% with SEQ ID NO: 80, 81, 82 or 83, respectively, 98% or 99% sequence identity;
  • a second chain whose amino acid sequence is set forth in SEQ ID NO:57, or has at least 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:57.
  • the aforementioned bispecific antigen binding molecule is a 4-peptide structure in the form of an IgG-scFv comprising two identical first chains and two identical second chains, wherein:
  • the amino acid sequence of the first chain is as set forth in SEQ ID NO: 80, 81, 82 or 83, or has at least 95%, 96%, 97%, 98 with SEQ ID NO: 80, 81, 82 or 83, respectively % or 99% sequence identity; and
  • amino acid sequence of the second chain is as set forth in SEQ ID NO:57, or has at least 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:57.
  • the aforementioned bispecific antigen binding molecule is a 4-peptide structure in the form of an IgG-scFv comprising two identical first chains and two identical second chains, wherein:
  • the aforementioned bispecific antigen binding molecule is a 4-peptide structure in the form of an IgG-scFv comprising two identical first chains and two identical second chains, wherein:
  • amino acid sequence of the first chain is set forth in SEQ ID NO:80; and the amino acid sequence of the second chain is set forth in SEQ ID NO:57;
  • amino acid sequence of the first chain is shown in SEQ ID NO:81; and the amino acid sequence of the second chain is shown in SEQ ID NO:57;
  • amino acid sequence of the first chain is shown in SEQ ID NO: 82; and the amino acid sequence of the second chain is shown in SEQ ID NO: 57; or
  • amino acid sequence of the first chain is shown in SEQ ID NO:83; and the amino acid sequence of the second chain is shown in SEQ ID NO:57.
  • the aforementioned bispecific antigen binding molecules have a 4-peptide structure in the form of a Crossmab.
  • the bispecific antigen binding molecule in the form of a Crossmab comprises:
  • the aforementioned bispecific antigen binding molecule comprises:
  • the constant domain CL of the first light chain is replaced with the constant domain CH1 of the first heavy chain;
  • the constant domain CL of the second light chain is replaced with the constant domain CH1 of the second heavy chain;
  • the light chain variable region VL of the first light chain is replaced with the heavy chain variable region VH of the first heavy chain;
  • the light chain variable region VL of the second light chain is replaced with the heavy chain variable region VH of the second heavy chain.
  • the aforementioned bispecific antigen binding molecule comprises:
  • the aforementioned bispecific antigen binding molecule comprises:
  • the aforementioned bispecific antigen binding molecule comprises:
  • the constant domain CL of the first light chain is replaced with the constant domain CH1 of the first heavy chain;
  • the constant domain CL of the second light chain is replaced with the constant domain CH1 of the second heavy chain;
  • the light chain variable region VL of the first light chain is replaced with the heavy chain variable region VH of the first heavy chain;
  • the light chain variable region VL of the second light chain is replaced with the heavy chain variable region VH of the second heavy chain.
  • the aforementioned bispecific antigen binding molecule comprises:
  • bispecific antigen binding molecules in the aforementioned bispecific antigen binding molecules:
  • the first light chain comprises the light chain variable region of SEQ ID NO:17
  • the first heavy chain comprises the heavy chain variable region of SEQ ID NO:33;
  • the first light chain comprises the light chain variable region of SEQ ID NO:40, and the first heavy chain comprises the heavy chain variable region of SEQ ID NO:45;
  • the second light chain comprises the light chain variable region of SEQ ID NO:56
  • the second heavy chain comprises the heavy chain variable region of SEQ ID NO:58.
  • the aforementioned bispecific antigen binding molecule wherein:
  • the first light chain comprises the amino acid sequence of SEQ ID NO: 85, or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO: 85, and
  • the first heavy chain comprises the amino acid sequence of SEQ ID NO:84, or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:84; or
  • the first light chain comprises the amino acid sequence of SEQ ID NO:88, or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:88, and
  • the first heavy chain comprises the amino acid sequence of SEQ ID NO:87, or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:87;
  • the first light chain comprises the amino acid sequence of SEQ ID NO: 57, or an amino acid sequence with at least 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO: 57;
  • the first heavy chain comprises the amino acid sequence of SEQ ID NO:86, or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:86.
  • SEQ ID NO:86 amino acid sequence of SEQ ID NO:86, or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:86.
  • amino acid sequence of the first light chain is shown in SEQ ID NO: 85, or has at least 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO: 85, and
  • amino acid sequence of the first heavy chain is set forth in SEQ ID NO: 84, or has at least 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO: 84; or
  • amino acid sequence of the first light chain is shown in SEQ ID NO: 88, or has at least 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO: 88, and
  • amino acid sequence of the first heavy chain is set forth in SEQ ID NO:87, or has at least 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:87;
  • amino acid sequence of the second light chain is set forth in SEQ ID NO:57, or has at least 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:57;
  • amino acid sequence of the second heavy chain is set forth in SEQ ID NO:86, or has at least 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:86.
  • bispecific antigen binding molecules in the aforementioned bispecific antigen binding molecules:
  • amino acid sequence of the first light chain is shown in SEQ ID NO: 85
  • amino acid sequence of the first heavy chain is shown in SEQ ID NO: 84;
  • amino acid sequence of the second light chain is shown in SEQ ID NO: 57, and the amino acid sequence of the second heavy chain is shown in SEQ ID NO: 86; or
  • amino acid sequence of the first light chain is shown in SEQ ID NO: 88
  • amino acid sequence of the first heavy chain is shown in SEQ ID NO: 87;
  • amino acid sequence of the second light chain is shown in SEQ ID NO:57, and the amino acid sequence of the second heavy chain is shown in SEQ ID NO:86.
  • the present disclosure provides an antibody that specifically binds ANG-2.
  • the aforementioned antibody that specifically binds to ANG-2 comprises:
  • a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 as set forth in SEQ ID NO:7, SEQ ID NO:38 and SEQ ID NO:39, respectively, and
  • a light chain variable region comprising LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12, respectively; wherein:
  • sequence of SEQ ID NO: 38 is: TINX 1 X 2 SSYTYYPDNVKG;
  • sequence of SEQ ID NO: 39 is: X 3 X 4 ATGX 5 FDY
  • X 1 is D or E
  • X 2 is D or N
  • X 3 is D or N
  • X 4 is E or Q
  • X 5 is C, S or V;
  • a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 as set forth in SEQ ID NO: 7, SEQ ID NO: 13 and SEQ ID NO: 14, respectively, and
  • a light chain variable region comprising LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 15, SEQ ID NO: 11 and SEQ ID NO: 16, respectively.
  • the aforementioned antibody that specifically binds to ANG-2 comprises:
  • a heavy chain variable region comprising HCDR1 as set forth in SEQ ID NO:7, HCDR2 as set forth in SEQ ID NO:8, 22 or 24, and HCDR2 as set forth in SEQ ID NO:9, 23, 25, 26 or 27 HCDR3;
  • a light chain variable region comprising LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12, respectively.
  • the aforementioned antibody that specifically binds to ANG-2 comprises:
  • a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 as set forth in SEQ ID NO:7, SEQ ID NO:22 and SEQ ID NO:23, respectively;
  • a light chain variable region comprising LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12, respectively;
  • a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 as set forth in SEQ ID NO:7, SEQ ID NO:24 and SEQ ID NO:23, respectively;
  • a light chain variable region comprising LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12, respectively;
  • a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 as set forth in SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 25, respectively;
  • a light chain variable region comprising LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12, respectively;
  • a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 as set forth in SEQ ID NO:7, SEQ ID NO:8 and SEQ ID NO:26, respectively;
  • a light chain variable region comprising LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12, respectively;
  • a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 as set forth in SEQ ID NO:7, SEQ ID NO:22 and SEQ ID NO:25, respectively;
  • a light chain variable region comprising LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12, respectively;
  • a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 as set forth in SEQ ID NO:7, SEQ ID NO:22 and SEQ ID NO:26, respectively;
  • a light chain variable region comprising LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12, respectively;
  • a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 as set forth in SEQ ID NO:7, SEQ ID NO:24 and SEQ ID NO:25, respectively;
  • a light chain variable region comprising LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12, respectively;
  • h a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 as set forth in SEQ ID NO:7, SEQ ID NO:24 and SEQ ID NO:26, respectively;
  • a light chain variable region comprising LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12, respectively;
  • a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 as set forth in SEQ ID NO:7, SEQ ID NO:8 and SEQ ID NO:23, respectively;
  • a light chain variable region comprising LCDR1, LCDR2 and LCDR3 as set forth in SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12, respectively; or
  • k a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 as set forth in SEQ ID NO:7, SEQ ID NO:22 and SEQ ID NO:27, respectively;
  • a light chain variable region comprising LCDR1, LCDR2 and LCDR3 as set forth in SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12, respectively; or
  • l a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 as set forth in SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9, respectively;
  • a light chain variable region comprising LCDR1, LCDR2 and LCDR3 as set forth in SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12, respectively.
  • the aforementioned antibodies that specifically bind ANG-2 bind to human ANG-2 with a dissociation equilibrium constant equal to or less than 10-7 M, in some embodiments, with a dissociation equilibrium constant equal to or less than 10-8 M or 10 -9 M dissociation equilibrium constant for human ANG-2 binding.
  • the aforementioned antibodies that specifically bind ANG-2 are cross-binding to monkey ANG-2.
  • the aforementioned antibodies that specifically bind ANG-2 block the binding of ANG-2 to the ANG-2 receptor Tie2 with an IC50 of less than 24.82 nM, less than 20 nM, less than 15 nM, less than 10 nM, or less than 5 nM, wherein the The blocking activity was detected by the ELISA assay described in Test Example 2.
  • the aforementioned antibodies that specifically bind ANG-2 inhibit ANG-2-induced Tie2 phosphorylation in Tie2-transfected CHO cells with IC50s of less than 21.27 nM, less than 15 nM, less than 10 nM, or less than 8 nM.
  • the aforementioned antibody that specifically binds to ANG-2 is a murine antibody, a chimeric antibody, a humanized antibody or a fully human antibody.
  • the aforementioned antibody that specifically binds ANG-2 wherein the antibody comprises a framework region, wherein:
  • the heavy chain framework region comprises one or more amino acid backmutations selected from 44R, 77S or 84S; and/or
  • the light chain framework region comprises one or more amino acid back mutations selected from IN, 43S, 68A, 85D, 87H; or
  • the heavy chain framework region comprises one or more amino acid back mutations selected from 2L, 44R, 74V, 82AS or 83K; and/or
  • the light chain framework region comprises one or more amino acid backmutations selected from IN, 43S, 46V, 68A, 85D, 87H.
  • the aforementioned antibody that specifically binds to ANG-2 comprises:
  • a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 7, SEQ ID NO: 38 and SEQ ID NO: 39, respectively, and whose heavy chain framework region comprises 44R, 77S or one or more amino acid backmutations in 84S;
  • a light chain variable region comprising LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12 respectively; and its light chain framework region comprises a light chain framework region selected from the group consisting of 1N, 43S, 68A One or more amino acid back mutations in , 85D, 87H; wherein:
  • sequence of SEQ ID NO: 38 is: TINX 1 X 2 SSYTYYPDNVKG;
  • sequence of SEQ ID NO: 39 is: X 3 X 4 ATGX 5 FDY
  • X 1 is D or E
  • X 2 is D or N
  • X 3 is D or N
  • X 4 is E or Q
  • X 5 is C, S or V; or
  • a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 7, SEQ ID NO: 13 and SEQ ID NO: 14, respectively, and whose heavy chain framework region comprises a heavy chain framework region selected from the group consisting of 2L, 44R one or more amino acid backmutations in , 74V, 82AS or 83K; and
  • a light chain variable region comprising LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 15, SEQ ID NO: 11 and SEQ ID NO: 16, respectively, and the light chain framework region thereof comprises a light chain framework region selected from the group consisting of IN, 43S, 46V , 68A, 85D, 87H one or more amino acid backmutations.
  • the aforementioned antibody that specifically binds to ANG-2 comprises:
  • a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 shown in SEQ ID NO: 7, SEQ ID NO: 22 and SEQ ID NO: 26, respectively, and whose heavy chain framework region comprises a heavy chain framework region selected from 44R, 77S or 84S one or more amino acid backmutations in;
  • a light chain variable region comprising LCDR1, LCDR2 and LCDR3 shown in SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12, respectively, and the light chain framework region comprising a light chain framework region selected from the group consisting of IN, 43S, 68A, One or more amino acid backmutations in 85D, 87H.
  • the aforementioned antibody that specifically binds to ANG-2 comprises:
  • a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:20, or an amino acid sequence comprising one or more amino acid backmutations selected from 44R, 77S or 84S in the amino acid sequence of SEQ ID NO:20;
  • a light chain variable region comprising the amino acid sequence of SEQ ID NO: 17, or comprising one or more amino acid backmutations selected from the group consisting of IN, 43S, 68A, 85D, 87H in the amino acid sequence of SEQ ID NO: 17 amino acid sequence.
  • the aforementioned antibody that specifically binds to ANG-2 comprises:
  • a heavy chain variable region the amino acid sequence of which is set forth in SEQ ID NO: 20, or comprising one or more amino acid back mutations selected from 44R, 77S or 84S in the amino acid sequence of SEQ ID NO: 20;
  • a light chain variable region the amino acid sequence of which is shown in SEQ ID NO: 17, or the amino acid sequence of SEQ ID NO: 17 comprising one or more amino acid restorations selected from the group consisting of 1N, 43S, 68A, 85D, 87H mutation.
  • the aforementioned antibody that specifically binds to ANG-2 comprises:
  • a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 33;
  • a light chain variable region comprising the amino acid sequence of SEQ ID NO: 17, or comprising one or more amino acid backmutations selected from the group consisting of IN, 43S, 68A, 85D, 87H in the amino acid sequence of SEQ ID NO: 17 amino acid sequence.
  • the aforementioned antibody that specifically binds to ANG-2 comprises:
  • a light chain variable region the amino acid sequence of which is shown in SEQ ID NO: 17, or the amino acid sequence of SEQ ID NO: 17 comprising one or more amino acid restorations selected from the group consisting of 1N, 43S, 68A, 85D, 87H mutation.
  • the aforementioned antibody that specifically binds to ANG-2 comprises:
  • a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 44, or comprising one or more amino acid backmutations selected from 2L, 44R, 74V, 82AS or 83K in the amino acid sequence of SEQ ID NO: 44 the amino acid sequence of ;
  • a light chain variable region comprising the amino acid sequence of SEQ ID NO: 40, or comprising one or more amino acid reversions selected from IN, 43S, 46V, 68A, 85D, 87H in the amino acid sequence of SEQ ID NO: 40 Mutated amino acid sequence.
  • the aforementioned antibody that specifically binds to ANG-2 comprises:
  • Heavy chain variable region the amino acid sequence of which is shown in SEQ ID NO: 44, or comprises one or more amino acids selected from 2L, 44R, 74V, 82AS or 83K in the amino acid sequence of SEQ ID NO: 44 back mutation;
  • a light chain variable region the amino acid sequence of which is shown in SEQ ID NO: 40, or one or more selected from the group consisting of 1N, 43S, 46V, 68A, 85D, 87H in the amino acid sequence of SEQ ID NO: 40 Amino acid backmutation.
  • the aforementioned antibody that specifically binds to ANG-2 comprises:
  • a heavy chain variable region comprising the amino acid sequence of any one of SEQ ID NO: 3, 20, 21, 28-37, 72 or 73, or comprising the amino acid sequence of SEQ ID NO: 3, 20, 21, 28-37, 72 or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity, respectively, in any of the 73 sequences; and/or
  • a light chain variable region comprising the amino acid sequence of SEQ ID NO: 4, 17, 18, 19 or 74, or comprising at least 95%, 96%, 97 and SEQ ID NO: 4, 17, 18, 19 or 74, respectively %, 98% or 99% sequence identity of amino acid sequences; or
  • a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 5, 44, 45, 46 or 75, or comprising at least 95%, 96% with SEQ ID NO: 5, 44, 45, 46 or 75, respectively , amino acid sequences of 97%, 98% or 99% sequence identity;
  • a light chain variable region comprising the amino acid sequence of SEQ ID NO: 6, 40, 41, 42, 43 or 76, or comprising at least 95% with SEQ ID NO: 6, 40, 41, 42, 43 or 76, respectively, Amino acid sequences with 96%, 97%, 98% or 99% sequence identity.
  • the aforementioned antibody that specifically binds to ANG-2 comprises:
  • a heavy chain variable region the amino acid sequence of which is shown in any one of SEQ ID NO: 3, 20, 21, 28-37, 72 or 73, or the same as SEQ ID NO: 3, 20, 21, 28-37 , 72 or 73 have at least 95%, 96%, 97%, 98% or 99% sequence identity, respectively;
  • a light chain variable region the amino acid sequence of which is shown in SEQ ID NO: 4, 17, 18, 19 or 74, or at least 95%, 96% with SEQ ID NO: 4, 17, 18, 19 or 74, respectively, 97%, 98% or 99% sequence identity; or
  • a heavy chain variable region the amino acid sequence of which is shown in SEQ ID NO: 5, 44, 45, 46 or 75, or at least 95% with SEQ ID NO: 5, 44, 45, 46 or 75, respectively, 96 %, 97%, 98% or 99% sequence identity;
  • a light chain variable region the amino acid sequence of which is set forth in SEQ ID NO: 6, 40, 41, 42, 43 or 76, or at least 95% with SEQ ID NO: 6, 40, 41, 42, 43 or 76, respectively , 96%, 97%, 98% or 99% sequence identity.
  • the aforementioned antibody that specifically binds to ANG-2 comprises:
  • the aforementioned antibody that specifically binds to ANG-2 comprises:
  • the aforementioned antibody that specifically binds to ANG-2 comprises:
  • the aforementioned antibody that specifically binds to ANG-2 comprises:
  • a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:33, and a light chain variable region comprising the amino acid sequence of SEQ ID NO:17; or
  • the heavy chain variable region comprising the amino acid sequence of SEQ ID NO:45
  • the light chain variable region comprising the amino acid sequence of SEQ ID NO:40.
  • the aforementioned antibody that specifically binds to ANG-2 comprises:
  • the aforementioned antibody that specifically binds ANG-2 comprises a constant region.
  • the heavy chain constant regions are selected from the group consisting of human IgGl, IgG2, IgG3 and IgG4 constant regions and conventional variants thereof, and the light chain constant regions are selected from the group consisting of human antibody kappa and lambda chain constant regions and conventional variants thereof Variants.
  • the aforementioned antibody that specifically binds ANG-2 comprises the heavy chain constant region of SEQ ID NO:47 and the light chain constant region of SEQ ID NO:48.
  • the aforementioned antibody that specifically binds to ANG-2 comprises:
  • a heavy chain comprising the amino acid sequence of SEQ ID NO: 49, 51 or 52, or comprising at least 95%, 96%, 97%, 98% or 99% of the sequence with SEQ ID NO: 49, 51 or 52, respectively identical amino acid sequences, and/or
  • a light chain comprising the amino acid sequence of SEQ ID NO:50, or comprising an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:50; or
  • a heavy chain comprising the amino acid sequence of SEQ ID NO: 53 or 55, or comprising amino acids having at least 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 53 or 55, respectively sequence, and/or
  • a light chain comprising the amino acid sequence of SEQ ID NO:54, or comprising an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:54.
  • the aforementioned antibody that specifically binds to ANG-2 comprises:
  • a heavy chain whose amino acid sequence is set forth in SEQ ID NO: 49, 51 or 52, or has at least 95%, 96%, 97%, 98% or 99% of the amino acid sequence from SEQ ID NO: 49, 51 or 52, respectively sequence identity, and/or
  • a light chain whose amino acid sequence is set forth in SEQ ID NO:54, or has at least 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:54.
  • the aforementioned antibody that specifically binds to ANG-2 comprises:
  • a heavy chain comprising the amino acid sequence of SEQ ID NO: 49, 51 or 52, and
  • a light chain comprising the amino acid sequence of SEQ ID NO: 50; or
  • a light chain comprising the amino acid sequence of SEQ ID NO:54.
  • the aforementioned antibody that specifically binds to ANG-2 comprises:
  • a heavy chain the amino acid sequence of which is set forth in SEQ ID NO: 49, 51 or 52, and
  • the present disclosure provides an isolated antibody that competes with the aforementioned antibody that specifically binds ANG-2 for binding to human ANG-2.
  • the present disclosure provides a nucleic acid molecule encoding a bispecific antigen binding molecule according to the foregoing, or an antibody that specifically binds ANG-2 as described above.
  • the present disclosure provides a vector comprising the aforementioned nucleic acid molecule.
  • the present disclosure provides a host cell comprising the aforementioned vector.
  • the present disclosure provides a pharmaceutical composition
  • a pharmaceutical composition comprising an effective amount of the aforementioned bispecific antigen binding molecule, or the aforementioned antibody that specifically binds ANG-2, or the aforementioned nucleic acid molecule, and one or more Various pharmaceutically acceptable carriers, diluents or excipients.
  • the therapeutically effective amount is 0.1-3000 mg of a bispecific antigen binding molecule or antibody that specifically binds ANG-2 as previously described in a unit dose of the composition.
  • the present disclosure provides a method of producing the aforementioned bispecific antigen binding molecule, or the aforementioned antibody that specifically binds ANG-2, the method comprising culturing the aforementioned host cell to express the bispecific Antigen-binding molecules or antibodies that specifically bind to ANG-2.
  • the present disclosure provides a method of preventing or treating cancer or angiogenic eye disease, the method comprising administering to a subject a therapeutically effective amount of the aforementioned bispecific antigen binding molecule, or the aforementioned specificity An antibody that binds to ANG-2, or the aforementioned nucleic acid molecule, or the aforementioned pharmaceutical composition.
  • the aforementioned method of treatment wherein the cancer is selected from the group consisting of breast cancer, adrenal tumor, fallopian tube cancer, squamous cell carcinoma, ovarian cancer, gastric cancer, colorectal cancer, non-small cell lung cancer, bile duct cancer, bladder cancer cancer, pancreatic cancer, skin cancer and liver cancer.
  • the angiogenic eye disease is selected from the group consisting of neovascular glaucoma, age-related macular degeneration (AMD), diabetic macular edema, corneal neovascularization, corneal transplant neovascularization, corneal transplant rejection, Retinal/choroidal neovascularization, angle neovascularization (iris redness), ocular neovascular disease, vascular restenosis, and arteriovenous malformations (AVMs).
  • the aforementioned cancer or angiogenic eye disease is associated with VEGF or ANG-2.
  • the present disclosure provides the aforementioned bispecific antigen binding molecules, or the aforementioned antibodies that specifically bind ANG-2, or the aforementioned nucleic acid molecules, or the aforementioned pharmaceutical compositions in preparation for the treatment or prevention of cancer Or the use in the medicine of angiogenic eye disease.
  • the aforementioned use wherein the cancer is selected from the group consisting of breast cancer, adrenal tumor, fallopian tube cancer, squamous cell carcinoma, ovarian cancer, gastric cancer, colorectal cancer, non-small cell lung cancer, bile duct cancer, bladder cancer , pancreatic cancer, skin cancer and liver cancer.
  • the angiogenic eye disease is selected from the group consisting of neovascular glaucoma, age-related macular degeneration (AMD), diabetic macular edema, corneal neovascularization, corneal transplant neovascularization, corneal transplant rejection, Retinal/choroidal neovascularization, angle neovascularization (iris redness), ocular neovascular disease, vascular restenosis, and arteriovenous malformations (AVMs).
  • the aforementioned cancer or angiogenic eye disease is associated with VEGF or ANG-2.
  • the present disclosure provides the aforementioned bispecific antigen binding molecules, or the aforementioned antibodies that specifically bind ANG-2, or the aforementioned nucleic acid molecules, or the aforementioned pharmaceutical compositions for use as a medicament.
  • the present disclosure provides the aforementioned bispecific antigen binding molecules, or the aforementioned antibodies that specifically bind ANG-2, or the aforementioned nucleic acid molecules, or the aforementioned pharmaceutical compositions for use as a medicament, useful as Treat or prevent cancer or angiogenic eye disease.
  • the cancer is selected from the group consisting of breast cancer, adrenal tumor, fallopian tube cancer, squamous cell carcinoma, ovarian cancer, gastric cancer, colorectal cancer, non-small cell lung cancer, bile duct cancer, bladder cancer, pancreatic cancer, Skin cancer and liver cancer.
  • the angiogenic eye disease is selected from the group consisting of neovascular glaucoma, age-related macular degeneration (AMD), diabetic macular edema, corneal neovascularization, corneal transplant neovascularization, corneal transplant rejection, Retinal/choroidal neovascularization, angle neovascularization (iris redness), ocular neovascular disease, vascular restenosis, and arteriovenous malformations (AVMs).
  • the aforementioned cancer or angiogenic eye disease is associated with VEGF or ANG-2.
  • Figure 1 shows the results of bispecific antibodies inhibiting the binding of ANG-2 to Tie2
  • Figure 2 shows the results of bispecific antibody inhibition-induced Tie2 phosphorylation
  • Figure 3 shows that the bispecific antibody significantly inhibited VEGF-induced increase in the level of intracellular phosphorylated VEGFR in HUVECs
  • FIG. 4 shows that bispecific antibodies can significantly inhibit the proliferation of HUVECs induced by VEGF
  • FIG. 5 shows that all of the bispecific antibodies of the present disclosure significantly inhibit PC-3 tumor growth in mice
  • FIG. 6 shows that all of the bispecific antibodies of the present disclosure can significantly inhibit H460 transplanted tumor growth in mice
  • FIG. 7 shows that bispecific antibodies of the present disclosure can significantly inhibit A431 transplanted tumor growth in mice
  • FIG. 8A and 8B show the inhibition results of rhesus monkey choroidal neovascularization by the bispecific antibody of the present disclosure; wherein FIG. 8A shows the improvement rate of the bispecific antibody of the present disclosure on the fluorescent leakage area in the eye of the rhesus monkey , Figure 8B shows that the bispecific antibodies of the present disclosure can significantly reduce the number of fluorescent spots in the eyes of rhesus monkeys;
  • Figure 9 shows that the bispecific antibodies of the present disclosure can significantly reduce the expression of VEGF in the aqueous humor of rhesus monkey eyes.
  • Figures 10A and 10B show the molecular structures of exemplary bispecific antibodies in the present disclosure
  • Figure 10A shows the structure of an IgG-scFv type bispecific antibody
  • Figure 10B shows the structure of a Crossmab type bispecific antibody.
  • 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 the naturally occurring amino acids.
  • Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that have been modified later, such as hydroxyproline, gamma-carboxyglutamic acid, and O-orthophosphoserine.
  • Amino acid analogs are those that have the same basic chemical structure as a naturally occurring amino acid (i.e., a hydrogen-bonded alpha carbon, carboxyl, amino, and R groups, e.g., homoserine, norleucine, methionine sulfoxide, methionine amino acid methylsulfonium).
  • 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 compound having a structure that differs from the general chemical structure of amino acids but functions in a similar manner to naturally occurring amino acids.
  • ANG-2 refers to Angiopoietin-2 (ANGPT2 or ANG2), which is described, for example, in Maisonpierre, P.C. et al., Science 277 (1997) 55-60 and Cheung, A.H. et al., Genomics 48 (1998) 389-91.
  • Angiopoietin-1 and -2 were found to be ligands for Tie, a family of tyrosine kinases selectively expressed within the vascular endothelium, Yancopoulos, G.D. et al., Nature 407 (2000) 242-48.
  • angiopoietin-3 and -4 may represent counterparts in a broad region of the same genetic locus in mice and humans.
  • ANG1 and ANG2 were originally identified in tissue culture experiments as agonists and antagonists, respectively (for ANG1, see, Davis, S. et al., Cell 87 (1996) 1161-69; for ANG2, see Maisonpierre, PC, et al., Science 277 (1997) 55-60).
  • angiopoietins primarily bind Tie2, while both ANG1 and 2 bind Tie2 with an affinity of 3 nM (Kd), Maisonpierre, P.C. et al., Science 277 (1997) 55-60.
  • VEGF refers to human vascular endothelial growth factor (VEGF/VEGF-A), which is described, for example, in Leung, DW et al., Science 246 (1989) 1306-9; Keck, PJ et al., Science 246 (1989) 1309-12 and Connolly, DT et al., J. Biol. Chem. 264 (1989) 20017-24.
  • VEGF is involved in the regulation of normal and abnormal angiogenesis and neovascularization associated with tumors and intraocular disorders (Ferrara, N., Endocr. Rev. 18 (1997) 4-25; Berkman, RA, J. Clin. Invest.
  • VEGF is a homodimeric glycoprotein that promotes mitogenicity to endothelial cells.
  • an "antigen-binding molecule” as used in this disclosure is used herein in the broadest sense to refer to a molecule that specifically binds an antigen
  • antigen-binding molecules are antibodies, antibody fragments, antibody fusion proteins, or fusion proteins.
  • bispecific antigen binding molecules herein include bispecific antibodies and antibody fusion proteins.
  • a bispecific antibody or antibody fusion protein herein can comprise a first chain, the first chain being the heavy chain of an antibody or a polypeptide comprising an antibody heavy chain, and a second chain being the antibody the light chain or a polypeptide comprising the light chain of an antibody.
  • the bispecific antigen binding molecules herein have the basic structure of a full-length antibody, consisting of two identical first chains and two identical second chains through interchain disulfides.
  • a "bispecific antigen-binding molecule” as used herein refers to an antigen-binding molecule capable of simultaneously binding two antigens or antigenic determinants, comprising a first antigen-binding domain that binds the first antigen or antigenic determinant and a second antigen-binding domain that binds the first antigen or antigenic determinant.
  • the second antigen-binding domain of a secondary antigen or antigenic determinant is a secondary antigen or antigenic determinant.
  • the first antigen-binding domain specifically binds ANG-2 and the second antigen-binding domain specifically binds VEGF; alternatively, in some embodiments of the present disclosure, the first antigen-binding domain specifically binds Binding to VEGF, the second antigen binding domain specifically binds ANG-2.
  • the bispecific antigen binding molecules described in the present disclosure are bispecific bivalent antibodies or bispecific tetravalent antibodies.
  • bispecific antigen-binding molecules can be divided into two categories according to different structures: bispecific antigen-binding molecules containing an Fc fragment and bispecific antigen-binding molecules without an Fc fragment.
  • Fc fragment-containing bispecific antigen-binding molecular structures include, but are not limited to, TrioMab, Crossmab/KIH, DVD-Ig, IgG-scFv, FIT-Ig, mAb-Trap, and the like.
  • Bispecific antigen binding molecular structures without Fc fragments include, but are not limited to, BiTE, DART, TandAb, ImmTAC, TriKE, and the like.
  • a “Crossmab” as described in the present disclosure is an IgG-like bispecific antigen-binding molecule structure in the presence of an Fc region that results in VH-VL and The molecular structure of the interface between CH1-CL changes. Due to the principle of mutual exclusion, that is, the mutual exclusion of VH and VH, and the mutual exclusion of CL and CL, the exchanged antibody light chain is not prone to mismatch with the heavy chain of the unmodified antibody, resulting in the correct pairing of the light and heavy chains.
  • the bispecific antigen binding molecules of the present disclosure are in the form of a Crossmab, a tetrapeptide structure consisting of a first heavy chain, a first light chain, a second heavy chain, and a second light chain, wherein the first The heavy chain and the first light chain constitute the first antigen binding domain that binds ANG-2, the second heavy chain and the second light chain constitute the second antigen binding domain that binds VEGF, and the CH1 of the first heavy chain and the first light chain CLs are exchanged with each other.
  • IgG-scFv as described in this disclosure is an IgG-like bispecific antigen-binding molecule structure in the presence of an Fc region, wherein the scFv of one antigen-binding domain is fused to the C-terminus of the IgG heavy or light chain of the other antigen-binding domain, To form an IgG-scFv bispecific antigen-binding molecule.
  • the bispecific antigen binding molecules of the present disclosure are in the form of IgG-scFv.
  • the scFv that specifically binds ANG-2 is linked directly or through a linker to the C-terminus of the heavy chain of an antibody that specifically binds VEGF to form a bispecific antigen binding molecule in the form of an IgG-scFv.
  • valency refers to the presence of a defined number of binding sites in an antigen-binding molecule.
  • bivalent refers to the presence of two, four and six binding sites, respectively, in an antigen-binding molecule.
  • hexavalent refers to the presence of two, four and six binding sites, respectively, in an antigen-binding molecule.
  • first antigen-binding domain capable of specifically binding to ANG-2 or “the second antigen-binding domain capable of specifically binding to VEGF” in the present disclosure refers to an antigen-binding molecule comprising all or part of ANG-2 or VEGF specific binding region.
  • the antigen binding domain may comprise one or more antibody variable regions.
  • the antigen binding domain capable of specifically binding an antigen comprises an antibody light chain variable region and an antibody heavy chain variable region, which can form scFv, Fab and other configurations.
  • antibody fusion protein refers to a biologically active fusion protein formed by linking a target protein (polypeptide) with an antibody, and the fusion protein has the biological activity and immunoglobulin activity of the linked protein.
  • antibody is used in the broadest sense and encompasses a variety of antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (eg, bispecific antibodies), and antibody fragments so long as they exhibit the desired Antigen binding activity.
  • Native antibody refers to naturally-occurring immunoglobulin molecules with different structures.
  • native IgG antibodies are heterotetrameric glycoproteins of approximately 150,000 Daltons composed of two identical light chains and two identical heavy chains that are disulfide-bonded. From N to C-terminus, each heavy chain has a variable domain (VH), also known as a variable heavy domain or heavy chain variable domain, followed by three constant domains (CH1, CH2, and CH3).
  • VH variable domain
  • CH1, CH2, and CH3 constant domains
  • each light chain has a variable region (VL), also known as a variable light domain, or light chain variable domain, followed by a constant light (CL) domain.
  • VH variable domain
  • CL constant light
  • Antibody light chains include two types, kappa ( ⁇ ) and lambda ( ⁇ ), according to their constant domain amino acid sequences.
  • antibodies can be divided into five categories, or antibody isotypes, namely IgM, IgD, IgG, IgA and IgE, and their corresponding heavy chains are ⁇ chains respectively , delta chains, gamma chains, alpha chains, and epsilon chains.
  • the same type of Ig can be divided into different subclasses according to the difference in the amino acid composition of the hinge region and the number and position of disulfide bonds in the heavy chain.
  • IgG can be divided into IgG1, IgG2, IgG3, and IgG4.
  • Each of the five classes of Ig can have a kappa chain or a lambda chain.
  • Each heavy chain consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region.
  • the heavy chain constant region consists of three domains (CH1, CH2 and CH3).
  • Each light chain consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region.
  • the light chain constant region contains one domain, CL.
  • the VH and VL regions can be further subdivided into hypervariable regions, termed complementarity determining regions (CDRs), interspersed with more conserved regions termed framework regions (FRs).
  • CDRs complementarity determining regions
  • Each light chain contains 3 CDR regions: LCDR1, LCDR2, and LCDR3; each heavy chain contains 3 CDR regions: HCDR1, HCDR2, and HCDR3.
  • Each VH and VL consists of three CDRs and four FRs arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the variable regions of the heavy and light chains contain binding domains that interact with the antigen.
  • the constant regions of the antibodies can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (eg, effector cells) and the first component (Clq) of the classical complement system.
  • CDRs complementarity determining regions
  • HCDR1, HCDR2, HCDR3 three CDRs
  • LCDR1, LCDR2, LCDR3 three CDRs
  • the amino acid sequence boundaries of CDRs can be determined by various well-known schemes, for example: the "Kabat” numbering convention (see Kabat et al. (1991), “Sequences of Proteins of Immunological Interest", 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD), the "Chothia” numbering scheme (see Martin, ACR.
  • the CDR amino acid residues in the heavy chain variable domain (VH) are numbered 31-35 (HCDR1), 50-65 (HCDR2) and 95-102 (HCDR3); light
  • the CDR amino acid residues in the chain variable domain (VL) are numbered 24-34 (LCDR1), 50-56 (LCDR2) and 89-97 (LCDR3).
  • CDR amino acids in VH are numbered 26-32 (HCDR1), 52-56 (HCDR2), and 95-102 (HCDR3); and amino acid residues in VL are numbered 24-34 (LCDR1), 50- 56 (LCDR2) and 89-97 (LCDR3).
  • the CDRs are defined by amino acid residues 26-35 (HCDR1), 50-65 (HCDR2) and 95-102 (HCDR3) in human VH and amino acid residues 24- 34 (LCDR1), 50-56 (LCDR2) and 89-97 (LCDR3).
  • CDR amino acid residue numbers in VH are approximately 27-38 (CDR1), 56-65 (CDR2), and 105-117 (CDR3)
  • CDR amino acid residues in VL are approximately 27-38 (CDR1 ), 56-65 (CDR2) and 105-117 (CDR3).
  • the CDR regions of antibodies can be determined using the program IMGT/DomainGap Align.
  • CDR amino acids in VH are numbered 26-35 (HCDR1), 50-58 (HCDR2), and 95-102 (HCDR3); and amino acid residues in VL are numbered 24-34 (LCDR1), 50- 56 (LCDR2) and 89-97 (LCDR3).
  • antibody framework or "FR region” refers to the portion of a variable domain VL or VH that serves as a scaffold for the antigen binding loops (CDRs) of the variable domain. Essentially, it is a variable domain without CDRs.
  • Antibody constant region domain refers to domains derived from the constant regions of the light and heavy chains of antibodies, including CL and CH1, CH2, CH3 and CH4 domains derived from different classes of antibodies.
  • the constant regions of the present disclosure also include "conventional variants" of the human antibody heavy chain constant regions and human antibody light chain constant regions, which refer to the human-derived variable regions disclosed in the prior art that do not alter the structure and function of the antibody variable regions Variants of the heavy chain constant region or light chain constant region of Technically known YTE mutations, L234A and/or L235A mutations, S228P mutations, and/or mutations that obtain a knob-into-hole structure (giving the antibody heavy chain a combination of knob-Fc and hole-Fc) that have been confirmed The antibody has new properties, but does not change the function of the variable region of the antibody.
  • antibody fragment refers to a molecule other than an intact antibody that comprises a portion of the intact antibody that binds to the antigen to which the intact antibody binds.
  • antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2, Fd, dAb; camelid VHH domains; diabodies; linear antibodies; ); and multispecific antibodies formed from antibody fragments.
  • a “single-chain variable fragment (scFv)”, also referred to as a “single-chain antibody,” is a fusion protein of the variable heavy (VH) and light chain (VL) domains of an antibody linked by a linker.
  • linkers are short polypeptides of 10 to 25 amino acids and are typically rich in glycine for flexibility, and serine or threonine for solubility, and can link the N-terminus of VH to the C-terminus of VL, or vice versa Of course. This protein retains the specificity of the original antibody despite the removal of the constant region and the introduction of linkers.
  • Linker refers to a polypeptide sequence used to connect polypeptides (such as protein domains), usually with a certain flexibility, and the use of the linker will not cause the loss of the original structure and function of the polypeptide.
  • Fc region is used herein to define the C-terminal region of an immunoglobulin heavy chain, which contains at least part of the constant region.
  • the term includes native sequence Fc regions and variant Fc regions.
  • the C-terminal lysine (Lys447) of the Fc region may or may not be present.
  • the numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also known as the EU index.
  • binding site or "antigen binding site” refers to the region of an antibody molecule that actually binds to an antigen.
  • the term “antigen binding site” comprises an antibody heavy chain variable domain (VH) and an antibody light chain variable domain (VL), or only the antibody heavy chain variable domain or the light chain variable domain.
  • a “chimeric” antibody refers to an antibody in which a portion of the heavy and/or light chain is derived from a particular source or species, and the remainder of the heavy and/or light chain 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 accomplished, for example, by retaining the non-human CDR regions and replacing the rest of the antibody with their human counterparts (ie, the constant regions and framework portions of the variable regions). See, eg, Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855, 1984; Morrison and Oi, Adv. Immunol. [Progress in Immunology], 44:65 -92, 1988; Verhoeyen et al., Science, 239:1534-1536, 1988; Padlan, Molec. Immun.
  • non-human antibodies are humanized to reduce immunogenicity to humans while retaining the specificity and affinity of the parental non-human antibody.
  • humanized antibodies comprise one or more variable domains, wherein HVRs (heavy chain variable regions), eg, CDRs (or portions thereof), are derived from non-human antibodies, and FRs (or portions thereof) are derived from human antibody sequences derivative.
  • the humanized antibody will also comprise at least a portion of a human constant region.
  • some FR residues in a humanized antibody are replaced with corresponding residues from a non-human antibody (eg, an antibody from which the HVR residues are derived), eg, to restore or improve antibody specificity or affinity.
  • human antibody is intended to include antibodies having variable regions in which both the framework and CDR regions are derived from sequences of human origin. Furthermore, if the antibody contains a constant region, the constant region is also derived from such human sequences, eg, human germline sequences or mutant forms of human germline sequences. Human antibodies of the present disclosure can include amino acid residues not encoded by human sequences (eg, mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo).
  • full-length antibody “intact antibody” and “whole antibody” are used interchangeably herein to refer to an antibody having a substantially similar structure to that of a native antibody or having a heavy chain containing an Fc region as defined herein. antibody.
  • antigen refers to a molecule or molecular portion capable of being bound by a selective binding agent such as an antigen-binding protein (including, for example, an antibody) and otherwise capable of being used in an animal to generate an antibody capable of binding the antigen.
  • a selective binding agent such as an antigen-binding protein (including, for example, an antibody) and otherwise capable of being used in an animal to generate an antibody capable of binding the antigen.
  • Antigens can have one or more epitopes capable of interacting with different antigen binding proteins (eg, antibodies).
  • epitope refers to an area or region on an antigen that is capable of specific binding by an antibody or antigen-binding fragment thereof.
  • Epitopes may be formed from contiguous strings of amino acids (linear epitopes) or comprise non-contiguous amino acids (conformational epitopes), eg, brought into steric proximity due to folding of the antigen (ie, tertiary folding of the antigen by a proteinaceous nature).
  • a conformational epitope differs from a linear epitope in that in the presence of a denaturing solvent, binding of the antibody to the conformational epitope 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 antigens (see Prot. Sci. 9 (2000) 487-496), and cross-blocking (see “Antibodies,” Harlow and Lane (Cold Spring Harbor Press, Cold Spring Harbor., NY)).
  • an "antibody that binds the same epitope" as a reference antibody refers to an antibody that blocks binding of the reference antibody to its antigen by 50% or more in a competition assay, and in contrast, a reference antibody that blocks the binding of the reference antibody to its antigen in a competition assay Binding to its antigen is blocked by 50% or more. Also for example, to determine whether a test antibody binds to the same epitope as a reference antibody, the reference antibody is allowed to bind to the antigen under saturating conditions. After removal of excess reference antibody, the ability of the test antibody to bind the antigen is assessed.
  • test antibody If the test antibody is able to bind the antigen after saturation binding of the reference antibody, then it can be concluded that the test antibody binds to different epitopes than the reference antibody. However, if the test antibody is unable to bind the antigen after saturation binding of the reference antibody, then the test antibody can bind to the same epitope as the reference antibody. To confirm whether the antibody to be tested binds to the same epitope or is only hindered by steric reasons, routine experiments (eg, peptide mutation and use of ELISA, RIA, surface plasmon resonance, flow cytometry, or any other available in the art can be used) Binding assays for quantitative or qualitative antibody binding assays). This assay should be performed in both settings, i.e. both antibodies as saturating antibodies. If, in both settings, only the first (saturating) antibody is able to bind the antigen, then it can be concluded that the test antibody and the reference antibody compete for binding to the antigen.
  • an antibody is 1-fold, 5-fold, 10-fold, 20-fold
  • Two antibodies are considered to bind the same or overlapping epitope if they inhibit binding of the other antibody by at least 50%, at least 75%, at least 90%, or even 99% or more by a fold or 100-fold excess.
  • “competition" between antibodies and/or antigen-binding fragments thereof means that two antibodies (or binding fragments thereof) bind to the same or overlapping epitopes (eg, as determined by competitive binding, as determined by the art determined by any method known to the skilled person). If the competing antibody or antigen-binding fragment thereof binds the same epitope or overlapping epitope as the antibody or antigen-binding fragment of the present disclosure, the antibody or antigen-binding fragment thereof also binds to the antibody or antigen-binding fragment of the present disclosure" compete”.
  • a competing antibody or antigen-binding fragment thereof may also include the following: (i) a competing antibody or antigen-binding fragment thereof that sterically blocks the binding of an antibody or antigen-binding fragment of the present disclosure to its target (eg, if the The competing antibodies bind adjacent, non-overlapping and/or the same epitope and physically prevent the antibody or antigen-binding fragment of the present disclosure from binding to its target); and/or (ii) bind different, non-overlapping epitopes A competing antibody or antigen-binding fragment thereof that is in position and induces a conformational change in the antigen such that the antigen no longer binds the antibody or antigen-binding fragment of the present disclosure in a manner that it would have without the conformational change.
  • binds means that an antibody binds to an antigen or epitope within that antigen with a higher affinity than for other antigens or epitopes.
  • antibodies are prepared at about 1 ⁇ 10-7M or less (eg, about 1 ⁇ 10-8M or less, about 1 ⁇ 10-9M or less, about 1 ⁇ 10-10M or less, about 1 ⁇ 10-
  • An equilibrium dissociation constant (KD) of 11 M or less, or about 1 x 10-12 M or less) binds an antigen or an epitope within an antigen, typically the KD is the antibody binding to a non-specific antigen (eg, BSA, casein) of at least one percent of the KD.
  • a non-specific antigen eg, BSA, casein
  • antibodies that specifically bind to an antigen or an epitope within an antigen may be cross-reactive to other related antigens, for example, to those from other species (homologous) such as humans or monkeys, eg, Macaca fascicularis (cynomolgus, cyno), chimpanzees (Pan troglodytes) (chimpanzee, chimp) or marmosets (Callithrix jacchus) (common marmoset, marmoset) are cross-reactive.
  • homologous such as humans or monkeys, eg, Macaca fascicularis (cynomolgus, cyno), chimpanzees (Pan troglodytes) (chimpanzee, chimp) or marmosets (Callithrix jacchus) (common marmoset, marmoset) are cross-reactive.
  • Affinity refers to the strength of the sum of all non-covalent interactions between a single binding site of a molecule (eg, an antibody) and its binding partner (eg, an antigen).
  • the affinity of a molecule X for its partner Y can generally be expressed in terms of the dissociation constant (KD). Affinity can be measured by common methods known in the art, including those described herein.
  • kassoc or “ka” is intended to refer to the rate of association of a particular antibody-antigen interaction
  • kdis or “kd” as used herein is intended to refer to the dissociation of a particular antibody-antigen interaction rate.
  • KD is intended to refer to the dissociation constant, which is obtained from the ratio of kd to ka (ie, kd/ka) and expressed as molar concentration (M).
  • M molar concentration
  • the KD value of an antibody can be determined using methods well established in the art. Methods for determining antibody KD include measuring surface plasmon resonance using biosensing systems such as systems, or measuring affinity in solution by solution equilibrium titration (SET).
  • nucleic acid is used interchangeably herein with the term “polynucleotide” and refers to deoxyribonucleotides or ribonucleotides and polymers thereof in single- or double-stranded form.
  • the term encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or linkages that are synthetic, naturally occurring and non-naturally occurring, have binding properties similar to the reference nucleic acid, and are Metabolized in a manner similar to the reference nucleotide.
  • Examples of such analogs include, but are not limited to, phosphorothioates, phosphoramidates, methylphosphonates, chiral-methylphosphonates, 2-O-methylribonucleotides, peptide-nucleic acid (PNA) ).
  • nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (eg, degenerate codon substitutions) and complementary sequences as well as explicitly indicated sequences.
  • degenerate codon substitutions can be obtained by generating sequences in which one or more selected (or all) codons are substituted at the third position by mixed bases and/or deoxy Inosine residue substitution (Batzer et al., Nucleic Acid Res. 19:5081, 1991; Ohtsuka et al., J. Biol. Chem. 260:2605-2608, 1985; and Rossolini et al. Human, Mol. Cell. Probes [Molecular and Cell Probes] 8:91-98, 1994).
  • sequence identity means that when two sequences are optimally aligned, gaps are introduced as necessary to obtain the maximum percent sequence identity, and any conservative substitutions are not considered to be part of the sequence identity, the two sequences The degree (percent) to which amino acids/nucleic acids are identical at equivalent positions.
  • alignment can be achieved in a variety of ways that are within the skill in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR). )software.
  • parameters suitable for measuring alignment including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.
  • Constantly modified variants or “conservative substitutions” refer to the replacement of amino acids in a protein with other amino acids having similar characteristics (e.g., charge, side chain size, hydrophilicity/hydrophobicity, backbone structure, rigidity, etc.), It is often possible to make such changes without altering the biological activity of the protein.
  • Those skilled in the art are aware that, in general, single amino acid substitutions in non-essential regions of a polypeptide do not substantially alter biological activity (see, e.g., Watson et al., (1987) Molecular Biology of the Gene, The Benjamin/Cummings Pub. Co., p. 224 (4th Ed.)).
  • substitution of structurally or functionally similar amino acids is unlikely to disrupt biological activity. Exemplary conservative substitutions are shown in the table below.
  • nucleic acid sequences refers to those nucleic acids encoding the same or substantially the same amino acid sequence, or in the case of the nucleic acid not encoding an amino acid sequence, substantially the same the sequence of. Due to the degeneracy of the genetic code, any given protein can be encoded by multiple functionally identical nucleic acids. For example, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at each position where a codon specifies an alanine, the codon can be changed to any of the corresponding codons described without changing the encoded polypeptide. Such nucleic acid variations are "silent variations," which are one type of conservatively modified variation.
  • Every nucleic acid sequence herein that encodes a polypeptide also describes every possible silent variation of the nucleic acid.
  • the skilled artisan will recognize that every codon in a nucleic acid (except AUG--usually the only codon for methionine; and TGG--usually the only codon for tryptophan) can be modified to produce function the same molecule.
  • each silent variation of the nucleic acid encoding the polypeptide is implied in each such sequence.
  • vector means a polynucleotide molecule capable of transporting another polynucleotide to which it is 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 or AAV2 adeno-associated viral vector
  • Certain vectors are capable of autonomous replication in the host cell into which they are introduced (eg, bacterial vectors with bacterial origins of replication and episomal mammalian vectors). Other vectors (eg, non-episomal mammalian vectors) can integrate into the genome of the host cell upon introduction into the host cell, thereby replicating together with the host genome.
  • expression vector refers to a device suitable for transforming a host cell and containing one or more heterologous coding regions that direct and/or control (along with the host cell) the expression of which is operably linked to it.
  • Expression constructs can include, but are not limited to, sequences that affect or control transcription, translation, and when introns are present, RNA splicing of the coding region to which they are operably linked.
  • operably linked means that the components to which the term applies are in a relationship that allows them to perform their inherent functions under suitable conditions.
  • a control sequence in a vector that is "operably linked" to a protein-coding sequence is linked thereto such that expression of the protein-coding sequence is achieved under conditions compatible with the transcriptional activity of the control sequence.
  • host cell refers to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells.
  • Host cells include “transformants” and “transformed cells,” which include the primary transformed cell and progeny derived therefrom without regard to the number of passages.
  • the progeny may not be identical in nucleic acid content to the parent cell, but may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected in the original transformed cell are included herein.
  • Host cells include prokaryotic and eukaryotic host cells, wherein eukaryotic host cells include, but are not limited to, mammalian cells, insect cell lines (eg, Spodoptera frugiperda or Trichoplusia ni), amphibians cells, bacterial cells, plant cells and fungal cells.
  • eukaryotic host cells include, but are not limited to, mammalian cells, insect cell lines (eg, Spodoptera frugiperda or Trichoplusia ni), amphibians cells, bacterial cells, plant cells and fungal cells.
  • Mammalian host cells include human, mouse, rat, canine, monkey, porcine, goat, bovine, equine and hamster cells, including but not limited to Chinese hamster ovary (CHO) cells, NSO, SP2 cells, HeLa cells, baby hamsters Kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (eg, Hep G2), A549 cells, 3T3 cells and HEK-293 cells.
  • CHO Chinese hamster ovary
  • NSO Chinese hamster ovary
  • SP2 cells HeLa cells
  • BHK baby hamsters Kidney
  • COS monkey kidney cells
  • human hepatocellular carcinoma cells eg, Hep G2
  • 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 thermolerans, Pichia salictaria), Pichia guercuum, Pichia pijperi, Pichia stiptis, Pichia methanolica, Pichia, Saccharomyces cerevisiae, Saccharomyces cerevisiae , Hansenula polymorpha, Kluyveromyces, Kluyveromyces lactis, Candida albicans, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Trichoderma reesei, Chrysosporium lucknowense, Fusarium
  • the host cell is a non-human cell.
  • the expressions "cell”, “cell line” and “cell culture” are used interchangeably and all such designations include progeny.
  • the words “transformants” and “transformed cells” include primary subject cells and cultures derived therefrom, regardless of the number of passages. It will also be appreciated that not all progeny will have exactly the same DNA content due to intentional or unintentional mutations. Mutant progeny that have the same function or biological activity as the original transformed cells from which they were screened are included.
  • “Pharmaceutical composition” means a mixture comprising one or more of the antibodies or antigen-binding fragments thereof described herein and other chemical components, such as physiological/pharmaceutically acceptable carriers and excipients.
  • pharmaceutically acceptable carrier means any solvent, dispersion medium, coating, antibacterial and antifungal agents, isotonic and absorption enhancing or delaying agents, and the like that are physiologically compatible.
  • pharmaceutically acceptable carriers are water, saline, phosphate buffered saline, acetate buffer with sodium chloride, dextrose, glycerol, polyethylene glycol, ethanol, and the like, and combinations thereof.
  • isotonic agents such as sugars, polyols (eg, mannitol, sorbitol) or sodium chloride in the composition.
  • pharmaceutically acceptable substances are surfactants, wetting agents or minor amounts of auxiliary substances such as wetting or emulsifying agents, preservatives or buffers, which enhance the shelf-life or effectiveness of the antibody.
  • compositions of the present disclosure can be administered by various methods known in the art.
  • the route and/or mode of administration will vary depending on the desired result.
  • administration may be intravitreal, intravenous, intramuscular, intraperitoneal or subcutaneous or near the target site.
  • a pharmaceutically acceptable carrier should be suitable for intravitreal, intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (eg, by injection or infusion).
  • the active compound ie, antibodies, bispecific and multispecific molecules
  • subject 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.
  • cyno or “cynomolgus” refers to a cynomolgus monkey (Macaca fascicularis).
  • the individual or subject is a human.
  • administering when applied to animals, humans, experimental subjects, cells, tissues, organs, or biological fluids, refer to exogenous drugs, therapeutic agents, diagnostic agents, or compositions Contact with animals, humans, 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 serous fluid, plasma, lymph, urine, saliva, cystic fluid, tears, feces, sputum, mucosal secretions of secretory tissues and organs, vaginal secretions, ascites , fluids in the pleura, pericardium, peritoneum, peritoneal cavity and other body cavities, fluids 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 conditions culture medium), lavage fluid, etc., tissue biopsy samples, fine needle aspiration, surgically resected tissue, organ cultures or cell cultures.
  • biological fluids such as blood, serum and serous fluid, plasma, lymph, urine, saliva, cystic fluid, tears, feces, sputum, mucosal secretions of
  • Treatment/treatment refers to clinical interventions that attempt to alter the natural course of the individual being treated, and may be performed for prophylaxis or during the course of clinical pathology. Desired effects of treatment include, but are not limited to, preventing the occurrence or recurrence of the disease, alleviating symptoms, alleviating/reducing any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, ameliorating or alleviating the disease state, and remission or amelioration of the disease. Prognosis.
  • the antibodies 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 underlying causes, prevent the appearance of symptoms and/or their underlying causes, and/or ameliorate or ameliorate impairments caused by or associated with a disease state (eg lung disease).
  • the effective amount is a therapeutically effective amount or a prophylactically effective amount.
  • a “therapeutically effective amount” is sufficient to treat a disease state or symptom, particularly a state or symptom associated with the disease state, or to otherwise prevent, retard, delay or reverse the disease state or any other irreversible disorder in any way associated with the disease state Amount of progression of desired symptoms.
  • a “prophylactically effective amount” is an amount that, when administered to a subject, will have a predetermined preventive 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 with the administration of a single dose, and may occur only after a series of doses have been administered.
  • a therapeutically or prophylactically effective amount can be administered in one or more administrations.
  • a “therapeutically effective amount” and a “prophylactically effective amount” may vary depending on factors such as the individual's disease state, age, sex and weight, 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 in a patient.
  • cancer and “cancerous” refer to or describe a physiological disorder that is typically characterized by unregulated cell growth in mammals. Benign and malignant cancers are included in this definition. "Early stage cancer” or “early stage tumor” refers to cancer that is either non-invasive or metastatic, or classified as stage 0, stage I, or stage II cancer.
  • cancers include, but are not limited to, lymphomas, blastomas (including medulloblastoma and retinoblastoma), sarcomas (including liposarcoma and synovial cell sarcoma), neuroendocrine tumors (including carcinoid tumors, gastrin tumor and islet cell carcinoma), mesothelioma, Schwann cell tumor (including acoustic neuroma), meningioma, adenocarcinoma, melanoma, adrenal tumor, and leukemic or lymphoid malignancies.
  • lymphomas including blastomas (including medulloblastoma and retinoblastoma), sarcomas (including liposarcoma and synovial cell sarcoma), neuroendocrine tumors (including carcinoid tumors, gastrin tumor and islet cell carcinoma), mesothelioma, Schwann cell tumor (including acoustic neuroma
  • squamous cell carcinoma eg, epithelial squamous cell carcinoma
  • lung cancer includes small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), adenocarcinoma of the lung, and squamous cell carcinoma of the lung ; Peritoneal cancer; Hepatocellular carcinoma; Gastric or stomach cancer including gastrointestinal cancer; Pancreatic cancer; Glioblastoma; Cervical cancer; Ovarian cancer; hepatoma); breast cancer (including metastatic breast cancer); colon cancer; rectal cancer; colorectal cancer; endometrial or uterine cancer; salivary gland cancer; kidney cancer (kidney or renal cancer); prostate cancer; vulvar cancer; thyroid cancer; anal cancer; penile cancer; testicular cancer; esophageal cancer; bile duct tumors; fallopian tube cancer; ovarian cancer; bile duct cancer; bladder cancer; pancreatic cancer; skin cancer; and head
  • the sequences encoding the extracellular domain of human ANG-2 with human IgG1-Fc tag and human ANG-2 receptor Tie2 were inserted into the phr vector, constructed into an expression plasmid, and then transfected into HEK293.
  • the specific transfection steps are as follows: the day before, HEK293E cells were inoculated in freestyle expression medium (containing 1% FBS, Gibco, 12338-026) at 1 ⁇ 10 6 /mL, placed on a 37-degree constant temperature shaker (120 rpm) and continued to culture for 24 Hour.
  • transfection plasmid and transfection reagent PEI were slowly added to 200 mL of HEK293E cells, and cultured in a shaker at 8% CO 2 , 120 rpm, and 37°C.
  • feed medium (20 mM glucose + 2 mM L-glutamate) was supplemented with 10% volume by volume.
  • cell supernatant was collected by centrifugation at 4500 rpm for 10 minutes, and the recombinant ANG-2 and Tie2 receptor proteins were purified according to the method described in Example 2.
  • the amino acid sequence of human ANG-2 is shown in SEQ ID NO: 1, and the amino acid sequence of Tie2 extracellular region Fc fusion protein is shown in SEQ ID NO: 2.
  • the horizontal line part is the full-length sequence of ANG-2 protein, the dotted line is the linker, and the italic part is the human IgG1Fc tag.
  • the underlined part is the extracellular region of Tie2, and the italicized part is the human IgG1 Fc tag.
  • the supernatant samples of cells expressing antibodies or huANG-2-Fc and huTie2-Fc were centrifuged at high speed to remove impurities, and purified by Protein A column.
  • the column was rinsed with PBS until the A280 reading dropped to baseline.
  • the target protein was eluted with 100 mM acetic acid pH 3.5 and neutralized with 1 M Tris-HCl, pH 8.0.
  • the eluted samples were properly concentrated and further purified by gel chromatography Superdex200 (GE) equilibrated with PBS, and the obtained proteins were identified as correct by electrophoresis, peptide map, and LC-MS, and then separated for use.
  • the present disclosure constructed a CHO-K1/Tie2 cell line expressing Tie2.
  • the full-length human Tie2 gene was cloned into the mammalian cell expression vector pBABE, and the three plasmids pVSV-G, pGag-pol and pBABE-Tie2 were used to co-transfect HEK293T cells (ATCC, CRL-3216) to package the virus for 48 hours. Afterwards, the virus was collected to infect CHOK1 cells (ATCC, CRL-9618). After 72 hours of infection, pressurized screening with 10 ⁇ g/mL puromycin. After the clones were expanded and grown, the cells were digested to detect the expression level by FACS, and the positive rate was about 40%, and then the monoclonal cells were sorted to obtain the monoclonal expressing human Tie2. 1B11.
  • the present disclosure prepares a monoclonal antibody against human ANG-2 through hybridoma technology, the obtained antibody has a high affinity with human ANG-2, and can cross-react with cynomolgus monkey ANG-2, and can block ANG-2 Binds to its receptor and inhibits ANG-2-induced phosphorylation of Tie2.
  • mice were immunized with recombinant protein huANG-2-Fc (100/50/50 ⁇ g) in TiterMax/Alum/CpG adjuvant. Specific immune responses to ANG-2 were determined by ELISA and ligand receptor blockade assays for serum titers. Mice with better specific immune response were selected, and after being sacrificed, spleen cells were taken and fused with myeloma cells.
  • the primary screening was performed with an ELISA binding assay for human and murine ANG-2, an assay that blocks binding of human ANG-2 to its receptor Tie2.
  • an ELISA binding assay for human and murine ANG-2 an assay that blocks binding of human ANG-2 to its receptor Tie2.
  • the cell supernatants were subjected to an ELISA binding assay against human and murine ANG-2, an ELISA-based receptor blocking assay that blocks ANG-2 binding to its receptor Tie2 Rescreen.
  • hybridoma clones were obtained for antibody production and purified by affinity method.
  • the active monoclonal hybridoma cell lines HR54 and CP33 were screened out, and the logarithmic growth phase hybridoma cells were collected respectively, RNA was extracted with NucleoZol (MN) (according to the kit instructions), and reverse transcription (PrimeScript TM Reverse Transcriptase) was performed. , Takara, cat#2680A).
  • the cDNA obtained by reverse transcription was amplified by PCR using mouse Ig-Primer Set (Novagen, TB326 Rev. B 0503) and then sent to a sequencing company for sequencing.
  • the mouse anti-ANG-2 antibodies were obtained by sequencing: HR54 and CP33, and the amino acid sequences of their variable regions are as follows:
  • the dashed lines represent CDRs, the italicized parts represent linkers, and the rest are FRs.
  • amino acid sequences of the CDR regions obtained according to the Kabat numbering rules are shown in the following table:
  • the light and heavy chain variable regions of the above-mentioned murine antibodies are combined with the light and heavy chain constant regions of human antibodies (heavy chain constant regions shown in SEQ ID NO: 47 and light chain constant regions shown in SEQ ID NO: 48). ) were connected to form a chimeric antibody, the chimeric antibody corresponding to the HR54 clone was named CHR54, and other antibodies were analogous.
  • Humanization of murine monoclonal antibodies can be carried out according to the methods disclosed in many literatures in the art. Briefly, on the basis of the obtained murine antibody VH/VL CDR canonical structures, the homologous sequences of the light chain variable region (VL) and heavy chain variable region (VH) were searched from the human germline database, The germline with high FR homology is selected as the template, the CDR region of the mouse antibody is transplanted to the human template, and some amino acids in the FR region are back-mutated, and the constant region of the mouse antibody is replaced with a human constant region to obtain The final humanized molecule.
  • VL light chain variable region
  • VH heavy chain variable region
  • the humanized VH template of HR54 is IGHV3-7*01, and the humanized VL template is IGKV4-1*01.
  • the CDRs of HR54 mouse antibody were transplanted to the human template, and the amino acids in the framework region were backmutated.
  • the specific reversion mutations are as follows:
  • Grafted represents the CDR of murine antibody implanted into the human germline FR region sequence
  • N84S represents the mutation of the 84th N back to S according to the Kabat numbering system.
  • variable region sequence of the mouse antibody HR54 humanized antibody is as follows:
  • the dashed lines represent CDRs, the italicized parts represent linkers, and the rest are FRs.
  • variable region of the heavy chain is linked with the constant region shown in SEQ ID NO: 47 to form an antibody heavy chain, and the variable region of the light chain is connected with the constant region shown in SEQ ID NO: 48 to form an antibody heavy chain.
  • the constant region is connected to form the antibody light chain, and the obtained humanized antibody of HR54 (huHR54), the variable regions of the heavy and light chains of different huHR54 antibodies are shown in the following table.
  • huHR54-01 antibody its heavy chain variable region is huHR54VH1
  • light chain variable region is huHR54VL1
  • heavy chain constant region is SEQ ID NO: 47
  • light chain constant region is SEQ ID NO: 48, and so on.
  • streptavidin (abcam, ab123480) at a concentration of 1 ng/ ⁇ L, 100 ⁇ L per well, overnight at 4°C, remove the supernatant, add 250 ⁇ L of 5% nonfat dry milk to block for 1 hour at 37°C, and wash with a plate washer. board 3 times. Add 0.5ng/ ⁇ L biotin-hAng2-His (sinobiologican, 10691-H07H), and incubate at 37°C for 1 hour. Wash the plate 3 times with a plate washer, add 100 ⁇ L of 1:1 diluted phage supernatant, and incubate at 37°C for 1 hour.
  • the HCDR2 of the huHR54-04 antibody was mutated with D52A E or D53N, and the HCDR3 was mutated with D95N, E96Q, C100S or C100V.
  • the CDR sequences of the huHR54-04 mutant obtained after the mutation were as follows:
  • mutated heavy chain variable region of huR54 is as follows:
  • the dashed lines represent CDRs, the italicized parts represent linkers, and the rest are FRs.
  • the heavy chain variable region and heavy chain constant region of the above-mentioned huHR54-04 mutant were combined to form a complete heavy chain sequence, and then recombined with the light chain of the huHR54-04 humanized antibody to obtain a full-length antibody.
  • the variable region of the heavy chain is linked with the constant region shown in SEQ ID NO: 47 to form an antibody heavy chain, and the variable region of the light chain is connected with the constant region shown in SEQ ID NO: 48 to form an antibody heavy chain.
  • the constant regions are connected to form the antibody light chain, and the combination of the heavy and light chain variable regions of the obtained humanized antibody is shown in the following table.
  • huHR54-07 antibody its heavy chain variable region is huHR54VH2a, light chain variable region is huHR54VL1, heavy chain constant region is SEQ ID NO: 47, light chain constant region is SEQ ID NO: 48, and other analogies.
  • the CDR regions of the huHR54 humanized antibody can be shown in the following table:
  • X 1 is D or E
  • X 2 is D or N
  • X 3 is D or N
  • X 4 is E or Q
  • X 5 is C, S or V.
  • the humanized VH template of CP33 is IGHV3-7*01, and the VL template is IGKV4-1*01.
  • the CDRs of CP33 are transplanted to the human template, and the amino acids in the framework region are back-mutated. The specific back-mutations are shown in the following table. :
  • variable region sequence of the mouse antibody CP33 humanized antibody is as follows:
  • the dashed lines represent CDRs, the italicized parts represent linkers, and the rest are FRs.
  • variable region of the heavy chain is linked with the constant region shown in SEQ ID NO: 47 to form an antibody heavy chain
  • variable region of the light chain is connected with the constant region shown in SEQ ID NO: 48 to form an antibody heavy chain.
  • the constant regions are connected to form the antibody light chain chain, and the obtained humanized antibody of CP33 (huCP33), the variable regions of the heavy and light chains of different huCP33 antibodies are shown in the following table.
  • huCP33-01 antibody its heavy chain variable region is huCP33VH1
  • light chain variable region is huCP33VL1
  • heavy chain constant region is SEQ ID NO: 47
  • light chain constant region is SEQ ID NO: 48, and so on.
  • the heavy and light chain constant regions of the antibodies of the exemplary antibodies are as follows:
  • exemplary huHR54 full-length antibodies and huCP33 full-length antibodies are as follows: (a) huHR54-01 antibody
  • the dashed lines represent CDRs, the rest are FRs, and the dashed-dotted lines represent constant regions.
  • the present disclosure constructs bispecific antibodies against VEGF and ANG-2 in different formats, including but not limited to IgG-scFv, DVD and crosmab formats.
  • Anti-VEGF antibody can be any antibody against VEGF currently known, such as Avastin (Avastin), RAZUMAB (Axxiom Inc), GNR-011 (Affitech A/S), R-TPR-024 (Reliance Life Sciences Grou) , Ramucirumab (ramucirumab, ImClone Systems), etc.
  • An exemplary antibody is the Genentech marketed Fab antibody ranibizumab (Lucentis), whose light chain variable region sequence is shown in SEQ ID NO: 56 (see WO1998045332 or CAS Registry Number: 347396-82-1).
  • the heavy chain of the anti-VEGF antibody is a complete IgG1 heavy chain that combines the heavy chain variable region of ranibizumab (sequence shown in SEQ ID NO: 58) with various human IgG1 constant regions. Its specific sequence is as follows:
  • Ranibizumab heavy chain variable region + IgG1 constant region (herein referred to as Ranibizumab heavy chain 1)
  • Ranibizumab heavy chain variable region + IgG1 constant region variant (herein referred to as Ranibizumab heavy chain 2)
  • the dashed lines represent CDRs, the italicized parts represent linkers, and the rest are FRs.
  • the heavy chain variable region and light chain variable region of the anti-ANG-2 antibody obtained by screening were connected by a linker to obtain an anti-ANG-2 single-chain antibody.
  • the linker can be any linker known in the art, and an exemplary linker is (GGGGS)n, where n is an integer from 1 to 10. Exemplary ANG-2 single chain antibody sequences are shown below:
  • the dashed lines represent CDRs, the italicized parts represent linkers, and the rest are FRs.
  • cysteine mutations were introduced into the heavy and light chain variable regions of the anti-ANG-2 single-chain antibody, and the exemplary sequences are as follows:
  • the dashed lines represent CDRs, and the rest are FRs.
  • sequence of the anti-ANG-2 single chain antibody is as follows:
  • the dashed lines represent CDRs, the italicized parts represent linkers, and the rest are FRs.
  • the anti-ANG-2 single-chain antibody is directly (through a peptide bond) or indirectly (through a linker) linked to the heavy or light chain of the anti-VEGF antibody by genetic recombination technology, and expressed through the 293 expression system to obtain a bispecific antibody .
  • the N-terminus of the anti-ANG-2 single chain antibody can be linked to the C-terminus of the heavy chain of the anti-VEGF antibody through a linker (eg (GG)n, where n is 1-20) to obtain a bispecific antibody , whose sequence is as follows:
  • the wavy underlined part represents the HCDR of the anti-VEGF antibody
  • the dotted underlined part represents the constant region
  • the double underlined part represents the linker
  • the italicized part represents ANG2-scFv
  • the underlined part represents the CDR of ANG2-scFv.
  • the dotted underline represents the constant region
  • the dashed portion represents the CDR.
  • the wavy underlined part represents the HCDR of the anti-VEGF antibody
  • the dotted underlined part represents the constant region
  • the double underlined part represents the linker
  • the italicized part represents ANG2-scFv
  • the underlined part represents the CDR of ANG2-scFv.
  • the dotted underline represents the constant region
  • the dashed portion represents the CDR.
  • the wavy underlined part represents the HCDR of the anti-VEGF antibody
  • the dotted underlined part represents the constant region
  • the double underlined part represents the linker
  • the italicized part represents ANG2-scFv
  • the underlined part represents the CDR of ANG2-scFv.
  • the dotted underline represents the constant region
  • the dashed portion represents the CDR.
  • the wavy underlined part represents the HCDR of the anti-VEGF antibody
  • the dotted underlined part represents the constant region
  • the double underlined part represents the linker
  • the italicized part represents ANG2-scFv
  • the underlined part represents the CDR of ANG2-scFv.
  • the dotted underline represents the constant region
  • the dashed portion represents the CDR.
  • the present disclosure constructs a bispecific antibody in the form of Crossmab, and the sequence of an exemplary bispecific antibody in the form of Crossmab is as follows:
  • the dotted underline represents the constant region, and the dashed portion represents the CDR.
  • the dotted underline represents the constant region
  • the dashed portion represents the CDR.
  • a bispecific antibody molecule with a purity of >98% can be obtained by using proteinA affinity chromatography to purify.
  • This disclosure also uses RG7716, Avastin, and RG7221 (vanucizumab) as control molecules, the sequences of which are shown below:
  • NC negative control
  • VEGF/ANG-2 bispecific antibody The affinity of the VEGF/ANG-2 bispecific antibody to human, monkey and mouse VEGF and ANG-2 was determined using a Biacore T200 (GE).
  • Antibodies were affinity captured with Protein A biosensor chip, and then the antigens human VEGF (R&D, 293-VE), monkey VEGF (sinobiological, 11066), mouse VEGF (sinobiological, 51059), and human ANG-2 were passed on the chip surface. (sinobiological, 10691-H08H), monkey ANG-2 (sinobiological, 90026-C07H), mouse ANG-2 (sinobiological, 50298-M07H). Reaction signals were detected in real time with a Biacore T200 instrument, and binding and dissociation curves were obtained.
  • Bispecific Antibody 1, Bispecific Antibody 5, Bispecific Antibody 2 and Bispecific Antibody 3 had high affinity to human and monkey VEGF and ANG-2.
  • Test Example 2 ELISA-based antibody blocking experiment of ANG-2 binding to Tie2 receptor
  • ANG-2 binds to the ANG-2 receptor Tie2 on the surface of vascular endothelial cells, triggers the phosphorylation of Tie2 intracellular tyrosine kinase, and then transmits signals to cause peripheral cells to fall off from vascular endothelial cells, leaving blood vessels in a state of instability and easy proliferation. . Therefore, blocking the binding of ANG-2 to Tie2 by antibodies can make blood vessels more stable and inhibit the formation of new blood vessels. The results of this experiment indicated that the bispecific antibody could block the binding of ANG-2 to the extracellular domain of recombinantly expressed Tie2 protein.
  • huANG-2-Fc (SEQ ID NO: 1, bio-huANG-2-Fc, final concentration of 0.15 ⁇ g/mL) and 50 ⁇ L of the antibody to be tested (initial concentration of 10 ⁇ g/mL, 3-fold ratio dilution) ), incubate at 37°C for 15 minutes after mixing, add to the ELISA plate, and incubate at 37°C for 1 hour. After the incubation, the reaction solution in the ELISA plate was discarded. After washing the plate with PBST for 5 times, 100 ⁇ L/well of 1:4000 diluted streptavidin-peroxidase polymer (Sigma, S2438-250UG) was added.
  • the coating solution was removed, and 250 ⁇ L/well of 1% BSA+0.05% NaN 3 blocking solution was used for blocking at room temperature for 2 hours.
  • 50 ⁇ L of the culture supernatant was discarded, 50 ⁇ L of the prepared antigen-antibody mixture was added, and the cells were incubated at 37°C for 10 minutes.
  • lysis buffer ((1 ⁇ lysis buffer + 10 ⁇ g/mL Leupeptin hemisulfate (Tocris, Cat#1167) + 10.0 ⁇ g/mL APROTININ, Sigma, Cat#SRE0050)) on ice for 10-15 minutes, centrifuge at 4000g for 5 minutes . The cell lysate was collected, added to the blocked ELISA plate, and incubated at room temperature for 2 h.
  • the plate was washed 5 times with PBST, the secondary antibody anti-PY-HRP (R&D Systems, Cat#DYC2720E) diluted 1:1000 was added, and incubated at room temperature for 1-2 hours.
  • the plate was washed 5 times with PBST, the color was developed with TMB for 15-30 minutes, and the color development was stopped with 1M H 2 SO 4 .
  • OD450 was read with Versa Max microplate reader and IC50 was calculated.
  • VEGF binds to VEGFR on vascular endothelial cells, phosphorylates the intracellular region of VEGFR, and promotes endothelial cell proliferation to form new blood vessels, thereby promoting tumor cell growth and metastasis. This experiment was used to identify bispecific antibodies that prevent VEGF-induced phosphorylation of VEGFR.
  • HUVEC cells (PromoCell/Miaotong Bio, C-12205) were digested, and the cell density was adjusted to contain 1.5 ⁇ 10 5 cells per 500 ⁇ L with complete medium, and added to a 24-well plate, 500 ⁇ L per well. After overnight incubation in a 37°C incubator, the medium was discarded, washed with 500 ⁇ L of ice DPBS (Gibco, 14190-250), and 200 ⁇ L of minimal medium containing 0.1% BSA was added to each well for starvation for 30 minutes.
  • ice DPBS Gibco, 14190-250
  • Test antibody negative control antibody were diluted with minimal medium to 10 nM, 1 nM and 0.1 nM (20 nM, 2 nM and 0.2 nM for RG7221 and bispecific antibody 5), PBS was used as control (ctrl).
  • VEGF R&D system, Cat#293-VE
  • VEGF was diluted to 400 ng/mL with minimal medium. Take an equal volume of diluted VEGF and antibody, mix them, add 200 ⁇ L to the corresponding well of the culture plate, and incubate at 37°C for 5 minutes.
  • 4X Lysis Buffer # 1 (cisbio, 63ADK041PEG) was diluted to 1X with ddH2O.
  • the blocking solution was diluted 100 times with 1 ⁇ lysis buffer to prepare a lysis solution. Take out the cell culture plate, discard the medium in the cell culture plate, add 500 ⁇ L of ice-cold PBS, and then discard the PBS after slight shaking. Immediately add 50 ⁇ L of the prepared lysate, place on a shaker and incubate at room temperature for 30 minutes. Centrifuge at 2400g for 10 minutes and collect the supernatant. p-VEGFR in the supernatant was detected using Phospho-VEGFR2 (Tyr1175) kit (cisbio, 63ADK041PEG).
  • the detection method is to take 10 ⁇ L of phospho-VEGFR2(Tyr1175)d2 antibody, add 200 ⁇ L of detection buffer, and prepare a working solution. Take 10 ⁇ L of phospho-VEGFR2 (Tyr1175) Cryptate antibody, add 200 ⁇ L detection buffer, and prepare a working solution. Mix equal volumes of d2 antibody working solution and Cryptate antibody working solution, add 16 ⁇ L of cell lysate and 4 ⁇ L of d2 antibody and Cryptate antibody mixture to HTRF96-well microplate, seal with sealing film, and centrifuge the microplate.
  • VEGF binds to VEGFR on HUVEC, phosphorylates the intracellular region of VEGFR, and promotes the proliferation of HUVEC. This experiment is used to identify bispecific antibodies that can prevent the proliferation of HUVEC induced by VEGF.
  • HUVEC cells in logarithmic growth phase were digested with 0.08% trypsin, about 1-2 minutes at room temperature, and stopped by adding 10% FBS.
  • the above-digested HUVECs were collected, centrifuged at 800 rpm/min for 5 minutes, washed three times with PBS to remove cytokines that stimulate HUVEC proliferation in the medium (800 rpm/min, centrifuged for 5 minutes).
  • the HUVEC cells were resuspended in 6% FBS medium, and after cell counting, 4000 cells/50 ⁇ L/well were seeded in a white 96-well cell culture plate, and cultured in an incubator for 2 hours.
  • the initial concentration of VEGF was adjusted to 300 ng/mL, and 120 ⁇ L/well was added to a sterile 96-well plate.
  • Gradient dilution of the antibody to be tested the initial concentration of the antibody is 600 nM, and the gradient is diluted by 4 times.
  • the diluted antibody is added in an equal volume to the above 96-well plate, and incubated at room temperature for 30 min. Add 100 ⁇ L/well of the incubated antibody and VEGF mixture to the adherent HUVEC cells, and culture in the incubator for 5 days. (G7573, PROMEGA), 50 ⁇ L/well, incubated at room temperature for 10 min in the dark, and detected by Cytation5 cell imager Luminescence program. The results are shown in Figure 4.
  • Test Example 6 Pharmacological effects of bispecific antibodies on mouse subcutaneous xenograft model of prostate cancer cells
  • mice 5 ⁇ 10 6 PC-3 cells (ATCC) were inoculated subcutaneously in the right flank of Balb/c nude mice.
  • the day of grouping was defined as day 0, and equimolar amount of each antibody was intraperitoneally injected on the day of grouping, twice a week, for a total of 6 times.
  • Tumor volumes, animal weights were monitored and data recorded twice weekly.
  • Tumor-bearing animals were euthanized as experimental endpoints when tumor volume exceeded 1000 mm 3 or when most tumors ruptured or lost 20% of body weight. All data were graphed and statistically analyzed using Excel and GraphPad Prism 5 software.
  • V tumor volume
  • T/C(%) (T-T0)/(C-C0) ⁇ 100, where T and C are the tumor volumes of the treatment group and the control group at the end of the experiment; T0 and C0 are the tumor volumes at the beginning of the experiment. tumor volume.
  • TGI (%) 1-T/C (%).
  • the grouping situation and dosing schedule are shown in Table 18, and the tumor growth curve and tumor inhibition rate are shown in Figure 5 and Table 19.
  • Dosage (mg/kg) dosing regimen 1 PBS (vehicle) 3 i.p.BIW*3 weeks 2 Avastin 3 i.p.BIW*3 weeks 3 RG7221 6 i.p.BIW*3 weeks 4 Bispecific Antibody 5 6 i.p.BIW*3 weeks 5 Bispecific Antibody 2 3.75 i.p.BIW*3 weeks
  • bispecific antibody 5 and bispecific antibody 2 of the present disclosure can significantly inhibit PC-3 tumor growth.
  • the antitumor effect of bispecific antibody 5 was significantly better than that of Avastin and RG7221.
  • Test Example 7 The efficacy of the bispecific antibody on the subcutaneous xenograft model of the highly metastatic non-small cell lung cancer H460-Luc cell line BALB/c nude mice
  • This experiment evaluated the effect of ANG-2/VEGF bispecific antibody in inhibiting the growth and metastasis of human non-small cell lung cancer H460 xenograft after intraperitoneal injection.
  • mice Female BALB/c nude mice, 4-5 weeks, 18-20 g, were purchased from Shanghai Lingchang Biotechnology Co., Ltd.
  • Tumor volumes were measured twice a week for 3 consecutive weeks after grouping. Tumor volume (V) was calculated as follows:
  • V (length ⁇ width2)/ 2 .
  • the relative tumor volume (RTV) per mouse was calculated as:
  • Vt/V0 where Vt is the measured volume per day and V0 is the volume at the start of treatment.
  • Results will be presented as mean ⁇ S.E.M. Comparisons between two groups will be tested with Dunnett's multiple comparisons test. Differences were considered statistically significant if p ⁇ 0.05.
  • FIG. 6 shows that both bispecific antibody 1 and bispecific antibody 2 antibodies of the present disclosure can significantly inhibit tumor growth.
  • Test Example 8 Pharmacological effects of bispecific antibodies on mouse subcutaneous xenograft model of human skin cancer cells
  • A431 cells 2 ⁇ 10 6 cells/mouse/100 ⁇ L were inoculated subcutaneously in the right flank of Balb/c nude mice.
  • the mice were randomly divided into 3 groups: Vehicle (PBS), bispecific antibody 2 4mpk and bispecific antibody 5 6mpk, 8 in each group.
  • the day of grouping was defined as day 0, and each antibody was intraperitoneally injected on the day of grouping, twice a week, for a total of 6 times, and the tumor volume and animal weight were monitored twice a week and the data were recorded.
  • Tumor-bearing animals were euthanized as experimental endpoints when tumor volume exceeded 1000 mm 3 or when most tumors ruptured or lost 20% of body weight. All data were graphed and statistically analyzed using Excel and GraphPad Prism 5 software. The tumor volume (V) was calculated as:
  • V 1/2 ⁇ a ⁇ b 2 , where a and b represent length and width, respectively.
  • T/C(%) (T-T0)/(C-C0) ⁇ 100, where T and C are the tumor volumes of the treatment group and the control group at the end of the experiment; T0 and C0 are the tumor volumes at the beginning of the experiment. tumor volume.
  • TGI (%) 1-T/C (%).
  • the grouping and dosing schedule are shown in Table 21, and the tumor growth curve is shown in Figure 7.
  • Test Example 9 Test of Bispecific Antibodies on Laser-induced Choroidal Neovascularization in Rhesus Monkeys
  • Lucentis group, RG7716 group, and bispecific antibody group 3 were given 50 ⁇ L Lucentis at a concentration of 1.92 mg/mL, 5.84 mg/mL RG7716 at 96 ⁇ g, 292 ⁇ g, and 396 ⁇ g/eye, respectively. 7.92 mg/mL of bispecific antibody 3, the vehicle control group was given an equal volume of solvent.
  • intraocular pressure examination, fundus color photography, fluorescein fundus angiography and optical coherence tomography (OCT) were performed to observe the inhibition of choroidal neovascularization by antibodies.
  • Lucentis group, RG7716 group, and bispecific antibody 3 groups all had a certain degree of reduction in the area of fluorescent spots at 7, 14, and 28 days after administration.
  • the improvement rate of fluorescence leakage area and the reduction of fluorescein leakage area in each group were all excellent.
  • the number of grade 4 fluorescent spots in each group was significantly lower than that in the vehicle control group.
  • the 2 ⁇ M bispecific antibody 3 groups, the 2 ⁇ M ranibizumab group, and the 2 ⁇ M RG7716 group could both reduce the area of fluorescent leakage and the number of fluorescent spots. Results See Figures 8A and 8B.
  • Aqueous VEGF Aqueous VEGF
  • VEGF vascular endothelial growth factor
  • bispecific antibody 3 has obvious inhibitory effect on monkey CNV at the dose of 396 ⁇ g/eye.

Abstract

涉及双特异性抗原结合分子，其包含特异性结合ANG-2的第一抗原结合域和特异性结合VEGF的第二抗原结合域。同时，也涉及特异性结合ANG-2的单克隆抗体，以及抗体的制备和应用。

Description

特异性结合VEGF和ANG-2的双特异性抗原结合分子 技术领域
本披露属于生物制药领域，具体地，本披露涉及抗ANG-2抗体以及抗ANG-2和抗VEGF的双特异性抗原结合分子的制备和应用。
背景技术
这里的陈述仅提供与本披露有关的背景信息，而不必然地构成现有技术。
新血管的生成为肿瘤细胞提供氧气和养料，使得肿瘤细胞获得生长优势，从无血管的慢速生长期到有血管的快速增长期。因此，通过抑制血管生成来抑制肿瘤的生长是一个较有潜力的有效策略。在众多促进血管生成的相关因子中，血管内皮生长因子VEGF是非常关键和重要的促进血管生成的因子。VEGF可以通过与VEGF受体结合促进细胞的增殖、迁移、增加血管通透性等促进肿瘤细胞的新生血管生成。因此通过阻断VEGF可抑制肿瘤血管的生成，进而达到抑制肿瘤生长和转移的目的。临床上有很多通过不同策略阻断VEGF的生物制剂，诸如针对VEGF的单抗阿瓦斯汀(Avastin)，中和VEGF的可溶性VEGF受体，针对VEGF受体的单抗等都显示出较好的活性。
但肿瘤血管的生成是由众多分子、多信号通路参与的复杂过程，通过阻断一条通路仍不能达到完全抑制肿瘤的目的，需要同时阻断其他血管生成相关因子。
Tie2是第二个被鉴定出的血管内皮细胞特异的酪氨酸激酶受体，其与配体血管生成素-1(ANG1)和血管生成素-2(ANG2)的结合对于血管生成也起着重要作用。ANG1与ANG2都结合Tie2，其中ANG1支持内皮细胞(EC)存活并促进血管的完整性和稳定性，而ANG2具有相反效应，可以使周边细胞从内皮细胞脱落下来，导致内皮细胞通透性提高，使VEGF发挥促进新生血管形成的作用。ANG2和VEGF在肿瘤的血管形成过程中互补协调，共同作用。因此，同时阻断VEGF和ANG2可以更有效抑制血管的生成，促进血管的正常化，达到抑制肿瘤生长和转移的目的。
目前，专利申请WO1998045332、WO2007095338A2、WO201004058、CN102250247A、WO2011117329等中公开了抗ANG-2和VEGF的双特异性抗体或VEGF抗体，但仍有待于开发新的高效的抗ANG-2和VEGF的双特异性抗体。
发明内容
本披露提供了一种靶向ANG-2和VEGF的双特异性抗体，其在体外，具有更好的阻断ANG-2与其受体Tie2结合的阻断活性，并可显著抑制VEGF引起的HUVEC细胞内磷酸化VEGFR水平的升高，抑制VEGF引起的HUVEC细胞的增殖。除此之外，本披露中的双特异性抗体，在小鼠体内具有更优异的抑制肿瘤生 长的效果，并可用于治疗眼部疾病，如AMD。
在一些实施方案中，本披露提供一种双特异性抗原结合分子，其包含特异性结合ANG-2的第一抗原结合域和特异性结合VEGF的第二抗原结合域，其中所述的特异性结合ANG-2的第一抗原结合域包含：
i)重链可变区，其包含分别如SEQ ID NO：7，SEQ ID NO：38和SEQ ID NO：39所示的HCDR1，HCDR2和HCDR3，和
轻链可变区，其包含分别如SEQ ID NO：10，SEQ ID NO：11和SEQ ID NO：12所示的LCDR1，LCDR2和LCDR3；其中：
SEQ ID NO：38的序列为：TINX 1X 2SSYTYYPDNVKG；
SEQ ID NO：39的序列为：X 3X 4ATGX 5FDY
其中，X 1为D或E，X 2为D或N，X 3为D或N，X 4为E或Q，X 5为C，S或V；
或
ii)重链可变区，其包含分别如SEQ ID NO：7，SEQ ID NO：13和SEQ ID NO：14所示的HCDR1，HCDR2和HCDR3，和
轻链可变区，其包含分别如SEQ ID NO：15，SEQ ID NO：11和SEQ ID NO：16所示的LCDR1，LCDR2和LCDR3。
在一些实施方案中，前述的双特异性抗原结合分子，其中所述的特异性结合ANG-2的第一抗原结合域包含：
重链可变区，其包含如SEQ ID NO：7所示的HCDR1，如SEQ ID NO：8、22或24所示的HCDR2，和如SEQ ID NO：9、23、25、26或27所示的HCDR3；和
轻链可变区，其包含分别如SEQ ID NO：10，SEQ ID NO：11和SEQ ID NO：12所示的LCDR1，LCDR2和LCDR3。
在一些实施方案中，前述的双特异性抗原结合分子，其中所述的特异性结合ANG-2的第一抗原结合域包含：
a)重链可变区，其包含分别如SEQ ID NO：7，SEQ ID NO：22和SEQ ID NO：23所示的HCDR1，HCDR2和HCDR3；和
轻链可变区，其包含分别如SEQ ID NO：10，SEQ ID NO：11和SEQ ID NO：12所示的LCDR1，LCDR2和LCDR3；
b)重链可变区，其包含分别如SEQ ID NO：7，SEQ ID NO：24和SEQ ID NO：23所示的HCDR1，HCDR2和HCDR3；和
轻链可变区，其包含分别如SEQ ID NO：10，SEQ ID NO：11和SEQ ID NO：12所示的LCDR1，LCDR2和LCDR3；
c)重链可变区，其包含分别如SEQ ID NO：7，SEQ ID NO：8和SEQ ID NO：25所示的HCDR1，HCDR2和HCDR3；和
轻链可变区，其包含分别如SEQ ID NO：10，SEQ ID NO：11和SEQ ID NO：12所示的LCDR1，LCDR2和LCDR3；
d)重链可变区，其包含分别如SEQ ID NO：7，SEQ ID NO：8和SEQ ID NO：26所示的HCDR1，HCDR2和HCDR3；和
轻链可变区，其包含分别如SEQ ID NO：10，SEQ ID NO：11和SEQ ID NO：12所示的LCDR1，LCDR2和LCDR3；
e)重链可变区，其包含分别如SEQ ID NO：7，SEQ ID NO：22和SEQ ID NO：25所示的HCDR1，HCDR2和HCDR3；和
轻链可变区，其包含分别如SEQ ID NO：10，SEQ ID NO：11和SEQ ID NO：12所示的LCDR1，LCDR2和LCDR3；
f)重链可变区，其包含分别如SEQ ID NO：7，SEQ ID NO：22和SEQ ID NO：26所示的HCDR1，HCDR2和HCDR3；和
轻链可变区，其包含分别如SEQ ID NO：10，SEQ ID NO：11和SEQ ID NO：12所示的LCDR1，LCDR2和LCDR3；
g)重链可变区，其包含分别如SEQ ID NO：7，SEQ ID NO：24和SEQ ID NO：25所示的HCDR1，HCDR2和HCDR3；和
轻链可变区，其包含分别如SEQ ID NO：10，SEQ ID NO：11和SEQ ID NO：12所示的LCDR1，LCDR2和LCDR3；
h)重链可变区，包含分别如SEQ ID NO：7，SEQ ID NO：24和SEQ ID NO：26所示的HCDR1，HCDR2和HCDR3；和
轻链可变区，其包含分别如SEQ ID NO：10，SEQ ID NO：11和SEQ ID NO：12所示的LCDR1，LCDR2和LCDR3；
j)重链可变区，其包含分别如SEQ ID NO：7，SEQ ID NO：8和SEQ ID NO：23所示的HCDR1，HCDR2和HCDR3；和
轻链可变区，其包含分别如SEQ ID NO：10，SEQ ID NO：11和SEQ ID NO：12所示的LCDR1，LCDR2和LCDR3；或
k)重链可变区，其包含分别如SEQ ID NO：7，SEQ ID NO：22和SEQ ID NO：27所示的HCDR1，HCDR2和HCDR3；和
轻链可变区，其包含分别如SEQ ID NO：10，SEQ ID NO：11和SEQ ID NO：12所示的LCDR1，LCDR2和LCDR3；或
l)重链可变区，其包含分别如SEQ ID NO：7，SEQ ID NO：8和SEQ ID NO：9所示的HCDR1，HCDR2和HCDR3；和
轻链可变区，其包含分别如SEQ ID NO：10，SEQ ID NO：11和SEQ ID NO：12所示的LCDR1，LCDR2和LCDR3。
在一些实施方式中，前述双特异性抗原结合分子以等于或小于10 -7M解离平衡常数与人ANG-2结合，在一些实施方式中，以等于或小于10 -8M或10 -9M解离 平衡常数与人ANG-2结合。
在一些实施方式中，前述双特异性抗原结合分子以等于或小于10 -7M解离平衡常数与人VEGF结合，在一些实施方式中，以等于或小于10 -8M、10 -9M、10 -10M、10 -11M、10 -12M或10 -13M解离平衡常数与人VEGF结合。
在一些实施方案中，前述双特异性抗原结合分子与猴的ANG-2和VEGF可交叉结合。
在一些实施方案中，前述的双特异性抗原结合分子以小于24.82nM，小于20nM，小于15nM，小于10nM，小于5nM，小于3nM，小于1nM，或小于0.5nM的IC50阻断ANG-2与ANG-2受体Tie2结合，其中所述的阻断活性通过测试例2所述的ELISA实验检测。
在一些实施方案中，前述的双特异性抗原结合分子以小于21.27nM，小于15nM，小于10nM，小于8nM或小于5nM的IC50抑制经Tie2转染的CHO细胞中ANG-2诱导的Tie2磷酸化。
在一些实施方案中，前述的双特异性抗原结合分子可显著抑制VEGF引起的HUVEC胞内磷酸化VEGFR水平升高。
在一些实施方案中，前述的双特异性抗原结合分子以小于18nM，小于10nM，或小于5nM的IC50抑制VEGF诱导的HUVEC细胞增殖。
在一些实施方案中，前述的双特异性抗原结合分子，其中所述的特异性结合ANG-2的第一抗原结合域包含：
i)重链可变区，包含SEQ ID NO：3、20、21、28-37、72或73中任一氨基酸序列，或包含与SEQ ID NO：3、20、21、28-37、72或73中任一序列分别具有至少95％，96％，97％，98％或99％的序列同一性的氨基酸序列；和/或
轻链可变区，包含SEQ ID NO：4、17、18、19或74的氨基酸序列，或包含与SEQ ID NO：4、17、18、19或74分别具有至少95％，96％，97％，98％或99％的序列同一性的氨基酸序列；或
ii)重链可变区，包含SEQ ID NO：5、44、45、46或75的氨基酸序列，或包含与SEQ ID NO：5、44、45、46或75分别具有至少95％，96％，97％，98％或99％的序列同一性的氨基酸序列；和/或
轻链可变区，包含SEQ ID NO：6、40、41、42、43或76的氨基酸序列，或包含与SEQ ID NO：6、40、41、42、43或76分别具有至少95％，96％，97％，98％或99％的序列同一性的氨基酸序列。
在一些实施方案中，前述的双特异性抗原结合分子，其中所述的特异性结合ANG-2的第一抗原结合域包含：
i)重链可变区，其氨基酸序列如SEQ ID NO：3、20、21、28-37、72或73中任一所示，或与SEQ ID NO：3、20、21、28-37、72或73分别具有至少95％，96％，97％，98％或99％的序列同一性；和/或
轻链可变区，其氨基酸序列如SEQ ID NO：4、17、18、19或74所示，或与SEQ ID NO：4、17、18、19或74分别具有至少95％，96％，97％，98％或99％的序列同一性；或
ii)重链可变区，其氨基酸序列如SEQ ID NO：5、44、45、46或75所示，或与SEQ ID NO：5、44、45、46或75分别具有至少95％，96％，97％，98％或99％的序列同一性；和/或
轻链可变区，其氨基酸序列如SEQ ID NO：6、40、41、42、43或76所示，或与SEQ ID NO：6、40、41、42、43或76分别具有至少95％，96％，97％，98％或99％的序列同一性。
在一些实施方案中，前述的双特异性抗原结合分子，其中所述的特异性结合ANG-2的第一抗原结合域包含：
a)重链可变区，包含SEQ ID NO：3的氨基酸序列；和轻链可变区，包含SEQ ID NO：4的氨基酸序列；
b)重链可变区，包含SEQ ID NO：20或21的氨基酸序列；和轻链可变区，包含SEQ ID NO：17、18或19的氨基酸序列；
c)重链可变区，包含SEQ ID NO：28-37中任一所示的氨基酸序列；和轻链可变区，包含SEQ ID NO：17的氨基酸序列；
d)重链可变区，包含SEQ ID NO：72或73的氨基酸序列；和轻链可变区，包含SEQ ID NO：74的氨基酸序列；
e)重链可变区，包含SEQ ID NO：5的氨基酸序列；和轻链可变区，包含SEQ ID NO：6的氨基酸序列；
f)重链可变区，包含SEQ ID NO：44、45或46的氨基酸序列；和轻链可变区，包含SEQ ID NO：40、41、42或43的氨基酸序列；或
g)重链可变区，包含SEQ ID NO：75的氨基酸序列；和轻链可变区，包含SEQ ID NO：76的氨基酸序列。在一些实施方案中，前述的双特异性抗原结合分子，其中所述的特异性结合ANG-2的第一抗原结合域包含：
a)如SEQ ID NO：3所示的重链可变区；和如SEQ ID NO：4所示的轻链可变区；
b)如SEQ ID NO：20或21所示的重链可变区；和如SEQ ID NO：17、18或19所示的轻链可变区；
c)如SEQ ID NO：28-37中任一所示的重链可变区；和如SEQ ID NO：17所示的轻链可变区；
d)如SEQ ID NO：72或73所示的重链可变区；和如SEQ ID NO：74所示的轻链可变区；
e)如SEQ ID NO：5所示的重链可变区；和如SEQ ID NO：6所示的轻链可变区；
f)如SEQ ID NO：44、45或46所示的重链可变区；和如SEQ ID NO：40、41、42或43所示的轻链可变区；或
g)如SEQ ID NO：75所示的重链可变区；和如SEQ ID NO：76所示的轻链可变区。
在一些实施方案中，前述的双特异性抗原结合分子，其中所述的特异性结合ANG-2的第一抗原结合域包含：
a)SEQ ID NO：20、21或33的重链可变区和SEQ ID NO：17的轻链可变区；
b)SEQ ID NO：44或45的重链可变区和SEQ ID NO：40的轻链可变区；
c)SEQ ID NO：72或73的重链可变区和SEQ ID NO：74的轻链可变区；或
d)SEQ ID NO：75的重链可变区和SEQ ID NO：76的轻链可变区。
在一些实施方案中，前述的双特异性抗原结合分子，其中所述的特异性结合ANG-2的第一抗原结合域包含：
重链可变区，包含SEQ ID NO：33的氨基酸序列，和轻链可变区，包含SEQ ID NO：17的氨基酸序列；或
重链可变区，包含SEQ ID NO：45的氨基酸序列，和轻链可变区，包含SEQ ID NO：40的氨基酸序列。在一些实施方案中，前述的双特异性抗原结合分子，其中所述的特异性结合ANG-2的第一抗原结合域包含：
如SEQ ID NO：33所示的重链可变区，和如SEQ ID NO：17所示的轻链可变区；或
如SEQ ID NO：45所示的重链可变区，和如SEQ ID NO：40所示的轻链可变区。
在一些实施方案中，前述的双特异性抗原结合分子，其中所述特异性结合VEGF的第二抗原结合域包含：
重链可变区，包含分别如SEQ ID NO：61，SEQ ID NO：62和SEQ ID NO：63所示的HCDR1，HCDR2和HCDR3；和
轻链可变区，包含分别如SEQ ID NO：64，SEQ ID NO：65和SEQ ID NO：66所示的LCDR1，LCDR2和LCDR3。
在一些实施方案中，前述的双特异性抗原结合分子，其中所述特异性结合VEGF的第二抗原结合域包含：
重链可变区，包含SEQ ID NO：58的氨基酸序列，和轻链可变区，包含SEQ ID NO：56的氨基酸序列。在一些实施方案中，前述的双特异性抗原结合分子，其中所述特异性结合VEGF的第二抗原结合域包含：
如SEQ ID NO：58所示的重链可变区，和如SEQ ID NO：56所示的轻链可变区。
在一些实施方案中，前述的双特异性抗原结合分子，其中所述特异性结合人VEGF的第二抗原结合域进一步包含重链恒定区和轻链恒定区。
在一些实施方案中，前述的双特异性抗原结合分子，其中所述特异性结合人VEGF的第二抗原结合域的重链恒定区包含L234A，L235A，I253A，H310A和H435A突变。
在一些实施方案中，前述的双特异性抗原结合分子，其中所述特异性结合人VEGF的第二抗原结合域的重链恒定区为IgG1恒定区，并包含L234A，L235A，I253A，H310A和H435A突变(EU编号)。
在一些实施方案中，前述的双特异性抗原结合分子，其中所述的特异性结合VEGF的第二抗原结合域包含：
重链，包含SEQ ID NO：59或60的氨基酸序列，或与SEQ ID NO：59或60具有至少95％，96％，97％，98％或99％的序列同一性的氨基酸序列；和
轻链，包含SEQ ID NO：57的氨基酸序列，或与SEQ ID NO：57具有至少95％，96％，97％，98％或99％的序列同一性的氨基酸序列。在一些实施方案中，前述的双特异性抗原结合分子，其中所述的特异性结合VEGF的第二抗原结合域包含：
重链，其氨基酸序列如SEQ ID NO：59或60所示，或与SEQ ID NO：59或60具有至少95％，96％，97％，98％或99％的序列同一性；和
轻链，其氨基酸序列如SEQ ID NO：57所示，或与SEQ ID NO：57具有至少95％，96％，97％，98％或99％的序列同一性。
在一些实施方案中，前述的双特异性抗原结合分子，其中所述的特异性结合VEGF的第二抗原结合域包含：
抗原结合分子，其中所述的特异性结合VEGF的第二抗原结合域包含：
如SEQ ID NO：59所示的重链；和
如SEQ ID NO：57所示的轻链。
在一些实施方案中，前述的双特异性抗原结合分子，其中所述的特异性结合VEGF的第二抗原结合域包含：
如SEQ ID NO：60所示的重链；和
如SEQ ID NO：57所示的轻链。
前述的双特异性抗原结合分子可具有本领域中公知的任意的双特异性抗体的分子结构。具体地，前述的双特异性抗原结合分子可以具有含Fc片段的双特异性抗体的分子结构，或者不含Fc片段的双特异性抗体的分子结构。更具体地，所述含Fc片段的双特异性抗体的分子结构包括但不限于TrioMab、Crossmab/KIH、DVD-Ig、IgG-scFv、FIT-Ig、mAb-Trap。不含Fc片段的双特异性抗体的分子结构包括但不限于BiTE、DART、TandAb、ImmTAC、TriKE。
在一些实施方案中，前述双特异性抗原结合分子是IgG-scFv形式。
在IgG-scFv形式的双特异性抗体示例性的实施方案中，所述的特异性结合ANG-2的第一抗原结合域为抗ANG-2单链抗体(scFv)，所述特异性结合VEGF 的第二抗原结合域为全长IgG抗体，其中所述抗ANG-2单链抗体直接地或通过连接子与特异性结合VEGF的全长IgG抗体重链或轻链的C端连接。
在一些实施方案中，前述的双特异性抗原结合分子，其中所述的特异性结合ANG-2的第一抗原结合域为抗ANG-2单链抗体。
在一些实施方案中，前述的双特异性抗原结合分子，其中所述的特异性结合ANG-2的第一抗原结合域为抗ANG-2单链抗体，其包含：
a)SEQ ID NO：20、21或33的重链可变区和SEQ ID NO：17的轻链可变区；
b)SEQ ID NO：44或45的重链可变区和SEQ ID NO：40的轻链可变区；
c)SEQ ID NO：72或73的重链可变区和SEQ ID NO：74的轻链可变区；或
d)SEQ ID NO：75的重链可变区和SEQ ID NO：76的轻链可变区。
在一些实施方案中，前述的双特异性抗原结合分子，其中所述的抗ANG-2单链抗体包含SEQ ID NO：67、68、69、70、71、77、78或79的氨基酸序列。
在一些实施方案中，前述的双特异性抗原结合分子，其中所述的特异性结合ANG-2的第一抗原结合域直接，或通过连接子连接至所述的特异性结合VEGF的第二抗原结合域。
在一些实施方案中，前述的双特异性抗原结合分子，其中所述的特异性结合ANG-2的第一抗原结合域直接，或通过连接子连接至所述的特异性结合VEGF的第二抗原结合域的重链或轻链。
在一些实施方案中，前述的双特异性抗原结合分子，其中所述的抗ANG-2单链抗体的N端通过连接子，连接至所述的特异性结合VEGF的第二抗原结合域的重链C-端。
在一些实施方案中，前述的双特异性抗原结合分子，其中所述的连接子为(GG) n，其中n为1-20中的整数。
在一些实施方案中，前述的双特异性抗原结合分子，其包含：
i)第一条链，包含SEQ ID NO：80、81、82或83的氨基酸序列，或与SEQ ID NO：80、81、82或83分别具有至少95％，96％，97％，98％或99％的序列同一性的氨基酸序列；和
ii)第二条链，其包含SEQ ID NO：57的氨基酸序列，或与SEQ ID NO：57具有至少95％，96％，97％，98％或99％的序列同一性的氨基酸序列。
在一些实施方案中，前述的双特异性抗原结合分子为具有IgG-scFv形式的4肽结构，其包含两条相同的第一条链和两条相同的第二条链，其中：
i)第一条链，包含SEQ ID NO：80、81、82或83的氨基酸序列，或与SEQ ID NO：80、81、82或83分别具有至少95％，96％，97％，98％或99％的序列同一性的氨基酸序列；和
ii)第二条链，其包含SEQ ID NO：57的氨基酸序列，或与SEQ ID NO：57具有至少95％，96％，97％，98％或99％的序列同一性的氨基酸序列。
在一些实施方案中，前述的双特异性抗原结合分子包含：
i)第一条链，其氨基酸序列如SEQ ID NO：80、81、82或83所示，或与SEQ ID NO：80、81、82或83分别具有至少95％，96％，97％，98％或99％的序列同一性；和
ii)第二条链，其氨基酸序列如SEQ ID NO：57所示，或与SEQ ID NO：57具有至少95％，96％，97％，98％或99％的序列同一性。
在一些实施方案中，前述的双特异性抗原结合分子为具有IgG-scFv形式的4肽结构，其包含两条相同的第一条链和两条相同的第二条链，其中：
i)第一条链的氨基酸序列如SEQ ID NO：80、81、82或83所示，或与SEQ ID NO：80、81、82或83分别具有至少95％，96％，97％，98％或99％的序列同一性；和
ii)第二条链的氨基酸序列如SEQ ID NO：57所示，或与SEQ ID NO：57具有至少95％，96％，97％，98％或99％的序列同一性。
在一些实施方案中，前述的双特异性抗原结合分子为具有IgG-scFv形式的4肽结构，其包含两条相同的第一条链和两条相同的第二条链，其中：
i)第一条链包含SEQ ID NO：80的氨基酸序列；和第二条链，其包含SEQ ID NO：57的氨基酸序列；
ii)第一条链，包含SEQ ID NO：81的氨基酸序列；和第二条链，其包含SEQ ID NO：57的氨基酸序列；
iii)第一条链，包含SEQ ID NO：82的氨基酸序列；和第二条链，其包含SEQ ID NO：57的氨基酸序列；或
iv)第一条链，包含SEQ ID NO：83的氨基酸序列；和第二条链，其包含SEQ ID NO：57的氨基酸序列。
在一些实施方案中，前述的双特异性抗原结合分子为具有IgG-scFv形式的4肽结构，其包含两条相同的第一条链和两条相同的第二条链，其中：
i)第一条链的氨基酸序列如SEQ ID NO：80所述；和第二条链的氨基酸序列如SEQ ID NO：57所示；
ii)第一条链的氨基酸序列如SEQ ID NO：81所示；和第二条链的氨基酸序列如SEQ ID NO：57所示；
iii)第一条链的氨基酸序列如SEQ ID NO：82所示；和第二条链的氨基酸序列如SEQ ID NO：57所示；或
iv)第一条链的氨基酸序列如SEQ ID NO：83所示；和第二条链的氨基酸序列如SEQ ID NO：57所示。
在一些实施方案中，前述的双特异性抗原结合分子具有Crossmab形式的4肽结构。
在一些实施方案中，所述Crossmab形式的双特异性抗原结合分子包含：
a)特异性结合ANG-2的第一全长抗体的第一轻链和第一重链；
b)特异性结合VEGF的第二全长抗体的第二轻链和第二重链；其中恒定结构域CL和CH1相互替换。
在一些实施方案中，前述的双特异性抗原结合分子包含：
a)特异性结合ANG-2的第一全长抗体的第一轻链和第一重链；和
b)特异性结合VEGF的第二全长抗体的第二轻链和第二重链；其中：
所述第一轻链的恒定结构域CL与第一重链的恒定结构域CH1相互替换；
所述第二轻链的恒定结构域CL与第二重链的恒定结构域CH1相互替换；
所述第一轻链的轻链可变区VL与第一重链的重链可变区VH相互替换；或
所述第二轻链的轻链可变区VL与第二重链的重链可变区VH相互替换。
在一些实施方案中，前述的双特异性抗原结合分子包含：
a)特异性结合ANG-2的第一全长抗体的第一轻链和第一重链；和
b)特异性结合VEGF的第二全长抗体的第二轻链和第二重链；并且，
其中所述第一轻链的恒定结构域CL与第一重链的恒定结构域CH1相互替换。
在一些实施方案中，前述的双特异性抗原结合分子包含：
a)特异性结合ANG-2的第一轻链和第一重链；
b)特异性结合VEGF的第二轻链和第二重链；其中恒定结构域CL和CH1相互替换。
在一些实施方案中，前述的双特异性抗原结合分子包含：
a)特异性结合ANG-2的第一轻链和第一重链；和
b)特异性结合VEGF的第二轻链和第二重链；其中：
所述第一轻链的恒定结构域CL与第一重链的恒定结构域CH1相互替换；
所述第二轻链的恒定结构域CL与第二重链的恒定结构域CH1相互替换；
所述第一轻链的轻链可变区VL与第一重链的重链可变区VH相互替换；或
所述第二轻链的轻链可变区VL与第二重链的重链可变区VH相互替换。
在一些实施方案中，前述的双特异性抗原结合分子包含：
a)特异性结合ANG-2的第一轻链和第一重链；和
b)特异性结合VEGF的第二轻链和第二重链；并且，
其中所述第一轻链的恒定结构域CL与第一重链的恒定结构域CH1相互替换。
在一些实施方案中，在前述的双特异性抗原结合分子中：
a)所述第一轻链包含SEQ ID NO：17的轻链可变区，和所述第一重链包含SEQ ID NO：33的重链可变区；或
所述第一轻链包含SEQ ID NO：40的轻链可变区，和所述第一重链包含SEQ ID NO：45的重链可变区；
和
b)所述第二轻链包含SEQ ID NO：56的轻链可变区，和所述第二重链包含 SEQ ID NO：58的重链可变区。
在一些实施方案中，前述的双特异性抗原结合分子，其中：
a)所述的第一轻链包含SEQ ID NO：85的氨基酸序列，或与SEQ ID NO：85具有至少95％，96％，97％，98％或99％的序列同一性的氨基酸序列，和
所述第一重链包含SEQ ID NO：84氨基酸序列，或与SEQ ID NO：84具有至少95％，96％，97％，98％或99％的序列同一性的氨基酸序列；或
所述的第一轻链包含SEQ ID NO：88的氨基酸序列，或与SEQ ID NO：88具有至少95％，96％，97％，98％或99％的序列同一性的氨基酸序列，和
所述第一重链包含SEQ ID NO：87氨基酸序列，或与SEQ ID NO：87具有至少95％，96％，97％，98％或99％的序列同一性的氨基酸序列；和
b)所述的第一轻链包含SEQ ID NO：57的氨基酸序列，或与SEQ ID NO：57具有至少95％，96％，97％，98％或99％的序列同一性的氨基酸序列；和
所述第一重链包含SEQ ID NO：86的氨基酸序列，或与SEQ ID NO：86具有至少95％，96％，97％，98％或99％的序列同一性的氨基酸序列。在一些实施方案中，在前述的双特异性抗原结合分子中：
a)所述的第一轻链的氨基酸序列如SEQ ID NO：85所示，或与SEQ ID NO：85具有至少95％，96％，97％，98％或99％的序列同一性，和
所述第一重链的氨基酸序列如SEQ ID NO：84所示，或与SEQ ID NO：84具有至少95％，96％，97％，98％或99％的序列同一性；或
所述的第一轻链的氨基酸序列如SEQ ID NO：88所示，或与SEQ ID NO：88具有至少95％，96％，97％，98％或99％的序列同一性，和
所述第一重链的氨基酸序列如SEQ ID NO：87所示，或与SEQ ID NO：87具有至少95％，96％，97％，98％或99％的序列同一性；和
b)所述第二轻链的氨基酸序列如SEQ ID NO：57所示，或与SEQ ID NO：57具有至少95％，96％，97％，98％或99％的序列同一性；和
所述第二重链的氨基酸序列如SEQ ID NO：86所示，或与SEQ ID NO：86具有至少95％，96％，97％，98％或99％的序列同一性。
在一些实施方案中，在前述的双特异性抗原结合分子中：
i)所述的第一轻链的氨基酸序列如SEQ ID NO：85所示，和所述第一重链的氨基酸序列如SEQ ID NO：84所示；和
所述的第二轻链的氨基酸序列如SEQ ID NO：57所示，和所述第二重链的氨基酸序列如SEQ ID NO：86所示；或
ii)所述的第一轻链的氨基酸序列如SEQ ID NO：88所示，和所述第一重链的氨基酸序列如SEQ ID NO：87所示；和
所述的第二轻链的氨基酸序列如SEQ ID NO：57所示，和所述第二重链的氨基酸序列如SEQ ID NO：86所示。
在一些实施方案中，本披露提供一种特异性结合ANG-2的抗体。
在一些实施方案中，前述的特异性结合ANG-2的抗体，其包含：
i)重链可变区，包含分别如SEQ ID NO：7，SEQ ID NO：38和SEQ ID NO：39所示的HCDR1，HCDR2和HCDR3，和
轻链可变区，包含分别如SEQ ID NO：10，SEQ ID NO：11和SEQ ID NO：12所示的LCDR1，LCDR2和LCDR3；其中：
SEQ ID NO：38的序列为：TINX 1X 2SSYTYYPDNVKG；
SEQ ID NO：39的序列为：X 3X 4ATGX 5FDY
其中，X 1为D或E，X 2为D或N，X 3为D或N，X 4为E或Q，X 5为C，S或V；
或
ii)重链可变区，包含分别如SEQ ID NO：7，SEQ ID NO：13和SEQ ID NO：14所示的HCDR1，HCDR2和HCDR3，和
轻链可变区，包含分别如SEQ ID NO：15，SEQ ID NO：11和SEQ ID NO：16所示的LCDR1，LCDR2和LCDR3。
在一些实施方案中，前述的特异性结合ANG-2的抗体，其包含：
重链可变区，包含如SEQ ID NO：7所示的HCDR1，如SEQ ID NO：8、22或24所示的HCDR2，和如SEQ ID NO：9、23、25、26或27所示的HCDR3；和
轻链可变区，包含分别如SEQ ID NO：10，SEQ ID NO：11和SEQ ID NO：12所示的LCDR1，LCDR2和LCDR3。
在一些实施方案中，前述的特异性结合ANG-2的抗体，其包含：
a)重链可变区，其包含分别如SEQ ID NO：7，SEQ ID NO：22和SEQ ID NO：23所示的HCDR1，HCDR2和HCDR3；和
轻链可变区，其包含分别如SEQ ID NO：10，SEQ ID NO：11和SEQ ID NO：12所示的LCDR1，LCDR2和LCDR3；
b)重链可变区，其包含分别如SEQ ID NO：7，SEQ ID NO：24和SEQ ID NO：23所示的HCDR1，HCDR2和HCDR3；和
轻链可变区，其包含分别如SEQ ID NO：10，SEQ ID NO：11和SEQ ID NO：12所示的LCDR1，LCDR2和LCDR3；
c)重链可变区，其包含分别如SEQ ID NO：7，SEQ ID NO：8和SEQ ID NO：25所示的HCDR1，HCDR2和HCDR3；和
轻链可变区，其包含分别如SEQ ID NO：10，SEQ ID NO：11和SEQ ID NO：12所示的LCDR1，LCDR2和LCDR3；
d)重链可变区，其包含分别如SEQ ID NO：7，SEQ ID NO：8和SEQ ID NO：26所示的HCDR1，HCDR2和HCDR3；和
轻链可变区，其包含分别如SEQ ID NO：10，SEQ ID NO：11和SEQ ID NO：12所示的LCDR1，LCDR2和LCDR3；
e)重链可变区，其包含分别如SEQ ID NO：7，SEQ ID NO：22和SEQ ID NO：25所示的HCDR1，HCDR2和HCDR3；和
轻链可变区，其包含分别如SEQ ID NO：10，SEQ ID NO：11和SEQ ID NO：12所示的LCDR1，LCDR2和LCDR3；
f)重链可变区，其包含分别如SEQ ID NO：7，SEQ ID NO：22和SEQ ID NO：26所示的HCDR1，HCDR2和HCDR3；和
轻链可变区，其包含分别如SEQ ID NO：10，SEQ ID NO：11和SEQ ID NO：12所示的LCDR1，LCDR2和LCDR3；
g)重链可变区，其包含分别如SEQ ID NO：7，SEQ ID NO：24和SEQ ID NO：25所示的HCDR1，HCDR2和HCDR3；和
轻链可变区，其包含分别如SEQ ID NO：10，SEQ ID NO：11和SEQ ID NO：12所示的LCDR1，LCDR2和LCDR3；
h)重链可变区，包含分别如SEQ ID NO：7，SEQ ID NO：24和SEQ ID NO：26所示的HCDR1，HCDR2和HCDR3；和
轻链可变区，其包含分别如SEQ ID NO：10，SEQ ID NO：11和SEQ ID NO：12所示的LCDR1，LCDR2和LCDR3；
j)重链可变区，其包含分别如SEQ ID NO：7，SEQ ID NO：8和SEQ ID NO：23所示的HCDR1，HCDR2和HCDR3；和
轻链可变区，其包含分别如SEQ ID NO：10，SEQ ID NO：11和SEQ ID NO：12所示的LCDR1，LCDR2和LCDR3；或
k)重链可变区，其包含分别如SEQ ID NO：7，SEQ ID NO：22和SEQ ID NO：27所示的HCDR1，HCDR2和HCDR3；和
轻链可变区，其包含分别如SEQ ID NO：10，SEQ ID NO：11和SEQ ID NO：12所示的LCDR1，LCDR2和LCDR3；或
l)重链可变区，其包含分别如SEQ ID NO：7，SEQ ID NO：8和SEQ ID NO：9所示的HCDR1，HCDR2和HCDR3；和
轻链可变区，其包含分别如SEQ ID NO：10，SEQ ID NO：11和SEQ ID NO：12所示的LCDR1，LCDR2和LCDR3。
在一些实施方式中，前述的特异性结合ANG-2的抗体以等于或小于10 -7M解离平衡常数与人ANG-2结合，在一些实施方式中，以等于或小于10 -8M或10 -9M解离平衡常数与人ANG-2结合。
在一些实施方案中，前述的特异性结合ANG-2的抗体与猴的ANG-2可交叉结合。
在一些实施方案中，前述的特异性结合ANG-2的抗体以小于24.82nM，小于 20nM，小于15nM，小于10nM或小于5nM的IC50阻断ANG-2与ANG-2受体Tie2结合，其中所述的阻断活性通过测试例2所述的ELISA实验检测。
在一些实施方案中，前述的特异性结合ANG-2的抗体以小于21.27nM，小于15nM，小于10nM或小于8nM的IC50抑制经Tie2转染的CHO细胞中ANG-2诱导的Tie2磷酸化。
在一些实施方案中，前述的特异性结合ANG-2的抗体，其为鼠源抗体，嵌合抗体，人源化抗体或全人抗体。
在一些实施方案中，前述的特异性结合ANG-2的抗体，其中所述的抗体包含框架区，其中：
i)所述重链框架区包含选自44R、77S或84S中的一个或更多个氨基酸回复突变；和/或
所述轻链框架区包含选自1N、43S、68A、85D、87H中的一个或更多个氨基酸回复突变；或
ii)所述重链框架区包含选自2L、44R、74V、82AS或83K中的一个或更多个氨基酸回复突变；和/或
所述轻链框架区包含选自1N、43S、46V、68A、85D、87H中的一个或更多个氨基酸回复突变。
在一些实施方案中，前述的特异性结合ANG-2的抗体，其包含：
i)重链可变区，其包含分别如SEQ ID NO：7，SEQ ID NO：38和SEQ ID NO：39所示的HCDR1，HCDR2和HCDR3，且其重链框架区包含选自44R、77S或84S中的一个或更多个氨基酸回复突变；和
轻链可变区，其包含分别如SEQ ID NO：10，SEQ ID NO：11和SEQ ID NO：12所示的LCDR1，LCDR2和LCDR3；且其轻链框架区包含选自1N、43S、68A、85D、87H中的一个或更多个氨基酸回复突变；其中：
SEQ ID NO：38的序列为：TINX 1X 2SSYTYYPDNVKG；
SEQ ID NO：39的序列为：X 3X 4ATGX 5FDY
其中，X 1为D或E，X 2为D或N，X 3为D或N，X 4为E或Q，X 5为C，S或V；或
ii)重链可变区，其包含分别如SEQ ID NO：7，SEQ ID NO：13和SEQ ID NO：14所示的HCDR1，HCDR2和HCDR3，且其重链框架区包含选自2L、44R、74V、82AS或83K中的一个或更多个氨基酸回复突变；和
轻链可变区，其包含分别如SEQ ID NO：15，SEQ ID NO：11和SEQ ID NO：16所示的LCDR1，LCDR2和LCDR3，且其轻链框架区包含选自1N、43S、46V、68A、85D、87H中的一个或更多个氨基酸回复突变。
在一些实施方案中，前述的特异性结合ANG-2的抗体，其包含：
重链可变区，其包含分别如SEQ ID NO：7，SEQ ID NO：22和SEQ ID NO： 26所示的HCDR1，HCDR2和HCDR3，且其重链框架区包含选自44R、77S或84S中的一个或更多个氨基酸回复突变；和
轻链可变区，其包含分别如SEQ ID NO：10，SEQ ID NO：11和SEQ ID NO：12所示的LCDR1，LCDR2和LCDR3，且轻链框架区包含选自1N、43S、68A、85D、87H中的一个或更多个氨基酸回复突变。
在一些实施方案中，前述的特异性结合ANG-2的抗体，其包含：
重链可变区，包含SEQ ID NO：20的氨基酸序列，或在SEQ ID NO：20的氨基酸序列中包含选自44R、77S或84S中的一个或更多个氨基酸回复突变的氨基酸序列；和
轻链可变区，包含SEQ ID NO：17的氨基酸序列，或在SEQ ID NO：17的氨基酸序列中包含选自1N、43S、68A、85D、87H中的一个或更多个氨基酸回复突变的氨基酸序列。
在一些实施方案中，前述的特异性结合ANG-2的抗体，其包含：
重链可变区，其氨基酸序列如SEQ ID NO：20所示，或在SEQ ID NO：20的氨基酸序列中包含选自44R、77S或84S中的一个或更多个氨基酸回复突变；和
轻链可变区，其氨基酸序列如SEQ ID NO：17所示，或在SEQ ID NO：17的氨基酸序列中包含选自1N、43S、68A、85D、87H中的一个或更多个氨基酸回复突变。
在一些实施方案中，前述的特异性结合ANG-2的抗体，其包含：
重链可变区，包含SEQ ID NO：33的氨基酸序列；和
轻链可变区，包含SEQ ID NO：17的氨基酸序列，或在SEQ ID NO：17的氨基酸序列中包含选自1N、43S、68A、85D、87H中的一个或更多个氨基酸回复突变的氨基酸序列。
在一些实施方案中，前述的特异性结合ANG-2的抗体，其包含：
SEQ ID NO：33的重链可变区；和
轻链可变区，其氨基酸序列如SEQ ID NO：17所示，或在SEQ ID NO：17的氨基酸序列中包含选自1N、43S、68A、85D、87H中的一个或更多个氨基酸回复突变。
在一些实施方案中，前述的特异性结合ANG-2的抗体，其包含：
重链可变区，包含SEQ ID NO：44的氨基酸序列，或在SEQ ID NO：44的氨基酸序列中包含选自2L、44R、74V、 82AS或83K中的一个或更多个氨基酸回复突变的氨基酸序列；和
轻链可变区，包含SEQ ID NO：40的氨基酸序列，或在SEQ ID NO：40的氨基酸序列中包含选自1N、43S、46V、68A、85D、87H中的一个或更多个氨基酸回复突变的氨基酸序列。
在一些实施方案中，前述的特异性结合ANG-2的抗体，其包含：
重链可变区，其氨基酸序列如SEQ ID NO：44所示，或在SEQ ID NO：44的氨基酸序列中包含选自2L、44R、74V、 82AS或83K中的一个或更多个氨基酸回复突变；和
轻链可变区，其氨基酸序列如SEQ ID NO：40所示，或在SEQ ID NO：40的氨基酸序列中包含选自1N、43S、46V、68A、85D、87H中的一个或更多个氨基酸回复突变。
在一些实施方案中，前述的特异性结合ANG-2的抗体，其包含：
i)重链可变区，包含SEQ ID NO：3、20、21、28-37、72或73中任一氨基酸序列，或包含与SEQ ID NO：3、20、21、28-37、72或73中任一序列分别具有至少95％，96％，97％，98％或99％的序列同一性的氨基酸序列；和/或
轻链可变区，包含SEQ ID NO：4、17、18、19或74的氨基酸序列，或包含与SEQ ID NO：4、17、18、19或74分别具有至少95％，96％，97％，98％或99％的序列同一性的氨基酸序列；或
ii)重链可变区，包含SEQ ID NO：5、44、45、46或75的氨基酸序列，或包含与SEQ ID NO：5、44、45、46或75分别具有至少95％，96％，97％，98％或99％的序列同一性的氨基酸序列；和/或
轻链可变区，包含SEQ ID NO：6、40、41、42、43或76的氨基酸序列，或包含与SEQ ID NO：6、40、41、42、43或76分别具有至少95％，96％，97％，98％或99％的序列同一性的氨基酸序列。
在一些实施方案中，前述的特异性结合ANG-2的抗体，其包含：
i)重链可变区，其氨基酸序列如SEQ ID NO：3、20、21、28-37、72或73中任一所示，或与SEQ ID NO：3、20、21、28-37、72或73分别具有至少95％，96％，97％，98％或99％的序列同一性；和/或
轻链可变区，其氨基酸序列如SEQ ID NO：4、17、18、19或74所示，或与SEQ ID NO：4、17、18、19或74分别具有至少95％，96％，97％，98％或99％的序列同一性；或
ii)重链可变区，其氨基酸序列如SEQ ID NO：5、44、45、46或75所示，或与SEQ ID NO：5、44、45、46或75分别具有至少95％，96％，97％，98％或99％的序列同一性；和/或
轻链可变区，其氨基酸序列如SEQ ID NO：6、40、41、42、43或76所示，或与SEQ ID NO：6、40、41、42、43或76分别具有至少95％，96％，97％，98％或99％的序列同一性。
在一些实施方案中，前述的特异性结合ANG-2的抗体，其包含：
a)重链可变区，包含SEQ ID NO：3的氨基酸序列；和轻链可变区，包含SEQ ID NO：4的氨基酸序列；
b)重链可变区，包含SEQ ID NO：20或21的氨基酸序列；和轻链可变区， 包含SEQ ID NO：17、18或19的氨基酸序列；
c)重链可变区，包含SEQ ID NO：28-37中任一所示的氨基酸序列；和轻链可变区，包含SEQ ID NO：17的氨基酸序列；
d)重链可变区，包含SEQ ID NO：72或73的氨基酸序列；和轻链可变区，包含SEQ ID NO：74的氨基酸序列；
e)重链可变区，包含SEQ ID NO：5的氨基酸序列；和轻链可变区，包含SEQ ID NO：6的氨基酸序列；
f)重链可变区，包含SEQ ID NO：44、45或46的氨基酸序列；和轻链可变区，包含SEQ ID NO：40、41、42或43的氨基酸序列；或
g)重链可变区，包含SEQ ID NO：75的氨基酸序列；和轻链可变区，包含SEQ ID NO：76的氨基酸序列。
在一些实施方案中，前述的特异性结合ANG-2的抗体，其包含：
a)SEQ ID NO：3的重链可变区；和SEQ ID NO：4的轻链可变区；
b)SEQ ID NO：20或21的重链可变区；和SEQ ID NO：17、18或19的轻链可变区；
c)SEQ ID NO：28-37中任一所示的重链可变区；和SEQ ID NO：17的轻链可变区；
d)SEQ ID NO：72或73的重链可变区；和SEQ ID NO：74的轻链可变区；
e)SEQ ID NO：5的重链可变区；和SEQ ID NO：6的轻链可变区；
f)SEQ ID NO：44、45或46的重链可变区；和SEQ ID NO：40、41、42或43的轻链可变区；或
g)SEQ ID NO：75的重链可变区；和SEQ ID NO：76的轻链可变区。
在一些实施方案中，前述的特异性结合ANG-2的抗体，其包含：
a)SEQ ID NO：20、21或33的重链可变区和SEQ ID NO：17的轻链可变区；
b)SEQ ID NO：44或45的重链可变区和SEQ ID NO：40的轻链可变区；
c)SEQ ID NO：72或73的重链可变区和SEQ ID NO：74的轻链可变区；或
d)SEQ ID NO：75的重链可变区和SEQ ID NO：76的轻链可变区。
在一些实施方案中，前述的特异性结合ANG-2的抗体，其包含：
重链可变区，包含SEQ ID NO：33的氨基酸序列，和轻链可变区，包含SEQ ID NO：17的氨基酸序列；或
重链可变区，包含SEQ ID NO：45的氨基酸序列，和轻链可变区，包含SEQ ID NO：40的氨基酸序列。
在一些实施方案中，前述的特异性结合ANG-2的抗体，其包含：
SEQ ID NO：33的重链可变区，和SEQ ID NO：17的轻链可变区；或
SEQ ID NO：45的重链可变区，和SEQ ID NO：40的轻链可变区。
在一些实施方案中，前述的特异性结合ANG-2的抗体，其包含恒定区。在另外一些实施方案中，所述重链恒定区选自人IgG1、IgG2、IgG3和IgG4恒定区及其常规变体，所述轻链恒定区选自人抗体κ和λ链恒定区及其常规变体。
在一些实施方案中，前述的特异性结合ANG-2的抗体，其包含SEQ ID NO：47的重链恒定区和SEQ ID NO：48的轻链恒定区。
在一些实施方案中，前述的特异性结合ANG-2的抗体，其包含：
a)重链，包含SEQ ID NO：49、51或52的氨基酸序列，或包含与SEQ ID NO：49、51或52分别具有至少95％，96％，97％，98％或99％的序列同一性的氨基酸序列，和/或
轻链，包含SEQ ID NO：50的氨基酸序列，或包含与SEQ ID NO：50具有至少95％，96％，97％，98％或99％的序列同一性的氨基酸序列；或
b)重链，包含SEQ ID NO：53或55的氨基酸序列，或包含与SEQ ID NO：53或55分别具有至少95％，96％，97％，98％或99％的序列同一性的氨基酸序列，和/或
轻链，包含SEQ ID NO：54的氨基酸序列，或包含与SEQ ID NO：54具有至少95％，96％，97％，98％或99％的序列同一性的氨基酸序列。
在一些实施方案中，前述的特异性结合ANG-2的抗体，其包含：
a)重链，其氨基酸序列如SEQ ID NO：49、51或52所示，或与SEQ ID NO：49、51或52分别具有至少95％，96％，97％，98％或99％的序列同一性，和/或
轻链，其氨基酸序列如SEQ ID NO：50所示，或与SEQ ID NO：50具有至少95％，96％，97％，98％或99％的序列同一性；或
b)重链，其氨基酸序列如SEQ ID NO：53或55所示，或与SEQ ID NO：53或55分别具有至少95％，96％，97％，98％或99％的序列同一性，和/或
轻链，其氨基酸序列如SEQ ID NO：54所示，或与SEQ ID NO：54具有至少95％，96％，97％，98％或99％的序列同一性。
在一些实施方案中，前述的特异性结合ANG-2的抗体，其包含：
a)重链，包含SEQ ID NO：49、51或52的氨基酸序列，和
轻链，包含SEQ ID NO：50的氨基酸序列；或
b)重链，包含SEQ ID NO：53或55的氨基酸序列，和
轻链，包含SEQ ID NO：54的氨基酸序列。
在一些实施方案中，前述的特异性结合ANG-2的抗体，其包含：
a)重链，其氨基酸序列如SEQ ID NO：49、51或52所示，和
轻链，其氨基酸序列如SEQ ID NO：50所示；或
b)重链，其氨基酸序列如SEQ ID NO：53或55所示，和
轻链，其氨基酸序列如SEQ ID NO：54所示。
在一些实施方案中，本披露提供一种分离的抗体，其与前述的特异性结合ANG-2的抗体竞争结合人ANG-2。
在一些实施方案中，本披露提供一种核酸分子，其编码根据前述的双特异性抗原结合分子，或前述的特异性结合ANG-2的抗体。
在一些实施方案中，本披露提供一种载体，其包含前述的核酸分子。
在一些实施方案中，本披露提供一种宿主细胞，其包含前述的载体。
在一些实施方案中，本披露提供一种药物组合物，其包含有效量的前述双特异性抗原结合分子，或前述特异性结合ANG-2的抗体，或前述的核酸分子，以及一种或更多种药学上可接受的载体、稀释剂或赋形剂。在一些实施方案中，所述治疗有效量为单位剂量的组合物中含有0.1-3000mg的如前所述的双特异性抗原结合分子或特异性结合ANG-2的抗体。
在一些实施方案中，本披露提供一种生产前述的双特异性抗原结合分子，或前述的特异性结合ANG-2的抗体的方法，所述方法包括培养前述的宿主细胞以表达所述双特异性抗原结合分子或特异性结合ANG-2的抗体。
在一些实施方案中，本披露提供一种预防或治疗癌症或血管生成性眼病的方法，所述方法包括向受试者施用治疗有效量的前述的双特异性抗原结合分子，或前述的特异性结合ANG-2的抗体，或前述的核酸分子，或前述的药物组合物。
在一些实施方案中，前述的治疗方法，其中所述的癌症选自乳腺癌、肾上腺肿瘤、输卵管癌、鳞状细胞癌、卵巢癌、胃癌、结直肠癌、非小细胞肺癌、胆管癌、膀胱癌、胰腺癌、皮肤癌和肝癌。在一些实施方案中，其中所述的血管生成性眼病选自新生血管性青光眼、年龄相关的黄斑变性(AMD)、糖尿病性黄斑水肿、角膜新血管生成、角膜移植新血管生成、角膜移植排斥、视网膜/脉络膜新血管生成、房角的新血管生成(虹膜发红)、眼部新生血管性疾病、血管再狭窄和动静脉畸形(AVM)。在一些实施方案中，前述的癌症或血管生成性眼病与VEGF或ANG-2相关。
在一些实施方案中，本披露提供前述的双特异性抗原结合分子，或前述的特异性结合ANG-2的抗体，或前述的核酸分子，或前述的药物组合物在制备用于治疗或预防癌症或血管生成性眼病的药物中的用途。
在一些实施方案中，前述的用途，其中所述的癌症选自乳腺癌、肾上腺肿瘤、输卵管癌、鳞状细胞癌、卵巢癌、胃癌、结直肠癌、非小细胞肺癌、胆管癌、膀胱癌、胰腺癌、皮肤癌和肝癌。在一些实施方案中，其中所述的血管生成性眼病选自新生血管性青光眼、年龄相关的黄斑变性(AMD)、糖尿病性黄斑水肿、角膜新血管生成、角膜移植新血管生成、角膜移植排斥、视网膜/脉络膜新血管生成、房角的新血管生成(虹膜发红)、眼部新生血管性疾病、血管再狭窄和动静脉畸形(AVM)。在一些实施方案中，前述的癌症或血管生成性眼病与VEGF或ANG-2相关。
在在一些实施方案中，本披露提供用作药物的前述的双特异性抗原结合分子，或前述的特异性结合ANG-2的抗体，或前述的核酸分子，或前述的药物组合物。
在一些实施方案中，本披露提供用作药物的前述的双特异性抗原结合分子，或前述的特异性结合ANG-2的抗体，或前述的核酸分子，或前述的药物组合物，可用作治疗或预防癌症或血管生成性眼病。在一些实施方案中，其中所述的癌症选自乳腺癌、肾上腺肿瘤、输卵管癌、鳞状细胞癌、卵巢癌、胃癌、结直肠癌、非小细胞肺癌、胆管癌、膀胱癌、胰腺癌、皮肤癌和肝癌。在一些实施方案中，其中所述的血管生成性眼病选自新生血管性青光眼、年龄相关的黄斑变性(AMD)、糖尿病性黄斑水肿、角膜新血管生成、角膜移植新血管生成、角膜移植排斥、视网膜/脉络膜新血管生成、房角的新血管生成(虹膜发红)、眼部新生血管性疾病、血管再狭窄和动静脉畸形(AVM)。在一些实施方案中，前述的癌症或血管生成性眼病与VEGF或ANG-2相关。
附图说明
图1示出双特异性抗体抑制ANG-2与Tie2的结合的结果；
图2示出双特异性抗体抑制诱导的Tie2磷酸化结果；
图3示出双特异性抗体显著抑制VEGF引起的HUVEC胞内磷酸化VEGFR水平升高；
图4示出双特异性抗体可以显著抑制VEGF引起的HUVEC的增殖；
图5示出本披露中的双特异性抗体均可显著抑制小鼠体内的PC-3肿瘤生长；
图6示出本披露中的双特异性抗体均可显著抑制小鼠体内的H460移植肿瘤生长；
图7示出本披露中的双特异性抗体可显著抑制小鼠体内的A431移植肿瘤生长；
图8A和图8B示出本披露的双特异性抗体恒河猴脉络膜新生血管的抑制结果；其中图8A示出，本披露中的双特异性抗体对恒河猴眼中荧光渗漏面积的改善率，图8B示出本披露中的双特异性抗体可显著减少恒河猴眼中的荧光斑点数；
图9示出本披露中的双特异性抗体可显著降低恒河猴眼睛房水中VEGF的表达量。
图10A和图10B示出本披露中示例性的双特异性抗体的分子结构；图10A显示IgG-scFv类型双特异性抗体的结构；图10B显示Crossmab类型双特异性抗体的结构。
具体实施方式
术语
为了更容易理解本披露，以下具体定义了某些技术和科学术语。除非在本文中另有明确定义，本文使用的所有其它技术和科学术语都具有本披露所属领域的 一般技术人员通常理解的含义。
本披露所用氨基酸三字母代码和单字母代码如J.biol.chem,243,p3558(1968)中所述。
术语“氨基酸”是指天然存在的和合成的氨基酸，以及以与天然存在的氨基酸类似的方式起作用的氨基酸类似物和氨基酸模拟物。天然存在的氨基酸是由遗传密码编码的那些氨基酸，以及后来修饰的那些氨基酸，例如羟脯氨酸、γ-羧基谷氨酸和O-邻磷酸丝氨酸。氨基酸类似物是指与天然存在的氨基酸具有相同基本化学结构(即与氢结合的α碳、羧基、氨基和R基团，例如高丝氨酸、正亮氨酸、甲硫氨酸亚砜、甲硫氨酸甲基锍)的化合物。此类类似物具有修饰的R基团(例如，正亮氨酸)或修饰的肽骨架，但保留与天然存在的氨基酸相同的基本化学结构。氨基酸模拟物是指具有如下结构的化合物，该结构与氨基酸的一般化学结构不同但是以与天然存在的氨基酸类似的方式起作用。
术语“ANG-2”指血管生成素-2(ANGPT2或ANG2)，其记载于例如Maisonpierre,P.C.等,Science 277(1997)55-60和Cheung,A.H.等,Genomics 48(1998)389-91。发现血管生成素-1和-2为Tie(即，一种在血管内皮内选择性表达的酪氨酸激酶家族)的配体，Yancopoulos,G.D.等,Nature 407(2000)242-48。血管生成素家族有四种确定的成员，血管生成素-3和-4(ANG-3和ANG-4)可以代表小鼠和人中相同基因基因座的广泛区域的对应物。Kim,I.等,FEBS Let,443(1999)353-56；Kim,I.等,J Biol Chem 274(1999)26523-28。ANG1和ANG2最初是在组织培养实验中分别作为激动剂和拮抗剂鉴定的(对于ANG1，参见，Davis,S.等,Cell 87(1996)1161-69；对于ANG2，参见Maisonpierre,P.C.等,Science 277(1997)55-60)。所有已知的血管生成素主要结合Tie2，而ANG1和2两者都以3nM(Kd)的亲和力结合Tie2，Maisonpierre,P.C.等,Science 277(1997)55-60。
术语“VEGF”指人血管内皮生长因子(VEGF/VEGF-A)，其记载于例如Leung,D.W.等,Science 246(1989)1306-9；Keck,P.J.等,Science 246(1989)1309-12和Connolly，D.T.等,J.Biol.Chem.264(1989)20017-24。VEGF参与调节正常的和异常的血管发生和与肿瘤和眼内病症有关的新血管化(Ferrara,N.,Endocr.Rev.18(1997)4-25；Berkman,R.A.,J.Clin.Invest.91(1993)153-159；Brown,L.F.等,HumanPathol.26(1995)86-91；Brown,L.F.等,Cancer Res.53(1993)4727-4735；Mattern,J.等,Brit.J.Cancer.73(1996)931-934；及Dvorak,H.F.等,Am.J.Pathol.146(1995)1029-1039)。VEGF是一种同二聚体糖蛋白，VEGF可促进对内皮细胞的促有丝分裂。
本披露所述的“抗原结合分子”在本文中以最广意义指特异性结合抗原的分子，抗原结合分子的例子是抗体、抗体片段、抗体融合蛋白或融合蛋白。示例性的，本文中的双特异性抗原结合分子包含双特异性抗体和抗体融合蛋白。示例性的，本文的双特异性抗体或抗体融合蛋白可以包含第一条链与第二条链，该第一 条链是抗体的重链或包含抗体重链的多肽，第二条链是抗体的轻链或包含抗体轻链的多肽。示例性的，在一些实施方案中，本文的双特异性抗原结合分子具有全长抗体的基本结构，是由两条相同的第一条链和两条相同的第二条链通过链间二硫键连接而成的四肽链结构；其中，第一条链包含抗VEGF抗体重链和抗ANG-2的单链抗体，第二条链包含抗VEGF抗体的轻链。
如本披露所述的“双特异性抗原结合分子”是指能同时结合两个抗原或抗原决定簇的抗原结合分子，其包含结合第一抗原或抗原决定簇的第一抗原结合域和结合第二抗原或抗原决定簇的第二抗原结合域。在本披露的一些实施方案中，第一抗原结合域特异性结合ANG-2，第二抗原结合域特异性结合VEGF；或者，在在本披露的一些实施方案中，第一抗原结合域特异性结合VEGF，第二抗原结合域特异性结合ANG-2。
在一些实施方案中，本披露所述的双特异性抗原结合分子为双特异性二价抗体或双特异性四价抗体。
在本领域中公知，根据不同结构可将双特异性抗原结合分子分为2大类：含Fc片段的双特异性抗原结合分子与不含Fc片段的双特异性抗原结合分子。含Fc片段的双特异性抗原结合分子结构包括但不限于TrioMab、Crossmab/KIH、DVD-Ig、IgG-scFv、FIT-Ig、mAb-Trap等。不含Fc片段的双特异性抗原结合分子结构包括但不限于BiTE、DART、TandAb、ImmTAC、TriKE等。
如本披露所述的“Crossmab”是存在Fc区域的IgG样双特异性抗原结合分子结构，其通过在第一抗原结合域的Fab区内交换重链和轻链结构，从而导致VH-VL和CH1-CL之间界面的分子结构发生变化。经过交换的抗体轻链由于相互排斥的原理，即VH与VH相互排斥、CL与CL相互排斥，不易与未改造抗体的重链发生错配，从而产生轻重链的正确配对。
在另一些实施方案中，本披露的双特异性抗原结合分子是Crossmab形式，由第一重链，第一轻链，第二重链和第二轻链组成的四肽结构，其中，第一重链和第一轻链组成结合ANG-2的第一抗原结合域，第二重链和第二轻链组成结合VEGF的第二抗原结合域，并且第一重链的CH1与第一轻链的CL互相交换。
如本披露所述的“IgG-scFv”是存在Fc区域的IgG样双特异性抗原结合分子结构，其中一个抗原结合域的scFv融合到另一个抗原结合域IgG重链或轻链的C端，以构成IgG-scFv双特异性抗原结合分子。
在另一些实施方案中，本披露的双特异性抗原结合分子是IgG-scFv形式。在示例性的实施方案中，特异性结合ANG-2的scFv直接地或通过连接子与特异性结合VEGF的抗体重链C端连接，构成IgG-scFv形式双特异性抗原结合分子。
术语“价”指抗原结合分子中存在规定数目的结合位点。因此，术语“二价”、“四价”和“六价”分别指抗原结合分子中存在两个结合位点、四个结合位点和六个结合位点。
本披露所述的“能够特异性结合ANG-2的第一抗原结合域”或“能够特异性结合VEGF的第二抗原结合域”指抗原结合分子中包含与ANG-2或VEGF的全部或部分特异性结合的区域。例如，所述抗原结合域可以包含一个或多个抗体可变区。特别的，能够特异性结合抗原的抗原结合域包含抗体轻链可变区和抗体重链可变区，其可以形成scFv、Fab等构型。
术语“抗体融合蛋白”是指将目的蛋白质(多肽)与抗体连接形成的具有生物活性的融合蛋白，所述的融合蛋白具有所连接的蛋白质的生物学活性以及免疫球蛋白活性。
术语“抗体”以最广义使用，并且涵盖各种抗体结构，包括但不限于单克隆抗体，多克隆抗体，多特异性抗体(例如双特异性抗体)，和抗体片段，只要它们展现出期望的抗原结合活性。
“天然抗体”指具有不同结构的天然存在的免疫球蛋白分子。例如，天然IgG抗体是约150,000道尔顿的异四聚糖蛋白，由二硫化物键合的两条相同轻链和两条相同重链构成。从N至C端，每条重链具有一个可变区(VH)，又称作可变重域或重链可变域，接着是三个恒定域(CH1,CH2,和CH3)。类似地，从N至C端，每条轻链具有一个可变区(VL)，又称作可变轻域，或轻链可变域，接着是一个恒定轻(CL)域。根据其恒定域氨基酸序列，抗体轻链包括两种类型，卡帕(κ)和拉姆达(λ)。根据抗体重链恒定区的氨基酸组成和排列顺序不同，可将抗体分为五类，或称为抗体同种型，即IgM、IgD、IgG、IgA和IgE，其相应的重链分别为μ链、δ链、γ链、α链、和ε链。同一类Ig根据其铰链区氨基酸组成和重链二硫键的数目和位置的差别，又可分为不同的亚类，如IgG可分为IgG1、IgG2、IgG3、IgG4。五类Ig中每类Ig都可以有κ链或λ链。
抗体重链和轻链靠近N端的约110个氨基酸的序列变化很大，为可变区；靠近C端的其余氨基酸序列相对稳定，为恒定区。每条重链由重链可变区(本文中缩写为VH)和重链恒定区组成。重链恒定区由三个结构域(CH1、CH2和CH3)组成。每条轻链由轻链可变区(本文中缩写为VL)和轻链恒定区组成。轻链恒定区包含一个结构域，CL。VH和VL区可进一步细分为高变区，称为互补性决定区(CDR)，其间穿插有称为框架区(FR)的较保守区。每条轻链的包含3个CDR区：LCDR1、LCDR2、和LCDR3；每条重链的包含3个CDR区：HCDR1、HCDR2和HCDR3。每个VH和VL由从氨基末端排到羧基末端按以下顺序排列的三个CDR和四个FR构成：FR1、CDR1、FR2、CDR2、FR3、CDR3、FR4。重链和轻链的可变区含有与抗原相互作用的结合结构域。抗体的恒定区可以介导免疫球蛋白与宿主组织或因子(包括免疫系统的各种细胞(例如，效应细胞)和经典补体系统的第一组分(Clq))的结合。
术语“互补决定区”、“CDR”或“高变区”是指可变结构域内主要促成抗原结合的区域。通常，每个重链可变区中存在三个CDR(HCDR1、HCDR2、HCDR3)， 每个轻链可变区中存在三个CDR(LCDR1、LCDR2、LCDR3)。可以各种公知方案来确定CDR的氨基酸序列边界，例如：“Kabat”编号规则(参见Kabat等(1991)，“Sequences of Proteins of Immunological Interest”，第5版，Public Health Service，National Institutes of Health，Bethesda，MD)、“Chothia”编号规则(参见Martin,ACR.Protein Sequence and Structure Analysis of Antibody Variable Domains[J].2001)和ImMunoGenTics(IMGT)编号规则(Lefranc，M.P.等，Dev.Comp.Immunol.，27，55-77(2003)；Front Immunol.2018 Oct 16；9:2278等)。包括例如Kabat编号和IMGT独特编号系统在内的编号系统之间的关系是本领域技术人员熟知的。
例如，对于经典格式，遵循Kabat规则，所述重链可变域(VH)中的CDR氨基酸残基编号为31-35(HCDR1)、50-65(HCDR2)和95-102(HCDR3)；轻链可变域(VL)中的CDR氨基酸残基编号为24-34(LCDR1)、50-56(LCDR2)和89-97(LCDR3)。
遵循Chothia规则，VH中的CDR氨基酸编号为26-32(HCDR1)、52-56(HCDR2)和95-102(HCDR3)；并且VL中的氨基酸残基编号为24-34(LCDR1)、50-56(LCDR2)和89-97(LCDR3)。
通过组合Kabat和Chothia两者的CDR定义，CDR由人VH中的氨基酸残基26-35(HCDR1)、50-65(HCDR2)和95-102(HCDR3)和人VL中的氨基酸残基24-34(LCDR1)、50-56(LCDR2)和89-97(LCDR3)构成。
遵循IMGT规则，VH中的CDR氨基酸残基编号大致为27-38(CDR1)、56-65(CDR2)和105-117(CDR3)，VL中的CDR氨基酸残基编号大致为27-38(CDR1)、56-65(CDR2)和105-117(CDR3)。遵循IMGT规则，抗体的CDR区可以使用程序IMGT/DomainGap Align确定。
遵循AbM规则，VH中的CDR氨基酸编号为26-35(HCDR1)、50-58(HCDR2)和95-102(HCDR3)；并且VL中的氨基酸残基编号为24-34(LCDR1)、50-56(LCDR2)和89-97(LCDR3)。
除非另有说明，本披露实施例中的可变区和CDR序列均适用“Kabat”编号规则。
术语“抗体框架”或“FR区”，是指可变结构域VL或VH的一部分，其用作该可变结构域的抗原结合环(CDR)的支架。从本质上讲，其是不具有CDR的可变结构域。
“抗体恒定区结构域”指来源于抗体的轻链和重链的恒定区的结构域，包括CL和来源于不同类抗体的CH1、CH2、CH3和CH4结构域。本披露的恒定区还包括所述人抗体重链恒定区和人抗体轻链恒定区的“常规变体”，其指现有技术已公开的来源于人的不改变抗体可变区结构和功能的重链恒定区或轻链恒定区的变体，示例性变体包括对重链恒定区进行定点改造和氨基酸替换的IgG1、IgG2、IgG3或IgG4重链恒定区变体，具体替换如现有技术已知的YTE突变，L234A和/ 或L235A突变，S228P突变，和/或获得knob-into-hole结构的突变(使得抗体重链具有knob-Fc和hole-Fc组合)，这些突变已被证实使得抗体具有新的性能，但不改变抗体可变区的功能。
“抗体片段”指不同于完整抗体的分子，其包含完整抗体的部分，所述部分与完整抗体所结合的抗原相结合。抗体片段的实例包括但不限于Fv、Fab、Fab’、Fab’-SH、F(ab′)2、Fd、dAb；驼类VHH结构域；双抗体；线性抗体；单链抗体分子(例如scFv)；以及由抗体片段形成的多特异性抗体。
“单链可变片段(scFv)”，也称“单链抗体”，是通过接头连接的抗体的重链可变区(VH)和轻链可变区(VL)的融合蛋白。特别地，接头是10至25个氨基酸的短多肽且通常富含有关柔性的甘氨酸，以及有关溶解性的丝氨酸或苏氨酸，并且可将VH的N端与VL的C端连接，或反之亦然。尽管去除了恒定区并引入了接头，但这种蛋白质保留了原始抗体的特异性。关于scFv片段的综述，见例如Pluckthün,于ThePharmacology of Monoclonal Antibodies,第113卷,Rosenburg和Moore编,(Springer-Verlag,New York),第269-315页(1994)；还可见WO 93/16185；和美国专利No.5,571,894和5,587,458。
“接头”或“连接子”指用于连接多肽(如蛋白质结构域)的多肽序列，通常具有一定的柔性，接头的使用不会使多肽原有结构和功能丧失。
术语“Fc区”在本文中用于定义免疫球蛋白重链的C端区域，其含有恒定区的至少部分。该术语包括天然序列Fc区和变体Fc区。Fc区的C末端赖氨酸(Lys447)可以存在或不存在。除非本文另有说明，Fc区或恒定区中的氨基酸残基编号根据EU编号系统，也称为EU索引。
术语“结合位点”或“抗原结合位点”指抗体分子与抗原实际结合的区域。术语“抗原结合位点”包含抗体重链可变域(VH)和抗体轻链可变域(VL)，或仅包含抗体重链可变域或轻链可变结构域。
“嵌合”抗体指其中的重链和/或轻链的一部分自特定的来源或物种衍生，而重链和/或轻链的剩余部分自不同来源或物种衍生的抗体。
术语“人源化”抗体是保留非人抗体的反应性同时在人中具有较低免疫原性的抗体。例如，这可以通过保留非人CDR区并用其人对应物(即，恒定区以及可变区的框架部分)替换抗体的其余部分来实现。参见，例如，Morrison等人,Proc.Natl.Acad.Sci.USA[美国国家科学院院刊],81:6851-6855,1984；Morrison和Oi,Adv.Immunol.[免疫学进展],44:65-92,1988；Verhoeyen等人,Science[科学],239:1534-1536,1988；Padlan,Molec.Immun.[分子免疫学],28:489-498,1991；以及Padlan,Molec.Immun.[分子免疫学],31:169-217,1994。人工程化技术的其他实例包括但不限于在US 5,766,886中披露的Xoma技术。
通常，将非人抗体人源化以降低对人的免疫原性，同时保留亲本非人抗体的特异性和亲和力。一般地，人源化抗体包含一个或多个可变域，其中HVR(重链 可变区)，例如CDR(或其部分)自非人抗体衍生，而FR(或其部分)自人抗体序列衍生。任选地,人源化抗体还会至少包含人恒定区的一部分。在一些实施方案中，将人源化抗体中的一些FR残基用来自非人抗体(例如衍生HVR残基的抗体)的相应残基替代，例如以恢复或改善抗体特异性或亲和力。
术语“人抗体”旨在包括具有可变区的抗体，其中框架区和CDR区两者均衍生自人来源的序列。此外，如果抗体含有恒定区，则恒定区还衍生自此类人序列，例如人种系序列或突变形式的人种系序列。本披露的人抗体可以包括不是由人序列编码的氨基酸残基(例如，通过在体外随机诱变或位点特异性诱变或通过在体内体细胞突变来引入的突变)。
术语“全长抗体”，“完整抗体”和“全抗体”在本文中可互换使用，指与天然抗体结构具有基本上类似的结构或者具有含有如本文中所限定的Fc区的重链的抗体。
术语“抗原”是指能够由诸如抗原结合蛋白(包括例如抗体)的选择性结合剂结合且另外能够用于动物中以产生能够结合该抗原的抗体的分子或分子部分。抗原可具有一个或多个能够与不同的抗原结合蛋白(例如抗体)相互作用的表位。
术语“表位”指能够与抗体或其抗原结合片段特异性结合的抗原上的区域(area或region)。表位可以自连续氨基酸串(线性表位)形成或包含非连续氨基酸(构象表位)，例如因抗原的折叠(即通过蛋白质性质的抗原的三级折叠)而变成空间接近。构象表位和线性表位的差别在于：在变性溶剂的存在下，抗体对构象表位的结合丧失。例如，表位包含处于独特空间构象的至少3，至少4，至少5，至少6，至少7，或8-10个氨基酸。
筛选结合特定表位的抗体(即那些结合相同表位的)可以使用本领域例行方法来进行，例如但不限于丙氨酸扫描,肽印迹(见Meth.Mol.Biol.248(2004)443-463),肽切割分析，表位切除，表位提取，抗原的化学修饰(见Prot.Sci.9(2000)487-496),和交叉阻断(见“Antibodies,”Harlow and Lane(Cold Spring Harbor Press,Cold Spring Harb.,NY))。
还可以使用竞争性结合来鉴定抗体是否与参照抗抗体结合相同表位或竞争结合。例如，与参照抗体“结合相同表位的抗体”指在竞争测定法中将参照抗体对其抗原的结合阻断50％或更多的抗体，且相反，参照抗体在竞争测定法中将该抗体对其抗原的结合阻断50％或更多。还例如，为了测定待测抗体是否与参照抗体结合相同表位，在饱和条件下容许参照抗体结合抗原。在去除过量的参照抗体后，评估待测抗体结合抗原的能力。如果该待测抗体能够在参照抗体的饱和结合之后结合抗原，那么可以得出结论，该待测抗体与参照抗体结合不同表位。但是，如果该待测抗体在参照抗体的饱和结合之后不能够结合抗原，那么该待测抗体可结合与参照抗体结合相同的表位。为了确认待测抗体是否结合相同表位或仅仅受到空间原因阻碍结合，可以使用例行实验(例如，肽突变和使用ELISA、RIA、表面 等离子共振、流式细胞术或本领域可获得的任何其它定量或定性抗体结合测定的结合分析)。这种测定法应当以两种设置中进行，即两种抗体均作为饱和抗体。如果在两种设置中，均只有第一(饱和)抗体能够结合抗原，那么可以得出结论，该待测抗体和参照抗体竞争结合该抗原。
在一些实施方案中，如在竞争性结合测定中所测量的(参见例如，Junghans等人，Cancer Res.50(1990)1495-1502)，如果一个抗体的1倍、5倍、10倍、20倍或100倍过量抑制另一个抗体的结合至少50％、至少75％、至少90％或甚至99％或更多，则认为两个抗体结合相同或重叠的表位。
如此处所述，抗体和/或其抗原结合片段之间的“竞争”意味着两种抗体(或其结合片段)结合相同或重叠的表位(例如，如通过竞争性结合测定，通过本领域技术人员所熟知的任一方法所确定的)。如果所述竞争性抗体或其抗原结合片段与本披露的抗体或抗原结合片段结合相同的表位或重叠的表位，则该抗体或其抗原结合片段还与本披露的抗体或抗原结合片段“竞争”。如本文所使用的，竞争性抗体或其抗原结合片段还可包括如下：(i)空间阻断本披露的抗体或抗原结合片段结合其靶标的竞争性抗体或其抗原结合片段(例如，如果所述竞争性抗体结合临近的、非重叠的和/或相同表位并且以物理的方式防止本披露的抗体或抗原结合片段结合其靶标)；和/或(ii)结合不同的、非重叠的表位并且诱导抗原发生如下构象变化的竞争性抗体或其抗原结合片段，所述构象变化使得所述抗原不再以无该构象变化时具有的方式结合本披露的抗体或抗原结合片段。
“特异性地结合”、“特异性结合”或“结合”是指抗体以比针对其他抗原或表位更高的亲和力结合至某个抗原或该抗原内的表位。通常，抗体以约1×10-7M或更小(例如约1×10-8M或更小、约1×10-9M或更小、约1×10-10M或更小、约1×10-11M或更小，或者约1×10-12M或更小)的平衡解离常数(KD)结合抗原或抗原内的表位，通常KD为该抗体结合至非特异性抗原(例如BSA、酪蛋白)的KD的至少百分之一。可使用标准程序来测量KD。然而，特异性结合至抗原或抗原内的表位的抗体可能对其它相关的抗原具有交叉反应性，例如，对来自其它物种(同源)(诸如人或猴，例如食蟹猕猴(Macaca fascicularis)(cynomolgus，cyno)、黑猩猩(Pan troglodytes)(chimpanzee，chimp)或狨猴(Callithrix jacchus)(commonmarmoset，marmoset)的相同抗原具有交叉反应性。
“亲和力”指分子(例如抗体)的单一结合位点与其结合配偶体(例如抗原)之间全部非共价相互作用总和的强度。分子X对其配偶体Y的亲和力通常可以用解离常数(KD)来表述。亲和力可以通过本领域知道的常用方法来测量，包括本文中所描述的方法。
术语“kassoc”或“ka”意在是指特定抗体-抗原相互作用的缔合速率，而如本文所使用的术语“kdis”或“kd”意在是指特定抗体-抗原相互作用的解离速率。如本文所使用的，术语“KD”意在是指解离常数，其获得自kd与ka的比率(即 kd/ka)并且表示为摩尔浓度(M)。可以使用本领域良好建立的方法测定抗体的KD值。用于测定抗体KD的方法包括使用生物传感系统例如系统测量表面等离子体共振，或通过溶液平衡滴定法(SET)测量溶液中的亲和力。
术语“核酸”在本文中可与术语“多核苷酸”互换使用，并且是指呈单链或双链形式的脱氧核糖核苷酸或核糖核苷酸及其聚合物。所述术语涵盖含有已知核苷酸类似物或修饰的骨架残基或连接的核酸，所述核酸是合成的、天然存在的和非天然存在的，具有与参考核酸相似的结合特性，并且以类似于参考核苷酸的方式代谢。此类类似物的实例包括但不限于硫代磷酸酯、氨基磷酸酯、甲基膦酸酯、手性-甲基膦酸酯、2-O-甲基核糖核苷酸、肽-核酸(PNA)。
除非另有说明，否则特定的核酸序列还隐含地涵盖其保守修饰的变体(例如，简并密码子取代)和互补序列以及明确指明的序列。具体地，如下详述，简并密码子取代可以通过产生如下序列而获得，在这些序列中，一个或多个所选的(或全部)密码子的第三位被混合碱基和/或脱氧肌苷残基取代(Batzer等人,Nucleic Acid Res[核酸研究].19:5081,1991；Ohtsuka等人,J.Biol.Chem[生物化学杂志].260:2605-2608,1985；和Rossolini等人,Mol.Cell.Probes[分子与细胞探针]8:91-98,1994)。
序列“同一性”指，当对两条序列进行最佳比对时，必要时引入间隙，以获取最大序列同一性百分比，且不将任何保守性取代视为序列同一性的一部分，两条序列的氨基酸/核酸在等价位置相同的程度(百分比)。为测定序列同一性百分比，比对可以通过属于本领域技术的范围内的多种方式来实现，例如使用公开可得到的计算机软件，诸如BLAST、BLAST-2、ALIGN、ALIGN-2或Megalign(DNASTAR)软件。本领域技术人员可确定适用于测量比对的参数，包括在所比较的序列全长上达成最大比对所需的任何算法。
“保守修饰的变体”或“保守性取代”指使用具有相似特征(例如，电荷、侧链尺寸、亲水性/疏水性、骨架构型和刚性等)的其他氨基酸置换蛋白中的氨基酸，使得通常可以做出这样的变化而不改变蛋白的生物活性。本领域技术人员知晓，在通常情况下，在多肽的非必需区域中的单氨基酸置换基本上不改变生物活性(参见例如，Watson等，(1987)Molecular Biology of the Gene,The Benjamin/Cummings Pub.Co.,p.224(4th Ed.))。此外，结构或功能上类似的氨基酸的置换不太可能破坏生物活性。示例性的保守性置换如下表中所示。
表1.示例性的保守性氨基酸置换
原始残基 保守性取代
Ala(A) Gly；Ser
Arg(R) Lys；His
Asn(N) Gln；His
Asp(D) Glu；Asn
Cys(C) Ser；Ala
Gln(Q) Asn
Glu(E) Asp；Gln
Gly(G) Ala
His(H) Asn；Gln
Ile(I) Leu；Val
Leu(L) Ile；Val
Lys(K) Arg；His
Met(M) Leu；Ile；Tyr
Phe(F) Tyr；Met；Leu
Pro(P) Ala
Ser(S) Thr
Thr(T) Ser
Trp(W) Tyr；Phe
Tyr(Y) Trp；Phe
Val(V) Ile；Leu
术语“保守修饰的变体”当适用核酸序列时，保守修饰的变体是指那些编码相同或基本上相同的氨基酸序列的核酸，或在该核酸不编码氨基酸序列的情况下，是指基本相同的序列。由于遗传密码的简并性，任何给定的蛋白质均可以由多个功能相同的核酸编码。例如，密码子GCA、GCC、GCG和GCU都编码氨基酸丙氨酸。因此，在密码子指定丙氨酸的每个位置，该密码子可以改变为任何所述相应密码子而不改变编码的多肽。此类核酸变异是“沉默变异”，它们是保守修饰变异中的一种。本文中编码多肽的每个核酸序列也描述了核酸的每种可能的沉默变异。技术人员将认识到，核酸中的每个密码子(除了AUG--通常是甲硫氨酸的唯一密码子；和TGG--通常是色氨酸的唯一密码子)均可以被修饰以产生功能相同的分子。因此，在每个所述序列中均隐含了编码多肽的核酸的每一种沉默变异。
术语“载体”意指能够转运与其连接的另一多核苷酸的多核苷酸分子。一种类型的载体是“质粒”，其是指环状双链DNA环，其中可以连接附加的DNA区段。另一种类型的载体是病毒载体，例如腺相关病毒载体(AAV或AAV2)，其中另外的DNA区段可以连接到病毒基因组中。某些载体能够在引入它们的宿主细胞中自主复制(例如，具有细菌复制起点的细菌载体和附加型哺乳动物载体)。其他载体(例如，非附加型哺乳动物载体)可以在引入宿主细胞中后整合到宿主细胞的基因组中，从而与宿主基因组一起复制。
术语“表达载体”或“表达构建体”是指适用于对宿主细胞进行转化且含有指导及/或控制(连同宿主细胞一起)与其可操作地连接的一个或多个异源编码区的表达的核酸序列的载体。表达构建体可以包括但不限于影响或控制转录、翻译且在存在内含子时影响与其可操作地连接的编码区的RNA剪接的序列。
如本文中所使用，“可操作地连接”意指该术语所适用的组分呈允许其在适合条件下执行其固有功能的关系。举例而言，载体中与蛋白质编码序列“可操作地 连接”的控制序列是与其连结，从而在与该控制序列的转录活性兼容的条件下达成该蛋白质编码序列的表达。
术语“宿主细胞”，“宿主细胞系”和“宿主细胞培养物”可互换使用，并且指已经导入外源核酸的细胞，包括此类细胞的后代。宿主细胞包括“转化体”和“经转化的细胞”，其包括原代的经转化的细胞及自其衍生的后代而不考虑传代的次数。后代在核酸内容物上可以与亲本细胞不完全相同，而是可以含有突变。本文中包括具有与在初始转化细胞中筛选或选择的相同功能或生物学活性的突变体后代。宿主细胞包括原核和真核宿主细胞，其中真核宿主细胞包括但不限于哺乳动物细胞、昆虫细胞系(例如，草地贪夜蛾(Spodoptera frugiperda)或粉纹夜蛾(Trichoplusia ni))、两栖动物细胞、细菌细胞、植物细胞和真菌细胞。哺乳动物宿主细胞包括人、小鼠、大鼠、犬、猴、猪、山羊、牛、马和仓鼠细胞，包括但不限于中国仓鼠卵巢(CHO)细胞、NSO、SP2细胞、HeLa细胞、幼仓鼠肾(BHK)细胞、猴肾细胞(COS)、人肝细胞癌细胞(例如，Hep G2)、A549细胞、3T3细胞和HEK-293细胞。真菌细胞包括酵母和丝状真菌细胞，包括例如巴氏毕赤酵母(Pichiapastoris)、芬兰毕赤酵母(Pichia finlandica)、海藻毕赤酵母(Pichia trehalophila)、科克拉马毕赤酵母(Pichia koclamae)、膜状毕赤酵母(Pichia membranaefaciens)、小毕赤酵母(Pichia minuta)(Ogataea minuta、Pichia lindneri)、仙人掌毕赤酵母(Pichiaopuntiae)、耐热毕赤酵母(Pichia thermotolerans)、柳毕赤酵母(Pichia salictaria)、Pichia guercuum、皮杰普毕赤酵母(Pichia pijperi)、具柄毕赤酵母(Pichia stiptis)、甲醇毕赤酵母(Pichia methanolica)、毕赤酵母属、酿酒酵母(Saccharomycescerevisiae)、酿酒酵母属、多形汉逊酵母(Hansenula polymorpha)、克鲁维酵母属、乳酸克鲁维酵母(Kluyveromyces lactis)、白色念珠菌(Candida albicans)、构巢曲霉(Aspergillus nidulans)、黑曲霉(Aspergillus niger)、米曲霉(Aspergillus oryzae)、里氏木霉(Trichoderma reesei)、勒克氏菌(Chrysosporium lucknowense)、镰刀菌属(Fusarium sp.)、禾谷镰刀菌(Fusarium gramineum)、菜镰刀菌(Fusarium venenatum)、小立碗藓(Physcomitrella patens)和粗糙脉孢菌(Neurospora crassa)。毕赤酵母属、任何酿酒酵母属、多形汉逊酵母(Hansenula polymorpha)、任何克鲁维酵母属、白色念珠菌(Candida albicans)、任何曲霉属、里氏木霉(Trichoderma reesei)、勒克霉菌(Chrysosporium lucknowense)、任何镰刀菌属、解脂耶氏酵母(Yarrowia lipolytica)和粗糙脉孢菌(Neurospora crassa)。在一些实施方案中，所述宿主细胞是非人细胞。
如在本申请中所使用的，表述“细胞”、“细胞系”和“细胞培养物”可以互换使用，并且所有这样的名称均包括子代。因而，词语“转化体”和“转化的细胞”包括原代受试者细胞和来源于其的培养物，而与传代的次数无关。还应理解的是，由于有意或无意的突变，使得并非所有子代均具有完全相同的DNA内容物。包括与筛选出其的原始转化细胞具有相同功能或生物活性的突变子代。
“任选”或“任选地”意味着随后所描述地事件或环境可以但不必发生，该 说明包括该事件或环境发生或不发生的场合。
“药物组合物”表示含有一种或多种本文所述的抗体或其抗原结合片段与其他化学组分的混合物，所述其他组分例如生理学/可药用的载体和赋形剂。
术语“药学上可接受的载体”意指生理学上相容的任何溶剂、分散介质、涂层、抗细菌和抗真菌剂、等渗和吸收增强或延迟剂等。药学上可接受的载体的一些实例为水、盐水、磷酸盐缓冲盐水、具有氯化钠的乙酸盐缓冲液、右旋糖、甘油、聚乙二醇、乙醇等以及其组合。在许多情况下，组合物中优选包含等渗剂，例如糖、多元醇(例如甘露醇、山梨糖醇)或氯化钠。药学上可接受的物质的其他实例为表面活性剂、湿润剂或少量辅助物质例如湿润剂或乳化剂、防腐剂或缓冲剂，其增强抗体的保质期或有效性。
本披露的药物组合物可通过本领域已知的各种方法施与。施与途径和/或方式根据所希望的结果而变化。优选地，施与可以是玻璃体内、静脉内、肌肉内、腹膜内或皮下或在靶标部位附近施与。药学上可接受的载体应适合于玻璃体内、静脉内、肌肉内、皮下、肠胃外、脊柱或表皮施与(例如，通过注射或输注)。根据施与途径，活性化合物(即抗体，双特异性和多特异性分子)可以包被在材料中以保护化合物免受酸和可能使化合物失活的其他自然条件的作用。
术语“受试者”包括人类和非人类动物。非人动物包括所有脊椎动物(例如哺乳动物和非哺乳动物)例如非人灵长类(例如，食蟹猴)、绵羊、狗、牛、鸡、两栖动物和爬行动物。除非指出时，否则所述术语“患者”或“受试者”在本文中可互换地使用。如本文所使用的，术语“食蟹猴(cyno)”或“食蟹猴(cynomolgus)”是指食蟹猴(Macaca fascicularis)。在某些实施方案中，个体或受试者是人。
“施用”、“给予”和“处理”，当其应用于动物、人、实验受试者、细胞、组织、器官或生物流体时，是指外源性药物、治疗剂、诊断剂或组合物与动物、人、受试者、细胞、组织、器官或生物流体的接触。
“样本”是指从受试者分离的类似流体、细胞、或组织的采集物，以及存在于受试者体内的流体、细胞或组织。示例性样本为生物流体，诸如血液、血清和浆膜液、血浆、淋巴液、尿液、唾液、囊液、泪液、排泄物、痰、分泌组织和器官的粘膜分泌物、阴道分泌物、腹水、胸膜、心包、腹膜、腹腔和其它体腔的流体、由支气管灌洗液收集的流体、滑液、与受试者或生物来源接触的液体溶液，例如细胞和器官培养基(包括细胞或器官条件培养基)、灌洗液等，组织活检样本、细针穿刺、手术切除的组织、器官培养物或细胞培养物。
“治疗/处理”(及其语法变型)指试图改变所治疗个体的天然过程的临床干预，并且可以为了预防或者在临床病理学的过程期间实施。治疗的期望效果包括但不限于预防疾病的发生或再发生，减轻症状，减轻/减少疾病的任何直接或间接病理后果，预防转移，降低疾病进展速率，改善或减轻疾病状态，和消退或改善的预后。在一些实施方案中,使用本披露的抗体来延迟疾病的形成或减缓疾病的进展。
“有效量”一般是足以降低症状的严重程度及/或频率、消除这些症状及/或潜在病因、预防症状及/或其潜在病因出现及/或改良或改善由疾病状态引起或与其相关的损伤(例如肺病)的量。在一些实施例中，有效量是治疗有效量或预防有效量。“治疗有效量”是足以治疗疾病状态或症状、尤其与该疾病状态相关的状态或症状，或者以其他方式预防、阻碍、延迟或逆转该疾病状态或以任何方式与该疾病相关的任何其他不理想症状的进展的量。“预防有效量”是当给予受试者时将具有预定预防效应，例如预防或延迟该疾病状态的发作(或复发)，或者降低该疾病状态或相关症状的发作(或复发)可能性的量。完全治疗或预防效应未必因给予一个剂量便发生，而且可能仅在给予一系列剂量之后发生。因而，治疗或预防有效量可以一次或多次给予的方式给予。“治疗有效量”和“预防有效量”可取决于以下因素变化：诸如个体的疾病状态、年龄、性别和体重，以及治疗剂或治疗剂组合在个体中引发期望的应答的能力。有效治疗剂或治疗剂组合的示例性指标包括例如患者改善的健康状况。
术语“癌(症)”和“癌(性)的”指向或描述哺乳动物中典型的以不受调节的细胞生长为特征的生理疾患。此定义中包括良性和恶性癌症。“早期癌症”或“早期肿瘤”指非侵入性的或转移性的，或者归为0期、I期、或II期癌症的癌症。癌症的例子包括但不限于淋巴瘤、母细胞瘤(包括髓母细胞瘤和视网膜母细胞瘤)、肉瘤(包括脂肪肉瘤和滑膜细胞肉瘤)、神经内分泌肿瘤(包括类癌瘤、胃泌素瘤和胰岛细胞癌)、间皮瘤、施旺氏细胞瘤(包括听神经瘤)、脑膜瘤、腺癌、黑素瘤、肾上腺肿瘤、和白血病或淋巴样恶性肿瘤。此类癌症的更具体例子包括但不限于鳞状细胞癌(例如上皮鳞状细胞癌)；肺癌包括小细胞肺癌(SCLC)、非小细胞肺癌(NSCLC)、肺的腺癌和肺的鳞癌；腹膜癌；肝细胞癌；胃癌(gastric or stomach cancer)包括胃肠癌；胰腺癌；成胶质细胞瘤；宫颈癌；卵巢癌；肝癌(liver cancer or hepatic carcinoma)、膀胱癌；肝瘤(hepatoma)；乳腺癌(包括转移性乳腺癌)；结肠癌；直肠癌；结肠直肠癌；子宫内膜癌或子宫癌；唾液腺癌；肾癌(kidney or renal cancer)；前列腺癌；外阴癌；甲状腺癌；肛门癌；阴茎癌；睾丸癌；食道癌；胆管肿瘤；输卵管癌；卵巢癌；胆管癌；膀胱癌；胰腺癌；皮肤癌；及头和颈癌和多发性骨髓瘤。
实施例和测试例
以下结合实施例和测试例进一步描述本披露，但这些实施例和测试例并非限制着本披露的范围。本披露实施例或测试例中未注明具体条件的实验方法，通常按照常规条件，如冷泉港的抗体技术实验手册，分子克隆手册；或按照原料或商品制造厂商所建议的条件；未注明具体来源的试剂材料，为市场购买获得。
实施例1.ANG-2和ANG-2受体Tie2的表达
编码带人IgG1-Fc标签的人ANG-2和人ANG-2受体Tie2胞外区序列插入phr载体中，构建成表达质粒，然后转染HEK293。具体转染步骤为：前一天将HEK293E细胞以1×10 6/mL接种于freestyle表达培养基(含有1％FBS，Gibco，12338-026)，放置于37度恒温摇床(120rpm)继续培养24小时。24小时后，将转染质粒和转染试剂PEI混合物缓慢加入200mL HEK293E的细胞中，放入8％CO 2、120rpm、37℃的摇床中培养。转染第3天，补充10％体积的补料培养基(20mM葡萄糖+2mM L-谷氨酸)。待转染第6天，取样4500rpm离心10分钟收集细胞上清，将重组的ANG-2和Tie2受体蛋白按照实施例2所述的方法进行纯化。
其中人ANG-2氨基酸序列如SEQ ID NO：1所示，Tie2胞外区Fc融合蛋白氨基酸序列如SEQ ID NO：2所示。
相关序列如下所示：
(1)带人Fc标签的人ANG-2氨基酸序列(huANG-2-Fc)
Figure PCTCN2021119017-appb-000001
注释：横线部分为ANG-2蛋白全长序列，点线为接头，斜体部分为人IgG1Fc标签。
(2)带人Fc标签的Tie2氨基酸序列(huTie2-Fc)
Figure PCTCN2021119017-appb-000002
Figure PCTCN2021119017-appb-000003
注释：下划线部分为Tie2的胞外区，斜体部分为人IgG1Fc标签。
实施例2.Protein A亲和层析纯化带Fc标签的重组蛋白或抗体
将表达抗体或huANG-2-Fc、huTie2-Fc的细胞上清样品高速离心去除杂质，通过Protein A柱进行纯化。用PBS冲洗柱子，至A280读数降至基线。用100mM乙酸pH 3.5洗脱目的蛋白，用1M Tris-HCl，pH8.0中和。洗脱样品适当浓缩后利用PBS平衡好的凝胶层析Superdex200(GE)进一步纯化，将得到的蛋白经电泳，肽图，LC-MS鉴定为正确后分装备用。
实施例3.表达重组ANG-2受体Tie2的细胞系的构建和鉴定
为筛选有功能的抗体，本披露构建了表达Tie2的CHO-K1/Tie2细胞株。
将人Tie2全长基因克隆到哺乳动物细胞表达载体pBABE上，用pVSV-G，pGag-pol和pBABE-Tie2三种质粒共同转染HEK293T细胞(ATCC,CRL-3216)包装病毒，转染48小时后，收集病毒感染CHOK1细胞(ATCC,CRL-9618)。感染72小时后用10μg/mL嘌呤霉素加压筛选，待克隆团扩增生长后，消化细胞用FACS检测表达量，阳性率约40％，然后分选单克隆细胞，得到表达人Tie2的单克隆1B11。
实施例4.抗人ANG-2杂交瘤抗体的筛选和鉴定
本披露通过杂交瘤技术制备了针对人ANG-2的单克隆抗体，所得抗体与人ANG-2以较高的亲和力，并且可以与食蟹猴ANG-2有交叉反应，能够阻断ANG-2与其受体结合，且可以抑制ANG-2诱导的Tie2的磷酸化。
用重组蛋白huANG-2-Fc(100/50/50μg)，与TiterMax/Alum/CpG佐剂免疫SJL小鼠。ANG-2的特异性免疫反应通过检测血清效价的ELISA和配体受体阻断实验 来测定。选取有较好特异性免疫反应的小鼠，处死后，取脾细胞，与骨髓瘤细胞融合。
初次筛选用针对人和鼠ANG-2的ELISA结合实验，阻断人ANG-2结合其受体Tie2的实验进行筛选。当将杂交瘤细胞转移到24孔板后，用针对人和鼠ANG-2的ELISA结合实验，基于ELISA的阻断ANG-2结合其受体Tie2的受体阻断实验对其细胞上清进行复筛。筛选出的阳性克隆经过两轮亚克隆后，得到杂交瘤克隆，用于抗体生产，并通过亲和方法纯化。
筛选出活性好的单克隆杂交瘤细胞株HR54和CP33，分别收集对数生长期杂交瘤细胞，用NucleoZol(MN)提取RNA(按照试剂盒说明书步骤)，并进行反转录(PrimeScript TM Reverse Transcriptase，Takara,cat#2680A)。将反转录得到的cDNA采用鼠Ig-Primer Set(Novagen,TB326 Rev.B 0503)进行PCR扩增后送测序公司测序。经测序得到鼠源抗ANG-2抗体：HR54和CP33，其可变区氨基酸序列如下：
>HR54鼠源重链可变区序列：
Figure PCTCN2021119017-appb-000004
>HR54鼠源轻链可变区序列：
Figure PCTCN2021119017-appb-000005
>CP33鼠源重链可变区序列：
Figure PCTCN2021119017-appb-000006
>CP33鼠源轻链可变区序列：
Figure PCTCN2021119017-appb-000007
其中，划线表示CDR，斜体部分表示连接子，其余部分为FR。
根据Kabat编号规则获得的CDR区氨基酸序列如下表所示：
表2.杂交瘤克隆来源抗体重链及轻链的CDR区序列
Figure PCTCN2021119017-appb-000008
Figure PCTCN2021119017-appb-000009
将上述鼠源抗体的轻重链可变区与人源抗体的轻、重链恒定区(如SEQ ID NO：47所示的重链恒定区和如SEQ ID NO：48所示的轻链恒定区)连接后形成嵌合抗体，HR54克隆对应的嵌合抗体命名为CHR54，其他抗体类推。
实施例5.抗人ANG-2单克隆抗体的人源化
鼠源单克隆抗体人源化，可以根据本领域许多文献公示的方法进行。简言之，在所获得的鼠源抗体VH/VL CDR典型结构的基础上，从人源germline数据库中搜索轻链可变区(VL)和重链可变区(VH)的同源序列，选取FR同源性高的germline作为模板，将鼠源抗体的CDR区移植到人源模板上，并对FR区的某些氨基酸进行回复突变，将鼠源抗体的恒定区替换为人恒定区，得到最终的人源化分子。
5.1 HR54鼠源抗体的人源化
HR54的人源化VH模板为IGHV3-7*01，人源化VL的模板为IGKV4-1*01，将HR54鼠源抗体的CDR移植到人源模板上，并对框架区氨基酸进行回复突变，具体回复突变如下表：
表3.HR54抗体的回复突变
Figure PCTCN2021119017-appb-000010
注：Grafted代表鼠抗体CDR植入人种系FR区序列，N84S表示依照Kabat编号系统，将84位N突变回S。
鼠源抗体HR54人源化抗体的可变区序列如下：
>huHR54VL1(hu HR54 VL-CDR grafted)
Figure PCTCN2021119017-appb-000011
>huHR54VL2
Figure PCTCN2021119017-appb-000012
>huHR54VL3
Figure PCTCN2021119017-appb-000013
>huHR54VH1(huHR54 VH-CDR Grafted)
Figure PCTCN2021119017-appb-000014
>huHR54VH2
Figure PCTCN2021119017-appb-000015
其中，划线表示CDR，斜体部分表示连接子，其余部分为FR。
将上述轻、重链可变区与人种系轻链、重恒定区序列组合形成最终的完整轻、重链序列，进而得到全长序列的抗体。在本披露中如无明确说明时，形成全长抗体时重链可变区与SEQ ID NO：47所示恒定区连接形成抗体重链，轻链可变区与SEQ ID NO：48所示的恒定区连接形成抗体轻链链，得到的HR54的人源化抗体(huHR54)，不同huHR54抗体的重、轻链可变区见下表。
表4.huHR54抗体的可变区
可变区 huHR54VH1 huHR54VH2
huHR54VL1 huHR54-01 huHR54-04
huHR54VL2 huHR54-02 huHR54-05
huHR54VL3 huHR54-03 huHR54-06
注：huHR54-01抗体，其重链可变区为huHR54VH1、轻链可变区为huHR54VL1，重链恒定区为SEQ ID NO：47，轻链恒定区为SEQ ID NO：48，其他类推。
通过ELISA实验检测HR54人源化抗体或嵌合抗体与人ANG-2的结合活性。方法如下：
用链霉亲和素(abcam,ab123480)，浓度1ng/μL，每孔100μL，4℃过夜包被平板后，除去上清液，加250μL 5％脱脂奶粉37℃封闭1小时，洗板机洗板3遍。加0.5ng/μL生物素-hAng2-His(sinobiologican,10691-H07H)，37℃孵育1小时。洗板机洗板3遍，加100μL1:1稀释的噬菌体上清液，37℃孵育1小时。洗板机洗板3遍，每孔加100μL 1:10000稀释的抗M13-HRP(GE,27-9421-01)，37℃孵育1小时。洗板机洗板3遍，每孔加100μL TMB显色。5-10分钟后每孔加100μL 1M H 2SO 4终止显色，酶标仪测OD450值。其结果如下表所示：
表5.抗体与人ANG-2结合ELISA结果
抗体 EC50(nM) 抗体 EC50(nM)
huHR54-01 0.16 huHR54-04 0.27
huHR54-02 0.23 huHR54-05 0.27
huHR54-03 0.27 huHR54-06 0.32
ChHR54 0.33    
结果显示，本披露中的HR54嵌合抗体和人源化抗体均可与人ANG-2特异性结合。
5.2 huHR54人源化抗体的改造
对huHR54-04抗体的HCDR2进行D 52AE或D53N突变，对HCDR3进行D95N、E96Q、C100S或C100V突变，突变后得到的huHR54-04突变体的CDR序列如下：
表6.huHR54突变体的CDR序列
Figure PCTCN2021119017-appb-000016
Figure PCTCN2021119017-appb-000017
Figure PCTCN2021119017-appb-000018
示例性的，huR54的突变后的重链可变区如下：
>huHR54VH2a
Figure PCTCN2021119017-appb-000019
>huHR54VH2b
Figure PCTCN2021119017-appb-000020
>huHR54VH2c
Figure PCTCN2021119017-appb-000021
>huHR54VH2d
Figure PCTCN2021119017-appb-000022
>huHR54VH2e
Figure PCTCN2021119017-appb-000023
>huHR54VH2f
Figure PCTCN2021119017-appb-000024
>huHR54VH2g
Figure PCTCN2021119017-appb-000025
Figure PCTCN2021119017-appb-000026
>huHR54VH2k
Figure PCTCN2021119017-appb-000027
>huHR54VH2m
Figure PCTCN2021119017-appb-000028
>huHR54VH2n
Figure PCTCN2021119017-appb-000029
其中，划线表示CDR，斜体部分表示连接子，其余部分为FR。
将上述huHR54-04突变体的重链可变区与重链恒定区组合形成完整重链序列，再与huHR54-04人源化抗体的轻链重组，进而得到全长抗体。在本披露中如无明确说明时，形成全长抗体时重链可变区与SEQ ID NO：47所示恒定区连接形成抗体重链，轻链可变区与SEQ ID NO：48所示的恒定区连接形成抗体轻链，得到的人源化抗体的重、轻链可变区组合见下表。
表7.huHR54a抗体的可变区组合
突变体VH huHR54VL1
HuHR54VH2a huHR54-07
HuHR54VH2b huHR54-08
HuHR54VH2c huHR54-09
HuHR54VH2d huHR54-10
HuHR54VH2e huHR54-11
HuHR54VH2f huHR54-12
HuHR54VH2g huHR54-13
HuHR54VH2k huHR54-14
HuHR54VH2m huHR54-15
HuHR54VH2n huHR54-16
注：huHR54-07抗体，其重链可变区为huHR54VH2a、轻链可变区为huHR54VL1，重链恒定区为SEQ ID NO：47，轻链恒定区为SEQ ID NO：48，其 他类推。
通过ELISA实验检测huHR54抗体突变体与人ANG-2的结合活性，其结果如下表所示：
表8.huHR54抗体突变体与人ANG-2的结合活性
抗体 EC50(nM)
huHR54-07 0.1996
huHR54-08 0.1558
huHR54-09 0.2225
huHR54-10 0.1589
huHR54-11 0.141
huHR54-12 0.1593
huHR54-13 0.1756
huHR54-14 0.2165
huHR54-15 0.1546
huHR54-16 0.2912
huHR54-04 0.2247
结果显示，在huHR54抗体的HCDR2，HCDR3上的点突变，并不会影响huHR54抗体与人ANG-2的结合活性。
huHR54人源化抗体的CDR区可如下表：
表9.huHR54人源化抗体的CDR区
Figure PCTCN2021119017-appb-000030
其中X 1为D或E，X 2为D或N，X 3为D或N，X 4为E或Q，X 5为C，S或V。
5.3 CP33鼠源抗体的人源化
CP33的人源化VH模板为IGHV3-7*01，VL的模板为IGKV4-1*01，将CP33的CDR移植到人源模板上，并对框架区氨基酸进行回复突变，具体回复突变见下表：
表10.CP33人源化抗体的回复突变
Figure PCTCN2021119017-appb-000031
Figure PCTCN2021119017-appb-000032
注：Grafted代表鼠抗体CDR植入人种系FR区序列，N84S表示依照Kabat编号系统，将84位N突变回S。
鼠源抗体CP33人源化抗体可变区序列如下：
>huCP33VL1(hu CP33 VL-CDR grafted)
Figure PCTCN2021119017-appb-000033
>huCP33VL2
Figure PCTCN2021119017-appb-000034
>huCP33VL3
Figure PCTCN2021119017-appb-000035
>huCP33VL4
Figure PCTCN2021119017-appb-000036
>huCP33VH1(huCP33VH-CDR Grafted)
Figure PCTCN2021119017-appb-000037
>huCP33VH2
Figure PCTCN2021119017-appb-000038
Figure PCTCN2021119017-appb-000039
>huCP33VH3
Figure PCTCN2021119017-appb-000040
其中，划线表示CDR，斜体部分表示连接子，其余部分为FR。
将上述轻、重链可变区与人轻链、重恒定区序列组合形成最终的完整轻、重链序列，进而得到全长序列的抗体。在本披露中如无明确说明时，形成全长抗体时重链可变区与SEQ ID NO：47所示恒定区连接形成抗体重链，轻链可变区与SEQ ID NO：48所示的恒定区连接形成抗体轻链链，得到的CP33的人源化抗体(huCP33)，不同huCP33抗体的重、轻链可变区见下表。
表11.huCP33抗体可变区
可变区 huCP33VH1 huCP33VH2 huCP33VH3
huCP33VL1 huCP33-01 huCP33-05 huCP33-09
huCP33VL2 huCP33-02 huCP33-06 huCP33-10
huCP33VL3 huCP33-03 huCP33-07 huCP33-11
huCP33VL3 huCP33-04 huCP33-08 huCP33-12
注：huCP33-01抗体，其重链可变区为huCP33VH1、轻链可变区为huCP33VL1，重链恒定区为SEQ ID NO：47，轻链恒定区为SEQ ID NO：48，其他类推。
通过ELISA实验检测huCP33抗体及嵌合抗体与人ANG-2的结合活性，其结果如下表所示：
表12.ANG-2抗体与人ANG-2结合ELISA检测结果
抗体 EC50(nM) 抗体 EC50(nM) 抗体 EC50(nM)
huCP33-01 0.17 huCP33-05 0.21 huCP33-09 0.22
huCP33-02 0.23 huCP33-06 0.32 huCP33-10 0.30
huCP33-03 0.21 huCP33-07 0.25 huCP33-11 0.30
huCP33-04 0.22 huCP33-08 0.36 huCP33-12 0.24
ChCP33 0.47        
结果显示，CP33人源化抗体与人ANG-2具有亲和力与ChCP33相当，均可特异性识别人ANG-2。
5.4.抗ANG-2抗体的恒定区及全长抗体序列
示例性的抗体的抗体的重、轻链恒定区如下：
重链恒定区：
Figure PCTCN2021119017-appb-000041
Figure PCTCN2021119017-appb-000042
轻链恒定区：
Figure PCTCN2021119017-appb-000043
示例性的huHR54全长抗体和huCP33全长抗体的序列如下：(a)huHR54-01抗体
huHR54-01重链：
Figure PCTCN2021119017-appb-000044
huHR54-01轻链：
Figure PCTCN2021119017-appb-000045
(b)huHR54-04抗体
huHR54-04重链：
Figure PCTCN2021119017-appb-000046
Figure PCTCN2021119017-appb-000047
huHR54-04轻链：
Figure PCTCN2021119017-appb-000048
(c)huHR54-12抗体
huHR54-12重链：
Figure PCTCN2021119017-appb-000049
huHR54-12轻链：
Figure PCTCN2021119017-appb-000050
(d)huCP33-01抗体
huCP33-01抗体重链：
Figure PCTCN2021119017-appb-000051
Figure PCTCN2021119017-appb-000052
huCP33-01抗体轻链：
Figure PCTCN2021119017-appb-000053
(e)huCP33-05抗体
huCP33-05重链：
Figure PCTCN2021119017-appb-000054
huCP33-05轻链：
Figure PCTCN2021119017-appb-000055
其中，划线表示CDR，其余部分为FR，点划线部分为恒定区。
实施例6.抗VEGF/ANG-2双特异性抗体的制备及鉴定
本披露构建了针对VEGF和ANG-2的不同形式的双特异性抗体，包括但不限于IgG-scFv、DVD和crosmab形式。
抗VEGF抗体可以是目前已知的针对VEGF的任何抗体，比如阿瓦斯汀(Avastin)、RAZUMAB(Axxiom Inc)、GNR-011(Affitech A/S)、R-TPR-024(Reliance Life Sciences Grou)、雷莫芦单抗(ramucirumab，ImClone Systems)等。
示例性的抗体为基因泰克已上市Fab抗体雷珠单抗(Lucentis)，其轻链可变区序列如SEQ ID NO：56所示(参见WO1998045332或CAS Registry Number:347396-82-1)。抗VEGF抗体的重链是将雷珠单抗的重链可变区(序列如SEQ ID NO：58所示)与不同的人IgG1恒定区组合成完整IgG1重链。其具体序列如下：
(1)雷珠单抗的轻链可变区
Figure PCTCN2021119017-appb-000056
(2)雷珠单抗的轻链
Figure PCTCN2021119017-appb-000057
(3)雷珠单抗的重链可变区
Figure PCTCN2021119017-appb-000058
(4)雷珠单抗的重链可变区+IgG1恒定区(本文称为雷珠单抗重链1)
Figure PCTCN2021119017-appb-000059
(5)雷珠单抗的重链可变区+IgG1恒定区变体(本文称为雷珠单抗重链2)
Figure PCTCN2021119017-appb-000060
Figure PCTCN2021119017-appb-000061
其中，划线表示CDR，斜体部分表示连接子，其余部分为FR。
表13.雷珠单抗CDR区序列
Figure PCTCN2021119017-appb-000062
为了制备IgG-scFv构型的双特异性抗体，将筛选得到的抗ANG-2抗体的重链可变区和轻链可变区通过连接子连接，得到抗ANG-2单链抗体。其中，连接子可以是本领域公知任何连接子，示例性的连接子为(GGGGS)n，其中n为1-10中的整数。示例性的ANG-2单链抗体序列如下所示：
huHR54-01-scFv
Figure PCTCN2021119017-appb-000063
huHR54-04-scFv
Figure PCTCN2021119017-appb-000064
huHR54-12-scFv：
Figure PCTCN2021119017-appb-000065
Figure PCTCN2021119017-appb-000066
huCP33-01-scFv：
Figure PCTCN2021119017-appb-000067
huCP33-05-scFv：
Figure PCTCN2021119017-appb-000068
其中，划线表示CDR，斜体部分表示连接子，其余部分为FR。
此外，在抗ANG-2的单链抗体的重、轻链可变区中引入半胱氨酸突变，示例性的序列如下：
huHR54VH2-C
Figure PCTCN2021119017-appb-000069
huHR54VH2f-C
Figure PCTCN2021119017-appb-000070
huHR54VL1-C
Figure PCTCN2021119017-appb-000071
huCP33VH2-C
Figure PCTCN2021119017-appb-000072
huCP33VL1-C
Figure PCTCN2021119017-appb-000073
其中，划线表示CDR，其余部分为FR。
示例性的，抗ANG-2单链抗体的序列如下：
huHR54-04-scFv-CC:
Figure PCTCN2021119017-appb-000074
huHR54-12-scFv-CC
Figure PCTCN2021119017-appb-000075
huCP33-05-scFv-CC
Figure PCTCN2021119017-appb-000076
其中，划线表示CDR，斜体部分表示连接子，其余部分为FR。
通过基因重组技术将抗ANG-2单链抗体直接地(通过肽键)或间接地(通过接头)连接至抗VEGF抗体的重链或轻链，通过293表达系统进行表达，得到双 特异性抗体。
示例性的，可将抗ANG-2单链抗体的N-端通过接头(如(GG)n，其中n为1-20)连接至抗VEGF抗体的重链C-端，得到双特异性抗体，其序列如下所示：
(1)双特异抗体1
>双特异性抗体1第一条链：
Figure PCTCN2021119017-appb-000077
其中，波浪下划线部分表示抗VEGF抗体的HCDR，点下划线表示恒定区，双下划线表示连接子，斜体部分表示ANG2-scFv，划线部分表示ANG2-scFv的CDR。
>双特异抗体1第二条链：
Figure PCTCN2021119017-appb-000078
其中，点下划线表示恒定区，划线部分表示CDR。
(2)双特异性抗体2
>双特异性抗体2第一条链：
Figure PCTCN2021119017-appb-000079
Figure PCTCN2021119017-appb-000080
其中，波浪下划线部分表示抗VEGF抗体的HCDR，点下划线表示恒定区，双下划线表示连接子，斜体部分表示ANG2-scFv，划线部分表示ANG2-scFv的CDR。
>双特异性抗体2第二条链：
Figure PCTCN2021119017-appb-000081
其中，点下划线表示恒定区，划线部分表示CDR。
(3)双特异性抗体3
>双特异抗体3第一条链：
Figure PCTCN2021119017-appb-000082
Figure PCTCN2021119017-appb-000083
其中，波浪下划线部分表示抗VEGF抗体的HCDR，点下划线表示恒定区，双下划线表示连接子，斜体部分表示ANG2-scFv，划线部分表示ANG2-scFv的CDR。
>双特异抗体3第二条链：
Figure PCTCN2021119017-appb-000084
其中，点下划线表示恒定区，划线部分表示CDR。
(4)双特异性抗体4
>双特异性抗体4第一条链：
Figure PCTCN2021119017-appb-000085
其中，波浪下划线部分表示抗VEGF抗体的HCDR，点下划线表示恒定区，双下划线表示连接子，斜体部分表示ANG2-scFv，划线部分表示ANG2-scFv的CDR。
>双特异性抗体4第二条链：
Figure PCTCN2021119017-appb-000086
Figure PCTCN2021119017-appb-000087
其中，点下划线表示恒定区，划线部分表示CDR。
同时，利用筛选得到的抗ANG-2抗体和VEGF抗体，本披露构建了Crossmab形式的双特异性抗体，示例性的Crossmab形式的双特异性抗体的序列如下：
(5)双特异性抗体5
>双特异性抗体5第一重链：抗ANG-2重链
Figure PCTCN2021119017-appb-000088
>双特异性抗体5第一轻链：抗ANG-2轻链
Figure PCTCN2021119017-appb-000089
>双特异性抗体5第二重链：抗VEGF重链
Figure PCTCN2021119017-appb-000090
>双特异性抗体5第二轻链：抗VEGF轻链
Figure PCTCN2021119017-appb-000091
Figure PCTCN2021119017-appb-000092
其中，点下划线表示恒定区，划线部分表示CDR。
(6)双特异性抗体6
>双特异性抗体6第一重链：抗ANG-2重链
Figure PCTCN2021119017-appb-000093
>双特异性抗体6第一轻链：抗ANG-2轻链
Figure PCTCN2021119017-appb-000094
>双特异性抗体6第二重链：抗VEGF重链
Figure PCTCN2021119017-appb-000095
>双特异性抗体6第二轻链：抗VEGF轻链
Figure PCTCN2021119017-appb-000096
Figure PCTCN2021119017-appb-000097
其中，点下划线表示恒定区，划线部分表示CDR。
按照实施例2中的纯化方式，使用proteinA亲和层析纯化即可得到纯度>98％的双特异性抗体分子。
本披露还使用RG7716，阿瓦斯汀和RG7221(vanucizumab)作为对照分子，其序列如下所示：
>RG7716抗-ANG-2重链:
Figure PCTCN2021119017-appb-000098
>RG7716抗ANG-2轻链:
Figure PCTCN2021119017-appb-000099
>RG7716抗VEGF重链
Figure PCTCN2021119017-appb-000100
Figure PCTCN2021119017-appb-000101
>RG7716抗VEGF轻链
Figure PCTCN2021119017-appb-000102
>阿瓦斯汀重链
Figure PCTCN2021119017-appb-000103
>阿瓦斯汀轻链
Figure PCTCN2021119017-appb-000104
>RG7221抗ANG2重链
Figure PCTCN2021119017-appb-000105
>RG7221抗ANG2轻链
Figure PCTCN2021119017-appb-000106
>RG7221抗VEGF重链
Figure PCTCN2021119017-appb-000107
>RG7221抗VEGF轻链
Figure PCTCN2021119017-appb-000108
另外，本披露中使用的阴性对照(NC)是指针对HIV的一个IgG单克隆抗体。
体外活性生物学评价
测试例1.Biacore测定VEGF/ANG-2双特异性抗体与不同种属VEGF/ANG-2的亲和力
用Biacore T200(GE)测定VEGF/ANG-2双特异性抗体与人、猴和小鼠VEGF和ANG-2的亲和力。
用Protein A生物传感芯片亲和捕获抗体，然后于芯片表面流经抗原人VEGF(R&D，293-VE)，猴VEGF(sinobiological，11066)，小鼠VEGF(sinobiological,51059)，人ANG-2(sinobiological，10691-H08H)，猴ANG-2(sinobiological,90026-C07H)，小鼠ANG-2(sinobiological，50298-M07H)。用Biacore T200仪器实时检测反应信号，获得结合和解离曲线。在每个实验循环解离完成后，用10mM甘氨酸-盐酸再生溶液(pH 1.5)将生物传感芯片洗净再生。用BIA评估版本4.1， GE软件以(1:1)Langmuir模型拟合数据，得出亲和力数值，如表14所示。
表14.双特异性抗体与不同种属ANG-2的亲和力
Figure PCTCN2021119017-appb-000109
结果显示，双特异性抗体1，双特异性抗体5，双特异性抗体2和双特异性抗体3与人和猴的VEGF和ANG-2均有较高的亲和力。
测试例2.基于ELISA的抗体阻断ANG-2结合Tie2受体实验
ANG-2与血管内皮细胞表面的ANG-2受体Tie2结合，引发Tie2细胞内酪氨酸激酶发生磷酸化，继而传导信号使外周细胞从血管内皮细胞脱落，使血管处于不稳定易于增殖的状态。因此，通过抗体阻断ANG-2与Tie2的结合可以使血管更稳定，抑制新生血管的生成。本实验鉴定结果表明双特异性抗体可以阻断ANG-2结合到重组表达的Tie2蛋白胞外区。
具体方法：用Tie2-Fc(SEQ ID NO：2，3μg/mL溶于PBS，100μL/孔，)包被ELISA板，4℃包被过夜，去除包被液后，加入用PBS稀释的5％脱脂牛奶封闭液200μL/孔，37℃孵育箱孵育2小时进行封闭。封闭结束后，弃去封闭液，并用PBST缓冲液(含0.05％吐温-20的PBS，pH7.4)洗板5次后，加入50μL用1％BSA稀释生物素标记试剂盒(东仁化学，LK03)标记的huANG-2-Fc(SEQ ID NO：1，bio-huANG-2-Fc，终浓度为0.15μg/mL)和50μL待测抗体(始浓度为10μg/mL，3倍比稀释)，混匀后37℃孵育15分钟，加至ELISA板，37℃孵育1小时。孵育结束后，弃去酶标板中的反应液，用PBST洗板5次后，加入100μL/孔1:4000稀释的链霉亲和素-过氧化物酶聚合物(Sigma,S2438-250UG)于37℃孵育1小时。用PBST洗板5次后，加入100μL/孔TMB显色底物(KPL，52-00-03)，于室温孵育3-10分钟，加入100μL/孔1M H 2SO 4终止反应，用NOVOStar酶标仪在450nm处读取吸收值，计算抗体阻断ANG-2与Tie2结合的IC50值。
结果显示，双特异性抗体均可较强地抑制ANG-2与Tie2的结合(见表15和 图1)。
表15.双特异性抗体抑制ANG-2与Tie2的结合结果
Figure PCTCN2021119017-appb-000110
测试例3.双特异性抗体抑制ANG-2诱导的Tie2磷酸化
将过表达huTie2的稳转株CHO-Tie2#1B11-1消化重悬后，用完全培养基调整密度为2.5×10 5细胞/mL，100μL/孔铺入96孔细胞培养板，即2.5×10 4细胞/孔。4～5小时后换液(DME/F-12，HyClone，Cat#SH30023.01+0.1％BSA+20μg/mL嘌呤霉素，Gibco，Cat#A1113803)，饥饿过夜。4.0μg/mL，100μL/孔抗huTie2 capture(R&D Systems，Cat#DYC2720E)包板，常温过夜。去除包被液，250μL/孔1％BSA+0.05％NaN 3封闭液，常温封闭2小时。将25μL huANG-2-Fc(终浓度2.5μg/mL)和25μL待测抗体(3倍比稀释抗体，最高浓度50.0nM)等体积混匀，37℃孵育15分钟后，再加入Na 3VO 4(1.0mM，Sigma，Cat#S6508)，混匀。过夜培养的细胞，弃去50μL培养上清后，加入50μL准备好的抗原抗体混合液，37℃孵育10分钟。200μL/孔洗液(PBS+2.0mM Na3VO4)洗两次。加入90μL裂解液((1×裂解缓冲液+10μg/mL Leupeptin hemisulfate(Tocris，Cat#1167)+10.0μg/mL APROTININ，Sigma，Cat#SRE0050))冰上裂解10～15分钟，4000g离心5分钟。收集细胞裂解液，加入已封闭好的ELISA板中，常温孵育2h。PBST洗板5次，加入1：1000稀释的二抗抗PY-HRP(R&D Systems，Cat#DYC2720E)，常温孵育1～2小时。PBST洗板5次，TMB显色15～30分钟，1M H 2SO 4终止显色。用Versa Max酶标仪读取OD450，计算IC50。
结果(见图2)显示，双特异性抗体显示出很强的抑制ANG-2介导的Tie2磷酸化的能力。
表16.双特异性抗体抑制诱导的Tie2磷酸化结果
Figure PCTCN2021119017-appb-000111
测试例4.双特异性抗体抑制VEGF诱导的VEGFR的磷酸化
VEGF与血管内皮细胞上的VEGFR结合，使VEGFR胞内区生磷酸化，促进内皮细胞增殖形成新的血管，进而促进肿瘤细胞的生长和转移。本实验用来鉴定双特异性抗体可以阻止VEGF诱导的VEGFR的磷酸化。
具体为：将HUVEC细胞(PromoCell/妙通生物，C-12205)消化后用完全培 养基将细胞密度调整为每500μL中含1.5×10 5个细胞，加到24孔板中，每孔500μL。在37℃培养箱中培养过夜后，弃去培养基，用500μL冰DPBS(Gibco，14190-250)洗一遍，每孔加200μL含0.1％BSA的基本培养基饥饿培养30分钟。将待测抗体、阴性对照抗体用基本培养基稀释至10nM、1nM和0.1nM(RG7221和双特异性抗体5为20nM、2nM和0.2nM)，使用PBS作为对照(ctrl)。将VEGF(R&D system，Cat#293-VE)用基本培养基稀释至400ng/mL。取等体积稀释好的VEGF和抗体混匀后取200μL加到培养板对应的孔中，37℃孵育5分钟。将4×裂解缓冲液#1(cisbio，63ADK041PEG)用dd H 2O稀释成1×。将封闭液用1×裂解缓冲液稀释100倍配制成裂解液。取出细胞培养板，弃去细胞培养板中的培养基，加入冰PBS 500μL，轻微晃动后，弃去PBS。立即加入50μL配制好的裂解液，放在振荡器上，室温孵育30分钟。2400g离心10分钟，收集上清。使用Phospho-VEGFR2(Tyr1175)试剂盒(cisbio，63ADK041PEG)检测上清中p-VEGFR。检测方法为取10μL磷酸-VEGFR2(Tyr1175)d2抗体，加入200μL检测缓冲液，配制成工作液。取10μL磷酸-VEGFR2(Tyr1175)Cryptate抗体，加入200μL检测缓冲液，配制成工作液。将d2抗体工作液与Cryptate抗体工作液等体积混合，在HTRF96孔微孔板中，加入16μL细胞裂解液，加入4μL d2抗体与Cryptate抗体混合液，用封板膜封好，微孔板离心机离心1分钟，室温避光孵育4-24小时，用PHERAstar多功能酶标仪读取340nm波长激发，665nm和620nm波长发射的荧光值。
数据处理：比＝信号665nm/信号620nm×10000，用Graphpad Prism 5绘制柱状图。结果如图3所示。
结果表明，双特异性抗体可以显著抑制VEGF引起的HUVEC胞内磷酸化VEGFR水平升高。
测试例5.双特异性抗体抑制VEGF诱导的HUVEC的增殖
VEGF与HUVEC上的VEGFR结合，使VEGFR胞内区发生磷酸化，促进HUVEC增殖，本实验用来鉴定双特异性抗体可以阻止VEGF诱导的HUVEC的增殖。
具体方法如下：
将对数生长期的HUVEC细胞用0.08％胰酶消化，室温大约1-2分钟，加10％FBS终止。收集上述消化后的HUVEC，800rpm/分钟，离心5分钟，用PBS洗三遍除去培养基中刺激HUVEC增殖的细胞因子(800rpm/分钟，离心5分钟)。将HUVEC细胞重悬于6％FBS培养基中，细胞计数后，按照4000细胞/50μL/孔接种在白色96孔细胞培养板中，培养箱中培养2小时。将VEGF起始浓度调整为300ng/mL，120μL/孔加入无菌96孔板中。将待测抗体梯度稀释：抗体起始浓度为600nM，4倍比进行梯度稀释，将稀释好的抗体等体积加入上述96孔板中，室温孵育30min。将孵育好的抗体、VEGF混合物100μL/孔加入贴壁后的HUVEC细胞 中，培养箱中培养5天，培养结束后加入
Figure PCTCN2021119017-appb-000112
(G7573,PROMEGA)，50μL/孔，室温避光孵育10min，用Cytation5细胞成像仪Luminescence程序检测。结果如图4所示。
结果显示，双特异性抗体可以显著抑制VEGF引起的HUVEC的增殖。
表17.双特异性抗体抑制HUVEC的增殖结果
Figure PCTCN2021119017-appb-000113
体内活性生物学评价
测试例6：双特异性抗体在前列腺癌细胞小鼠皮下移植瘤模型上的药效
将5×10 6PC-3细胞(ATCC)接种于Balb/c裸鼠右肋部皮下，当荷瘤小鼠肿瘤体积达到100mm 3左右时，将小鼠分别随机分为6组，每组8只。将分组当天定义第0天，分组当日开始腹腔注射等摩尔量的各抗体，每周2次，共给药6次。每周2次监测肿瘤体积、动物重量并记录数据。当肿瘤体积超过1000mm 3或多数肿瘤出现破溃或体重下降20％时，将荷瘤动物进行安乐死作为实验终点。所有数据使用Excel和GraphPad Prism 5软件进行作图及统计分析。
肿瘤体积(V)计算公式为：V＝1/2×a×b 2，其中a、b分别表示长、宽。
相对肿瘤增殖率T/C(％)＝(T-T0)/(C-C0)×100，其中T、C为实验结束时治疗组和对照组的肿瘤体积；T0、C0为实验开始时的肿瘤体积。
抑瘤率TGI(％)＝1-T/C(％)。
分组情况和给药方案如表18所示，肿瘤生长曲线和抑瘤率见图5和表19。
表18.分组、给药方案
组别 受试物 剂量(mg/kg) 给药方案
1 PBS(溶媒) 3 i.p.BIW*3周
2 阿瓦斯汀 3 i.p.BIW*3周
3 RG7221 6 i.p.BIW*3周
4 双特异性抗体5 6 i.p.BIW*3周
5 双特异性抗体2 3.75 i.p.BIW*3周
表19.抑瘤率结果
抗体 抑瘤率(％)
溶媒 /
阿瓦斯汀 8
RG7221 23
双特异性抗体5 51
双特异性抗体2 27
结果显示，本披露中的双特异性抗体5和双特异性抗体2均可显著抑制PC-3肿瘤的生长。双特异性抗体5的抑瘤效果显著优于与阿瓦斯汀单抗，以及RG7221双抗。
测试例7双特异性抗体在高转移非小细胞肺癌H460-Luc细胞株BALB/c裸鼠皮下移植瘤模型上的药效
本实验评价ANG-2/VEGF双特异抗体以腹腔注射给药后抑制人非小细胞肺癌H460移植瘤生长和转移的效果。
BALB/c裸鼠雌性，4-5周，18-20克，购自上海灵畅生物科技有限公司。人非小细胞肺癌H460-Luc(稳定转染荧光素酶基因)细胞在添加10％FBS的RPMI 1640培养基中培养。细胞连续培养5代，接种于小鼠皮下。接种前用3-4％异氟烷将小鼠麻醉。将约1×10 6个H460-Luc细胞重悬于无血清培养基和基质胶(Matrigel)混悬液中(培养基：Matrigel＝50％：50％)，通过皮下注射接种小鼠，接种体积为200μL。当肿瘤生长到平均约100-150mm 3左右时，随机分成3组，每组8只。分组给药当天定义为第0天。分组情况和给药方案如表20所示。
表20.分组、给药方案及抑瘤率
Figure PCTCN2021119017-appb-000114
分组后每周测量肿瘤体积两次，连续3周。肿瘤体积(V)的计算方法如下：
V＝(长×宽 2)/2。
每只小鼠相对肿瘤体积(RTV)的计算方法是：
RTV＝Vt/V0，其中Vt为每天的测量体积，V0为治疗开始时的体积。
实验结束时，所有荷瘤动物拍照，将所有肿瘤取出，称重并拍照。
统计分析
结果将以平均值±S.E.M的方式呈现。两组间比较将用Dunnett多重比较检验进行检验。如果p<0.05则认为有统计学显著性差异。
测试结果如图6所示。
图6显示，本披露双特异性抗体1和双特异性抗体2抗体均可显著抑制肿瘤的生长。
测试例8：双特异性抗体在人皮肤癌细胞小鼠皮下移植瘤模型上的药效
将A431细胞(ATCC)2×10 6细胞/小鼠/100μL接种于Balb/c裸鼠右肋部皮下， 当荷瘤小鼠肿瘤体积达到100mm 3左右时将小鼠分别随机分为3组：载剂(PBS)、双特异性抗体2 4mpk和双特异性抗体5 6mpk，每组8只。将分组当天定义第0天，分组当日开始腹腔注射各抗体，每周2次，共给药6次，每周2次监测肿瘤体积、动物重量并记录数据。当肿瘤体积超过1000mm 3或多数肿瘤出现破溃或体重下降20％时，将荷瘤动物进行安乐死作为实验终点。所有数据使用Excel和GraphPad Prism 5软件进行作图及统计分析。肿瘤体积(V)计算公式为：
V＝1/2×a×b 2，其中a、b分别表示长、宽。
相对肿瘤增殖率T/C(％)＝(T-T0)/(C-C0)×100，其中T、C为实验结束时治疗组和对照组的肿瘤体积；T0、C0为实验开始时的肿瘤体积。
抑瘤率TGI(％)＝1-T/C(％)。
分组情况和给药方案如表21所示，肿瘤生长曲线如图7所示。
表21.分组、给药方案
组别 受试物 剂量(mg/kg) 给药方案
1 PBS 3 i.p.BIW*3周
2 双特异性抗体5 6 i.p.BIW*3周
3 双特异性抗体2 4 i.p.BIW*3周
结果见图7，显示本披露中的双特异性抗体2和双特异性抗体5均可显著抑制A431肿瘤的生长。
测试例9.双特异性抗体对激光致恒河猴脉络膜新生血管抑制功能的测试
通过眼玻璃体注射给药，检测对恒河猴激光致脉络膜新生血管渗漏和生长的影响，以验证双特异性抗体可玻璃体注射用于老年性黄斑变性(Age-related macular degeneration，AMD)等疾病的治疗。具体方法如下：
通过激光围绕恒河猴眼底黄斑中心凹光凝，诱导眼底脉络膜血管新生，建立与人类脉络膜新生血管类似的动物模型。光凝前及光凝后20天进行荧光素眼底血管造影判定造模情况，选择造模成功的16只恒河猴(四川格林豪斯生物科技有限公司，生产许可证号：SCXK(川)2014-013，实验动物质量合格证编号：No：0022202)，将恒河猴随机分为溶媒对照组、雷珠单抗-IgG1(Lucentis，96μg，2μM)组、RG7716(292μg，2μM)组、双特异性抗体3(396μg，2μM)组，共4组，每组4只猴。
光凝后21天，Lucentis组、RG7716组、双特异性抗体3组，分别按96μg、292μg、396μg/眼，双眼玻璃体注射给予50μL浓度为1.92mg/mL的Lucentis，5.84mg/mL的RG7716，7.92mg/mL的双特异性抗体3，溶媒对照组给与等体积溶剂。各组动物于给药后7、14、28天进行眼压检查、眼底彩色照相、荧光素眼底血管造影检查、光学相干断层扫描(OCT)，观察抗体对脉络膜新生血管的抑制情况。于给药后28天取房水100～200μL，分装100μL进行房水VEGF测定。于给药后29天实施安乐死后每组取3只眼球进行HE染色组织学检查。结果如下：
AMD造模
激光造模后20天，所纳入试验的16只猴双眼眼底彩色照相均可见黄斑周围各9个激光斑，眼底黄斑周围均可见有激光斑呈高荧光，明显的荧光素渗漏，且渗漏超过光斑边缘，激光造模后20天(给药前)，溶媒对照组、Lucentis组、RG7716组、双特异性抗体3组4级荧光斑点数分别为46、42、40、45个。上述改变类似临床脉络膜新生血管(Choroidal Neovascularization，CNV)改变，提示造模成功。
荧光造影检查
Lucentis组、RG7716组、双特异性抗体3组在给药后7、14、28天荧光斑面积均有一定程度的缩小，各组荧光渗漏面积改善率、荧光素渗漏面积减少量均优于溶媒对照组，各组4级荧光斑点数与溶媒对照组相比明显降低。
28天后，给药量为2μM双特异性抗体3组与给药量2μM的雷珠单抗组和给药量为2μM的RG7716组，均可降低荧光渗漏面积，并减少荧光斑点数，结果见图8A和8B。
房水VEGF
Lucentis组、RG7716组、双特异性抗体3组在给药后28天房水VEGF表达量均明显低于溶媒对照组。双特异性抗体3组房水VEGF表达量均明显低于Lucentis组和RG7716组，结果见图9。
综上所述，在本试验条件下，激光CNV模型的恒河猴经双眼玻璃体单次注射给予396μg/眼剂量的双特异性抗体3，经视网膜血管荧光造影、光学相干断层成像、房水VEGF及眼组织病理学检查，双特异性抗体3在396μg/眼剂量下对猴CNV具有明显的抑制作用。

Claims (32)

  1. 一种双特异性抗原结合分子，其包含特异性结合ANG-2的第一抗原结合域和特异性结合VEGF的第二抗原结合域，其中所述的特异性结合ANG-2的第一抗原结合域包含：
    i)重链可变区，其包含分别如SEQ ID NO：7，SEQ ID NO：38和SEQ ID NO：39所示的HCDR1，HCDR2和HCDR3，和
    轻链可变区，其包含分别如SEQ ID NO：10，SEQ ID NO：11和SEQ ID NO：12所示的LCDR1，LCDR2和LCDR3；其中：
    SEQ ID NO：38的序列为：TINX 1X 2SSYTYYPDNVKG；
    SEQ ID NO：39的序列为：X 3X 4ATGX 5FDY；
    其中，X 1为D或E，X 2为D或N，X 3为D或N，X 4为E或Q，X 5为C，S或V；
    或
    ii)重链可变区，其包含分别如SEQ ID NO：7，SEQ ID NO：13和SEQ ID NO：14所示的HCDR1，HCDR2和HCDR3，和
    轻链可变区，其包含分别如SEQ ID NO：15，SEQ ID NO：11和SEQ ID NO：16所示的LCDR1，LCDR2和LCDR3。
  2. 根据权利要求1所述的双特异性抗原结合分子，其中所述的特异性结合ANG-2的第一抗原结合域包含：
    重链可变区，其包含如SEQ ID NO：7所示的HCDR1，如SEQ ID NO：8、22或24所示的HCDR2，和如SEQ ID NO：9、23、25、26或27所示的HCDR3；和
    轻链可变区，其包含分别如SEQ ID NO：10，SEQ ID NO：11和SEQ ID NO：12所示的LCDR1，LCDR2和LCDR3。
  3. 根据权利要求2所述的双特异性抗原结合分子，其中所述的特异性结合ANG-2的第一抗原结合域包含：
    轻链可变区，其包含分别如SEQ ID NO：10，SEQ ID NO：11和SEQ ID NO：12所示的LCDR1，LCDR2和LCDR3；和
    重链可变区，其选自a)-l)中任一项所示的重链可变区：
    a)重链可变区，包含分别如SEQ ID NO：7，SEQ ID NO：22和SEQ ID NO：23所示的HCDR1，HCDR2和HCDR3；
    b)重链可变区，包含分别如SEQ ID NO：7，SEQ ID NO：24和SEQ ID NO：23所示的HCDR1，HCDR2和HCDR3；
    c)重链可变区，包含分别如SEQ ID NO：7，SEQ ID NO：8和SEQ ID NO：25所示的HCDR1，HCDR2和HCDR3；
    d)重链可变区，包含分别如SEQ ID NO：7，SEQ ID NO：8和SEQ ID NO：26所示的HCDR1，HCDR2和HCDR3；
    e)重链可变区，包含分别如SEQ ID NO：7，SEQ ID NO：22和SEQ ID NO：25所示的HCDR1，HCDR2和HCDR3；
    f)重链可变区，包含分别如SEQ ID NO：7，SEQ ID NO：22和SEQ ID NO：26所示的HCDR1，HCDR2和HCDR3；
    g)重链可变区，包含分别如SEQ ID NO：7，SEQ ID NO：24和SEQ ID NO：25所示的HCDR1，HCDR2和HCDR3；
    h)重链可变区，包含分别如SEQ ID NO：7，SEQ ID NO：24和SEQ ID NO：26所示的HCDR1，HCDR2和HCDR3；
    j)重链可变区，包含分别如SEQ ID NO：7，SEQ ID NO：8和SEQ ID NO：23所示的HCDR1，HCDR2和HCDR3；
    k)重链可变区，包含分别如SEQ ID NO：7，SEQ ID NO：22和SEQ ID NO：27所示的HCDR1，HCDR2和HCDR3；
    l)重链可变区，包含分别如SEQ ID NO：7，SEQ ID NO：8和SEQ ID NO：9所示的HCDR1，HCDR2和HCDR3。
  4. 根据权利要求1至3中任一项所述的双特异性抗原结合分子，其中所述的特异性结合ANG-2的第一抗原结合域包含：
    i)重链可变区，包含SEQ ID NO：3、20、21、28-37、72或73中任一氨基酸序列，或包含与SEQ ID NO：3、20、21、28-37、72或73中任一序列分别具有至少95％，96％，97％，98％或99％的序列同一性的氨基酸序列；和/或
    轻链可变区，包含SEQ ID NO：4、17、18、19或74的氨基酸序列，或包含与SEQ ID NO：4、17、18、19或74分别具有至少95％，96％，97％，98％或99％的序列同一性的氨基酸序列；或
    ii)重链可变区，包含SEQ ID NO：5、44、45、46或75的氨基酸序列，或包含与SEQ ID NO：5、44、45、46或75分别具有至少95％，96％，97％，98％或99％的序列同一性的氨基酸序列；和/或
    轻链可变区，包含SEQ ID NO：6、40、41、42、43或76的氨基酸序列，或包含与SEQ ID NO：6、40、41、42、43或76分别具有至少95％，96％，97％，98％或99％的序列同一性的氨基酸序列。
  5. 根据权利要求4所述的双特异性抗原结合分子，其中所述的特异性结合ANG-2的第一抗原结合域包含：
    a)重链可变区，包含SEQ ID NO：3的氨基酸序列；和轻链可变区，包含SEQ ID NO：4的氨基酸序列；
    b)重链可变区，包含SEQ ID NO：20或21的氨基酸序列；和轻链可变区，包含SEQ ID NO：17、18或19的氨基酸序列；
    c)重链可变区，包含SEQ ID NO：28-37中任一所示的氨基酸序列；和轻链可变区，包含SEQ ID NO：17的氨基酸序列；
    d)重链可变区，包含SEQ ID NO：72或73的氨基酸序列；和轻链可变区，包含SEQ ID NO：74的氨基酸序列；
    e)重链可变区，包含SEQ ID NO：5的氨基酸序列；和轻链可变区，包含SEQ ID NO：6的氨基酸序列；
    f)重链可变区，包含SEQ ID NO：44、45或46的氨基酸序列；和轻链可变区，包含SEQ ID NO：40、41、42或43的氨基酸序列；或
    g)重链可变区，包含SEQ ID NO：75的氨基酸序列；和轻链可变区，包含SEQ ID NO：76的氨基酸序列。
  6. 根据权利要求1至5中任一项所述的双特异性抗原结合分子，其中所述的特异性结合VEGF的第二抗原结合域包含：
    重链可变区，包含分别如SEQ ID NO：61，SEQ ID NO：62和SEQ ID NO：63所示的HCDR1，HCDR2和HCDR3；和
    轻链可变区，包含分别如SEQ ID NO：64，SEQ ID NO：65和SEQ ID NO：66所示的LCDR1，LCDR2和LCDR3。
  7. 根据权利要求6所述的双特异性抗原结合分子，其中所述特异性结合VEGF的第二抗原结合域包含：
    重链可变区，包含SEQ ID NO：58的氨基酸序列，和
    轻链可变区，包含SEQ ID NO：56的氨基酸序列。
  8. 根据权利要求7所述的双特异性抗原结合分子，其中所述的特异性结合人VEGF的第二抗原结合域进一步包含重链恒定区和轻链恒定区；优选地，其中所述的重链恒定区包含L234A，L235A，I253A，H310A和H435A突变。
  9. 根据权利要求1至8中任一项所述的双特异性抗原结合分子，其中所述的特异性结合VEGF的第二抗原结合域包含：
    重链，包含SEQ ID NO：59或60的氨基酸序列，或与SEQ ID NO：59或60具有至少95％，96％，97％，98％或99％的序列同一性的氨基酸序列；和
    轻链，包含SEQ ID NO：57的氨基酸序列，或与SEQ ID NO：57具有至少 95％，96％，97％，98％或99％的序列同一性的氨基酸序列。
  10. 根据权利要求9所述的双特异性抗原结合分子，其中所述的特异性结合ANG-2的第一抗原结合域为抗ANG-2单链抗体；优选地，其中所述的抗ANG-2单链抗体包含：
    a)SEQ ID NO：20、21或33的重链可变区和SEQ ID NO：17的轻链可变区；
    b)SEQ ID NO：44或45的重链可变区和SEQ ID NO：40的轻链可变区；
    c)SEQ ID NO：72或73的重链可变区和SEQ ID NO：74的轻链可变区；或
    d)SEQ ID NO：75的重链可变区和SEQ ID NO：76的轻链可变区。
  11. 根据权利要求10所述的双特异性抗原结合分子，其中所述的抗ANG-2单链抗体包含SEQ ID NO：67、68、69、70、71、77、78或79的氨基酸序列。
  12. 根据权利要求11所述的双特异性抗原结合分子，其中所述的特异性结合ANG-2的第一抗原结合域直接，或通过连接子连接至所述的特异性结合VEGF的第二抗原结合域；
    优选地，其中所述的抗ANG-2单链抗体的N端通过连接子连接至所述的特异性结合VEGF的第二抗原结合域的重链C端；
    更优选地，其中所述的连接子为(GG) n，其中n为1-20中的整数。
  13. 根据权利要求1至12中任一项所述的双特异性抗原结合分子，其包含：
    i)第一条链，包含SEQ ID NO：80、81、82或83的氨基酸序列，或与SEQ ID NO：80、81、82或83分别具有至少95％，96％，97％，98％或99％的序列同一性的氨基酸序列；和
    ii)第二条链，其包含SEQ ID NO：57的氨基酸序列，或与SEQ ID NO：57具有至少95％，96％，97％，98％或99％的序列同一性的氨基酸序列；优选地，其包含两条相同的第一条链和第二条链，其中：
    第一条链，包含SEQ ID NO：80、81、82或83的氨基酸序列；和
    第二条链，其包含SEQ ID NO：57的氨基酸序列。
  14. 根据权利要求1至8中任一项所述的双特异性抗原结合分子，其包含：
    a)特异性结合ANG-2的第一轻链和第一重链；
    b)特异性结合VEGF的第二轻链和第二重链；其中：
    恒定结构域CL和CH1相互替换；优选地，
    其中所述第一轻链的恒定结构域CL与第一重链的恒定结构域CH1相互替换。
  15. 根据权利要求14所述的双特异性抗原结合分子，其中：
    a)所述第一轻链包含SEQ ID NO：17的轻链可变区，和所述第一重链包含SEQ ID NO：33的重链可变区；或
    所述第一轻链包含SEQ ID NO：40的轻链可变区，和所述第一重链包含SEQ ID NO：45的重链可变区；
    和
    b)所述第二轻链包含SEQ ID NO：56的轻链可变区，和所述第二重链包含SEQ ID NO：58的重链可变区。
  16. 根据权利要求15所述的双特异性抗原结合分子，其中：
    a)所述的第一轻链包含SEQ ID NO：85的氨基酸序列，或与SEQ ID NO：85具有至少95％，96％，97％，98％或99％的序列同一性的氨基酸序列，和
    所述第一重链包含SEQ ID NO：84氨基酸序列，或与SEQ ID NO：84具有至少95％，96％，97％，98％或99％的序列同一性的氨基酸序列；或
    所述的第一轻链包含SEQ ID NO：88的氨基酸序列，或与SEQ ID NO：88具有至少95％，96％，97％，98％或99％的序列同一性的氨基酸序列，和
    所述第一重链包含SEQ ID NO：87氨基酸序列，或与SEQ ID NO：87具有至少95％，96％，97％，98％或99％的序列同一性的氨基酸序列；和
    b)所述的第二轻链包含SEQ ID NO：57的氨基酸序列，或与SEQ ID NO：57具有至少95％，96％，97％，98％或99％的序列同一性的氨基酸序列；和
    所述第二重链包含SEQ ID NO：86的氨基酸序列，或与SEQ ID NO：86具有至少95％，96％，97％，98％或99％的序列同一性的氨基酸序列。
  17. 一种特异性结合ANG-2的抗体，其包含：
    i)重链可变区，包含分别如SEQ ID NO：7，SEQ ID NO：38和SEQ ID NO：39所示的HCDR1，HCDR2和HCDR3，和
    轻链可变区，包含分别如SEQ ID NO：10，SEQ ID NO：11和SEQ ID NO：12所示的LCDR1，LCDR2和LCDR3；其中：
    SEQ ID NO：38的序列为：TINX 1X 2SSYTYYPDNVKG；
    SEQ ID NO：39的序列为：X 3X 4ATGX 5FDY；
    其中，X 1为D或E，X 2为D或N，X 3为D或N，X 4为E或Q，X 5为C，S或V；
    或
    ii)重链可变区，包含分别如SEQ ID NO：7，SEQ ID NO：13和SEQ ID NO：14所示的HCDR1，HCDR2和HCDR3，和
    轻链可变区，包含分别如SEQ ID NO：15，SEQ ID NO：11和SEQ ID NO： 16所示的LCDR1，LCDR2和LCDR3。
  18. 根据权利要求17所述的特异性结合ANG-2的抗体，其包含：
    重链可变区，包含如SEQ ID NO：7所示的HCDR1，如SEQ ID NO：8、22或24所示的HCDR2，和如SEQ ID NO：9、23、25、26或27所示的HCDR3；和
    轻链可变区，包含分别如SEQ ID NO：10，SEQ ID NO：11和SEQ ID NO：12所示的LCDR1，LCDR2和LCDR3。
  19. 根据权利要求18所述的特异性结合ANG-2的抗体，其包含：
    轻链可变区，包含分别如SEQ ID NO：10，SEQ ID NO：11和SEQ ID NO：12所示的LCDR1，LCDR2和LCDR3；和
    如下a)-l)中任一项所示的重链可变区：
    a)重链可变区，包含分别如SEQ ID NO：7，SEQ ID NO：22和SEQ ID NO：23所示的HCDR1，HCDR2和HCDR3；
    b)重链可变区，包含分别如SEQ ID NO：7，SEQ ID NO：24和SEQ ID NO：23所示的HCDR1，HCDR2和HCDR3；
    c)重链可变区，包含分别如SEQ ID NO：7，SEQ ID NO：8和SEQ ID NO：25所示的HCDR1，HCDR2和HCDR3；
    d)重链可变区，包含分别如SEQ ID NO：7，SEQ ID NO：8和SEQ ID NO：26所示的HCDR1，HCDR2和HCDR3；
    e)重链可变区，包含分别如SEQ ID NO：7，SEQ ID NO：22和SEQ ID NO：25所示的HCDR1，HCDR2和HCDR3；
    f)重链可变区，包含分别如SEQ ID NO：7，SEQ ID NO：22和SEQ ID NO：26所示的HCDR1，HCDR2和HCDR3；
    g)重链可变区，包含分别如SEQ ID NO：7，SEQ ID NO：24和SEQ ID NO：25所示的HCDR1，HCDR2和HCDR3；
    h)重链可变区，包含分别如SEQ ID NO：7，SEQ ID NO：24和SEQ ID NO：26所示的HCDR1，HCDR2和HCDR3；
    j)重链可变区，包含分别如SEQ ID NO：7，SEQ ID NO：8和SEQ ID NO：23所示的HCDR1，HCDR2和HCDR3；
    k)重链可变区，包含分别如SEQ ID NO：7，SEQ ID NO：22和SEQ ID NO：27所示的HCDR1，HCDR2和HCDR3；和
    l)重链可变区，包含分别如SEQ ID NO：7，SEQ ID NO：8和SEQ ID NO：9所示的HCDR1，HCDR2和HCDR3。
  20. 根据权利要求17至19中任一项所述的特异性结合ANG-2的抗体，其为鼠源抗体，嵌合抗体，人源化抗体或全人抗体。
  21. 根据权利要求20所述的特异性结合ANG-2的抗体，其中所述的抗体包含框架区，其中：
    i)所述重链框架区包含选自44R、77S和84S中的一个或更多个氨基酸回复突变；和/或
    所述轻链框架区包含选自1N、43S、68A、85D和87H中的一个或更多个氨基酸回复突变；或
    ii)所述重链框架区包含选自2L、44R、74V、82AS和83K中的一个或更多个氨基酸回复突变；和/或
    所述轻链框架区包含选自1N、43S、46V、68A、85D和87H中的一个或更多个氨基酸回复突变。
  22. 根据权利要求20所述的特异性结合ANG-2的抗体，其包含：
    i)重链可变区，包含SEQ ID NO：3、20、21、28-37、72或73中任一氨基酸序列，或包含与SEQ ID NO：3、20、21、28-37、72或73中任一序列分别具有至少95％，96％，97％，98％或99％的序列同一性的氨基酸序列；和/或
    轻链可变区，包含SEQ ID NO：4、17、18、19或74的氨基酸序列，或包含与SEQ ID NO：4、17、18、19或74分别具有至少95％，96％，97％，98％或99％的序列同一性的氨基酸序列；或
    ii)重链可变区，包含SEQ ID NO：5、44、45、46或75的氨基酸序列，或包含与SEQ ID NO：5、44、45、46或75分别具有至少95％，96％，97％，98％或99％的序列同一性的氨基酸序列；和/或
    轻链可变区，包含SEQ ID NO：6、40、41、42、43或76的氨基酸序列，或包含与SEQ ID NO：6、40、41、42、43或76分别具有至少95％，96％，97％，98％或99％的序列同一性的氨基酸序列。
  23. 根据权利要求22所述的特异性结合ANG-2的抗体，其包含：
    a)重链可变区，包含SEQ ID NO：3的氨基酸序列；和轻链可变区，包含SEQ ID NO：4的氨基酸序列；
    b)重链可变区，包含SEQ ID NO：20或21的氨基酸序列；和轻链可变区，包含SEQ ID NO：17、18或19的氨基酸序列；
    c)重链可变区，包含SEQ ID NO：28-37中任一所示的氨基酸序列；和轻链可变区，包含SEQ ID NO：17的氨基酸序列；
    d)重链可变区，包含SEQ ID NO：72或73的氨基酸序列；和轻链可变区， 包含SEQ ID NO：74的氨基酸序列；
    e)重链可变区，包含SEQ ID NO：5的氨基酸序列；和轻链可变区，包含SEQ ID NO：6的氨基酸序列；
    f)重链可变区，包含SEQ ID NO：44、45或46的氨基酸序列；和轻链可变区，包含SEQ ID NO：40、41、42或43的氨基酸序列；或
    g)重链可变区，包含SEQ ID NO：75的氨基酸序列；和轻链可变区，包含SEQ ID NO：76的氨基酸序列。
  24. 根据权利要求23所述的特异性结合ANG-2的抗体，其进一步包含恒定区；优选地，其包含SEQ ID NO：47的重链恒定区和/或SEQ ID NO：48的轻链恒定区。
  25. 根据权利要求17至24中任一项所述的特异性结合ANG-2的抗体，其包含：
    a)重链，包含SEQ ID NO：49、51或52的氨基酸序列，或包含与SEQ ID NO：49、51或52分别具有至少95％，96％，97％，98％或99％的序列同一性的氨基酸序列，和/或
    轻链，包含SEQ ID NO：50的氨基酸序列，或包含与SEQ ID NO：50具有至少95％，96％，97％，98％或99％的序列同一性的氨基酸序列；或
    b)重链，包含SEQ ID NO：53或55的氨基酸序列，或包含与SEQ ID NO：53或55分别具有至少95％，96％，97％，98％或99％的序列同一性的氨基酸序列，和/或
    轻链，包含SEQ ID NO：54的氨基酸序列，或包含与SEQ ID NO：54具有至少95％，96％，97％，98％或99％的序列同一性的氨基酸序列。
  26. 一种分离的特异性结合ANG-2的抗体，其与权利要求1至16中任一项所述的双特异性抗原结合分子，或权利要求17至25中任一项所述的特异性结合ANG-2的抗体竞争结合人ANG-2。
  27. 核酸分子，其编码根据权利要求1至16中任一项所述的双特异性抗原结合分子，或权利要求17至26中任一项所述的特异性结合ANG-2的抗体。
  28. 载体，其包含根据权利要求27所述的核酸分子。
  29. 宿主细胞，其包含根据权利要求28所述的载体。
  30. 一种药物组合物，其包含治疗有效量的根据权利要求1至16中任一项所述的双特异性抗原结合分子，或根据权利要求17至26中任一项所述的特异性结合ANG-2的抗体，或根据权利要求27所述的核酸分子，以及一种或更多种药学上可接受的载体、稀释剂或赋形剂。
  31. 一种生产根据权利要求1至16中任一项所述的双特异性抗原结合分子，或根据权利要求17至26中任一项所述的特异性结合ANG-2的抗体的方法，所述方法包括培养根据权利要求29所述的宿主细胞，以表达所述双特异性抗原结合分子或特异性结合ANG-2的抗体。
  32. 一种预防或治疗癌症或血管生成性眼病的方法，所述方法包括向受试者施用治疗有效量的根据权利要求1至16中任一项所述的双特异性抗原结合分子，或根据权利要求17至26中任一项所述的特异性结合ANG-2的抗体，或根据权利要求27所述的核酸分子，或根据权利要求30所述的药物组合物；优选地，其中所述的癌症选自乳腺癌、肾上腺肿瘤、输卵管癌、鳞状细胞癌、卵巢癌、胃癌、结直肠癌、非小细胞肺癌、胆管癌、膀胱癌、胰腺癌、皮肤癌和肝癌；其中所述的血管生成性眼病选自新生血管性青光眼、年龄相关的黄斑变性(AMD)、糖尿病性黄斑水肿、角膜新血管生成、角膜移植新血管生成、角膜移植排斥、视网膜/脉络膜新血管生成、房角的新血管生成(虹膜发红)、眼部新生血管性疾病、血管再狭窄和动静脉畸形(AVM)；更优选地，其中所述的癌症或血管生成性眼病与VEGF或ANG-2相关。
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